骨髓基质细胞移植修复半月板无血运区损伤的实验研究

目的比较自体与同种异体骨髓基质细胞移植对半月板无血运区损伤修复的影响。方法 40只成年新西兰大白兔随机平均分为 A、 B两组。 A组兔的骨髓基质细胞 (MSC)经体外培养后与纤维蛋白凝胶 (FG)混合,自体移植于其一侧的膝关节半月板缺损区,即 FG＋自体 MSC（自体移植组）;另一侧单纯植入 FG(FG植入组 )。于 B组兔的一侧膝关节半月板缺损区移植 FG＋同种异体 MSC(异体移植组 ),另一侧缺损不予修复 (空白对照组 )。分别于术后第 1、 2、 3个月取材,观察半月板损伤部位的组织形态学变化。结果 (1)自体移植组 :术后 1个月缺损区可见纤维组织,内有大量成纤维细胞;术后 2个月见大量软骨细胞并有胶原纤维形成;术后 3个月损伤区呈纤维软骨愈合。 (2)空白对照组 :术后 1～ 3个月缺损区始终未愈合。 (3)单纯 FG植入组 :术后 1～ 3个月缺损区可见纤维组织,内有少量成纤维细胞,没有软骨细胞生长,呈瘢痕样愈合。 (4)同种异体移植组 :与自体移植组所见大致相同,但有 3侧缺损区可见大量淋巴细胞浸润,胶原纤维少。结论骨髓基质细胞移植可促进半月板无血运区损伤的愈合,同种异体骨髓基质细胞移植修复半月板无血运区损伤发生免疫排斥反应的机率较低。     

骨髓基质干细胞复合藻酸钙凝胶修复全层半月板无血运区缺损

目的 观察骨髓基质干细胞复合藻酸钙凝胶注射式修复全层半月板无血运区缺损的效果.方法 2008年6月至2009年2月制造成年山羊半月板前角无血运区全层缺损模型.以自体骨髓基质干细胞复合可注射藻酸钙凝胶修复半月板缺损(Ⅰ组),同时设立单纯载体组(Ⅱ组)和空白对照组(Ⅲ组).术后4、8、16周处死动物,行大体观察,组织学观察、电镜观察和MRI检查并比较修复效果.结果 Ⅰ组缺损完全被修复组织填充,结合紧密,与正常半月板组织相似,在4～16周效果逐渐改善,大体观察优于其他各组.光镜见细胞随凝胶纤维排列分布,载体纤维间隙大多为细胞分泌基质所充填,细胞排列密集,基质分布均匀.透射电镜见Ⅰ组细胞呈软骨细胞样形态,细胞突起较多,细胞器丰富,细胞为不同走向排列的纤维包绕.MRI检查发现Ⅰ组修复效果较好.结论 骨髓基质干细胞复合藻酸钙凝胶注射可有效地修复全层半月板无血运区缺损.     

微量直流电刺激半月板无血运区裂伤愈合的实验研究

目的　观察微量直流电刺激犬半月板无血运区裂伤修复的组织学特征。方法　将２０ 条成年杂种犬双后肢膝外侧半月板无血运区造成纵形裂伤,每条犬后肢一侧为刺激组,对侧为对照组,用自制微电流刺激器刺激２ 、４ 、８ 、１２ 周后分别取材,行光镜及电镜观察。结果　电刺激２ 周半月板裂伤出现部分愈合,１２ 周完全愈合,而自身对照组均未愈合。结论　电刺激可诱导裂缘纤维软骨细胞分裂增殖;促进毛细血管趋向性增生、胶原合成及排列。半月板无血运区组织细胞并非惰性细胞,采用微量直流电刺激对半月板无血运区裂伤具有明显的促进修复作用。     

电刺激半月板无血区愈合的组织学特征

半月板无血运区的损伤是不能自然愈合的，如何能使半月板无血区愈合已经成为骨科临床上的一大难题。我们根据微电流刺激骨生长的理论，采用自行设计的微量直流电刺激器，用２０只成年杂种狗作为实验动物，在每只狗双后肢膝外侧半月板无血运区造成纵形裂伤，随机分为实验组和对照组。１２周后实验组均出现愈合，自身对照组均未愈合。本文对电刺激半月板愈合光镜、电镜组织学特征进行了观察及讨论，认为半月板无血运区组织细胞并非惰性细胞，它们具备有潜在的再生能力，而电刺激具有诱导软骨细胞分化、增殖作用，并能促进毛细血管趋向性增生和胶原合成及排列。     

纤维蛋白粘合剂治疗半月板无血运区损伤的实验研究

目的　研究纤维蛋白粘合剂 (FS)对半月板无血运区损伤愈合的作用。　方法　选用健康成年青紫兰兔2 4只 ,首先在兔半月板上造成统一的无血运区损伤模型。随机将兔分成 2组 ,分别为空白对照组、FS治疗组。术后 2周、6周、12周分批处死动物 ,进行大体形态观察和组织学检查。　结果　空白对照组半月板裂口均未见愈合 ,FS治疗组能形成疤痕组织愈合。　结论　应用FS治疗半月板无血运区的损伤是一条有效的治疗途径     

结缔组织生长因子促进半月板无血管区损伤愈合

目的：探讨结缔组织生长因子（CTGF）对纤维软骨细胞外基质分泌、VEGF表达及促进半月板无血管区的损伤修复中的作用。方法：将新西兰大白兔半月板白区,经胶原酶消化、离心分离后,提取半月板纤维软骨细胞,培养至第2代。用流式细胞仪鉴定该细胞表面CD31,CD44,CD45和CD105标志物,并用Ⅱ型胶原抗体做免疫细胞化学鉴定,以证明所培养、传代的细胞是纤维软骨细胞。分别将细胞培养在浓度100 ng/ml的CTGF培养基中3、14 d后,通过实时定量聚合酶链反应,检测Ⅰ型胶原、Ⅱ型胶原和VEGF基因的表达变化情况。造模,在兔半月板中央区,制作长3 mm的纵行撕裂。将45只大白兔随机分为3组,处理方式为：半月板单纯缝合术,缝合术加填充PBS-纤维蛋白胶,缝合术加填充1．5滋g CTGF-纤维蛋白胶。术后第1、4、10周用荧光免疫组织化学分析法显示Ⅰ型、Ⅱ型胶原和VEGF在损伤处的表达与分布情况,直观撕裂处的愈合情况。结果：体外实验第14天,定量RT-PCR结果显示,100 ng/ml CTGF组中的Ⅰ型、Ⅱ型胶原和VEGF mRNA表达比PBS对照组,明显增加。体内实验的荧光免疫组织化学结果显示,在术后第10周,CTGF治疗组中的Ⅰ型、Ⅱ型胶原和VEGF已完全填充撕裂损伤处。 PBS-蛋白胶组中,损伤处仍有明显裂隙。结论：CTGF可促进半月板无血管区重要的细胞外基质（Ⅰ型、Ⅱ型胶原）的合成,同时损伤处VEGF的表达活性明显增强,有利于促进无血管区半月板撕裂损伤的愈合。     

关节软骨和半月板组织碎块移植研究进展

关节软骨和半月板无血运区损伤后不能自然愈合,软骨组织碎块移植为之修复提供了新思路。体外和体内实验研究均证实,关节软骨及半月板无血运区切成碎块后细胞能有效地从碎块组织释放,与周围支架材料复合或直接导致组织修复。自体软骨植入系统（CAIS）及同种畀体幼年关节软骨碎块移植物（DeNovoNT）,已应用于,临床。该文就关节软骨及半月板组织碎块移植的基础研究及临床应用研究及其进展作一综述。     

用单克隆抗体和免疫毒素体外净化骨髓行自体骨髓移植

采取４例处缓解期的普通型急性淋巴细胞白血病患者的骨髓，用补体介导细胞毒法体外净化后进行自体骨髓移植，其中３例分别于移植后４２、３５和１２个月仍处于完全缓解；１例移植后４个月复发。对另外３例经化疗达完全缓解的未分化型（２例）和普通型（１例）急性淋巴细胞白血病患者采取骨髓，用免疫毒素法体外净化后行自体骨髓移植，２例分别于移植后１４和６个月仍处于完全缓解，１例移植后５个月，因患急性重型肝炎死亡，但骨髓仍     

滑膜植入促进半月板再生的实验研究

本实验选用２３只家兔为实验动物，分半月板单纯切除对照与滑膜植入实验两组，对照组即将半月板切除后不作特殊处理；实验组则在半月板切除后，取其相邻部位带蒂滑膜瓣置入原半月板处。术后分别于３、６、１２及周取材，标本行光镜及电镜观察，３只动物行相应部位游离滑膜瓣，行嗜银染色。结果发现：实验侧再生半月板较对照侧在形态和质地方面明显优竽对照者。６周时，细胞开始软骨细胞转化，１２周时出现较成熟的软骨细胞，并在半月     

兔半月板纤维软骨细胞在纤维蛋白凝胶中体外培养的生物学特性

目的：研究兔半月板纤维软骨细胞的分离、培养及在纤维蛋白凝胶中三维培养时的生物学特性。方法：(1)分离、培养兔半月板纤维软骨细胞；(2)制备纤维软骨细胞与纤维蛋白凝胶复合物，在纤维蛋白中培养细胞；(3)通过倒置相差显微镜，HE染色、电镜检查作形态学观察；(4)通过测定生长曲线及ABPAS染色、Ⅰ、Ⅱ型胶原免疫组化检查，了解细胞生长特性及软骨基质的分泌情况。结果：(1)单层培养的纤维软骨细胞呈线形，部分呈多角形，在纤维蛋白中的纤维软骨细胞为圆形，表面细胞呈多角形；(2)细胞在纤维蛋白中培养时，对数生长期明显延长；(3)AB-PAS染色( )，Ⅰ、Ⅱ型胶原免疫组化检查( )。结论：兔半月板纤维软骨细胞在纤维蛋白凝胶中能够增殖，保持其表型和分泌胞外基质。     

Augmented meniscal healing with free synovial autografts: an organ culture model

We examined whether or not free synovial autografts can augment the healing of lesions artificially created in the human knee meniscus in an organ culture model. The effectiveness of free synovial autografts was also compared with that of fibrin glue (Tisseel). In the control group, the accumulation of cellular components increased at the edge of a 4-mm meniscal defect following culture, although no cells were found to invade the defect. In the fibrin glue group, an increased migration of cells into fibrin glue packed into the defect was observed until 4 weeks following cultivation. However, newly formed collagen produced by the cells in the fibrin glue was minimal even 4 weeks after cultivation. In contrast, in the synovial group, newly formed collagen production, as well as cellular proliferation, increased in the grafted synovium with time after cultivation. By 4 weeks, the gap between the edge of the defect and the grafted synovium was bridged by newly formed collagen tissue, indicating improved histologic repair. Our results suggest that free autogenous synovial tissue grafts in meniscal lesions are superior to fibrin glue, and that interpositional synovial autografts to augment repair of meniscal tears in the avascular zone may be clinically useful.     

角质细胞与成纤维细胞混合移植修复裸鼠全层皮肤缺损的初步报告

目的：利用组织工程原理探讨修复全层皮肤缺损的理想方式。方法：以裸鼠为动物模型,在皮肤全层缺损区域分别移植纤维蛋白胶(n=10),纤维蛋白胶角质细胞悬液（n=10）,纤维蛋白胶成纤维细胞悬液（n=10）以及纤维蛋白胶角质细胞成纤维细胞悬液（n=10）,术后每天对伤口进行大体观察,第5,7,10,14,21,35d,分别取材活检行组织学及免疫组织化学检查。结果：移植有角质细胞组（2和4组）的创面愈合快,术后10d组织学提示创面完全上皮化,抗人特异性HLA－1型抗原、抗involucrin染色和抗Ⅶ型胶原染色阳性证明新生上皮由移植的人角质细胞形成,抗involucrin染色阳性又证明角质细胞分化成熟有角质层形成,抗Laminin染色、抗Ⅶ型胶原染色阳性提示早期基底膜形成。组织学检查提示第4组新生上皮有许多类似皮钉样结构。结论：培养的角质细胞,成纤维细胞结合纤维蛋白胶移植到创面上后,可以形成复层分化良好、接近正常结构和功能的新生成肤组织。     

比目鱼肌内侧半肌、胫骨骨膜复合瓣逆行移位的临床研究

目的 探讨治疗胫骨中、下段骨病损合并局部软组织缺失的新手术。方法 通过对３５侧成人下肢标本解剖观测比目鱼肌血供和比邻关系,创作比目鱼肌内侧半肌、胫骨骨膜复合瓣逆行移位以修复胫骨中下段骨不连、骨缺损或新鲜粉碎性骨折合并局部软组织缺失,为８例患者施行手术。结果 临床疗效满意。８例中小腿创面完全愈合,骨病损分别于术后３～６个月愈者６例,６～９个月愈合者２例。结论 此手术操作简便易行、效果可靠,是治疗胫骨     

骨髓间充质干细胞分化为血管内皮细胞的实验研究

目的：探讨骨髓间充质干细胞（MSCs)分化为肉芽组织中血管内皮细胞的可能性。及其参与创面修复的可能机制。方法：抽取小型香猪的骨髓，经密度梯度离心分离、纯化MSCs，体外培养扩增后，应用溴脱氧尿嘧啶（BrdU)标记技术标记细胞。于已抽取骨髓的小型香猪背部制作全层皮肤缺损创面，即刻以纤维蛋白胶为载体，将已标记的MSC回植到供体动物创面上。术后2、4、6、8、12周切取创面组织，行BrdU和因子Ⅷ（FⅧ）免疫组织化学染色，进行对比观察。结果：BrdU阳性的MSCs多聚集在创面肉芽组织中的小血管周围，且有个别血管内皮细胞也呈现BrdU 阳性。部分BrdU阳性的MSCs胞浆中亦有FⅧ表达。结论：创面愈合过程中，MSCs与肉芽组织中小血管的形成密切相关。在创面微环境下，MSCs可分化为血管内皮细胞，并参与创面修复。     

A comparative histologic analysis of tissue-engineered bone using platelet-rich plasma and platelet-enriched fibrin glue.

OBJECTIVE: The aim of this study was to compare the effects of platelet-rich plasma (PRP) and platelet-enriched fibrin glue on bone formation in bone tissue engineering. STUDY DESIGN: PRP was mixed with bone marrow mesenchymal stem cells and bone morphogenetic protein-2 (BMP-2), and the composites were injected into the subcutaneous space on the dorsum of nude mice. On the contralateral side of the dorsum, platelet-enriched fibrin glue/bone marrow mesenchymal stem cells/BMP-2 composites were injected. Bone formation was evaluated after 12 weeks. RESULTS: The volumes of subcutaneous nodules formed in nude mice were 55 +/- 18 microL at the PRP/bone marrow mesenchymal stem cells/BMP-2 sites and 135 +/- 27 microL at the platelet-enriched fibrin glue/bone marrow mesenchymal stem cells/BMP-2 sites. Histomorphometric analysis demonstrated that the nodules contained 14.9 +/- 4.1% newly formed bone when using PRP and 19.8 +/- 3.6% newly formed bone when using platelet-enriched fibrin glue. CONCLUSION: The results indicated that the osteogenic characteristics of platelet-enriched fibrin glue are superior to PRP in bone tissue engineering.     

Meniscal repair using an exogenous fibrin clot. An experimental study in dogs

To evaluate the ability of a fibrin clot to stimulate and support a reparative response in the avascular portion of the meniscus, two-millimeter-diameter full-thickness lesions in the avascular portion of the medial meniscus of twelve adult dogs were filled with an exogenous fibrin clot that had been prepared from each animal. The healing response was then examined using histology and autoradiography with 35SO4 at intervals from one week to six months. The defects that had been filled with a fibrin clot healed through a proliferation of fibrous connective tissue that eventually modulated into fibrocartilaginous tissue. The fibrin clot appeared to act as a chemotactic and mitogenic stimulus for reparative cells and to provide a scaffolding for the reparative process. The origin of these reparative cells was not determined in this study, but they were thought to arise from the synovial membrane as well as the adjacent meniscal tissue. Control defects remained empty. While the reparative tissue was grossly and histologically different from the normal adjacent meniscal tissue, it was morphologically similar to the reparative tissue that was previously observed in the vascular area of the meniscus. CLINICAL RELEVANCE: The ability of an exogenous fibrin clot to stimulate and support a reparative response in the avascular portion of the meniscus may represent a potential method of avascular meniscal repair.     

可注射型生物蛋白胶包埋骨髓基质细胞工程化组织的体外实验

目的构建可注射型生物蛋白胶包埋骨髓基质细胞的工程化组织,体外培养并研究其生物学特性,探讨将可注射型生物蛋白胶作为组织工程支架用于临床的实验基础。方法体外培养浇铸有骨髓基质细胞的生物蛋白胶,通过倒置相差显微镜、激光共聚焦显微镜观察载体内细胞生长及载体降解情况,5-溴脱氧尿苷(5-Bromodeocyuridine,BrdU)掺入标记后免疫组化等方法研究可注射型载体内包埋细胞的增殖情况。结果骨髓基质细胞包埋于生物蛋白胶内能很好地存活并增殖,2d后细胞呈典型的成纤维细胞形态;6d后生物蛋白胶边缘部分开始降解,细胞脱落至培养板;体外培养14d,细胞生长良好,大部分生物胶降解,脱落的细胞增多,贴壁生长的细胞形态正常;3周后生物蛋白胶完全降解。结论生物蛋白胶聚合后包埋的种子细胞能够正常增殖,生物蛋白胶是一种理想的适用于微创方法修复组织的可注射型组织工程培养和移植的支架。     

Viability of allogeneic bone marrow stromal cells following local delivery into patella tendon in rabbit model

Bone marrow stromal cells are potentially useful for tendon repair and regeneration. To provide lasting benefits, the seeded cells must survive implantation at local tendon sites. Our objective was to determine the in vivo fate of allogeneic bone marrow stromal cells (bMSCs) at different time points after implantation into patella tendon defects (i.e., at 2, 3, 5, and 8 weeks). The protocol involved the labeling of bMSCs with green fluorescent protein (GFP) or carboxyfluorescein diacetate (CFDA) before implantation. A window defect (5 x 5 mm) was created at the central portion of rabbit patella tendon and subsequently treated with GFP- or CFDA-marked bMSCs. The marked bMSCs were loaded into the window defect with fibrin glue. Upon sacrifice of the rabbits at the different time points, the implant site of the patellar tendon was immediately retrieved and the viability of the labeled cells was assessed under confocal microscopy. The results showed that the seeded bMSCs remained viable within the tendon wound site for at least 8 weeks after implantation. The cell morphology was changed from a round shape at 2 weeks to a spindle shape at 5 weeks after implantation. This study demonstrated that the bMSCs remained viable for prolonged periods after implantation and therefore have the potential to influence the formation and remodeling of neotendon tissue after tendon repair.     

Formation and maturation of the murine meniscus

Meniscal injuries are commonplace, but current surgical repair procedures do not prevent degenerative joint changes that occur after meniscal injury and often lead to osteoarthritis. Successful tissue regeneration in adults often recapitulates events that occur during embryogenesis, suggesting that understanding the regulatory pathways controlling these early processes may provide clues for developing strategies for tissue repair. While the mouse is now widely used to study joint diseases, detailed knowledge of the basic biology of murine meniscus is not readily available. Here, we examine meniscal morphogenesis in mice from embryonic day 13.5 (E13.5) to 6 months of age using histology, in situ hybridization, and immunohistochemistry. We find that the meniscus is a morphologically distinct structure at E16 when it begins to regionalize. At birth, the meniscus has a distinguishable inner, avascular, round chondrocyte cell region, an outer, vascularized, fibroblast cell region, and a surface superficial zone. Maturation begins at 2 weeks of age when the meniscus expresses type I collagen, type II collagen, type X collagen, and MMP‐13 in specific patterns. By 4 weeks of age, small areas of ossification are detected in the anterior meniscal horn, a common feature seen in rodents. Maturation appears complete at 8 weeks of age, when the meniscus resembles the adult structure complete with ossifying tissue that contains bone marrow like areas. Our results provide, the first systematic study of mouse meniscal development and will be a valuable tool for analyzing murine models of knee joint formation and disease. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:1683–1689, 2017.