转化生长因子β1和骨形成蛋白2体外诱导成牙本质细胞分化

目的 观察转化生长因子β1（transforming growth factorβ1,TGF-β1）和骨形成蛋白2（bone morphogenetic protein2,BMP2)对体外培养的鼠牙乳头成牙本质细胞分化的影响。方法 取17d胎龄小鼠下颌第一磨牙牙胚，胰蛋白酶消化分离牙乳头，置半固态培养基培养6d，半固态培养基中加入重组TGF-β1或BMP2与肝素，组织学观察。结果 TGF-β1或BMP2加肝素可诱导牙乳头周边细胞发生极化，并分泌胞外基质。TGF-β1或BMP2单独加入时未见细胞极化，但基质分泌增加。结论 TGF-β1和BMP2均能诱导成牙本质细胞的细胞学分化和分泌功能。     

转化生长因子β1对体外成牙本质细胞分化的影响

目的：观察转化生长因子β1（transforming growth factorβ1，TGF－β1）对体外培养鼠牙乳头成本本质细胞分化的影响。方法：取17d胎龄小鼠下颌第一磨牙牙胚，胰腺蛋白消化分离牙乳头，置半固态培养基培养6d，半固态培养基中加入重组转化生长因子β1和肝素，组织学观察。结果：TGF－β1加肝素可诱导牙乳头周边细胞发生极化，并分泌胸外基质，单独加入TGF－β1未见细胞极化，但基质分泌增加，结论：TGF－β1能诱导前成牙本质细胞的细胞学和功能上分化。     

Effect of mineral trioxide aggregate cements on transforming growth factor beta1 and bone morphogenetic protein production by human fibroblasts in vitro

The aim of this study was to evaluate and compare the effects of two commercial mineral trioxide aggregate (MTA) cements (ProRoot MTA and MTA Angelus) on transforming growth factor (TGF)-beta1 and bone morphogenetic protein (BMP)-2 levels produced by cultured human gingival fibroblasts (HGFs). Human gingival tissues were obtained from individuals with healthy periodontium. HGFs were grown at 37 degrees C in humidified atmosphere of 5% CO(2) in Dulbecco's modified Eagle's medium, supplemented with 10% fetal calf serum, penicillin, and streptomycin. After 24 and 72 hours of exposure to the MTA products, HGF viability was determined by using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay. TGF-beta1 and BMP-2 levels in cell-free culture media were determined by enzyme-linked immunosorbent assay. Cell viability of the test groups was significantly lower than that of control at 24 and 72 hours (p < 0.05) but showed an increase at 72 hours (p < 0.05). Both test groups showed increased TGF beta-1 levels at 72 hours (p < 0.05), whereas the MTA Angelus group displayed higher TGF beta-1 levels than control and ProRoot MTA groups at 24 and 72 hours (p < 0.05). At 24 hours, BMP-2 levels of the ProRoot group were significantly higher than that of MTA Angelus (p < 0.05). Both test materials increased the BMP-2 levels within time (p < 0.05) and displayed similar levels at 72 hours (p > 0.05). These results suggest that both MTA products are capable of stimulating HGF to produce BMP-2, whereas the stimulatory effect for TGF beta-1 is material dependent.     

Effects of fibroblast growth factor 2 on osteoblastic proliferation and differentiation by regulating bone morphogenetic protein receptor expression

Fibroblast growth factors (FGFs) are known to play a critical role in bone growth and development, affecting both osteogenesis and chondrogenesis. Fibroblast growth factor 2 (FGF-2) is produced intracellularly by osteoblasts and secreted into the surrounding matrix in bone.The dose-dependent effects of FGF-2 were tested to examine the relationship between FGF-2 and osteoblast proliferation and differentiation. Tests used included a cell viability test, an alkaline phosphatase activity test, and a Western blot analysis.Cultures growing in the presence of FGF-2 showed an increased value for 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay and a decreased value for alkaline phosphatase activity. Results of the Western blot analysis showed that the addition of FGF-2 seems to decrease osteocalcin and bone morphogenetic protein receptor IA.These data show that FGF-2 in the tested dosage within MC3T3-E1 cells seems to affect proliferation and differentiation. Results of the Western blot analysis may add some possible mechanisms, and it may be suggested that treatment of FGF-2 may have an influence on the expression of bone morphogenetic protein receptors in osteoprecursor cells. Further elucidation of the mechanisms related to this mechanism within the in vivo model may be necessary to ascertain greater detail.     

碱性成纤维细胞生长因子和骨形成蛋白2在牙源性细胞分化中的表达及意义

目的研究人牙髓细胞(DPCs)和牙周韧带细胞(PDLCs)矿化过程中碱性成纤维细胞生长因子(FGF2)和骨形成蛋白2(BMP2)的表达变化,以及FGF2和BMP2在牙髓牙周损伤修复动物模型中的分布,探讨FGF和BMP信号通路在DPCs和PDLCs向成牙本质/成骨样细胞分化及牙髓牙周组织损伤修复中的调控作用。方法酶消化法分离培养DPCs和PDLCs,取诱导前及矿化诱导14d的DPCs和PDLCs,实时荧光定量反转录聚合酶链反应检测FGF2和BMP2的表达变化并进行统计学分析。建立大鼠牙髓牙周联合损伤动物模型,HE染色观察4周后牙髓牙本质复合体及牙周组织修复情况,免疫荧光检测FGF2和BMP2在新生牙髓及牙周组织的表达和分布。结果 DPCs和PDLCs经矿化诱导,FGF2mRNA表达下调,BMP2mRNA表达上调,与对照组间差异有统计学意义(P<0.05)。免疫荧光示FGF2在新生的牙周韧带组织强表达,在新生牙髓及正常牙髓牙周组织弱表达,BMP2蛋白在损伤处的新生牙髓及牙周韧带组织细胞浆强表达,FGF2和BMP2在正常牙髓及牙周韧带组织均无表达。结论 FGF2和BMP2在DPCs和PDLCs体外成牙本质/成骨分化中可能具备独立的生物学功能,在体内牙齿损伤修复过程中可能存在协同调控作用。     

Transforming growth factor beta2 regulates growth and differentiation of pulp cells via ALK5/Smad2/3

Transforming growth factor beta (TGF-beta) may regulate the biological activities of dental pulp cells. We found that human dental pulp cells expressed TGF-beta1, TGF-beta2, and a little amount of TGF-beta3 messenger RNA (mRNA). The exposure of pulp cells to TGF-beta2 induced the phosphorylation of Smad2/3, Smad1/5/8, and extracellular regulated-kinase 1/2 (ERK1/2) as observed by Western blotting. Exposure to TGF-beta2 decreased the alkaline phosphatase (ALP) mRNA expression and enzyme activity. Pretreatment of pulp cells with SB431542 (an inhibitor of TGF-beta ALK-4, ALK-5, and ALK-7 receptors) but not U0126 (a MEK1 inhibitor) prevented the inhibition of viable cell number, ALP activity, and mRNA expression by TGF-beta2 in dental pulp cells. These results suggest that TGF-beta may affect the growth and differentiation of dental pulp cells via an autocrine fashion by activation of the ALK/Smad2/3-signal transduction pathways. TGF-beta2 possibly regulates the differentiation of pulp cell at specific stages synergistically with other factors.     

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.     

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     

Platelet-derived growth factor and bone morphogenetic protein in the healing of mandibular fractures in rats

We studied the effects of platelet-derived growth factor-B (PDGF-B) and bone morphogenetic protein-2 (BMP-2) during the healing of mandibular closed fractures in rats by immunohistochemical methods. Unilateral closed fractures were created in the mandibles of thirty 12-week-old rats. BMP-2 was expressed during all stages of healing, but PDGF-B was expressed mainly in the early and middle stages, and not in the later stage of the healing process. We conclude that PDGF-B was associated with the proliferation and migration of primitive mesenchymal cells. BMP-2 was related to the differentiation of mesenchymal cells into osteoblasts and chondroblasts. PDGF-B and BMP-2 both have distinct regulatory effects on the healing of fractures.     

Failure to induce supracrestal bone growth between and around partially inserted titanium implants using bone morphogenetic protein (BMP): an experimental study in dogs

The effect of bone morphogenetic protein on supracrestal bone growth around partially inserted implants in a dog model is described. The lower premolar teeth (P1, P2, P3 and P4) were extracted on both sides of the mandible in six dogs. At a surgical exposure 12 weeks later, two 10-mm turned titanium implants were partially inserted, approximately 15 mm apart, in the areas of the P1 and P3 in each side of the mandible, allowing five threads to protrude from the bone crest. A titanium mesh was fastened to the coronal aspect of the two fixtures and the space beneath the mesh was filled with bone morphogenetic protein (S300 BMP) in combination with an insoluble bone matrix carrier, or with the carrier alone. The mesh was covered with an ePTFE membrane. Thus, a space for potential bone formation was created between the two implants. The surgical flaps were coronally positioned and secured with vertical mattress sutures. After 16 weeks of healing, biopsy specimens were retrieved and examined histologically. Bone was not formed around the protruding implants or in the created space between the implants in any case. The carrier was incompletely resorbed. We conclude that supracrestal bone growth beyond the crestal limit with or without BMP in such a large space as in this experimental design may not be possible.     

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.     

Effects of epidermal growth factor and/or nerve growth factor on Malassez’s epithelial rest cells in vitro: expression of mRNA for osteopontin,bone morphogenetic protein 2 and vascular endothelial growth factor

Yamawaki K, Matsuzaka K, Kokubu E, Inoue T. Effects of epidermal growth factor and/or nerve growth factor on Malassez’s epithelial rest cells in vitro: expression of mRNA for osteopontin, bone morphogenetic protein 2 and vascular endothelial growth factor. J Periodont Res 2010; 45: 421–427. © 2010 John Wiley & Sons A/S Background and Objective: Malassez’s epithelial rest (MER) cells are involved in the maintenance and homeostasis of the periodontal ligament (PDL). The purpose of this study was to determine the effects of epidermal growth factor (EGF) and/or nerve growth factor (NGF) in vitro on these functions of MER cells. Material and Methods: MER cells from porcine PDL were incubated for 3 or 9 h after the addition of EGF and/or NGF to final concentrations of 10 ng/mL. Cells cultured without those growth factors were used as controls. The expression of mRNA for osteopontin, bone morphogenetic protein 2 (BMP‐2) and vascular endothelial growth factor (VEGF) was analyzed using quantitative RT‐PCR. Results: There was a decrease in the expression of osteopontin mRNA by MER cells treated for 9 h with NGF and the level of mRNA expressed was lower than that of the control and EGF‐treated groups. The expression of BMP‐2 mRNA by MER cells treated with NGF for 9 h also decreased, and was lower than that of the control and EGF‐treated groups. The expression of VEGF mRNA by MER cells treated with EGF for 3 or 9 h was higher than in the control and NGF‐treated groups. The expression of VEGF mRNA was lower in MER cells treated with NGF for 3 and 9 h than in the control and EGF‐treated groups, and decreased from 3 to 9 h of treatment. EGF stimulated MER cells to secrete VEGF, which suggests that EGF plays an important role in maintaining the homeostasis of the PDL. NGF acts on MER cells to inhibit calcification in the PDL. Furthermore, in the EGF+NGF‐treated MER cells, expression of mRNA for BMP‐2 and VEGF was similar to that of the NGF‐treated group, but cell proliferation and expression of osteopontin mRNA were similar to that of the EGF‐treated group. Conclusion: EGF and NGF play important roles in maintaining the PDL.     

A quantitative immunohistochemical analysis of bone morphogenetic protein (BMP) in osteosarcoma of jaw]

The monoclonal antibody against bone morphogenetic protein (BMP-McAb) was first used for demonstration of bone morphogenetic protein (BMP) in 13 patients with osteosarcoma. Using avidin-biotin complex method (ABC), we demonstrated that BMP mainly existed in the tumor cell cytoplasm and tumorous osteoblast with positive staining in 10 out of 13 osteosarcoma patients. Using this staining method, we can not only differentiate osteosarcoma from fibrosarcoma (all are negative) and other non-osteogenic tumors, but also further classify osteosarcoma according to the BMP content and distribution by means of quantitative histological analysis. The BMP quantity of osteosarcoma with the patients' clinical situation will be useful in clinical diagnosis, treatment and prognosis. The relationship between BMP and the formation of the tumorous bone, and the relation between BMP and the process of osteosarcoma are discussed.     

Distribution of bone morphogenetic protein in human bone and tooth germs: analysis of specificity of monoclonal antibody against bone morphogenetic protein]

A cell strain stably secreting monoclonal antibody against BMP was obtained by hybridoma technique. The monoclonal antibody specifically reacted with the cells of human bone and tooth germs on paraffin embedded tissue sections. Immunohistochemical staining shows that BMP is distributed along collagen fibres in normal bone, also exist in osteoid tissue of new bone, in osteoblasts and in the cells of bone marrow. BMP may be found in human tooth germs such as in predentin, in the cells of outer and inner enamel epithelium, in the cells of dental sac generating alveolar bone. The results demonstrate that generation and growth of human bone and development of tooth germs have relation to BMP. The results at the molecular level prove that some antigenic determinants of human and bovine BMP are the same. BMP function activity inhibition test suggests that this antibody may block the function group of BMP. The ability of the monoclonal antibody to detect antigen and to inhibit generation of new bone makes it potentially useful in purification of BMP and in treating osteosarcoma and other bone tumors.     

Immunohistochemical analysis of bone morphogenetic protein (BMP) in osteosarcoma

The monoclonal antibody against bovine bone morphogenetic protein (bBMP-McAb) was first used for demonstration of bone morphogenetic protein (BMP) in osteosarcoma. The avidin-biotin complex method (ABC) demonstrated that of the 18 osteosarcomas, 15 stained positive, while all 6 fibrosarcomas were negative. The results showed that BMP mainly exists in the tumor cell plasma and some tumor-like bone tissues. Using this staining method, we can not only differentiate osteosarcoma from fibrosarcoma and other non-bone-derived tumors, but also classify osteosarcoma according to the content and distribution of BMP and the patient's clinical situation, thus providing a scientific basis for clinical treatment.     

Prefabrication of vascularized bone flap using bone morphogenetic protein (BMP)

The purpose of this study was to produce newly vascularized bone, and to form the bone into the desired shape. Silicone molds given the shape of a cylinder were used to deliver collagen sheets impregnated with or without rhBMP-2. The superficial inferior epigastric neurovascular bundle in a Wistar rat was identified and sandwiched between the silicone molds. The molds containing collagen sheets impregnated with 10 micrograms of rhBMP-2 were used for the experimental purpose, and those without rhBMP-2 were used as a control. In some of the experimental molds, the neurovascular bundle was ligated at both proximal and distal sites of the molds. After 2 and 4 weeks operation, bone formation was detected macroscopically, radiologically, and histologically. As a result, newly formed bone was observed in all experimental sites without the ligation, and the shape of the bone was exactly the same shape as the silicone mold. Newly formed bone was supplied by the superficial inferior epigastric artery, whereas newly formed bone was not observed in the control and the ligated flaps. Newly vascularized and shaped bone was created by applying an osteoinductive factor to a neurovascular bundle. This study demonstrates that this experimental model could be a therapeutically potential approach for effective bone reconstruction.