骨形态发生蛋白4（BMP4）基因表达在骨折愈合过程中的定位研究

创伤后，骨组织的再生修复十分完全，其修复常无瘢痕残留。骨组织之所以有如此完美的愈合能力，是因为骨骼组织内存在有成骨因子，即骨生长因子。骨形态发生蛋白（ｂｏｎｅｍｏｒｐｈｏｇｅｎｅｔｉｃｐｒｏｔｅｉｎ，ＢＭＰ）因其直接诱导软组织成骨而受重视。本文应用ＢＭＰ４ｃＤＮＡ探针检测骨折愈合过程中外骨痂内ＢＭＰ４基因表达的定位与分布，探讨ＢＭＰ４基因表达在闭合性骨折愈合外骨痂形成中的作用。用６４只健康ＳＤ大鼠制备闭合性胫骨骨折动物模型。分别于骨折后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ＢＭＰ４ｃＤＮＡ探针进行原位核酸分子杂交。大鼠骨折后１２小时～３天，骨折周围血肿内细胞及肌肉中新出现的间充质细胞内ＢＭＰ４ｍＲＮＡ表达检测为阳性信号。表明创伤激活ＢＭＰ４ｍＲＮＡ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。     

复合骨形态发生蛋白胎儿骨修复长骨缺损的实验研究

寻找满意的骨形态发生蛋白（ＢＭＰ）的载体一直是应用ＢＭＰ修复骨缺损研究中的一个重要课题，为探讨胎儿骨（ＦＢ）能否成为ＢＭＰ载体，制备了复合ＢＭＰ胎儿骨（ＢＭＰ／ＦＢ），将其植入兔桡骨中段１５ｍｍ人工缺损处，以单纯ＦＢ植入、异体骨（ＡＬＢ）植入、空白作为对照，通过Ｘ线摄片，光镜观察，电子探针钙磷元素测定等方法了解各组骨缺损修复速度和愈合程度。结果发现ＢＭＰ／ＦＢ植入侧４周时骨缺损处有大量间充质细胞聚集，８周形成骨小梁，ＦＢ基本被吸收，１２周出现部分板层骨，钙磷值达到正常桡骨皮质骨水平，１６周骨细胞成熟，髓腔再通，外观塑形较好，在不同时间点骨缺损修复程度，ＢＭＰ／ＦＢ组明显优于ＦＢ组，ＦＢ组优于ＡＬＢ组，空白组骨缺损则被纤维组织，脂肪组织等填充。实验证明ＦＢ是一种良好的ＢＭＰ载体。     

带旋髂深血管蒂髂骨瓣和BMP人工骨移植治疗股骨头缺血性坏死

目的探讨带旋髂深血管蒂髂骨瓣和骨形态发生蛋白(bone morphogenetic prote in,BMP)人工骨移植治疗股骨头缺血性坏死的方法及临床疗效。方法2000年1月~2006年8月采用带旋髂深血管蒂髂骨瓣联合BMP人工骨移植治疗股骨头缺血性坏死25例(29髋),左髋18例,右髋11例。按F icat分期:Ⅰ期8例(10髋),Ⅱ期14例(16髋),Ⅲ期3例(3髋)。结果术后平均随访37.6(24~60)个月。移植骨瓣与股骨颈均愈合,平均愈合时间3.6(3~8)个月。按Harris评分,优15例16髋,良7例10髋,可3例3髋,优良率89.7%。结论带旋髂深血管蒂髂骨瓣和BMP人工骨移植能有效重建股骨头的血运、清除死骨、降低髓腔压力、支撑股骨头关节软骨面和BMP能诱导成骨的特点,能有效治疗股骨头缺血性坏死。     

成年狗股骨微波灭活骨缺损模型的建立及其修复

目的：建立成年狗股骨经微波灭活造成骨缺损的实验动物模型，并用复合骨形成蛋白材料修复。方法：用自制的骨肿瘤微波治疗仪，以1．5kHz，70W功能加热至50℃-55℃，维持20min，造成狗股骨中段骨缺损，术后不同时段观察愈合情况，制备复合骨形成蛋白材料，经评价后植入骨缺损处修复并观察结果。结果：在保持狗股骨连续性的前提下，长2.5cm，宽1.0cm的骨缺损9个月的无愈合倾向。复合骨形成蛋白材料具有成骨诱导活性和生物相容性，有粘合性和可塑型性，具备有利于新生血管长入的空间，可与宿主骨“生物铆定”。结论：成年狗股骨微波赞赏成的骨缺损实验模型，长2.5cm，宽1.0cm9个月不能自愈，适宜于各种骨修复材料填充。复合骨形成蛋白材料修复微波灭活骨缺损效果良好，可作修复骨缺损的骨替代材料。     

带血供骨瓣移位治疗青壮年股骨颈骨折

目的:探讨青壮年股骨颈骨折的治疗方法,以减少骨折不愈合和股骨头坏死等并发症。方法:应用带血供骨瓣转移加压螺纹钉内固定治疗青壮年股骨颈骨折36例,其中带肌蒂骨瓣22例,带血管蒂骨瓣14例。结果:28例得到2～8年随访,按照中华医学会骨坏死学推荐的疗效评价标准。优21例,良5例,差2例,优良率为92.86％、结论:带血供 骨瓣移位治疗青壮年股骨颈骨折可促进骨折愈合、降低股骨头坏死妁发生率     

Differential Bone Turnover in an Angulated Fracture Model in the Rat

We have developed a simple rat model of angulated tibial fracture which elicits substantial differences in bone formation and resorption within the same bone. In 35 rats the right mid-tibia was manually fractured and fixed with an intramedullary 17-gauge cannula needle. Twenty tibias were fixed in anterior angulation (27 ± 5°) and 15 in posterior angulation (31 ± 5°). Serial X-rays were taken over a 12-week period. All fractures healed completely within five weeks. In both groups, bone thickness was already significantly greater on the concave side than on the convex side at week 3 and remained so until the end of the experiment. The thickness on the convex side decreased dramatically within 3 to 5 weeks and gradually thereafter. For morphological analysis of bone mineralization, 3 rats from each group were given calcein and alizarin red injected at different time points up to 14 weeks. Maximum new bone formation was noted within the first 3 weeks. Over the ensuing weeks, new bone formation remained intense on the concave side, but it was virtually absent on the convex side. These results show that angulated fracture deformity reproducibly exhibits differential bone turnover, which can be exploited in research on local regulatory factors. To exemplify the utility of the model, an immunohistochemical study on two local markers was done. Callus tissue of five rats in the anterior angulation group at week 3 post-fracture was stained for the cytokine IL-1, a stimulator of bone resorption, and the neuropeptide CGRP, an inhibitor of resorption, showing clear differences in positive staining between the concave and convex sides. Our in-vivo model offers a means of analyzing morphologically and quantitatively the differential expression and action of factors involved in local bone turnover.     

Distinct and Overlapping Patterns of Localization of Bone Morphogenetic Protein (BMP) Family Members and a BMP Type II Receptor During Fracture Healing in Rats

Bone morphogenetic proteins (BMPs) and their receptors (BMPRs) are thought to play an important role in bone morphogenesis. The purpose of this study was to determine the locations of BMP-2/-4, osteogenic protein-1 (OP-1, also termed BMP-7), and BMP type II receptor (BMPR-II) during rat fracture healing by immunostaining, and thereby elucidate the possible roles of the BMPs and BMPR-II in intramembranous ossification and endochondral ossification. In the early stage of fracture repair, the expression of BMP-2/-4 and OP-1 was strongly induced in the thickened periosteum near the fracture ends, and coincided with an enhanced expression of BMPR-II. On day 7 after fracture, staining for BMP-2/-4 and OP-1 immunostaining was increased in various types of chondrocytes, and was strong in fibroblast-like spindle cells and proliferating chondrocytes in endochondral bone. On day 14 after fracture, staining with OP-1 antibody disappeared in proliferating and mature chondrocytes, while BMP-2/-4 staining continued in various types of chondrocytes until the late stage. In the newly formed trabecular bone, BMP-2/-4 and OP-1 were present at various levels. BMPR-II was actively expressed in both intramembranous ossification and endochondral ossification. Additionally, immunostaining for BMP-2/-4 and OP-1 was observed in multinucleated osteoclast-like cells on the newly formed trabecular bone, along with BMPR-II. In reference to our previous study of BMP type I receptors (BMPR-IA and BMPR-IB), BMPR-II was found to be co-localized with BMPR-IA and BMPR-IB. BMP-2/-4 and OP-1 antibodies exhibited distinct and overlapping immunostaining patterns during fracture repair. OP-1 may act predominantly in the initial phase of endochondral ossification, while BMP-2/-4 acts throughout this process. Thus, these findings suggested that BMPs acting through their BMP receptors may play major roles in modulating the sequential events leading to bone formation.     

四种可溶性载体对骨形态发生蛋白在小鼠体内诱导成骨活性的影响

以四种可溶性物质作为牛骨形态发生蛋白（ｂＢＭＰ）的载体，通过实验观察哪种物质是ｂＢＭＰ的有效缓释载体。将聚乙烯吡咯烷酮（ＰＶＰ）、葡聚糖、羟乙基淀粉（ＨＥＳ）、甘露醇分别与等量ｂＢＭＰ复合后，注射入小鼠股部肌肉，观察组织学成骨及测定标本内钙含量。结果，ｂＢＭＰ／ＰＶＰ有良好成骨，吸收较快，未见炎症及排斥反应，其混悬液较稳定，具有良好的适针性及可注射性，ｂＢＭＰ／甘露醇只有很低成骨率。其余各组未见成骨。ＰＶＰ对ｂＢＭＰ具有满意助溶、助悬及缓释载体作用，对ｂＢＭＰ活性无损害，生物相容性好。     

股方肌蒂骨瓣移植及内固定术治疗陈旧性股骨颈骨折

作者报告股方肌蒂骨瓣移植及内固定术，治疗陈旧性股骨颈骨折１８例，获得３年以上随访者１５例，其骨折愈合率９３．３％，优良率７３．３％。股方肌蒂骨瓣是有肌蒂与血管蒂双重供血的肌蒂骨瓣，具有血循环丰富、操作方便，易于推广等优点。     

Characterization of a Femoral Segmental Nonunion Model in Laboratory Rats: Report of a Novel Surgical Technique

The literature is lacking conclusive results regarding the exact mechanism of maximizing the fracture healing stages with minimal traumatic side effects. This observation mandates the development of a novel surgical procedure using small animals as a model to study fracture healing in the presence of osteoinductive agents. Previously, stabilization of osteotomies in small animal models has mainly been accomplished using Kirschner wires, but the rat's tremendous ability to heal an osteotomy stabilized by this method has masked the effects of osteoinductive agents. Thus, this study proposes using a modified 20-hole, 1.5-mm stainless-steel plate to stabilize a 5-mm segmental defect. Thirty of 32 adult male rats were fully weight-bearing within 2 days and were followed over a 15-week period. Two animals showed evidence of fixation failure due to technical error, and the animals were humanely sacrificed. At the end of the study, the fractures were stable with significantly less bone formation evident when compared to controls (p <. 001). Therefore, this technique can effectively be used to evaluate compounds that will enhance bone formation and allows for stable fixation of the control with minimal callus formation or bony ingrowth. The goal of this article is to allow other investigators to reproduce this technique as well as outline the advantages and disadvantages of this novel plating technique versus the former Kirschner wire technique for the study of osteoinductive agents using small animals as a model.     

两种内固定后兔骨痂中成骨细胞内骨形态生成蛋白含量的比较

目的：比较钢板和矩形髓内钉固定对兔胫腓骨骨折成骨细胞内骨形态生成蛋白（BMP）含量的影响。方法：新西兰兔24只，对同一动物的两侧胫骨分别用矩形髓内钉和4孔不锈钢钢板固定，并分3批于固定后14、21、28d处死。用兔疫组化方法检测骨痂中成骨细胞中BMP的分布，用计算机图像分析系统将结果进行定量分析。结果：矩形髓内钉组骨痂中成骨细胞内BMP含量高于钢板组（P＜0．05）。结论：矩形髓内钉内固定胫骨后骨痂含量增加可能与矩形髓内钉的微动有关，可能有利于骨折愈合。     

Systemic Injection of CK2.3, a Novel Peptide Acting Downstream of Bone Morphogenetic Protein Receptor BMPRIa,Leads to Increased Trabecular Bone Mass

Bone Morphogenetic Protein 2 (BMP2) regulates bone integrity by driving both osteogenesis and osteoclastogenesis. However, BMP2 as a therapeutic has significant drawbacks. We have designed a novel peptide CK2.3 that blocks the interaction of Casein Kinase 2 (CK2) with Bone Morphogenetic Protein Receptor type Ia (BMPRIa), thereby activating BMP signaling pathways in the absence of ligand. Here, we show that CK2.3 induced mineralization in primary osteoblast cultures isolated from calvaria and bone marrow stromal cells (BMSCs) of 8 week old mice. Further, systemic tail vein injections of CK2.3 in 8 week old mice resulted in increased bone mineral density (BMD) and mineral apposition rate (MAR). In situ immunohistochemistry of the femur found that CK2.3 injection induced phosphorylation of extracellular signal‐related kinase (ERK), but not Smad in osteocytes and osteoblasts, suggesting that CK2.3 signaling occurred through Smad independent pathway. Finally mice injected with CK2.3 exhibited decreased osteoclast differentiation and osteoclast activity. These data indicate that the novel mimetic peptide CK2.3 activated BMPRIa downstream signaling to enhance bone formation without the increase in osteoclast activity that accompanies BMP 2 stimulation.© 2014 Orthopaedic Research Society. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:208–215, 2015.

     

Comparison of Two Different Fixation Techniques for a Segmental Defect in a Rat Femur Model

Studies have attempted to identify the osteogenic effects of bone morphogenetic proteins using a rat femur model, which commonly involves the creation of a critical size defect followed by internal fixation of the femur. Among the most familiar fixation methods are either plating or intramedullary placement of a Kirschner wire (K-wire). There are advantages and disadvantages to each method; however, this study attempts to identify the best method by exploring the histological effects of each technique. The experiment involved two groups with no added treatment: Group P (plate fixation method) and Group K (K-wire fixation method). The animals were allowed a 4-week interval for the femurs to heal, and proximal, distal, and two midshaft cuts were examined under high-power microscopy after the fixation apparatus was removed. Group K exhibited a peculiar fibrotic healing pattern that followed the shaft of the then vacated K-wire and there was minimal new viable bone formation. Group P, however, exhibited a more natural ingrowth of newly formed bone that began at the proximal and distal cuts and proceeded centrally into the core of the defect. Due to the fibrotic tissue in Group K, this study shows that the model is insufficient due to the micromotion created and thus supports plating of critical defects as the fixation method of choice due to the creation of a stable healing environment.     

DHS结合空心螺钉植骨内固定治疗青壮年股骨颈骨折

目的探讨DHS结合空心钉有限切开植骨内固定治疗青壮年股骨颈骨折的临床疗效及意义。方法回顾性分析我院2007年7月~2010年9月收治的29例青壮年股骨颈骨折患者的临床资料,均采用DHS结合空心钉行有限切开植骨内固定。结果本组29例患者均获随访,平均时间32.5(24~65)个月。无螺钉松动、退钉或断裂发生,1例发生延迟愈合,2例发生股骨头缺血性坏死,其余26例患者骨折均愈合,无肢体短缩、髋内翻畸形等其他并发症,骨折愈合时间平均4.5(3~17)个月。按Harris髋关节功能评分标准评定疗效,优22例(75.9%),良5例(17.2%),差2例(6.9%),优良率为93.1%。结论牵引床闭合复位DHS结合空心钉有限切开植骨内固定治疗青壮年股骨颈骨折,疗效肯定,解剖复位、坚强固定是获得满意疗效的关键,且可减少并发症的发生。     

Effects of OP-1 and PTH in a new experimental model for the study of metaphyseal bone healing.

The purpose of this study was to establish a reliable model of metaphyseal bone healing and to use this model to investigate the effect of recombinant human osteogenic protein 1 (rhOP-1; BMP-7) and parathyroid hormone fragment (PTH 1-34) on healing. A wedge-shaped osteotomy was created in the distal tibia of 16-week-old female New Zealand White rabbits (n = 20) and was bridged with a custom-made external fixator. Five experimental groups of four animals each were investigated. In groups 1-4 the osteotomy gap was filled with tricalcium phosphate (TCP), and the gap was left unfilled in group 5 ("normal healing"). In group 1, 200 microg OP-1 was mixed in with the TCP. Groups 2 and 3 received daily subcutaneous injections of 10 and 40 microg/kg PTH, respectively, beginning on postoperative day 1. Radiographs were taken weekly. Following sacrifice on postoperative day 28, peripheral quantitative computed tomography (pQCT), histology, and mechanical testing (axial compression and torsion) were performed. Only one animal failed to complete the full 4-week time course, and no infections were encountered. Bone healing occurred in all animals. OP-1 stimulated bone formation locally, while the lower dose of PTH enhanced bone formation systemically (p < 0.05). Tibiae treated with OP-1 exhibited higher torsional strength (p = 0.04) than those in the normal healing group. These results indicate that a reliable and reproducible surgical model of metaphyseal healing has been established. In addition, differences in systemic versus local effects of PTH and OP-1 in accelerating metaphyseal fracture healing were found.     

骨髓间充质干细胞复合骨形态发生蛋白和纤维蛋白修复节段性骨缺损

目的　用自体骨髓间充质干细胞 (MSCs)、骨形态发生蛋白 (BMP)和纤维蛋白的复合物修复骨缺损。方法　抽取兔自体骨髓并分离和大量培养MSCs。在 12只MSCs供体兔的两侧桡骨中段造成 1 5cm缺损 ,一侧植入自体MSCs、 15mgBMP和纤维蛋白的复合物 (B +M +F) ,另一侧植入自体MSCs与纤维蛋白的复合物 (M +F)。另一组等数量实验兔造成相同缺损后 ,一侧植入 15mgBMP与纤维蛋白的复合物 (B +M ) ,另一侧留作空白对照。术后第 2 ,4 ,8周做放射学、组织学和ECT检查。结果　B +M +F组在术后 2周即产生填满缺损区的骨痂影 ,8周时骨缺损得到良好修复。B +M组所产生骨痂量和修复效果均不如B +M +F组 ,但优于M +F组。结论　用自体骨髓间充质干细胞复合骨形态发生蛋白和纤维蛋白修复骨缺损是一种切实可行和有效的方法。     

Augmentation of fracture healing by hydroxyapatite/collagen paste and bone morphogenetic protein‐2 evaluated using a rat femur osteotomy model

In fracture treatment, biological bone union generally depends on the bone's natural fracture healing capacity, even in surgically treated cases. Hydroxyapatite/collagen composite (HAp/Col) has high osteoconductivity and stimulates osteogenic progenitors. Furthermore, it has the potent capacity to adsorb bone morphogenetic proteins (BMPs). In this study, we prepared an injectable HAp/Col paste and evaluated its augmentation of bone union. Furthermore, the effect of HAp/Col paste combined with BMP‐2 was also evaluated. We used a rat femur osteotomy model with a defect size of 1 mm. Male Wistar rats were assigned to one of the following four groups; a control group without any implant, a HAp/Col implant group, a group that received an absorbable collagen sponge (ACS) implant impregnated with BMP‐2 (1 μg), and a group that received a HAp/Col implant impregnated with BMP‐2 implant. Micro‐CT analysis, three‐point bending tests, and histological evaluation were performed. Bone union was achieved in two of eight cases in the HAp/Col group, five of eight cases in the ACS + BMP‐2 group, and all cases in the HAp/Col + BMP‐2 group at 8 weeks post‐surgery. The control group did not achieve bone union. In addition, in the HAp/Col + BMP‐2 group, the biomechanical strength of the fused femurs was comparable to that of the contralateral intact femur; the ratio of the mechanical load at the breaking point of the osteotomy side relative to that of the contralateral side was 1.00 ± 0.151 (SD). These results indicate that HAp/Col paste with or without BMP‐2 augments bone union. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:129–137, 2018.     

Mechanical Properties of Bone in a Paraplegic Rat Model

Abstract

Pathologic fractures may occur with minimal trauma after spinal cord injury (SCI) because of osteoporosis. Rats were evaluated to determine whether they could be used as an SCI animal model. Male Sprague-Dawley rats underwent spinal cord transection at the ninth thoracic vertebrae. Control rats underwent a sham procedure. Mechanical testing of the humeral shaft, femoral shaft, tibial shaft, femoral neck, distal femur, and proximal tibia was performed separately at 0, 8, and 24 weeks after surgery. At 24 weeks, significant differences between SCI and control rats were found in maximum torque needed to produce failure in the femoral shaft (63 percent of control, p < 0.05) and tibial shaft (63 percent, p < 0.01), and in compressive load to produce failure in cross-sectional specimens of the distal femur (51 percent, p < 0.05) and proximal tibia (50 percent, p < 0.01 ). No differences were found in the maximum torque needed to produce failure of the humeral shaft (106 percent, p = 0.77) between SCI and control rats. Reductions in relative bone strength in SCI rats at 24 weeks were similar in magnitude to bone mineral density changes reported in humans with chronic paraplegia. Thus, Sprague-Dawley rats appear to be good animal models in which to evaluate changes in bone strength following SCI. U Spinal Cord Med 1998; 21:302-308)     

Effects of a Single Percutaneous Injection of Basic Fibroblast Growth Factor on the Healing of a Closed Femoral Shaft Fracture in the Rat

Recently, bioactive agents to stimulate bone formation have been available in the orthopedic field. We have shown previously that a single, local injection of basic fibroblast growth factor (bFGF) contributes to the formation of a larger cartilage (soft callus) but does not promote replacement of the cartilage by osseous tissue during experimental closed femoral fracture healing. Aiming at a clinical application, the present study was undertaken to clarify the effects of locally injected bFGF on bone (hard callus) formation and the mechanical properties of the callus in closed fracture healing in rats. Immediately after fracture, a carrier (200 μL of fibrin gel) containing 100 μg of bFGF or carrier alone was applied to the fracture site. At days 42 and 56 postfracture, the bone union rate, bone mineral density (BMD), and mechanical properties (strength and stiffness) of the callus were evaluated. Unexpectedly, with the exception of reduced stiffness in the FGF-injected callus at day 56, none of these parameters showed a significant difference between the control and the FGF-injected groups. Furthermore, the temporal expression pattern of OPN mRNA during healing was very similar between groups. We conclude that, in the healing of closed fractures of long bones, administration of bFGF forms a larger callus but does not necessarily accelerate the healing process.     

三种假体置换治疗老年股骨颈骨折疗效观察

目的　比较不同种类假体置换对老年人股骨颈骨折的治疗效果。方法　于 1987年 1月至 1998年 1月 ,对 2 0 9例 6 0岁以上老年人股骨颈骨折 ,采用单极股骨头、双极股骨头及全髋关节置换术。结果　 2 0 9例术后 1～ 3周均恢复行走活动 ,无 1例死亡。 187例获 1 5～ 8年随访 ,疗效按 1980年第一届全国骨科学术会议拟定的髋关节置换效果评定标准 ,满意率 (6～ 4级 ) :单极股骨头置换 90 6 9% ,双极股骨头置换 96 77% ,全髋置换 95 0 0 %。 187例X线片检查 ,髋臼完好 110例 ,髋臼外缘增生 75例 ,髋臼磨损人工股骨头中心性脱位 2例 ,假体柄下沉 >2mm 3例 ,假体透光区域 >2mm 10例。结论　人工股骨头或全髋关节置换术是治疗老年人股骨颈骨折的有效方法 ,可促使老年人早期活动 ,提高生活质量。本组资料表明三种类型假体置换无明显差异。对高龄及活动量不大不强求全髋关节置换 ,人工股骨头尤其是双极关置换也可望达到良好疗效