CS/PVA凝胶负载Ad-hTGF-β1转染的BMSCs移植修复兔关节软骨缺损

目的 探讨温敏型CS/PVA凝胶负载Ad-hTGF-β1转染的骨髓间充质干细胞(BMSCs)移植修复兔关节软骨缺损的实验效果.方法 体外分离培养兔BMSCs,在Ad-hTGF-β1转染1周后,用细胞免疫化学方法检测hTGF-β1在细胞内的表达.用24只成年新西兰大白兔制造关节软骨缺损模型,双侧后肢均用于实验,动物模型随机分为4组,各组动物6只.A组:凝胶复合转染BMSCs修复组;B组:凝胶复合未转染BMSCs修复组;C组:凝胶修复组;D组:空白对照组.在术后16周时处死动物取材,通过大体标本和组织学染色观察评价各组修复效果,按照改良Pinoda法评分,对各组修复效果进行统计学分析.结果 免疫组化证实体外培养的BMSCs在Ad-hTGF-β1转染后表达hTGF-β1蛋白,阳性率为85.4%.术后16周取材见凝胶复合转染细胞组关节软骨缺损部位为软骨样组织填充,组织学观察见再生的软骨组织细胞排列及细胞密度与正常软骨相似,Ⅱ型胶原免疫组化阳性,Pineda评分同其它各组相比差异有统计学意义(P＜0.05).结论 CS/PVA凝胶作为一种温敏型可注射支架材料,其负载hTGF-β1转染的BMSCs移植可用于兔关节软骨缺损修复.

Abstract：

Objective To investigate the experiment effects of rabbit joint articular cartilage defects repaired by thermosensitive CS/PVA composite hydrogel engineered hTGF-β1 transfected bone marrow mesenchymal stem cells. Methods Bone marrow mesenchymal stem cells were isolated and cultured in vitro. The positive rate of transfection was defected by cell immunohistochemistry methods after Ad-hTGF-β1 transfected for 1 week. Twenty-four adult New Zealand white rabbits with full articular cartilage defects were randomly divided into 4 groups, each group had 6 animals, both hind limbs were used in the experiment. Group A: hydrogel combined with transfected cells; Group B: hydrogel combined with untransfected cells; Group C: hydrogel group; Group D: blank control group. Specimens and histological observation were used to evaluate the repair effect after 16 weeks according to Pineda's score. Results The positive rate of hTGF-β1 expression in BMSCs was about 85.4% after transfection. After 16 weeks the defects of group A were repaired by cartilage-like tissue, the cell arrangement and densities of regenerated cartilage were similar to normal cartilage, type Ⅱ collagen immunohistochemistry were positive. There was a significant difference in Pineda's score compaired with other groups (P ＜ 0.05). Conclusion Rabbit articiular cartilage defects could be repaired by CS/PVA hydrogel engineered hTGF-β1-transfected bone marrow mesenchymal stem cells.     

Repairing articular cartilage defects with tissueengineering cartilage in rabbits

Objective: To investigate the effect of cancellous bone matrix gelatin ( BMG ) engineered with allogeneic chondrocytes in repairing articular cartilage defects in rabbits. Methods: Chondrocytes were seeded onto three-dimensional cancellous BMG and cultured in vitro for 12 days to prepare BMG-chondrocyte complexes. Under anesthesia with 2.5% pentobarbital sodium (1ml/kg body weight), articular cartilage defects were made on the right knee joints of 38 healthy New Zealand white rabbits (regardless of sex, aged 4-5 months and weighing 2. 5-3 kg) and the defects were then treated with 2. 5% trypsin. Then BMG-chondrocyte complex ( Group A, n = 18 ), BMG (Group B, n = 10), and nothing (Group C, n = 10) were implanted into the cartilage defects, respectively. The repairing effects were assessed by macroscopic, histologic, transmission electron microscopic ( TEM ) observation, immunohistochemical examination and in situ hybridization detection, respectively, at 2, 4, 8, 12 and 24 weeks after operation. Results: Cancellous BMG was degraded within 8 weeks after operation. In Group A, lymphocyte infiltration was observed around the graft. At 24 weeks after operation, the cartilage defects were repaired by cartilage tissues and the articular cartilage and subchondral bone were soundly healed. Proteoglycan and type II collagen were detected in the matrix of the repaired tissues by Safranin-O staining and immunohistochemical staining, respectively. In situ hybridization proved gene expression of type II collagen in the cytoplasm of chondrocytes in the repaired tissues. TEM observation showed that chondrocytes and cartilage matrix in repaired tissues were almost same as those in the normal articular cartilage. In Group B, the defects were repaired by cartilage-fibrous tissues. In Group C, the defects were repaired only by fibrous tissues. Conclusions: Cancellous BMG can be regarded as the natural cell scaffolds for cartilage tissue engineering. Articular cartilage defects can be repaired by cancellous BMG engineered with allogeneic chondrocytes. The nature of repaired tissues is closest to the normal cartilage. Local administration of trypsin can promote the adherence of repaired tissues to host tissues. Transplantation of allogeneic chondrocytes has immunogenicity, but the immune reaction is weak.     

自体软骨碎屑回植对肋软骨修复再生的影响

Objective To investigate the influence on costal cartilage reparative regeneration by replanting the small blocks of autogeneic cartilage into the perichondrial pocket at the donor-site. Methods 16 rabbits (8-10 weeks old, 1.8-2. 2 kg) were randomly divided into four groups as three experimental groups and one control group. The 1.5 cm in length of costal cartilage defect was made in experimental groups with the perichondrium and costochondral junction left completely intact. The cartilage defect was cloesd by 3 methods as suturation directly, or replanting the small blocks of autogeneic cartilage, or plugging bioprotein jelly after cartilage replanting. Each experimental group was handled with two methods in two sides of costal cartilage. No operation was performed in control group. All the rabbits were sacrificed 16 weeks after operation. The appearance of thoracic cage and new-formed tissue at the defect site were examined grossly. Haematoxylin-eosin staining was performed to evaluate the characteristics of new-formed tissues and biomechanical detection was used to measure intension of new-formed tissues. Results The appearance of thoracic cage was normal in every experimental group. Histological study showed that the defect was filled with abundant fibrous tissue in each group. The chipping of cartilage survived effectively with little proliferation. Biomechanical detection showed that the intension of new-formed tissue in the non replanted group [( 193. 92 ± 41. 41 ) N] was obviously less than that in the replanted group [( 318. 88 ±28. 28)N] ,or bioprotein jelly group[(301. 00 ±39. 52) N] , or control group[(300. 54 ±38. 35) N] (P <0. 01). Furthermore, there was no statistical difference between the latter three groups ( P > 0. 05 ) . Conclusions Although replanting the chipping of cartilage can' t promote reparative regeneration of hyaline cartilage, it can definitively strengthen the intensity of new-formed tissue, reinforce thoracic stability. It may also indirectly decrease the incidence rate of postoperative chest wall deformity.     

自体软骨碎屑回植对肋软骨修复再生的影响

Objective To investigate the influence on costal cartilage reparative regeneration by replanting the small blocks of autogeneic cartilage into the perichondrial pocket at the donor-site. Methods 16 rabbits (8-10 weeks old, 1.8-2. 2 kg) were randomly divided into four groups as three experimental groups and one control group. The 1.5 cm in length of costal cartilage defect was made in experimental groups with the perichondrium and costochondral junction left completely intact. The cartilage defect was cloesd by 3 methods as suturation directly, or replanting the small blocks of autogeneic cartilage, or plugging bioprotein jelly after cartilage replanting. Each experimental group was handled with two methods in two sides of costal cartilage. No operation was performed in control group. All the rabbits were sacrificed 16 weeks after operation. The appearance of thoracic cage and new-formed tissue at the defect site were examined grossly. Haematoxylin-eosin staining was performed to evaluate the characteristics of new-formed tissues and biomechanical detection was used to measure intension of new-formed tissues. Results The appearance of thoracic cage was normal in every experimental group. Histological study showed that the defect was filled with abundant fibrous tissue in each group. The chipping of cartilage survived effectively with little proliferation. Biomechanical detection showed that the intension of new-formed tissue in the non replanted group [( 193. 92 ± 41. 41 ) N] was obviously less than that in the replanted group [( 318. 88 ±28. 28)N] ,or bioprotein jelly group[(301. 00 ±39. 52) N] , or control group[(300. 54 ±38. 35) N] (P <0. 01). Furthermore, there was no statistical difference between the latter three groups ( P > 0. 05 ) . Conclusions Although replanting the chipping of cartilage can' t promote reparative regeneration of hyaline cartilage, it can definitively strengthen the intensity of new-formed tissue, reinforce thoracic stability. It may also indirectly decrease the incidence rate of postoperative chest wall deformity.     

Experimental study on mechanism of vertebral osteophyte formation

Objective:The purpose of this experimental study was to explore the mechanism of the vertebral osteophyte formation.Methods:An experimental model of cervical spondylosis in rabbits was established by resection of the cervical supraspinous and interspinous ligaments and detachment of the posterior paravertebral muscles from cervical vertebrae.Because of individual difference,The natural development procedure of the vertebral osteophyte formation could be seen with a microscope by dynamic observation.Results:The cartilage end-plate was divided into a growth cartilage layer and an articular cartilage layer.Vertebrae and discs from the 3-month control group rabbits showed normal structure.The changes of cartilage plates from the 3-monthe experimental group and the 8-month control group animals showed proliferation in peripheral articular cartilage.The osteophytes from the 8-monthe experimental group animals could be seen.The osteophyte obviously arised from proliferation,calcification and ossification of the peripheral articular cartilage.Conclusions: The vertebral osteophyte arises from proliferation of peripheral articular cartilage which undergoes cartilaginous osteophyte,and then changes into bony osteophyte through an endochondrqal calcification and ossification.     

Autogenous standard versus inside-out vein graft to repair facial nerve in rabbits

Objective： To evaluate autogenous vein grafts and inside-out vein grafts as conduits for the defects repair in the rabbit facial nerves. Methods： The 10 nun segments of buccal division of facial nerve were transected for 48 rabbits in this study. Then the gaps were immediately repaired by autogenous vein grafts or inside-out vein grafts in different groups. All the animals underwent the whisker movement test and electrophysiologic test during the following 16 weeks at different time points postoperatively. Subsequently, the histological examination was performed to observe the facial nerve regeneration morphologically. Results： At 8 weeks after operation, the facial nerve regeneration has significant difference between the experimental group and the control group in electrophysiologic test and histological observation. However, at the end of this study, 16 weeks after operation, there was no signifi- cant difference between inside-out vein grafts and standard vein grafts in enhancing peripheral nerve regeneration. Conclusion： This study suggest that both kinds of vein grafts play positive roles in facial nerve regeneration after being repaired immediately, but the autogenous inside-out vein grafts might accelerate and facilitate axonal regeneration as compared with control.     

膝关节骨关节炎滑液和软骨骨桥蛋白水平与其病变程度的相关性

Objective To examine osteopontin (OPN) levels in both synovial fluid and articular cartilage of patients with primary knee osteoarthritis (OA) and to investigate their relationship with severity of the disease. Methods Fifty patients with knee OA and 10 healthy controls were enrolled in this study.There were 15 males and 35 females with an average age of 61.8±7.4 years in OA group. The control group included 4 males and 6 females with an average age of 63.2±6.0 years. Mankin score were taken to determine the disease severity of the affected knee. The radiographic grading of OA in the knee was performed using the Kellgren-Lawrence criteria. OPN levels in synovial fluid were measured using enzyme-linked immunosorbent assay. OPN levels in articular cartilage were assessed by immunohistochemical methods. Results Compared to healthy controls, the knee OA patients had higher OPN concentration in synovial fluid ([4519.60±1830.37] pg/ml vs. [1179.70±303.39] pg/ml) and articular cartilage([0.60±0.06] vs. [0.43 ±0.07]). In addition, synovial fluid OPN levels showed a positive correlation with articular cartilage OPN levels (r=0.411,P=0.003). Subsequent analysis showed that the OPN levels in synovial fluid significantly had been correlated with severity of disease using Kellgren-Lawrence criteria (r=0.581, P＜ 0.001). Furthermore,the levels of OPN in the articular cartilage also were correlated with disease severity using Mankin score (r=0.675, P＜ 0.001).Conclusion The data suggest that OPN in synovial fluid and articular cartilage is related to progressive joint damage and could be a predictive biomarker respect to disease severity and progression in knee OA.     

膝关节骨关节炎滑液和软骨骨桥蛋白水平与其病变程度的相关性

Objective To examine osteopontin (OPN) levels in both synovial fluid and articular cartilage of patients with primary knee osteoarthritis (OA) and to investigate their relationship with severity of the disease. Methods Fifty patients with knee OA and 10 healthy controls were enrolled in this study.There were 15 males and 35 females with an average age of 61.8±7.4 years in OA group. The control group included 4 males and 6 females with an average age of 63.2±6.0 years. Mankin score were taken to determine the disease severity of the affected knee. The radiographic grading of OA in the knee was performed using the Kellgren-Lawrence criteria. OPN levels in synovial fluid were measured using enzyme-linked immunosorbent assay. OPN levels in articular cartilage were assessed by immunohistochemical methods. Results Compared to healthy controls, the knee OA patients had higher OPN concentration in synovial fluid ([4519.60±1830.37] pg/ml vs. [1179.70±303.39] pg/ml) and articular cartilage([0.60±0.06] vs. [0.43 ±0.07]). In addition, synovial fluid OPN levels showed a positive correlation with articular cartilage OPN levels (r=0.411,P=0.003). Subsequent analysis showed that the OPN levels in synovial fluid significantly had been correlated with severity of disease using Kellgren-Lawrence criteria (r=0.581, P＜ 0.001). Furthermore,the levels of OPN in the articular cartilage also were correlated with disease severity using Mankin score (r=0.675, P＜ 0.001).Conclusion The data suggest that OPN in synovial fluid and articular cartilage is related to progressive joint damage and could be a predictive biomarker respect to disease severity and progression in knee OA.     

新型低弹性β钛合金关节表面微假体治疗犬膝关节软骨全层缺损的实验研究

目的 评价新犁低弹性β钛合金关节表面微假体治疗犬膝关节软骨面全层缺损的可行性与效果.方法 取8只成年家犬,制备舣侧股骨髁直径为7 mm的软骨面全层缺损,选用两种弹性模量钛合金材料制备关节表而微假体并植入软骨缺损区,左侧为低弹性模量组(弹性模量为42 GPa),右侧为高弹性模量组(弹性模量为110 GPa).3个月后取材,行X线片、显微CT扫描、骨计量学分析和组织学检测观察两组关节表面微假体的稳定性及周边骨与软骨的生长情况.结果 X线片示关节表面微假体钉帽周围软骨生长良好,完全填充钉帽周边软骨间隙,关节软骨面平整.显微CT三维重建及骨计量学分析显示低弹性模量组骨体积分数、骨小梁厚度、骨小梁数目及骨组织密度平均分别为0.389%±0.025%、(0.049±0.002)μm、(8.9±0.4)mm1、(652.7±12.6)mg/mm3,而高弹性模量组平均分别为0.253%±0.024%、(0.038 ±0.002)μm、(5.9±0.4)mm-1、(595.2±7.6)mg/mm3,以上指标两组间比较差异均有统计学意义(P<0.05).组织学观察显示低弹性模量组微关节表面假体固定钉周围有更多的新生骨小梁包绕,钉骨结合牢固,界面成骨量明显多于高弹性模量组.结论 新型低弹性β钛合金关节表面微假体治疗关节软骨面缺损是可行的,其低弹性模量的特点更有利干钉骨界面骨的形成.

Abstract：

Objective To assess the effect of a new β Ti alloy mini-prosthesis with low elastic modulus on resurfacing the full-thickness Surface defect of the knee joint in dogs.Methods A full-thickness osteochondral defect of 7 mm in diameter was created at the medial femoral condyle of both hind limbs in 8 healthy adult dogs.The titanium alloy mini-prostheses with high-modulus of elasticity were implanted on the right side(control group)and those with low-modulus of elasticity on the left side(experiment group)to repair the cartilage defects.After 3 months,all the 8 dogs were sacrificed to harvest the specimens.Radiographic,histologic and micro-CT examinations were conducted to observe stability of the mini-prostheses and growth of the bone and cartilage surrounding the implants. Results Radiographic evaluation revealed no indications of device disassembly or subsidence.Bone trabeculae surrounding anchoring screws in the experiment group was visibly more and denser compared with the control group.Micro-CT data revealed that the bone volume fraction,trabecular thickness,trabecular number and tissue mineral density were 0.389%±0.025%,0.049±0.002 μm,8.9±0.4 mm-1 and 652.7 ±12.6 mg/mm3 respectively in the experiment group,compare with 0.253%±0.024%,0.038±0.002 μm,5.9±0.4 mm-1 and 595.2±7.6 mg/mm3 in the control group,with statistically significant differences between the 2 groups(P<0.05).Cartilage around the cap grew well and the surface remained smooth.Periphery of the resurfacing implant was covered by an extension of the superficial cartilage emanating from the defect margins. Conclusions The new β Ti alloy mini-prosthesis with low elastic modulus is favorable toward resurfacing the articular surface defect.The low-modulus of elasticity can improve new bone formation on the implant-bone interface.     

软骨细胞与骨髓基质细胞共培养体外构建软骨组织的实验研究

Objective To investigate the feasibility of chondrogenesis in vitro with bone marrow stromal cells (BMSCs) induced by the co-cultured chondrocytes. Methods The BMSCs and chondrocytes were separated from pig and cultured. The supernatant of chondrocytes was used as the inducing solution for BMSCs from the 2nd generation. 7 days later, samples were taken and underwent immunohistochemistry and RT-PCR for detection of the expression of specific type Ⅱ cartilage collagen,type Ⅱ collagen and aggrecan mRNA. The cultured BMSCs and chondrocytes were mixed at a ratio of 8:2(BMSC: cartilage cell) and were inoculated into a polyglycolic acid/polylactic acid (PGA/PLA) scaffold at the final concentration of 5.0 × 107/ml. The cartilage cells and BMSCs were also inoculated seperately at the same concentration as the positive and negative control. Pure cartilage cells at 20% of the abovementioned concentration (1.0 × 107/ml) were used as the low concentration cartilage cell control group. Samples were collected 8 weeks later. General observations, wet weight, glycosaminoglycans (GAGs) determination and histological and immunohistochemistry examinations were performed. Results The expression of type Ⅱ collagen, type Ⅱ collagen and aggrecan mRNA were positive in induced BMSCs.In the co-cultured group and the positive control group, pure mature cartilage was formed after 8 weeks of culture in vitro, and the size and shape of the scaffold were maintained. The newly formed cartilage in the two groups were almost the same in appearance and histological properties. The immunohistochemistry results indicated that the cartilage cells of the two groups all expressed ample cartilage-specific type Ⅱ collagen. The average wet weight and GAG content in the co-cultured group reached more than 70% of those in positive control group. Only an extremely small amount of immature cartilage tissues was formed in local regions in pure BMSC group, and the scaffold was obviously shrunk and deformed. Although the wet weight of newly generated cartilage tissue in the low concentration cartilage cell group reached 30% of that in positive control group, the scaffold was obviously shrunken and deformed. Only regional and discontinuous cartilage tissues were formed, and the amount of newly formed cartilage was obviously less than that in the co-culture group and the positive control group. Conclusions Chondrocytes can provide a micro-environment for the formation of cartilage, and also effectively induce BMSC to differentiate into chondrocytes and form tissue-engineered cartilage in vitro.     

胶原复合梯度TCP修复关节软骨的形态学观察

目的采用新型双向三维可降解生物活性材料胶原复合梯度TCP（collagen complexTCP，Col／TCP）对兔关节软骨缺损进行修复，并对再生软骨进行组织形态学观察。方法取30只成年大白兔，体重2．0～2．5kg，雌雄不限，于双侧股骨外侧髁制作关节软骨缺损模型。于右侧植入Col／TCP修复缺损，作为实验组，左侧不予处理作为对照组。术后4、6、8、12和24周分别处死6只动物，取股骨外侧髁关节面行大体、组织学、透射电镜及Ⅱ型胶原免疫组织化学染色观察。采用Wakitanifa法软骨组织形态学评分评价修复组织质量。结果大体观察：实验组术后4周，缺损区由白色组织完全充填，表面较光滑，有光泽；12周，修复关节软骨组织与周围正常软骨基本一致，且与关节下骨结合紧密；24周，再生软骨未见明显退变。对照组观察期内均未见软骨组织形成，缺损由纤维组织填充，修复组织表面粗糙，与正常组织界线清楚。实验组术后4、6、8、12和24周组织学评分分别为（7．60&#177;0．98）、（5．69&#177;0．58）、（4．46&#177;0．85）、（4．35&#177;0．12）、（4．41&#177;0．58）分，对照组分别为（10．25&#177;1．05）、（9．04&#177;0．96）、（8．96&#177;0．88）、（8．88&#177;0．68）、（8．66&#177;0．54）分；Ⅱ型胶原含量实验组分别为0．28％&#177;0．01％、0．59％&#177;0．03％、0．68％&#177;0．02％、0．89％&#177;0．02％和0．90％&#177;0．01％，对照组为0．08％&#177;0．02％、0、09％&#177;0．04％、0．11％&#177;0．03％、0．25％&#177;0．03％和0．29％&#177;0．01％；两组各指标比较差异均有统计学意义（P〈0．05）。透射电镜观察实验组可见典型软骨细胞，而对照组为粗大胶原纤维，细胞少见。结论双向三维可降解生物活性材料Col／TCP在动物体内可诱导关节软骨缺损后的软骨修复。     

骨髓基质细胞与几丁质复合移植修复关节软骨缺损的实验研究

目的 :探讨骨髓基质细胞 (MSCs)与几丁质复合移植对关节软骨缺损的修复效果。方法 :分离兔骨髓基质细胞并体外培养增殖后 ,与几丁质无纺网复合培养 ;制作兔膝关节软骨全层缺损模型 ,分别用MSCs 几丁质复合物移植、单纯几丁质移植及空白对照组 ,术后第 4、 8、 12、 16周处死动物 ,大体观察并做组织形态学观察。结果 :几丁质 MSCs组术后 16周关节软骨缺损其修复组织表面与正常软骨完全相同 ,软骨及软骨下骨修复 ;单纯几丁质移植组为透明软骨修复 ,表面不平整 ,细胞排列不规则 ,软骨下骨基本修复 ;空白对照组术后各期均为纤维组织修复。结论 :MSCs与几丁质复合移植对关节软骨缺损有较好的修复效果     

异体软骨细胞复合Pluronic修复关节软骨缺损

目的 探讨运用同种异体软骨细胞复合Pluronic修复关节软骨缺损的可行性，并应用^3H—TdR放射自显影方法鉴别软骨缺损修复的细胞来源。方法 取同种异体软骨细胞体外培养至第2代，用^3H—TdR标记后复合Pluronic植入兔关节软骨缺损区作为实验组，并采用单纯Pluronic植入作为材料对照组，不作任何处理组为空白对照组，分别于4、8及16周取材，观察其修复效果，并应用放射自显影方法鉴别修复组织的细胞来源。结果 实验组术后8周，缺损表面可见新生软骨形成，术后16周缺损完全修复，表面光滑，与周围界限模糊，放射自显影证实所修复组织的细胞来源于植入细胞。材料对照组及空白对照组缺损均未见明显修复。结论 ①同种异体软骨细胞复合Pluronic修复关节软骨缺损是可行的；②^3H—TdR标记细胞可作为鉴别细胞来源的一种简便可行的方法。     

A new biotechnology for articular cartilage repair: subchondral implantation of a composite of interconnected porous hydroxyapatite, synthetic polymer (PLA-PEG), and bone morphogenetic protein-2 (rhBMP-2)

OBJECTIVE: Articular cartilage repair remains a major obstacle in tissue engineering. We recently developed a novel tool for articular cartilage repair, consisting of a triple composite of an interconnected porous hydroxyapatite (IP-CHA), recombinant human bone morphogenetic protein-2 (rhBMP-2), and a synthetic biodegradable polymer [poly-d,l-lactic acid/polyethylene glycol (PLA-PEG)] as a carrier for rhBMP-2. In the present study, we evaluated the capacity of the triple composite to induce the regeneration of articular cartilage. METHODS: Full-thickness cartilage defects were created in the trochlear groove of 52 New Zealand White rabbits. Sixteen defects were filled with the bone morphogenetic protein (BMP)/PLA-PEG/IP-CHA composite (group I), 12 with PLA-PEG/IP-CHA (group II), 12 with IP-CHA alone (group III), and 12 were left empty (group IV). The animals were killed 1, 3, and 6 weeks after surgery, and the gross appearance of the defect sites was assessed. The harvested tissues were examined radiographically and histologically. RESULTS: One week after implantation with the BMP/PLA-PEG/IP-CHA composite (group I), vigorous repair had occurred in the subchondral defect. It contained an agglomeration of mesenchymal cells which had migrated from the surrounding bone marrow either directly, or indirectly via the interconnecting pores of the IP-CHA scaffold. At 6 weeks, these defects were completely repaired. The regenerated cartilage manifested a hyaline-like appearance, with a mature matrix and a columnar organization of chondrocytes. CONCLUSIONS: The triple composite of rhBMP-2, PLA-PEG, and IP-CHA promotes the repair of full-thickness articular cartilage defects within as short a period as 3 weeks in the rabbit model. Hence, this novel cell-free implant biotechnology could mark a new development in the field of articular cartilage repair.     

髋臼后壁重建后关节软骨修复的实验研究

目的 研究髋臼后壁重建区臼面软骨修复的组织学特点,初步探讨软骨柱播种在其修复中的作用。方法 选取12只家犬共24髋,随机分为3组,均采用后壁截骨法建立髋臼后壁60°弧1/2缺损的动物模型,两侧髋臼后壁缺损区分别选用不同的重建方法：A组：解剖重建+软骨柱播种,B组：解剖重建,C组：普通重建。术后12周取材进行大体形态、光镜观察,参照Pineda标准对新生组织进行评分。结果 A组中以成熟、新生类透明软骨修复缺损,B组为类纤维软骨修复,C组为纤维肉芽组织或板层骨修复。A、B、C组关节软骨修复Pineda总评分平均分别为(5.1±0.1)、(6.5±0.2)、(12.3±0.2)分,差异有统计学意义(F=3.157,P=0.000),3组间两两比较差异均有统计学意义(P＜0.05)。结论 解剖重建促使部分软骨柱集中在缺损区,其修复组织具备部分关节软骨的组织特性,能够较好地替代关节软骨。     

骨形态形成蛋白复合纤维蛋白载体修复全厚关节软骨缺损的实验研究

目的：用骨形态形成蛋白（BMP）复合纤维蛋白载体修复创伤性全厚关节软骨缺损,方法：60只新西兰家兔,体重2．5－3kg,雌雄不限,随机分为5组,每侧股骨髌髁关节面低速电钻钻一直径为4mm全厚关节软骨缺损,一侧缺损填充BMP／FS,对照侧缺损填充单纯FS,单纯BMP和空白组,膝关节不做固定,允许笼中自由活动,术后2,4,8,12周空气栓塞分批处死动物,大体观,组织学切片HE染色,S－100蛋白免疫组化染色和透射电镜观察实验结果,结果：术后4周,BMP／HF填充的部分关节软骨缺损由类透明软骨修复,术后8周,实验组缺损大部分由类透明软骨修复,而对照组则由纤维软骨或纤维组织修复,术后12周,实验组修复组织主要是透明软骨或类透明软骨,修复面较平整光滑,与周围组织愈合良好,但部分修复软骨面变薄,纤维化。结论：BMP／FS复合物促进了关节软骨的早期修复,并且最终的修复组织更接受正常的关节软骨,但术后12周修复的关节软骨出现退行性改变。     

Allograft of cultured chondrocytes into articular cartilage defects in rabbits--experimental study of the repair of articular cartilage injuries

Articular cartilage defects were created by dill holes, 2 mm wide and 3 mm deep, through the articular cartilage into the subchondral bone in the patellar groove of the femur in mature rabbits. The defects received graft of cultured chondrocytes and the matrix obtained from the primary culture of chondrocytes isolated from the articular cartilage or auricular cartilage in immature rabbits. The isolated cells were cultured for 10 to 14 days. For graft, the cultured chondrocytes together with the matrix were detached from the culture chamber using rubber policemen and centrifuged. The repair of the grafted defects or defects without graft (control) was histologically studied 2 to 12 weeks after operation. The defects without the graft were progressively filled with fibrous tissue containing spindle shaped cells, fibers perpendicular to the surface, and matrix showing weak metachromasia with toluidin blue at 8 weeks. The defects received articular cartilage cell graft were occupied by new cartilage tissue consisting colonylike crumps of chondrocytes 2 weeks after operation. The crumps showed strong metachromasia with toluidin blue and strong stainability for safranin-O. By 4-8 weeks, the defects were filled with homogeneous cartilage. At 12 weeks, arrangement of the chondrocytes of the superficial layer of the new cartilage became columnar as seen in the normal articular cartilage. The defects received elastic cartilage cell graft were filled by reformed cartilage with chondrocytes surrounded by elastic fibers 2-12 weeks after operation. The results indicate that allograft of cultured chondrocytes with matrix into the articular cartilage defects accerated the repair process of the defects by formation of the new cartilage derived from the grafted chondrocytes.     

同种异体软骨细胞移植术后关节软骨蛋白多糖的测定

目的 应用Pluronic F-127负载同种异体软骨细胞移植修复兔全厚关节软骨损伤,对于新生的修复组织进行基质蛋白多糖含量测定,以探讨此方法修复全厚关节软骨损伤的可行性.方法 取3个月龄新西兰大白兔关节软骨细胞体外培养扩增,与20%Plurortic F-127凝胶混合.选27只健康同种成年大白兔,人为造成双侧膝关节软骨缺损.实验组软骨缺损处植入培养的软骨细胞/Pluronic F-127混合物,对照组缺损处单纯注入Pluronic F-127凝胶和空白对照.然后,对修复组织进行大体观察及蛋白多糖含量测定.结果 移植的软骨细胞-载体复合物中的软骨细胞能良好地生长,12周时再生组织与周围正常软骨组织外观相似,界限模糊.实验组与对照组各时期蛋白多糖含量均有非常显著性差异,实验组不同时期的蛋白多糖含量之间均有显著性差异,实验组12周时蛋白多糖含量与正常软骨组织无显著性差异.结论 Pluronic F-127负载同种异体软骨细胞移植是治疗关节软骨缺损的有效方法.     

应用骨形态发生蛋白(BMP)修复关节软骨缺损的实验研究

目的探讨关节软骨全层缺损应用骨形态发生蛋白修复的效果。方法于2004年5月至2005年12月,30只新西兰种成年兔随机分为A,B,C三组,每只兔子左膝股骨髁间凹做一大小为4mm×5mm×2.5mm的全层关节软骨缺损。A,B组缺损内分别填充骨形态发生蛋白/纤维蛋白胶(BMP/FG)及FG,C组为空白。术后28周对缺损修复情况行大体形态、组织学和电镜观察。结果BMP/FG组,缺损组织以透明软骨修复,接近正常组织,而FG组和空白组则以纤维组织修复为主。结论BMP/FG能较好的完成关节骨软骨全层缺损的修复,并随着时间的延长修复的软骨越接近正常软骨,但修复软骨缺损的组织与邻近正常软骨组织连接性仍不是十分理想。