Recombinant Human Bone Morphogenetic Protein-2 Research Articles

An Innovative Design to Enhance Osteoinductive Efficacy and Biomechanical Behavior of a Titanium Dental Implant

The present study investigated the in vivo bone-forming efficacy of an innovative titanium (Ti) dental implant combined with a collagen sponge containing recombinant human bone morphogenetic protein-2 (BMP-2) in a pig model. Two different concentrations of BMP-2 (20 and 40 µg/mL) were incorporated into collagen sponges and placed at the bottom of Ti dental implants. The investigated implants were inserted into the edentulous ridge at the canine–premolar regions of Lanyu small-ear pigs, which were then euthanized at weeks 1, 2, 4, 8, and 12 post-implantation. Specimens containing the implants and surrounding bone tissue were collected for histological evaluation of their bone-to-implant contact (BIC) ratios and calculation of maximum torques using removal torque measurement. Analytical results showed that the control and BMP-2-loaded implants presented good implant stability and bone healing for all testing durations. After 1 week of healing, the BMP-2-loaded implants with a concentration of 20 µg/mL exhibited the highest BIC ratios, ranging from 58% to 76%, among all groups (p = 0.034). Additionally, they also possessed the highest removal torque values (50.1 ± 1.3 N-cm) throughout the 8-week healing period. The BMP-2-loaded implants not only displayed excellent in vivo biocompatibility but also presented superior osteoinductive performance. Therefore, these findings demonstrate that BMP-2 delivered through a collagen sponge can potentially enhance the early-stage osseointegration of Ti dental implants.

Comparative Studies of Bone Graft and Orthobiologics for Foot Ankle Arthrodesis: A Critical Review.

Graft materials available to supplement hindfoot and ankle arthrodesis procedures include autologous (autograft) or allogeneic bone graft (allograft) but also bone graft substitutes such as demineralized bone matrix, calcium sulfate, calcium phosphate, and tricalcium phosphate/hydroxyapatite. In addition, biologic agents, such as recombinant human bone morphogenetic protein-2 or recombinant human platelet derived growth factor-BB (rhPDGF-BB), and preparations, including platelet-rich plasma or concentrated bone marrow aspirate, have been used to facilitate bone healing in ankle or hindfoot arthrodesis. The purpose of this review was to summarize the available clinical evidence surrounding the utilization and efficacy of the above materials and biological agents in ankle or hindfoot arthrodesis procedures, with emphasis on the quality of the existing evidence to facilitate clinical decision making.

Regulating the bioactivity of non-glycosylated recombinant human bone morphogenetic protein-2 to enhance bone regeneration

Recombinant human bone morphogenetic protein-2 (rhBMP-2) is the predominant growth factor that effectively induces osteogenic differentiation in orthopedic procedures. However, the bioactivity and stability of rhBMP-2 are intrinsically associated with its sequence, structure, and storage conditions. In this study, we successfully determined the amino acid sequence and protein secondary structure model of non-glycosylated rhBMP-2 expressed by an E. coli expression system through X-ray crystal structure analysis. Furthermore, we observed that acidic storage conditions enhanced the proliferative and osteoinductive activity of rhBMP-2. Although the osteogenic activity of non-glycosylated rhBMP-2 is relatively weaker compared to glycosylated rhBMP-2; however, this discrepancy can be mitigated by incorporating exogenous chaperone molecules. Overall, such information is crucial for rationalizing the design of stabilization methods and enhancing the bioactivity of rhBMP-2, which may also be applicable to other growth factors.

Failure Rates Based on Alveolar Cleft Volume: An Analysis of the Critical-Sized Defect for Alveolar Bone Grafting.

Alveolar bone grafting (ABG) using iliac crest bone graft (ICBG) is best practice for children with complete cleft lip and palate. With the advent of recombinant human bone morphogenetic protein (rhBMP-2) and demineralized bone matrix (DBM), excellent results can still be achieved while avoiding donor-site morbidity. This study aims to determine the critical-sized defects by analyzing graft failure rates for ICBG and rhBMP-2/DBM to guide surgeons performing ABG. A retrospective review was conducted evaluating patients who underwent ABG from 2016-2022. Patients with preoperative and postoperative cone beam computed tomography (CBCT) imaging were included. Volumetric defect sizes were calculated using preoperative imaging. Graft success criteria were based on both clinical and radiographic outcomes. Logistic regressions analyzed graft failure rates to identify an optimal cutoff, which defined the critical-sized defect. Ninety-three patients were included. Bone graft cohorts included ICBG (n=30) and rhBMP-2/DBM (n=63). The critical-sized defects were calculated to be 810 mm 3 and 885 mm 3 for ICBG and rhBMP-2/DBM, respectively. There were significantly higher graft failure rates beyond the critical size compared to below for both ICBG (71.4% vs. 0.0%; p<0.001) and rhBMP-2/DBM (65.0% vs. 14.0%; p<0.001). This study identified critical-sized defects based on alveolar cleft volume for ICBG or rhBMP-2/DBM with higher graft failure rates beyond the predicted thresholds. Distinct ranges in cleft volume were identified where patients might benefit from each select graft option.

Mesenchymal Stem Cell Exosomes Enhance Posterolateral Spinal Fusion in a Rat Model.

Spinal fusion, a common surgery performed for degenerative lumbar conditions, often uses recombinant human bone morphogenetic protein 2 (rhBMP-2) that is associated with adverse effects. Mesenchymal stromal/stem cells (MSCs) and their extracellular vesicles (EVs), particularly exosomes, have demonstrated efficacy in bone and cartilage repair. However, the efficacy of MSC exosomes in spinal fusion remains to be ascertained. This study investigates the fusion efficacy of MSC exosomes delivered via an absorbable collagen sponge packed in a poly Ɛ-caprolactone tricalcium phosphate (PCL-TCP) scaffold in a rat posterolateral spinal fusion model. Herein, it is shown that a single implantation of exosome-supplemented collagen sponge packed in PCL-TCP scaffold enhanced spinal fusion and improved mechanical stability by inducing bone formation and bridging between the transverse processes, as evidenced by significant improvements in fusion score and rate, bone structural parameters, histology, stiffness, and range of motion. This study demonstrates for the first time that MSC exosomes promote bone formation to enhance spinal fusion and mechanical stability in a rat model, supporting its translational potential for application in spinal fusion.

Injectable bioactive poly(propylene fumarate) and polycaprolactone based click chemistry bone cement for spinal fusion in rabbits.

Degenerative spinal pathology is a widespread medical issue, and spine fusion surgeries are frequently performed. In this study, we fabricated an injectable bioactive click chemistry polymer cement for use in spinal fusion and bone regrowth. Taking advantages of the bioorthogonal click reaction, this cement can be crosslinked by itself eliminating the addition of a toxic initiator or catalyst, nor any external energy sources like UV light or heat. Furthermore, nano-hydroxyapatite (nHA) and microspheres carrying recombinant human bone morphogenetic protein-2 (rhBMP-2) and recombinant human vascular endothelial growth factor (rhVEGF) were used to make the cement bioactive for vascular induction and osteointegration. After implantation into a rabbit posterolateral spinal fusion (PLF) model, the cement showed excellent induction of new bone formation and bridging bone, achieving results comparable to autograft control. This is largely due to the osteogenic properties of nano-hydroxyapatite (nHA) and the released rhBMP-2 and rhVEGF growth factors. Since the availability of autograft sources is limited in clinical settings, this injectable bioactive click chemistry cement may be a promising alternative for spine fusion applications in addressing various spinal conditions.

Dysphagia After Anterior Cervical Spine Surgery: Pathophysiology, Diagnosis, and Management.

Anterior cervical spine surgery (ACSS) is a surgical intervention widely used for a myriad of indications including degenerative, oncologic, inflammatory, traumatic, and congenital spinal conditions. A primary concern for surgeons performing ACSS is the postoperative development of oropharyngeal dysphagia. Current literature reports a wide incidence of this complication ranging from 1 to 79%. Dysphagia after ACSS is multifactorial, with common risk factors being prolonged duration of operation, revision surgeries, multilevel surgeries, and use of recombinant human bone morphogenetic protein-2. Many technical strategies have been developed to reduce the risk of postoperative dysphagia, including the development of low-profile implants and retropharyngeal local steroid application. In this article, we review the most recent literature regarding the epidemiology and pathophysiology, diagnostic criteria, risk factors, and management of dysphagia after ACSS.

Does Bone Morphogenetic Protein Use Reduce Pseudarthrosis Rates in Single-Level Transforaminal Lumbar Interbody Fusion Surgeries?

Recombinant human bone morphogenetic protein 2 (rhBMP-2, or BMP for short) is a popular biological product used in spine surgeries to promote fusion and avoid the morbidity associated with iliac crest autograft. BMP's effect on pseudarthrosis in transforaminal lumbar interbody fusion (TLIF) remains unknown. To assess the rates of pseudarthrosis in single-level TLIF with and without concurrent use of BMP. This was a retrospective cohort study conducted at a single academic institution. Adults undergoing primary single-level TLIF with a minimum of 1 year of clinical and radiographic follow-up were included. BMP use was determined by operative notes at index surgery. Non-BMP cases with iliac crest bone graft were excluded. Pseudarthrosis was determined using radiographic and clinical evaluation. Bivariate differences between groups were assessed by independent t test and χ 2 analyses, and perioperative characteristics were analyzed by multiple logistic regression. One hundred forty-eight single-level TLIF patients were included. The mean age was 59.3 years, and 52.0% were women. There were no demographic differences between patients who received BMP and those who did not. Pseudarthrosis rates in patients treated with BMP were 6.2% vs 7.5% in the no BMP group (P = 0.756). There was no difference in reoperation for pseudarthrosis between patients who received BMP (3.7%) vs those who did not receive BMP (7.5%, P = 0.314). Patients who underwent revision surgery for pseudarthrosis more commonly had diabetes with end-organ damage (revised 37.5% vs not revised 1.4%, P < 0.001). Multiple logistic regression analysis demonstrated no reduction in reoperation for pseudarthrosis related to BMP use (OR 0.2, 95% CI 0.1-3.7, P = 0.269). Diabetes with end-organ damage (OR 112.6,95% CI 5.7-2225.8, P = 0.002) increased the risk of reoperation for pseudarthrosis. BMP use did not reduce the rate of pseudarthrosis or the number of reoperations for pseudarthrosis in single-level TLIFs. Diabetes with end-organ damage was a significant risk factor for pseudarthrosis. BMP is frequently used "off-label" in transforaminal lumbar interbody fusion; however, little data exists to demonstrate its safety and efficacy in this procedure.

Efficacy of hydroxyapatite and fibrin sealant as carriers for bone morphogenetic protein-2 in maxillary sinus floor augmentation: a retrospective study

The aim of this retrospective study was to assess the efficacy of recombinant human bone morphogenetic protein-2 (rhBMP-2) with hydroxyapatite (HA) granules and fibrin sealant (FS) in maxillary sinus floor augmentation (MSFA), with a focus on the volume change. Fifty-two of 137 patients who underwent MSFA with rhBMP-2/HA grafting between June 2016 and December 2022 met the study inclusion criteria; 25 had received rhBMP-2/HA without FS and 27 had received rhBMP-2/HA with FS. Computed tomography (CT) images were obtained preoperatively, immediately following the operation, and at 6 months postoperative. These images were three-dimensionally reconstructed to measure the volumetric and height changes following MSFA. The mean ± standard deviation percentage of volumetric change at 6 months was 48.75 ± 37.44% in the group with FS and 29.77 ± 13.42% in the group without FS (P = 0.019). The vertical height measured at a specific site of the grafted area showed a mean percentage change at 6 months of 4.05 ± 12.08% in the group with FS and 6.07 ± 10.15% in the group without FS (P = 0.518). The additional use of FS as a carrier for rhBMP-2/HA in MSFA was found to improve surgical convenience and bone regeneration ability.

Transpedicular injection of rhBMP-2 with β-tricalcium phosphate to reduce the proximal junctional kyphosis after adult spinal deformity correction: preliminary study

The aim of this preliminary study was to assess the impact of injecting recombinant human bone morphogenetic protein-2 (rhBMP-2) with β-tricalcium phosphate (β-TCP) carrier into the uppermost instrumented vertebra (UIV) during surgery to prevent the development of proximal junctional kyphosis (PJK) and proximal junctional failure (PJF). The 25 patients from study group had received 0.5 mg rhBMP-2 mixed with 1.5 g β-TCP paste injection into the UIV during surgery. The control group consisted of 75 patients who underwent surgery immediately before the start of the study. The incidences of PJK and PJF were analyzed as primary outcomes. Spinopelvic parameters and patient-reported outcomes were analyzed as secondary outcomes. Hounsfield unit (HU) measurements were performed to confirm the effect of rhBMP-2 with β-TCP on bone formation at preoperative and postoperative at computed tomography. PJK and PJF was more occurred in control group than study group (p = 0.02, 0.29, respectively). The HU of the UIV significantly increased 6 months after surgery. And the increment at the UIV was also significantly greater than that at the UIV-1 6 months after surgery. Injection of rhBMP-2 with β-TCP into the UIV reduced PJK and PJF rates 6 months after surgery with new bone formation.

Bioactive Moldable Click Chemistry Polymer Cement with Nano-Hydroxyapatite and Growth Factor-Enhanced Posterolateral Spinal Fusion in a Rabbit Model.

Spinal injuries or diseases necessitate effective fusion solutions, and common clinical approaches involve autografts, allografts, and various bone matrix products, each with limitations. To address these challenges, we developed an innovative moldable click chemistry polymer cement that can be shaped by hand and self-cross-linked in situ for spinal fusion. This self-cross-linking cement, enabled by the bioorthogonal click reaction, excludes the need for toxic initiators or external energy sources. The bioactivity of the cement was promoted by incorporating nanohydroxyapatite and microspheres loaded with recombinant human bone morphogenetic protein-2 and vascular endothelial growth factor, fostering vascular induction and osteointegration. The release kinetics of growth factors, mechanical properties of the cement, and the ability of the scaffold to support in vitro cell proliferation and differentiation were evaluated. In a rabbit posterolateral spinal fusion model, the moldable cement exhibited remarkable induction of bone regeneration and effective bridging of spine vertebral bodies. This bioactive moldable click polymer cement therefore presents a promising biomaterial for spinal fusion augmentation, offering advantages in safety, ease of application, and enhanced bone regrowth.

Evaluation of the Fusion Rate and Safety of Escherichia coli-Derived rhBMP-2 in Transforaminal Lumbar Interbody Fusion for Patients with Degenerative Lumbar Disease: A Prospective, Multicenter, Single-Arm Trial.

Background: Few studies have documented the viability of E. coli-derived recombinant human bone morphogenetic protein-2 (rhBMP-2) in transforaminal lumbar interbody fusion (TLIF). This study aimed to assess the safety and fusion rate of rhBMP-2 in TLIF. Methods: The study was conducted as a prospective, multicenter, single-arm trial, and 30 patients needing one- or two-level TLIF were enrolled. Fusion rate was assessed using the 12-month interbody fusion rate on CT. Postoperative problems, including seroma, radiculitis, and ectopic bone formation, which have been documented as risks associated with rhBMP-2 in prior studies, were recorded. Results: The study demonstrated fusion outcomes in all instances at 52 and 104 weeks post-surgery. Significant improvements were observed in clinical outcomes, with ODI, SF-36, and VAS scores, all achieving statistical significance (p < 0.0001). No perioperative adverse events requiring reoperation were reported, and there were no incidences of seroma, radiculitis, cage migration, grafted bone extrusion, postoperative neurologic deficit, or deep wound infection. Conclusions: The study demonstrates the high safety and efficacy in inducing bone fusion of E. coli-derived rhBMP-2 in TLIF, with a notable absence of adverse postoperative complications. Trial registration: This study protocol was registered at Korea Clinical Research Information Service (number identifier: KCT0004738) on July 2020.

The Evolution and Application of a Novel DNA Aptamer Targeting Bone Morphogenetic Protein 2 for Bone Regeneration.

Recombinant human bone morphogenetic protein 2 (rhBMP-2) is an FDA-approved growth factor for bone regeneration and repair in medical practice. The therapeutic effects of rhBMP-2 may be enhanced through specific binding to extracellular matrix (ECM)-like scaffolds. Here, we report the selection of a novel rhBMP-2-specific DNA aptamer, functionalization of the aptamer in an ECM-like scaffold, and its application in a cellular context. A DNA aptamer BA1 was evolved and shown to have high affinity and specificity to rhBMP-2. A molecular docking model demonstrated that BA1 was probably bound to rhBMP-2 at its heparin-binding domain, as verified with experimental competitive binding assays. The BA1 aptamer was used to functionalize a type I collagen scaffold, and fraction ratios were optimized to mimic the natural ECM. Studies in the myoblast cell model C2C12 showed that the aptamer-enhanced scaffold could specifically augment the osteo-inductive function of rhBMP-2 in vitro. This aptamer-functionalized scaffold may have value in enhancing rhBMP-2-mediated bone regeneration.

Use of Recombinant Human Bone Morphogenetic Protein-2 After Anterior Cervical Corpectomy and Fusion for the Treatment of Vertebral Osteomyelitis

To report the operative outcomes after treating vertebral osteomyelitis patients with an anterior cervical corpectomy and fusion procedure using recombinant human bone morphogenetic protein-2 as graft material. A retrospective review of electronic medical records of 26 adult patients who underwent an anterior cervical corpectomy and fusion procedure for cervical osteomyelitis using rhBMP-2 at the University of Puerto Rico University District Hospital was performed. Indication, preoperative laboratory results, levels of corpectomy, preoperative ASIA score, complications, fusion evaluation at 12 months, and ASIA score at 12 months was reviewed. For the cohort of patients, mean age was 47±13 years and 65% were male. Spinal instability was present in 54%. The levels of corpectomy were: one-level in 2 cases, two-level in 15 cases, three-level in 8 cases and five-level in 1 case. 4 patients had complications and of these, 2 experienced dysphagia. The fusion rate was 100% and no reoperations were performed. An improvement in ASIA score was seen for 54% patients at 12 months follow up. This study demonstrates a fusion rate of 100% with no reoperations reported. Recombinant human bone morphogenetic protein-2 could be considered and further researched as grafting material for anterior cervical corpectomy and fusion procedures in cervical osteomyelitis patients.

Osteobiologics in degenerative spine disease: current clinical applications of recombinant human bone morphogenetic proteins—a narrative review

Osteobiologics in degenerative spine disease: current clinical applications of recombinant human bone morphogenetic proteins—a narrative review

Association between structural rib autograft and the rate of arthrodesis in children undergoing occiput-C2 instrumentation and fusion.

The purpose of this study was to identify factors associated with fusion success among pediatric patients undergoing occiput-C2 rigid instrumentation and fusion. The Pediatric Spine Study Group registry was queried to identify patients ≤ 21 years of age who underwent occiput-C2 posterior spinal rigid instrumentation and fusion and had a 2-year minimum clinical and radiographic (postoperative lateral cervical radiograph or CT scan) follow-up. Fusion failure was defined clinically if a patient underwent hardware revision surgery > 30 days after the index procedure or radiographically by the presence of hardware failure or screw haloing on the most recent follow-up imaging study. Univariate comparisons and multivariable logistic regression analyses were subsequently performed. Seventy-six patients met inclusion criteria. The median age at surgery was 9 years (range 1.5-17.2 years), and 51% of the cohort was male. Overall, 75% of patients had syndromic (n = 41) or congenital (n = 15) etiologies, with the most frequent diagnoses of Down syndrome (28%), Chiari malformation (13%), and Klippel-Feil syndrome (12%). Data were available to determine if there was a fusion failure in 97% (74/76) of patients. Overall, 38% (28/74) of patients had fusion failure (95% CI 27%-50%). Univariate analysis demonstrated that use of a rigid cervical collar postoperatively (p = 0.04) and structural rib autograft (p = 0.02) were associated with successful fusion. Multivariable logistic regression analysis determined that patients who had rib autograft used in surgery had a 73% decrease in the odds of fusion failure (OR 0.27, 95% CI 0.09-0.82; p = 0.02). Age, etiology including Down syndrome, instrumentation type, unilateral instrumentation, use of recombinant human bone morphogenetic protein, and other variables did not influence the risk for fusion failure. In this multicenter, multidisciplinary, international registry of children undergoing occiput-C2 instrumentation and fusion, fusion failure was seen in 38% of patients, a higher rate than previously reported in the literature. The authors' data suggest that postoperative immobilization in a rigid cervical collar may be beneficial, and the use of structural rib autograft should be considered, as rib autograft was associated with a 75% higher chance of successful fusion.

Goat mammary epithelial cells provide a better expression system for production of recombinant human bone morphogenetic protein 2 compared to Chinese hamster ovarian cells.

Bone Morphogenetic Protein 2 (BMP2), a member of the Transforming Growth Factor-β (TGF-β) super family of proteinsand is instrumental in the repair of fractures. The synthesis of BMP2 involves extensive post-translational processing and several studies have demonstrated the abysmally low production of rhBMP2 in eukaryotic systems, which may be due to the short half-life of the bioactive protein. Consequently, production costs of rhBMP2 are quite high, limiting its availability to the general populace. Therefore, there is an urgent need to identify better in-vitro systems for large scale production of rhBMP2. In the present study, we have carried out a comparative analysis of rhBMP2 production by the conventionally used Chinese Hamster ovarian cells (CHO) and goat mammary epithelial cells (GMEC), upon transfection with appropriate construct. Udder gland cells are highly secretory, and we reasoned that such cells may serve as a better in-vitro model for large scale production of rhBMP2. Our results indicated that the synthesis and secretion of bioactive rhBMP2 by goat mammary epithelial cells was significantly higher as compared to that by CHO-K1 cells. Our results provide strong evidence that GMECs may serve as a better alternative to other mammalian cells used for therapeutic protein production.

In Vivo Osteo-organoid Approach for Harvesting Therapeutic Hematopoietic Stem/Progenitor Cells.

Hematopoietic stem cell transplantation (HSCT) requires a sufficient number of therapeutic hematopoietic stem/progenitor cells (HSPCs). To identify an adequate source of HSPCs, we developed an in vivo osteo-organoid by implanting scaffolds loaded with recombinant human bone morphogenetic protein-2 (rhBMP-2) into an internal muscle pouch near the femur in mice. After 12 weeks of implantation, we retrieved the in vivo osteo-organoids and conducted flow cytometry analysis on HPSCs, revealing a significant presence of HSPC subsets within the in vivo osteo-organoids. We then established a sublethal model of hematopoietic/immune system injury in mice through radiation and performed hematopoietic stem cell transplantation (HSCT) by injecting the extracted osteo-organoid-derived cells into the peripheral blood of radiated mice. The effect of hematopoietic recovery was evaluated through hematological, peripheral blood chimerism, and solid organ chimerism analyses. The results confirmed that in vivo osteo-organoid-derived cells can rapidly and efficiently reconstruct damaged peripheral and solid immune organs in irradiated mice. This approach holds potential as an alternative source of HSPCs for HSCT, offering benefits to a larger number of patients.

Mesenchymal stem cells enhance targeted bone growth from injectable hydrogels with BMP-2 peptides.

Osteoporosis is the most common chronic metabolic bone disease, and the prevalence of osteoporotic fractures is rapidly increasing with the aging population. While bisphosphonates can reduce bone loss and risk of fracture, these drugs are systemic, rely on long-term use, and patient compliance is low. Recombinant human bone morphogenetic protein-2 (BMP-2) is an FDA-approved protein that can offer a more targeted therapeutic than systemic treatments. DWIVA is a peptide sequence corresponding to the wrist epitope of BMP-2, and DWIVA-functionalized hydrogels feature osteoinductive propertiesin vitro and in vivo. This study reports that self-forming DWIVA-functionalized hydrogels injected into the intramedullary canal of rat femurs induce a local increase in trabecular bone in as little as 2 weeks. Increases in bone volume, trabecular thickness, and trabeculae count from DWIVA-laden hydrogels persist for at least 4 weeks, and the inclusion of mesenchymal stem cells (MSCs) significantly enhances the development of mineralized bone. Histological analysis of decalcified femurs also shows that hydrogel injections containing DWIVA peptide and MSCs stimulate unmineralized bone tissue formation and induce an increased count of osteoblasts and osteoclasts at the injection site after 4 weeks. Overall, the MSC-laden DWIVApeptide-functionalized hydrogels presented rapidly induce targeted bone formation and have the potential to form nascent bone within bones in jeopardy of an osteoporotic fracture such as the femur.

Effect of recombinant human bone morphogenetic protein-2 and osteoprotegerin-Fc in MC3T3-E1 cells.

We compared the osteoblastogenesis by serially administrating recombinant human bone morphogenetic protein-2 (rhBMP-2) and osteoprotegerin-immunoglobulin Fc segment complex (OPG-Fc). The MC3T3-E1 preosteoblast cell line was differentiated for 1, 3, and 7 days with a treatment of OPG-Fc in 10~200 ng/mL concentration and the cell viability was evaluated by Cell Counting Kit-8 analysis. The level of differentiation from MC3T3-E1 cells to osteoblasts was determined by alkaline phosphatase activity. The level of runt domain-containing transcription factor 2 (Runx2) and osteopontin (OPN) manifestation, involved in osteoblast differentiation, was examined by real-time polymerase chain reaction and western blotting. During MC3T3-E1 cell differentiation, the differentiation level was high with 1-day treatment using 100 ng/mL OPG-Fc. The treatment with 50 ng/mL rhBMP-2 for 7 days, followed by 1-day treatment with 100 ng/mL OPG-Fc produced the highest differentiation level, which was approximately 5.3 times that of the control group (p<0.05). The expression of Runx2 mRNA significantly increased, reaching 2.5 times the level of the control group under the condition of 7-day treatment with rhBMP-2 and 1-day treatment with OPG-Fc (p<0.001). The expression of Runx2 protein significantly increased to approximately 5.7 times that of the control group under the condition of 7-day treatment with rhBMP-2, followed by 1-day treatment with OPG-Fc (p<0.01). The expression of OPN protein showed no change from that of the control group under various conditions of rhBMP-2 and OPG-Fc combinations. These results imply that the treating preosteoblasts with rhBMP-2 first and then with OPG-Fc increased osteoblast differentiation efficacy.