Nanostructured biomaterials for regenerative medicine: Clinical perspectives

Nanostructured biomaterials for regenerative medicine: Clinical perspectives

3

Bin Zhanga, Jie Huanga, Roger Narayanb a Department of Mechanical Engineering, University College London, London, United Kingdom, bUNC/NCSU Joint Department of Biomedical Engineering, Raleigh, NC, United States

3.1 Introduction Regenerative medicine holds great promise for developing biological substitutes, which can replace or regenerate human tissues [1]. Mimicking native tissue environment using bioengineered scaffolds, cells, and growth factors is a promising strategy to overcome the limitation of tissue volume supply [2, 3], the risk of immune reaction and disease transmission issue [4–6] in autografts and allografts clinical operations. Biomimetic tissue including hard tissue, mainly bone, and soft tissues, such as cartilage, blood vessels, skin, tendon, muscle, and heart have been successfully engineered and implanted in vivo [7–13]. Those bioengineered tissues serve as a template to guide cell organization and growth and allow diffusion of nutrients to transplanted cells, which ultimately leads to the generation of complex architectures which mimic native tissues. Recent advances in bioengineered tissue mimic the characteristics of natural tissue at the nanometre scale, have shown the benefits for cell attachment, proliferation, differentiation, and matrix deposition in vitro and induction in vivo [14]. Since cells dynamically interact with their local environment at the nanoscale [15], it is necessary to control the properties of engineered tissues at nanoscale lengths for more analogous to natural tissues. Some studies have focused on (a) use of nanostructured materials (e.g., incorporation of nanoparticles in a polymer matrix to mimic the nanocomposite architecture of natural tissue) and (b) manipulation of the mechanical properties (e.g., Young’s modulus and strength) of the scaffolds. Also, nanostructured biomaterials can decrease inflammatory response and increase wound healing in comparison to conventional biomaterials, possibly due to their high surface energy affecting protein adsorption and cell adhesion [16, 17]. However, despite the significance of nanoscale material on a cellular response, how nanostructure properties (e.g., mechanical property, surface energy, and surface roughness) regulate the behaviors of cells in vitro or in vivo are yet to be clearly understood. In this review, the role of nanostructured ceramics, metals, polymers, and their nanocomposites in tissue regeneration and regenerative medicine is considered. Also, the clinically approved nanostructured products for regenerative tissue applications are summarized. Nanostructured Biomaterials for Regenerative Medicine. https://doi.org/10.1016/B978-0-08-102594-9.00003-6 © 2020 Elsevier Ltd. All rights reserved.

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Nanostructured Biomaterials for Regenerative Medicine

3.2 Ceramic nanostructures There is a high degree of demand for biomaterials to repair large defects of hard tissue (i.e., bone and tooth) [18]. Bone tissue has self-healing functions. However, it is difficult to restore a large bone defect size beyond 2–2.5 times of the affected bone diameter without using a graft or scaffold [19]. The bone tissue comprises an organic and inorganic compound. On the other hand, bone is mainly made up of the inorganic mineral crystallites hydroxyapatite (HA) at nano-sized scale [20, 21]. These crystallites are deposited and arranged in parallel to the collagen fibers thus providing the rigidity and stiffness to the hard tissue [22]. Fig. 3.1 shows the macrostructure, microstructure, and nanostructure of bone tissue. The ratio of organic to inorganic phases in bone tissue affects the mechanical property [23]. The collagen fibers retain similar characteristics to polymers, which give bone tissue the flexural resilience and toughness by decreasing the brittleness of the mineralized phase HA. Conversely, the mineral crystallites HA provides bone tissue the mechanical strength and stiffness.

3.2.1 Nano-hydroxyapatite Calcium phosphates, especially hydroxyapatites [HA, Ca10(PO4)6(OH)2] has already been successfully used as bone graft due to its similarity with the mineral crystal of bone. Robinson [24] reported an average crystal size in human bone tissue are roughly 50 nm long, 25 nm wide, and 2–5 nm, thick. These HA nanoparticles constitute approximately 70% of the native bone. Nano-sized hydroxyapatite (nano-HA) in the rod

Osteocytes

Osteon: 100–500 µm

Blood vessels

Collagen fibril

(A)

Macrostructure

(B)

Collagen fiber

Nano hydroxyapatite

Collagen molecule

g Microstructure

(C)

Nanostructure

Fig. 3.1  The macro, micro, and nanostructure of the bone tissue. (A) At the macrostructure level, bone consists of cortical bone and cancellous bone. (B) At the microstructure level, there are many repeated osteon units in cortical bone. In the osteons, the blood vessels and nerves are surrounded by concentric layers of collagen fibers. (C) Collagen fibers consist of repeating individual collagen fibrils (30–300 nm), and the collagen fibril is composed of a single collagen molecular species. Nanohydroxyapatite distribute in the collagen fibers and increase the stiffness of the bone. This figure is modified based on T. Gong, J. Xie, J. Liao, T. Zhang, S. Lin, Y. Lin. Nanomaterials and bone regeneration. Bone Res. 3 (2015) 15029.

Nanostructured biomaterials for regenerative medicine: Clinical perspectives49

or plate shape is commonly used in bioengineered tissue to mimic bone minerals, as shown in Fig. 3.2. Since Levitt et al. [26] developed apatite bioceramics and suggested the possibility of medical applications, HA has been widely used for clinical applications. HA material in dense or porous forms have been clinically used for alveolar ridge augmentation [27], the increment of spine fusion and repair of bone defects [28–30]. Many studies indicate that involving nano-sized HA could resemble bone minerals and better osteoconductivity would be achieved. Since engineered nano-architecture features a high surface area to volume ratio, it can systematically expose cells to multiple biological components with different functionalities [31]. Webster et al. [17] have shown the enhanced osteoblast adhesion and protein adsorption in vitro on the nano-size HA (less than 100 nm) compared to the traditional

Fig. 3.2  Transmission electron microscopy of (A) plate-shaped nano-HA in clusters; (B) singular plate-shaped nano-HA (both side lengths are 20 and 50 nm, separately); (C) roundshaped nano-HA in clusters; (D) singular round-shaped nano-HA (diameter is approximately 20 nm) [25].

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Nanostructured Biomaterials for Regenerative Medicine

micro-size HA. Liao et al. [32] indicated nano-sized HA influence the formation of adsorbed vitronectin which can subsequently mediate osteoblast adhesion in  vitro. Goené et  al. [33] investigated the influence of nano-HA treatment to the titanium implant surface with a clinical trial, which has shown that there was an increase in the extent of bone development in human maxillae after 4 weeks of healing. Although there is the ongoing discussion about the safety concern associated with nano-sized HA as it is likely that nanoparticles will transport across cell membranes to interact with DNA and RNA, it is not clear yet that the health consequences [34]. Scheel and Hermann [35] indicated there is no clear evidence that nano-sized HA is toxic. Furthermore, Kalia et al. [25] compared to the plate and round shapes of nano-HA in vitro, which demonstrated that the round shape nano-HA had relative higher alkaline phosphatase activity and matrix vesicle release than the plate-shaped nano-HA. The results indicated that the shape of nano-HA could be related to osteoblast viability and activity. Jang et al. [36] assembled a nanochannel network with nano-HA material, which provided both sufficient mechanical strength and efficient nutrient supply for bone cell growth and differentiation in  vitro. Further, in  vivo/clinical trials are required to establish the efficacy of the structure of nano-HA. Regardless of HA nanoparticles, another inorganic phase of human bone tissue is whitlockite nanocrystallites [WH: Ca18Mg2(HPO4)2(PO4)12]. These two crystals are distributed in different ratios depending on the regions of bone tissue, implying that HA and WH nanoparticles have distinguished biological roles [37]. Jang et al. [38] synthesized HA and WH nanoparticles, to mimic the inorganic composition of bone. The synthesis substance had the enhanced proliferation of bone cells and induced rapid regeneration of bone tissues in vitro and in vivo, compared with pure HA nanoparticles [39]. Thus, controlling HA and whitlockite nanoparticles composition distribution at the nanoscale is important for mimicking native bone tissue.

3.2.2 Nano-bioactive glass In addition to being osteoconductive and biocompatible, bioactive glass has also been investigated for use as a bone substitute. The 45S5 bioactive glass composition was invented by Hench et al. [40], which could release active biological ions and sequentially bond to living bone tissue. Merwin [41] induced the first application of 45S5 bioactive glass to replace the small bones in the middle ear for curing conductive hearing loss. Since then, 45S5 bioactive glass has the approval of the US Food and Drug Administration (FDA) and is successfully implanted in thousands of patients, mainly in bone tissue engineering application [42]. Bioactive glasses have been used as a coating in biomedical devices, dental fillers, tissue engineering scaffolds, and drug-delivery system [43–46]. Also, various bioactive glass compositions have been proposed, which contain no sodium or have additional elements incorporated in the network of silicate structure. For instance, the incorporation of silver [47] and zinc [48] in the silicate network, have been investigated to develop antibacterial materials. Waltimo et al. [49] have shown the stronger antimicrobial effect of nanoparticles of 45S5 (20–60 nm) than micro-sized particles by killing more Enterococcus faecalis. One

Nanostructured biomaterials for regenerative medicine: Clinical perspectives51

of the possible reasons is the reduction in size to nanometer scale can increase the surface area of nanoscale bioactive glasses, which allows a faster silica release and solution pH elevation. There are also other advantages of reducing the particles into nanosize, such as produce thin bioactive coating and reinforce polymeric nanofibers [16]. Bioactive glass exhibit potential benefit in comparison to HA regarding bioactivity. For instance, Wheeler et  al. [50] shown that the dissolution products from bioactive glasses upregulate the expression of genes that control osteogenesis in  vivo. However, the clinical application of bioactive glass is still lag than HA regarding commercial success. The possible reason can find from the literature that they generally performed poorly regarding mechanical strength and fracture toughness, therefore, not suitable for all grafting applications. Further, the development of apatite-wollastonite ­glass-ceramic was shown to possess a higher mechanical strength but lower fracture toughness than human cortical bone [51]. They could not involve cyclic loads with the host bone. Thus, the development of tougher scaffolds is required that still have all the bioactive properties of bioglass 45S5. The possible engineering solution is the development of composite materials [52], for example, incorporation of bioglass nanoparticles with a polymer, as discussed in detail further.

3.3 Polymeric nanostructures It is well known that native extracellular matrix (ECM) is the nanoscale dimensions of the physical structure, which is mainly composed of collagen fibers, between 50 and 200 nm in diameter [53]. Collagen fibers would regulate cell attachment, proliferation, and differentiation, and thus the design of engineered tissue scaffold should close mimic this structure. The polymeric nanofibers nonwoven scaffold is among the most promising biomaterials for the native fibrillar ECM. There are various polymeric materials have been nanofibrous, which mainly classified as natural materials, such as collagen, silk, alginate and chitosan, and synthetic polymers, such as poly (lactic acid) (PLA), poly (ethylene oxide) (PEO), and poly (ɛ-caprolactone) (PCL).

3.3.1 Fabrication of polymeric nanostructures Various methods have been applied to fabricate polymeric nanostructures, such as electrospinning, phase separation, and molecular self-assembly. The principle of electrospinning is applied an electrical field between the metallic nozzle and collector, to draw a thin liquid polymer solution. The fabricated polymer fibers can be produced in a range of nanometers to micrometers [54]. A variety of polymeric biomaterials, such as PEO, PLA, PCL, silk, chitosan, collagen, and alginate have been applied to form polymeric nanofibers using electrospinning [55–61]. In general, the minimum diameter of 9 nm fibers can be achieved using electrospinning, which can mimic the nanostructure of natural collagen fibers [59]. However, it is difficult to create 3D porous scaffolds using electrospinning. Moroni et al. [62] have applied electrospinning to combine with 3D fiber deposition to fabricate 3D scaffolds, but further investigation is required to optimize the scaffold structure.

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Phase separation, as another method, is frequently applied to fabricate 3D engineered tissue scaffolds. The porous polymer scaffolds can be manufactured by removing the solvent from the polymer solutions through free-drying [63]. Polymer scaffolds fabricated by the method of phase separation, have a sponge-like porous morphology. The micro or nanoscale polymer fibers can be obtained by controlling the solvent, polymer concentration, gelation time, and temperature. Yang et al. [64] fabricated poly(l-lactic acid) (PLLA) nanostructured porous scaffold using phase separation methods, with the minimum nanofibers diameter as 50 nm, which is comparable to those collagen fibers in the native ECM. The nanostructured fibers can also be fabricated by molecular self-assembly which is mediated by noncovalent bonds (e.g., hydrogen bonding as well as electrostatic, hydrophobic, and van der Waals interactions) [65, 66]. The fiber diameter can be reached less than 6 nm by molecular self-assembly, which is smaller than those fabricated using electrospinning and phase separation [67]. However, the mechanical properties of self-assembled scaffolds should be improved before using in tissue engineering application [68]. As Fig. 3.3 shown, cell spread as two-dimensional (2D) structure when seeding on microporous or microfibrous scaffolds. While, when cell cultured on nanostructured scaffolds, cells can make higher zone contacts which would lead to improved cell attachment and cellular interactions. The possible reason is that the higher surface area to volume ratio for the adsorption of proteins and binding of ligands [69]. Micropore scaffold

Microfiber scaffold

Cell binding

Scaffold architecture

Nanofiber scaffold

(A)

(B)

(C)

Fig. 3.3  Cells flatten and spread to microporous (A) or microfibrous scaffold (B), while cell exhibited more binding sites to nanofiber scaffold (C) [69].

Nanostructured biomaterials for regenerative medicine: Clinical perspectives53

3.3.2 Functionalized polymeric nanostructures 3.3.2.1 Controlling structures of polymeric nanostructure Cells are regularly oriented in native tissues, which is vital for tissue function. Thus, it is essential to control the orientation of the cell in tissue engineered scaffolds to mimic native tissue. In general, nanofibers fabricated on a static platform using electrospinning have a randomly oriented nonwoven fiber matrix. However, if the nanofibers are collected with a rotating collector, the aligned nanofiber matrix can be produced [70]. As shown in Fig.  3.4, Teixeira et  al. [71] found that human corneal epithelial cells could align and elongate along with the nanostructure substrate. Thus, the application of aligned polymer nanofibers can control cell orientation. Table 3.1 selectively summarized the functional nanopolymers for the application of tissue regeneration in vitro and in vivo. Those functional nanomaterials have successfully generated similar or even better tissue functions to stimulate cells to repair tissues. For instance, Dalby et al. [72] used nanoscale polymethylmethacrylate (PMMA) to stimulate stem cells toward osteogenic differentiation in  vitro without osteogenic supplements; these cells showed similar levels of the bone mineral to those of cells cultured with osteogenic media. However, compared with cells treated with osteogenic media, the topographically treated stem cells have a distinct osteogenic differentiation. McMurray et  al. [84] indicated that nanoscale surface topographies could determine cell fate and functions. Nanoscale square lattice symmetry patterns can promote the growth of stem cells and the retention of multipotency in vitro. Similarly, Badrossamay et al. [76] successfully controlled heart tissue constructs in vivo to mimic the ECM structure of myocardial tissue and the induced alignment of rat v­ entricular

(A)

(B)

10 µm 500 nm

Fig. 3.4  SEM images of cells cultured on the nanostructured substrate. (A) Human corneal epithelial cells aligned along the patterns of nanotopography. (B) Filopodia extend along the patterns of grooves and ridges with nano feature dimensions. This figure is modified based on A.I. Teixeira, G.A., Abrams, P.J. Bertics, C.J. Murphy, P.F. Nealey. Epithelial contact guidance on well-defined micro-and nanostructured substrates. J. Cell Sci. 116 (2003) 1881–1892.

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Table 3.1  Functional nanopolymers for use in tissue regeneration Nanopolymers

Functionality

Results obtained

Bone

Nanofibrous PMMA with the various surface pattern

Modulate stem cell differentiation

Biodegradable poly(lactideco-glycolide) nanoparticles with osteogenesis related growth factors Peptide amphiphilic nanofibers functionalized with glycosaminoglycans (GAG) molecules Glycopeptide nanofibers selfassembled supramolecular GAGs

Nanoparticles could be as delivery carriers for growth factors

A topographically treated stem cell has a distinct osteogenesis differentiation The hMSCs transfection of PLGA nanoparticles significantly enhanced osteogenesis Enhanced aggregation of stem cells and deposition of cartilage-specific matrix elements Induced chondrogenic differentiation of MSCs and enhanced formation of hyaline-like cartilage Induced alignment of rat ventricular myocytes along with the nanofibers Improved vascularization of the scaffold with upregulation of gene expression related to ECM remodeling, after implanted

Cartilage

Heart

Highly aligned hybrid polymer-protein nanofiber Nanofibrous collagen scaffold

Mimicking composition, structure moreover, the function of the ECM, and chondrogenic differentiation Mimicking bioactive functions of natural cartilage tissue Controlled nanoscale surface topographies mimicking the function of myocardial tissue Mimicking composition of myocardial connective stroma and delivery of cardiomyocytes

Experimental types

References

In vitro

[72]

In vitro and in vivo

[73]

In vitro

[74]

In vitro and in vivo

[75]

In vivo

[76]

In vitro and in vivo

[8]

Nanostructured Biomaterials for Regenerative Medicine

Tissue

Tendon Skin

Vessel

Mimicking composition and structure of skeletal muscle basal lamina

Tenascin-C was incorporated into self-assembling nanofibers formed by peptide amphiphiles (PAs) Laminin mimetic peptide nanofibers

Facilitating the regeneration of skeletal muscle

Electrospun aligned poly-llactic acid nanofibers 3D PCL/collagen multilayered nanofibrous scaffold Multilayer nanofilm composed of hyaluronic acid and poly-l-lysine

Mimicking composition and structure of tendon tissue Mimicking composition of skin tissue with multiple types of cells Mimicking epidermal-dermal composition and structure of skin at the nanometre scale

Ar plasma-treated nanostructured surface of polymers Nanopores in the vessel wall mimicking a vascular bed

The surface roughness of the polymers changes with the plasma treatment Mimicking composition of vessel tissue

Enhanced cellular gene expression related to the skeletal muscle-specific marker Promoted satellite cell activation, accelerated myofibrillar regeneration, and reduced the time for muscle tissue repairing Upregulated tendon-specific genes Produced skin tissues with bilayer-epidermal and dermal layers Promoted adhesion of adhesion of keratinocytes, enhancing epidermal protective barrier function of the skin The increase of vascular smooth muscle cells cell adhesion Enhanced permeability and intercellular interference

In vitro

[77]

In vivo

[78]

In vitro and in vivo In vivo

[79] [80]

In vitro

[81]

In vitro

[82]

In vitro and in vivo

[83]

Nanostructured biomaterials for regenerative medicine: Clinical perspectives55

Muscle

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Nanostructured Biomaterials for Regenerative Medicine

myocytes along with the nanofiber. Thus, nanoscale structural cues can further control the function of tissue constructs. Aligned nanofibers are especially useful in guiding cellular orientation to mimic the anisotropy of natural tissues, including heart, nerve, tendon, skin, and blood vessels. Yin et al. [79] seeded human tendon progenitor cells on aligned PLLA nanofibers that recapitulated parallel collagen fibers in the tendon. These cells expressed a higher level of tendon specific genes compared with cells grown on random fibers in  vitro. Also, in vivo experiments, there were spindle-shaped cells formed on the aligned nanofibers and tendon-like tissue. Monteiro et al. [81] developed multilayer nanofilm composed of hyaluronic acid and poly-l-lysine on top of a hyaluronic acid scaffold by using layer-by-layer assembly for mimicking epidermal-dermal composition and structure of the skin at the nanometre scale. The results showed the promoting adhesion of keratinocytes, enhancing the epidermal protective barrier function of the skin. Further in vivo experiments are needed to establish an effective combination of hierarchical structures for the multifunction for tissue regeneration. Nanochannels in natural tissues are also vital for maintaining the activity of cells, as they provide transport paths for oxygen and nutrients [85]. Zhang et al. [83] incorporated nanochannel in vascularized cardiac tissue constructs and bone scaffolds in vivo. They used self-assembled and porogen methods to enhance permeability and permit cellular crosstalk while maintaining mechanical properties. Core-shell nanofiber structure can be fabricated using modified electrospinning technology. This technique uses a designed nozzle containing a core opening and a surrounding annular opening. This technique is commonly applied to embed growth factors [86] into the core of biodegradable polymer nanofibers, producing polymer nanofibers able to release growth factor. Also, the core-shell structure can be used to form nanofibers with a natural polymer as the shell material and a synthetic polymer as the core material, which would improve the mechanical strength [87]. He et al. [88] fabricated PCL/zein core/shell nanofibers, and the results indicated the encapsulation of natural zein resulted in enhanced cell adhesion and proliferation in  vitro. Thus, the core-shell structure approach also solves the problem of poor biocompatibility of synthetic polymer nanofibers, as the biocompatibility of synthetic polymer is not as good as the natural polymer. More in vivo experiments are necessary to investigate the advantages of mechanical strength and biocompatibility in the core-shell nanofiber structures.

3.3.2.2 Surface modification of polymeric nanostructures Various synthetic biodegradable polymers have been utilized as tissue engineered scaffolds; however, one of the disadvantages is lacking biological recognition on their surface [89]. Thus, it is possible to promote cell-scaffold interaction by modifying the scaffold surface to obtain the desired characteristics. Common techniques for surface modification are plasma treatment [82, 90], laser treatment [91, 92], nanoparticle modification [93–95], and self-assembly [96]. Plasma treatment leads to changes in surface roughness of the polymers, which could affect the biological response. Reznickova et al. [82] applied Ar plasma-treated

Nanostructured biomaterials for regenerative medicine: Clinical perspectives57

low-density polyethylene, high-density polyethylene, and ultra-high-molecular-weight polyethylene surfaces with different nanomorphologies. The in  vitro results proved there was a significant increase of vascular smooth muscle cells cell adhesion, compared with the untreated polymers. Laser treatment can generate periodic surface nanostructures. Rebollar et al. [92] modified the surface of polystyrene foils by laser, and the results indicate that the presence of nanostructures on the surface can guide cell alignment. Also, the changes in surface chemistry and morphology have a positive influence on human embryonic kidney cells proliferation, compared with the untreated one. The chemical bonds are broken by laser radiation, which leads to highly reactive radicals reacting with the surrounding atmosphere on the surface. This approach leads to forming new functional groups, such as the ­oxygen-containing group, and the amino group [91]. The presence of amino acid and oxygen functional groups has been proved directly proportional to the spreading and adhesion of seeded cells [97]. However, plasma and laser treatment mostly focuses on 2D film surfaces, and it is difficult for the modification of 3D scaffold surface. Liu et al. [96] developed electrostatic layer-by-layer self-assembly technique to modify 3D nanofibrous PLLA scaffolds with gelatin. Cell proliferation was distributed more effectively and uniformly throughout the surface-modified PLLA nanofibers scaffolds, compare to the control scaffold. Another method for modifying the polymer surface is the incorporation of ceramic [94], noble metal nanoparticles [95, 98], and carbon-based nanotubes [93] on top of the scaffold surface. The electrospinning techniques could be applied to modify the scaffold surface, but it does not suit for 3D scaffold surface [99]. To obtain a uniform apatite layer on the 3D scaffold surface for bonding with living tissue, the polymer scaffold can be soaked in simulated body fluid (SBF) to allow apatite crystals to grow onto the scaffold surface [100].

3.3.2.3 Bulk modification of polymeric nanostructures Regardless of the surface modification of polymeric nanostructures, the ceramic nanoparticles can be directly mixed into polymers during processing. Thus, the incorporated ceramic nano-sized particles remain locked inside the polymeric scaffold structure. By bulk modification of polymeric nanostructures, the mechanical strength of nanocomposite could improve compared with the pure polymers. However, most of the ceramic particle is within the polymer scaffold, rather than on top of the surface. The cell-scaffold interaction should occur at the scaffold surface. Thus, the no exposed ceramic nanoparticles is a waste [68]. Moreover, the polymer can be functionalized by mixing with growth factors, and the biocompatibility of polymer can be improved [101, 102]. Although the presence of growth factors is of crucial importance to trigger healing for tissue regeneration, one of the limitations to growth factor therapy is insufficient local retention and require large quantities due to the fast inactivation of growth factors [103]. Nanoscale delivery systems have attracted a great deal of attention by researchers in the field of regenerative medicine based on their unique features, such as high s­ urface

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Nanostructured Biomaterials for Regenerative Medicine

area and easiness of surface functionalization, which can promote the adsorption of growth factors or relevant gene materials [104–106]. For instance, Kong et  al. [107] incorporated chitosan nanospheres into mineralized collagen coatings in vivo to improve rhBMP-2 loading and morphogen releasing based on the good affinity of chitosan for proteins and the large surface area of nanospheres. Also, Kumar et al. [108] developed an injectable, self-assembled peptide-based nanofibrous hydrogel that contains peptides for proangiogenic moieties, which can rapidly form mature vascular networks and induce tissue integration after subcutaneous delivery in vivo via a syringe needle. Also, Park et al. [73] used biodegradable PLGA nanoparticles as carriers loaded with runt-related transcription factor 2 (RUNX2) protein and coated with bone morphogenetic protein 2 (BMP2) plasmid DNA (pDNA), delivery to hMSCs in vitro and in vivo. The hMSCs transfection of PLGA nanoparticles significantly enhanced osteogenesis. These examples proved that nanoparticles can be exploited as delivery carriers for growth factors.

3.4 Metal nanostructures Since Faraday [109] firstly identified metallic nanoparticles in an aqueous solution, noble metal nanoparticles have been extensively studied as there is a significant difference with its bulk counterparts in the respects of physical, chemical, and biological properties [110]. The unique characteristics are high energy atoms located on the particle surface area [111], a high ratio of surface area to volume, high surface energy, and electron storage capacity [112]. Silver (Ag) and gold (Au) represent pure metallic nanoparticles, while iron oxide nanoparticles (IONPs), titanium oxide (TiO2), and zinc oxide (ZnO) are metal oxide nanoparticles, which have been used for tissue engineering [113–119]. These applications can be achieved by directly adding nanoparticles into culture media, coating or incorporating with other materials as composites. For instance, silver nanoparticles show antimicrobial activity while gold nanoparticles show good biocompatibility and rarely induce an allergic response. They can be seen as good candidates for various biomedical applications. The nanoparticle size, shape, stiffness, and surface property are essential for internalization into the cells. Ko et al. [117] indicated that smaller size gold nanoparticles (30–50 nm) have higher uptake and more cellular internalization, compared to bigger size nanoparticles (50–200 nm). Zhang et al. [119] indicated that nanoparticles possessing a positive charge could avoid lysosomal degradation, compared with the negative or neutral charges. Thus, the nanoparticle surface charge is related to cellular internalization rates. Zhang et al. [120] also mentioned that spherical-shaped nanoparticles had higher uptake and more cellular internalization, compared to 2D disk-shaped nanoparticles. Further, Li et al. [118] in vitro compared spherical-shaped and rod-shaped gold nanoparticles, and the spherical-shaped nanoparticles showed higher uptake rate than that of rod-shaped nanoparticles. There is evidence in the literature that modifying surface approaches, such as coating, can increase the bioactivity of metal or polymer surface. The commonly applied approach is ceramic coating using plasma spray method, for example, nano-HA [121–123]. However, HA coatings are prone to degradation over a short period [124]. Thus the metal nanoparticle coating can be an alternative solution.

Nanostructured biomaterials for regenerative medicine: Clinical perspectives59

Augustine et  al. [114] shown ZnO nanoparticles could generate reactive oxygen species which enhanced angiogenesis through growth factor-mediated mechanisms. The results showed fibroblast growth factor and vascular endothelial growth factor upregulated due to the incorporation of ZnO nanoparticles. IONPs has the potential for use in tissue engineering due to its specific physical properties and excellent biocompatibility, epically IONPs have been widely investigated for magnetic hyperthermia [125]. Also, IONPs can be possibly used for targeting growth factors (e.g., nerve growth factor) to the desired location within the body using an external magnetic field [126]. TiO2 nanoparticles have been clinically applied for the coating material since it has good bactericidal activity but has the nontoxic property [127, 128]. The membrane of microorganisms can be damaged since the formation of superoxide anions (O2−), hydroxyl radicals (OH−), and hydrogen peroxide (H2O2) [129, 130]. For instance, Shiraishi et  al. [131] shown TiO2 film had a strong bactericidal effect on Staphylococcus aureus which is one of the primary bacteria causing pin (stainless steel) site infection. Koseki et al. [132] in vivo evaluated the plasma spray TiO2 coating in inhibition of infection when using percutaneous external fixation pins. There was less infection in the TiO2-coated pin group than the control pin group. Dr. Tadashi Kokubo’s group successfully used the sol-gel method to form thin uniform titanium oxide layers on polymer materials (e.g., polyethylene terephthalate [133] and polyetheretherketone (PEEK) [134]). They also demonstrated that the temperature used in the sol-gel coating method is significantly lower than the plasma spray technique and thus does not exceed the glass transition temperature of most polymers. The in vitro experimental results show that adding a sol-gel-derived TiO2 layer can positively affect the formation of apatite in SBF. Shimizu et al. [135] pretreated PEEK with dilute HCl acid before the sol-gel coating. Both in vitro and in vivo results posttreatment with acid was proved to confer the apatite-forming ability to the surface, compare to the untreated one. Moreover, chemical and heat treatments were used with metal implants; this is a commonly used method to modify the implant surface of Ti metal. For instance, NaOH and heat treatment was applied to the Ti metal implant to form sodium titanate with nanoscale needle-like network structure on the surface. The treated Ti metal formed an apatite layer on their surfaces in SBF and tightly bonded to living bone. These treatments have been successfully applied in the clinic since 2007 [136]. More recently, Kokubo and Yamaguchi [51] directly modified the titanium surface to form nanometer-scale roughness with heat treatment after exposure to acid (HCl) and alkali (NaOH) solution, which conferred good bone bonding ability. The formed rutile induces a gel layer on the titanium surface. They also demonstrated that the titania layer is vital for forming bone-like apatite on the implant surface, which would bond to living bone. This treatment method has already been applied to porous Ti metal for spinal fusion device in clinical trials [137, 138]. Divakarla et  al. [139] modified the surface of the commercial titanium alloy GUMMETAL (Ti59Nb36Ta2Zr3O0.3) with alkali and heat treatments. The metabolic activity of bone marrow stem cells on the surface treated commercial alloy was increased in comparison to control one.

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3.5 Composite nanostructures 3.5.1 The applications of polymer/hydroxyapatite nanocomposites Nanocomposite structures are used widely, as they can enhance the mechanical strength of hybrid organic/inorganic composites, and thus influence cellular proliferation and differentiation. Table 3.2 shows functional polymer/ceramic nanocomposites for the application of tissue regeneration. To mimic the organization of bone tissue that is composed of inorganic minerals and organic collagen matrix, silicate nanoparticles were incorporated into organic materials, enhancing mechanical properties, and further promoting cellular proliferation. Chae et al. [94] successfully fabricated HA/alginate nanocomposite fibrous scaffolds using electrospinning for mimicking mineralized collagen fibers in bone tissue, as shown in Fig.  3.5. The results showed that osteoblasts were a more stable attachment on HA nanoparticles/alginate scaffolds than pure alginate scaffold. The osteoblasts were round-shape on the pure alginate scaffold, whereas they presented spindle-shape on the HAp/alginate scaffolds. Stiffness is another key parameter for altering cell growth and differentiation. Alakpa et al. [150] fabricated supramolecular nanofiber hydrogels and controlled their stiffness to direct the differentiation of stem cells in  vitro without any biochemical functionalization. In developing regenerative biomaterials for hard tissue engineering (i.e., engineering of bone tissue), one material alone (either an organic polymer or an inorganic bioceramic) cannot meet the requirement. For closely mimic the characteristics of bone tissue at the nanometre scale, HA nanoparticles have been combined with natural and synthetic polymers [151–153]. Wang et al. [153] have shown the HA/ polyamide nanocomposite scaffolds had better biocompatibility and extensive osteoconductivity with host bone, comparing the pure polyamide scaffolds, at the preliminary period after implantation in vivo. Frohbergh et al. [151] in vitro indicated there are a higher expression and enzymatic activity of alkaline phosphatase (osteogenic marker) on the HA/chitosan nanocomposite scaffolds than the pure chitosan scaffolds. Also, there was an enhanced osteoinductivity of the HA/chitosan nanocomposite scaffolds due to the higher rate of osteonectin mRNA expression in composite scaffolds. Ribeiro et al. [152] have shown that the compression modulus of nano-HA/silk fibroin composite scaffolds was improved with an increasing nano-HA concentration. The alkaline phosphatase activities of osteoblastic cells in vitro were enhanced by the incorporation of nano-HA in the silk fibroin matrix.

3.5.2 The applications of polymer/bioactive glass nanocomposites Bioactive glass/biodegradable polymer composite materials have emerged with main applications of composite coating for the implant [154] and tissue engineering scaffolds [146, 155, 156]. In particular, recent studies indicate the application of

Tissue Bone tissue

Skull tissue Dental tissue

Polymer/ceramic nanocomposites

Experimental type

Functionality

Results obtained

PCL/nano-HA composite

Mimicking bone graft structure and composition

In vitro and in vivo

[140]

PCL/bioactive glass nanocomposite

Mimicking bone graft structure and composition

In vitro and in vivo

[141]

The nanocomposite of poly(ethylene oxide) and laponite nanoparticles A nano-HA—pullulan/dextran polysaccharide composite

Mimicking bone mineralization

Better attachment and proliferation, and higher alkaline phosphatase activity and calcium content on the PCL/nano-HA than the PCL/micro-HA The bioactivity and mechanical stability were higher in PCL/bioactive glass nanocomposite than PCL/bioglass microparticles composite Induced increasing mechanical strength, and enhancing cellular activities and mineralization The nano-HA matrix induced a higher mineralized tissue than the one without nano-HA

In vitro

[142]

In vitro and in vivo

[143]

Improved alkaline phosphatase activity and human osteoblasts proliferation with increasing nano-bioglass content Improved mechanical properties of the scaffold and increased osteoconductivity The scaffolds presented better cell viability and increased the formation of hydroxyapatite once immersion in SBF Extensive mineralization in SBF in the PCL/ nano-HA and the content of nano-HA affect antimicrobial activity

In vitro

[144]

In vitro and in vivo In vitro

[145]

In vitro

[147]

Dextran hydrogels incorporated with bioactive glass nanoparticles Chitosan/nano-HA scaffolds Nanobioglass into the chitosan gelatin to fabricate composite scaffolds PCL/nano-HA and loaded with amoxicillin

Mimicking bone tissue stimulating bone cell differentiation Mimicking bone

Mimicking skull tissue Mimicking alveolar bone tissue Mimicking dental tissue

References

[146]

Continued

Nanostructured biomaterials for regenerative medicine: Clinical perspectives61

Table 3.2  Functional polymer/ceramic nanocomposites for the application of tissue regeneration

62

Table 3.2  Functional polymer/ceramic nanocomposites for the application of tissue regeneration—Cont’d Tissue Calcified cartilage

Polymer/ceramic nanocomposites Self-assembled peptide amphiphile nanofibrous matrices to induce biomimetic nucleation of hydroxyapatite crystals

HA/alginate nanocomposite fibrous scaffolds

References

Promoted new bone formation in a rat femoral defect

In vivo

[148]

Induced continuous deposition of lamellar bone tissue while maintaining osteoblast activity

In vivo

[149]

Osteoblasts attached on HA/alginate scaffolds are more stable than on pure alginate

In vitro

[94]

Results obtained

Mimicking bone mineralization with collagen fiber structure and nucleation of hydroxyapatite crystals Mimicking natural bone tissue based on the inorganic materials and natural polymers Mimicking mineralized collagen fibers

Nanostructured Biomaterials for Regenerative Medicine

HA composites sponge with concentrated collagen nanofibres

Experimental type

Functionality

Nanostructured biomaterials for regenerative medicine: Clinical perspectives63

Fig. 3.5  Microscopy images of synthesized HA nanoparticles/alginate scaffolds. This figure is modified based on T. Chae, H. Yang, V. Leung, F. Ko, T. Troczynski. Novel biomimetic hydroxyapatite/alginate nanocomposite fibrous scaffolds for bone tissue regeneration. J. Mater. Sci.: Mater. Med. 24, (2013) 1885-1894.

n­ ano-sized bioactive glass particles in composites could improve the performance for biomedical applications in tissue engineering [17, 157]. Couto et al. [154] developed chitosan and bioactive glass nanoparticle multilayer coatings for the application on prosthetic devices. The bioglass improved bioactivity for the organic-inorganic structure, and the chitosan provided viscoelastic properties of the coating. The in vitro results showed that the multilayer coating induced apatite formation. Apart from the use of polymer/bioactive glass nanoparticle for surface coatings, it has been utilized as a tissue scaffold material. Peter et al. [146] have shown protein adsorption was increased with the addition of bioactive glass nanoparticle into the ­chitosan-gelatin

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Nanostructured Biomaterials for Regenerative Medicine

to fabricate composite scaffolds fabricated with the sol-gel method. de Oliveira et al. [155] developed polyurethane/bioactive glass nanoparticle scaffolds, and the scaffolds present good cell viability and hydroxyapatite layer formation upon immersion in SBF. Ji et al. [156] indicated that the elastic modulus of the bioactive glass/PCL nanocomposite was improved by an increase of the bioactive glass nanoparticle content. Comparing with pure PCL polymer, the hydrophilic property, degradation behavior, and mineralization behavior were all improved with the addition of the bioactive glass nanoparticle. The polymer/bioactive glass nanocomposites also have the improved roughness, wettability, and surface area, which could promote bone regeneration through the increased nutrient exchange, protein adsorption, and porosity [16, 158, 159]. In summary, nanostructured ceramics, especially nano-HA, are popular as coatings, filler materials, and incorporation with other materials as nanocomposites to be used as bone substitutes. The high surface fraction in ceramic nanoparticles would increase osteoblast functions, such as adhesion, proliferation, and differentiation. Nanocomposite scaffolds also provide a support structure for cells, and thus if changing the tissue at the nanoscale level would affect cell-scaffold adhesion, interaction, and cellular function.

3.5.3 The application of polymer/metal nanocomposites There are increasing studies about the use of metal nanoparticles and polymer as the composites to apply in the field of regenerative medicine. With the optimized combination of polymer and metal nanoparticles, the desired property of nanocomposite can be obtained. Silver nanoparticles have been commonly used in tissue engineering due to its capability to release silver ions which in turn leads to an antibacterial activity [95, 98]. However, silver nanoparticles are easily aggregated due to the high surface free energy, and also they can be oxidized or contaminated in the air. Thus, these difficulties in processing have restricted its application [160]. The valid solution to solve the problem is the incorporation of silver nanoparticles into the biodegradable polymer [113, 161]. Also, by varying the concentration of silver nanoparticles in a certain range, the surface morphology of polymer/silver nanocomposite would change, and further ­affect the roughness and wettability on the nanocomposite surface. These characteristics can influence the bacterial adhesion on the nanocomposite surface [162, 163]. Moreover, the incorporation of gene materials at the local site can sustainably produce growth factor with gene transfection. For instance, Tandon et al. [164] demonstrated that polyethylenimine (PEI)-conjugated gold nanoparticles was an efficient carrier for mediating BMP7 gene delivery in vivo, which also modulated wound healing and inhibits fibrosis. Also, the mechanical properties of the polymer can be improved by incorporating of metal nanoparticles, due to the characteristics of nanostructured metal (e.g., high modulus and large surface area) [165]. Chatterjee et al. [166] indicated that the mechanical property of nanocomposites can be achieved by increasing the interaction area between the polymer matrix and nanoparticles. However, some of these interactions also lead to toxicity, which can be a serious problem for tissue regeneration. Thus, in-depth investigations of nanomaterials in vivo are required.

Nanostructured biomaterials for regenerative medicine: Clinical perspectives65

3.5.4 Other nanostructured composite biomaterials With adding nanoparticles, many studies have proved that a nanostructured composite can promote osteogenesis in the absence of growth factors. Gaharwar et al. [167] indicated the adhesion, spreading, and proliferation of MC3T3-E1 mouse preosteoblast cells can be modified by varying the concentration of laponite nanoparticles in laponite-PEO nanocomposites. The average diameter and thickness of the laponite nanoparticles were 25–30 nm, and 1 nm, respectively. Laponite nanoparticles also influence the differentiation of preosteoblast cells as there was increased mineralized phosphate producing on the nanocomposite surfaces. Wu et al. [168] indicated that adding laponite, the composite hydrogel had higher elongation and improved toughness comparing with pure hydrogel. Xavier et al. [169] studied the cellular response to the nano-laponite/collagen-based hydrogel film. The nanocomposite hydrogels could promote osteogenesis in vitro without the involvement of osteoinductive growth factor. There were an increased in alkaline phosphatase activity and the formation of a mineralized matrix by adding the nano-laponite to the collagen-based hydrogels. Moreover, the Kalpana S. Katti’s group modified nanoclay with amino acids to mineralize HA mimicking biomineralization in bone [170]. In Ambre et al. [171] work, the modified nanoclay was incorporated into chitosan/polygalacturonic acid films; hMSCs were seeded on the films to investigate the cellular response. The in  vitro results indicated there is the formation of mineralized nodules on the chitosan/polygalacturonic acid films without adding osteogenic supplements used for hMSCs differentiation. Viability and differentiation assays results show that the composite scaffolds were favorable for the viability and differentiation of hMSCs. Further, Ambre et al. [172] incorporated modified nanoclay in polycaprolactone to form PCL/nano-HA clay films; the cellular response of the composite film was examined. The results indicated that hMSCs composed mineralized ECM without the use of the osteogenic supplement. Also, PCL/nano-HA clay films had significantly increased in elastic moduli nanomechanical properties. Liao et al. [173] developed polyethylene glycol/graphene oxide nanocomposite scaffold for mimicking cartilage engineering, and the results show the improvement of mechanical properties and electrical conductivity of scaffold by graphene oxide, which leads to enhanced regeneration of cartilage tissue. Ahadian et al. [93] incorporated aligned carbon nanotubes into hybrid hydrogel scaffold, and there are tunable and anisotropic mechanical and electrical characteristics, which lead to enhanced cardiac differentiation of embryoid bodies with increased beating activity.

3.6 The clinical products for tissue regeneration based on nanotechnologies In 2014, the US FDA defined nanotechnology products as those which have at least one dimension between 1 and 100 nm in size [85]. The development progress of regenerative medicine nanostructured products for tissue regeneration in the clinic, starting with the basic concept product, then in vitro and in vivo studies, and culminating with clinical investigations and commercialization. Table 3.3 summarizes the selected nanostructured medicine products that have obtained from the FDA and applied in clinics.

Product/company Nanostructured polymers Regranex/Smith & Nephew, Inc.

HemCom/Medical Technologies, Inc. Nanostructured metal Acticoat/Smith and Nephew, Inc.

NanOss/Angstrom Medica, Inc.

Indications for use

Nanoparticle advantage and clinical outcomes

Year approved

References

Sodium carboxymethylcellulose gel, containing growth factor Chitosan acetate

Diabetic foot ulcers

Low concentrations of growth factors in nanoparticles minimize side defect

1997

([174], [175])

External wound healing

More effective in reducing bacterial luminescence

2008

[176]

Nanocrystalline silver

External wound healing

Prevents bacterial infection and improves wound healing

2009

[177]

Hydroxyapatite nanocrystalline

The filler is injected into a bone void or defect

2004

([178], [179])

Hydroxyapatite nanocrystals

Filler, osteoconductive, resorbable bone graft for osseous defects

This filler facilitates bone regeneration, based on its bone mimetic chemical composition and crystalline structures HA nanoparticles mimic the microstructure and the composition of bone and have higher mechanical properties and osteoconductive effects

2005

([180], [181])

The filler can control timed the release of calcium sulfate that supports bone augmentation

2006

[182]

Nanostructured composites BoneGen TR/BioLok Calcium sulfate-based International, Inc. nanocomposite

Filler, oral surgery, periodontics, endodontics, implantology

Nanostructured Biomaterials for Regenerative Medicine

Nanostructured ceramics Ostim/Osartis, Inc.

Nanostructured material descriptions

66

Table 3.3  Selective FDA approved nanostructured products for the application of tissue regeneration in the clinic

2008

[183]

2009

[184]

2010

[184]

This filler is controlled to be degraded over the time, stimulating bone regeneration There are increased alkaline activity and collagen production

2011

[185]

2013

([186], [178])

Implant, spinal fusion procedures

This implant has a nanotubeenhanced surface which can promote bone regeneration around the implant

2014

[85]

Porous 3D composite construct as bone boid filler

This filler has interconnected porosity mimic human cancellous bone to promote tissue interaction and regeneration

2014

[183]

Type I collagen fibers with nano-HA particles

Spinal surgery/bone void filler

EquivaBone/ETEX Corporation

Paste composed of a bone matrix and nanocrystalline hydroxyapatite

Bone void filler in spinal and trauma surgery

Beta-BSM/ETEX Corporation

Synthetic calcium phosphate bone graft material in a nanocrystalline matrix Calcium sulfate hemihydrate based nanocomposite Type I collagen fibers with Mg-HA nanoparticles Implant composed of a highly porous titanium a scaffold that is integrated with a PEEK core Hydroxyapatite nanogranules suspended in a collagen-based foam matrix

Injectable bone substitute material for orthopedic trauma and bone void filler Filler, osseous defects

NanoGen/Orthogen, LLC

RegenOss/JRI orthopaedics FortiCore/Nanovis, Inc.

nanOss Bioactive 3D bone void filler/Pioneer Surgical Technology, Inc.

Filler acetabular defects and spinal fusion

Nanostructured biomaterials for regenerative medicine: Clinical perspectives67

The filler is resorbed and remodeled into the new bone as part of the natural healing process This scaffold has osteoconductive effect by providing hydroxyapatite nanocrystalline and osteoinductive growth factors This filler has osteoconductive properties based on the bone mimetic chemical structure

Healos/DePuy synthes spine

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Nanostructured Biomaterials for Regenerative Medicine

Polymer nanoparticles can be used as carriers loaded with drugs or biological molecules, which is a promising tool for skin regeneration and wound healing. Regranex, becaplermin gel, was first approved by the FDA for the healing of diabetic neuropathic foot ulcers. It is sodium carboxymethylcellulose-based gel containing recombinant platelet-derived growth factor which is beneficial in wound healing [187]. However, a side effect has been reported (e.g., carcinogenic effects), which could be minimized by the application of low concentrations of growth factors in nanoparticles [174]. HemCom, a chitosan-based hemostatic agent, has been widely used for the control of severely bleeding wounds [176]. Biological molecules, such as corticosteroids [188] and antioxidants [189] have been loaded into chitosan nanoparticles in  vitro and in vivo for promoting wound healing; however, further clinical study is required. Silver nanoparticles are the only metal nanoparticles used for wound healing in the clinical market due to their low systemic toxicity, antibacterial activity, biocompatibility, and low cost [190]. Acticoat, nanocrystalline silver wound dressing, has been clinically used for topical treatment of infected burns, open wounds, and chronic ulcers [174, 177]. Also, silver nanoparticles-based drug-delivery systems can improve antibacterial efficacy by directly targeting antimicrobial agents to the site of infection [191]. Most of the current nanotechnology-based regenerative medicine products are made for bone tissue regeneration, fillers, and osseous defects. Bioceramic, mainly nano-sized HA has been used in commercial products (e.g., nanOss, Ostim, Cerabone, and BoneSave) [179, 181] and have gained the requisite regulatory approval. Recently, some nanoparticle-based composite biomaterials also have been obtained from the FDA. For instance, Healos is nanocomposite of type I collagen fibers and nonsintered calcium phosphate mineral nanoparticle. However, Kraiwattanapong et al. [192] indicated Healos was ineffective as a bone graft substitute when combined with autogenous bone marrow in rabbit’s body. The composition of RegenOss is the type I collagen fibers coated with Mg-HA nanoparticles. It has been shown to be biocompatible and was resorbed 90 days after implantation in sheep [193].

3.7 Conclusion and future perspective In this review, we highlighted leading edge nanostructured materials that mimic the composition and structure of the hard and soft tissue. Based on recent advances in nanotechnologies, bioengineered scaffolds are becoming more similar to natural tissue, thus enabling the recovery of damaged tissue. To mimic the organization of hard tissue (e.g., bone, calcified cartilage, and dental tissue) that is composed of inorganic minerals and an organic collagen matrix, ceramic nanoparticles were incorporated into polymer materials, enhancing the mechanical properties of the polymer and further promoting cellular proliferation. The increased roughness and the ratio of area to volume can be achieved, which are beneficial for the enhancement of protein adsorption. In addition, HA and WH are mainly two bone crystal nanoparticles, which are distributed in different ratios depending on the regions of bone tissue. By controlling their composition distribution to mimic native bone

Nanostructured biomaterials for regenerative medicine: Clinical perspectives69

tissue at the nanoscale can enhance the proliferation of bone cells and induced rapid regeneration of bone tissues. Various nanotechnologies, such as electrospinning, nanolithography, self-assembly, phase separation method, have been developed, to fabricate nanocomposite scaffolds. The nanocomposites have better mechanical and biological property, compared with either sole using polymer or nanostructured ceramic materials. However, the most common method for a bone substitute is mainly ceramic filler/paste form. This could be due to the absence of understanding of biological components in bioengineered tissue fabricated using various nanotechnologies. Moreover, some studies focus on the application of nanostructured material to closely mimic the soft tissue. By the surface modification of vascular polymer graft surface, the nanometer surface roughness could improve endothelial cell functions. Laser treatment, one of the surface modification methods, can be used to develop nanometer surface morphology. The modification process also involves the change of surface chemistry, such as the formation of new oxygen-containing groups and amino groups, which can facilitate better cell adhesion. Nanopatterns also play an essential role in directing various cellular behaviors, and control cell functions. The alignment of nanofibers in bioengineered tissue can guide cell movement and orientation, which can mimic the heart, tendon, and blood vessels tissues. Furthermore, the growth factor can regulate cellular migration, differentiation, and proliferation, and it is vital for the growth factor located in the site of injury for the repairing process. Due to the size and surface chemistries, the nanostructured biomaterials can be the delivery carriers for growth factors, peptides, and gene materials. Some studies demonstrated there were improved transport properties and more efficient delivery of growth factors to target sites. Also, the incorporation of laponite nanoparticles in nanocomposite can promote osteogenesis without growth factors. Although there are many positive results about the application of nanostructured biomaterials in soft tissue, the majority of the developed nanomaterials have not been utilized for soft tissue regeneration in a clinical setting. The main challenge is to scale up the formation of soft tissue from the nanoscale to macroscale for the tissue repair. The use of nanotechnology to create bioengineered scaffolds to closely mimic natural tissues in regenerative medicine has received increased attention over the years. It is ideally able to design the bioengineered scaffold to match with the natural tissue regarding integral structure, material composition, and surface morphology. However, the biological environment in vivo is dynamic, and the bioengineered scaffold is unlikely to sustain tissue growth with time. Although many nanoparticles have a positive performance effect on cellular internalization rates and have been utilized in regenerative medicine, there are still safety concerns about the use of these nanomaterials. The mechanisms have not yet been clarified about if the nanoparticles can cause toxic effects by crossing cell barriers, which will need to be explored further. These problems could be addressed thorough investigating the physicochemical characterization of nanomaterials. Based on the understanding of the effectiveness and safety of nanomaterials, proper in vivo studies should be pursued for clinical translation of nanomaterials.

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Despite these shortcomings, the use of nanostructured materials can be used to precisely control bioengineered scaffold structures, and positively affect the release of bioactive factor(s) that are vital for tissue regeneration. There will be a continuing trend in (a) the use of nanostructured biomaterials and (b) the approval of nanostructured products by the FDA. The study of nanostructured material in a clinical stage has more growth potential and overcome current challenges in regenerative medicine to heal damaged tissues.

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