Modification of the Micro Arc-oxidized Ti Surface for Implant Applications

Surface treatments applied to titanium and its alloys for implant applications are important for the development of bio properties.In this study,first an oxide layer was formed on the surface of the titanium plate by micro arc oxidation,and then both calcium phosphate and calcium phosphate/chitosan accumulation were performed for different samples by the sol-gel method.FE-SEM/EDS examinations,XRD,FTIR and thermal analysis were performed for these micro arc-oxidized,cal-cium phosphate-coated and calcium phosphate/chitosan-coated surfaces.The surface roughnesses for these surfaces were measured between 10 μm and 100 μm,suitable for bone development on the surface.The effect of chitosan addition on the calcium phosphate-coated surface on apatite formation ability and antibacterial properties was investigated.Although the addition of chitosan slows down the formation of apatite,it ensured that the coating had antibacterial properties.The calcium phosphate/chitosan biocomposite obtained can be recommended for dental and orthopedic implants.     

纳米羟基磷灰石/胶原复合材料制备方法研究

研究了在脱钙骨基质内原位沉积纳米羟基磷灰石的电化学方法,探讨了影响沉积的实验因素和条件.并利用红外光谱和X衍射表征无机相的组成,透射电子显微镜观测晶体的形态和尺寸,光学显微镜观察无机相分布,灰化法测定无机成分含量.结果表明,电化学方法可以制备出纳米羟基磷灰石/胶原复合材料,其无机成分为53 9±3.2%,并且无机相的组成、分布、性质与自然骨非常一致,是纳米复合材料.     

Physicochemical characterization of casein phosphopeptide-amorphous calcium phosphate nanocomplexes

Milk caseins stabilize calcium and phosphate ions and make them available to the neonate. Tryptic digestion of the caseins yields phosphopeptides from their polar N-terminal regions that contain clusters of phosphorylated seryl residues. These phosphoseryl clusters have been hypothesized to be responsible for the interaction between the caseins and calcium phosphate that lead to the formation of casein micelles. The casein phosphopeptides stabilize calcium and phosphate ions through the formation of complexes. The calcium phosphate in these complexes is biologically available for intestinal absorption and remineralization of subsurface lesions in tooth enamel. We have studied the structure of the complexes formed by the casein phosphopeptides with calcium phosphate using a range of physicochemical techniques including x-ray powder diffraction, scanning electron microscopy, transmission electron microscopy, and equilibrium binding analyses. The amorphous nature of the calcium phosphate phase was confirmed by two independent methods: x-ray powder diffraction and selected area diffraction. In solution, the ion activity product of a basic amorphous calcium phosphate phase was the only ion product that was a function of bound phosphate independent of pH, consistent with basic amorphous calcium phosphate being the phase stabilized by the casein phosphopeptides. Detailed investigations of calcium and calcium phosphate binding using a library of synthetic homologues and analogues of the casein phosphopeptides have revealed that although the fully phosphorylated seryl-cluster motif is pivotal for the interaction with calcium and phosphate, other factors are also important. In particular, calcium binding and calcium phosphate stabilization by the peptides was influenced by peptide net charge, length, and sequence.     

Phosphorylation of phosphophoryn is crucial for its function as a mediator of biomineralization

Phosphoproteins of the organic matrix of bone and dentin have been implicated as regulators of the nucleation and growth of the inorganic Ca-P crystals of vertebrate bones and teeth. One such protein identified in the dentin matrix is phosphophoryn (PP). It is highly acidic in nature because of a high content of aspartic acid and phosphate groups on serines. The 244-residue carboxyl-terminal domain of rat PP, predominantly containing the aspartic acid-serine repeats, has been cloned, and the corresponding protein has been expressed recombinantly in Escherichia coli. This portion of PP, named DMP2 (dentin matrix protein 2), is not phosphorylated by the bacteria and thus provided a means to study the function of the phosphate groups, the major post-translational modification of native PP. The recombinant DMP2 (rDMP2) possessed much lower calcium binding capacity than native PP. Small angle x-ray scattering experiments demonstrated that PP folds to a compact globular structure upon calcium binding, whereas rDMP2 maintained an unfolded structure. In vitro nucleation experiments showed that PP could nucleate plate-like apatite crystals in pseudophysiological buffer, whereas rDMP2 failed to mediate the transformation of amorphous calcium phosphate to apatite crystals under the same experimental conditions. Collagen binding experiments demonstrated that PP favors the formation of collagen aggregates, whereas in the presence of rDMP2 thin fibrils are formed. Overall these results suggested that the phosphate moieties in phosphophoryn are important for its function as a mediator of dentin biomineralization.     

Stress chaperone GRP-78 functions in mineralized matrix formation

Mineralized matrix formation is a well orchestrated event requiring several players. Glucose-regulated protein-78 (GRP-78) is an endoplasmic reticulum chaperone protein that has been implicated in functional roles ranging from involvement in cancer biology to serving as a receptor for viruses. In the present study we explored the role of GRP-78 in mineralized matrix formation. Differential expression of GRP-78 mRNA and protein was observed upon in vitro differentiation of primary mouse calvarial cells. An interesting observation was that GRP-78 was identified in the secretome of these cells and in the bone matrix, suggesting an extracellular function during matrix formation. In vitro nucleation experiments under physiological concentrations of calcium and phosphate ions indicated that GRP-78 can induce the formation of calcium phosphate polymorphs by itself, when bound to immobilized type I collagen and on demineralized collagen wafers. We provide evidence that GRP-78 can bind to DMP1 and type I collagen independent of each other in a simulated extracellular environment. Furthermore, we demonstrate the cell surface localization of GRP-78 and provide evidence that it functions as a receptor for DMP1 endocytosis in pre-osteoblasts and primary calvarial cells. Overall, this study represents a paradigm shift in the biological function of GRP-78.     

The effect of autologous bone marrow stromal cells differentiated on scaffolds for canine tibial bone reconstruction

Bone marrow contains mesenchymal stem cells that form many tissues. Various scaffolds are available for bone reconstruction by tissue engineering. Osteoblastic differentiated bone marrow stromal cells (BMSC) promote osteogenesis on scaffolds and stimulate bone regeneration. We investigated the use of cultured autologous BMSC on different scaffolds for healing defects in tibias of adult male canines. BMSC were isolated from canine humerus bone marrow, differentiated into osteoblasts in culture and loaded onto porous ceramic scaffolds including hydroxyapatite 1, hydroxyapatite gel and calcium phosphate. Osteoblast differentiation was verified by osteonectine and osteocalcine immunocytochemistry. The scaffolds with stromal cells were implanted in the tibial defect. Scaffolds without stromal cells were used as controls. Sections from the defects were processed for histological, ultrastructural, immunohistochemical and histomorphometric analyses to analyze the healing of the defects. BMSC were spread, allowed to proliferate and differentiate to osteoblasts as shown by alizarin red histochemistry, and osteocalcine and osteonectine immunostaining. Scanning electron microscopy showed that BMSC on the scaffolds were more active and adhesive to the calcium phosphate scaffold compared to the others. Macroscopic bone formation was observed in all groups, but scaffolds with stromal cells produced significantly better results. Bone healing occurred earlier and faster with stromal cells on the calcium phosphate scaffold and produced more callus compared to other scaffolds. Tissue healing and osteoblastic marker expression also were better with stromal cells on the scaffolds. Increased trabecula formation, cell density and decreased fibrosis were observed in the calcium phosphate scaffold with stromal cells. Autologous cultured stromal cells on the scaffolds were useful for healing of canine tibial bone defects. The calcium phosphate scaffold was the best for both cell differentiation in vitro and bone regeneration in vivo. It may be possible to improve healing of bone defects in humans using stem cells from bone marrow.     

Cuttlefish bone scaffold for tissue engineering: a novel hydrothermal transformation, chemical-physical, and biological characterization

Purpose: Natural resources are receiving growing interest because of their possible conversion from a cheap and easily available material into a biomedical product. Cuttlefish bone from Sepia Officinalis was investigated in order to obtain an hydroxyapatite porous scaffold using hydrothermal transformation. Methods: Complete conversion of the previous calcium carbonate (aragonite) phase into a calcium phosphate (hydroxyapatite) phase was performed with an hydrothermal transformation at 200 °C (~ 15 atm), for four hours, with an aqueous solution of KH2PO4 in order to set the molar ratio Ca/P = 10/6 in a reactor (Parr 4382). The complete conversion was then analyzed by TGA, ATR-FTIR, x-ray diffraction, and SEM. Moreover, the material was biologically investigated with MC3T3-E1 in static cultures, using both osteogenic and maintenance media. The expression of osteogenic markers as ALP and osteocalcin and the cell proliferation were investigated. Results: Cuttlefish bone has been successfully transformed from calcium carbonate into calcium phosphate. Biological characterization revealed that osteogenic markers are expressed using both osteogenic and maintenance conditions. Cell proliferation is influenced by the static culture condition used for this three-dimensional scaffold. Conclusions: The new scaffold composed by hydroxyapatite and derived for a natural source presents good biocompatibility and can be used for further investigations using dynamic cultures in order to improve cell proliferation and differentiation for bone tissue engineering.     

Surgically induced uremia in rats I: Effect on bone strength and metabolism

During the course of chronic renal failure (CRF) in man, renal osteodystrophy (osteitis fibrosa and/or osteomalacia) gradually develops. The present study aimed to establish a similar type of CRF leading to renal osteodystrophy in rats.During progressive CRF development over 225 days after 5/6 nephrectomy, the following serum variables were measured: creatinine, immunoreactive parathryoid hormone (iPTH), 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), a25-hydroxyvitamin D₃, (25(OH)D₃), alkaline phosphatase, albumin, phosphate, urea nitrogen, total calcium, and other blood electrolytes. Subsequent to sacrifice, mechanical properties of the rat femur, bone histomorphometry (osteoid and eroded surfaces) and bone contents of calcium, phosphate and hydroxyproline were also examined.Serum creatinine in rats with CRF gradually escalated by some 70%, while circulating 1,25(OH)₂D₃ was reduced beneath detection level. Total plasma calcium and phosphate concentrations were, however, almost unchanged indicating that PTH-induced bone remodeling due to moderate hyperparathyroidism sustained calcium homeostasis. Alkaline phosphatase levels were reduced by some 50%, which reflects chronically impeded bone formation. Bone histomorphometry assessment revealed substantial elevation of resorption with moderate accompanying fibrosis in about 70% of afflicted animals. Bone calcium, phosphate and hydroxpyroline contents remained unaltered. However, hydroxoproline/calcium ratio was marginally reduced. These results, together with altered mechanical bending stress characteristics and diminished diaphysis cross section area, confirm development of mixed bone lesions in the uremic animals.Our results are compatible with the early development of CRF in man. The established rat model is therefore useful in elucidating the precipitation and early treatment of renal osteodystrophy in humans.     

Effects of fibronectin on hydroxyapatite formation.

There is increasing evidence that noncollagenous matrix proteins initiate bone mineralization in vivo. Fibronectin, which is present during the early phases of mineralization, may contribute to this process in bone tissues. In this context, the mineralization potential of fibronectin was tested in an agarose gel precipitation system and a metastable calcium phosphate solution. The protein inhibited the precipitation of calcium phosphate crystals in solution but had no apparent effect in gel. Conversely, fibronectin stimulated crystal formation when apatite powder was used to seed crystal growth in gel. Although these results in vitro do not clearly indicate that fibronectin is involved in the mineralization process, they are consistent with in vivo events. Free fibronectin (e.g. in biological fluids) could inhibit crystal growth but might also activate the mineralization process when absorbed on apatite powder in a bone environment and areas of ectopic mineralization.     

THE EVOLUTION OF BONE

Vertebrates are practically unique among the Metazoa in their possession of a skeleton made from calcium phosphate rather than calcium carbonate. Interpretation of the origin of a phosphatic skeleton in early vertebrates has previously centered primarily on systemic requirements for phosphate and/or calcium storage or excretion. These interpretations afford no anatomical or physiological advantage(s) that would not have been equally valuable to many invertebrates. We suggest the calcium phosphate skeleton is distinctly advantageous to vertebrates because of their relatively unusual and ancient pattern of activity metabolism: intense bursts of activity supported primarily by rapid intramuscular formation of lactic acid. Bursts of intense activity by vertebrates are followed by often protracted periods of marked systemic acidosis. This postactive acidosis apparently generates slight skeletal dissolution, associated with simultaneous vascular hypercalcemia. A variety of apparently unrelated histological features of the skeleton in a number of vertebrates may minimize this postactive hypercalcemia. We present new data that suggest that postactive skeletal dissolution would be significantly exacerbated if bone were composed of calcium carbonate rather than calcium phosphate. The former is far less stable both in vivo and in vitro than is calcium hydroxyapatite, under both resting and postactive physiological conditions.     

Free-standing nanogold membranes as supports for the growth of calcium phosphate crystals

Current strategies for bone tissue regeneration focus on the development of implantable matrices that mimic biological tissues. Inorganic composites are of special interest for bone substitute applications. It is necessary to create an artificial three-dimensional scaffold-like porous material with certain geometrical structure to induce bone growth. We report here the growth of calcium phosphate crystals on free-standing carboxylic acid functionalized gold nanoparticle membranes. The gold nanoparticle membrane is synthesized by the spontaneous reduction of aqueous chloroaurate ions by a diamine molecule at a liquid-liquid interface. This membrane is robust and malleable, and most importantly, the gold nanoparticles in the membrane may be functionalized with suitable ligands. In this study, the amino acids aspartic acid and cysteine together with an aromatic bifunctional molecule, anthranilic acid, were used to modify the surface of the gold nanoparticles in the membrane. The free carboxylic acid groups on the gold nanoparticles further to functionalization with these molecules were then used to bind Ca(2+) ions and reacted with phosphate ions to yield calcium phosphate. The nature of the nanogold surface modifier directed the formation of either crystalline hydroxyapatite or amorphous calcium phosphate. The nanogold membrane thus suggests potential biomedical application as biocompatible implants and grafts.     

Ion beam treatment of titanium surfaces for enhancing deposition of hydroxyapatite from solution

Surface coating with hydroxyapatite (HA) is a common way to improve the osseointegration of orthopaedic and dental titanium (Ti)-based materials. The main problems with current techniques are changes in composition during heating and poor adhesion to the surface. An alternative method is deposition of HA onto an activated surface out of a solution. The present work studies the surface treatment involving ion implantation of Na into Ti to induce a modification in chemistry and morphology, showing sodium titanate (Na(2)TiO(3)) incorporated within the surface layer with concentration, depth distribution, and morphology depending on the parameters of the ion implantation. Such ion-implanted Ti surfaces actively induce heterogeneous precipitation of HA from a simulated body fluid containing physiological concentrations of calcium and phosphate ions. This is compared with the activation by NaOH etching. The growth of bone forming cells on the pure Na implanted surface is oriented without an increased bone formation. Cell growth on the NaOH etched surface is reduced. After deposition of HA on both surfaces cell the growth pattern was improved.     

Ectopic Osteoid and Bone Formation by Three Calcium-Phosphate Ceramics in Rats,Rabbits and Dogs

Calcium phosphate ceramics with specific physicochemical properties have been shown to induce de novo bone formation upon ectopic implantation in a number of animal models. In this study we explored the influence of physicochemical properties as well as the animal species on material-induced ectopic bone formation. Three bioceramics were used for the study: phase-pure hydroxyapatite (HA) sintered at 1200°C and two biphasic calcium phosphate (BCP) ceramics, consisting of 60 wt.% HA and 40 wt.% TCP (β-Tricalcium phosphate), sintered at either 1100°C or 1200°C. 108 samples of each ceramic were intramuscularly implanted in dogs, rabbits, and rats for 6, 12, and 24 weeks respectively. Histological and histomorphometrical analyses illustrated that ectopic bone and/or osteoid tissue formation was most pronounced in BCP sintered at 1100°C and most limited in HA, independent of the animal model. Concerning the effect of animal species, ectopic bone formation reproducibly occurred in dogs, while in rabbits and rats, new tissue formation was mainly limited to osteoid. The results of this study confirmed that the incidence and the extent of material-induced bone formation are related to both the physicochemical properties of calcium phosphate ceramics and the animal model.     

Synthesis and in vitro behavior of β-TCP zirconia/polymeric biocomposites for bio-applications

Novel biocomposites were fabricated by impregnating β-tricalcium phosphate (β-TCP)/zirconia particles into the polymers matrix. The composite materials were characterized using thermo-gravimetric analysis (TGA), X-ray diffraction (XRD), Fourier Transform Infrared (FT-IR) analyzes and Scanning Electron Microscopy (SEM). The results confirmed the conversion of hydroxyapatite (HA) to β-TCP at a sintering temperature of 1150 °C with or without zirconia powder. The in vitro behavior was assessed via measurement of calcium and phosphorus ions in SBF (simulated body fluid). FT-IR and SEM of the composites were performed pre and post immersion in SBF. The results prove that the bone like apatite layer formation was enhanced on the β-TCP-Z20/polymeric composite surface more than that on the β-TCP-Z10/polymeric composite. Therefore, the data confirmed that zirconia plays an important role in the enhancement of the apatite formation. The conclusions proved that the β-TCP-Z20/polymeric biocomposites, containing 20% of zirconia, are promising for bone remodeling applications.     

Association of a synthetic bone graft and bone marrow cells as a composite biomaterial

Porous calcium phosphates have osteoconductive properties. The aim of this study was to obtain synthetic calcium phosphate bone graft substitute. X-ray diffraction was employed to investigate the formation of the beta-tricalcium phosphate (β-TCP) phase. We evaluated the effects of bone marrow on the osteoconductivity and mechanical properties of synthetic bone graft (SG). SG cylinders loaded with bone marrow (SGBM) and SG alone were implanted into rabbits femoral condyle bone defects. Histological examinations revealed the resorption of the SG, trabecular bone with osteoblasts and osteoid substance around the implants, and colonization inside the porous β-TCP by newly formed bone. Histomorphometry conducted after three months revealed the osteoid surface to be higher in SGBM than SG (p < 0.05). The compressive strengths of SG and SGBM were significantly higher than the anatomic control at all time periods. The elastic modulus of SBG and SGBM became weaker after implantation. The present results indicate that gB-TCP is a good matrix for bone marrow, which contributes osteoinductive properties in an orthotopic. The composite biomaterial may be useful in reconstructive bone surgery.     

In vitro biocompatibility of chitosan/hyaluronic acid-containing calcium phosphate bone cements

The need for bone repair has increased as the population ages. In this research, calcium phosphate cements, with and without chitosan (CS) and hyaluronic acid (HA), were synthesized. The composition and morphological properties of cements were evaluated by X-ray diffraction and scanning electron microscopy. The acellular in vitro bioactivity revealed that different apatite morphologies were formed on the surfaces of cements after soaking in simulated body fluid. The in vitro osteoblastic cell biocompatibility of in situ forming cements was evaluated and compared with those of conventional calcium phosphate cements (CPCs). The viability and growth rate of the cells were similar for all CPCs, but better alkaline phosphatase activity was observed for CPC with CS and HA. Calcium phosphate cements supported attachment of osteoblastic cells on their surfaces. Spindle-shaped osteoblasts with developed cytoplasmic membrane were found on the surfaces of cement samples after 7 days of culture. These results reveal the potential of the CPC–CS/HA composites to be used in bone tissue engineering.     

Evidence for a serum factor that initiates the re-calcification of demineralized bone

The present studies show for the first time that demineralized bone re-calcifies rapidly when incubated at 37 degrees C in rat serum: re-calcification can be demonstrated by Alizarin Red and von Kossa stains, by depletion of serum calcium, and by uptake of calcium and phosphate by bone matrix. Re-calcification is specific for the type I collagen matrix structures that were calcified in the original bone, with no evidence for calcification in periosteum or cartilage. Re-calcification ceases when the amount of calcium and phosphate introduced into the matrix is comparable to that present in the original bone prior to demineralization, and the re-calcified bone is palpably hard. Re-calcified bone mineral is comparable to the original bone mineral in calcium to phosphate ratio and in Fourier transform infrared and x-ray diffraction spectra. The serum activity responsible for re-calcification is sufficiently potent that the addition of only 1.5% serum to Dulbecco's modified Eagle's medium causes bone re-calcification. This putative serum calcification factor has an apparent molecular mass of 55-150 kDa and is inactivated by trypsin or chymotrypsin. The serum calcification factor must act on bone for 12 h before re-calcification can be detected by Alizarin Red or von Kossa staining and before the subsequent growth of calcification will occur in the absence of serum. The speed, matrix-type specificity, and extent of the serum-induced re-calcification of demineralized bone suggest that the serum calcification factor identified in these studies may participate in the normal calcification of bone.     

Experimental calcification of HEMA-based hydrogels in the presence of albumin and a comparison to the in vivo calcification

The precipitation patterns and characteristics of calcium phosphate (CaP) phases deposited on HEMA-based hydrogels upon incubation in simulated body fluid (SBF-2) containing a protein (human serum albumin) have been investigated in relation to the calcification in an organic-free medium (SBF-1) and to that occurring after subcutaneous implantation in rats. In SBF-2, the deposits occurred exclusively as a peripheral layer on the surface of the hydrogels and consisted mainly of "precipitated hydroxyapatite", a species deficient in calcium and hydroxyl ions, similarly to the deposits formed on the implanted hydrogels, where the deposited layer was thicker. In SBF-1, the deposits were mainly of brushite type. There was no evidence that albumin penetrated the interstices of hydrogels. As the X-ray diffraction patterns of the CaP deposits generated in SBF-2 showed a similar nature with those formed on the implanted hydrogel, it was concluded that the calcification in SBF-2 can mimic to a reliable extent the calcification process taking place in a biological environment.     

Arginine vasopressin increases the rate of protein synthesis in isolated perfused adult rat heart via the V1 receptor

The effect of daidzein on cortical bone in vitro was investigated. Femoral-diaphyseal tissues obtained from elderly female rats were cultured for 24 h in Dulbecco's modified Eagle's medium (high glucose, 4.5%) supplementation with antibiotics and bovine serum albumin. The experimental cultures contained 10-7 to 10-5 M daidzein. The presence of daidzein (10-6 and 10-5 M) caused a significant increase of alkaline phosphatase activity, deoxyribonucleic acid (DNA) and calcium contents in bone tissues. This effect was equal to that of genistein (10-6 and 10-5 M). Daidzein (10-5 M) or genistein (10-5 M)-induced increase of calcium content and alkaline phosphatase activity in bone tissues was completely prevented by cycloheximide (10-6 M), an inhibitor of protein synthesis. Anabolic effect of daidzein and genistein on bone components was equal to that of 17-estradiol (10-8 M). The effect of isoflavohoids was not enhanced by the addition of 17-estradiol. The combination of daidzein and genistein did not have an additive effect. These findings indicate that daidzein has an anabolic effect on bone metabolism in tissue culture in vitro, and that this effect is equal to genistein effect. Isoflavonoids may stimulate bone formation and mineralization.     

Bioactivity studies and adhesion of human osteoblast (hFOB) on silicon-biphasic calcium phosphate material

Surface reactivity of bioactive ceramics contributes in accelerating bone healing by anchoring osteoblast cells and the connection of the surrounding bone tissues. The presence of silicon (Si) in many biocompatible and bioactive materials has been shown to improve osteoblast cell adhesion, proliferation and bone regeneration due to its role in the mineralisation process around implants. In this study, the effects of Si-biphasic calcium phosphate (Si-BCP) on bioactivity and adhesion of human osteoblast (hFOB) as an in vitro model have been investigated. Si-BCP was synthesised using calcium hydroxide (Ca(OH)₂) and phosphoric acid (H₃PO₄) via wet synthesis technique at Ca/P ratio 1.60 of material precursors. SiO₂ at 3 wt% based on total precursors was added into apatite slurry before proceeding with the spray drying process. Apatite powder derived from the spray drying process was pressed into discs with Ø 10 mm. Finally, the discs were sintered at atmospheric condition to obtain biphasic hydroxyapatite (HA) and tricalcium phosphate (TCP) peaks simultaneously and examined by XRD, AFM and SEM for its bioactivity evaluation. In vitro cell viability of L929 fibroblast and adhesion of hFOB cell were investigated via AlamarBlue® (AB) assay and SEM respectively. All results were compared with BCP without Si substitution. Results showed that the presence of Si affected the material’s surface and morphology, cell proliferation and cell adhesion. AFM and SEM of Si-BCP revealed a rougher surface compared to BCP. Bioactivity in simulated body fluid (SBF) was characterised by pH, weight gain and apatite mineralisation on the sample surface whereby the changes in surface morphology were evaluated using SEM. Immersion in SBF up to 21 days indicated significant changes in pH, weight gain and apatite formation. Cell viability has demonstrated no cytotoxic effect and denoted that Si-BCP promoted good initial cell adhesion and proliferation. These results suggest that Si-BCP’s surface roughness (164 nm) was significantly higher than BCP (88 nm), thus enhancing the adhesion and proliferation of the osteoblast.