骨形成蛋白介导的基因治疗研究进展

基因治疗是指通过导入基因的功能片段改善机体生理状况或者治疗疾病.利用基因治疗可在骨缺损或骨折局部释放骨形成蛋白（bone morphogenetic proteins,BMP）,并且无需异体载体,方法包括体内法和离体法.BMP基因治疗可以促进骨和软骨形成、脊柱融合、颌面骨和牙齿修复、肌腱韧带形成,此外对椎间盘退变性疾病也可采用BMP基因治疗.总之BMP基因治疗方法经济有效,是有前景的治疗手段.     

不同植骨材料及骨质疏松对腰后路椎间植骨成功率的影响

背景：对于骨质疏松性腰椎疾病患者,在实施后路腰椎间融合治疗时受骨密度等因素的影响会降低植骨融合率,但关于不同植骨材料及骨质疏松对后路腰椎间融合的影响,目前尚无全面的报道。目的：分析不同植骨材料及骨质疏松对腰后路椎间植骨成功率的影响。 方法：回顾性分析227例行腰椎后路椎间融合治疗患者的临床资料,按照植骨材料的不同分为自体髂骨组(n=121)和融合器联合自体髂骨组(n=65)、同种异体骨组(n=41),3组中骨质疏松患者分别有20,22,6例,治疗后随访24个月,对比3组植骨融合情况、融合时间、椎间高度变化及内固定失败等不良事件的发生情况。结果与结论：同种异体骨组内固定失败率、植骨融合时间、椎间高度丢失高于自体髂骨组和融合器联合自体髂骨组(P < 0.05),植骨融合率低于自体髂骨组和融合器联合自体髂骨组(P < 0.05),自体髂骨组和融合器联合自体髂骨组各指标比较差异无显著性意义。在骨质疏松患者中,同种异体骨组内固定失败率、植骨融合时间高于自体髂骨组和融合器联合自体松质骨组(P < 0.05),椎间高度丢失及植骨融合率低于自体髂骨组和融合器联合自体髂骨组(P < 0.05);自体髂骨组融合时间短于融合器联合自体髂骨组(P < 0.05),融合率高于融合器联合自体髂骨组(P < 0.05)。表明在后路腰椎椎间融合过程中,使用自体髂骨或融合器联合自体髂骨均可以获得较高的植骨融合率,内固定失败情况较少;对于合并骨质疏松患者,予以自体髂骨块植骨可以获得更好的融合效果。中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

经椎弓根入路不同植骨材料内固定治疗胸椎结核

背景：对于脊柱结核病灶清除后的骨质缺损,可以选择自体髂骨、自体肋骨、钛网加同种异体骨等多种植骨材料。 目的：对比椎弓根入路不同植骨材料内固定治疗胸椎结核的效果。 方法：纳入40例胸椎结核患者,其中18例伴有截瘫,15例存在后凸畸形,经正规抗结核治疗2-4周行后路椎弓根系统内固定,一期经椎弓根入路病灶清除植骨融合内固定,根据植骨材料的不同分为自体髂骨组、自体肋骨组、钛网加同种异体骨组。治疗后随访24个月,观察病灶愈合、植骨融合、截瘫恢复、后凸畸形矫正及不良反应发生率。 结果与结论：自体髂骨组植骨融合时间短于自体肋骨组、钛网加同种异体骨组(P < 0.05),后两组间植骨融合时间比较差异无显著性意义。3组均未发生植骨块及钛网脱落、断裂与移位,无骨不连及假关节等现象,无结核病灶复发。截瘫与脊柱后凸畸形患者经3-6个月的对症治疗,肌力基本恢复,脊柱后凸畸形基本矫正。表明自体髂骨治疗效果最好,自体肋骨、钛网加同种异体骨治疗效果相当。 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

重组人骨形态发生蛋白2复合骨在腰椎融合中的效果

背景：诸多研究已证实重组人骨形态发生蛋白2在骨形成及骨折愈合中发挥十分重要的作用,但单纯予以重组人骨形态发生蛋白2植入容易出现扩散和降解,无法对新骨形成予以持续性的影响。 目的：观察重组人骨形态发生蛋白2复合骨在兔腰椎中的融合效果。 方法：取30只新西兰大白兔,构建后路腰椎横突间植骨融合模型,随机均分为3组,分别在L5-6横突间植入自体髂骨、同种异体骨及重组人骨形态发生蛋白2复合骨(重组人骨形态发生蛋白2与同种异体骨复合物),植入后6周,进行大体观察、X射线检查及组织学观察。 结果与结论：重组人骨形态发生蛋白2复合骨组融合率、新生骨组织在总面积中所占百分比高于自体髂骨组、同种异体骨组(P < 0.05);重组人骨形态发生蛋白2复合骨组、自体髂骨组拉伸强度大于同种异体骨组(P < 0.05),前两组拉伸强度无差异。X射线显示3组植骨区均可见骨痂形成;组织学显示,自体髂骨组移植物已消失,形成大量软骨,有少量骨小梁,并有一定编织骨形成;同种异体骨组有较多的纤维组织包裹材料,有骨岛形成,有数量较少的骨小梁及软骨形成;重组人骨形态发生蛋白2复合骨组存在大量骨小梁和少量软骨,形成编织骨且有皮质骨形成。表明重组人骨形态发生蛋白2复合骨在兔腰椎中可以获得良好的融合效果。 中国组织工程研究杂志出版内容重点：生物材料;骨生物材料; 口腔生物材料; 纳米材料; 缓释材料; 材料相容性;组织工程     

重组人骨形态发生蛋白2联合自体骨行椎间植骨融合治疗胸腰椎结核

文题释义：重组人骨形态发生蛋白2：是一种利用基因重组技术在体外克隆出来的蛋白,属于骨形态发生蛋白家族中活性最强的成员,是骨形成中重要的调控因子。它能够将体内未分化的间充质干细胞定向诱导分化为骨细胞,形成新生骨和软骨,促进骨组织修复。 椎间植骨融合术：是临床上重建和保持脊柱稳定性的手术方式,它不但能够恢复脊柱的稳定性,避免因脊柱不稳带来的疼痛,而且能维持椎体和椎间孔的高度,降低神经根压迫的风险,能够起保护神经功能和保证远期疗效作用。 背景：重组人骨形态发生蛋白2(recombinant human bone morphogenetic protein-2, rhBMP-2)联合自体骨行椎间植骨融合术治疗腰椎滑脱、椎管狭窄、椎间盘突出等脊柱退行性变疾病的疗效和安全性已经得到认可,但对于治疗脊柱结核等脊柱感染性疾病的疗效和安全性少有临床研究。 目的：评价rhBMP-2联合自体骨行椎间植骨融合术对脊柱结核的临床疗效及其安全性。 方法：回顾性分析2010年11月至2018年5月广州中医药大学第一附属医院收治的胸腰椎结核患者的临床资料,所有患者均采用后路经椎弓根螺钉内固定+植骨融合术,根据植骨时是否使用rhBMP-2将其分为2组,试验组33例采用1 mg rhBMP-2联合自体骨植入大小适宜的支撑体中,对照组35例单纯用自体骨植入支撑体。术后随访1年以上,并对术前、术后疼痛目测类比评分、脊髓损伤ASIA分级、围术期并发症以及融合率进行统计学分析。研究通过广州中医药大学第一附属医院伦理委员会的批准,患者对治疗方案均知情同意。 结果与结论：①所有患者均完成1年以上的随访,随访中未发现内固定断裂移动和椎体的明显塌陷;②两组手术时间、术中出血量、住院时间、围术期并发症比例差异无显著性意义(P > 0.05);③两组术后1周和术后1年的目测类比评分、ASIA分级与术前相比,有显著改善(P < 0.05),但两组之间对比差异无显著性意义(P > 0.05);④在融合率方面,试验组术后6个月的融合率明显高于对照组(P < 0.05),然而,两组术后1年的融合率比较却无统计学差异(P > 0.05)。提示：rhBMP-2联合自体骨行植骨融合术治疗胸腰椎结核能在短期内加快骨性融合,具有良好的疗效和安全性。 ORCID: 0000-0002-3809-4431(翁汭) 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

颈椎同种异体骨笼椎间融合的组织学研究

目的：研究颈椎同种异体骨笼在颈椎融合过程中的组织学变化和融合效果。方法：采用12只绵羊行C3,4 C4,5椎间盘摘除后，分别植入自体髂骨与同种异体骨笼，于术后4、12、24周分别处死动物，行大体标本观察、光镜观察和扫描电镜观察。结果：同种骨笼植入后4周骨笼轮廓完整，周边出现吸收腔，腔壁表面有薄层新骨形成。12周，笼壁与椎体之间界线消失，大部分的骨陷窝中有骨细胞，新骨与死骨交织．成骨活动仍在进行，爬行替代尚未结束。24周时，上、下椎体完全骨性连接，骨笼被新骨代替，完成骨性融合。结论：同种异体骨笼充填自体松质骨植入颈椎间后能获得满意的脊柱融合效果，并可以逐步被新骨取代，是一种理想的椎间植人物。     

骨形态发生蛋白7局部基因治疗和组织工程化骨对大鼠脊柱融合的影响

目的 评价腺病毒介导的人骨形态发生蛋白7 (Ad-hBMP-7)基因转染的骨髓间充质干细胞(BMSCs)和纳米羟基磷灰石/胶原复合材料(NHAC)复合后对脊柱融合的影响. 方法 抽取大鼠骨髓获得BMSCs,应用Ad-hBMP-7转染大鼠BMSCs,将NHAC材料和密度为2×105/ml的Ad-BMP-7转染大鼠的BMSCs细胞孵育2h后备用;Wistar大鼠56只,随机分成4组,建立脊柱后外侧融合模型,分别植入自体髂骨(A组)、NHAC材料(B组)、NHAC材料和BMSCs(C组)以及Ad-hBMP-7-BMSCs与NHAC材料混合物(D组).每组行RT-PCR 及Western blotting检测,8周和12周后分别行X线摄片,并对标本进行手触检测硬度、生物力学检测和组织学观察. 结果 D组10只大鼠的脊柱L4/5节段全部融合;A组12周0例融合;B组12周0例融合;C组12周3例融合.D组同A、B、C组结果 相比均有显著性差异( P <0.001). 结论 BMP-7局部基因治疗和组织工程化骨能促进脊柱融合.     

骨移植替代材料在脊柱外科中的应用

脊柱手术中应用骨移植材料促进骨融合、增强脊柱的稳定性。自体骨移植虽然是脊柱融合的金标准，但自体骨移植数量有限、取骨延长手术时间、增加取骨区的并发症。因此多年来脊柱外科医生和生物骨材料学者一直致力于自体骨移植替代材料的研究，目前脊柱外科应用的自体骨移植的替代物主要分为天然材料和合成材料两种，虽然目前这些替代物没有一种同时具有自体骨移植的三种基本特性(成骨性、骨传导性和骨诱导性)，     

矿化胶原基材料与BMP－2复合用于兔腰椎横突间融合

目的 用兔的脊柱后路横突间融合模型来评价一种新型的仿生矿化胶原基质：nHAC／PLA复合或不复合生长因子rhBMP-2的骨形成能力。方法64只兔子分为4组：自体髂骨，nHAC／PLA，自体骨 nHAC／PLA，nHAC／PLA BMP-2。术后6周和10周观察。结果自体骨 nHAC／PLA和nHAC／PLA BMP-2与自体骨移植的效果相当。结论nHAC／PLA BMP-2可代替自体骨移植用于骨科手术中。     

负载重组人骨形态发生蛋白2的纳米羟基磷灰石/丝素蛋白复合材料重建脊柱稳定性北大核心

背景:随着骨修复支架在医学领域的广泛应用,具有良好生物学性能的纳米羟基磷灰石和丝素蛋白成为近年来的研究热点。目的:探讨负载重组人骨形态发生蛋白2的纳米羟基磷灰石/丝素蛋白复合材料重建兔脊柱节段初始稳定性的可行性。方法:将36只雄性新西兰兔随机分为3组,均制备脊柱失稳模型。自体骨组在L4、L5椎间植入自体髂骨,n HA/SF组将纳米羟基磷灰石/丝素蛋白复合材料植入椎间隙进行椎间融合,rhB MP-2/nH A/SF组将负载重组人骨形态发生蛋白2的纳米羟基磷灰石/丝素蛋白复合材料植入椎间隙进行椎间融合。术后12周,X射线观察手术节段的融合情况,然后处死实验动物进行大体观察。使用电子拉力机测试融合节段的稳定性。组织学观察手术部位的植骨融合情况。结果与结论:(1)术后各组标本融合部位质地均较硬。自体骨组融合迹象明显,rhB MP-2/nH A/SF组次之,n HA/SF组未见融合迹象;(2)自体骨组椎体与髂骨植骨块分界处有大量骨小梁长入,nH A/SF组分界处有少量骨小梁长入,rh BMP-2/nH A/SF组分界处骨小梁较多。经X射线评估分别有10只、3只和9只符合融合标准;(3)rh BMP-2/n HA/SF组脊柱活动度与自体骨组间比较差异无显著性意义,rh BMP-2/nH A/SF组脊柱活动度优于nH A/SF组,差异有显著性意义(P<0.05);(4)自体骨组髂骨周边可见骨性连接,n HA/SF组材料周边可见骨性连接,未见成熟骨组织,rh BMP-2/nH A/SF组周边有大量新生毛细血管,并渗入组织内部;(5)结果表明,负载重组人骨形态发生蛋白2的纳米羟基磷灰石/丝素蛋白复合材料具有良好的生物相容性、力学性能和骨诱导能力,能在较短时间内重建兔脊柱稳定性。     

Evaluation of DBM/AM composite as a graft substitute for posterolateral lumbar fusion

Demineralized bone matrix (DBM) has been investigated as a bone graft substitute for spinal fusion with less morbidity. Various carriers have been added to DBM to enhance its handling characteristics. This study investigates the spinal fusion induced by a composite of DBM and acellular dermal matrix (AM) in comparison with autologous bone in an athymic rat spinal fusion model. Single-level intertransverse process fusions were performed in 60 athymic nude rats grafted with 2 mL/kg of DBM/AM composite, AM alone, or autologous bone. Fusion was assessed at 6 weeks by radiography, manual palpation, and histology. At 6 weeks, 70% of the animals from the DBM/AM composite group exhibited complete spine fusion, whereas 35% from the autologous bone group and 20% from AM group showed bridging with some gaps. The DBM/AM composite induced a significantly higher fusion rate than both the autologous bone and AM groups (p < 0.001) in all measured parameters. The current study demonstrated that using DBM/AM composite can have more robust fusion than autologous bone at 6 weeks in an athymic rat spinal fusion model.     

颈椎解剖与钛质网笼生物力学研究进展

钛质网笼作为颈前路椎体次全切除减压融合术（anterior cervical corpectomy and fusion,ACCF）中使用的融合器之一,不仅能够在术中即刻重建颈椎稳定性、维持椎间高度及生理曲度,而且可以避免自体取骨造成供骨区并发症的问题,故成为ACCF术中较为常用的内植物。但传统钛笼多存在应力遮挡、钛笼下沉等问题,在一定程度上影响了手术效果,甚至导致严重术后并发症,需要行翻修手术。目前已有多种新型钛笼问世,一定程度上解决了传统钛笼带来的问题。生物力学评价及其测试方法是评判一款新型脊柱融合器能否应用于临床必不可少的过程。综述颈椎解剖相关生物力学、传统钛笼与新型钛笼生物力学特性的相关研究,为传统钛笼的改进以及新型钛笼的研发提供新思路。     

Bone tissue engineering and spinal fusion: the potential of hybrid constructs by combining osteoprogenitor cells and scaffolds

In this paper, we discuss the current knowledge and achievements on bone tissue engineering with regard to spinal fusion and highlight the technique that employs hybrid constructs of porous scaffolds with bone marrow stromal cells. These hybrid constructs potentially function in a way comparable to the present golden standard, the autologous bone graft, which comprises besides many other factors, a construct of an optimal biological scaffold with osteoprogenitor cells. However, little is known about the role of the cells in autologous grafts, and especially survival of these cells is questionable. Therefore, more research will be needed to establish a level of functioning of hybrid constructs to equal the autologous bone graft. Spinal fusion models are relevant because of the increasing demand for graft material related to this procedure. Furthermore, they offer a very challenging environment to further investigate the technique. Anterior and posterolateral animal models of spinal fusion are discussed together with recommendations on design and assessment of outcome parameters.     

Bone tissue engineering for spine fusion: an experimental study on ectopic and orthotopic implants in rats

Alternatives to the use of autologous bone as a bone graft in spine surgery are needed. The purpose of this study was to examine tissue-engineered bone constructs in comparison with control scaffolds without cells in a posterior spinal implantation model in rats. Syngeneic bone marrow cells were cultured in the presence of bone differentiation factors and seeded on porous hydroxyapatite particles. Seven rats underwent a posterior surgical approach, in which scaffolds with (five rats) or without cells (two rats) were placed on both sides of the lumbar spine. In addition, separate scaffolds were inserted intramuscularly and subcutaneously during the surgical procedure. After 4 weeks, all rats were killed and examined radiographically, by manual palpation of the excised spine and histologically for signs of bone formation or spine fusion. All rats that received cell-seeded scaffolds showed newly formed bone in all three locations, whereas none of the locations in the control rats showed bone formation. The results of this study support the concept of developing tissue-engineering techniques in posterior spine fusion as an alternative to autologous bone.     

An in vivo Biocompatibility Assessment of MEMS Materials for Spinal Fusion Monitoring

The site-specific biocompatibility of silicon chips and commercially available silicon pressure sensor die were evaluated after implantation in caprine (goat) spine. Surgical procedures were developed to insert silicon chips into the nucleus pulposus regions of the lumbar discs and pressure sensors into autologous bone grafts for cervical spine fusion. After a six-month implantation period, the animal was sacrificed and the spinal segments were meticulously harvested and analyzed for local tissue response via gross examination and histological techniques. Gross examination of cervical and lumbar spinal segments after harvest and dissection did not reveal any visible signs of adverse reactions to the MEMS materials. Furthermore, the surrounding tissues and musculature for both spinal regions were devoid of necrosis. Histological analysis of compromised spinal segments did not reveal evidence of any adverse foreign body response by the caprine spinal tissue to the implanted MEMS materials. These preliminary results support the further development of a spinal fusion monitoring system based on implantable MEMS sensors.     

自体骨髓富集间质干细胞与羟基磷灰石磷酸三钙材料复合在脊柱融合中的应用

BACKGROUND: The use of bone graft materials can promote bone fusion and enhance the stability of the spine during the spinal fusion. OBJECTIVE: To investigate the effect of autologous bone marrow mesenchymal stem cells with hydroxyapatite/ tricalcium phosphate in the spinal fusion. METHODS: A retrospective analysis of clinical data of 64 patients with spinal fusion was carried out, and these patients were divided into two groups (n=32 per group): control group undergoing autogenous iliac bone grafting and observation group undergoing autologous bone marrow mesenchymal stem cells combined with hydroxyapatite/tricalcium phosphate. All patients were followed up for 12 months, and their recovery conditions about low back pain, spinal fusion and vertebral reset were assessed. RESULTS AND CONCLUSION: The low-back outcome scale scores and excellent rate, Lenke grading and Cobb angle had insignificant differences between the two groups after treatment (P > 0.05). No infection, inflammation and skin irritation occurred in the two groups. The coagulation function, renal function and inflammatory factor levels were at normal levels in all the patients, and there was no difference between the two groups (P > 0.05). These findings indicate that autologous bone marrow mesenchymal stem cells combined with hydroxyapatite/tricalcium phosphate can achieve clinical outcomes equivalent to the autologous iliac bone grafting.      

富血小板纤维蛋白新生诱导骨的组织学观察

BACKGROUND: In previous experiments, we have confirmed that platelet rich fibrin has the ability of osteoinduction, and have conducted a preliminary study on its microstructure and biomechanics. However, little is reported on its histology research. OBJECTIVE: To compare the histological changes after implanting platelet-rich fibrin, Bio-Oss and autologous bone and to analyze the pros and cons of platelet-rich fibrin implantation for repair of bone defects. METHODS: As previously reported, animal models of critical bone defects were established respectively on the bilateral femoral condyles of 12 beagle dogs. Then, platelet-rich fibrin, Bio-Oss+collagen membrane (Bio-Oss group) and autologous bone (autologous bone group)+collagen membrane were respectively implanted. At 3, 6, 8 and 12 months, one experimental dog from each group was killed, respectively, and histological observation was performed. Another beagle dog as blank control was enrolled to establish the animal model of critical bone defects, with no implantation. RESULTS AND CONCLUSION: At 3, 6, 8 and 12 months after implantation, there were significant differences in the new bone formation speed and amount between the platelet-rich fibrin group, Bio-Oss group and autologous bone group. These three kinds of bone grafts all had osteoinductive ability to different extents. In the platelet-rich fibrin group, the osteogenic effects were better at 3 and 6 months, and the new bone was similar to natural one; in the autologuos bone group, bone necrosis was noticeable at 3 and 6 months, but the osteogenic effects became better at 8 months, and the new bone was similar to natural one at 12 months; in the Bio-oss group, the osteogenic effects were similar to those in the platelet-rich fibrin group, but the residual of Bio-oss was visible at 12 months; in the blank control group, no bone formed at 3 months, indicating the animal model of critical bone defects was made successfully. In brief, the platelet-rich fibrin has good osteoinductive ability, with shorter time and better quality.       

A highly functional mini-dystrophin/GFP fusion gene for cell and gene therapy studies of Duchenne muscular dystrophy

A promising approach for treating Duchenne muscular dystrophy (DMD) is by autologous cell transplantation of myogenic stem cells transduced with a therapeutic expression cassette. Development of this method has been hampered by a low frequency of cellular engraftment, the difficulty of tracing transplanted cells, the rapid loss of autologous cells carrying marker genes that are unable to halt muscle necrosis and the difficulty of stable transfer of a large dystrophin gene into myogenic stem cells. We engineered a 5.7 kb miniDys-GFP fusion gene by replacing the dystrophin C-terminal domain (DeltaCT) with an eGFP coding sequence and removing much of the dystrophin central rod domain (DeltaH2-R19). In a transgenic mdx(4Cv) mouse expressing the miniDys-GFP fusion protein under the control of a skeletal muscle-specific promoter, the green fusion protein localized on the sarcolemma, where it assembled the dystrophin-glycoprotein complex and completely prevented the development of dystrophy in transgenic mdx(4Cv) muscles. When myogenic and other stem cells from these mice were transplanted into mdx(4Cv) recipients, donor cells can be readily identified in skeletal muscle by direct green fluorescence or by using antibodies against GFP or dystrophin. In mdx(4Cv) mice reconstituted with bone marrow cells from the transgenic mice, we monitored engraftment in various muscle groups and found the number of miniDys-GFP(+) fibers increased with time. We suggest that these transgenic mdx(4Cv) mice are highly useful for developing autologous cell therapies for DMD.     

自体骨髓细胞移植和心肌血管新生

治疗性血管生成是治疗缺血性心脏病的新方法,而自体骨髓细胞移植为血管生治疗开辟了新的途径并受到广泛的关注.本文结合最近几年内的相关研究,对于骨髓细胞移植与促血管生成的有关细胞学基础、相关机制作了分析,同时对于骨髓细胞移植用于促心肌血管生成治疗的有关动物和临床实验作了回顾和评价.