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Proteins, peptides and peptidomimetics as active agents in implant surface functionalization
Journal Pre-proof Proteins, peptides and peptidomimetics as active agents in implant surface functionalization
Przemyslaw Jurczak, Julia Witkowska, Sylwia RodziewiczMotowidlo, Slawomir Lach PII:
S0001-8686(19)30371-9
DOI:
https://doi.org/10.1016/j.cis.2019.102083
Reference:
CIS 102083
To appear in:
Advances in Colloid and Interface Science
Revised date:
9 December 2019
Please cite this article as: P. Jurczak, J. Witkowska, S. Rodziewicz-Motowidlo, et al., Proteins, peptides and peptidomimetics as active agents in implant surface functionalization, Advances in Colloid and Interface Science(2019), https://doi.org/ 10.1016/j.cis.2019.102083
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© 2019 Published by Elsevier.
Journal Pre-proof Proteins, peptides and peptidomimetics as active agents in implant surface functionalization
Przemyslaw Jurczak1†, Julia Witkowska1, Sylwia Rodziewicz-Motowidlo1, Slawomir Lach1†* 1
Department of Biomedical Chemistry, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland. † These authors contributed equally to this work. * Corresponding author. Email: [email protected] Abstract
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The recent impact of implants on improving the human life quality has been enormous. During the past two decades we witnessed major advancements in both material and structural development of implants. They were driven mainly by the increasing patients’ demand and the need to address the major issues that come along with the initially underestimated complexity of the bone-implant interface. While both, the materials and design of implants reached a certain, balanced state, recent years brought a shift in focus towards the bone-implant interface as the weakest link in the increasing implant long-term usability. As a result, several approaches were developed. They aimed at influencing and enhancing the implant osseointegration and its proper behavior when under load and stress. With this review, we would like to discuss the recent advancements in the field of implant surface modifications, emphasizing the importance of chemical methods, focusing on proteins, peptides and peptidomimetics as promising agents for titanium surface coatings.
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functionalization
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Keywords: implants; osteointegration; osseointegration; proteins; peptides; peptidomimetics; surface
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Contents of the paper: 1.
Introduction ..........................................................................................................................................3
2.
Proteins .................................................................................................................................................4 2.1.
Elastin-like protein modifications .................................................................................................5
2.2.
Type I collagen modifications .......................................................................................................5
2.3.
Fibronectin and vitronectin modifications....................................................................................8
2.4.
Bone sialoprotein modifications ...................................................................................................9
2.5.
Growth factor modifications ...................................................................................................... 10
2.5.1.
Vascular endothelial growth factor ................................................................................... 10
2.5.2.
Bone morphogenic proteins .............................................................................................. 11
3.
2.5.2.2.
Bone morphogenic protein 7 ..................................................................................... 13
2.5.2.3.
Bone morphogenic protein 9 ..................................................................................... 13
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Bone morphogenic protein 2 ..................................................................................... 11
Albumin modifications ............................................................................................................... 14
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2.6.
2.5.2.1.
Peptides ............................................................................................................................................. 14 Integrin-binding peptide modifications ..................................................................................... 14
3.2.
Bone morphogenic protein-derived peptide modifications ...................................................... 16
3.3.
Osteogenic growth peptide modifications ................................................................................ 17
3.4.
Osteopontin-derived peptides ................................................................................................... 18
3.5.
Parathyroid hormone-derived peptides .................................................................................... 19
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3.1.
Peptidomimetics ................................................................................................................................ 20
5.
Summary ............................................................................................................................................ 22
6.
Authors contributions ........................................................................................................................ 23
7.
Declaration of interest ....................................................................................................................... 23
8.
Acknowledgements............................................................................................................................ 23
9.
References ......................................................................................................................................... 24
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10.
Tables ............................................................................................................................................. 41
11.
Graphical Abstract.......................................................................................................................... 48
12.
Figures and Associated Captions ................................................................................................... 49
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1. Introduction
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We live in times when each individual can benefit from science and technology in every aspect of her of his existence. Starting with goods and food manufacturing, followed by the computer revolution, advancements in the field of medicine and much more. From the perspective of an individual, one of the most important accomplishments of the last 50 years was the global increase of human lifespan and the life expectancy at birth – reaching the average of 71 years for those born in 2015[1]. Nevertheless, a longer life has its own burdens. Aging combined with exposure to environmental factors causes the increased risks of ageassociated diseases[2]. Alongside hypertension, diabetes, Alzheimer’s and Parkinson’s diseases, bone-related disorders are considered one of the “key conditions” associated with age. Such conditions as osteopenia and osteoporosis leave the suffering person with weakened joints and bone structure, prone to fractures associated with an increased mortality rate[3]. The most common way of dealing with these issues are implantation procedures. In 2010, the amount of knee and hip replacement surgeries performed in the United States surpassed 1 million[4,5], with further predictions of a 7-fold increase for hip and 2-fold increase for knee related primary procedures in 2030[6]. The data presented by the OECD[7] clearly shows the hip and knee related surgery trends (Figure 1), with a 30 % increase of the rate of hip replacement and almost a double increase of the knee replacement surgeries.
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Similarly, an increase in the number of the procedures is also expected in Europe[8]. While the numbers state that implant surgeries are common and suggest that the demand for them will grow, doctors and patients are facing an important issue – incomplete post-implantation recovery. Despite its outstanding regenerative abilities, the bone tissue does not always heal completely, preventing successful fixation of the implant and eventually leading to implant failure and revision surgeries[9], adding in the end to the economic burden[10,11].
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Several factors govern the success of an implant fixation procedure. They involve patient’s profile (e.g. age, health, activity habits), characteristics of the implantation site (e.g. localization, vascularization, possible infection), properties of the implant and the surgical procedure alone[12] (Figure 2).
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In general, the aseptic loosening of an implant and bacterial infections are being highlighted as the main two reasons for implant failure[13–15]. The high predisposition for infections in post-implant patients is caused by lowered immune system efficacy around the implant area and the ability of bacteria to adhere to solid substrates[16]. Once present, bacteria quickly multiply and cover the implant surface with a biofilm, which is highly resistant to the immune system actions or conventional antibiotics (e.g. Staphylococcus aureus or Staphylococcus epidermidis). The possibility of spreading of the infection to other tissues contributes significantly to poor clinical outcomes of implant associated procedures[17]. Currently, the most promising strategies tackling the problem of implant related infections have been reported and reviewed by Mas-Moruno et al[18]. Aseptic loosening[19,20] is associated with a poor level of connection between the periimplant bone and the implant surface. Establishing a connection between the bone and the implant, also known as osseointegration (or osteointegration)[21], lies at the foundation of long-term implant usage. The success of osseointegration is complimented by the mechanical interlink between the implant and the bone. By extension, it depends on the quality and quantity of interactions on the bone-implant interface[22]. Currently there exists a variety of materials used for implant manufacturing with titanium and its alloys being the most 3
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recognizable and the most widely used[23,24]. The options such as magnesium[25,26] or zirconium are actively explored[27,28]. But it is not just the type of material and its general physical properties/biocompatibility that matter, it is the surface characteristics, that add greatly to the processes happening at the bone-implant interface. It has been established that wettability[29], surface chemistry[30], roughness and presence of nanostructures[31] and other functionalities[32], each to a different extent, determine if and how the host cells will bind to the implant. Based on the increasing amount of information on the processes governing bone tissue formation, multitude of passive/active coatings were developed with the aim to either act as preventive measure against bacteria or to improve osseointegration, and ideally, exhibit overlap of these two effects. Several excellent reviews on these topics are available[18,33,34].
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We would like to devote this review to proteins, peptides and peptidomimetics as molecules for surface modifications, emphasizing their role in the strategies aimed towards mitigating issues present in today’s dental and orthopaedical implantology. In our understanding, the development of these three classes of molecules will have the major impact on modern implantology. Since modifications of implant surfaces eliciting antibacterial properties are thoroughly reviewed in literature[35–39], in this review we will focus exclusively on implant surface medications improving osseointegration processes.
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2. Proteins
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The use of proteins was one the first steps towards solving the problems with osseointegration in implantology. Compared to solutions described later in this text (the use of peptides and peptidomimetics) the use of proteins as implant coatings should, at least theoretically, have given a significantly higher probability of successful bone-implant integration. This was due to (i) a high level of recognition of the protein-covered implant surface as a “host-type” tissue – a biomimetic factor and (ii), incorporation of specific biological cues that can further alter the response of the bone tissue around the implant. This way, the processes of attaching the new bone cells to the implant and promoting the process of bone regeneration (increased pace of bone cells growth and differentiation) were facilitated. Extensive research has been dedicated to proteins used for implant surface modifications. However, this rather small group of studied molecules was limited to genetically engineered proteins (e.g. elastin-like protein), type I collagen, fibronectin, vitronectin, bone sialoprotein, growth factors (e.g. vascular endothelial growth factor (VEGF), BMP2, BMP7, BMP9) and albumin. This section of our review highlights the selected examples of use of the proteins in implant engineering. (Table 1).
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Elastin-like protein modifications
The elastin-like protein (ELP) is a flexible, biologically stable synthetic biopolymer[63]. Its structure was designed based on tropoelastin, a basic building block of the elastin protein – the main component of the elastic fibers occurring in a range of tissues present in the organism[64]. One of the many functions of ELP is the formation of a sturdy hydrogel scaffold which promotes the growth of human mesenchymal stem cells and facilitates bone regeneration[40].
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The ELP coatings were the subject of two major studies performed by Raphel et al.[41,65]. In the first study, the group focused on different ways of photo-processing of the diazerine modified ELP (ELP-D), the stability of the photo-crosslinked ELP-D biomaterial and the effect of the photo-crosslinked ELP-D film on the human adipose-derived stem cells (hASC)[41]. As a result, they were able to create stable 2D films and 3D scaffolds with the use of versatile photoprocessing methods. Both structures exhibited relatively high mechanic resistance. Additionally, the ELP-D also showed high cytocompatibility towards hASC cells. It caused rapid adherence and larger spread area of cells in comparison to control, thus showing a potential to be applied in tissue engineering.
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In the second study Raphel et al. used the designed ELP as a coating for titanium-based implants[65]. The ELP containing the RGD cell-adhesive sequence was covalently bound to the implant surface via UV-mediated crosslinking (Figure 3).
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Further, authors showed that the designed coating withstood the procedures simulating dental screw and hip replacement implantations. Raphel et al. inserted the dental screws into a 10 PCF polyurethane foam mimicking bone properties and press fitted rods mimicking hip prostheses into mouse cadaver femurs. The ELP coating promoted rapid adhesion of osteoblast-like cells (MG63) in comparison to a bare titanium surface. The coating promoted also faster osteogenic differentiation and mineral deposition on the implant surface – determined based on early increase in alkaline phosphatase (ALP) activity. The in vivo studies of rat femur and tibia showed an increased bone-implant contact area and interfacial strength in favor of the cell-adhesive ELP coated implants, suggesting that the ELP-RGD coating has the potential to prevent aseptic loosening of implants. Owing to its osseoinductive properties, ELP, especially in combination with the RGD peptide, seems to be a promising direction for implant surface engineering. It may potentially be one of standard components of clinically approved implant coatings, next to a range of proteins e.g. type I collagen. 2.2.
Type I collagen modifications
Type I collagen is a potentially promising molecule to be used as an implant coating. It plays a crucial role in the bone structure, where it serves as one of the structural proteins[67] and as a mediator of osteoblastic cell functions (adhesion, differentiation and extracellular matrix secretion)[68,69]. Utilized in implant coatings, it has also been shown to possess a significant influence on peri-implant bone regeneration[70,71]. The latest data on type I collagen titanium implant modifications was presented by i.e. Bae et al.[43], Ao et al.[42,45,46], Marin-Pareja et al.[44] Truc et al.[47]. In most of these cases collagen was bound to the implant
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surface covalently, via crosslinking agents[42–44] or by electrochemical deposition[47]. Additionally, Marin-Pareja et al.[44] also immobilized collagen by physical adsorption.
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Bae et al. compared two different ways of covalent immobilization of type I collagen on the titanium surface – crosslinking with gamma-irradiation and crosslinking with glutaraldehyde[43]. The coated titanium implants were studied in vitro using human mesenchymal stem cells (hMSC) and in vivo in a rat model. The in vitro test showed the highest osteogenic differentiation and osteogenesis related gene expression levels for implants crosslinked with gamma-irradiation in comparison to the glutaraldehyde crosslinked and noncoated implants. The in vivo results did not indicate significant differences between differently crosslinked surfaces, with both samples providing the same level of osseointegration. Considering the cytotoxic effects associated with the use of glutaraldehyde and other chemical reagents associated with the crosslinking procedure, crosslinking with gamma-irradiation is a more promising method for immobilizing collagen type I on titanium surfaces.
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Ao et al.[42] focused on in vitro migration of hMSC into the surface of the implant, in vivo peri-implant bone regeneration and osseointegration itself. The presented results indicate that the hMSC cells, which play an important role in osseointegration and pose multidirectional differentiation potency[72], migrated into the pores in the surface of the collagen coated titanium. This is attributed to one of the natural functions of type I collagen, its ability to form scaffolds allowing for effective attachment and migration of hMSC cells[73]. The increased cell migration on the surface of collagen coated titanium in comparison to bare titanium (in vitro studies) presented by Ao et al., correlated with the increased pace of osteoconduction and osseointegration – confirmed by in vivo studies on rabbit specimens[42]. Importantly, the increased pace of bone formation and incorporation of bone cells into the implant surface should potentially shorten the time of the integration of the implant into the bone structure and decrease the possibility of implant’s loosening.
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In another study Ao et al. presented a composite collagen/hyaluronic acid coating aimed at titanium surfaces[46]. The coating was built by covering the metal surface with several subsequent layers of type I collagen and hyaluronic acid. The osteogenic properties of this coating in vitro were verified in the previous study, showing excellent biological properties and promotion of attachment, spreading, proliferation and differentiation of hMSC cells[45]. The present work focused on in vivo studies of the coating in a rabbit model with femur condyle defect. Two different methods of the coating preparation were studied: in the first method the coating layers were bound covalently, and in the second method via physical adsorption. The results of the study showed that the covalently bound multilayer was more stable. This was critical for osteogenesis, osteoinduction, osseointegration and aseptic loosening of the coated implants. The studies performed by Marin-Pareja et al.[44] and Truc et al.[47] focused specifically on dental implants, which are typically exposed to high pressure during chewing, thus being prone to potential failure. Since dental implants require not only osseointegration but also development of necessary soft tissue covering the transmucosal section of the implant, focusing on factors responsible for tissue development is a reasonable strategy.
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Marin-Pareja et al.[44] studied the influence of the type I collagen coating on the human dermal fibroblasts (HDF), cells responsible for extracellular matrix synthesis and connective tissue formation, crucial for the wound healing process. The research focused on (i) two different methods of titanium surface treatment: acid, usually Piranha solution, etching and oxygen plasma treatment, and (ii), two different methods of immobilization of collagen on the titanium surface. It was also verified how these factors and their combinations influence collagen itself and the collagen-HDF cells interplay. As it appears, both, surface treatment and immobilization methods, influenced the stability of collagen on the implant surface. The greatest stability of the coating was achieved by covalent binding of collagen to the relatively smooth (nano-roughness 17.9±4.8 nm), plasma-treated surface which was also more biocompatible and provided better adhesion and activation of the fibroblasts. This effect was most probably caused by the fact that the increase in roughness of the surface is accompanied by the increase of its hydrophobicity/decrease of wettability[74]. Compared to plasma treatment, use of Piranha yielded a surface with much higher roughness value (144.5±5.1 nm), increased hydrophobicity and effectively reduced ability of the surface to support fibroblast attachment. Cell proliferation was not influenced by the surface treatment or the type of collagen binding. However, it was significantly increased in the presence of the collagen coating.
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The study results presented by Truc et al.[47] focused on in vitro interactions between the type I collagen coated implant and mouse fibroblast L929 cell line. Authors verified bioactive properties of the implant surface towards the cells and showed that the presence of the collagen coating increased the cell adhesion and cell spreading rate in comparison to a bare titanium implant. The coating also increased the viability and proliferation of the cells. Additionally, Truc et al.[47] optimized the method of electrochemical deposition of type I collagen on the titanium surface which became an attractive, much faster alternative to the previously used dipping and plasma-polarization methods[75,76].
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Collagen composites are an interesting group of materials. An example, collagen/calcium phosphate nanocomposites are generally considered a good choice for hard tissue engineering due to their structural and compositional similarity to mammalian bone tissue[77]: mammalian bones contain ca. 70 vol % of hydroxyapatite, with the rest being water and matrix - 90 wt. % of which is collagen. Collagen/calcium phosphate coating for titanium surfaces was presented by Neacsu et al.[48]. Additionally, to increase the potential of the composite to promote osseointegration processes the authors added Zn2+ ions to its structure, yielding Zn2+ substituted calcium phosphate. Neacsu et al. focused on the comparison of the two methods of titanium coating, namely spin coating and matrix-assisted pulsed laser evaporation (MAPLE). For both methods the results of the in vitro study showed the synergic effect of type I collagen and Zn2+ substituted calcium phosphate: the composite stimulated the mineralization process in Simulated Body Fluid (SBF). However, the MAPLE method did not produce secondary compounds that alter cell viability or normal cell cycle phases. The method also allowed to obtain high uniformity of the coating and preserved collagen structure, improving homogeneity and morphology of the surface, giving the MAPLE method an advantage over spin coating. Taking into account the synergistic effect exhibited by type I collagen towards integration of the titanium implants with both, bone[42] and soft tissue[44,47], the collagen coating
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approach towards implant surface engineering appears to be a very promising strategy, especially in the field of dental implants. 2.3.
Fibronectin and vitronectin modifications
Fibronectin and vitronectin are glycoproteins possessing numerous functions, with the most relevant, from the perspective of this review, being involvement of these proteins in cell adhesion and migration[49]. Considering their functions, these proteins are readily used in implant coatings, where they increase the implant bioactive properties and accelerate the process of bone osseointegration. Use of fibronectin is much more common, most probably due to its higher biocompatibility and more significant cell response in comparison to vitronectin[78].
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One of the studies focusing on both, fibronectin and vitronectin, as potential coatings for the titanium Ti6Al4V alloy (the most common alloy used in implant manufacture) were performed by Felgueiras et al.[78]. The proteins were adsorbed to a sodium styrene sulfonate (poly(NASS)) grafted titanium alloy surface and, as a control sample, to an ungrafted alloy. The influence of both samples on bone cells was verified with the use of mouse calvaria-derived osteoblast-like cell line (MC3T3-E1). The results show that the grafting procedure increased the level of adhesion of the MC3T3-E1 cells to the titanium surface. The increase of adhesive properties occurs due to the presence of the SO3- groups of the poly(NaSS) bound to the titanium surface[79]. Furthermore, and most importantly, the presence of the fibronectin and vitronectin adsorbed to the surface increased the adhesion level of MC3T3-E1 even further. Though, it should be stressed that the adhesion was much stronger in the presence of fibronectin in comparison to vitronectin. Similar trend was observed in the case of cell spreading and morphology, again favoring grafted surfaces with adsorbed proteins. However, the presence of vitronectin on the grafted and ungrafted surfaces caused some of the cells to exhibit rounded morphology, while the morphology on fibronectin treated surface was polygonal, the same as during the natural osteoblast maturation processes[80]. Such result may indicate a higher degree of influence of fibronectin on the early osteoblastic differentiation. An extensive study of fibronectin based coatings for titanium implants was also performed by Chang et al.[50,51]. They started with an in vitro analysis of the fibronectin-modified surface[50]. Utilizing the glow discharge plasma (GDP) allowed at the same time for (i) cleaning the implant surface, and (ii), incorporation of amine groups (-NH2) into the surface. The -NH2 groups were used in the next step as attachment points for the fibronectin molecules. The results showed that the GDP treatment of the implant surface increased the pace of the MG-63 cell line growth and proliferation in comparison to the untreated control. Further, addition of fibronectin to the GDP treated surface increased the cell viability in comparison to the GDP treatment only. These results indicate that the fibronectin grafting combined with GDP treatment modifies the properties of the titanium implant surface so that by extension, it may reduce the time of osseointegration of the implant. Therefore the authors decided to continue with a follow-up in vivo trial[51], with the intention to evaluate the bone integration efficiency of plain and GDP treated implant versus a GDP treated fibronectin grafted one. The implant stability and bone-implant integration were evaluated utilizing resonance frequency analysis (RFA), histologic evaluation and microcomputed tomography (micro-CT). The RFA results 8
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showed that the level of stability was greater in case of fibronectin coated GDP treated implant in comparison to the plain, GDP treated one, especially shortly after the implantation. In both cases, the histological analysis showed that the bone regeneration proceeded during the experiment. Maturing of the bone was faster in the case of fibronectin coated implant though. The micro-CT analysis revealed greater bone formation in the case of fibronectin coated implant 2 weeks after the implantation. All above outlined results show that the applied surface modifications increased implant stability. Additionally, coating of the implant with fibronectin provided further benefits involving shortened treatment time and fast implant provisionalization.
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The possibility of using fibronectin as implant surface modification agent was also explored by Cho et al.[81], though their approach was based on a fibronectin derived oligopeptide F20, of the following sequence PHSRNSITGTNLTPGYTITVYAVTGRGD[52], in which the main twenty amino acid long fibronectin derived fragment, in the middle, has been terminated with the RGD motif on one end, while the other end has been equipped with RGD-synergic sequence (PHSRN), required for optimal cell activity. Authors of the study hoped to achieve superior osseointegration of dental implants with the help of the F20 molecule. Its efficiency was verified by a range of different techniques, such as scanning electron microscopy (SEM), confocal laser scanning microscopy (CLASM), picogreen assay, real-time PCR and immunoblot with the focus on adhesion, proliferation and differentiation of the stromal cell line ST2. The data obtained from the study showed that the F20 coating promoted all three processes. The positive study results combined with a simple coating procedure (direct adhesion on the implant surface) showed once again that fibronectin is a promising candidate for a titanium implant coating.
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Another study which confirmed the improved cell adhesion, proliferation and spreading on the fibronectin coated surface was performed by Jian et al.[82]. This study was performed on gingival fibroblasts with fibronectin covalently attached to the micro-grooved silanized surface of the titanium implant. Authors showed that micro-grooving of the titanium surface increased the strength of the cell adhesion, with its further increase in the case when fibronectin was applied on the surface of the implant. Considering the above-described data, glycoproteins with natural functions spanning over cell adhesion, growth, differentiation and migration, e.g. fibronectin and vitronectin[83,84], have tremendous potential in titanium implant coating engineering. However, as the data[78] and frequency of application suggest, fibronectin exhibits much higher bioactive properties compared to vitronectin. 2.4.
Bone sialoprotein modifications
Bone sialoprotein is a component of mineralized tissues (e.g. bone, dentin)[54]. It is found in the bone extracellular organic matrix (ECM)[53]. This non-collagenous protein has been shown to enhance bone formation when coated on titanium implants[53] however, its primary in vivo functions are not entirely clear[54]. Here we summarize two papers regarding the bone sialoprotein as titanium implant coatings.
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The coating presented by Im et al.[85] contained the human bone sialoprotein (bone sialoprotein II) covalently attached to the silanized surface of micro-grooved titanium implant (Figure 4). Authors of the study verified the influence of micro-grooving and surface coating on human bone marrow-derived mesenchymal stem cells (MSC) by monitoring the expression of the main osteogenic transcription factors (ARF4, FRA1, RUNX2, OSX). They also monitored the differentiation and spreading of the cells with SEM. In response to the micro-grooving of the titanium surface both, differentiation and spreading of bone marrow-derived MSC increased, in comparison to the smooth titanium surface. The application of bone sialoprotein II (BSPII) coating increased the bioactive properties of the micro-grooved implant surface, significantly improving differentiation and spreading of the cells. In both cases the cells exhibited a tendency to orientate themselves perpendicularly to the surface micro-groves. In case of the transcription factor expression, the comparative analysis of time-dependent expression of ARF4, FRA1, RUNX2 and OSX coding genes, showed the greatest upregulation in favor of the micro-grooved, BSPII coated surface, in comparison to micro-grooved and smooth surfaces.
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Similarly to the above described study, Baranowski et al.[53] also tried to verify the influence of bone sialoprotein (human recombinant; BSP) coating of titanium implants on primary human osteoblasts (hOb) and L929 fibroblasts. The BSP was attached to the activated, piranha treated, titanium surface by either covalent coupling through silanization or physisorption. Even though during the first few hours after exposing the cells to the titanium surface, their proliferation was delayed, reaching normal levels after 24 hours, the covalent coupling with BSP was favored by the cells. This preference was manifested by the increase of the cell proliferation, differentiation, expression of RUNX2 (osteoblast-specific transcription factor[86]) and osteopontin (OPN; component of extracellular matrix of bone and dentin[87]) genes which influence mineralization of ECM. Additionally, the increase of the concentration of BSP in the coating positively affected the cell migration. Suggesting that coatings containing this protein show a potential to increase the pace of implant-bone integration. However, even though the bone sialoprotein exhibits increased bioactive properties, the earlier described fibronectin displays even higher protein-bone biocompatibility, especially in case of stimulating OSX gene expression[85]. Therefore, when considering the use of BSP as a coating, fibronectin should also be taken under consideration as a molecule with potentially better influence on bone cells. 2.5.
Growth factor modifications
Growth factors are one of the developing branches in engineering of implant coatings. Different growth factors showed potential to increase the early osseointegration and implantbone compatibility. Among many the most promising examples involve VEGF[58], BMP2[60], BMP7[61] and BMP9[88]. 2.5.1.
Vascular endothelial growth factor
The vascular endothelial growth factor (VEGF), a growth factor stimulating formation of blood vessels[55], was studied by Guang et al.[56] and Izquierdo-Barba et al.[89], who used VEGF based coatings and coatings combining VEGF and silicon substituted hydroxyapatite respectively. Both studies showed a positive influence of the growth factor on bone regeneration processes. 10
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The in vivo studies performed by Guang et al.[56] on rat specimens showed that VEGF promotes proliferation of osteoblasts and endothelial cells. In both cases, the levels of protein markers characteristic for processes necessary for successful bone tissue repair – osteocalcin (OCN), an osteoblast-specific protein linked to bone formation[90], and CD31, protein involved in angiogenesis processes[91], have increased in comparison to the control sample. The results of a supporting in vitro study indicate that VEGF induced also the proliferation of primary rat osteoblasts, vasculogenesis and increased the activity of ALP.
Bone morphogenic proteins
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The studies on VEGF performed by Izquierdo-Barba et al.[89] required converting the surface of a microporous titanium scaffold with silicon substituted hydroxyapatite (SiHA). Further, the modified surface was coated with VEGF to explore the possibility of the synergistic effect exhibited by the simultaneous presence of SiHA and VEGF. The growth factor was adsorbed to the surface noncovalently. At first, the in vitro studies showed that the adsorption of VEGF to SiHA coated titanium was notably higher in comparison to bare titanium. Further experiments also showed that the VEGF coating stimulates proliferation of endothelial cells regardless of the SiHA presence. This also correlates with the data presented by Guang et al.[56]. On the other hand, the pre-osteoblasts (MC3T3-E1 cells) preferred to grow on the SiHA/VEGF coated implants. To support the in vitro results, authors performed the in vivo study in an osteoporotic sheep model. They implanted the titanium scaffolds in different parts of sheep bones (proximal tibia epiphysis, medial epicondyle of femur and greater tuberosity of humerus). The histological analysis of the bone segments containing defects showed that neither of the used coatings (SiHA or VEGF) enhanced the osteogenesis separately. However, when combined, the molecules exhibited a synergic effect and increased the osteogenesis, size of trabeculae and degree of angiogenesis.
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Bone morphogenic proteins (BMPs) are a group of signaling molecules[92], originally discovered as factors promoting osteogenesis. However, later studies showed that BMPs serve important functions in formation and maintenance of such organs as cartilage, blood vessels, muscle and kidney[93,94]. Even though as many as fifteen BMPs has been discovered and studied, only BMP2 and BMP7 successfully completed clinical trials and are approved for clinical application[59]. 2.5.2.1. Bone morphogenic protein 2 The bone morphogenic protein 2 (BMP2), is responsible for regulation of many processes leading to bone formation and regeneration[94]. Therefore, it is a popular target in implant coating engineering. BMP2 has been successfully used as a separate titanium surface coating[57] and in combination with other molecules (e.g. GDF5 – one of growth factors playing a leading role in bone regeneration[95]). One of BMP2-based multicomponent coatings was designed by Yang et al.[95]. This double layer titanium implant coating contained BMP2 and GDF5 growth factors separated by heparin layer (Ti-BMP2-GDF5; Figure 5, left). The coating was designed in a manner allowing for slow release of the growth factors from the surface of the implant. It was possible due to the fact that many growth factors, BMP2 and GDF5 including, bind heparin[96,97] non-covalently[98]. The positively charged proteins in combination with high negative charge of heparin allows for 11
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their slow and sustained release[99,100] (Figure 5, right). Yang et al. verified the influence of the double coating on the bone regeneration processes in comparison to bare Ti, Ti-BMP2 and Ti-GDF5 coatings. The in vitro studies on MC3T3-E1 cells cultured on Ti-BMP2-GDF5 coating showed significantly increased proliferation and ALP activity, resulting in calcium deposition and increased expression of osteocalcin (OCN), type I collagen (COL1) and ALP mRNA in comparison to Ti, Ti-BMP2 and Ti-GDF5. The follow-up in vivo studies correlated with the in vitro data showing increase of bone formation at the bone-implant interface, indicating enhanced bone remodeling and regeneration processes especially in case of the Ti-BMP2-GDF5 coating.
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BMP2 was also used as a titanium implant coating by Böhrnsen et al.[60], however, they used a specific method of anchoring the protein to the implant surface. The method involved covering the surface of both titanium and BMP2 with oligonucleotides. The oligonucleotides were later hybridized forming a link between the protein and the implant. This method of protein-implant binding allowed for slow release of the biologically active BMP2 growth factor to the environment. The analysis of such implants placed in rat tibiae showed increased expression of ALP, however the expression of RUNX2 transcription factor, responsible for fast osteogenic differentiation, was not significantly influenced by the implant coating. The data presented by Böhrnsen et al. indicates that the BMP2 coating increases the osteogenic differentiation around titanium implants shortly after implantation (confirmed by enhanced ALP expression). The RUNX2 does not seem to be the best marker to evaluate osteogenic differentiation, due to the potentially diverse involvement of this transcription factor in cellular differentiation processes. Anchoring the BMP2 to titanium through oligonucleotides allowed to link significantly more proteins to the implant surface in comparison to samples without oligonucleotide conjugation. Comparison to the previous data[101] suggests that it is the amount of the growth factor presented on the surface of the implant, rather than the concentration of the released growth factor is responsible for the positive effect of the engineered coating on bone regeneration processes.
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Contrary to the two previously described studies, Youn et al.[57] used covalent immobilization during the BMP2-based coting preparation. The authors deposited poly glycidyl methacrylate (pGMA) on the surface of the titanium implant by means of initiated chemical vapor deposition (iCVD) and in the next step, they covalently bound BMP2 to pGMA. This method allowed to immobilize significantly higher amount of BMP2 to the titanium surface in comparison to direct adsorption of the growth factor to bare titanium. The osteogenic properties of the coating were verified against human adipose-derived stem cells (hASC). The BMP2 coating showed high biocompatibility to the cells and caused the increase of osteogenic gene expression (OCN, RUNX2, COL1), ALP activity and calcium deposition. Despite the fact that covalent binding does not allow the release of BMP2 to surrounding tissues, it seems, surprisingly, that the good osteogenic properties of the BMP2 coating designed by Youn et al. occur as a result of this firm binding of the BMP2 to the titanium surface. This indicates that the method of active agent immobilization plays a significant role in improving its osteogenic properties of the coating.
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2.5.2.2. Bone morphogenic protein 7
Bone morphogenic protein 7 (BMP7) is another example of a protein involved in bone formation processes. Similarly, to other BMPs, it plays an important role in transforming MSCs into bone tissue and cartilage. As mentioned before, BMP2 and BMP7 are the only two in the group of morphogenic proteins approved for clinical applications[59], thus gathering relatively high interest in the field of bone regeneration and implantology[102–104].
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An interesting approach to implant surface modification with BMP7 was presented by AlJarsha et al.[61]. The combination of the properties exhibited by BMP7, poly(ethyl acrylate) (PEA) and fibronectin allowed them to obtain a system enabling a dramatic decrease of BMP7 concentrations while keeping the osteogenic properties of the coated implant. Previous studies[105] show that combining the PEA and fibronectin causes unfolding of the protein and its rearrangement into specific nanonetworks. This change of protein conformation facilitated the access to the growth factor binding domains FNIII12-14. Jarsha et al. used the synergic properties of PEA and fibronectin and combined them with osteogenic potential of BMP7, which was attached to the exposed growth factor binding domains of fibronectin. The decrease of the GF concentration eliminated the problem of side effects caused by the need of high growth (GF) factor dosage (used when bare titanium is coated with the GF) and its relatively high clearance from the organism. The results of the study show that it is possible to create an environment with low amount of osteogenic factor, which will allow the promotion of bone forming cells – in this case differentiation of hMCS towards osteogenic lineage. The presented GF delivery system shows a potential for better control over the concentration of GF at the target sites.
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2.5.2.3. Bone morphogenic protein 9
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Similarly, to BMP2, bone morphogenic protein 9 (BMP9) takes part in processes leading to bone formation and regeneration. BMP9 is one of the morphogenic proteins exhibiting relatively high osteogenic potential[88], with some studies showing BMP9 exhibiting even higher osteogenic properties than BMP2[106,107]. Nevertheless, in terms of osteogenic potential, BMP9 is still one of the less explored proteins among BMPs. Souza et al.[88] performed a preliminary in vitro study, in which BMP9 was mixed into an osteogenic medium instead of being immobilized on the implant surface. MC3T3-E1 preosteoblastic cell line were seeded on the titanium discs and incubated in the BMP-containing medium. The results of this study showed that BMP9 enhanced the osteoblast differentiation, meaning that this growth factor boosts bone regeneration processes, suggesting that linking BMP9 to the surface of titanium implants may be a good direction for the development of the BMP9-based implant coatings. Considering its supposedly high osteogenic properties, the BMP9 protein may be an even better implant coating agent than the commonly studied BMP2. The described growth factors comprise a large group of proteins exhibiting various properties and influencing different types of cells (e.g. endothelial cells or osteoblasts). Even though some of them occur more commonly than the others in studies dedicated to novel implant coatings, they all seem, at least to some degree, to positively influence bone regeneration. The growth factors are often immobilized on the implant surface in a way that facilitates the factors release or are used in media in which implants are immersed (no immobilization). In both cases, enhancement of differentiation of cells grown on the implants 13
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surface is visible, however it seems that permanent immobilization of the growth factors results in better osteogenic properties[57], presenting covalent binding of growth factors to the titanium implant surface as a more favorable direction of exploration for implant coating engineering. 2.6.
Albumin modifications
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When considering materials used for implant preparation titanium is one of the most commonly used due to its high corrosion resistance, good mechanical properties and biological biocompatibility[108]. However, in recent years the concern regarding release of metal from implants increases and the process leading to the degradation of implant surface are mainly unknown[62]. Here albumin comes with help. This protein may be immobilized on the surface of titanium implants and effectively prevent corrosion even under simulated inflammatory conditions[109]. It was also proved that albumin based coatings facilitate (i) protein adsorption to, and (ii) calcium phosphate (naturally present in bone mineral and tooth enamel) accumulation on the surface of the implant[109]. To verify further, potential biomedical applications for albumin as an implant coating agent combined with extensive studies regarding cellular growth and differentiation are necessary. On the other hand, albumin coating may potentially serve as a future multifunctional system, enhancing bone regeneration and preventing implant corrosion at the same time.
3. Peptides
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Parallel to the studies on protein-implant coatings, functionalization of implant surfaces based on peptides is being developed. The peptides are often derived from proteins which exhibit osteogenic properties themselves, such as type I collagen[110] or osteopontin (OPN)[111,112]. Peptides derived from bone-related growth factors (e.g. BMPs[113,114], OGPs[115]) and peptide/protein hormones with recognized anabolic effects related to bone metabolism (e.g. parathyroid hormone; PTH)[116,117] were also suggested (Figure 6).
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Additionally, some peptides, unrelated to proteins enhancing bone regeneration, can also function as building blocks for new biomaterials. These peptides, named self-assembling (SA) peptides, spontaneously aggregate into nanofibers and create hydrogels that can be used to culture cells in three-dimensional biomaterial scaffolds[118]. The main advantages of using peptides in regenerative medicine involve (i) relatively easy synthesis, with the use of liquid- or solid-phase synthesis procedures in a cost-effective manner, and (ii), relatively small size, minimizing their immunogenic properties in comparison to proteins. In this section we focused on osteoinductive peptides, which can promote cell adhesion, proliferation, differentiation and angiogenesis. 3.1.
Integrin-binding peptide modifications
The arginylglycylaspartic acid (RGD) is the most widely studied adhesive peptide in implant engineering. This motif was identified by Ruoslahti and Pierschbacher in the early 1980s[141]. The RGD sequence was discovered in the cell binding domain of fibronectin where it functions as the minimal recognition sequence required for cell attachment[141]. RGD is often used in conjunction with other cell-adhesive molecules like integrins, cadherins or extracellular matrix 14
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proteins to mimic cell interactions and to enhance cell adhesion[142]. Although the RGD sequence alone is capable of acting as a ligand for integrins, a high-affinity recognition requires both the RGD and PHSRN sequence[143]. Since both these sequences interact with the same integrin, the spatial configuration of RGD and PHSRN is crucial to assure correct binding. Because of this, designing a peptide containing RGD and PHRSN requires a careful consideration of the length and nature of the spacer to assure their synergistic effect[144]. Here, we describe different studies performed regarding the RGD sequence. They involved covering implant surfaces with RGD only and in combinations with other peptides and indicate that immobilizing RGD on titanium and other biomaterial surfaces is a useful approach for promoting bone regeneration.
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One of the strategies of the RGD-based coating development involved the structural modification of the peptide. Heller et al. studied the differences in the osteogenic properties between linear and cyclic form of the RGD peptide (L-RGD and C-RGD respectively)[119]. Both peptides were immobilized covalently, via silanization on the titanium surface. The results showed only subtle differences between the hipbone derived human osteoblast adherence, proliferation and differentiation when cultured in vitro on L-RGD or C-RGD coated surfaces. On the other hand, when studied in vivo, C-RGD caused a significant increase of vertical bone apposition indicating faster bone regeneration, showing that not only the peptide sequence but also its structure may influence the osseointegration processes.
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Another strategy for peptide-based implant coating was presented by Pagel et al. who combined three peptides exhibiting different properties to facilitate cell adhesion[120]. The combined peptide contained a cyclic form of the RGD peptide (C-RGD), bone sialoprotein derived heparin binding FHRRIKA peptide (HBP) and a peptide containing L-3,4dihydroxylphenylalanine (DOPA), which helped to bind the construct covalently to the titanium surface. The osteogenic properties of the coating were demonstrated using human sarcoma osteogenic (SaOS-2) cells. The results of the study showed that the presence of both integrinbinding RGD and heparin-binding HBP increased spreading, proliferation, viability, and the formation of actin cytoskeleton and focal contacts of the SaOS-2 cells. The peptide-based titanium implant surface coatings were also utilized to increase the potential for cell adhesion and at the same time improve bacteriostatic and bactericidal properties of implants[121]. Such coating construct was presented by Hoyos-Nogués et al. In the first step, the titanium surface was coated with PEG to prevent bacterial adhesion (Figure 7). In the second step, two peptides have been attached to the surface: an antimicrobal peptide (AMP) LF1-11 (the first eleven residues of the N-terminal part of the lactoferrin, responsible for antibacterial properties of the protein) and the RGD peptide serving as a factor facilitatiang cell adhesion. The obtained data showed that the presented coating increased attachment and spreading of SaOS-2 cells preventing at the same time bacteria (S. sanguinis) growth on the coated surface. This strategy of titanium implant modification shows that it is possible to functionalize the implant surface in a way, that will facilitate the osseogenic processes after implantation and, at the same time, prevent bacterial growth on the boneimplant interface. 15
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Some strategies of peptide-based implant modifications require functionalization of other biomaterials (e.g. nanotubes, alginate microspheres, hydrogels, etc.) which can be attached to the titanium implant surface or utilized as implants on their own. Cao et al. showed that TiO2 nanotube layers modified with RGD via covalent binding increased the rat bone marrow stromal cells adhesion and osteogenic gene expression, in comparison to nonfunctionalized nanotubes[122]. Another study regarding the RGD peptide was performed by Evangelista et al.[123]. Here, pre-osteoblastic MC3T3 cells were immobilized within RGD-coupled alginate microspheres. The use of CLASM and transmission electron microscopy imaging allowed to determine that the RGD promotes the adhesion and differentiation of the MC3T3 cells. A slightly different approach towards supplying the active agents was undertaken by Zhu et al., who applied RGD and HAV (His-Ala-Val) peptides on hyaluronic acid (HA) hydrogels[124]. HAV is a conserved motif of type I cadherins that promotes the chondrogenesis of encapsulated human mesenchymal stem cells (hMSCs)[145]. The data obtained from the study showed that the biofunctionalized hydrogel enhances the expression of the osteogenic marker genes and promotes ALP activity, type I collagen deposition and matrix mineralization under in vitro and in vivo conditions.
Bone morphogenic protein-derived peptide modifications
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The triple helical peptide containing the GFOGER sequence, derived from the type I collagen is another, slightly less popular example of a peptide capable of binding integrins[146]. Reyes et al. studied titanium implant coatings based on GFOGER, a collagen-mimicking peptide which selectively binds the α2β1 integrin[110]. Authors verified the influence of the coating on bone marrow stromal cells, (in vitro) and on implant osseointegration processes (in vivo), in a rat cortical bone model. The data obtained from the study showed that the coating increased integrin-mediated cell adhesion and osteoblast differentiation in vitro and significantly increased osseointegration of the coated titanium implants in vivo.
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As mentioned before, the BMPs are a group of signaling molecules[92]. They represent the main osteoinductive factors involved in skeleton development, bone formation and bone remodeling in mammalian organisms. The BMPs contain two sequences essential for their proper activity, which are referred to as the “wrist” and the “knuckle” epitopes and preferentially interact with BMP type I and BMP type II receptors present on the cell surface[126]. One of the modifications of titanium implants based on the BMP2 derived “knuckle” peptide (K-peptide) was presented by Ma et al.[114]. The authors developed a peptide-based coating where they immobilized the K-peptide on the surface of TiO2 nanotubes with the use of dopamine and further applied real-time PCR, histological analysis and fluorescence microscopy to verify the osteogenic properties of the coating when implanted into New Zealand White rabbit tibiae. Obtained data shows that the K-peptide coating increased the expression level of osteogenic genes (ALP, COL1, OSX, TRAP) and promoted early stability of the implant after implantation via increased pace of bone deposition around the implant.
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Another coating based on BMP2-derrived peptide (KIPKASSVPTELSAISTL) was designed by Cai et al.[113] who combined the osteoconductive properties of hydroxyapatite and osteoinductive properties of BMP2-derrived peptide. For this purpose, the surface of titanium was covered with polydopamine film to induce the formation of hydroxyapatite. Next, the BMP2-derrived peptide was absorbed into the hydroxyapatite. The data from in vitro tests of the coating showed enhanced cellular adhesion, ALP activity and expression of osteogenic markers. The coating promoted human osteosarcoma (MG63) cell differentiation contributing to increased pace of bone growth.
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The BMP-derived peptides may also be bound to other biomaterials e.g. hydrogels which can potentially be adsorbed to the surface of titanium or other material-based implants forming an advanced coating construct. An idea of incorporating peptides into a hydrogel was applied by Suzuki et al. who originally described a twenty amino acid long sequence (NSVNSKIPKACCVPTELSAI) corresponding to the residues 68-87 within the BMP2 protein[125]. Further, extensive studies by Saito et al. showed that the peptide corresponding to residues 7392 of BMP2, with Cys78 and Cys79 substituted by serine and Met89 by threonine (KIPKASSVPTELSAISTLYL), allowed higher induction of ALP activity when compared to the 68-87 peptide (proved on the example of C3H10T1/2 murine fibroblasts)[126]. Authors also showed that the alginate gels conjugated with 73-92 peptide, prolonged ectopic calcification but contrary to BMP2 protein did not promote the formation of trabecular bone structures[127], however significantly promoted repair of rat tibial bone defects[128].
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The 73-92 BMP2 peptide was also studied in combination with the RGD sequence[129,130] and as a combination of three active peptides, including RGD and an osteopontin-derived (OPD) angiogenic peptide[131]. The properties of these peptides were studied while being either anchored to aminoalkylsilane functionalized glass surface or covalently attached to a hydrogel. All these studies revealed synergistic enhancement of osteogenic and vasculogenic differentiation of bone marrow stromal (BMS) cells on the functionalized surfaces. Many studies have shown that not only BMP2, but also BMP4, BMP6, BMP7 and BMP9derrived peptides can regulate osteogenic differentiation of MSCs and bone regeneration[147– 150]. 3.3.
Osteogenic growth peptide modifications
Osteogenic growth peptide (OGP) is a native 14-amino acid peptide (ALKRQGRTLYGFGG) with a primary structure identical to the C-terminus of the H4 histone[115]. The proteolytic cleavage of C-terminal part of OGP generates the YGFGG pentapeptide, known as OGP10–14. This pentapeptide is the minimal OGP-derived sequence which maintains full activity of the OGP. Both peptides, OGP and OGP10–14, play an important role in bone regeneration, ALP activity and matrix mineralization of osteoblast-lineage cells[115,151]. The response of MC3T3-E1 osteoblast cell to OGP and OGP10–14 was studied by More et al.[132]. The study did not focus on coating any particular implant material. Instead it aimed at monitoring of the influence of concentration of the active agent on the osteoblast behavior. 17
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Therefore, both peptides were coupled to oxidized self-assembled monolayer (SAM) to form peptide gradient surfaces. The data obtained from the study showed that OGP peptide induced enhancement of initial MC3T3-E1 cell attachment and their short-term proliferation rate at low densities. The shorter OGP10–14 also showed a potential to stimulate the proliferation of osteoblasts. Generally, the comparison with previously studied growth factors[152–155] shows that the effect of immobilization of OGP is rather short-lived.
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The application of OGP as a surface functionalization agent was studied by Chen et al. who designed an implant coating exhibiting synergic properties of the OGP peptide and fibronectin[133]. The peptide and protein were coprecipitated on the titanium coated with mineralized calcium chloride. The influence of the coating on cell behavior was verified using rat mesenchymal stem cells (rMSC). The data obtained from the study showed the highest cell attachment, proliferation and osteogenic differentiation when the OGP and fibronectin were immobilized on the titanium surface simultaneously. However, the cellular response was also observed on implants coated with each of the molecules separately, showing that the OGP peptide exhibits osteogenic properties. In another study Chen et al. utilized the coprecipitation method to incorporate the OGP peptide into the layer of calcium phosphate mineralized on the titanium surface[134]. MSC cells grown on the mineral-OGP coating construct exhibited significant increase of adhesion, proliferation and activity of ALP.
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Another OGP-based titanium coating was presented by Lai et al.[135] . Authors initially modified the titanium surface with nanotubes obtained via anodization and further immobilized the OGP peptide on the nanotubes with the use of polydopamine to verify if such surface modification exhibits osteoinductive properties. The successful immobilization of the peptide on the surface was monitored by SEM, AFM, XPS and contact angle measurements. The in vitro experiments performed on rat cranial osteoblasts showed that the OGP functionalized nanotube surface promotes the cell spreading and differentiation indicating the studies on OGP peptide as a promising direction for implant engineering. A motif containing OGP-derived sequence, called ALK (ALKRQGRTLYGF) for the purpose of the study, was also used for functionalization of the RADA16 self-assembling hydrogel peptide scaffold[136]. The results of the study showed that the ALK-functionalized scaffold significantly increased proliferation of MC3T3-E1 mouse preosteoblast in comparison to the pure RADA16 peptide scaffold. ALP activity and OCN expression were also increased. The hydrogel functionalization is one of the emerging branches in the development of biocompatible implants. Due to their properties they can be used multidirectionally, as a stand-alone implant material or as coating constructs immobilized on other implant surfaces. 3.4.
Osteopontin-derived peptides
The phosphoprotein osteopontin (OPN), together with bone sialoprotein belongs to the SIBLING (Small Integrin-Binding Ligand, N-linked Glycoprotein) family. Similarly, to all the family members the OPN is expressed in bone tissue and secreted in the extracellular matrix during the processes of osteoid formation and mineralization[156]. The data on functionalization of 18
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implant surfaces with OPN-derived peptides is rather scarce, however the osteogenic properties of the peptides cannot be denied. One of the few studies focusing on functionalization of the implant surface with OPNderived peptides was performed by Fiorellini et al.[112] Authors of the study immobilized the OPN-derived peptide on a nanocoated titanium plasma sprayed (TPS) surface of a dental implant. The osteogenic properties of the coating were verified in an in vivo study in a canine model. The histomorphometric analysis of the implants after extraction showed that the coating accelerated the early stages of osseointegration and bone healing around implants in comparison to uncoated control. This result indicates that the OPN-derived peptide-based coating may shorten the time of treatment and lower the risk of implantation failure.
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Compared to the direct implant surface coating, functionalization of hydrogels (which can be later used as surface coatings) with OPN-derived peptides seems to be more frequently performed. The cell-stimulating properties of one of the peptides were presented by Hamada et al.[111]. In their in vitro study authors found that OPN-derived peptide SVVYGLR stimulates the angiogenesis and migration of endothelial cell lines in 3D collagen gels much stronger than commonly studied VEGF. The peptide was also shown to enhance the adhesion and proliferation of MSCs through the αvβ3 integrin, while suppressing osteoclastogenesis[137]. Noteworthy, it has also been shown that the OPN-derived peptides can induce blood vessel formation in vivo, making them potentially interesting when considering, scaffolds incorporating this biomolecule[157].
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The effect of OPN-derived peptide (DVDVPDGRGDSLAYG) was also studied by Shin et al.[138]. In this study the peptide was linked to oligo(poly(ethylene glycol) fumarate) hydrogels and the authors monitored the modulation of rat MSCs on the modified hydrogel surface. The results obtained from the study showed an increase of attachment strength of the MSCs to the surface. Shin et al. tested also the peptide with the scrambled sequence and demonstrated that the RGD sequence plays the critical role in cell attachment to the biomaterial, indicating the integrin-depending binding[138]. In another work, Shin et al. cultured osteoblasts on the modified hydrogel with the same OPN-derived peptide and observed increase in the levels of ALP activity, secreted osteopontin and ECM mineralization[139]. Another fragment of human OPN was investigated by Lee et al.[140]. The amino acid sequence of the peptide corresponded to the residues 150–177 of human OPN, the region presenting the highest binding affinity to collagen. During the study the peptide was immobilized in alginate hydrogels. Human bone marrow stromal cells (hBMSCs) adhesion, spreading and proliferation on peptide-immobilized gel were significantly increased as compared with control surface. The modified alginate hydrogel induced also new bone formation in the rabbit calvaria defect model. 3.5.
Parathyroid hormone-derived peptides
Parathyroid hormone (PTH) is an 84-amino acid long hormone that regulates calcium homeostasis and plays an important role in bone remodeling[158]. The N-terminal 34 residues 19
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of PTH are responsible for most of its bioactivity[159]. This peptide is known as teriparatide and is used in the treatment of postmenopausal osteoporosis. A few studies have addressed the influence of PTH1-34 on bone regeneration.
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Yewle et al.[117] used an oxidized form of PTH1-34 covalently conjugated to hydrazine bisphosphonates (HBPs), which was used as drug delivery system. The conjugation of the peptide to HBPs increased its affinity to the bone tissue and improved its interactions with preosteoblastic (MC3T3-E1) cells. In another work, Arrighi et al. demonstrated the beneficial effects of PTH locally delivered into bone defects[116]. Authors modified fibrin matrices with PTH1-34, using a plasmin-sensitive substrate sequence as a linker. In vivo, the enzymatic cleavage of the linker results in the release of PTH1-34 in situ. These studies, performed in sheep bone defect model, showed dose dependent bone formation with the evidence of both osteoconductive and osteoinductive mechanisms.
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To our knowledge the studies on PTH-based coatings are scarce[160] and coatings of titanium implants based on PTH 1-34 were not yet designed. However, the PTH 1-34 is readily studied as a drug supplementing the post-implantation recoalescence, where it is used as an agent accelerating bone-implant integration[161,162]. The above described studies regarding the influence of the peptide bone regeneration indicate that the peptide may potentially be a promising factor to be used in implant coatings promoting osseointegration processes.
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4. Peptidomimetics
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Peptidomimetics were developed to mitigate the typical peptide related issues (e.g. susceptibility to protein degradation, poor absorption, pH and salt sensitivity or undesired effects due to interactions with several receptors) while at the same time retaining the activity and selectivity profile of the original “parent” peptide. The success of peptidomimetics is dependent on the understanding of the modus operandi of the parent peptide. This further requires tedious research which covers kinetic studies and solving crystal structures of the peptides and their complexes. Additionally, advances in bioinformatics are often utilized to simulate the active site of the peptide (ligand) while attached to the target molecule (often a receptor). These results serve as the base to design first peptidomimetics which go further through a cycle of structure-activity relationship studies and optimization to yield a compound exhibiting the best possible ratio of desired to side effects. Probably the most explored group of peptidomimetics is based on the structure of the RGD peptide[163,164], described in detail in Section 3.1, of the sequence Arg-Gly-Asp, known for its affinity towards integrins[141]. Among many integrins, the α5β1 and αvβ3 subtypes have been carefully studied and recognized as key mediators of cell adhesion, differentiation, tumor growth and angiogenesis. Several follow up studies have shed more light on their properties, placing the α5β1 subtype in a more favorable position as being responsible for induction of osteogenesis, specifically upregulation of osteogenic marker expression and the activity of ALP[165–167], and most importantly in the context of this review, osseointegration of implants[164,168,169]. The library of nonpeptidic compounds capable of affecting the mentioned integrins, α5β1 and αvβ3, has grown over the years, mostly in response to understanding of key structure-activity dependencies between the RGD peptide and the 20
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integrins[170]. Table 3 lists several recognized examples of such nonpeptidic compounds aimed towards the αvβ3 integrin. Several of these compounds exhibited additional properties, e.g. Compound 1 and 2 exhibited activity towards the αIIbβ3 receptor, though with good selectivity towards αvβ3; Compound 3 was selective against α5β1, while Compound 4 exhibited high affinity towards both αvβ3 and αIIbβ3 receptors. Compound 5, one of the most potent presented here, had only modest selectivity over the αIIbβ3 receptor and much higher when compared to the αvβ3 and α5β1 integrins. Furthermore, several of the mentioned compounds have been evaluated in order to assess their additional pharmacological properties. This was motivated by the fact that the integrin family is involved in numerous important physiological processes, e.g. αvβ3 mediated migration/proliferation of endothelial[171] or muscle[172] cells, and this renders them as therapeutic targets of increasing interest[173]. As an examples, Compound 5 (Table 3) has been evaluated towards angiogenesis and tumorigenesis[174] and it has been shown to decrease blood vessel formation in a mouse matrigel model with the value of ED50 = 0.055 µg kg-1 day-1 and also to inhibit tumor growth on the level of ca. 20%; Compound’s 7 (Table 3) effect on restenosis, the loss of blood vessel lumen, has been intensively studied[175]. It has been found to decrease the neointimal area by 51% in a pig model. While the above-mentioned examples were shown to be promising, further effort has been placed towards assuring higher affinity towards either the α5β1 or αvβ3 integrin. Smallheer et al. focused on utilizing heterocyclic moieties as means of introducing conformational constraints into the designed compounds[183]. One of such compounds (Compound 1; Table 4) has a spirocyclic scaffold incorporating an isoxazoline ring, and showed excellent affinity towards the α5β1 integrin and selectivity when compared to αvβ3 with additional effects on inhibition of angiogenesis[184], tumor cell proliferation[185] and glioblastoma cell apoptosis[186]. Availability of crystal structures followed by docking studies[187,188] led to the development of several potent peptidomimetics. In their works, Heckermann et al.[189] gave several examples of novel integrin-oriented peptidomimetics. The authors thoroughly analyzed activities of several compounds and supported their results with docking studies for optimal design of several analogs. Two of the most prominent examples of these developed compounds, presented in Table 4 (Compounds 2a and 2b) exhibit drastically different selectivity towards either the α5β1 or αvβ3 integrin, with activities in the nanomolar range. This study gives several further examples which underline the importance of structureactivity relationship studies and show how subtle changes in the structure of a compound can tremendously affect its activity. Similar process has been explored by Stragies et al.[190]. In this work, authors reported a library of compounds which activities have been evaluated with respect to the five integrins: α5β1, αvβ3, αvβ5, αIIbβ3 and α3β1. Understanding of crucial interactions followed by conformational analysis yielded a family of α5β1 antagonists (e.g. Compound 3; Table 4), with preferred selectivity over αvβ3 integrin. Compound 4 (Table 4) has been developed by Hackerman et al.[191] on the base of a tyrosine scaffold, already successfully employed against the αIIbβ3 receptor. The resulting peptidomimetic exhibits high activity towards the α5β1 integrin (IC50 = 0.86 nM) and superb selectivity over the αvβ3 integrin (IC50 = 9600 nM). Compound 5 (Table 4) is an example of benzazepine Gly-Asp mimetic, developed by Miller et al.[192], highly potent and orally active against the αvβ3 integrin (KI = 0.9 nM). Interestingly, this compound was also active in models of bone resorption and osteoporosis. An in vitro human osteoclast resorption assay[193] showed inhibition of osteoclast mediated bone resorption (IC50 = 29 nM) and inhibition of parathyroid hormone21
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stimulated calcemic response in a rat model[194] with EC50 = 35 µM. Neubauer et al.[195] prepared several compounds which comprise a β-homotyrosine, shown to be essential for selectivity against αIIbβ3 and α5β1[189], and a methoxypyridine subunit, acting as an arginine mimic, establishing interactions with (αv)-Asp218 (Figure 8). Compounds 6(a) and 6(b), the most potent examples presented in Table 4, both showed subnanomolar activity against the αvβ3 integrin (Compound 6(a): IC50 = 0.86 nM, Compound 6(b): IC50 = 0.65 nM) with low affinity towards the α5β1 receptor. Jumar et al.[196] evaluated properties of Compound 7 (Table 3), a very potent αvβ3 antagonist, with affinity IC50 = 3.2 nM, good selectivity against the α5β1 receptor and ability to inhibit fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF) induced endothelial cell proliferation in vitro.
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The examples mentioned in Tables 3 and 4 are just a fraction of many developed and evaluated peptidomimetics. While the compounds are exhibiting very high affinities and selectivities, the number of actual examples of their application as surface functionalization agents, to-date, is surprisingly small. Examples of peptidomimetics with corresponding functionalized surfaces type and properties are listed in Table 5. The first type of surfaces being functionalized with integrin oriented peptidomimetics were based on poly(ethylene terephthalate) (PET) which is extensively used in medical devices for vascular grafts and cruciate ligament prosthesis[197]. Compounds 1-3 (Table 5) are examples used to functionalize PET surface. The bioactive part of the molecule has been attached to the surface by a spacer/anchor unit. The activity of such constructs has been compared to a surface functionalized with the RGD peptide or its analogs. In the case of Compound 1, the functionalized surface had similar effect on supporting adenocarcinoma epithelial cells (CaCo2) adhesion as the control, comprising PET with RGDS peptide. Comparison to fibronectin and vitronectin coated PET showed that in both cases the adhesion results were not in favor of the peptidomimetic coated PET[198,199]. Compound 2, an αvβ3 antagonist, immobilized on PET also, similarly to Compound 1, promoted adhesion of CaCo2 cells, though performed worse compared to vitronectin[200,201]. Compound 3, very active against the αvβ3 receptor (IC50 = 0.7 nM) and selective with respect to the αIIbβ3 integrin, improved adhesion of human endothelial cells, showing better results compared to the RGDS peptide[202,203]. Functionalization of a titanium-based surfaces is showcased in the following examples. Compound 4, active against αvβ3 (IC50 = 0.72 nM), has been shown to affect adhesion of MC3T3 osteoblasts in a mouse model[204]. Compounds 5 and 6 present a very special case. Both of these compounds exhibit very good affinity and selectivity towards particular integrins: the former is active against the αvβ3 receptor and selective with respect to α5β1 while the latter shows a reversed pattern[205,206]. This allowed the simultaneous use of the compounds and so targeting both receptors at the same time. This combination showed to be very promising in cell differentiation assays and was further studied in vitro and in vivo[207].
5. Summary In this review we approached the topic of implant surface functionalization with various agents, with particular focus on proteins, peptides and peptidomimetics. We have shown that these biomolecules can be successfully immobilized at a surface and through their distinct physico-chemical properties, can change its characteristics in a desired way. While this statement is very optimistic, each stage, starting from biological recognition and understanding 22
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of the role of a given biomolecule, through synthesis or isolation, finishing with surface functionalization, was accompanied by numerous challenges and while many successes have been underlined in this review, definitely there is still a lot of work to be done. We have witnessed how large and complex proteins have been gradually substituted with peptides and finally with peptidomimetics. The initially used fibronectin and osteopontin proteins, complex and difficult to work with, have been “reduced” and their most potent part extracted in the form of peptides. These have been put to the challenge of sufficient surface functionalization while retaining the crucial activity. The most widely known, so-called, RGD peptide has long stood as the molecule of choice in surface functionalization of implants due to its outstanding affinity towards integrins[141] and ease of synthesis[208]. Understanding the fragility of such peptides, especially when they are used in harsh in vivo conditions, led to the development of peptidomimetics, for which most of the peptide-related limitations do not apply. Peptidomimetics exhibit high potency and target binding affinities, combined with excellent selectivity and stability to enzymatic degradation or temperature and pH fluctuations. Following the path of the RGD peptide, most of today’s peptidomimetics also display the same modus operandi. Among many, works published by Biltresse et al.[200], Maglot et al.[185], Rerat et al.[202], Nuebauer et al.[195] and Fraioli et al.[207] clearly underline the trends and importance of integrin targeting peptidomimetics. Further works by Mas-Moruno et al. pinpoint the status of peptidomimetics[209] and their significance as surface coating agents[18] in an excellent way.
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Given the current socio-economic factors, increasing life expectancy and prolonged activity, the development of medical implants and related technologies will be an area of increasing importance. We are already witnessing a shift towards new type of implant materials. Magnesium and its alloys[210,211], cobalt–chromium alloys[212,213], groups of ceramic[214,215] and composite materials[216] – all these options are being explored extensively and are bound to face, among many others, the problem of implant osseointegration. This paradigm shift in implant material development will require a thorough compatibility reevaluation of all the currently used coating strategies along with forging of new appropriate solutions. Therefore, we may expect that as a result of a marriage between biology, biochemistry and material engineering, the research on implant surface functionalization will intensify, and new biomolecules and targets will be developed in the near future.
6. Authors contributions SL was responsible for conceptualization, figure design and preparation. PJ and SL were responsible for data collection, original manuscript writing, review and editing. JW was responsible for original manuscript writing. SRM was responsible for revision support.
7. Declaration of interest None.
8. Acknowledgements We would like to thank Polish National Agency for Academic Exchange for the financial support within the “Polish Returns” programme (PPN/PPO/2018/1/00071/DEC/1) and National 23
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Center for Research and Development, TECHMATSTRATEG2/406384/7/NCBR/2019).
Poland
(grant
No.
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10. Tables
Table 1. Summary of proteins used for implant surface modification. In vitro study
In vivo study
Human adiposederived stem cells (hASC)
Human mesenchymal stem cells (hMSC)
Adhesion (MG63 and hasc), differentiation Femoral and tibial (hasc only), mineral deposition (from MG63 bone (rat) and hasc) in vitro ↗; bone-implant contact and interfacial strength in vivo ↗ Effective attachment and migration in vitro; Femoral bone osteoconduction and osseointegration in vivo (rabbit) ↗
hMSC
Tibial bone (rat)
Human dermal fibroblasts (HDF)
HeLa cell culture
-
MC3T3-E1 preosteoblasts
Mandibular premolar area (dog)
MG-63
ST2 stromal cell line
-
Human gingival fibroblasts (HGF)
-
Jo
Bone sialoprotein Primary human osteoblasts (hOb), L929 mouse fibroblasts
BMP2
-
Covalently via gamma irradiation or glutaraldehyde Physical adsorption, covalently via silanization
[43] [44]
Covalent binding or physical adsorption
[45,46]
[50] [51]
Proliferation, adhesion, differentiation and ALP activity ↗
Physical adsorption
[51]
Proliferation, adhesion, and differentiation ↗
Covalently via silanization
[52]
Osteogenic activity and osteoblastic differentiation ↗; TF4, FRA1, RUNX2, OSX gene expression ↗
Covalently via silanization
[53]
Cell migration, calcium deposition ↗; upregulation of RUNX2 expression
Physical adsorption, covalently via silanization
[54]
Physical adsorption
[55]
Proliferation, vasculogenesis and ALP activity in vitro ↗; proliferation of osteoblasts and endothelial cells in vivo ↗ Proliferation of endothelial cells in vitro ↗; ossification ↗, larger trabeculae, angiogenesis degree in vivo ↗
Tibial bone (rat)
-
[42]
Covalently via amine groups present after glow plasma discharge treatment
C2C12 mouse muscle myoblasts
MC3T3-E1 preosteoblasts
Covalently via silanization
Adhesion, migration, and proliferation in vitro ↗; stability of implants in vivo ↗
Femoral and tibial Proliferation ↗, bone formation and bone (rabbit) osseointegration in vivo ↗
BMP9
[41]
[49]
MC3T3-E1 preosteoblasts
-
Covalently via photoactive crosslinking
Physical adsorption
Tibial bone epiphysis (sheep)
hMSC
[40]
[48]
Endothelial cells EC2; MC3T3-E1 preosteoblasts
BMP7
Covalently via photoactive crosslinking
Via spin coating or MAPLE
Tibial bone (rat)
-
Ref.
[47]
Primary rat osteoblasts (ROB)
hASC
Immobilization method
Covalently via electrodeposition
-p
-
re
L929 mouse fibroblasts
ro
Femoral bone (rabbit)
hMSC
VEGF
Adhesion, proliferation and activation of fibroblasts ↗ Attachment spreading, proliferation and differentiating in vitro ↗; implant stability in vivo ↗ Adhesion, spreading, viability and proliferation ↗ high coating uniformity; preserved collagen structure improving homogeneity and morphology of the surface Adhesion and spreading (much stronger in case of fibronectin) ↗
-
hMSC
Fibronectin; Vitronectin
Proliferation and osteogenic differentiation in vitro ↗; fine osteointegration in vivo
of
Human bone carcinoma MG-63 line (MG-63), hASC
lP
Type I collagen
Cytocompatibility, rapid adherence, spread area ↗
-
na
ELP
Influence on cells/tissue
ur
Protein
ALP activity in vitro and in vivo ↗ Osteogenic differentiation and calcium deposition ↗; expression of OCN, RUNX2 and COL1 ↗; ALP activity ↗ Enhanced osteogenic differentiation; calcium deposition and expression of OCN and OPN ↗ Osteoblast differentiation ↗; expression of RUNX2, OSX, OC and BSP genes ↗
Noncovalent adsorption a to the surface of SiHA coated titanium surface Noncovalent electrostatic binding with heparin linking layer Hybridization with short nano-anchored oligomers Covalent binding with b pGMA as a linker
[56]
[57] [58] [59]
Covalent binding with fibronectin as a linker Protein not immobilized; cells grown on the titanium surface
[60] [61]
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Albumin
-
b
Alternating AC-EPD
c
[62]
c
ur
na
lP
re
-p
ro
of
- silicon substituted hydroxyapatite, - glycidyl methacrylate, - current electrophoretic deposition; ↗ - increase
Jo
a
-
Studies on living cells not performed; Facilitates protein adsorption and calcium phosphates accumulation ↗ on the implant surface (incubated in media simulating body fluids); coating improves also implant corrosion resistance
submerged in proteincontaining medium
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Table 2. Summary of peptides used for implant surface modification. Peptide
In vitro study
In vivo study
Hipbone-derived human osteoblast
Tibial bone (rabbit)
Human sarcoma osteogenic (SaOS-2)
-
SaOS-2
-
Rat bone marrow stromal cells
-
MC3T3 preosteoblasts
-
Influence on cells/tissue No significant increase of osseogenic properties over linear RGD in vitro; vertical bone apposition in vivo ↗ Spreading, proliferation, viability, and the formation of actin cytoskeleton and focal contacts of the SaOS-2 cells ↗ Attachment and spreading ↗
RGD
MG63
BMP268-87 BMP273-92
Calf muscle (rat) Calf muscle (rat) Calf muscle (rat)
C3H10T1/2 murine fibroblasts -
Tibial bone (rat)
-
ur
BMP273-92
Bone marrow stromal cells (BMS) Human bone marrow mesenchymal stem cells (hBMSC)
-
Jo
BMP273-92
-
BMP273-92
BMP273-92
Tibial bone (rabbit)
-
of
ro
Adhesion, RUNX2, OCN and BSP expression, ALP activity, calcium deposition in vitro ↗; Bone formation and osseointegration in vivo ↗
-p
Cortical bone (rat)
Expression of ALP, COL1, OSX, TRAP and pace of bone deposition ↗ Adhesion, ALP activity and expression of RUNX2, OPN, OC ↗ Calcification and trabecular bone formation ↗ ALP activity in vitro ↗; calcification in vivo ↗
re
Rat bone marrow stromal cells
lP
BMP274-91
Subcutaneous pockets on the back (mouse)
na
Type I collagenderived peptide (GFOGER) "Knuckle” peptide
hMSC
Adhesion and expression of BMP-2, RUNX2, BSP and OPN ↗ Adhesion, differentiation, ALP activity, OCN expression ↗ Osteogenic markers’ expression, ALP activity, type I collagen deposition and matrix mineralization in vitro and in vivo ↗
Prolonged ectopic calcification Differentiation of osteoblast precursor cells into osteoblasts, activation of osteoblasts to promote the repair of bone defects ↗ Osteogenic differentiation and mineralization ↗
-
Differentiation, expression of RUNX2 ↗
BMP273-92
Bone marrow stromal cells (BMS)
-
Differentiation, ALP activity, mineralization, expression of OPN and vasculogenic markers ↗
OGP and OGP10–14
MC3T3-E1 preosteoblasts
-
Attachment and proliferation ↗
OGP
Rat mesenchymal stem cells (rMSC)
-
Attachment, proliferation and osteogenic differentiation ↗
OGP
rMSC
-
Adhesion, proliferation and ALP activity ↗
OGP
Rat cranial osteoblasts
-
Spreading, differentiation, RUNX2, ALP, COL I, OPN, OCN expression ↗
MC3T3-E1 preosteoblasts
-
Proliferation, APL activity, OCN expression ↗
-
Mandibular premolars and first molar
Early stages of osseointegration and bone healing around implants ↗
OGPderived ALK motif OPN/OPNderived OC-1016
Immobilization method
Ref.
Covalently via silanization
[119]
Covalently via DOPA containing, mussel derived peptide Covalently via electrodeposited PEG Covalently via silanization Covalently with the use of EDC
[120]
[121] [122] [123]
Covalently via methacrylate groups
[124]
Physical adsorption
[110]
Covalently via polydopamine
[114]
Physical adsorption
[113]
Covalently with the use of EDC Covalently with the use of EDC Covalently with the use of EDC
[125] [126] [127]
Covalently with the use of EDC
[128]
Covalently via click chemistry
[129]
Covalently via silanization
[130]
Covalently via oxyme ligation and click chemistry Covalently via click chemistry Physical adsorption or coprecipitated on the titanium coated with mineralized calcium chloride Coprecipitated as in the example above Covalently via polydopamine Direct peptide coupling via Cterminus -
[131] [132]
[133]
[134] [135] [136]
[112]
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(dog)
OPN171-177
Transformed rat lung endothelial cells (TRLEC) Human periodontal ligament fibroblasts (hPLF), primary human gingival fibroblast (hGF), human umbilical vein endothelial cells (HUVEC), RAW264.7 murine macrophage-like cells, murine bone marrow monocyte/macrophage (BMM), (CHO)-K1 chinese hamster ovary cells, hMSC
OPN137-151
rMSC
OPN150–177
hBMSC
PTH1-34
MC3T3-E1 preosteoblasts
-
Calvarial bone (rat)
Calvarial bone (rabbit)
Angiogenesis, activation and migration ↗
Immobilized on PEG resin
[111]
Adhesion, proliferation in vitro ↗; new bone formation in vivo ↗; suppressed osteoclastogenesis in vivo
Grafting on PEG-PS copolymer
[137]
Attachment, migration, differentiation ALP and OPN expression and mineralization ↗ Adhesion, spreading and proliferation in vitro ↗; new bone formation in vivo ↗ Cell interactions at the bone surface, mineral binding affinity of PTH ↗
of
OPN171-177
-
Femoral and High matrix stability and bioactivity in humeral bone vitro; healing potential in vivo ↗ (sheep) EDC - 1-ethyl-(dimethylaminopropyl)carbodiimide, PEG - polyethylene glycol, PS – polystyrene, ↗ - increase
[140] [117] [116]
ur
na
lP
re
-p
ro
UMR-106 cells
[138] [139]
Jo
PTH1-34
Covalently via crosslinking reaction Covalently with the use of EDC Covalently via Nterminus Covalently; plasminsensitive substrate sequence as a linker
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Table 3. Examples of αvβ3 integrin antagonists. Structure
IC50/KI [nM]
Ref.
1
αvβ3 KI = 2
[176]
2
αvβ3 IC50 = 20
[177]
of
Compound
αvβ3 IC50 = 1.1
[178]
αvβ3 IC50 = 0.52
[179]
αvβ3 IC50 = 0.057
[180]
αvβ3 KI = 3.5
[181]
αvβ3 IC50 = 0.56
[182]
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7
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Mes = 2,4,6-trimethylphenyl, Cbz = benzyloxycarbonyl
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Table 4. Examples of α5β1 and αvβ3 antagonists.
IC50 α5β1 [nM]
IC50 αvβ3 [nM]
Ref.
1
0.18
49
[183]
2
264 (a) 0.7 (b)
1.2 (a) 279 (b)
[189]
Structure
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Compound
0.54
~30000
[190]
9600
[191]
[K ] I
[192]
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3
0.86
[K ] I
1000
0.9
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4
6
126.5 (a) 108 (b)
0.86 (a) 0.65 (b)
[195]
7
421
3.2
[196]
Mes = 2,4,6-trimethylphenyl, Cbz = benzyloxycarbonyl [KI] – affinity given as a KI value
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Table 5. Examples of peptidomimetics used in implant surface coating. Surface type
Effect
Ref.
1
PET
- Promoting adhesion of CaCo2 cells, - < FN
[198,199]
2
PET
- Promoting adhesion of CaCo2 cells, - < VN
[200,201]
Structure
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Compound
[202,203]
- Improve adhesion of mouse MC3T3 osteoblasts
[204]
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- Improve adhesion of human endothelial cells, - > RGDS
PET
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4
6
Ti6Al4V
- Selective cell adhesion in fibroblast cell lines, Ti6Al4V attachment, spreading TiO2 and proliferation of
[205,206]
sarcoma osteogenic SaOS-2 cells
- Selective cell adhesion in fibroblast cell lines, Ti6Al4V attachment, spreading and proliferation of TiO2
[205,206]
sarcoma osteogenic SaOS-2 cells
Spacer/anchor units are depicted in red, FN = fibronectin, VN = vitronectin, RGDS = Arg-Gly-Asp-Se peptide
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11. Graphical Abstract
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Figure 1. Knee and hip replacement surgeries in selected OECD countries. In most of the OECD members, the number of surgeries has increased rapidly since year 2000 both for knee (left) and hip (right) replacement.
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Figure 2. Summary of factors contributing to overall implant success.
Figure 3. Covalent binding of the RGD-containing ELP to the titanium implant surface. The presence of the diazirine moiety in the RGD-ELP molecule, allows generation of a carbene species after UV exposure, which interpose into the O-H bonds on the titanium surface. The amount of hydroxyl groups increases after the exposure of the titanium surface to UV light[66].
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Figure 4. The process of immobilization of proteins on a titanium surface via silanization. The first step involves treatment of the activated titanium with aminopropyltriethoxysilane (APTES). This facilitates positioning of the amine group on the surface of the metal, in such a way, that the appropriate protein can be bound to them in the second step.
Figure 5. The process of coating titanium surfaces with growth factors. For this purpose, either heparin (left) or poly-L-lysine (right) are used to anchor the growth factor to the activated titanium surface.
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Figure 6. Peptide based strategies for bone tissue engineering.
Figure 7. Various strategies used in engineering of implant coatings. Bare titanium is prone to bacterial film formation. Antiadhesive agents, bactericidal peptides and active agent release are the most common approaches addressing typical issues related to implant use. Novel approaches comprise multi-functioning constructs combining several factors, focusing on antiadhesive and bactericidal properties or facilitating eukaryotic cell adhesion (e.g. RGD peptide).
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Figure 8. Binding modes for α5β1 integrin of a) compound 6(a) and b) compound 6(b). Compounds 6(a) and (b) are shown as yellow and green sticks respectively, the α5 and β1 subunits are depicted as red and blue. The metal cation is represented as a magenta sphere. Figure reproduced with permission from ref. [195]. Copyright 2014 American Chemical Society.
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Highlights:
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Proteins, peptides, peptidomimetics as implant surface functionalization agents Methods for implant surface functionalization Broad biological effect evaluation, including both in vitro and in vivo studies
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Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
Przemyslaw Jurczak, Julia Witkowska, Sylwia RodziewiczMotowidlo, Slawomir Lach PII:
S0001-8686(19)30371-9
DOI:
https://doi.org/10.1016/j.cis.2019.102083
Reference:
CIS 102083
To appear in:
Advances in Colloid and Interface Science
Revised date:
9 December 2019
Please cite this article as: P. Jurczak, J. Witkowska, S. Rodziewicz-Motowidlo, et al., Proteins, peptides and peptidomimetics as active agents in implant surface functionalization, Advances in Colloid and Interface Science(2019), https://doi.org/ 10.1016/j.cis.2019.102083
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© 2019 Published by Elsevier.
Journal Pre-proof Proteins, peptides and peptidomimetics as active agents in implant surface functionalization
Przemyslaw Jurczak1†, Julia Witkowska1, Sylwia Rodziewicz-Motowidlo1, Slawomir Lach1†* 1
Department of Biomedical Chemistry, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland. † These authors contributed equally to this work. * Corresponding author. Email: [email protected] Abstract
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The recent impact of implants on improving the human life quality has been enormous. During the past two decades we witnessed major advancements in both material and structural development of implants. They were driven mainly by the increasing patients’ demand and the need to address the major issues that come along with the initially underestimated complexity of the bone-implant interface. While both, the materials and design of implants reached a certain, balanced state, recent years brought a shift in focus towards the bone-implant interface as the weakest link in the increasing implant long-term usability. As a result, several approaches were developed. They aimed at influencing and enhancing the implant osseointegration and its proper behavior when under load and stress. With this review, we would like to discuss the recent advancements in the field of implant surface modifications, emphasizing the importance of chemical methods, focusing on proteins, peptides and peptidomimetics as promising agents for titanium surface coatings.
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Keywords: implants; osteointegration; osseointegration; proteins; peptides; peptidomimetics; surface
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Contents of the paper: 1.
Introduction ..........................................................................................................................................3
2.
Proteins .................................................................................................................................................4 2.1.
Elastin-like protein modifications .................................................................................................5
2.2.
Type I collagen modifications .......................................................................................................5
2.3.
Fibronectin and vitronectin modifications....................................................................................8
2.4.
Bone sialoprotein modifications ...................................................................................................9
2.5.
Growth factor modifications ...................................................................................................... 10
2.5.1.
Vascular endothelial growth factor ................................................................................... 10
2.5.2.
Bone morphogenic proteins .............................................................................................. 11
3.
2.5.2.2.
Bone morphogenic protein 7 ..................................................................................... 13
2.5.2.3.
Bone morphogenic protein 9 ..................................................................................... 13
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Bone morphogenic protein 2 ..................................................................................... 11
Albumin modifications ............................................................................................................... 14
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2.5.2.1.
Peptides ............................................................................................................................................. 14 Integrin-binding peptide modifications ..................................................................................... 14
3.2.
Bone morphogenic protein-derived peptide modifications ...................................................... 16
3.3.
Osteogenic growth peptide modifications ................................................................................ 17
3.4.
Osteopontin-derived peptides ................................................................................................... 18
3.5.
Parathyroid hormone-derived peptides .................................................................................... 19
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Peptidomimetics ................................................................................................................................ 20
5.
Summary ............................................................................................................................................ 22
6.
Authors contributions ........................................................................................................................ 23
7.
Declaration of interest ....................................................................................................................... 23
8.
Acknowledgements............................................................................................................................ 23
9.
References ......................................................................................................................................... 24
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10.
Tables ............................................................................................................................................. 41
11.
Graphical Abstract.......................................................................................................................... 48
12.
Figures and Associated Captions ................................................................................................... 49
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1. Introduction
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We live in times when each individual can benefit from science and technology in every aspect of her of his existence. Starting with goods and food manufacturing, followed by the computer revolution, advancements in the field of medicine and much more. From the perspective of an individual, one of the most important accomplishments of the last 50 years was the global increase of human lifespan and the life expectancy at birth – reaching the average of 71 years for those born in 2015[1]. Nevertheless, a longer life has its own burdens. Aging combined with exposure to environmental factors causes the increased risks of ageassociated diseases[2]. Alongside hypertension, diabetes, Alzheimer’s and Parkinson’s diseases, bone-related disorders are considered one of the “key conditions” associated with age. Such conditions as osteopenia and osteoporosis leave the suffering person with weakened joints and bone structure, prone to fractures associated with an increased mortality rate[3]. The most common way of dealing with these issues are implantation procedures. In 2010, the amount of knee and hip replacement surgeries performed in the United States surpassed 1 million[4,5], with further predictions of a 7-fold increase for hip and 2-fold increase for knee related primary procedures in 2030[6]. The data presented by the OECD[7] clearly shows the hip and knee related surgery trends (Figure 1), with a 30 % increase of the rate of hip replacement and almost a double increase of the knee replacement surgeries.
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Similarly, an increase in the number of the procedures is also expected in Europe[8]. While the numbers state that implant surgeries are common and suggest that the demand for them will grow, doctors and patients are facing an important issue – incomplete post-implantation recovery. Despite its outstanding regenerative abilities, the bone tissue does not always heal completely, preventing successful fixation of the implant and eventually leading to implant failure and revision surgeries[9], adding in the end to the economic burden[10,11].
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Several factors govern the success of an implant fixation procedure. They involve patient’s profile (e.g. age, health, activity habits), characteristics of the implantation site (e.g. localization, vascularization, possible infection), properties of the implant and the surgical procedure alone[12] (Figure 2).
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In general, the aseptic loosening of an implant and bacterial infections are being highlighted as the main two reasons for implant failure[13–15]. The high predisposition for infections in post-implant patients is caused by lowered immune system efficacy around the implant area and the ability of bacteria to adhere to solid substrates[16]. Once present, bacteria quickly multiply and cover the implant surface with a biofilm, which is highly resistant to the immune system actions or conventional antibiotics (e.g. Staphylococcus aureus or Staphylococcus epidermidis). The possibility of spreading of the infection to other tissues contributes significantly to poor clinical outcomes of implant associated procedures[17]. Currently, the most promising strategies tackling the problem of implant related infections have been reported and reviewed by Mas-Moruno et al[18]. Aseptic loosening[19,20] is associated with a poor level of connection between the periimplant bone and the implant surface. Establishing a connection between the bone and the implant, also known as osseointegration (or osteointegration)[21], lies at the foundation of long-term implant usage. The success of osseointegration is complimented by the mechanical interlink between the implant and the bone. By extension, it depends on the quality and quantity of interactions on the bone-implant interface[22]. Currently there exists a variety of materials used for implant manufacturing with titanium and its alloys being the most 3
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recognizable and the most widely used[23,24]. The options such as magnesium[25,26] or zirconium are actively explored[27,28]. But it is not just the type of material and its general physical properties/biocompatibility that matter, it is the surface characteristics, that add greatly to the processes happening at the bone-implant interface. It has been established that wettability[29], surface chemistry[30], roughness and presence of nanostructures[31] and other functionalities[32], each to a different extent, determine if and how the host cells will bind to the implant. Based on the increasing amount of information on the processes governing bone tissue formation, multitude of passive/active coatings were developed with the aim to either act as preventive measure against bacteria or to improve osseointegration, and ideally, exhibit overlap of these two effects. Several excellent reviews on these topics are available[18,33,34].
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We would like to devote this review to proteins, peptides and peptidomimetics as molecules for surface modifications, emphasizing their role in the strategies aimed towards mitigating issues present in today’s dental and orthopaedical implantology. In our understanding, the development of these three classes of molecules will have the major impact on modern implantology. Since modifications of implant surfaces eliciting antibacterial properties are thoroughly reviewed in literature[35–39], in this review we will focus exclusively on implant surface medications improving osseointegration processes.
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2. Proteins
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The use of proteins was one the first steps towards solving the problems with osseointegration in implantology. Compared to solutions described later in this text (the use of peptides and peptidomimetics) the use of proteins as implant coatings should, at least theoretically, have given a significantly higher probability of successful bone-implant integration. This was due to (i) a high level of recognition of the protein-covered implant surface as a “host-type” tissue – a biomimetic factor and (ii), incorporation of specific biological cues that can further alter the response of the bone tissue around the implant. This way, the processes of attaching the new bone cells to the implant and promoting the process of bone regeneration (increased pace of bone cells growth and differentiation) were facilitated. Extensive research has been dedicated to proteins used for implant surface modifications. However, this rather small group of studied molecules was limited to genetically engineered proteins (e.g. elastin-like protein), type I collagen, fibronectin, vitronectin, bone sialoprotein, growth factors (e.g. vascular endothelial growth factor (VEGF), BMP2, BMP7, BMP9) and albumin. This section of our review highlights the selected examples of use of the proteins in implant engineering. (Table 1).
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Elastin-like protein modifications
The elastin-like protein (ELP) is a flexible, biologically stable synthetic biopolymer[63]. Its structure was designed based on tropoelastin, a basic building block of the elastin protein – the main component of the elastic fibers occurring in a range of tissues present in the organism[64]. One of the many functions of ELP is the formation of a sturdy hydrogel scaffold which promotes the growth of human mesenchymal stem cells and facilitates bone regeneration[40].
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The ELP coatings were the subject of two major studies performed by Raphel et al.[41,65]. In the first study, the group focused on different ways of photo-processing of the diazerine modified ELP (ELP-D), the stability of the photo-crosslinked ELP-D biomaterial and the effect of the photo-crosslinked ELP-D film on the human adipose-derived stem cells (hASC)[41]. As a result, they were able to create stable 2D films and 3D scaffolds with the use of versatile photoprocessing methods. Both structures exhibited relatively high mechanic resistance. Additionally, the ELP-D also showed high cytocompatibility towards hASC cells. It caused rapid adherence and larger spread area of cells in comparison to control, thus showing a potential to be applied in tissue engineering.
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In the second study Raphel et al. used the designed ELP as a coating for titanium-based implants[65]. The ELP containing the RGD cell-adhesive sequence was covalently bound to the implant surface via UV-mediated crosslinking (Figure 3).
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Further, authors showed that the designed coating withstood the procedures simulating dental screw and hip replacement implantations. Raphel et al. inserted the dental screws into a 10 PCF polyurethane foam mimicking bone properties and press fitted rods mimicking hip prostheses into mouse cadaver femurs. The ELP coating promoted rapid adhesion of osteoblast-like cells (MG63) in comparison to a bare titanium surface. The coating promoted also faster osteogenic differentiation and mineral deposition on the implant surface – determined based on early increase in alkaline phosphatase (ALP) activity. The in vivo studies of rat femur and tibia showed an increased bone-implant contact area and interfacial strength in favor of the cell-adhesive ELP coated implants, suggesting that the ELP-RGD coating has the potential to prevent aseptic loosening of implants. Owing to its osseoinductive properties, ELP, especially in combination with the RGD peptide, seems to be a promising direction for implant surface engineering. It may potentially be one of standard components of clinically approved implant coatings, next to a range of proteins e.g. type I collagen. 2.2.
Type I collagen modifications
Type I collagen is a potentially promising molecule to be used as an implant coating. It plays a crucial role in the bone structure, where it serves as one of the structural proteins[67] and as a mediator of osteoblastic cell functions (adhesion, differentiation and extracellular matrix secretion)[68,69]. Utilized in implant coatings, it has also been shown to possess a significant influence on peri-implant bone regeneration[70,71]. The latest data on type I collagen titanium implant modifications was presented by i.e. Bae et al.[43], Ao et al.[42,45,46], Marin-Pareja et al.[44] Truc et al.[47]. In most of these cases collagen was bound to the implant
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surface covalently, via crosslinking agents[42–44] or by electrochemical deposition[47]. Additionally, Marin-Pareja et al.[44] also immobilized collagen by physical adsorption.
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Bae et al. compared two different ways of covalent immobilization of type I collagen on the titanium surface – crosslinking with gamma-irradiation and crosslinking with glutaraldehyde[43]. The coated titanium implants were studied in vitro using human mesenchymal stem cells (hMSC) and in vivo in a rat model. The in vitro test showed the highest osteogenic differentiation and osteogenesis related gene expression levels for implants crosslinked with gamma-irradiation in comparison to the glutaraldehyde crosslinked and noncoated implants. The in vivo results did not indicate significant differences between differently crosslinked surfaces, with both samples providing the same level of osseointegration. Considering the cytotoxic effects associated with the use of glutaraldehyde and other chemical reagents associated with the crosslinking procedure, crosslinking with gamma-irradiation is a more promising method for immobilizing collagen type I on titanium surfaces.
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Ao et al.[42] focused on in vitro migration of hMSC into the surface of the implant, in vivo peri-implant bone regeneration and osseointegration itself. The presented results indicate that the hMSC cells, which play an important role in osseointegration and pose multidirectional differentiation potency[72], migrated into the pores in the surface of the collagen coated titanium. This is attributed to one of the natural functions of type I collagen, its ability to form scaffolds allowing for effective attachment and migration of hMSC cells[73]. The increased cell migration on the surface of collagen coated titanium in comparison to bare titanium (in vitro studies) presented by Ao et al., correlated with the increased pace of osteoconduction and osseointegration – confirmed by in vivo studies on rabbit specimens[42]. Importantly, the increased pace of bone formation and incorporation of bone cells into the implant surface should potentially shorten the time of the integration of the implant into the bone structure and decrease the possibility of implant’s loosening.
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In another study Ao et al. presented a composite collagen/hyaluronic acid coating aimed at titanium surfaces[46]. The coating was built by covering the metal surface with several subsequent layers of type I collagen and hyaluronic acid. The osteogenic properties of this coating in vitro were verified in the previous study, showing excellent biological properties and promotion of attachment, spreading, proliferation and differentiation of hMSC cells[45]. The present work focused on in vivo studies of the coating in a rabbit model with femur condyle defect. Two different methods of the coating preparation were studied: in the first method the coating layers were bound covalently, and in the second method via physical adsorption. The results of the study showed that the covalently bound multilayer was more stable. This was critical for osteogenesis, osteoinduction, osseointegration and aseptic loosening of the coated implants. The studies performed by Marin-Pareja et al.[44] and Truc et al.[47] focused specifically on dental implants, which are typically exposed to high pressure during chewing, thus being prone to potential failure. Since dental implants require not only osseointegration but also development of necessary soft tissue covering the transmucosal section of the implant, focusing on factors responsible for tissue development is a reasonable strategy.
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Marin-Pareja et al.[44] studied the influence of the type I collagen coating on the human dermal fibroblasts (HDF), cells responsible for extracellular matrix synthesis and connective tissue formation, crucial for the wound healing process. The research focused on (i) two different methods of titanium surface treatment: acid, usually Piranha solution, etching and oxygen plasma treatment, and (ii), two different methods of immobilization of collagen on the titanium surface. It was also verified how these factors and their combinations influence collagen itself and the collagen-HDF cells interplay. As it appears, both, surface treatment and immobilization methods, influenced the stability of collagen on the implant surface. The greatest stability of the coating was achieved by covalent binding of collagen to the relatively smooth (nano-roughness 17.9±4.8 nm), plasma-treated surface which was also more biocompatible and provided better adhesion and activation of the fibroblasts. This effect was most probably caused by the fact that the increase in roughness of the surface is accompanied by the increase of its hydrophobicity/decrease of wettability[74]. Compared to plasma treatment, use of Piranha yielded a surface with much higher roughness value (144.5±5.1 nm), increased hydrophobicity and effectively reduced ability of the surface to support fibroblast attachment. Cell proliferation was not influenced by the surface treatment or the type of collagen binding. However, it was significantly increased in the presence of the collagen coating.
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The study results presented by Truc et al.[47] focused on in vitro interactions between the type I collagen coated implant and mouse fibroblast L929 cell line. Authors verified bioactive properties of the implant surface towards the cells and showed that the presence of the collagen coating increased the cell adhesion and cell spreading rate in comparison to a bare titanium implant. The coating also increased the viability and proliferation of the cells. Additionally, Truc et al.[47] optimized the method of electrochemical deposition of type I collagen on the titanium surface which became an attractive, much faster alternative to the previously used dipping and plasma-polarization methods[75,76].
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Collagen composites are an interesting group of materials. An example, collagen/calcium phosphate nanocomposites are generally considered a good choice for hard tissue engineering due to their structural and compositional similarity to mammalian bone tissue[77]: mammalian bones contain ca. 70 vol % of hydroxyapatite, with the rest being water and matrix - 90 wt. % of which is collagen. Collagen/calcium phosphate coating for titanium surfaces was presented by Neacsu et al.[48]. Additionally, to increase the potential of the composite to promote osseointegration processes the authors added Zn2+ ions to its structure, yielding Zn2+ substituted calcium phosphate. Neacsu et al. focused on the comparison of the two methods of titanium coating, namely spin coating and matrix-assisted pulsed laser evaporation (MAPLE). For both methods the results of the in vitro study showed the synergic effect of type I collagen and Zn2+ substituted calcium phosphate: the composite stimulated the mineralization process in Simulated Body Fluid (SBF). However, the MAPLE method did not produce secondary compounds that alter cell viability or normal cell cycle phases. The method also allowed to obtain high uniformity of the coating and preserved collagen structure, improving homogeneity and morphology of the surface, giving the MAPLE method an advantage over spin coating. Taking into account the synergistic effect exhibited by type I collagen towards integration of the titanium implants with both, bone[42] and soft tissue[44,47], the collagen coating
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approach towards implant surface engineering appears to be a very promising strategy, especially in the field of dental implants. 2.3.
Fibronectin and vitronectin modifications
Fibronectin and vitronectin are glycoproteins possessing numerous functions, with the most relevant, from the perspective of this review, being involvement of these proteins in cell adhesion and migration[49]. Considering their functions, these proteins are readily used in implant coatings, where they increase the implant bioactive properties and accelerate the process of bone osseointegration. Use of fibronectin is much more common, most probably due to its higher biocompatibility and more significant cell response in comparison to vitronectin[78].
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One of the studies focusing on both, fibronectin and vitronectin, as potential coatings for the titanium Ti6Al4V alloy (the most common alloy used in implant manufacture) were performed by Felgueiras et al.[78]. The proteins were adsorbed to a sodium styrene sulfonate (poly(NASS)) grafted titanium alloy surface and, as a control sample, to an ungrafted alloy. The influence of both samples on bone cells was verified with the use of mouse calvaria-derived osteoblast-like cell line (MC3T3-E1). The results show that the grafting procedure increased the level of adhesion of the MC3T3-E1 cells to the titanium surface. The increase of adhesive properties occurs due to the presence of the SO3- groups of the poly(NaSS) bound to the titanium surface[79]. Furthermore, and most importantly, the presence of the fibronectin and vitronectin adsorbed to the surface increased the adhesion level of MC3T3-E1 even further. Though, it should be stressed that the adhesion was much stronger in the presence of fibronectin in comparison to vitronectin. Similar trend was observed in the case of cell spreading and morphology, again favoring grafted surfaces with adsorbed proteins. However, the presence of vitronectin on the grafted and ungrafted surfaces caused some of the cells to exhibit rounded morphology, while the morphology on fibronectin treated surface was polygonal, the same as during the natural osteoblast maturation processes[80]. Such result may indicate a higher degree of influence of fibronectin on the early osteoblastic differentiation. An extensive study of fibronectin based coatings for titanium implants was also performed by Chang et al.[50,51]. They started with an in vitro analysis of the fibronectin-modified surface[50]. Utilizing the glow discharge plasma (GDP) allowed at the same time for (i) cleaning the implant surface, and (ii), incorporation of amine groups (-NH2) into the surface. The -NH2 groups were used in the next step as attachment points for the fibronectin molecules. The results showed that the GDP treatment of the implant surface increased the pace of the MG-63 cell line growth and proliferation in comparison to the untreated control. Further, addition of fibronectin to the GDP treated surface increased the cell viability in comparison to the GDP treatment only. These results indicate that the fibronectin grafting combined with GDP treatment modifies the properties of the titanium implant surface so that by extension, it may reduce the time of osseointegration of the implant. Therefore the authors decided to continue with a follow-up in vivo trial[51], with the intention to evaluate the bone integration efficiency of plain and GDP treated implant versus a GDP treated fibronectin grafted one. The implant stability and bone-implant integration were evaluated utilizing resonance frequency analysis (RFA), histologic evaluation and microcomputed tomography (micro-CT). The RFA results 8
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showed that the level of stability was greater in case of fibronectin coated GDP treated implant in comparison to the plain, GDP treated one, especially shortly after the implantation. In both cases, the histological analysis showed that the bone regeneration proceeded during the experiment. Maturing of the bone was faster in the case of fibronectin coated implant though. The micro-CT analysis revealed greater bone formation in the case of fibronectin coated implant 2 weeks after the implantation. All above outlined results show that the applied surface modifications increased implant stability. Additionally, coating of the implant with fibronectin provided further benefits involving shortened treatment time and fast implant provisionalization.
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The possibility of using fibronectin as implant surface modification agent was also explored by Cho et al.[81], though their approach was based on a fibronectin derived oligopeptide F20, of the following sequence PHSRNSITGTNLTPGYTITVYAVTGRGD[52], in which the main twenty amino acid long fibronectin derived fragment, in the middle, has been terminated with the RGD motif on one end, while the other end has been equipped with RGD-synergic sequence (PHSRN), required for optimal cell activity. Authors of the study hoped to achieve superior osseointegration of dental implants with the help of the F20 molecule. Its efficiency was verified by a range of different techniques, such as scanning electron microscopy (SEM), confocal laser scanning microscopy (CLASM), picogreen assay, real-time PCR and immunoblot with the focus on adhesion, proliferation and differentiation of the stromal cell line ST2. The data obtained from the study showed that the F20 coating promoted all three processes. The positive study results combined with a simple coating procedure (direct adhesion on the implant surface) showed once again that fibronectin is a promising candidate for a titanium implant coating.
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Another study which confirmed the improved cell adhesion, proliferation and spreading on the fibronectin coated surface was performed by Jian et al.[82]. This study was performed on gingival fibroblasts with fibronectin covalently attached to the micro-grooved silanized surface of the titanium implant. Authors showed that micro-grooving of the titanium surface increased the strength of the cell adhesion, with its further increase in the case when fibronectin was applied on the surface of the implant. Considering the above-described data, glycoproteins with natural functions spanning over cell adhesion, growth, differentiation and migration, e.g. fibronectin and vitronectin[83,84], have tremendous potential in titanium implant coating engineering. However, as the data[78] and frequency of application suggest, fibronectin exhibits much higher bioactive properties compared to vitronectin. 2.4.
Bone sialoprotein modifications
Bone sialoprotein is a component of mineralized tissues (e.g. bone, dentin)[54]. It is found in the bone extracellular organic matrix (ECM)[53]. This non-collagenous protein has been shown to enhance bone formation when coated on titanium implants[53] however, its primary in vivo functions are not entirely clear[54]. Here we summarize two papers regarding the bone sialoprotein as titanium implant coatings.
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The coating presented by Im et al.[85] contained the human bone sialoprotein (bone sialoprotein II) covalently attached to the silanized surface of micro-grooved titanium implant (Figure 4). Authors of the study verified the influence of micro-grooving and surface coating on human bone marrow-derived mesenchymal stem cells (MSC) by monitoring the expression of the main osteogenic transcription factors (ARF4, FRA1, RUNX2, OSX). They also monitored the differentiation and spreading of the cells with SEM. In response to the micro-grooving of the titanium surface both, differentiation and spreading of bone marrow-derived MSC increased, in comparison to the smooth titanium surface. The application of bone sialoprotein II (BSPII) coating increased the bioactive properties of the micro-grooved implant surface, significantly improving differentiation and spreading of the cells. In both cases the cells exhibited a tendency to orientate themselves perpendicularly to the surface micro-groves. In case of the transcription factor expression, the comparative analysis of time-dependent expression of ARF4, FRA1, RUNX2 and OSX coding genes, showed the greatest upregulation in favor of the micro-grooved, BSPII coated surface, in comparison to micro-grooved and smooth surfaces.
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Similarly to the above described study, Baranowski et al.[53] also tried to verify the influence of bone sialoprotein (human recombinant; BSP) coating of titanium implants on primary human osteoblasts (hOb) and L929 fibroblasts. The BSP was attached to the activated, piranha treated, titanium surface by either covalent coupling through silanization or physisorption. Even though during the first few hours after exposing the cells to the titanium surface, their proliferation was delayed, reaching normal levels after 24 hours, the covalent coupling with BSP was favored by the cells. This preference was manifested by the increase of the cell proliferation, differentiation, expression of RUNX2 (osteoblast-specific transcription factor[86]) and osteopontin (OPN; component of extracellular matrix of bone and dentin[87]) genes which influence mineralization of ECM. Additionally, the increase of the concentration of BSP in the coating positively affected the cell migration. Suggesting that coatings containing this protein show a potential to increase the pace of implant-bone integration. However, even though the bone sialoprotein exhibits increased bioactive properties, the earlier described fibronectin displays even higher protein-bone biocompatibility, especially in case of stimulating OSX gene expression[85]. Therefore, when considering the use of BSP as a coating, fibronectin should also be taken under consideration as a molecule with potentially better influence on bone cells. 2.5.
Growth factor modifications
Growth factors are one of the developing branches in engineering of implant coatings. Different growth factors showed potential to increase the early osseointegration and implantbone compatibility. Among many the most promising examples involve VEGF[58], BMP2[60], BMP7[61] and BMP9[88]. 2.5.1.
Vascular endothelial growth factor
The vascular endothelial growth factor (VEGF), a growth factor stimulating formation of blood vessels[55], was studied by Guang et al.[56] and Izquierdo-Barba et al.[89], who used VEGF based coatings and coatings combining VEGF and silicon substituted hydroxyapatite respectively. Both studies showed a positive influence of the growth factor on bone regeneration processes. 10
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The in vivo studies performed by Guang et al.[56] on rat specimens showed that VEGF promotes proliferation of osteoblasts and endothelial cells. In both cases, the levels of protein markers characteristic for processes necessary for successful bone tissue repair – osteocalcin (OCN), an osteoblast-specific protein linked to bone formation[90], and CD31, protein involved in angiogenesis processes[91], have increased in comparison to the control sample. The results of a supporting in vitro study indicate that VEGF induced also the proliferation of primary rat osteoblasts, vasculogenesis and increased the activity of ALP.
Bone morphogenic proteins
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The studies on VEGF performed by Izquierdo-Barba et al.[89] required converting the surface of a microporous titanium scaffold with silicon substituted hydroxyapatite (SiHA). Further, the modified surface was coated with VEGF to explore the possibility of the synergistic effect exhibited by the simultaneous presence of SiHA and VEGF. The growth factor was adsorbed to the surface noncovalently. At first, the in vitro studies showed that the adsorption of VEGF to SiHA coated titanium was notably higher in comparison to bare titanium. Further experiments also showed that the VEGF coating stimulates proliferation of endothelial cells regardless of the SiHA presence. This also correlates with the data presented by Guang et al.[56]. On the other hand, the pre-osteoblasts (MC3T3-E1 cells) preferred to grow on the SiHA/VEGF coated implants. To support the in vitro results, authors performed the in vivo study in an osteoporotic sheep model. They implanted the titanium scaffolds in different parts of sheep bones (proximal tibia epiphysis, medial epicondyle of femur and greater tuberosity of humerus). The histological analysis of the bone segments containing defects showed that neither of the used coatings (SiHA or VEGF) enhanced the osteogenesis separately. However, when combined, the molecules exhibited a synergic effect and increased the osteogenesis, size of trabeculae and degree of angiogenesis.
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Bone morphogenic proteins (BMPs) are a group of signaling molecules[92], originally discovered as factors promoting osteogenesis. However, later studies showed that BMPs serve important functions in formation and maintenance of such organs as cartilage, blood vessels, muscle and kidney[93,94]. Even though as many as fifteen BMPs has been discovered and studied, only BMP2 and BMP7 successfully completed clinical trials and are approved for clinical application[59]. 2.5.2.1. Bone morphogenic protein 2 The bone morphogenic protein 2 (BMP2), is responsible for regulation of many processes leading to bone formation and regeneration[94]. Therefore, it is a popular target in implant coating engineering. BMP2 has been successfully used as a separate titanium surface coating[57] and in combination with other molecules (e.g. GDF5 – one of growth factors playing a leading role in bone regeneration[95]). One of BMP2-based multicomponent coatings was designed by Yang et al.[95]. This double layer titanium implant coating contained BMP2 and GDF5 growth factors separated by heparin layer (Ti-BMP2-GDF5; Figure 5, left). The coating was designed in a manner allowing for slow release of the growth factors from the surface of the implant. It was possible due to the fact that many growth factors, BMP2 and GDF5 including, bind heparin[96,97] non-covalently[98]. The positively charged proteins in combination with high negative charge of heparin allows for 11
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their slow and sustained release[99,100] (Figure 5, right). Yang et al. verified the influence of the double coating on the bone regeneration processes in comparison to bare Ti, Ti-BMP2 and Ti-GDF5 coatings. The in vitro studies on MC3T3-E1 cells cultured on Ti-BMP2-GDF5 coating showed significantly increased proliferation and ALP activity, resulting in calcium deposition and increased expression of osteocalcin (OCN), type I collagen (COL1) and ALP mRNA in comparison to Ti, Ti-BMP2 and Ti-GDF5. The follow-up in vivo studies correlated with the in vitro data showing increase of bone formation at the bone-implant interface, indicating enhanced bone remodeling and regeneration processes especially in case of the Ti-BMP2-GDF5 coating.
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BMP2 was also used as a titanium implant coating by Böhrnsen et al.[60], however, they used a specific method of anchoring the protein to the implant surface. The method involved covering the surface of both titanium and BMP2 with oligonucleotides. The oligonucleotides were later hybridized forming a link between the protein and the implant. This method of protein-implant binding allowed for slow release of the biologically active BMP2 growth factor to the environment. The analysis of such implants placed in rat tibiae showed increased expression of ALP, however the expression of RUNX2 transcription factor, responsible for fast osteogenic differentiation, was not significantly influenced by the implant coating. The data presented by Böhrnsen et al. indicates that the BMP2 coating increases the osteogenic differentiation around titanium implants shortly after implantation (confirmed by enhanced ALP expression). The RUNX2 does not seem to be the best marker to evaluate osteogenic differentiation, due to the potentially diverse involvement of this transcription factor in cellular differentiation processes. Anchoring the BMP2 to titanium through oligonucleotides allowed to link significantly more proteins to the implant surface in comparison to samples without oligonucleotide conjugation. Comparison to the previous data[101] suggests that it is the amount of the growth factor presented on the surface of the implant, rather than the concentration of the released growth factor is responsible for the positive effect of the engineered coating on bone regeneration processes.
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Contrary to the two previously described studies, Youn et al.[57] used covalent immobilization during the BMP2-based coting preparation. The authors deposited poly glycidyl methacrylate (pGMA) on the surface of the titanium implant by means of initiated chemical vapor deposition (iCVD) and in the next step, they covalently bound BMP2 to pGMA. This method allowed to immobilize significantly higher amount of BMP2 to the titanium surface in comparison to direct adsorption of the growth factor to bare titanium. The osteogenic properties of the coating were verified against human adipose-derived stem cells (hASC). The BMP2 coating showed high biocompatibility to the cells and caused the increase of osteogenic gene expression (OCN, RUNX2, COL1), ALP activity and calcium deposition. Despite the fact that covalent binding does not allow the release of BMP2 to surrounding tissues, it seems, surprisingly, that the good osteogenic properties of the BMP2 coating designed by Youn et al. occur as a result of this firm binding of the BMP2 to the titanium surface. This indicates that the method of active agent immobilization plays a significant role in improving its osteogenic properties of the coating.
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2.5.2.2. Bone morphogenic protein 7
Bone morphogenic protein 7 (BMP7) is another example of a protein involved in bone formation processes. Similarly, to other BMPs, it plays an important role in transforming MSCs into bone tissue and cartilage. As mentioned before, BMP2 and BMP7 are the only two in the group of morphogenic proteins approved for clinical applications[59], thus gathering relatively high interest in the field of bone regeneration and implantology[102–104].
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An interesting approach to implant surface modification with BMP7 was presented by AlJarsha et al.[61]. The combination of the properties exhibited by BMP7, poly(ethyl acrylate) (PEA) and fibronectin allowed them to obtain a system enabling a dramatic decrease of BMP7 concentrations while keeping the osteogenic properties of the coated implant. Previous studies[105] show that combining the PEA and fibronectin causes unfolding of the protein and its rearrangement into specific nanonetworks. This change of protein conformation facilitated the access to the growth factor binding domains FNIII12-14. Jarsha et al. used the synergic properties of PEA and fibronectin and combined them with osteogenic potential of BMP7, which was attached to the exposed growth factor binding domains of fibronectin. The decrease of the GF concentration eliminated the problem of side effects caused by the need of high growth (GF) factor dosage (used when bare titanium is coated with the GF) and its relatively high clearance from the organism. The results of the study show that it is possible to create an environment with low amount of osteogenic factor, which will allow the promotion of bone forming cells – in this case differentiation of hMCS towards osteogenic lineage. The presented GF delivery system shows a potential for better control over the concentration of GF at the target sites.
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2.5.2.3. Bone morphogenic protein 9
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Similarly, to BMP2, bone morphogenic protein 9 (BMP9) takes part in processes leading to bone formation and regeneration. BMP9 is one of the morphogenic proteins exhibiting relatively high osteogenic potential[88], with some studies showing BMP9 exhibiting even higher osteogenic properties than BMP2[106,107]. Nevertheless, in terms of osteogenic potential, BMP9 is still one of the less explored proteins among BMPs. Souza et al.[88] performed a preliminary in vitro study, in which BMP9 was mixed into an osteogenic medium instead of being immobilized on the implant surface. MC3T3-E1 preosteoblastic cell line were seeded on the titanium discs and incubated in the BMP-containing medium. The results of this study showed that BMP9 enhanced the osteoblast differentiation, meaning that this growth factor boosts bone regeneration processes, suggesting that linking BMP9 to the surface of titanium implants may be a good direction for the development of the BMP9-based implant coatings. Considering its supposedly high osteogenic properties, the BMP9 protein may be an even better implant coating agent than the commonly studied BMP2. The described growth factors comprise a large group of proteins exhibiting various properties and influencing different types of cells (e.g. endothelial cells or osteoblasts). Even though some of them occur more commonly than the others in studies dedicated to novel implant coatings, they all seem, at least to some degree, to positively influence bone regeneration. The growth factors are often immobilized on the implant surface in a way that facilitates the factors release or are used in media in which implants are immersed (no immobilization). In both cases, enhancement of differentiation of cells grown on the implants 13
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surface is visible, however it seems that permanent immobilization of the growth factors results in better osteogenic properties[57], presenting covalent binding of growth factors to the titanium implant surface as a more favorable direction of exploration for implant coating engineering. 2.6.
Albumin modifications
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When considering materials used for implant preparation titanium is one of the most commonly used due to its high corrosion resistance, good mechanical properties and biological biocompatibility[108]. However, in recent years the concern regarding release of metal from implants increases and the process leading to the degradation of implant surface are mainly unknown[62]. Here albumin comes with help. This protein may be immobilized on the surface of titanium implants and effectively prevent corrosion even under simulated inflammatory conditions[109]. It was also proved that albumin based coatings facilitate (i) protein adsorption to, and (ii) calcium phosphate (naturally present in bone mineral and tooth enamel) accumulation on the surface of the implant[109]. To verify further, potential biomedical applications for albumin as an implant coating agent combined with extensive studies regarding cellular growth and differentiation are necessary. On the other hand, albumin coating may potentially serve as a future multifunctional system, enhancing bone regeneration and preventing implant corrosion at the same time.
3. Peptides
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Parallel to the studies on protein-implant coatings, functionalization of implant surfaces based on peptides is being developed. The peptides are often derived from proteins which exhibit osteogenic properties themselves, such as type I collagen[110] or osteopontin (OPN)[111,112]. Peptides derived from bone-related growth factors (e.g. BMPs[113,114], OGPs[115]) and peptide/protein hormones with recognized anabolic effects related to bone metabolism (e.g. parathyroid hormone; PTH)[116,117] were also suggested (Figure 6).
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Additionally, some peptides, unrelated to proteins enhancing bone regeneration, can also function as building blocks for new biomaterials. These peptides, named self-assembling (SA) peptides, spontaneously aggregate into nanofibers and create hydrogels that can be used to culture cells in three-dimensional biomaterial scaffolds[118]. The main advantages of using peptides in regenerative medicine involve (i) relatively easy synthesis, with the use of liquid- or solid-phase synthesis procedures in a cost-effective manner, and (ii), relatively small size, minimizing their immunogenic properties in comparison to proteins. In this section we focused on osteoinductive peptides, which can promote cell adhesion, proliferation, differentiation and angiogenesis. 3.1.
Integrin-binding peptide modifications
The arginylglycylaspartic acid (RGD) is the most widely studied adhesive peptide in implant engineering. This motif was identified by Ruoslahti and Pierschbacher in the early 1980s[141]. The RGD sequence was discovered in the cell binding domain of fibronectin where it functions as the minimal recognition sequence required for cell attachment[141]. RGD is often used in conjunction with other cell-adhesive molecules like integrins, cadherins or extracellular matrix 14
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proteins to mimic cell interactions and to enhance cell adhesion[142]. Although the RGD sequence alone is capable of acting as a ligand for integrins, a high-affinity recognition requires both the RGD and PHSRN sequence[143]. Since both these sequences interact with the same integrin, the spatial configuration of RGD and PHSRN is crucial to assure correct binding. Because of this, designing a peptide containing RGD and PHRSN requires a careful consideration of the length and nature of the spacer to assure their synergistic effect[144]. Here, we describe different studies performed regarding the RGD sequence. They involved covering implant surfaces with RGD only and in combinations with other peptides and indicate that immobilizing RGD on titanium and other biomaterial surfaces is a useful approach for promoting bone regeneration.
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One of the strategies of the RGD-based coating development involved the structural modification of the peptide. Heller et al. studied the differences in the osteogenic properties between linear and cyclic form of the RGD peptide (L-RGD and C-RGD respectively)[119]. Both peptides were immobilized covalently, via silanization on the titanium surface. The results showed only subtle differences between the hipbone derived human osteoblast adherence, proliferation and differentiation when cultured in vitro on L-RGD or C-RGD coated surfaces. On the other hand, when studied in vivo, C-RGD caused a significant increase of vertical bone apposition indicating faster bone regeneration, showing that not only the peptide sequence but also its structure may influence the osseointegration processes.
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Another strategy for peptide-based implant coating was presented by Pagel et al. who combined three peptides exhibiting different properties to facilitate cell adhesion[120]. The combined peptide contained a cyclic form of the RGD peptide (C-RGD), bone sialoprotein derived heparin binding FHRRIKA peptide (HBP) and a peptide containing L-3,4dihydroxylphenylalanine (DOPA), which helped to bind the construct covalently to the titanium surface. The osteogenic properties of the coating were demonstrated using human sarcoma osteogenic (SaOS-2) cells. The results of the study showed that the presence of both integrinbinding RGD and heparin-binding HBP increased spreading, proliferation, viability, and the formation of actin cytoskeleton and focal contacts of the SaOS-2 cells. The peptide-based titanium implant surface coatings were also utilized to increase the potential for cell adhesion and at the same time improve bacteriostatic and bactericidal properties of implants[121]. Such coating construct was presented by Hoyos-Nogués et al. In the first step, the titanium surface was coated with PEG to prevent bacterial adhesion (Figure 7). In the second step, two peptides have been attached to the surface: an antimicrobal peptide (AMP) LF1-11 (the first eleven residues of the N-terminal part of the lactoferrin, responsible for antibacterial properties of the protein) and the RGD peptide serving as a factor facilitatiang cell adhesion. The obtained data showed that the presented coating increased attachment and spreading of SaOS-2 cells preventing at the same time bacteria (S. sanguinis) growth on the coated surface. This strategy of titanium implant modification shows that it is possible to functionalize the implant surface in a way, that will facilitate the osseogenic processes after implantation and, at the same time, prevent bacterial growth on the boneimplant interface. 15
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Some strategies of peptide-based implant modifications require functionalization of other biomaterials (e.g. nanotubes, alginate microspheres, hydrogels, etc.) which can be attached to the titanium implant surface or utilized as implants on their own. Cao et al. showed that TiO2 nanotube layers modified with RGD via covalent binding increased the rat bone marrow stromal cells adhesion and osteogenic gene expression, in comparison to nonfunctionalized nanotubes[122]. Another study regarding the RGD peptide was performed by Evangelista et al.[123]. Here, pre-osteoblastic MC3T3 cells were immobilized within RGD-coupled alginate microspheres. The use of CLASM and transmission electron microscopy imaging allowed to determine that the RGD promotes the adhesion and differentiation of the MC3T3 cells. A slightly different approach towards supplying the active agents was undertaken by Zhu et al., who applied RGD and HAV (His-Ala-Val) peptides on hyaluronic acid (HA) hydrogels[124]. HAV is a conserved motif of type I cadherins that promotes the chondrogenesis of encapsulated human mesenchymal stem cells (hMSCs)[145]. The data obtained from the study showed that the biofunctionalized hydrogel enhances the expression of the osteogenic marker genes and promotes ALP activity, type I collagen deposition and matrix mineralization under in vitro and in vivo conditions.
Bone morphogenic protein-derived peptide modifications
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The triple helical peptide containing the GFOGER sequence, derived from the type I collagen is another, slightly less popular example of a peptide capable of binding integrins[146]. Reyes et al. studied titanium implant coatings based on GFOGER, a collagen-mimicking peptide which selectively binds the α2β1 integrin[110]. Authors verified the influence of the coating on bone marrow stromal cells, (in vitro) and on implant osseointegration processes (in vivo), in a rat cortical bone model. The data obtained from the study showed that the coating increased integrin-mediated cell adhesion and osteoblast differentiation in vitro and significantly increased osseointegration of the coated titanium implants in vivo.
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As mentioned before, the BMPs are a group of signaling molecules[92]. They represent the main osteoinductive factors involved in skeleton development, bone formation and bone remodeling in mammalian organisms. The BMPs contain two sequences essential for their proper activity, which are referred to as the “wrist” and the “knuckle” epitopes and preferentially interact with BMP type I and BMP type II receptors present on the cell surface[126]. One of the modifications of titanium implants based on the BMP2 derived “knuckle” peptide (K-peptide) was presented by Ma et al.[114]. The authors developed a peptide-based coating where they immobilized the K-peptide on the surface of TiO2 nanotubes with the use of dopamine and further applied real-time PCR, histological analysis and fluorescence microscopy to verify the osteogenic properties of the coating when implanted into New Zealand White rabbit tibiae. Obtained data shows that the K-peptide coating increased the expression level of osteogenic genes (ALP, COL1, OSX, TRAP) and promoted early stability of the implant after implantation via increased pace of bone deposition around the implant.
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Another coating based on BMP2-derrived peptide (KIPKASSVPTELSAISTL) was designed by Cai et al.[113] who combined the osteoconductive properties of hydroxyapatite and osteoinductive properties of BMP2-derrived peptide. For this purpose, the surface of titanium was covered with polydopamine film to induce the formation of hydroxyapatite. Next, the BMP2-derrived peptide was absorbed into the hydroxyapatite. The data from in vitro tests of the coating showed enhanced cellular adhesion, ALP activity and expression of osteogenic markers. The coating promoted human osteosarcoma (MG63) cell differentiation contributing to increased pace of bone growth.
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The BMP-derived peptides may also be bound to other biomaterials e.g. hydrogels which can potentially be adsorbed to the surface of titanium or other material-based implants forming an advanced coating construct. An idea of incorporating peptides into a hydrogel was applied by Suzuki et al. who originally described a twenty amino acid long sequence (NSVNSKIPKACCVPTELSAI) corresponding to the residues 68-87 within the BMP2 protein[125]. Further, extensive studies by Saito et al. showed that the peptide corresponding to residues 7392 of BMP2, with Cys78 and Cys79 substituted by serine and Met89 by threonine (KIPKASSVPTELSAISTLYL), allowed higher induction of ALP activity when compared to the 68-87 peptide (proved on the example of C3H10T1/2 murine fibroblasts)[126]. Authors also showed that the alginate gels conjugated with 73-92 peptide, prolonged ectopic calcification but contrary to BMP2 protein did not promote the formation of trabecular bone structures[127], however significantly promoted repair of rat tibial bone defects[128].
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The 73-92 BMP2 peptide was also studied in combination with the RGD sequence[129,130] and as a combination of three active peptides, including RGD and an osteopontin-derived (OPD) angiogenic peptide[131]. The properties of these peptides were studied while being either anchored to aminoalkylsilane functionalized glass surface or covalently attached to a hydrogel. All these studies revealed synergistic enhancement of osteogenic and vasculogenic differentiation of bone marrow stromal (BMS) cells on the functionalized surfaces. Many studies have shown that not only BMP2, but also BMP4, BMP6, BMP7 and BMP9derrived peptides can regulate osteogenic differentiation of MSCs and bone regeneration[147– 150]. 3.3.
Osteogenic growth peptide modifications
Osteogenic growth peptide (OGP) is a native 14-amino acid peptide (ALKRQGRTLYGFGG) with a primary structure identical to the C-terminus of the H4 histone[115]. The proteolytic cleavage of C-terminal part of OGP generates the YGFGG pentapeptide, known as OGP10–14. This pentapeptide is the minimal OGP-derived sequence which maintains full activity of the OGP. Both peptides, OGP and OGP10–14, play an important role in bone regeneration, ALP activity and matrix mineralization of osteoblast-lineage cells[115,151]. The response of MC3T3-E1 osteoblast cell to OGP and OGP10–14 was studied by More et al.[132]. The study did not focus on coating any particular implant material. Instead it aimed at monitoring of the influence of concentration of the active agent on the osteoblast behavior. 17
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Therefore, both peptides were coupled to oxidized self-assembled monolayer (SAM) to form peptide gradient surfaces. The data obtained from the study showed that OGP peptide induced enhancement of initial MC3T3-E1 cell attachment and their short-term proliferation rate at low densities. The shorter OGP10–14 also showed a potential to stimulate the proliferation of osteoblasts. Generally, the comparison with previously studied growth factors[152–155] shows that the effect of immobilization of OGP is rather short-lived.
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The application of OGP as a surface functionalization agent was studied by Chen et al. who designed an implant coating exhibiting synergic properties of the OGP peptide and fibronectin[133]. The peptide and protein were coprecipitated on the titanium coated with mineralized calcium chloride. The influence of the coating on cell behavior was verified using rat mesenchymal stem cells (rMSC). The data obtained from the study showed the highest cell attachment, proliferation and osteogenic differentiation when the OGP and fibronectin were immobilized on the titanium surface simultaneously. However, the cellular response was also observed on implants coated with each of the molecules separately, showing that the OGP peptide exhibits osteogenic properties. In another study Chen et al. utilized the coprecipitation method to incorporate the OGP peptide into the layer of calcium phosphate mineralized on the titanium surface[134]. MSC cells grown on the mineral-OGP coating construct exhibited significant increase of adhesion, proliferation and activity of ALP.
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Another OGP-based titanium coating was presented by Lai et al.[135] . Authors initially modified the titanium surface with nanotubes obtained via anodization and further immobilized the OGP peptide on the nanotubes with the use of polydopamine to verify if such surface modification exhibits osteoinductive properties. The successful immobilization of the peptide on the surface was monitored by SEM, AFM, XPS and contact angle measurements. The in vitro experiments performed on rat cranial osteoblasts showed that the OGP functionalized nanotube surface promotes the cell spreading and differentiation indicating the studies on OGP peptide as a promising direction for implant engineering. A motif containing OGP-derived sequence, called ALK (ALKRQGRTLYGF) for the purpose of the study, was also used for functionalization of the RADA16 self-assembling hydrogel peptide scaffold[136]. The results of the study showed that the ALK-functionalized scaffold significantly increased proliferation of MC3T3-E1 mouse preosteoblast in comparison to the pure RADA16 peptide scaffold. ALP activity and OCN expression were also increased. The hydrogel functionalization is one of the emerging branches in the development of biocompatible implants. Due to their properties they can be used multidirectionally, as a stand-alone implant material or as coating constructs immobilized on other implant surfaces. 3.4.
Osteopontin-derived peptides
The phosphoprotein osteopontin (OPN), together with bone sialoprotein belongs to the SIBLING (Small Integrin-Binding Ligand, N-linked Glycoprotein) family. Similarly, to all the family members the OPN is expressed in bone tissue and secreted in the extracellular matrix during the processes of osteoid formation and mineralization[156]. The data on functionalization of 18
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implant surfaces with OPN-derived peptides is rather scarce, however the osteogenic properties of the peptides cannot be denied. One of the few studies focusing on functionalization of the implant surface with OPNderived peptides was performed by Fiorellini et al.[112] Authors of the study immobilized the OPN-derived peptide on a nanocoated titanium plasma sprayed (TPS) surface of a dental implant. The osteogenic properties of the coating were verified in an in vivo study in a canine model. The histomorphometric analysis of the implants after extraction showed that the coating accelerated the early stages of osseointegration and bone healing around implants in comparison to uncoated control. This result indicates that the OPN-derived peptide-based coating may shorten the time of treatment and lower the risk of implantation failure.
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Compared to the direct implant surface coating, functionalization of hydrogels (which can be later used as surface coatings) with OPN-derived peptides seems to be more frequently performed. The cell-stimulating properties of one of the peptides were presented by Hamada et al.[111]. In their in vitro study authors found that OPN-derived peptide SVVYGLR stimulates the angiogenesis and migration of endothelial cell lines in 3D collagen gels much stronger than commonly studied VEGF. The peptide was also shown to enhance the adhesion and proliferation of MSCs through the αvβ3 integrin, while suppressing osteoclastogenesis[137]. Noteworthy, it has also been shown that the OPN-derived peptides can induce blood vessel formation in vivo, making them potentially interesting when considering, scaffolds incorporating this biomolecule[157].
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The effect of OPN-derived peptide (DVDVPDGRGDSLAYG) was also studied by Shin et al.[138]. In this study the peptide was linked to oligo(poly(ethylene glycol) fumarate) hydrogels and the authors monitored the modulation of rat MSCs on the modified hydrogel surface. The results obtained from the study showed an increase of attachment strength of the MSCs to the surface. Shin et al. tested also the peptide with the scrambled sequence and demonstrated that the RGD sequence plays the critical role in cell attachment to the biomaterial, indicating the integrin-depending binding[138]. In another work, Shin et al. cultured osteoblasts on the modified hydrogel with the same OPN-derived peptide and observed increase in the levels of ALP activity, secreted osteopontin and ECM mineralization[139]. Another fragment of human OPN was investigated by Lee et al.[140]. The amino acid sequence of the peptide corresponded to the residues 150–177 of human OPN, the region presenting the highest binding affinity to collagen. During the study the peptide was immobilized in alginate hydrogels. Human bone marrow stromal cells (hBMSCs) adhesion, spreading and proliferation on peptide-immobilized gel were significantly increased as compared with control surface. The modified alginate hydrogel induced also new bone formation in the rabbit calvaria defect model. 3.5.
Parathyroid hormone-derived peptides
Parathyroid hormone (PTH) is an 84-amino acid long hormone that regulates calcium homeostasis and plays an important role in bone remodeling[158]. The N-terminal 34 residues 19
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of PTH are responsible for most of its bioactivity[159]. This peptide is known as teriparatide and is used in the treatment of postmenopausal osteoporosis. A few studies have addressed the influence of PTH1-34 on bone regeneration.
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Yewle et al.[117] used an oxidized form of PTH1-34 covalently conjugated to hydrazine bisphosphonates (HBPs), which was used as drug delivery system. The conjugation of the peptide to HBPs increased its affinity to the bone tissue and improved its interactions with preosteoblastic (MC3T3-E1) cells. In another work, Arrighi et al. demonstrated the beneficial effects of PTH locally delivered into bone defects[116]. Authors modified fibrin matrices with PTH1-34, using a plasmin-sensitive substrate sequence as a linker. In vivo, the enzymatic cleavage of the linker results in the release of PTH1-34 in situ. These studies, performed in sheep bone defect model, showed dose dependent bone formation with the evidence of both osteoconductive and osteoinductive mechanisms.
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To our knowledge the studies on PTH-based coatings are scarce[160] and coatings of titanium implants based on PTH 1-34 were not yet designed. However, the PTH 1-34 is readily studied as a drug supplementing the post-implantation recoalescence, where it is used as an agent accelerating bone-implant integration[161,162]. The above described studies regarding the influence of the peptide bone regeneration indicate that the peptide may potentially be a promising factor to be used in implant coatings promoting osseointegration processes.
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4. Peptidomimetics
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Peptidomimetics were developed to mitigate the typical peptide related issues (e.g. susceptibility to protein degradation, poor absorption, pH and salt sensitivity or undesired effects due to interactions with several receptors) while at the same time retaining the activity and selectivity profile of the original “parent” peptide. The success of peptidomimetics is dependent on the understanding of the modus operandi of the parent peptide. This further requires tedious research which covers kinetic studies and solving crystal structures of the peptides and their complexes. Additionally, advances in bioinformatics are often utilized to simulate the active site of the peptide (ligand) while attached to the target molecule (often a receptor). These results serve as the base to design first peptidomimetics which go further through a cycle of structure-activity relationship studies and optimization to yield a compound exhibiting the best possible ratio of desired to side effects. Probably the most explored group of peptidomimetics is based on the structure of the RGD peptide[163,164], described in detail in Section 3.1, of the sequence Arg-Gly-Asp, known for its affinity towards integrins[141]. Among many integrins, the α5β1 and αvβ3 subtypes have been carefully studied and recognized as key mediators of cell adhesion, differentiation, tumor growth and angiogenesis. Several follow up studies have shed more light on their properties, placing the α5β1 subtype in a more favorable position as being responsible for induction of osteogenesis, specifically upregulation of osteogenic marker expression and the activity of ALP[165–167], and most importantly in the context of this review, osseointegration of implants[164,168,169]. The library of nonpeptidic compounds capable of affecting the mentioned integrins, α5β1 and αvβ3, has grown over the years, mostly in response to understanding of key structure-activity dependencies between the RGD peptide and the 20
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integrins[170]. Table 3 lists several recognized examples of such nonpeptidic compounds aimed towards the αvβ3 integrin. Several of these compounds exhibited additional properties, e.g. Compound 1 and 2 exhibited activity towards the αIIbβ3 receptor, though with good selectivity towards αvβ3; Compound 3 was selective against α5β1, while Compound 4 exhibited high affinity towards both αvβ3 and αIIbβ3 receptors. Compound 5, one of the most potent presented here, had only modest selectivity over the αIIbβ3 receptor and much higher when compared to the αvβ3 and α5β1 integrins. Furthermore, several of the mentioned compounds have been evaluated in order to assess their additional pharmacological properties. This was motivated by the fact that the integrin family is involved in numerous important physiological processes, e.g. αvβ3 mediated migration/proliferation of endothelial[171] or muscle[172] cells, and this renders them as therapeutic targets of increasing interest[173]. As an examples, Compound 5 (Table 3) has been evaluated towards angiogenesis and tumorigenesis[174] and it has been shown to decrease blood vessel formation in a mouse matrigel model with the value of ED50 = 0.055 µg kg-1 day-1 and also to inhibit tumor growth on the level of ca. 20%; Compound’s 7 (Table 3) effect on restenosis, the loss of blood vessel lumen, has been intensively studied[175]. It has been found to decrease the neointimal area by 51% in a pig model. While the above-mentioned examples were shown to be promising, further effort has been placed towards assuring higher affinity towards either the α5β1 or αvβ3 integrin. Smallheer et al. focused on utilizing heterocyclic moieties as means of introducing conformational constraints into the designed compounds[183]. One of such compounds (Compound 1; Table 4) has a spirocyclic scaffold incorporating an isoxazoline ring, and showed excellent affinity towards the α5β1 integrin and selectivity when compared to αvβ3 with additional effects on inhibition of angiogenesis[184], tumor cell proliferation[185] and glioblastoma cell apoptosis[186]. Availability of crystal structures followed by docking studies[187,188] led to the development of several potent peptidomimetics. In their works, Heckermann et al.[189] gave several examples of novel integrin-oriented peptidomimetics. The authors thoroughly analyzed activities of several compounds and supported their results with docking studies for optimal design of several analogs. Two of the most prominent examples of these developed compounds, presented in Table 4 (Compounds 2a and 2b) exhibit drastically different selectivity towards either the α5β1 or αvβ3 integrin, with activities in the nanomolar range. This study gives several further examples which underline the importance of structureactivity relationship studies and show how subtle changes in the structure of a compound can tremendously affect its activity. Similar process has been explored by Stragies et al.[190]. In this work, authors reported a library of compounds which activities have been evaluated with respect to the five integrins: α5β1, αvβ3, αvβ5, αIIbβ3 and α3β1. Understanding of crucial interactions followed by conformational analysis yielded a family of α5β1 antagonists (e.g. Compound 3; Table 4), with preferred selectivity over αvβ3 integrin. Compound 4 (Table 4) has been developed by Hackerman et al.[191] on the base of a tyrosine scaffold, already successfully employed against the αIIbβ3 receptor. The resulting peptidomimetic exhibits high activity towards the α5β1 integrin (IC50 = 0.86 nM) and superb selectivity over the αvβ3 integrin (IC50 = 9600 nM). Compound 5 (Table 4) is an example of benzazepine Gly-Asp mimetic, developed by Miller et al.[192], highly potent and orally active against the αvβ3 integrin (KI = 0.9 nM). Interestingly, this compound was also active in models of bone resorption and osteoporosis. An in vitro human osteoclast resorption assay[193] showed inhibition of osteoclast mediated bone resorption (IC50 = 29 nM) and inhibition of parathyroid hormone21
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stimulated calcemic response in a rat model[194] with EC50 = 35 µM. Neubauer et al.[195] prepared several compounds which comprise a β-homotyrosine, shown to be essential for selectivity against αIIbβ3 and α5β1[189], and a methoxypyridine subunit, acting as an arginine mimic, establishing interactions with (αv)-Asp218 (Figure 8). Compounds 6(a) and 6(b), the most potent examples presented in Table 4, both showed subnanomolar activity against the αvβ3 integrin (Compound 6(a): IC50 = 0.86 nM, Compound 6(b): IC50 = 0.65 nM) with low affinity towards the α5β1 receptor. Jumar et al.[196] evaluated properties of Compound 7 (Table 3), a very potent αvβ3 antagonist, with affinity IC50 = 3.2 nM, good selectivity against the α5β1 receptor and ability to inhibit fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF) induced endothelial cell proliferation in vitro.
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The examples mentioned in Tables 3 and 4 are just a fraction of many developed and evaluated peptidomimetics. While the compounds are exhibiting very high affinities and selectivities, the number of actual examples of their application as surface functionalization agents, to-date, is surprisingly small. Examples of peptidomimetics with corresponding functionalized surfaces type and properties are listed in Table 5. The first type of surfaces being functionalized with integrin oriented peptidomimetics were based on poly(ethylene terephthalate) (PET) which is extensively used in medical devices for vascular grafts and cruciate ligament prosthesis[197]. Compounds 1-3 (Table 5) are examples used to functionalize PET surface. The bioactive part of the molecule has been attached to the surface by a spacer/anchor unit. The activity of such constructs has been compared to a surface functionalized with the RGD peptide or its analogs. In the case of Compound 1, the functionalized surface had similar effect on supporting adenocarcinoma epithelial cells (CaCo2) adhesion as the control, comprising PET with RGDS peptide. Comparison to fibronectin and vitronectin coated PET showed that in both cases the adhesion results were not in favor of the peptidomimetic coated PET[198,199]. Compound 2, an αvβ3 antagonist, immobilized on PET also, similarly to Compound 1, promoted adhesion of CaCo2 cells, though performed worse compared to vitronectin[200,201]. Compound 3, very active against the αvβ3 receptor (IC50 = 0.7 nM) and selective with respect to the αIIbβ3 integrin, improved adhesion of human endothelial cells, showing better results compared to the RGDS peptide[202,203]. Functionalization of a titanium-based surfaces is showcased in the following examples. Compound 4, active against αvβ3 (IC50 = 0.72 nM), has been shown to affect adhesion of MC3T3 osteoblasts in a mouse model[204]. Compounds 5 and 6 present a very special case. Both of these compounds exhibit very good affinity and selectivity towards particular integrins: the former is active against the αvβ3 receptor and selective with respect to α5β1 while the latter shows a reversed pattern[205,206]. This allowed the simultaneous use of the compounds and so targeting both receptors at the same time. This combination showed to be very promising in cell differentiation assays and was further studied in vitro and in vivo[207].
5. Summary In this review we approached the topic of implant surface functionalization with various agents, with particular focus on proteins, peptides and peptidomimetics. We have shown that these biomolecules can be successfully immobilized at a surface and through their distinct physico-chemical properties, can change its characteristics in a desired way. While this statement is very optimistic, each stage, starting from biological recognition and understanding 22
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of the role of a given biomolecule, through synthesis or isolation, finishing with surface functionalization, was accompanied by numerous challenges and while many successes have been underlined in this review, definitely there is still a lot of work to be done. We have witnessed how large and complex proteins have been gradually substituted with peptides and finally with peptidomimetics. The initially used fibronectin and osteopontin proteins, complex and difficult to work with, have been “reduced” and their most potent part extracted in the form of peptides. These have been put to the challenge of sufficient surface functionalization while retaining the crucial activity. The most widely known, so-called, RGD peptide has long stood as the molecule of choice in surface functionalization of implants due to its outstanding affinity towards integrins[141] and ease of synthesis[208]. Understanding the fragility of such peptides, especially when they are used in harsh in vivo conditions, led to the development of peptidomimetics, for which most of the peptide-related limitations do not apply. Peptidomimetics exhibit high potency and target binding affinities, combined with excellent selectivity and stability to enzymatic degradation or temperature and pH fluctuations. Following the path of the RGD peptide, most of today’s peptidomimetics also display the same modus operandi. Among many, works published by Biltresse et al.[200], Maglot et al.[185], Rerat et al.[202], Nuebauer et al.[195] and Fraioli et al.[207] clearly underline the trends and importance of integrin targeting peptidomimetics. Further works by Mas-Moruno et al. pinpoint the status of peptidomimetics[209] and their significance as surface coating agents[18] in an excellent way.
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Given the current socio-economic factors, increasing life expectancy and prolonged activity, the development of medical implants and related technologies will be an area of increasing importance. We are already witnessing a shift towards new type of implant materials. Magnesium and its alloys[210,211], cobalt–chromium alloys[212,213], groups of ceramic[214,215] and composite materials[216] – all these options are being explored extensively and are bound to face, among many others, the problem of implant osseointegration. This paradigm shift in implant material development will require a thorough compatibility reevaluation of all the currently used coating strategies along with forging of new appropriate solutions. Therefore, we may expect that as a result of a marriage between biology, biochemistry and material engineering, the research on implant surface functionalization will intensify, and new biomolecules and targets will be developed in the near future.
6. Authors contributions SL was responsible for conceptualization, figure design and preparation. PJ and SL were responsible for data collection, original manuscript writing, review and editing. JW was responsible for original manuscript writing. SRM was responsible for revision support.
7. Declaration of interest None.
8. Acknowledgements We would like to thank Polish National Agency for Academic Exchange for the financial support within the “Polish Returns” programme (PPN/PPO/2018/1/00071/DEC/1) and National 23
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Center for Research and Development, TECHMATSTRATEG2/406384/7/NCBR/2019).
Poland
(grant
No.
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10. Tables
Table 1. Summary of proteins used for implant surface modification. In vitro study
In vivo study
Human adiposederived stem cells (hASC)
Human mesenchymal stem cells (hMSC)
Adhesion (MG63 and hasc), differentiation Femoral and tibial (hasc only), mineral deposition (from MG63 bone (rat) and hasc) in vitro ↗; bone-implant contact and interfacial strength in vivo ↗ Effective attachment and migration in vitro; Femoral bone osteoconduction and osseointegration in vivo (rabbit) ↗
hMSC
Tibial bone (rat)
Human dermal fibroblasts (HDF)
HeLa cell culture
-
MC3T3-E1 preosteoblasts
Mandibular premolar area (dog)
MG-63
ST2 stromal cell line
-
Human gingival fibroblasts (HGF)
-
Jo
Bone sialoprotein Primary human osteoblasts (hOb), L929 mouse fibroblasts
BMP2
-
Covalently via gamma irradiation or glutaraldehyde Physical adsorption, covalently via silanization
[43] [44]
Covalent binding or physical adsorption
[45,46]
[50] [51]
Proliferation, adhesion, differentiation and ALP activity ↗
Physical adsorption
[51]
Proliferation, adhesion, and differentiation ↗
Covalently via silanization
[52]
Osteogenic activity and osteoblastic differentiation ↗; TF4, FRA1, RUNX2, OSX gene expression ↗
Covalently via silanization
[53]
Cell migration, calcium deposition ↗; upregulation of RUNX2 expression
Physical adsorption, covalently via silanization
[54]
Physical adsorption
[55]
Proliferation, vasculogenesis and ALP activity in vitro ↗; proliferation of osteoblasts and endothelial cells in vivo ↗ Proliferation of endothelial cells in vitro ↗; ossification ↗, larger trabeculae, angiogenesis degree in vivo ↗
Tibial bone (rat)
-
[42]
Covalently via amine groups present after glow plasma discharge treatment
C2C12 mouse muscle myoblasts
MC3T3-E1 preosteoblasts
Covalently via silanization
Adhesion, migration, and proliferation in vitro ↗; stability of implants in vivo ↗
Femoral and tibial Proliferation ↗, bone formation and bone (rabbit) osseointegration in vivo ↗
BMP9
[41]
[49]
MC3T3-E1 preosteoblasts
-
Covalently via photoactive crosslinking
Physical adsorption
Tibial bone epiphysis (sheep)
hMSC
[40]
[48]
Endothelial cells EC2; MC3T3-E1 preosteoblasts
BMP7
Covalently via photoactive crosslinking
Via spin coating or MAPLE
Tibial bone (rat)
-
Ref.
[47]
Primary rat osteoblasts (ROB)
hASC
Immobilization method
Covalently via electrodeposition
-p
-
re
L929 mouse fibroblasts
ro
Femoral bone (rabbit)
hMSC
VEGF
Adhesion, proliferation and activation of fibroblasts ↗ Attachment spreading, proliferation and differentiating in vitro ↗; implant stability in vivo ↗ Adhesion, spreading, viability and proliferation ↗ high coating uniformity; preserved collagen structure improving homogeneity and morphology of the surface Adhesion and spreading (much stronger in case of fibronectin) ↗
-
hMSC
Fibronectin; Vitronectin
Proliferation and osteogenic differentiation in vitro ↗; fine osteointegration in vivo
of
Human bone carcinoma MG-63 line (MG-63), hASC
lP
Type I collagen
Cytocompatibility, rapid adherence, spread area ↗
-
na
ELP
Influence on cells/tissue
ur
Protein
ALP activity in vitro and in vivo ↗ Osteogenic differentiation and calcium deposition ↗; expression of OCN, RUNX2 and COL1 ↗; ALP activity ↗ Enhanced osteogenic differentiation; calcium deposition and expression of OCN and OPN ↗ Osteoblast differentiation ↗; expression of RUNX2, OSX, OC and BSP genes ↗
Noncovalent adsorption a to the surface of SiHA coated titanium surface Noncovalent electrostatic binding with heparin linking layer Hybridization with short nano-anchored oligomers Covalent binding with b pGMA as a linker
[56]
[57] [58] [59]
Covalent binding with fibronectin as a linker Protein not immobilized; cells grown on the titanium surface
[60] [61]
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Albumin
-
b
Alternating AC-EPD
c
[62]
c
ur
na
lP
re
-p
ro
of
- silicon substituted hydroxyapatite, - glycidyl methacrylate, - current electrophoretic deposition; ↗ - increase
Jo
a
-
Studies on living cells not performed; Facilitates protein adsorption and calcium phosphates accumulation ↗ on the implant surface (incubated in media simulating body fluids); coating improves also implant corrosion resistance
submerged in proteincontaining medium
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Table 2. Summary of peptides used for implant surface modification. Peptide
In vitro study
In vivo study
Hipbone-derived human osteoblast
Tibial bone (rabbit)
Human sarcoma osteogenic (SaOS-2)
-
SaOS-2
-
Rat bone marrow stromal cells
-
MC3T3 preosteoblasts
-
Influence on cells/tissue No significant increase of osseogenic properties over linear RGD in vitro; vertical bone apposition in vivo ↗ Spreading, proliferation, viability, and the formation of actin cytoskeleton and focal contacts of the SaOS-2 cells ↗ Attachment and spreading ↗
RGD
MG63
BMP268-87 BMP273-92
Calf muscle (rat) Calf muscle (rat) Calf muscle (rat)
C3H10T1/2 murine fibroblasts -
Tibial bone (rat)
-
ur
BMP273-92
Bone marrow stromal cells (BMS) Human bone marrow mesenchymal stem cells (hBMSC)
-
Jo
BMP273-92
-
BMP273-92
BMP273-92
Tibial bone (rabbit)
-
of
ro
Adhesion, RUNX2, OCN and BSP expression, ALP activity, calcium deposition in vitro ↗; Bone formation and osseointegration in vivo ↗
-p
Cortical bone (rat)
Expression of ALP, COL1, OSX, TRAP and pace of bone deposition ↗ Adhesion, ALP activity and expression of RUNX2, OPN, OC ↗ Calcification and trabecular bone formation ↗ ALP activity in vitro ↗; calcification in vivo ↗
re
Rat bone marrow stromal cells
lP
BMP274-91
Subcutaneous pockets on the back (mouse)
na
Type I collagenderived peptide (GFOGER) "Knuckle” peptide
hMSC
Adhesion and expression of BMP-2, RUNX2, BSP and OPN ↗ Adhesion, differentiation, ALP activity, OCN expression ↗ Osteogenic markers’ expression, ALP activity, type I collagen deposition and matrix mineralization in vitro and in vivo ↗
Prolonged ectopic calcification Differentiation of osteoblast precursor cells into osteoblasts, activation of osteoblasts to promote the repair of bone defects ↗ Osteogenic differentiation and mineralization ↗
-
Differentiation, expression of RUNX2 ↗
BMP273-92
Bone marrow stromal cells (BMS)
-
Differentiation, ALP activity, mineralization, expression of OPN and vasculogenic markers ↗
OGP and OGP10–14
MC3T3-E1 preosteoblasts
-
Attachment and proliferation ↗
OGP
Rat mesenchymal stem cells (rMSC)
-
Attachment, proliferation and osteogenic differentiation ↗
OGP
rMSC
-
Adhesion, proliferation and ALP activity ↗
OGP
Rat cranial osteoblasts
-
Spreading, differentiation, RUNX2, ALP, COL I, OPN, OCN expression ↗
MC3T3-E1 preosteoblasts
-
Proliferation, APL activity, OCN expression ↗
-
Mandibular premolars and first molar
Early stages of osseointegration and bone healing around implants ↗
OGPderived ALK motif OPN/OPNderived OC-1016
Immobilization method
Ref.
Covalently via silanization
[119]
Covalently via DOPA containing, mussel derived peptide Covalently via electrodeposited PEG Covalently via silanization Covalently with the use of EDC
[120]
[121] [122] [123]
Covalently via methacrylate groups
[124]
Physical adsorption
[110]
Covalently via polydopamine
[114]
Physical adsorption
[113]
Covalently with the use of EDC Covalently with the use of EDC Covalently with the use of EDC
[125] [126] [127]
Covalently with the use of EDC
[128]
Covalently via click chemistry
[129]
Covalently via silanization
[130]
Covalently via oxyme ligation and click chemistry Covalently via click chemistry Physical adsorption or coprecipitated on the titanium coated with mineralized calcium chloride Coprecipitated as in the example above Covalently via polydopamine Direct peptide coupling via Cterminus -
[131] [132]
[133]
[134] [135] [136]
[112]
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(dog)
OPN171-177
Transformed rat lung endothelial cells (TRLEC) Human periodontal ligament fibroblasts (hPLF), primary human gingival fibroblast (hGF), human umbilical vein endothelial cells (HUVEC), RAW264.7 murine macrophage-like cells, murine bone marrow monocyte/macrophage (BMM), (CHO)-K1 chinese hamster ovary cells, hMSC
OPN137-151
rMSC
OPN150–177
hBMSC
PTH1-34
MC3T3-E1 preosteoblasts
-
Calvarial bone (rat)
Calvarial bone (rabbit)
Angiogenesis, activation and migration ↗
Immobilized on PEG resin
[111]
Adhesion, proliferation in vitro ↗; new bone formation in vivo ↗; suppressed osteoclastogenesis in vivo
Grafting on PEG-PS copolymer
[137]
Attachment, migration, differentiation ALP and OPN expression and mineralization ↗ Adhesion, spreading and proliferation in vitro ↗; new bone formation in vivo ↗ Cell interactions at the bone surface, mineral binding affinity of PTH ↗
of
OPN171-177
-
Femoral and High matrix stability and bioactivity in humeral bone vitro; healing potential in vivo ↗ (sheep) EDC - 1-ethyl-(dimethylaminopropyl)carbodiimide, PEG - polyethylene glycol, PS – polystyrene, ↗ - increase
[140] [117] [116]
ur
na
lP
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-p
ro
UMR-106 cells
[138] [139]
Jo
PTH1-34
Covalently via crosslinking reaction Covalently with the use of EDC Covalently via Nterminus Covalently; plasminsensitive substrate sequence as a linker
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Table 3. Examples of αvβ3 integrin antagonists. Structure
IC50/KI [nM]
Ref.
1
αvβ3 KI = 2
[176]
2
αvβ3 IC50 = 20
[177]
of
Compound
αvβ3 IC50 = 1.1
[178]
αvβ3 IC50 = 0.52
[179]
αvβ3 IC50 = 0.057
[180]
αvβ3 KI = 3.5
[181]
αvβ3 IC50 = 0.56
[182]
-p
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3
lP
re
4
7
ur Jo
6
na
5
Mes = 2,4,6-trimethylphenyl, Cbz = benzyloxycarbonyl
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Table 4. Examples of α5β1 and αvβ3 antagonists.
IC50 α5β1 [nM]
IC50 αvβ3 [nM]
Ref.
1
0.18
49
[183]
2
264 (a) 0.7 (b)
1.2 (a) 279 (b)
[189]
Structure
ro
of
Compound
0.54
~30000
[190]
9600
[191]
[K ] I
[192]
re
-p
3
0.86
[K ] I
1000
0.9
Jo
5
ur
na
lP
4
6
126.5 (a) 108 (b)
0.86 (a) 0.65 (b)
[195]
7
421
3.2
[196]
Mes = 2,4,6-trimethylphenyl, Cbz = benzyloxycarbonyl [KI] – affinity given as a KI value
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Table 5. Examples of peptidomimetics used in implant surface coating. Surface type
Effect
Ref.
1
PET
- Promoting adhesion of CaCo2 cells, - < FN
[198,199]
2
PET
- Promoting adhesion of CaCo2 cells, - < VN
[200,201]
Structure
of
Compound
[202,203]
- Improve adhesion of mouse MC3T3 osteoblasts
[204]
ro
- Improve adhesion of human endothelial cells, - > RGDS
PET
re
-p
3
Jo
5
ur
na
lP
4
6
Ti6Al4V
- Selective cell adhesion in fibroblast cell lines, Ti6Al4V attachment, spreading TiO2 and proliferation of
[205,206]
sarcoma osteogenic SaOS-2 cells
- Selective cell adhesion in fibroblast cell lines, Ti6Al4V attachment, spreading and proliferation of TiO2
[205,206]
sarcoma osteogenic SaOS-2 cells
Spacer/anchor units are depicted in red, FN = fibronectin, VN = vitronectin, RGDS = Arg-Gly-Asp-Se peptide
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11. Graphical Abstract
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Figure 1. Knee and hip replacement surgeries in selected OECD countries. In most of the OECD members, the number of surgeries has increased rapidly since year 2000 both for knee (left) and hip (right) replacement.
Jo
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Figure 2. Summary of factors contributing to overall implant success.
Figure 3. Covalent binding of the RGD-containing ELP to the titanium implant surface. The presence of the diazirine moiety in the RGD-ELP molecule, allows generation of a carbene species after UV exposure, which interpose into the O-H bonds on the titanium surface. The amount of hydroxyl groups increases after the exposure of the titanium surface to UV light[66].
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Figure 4. The process of immobilization of proteins on a titanium surface via silanization. The first step involves treatment of the activated titanium with aminopropyltriethoxysilane (APTES). This facilitates positioning of the amine group on the surface of the metal, in such a way, that the appropriate protein can be bound to them in the second step.
Figure 5. The process of coating titanium surfaces with growth factors. For this purpose, either heparin (left) or poly-L-lysine (right) are used to anchor the growth factor to the activated titanium surface.
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Figure 6. Peptide based strategies for bone tissue engineering.
Figure 7. Various strategies used in engineering of implant coatings. Bare titanium is prone to bacterial film formation. Antiadhesive agents, bactericidal peptides and active agent release are the most common approaches addressing typical issues related to implant use. Novel approaches comprise multi-functioning constructs combining several factors, focusing on antiadhesive and bactericidal properties or facilitating eukaryotic cell adhesion (e.g. RGD peptide).
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Figure 8. Binding modes for α5β1 integrin of a) compound 6(a) and b) compound 6(b). Compounds 6(a) and (b) are shown as yellow and green sticks respectively, the α5 and β1 subunits are depicted as red and blue. The metal cation is represented as a magenta sphere. Figure reproduced with permission from ref. [195]. Copyright 2014 American Chemical Society.
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Highlights:
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Proteins, peptides, peptidomimetics as implant surface functionalization agents Methods for implant surface functionalization Broad biological effect evaluation, including both in vitro and in vivo studies
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Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8