rhBMP2/7 Heterodimer: An Osteoblastogenesis Inducer of Not Higher Potency but Lower Effective Concentration Compared with rhBMP2 and rhBMP7 Homodimers

Abstract

Heterodimeric bone morphogenetic proteins (BMPs) were exhibited to be more potent than and thus potential substitutes for homodimeric BMPs whose clinical application is limited for the drawbacks resulted from their higher effective doses. This study aims to delineate the biofunctional characteristics of recombinant human BMP2/7 (rhBMP2/7) heterodimer in inducing osteoblastogenesis of MC3T3-E1 through in vitro time–course and dose–response studies. rhBMP2/7 heterodimer induced cell migration with a significantly lower optimal concentration and higher peak effect than the respective homodimers. rhBMP2/7 heterodimer induced cell differentiation with significantly lower threshold concentrations but similar maximum effects. On day 28, the area of calcium depositions induced by 50 ng/mL rhBMP2/7 was 12- or 38-fold more than that of 50 ng/mL rhBMP2 or 50 ng/mL rhBMP7, respectively. The results indicated that rhBMP2/7 heterodimer was an osteoblastogenesis inducer of not higher potency but lower effective concentration compared with rhBMP2 and rhBMP7 homodimers.

Introduction

The osseous restoration of voluminous bone defect remains a challenge in orthopedics, maxillofacial surgery, and implantology.^1,2 Bone tissue engineering including gene, cell, and cytokine therapies has been reported to substitute autografting and allografting whose clinical applications are hampered for their varieties of limitations.^3,4 Among the three therapies, cytokine therapy is advantageous in safety, feasibility, and potential for nearest clinical application over the other two.^1,5

Bone morphogenetic proteins (BMPs), a group of dimeric disulfide-linked polypeptide growth factors under transforming growth factor-β superfamily, are one of the paramount cytokines in promoting bone regeneration.⁶ However, the clinical effective doses of the homodimeric BMPs to induce bone formation are extremely high (e.g., up to milligrams),^7,8 which results in not only a substantial economic burden to patients but also a series of potential side effects, such as overstimulation of osteoclastic activity and ectopic bone formation in unintended area.^9,10

One alternative approach to solve the problem is to adopt more potent forms of BMPs. Previous studies showed that BMP heterodimers achieved several- or dozen-fold more effects than the respective homodimers in inducing alkaline phosphatase (ALP) activity and bone formation.^11–14 These findings suggested that BMP heterodimers were feasible and promising substitutes for homodimers. However, the biofunctional characteristics and mechanisms of BMP heterodimers on osteogenesis remain ambiguous. How BMP heterodimers affect the sequential osteoblastogenetic events, such as chemotactic migration, proliferation, differentiation, and mineralization, hereto, remains uncovered. Earlier studies confined the parameters to ALP activity, which is inadequate to reveal the characteristics of their biofunctional activities.^11,12 Recently, some conclusions were based on the conditioned medium of gene-transfected BMP producers instead of purified BMP heterodimers, which could not exclude the influence of other factors in the medium on cellular activities.^13,14 These studies mostly focused on the in vivo gene therapy that could only suggest their clinical efficacy instead of the functional characteristics of BMP heterodimers.

The parameters such as cell migration index, DNA content, ALP activity as early marker of differentiation, osteocalcin (OCN) as terminal marker of differentiation, and calcium deposition of cell culture were adopted to evaluate the biological activities of BMPs in previous studies.^13,15–17 Murine calvaria-derived MC3T3-E1 cell, a precursor of functionalized osteoblast, was also shown to be able to examine the potency of osteoinductive agents.^13,17

In this article, we wished to unveil the functional characteristics of BMP heterodimers by comparing osteoblastogenesis-inducing effects of recombinant human BMP2/7 (rhBMP2/7) heterodimer with rhBMP2 and rhBMP7 homodimers.

Materials and Methods

Cell culture

Preosteoblasts, MC3T3-E1 cell line (ATCC; Chinese Academy of Sciences, Shanghai, China), were cultured in α-Minimum essential medium (α-MEM) containing 10% fetal bovine serum (FBS) (Gibco™ Invitrogen, Grand Island, NY). The medium was changed every 3 days. Exponentially growing cells were plated at 1.5 × 10⁴ cells/well in 48-well plates for cell proliferation, at 4 × 10⁴ cells/cm² in 6-well plates for ALP activity assay, in 24-well plates for OCN detection, or in 48-well plates for alizarin red staining. After 24-h incubation, cells were subjected to a low-serum medium (2% FBS) for another 24 h and then followed by rhBMPs (R&D Systems, Inc., Minneapolis, MN) treatment.

rhBMP concentration selection test

To identify the concentration range for examining the biological activities of rhBMP2/7 heterodimer, a dose–response test of ALP assay was performed the same way as described in “ALP activity assay” section. The results showed that the ALP activity induced by rhBMP2/7 reached the maximum at 150–200 ng/mL and significantly decreased at 250 ng/mL. Besides, ALP activity induced by rhBMP2 or rhBMP7 reached a plateau stage at 200–400 ng/mL, respectively, with similar highest level. Consequently, the maximum concentration in this study was determined at 200 ng/mL.

Real-time cell migration monitoring

Cell migration of MC3T3-E1 induced by rhBMPs was continuously monitored using Real-Time Cell Electronic Sensing/Cell Invasion/Migration System (RT-CES/CIM™ System; ACEA Biosciences, San Diego, CA)^18,19 according to the manufacturer's instructions. Briefly, the bottoms of the upper chambers, the both sides, were coated with 0.01% bovine plasma fibronectin (Invitrogen, Carlsbad, CA), and 20 μL of 0.1% bovine serum albumin (BSA)–α-MEM, and 150 μL of 0.1% BSA serum-free medium containing various amounts of rhBMP2/7, rhBMP2, rhBMP7, or their 1:1 mixture (0 as negative control, 0.01, 0.1, 1, and 10 ng/mL)^15,16 were added into the upper and lower chambers, respectively. Confluent MC3T3-E1 cells were subjected to serum-free starvation for 4 h and then suspended in 0.1% BSA–α-MEM at a density of 12.5 × 10⁴ cells/mL followed by adding 80 μL of cell suspension into the RT-CIM upper chambers. Cell migration was continuously monitored for a period of 0–8 h, and data presented as cell index (CI) were collected every 15 min, chiefly indicating the numbers of cells that migrated to the lower side of the upper chambers.

Cell proliferation assay

To investigate cell proliferation of MC3T3-E1 cells in response to various concentrations (0 as control, 5, 10, 50, 100, 150, and 200 ng/mL) of different rhBMPs, the number of cells was determined by the fluorometric quantification of the amount of cellular DNA after stimulation for 1 day, 4 days, and 7 days using Quant-iT™ PicoGreen^® dsDNA Reagent and kits (Invitrogen™, Molecular Probes™, Eugene, OR) as previously described.²⁰ The fluorescent intensity of the cell lysate and dye mixed solution was measured using a fluorescence spectrometer (SPECTRAmax M2, Molecular Devices, Sunnyvale, CA, Ex 480 nm/Em 520 nm).

ALP activity assay

To determine the early differentiation of preosteoblasts stimulated by rhBMPs at different concentrations (0 as control, 5, 10, 50, 100, 150, and 200 ng/mL), ALP activity and protein content were measured after rhBMPs treatment on days 2, 4, and 7. ALP activity in the cell lysates (Sigma-Aldrich, St. Louis, MO) was determined using LabAssay™ ALP colorimetric assay kit (Wako Pure Chemicals, Osaka, Japan).¹¹ The cell number was estimated by determining total protein content that was measured at 570 nm using a commercial BCA Protein Assay kit (Beyotime, Shanghai, China). The values that presented ALP activity were expressed as μmol p-NP/mg total protein.

OCN expression assay

To assess the terminal differentiation of preosteoblasts stimulated by rhBMPs (concentrations set at 0 as control, 5, 10, 50, 100, 150, and 200 ng/mL), OCN secreted into the cell culture medium was determined. The cell supernatants were collected on days 4 and 7 and were centrifuged (10,000 rpm, 4°C, 5 min) before detection. The OCN concentrations of the supernatants were determined by ELISA using a mouse OCN EIA kit (Biomedical Technologies, Stoughton, MA).^13,20

Alizarin red staining

According to the results of above cellular events, we compared the mineralization possibility of MC3T3-E1 cells stimulated by 50 ng/mL rhBMPs. Quadruplicate cell cultures were prepared in the same way as described previously and then treated with mineralizing medium (10% FBS, 50μg/mL L-ascorbic acid, and 5 mM β-glycerophosphate; Sigma-Aldrich) containing 50 ng/mL rhBMPs. The medium was replaced every 3 days. After 3 and 4 weeks, mineralized nodules were determined by alizarin red staining (Sigma-Aldrich). Culture plates were photographed by NIS-Elements F2.20 (Nikon Eclipse 80i, Tokyo, Japan), and the calcified area was quantified using Image-Pro Plus 6.0 analysis.

Statistical analysis

Statistical comparisons between results obtained with the various concentrations of rhBMPs and different kinds of rhBMPs were made by one-way analysis of variance. Post hoc comparisons were made using Bonferroni corrections. The level of significance was set at p < 0.05. SPSS software (version 15, SPSS Inc., Chicago, IL) for a Windows computer system was employed for the statistical analysis.

Results

Chemotactic migration

CI of cell chemotactic migration for each rhBMP was in the same pattern which contained two stages: the initial steady-increase stage in the first 4–5 h and the following plateau stage (Fig. 1A–D). Cell started to migrate immediately after being installed in the system regardless of the presence of rhBMPs. The speeds of cell migration were enhanced to higher levels by different rhBMPs of certain concentrations than negative controls especially after 1–2 h. The chemotactic effects of rhBMPs lasted for 5 h after which relative cell migrations tapered. Despite the same pattern of CI, the optimal concentration (the lowest concentration to get the maximum effect) and the maximum value of chemotactic effects differed according to the rhBMP types. The chemotactic effect of rhBMP2/7 showed a bell-shaped, dose-dependent curve. The optimal concentration was 0.1 ng/mL, followed by 1 ng/mL (Fig. 1A). The effect dramatically decreased when the concentration decreased to 0.01 ng/mL or increased to 10 ng/mL. The maximum values of rhBMP2 and rhBMP7 were obtained around 1 ng/mL (Fig. 1B, C). Their decreasing trends were demonstrated when the concentration went either higher or lower. The chemotactic effect of 1:1 mixture of rhBMP2 and rhBMP7 increased slowly in the selected concentration range (Fig. 1D). The maximum fold of CI (CI normalized by CI of negative control group) of rhBMP2/7 (1.93 at 0.1 ng/mL) was significantly higher than that of rhBMP2 (1.48 at 1 ng/mL), rhBMP7 (1.31 at 1 ng/mL), or their 1:1 mixture (1.43 at 10 ng/mL) (Fig. 1E).

FIG. 1.

(A–D) Comparison of cell migration under different rhBMPs treatment. Real-time cell migration was continuously monitored using RT-CES/CIM™ System in a period of 8 h. The data presented as CI were collected every 15 min, chiefly indicating the numbers of cells that migrated. (E) Fold of CI at 5 h normalized by CI of control group (**p < 0.01, *p < 0.05, indicated significant increase vs. control group, ^a,b,cp < 0.05, indicated significant increase vs. rhBMP2, rhBMP7, or their 1:1 mixture group, respectively, n = 3). CI, cell index; rhBMPs, recombinant human bone morphogenetic proteins.

Cell proliferation

After stimulation for 1 day, there is no significant difference in cell proliferation among different BMPs of the selected concentrations.

On day 4 (Fig. 2), compared with the control group, the DNA contents increased significantly under the stimulation of rhBMP2/7 and rhBMP2 at the concentrations of 5–50 or 200 ng/mL. In contrast, rhBMP7 showed a stimulative effect on the proliferation of preosteoblasts in a broader range of concentration, from 5 to 200 ng/mL. The highest values of DNA content were obtained at 5 ng/mL for rhBMP2 or rhBMP2/7, at 200 ng/mL for rhBMP7, and at 50 ng/mL for the mixture of rhBMP2 and rhBMP7.

FIG. 2.

rhBMPs showed different optimal concentration with similar peak values on stimulating MC3T3-E1 proliferation. The cellular DNA contents were measured on days 1, 4, and 7. Values shown are means ± SD for quadruplicate cultures (*p < 0.05 indicated significant increase vs. control group). SD, standard deviation.

The DNA contents in all the rhBMP groups further increased on day 7. Compared with the respective control groups, the DNA contents were significantly increased by rhBMP2/7 at the concentrations of 5–10 ng/mL. For rhBMP2, the effective concentration range was 5–50 ng/mL. In accordance with the result on day 4, the DNA contents were increased by rhBMP7 in the selected concentration range from 5 to 200 ng/mL, with the peak value occurring at 100 ng/mL. The highest values of stimulative effects on cell proliferation were obtained at 5 ng/mL for rhBMP2 or rhBMP2/7, at 100 ng/mL for rhBMP7, and at 50 ng/mL for the mixture of rhBMP2 and rhBMP7. The mixture of rhBMP2 and rhBMP7 showed no synergistic effect. Besides, no significance was found among the highest values of the DNA contents in different rhBMP groups on day 4 or 7 (p > 0.05).

ALP activity

The minimum concentration for rhBMP2/7 heterodimer to exhibit a significant promoting effect on ALP was as low as 5 ng/mL, at which rhBMP2, rhBMP7, or their mixture remained ineffective (Fig. 3). The rhBMP homodimers started to exert their promotive effect on the ALP activities at the concentration of 50 ng/mL, at which the effect of rhBMP2/7 on stimulating the ALP activity has already reached a plateau (Fig. 3A–C). Therefore, the threshold concentration of rhBMP2/7 heterodimer was almost 1/10 of that of the homodimers. In addition, the levels of ALP activity induced by rhBMP2/7 on day 2 were about 1.4- to 2.4-fold of that of rhBMP2 or rhBMP7 at all the selected concentrations (Fig. 3A). After 4-day stimulation, the ALP activities further increased under the stimulation of rhBMP2/7 heterodimer at the concentrations of 5 and 10 ng/mL at which rhBMP2 and rhBMP7, remained ineffective (Fig. 3B). At the concentration of 50 ng/mL, the ALP activity stimulated by rhBMP2/7 was still significantly higher than rhBMP2, rhBMP7, and their 1:1 mixture. The ALP activities induced by rhBMP2/7 were stable in the concentration range of 50–200 ng/mL, at which the effects of rhBMP2, rhBMP7, and their 1:1 mixture showed a dose-dependent increasing curves. The advantages of rhBMP2/7 over the homodimers, thus, tapered with the increased concentrations. At the concentration of 100–200 ng/mL, rhBMP2/7 was only advantageous over rhBMP7. On day 7, the ALP levels induced by all rhBMPs were about 1.5-fold more than those of day 4 (Fig. 3C). The effect pattern of rhBMPs at the concentrations of 5 and 10 ng/mL was similar to that of day 4. The effect of rhBMP2 reached the same level of rhBMP2/7 at concentrations of 50–200 ng/mL. In contrast, rhBMP7 showed a weaker effect on inducing ALP activity of preosteoblast than rhBMP2/7 or rhBMP2 in the span of the given concentrations. The effect of the mixtures of rhBMP2 and rhBMP7 lied between the two homodimers.

FIG. 3.

ALP activities of MC3T3-E1 under different rhBMP treatments were determined by colorimetric assay on day 2 (A), day 4 (B), and day 7 (C). Values shown are means ± SD for triplicate wells in duplicate experiments normalized by total cellular protein (*p < 0.05 indicated significant increase vs. control group; ^a,b,cp < 0.05 indicated significant increase vs. rhBMP2, rhBMP7, or their 1:1 mixture group, respectively). ALP, alkaline phosphatase.

OCN expression

In accordance with its effect on the ALP activity, the threshold dose of rhBMP2/7 heterodimer to promote OCN expression (5 ng/mL) was also significantly lower than those of the rhBMP homodimers (50 ng/mL) (Fig. 4).

FIG. 4.

OCN expressions of MC3T3-E1 under different rhBMP treatments were determined by ELISA on day 4 (A) and day 7 (B). Values shown are means ± SD for quadruplicate wells (*p < 0.05 indicated significant increase vs. control group; ^a,b,cp < 0.05 indicated significant increase vs. rhBMP2, rhBMP7, or their 1:1 mixture group, respectively). OCN, osteocalcin.

After 4-day culture, the OCN expression level induced by rhBMP2/7 (5 ng/mL) increased with concentration until 50 ng/mL, after which its effect maintained stable (Fig. 4A). OCN expression showed a dose-dependent increasing curve for rhBMP2 and rhBMP7 from 50 ng/mL at which the effect of rhBMP2/7 was sixfold higher than the respective homodimers. The advantage of rhBMP2/7 decreased with concentration increase but maintained significant over the homodimers until the concentration of 200 ng/mL, at which there was no significant difference between rhBMP2/7 heterodimer and the respective homodimers (p > 0.05).

After culturing with rhBMPs for 7 days, the OCN expression increased to much higher levels, about twofold higher than those measured on day 4 (Fig. 4B). The effect of rhBMP2/7 on stimulating OCN was still significantly advantageous over rhBMP2 or rhBMP7 at the concentration range of 5–150 ng/mL. rhBMP2 increased the OCN expression significantly from 50 ng/mL and reached a plateau at the concentrations of 100–200 ng/mL. At 200 ng/mL, no significant difference could be found among the OCN levels induced by different rhBMPs.

Cell matrix mineralization

After 21-day culture, matrix mineralization of MC3T3-E1 was only found under the stimulation of 50 ng/mL rhBMP2/7 (Fig. 5). On day 28, calcium depositions were detected in all groups, with the significantly maximal level occurring in rhBMP2/7 group, which was 12-fold higher than that of rhBMP2, 38-fold higher than that of rhBMP7, or 26-fold higher than that of the mixture group. Besides, comparing the two time points, the area of calcified nodules was over three times at day 28 than that at day 21 for rhBMP2/7 group (Fig. 5B).

FIG. 5.

(A) The calcium depositions of cell culture were stained using alizarin red staining. (B) Quantitative calculation of calcified area in each rhBMP group. Values shown are means ± SD for quadruplicate wells (**p < 0.01, *p < 0.05 indicated significant increase vs. control group; ^a,b,cp < 0.05 indicated significant increase vs. rhBMP2, rhBMP7, or their 1:1 mixture group, respectively). Color images available online at www.liebertonline.com/ten.

Discussion

BMPs play pivotal roles in many body morphogenic events varying from the earliest embryonic patterning to organ development.²¹ The actions and activities of BMPs are dependent on the targeting cells and BMPs' resouces.^22,23 In this study, we adopted commercially available purified recombinant human BMP heterodimer and homodimers to compare their actions on a committed osteogenic cell type MC3T3-E1 by gauging a series of cellular events during osteoblastogenesis.

Infiltration of bone forming cells is an important event during the restoration of bone defect. Exogenous BMPs may be one of the dominant modulators on cell migration, especially when they are applied in an unphysiologically high amount for bone regeneration. Different BMP homodimers differed in their chemotactic effect to different targeting cell types.^15,16 BMP heterodimers can be even distinct from homodimers because they could exclusively assemble heteromeric type I receptors and thus pattern the dorsoventral axis of drosophila embryo while the homodimers could not.²⁴ It is, therefore, worthwhile monitoring the chemotactic effect of BMP heterodimers. Our results showed that the optimal concentration of rhBMP2/7 for chemotactic effect was significantly lower (1/10), and the maximum effect of the heterodimer was significantly higher than those of the respective homodimers. These results suggested that rhBMP2/7 heterodimer might play a more important role than homodimers in enhancing chemotactic migration and patterning the spatial interrelationship of osteogenic cells.

Consistent with the previous findings, we found that the threshold and the optimal concentrations of rhBMP2/7 heterodimer for each cellular event were significantly lower than the respective homodimers. However, our results showed that the maximum effects of rhBMP2/7 heterodimer on stimulating proliferation and differentiation were similar to instead of higher than those of rhBMP2 or rhBMP7 homodimers. In addition, the gene expression results of ALP and OCN on days 1, 4, and 7 determined by real-time reverse transcription (RT)–polymerase chain reaction also indicated that the final maximum levels stimulated by rhBMP2/7 heterodimer were similar to that by rhBMP2 homodimer (Supplemental Fig. S1, available online at www.liebertonline.com/ten). Although the novel findings were not in conflict with the previous definition higher potency for rhBMP2/7 heterodimer than the respective homodimers in the lower concentration range (0–50 ng/mL), the advantage of heterodimer in osteoinductive potency tapered in the higher concentration range (50–200 ng/mL). BMP2/7 heterodimer produced by transfected CHO cells was shown to be more potent than the BMP2 homodimer in stimulating osteogenic differentiation.²⁵ The inconsistency with our results may be due to the use of mammalian producer, conditioned medium instead of purified protein, and the single selected concentration. And this needs to be further clarified. Moreover, our results also exhibited that for stimulating cell migration (Fig. 1) and proliferation (Fig. 2), dose-dependent effects of rhBMPs, no matter heterodimer or homodimer, unanimously showed a dose-dependent, bell-shaped curve in which rhBMPs of either lower or higher concentrations than the optimal one would downregulate their effects. In the concentration selection test, the results also partially indicated that the ALP activities also analogously exhibited a bell-shaped, dose-dependent curve. These findings strongly suggest that the specificity of rhBMP2/7 heterodimer, as an osteoblastogenesis-inducer, is lower effective concentration instead of higher potency in comparison with the homodimers. In fact, the phenomenon that different BMPs affect in different concentration range (usually shown as different potencies) may generally occur to different heterodimers and homodimers.^14,26

By now, the molecular mechanisms accounting for the specificities of BMP remained unclear. One mechanism would be the various receptor complex created by the pairing combinations from three type I and three type II receptors.²⁷ On the other hand, this hypothesis is challenged by the phenomenon that several BMPs can use one receptor, and the absence of one receptor does not totally block signaling activated by specific BMPs.²⁸ A recent study that revealed different reliance of BMPs on different receptors,²⁹ made the mechanism more complete and reasonable. The source of the specificity of BMP2/7 heterodimer could also be its higher combined affinity for both the type I and type II receptors, and thus facilitating the formation of active signaling complexes endowed by the presence of both BMP2 and BMP7 moieties in the same ligand.²⁴ Another mechanism may be the complicated BMP antagonism network. BMP2/7 heterodimer was shown to induce a significant lower level Noggin, a BMP-induced competitive antagonist, and lower affinity to Noggin than the respective homodimers.³⁰ Another antagonist Cas-interacting zinc finger protein behaves on BMP2/7 heterodimer similarly to Noggin.^23,31

Similarly, the biological significance of the low-effective concentration specificity of heterodimer also remains unclear. Because the coexpression of two different BMP genes has been detected in developing limb and during fracture healing,^32,33 heterodimers might be a pivotal effecter on the site of less BMP producers where they could only provide a lower concentration of BMP homodimers, such as the beginning phase of fracture healing and embryonic development. For this hypothesis, our results provided several positive clues such as the significantly higher maximum level of rhBMP2/7 for chemotactic effect (the initial step of osteogenesis) or the significantly lower threshold of rhBMP2/7 for promoting proliferation and differentiation than the respective homodimers. Besides, it is also important to note that the ALP activities induced by rhBMP2/7 heterodimer were significantly higher than those of homodimers at concentrations of 100–200 ng/mL on day 2 (Fig. 3A) and the advantages tapered with time. On day 7, rhBMP2/7 was only advantageous over rhBMP7 in inducing the ALP activities. The differences of OCN expression between rhBMP2/7 and the respective homodimers at concentrations of 50–150 ng/mL were more significant on day 4 than on day 7. Mineralization induced by rhBMP2/7 was obviously observed on day 21, but not by the homodimers. These findings indicated that rhBMP2/7 heterodimer showed effects more rapidly and accelerated the process of differentiation and mineralization, and therefore suggested a pivotal role of BMP heterodimer in the beginning stage of osteogenic activities.

As we showed, although rhBMP2/7 heterodimer was a low-effective concentration inducer, its action also showed a concentration-dependent, bell-shaped curve for the cellular events that sequentially occurred with increasing concentrations. One event always 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 events. This finding is significant for designing the in vivo application pattern of rhBMPs that the appropriate level of BMPs should fit a certain stage of osteoblastogenesis. Overdose of BMPs in the initial stage may lead to the downregulation of cell migration, and thus suppress the accumulation of osteogenic cells as well as their proliferation. The delivering mode of BMPs was shown to influence the osteoconductivity of implant surfaces during the early phase of osseointegration.³⁴ Slow-released and low level of BMPs (low local concentration of BMP in initial stage of bone regeneration) is superior to burstly released ones in recruiting cells. We also observed fewer osteogenic activities in the initial stage when applying a higher amount of BMPs in an in vivo bony site (unpublished data). Although the overstimulation of osteoclastic activity was also believed to be one of the mechanisms for the less bone volume,³⁵ our findings strongly suggested that the suppression of osteoblastic activities by the locally transient high concentration of BMPs might also account for the attenuated bone formation. Therefore, caution must be especially taken to the application pattern of rhBMP2/7. However low its effective concentration is, the BMP heterodimer should also be delivered in a physiological-like manner. The principle was suitable not only for protein therapy but also for gene therapy. Controlling the level and duration of heterodimeric BMPs using the lower titer or reduced numbers of transduced cells or regulated expression system was also recommended to avoid bone overgrowth or immune overreaction in combinatorial gene therapy with BMP2/7.³⁶

Significantly lower effective dose of BMP heterodimers was also expected to bring fewer side effects than the homodimers. However, because the maximum capability of rhBMP2/7 in inducing osteogenic activities which is obtained in a relatively lower effective concentration range was similar to the homodimers, the capability of the heterodimer in inducing the side effects might also be similar to that of the homodimers in the same range. Consequently, further study should clarify this point before clinical application of BMP heterodimers.

One limitation in this study was that we only adopted one kind of osteogenic cell line, which might behave differently from the others such as bone marrow stromal cells. Moreover, a broader concentration range could be further studied to completely exhibit the functional characteristics of both heterodimers and homodimers. In addition, the bioactivities of the rhBMPs generated by Escherichia coli might be different from the ones generated by mammalian cells.

The specificity of lower effective concentration range conferred rhBMP2/7 heterodimer a promising clinical application potential over its respective homodimers. However, further studies must be performed to examine the potential side effects of rhBMP2/7 heterodimer before its in vivo application.

Footnotes

Acknowledgments

We thank Professor Ke, Y. for providing RT-CES/CIM system biosensor and also Fu, H. for technical assistance in Laboratory of Cell Signaling and Molecular Genetics, Zhejiang University. We also want to thank the Laboratory of Pathology for supplying ABI PRISM 7900HT Sequence Detection System and Song, K. for technical assistance. This work was supported by the International Team of Implantology Research Foundation (no. 547-2007).

Disclosure Statement

No competing financial interests exist.

This abstract has appeared in the IADR/AADR 87th General Session (April 1–4, 2009).

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