Bone regeneration with recombinant human bone morphogenetic protein 2: a systematic review

Aim

The aim of this work was to perform a systematic literature review on the clinical application of rhBMP-2 in bone reconstruction prior to placing implants.

Materials and Methods

A PUBMED search was made about the subject and nine clinical trials were selected according to strict inclusion criteria.

Results

Overall success rates of bone regeneration with rhBMP-2 was 81.4% and success of implants placed was 87.4%. Most frequent adverse events were pain, edema and erythema.

Conclusion

It was concluded that the treatment with rhBMP-2 foi satisfactory in most cases and the placement of dental implants in the bone regenerated with rhBMP-2 is feasible.

     

Effect of recombinant human bone morphogenetic protein-2 on bone regeneration and osseointegration of dental implants

Recombinant human bone morphogenetic protein-2 (rhBMP-2) induced bone regeneration and osseointegration was evaluated in bony defects created within the hollow chamber of endosseous dental implants in 14 foxhound dogs. Bilateral extractions of mandibular premolars were performed and surgical implantation of 104 hollow cylinder implants followed after 8 weeks of healing. Experimental implants had their hollow chamber filled with 20 microg of rhBMP-2 delivered with a bovine collagen carrier, whereas the control implants had their apical chamber left empty. Dogs were followed for 2, 4, 8 and 12 weeks. Histomorphometric evaluation and immunohistochemical analysis were performed. Minimal bone was regenerated at 2 weeks for both groups. At 4 weeks, bone fill averaged 23.48% for the rhBMP-2 and 5.98% for the control group (P<0.05). At 8 weeks, mean bone fill was 20.94% and 7.75% for the rhBMP-2 and the controls, respectively (P<0.05). At 12 weeks, mean bone fill was 31.39% and 24.31% for the rhBMP-2 and control implants, respectively (P>0.05). Bone-implant contact (BIC) increased for both groups over time and at 8 weeks the rhBMP-2 BIC value was 18.65% and for the control 7.22% (P<0.05). At 12 weeks, the BIC was 43.78% and 21.05% for the rhBMP-2 and the control group, respectively (P<0.05). Immunohistochemical staining for type II collagen was positive only for parts of the collagen carrier and formation of cartilaginous intermediate was not observed in any of the specimens. The results suggest that, in confined defects adjacent to dental implants, rhBMP-2 can induce bone regeneration in close apposition to the implant surface.     

Potential of recombinant human bone morphogenetic protein-2 in bone regeneration

Recombinant human bone morphogenetic protein (rhBMP-2) has been used as a bone substitute. This article describes the rhBMP-2 structure, mechanisms of action, carriers, advantages, safety, and recent clinical studies relevant to dentistry.     

Adenovirus-mediated recombinant human bone morphogenetic protein-7 expression promotes differentiation of human dental pulp cells

Recombinant human bone morphogenetic protein-7 (BMP-7) has been shown to stimulate new reparative dentin formation in animal models. However, little is known about whether BMP-7 could promote the odontoblast-like differentiation and the formation of mineralized nodules in human dental pulp cells. Here, we reported that the infection with adenovirus-BMP-7 (Ad-BMP-7), a BMP-7-expressing adenoviral vector, induced the expression of BMP-7 in primarily cultured human dental pulp cells in the long term with little effect on their proliferation and viability. Importantly, BMP-7 expression significantly increased alkaline phosphatase activity and induced the dentin sialophosphoprotein expression in a dose- and time-dependent manner, suggesting that BMP-7 promoted the odontoblast differentiation. Furthermore, BMP-7 expression stimulated the formation of many mineralized dentin-like calcified nodules. Our data suggest that Ad-BMP-7-mediated BMP-7 expression can promote the differentiation of human pulp cells into odontoblast-like cells and mineralization in vitro, which may provide insight for the design of new gene therapy for the pulp capping in the clinic.     

Dentin and dental pulp regeneration by the patient's endogenous cells

The goal of regenerative endodontics is to restore the functions of the dental pulp–dentin complex. Two approaches are being applied toward dental pulp–dentin regeneration: cell transplantation and cell homing. The majority of previous approaches are based on cell transplantation by delivering ex vivo cultivated cells toward dental pulp or dentin regeneration. Many hurdles limit the clinical translation of cell transplantation such as the difficulty of acquiring and isolating viable cells, uncertainty of what cells or what fractions of cells to use, excessive cost of cell manipulation and transportation, and the risk of immune rejection, pathogen transmission, and tumorigenesis is associated with ex vivo cell manipulation. In contrast, cell homing relies on induced chemotaxis of endogenous cells and therefore circumvents many of the difficulties that are associated with cell transplantation. An array of proteins, peptides, and chemical compounds that are yet to be identified may orchestrate endogenous cells to regenerate the dental pulp–dentin complex. Both cell transplantation and cell homing are scientifically valid approaches; however, cell homing offers a number of advantages that are compatible with the development of clinical therapies for dental pulp–dentin regeneration.     

人牙髓细胞诱导牙本质再生的实验观察

目的:观察人成体牙髓细胞体内诱导牙髓组织修复反应的能力.方法:在矿化诱导液作用下,将一定数量级的人牙髓细胞与β-TCP生物陶瓷颗粒进行复合,植入免疫缺陷鼠磨牙穿髓孔处,7 d、14 d后分别取材进行组织学观察.对照组采用氢氧化钙(Oycal)和空白对照组.结果:组织学观察表明,盖髓术后7 d,各组均出现了牙髓细胞向穿髓孔处迁移、聚集.术后14 d,牙髓细胞组炎症反应仅局限于穿髓孔处,有明显的修复性牙本质桥形成;Dycal组炎症反应涉及到少量冠髓,有部分矿化的纤维性屏障形成;空白对照组炎症反应涉及了大部分冠髓,仅有弥散的骨样牙本质形成.结论:在矿化诱导液作用下,牙髓细胞具有向成牙本质细胞样细胞定向分化的能力,将牙髓细胞植入鼠磨牙的穿髓孔处,显示其具有良好的维持牙髓活力和诱导修复性牙本质形成的能力.     

Expression of bone morphogenetic protein in the course of osteoinduction by recombinant human bone morphogenetic protein-2

To clarify the mechanism of osteoinduction by recombinant human bone morphogenetic protein-2 (rhBMP-2), we examined the time-course localization of bone morphogenetic proteins (BMPs) immunostained by an anti-BMP-2 monoclonal antibody after implantation of pellets consisting of rhBMP-2 and collagen in rat calf muscle pouch. On day 3 after implantation, BMP was detected in the entire lump, and the intensity of staining for BMP around the implant on day 7 was weaker than that on day 3. The staining for BMP decreased with time and the region of staining for BMP remained more centralized in the implant. On day 10 after implantation, BMP was observed in part of the newly induced cartilage, especially around chondrocytes. On day 14 after implantation, BMP was localized in the newly induced woven bone. On day 21, BMP staining was found in osteoblasts at the surface of the newly induced bone. Especially, the staining for BMP decreased from day 10 to day 21. These results indicate that the woven bone was replaced with mature lamellar bone from day 14 to day 21. The present findings suggest that rhBMP-2 plays an important role in osteoinduction, especially at the early stage.     

Bone repair following recombinant human bone morphogenetic protein-2 stimulated periodontal regeneration

BACKGROUND: Recombinant human bone morphogenetic protein-2 (rhBMP-2) in an absorbable sponge (ACS) carrier is currently being evaluated as candidate therapy for periodontal regeneration. The objective of this study was to characterize, in some detail, tissue reactions following surgical implantation of rhBMP-2/ACS into periodontal defects. METHODS: Four young adult, male beagle dogs with surgically induced, bilateral, critical size, supra-alveolar, mandibular premolar defects sequentially received rhBMP-2/ACS (rhBMP-2 at 0.2 mg/ml) in right and left jaw quadrants. After 4 or 8 weeks of healing, experimental teeth with surrounding tissues were harvested and processed for light and transmission electron microscopy. RESULTS: Surgical implantation of rhBMP-2/ACS into large supra-alveolar periodontal defects resulted in a variable tissue response without marked difference between 4- and 8-week observations. New bone, exceeding the volume of the normal alveolar process, had formed within 4 weeks. The regenerated bone tissue consisted of finely trabeculated woven bone. Marrow spaces exhibited a continuous lining of osteoblasts, osteoclasts, and resting cells. The marrow spaces contained numerous large, thin-walled vessels but were almost devoid of collagen fibrils or fibroblasts. Large voids (seromas) encountered in the newly formed bone were free of structured elements except for occasional aggregates of effete erythrocytes. A variety of tissue reactions were observed along the root surface including areas of resorption, areas of hard tissue deposition, and areas without resorptive or appositional activity. Ankylosis was a frequent observation, although areas showing characteristics of a periodontal ligament with a fine layer of acellular fiber cementum and occasional inserting Sharpey's fibers were also observed. Osteoblasts facing the root surface often appeared to be in a highly active state judged by their cuboidal shape, well-developed endoplasmic reticulum and numerous mitochondria, and the presence of an adjacent layer of preosteoblasts. Conspicuous bundles of wide collagen fibrils near the dentin surface as well as within the marrow spaces were considered to represent remnants of the ACS. These fibrils were associated with areas of mineralization as verified by examination of undecalcified specimens. CONCLUSIONS: rhBMP-2/ACS elicits a rapid osteoinductive process throughout the implant as well as along and onto the instrumented adjacent root surface. Lamellated trabecular bone was the predominant regenerated tissue. A typical cementum-periodontal ligament-alveolar bone relationship was a rare observation. The great variability in histological tissue response along the instrumented root surface indicates that the stimulus to hard tissue formation resided primarily in the rhBMP-2/ACS implant rather than in the root surface.     

Dentin regeneration using deciduous pulp stem/progenitor cells

Reparative dentin formation is essential for maintaining the integrity of dentin structure during disease or trauma. In this study, we investigated stem/progenitor cell-based tissue engineering for dentin regeneration in a large animal model. Porcine deciduous pulp stem/progenitor cells (PDPSCs) were mixed with a beta-tricalcium phosphate (β-TCP) scaffold for dentin regeneration. Different concentrations of PDPSCs were tested to determine the optimal density for dentin regeneration. Aliquots of 5×10(5) PDPSCs in 1 mL resulted in the highest number of cells attached to the scaffold and the greatest alkaline phosphatase activity. We labeled PDPSCs with green fluorescent protein (GFP) and used the optimal cell numbers mixed with β-TCP to repair pulp chamber roof defects in the premolars of swine. Four weeks after transplantation, GFP-positive PDPSCs were observed in PDPSC-embedded scaffold constructs. At 16 weeks after transplantation, the PDPSCs mixed with β-TCP significantly regenerated the dentin-like structures and nearly completely restored the pulp chamber roof defects. This study demonstrated that the PDPSC/scaffold construct was useful in direct pulp-capping and provides pre-clinical evidence for stem/progenitor cell-based dentin regeneration.     

Enhanced bone regeneration around dental implant with bone morphogenetic protein 2 gene and vascular endothelial growth factor protein delivery

Objective: To evaluate the synergistic effect of bone morphogenetic protein 2 (BMP‐2) and vascular endothelial growth factor (VEGF) on the repair of bone defects around dental implants. Material and methods: Five groups of scaffold were fabricated by a freeze‐drying method, including pure chitosan/collagen scaffold; scaffold loaded with adenoviruses expressing BMP‐2, adenoviruses expressing VEGF, both adenoviruses expressing BMP‐2 and VEGF, VEGF protein and adenovirus expressing BMP‐2. In vitro studies examined whether bone marrow stromal cells were responsive to these scaffolds over time. Bone formation capacity, bone‐to‐implant contact, as well as removal torque values were investigated in vivo. Differences between the various groups were statistically analyzed using the one‐way analysis of variance test. Results: The in vitro study revealed a burst and rapid release of VEGF with a sustained high‐level expression of BMP‐2 in scaffold combined with VEGF protein and adenoviruses expressing BMP‐2. Histomorphometry demonstrated that scaffolds expressing BMP‐2 enhanced more bone formation compared with other groups; VEGF alone is insufficient to promote bone formation. New bone formation in the bone defects around dental implants, bone‐to‐implant contact and mean peak removal torque showed statistically significant difference for the adenoviral vector encoding human bone morphogenetic protein 2 (Ad‐BMP‐2) and VEGF protein and adenovirus expressing BMP‐2 groups. Furthermore, scaffold combined with VEGF protein and Ad‐BMP‐2 represented the best outcomes in this model. Conclusions: A combination of BMP‐2 gene and VEGF protein could have a synergistic effect in promoting bone healing. To cite this article:
Luo T, Zhang W, Shi B, Cheng X, Zhang Y. Enhanced bone regeneration around dental implant with bone morphogenetic protein 2 gene and vascular endothelial growth factor protein delivery.
Clin. Oral Impl. Res. 23 , 2012 467–474.
doi: 10.1111/j.1600‐0501.2011.02164.x     

The effect of nicotine on osteoinduction by recombinant human bone morphogenetic protein 2

Nicotine, one of the constituents of tobacco, is known to have an adverse effect on human health. We sought to clarify the interaction between nicotine and recombinant human bone morphogenetic protein 2 (rhBMP-2) in terms of osteogenesis in vitro and osteoinduction in vivo. Nicotine did not inhibit or stimulate alkaline phosphatase (ALP) activity or the amount of osteocalcin in C2C12 cells in the presence of rhBMP-2 in vitro. Ectopic bone formation using a collagen sponge containing rhBMP-2 was evaluated with and without nicotine after 21 days using radiographic, histological, biochemical, and immunohistochemical analyses. ALP activity in the medium-dose group (2.2 ± 0.9 IU/mg protein; P = 0.047) and the high-dose group (2.0 ± 0.1 IU/mg protein; P = 0.03) was significantly lower than in the control group. The calcium content in the medium-dose group (35.4 ± 12.9 μg/mg tissue; P = 0.0099) and high-dose group (34.8 ± 10.5 μg/mg tissue; P = 0.006) was significantly lower than in the control group. The number of vascular endothelial growth factor-positive cells in the high-dose group (671.9 ± 57.3 cells/mm²; P = 0.03) was significantly lower than in the control group. Results showed that nicotine did not inhibit the stimulatory effect of rhBMP-2 in vitro, but a high dose of nicotine inhibited bone formation in vivo by adversely affecting vascularization.     

Bone induction by Escherichia coli -derived recombinant human bone morphogenetic protein-2 compared with Chinese hamster ovary cell-derived recombinant human bone morphogenetic protein-2

Most recombinant human bone morphogenetic protein (rhBMP) is currently obtained from Chinese hamster ovary (CHO) cells. If rhBMP with more activity could be derived from Escherichia coli (E. coli), a large quantity of rhBMP could be produced at low cost. The bone-inducing ability of an E. coli -derived rhBMP-2 (ErhBMP-2) variant with an N-terminal sequence was examined and compared with CHO cell-derived rhBMP-2 (CrhBMP-2). Two, 10, or 50 microgram of ErhBMP-2 or CrhBMP-2 was mixed with 3mg of atelopeptide type I collagen as the carrier, and specimens were implanted into the calf muscle pouches of Wistar rats (n= 5 in each group). Three weeks later, new bone had formed in all the ErhBMP-2-implanted and CrhBMP-2-implanted muscles. Radiographic and histological examinations showed that the bone induced by ErhBMP-2 had a large hollow bone matrix with more fatty marrow than the bone induced by CrhBMP-2. Quantitative analysis indicated that the activity of ErhBMP-2 was similar to that of CrhBMP-2, so ErhBMP-2 may be useful for inducing bone formation.     

Prefabrication of vascularized bone flap induced by recombinant human bone morphogenetic protein 2 (rhBMP-2)

An experimental model for the prefabrication of a vascularized bone flap was developed in this study. To form vascularized bone in the desired configuration and to increase the survival rate of the grafted bone, a muscle vascularized pedicle (MVP) was transformed into vascularized bone by the inducer recombinant human bone morphogenetic protein 2 (rhBMP-2). The muscle flap (8 x 8 mm) raised on saphenous vessels in the rat thigh was sandwiched between same-size collagen (Terudermis) sheets in the presence or absence of impregnated 25 microg of rhBMP-2 for the experimental group and the control group, respectively. The flaps were harvested 1, 2 and 3 weeks postoperatively. Bone transformation was detected by gross examination, radiology, and histologic testing. No evidence of muscle tissue transformation was found in control flaps, whereas all of the experimental flaps produced new bone. Saphenous vessels were observed to supply the new bone upon harvesting, and the newly formed vascularized bone showed good configuration with shape of the Terudermis sheet. This study indicates that this model of effective bone reconstruction could be potentially applied in a therapeutic setting.     

Dentin resorption and cementum-like tissue formation by bone morphogenetic protein application

BACKGROUND AND OBJECTIVE: Recent studies have shown that bone morphogenetic protein-2 (BMP-2) stimulates mineralization and osteoclast differentiation. Osteoclastic resorption by BMP-2 application may play an important role in the regulation of new cementum-like tissue formation on the dentin surfaces. Therefore, this study aimed to examine the effect of BMP-2 application on dentin resorption and cementum-like tissue formation at the dentin surfaces. MATERIAL AND METHODS: Seventy-two flat dentin blocks were prepared from rat roots and treated with 24% EDTA. Each block was assigned to group 0, group 100, or group 400, and immersed correspondingly in 0, 100, or 400 microg/ml BMP-2. The dentin blocks were then implanted into palatal connective tissue of rats, and specimens were prepared 2, 4 and 8 wk after surgery for histologic and histomorphometric analyses. RESULTS: BMP-2 caused a dose-dependent increase in dentin resorption by osteoclastic cells. New cementum-like tissue was randomly formed on parts of the nonresorbed and resorbed dentin surfaces in groups 100 and 400. Dentin resorption in groups 100 and 400 was significantly greater than group 0 (p < 0.01). However, at 8 wk, new cementum-like tissue formed in 41.8% of group 100, as compared with 16.2% of group 400 (p < 0.05). CONCLUSION: Dentin resorption was stimulated by a high dose of BMP-2, and cementum-like tissue was induced by a low dose of BMP-2, effectively suggesting that BMP-2 application, at an appropriate dose, to a dentin surface may enhance periodontal regeneration.     

Comparison of recombinant and purified human bone morphogenetic protein

Clinically, it would be more convenient to use recombinant than purified preparations of bone morphogenetic protein (BMP). Recently, recombinant human BMP (rhBMP) has attracted the attention of many investigators, but it has not been fully characterized. We examined the bone-inducing activity of rhBMP-2 and compared it with that of purified BMP derived from human bone matrix (phBMP). Two, 10, or 50 microg of rhBMP-2 or phBMP was mixed with 3 mg of atelopeptide type I collagen (carrier), and specimens were implanted in the calf muscles of Wistar rats (n=5 in each group). Four weeks later, new bone had formed in all the rhBMP-2- and phBMP-implanted muscles and was visible radiographically and histologically. The quantitative analysis indicated that the activity of rhBMP-2 was less than one tenth that of phBMP. It is necessary to find out why rhBMP-2 has fewer activities than phBMP.     

Inhibition of human dental pulp stem cell differentiation by Notch signaling

Notch signaling plays a critical role in development and cell fate specification. Notch receptors and ligands have been found to be expressed in dental epithelium or mesenchyme in the developing tooth, suggesting that Notch signaling may regulate odontogenesis. Post-natal human dental pulp stem cells (DPSCs) isolated from the dental pulp have characteristics of mesenchymal stem cells and can differentiate into odontoblasts. In this study, we examined whether Notch signaling regulated the odontoblastic differentiation of DPSCs. We found that over-expression of the Notch ligand, Jagged-1, activated the Notch signaling pathway in DPSCs. Jagged-1 inhibited the odontoblastic differentiation of DPSCs in vitro. Jagged-1-expressing DPSCs could not form mineralized tissues in vivo. Moreover, over-expression of the constitutively activated Notch1 intracellular domain (Notch-ICD) also inhibited odontoblastic differentiation of DPSCs. Taken together, our results demonstrate that Notch signaling can inhibit the odontoblastic differentiation of DPSCs.     

Acceleration effect of human recombinant bone morphogenetic protein-2 on differentiation of human pulp cells into odontoblasts

Predictable pulp capping procedures remain problematic, possibly because of the lack of appropriate stimulating factors for dentin formation. The present study examines the ability of one such stimulating factor, bone morphogenetic protein-2, to accelerate the differentiation of human dental pulp cells into odontoblasts. The number and morphology of cells between groups treated with 0 and 100 ng/ml of human recombinant bone morphogenetic protein-2 (rhBMP-2) did not significantly differ. However, ALPase activity (a marker for biomineralization) in the group stimulated with rhBMP-2 was more than double that of the control group. We then measured the expression of mRNA encoding dentin sialophosphoprotein (DSPP) as a marker of odontoblasts in rhBMP-2-stimulated human pulp cells using a quantitative polymerase chain reaction. The expression of DSPP mRNA in cells stimulated for 24 h by 1000 ng/ml of rhBMP-2 was approximately 20-fold and 5-fold higher than that by stimulated by 10 and 100 ng/ml, respectively. These findings show that rhBMP-2 promoted the differentiation of human dental pulp cells into odontoblasts but did not affect cell proliferation, suggesting that rhBMP-2 may have therapeutic utility in vital pulp therapy.     

脂多糖刺激人牙髓干细胞分泌的外泌体联合基质细胞衍生因子-1对牙髓再生的影响

目的探讨轻度炎症状态下人牙髓干细胞(human dental pulp stem cell,hDPSC)产生的外泌体与基质细胞衍生因子-1(stromal cell-derived factor-1,SDF-1)联合应用对牙髓组织再生的影响。方法分离培养hDPSC,脂多糖(lipopolysaccharide,LPS)刺激hDPSC,超速离心法提取hDPSC经LPS刺激后产生的外泌体(exosomes from lipopolysaccharide-stimulated hDPSC,L-EXO)和正常状态下分泌的外泌体(exosome from normal hDPSC,N-EXO),通过透射电镜和蛋白质印迹法鉴定提取物。将40只6~8周龄的SD大鼠通过随机数字表法分为S组(单独应用SDF-1)、L+S组(SDF-1与L-EXO联合应用)、N+S组(SDF-1与N-EXO联合应用)和空白对照组(根管内不植入任何物质),每组10只。以双侧下颌第一磨牙为实验牙,建立大鼠无髓根管模型,根据分组分别在根管内植入不同的内容物。植入后30 d过量麻醉处死所有大鼠,取大鼠双侧下颌骨组织,应用HE、Masson及免疫组织化学染色法进行组织学评价。结果HE染色结果显示,除空白对照组外,其他3组根管内均可见新生牙髓样组织,其中L+S组根管内新生组织的量及组织中的细胞数量最多,S组最少。Masson染色结果显示,L+S组矿化组织沿根管壁纵向排列,胶原纤维有序排列,N+S组呈无规律紊乱分布。定量分析各组新生血管面积,结果显示L+S组血管密度[(2.03±0.65)%]显著高于S组[(0.65±0.05)%]及N+S组[(1.06±0.38)%](F=5.879,P<0.05)。免疫组织化学结果显示,S组及L+S组的趋化因子受体4表达量显著低于N+S组(F=8.633,P<0.01)。结论hDPSC分泌的外泌体联合SDF-1可提高根管内新生组织的量和组织中的血管密度,L-EXO的作用较N-EXO强,并且新生组织中胶原纤维及矿化组织的排列更规律有序。