骨形态发生蛋白-7复合纤维蛋白对大鼠骨质疏松性椎体骨折愈合的影响

目的:观察以纤维蛋白为载体的骨形态发生蛋白-7(BMP-7)对骨质疏松性椎体骨折愈合的影响,探讨局部应用BMP-7纤维蛋白复合物治疗骨质疏松性椎体骨折的可行性。方法:雌性SPF级8月龄SD大鼠54只,随机选取36只,经去除卵巢建立骨质疏松模型并随机分为A、B组,每组18只;另18只行伪手术,在背部卵巢周围切除等体积脂肪,作为对照组(C组)。术后3个月所有动物采用L5椎体手术开窗刮除术区内松质骨方法建立骨折模型,A组骨折区内放入含BMP-724μg和纤维蛋白20mg的凝胶状混合物,B组和C组于同部位放入等体积小牛血清,盖好窗口骨瓣。建立骨折模型后4、6、8周,每组处死动物3只,取L5椎体切片行HE染色;6、8、12周时每组处死动物3只,行L5椎体力学性能测试(载荷能力、弹性模量和最大应变量)。结果:骨折后4周3组骨折区均有纤维骨痂形成,但B组纤维骨痂量少于A、C组;6周时A、C组有较多骨性骨痂形成,B组仅有微量骨性骨痂;8周时A、C组均为成熟小梁骨,骨折基本愈合,B组骨性骨痂量少,仍以纤维骨痂为主,尚有部分骨缺损存在;各时间点A组与C组相似。骨折后6、8、12周A、C组L5椎体的最大载荷、弹性模量及8、12周时的最大应变量均明显高于B组(P0.05或0.01),B组6周时的最大应变量与A、C组比较无显著性差异(P0.05),各时间点A组与C组比较无显著性差异(P0.05)。结论:局部应用以纤维蛋白为载体的BMP-7能明显促进大鼠骨质疏松性椎体骨折的愈合,纤维蛋白可作为BMP-7的载体。     

Use and efficacy of bone morphogenetic proteins in fracture healing

Purpose  

This review evaluates the application of bone morphogenetic proteins (BMPs) in delayed bone repair, aiming at a broad audience from clinicians to scientists. Next to an overview of the role of the different BMPs, their antagonists and their current applications, special attention is focused on new scientific developments improving the effects of BMP-based therapy for bone repair.     

骨形态发生蛋白5(BMP5)基因在骨折愈合中的定位研究

目的：应用骨形态发生蛋白5（BMP5）cDNA探针检测骨折愈合过程中外骨痂内BMP5基因表达的定位与分布,探讨BMP5基因表达在闭合性骨折愈合外骨痂形成中的作用。方法：以64只健康SD大鼠,制备闭合性胫骨骨折动物模型。骨折后分别于12小时,1,3,5,7,9,14及28天取材,取材后行恒冷切片,用地高辛素标记的BMP5 cDNA探针进行原位杂交,设立对照。结果：骨折后12小时及1天,骨折周围血肿内细胞及肌肉中新出现的间充质细胞内BMP5检测为阳性信号。结论：本实验表明创伤激活BMP5 cDNA的表达,并呈区域性参与骨折的修复,也说明骨折血肿及周围软组织在骨折愈合过程中具有非常重要的地位。     

Colocalization of noggin and bone morphogenetic protein-4 during fracture healing.

The regulation of callus formation during fracture repair involves the coordinate expression of growth factors and their receptors. This article describes the temporal and spatial expression of noggin gene, an antagonist to bone morphogenetic protein (BMP), during the fracture repair process. Noggin expression was examined by means of Northern blotting and in situ hybridization and compared with the expression pattern of BMP-4 in a model of fracture repair in adult mice. Expression levels of noggin messenger RNA (mRNA) were enhanced in the early phase of fracture callus formation. The localization of the noggin mRNA was similar to that of BMP-4 mRNA. Distinct noggin mRNA signals were located predominantly in cells lining the periosteum and the cortical endosteum near the fracture site at 2 days after fracture. At 5, 10, and 21 days after fracture, noggin mRNA was detected in the chondrocytes and osteoblasts in the newly formed callus. The pattern of localization was indistinguishable from that of BMP-4. These results suggest that the noggin/BMP-4 balance could be an important factor in the regulation of callus formation during fracture healing.     

Indomethacin-induced delayed fracture healing

A patient with an ankle joint fracture-dislocation was preoperatively treated with indomethacin. As in previous animal experiments, indomethacin was found to have an inhibitory effect on bone formation. It is suggested that indomethacin should be used with caution if periosteal fracture healing is anticipated.     

Immunolocalization and expression of bone morphogenetic proteins 2 and 4 in fracture healing

Recently, it has become increasingly evident that fracture healing involves a complex interaction of many local and systemic regulatory factors. The roles of some of these growth factors have been described; however, little is understood about the presence of the bone morphogenetic proteins in fracture repair, despite the fact that they are the most potent osteoinductive proteins known. This study defines and characterizes the physiologic presence, localization, and chronology of the bone morphogenetic proteins in fracture healing with an established rat fracture healing model. With use of a recently developed monoclonal antibody against bone morphogenetic proteins 2 and 4 developed with standard avidin-biotin complex/immunoperoxidase protocols, frozen undecalcified fracture calluses were analyzed semiquantitatively for the percentage of various types of fracture cells staining positively. During the early stages of fracture healing, only a minimum number of primitive cells stained positively in the fracture callus. As the process of endochondral ossification proceeded, the presence of bone morphogenetic proteins 2 and 4 increased dramatically, especially in the primitive mesenchymal and chondrocytic cells. While the cartilaginous component of the callus matured with a concomitant decrease in the number of primitive cells, there was a concomitant decrease in both the intensity and the number of positively staining cells. As osteoblasts started to lay down woven bone on the chondroid matrix, these osteoblastic cells exhibited strong positive staining. The intensity of this staining decreased, however, as lamellar bone replaced the primitive woven bone. A similar observation was noted for the areas of the callus undergoing intramembranous ossification. Initially, within several days after the fracture, periosteal cells and osteoblasts exhibited intense staining for bone morphogenetic proteins 2 and 4. As the woven bone was replaced with mature lamellar bone, this staining decreased. These data, and the awareness of the strong osteoinductive capacities of bone morphogenetic protein, suggest that bone morphogenetic proteins 2 and 4 are important regulators of cell differentiation during fracture repair.     

Bovine bone implant with bovine bone morphogenetic protein in healing a canine ulnar defect

Xenograft is considered an alternative material for bone transplantation, but its bone healing capacity is inferior compared to that of autografts and allografts. Here, we tested whether bone morphogenetic protein (BMP) addition enhances the suitability of demineralized xenogeneic bovine bone for bone grafting in dogs, and whether xenogeneic bone is a suitable carrier material for BMPs. The capacity of demineralized bovine bone implants, with and without native partially purified bovine BMP, to heal a 2-cm ulnar defect was determined in six dogs over a follow-up time of 20 weeks. No instances of bone union were seen, but there was slightly more bone formation in the xenografts with BMP, though the difference was not statistically significant. The ulnas treated with an implant with BMP were also mechanically stronger, but the difference was not significant. Computed tomography scans showed no differences in the implant area in bone density, bone mineral content, or bone cross-sectional area. It is concluded that native, partially purified BMP does not sufficiently improve the suitability of bovine demineralized xenografts as a bone substitute material for dog. Demineralized xenogeneic bone does not seem to be a feasible carrier material for BMP.     

Recombinant human bone morphogenetic protein‐7 enhances fracture healing in an ischemic environment

Ischemia predisposes orthopedic trauma patients to delayed fracture healing or nonunion. The goal of this study was to test the ability of bone morphogenetic protein 7 (BMP7) to stimulate fracture repair in an ischemic environment. Ischemic fractures were generated in male adult mice by resecting the femoral artery prior to the creation of a nonstabilized tibia fracture. Recombinant human BMP7 (rhBMP7, 50 µg) was injected into the fracture site immediately after surgery. At 7 days after injury, more tissue vascularization was observed in rhBMP7 treated fractures. Histomorphometric analyses revealed that rhBMP7 induced more cartilage at day 7, more callus and bone at days 14 and 28, and more adipose tissue and fibrous tissue at days 7, 14, and 28 compared to controls (n = 5/group/time). At day 28, all fractures treated with rhBMP7 (50 µg, n = 5) healed, whereas only three of five control fractures exhibited slight bony bridging. In addition, we found that rhBMP7 (both 10 and 50 µg) significantly increased the amount of cartilage compared to controls in stabilized fractures, confirming its chondrogenic effect. Lastly, using bone marrow transplantation, we determined that no donor‐derived osteocytes or chondrocytes were present in rhBMP7‐treated fractures, suggesting rhBMP7 did not recruit mesenchymal stem cells from the bone marrow to the fracture site. In conclusion, our results indicate that rhBMP7 is a promising treatment for fractures with severely disrupted blood supply. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:687–696, 2010     

Treatment of chronic nonunion of a sternal fracture with bone morphogenetic protein

Chronic nonunion of sternal fractures, up until now, has been treated by osteosynthetic plating with or without autologous bone grafting. A new technique is described involving the use of bone morphogenic protein to stimulate bone growth at the fracture site, thereby eliminating the need for bone grafting.     

Effects of delayed stabilization on fracture healing

Previous studies have revealed that delayed internal fixation can stimulate fracture callus formation and decrease the rate of nonunion. However, the effect of delayed stabilization on stem cell differentiation is unknown. To address this, we created fractures in mouse tibiae and applied external fixation immediately, at 24, 48, 72, or 96 h after injury. Fracture healing was analyzed at 10 days by histological methods for callus, bone, and cartilage formation, and the mechanical properties of the calluses were assessed at 14 days postinjury by tension testing. The results demonstrate that delaying stabilization for 24–96 h does not significantly affect the volume of the callus tissue (TV) and the new bone (BV) that formed by 10 days, or the mechanical properties of the calluses at 14 days, compared to immediate stabilization. However, delaying stabilization for 24–96 h induces 10–40× more cartilage in the fracture calluses compared with fractures stabilized immediately. These findings suggest that delaying stabilization during the early phase of fracture healing may not significantly stimulate bone repair, but may alter the mode of bone repair by directing formation of more cartilage. Fractures that are not rigidly stabilized form a significantly larger amount of callus tissue and cartilage by 10 days postinjury than fractures stabilized at 24–96 h, indicating that mechanical instability influences chondrocytes beyond the first 96 h of fracture healing. © 2007 Orthopaedic Research Society. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 25:1552–1558, 2007     

外源性TGF-β和BMP对兔尺骨骨折愈合作用的研究

目的：探讨外源性TGF—β和BMP对骨折愈合的作用。方法：用兔尺骨骨折模型，在骨折局部单独或联合应用TGF-β和BMP，通过X线片观察、生物力学、骨几何参数和骨痂钙含量测定对骨折愈合进行评估。结果：各骨生长因子治疗组的骨痂量、骨痂钙含量、骨愈合情况和骨折愈合后的力学强度明显优于对照组，TGF—β和BMP联合应用优于单用一种生长因子，单用TGF—β好于BMP。结论：在兔骨折周围局部应用外源性TGF-β和BMP，可促进骨折愈合，单用TGF—β的作用强于BMP，它们联合应用时，这种作用进一步增强，在促进骨折愈合方面具有协同作用。     

The use of bone morphogenetic protein 7 in fracture non‐unions

Objective: To assess the rate of clinical and radiological union with the use of bone morphogenetic protein 7 (BMP‐7) in a range of fractures. Methods: This case series retrospectively reviews a series of 16 fracture non‐unions in 13 patients. These patients were treated with the commercially available BMP‐7. Time to radiological and clinical union was assessed by serial out‐patient follow‐up. Results: At nine months post‐surgery in which BMP‐7 was added, 12 of 16 non‐unions had achieved clinical and radiographic union. Three patients required repeat grafting. The mean time to union was 5.1 ± 1.6 months after the application of BMP‐7. Conclusion: The use of the osteo‐inductive agent, BMP‐7 results in good clinical and radiological outcomes which are not restricted to tibial non‐unions.     

Osteoblast and osteocyte‐specific loss of Connexin43 results in delayed bone formation and healing during murine fracture healing

Connexin43 (Cx43) plays an important role in osteoblastic differentiation in vitro, and bone formation in vivo. Mice with osteoblast/osteocyte‐specific loss of Cx43 display decreased gap junctional intercellular communication (GJIC), bone density, and cortical thickness. To determine the role of Cx43 in fracture healing, a closed femur fracture was induced in Osteocalcin‐Cre+; Cx43^flox/flox (Cx43cKO) and Cre‐; Cx43^flox/flox (WT) mice. We tested the hypothesis that loss of Cx43 results in decreased bone formation and impaired healing following fracture. Here, we show that osteoblast and osteocyte‐specific deletion of Cx43 results in decreased bone formation, bone remodeling, and mechanical properties during fracture healing. Cx43cKO mice display decreased bone volume, total volume, and fewer TRAP+ osteoclasts. Furthermore, loss of Cx43 in mature osteoblasts and osteocytes results in a significant decrease in torsional rigidity between 21 and 35 days post‐fracture, compared to WT mice. These studies identify a novel role for the gap junction protein Cx43 during fracture healing, suggesting that loss of Cx43 can result in both decreased bone formation and bone resorption. Therefore, enhancing Cx43 expression or GJIC may provide a novel means to enhance bone formation during fracture healing. © 2012 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 31:147–154, 2012