Effect Of Bone Morphogenetic Protein Heterodimer On Osteoblastogenesis And Osteoclastogenesis In Vitro

The osseous restoration of voluminous bone defect remains a challenge in orthopedics, maxillofacial surgery and implantology. Bone tissue engineering including gene, cell, and cytokine therapies has been reported to substitute autografting and allografting. Among the three therapies, cytokine therapy is advantageous in safety, feasibility, and potential for nearest clinical application over the other two. Bone morphogenetic proteins (BMPs) a group of dimeric disulfide-linked polypeptide growth factors under transforming growth factor-P superfamily, are one of the paramount cytokines in promoting bone regeneration.Part 1:Direct Effect of RhBMP2/7 Heterodimer on Osteoblastogenesis in Vitro Compared to RhBMP2 and RhBMP7 HomodimersObjectives:The indication of BMPs for clinical applications is limited due to their high effective doses. BMP heterodimers were exhibited to be more potent than and thus were potential substitutes for BMP homodimers. However, the effect of BMP heterodimers on the sequential osteoblastogenetic events such as chemotactic migration, proliferation, differentiation and mineralization remains uncovered. This study aims to delineate the bio-functional characteristics of rhBMP2/7 heterodimer in inducing osteoblastogenesis of MC3T3-E1 in vitro.Materials and Methods:Time-course and dose-response studies were performed to compare the potencies of purified rhBMP2/7 heterodimer (R&D systems) versus rhBMP2 homodimer, rhBMP7 homodimer or their 1:1 mixture using a preosteoblast cell line (MC3T3-E1). Chemotactic effects were gauged using a real time cell electronic sensing/cell migration system (RT-CES/CIM system). Cell proliferation was examined by fluorometric quantification of cellular DNA content. Alkaline phosphatase (ALP) activities were investigated with a colorimetric assay kit. Osteocalcin (OCN) expressions were determined using an enzyme-linked immunosorbent assay (ELISA) kit. The gene expressions of ALP and OCN were detected by real-time RT-PCR analysis and normalized byβ-actin gene expression. Cell matrix mineralization was evaluated by alizarin red staining.Results:RhBMP2/7 heterodimer induced the sequential dose-dependent biological activities of preosteoblast in the same pattern as homodimers. The threshold and optimal concentrations of rhBMP2/7 were significantly lower (1/10-1/2) than rhBMP2 or rhBMP7 in inducing chemotaxis, proliferation, differentiation and matrix mineralization of MC3T3-E1. The maximum levels in chemotaxis induced by rhBMP2/7 were significantly higher than the respective rhBMPs homodimers. However, ALP activity and OCN expression induced by rhBMP2/7 heterodimer were similar to those induced by the homodimers. Besides, at a lower concentration range (5-100ng/ml), rhBMP2/7 heterodimer exhibited more advantages in the early period (dayl-day4) than the later period (day4-day7) of differentiation when compared to homodimers. ALP and OCN gene expression also showed similar results. Consistent with previous studies, the 1:1 mixture of rhBMP2 and rhBMP7 did not show synergistic effect. Regarding to cell matrix mineralization, on day21, calcium deposition could only be detected in the group of 50ng/ml rhBMP2/7. Moreover, on day28, areas of calcium depositions induced by 50ng/ml rhBMP2/7 were 12-or 38-fold more than those induced by 50ng/ml rhBMP2 or 50ng/ml rhBMP7 respectively. Similar to the homodimers, the rhBMP2/7-induced dose-dependent responses in chemotaxis, proliferation, ALP activities and gene expressions showed bell-shape curves, in which overdose resulted in a descending trend. These findings strongly suggested that the specificity of rhBMP2/7 heterodimer, as an osteoblastogenesis-inducer, was lower-effective-concentration instead of higher-potency in comparison to the homodimers. In addition, the action of rhBMP2/7 heterodimer during the in vitro osteoblastogenesis indicated that the cellular events sequentially occurred with the concentration increasing. One event booms with the former one tapering although there were broad overlaps. Only the specifically appropriate concentration of rhBMP2/7 heterodimer could exert an optimal promoting effect on certain cellular event.Conclusions:RhBMP2/7 heterodimer is an osteoblastogenesis-inducer that does not have a higher-potency but does have a lower-effective-concentration compared to rhBMP2 and rhBMP7 homodimers. This Specificity confers rhBMP2/7 heterodimer a promising clinical application potential over its respective homodimers. Besides, rhBMP2/7 should be delivered in a physiological-like manner to match the demand of each sequential osteoblastogenetic event.Part 2:Direct Effect of RhBMP2/7 Heterodimer on Rankl-stimulated Osteoclastogenesis in Vitro Compared to RhBMP2 and RhBMP7 Homodimers Objectives:Homodimeric recombinant human bone morphogenetic proteins (rhBMPs) can potentially over-stimulate osteoclastic activities for their high clinical effective doses. Heterodimeric BMPs could be ideal substitutes for their lower effective doses. However, the hypothesis has not been corroborated. We, hereby, explored the effects of rhBMP2/7 heterodimer on in vitro osteoclastogenesis in comparison to rhBMP2 and rhBMP7 homodimers.Materials and Methods:Time-course and dose-response studies were performed on osteoblastogenesis of RANKL(50ng/ml)-stimulated RAW264.7 cells. Cell proliferation and differentiation were monitored using DNA content, tartrate-resistant acid phosphatase (TRAP) activity and relative gene expressions of calcitonin receptor (calcr), capthesin K and TRAP (acp5). The area of TRAP positive multinucleated osteoclasts (TRAP+MNCs) and the calcium phosphate resorption pit formed by active osteoclasts were histomorphometrically determined and statistically analyzed by ANOVA(p<0.05).Results:After 3-day stimulation, the most effective concentration for stimulating preosteoclast proliferation was 150ng/ml for rhBMP2/7, lOng/ml for rhBMP2 or rhBMP7 with similar peak value. After 4-day stimulation, TRAP activity was increased by rhBMPs and the most stimulating effect occurred at 100-150ng/ml. Refer to relative gene expression, the peak value of acp5 occured when stimulated by 200ng/ml rhBMP2, while rhBMP2/7 and rhBMP7 had no significant effect. Interestingly, calcr expression decreased under the stimulation of rhBMPs, especially at 5ng/ml rhBMP2/rhBMP2/7, or 200ng/ml rhBMP7. No significant effect on cathepsin K expression could be detected for all the rhBMPs. The largest area of TRAP+MNCs was achieved by 150ng/ml rhBMP2 followed by 150ng/ml rhBMP2/7 or 100ng/ml rhBMP7. After 7-day stimulation, all the rhBMPs were shown to significantly promote pit formation except rhBMP7 at 150ng/ml. Of all the three different rhBMP groups, the largest pit area was observed at 150ng/ml rhBMP2 followed by 50ng/ml rhBMP2, 150ng/ml rhBMP2/7 and 5ng/ml rhBMP7. No synergistic effects were found between BMP2 and BMP7 homodimers for all parameters.Conclusions:Osteoclastogenesis could be specifically modulated by rhBMPs. RhBMP2/7 heterodimer of lowest effective concentration for osteogenesis resulted in less osteoclastic activities than the respective rhBMP homodimers.SummaryRhBMP2/7 heterodimer was proved in vitro to induce osteoblastogenesis at a concentration as low as 5ng/ml and could reach its effect plateau at 50ng/ml at which respective homodimers started to take effect in inducing osteoblastogenesis in vitro. Refer to rhBMP2/7's direct effect on rankl-stimulated osteoclastogenesis in vitro, a dose-dependent increasing manner was exhibited in the select concentration (5-200ng/ml) with peak level similar to respective homodimers.Thus, these two specificities conferred rhBMP2/7 heterodimer a promising application value in vivo to enhace bone regeneration in critical bone defect and peri-implant osteointergration.