A Vital Microscopic Description of the Effects of Electrical Stimulation of Bone Tissue

An implantable, titanium, optical chamber that allows vital microscopic observations of bone tissue during continuous electrical stimulation is described. Tissue reactions to direct current stimulation of a defined bone tissue compartment may be repeatedly observed and recorded on film for indefinite follow-up periods. In this study 5, 20 or 50 μA direct current were applied to chambers inserted into the rabbit tibia. The bone tissue was found to increase in volume after stimulation with 5 and 20 μA, while 50 μA caused bone resorption. Fat-cell concentration was independent of the current levels as used in the present study. Vital microscopy revealed no signs of acute microvascular alterations in the hours following onset of the current. Over an eleven-week follow-up, vessels close to bone borders were found to increase in size from 15–25 microns to 30–50 microns, independent of the level of stimulation. The amount of vessels increased after stimulation with 5 and 20 μA, while in the 50 μA group the vessels decreased to 50% or less of the vascular density observed before start of the stimulation.     

人骨形成蛋白3在昆虫杆状病毒表达系统中的表达、纯化及其诱骨活性检测

为了在昆虫杆状病毒系统中表达人骨形成蛋白 3(humanbonemorphogeneticproteoin3,hBMP3) ,检测表达产物的诱骨活性 .将hBMP3全长cDNA 1416bp克隆入转移载体K8中 ,再与病毒DNA经脂质体包裹后转染昆虫细胞Sf9.重组病毒经蓝白筛选后 ,用PCR方法扩增目的基因片段进行初步鉴定 .收集重组病毒的转染上清 ,肝素亲和层析纯化目的蛋白 .SDS PAGE和Western印迹进一步鉴定此蛋白 .体外培养MC3T3 E1细胞 ,经目的蛋白刺激后 ,检测细胞内碱性磷酸酶 (ALP)活性 .并将目的蛋白进行小鼠体内肌肉包埋实验 ,检测其异位诱骨活性 .结果显示 ,肝素亲和层析可以收集 1个高的洗脱峰 .SDS PAGE显示 ,非还原型样本为 32kD的二聚体蛋白带和少量 16kD单体蛋白带 ;还原型样本仅有相应大小的单体蛋白带 ,纯度达 80 % .Western印迹在相应位置呈阳性染色 .此蛋白刺激小鼠成纤维细胞 4 8h后 ,胞内ALP的活性增高 ,经rhBMP3作用后的细胞MTT染色减弱 .在小鼠股部肌肉内包埋蛋白样品 2～ 3周 ,组织学检测有成骨组织生成 .说明hBMP3在此套系统中获得了表达 .表达产物在体外可以刺激成骨细胞的分化 ,却抑制细胞的增殖 .小鼠体内异位诱骨实验进一步证实表达产物具有成骨活性     

Pseudoarthrosis Treatment with Cancellous Bone Grafts and Low-Frequency Pulsing Electromagnetic Fields

We report our experience in the use of cancellous bone grafts and stimulation with pulsing electromagnetic fields (PEMFs) in the treatment of acquired pseudarthrosis in 28 patients, including 17 patients with active infections. Stimulation was begun immediately after the grafting, and all patients healed in an average time of 4 months.     

Glucagon in the Treatment of Paget's Disease of Bone

Glucagon given by intravenous infusion at a dosage of 0·2 to 0·8 mg/hour to four patients with Paget''s disease of bone resulted in a dramatic fall in plasma alkaline phosphatase. This was associated with a fall in 24-hour urinary calcium and in total urinary hydroxyproline excretion and a marked relief of bone pain.Glucagon may induce these changes by three possible mechanisms: (1) by stimulating release of calcitonin; (2) by a direct action of the hormone on bone; and (3) by stimulation of certain bone pyrophosphatases, thus altering the local mechanisms controlling the rate of bone formation and resorption.     

Bone marrow autotransplantation as a factor modifying the process of hematopoietic tissue recovery in irradiated guinea pigs

Guinea pigs were subjected to myeloexfusion 30-90 min before gamma-irradiation with a superlethal dose of 8 Gy. Immediately after irradiation, the bone marrow (4 X 10(7) cells) was autotransplanted. The automyelotransplantation provided a pronounced stimulation of haemopoiesis and substantially shortened the time of its regeneration. The myeloexfusion in some of the exposed control animals had a beneficial effect in their resistance and blood system recovery.     

Bone densitometry: assessing the effects of growth hormone treatment in adults

Dual-energy X-ray absorptiometry (DXA) is the reference method for the measurement of bone mineral mass at different skeletal sites. It has been widely used in recent years to assess the effects of growth hormone (GH) treatment on bone metabolism. In normal individuals, bone mineral content (BMC) and density (BMD), as assessed using DXA, correlate with body size. Therefore, using DXA in patients with congenital GH deficiency (GHD), who have a smaller body frame, would be expected to result in lower bone mass. Thus, comparisons with reference data derived from populations of normal body size are invalid. The evaluation of the effects of GH administration should take into account the possible effects of GH on bone size, not only in children, but also in adults. The enlargement of bone, due to stimulation of the periosteal apposition, may partially mask an increase in BMC, resulting in little or no change in BMD. The ability of GH to affect bone area therefore requires analysis of the possible changes in bone area and BMC, as well as BMD. This issue has been poorly handled in the studies published to date. Lastly, the acceleration of bone turnover induced by GH leads to an increase in bone remodelling space, which in turn is associated with a reduction in BMC and BMD, independent of the net balance between breakdown and formation in each metabolic unit. This bone loss is completely reversible when the remodelling space returns to previous levels. This phenomenon must be taken into account when analysing the effects of GH treatment on bone mass, because a net gain in bone mass may be found in long-term GH treatment or after GH discontinuation, even if bone loss was evident during the first 6 months of treatment. In conclusion, the interpretation of bone density data in patients with GHD, and after GH administration, should take into account some of the methodological aspects of bone densitometry, as well as the specific actions of GH on bone metabolism and body composition.     

Analysis of Bone Remodeling Under Piezoelectricity Effects Using Boundary Elements

Piezoelectric materials exhibit a response to mechanical-electrical coupling,which represents an important contribution to the electrical-mechanical interaction in bone remodeling process.Therefore,the study of the piezoelectric effect on bone remodeling has high interest in applied biomechanics.The effects of mechano-regulation and electrical stimulation on bone healing are explained.The Boundary Element Method (BEM) is used to simulate piezoelectric effects on bones when sheafing forces are applied to collagen fibers to make them slip past each other.The piezoelectric fundamental solutions are obtained by using the Radon transform.The Dual Reciprocity Method (DRM) is used to simulate the particular solutions in time-dependent problems.BEM analysis showed the strong influence of electrical stimulation on bone remodeling.The examples discussed in this work showed that,as expected,the electrically loaded bone surfaces improved the bone deposition.BEM results confirmed previous findings obtained by using the Finite Element Method (FEM).This work opens very promising doors in biomechanics research,showing that mechanical loads can be replaced,in part,by electrical charges that stimulate strengthening bone density.The obtained results herein are in good agreement with those found in literature from experimental testing and/or other simulation approaches.     

多孔钛合金不同孔径大小对新骨长入的影响

目的:评价不同孔径多孔钛合金植入物在骨缺损区对新骨长入的影响。方法:采用电子束熔融(EBM)技术制备三种不同孔径(孔径分别为1.0 mm,2.0 mm,3.0 mm)的多孔钛合金材料,其孔隙率依次为73%,79%,86%。将18只家犬随机分为1.0 mm孔径材料组,2.0 mm孔径材料组,3.0 mm孔径材料组,每组6只。制备家犬双侧股骨外侧髁缺损模型,然后植入各孔径组材料,于术后4周,8周,12周分别行大体标本观察,X线片观察,组织形态学观察三组不同孔径材料与周围骨的整合情况及孔隙中的新骨长入情况。结果:通过大体标本观察和X线片观察显示,12周后三组材料均与周围紧密骨连接。其中1.0 mm孔径组材料中心明显成骨,2.0 mm孔径组和3.0 mm孔径组中心仍为较多白色组织填充。组织学观察显示,12周时2.0 mm孔径组和3.0 mm孔径组材料周围有骨质包绕,但中心空洞,基本无骨质形成。1.0 mm孔径组材料周围骨质包绕紧密,孔中新生骨形成较多,且有大量纤维母细胞和软骨细胞形成。各时间点1.0 mm孔径组新生骨面积百分比明显高于2.0 mm孔径组和3.0 mm孔径组,P<0.01,差异具有统计学意义。2.0 mm孔径组和3.0 mm孔径组相比,P>0.05,无显著差异。结论:孔径大小影响多孔钛合金材料的骨长入,适当孔径的设计将更有利于材料的传导成骨。     

Vitamin C Prevents Hypogonadal Bone Loss

Epidemiologic studies correlate low vitamin C intake with bone loss. The genetic deletion of enzymes involved in de novo vitamin C synthesis in mice, likewise, causes severe osteoporosis. However, very few studies have evaluated a protective role of this dietary supplement on the skeleton. Here, we show that the ingestion of vitamin C prevents the low-turnover bone loss following ovariectomy in mice. We show that this prevention in areal bone mineral density and micro-CT parameters results from the stimulation of bone formation, demonstrable in vivo by histomorphometry, bone marker measurements, and quantitative PCR. Notably, the reductions in the bone formation rate, plasma osteocalcin levels, and ex vivo osteoblast gene expression 8 weeks post-ovariectomy are all returned to levels of sham-operated controls. The study establishes vitamin C as a skeletal anabolic agent.     

Proteomic Analysis of Gingival Tissue and Alveolar Bone during Alveolar Bone Healing

Bone tissue regeneration is orchestrated by the surrounding supporting tissues and involves the build-up of osteogenic cells, which orchestrate remodeling/healing through the expression of numerous mediators and signaling molecules. Periodontal regeneration models have proven useful for studying the interaction and communication between alveolar bone and supporting soft tissue. We applied a quantitative proteomic approach to analyze and compare proteins with altered expression in gingival soft tissue and alveolar bone following tooth extraction. For target identification and validation, hard and soft tissue were extracted from mini-pigs at the indicated times after tooth extraction. From triplicate experiments, 56 proteins in soft tissue and 27 proteins in alveolar bone were found to be differentially expressed before and after tooth extraction. The expression of 21 of those proteins was altered in both soft tissue and bone. Comparison of the activated networks in soft tissue and alveolar bone highlighted their distinct responsibilities in bone and tissue healing. Moreover, we found that there is crosstalk between identified proteins in soft tissue and alveolar bone with respect to cellular assembly, organization, and communication. Among these proteins, we examined in detail the expression patterns and associated networks of ATP5B and fibronectin 1. ATP5B is involved in nucleic acid metabolism, small molecule biochemistry, and neurological disease, and fibronectin 1 is involved in cellular assembly, organization, and maintenance. Collectively, our findings indicate that bone regeneration is accompanied by a profound interaction among networks regulating cellular resources, and they provide novel insight into the molecular mechanisms involved in the healing of periodontal tissue after tooth extraction.Healthy dental gingival tissue and the alveolar bone that surrounds the teeth are essential for the proper function of teeth, as well as for a good appearance and good general health. Socket healing after tooth extraction is a useful experimental model for investigating the communication between gingival tissue and alveolar bone after tooth extraction. Preservation of the alveolar socket after tooth extraction requires the formation of a biological connection between the living and osseous tissue, which has to be created during the healing process. The success of such dental remodeling is dependent on the establishment of a soft tissue barrier that is able to shelter the underlying osseous structures and the osseo-integration of the soft tissue surrounding the alveolar bone. Understanding the processes governing soft and hard tissue healing and maintenance around the alveolar socket is paramount for oral health.Several studies have reported significant structural changes and bone reabsorption in fresh sockets following tooth extraction, with important dimensional changes in the surrounding alveolar bone (1–3). A reduction of alveolar bone may present problems after tooth extraction, especially in aged individuals in whom bone volume is important for both physiological and medical reasons. Although it has been shown that reduction defects in alveolar bone can be completely repaired using surgical techniques such as guided bone regeneration (4, 5), bone autograft, bone allograft, and xenograft (6, 7), these techniques are not broadly applicable (8). However, the introduction of biomimetic agents such as enamel matrix derivatives (9), platelet-rich plasma (10), platelet-derived growth factor (11, 12), and bone morphogenic proteins (BMPs)¹ (9) promises potentially better outcomes with bone regeneration treatments, although their efficacy remains controversial.The proteins present in bone are essential for all of the life processes ongoing in bone, and they are the most important final products of the homeostatic signaling pathways. Profiling those proteins is vital for a thorough understanding of bone biology. To date, proteome research on bone has been focused mainly on in vitro analysis of bone-forming cells (osteoblasts and osteoclasts) to determine which proteins are expressed under a given set of experimental conditions (13–16). Although important, such studies cannot identify the actual protein profile in oral alveolar bone. Recently, the extraction of proteins directly from skull bone for proteome analysis was reported (17, 18). The extracted proteins were first separated using two-dimensional gel electrophoresis, after which spots of interest were excised and the proteins were identified via mass spectrometry (MS). However, using two-dimensional gel electrophoresis to analyze extreme proteins (e.g. extremely basic or acidic, extremely small or large, extremely hydrophobic) is challenging. Shotgun proteomics, which is a method of high-throughput proteome analysis (19–21), avoids the intrinsic limitations of two-dimensional gel electrophoresis. Despite an interesting need for large-scale characterization of the bone proteome, one study has been reported to apply shotgun proteomics for proteome analysis of rat femur bone (22). However, they identified only 133 proteins, because they analyzed bone proteins using a one-step method without a demineralization stage. The other report showed only that bone proteins extracted from the skull bone of an adult beagle are carried using a demineralization step (23). There are no reports regarding the interaction between alveolar bone and soft tissue yet.The efficient extraction of bone proteins is a critical issue for proteome analysis (24). Because bone is largely mineralized, and therefore nearly solid, classical protein extraction methods used for soft tissues and cells may not be appropriate for bone. It is therefore necessary to develop methods to efficiently extract protein from bone. In earlier bone proteome analyses (17, 18, 22), the bones were first ground to powder, after which the proteins were extracted by means of incubating the powder in lysis buffer. However, mechanically breaking bones down into powder is laborious, especially for large animal bones. More important, large amounts of collagen and proteoglycans also are extracted, and this can impair the detection of low-abundance proteins and strongly affect isoelectric focusing (25). For the present study, we adopted an alternative method of demineralizing bone tissue and then investigated the efficiency of protein extraction from the demineralized bone tissue. This method was based on a recently reported sequential protein extraction protocol that was used to extract proteins from skull for comprehensive analysis of its proteome. Two-dimensional high-performance liquid chromatography–tandem mass spectrometry (LC-MS/MS) was then applied to analyze the protein extracts, enabling the identification of 2479 proteins (23). We employed a similar method to extract and identify proteins in tooth alveolar bone.Given that a large number of proteins are likely involved in the healing of bone, as well as of soft tissues, another goal of the present study was to examine protein expression and putative signaling during bone healing after tooth extraction. Here, we used nano-UPLC-MS^E-based label-free quantitative proteomics to analyze alveolar bone and the adjacent soft tissue. The environment surrounding healing bone would be expected to affect the specific signaling networks involved in bone regeneration. We suggest that determining the protein networks in alveolar bone and gingival tissue will enable improvement of the soft tissue interface, aspects of the hard tissue, and dental appearance during and after therapy.     

大鼠骨髓来源未成熟与成熟树突状细胞的对比研究

目的:对比培养大鼠骨髓来源的未成熟树突状细胞与成熟树突状细胞,并从形态学、表型及功能检测等多方面进行对比研究,为后续的实验做出基础研究。方法:大鼠脱臼法处死后取两侧胫骨、股骨,PBS冲洗骨髓腔收集骨髓细胞,经GM-CSF和IL-4刺激培养六天后,对比研究经LPS刺激组与未经LPS刺激培养组细胞状况。结果:①成熟树突状细胞悬浮生长,集落分散,扫描电镜下见其突起数目明显多于未成熟树突状细胞。②成熟树突状细胞高表达表面标记分子CD80、CD86、MHCⅡ,而未成熟树突状细胞均低表达。③成熟树突状细胞培养基上清中IL-12水平高,而未成熟树突状细胞培养基上清中IL-12水平低。④成熟树突状细胞具有强的刺激T细胞增殖能力,而未成熟树突状细胞基本不具有诱导T细胞增殖能力。结论:未成熟状态的树突状细胞具备致耐受原性,可抑制T细胞的应答,而成熟状态的树突状细胞由于获得了免疫刺激潜能从而会对炎性刺激做出反应。     

Increased Expression of Matrix Extracellular Phosphoglycoprotein (MEPE) in Cortical Bone of the Rat Tibia after Mechanical Loading: Identification by Oligonucleotide Microarray

Skeletal integrity in humans and animals is maintained by daily mechanical loading. It has been widely accepted that osteocytes function as mechanosensors. Many biochemical signaling molecules are involved in the response of osteocytes to mechanical stimulation. The aim of this study was to identify genes involved in the translation of mechanical stimuli into bone formation. The four-point bending model was used to induce a single period of mechanical loading on the right tibia, while the contra lateral left tibia served as control. Six hours after loading, the effects of mechanical loading on gene-expression were determined with microarray analysis. Protein expression of differentially regulated genes was evaluated with immunohistochemistry. Nine genes were found to exhibit a significant differential gene expression in LOAD compared to control. MEPE, Garnl1, V2R2B, and QFG-TN1 olfactory receptor were up-regulated, and creatine kinase (muscle form), fibrinogen-B beta-polypeptide, monoamine oxidase A, troponin-C and kinesin light chain-C were down-regulated. Validation with real-time RT-PCR analysis confirmed the up-regulation of MEPE and the down-regulation of creatine kinase (muscle form) and troponin-C in the loaded tibia. Immunohistochemistry showed that the increase of MEPE protein expression was already detectable six hours after mechanical loading. In conclusion, these genes probably play a role during translation of mechanical stimuli six hours after mechanical loading. The modulation of MEPE expression may indicate a connection between bone mineralization and bone formation after mechanical stimulation.     

Diverse Bone Morphogenetic Protein Expression Profiles and Smad Pathway Activation in Different Phenotypes of Experimental Canine Mammary Tumors

Background

BMPs are currently receiving attention for their role in tumorigenesis and tumor progression. Currently, most BMP expression studies are performed on carcinomas, and not much is known about the situation in sarcomas.

Methodology/Principal Findings

We have investigated the BMP expression profiles and Smad activation in clones from different spontaneous canine mammary tumors. Spindle cell tumor and osteosarcoma clones expressed high levels of BMPs, in particular BMP-2, -4 and -6. Clones from a scirrhous carcinoma expressed much lower BMP levels. The various clones formed different tumor types in nude mice but only clones that expressed high levels of BMP-6 gave bone formation. Phosphorylated Smad-1/5, located in the nucleus, was detected in tumors derived from clones expressing high levels of BMPs, indicating an active BMP signaling pathway and BMP-2 stimulation of mammary tumor cell clones in vitro resulted in activation of the Smad-1/5 pathway. In contrast BMP-2 stimulation did not induce phosphorylation of the non-Smad pathway p38 MAPK. Interestingly, an increased level of the BMP-antagonist chordin-like 1 was detected after BMP stimulation of non-bone forming clones.

Conclusions/Significance

We conclude that the specific BMP expression repertoire differs substantially between different types of mammary tumors and that BMP-6 expression most probably has a biological role in bone formation of canine mammary tumors.     

Bone remodelling around a cementless glenoid component

Post-operative change in the mechanical loading of bone may trigger its (mechanically induced) adaptation and hamper the mechanical stability of prostheses. This is especially important in cementless components, where the final fixation is achieved by the bone itself. The aim of this study is, first, to gain insight into the bone remodelling process around a cementless glenoid component, and second, to compare the possible bone adaptation when the implant is assumed to be fully bonded (best case scenario) or completely loose (worst case scenario). 3D finite element models of a scapula with and without a cementless glenoid component were created. 3D geometry of the scapula, material properties, and several physiological loading conditions were acquired from or estimated for a specific cadaver. Update of the bone density after implantation was done according to a node-based bone remodelling scheme. Strain energy density for different loading conditions was evaluated, weighted according to their frequencies in activities of daily life and used as a mechanical stimulus for bone adaptation. The average bone density in the glenoid increased after implantation. However, local bone resorption was significant in some regions next to the bone-implant interface, regardless of the interface condition (bonded or loose). The amount of bone resorption was determined by the condition imposed to the interface, being slightly larger when the interface was loose. An ideal screw, e.g. in which material fatigue was not considered, was enough to keep the interface micromotions small and constant during the entire bone adaptation simulation.     

ZAP-70 Promotes the Infiltration of Malignant B-Lymphocytes into the Bone Marrow by Enhancing Signaling and Migration after CXCR4 Stimulation

ZAP-70 in chronic lymphocytic leukemia (CLL) is associated with enhanced response to microenvironmental stimuli. We analyzed the functional consequences of ZAP-70 ectopic expression in malignant B-cells in a xenograft mouse model of disseminated B-cell leukemia. Mice injected with B-cells expressing ZAP-70 showed a prominently higher infiltration of the bone marrow. In vitro analysis of the response of malignant B-cells to CXCL12, the main attracting chemokine regulating trafficking of lymphocytes to the bone marrow, or to bone marrow stromal cells, revealed that ZAP-70 induces an increased response in terms of signaling and migration. These effects are probably mediated by direct participation of ZAP-70 in CXCL12-CXCR4 signaling since CXCR4 stimulation led to activation of ZAP-70 and downstream signaling pathways, such as MAPK and Akt, whereas ZAP-70 did not alter the expression of the CXCR4 receptor. In addition, subclones of primary CLL cells with high expression of ZAP-70 also showed increased migrative capacity toward CXCL12. Neutralization of CXCR4 with a monoclonal antibody resulted in impaired in vitro responses to CXCL12 and bone marrow stromal cells. We conclude that ZAP-70 enhances the migration of malignant B-cells into the supportive microenvironment found in the bone marrow mainly by enhancing signaling and migration after CXCR4 stimulation.     

微波高温灭活及自体髂骨异体骨粒复合骨水泥修复 治疗长骨骨巨细胞瘤骨缺损

目的:探讨微波高温灭活及自体髂骨、异体骨粒复合骨水泥修复骨巨细胞瘤病灶刮除后骨缺损的临床应用效果。方法:应用原位分离插入式微波天线高温灭活技术,自体髂骨、异体骨粒复合骨水泥修复21例长骨骨巨细胞瘤术后骨缺损,从手术技术、肿瘤复发情况、肢体关节功能等方面全面综合评价此方法临床应用效果。结果:21例患者均获得骨性愈合,无骨折及内固定断裂发生,2例复发,复发率9.8%;肢体关节功能优18例(85.7%)、良3例(14.3%)、中差0例。结论:微波高温能彻底杀灭肿瘤组织降低复发率,自体髂骨保证与近关节软骨下骨愈合,异体骨粒复合骨水泥能良好充填残余瘤腔、且具有良好的生物力学性能,以防发生关节软骨面塌陷。     

Bone regeneration: molecular and cellular interactions with calcium phosphate ceramics

Calcium phosphate bioceramics are widely used in orthopedic and dental applications and porous scaffolds made of them are serious candidates in the field of bone tissue engineering. They have superior properties for the stimulation of bone formation and bone bonding, both related to the specific interactions of their surface with the extracellular fluids and cells, ie, ionic exchanges, superficial molecular rearrangement and cellular activity.     

Bone ingrowth simulation for a concept glenoid component design

Glenoid component loosening is the major problem of total shoulder arthroplasty. It is possible that uncemented component may be able to achieve superior fixation relative to cemented component. One option for uncemented glenoid is to use porous tantalum backing. Bone ingrowth into the porous backing requires a degree of stability to be achieved directly post-operatively. This paper investigates the feasibility of bone ingrowth with respect to the influence of primary fixation, elastic properties of the backing and friction at the bone prosthesis interface. Finite element models of three glenoid components with different primary fixation configurations are created. Bone ingrowth into the porous backing is modelled based on the magnitude of the relative interface micromotions and mechanoregulation of the mesenchymal stem cells that migrated via the bonded part of the interface. Primary fixation had the most influence on bone ingrowth. The simulation showed that its major role was not to firmly interlock the prosthesis, but rather provide such a distribution of load, that would result in reduction of the peak interface micromotions. Should primary fixation be provided, friction has a secondary importance with respect to bone ingrowth while the influence of stiffness was counter intuitive: a less stiff backing material inhibits bone ingrowth by higher interface micromotions and stimulation of fibrous tissue formation within the backing.     

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.