Nanoceramics on osteoblast proliferation and differentiation in bone tissue engineering

Accepted Manuscript Title: Nanoceramics on Osteoblast Proliferation and Differentiation in Bone Tissue Engineering Authors: Sai Nievethitha S., Subhapradha N., Saravanan D., Selvamurugan N., Wei-Bor Tsai, Srinivasan N., Murugesan R., Moorthi A. PII: DOI: Reference:

S0141-8130(16)31814-1 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.01.089 BIOMAC 6996

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

28-9-2016 11-1-2017 18-1-2017

Please cite this article as: Sai Nievethitha S., Subhapradha N., Saravanan D., Selvamurugan N., Wei-Bor Tsai, Srinivasan N., Murugesan R., Moorthi A., Nanoceramics on Osteoblast Proliferation and Differentiation in Bone Tissue Engineering, International Journal of Biological Macromolecules http://dx.doi.org/10.1016/j.ijbiomac.2017.01.089 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Nanoceramics on Osteoblast Proliferation and Differentiation in Bone Tissue Engineering Sai Nievethitha S1, Subhapradha N1, Saravanan D4, Selvamurugan N2, Wei-Bor Tsai3, Srinivasan N1, Murugesan R1, Moorthi A1* 1. Faculty of Allied Health Sciences, Chettinad Academy of Research and Education, Chettinad Health City, Kelambakkam, Tamil Nadu, India 603 103. 2. Tissue Engineering and Cancer Research Laboratory, Department of Biotechnology, SRM University, Kattankulathur, Tamil Nadu, India 603 103. 3. Tissue Engineering and Drug Delivery Laboratory, Department of Biomedical Engineering, National Yang Ming University, Taipei, Taiwan, R.O.C-112. 4. Department of Animal Sciences, University of Illinois, Urbana Champaign, IL, USA, 61801.

*To whom Correspondence: Ambigapathi Moorthi, Ph.D. Assistant Professor, Department of Bionanotechnology Faculty of Allied Health Sciences, Chettinad Hospital and Research Institute, Kelambakkam, Chennai 603 103. Mobile: +91 9710071925 Email: [email protected], [email protected].

ABSTRACT Bone, a highly dynamic connective tissue, consist of a bioorganic phase comprising osteogenic cells and proteins which lies over an inorganic phase predominantly made of CaPO4 (biological apatite). Injury to bone can be due to mechanical, metabolic or inflammatory agents also owing pathological conditions like fractures, osteomyelitis, osteolysis or cysts may arise in enameloid, chondroid, cementum, or chondroid bone which forms the intermediate tissues of the body. Bone tissue engineering (BTE) applies bioactive 1

scaffolds, host cells and osteogenic signals for restoring damaged or diseased tissues. Various bioceramics used in BTE can be bioactive (like glass ceramics and hydroxyapatite bioactive glass), bioresorbable (like tricalcium phosphates) or bioinert (like zirconia and alumina). Limiting the size of these materials to nano-scale has resulted in a higher surface area to volume ratio thereby improving multi-functionality, solubility, surface catalytic activity, high heat and electrical conductivity. Nanoceramics have been found to induce osteoconduction, osteointegration, osteogenesis and osteoinduction. The present review aims at summarizing the interactions of nanoceramics and osteoblast/stem cells for promoting the proliferation and differentiation of the osteoblast cells by nanoceramics as superior bone substitutes in bone tissue engineering applications. Keywords:Osteoblast, Nanoceramics, Bioactive ceramics, Bone tissue engineering.

1. Introduction Bone is a dynamic connective tissue, which forms a supportive framework with an innate capacity of regeneration. It has a mineralized inorganic phase made of CaPO4, water, citrates, carbonates, magnesium, sodium and other trace elements which forms a scaffold for the bioorganic phase comprising mainly of collagen, non-collagenous proteins like osteocalcin (OC), osteonectin (ON), osteopontin (OP), thrombospondin, morphogenic proteins, bone sialoprotein (BSP), osteogenic cells (osteoblasts, osteocytes and osteoclasts) and other trace components (polysaccharides, cytokines and lipids)[1,2]. Damage to bone tissue will result into various pathological conditions like bone tumors, periodontitis, osteomyelitis, heavy fractures or osteochondral degenerative diseases (like arthrosis and osteoporosis) due to ageing [3,4]. Repair and regeneration processes can be enhanced using synthetic or natural grafts for rapid restoration of functions by promoting osteoblast migration, proliferation and differentiation [5]. Recovery in bone with large defects is absent unlike in case of small defects where inflammation, callus formation, and bone remodelling is executed. Metallic materials like cobalt chrome alloys, stainless steel, titanium and its alloys were used for internal fixation, but are brittle, immunogenic and non- bioresorbable. So the employment of bone harvested from humans, autografts (genetically identical individuals) or allografts (genetically non-identical individuals), consisting bone marrow cells, osteocytes and collagen matrix promotes healing. The limitations of the autografts are tuned with limited 2

availability, high costs and donor site complications like pain and infection. Also allografts will cause long term immunological rejection and increased incidence of disease transmission [5,6]. Hence, many approaches have been made for application of nanotechnology in regenerative medicine,

nanofiber scaffolding, modification of nanotopography of

nanoparticles and scaffolds. Bone tissue regeneration requiresscaffold with biocompatibility mimicking the natural bone extracellular matrix (ECM) niche, osteogenic cells andsignalling molecules for tissue specific differentiation and vascularisation of host bed [7,8]. Development of new bone on the defective region of bone requires various sequence of process like adhesion of osteogenic cells followed by their survival and multiplication. Osteoprogenitor cells or undifferentiated stem cells differentiate into osteoblast, which are integrated between the scaffold and host bone [5]. Applications of ceramics with decreased grain dimensions have been in use since 1998 correlated with increase in function of osteogenic cells [9]. Ceramics at nano-scale having size less than 100 nm promotes the activity of osteogenic cells by remodelling process [10]. Hence the present review aims at evaluating various interactions between nano-phased ceramics and osteogenic cells both in vivo and in vitroconditions. 2. Bioactive nanoceramics Bioactive glass nanoceramics (n-BGC) are SiO2–CaO–P2O5–Na2O based systems were developed in 1969 by Hench. Constituents of bioglass include sodium, silicone, magnesium, potassium, oxygen, phosphorous and calcium which are readily absorbed by the cells onto the biological fluid systems [11]. Enhanced functions of the ceramics are attributed to topographical features and surface chemistry. The ceramics possess better osteointegrative functions required for superior action on implants. Studies with 45S5 Bioglass showed that atomic structure influences the alkaline ion dissolution rates forming silanol bonds following polymerization [12,13]. This promotes the crystallization of ions as a layer of calcium deficient hydroxycarbonate apatite (HCA) at the bone–material interface enhancing protein adsorption onto the surface serving as substratum for attachment, differentiation and proliferation of stem cells. Lower crystallization of n-BGC increased mineralization in vitro [14]. The ECM is secreted in response with the nanoceramics onto the site of injury promotes the bone healing. Nanoceramics with smaller pore environment promote osteochondral formation followed by osteogenesis and vascularisation [15–17]. The interaction between different kinds of nanoceramics and osteoblasts are represented in Fig. 1. The dissolution products are found to up-regulate genes controlling osteogenesis also by 3

regulating the expression and secretion of vascular endothelial growth factor (VEGF) which promotes angiogenesis both in vitro and in vivo [18]. Moorthi et al. have reported that bioglass at nano scale stimulated the expression levels of cyclins C and E promoted the osteoblasts to enter in cell cycle activation state and also by reducing growth cycle passing G1 and S phases following a quick entry into G2 phase by the enhanced dissolution of silicon and calcium form the nanoceramics leading enhanced osteoblast proliferation [20,21]. Stimulation of osteoblast differentiation by nBGC treatment in mouse preosteoblastic cells was confirmed and calcium deposition was determined by von Kossa staining (Fig. 5).At the post-transcriptional level also, nBGC found to promote enhanced osteoblast differentiation by regulating the over-expression of microRNA-30c thereby promoting Runx2 (Runt- related transcription factor-2) expression [22]. BGC at nanoscale exhibits hydrophilicity which leads to more protein adsorption onto the surfaces and formation of focal adhesions turn into activation of necessary downstream signalling promotes enhanced cell proliferation [23,24]. Coating of calcium silicate (CaSiO3) (CS) over inert substrates leading release of SiO3 2- ions into the biological fluid results in its hydrolysis to HSiO3- ions along with Ca2+ ions triggers various cellular activities and promotes better cell attachment, proliferation and differentiation. This chemical gradient results in nucleation of tobermorite on the eroded surface of the coating which is further influenced by levels of silicon oxygen tetrahedron ([SiO4]4) [25]. Nanoscale formulation of 45S5 bioactive glass in simulated body fluid possesses antimicrobial activity due to the dissolution products released at alkalineenvironment [54, 55]. Doping of borate based bioactive glass with ions, like strontium, magnesium, gallium, iodine and silver, has a synergistic action against broad spectrum of microorganisms [56- 58]. A high surface area to volume ratio of these nanoparticles has increased the loading efficiency of therapeutic agents as demonstrated in different studies. Moreover the release kinetics of the therapeutic agent can be controlled by modulating the preparation parameters [59] Doxorubicin loaded bioactive glass nanospheres prepared via facile hydrothermal synthesis exhibited apH dependent release kinetics with effective targeting of osteosarcoma cells in vitro[60]. Another in vitro study stated the sustained release of vancomycin and 5fluoro uracil from lithium modified bioactive glass nanoparticles reduced the incidence of post-operative infections, osteomyelitis and tumours through diffusionbeyond 32 days [61]. An in vivo study in rabbits described the incorporation of teicoplanin in borate modified bioactive glass particles on chitosan for enhanced restoration of bone and clearing the chronic infection within a period of 12 weeks [62]. Loading efficiency can be further optimized based 4

on appropriate functionalization of the bioactive glass nanoparticles which affects the drug release profiles. Ipriflavone, a highly hydrophobic drug, was loaded on bioactive glass nanoparticles functionalized with different functional groups. In simulated body fluid, release of the drug from mercaptopropanol functionalized particles was high for prolonged period compared to phenyl-functionalized particles [63]. A similar approach has been made using lysozyme, with reactive oxygen species (ROS) based release kinetics, for functionalization, which has anti-tumor and anti-bacterial properties sparing the healthy cells from any cytotoxicity [64].An external stimulus like UV light has been used to induce release of drug from coumarin-modified bioactive glasses [65]. Genes like small intergering RNA (siRNA) for silencing certain gene functions and bioactive growth factors like VEGF to induce angiogenesis have also been delivered to the targeted site with similar strategies [66, 67]. 3. Bioresorbable Nanoceramics Completely resorbable ceramic is based mainly on its composition; however increased resorption rates might interfere with bone formation due to modified surface topography (concavity) of macropores and mechanical stability. Increased concentrations of calcium ions change the pH of microenvironment favouring formation of fibrous tissue [26].Bioresorbable nanoceramics are calcium phosphate based materials (CaP) which includes wide range of materials like hydroxyapatite (Ca10(PO4)6OH2–HA), calcium aluminate, tricalcium phosphate (Ca3(PO4)2)–TCP, -TCP), dicalcium phosphate dehydrate (CaHPO42H2O–brushite), calcium carbonate

(CaCO3–argonite)(-argonite),

(CaSO42H2O–plaster

of

paris),-octacalcium

phosphate (Ca8H2 (PO4)65H2O-OCP) and biphasic calcium phosphate (Ca10-xMx(PO4)6y(HPO4)yOH2-BCP)

[25,27]. They can be tuned in different way to vary in their shapes like

spheres, particles, needles/rods/wires/whiskers/fibres, sheets/flakes/disks/strips/platelets or 3D structures [28].The solubilization and resorption of the calcium phosphates are owing to pH, composition, temperature of the solutionas well as topography, particle size and pore size of the ceramic [29–31]. The freely available dissolute calcium and phosphate can be organized under three different categories (i) saturated stable zone (crystallization is not possible), (ii) supersaturated metastable zone (crystallization occurs post seeding) and (iii) supersaturated unstable zone (spontaneous crystallization is possible). Hence the exposure of nano-CaP (nCaP) to biological fluids serves the source of calcium and phosphate ions, due to 2+ 2− hydration, which are released in the form of PO3− 4 , Ca and HPO4 therefore regulates the

functions of osteogenic cells because according to thermodynamics, order of stability in DCDP, OCP, TCP and HA from the most to least soluble [32]. Doping nano ceramics with 5

ions like Mg2+ enhances rapid osteogenesis and remodelling due to reduced crystallinity [33]. For example, the controlled release of cobalt (Co) ions enhanced angiogenesis by upregulating secretion of VEGF and HIF-1α expression by the cells without any potential cytotoxicity [34]. Hence the local saturation of ceramics causes reprecipitation of leached ions and forms carbonated biological apatite. Therefore, the osteogenic/stem cells migrate and adhere to the apatite layer, which regulated by integrins, vitronectin and fibronectin (FN) resulting in expression of extracellular matrix components or osteoblast differentiation markers like ALP, collagen type I (Col-I), osteocalcin (OC) and necessary proteins followed by biomineralization [35,36]. Orchestration of the ceramics at nano scale will mediate the recruitment of osteogenic factors onto the materials surface will positively regulate the nucleation of CaP and osteoblast proliferation and differentiation. The nucleation process controlled by the nature of the proteins like isoelectric pH, conformation, concentration, composition and their activity on ceramic surface are triggered by the nanoscale ceramics [28]. Bioresorbable ceramics enhanced extracellular matrix secretion leading to the phosphorylation of various late and early differentiation markers of osteoblast like BSP, collagen, OC and ON triggers crystal nucleation. nCaP up regulated expression levels of BMP-2 (Bone Morphogenetic Protein-2), Runx2 and OP leading to proliferation and differentiation of osteoblast.In addition, the increased surface area contributed by the nano ceramic trigger enhanced leaching out of the calcium ionsthat promoted increased Ang-1 (Angiopoietin-1) and Ang-2 (Angiopoietin-2) expressions, which are responsible for structural integrity of blood vessels and are antagonists of each other. Ang-1 also influences osteogenesis, production and mineralization of OC and ALP activity [37]. Due to its brittle nature, composites of hydroxyapatite with naturally occurring or synthetic polymers increased its potential applications at load-bearing sites [68]. A composite of chitosan–gelatin–nano hydroxyapatite scaffolds supported better adhesion and proliferation of osteoblasts in vitro in stimulated body fluid [69].Peter and his co-workers explained the nano composite scaffold composed of chitosan/gelatin/nano BGC are nontoxic, biocompatible and suitable for alveolar bone tissue engineering [70].Combination of fucoidan, a naturally occurring polymer with hydroxyapatite helped in increased differentiation of adipocyte derived stem cells into osteoblasts with matrix mineralization compared to control (Fig. 2) [101]. A study by Xiong and his group (2014) on porous composite made of nano hydroxyapatite and polyamide 66 (PA66) gave promising results 6

both in vitro and in vivo. Clinical studies on patients who underwent resection of bone tumour over a period of 5 years gave satisfactory results with no abnormal blood investigations and other biochemical tests. Moreover the scaffold exhibited complete osseointegration on radiographic assessments. Preparations of composites with polymers containing nano hydroxyapatite and ions like silver, strontium, zirconium, tin, molybdenum, nickel, zinc and copper has further widened the applications of CaP ceramics. Saravanan et al (2011) prepared a highly porous composite scaffold comprising of chitosan, nano hydroxyapatite and silver nanoparticles with a mean pore size ranging from 50–100 μm, capable of supporting osteogenesis and vascularization, essential for optimal osseointegration of the implant along with antibacterial activity to prevent any post-operative infections. Another study by Sahithi et al (2010) combined nano hydroxyapatite, copper ions and polyethylene glycol 400 (PEG 400) which was more potent against Gram-positive strains than Gram-negative strains. Composites fabricated with chitosan comprising nHA and copper–zinc alloy nanoparticles exhibited high porosity and hydrophicility (Fig. 3) [74]. Nano hydroxyapatite has also found its uses as controlled drug delivery system. Zinc doped nano hydroxyapatite is a better candidate over nano hydroxyapatite in releasing Ciprofloxacin for treating bone related infections [75]. Various studies have reported fabrication of inorganic–organic composites that has demonstrated promising results including gentamicin loaded chitosan–nHA–ethyl cellulose microspheres, nHA–poly (lactic-co-glycolic acid) (PLGA) nanofiber composite loaded with amoxicillin or doxorubicin [76-78]. Nano hydroxyapatite containing toothpaste and mouth rinse formulations have also found to be effective in remineralizing the demineralized enamel [79, 80]. 4. Bioinert nanoceramics Bioinert nanoceramics includes titanium, alumina and zirconia based materials, which possess bioinertness, fracture toughness and high mechanical strength in a biological environment. Titanium and titanium based alloys play a major role in bone tissue reconstruction owing to corrosion resistance [38]. Anatase titania promotes deposition of HCA layer and has strong interactions between support and metal. The human osteoblast cultured onto the nanotubes titanium disks (NTD) up regulated bone related genes ENG, SP7, SPP1 and FOSL1 promoting osseointegration and persistant expression since the ENG, SP7 and FOSL1 genes are an indicator of osteodifferentiation of osteoblasts [39]. The β1 integrin mRNA expression was enhanced in the cells cultured with nanoporous titanium (30 nm size) which triggered enhanced osteoblast adhesion [40]. 7

For enhanced integration of the implant with the bone, titanium oxide implants have been modified with Ca2+ ions which form calcium chloride (CaCl2) under hydrated conditions, readily dissociating into Ca2+ and Cl− ions.The local Ca2+ ion gradient facilitated displacement of monovalent ions and water molecules from the surface of the implant and electro-statically interacted with the oxide layer increasing protein adsorption. These factors influenced the attachment and proliferation of osteoblasts and their differentiation [81]. Modification of titanium and their alloys by sand blasting; acid etching and heat treatment have enhanced the implant integration thereby accelerating the healing process [82–84]. High magnification images revealed the presence of nanopolymorphisms of the implant surface ranging from tuft, plate and nanonodule like structures [85]. Titanium alloys like Ti6Al4V and Ti–24Nb–4Zr–7.9Sn have been demonstrated as potential candidates for use as implants. Ti6Al4V substrates coated with a bilayer composite made of bioglass and yttria-stabilized zirconia possessed mechanical strength and resistance to corrosion [86]. Li et al (2016) modified Ti–24Nb–4Zr–7.9Sn substrates by anodic oxidation to improve their biocompatibility. Additives of barium titanate (BaTiO3) have been reported to inhibit crystal growth preventing aggregation of nanophase particles [88]. Self-ordered electrochemical anodization have been used to generate titania nanotube (TNT) arrays over titanium surfaces, a potential drug delivery candidate, which can be customized based on drug properties, dosage, and the disease to be treated [89]. TNT loaded with octenidine dihydrochloride (OCT)/ poly (lactic-co-glycolic acid) (PLGA) have shown to reduce post-operative infections attributing to its controllable drug release profile, osseointegration and sustained antibacterial potency [90]. Aluminium derivatives as oxides have been another potential candidate for application in dental and orthopaedic applications due to the desirable mechanical and biological properties. Monolithic alumina, being bioinert in nature, does not promote osseointegration resulting in implant failure over time. This is overcome by preparing porous ceramic coatings using anodisation [91]. Hence, the nanoscale topography and hydrophilicity of the implant surface has promoted enhanced adhesion of proteins increasing expression of molecules like VN and FN [92]. Another bioinert ceramic called zirconium at nano scale was found to enhance the apatite formation with polymeric scaffolds [41], triggering BMP2 signalling by enhancing the translocation of phosphorylated SMADs into nucleus promoting osteogenesis [42]. 8

Generally, the addition of oxide like Y2O3, MgO, CaO or CeO2 yielding multiphase particles called Partially Stabilized Zirconia (PSZ) with nano structured zirconium enhanced the interaction between inert phase and bone along with prevention of bioflim formation onto the implants [43]. Hence the zirconia–yittria ceramics, called Tetagonal Zirconia Polycrystals (TZP) or Yittria-stabilized zirconia (YZ) plays critical role in bone tissue engineering applications as artificial bone fillers in dental crowns, prostheses and joint heads for knee and hip, tibial plates and temporary supports [44–46]. Reorganization of actin filaments was linked to higher expression of α5β1integrin which is specific for FN present in the extracellular matrix. Also the binding of actin and integrins requires proteins like tensin, vinculin or talin which forms stress fibers and mediates the signalling involved in the proliferation of the osteoblast cells. [47]. In response to the bioinert ceramics, rough topography at nano scale up regulates of the early osteoblast differentiation markers like ALP and COL within a shorter time period in order to promote the differentiation and maturation of the osteoblast [46,48]. Also ceramics functionalized with alumina promoted precipitation of Ca2+ and subsequent formation of carbonated apatite thereby promoting the osteoblast attachment. [49]. Zirconia based nanoceramics have extensive use in bone tissue reconstruction owing to its high fracture toughness, biocompatibility, reduced wear rates and increased strength [50]. Interaction of various nanoceramics with osteoblasts and its actions are given in Table 1. Y-TZP under physiological conditions undergoes transformation from tetragonal phase to monoclinic phase increased the physical toughness of the implant. Substantial transformations results in roughening of the surface and cracking of the implant, termed aging. Aging of an implant is further influenced by factors like purity, density, topography, grain size and yttria content of the implant [93- 95, 97]. The controlled biodegradation and improved apatite deposition of chitosan/aginate/nano silica scaffold by nano silica (Fig. 4) would certainly advance bone formation which is crucial in BTE [96]. Pattnaik et al fabricated chitosan scaffolds comprising of zirconia nanoparticles and silicon dioxide, showed enhanced mineralization in SBF compared to chitosan-silicon dioxide composites. Osseointegration has been promoted using fiber laser irradiation for creating grooves and UV light for increasing hydrophilicity of the surface [98, 99]. Irradiation with diode laser and CO2 laser prior to implantation has also demonstrated anti-bacterial effects [100]. 5. Conclusion and Future perspectives

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Bioceramics are the most preferred implant materials in bone tissue engineering due to their biocompatibility, enhanced mechanical strength and negligible tissue reactions and composition orientation with the components of the bone. The structural orchestrations of bone substitute with conventional methods have brought great implications on BTE. Hence the structural modifications of ceramics at nanoscale have been found to tightly regulate adhesion, proliferation and differentiation of osteoblast/stem cells by admiring necessary cellular components onto the surface of the nano ceramics, thereby triggering better healing process of the defective bone at the site of the injury by enhanced interlocking mechanisms with the bone. Thus, tailoring ceramic materials at nanoscale that mimics the natural bone composition would offer sustainable advantages in regulating the osteoblast proliferation and differentiation which would bring great attention in bone tissue engineering. Further studies on interactions between cells and ceramics orchestrated at nano range mayelucidate the performance of nano ceramics in vivo. Improving the processing parameters and techniques currently used for synthesis the materials can further enhance the loading capacity of drugs and bio-active factors for enhancing anti-bacterial effects, vascularization and gene delivery along with decreasing activity of osteoclasts and other inhibitors Also approaches to construct three dimensional matrix with nanoceramics laid by cells in vitro can help in better integration with the host tissue thereby reducing any risks and maintaining reproducibility. Use of advanced techniques like in vitro tissue construction using appropriate growth factors and physico-chemical conditions and pre-designed scaffolds tailor made to the patient’s requirements can help enhance the integration of the scaffold with the host tissue. Better combinations of scaffolding materials and growth factors have to be researched for quick recovery. References 1. S. Dorozhkin, Calcium orthophosphate-containing biocomposites and hybrid biomaterials for biomedical applications, J. Funct. Biomater.6(2015) 708–832. 2. C. Rey, C. Combes, C. Drouet, M.J. Glimcher, Bone mineral: update on chemical composition and structure. Osteoporosis international: a journal established as result of cooperation between the European Foundation for Osteoporosis and the National Osteoporosis Foundation of the USA 20(2009) 1013–1021.

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101.

Young AT, Kang JH, Kang DJ, Venkatesan J, Chang HK, Bhatnagar I, Chang KY,

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Fig. 1. Schematic representation of interactions between nanoceramics and osteoblasts.

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Fig. 2: Alizarin red staining for visualizing matrix mineralization by adipose derived stem cells in presence of (A) DMEM medium, (B) osteogenic differentiation medium (ODM), (C) ODM and HA, (D) ODM and HS-fucoidan nanocomposite [101] The permission to reproduce obtained.

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Fig. 3. SEM photographs of the CS/nHAp/nCu–Zn scaffolds. (a) Surface morphology and pore dimension, (b) interconnecting pores and (c) the sizes of copper zinc alloy nanoparticles. The figure describing the pore size and the interconnecting pores in the scaffolds. This interconnected porous architecture may be essential to sustain cell passage, cell proliferation and vascularization deep inside the scaffolds [74].

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Fig. 4. Exogenous biomineralization of chitosan/alginate and chitosan/alginate/nano-silica scaffolds were studied by soaking the scaffolds for 7 days, and 21 days in SBF andmineral deposition was investigated by SEM. Fig. 4A: chitosan/alginate scaffolds exhibited lessermineral deposition of apatite layer in 7 days. Fig. 4C: chitosan/ alginate/nano-silica scaffolds deposited significantly more apatite crystals. Fig. 4D: chitosan/alginate/nano-silica scaffolds exhibit more pronounced and elevates the deposition of apatite crystals. It revealed more apatite crystals than chitosan/alginate scaffold (Fig. 4B) [96].

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Fig. 5. Effect of nBGC particles on mouse osteoblast differentiation and mineralization. Mouse preosteoblastic cells (MC3T3) were incubated with control or nBGC particles containing normal media or osteogenic media for 14 days. On every 3 days, the media were replaced with fresh media. At the end of 14th day, cells were fixed and subjected to von Kossa staining for determination of calcium deposits [19].

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Table 1 Interactions of nanoceramics with osteoblasts Nanoceramics Bioglass (n-BGC)

Mode of action Dissolution of alkaline ions enhances proliferation and differentiation of osteoblasts, angiogenesis and

Increased adhesion of osteoblasts and maturation

Hydroxy

Increased adhesion of osteoblasts followed by

apatite (n-HA)

differentiation

phosphate (ßTCP) Zinc aluminate

Titanium Yittria stabilized zirconia (YZ)

[18], [21]

mineralization

Silicon (Si)

ßTri- Calcium

References

Increased adhesion of osteoblasts, secretion of ECM proteins followed by enhanced proliferation Increased adhesion of osteoblasts and enhanced mineralization Increased adhesion of osteoblasts followed by proliferation Anchorage via filopods followed by maturation in short time

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[21], [28], [32] [50]

[37], [51]

[53]

[47], [48]

[46], [48]