Transglutaminase-mediated oligomerization promotes osteoblast adhesive properties of osteopontin and bone sialoprotein

Tissue transglutaminase (TG2) is a widely distributed, protein-crosslinking enzyme having a prominent role in cell adhesion as a β1 integrin co-receptor for fibronectin. In bone and teeth, its substrates include the matricellular proteins osteopontin (OPN) and bone sialoprotein (BSP). The aim of this study was to examine effects of TG2-mediated crosslinking and oligomerization of OPN and BSP on osteoblast cell adhesion. We show that surfaces coated with oligomerized OPN and BSP promote MC3T3-E1/C4 osteoblastic cell adhesion significantly better than surfaces coated with the monomeric form of the proteins. Both OPN and BSP oligomer-adherent cells showed more cytoplasmic extensions than those cells grown on the monomer-coated surfaces indicative of increased cell connectivity. Our study suggests a role for TG2 in promoting the cell adhesion function of two matricellular substrate proteins prominent in bone, tooth cementum and certain tumors.     

Osteocalcin induces chemotaxis, secretion of matrix proteins, and calcium-mediated intracellular signaling in human osteoclast-like cells

Osteocalcin, also called Bone Gla Protein (BGP), is the most abundant of the non-collagenous proteins of bone produced by osteoblasts. It consists of a single chain of 46-50 amino acids, according to the species, and contains three vitamin K-dependent gamma-carboxyglutamic acid residues (GLA), involved in its binding to calcium and hydroxylapatite. Accumulating evidences suggest its involvement in bone remodeling, its physiological role, however, is still unclear. In this study the adhesion properties and the biological effects of osteocalcin on osteoclasts have been analyzed using as an experimental model, human osteoclast-like cells derived from giant cell tumors of bone (GCT). Osteocalcin promoted adhesion and spreading of these cells, triggering the release of bone sialoprotein (BSP), osteopontin (OPN) and fibronectin (FN), that in turn induced the clustering in focal adhesions of beta 1 and beta 3 integrin chains. Spreading was dependent upon the synthesis of these proteins. In fact, when the cells were incubated in the presence of monensin during the adhesion assay, they still adhered but spreading did not occur, focal adhesions disappeared and BSP, OPN, and FN were accumulated in intracellular granules. Furthermore osteocalcin induced chemotaxis in a dose-dependent manner. The action of BGP on osteoclasts was mediated by an intracellular calcium increase due to release from thapsigargin-sensitive stores. These results provide evidences that BGP exerts a role in the resorption process, inducing intracellular signaling, migration and adhesion, followed by synthesis and secretion of endogenous proteins.     

Early expression of bone matrix proteins in osteogenic cell cultures.

Osteogenic cells express some matrix proteins at early culture intervals. The aim of this study was to determine if, and in what proportion, cells used for plating contain bone sialoprotein (BSP) and osteopontin (OPN), two matrix proteins associated with initial events in bone formation. Their pattern of expression, as well as that of fibronectin (FN) and type I pro-collagen, was also examined at 6 hr and at 1 and 3 days. The cells were obtained by enzymatic digestion of newborn rat calvariae, and grown on glass coverslips. Cytocentrifuge preparations of isolated cells and coverslips were processed for single or dual immunolabeling with monoclonal and/or polyclonal primary antibodies, followed by fluorochrome-conjugated antibodies. The cell labeling was mainly associated with perinuclear elements. OPN was also distinctively found at peripheral cytoplasmic sites. About 31% of isolated cells were OPN-positive and 18% were BSP-positive. After 1 day, almost 50% of cells were immunoreactive for OPN and for type I pro-collagen, and still less than 20% reacted for BSP. Approximately 7% exhibited peripheral staining for OPN. Almost all cells were associated with extracellular FN. However, only 15% showed intracellular labeling. These results indicate that an important proportion of cells used for plating contain BSP and OPN, a situation that should be taken into consideration in experimental analyses of osteoblast activity in vitro.     

Temporal studies on the tissue compartmentalization of bone sialoprotein (BSP), osteopontin (OPN), and SPARC protein during bone formation in vitro.

To study the role of noncollagenous proteins in bone formation, the synthesis and tissue distribution of BSP (bone sialoprotein), OPN (osteopontin) and SPARC (secreted protein acidic and rich in cysteine) were analyzed using pulse-chase and continuous labeling protocols during bone formation by cultures of rat calvarial cells. Following a 1 h labeling period with [35S]methionine or [35SO4], radiolabeled BSP was rapidly lost from the cells and appeared transiently in the culture medium and in a 4 M GuHCl extract (G1) of the mineralized tissue. Coinciding with the loss of BSP from these compartments, radiolabeled BSP increased in demineralizing, 0.5 M EDTA extracts (E) of the bone, in a subsequent GuHCl extract (G2), and in a bacterial collagenase digest (CD fraction) of the extracted tissue, over a 24 h chase period. In comparison, the 55 kDa form of OPN, with a small amount of the 44 kDa OPN, was secreted almost entirely into the culture medium. Most of the 44 kDa OPN, together with some 55 kDa OPN, accumulated rapidly in the E extract but could not be detected in either G extract or in the CD fraction. SPARC appeared transiently in the G1 extract, but was otherwise quantitatively secreted into the culture medium from where it was lost by complexing and/or degradation. When cultures were continuously labeled over a 12 day period with [35S]methionine, radiolabeled BSP and 44 kDa OPN accumulated in the E extract together with a small amount of SPARC. Some radiolabeled BSP also accumulated in the G2 extract. From the relative incorporation of [35SO4] over the same time period, a time-dependent loss in sulphate from the BSP was evident. Using a 24 h pulse-labeling protocol, the amount of radiolabeled BSP and OPN in the E extract and the BSP in the G2 extract were not altered significantly over a 12-day chase period. These studies demonstrate that the 44 kDa OPN and most of the BSP are rapidly bound to the hydroxyapatite crystals where they may regulate crystal formation and growth during bone formation. Some BSP is deposited in the osteoid and appears to become masked by the formation of hydroxyapatite, indicating a potential role for this protein in epitactic nucleation of hydroxyapatite crystal formation.     

Bone sialoprotein mRNA expression and ultrastructural localization in fetal porcine calvarial bone: comparisons with osteopontin

Summary Bone sialoprotein (BSP) and osteopontin (OPN) are two major non-collagenous proteins in bone that have similar biochemical properties and can mediate cell attachment through an RGD (Arg-Gly-Asp) motif that recognizes the vitronectin receptor. To facilitate evaluations of the biological functions of BSP and OPN in bone formation, affinity-purified rabbit polyclonal antibodies against porcine BSP and OPN were used, together with a high-resolution protein A-gold immunocytochemical technique to reveal the ultrastructural localization of these proteins in undermineralized sections of 50-day fetal porcine calvarial bone. In addition,³⁵S-labelled antisense riboprobes were prepared to demonstrate the cellular expression of BSP and OPN in the same tissues usingin situ hybridization. Immunolocalization for both BSP and OPN revealed the highest density of gold particles associated with electron-dense organic material found at the mineralization front and in ‘cement lines’. Labelling was also observed in the mineralized matrix over electron-dense material between collagen fibrils. In the osteoid of newly-formed bone, immunogold labelling for BSP and OPN was associated with loci of mineralization, which were often characterized by feathery clusters of fine needle-like crystals. Results ofin situ hybridization on the same tissues demonstrated that BSP mRNA expression was restricted to differentiated osteoblasts with particularly strong signals evident at sites ofde novo bone formation. More moderate expression of BSP was observed in ‘older’ osteoblasts and in some of the newly-entrapped osteocytes. Although expression of OPN mRNA was also observed in osteoblasts and osteocytes, the level of hybridization was similar for most bone cells and not markedly stronger than the signal observed in some stromal cells. While it is evident from these and other studies that both BSP and OPN are associated with bone formation, the differences observed in cellular expression indicate distinct roles for these proteins in bone formation.     

Factor H binding to bone sialoprotein and osteopontin enables tumor cell evasion of complement-mediated attack

Metastatic cancer cells, like trophoblasts of the developing placenta, are invasive and must escape immune surveillance to survive. Complement has long been thought to play a significant role in the tumor surveillance mechanism. Bone sialoprotein (BSP) and osteopontin (OPN, ETA-1) are expressed by trophoblasts and are strongly up-regulated by many tumors. Indeed, BSP has been shown to be a positive indicator of the invasive potential of some tumors. In this report, we show that BSP and OPN form rapid and tight complexes with complement Factor H. Besides its key role in regulating complement-mediated cell lysis, Factor H also appears to play a role when "hijacked" by invading organisms in enabling cellular evasion of complement. We have investigated whether BSP and OPN may play a similar role in tumor cell complement evasion by testing to see whether these glycoproteins could promote tumor cell survival. Recombinant OPN and BSP can protect murine erythroleukemia cells from attack by human complement as well as human MCF-7 breast cancer cells and U-266 myeloma cells from attack by guinea pig complement. The mechanism of this gain of function by tumor cell expression of BSP or OPN has been defined using specific peptides and antibodies to block BSP and OPN protective activity. The expression of BSP and OPN in tumor cells provides a selective advantage for survival via initial binding to alpha(V)beta(3) integrin (both) or CD44 (OPN) on the cell surface, followed by sequestration of Factor H to the cell surface and inhibition of complement-mediated cell lysis.     

The Impairment of Osteogenesis in Bone Sialoprotein (BSP) Knockout Calvaria Cell Cultures Is Cell Density Dependent

Bone sialoprotein (BSP) belongs to the "small integrin-binding ligand N-linked glycoprotein" (SIBLING) family, whose members interact with bone cells and bone mineral. BSP is strongly expressed in bone and we previously showed that BSP knockout (BSP-/-) mice have a higher bone mass than wild type (BSP+/+) littermates, with lower bone remodelling. Because baseline bone formation activity is constitutively lower in BSP-/- mice, we studied the impact of the absence of BSP on in vitro osteogenesis in mouse calvaria cell (MCC) cultures. MCC BSP-/- cultures exhibit fewer fibroblast (CFU-F), preosteoblast (CFU-ALP) and osteoblast colonies (bone nodules) than wild type, indicative of a lower number of osteoprogenitors. No mineralized colonies were observed in BSP-/- cultures, along with little/no expression of either osteogenic markers or SIBLING proteins MEPE or DMP1. Osteopontin (OPN) is the only SIBLING expressed in standard density BSP-/- culture, at higher levels than in wild type in early culture times. At higher plating density, the effects of the absence of BSP were partly rescued, with resumed expression of osteoblast markers and cognate SIBLING proteins, and mineralization of the mutant cultures. OPN expression and amount are further increased in high density BSP-/- cultures, while PHEX and CatB expression are differentiatlly regulated in a manner that may favor mineralization. Altogether, we found that BSP regulates mouse calvaria osteoblast cell clonogenicity, differentiation and activity in vitro in a cell density dependent manner, consistent with the effective skeletogenesis but the low levels of bone formation observed in vivo. The BSP knockout bone microenvironment may alter the proliferation/cell fate of early osteoprogenitors.     

Bone sialoprotein stimulates focal adhesion‐related signaling pathways: Role in migration and survival of breast and prostate cancer cells

Bone sialoprotein (BSP) is a secreted glycoprotein found in mineralized tissues however, BSP is aberrantly expressed in a variety of osteotropic tumors. Elevated BSP expression in breast and prostate primary carcinomas is directly correlated with increased bone metastases and tumor progression. In this study, the intracellular signaling pathways responsible for BSP‐induced migration and tumor survival were examined in breast and prostate cancer cells (MDA‐MB‐231, Hs578T and PC3). Additionally, the effects of exogenous TGF‐β1 and EGF, cytokines associated with tumor metastasis and present in high‐levels in the bone microenvironment, were examined in BSP‐expressing cancer cells. Expression of BSP but not an integrin‐binding mutant (BSP‐KAE) in tumor cell lines resulted in increased levels of α_v‐containing integrins and number of mature focal adhesions. Adhesion of cells to recombinant BSP or the expression of BSP stimulated focal adhesion kinase and ERK phosphorylation, as well as activated AP‐1‐family proteins. Activation of these pathways by BSP expression increased the expression of the matrix metalloproteinases MMP‐2, MMP‐9, and MMP‐14. The BSP‐mediated activation of the FAK‐associated pathway resulted in increased cancer cell invasion in a Matrigel‐coated Boyden‐chamber assay and increased cell survival upon withdrawal of serum. Addition of EGF or TGF‐β1 to the BSP‐expressing cell lines significantly increased ERK phosphorylation, AP‐1 activation, MMP‐2 expression, cell migration and survival compared to untreated cells expressing BSP. This study thus defines the cooperative mechanisms by which BSP can enhance specific factors associated with a metastatic phenotype in tumor cell lines, an effect that is increased by circulating TGF‐β1 and EGF. J. Cell. Biochem. 107: 1118–1128, 2009. © 2009 Wiley‐Liss, Inc.     

Synthesis and processing of bone sialoproteins during de novo bone formation in vitro.

Bone sialoprotein (BSP) and osteopontin (OPN) are sulphated and phosphorylated sialoglycoproteins that regulate the formation of hydroxyapatite crystals during de novo bone formation. To gain insights into the relationship between the synthesis and posttranslational modification of BSP and OPN and the mineralization of bone, pulse-chase studies were conducted on cultures of newly forming bone nodules produced by fetal rat calvarial cells in vitro. Cultures were pulse labelled with 35SO4, or with either 32PO4 or [gamma-32P]ATP to study intracellular and extracellular phosphorylation, respectively, and chased in isotope-free medium for various times up to 24 h. The presence of radiolabelled BSP and OPN was determined in the cells, in culture medium, and in various tissue compartments obtained by dissociative extraction with 4 M GuHCl (G1), 0.5 M EDTA (E), and again with 4 M GuHCl (G2) and a bacterial collagenase digestion of the demineralized collagenous tissue residue. With each isotope employed, radiolabelled BSP and OPN were detected in the E extract within the 1-h chase period and increased in amount with time. Similarly, 35SO4- and 32PO4-labelled BSP increased in the G2 extract, but OPN was not detected. In the G1 extract the 35SO4-labelled BSP decreased with chase time, whereas the 32PO4-labelled BSP increased. No differences were evident in the profiles of BSP labelled with 32PO4 or [gamma-32P]ATP. In the absence of beta-glycerophosphate, which is required for optimal mineralization of the bone nodules, 35SO4-labelled BSP was increased in the medium and G1 extract and decreased in the E extract and G2 extract after 3 h. In addition to differences in the tissue compartmentalization of BSP and OPN, these studies indicate that 35SO4 is lost from BSP during mineralization and that isoforms of BSP exist with a selective affinity for the organic and mineral phases. Moreover, the additional phosphorylation of BSP and OPN catalyzed by ectokinase activity does not appear to alter the distribution of these sialoproteins.     

Dentin matrix protein-1 isoforms promote differential cell attachment and migration

Dentin matrix protein-1 (DMP1), bone sialoprotein (BSP), and osteopontin (OPN) are three SIBLINGs (small integrin-binding ligand, N-linked glycoproteins) co-expressed/secreted by skeletal and active ductal epithelial cells. Although etiological mechanisms remain unclear, DMP1 is the only one of these three genes currently known to have mutations resulting in human disease, and yet it remains the least studied. All three contain the highly conserved integrin-binding tripeptide, RGD, and experiments comparing the cell attachment and haptotactic migration-enhancing properties of DMP1 to BSP and OPN were performed using human skeletal (MG63 and primary dental pulp cells) and salivary gland (HSG) cells. Mutation of any SIBLING's RGD destroyed all attachment and migration activity. Using its alphaVbeta5 integrin, HSG cells attached to BSP but not to DMP1 or OPN. However, HSG cells could not migrate onto BSP in a modified Boyden chamber assay. Expression of alphaVbeta3 integrin enhanced HSG attachment to DMP1 and OPN and promoted haptotactic migration onto all three proteins. Interchanging the first four coding exons or the conserved amino acids adjacent to the RGD of DMP1 with corresponding sequences of BSP did not enhance the ability of DMP1 to bind alphaVbeta5. For alphaVbeta3-expressing cells, intact DMP1, its BMP1-cleaved C-terminal fragment, and exon six lacking all post-translational modifications worked equally well but the proteoglycan isoform of DMP1 had greatly reduced ability for cell attachment and migration. The sequence specificity of the proposed BMP1-cleavage site of DMP1 was verified by mutation analysis. Direct comparison of the three proteins showed that cells discriminate among these SIBLINGs and among DMP1 isoforms.     

骨唾液酸蛋白基因沉默抑制乳腺癌细胞与骨基质粘附

旨在探索骨唾液酸蛋白 (Bone sialoprotein,BSP) 基因沉默对亲骨转移乳腺癌细胞 (MDA-MB-231BO) 与骨基质粘附能力的影响,为以BSP为靶点的乳腺癌骨转移预防和靶向治疗提供实验依据。体外检测BSP基因沉默对乳腺癌细胞与小鼠骨基质粘附能力的影响,MTS法检测细胞增殖能力;扫描电镜观察骨片表面肿瘤细胞粘附情况和骨吸收状况;ELISA法检测骨基质细胞粘附培养上清中TGF-β1和RANKL表达分泌量差异;左心室注射法构建裸鼠骨转移模型,检测不同细胞株在裸鼠体内转移能力。结果提示BSP     

MG63 osteoblast-like cells exhibit different behavior when grown on electrospun collagen matrix versus electrospun gelatin matrix

Electrospinning is a simple and efficient method of fabricating a non-woven polymeric nanofiber matrix. However, using fluorinated alcohols as a solvent for the electrospinning of proteins often results in protein denaturation. TEM and circular dichroism analysis indicated a massive loss of triple-helical collagen from an electrospun collagen (EC) matrix, and the random coils were similar to those found in gelatin. Nevertheless, from mechanical testing we found the Young's modulus and ultimate tensile stresses of EC matrices were significantly higher than electrospun gelatin (EG) matrices because matrix stiffness can affect many cell behaviors such as cell adhesion, proliferation and differentiation. We hypothesize that the difference of matrix stiffness between EC and EG will affect intracellular signaling through the mechano-transducers Rho kinase (ROCK) and focal adhesion kinase (FAK) and subsequently regulates the osteogenic phenotype of MG63 osteoblast-like cells. From the results, we found there was no significant difference between the EC and EG matrices with respect to either cell attachment or proliferation rate. However, the gene expression levels of OPN, type I collagen, ALP, and OCN were significantly higher in MG63 osteoblast-like cells grown on the EC than in those grown on the EG. In addition, the phosphorylation levels of Y397-FAK, ERK1/2, BSP, and OPN proteins, as well as ALP activity, were also higher on the EC than on the EG. We further inhibited ROCK activation with Y27632 during differentiation to investigate its effects on matrix-mediated osteogenic differentiation. Results showed the extent of mineralization was decreased with inhibition after induction. Moreover, there is no significant difference between EC and EG. From the results of the protein levels of phosphorylated Y397-FAK, ERK1/2, BSP and OPN, ALP activity and mineral deposition, we speculate that the mechanism that influences the osteogenic differentiation of MG63 osteoblast-like cells on EC and EG is matrix stiffness and via ROCK-FAK-ERK1/2.     

Structural characterization of human recombinant and bone-derived bone sialoprotein. Functional implications for cell attachment and hydroxyapatite binding.

Human bone sialoprotein (BSP) comprises 15% of the total noncollagenous proteins in bone and is thought to be involved in bone mineralization and remodeling. Recent data suggest a role for BSP in breast cancer and the development of bone metastases. We have produced full-length recombinant BSP in a human cell line and purified the protein from human bone retaining the native structure with proper folding and post-translational modifications. Mass spectrometry of bone-derived BSP revealed an average mass of 49 kDa and for recombinant BSP 57 kDa. The post-translational modifications contribute 30-40%. Carbohydrate analysis revealed 10 different complex-type N-glycans on both proteins and eight different O-glycans on recombinant BSP, four of those were found on bone-derived BSP. We could identify eight threonines modified by O-glycans, leaving the C terminus of the protein free of glycans. The recombinant protein showed similar secondary structures as bone-derived BSP. BSP was visualized in electron microscopy as a globule linked to a thread-like structure. The affinity for hydroxyapatite was higher for bone-derived BSP than for recombinant BSP. Cell adhesion assays showed that the binding of BSP to cells can be reversibly diminished by denaturation.     

The role of osteopontin in inflammatory processes

Osteopontin (OPN) is a matricellular protein that mediates diverse biological functions. OPN is involved in normal physiological processes and is implicated in the pathogenesis of a variety of disease states, including atherosclerosis, glomerulonephritis, cancer, and several chronic inflammatory diseases. Through interactions with several integrins, OPN mediates cell migration, adhesion, and survival in many cell types. OPN also functions as a Th1 cytokine, promotes cell-mediated immune responses, and plays a role in chronic inflammatory and autoimmune diseases. Besides its function in inflammation, OPN is also a regulator of biomineralization and a potent inhibitor of vascular calcification.     

Functional role of periostin in development and wound repair: implications for connective tissue disease

Integrity of the extracellular matrix (ECM) is essential for maintaining the normal structure and function of connective tissues. ECM is secreted locally by cells and organized into a complex meshwork providing physical support to cells, tissues, and organs. Initially thought to act only as a scaffold, the ECM is now known to provide a myriad of signals to cells regulating all aspects of their phenotype from morphology to differentiation. Matricellular proteins are a class of ECM related molecules defined through their ability to modulate cell-matrix interactions. Matricellular proteins are expressed at high levels during development, but typically only appear in postnatal tissue in wound repair or disease, where their levels increase substantially. Members of the CCN family, tenascin-C, osteopontin, secreted protein acidic rich in cysteine (SPARC), bone sialoprotein, thrombospondins, and galectins have all been classed as matricellular proteins. Periostin, a 90 kDa secreted homophilic cell adhesion protein, was recently added to matricellular class of proteins based on its expression pattern and function during development as well as in wound repair. Periostin is expressed in connective tissues including the periodontal ligament, tendons, skin and bone, and is also prominent in neoplastic tissues, cardiovascular disease, as well as in connective tissue wound repair. This review will focus on the functional role of periostin in tissue physiology. Fundamentally, it appears that periostin influences cell behaviour as well as collagen fibrillogenesis, and therefore exerts control over the structural and functional properties of connective tissues in both health and disease. Periostin is a novel matricellular protein with close homology to Drosophila fasciclin 1. In this review, the functional role of periostin is discussed in the context of connective tissue physiology, in development, disease, and wound repair.     

The Time-Dependent Manner of Sinusoidal Electromagnetic Fields on Rat Bone Marrow Mesenchymal Stem Cells Proliferation,Differentiation, and Mineralization

Electromagnetic fields (EMFs) are used clinically to promote fracture healing and slow down osteoporosis without knowledge of optimal parameters and underlying principles. In the present study, we investigate the effects of irritation for different durations with 15 Hz 1 mT sinusoidal EMFs (SEMFs) on rat bone marrow mesenchymal stem cells (BMSCs) proliferation, differentiation, and mineralization potentials. Our results show that SEMFs irritation promote rat BMSCs proliferation in a time-dependent manner, and the expression of osteogenic gen [Cbfa 1/RUNX2, bone sialoprotein (BSP), osteopontin (OPN)], alkaline phosphatase activity, and calcium deposition were enhanced after SEMFs treatment depending on the time duration of treatment. To determine the role of MEK/ERK signaling pathway, U0126, a MEK/ERK inhibitor was used. It can suppress rat BMSCs’ proliferation with or without SEMF exposure, and partly attenuate the expression of osteogenesis related proteins (RUNX2, BSP, OPN) which were improved by SEMF. This finding suggests that the effects of SEMF on rat BMSCs’ proliferation differentiation and mineralization are time duration dependent and MEK/ERK signaling pathway plays important role.     

Surface Functionalization of Orthopedic Titanium Implants with Bone Sialoprotein

Orthopedic implant failure due to aseptic loosening and mechanical instability remains a major problem in total joint replacement. Improving osseointegration at the bone-implant interface may reduce micromotion and loosening. Bone sialoprotein (BSP) has been shown to enhance bone formation when coated onto titanium femoral implants and in rat calvarial defect models. However, the most appropriate method of BSP coating, the necessary level of BSP coating, and the effect of BSP coating on cell behavior remain largely unknown. In this study, BSP was covalently coupled to titanium surfaces via an aminosilane linker (APTES), and its properties were compared to BSP applied to titanium via physisorption and untreated titanium. Cell functions were examined using primary human osteoblasts (hOBs) and L929 mouse fibroblasts. Gene expression of specific bone turnover markers at the RNA level was detected at different intervals. Cell adhesion to titanium surfaces treated with BSP via physisorption was not significantly different from that of untreated titanium at any time point, whereas BSP application via covalent coupling caused reduced cell adhesion during the first few hours in culture. Cell migration was increased on titanium disks that were treated with higher concentrations of BSP solution, independent of the coating method. During the early phases of hOB proliferation, a suppressive effect of BSP was observed independent of its concentration, particularly when BSP was applied to the titanium surface via physisorption. Although alkaline phosphatase activity was reduced in the BSP-coated titanium groups after 4 days in culture, increased calcium deposition was observed after 21 days. In particular, the gene expression level of RUNX2 was upregulated by BSP. The increase in calcium deposition and the stimulation of cell differentiation induced by BSP highlight its potential as a surface modifier that could enhance the osseointegration of orthopedic implants. Both physisorption and covalent coupling of BSP are similarly effective, feasible methods, although a higher BSP concentration is recommended.