BMP-7 gene transfer to inflamed ferret dental pulps

In vivo and ex vivo gene transfer are being developed for localized skeletal regeneration. These strategies for tissue regeneration were tested in an adult ferret model of vital pulp therapy. In this model a reversible pulpitis was induced first. Then after 3 d, the pulps were directly infected with recombinant virus or implanted with ex vivo transduced autologous dermal fibroblasts. The genome of the recombinant adenovirus contained a full-length cDNA encoding mouse bone morphogenetic protein (BMP)-7 (AdBMP7) or bacterial beta-galactosidase cDNA (AdlacZ). The BMP-7, but not lacZ, ex vivo transduced dermal fibroblasts induced reparative dentinogenesis with apparent regeneration of the dentin-pulp complex. In vivo infection with AdBMP-7 failed to produce reparative dentin in all cases. E. vivo gene transfer of BMP-7 may be an effective method for inducing dentin regeneration in teeth with reversible pulpitis.     

Treatment of inflamed ferret dental pulps with recombinant bone morphogenetic protein-7

Recombinant human BMP-7 (bone morphogenetic protein-7, osteogenic protein-1) is osteogenic, dentinogenic and cementogenic when implanted into the appropriate tissue in vivo. However, most studies characterizing the induction of these tissues have implanted BMP-7 into freshly surgerized, clinically healthy tissues. To determine if BMP-7 is dentinogenic in inflamed dental pulps, we applied BMP-7 to inflamed ferret pulps. A single application of 5 microg of a commercial preparation of lipopolysaccharide (LPS) from Salmonella typhimurium directly to the coronal pulp induced a reversible mixed inflammatory exudate of moderate intensity within 3 d. Treatment with a single application of 2.5, 7.5 or 25 microg recombinant human BMP-7/mg collagen (2 mg total mass/tooth) induced reparative dentinogenesis in controls but not LPS treated dental pulps. These data reveal that a single application of up to 50 microg/tooth of exogenous recombinant BMP-7 is insufficient to induce reparative dentinogenesis in ferret teeth with reversible pulpitis. Given that pulp cells in the inflamed tissues likely retain the capacity to respond to exogenous BMP-7, it is possible that insufficient active recombinant protein is available to induce tissue formation in experimentally inflamed dental pulps.     

重组人骨形成蛋白牙槽嵴内诱导成骨的实验研究

目的:探讨原核表达重组人骨形成蛋白-7牙槽嵴内诱导成骨的作用效果。方法:拔除大白兔的切牙建立动物模型,以明胶海绵作为载体,将原核表达的重组人骨形成蛋白-7与之复合后植入即刻拔除牙齿的牙槽窝内进行干预治疗,通过扫描电镜观察及钙含量测定,探讨原核表达重组人骨形成蛋白-7牙槽嵴内诱导成骨的作用效果。结果:扫描电镜观察显示:实验组的骨创愈合比对照组大约提前4~6周;钙含量测定显示实验组明显高于对照组,差异有统计学意义。结论:重组人骨形成蛋白-7具有良好的诱导牙槽嵴内成骨的效果。     

Expression of bone morphogenetic proteins in normal human intramembranous and endochondral bones

Bone morphogenetic proteins (BMPs) are growth and differentiation factors that have been purified and widely accepted to be the most important regulators in the processes of bone formation. The aim of this study was to identify the BMPs that are expressed in normal human bone, and to investigate the specific pattern of BMP2-BMP9 expression in normal human intramembranous and endochondral bone to maintain homeostasis, as well as in ex vivo primary cell culture of human osteoblasts from intramembranous and endochondral bone. Semi-quantitative RT-PCR indicated that 2 types of bone of different embryological origin have distinct patterns of BMP expression. BMP3, 4, 7 and 8 were strongly expressed in normal intramembranous bone compared to endochondral bone, whereas BMP2 and 5 were highly expressed in endochondral bone. The expression of BMP9 and BMP15 in human bone was identified for the first time. From the very similar expression patterns of BMPs in fresh normal bone and ex vivo osteoblastic cell culture, it can be proposed that the different proportions of BMPs in normal human intramembranous and endochondral bone needed to maintain normal homeostasis.     

Expression of BMP-2 and TGF-β1 mRNA during healing of the rabbit mandible

To identify the cell types which produce BMP and TGF-β during fracture healing and to elucidate the interactions between BMP and TGF-β in regulating cell proliferation and differeentiation at various stages, an experimental model of fracture healing in the rabbit mandible was established and the expression of BMP-2 and TGF-β1 mRNA was studied at different healing stages by in situ hybridization. The results showed that undifferentiated mesenchymal cells, differentiating osteoblasts and chondroblasts, had higher levels of BMP-2 mRNA at the stage of intramembranous bone formation and early chondrogenesis, while the level of TGF-β1 mRNA expression was closely associated with the active synthetic stage of osteoblasts and chondrocytes. These obserbvations suggest that both BMP and TGF-β are involved in the regulation of fracture healing, BMP may play an important role in bone induction and early chondrogenesis, while TGF-β regulates the proliferation and active synthetic ability of chondrocytes and osteoblasts.     

重组腺病毒-骨形成蛋白7对骨髓间充质干细胞骨向分化影响的实验观察

目的观察骨形成蛋白（bone morphogenetic protein，BMP）7重组腺病毒对大鼠骨髓间充质干细胞（mesenchymal stem cell，MSC）骨向分化的影响。方法构建重组腺病毒载体pAd-BMP-7，测定其滴度。应用pAd-BMP-7和空白载体pAdTrack—CMV分别转染MSC，检测外源基因转染效率，并应用RT-PCR、免疫细胞化学手段检测BMP-7的表达。将MSC分为3组，A组：转染pAd-BMP-7；B组：转染pAdTrack-CMV；C组：转染pAdTrack-CMV＋骨向分化液。观察其矿化结节形成，评价3组细胞骨向分化情况。结果重组腺病毒pad-BMP-7的滴度可达2．0×10^15 pfu／L；外源基因的转染效率为99％，表达时间持续5—7周，3周内表达水平较高。RT-PCR和免疫细胞化学检测证实了BMP-7在MSC中的有效表达。病毒转染后，A组和C组细胞均有矿化结节形成，其中A组矿化结节数目显著多于C组（P〈0．01）；B组无矿化结节形成。结论BMP-7重组腺病毒可有效转染大鼠骨髓MSC，并促进其骨向分化，转染的BMP-7得到了有效表达。     

Evaluation of 2 novel approaches for assessing the ability of demineralized freeze-dried bone allograft to induce new bone formation.

BACKGROUND: Because of the wide variation in the ability of human demineralized freeze-dried bone allograft (DFDBA) to reproducibly induce new bone formation, there is a need for a reliable measure of bone induction activity. In this study we examined an immature osteoprogenitor cell line for its potential utility in measuring the activity of DFDBA in vitro. METHODS: We characterized the response of 2T9 cells, an immature osteoprogenitor cell line derived from the calvariae of transgenic mice containing the SV40 T-antigen driven by the mouse bone morphogenetic protein (BMP)-2 promoter, to recombinant human BMP-2 by measuring alkaline phosphatase specific activity, osteocalcin production, and matrix mineralization. Responses were compared to those obtained with 1,25-(OH)2D3. In addition, 2T9 cells were cultured with active or inactive human DFDBA in the presence or absence of BMP-2. We also tested the hypothesis that radio-opacity of tissue following implantation of DFDBA in vivo correlates with the ability of human DFDBA to induce new bone. DFDBA from 9 different donors, stratified by age, were implanted subcutaneously in the thorax of 18 nude (nu/nu) mice. Tissue was harvested at 36 days postoperatively and examined histologically and biochemically for calcium and phosphorus uptake. RESULTS: 2T9 cells exhibited a dose- and time-dependent response to soluble BMP-2. Proliferation was decreased and alkaline phosphatase activity, osteocalcin production, and mineralized nodule formation were increased. The effects were dose- and time-dependent. Peak effects on alkaline phosphatase and osteocalcin were noted on day 8, whereas mineral deposition did not begin to occur until day 12. 1,25-(OH)2D3 did not regulate these effects unless used with BMP-2. When the cells were exposed to active or inactive DFDBA in the presence or absence of BMP-2, no effect on 2T9 cell differentiation was observed. This indicated that DFDBA released no soluble factors with bone inductive ability and that if any active factors were adsorbed to the DFDBA, they were inactivated. When DFDBA was implanted subcutaneously in the thorax of nude mice, there was no histologic evidence of new bone formation. However, there was a donor age-dependent decrease in Ca and P uptake of the implanted tissue, reflecting a donor age-dependent decrease in remineralization of DFDBA. CONCLUSIONS: These data indicate that cell culture assays like the one used in this study may not be appropriate indicators of bone induction ability by DFDBA since soluble factors may not be responsible for bone induction in vivo. Nonetheless, in vitro assays are still needed. While Ca and P uptake by DFDBA-implanted tissue in the present study correlated with the age-dependent decrease in bone induction at intramuscular sites in a previously reported study, these data show that early x-rays may actually detect remineralization and not new bone formation. Thus, assessment of bone induction ability may still depend on histologic analysis of animal models.     

Gene therapy of bone morphogenetic protein for periodontal tissue engineering

BACKGROUND: The reconstruction of lost periodontal support including bone, ligament, and cementum is a major goal of therapy. Bone morphogenetic proteins (BMPs) have shown much potential in the regeneration of the periodontium. Limitations of BMP administration to periodontal lesions include need for high-dose bolus delivery, BMP transient biological activity, and low bioavailability of factors at the wound site. Gene transfer offers promise as an alternative treatment strategy to deliver BMPs to periodontal tissues. METHODS: This study utilized ex vivo BMP-7 gene transfer to stimulate tissue engineering of alveolar bone wounds. Syngeneic dermal fibroblasts (SDFs) were transduced ex vivo with adenoviruses encoding either green fluorescent protein (Ad-GFP or control virus), BMP-7 (Ad-BMP-7), or an antagonist of BMP bioactivity, noggin (Ad-noggin). Transduced cells were seeded onto gelatin carriers and then transplanted to large mandibular alveolar bone defects in a rat wound repair model. RESULTS: Ad-noggin treatment tended to inhibit osteogenesis as compared to the control-treated and Ad-BMP-7-treated specimens. The osseous lesions treated by Ad-BMP-7 gene delivery demonstrated rapid chrondrogenesis, with subsequent osteogenesis, cementogenesis and predictable bridging of the periodontal bone defects. CONCLUSION: These results demonstrate the first successful evidence of periodontal tissue engineering using ex vivo gene transfer of BMPs and offers a new approach for repairing periodontal defects.     

Factors that modulate the effects of bone morphogenetic protein-induced periodontal regeneration: a critical review

The healing process initiated by a single molecular species of bone morphogenetic protein (BMP) such as BMP-2 or BMP-7 sets in motion a cascade of cellular events resulting in differentiation of progenitor cells into phenotypes involved in periodontal regeneration. For example, animal studies show that a single dose of recombinant human (rh) BMP-2 increases the rate of normal intramembranous bone formation and enhanced cementum formation during periodontal wound healing. However, the optimal effects of BMPs are modulated by a range of factors that need careful evaluation in clinical studies. These factors include the influence of root conditioning, occlusal loading, BMP dose, and the release characteristics of the carrier as well as the suitability of the model to evaluate the efficacy of BMPs. Each of these factors may affect the rate of BMP-induced osteogenesis and cementogenesis and subsequent periodontal ligament (PDL) formation during the early and late stages of periodontal wound healing. Although BMP-2 initiates stem cells along an osteogenic pathway, the dose may have to be of sufficient concentration to ensure other growth and differentiation factors do not redirect or retard the osteogenic potential of the cell. Understanding when to manipulate the cell's differentiation pathway with the application of single or multiple doses of BMPs at the appropriate concentration is required to optimize the effect of BMPs in periodontal wound healing. Therefore, different release profiles from the same carrier may be particularly important in tissues with mixed cell populations such as in the periodontium, where similar tissues like bone and cementum grow at different rates. Furthermore, treatment of intrabony defects with BMPs are likely to not only require appropriate temporal release of the BMP(s), but also a carrier that can serve as a template for new tissue formation providing space maintenance and supporting the mucoperiosteal flap. Many of these issues have not been adequately addressed from a periodontal standpoint; therefore the purpose of this review is to clarify our current understanding of the factors that are likely to modulate the effects of BMP-induced periodontal regeneration. Moreover, assessing the importance of these factors is essential prior to conducting expensive human clinical trials.     

Combinatorial gene therapy with BMP2/7 enhances cranial bone regeneration

BMP2/7 heterodimer expression by adenovirus can stimulate bone formation at subcutaneous sites. In the present study, we evaluate whether this approach will also promote healing of cranial defects. Adenovirus expressing BMP2 or BMP7 (AdBMP2, AdBMP7) was titrated to yield equivalent BMP protein levels after transduction into murine BLK cells. Analysis of conditioned medium showed that BMP2/7 heterodimers have enhanced ability to stimulate alkaline phosphatase and Smad 1,5,8 phosphorylation relative to equivalent amounts of BMP2 or BMP7 homodimers. To measure bone regeneration, we implanted virally transduced BLK cells into critical-sized calvarial defects generated in C57BL6 mice. AdBMP2/7-transduced cells were more effective in healing cranial defects than were cells individually transduced with AdBMP2 or BMP7. Dramatic increases in bone volume fraction, as measured by microCT, as well as fusion of regenerated bone with the defect margins were noted. Thus, the use of gene therapy to express heterodimeric BMPs is a promising potential therapy for healing craniofacial bones.     

Molded bone augmentation by a combination of barrier membrane and recombinant human bone morphogenetic protein-2

OBJECTIVES: To provide the histological background to a new method of local bone augmentation, we examined the events occurring beneath a barrier membrane applied with recombinant human bone morphogenetic protein-2 (rhBMP-2). MATERIALS AND METHODS: The effects on bone augmentation of rhBMP-2, applied with a membrane mold (BMP-Memb), over surgically-induced bone defects in rat calvaria were examined histologically, and the results compared with those from application of rhBMP-2 (BMP) alone, or of a molded membrane (Memb) alone. RESULTS: At postoperative week 2, the BMP group showed the most marked bone formation. However, the bone diminished in size by week 8. The Memb group showed slow but continuous bone formation by week 8. In the BMP-Memb group, bone filled the space in the mold at week 2, and this was maintained until week 8. Moreover, the soft tissue that had intervened between newly formed bone and the membrane in the Memb group was not evident in the BMP-Memb group, in which bone had formed directly on the membrane. CONCLUSIONS: The results suggest that the combination of rhBMP-2 and barrier membrane has advantages in producing and maintaining bone in the intended shape by inducing osteoblasts directly on the inner surface of the membrane.     

Effect of Emdogain enamel matrix derivative and BMP-2 on the gene expression and mineralized nodule formation of alveolar bone proper-derived stem/progenitor cells

The objective of this study was to evaluate the effect of Emdogain (Enamel Matrix Derivative, EMD) and Bone Morphogenetic Protein-2 (BMP-2), either solely or in combination, on the gene expression and mineralized nodule formation of alveolar bone proper-derived stem/progenitor cells.Stem/progenitor cells were isolated from human alveolar bone proper, magnetically sorted using STRO-1 antibodies, characterized flowcytometrically for their surface markers' expression, and examined for colony formation and multilineage differentiation potential. Subsequently, cells were treated over three weeks with 100 μg/ml Emdogain (EMD-Group), or 100 ng/ml BMP-2 (BMP-Group), or a combination of 100 ng/ml BMP-2 and 100 μg/ml Emdogain (BMP/EMD-Group). Unstimulated stem/progenitor cells (MACS⁺-Group) and osteoblasts (OB-Group) served as controls. Osteogenic gene expression was analyzed using RTq-PCR after 1, 2 and 3 weeks (N = 3/group). Mineralized nodule formation was evaluated by Alizarin-Red staining.BMP and EMD up-regulated the osteogenic gene expression. The BMP Group showed significantly higher expression of Collagen-I, III, and V, Alkaline phosphatase and Osteonectin compared to MACS⁺- and OB-Group (p < 0.05; Two-way ANOVA/Bonferroni) with no mineralized nodule formation.Under in-vitro conditions, Emdogain and BMP-2 up-regulate the osteogenic gene expression of stem/progenitor cells. The combination of BMP-2 and Emdogain showed no additive effect and would not be recommended for a combined clinical stimulation.     

Slow and continuous application of human recombinant bone morphogenetic protein via biodegradable poly(lactide-co-glycolide) foamspheres

Bone morphogenetic proteins (BMPs) are multifunctional cytokines that were originally identified as molecules that induce bone and cartilage formation in vivo. In order to increase the efficacy of this potent protein for application in medicine, a carrier system is needed to retain the BMP at the preferred site. Here we present and characterize a slow-release carrier system for pure human recombinant (rh)BMP. The large porous microspheres, called 'foamspheres', are biodegradable, because they consist of poly(lactide-co-glycolide) acids and release loaded rhBMP slowly and continuously. In vivo studies in rodents revealed that rhBMP-loaded foamspheres increased the thickness of the calvarial bone of rats by 222%. When the same amount of rhBMP was applied via a gelatine-based hydrogel, the increase in bone height was only 66%. Thus, the carrier system for rhBMP is an important factor for the efficacy of BMPs.     

Effects of occlusal loading on ankylosis, bone, and cementum formation during bone morphogenetic protein-2-stimulated periodontal regeneration in vivo.

BACKGROUND: The purpose of this study was to investigate the influence of occlusal loading on recombinant human bone morphogenetic protein-2 (BMP-2)-induced bone and cementum formation in a previously established rat model of periodontal regeneration during the early and late stages of wound healing. METHODS: 64 Wistar rats were divided into 8 groups and had surgically created fenestrated defects on the right side of the mandible involving the removal of bone and exposure of the first and second molar roots. Four groups had their right maxillary molars extracted 2 weeks prior to surgery. Ten microl of 100 ug/ml BMP-2 in a collagen membrane was placed in extracted (hypofunctional) and non-extracted (functional) groups (BMPe and BMPf, respectively) while control groups had collagen membrane only (CONe and CONf). Groups were sacrificed at 10 (BMPe, BMPf, CONe, CONf) or 35 days (BMP35e, BMP35f, CON35e, CON35f) postoperatively and tissues processed for histological examination. Transverse 5 microm sections were stained for identification of new bone, ankylosis and cementum formation. Results: At 10 days, CONe developed greater bone growth compared with CONf (P<0.05), while both BMP groups developed greater bone compared with controls. However, BMPe developed more ankylosis compared with both CONe and CONf while BMPf was significantly greater than CONf only (P<0.05). BMPf only developed significantly greater new cementum compared with controls. At 35 days, BMP35f developed greater bone growth compared with all other groups including BMP35e (P<0.05) and unlike results at 10 days, no differences were apparent between CON35f and CON35e. Unwanted bone growth beyond the defect margin anteriorly was significantly greater in BMP35f. Conclusions: Results suggest hypofunction stimulates early bone formation. Furthermore, hypofunction and BMP-2 increase the development of transient ankylosis. However, after wound healing is complete, function augments the early effects of BMP-2-induced new bone growth indicating remodeling to physiological levels does not occur. Finally, occlusal loading is both an important stimulus for remodeling and establishment of the periodontal ligament space during early wound healing as well as enhancing BMP-2-induced cementogenesis.     

牵张成骨整复猕猴腭裂模型成骨区骨形态发生蛋白-2的表达

目的观察牵张成骨术整复腭裂新骨形成的骨形态发生蛋白-2（BMP-2）表达分布规律。方法猕猴23只以外科方法建立腭裂动物模型。其中实验组动物21只,以牵张成骨术整复其腭部软硬组织缺损,至骨运送盘移动关闭裂隙后原位固定。分别于固定期第1、2、4、6、8、12及24周取材3只实验动物,标本行免疫组化染色,观察其不同时间的表达分布,并与实验对照组及空白对照组结果对比。结果免疫组化实验表明, BMP-2于新骨形成过程中主要存在于成骨细胞胞浆中,在牵张成骨早期新生骨小梁的周围存在大量成骨细胞,染色为强阳性;4~6周时,成骨细胞进一步增多,BMP-2表达也呈现强阳性,表明成骨过程到达高峰;8周时成骨细胞数量减少,BMP-2表达趋于减弱;至12周时,成骨过程已基本完成,免疫组化染色基本无着色。结论术后固定成骨期内,BMP-2的表达与分布是一个由弱变强,达到成骨高峰后,随着新骨改建成熟又逐渐趋弱的动态变化过程。     

Porcine sinus mucosa holds cells that respond to bone morphogenetic protein (BMP)-6 and BMP-7 with increased osteogenic differentiation in vitro

The aim of this in vitro study was to determine whether the sinus mucosa holds cells with an osteogenic potential. Frozen sections of sinus mucosa from three adult pigs were investigated for the expression of STRO-1, a marker of mesenchymal progenitor cells, and alkaline phosphatase activity, an enzyme expressed by cells committed to the osteogenic lineage and by mature osteoblasts. To determine their osteogenic potential, mucosa-derived cells were incubated with bone morphogenetic protein (BMP)-6 and BMP-7, and alkaline phosphatase activity, osteocalcin expression, and mineralization of the extracellular matrix was measured. We found sinus mucosa cells staining positive for STRO-1 and alkaline phosphatase activity. When sinus mucosa tissue was placed in culture, alkaline phosphatase positive cells grew out from the explants and further increased alkaline phosphatase activity in response to BMP-6 and BMP-7. The expression level of the osteoblast-specific extracellular matrix protein osteocalcin, and the amount of calcium accumulation within the extracellular matrix was also increased in response to BMPs. We conclude that the sinus mucosa holds mesenchymal progenitor cells and cells committed to the osteogenic lineage that can respond to BMP-6 and BMP-7 by an increase of their osteogenic differentiation.     

Epidermal growth factor and bone morphogenetic proteins upregulate osteoblast proliferation and osteoblastic markers and inhibit bone nodule formation

Objective

The aim of this study was to investigate the in vitro osteogenic activity of EGF in association with bone morphogenetic proteins BMP2 and BMP7.

Methods

SaOS-2 (osteoblast-like cell line from human osteosarcoma) were cultured in the presence of EGF and BMPs for various culture periods to assess (a) cell proliferation by MTT assay, (b) Runx2, alkaline phosphatase (ALP) and osteocalcin (OC) mRNA expression using quantitative RT-PCR and ELISA, and (c) bone tissue mineralization using Alizarin Red staining.

Results

EGF alone was able to stimulate osteoblast growth in a time-dependent manner. When mixed with BMP2, BMP7, and their combination, EGF greatly promoted osteoblast growth, compared to the BMP- and EGF-stimulated cells, suggesting a possible synergistic effect between EGF and BMPs on osteoblast growth. Stimulation with EGF, EGF/BMP2, and EGF/BMP2/BMP7 for 7 days upregulated Runx2 mRNA expression by the osteoblasts. EGF downregulated ALP mRNA expression, which was recovered when the BMP2/BMP7 combination was added to the osteoblast culture. Tested on OC mRNA expression, EGF had no effect and inhibited the enhancing effect of BMP2 and BMP7 on osteocalcin expression. The bone mineralization assay showed that EGF reduced both the number and size of the bone nodules. This reducing effect was observable even in the presence of BMP2 and BMP7.

Conclusion

This study demonstrated that EGF may act in the early phase to promote osteoblast growth and specific marker expression rather than the late phase involving cell differentiation/mineralization.     

Addition of bone morphogenetic protein type 2 to ascorbate and β-glycerophosphate supplementation did not enhance osteogenic differentiation of human adipose-derived stem cells

Bone morphogenetic protein type 2 (BMP-2) is a potent local factor, which promotes bone formation and has been used as an osteogenic supplement for mesenchymal stem cells.

Objectives

This study evaluated the effect of a recombinant BMP-2 as well as the endogenous BMP-4 and BMP-7 in the osteogenic differentiation of adipose-derived stem cells (ASCs) in medium supplemented with ascorbate and β-glycerophosphate.

Material and Methods

Human ASCs were treated with osteogenic medium in the presence (ASCs+OM+BMP-2) or absence (ASCs+OM) of BMP-2. The alkaline phosphatase (ALP) activity was determined and the extracellular matrix mineralization was evaluated by Von Kossa staining and calcium quantification. The expressions of BMP-4, BMP-7, Smad1, Smad4, and phosphorylated Smad1/5/8 were analyzed by western blotting. Relative mRNA expressions of Smad1, BMP receptor type II (BMPR-II), osteonectin, and osteocalcin were evaluated by qPCR. Results: ASCs+OM demonstrated the highest expression of BMP-4 and BMP-7 at days 21 and 7, respectively, the highest levels of BMPR-II mRNA expression at day 28, and the highest levels of Smad1 mRNA at days 14 and 28. ASCs+OM+BMP-2 demonstrated the highest levels of Smad1 mRNA expression at days 1, 7, and 21, the highest expression of Smad1 at day 7, the highest expression of Smad4 at day 14, the highest ALP activity at days 14 and 21, and expression of phosphorylated Smad1/5/8 at day 7. ASCs+OM and ASCs+OM+BMP2 showed similar ALP activity at days 7 and 28, similar osteonectin and osteocalcin mRNA expression at all time periods, and similar calcium depositions at all time periods.

Conclusions

We concluded that human ASCs expressed endogenous BMP-4 and BMP-7. Moreover, the supplementation of ASCs with BMP-2 did not increase the level of osteogenic markers in the initial (ALP activity), intermediate (osteonectin and osteocalcin), or final (calcium deposition) phases, suggesting that the exogenous addition of BMP-2 did not improve the in vitro osteogenesis process of human ASCs.     

Effect of aging on bone formation induced by recombinant human bone morphogenetic protein-2 combined with fibrous collagen membranes at subperiosteal sites

This study was designed to examine the effect of aging on bone formation induced by recombinant human bone morphogenetic protein-2 (rhBMP-2) combined with a fibrous collagen membrane (FCM). Implantation was done subperiosteally in bilateral palatal grooves in 34 male Wistar rats divided into three age groups: a 10-week-old group (10w group), a 30-week-old group (30w group) and a 70-week-old group (70w group). RhBMP-2-combined FCMs were implanted on the left palatal grooves as BMP-implanted sites (BMP site), while rhBMP-2 was not implanted on the right palatal grooves as control sites. The rats were sacrificed 6 weeks after implantation, and histometric evaluations were performed. New bone formation was observed in every site of each age group and the new bone was almost completely continuous with the original bone. The new bone volume (NBV) of the BMP site was significantly higher than that of the control site in each age group. The NBV of both the control and BMP sites were highest in the 10w group and lowest in the 70w group. The disparity of NBV between the control and BMP sites, which indicated the response to implanted BMP excluding the effect of skeletal growth and surgical stimulation, did not significantly differ among the age groups. These results indicate that rhBMP-2-combined FCM has the ability to induce new bone formation continuous with original bone even in senescent rats. Furthermore, it appeared that, in the case of palatal subperiosteal implantation, the responsiveness to implanted BMP was independent of age, although the total volume of newly formed bone declined with aging.