以软骨细胞外基质与脱细胞骨基质构建组织工程骨软骨双层支架及其性能的研究

目的 探讨采用软骨细胞外基质(CECM)与脱细胞骨基质(ACBM)为材料制作新型组织工程骨软骨双层支架的可行性,并检测其性能.方法 双层支架的骨部分以犬松质骨制备的ACBM为原料,软骨部分以人CECM为材料,采用冷冻冻干法制备CECM/ACBM双层支架并交联.测定支架孔隙率,采用四甲基偶氮唑盐(MTT)法分析支架浸提液毒性.分离培养犬骨髓基质干细胞(BMSCs),成软骨诱导后种植到支架上,倒置显微镜、电镜、Dead/Live荧光染色观察细胞在支架的生长、分化情况.结果 扫描电镜及Micro-CT观察显示支架内孔洞相互贯通呈海绵状,CECM部分孔径(155±34)μm,孔隙率为91.3%±2.0%;ACBM部分具有大然骨的孔径和空隙率,骨软骨部分结合良好.培养1～6 d不同浓度支架浸提液与对照培养液吸光度值比较羌异均无统计学意义(P＞0.05).倒置显微镜、电镜检查结果表明BMSCs在支架上黏附良好,细胞基质分泌增加,Dead/Live荧光染色表明双层支架内细胞均呈绿色.结论 CECM/ACBM骨软骨双层支架具备良好的孔径和孔隙率,骨、软骨两层间结合良好,无毒,生物相容性良好,可作为支架载体用于组织工程骨软骨复合体的构建.

Abstract：

Objective To fabricate a novel bilayered scaffold constructed with cartilage extracellular matrix (CECM) and acellular bone matrix (ACBM) for osteochondral tissue engineering.Methods The bone layer of the osteochondral scaffold was prepared using canine bone cancellous bone columns, and the cartilage layer was fabricated using CECM.After CECM microfilaments were decellularized, the biphasic scaffolds were fabricated by soaking the ACBM columns into cylindrical silicon moulds with a 30 g/L CECM suspension using simple freeze-drying method.After the scaffolds were cross-linked, the porosity was measureed.MTT test was also done to assess cytotoxicity of the scaffolds.Canine bone marrow-derived mesenchymal stem cells (BMSCs) were induced by chondrogenic medium and seeded into novel scaffold.Cell proliferation and differentiation were analyzed using inverted microscopy, scanning electron microscopy (SEM)and Dead/Live staining method.Results SEM and Micro-CT revealed a 3-D interconnected porous structure, with the CECM pore diameter of 155 ± 34 μm and the porosity of 91.3% ± 2.0%.Cytotoxicity testing with MTT revealed no significant difference in absorbance among different extracts, showing no cytotoxic effect of the scaffold on BMSCs.Inverted microscopy and SEM showed that the novel scaffold could provide a suitable 3-D environment to support the adhesion, proliferation and differentiation of BMSCs to chondroeytes in culture with chondrogenic medium.Confocal microscopy of cell-scaffold constructs revealed cells with green fluorescence.Conclusion Since the novel CECM/ACBM bilayered integrated osteochondral scaffold has good mircostructure, non-toxicity and good biocompatibility, it may be a suitable candidate as an alternative cell-carrier for osteochondral tissue engineering.     

关节软骨-骨一体化修复体修复全层关节软骨缺损的实验研究

目的 评价新型多级结构仿生型关节软骨-骨一体化修复体的生物相容性,使用一体化修复体对兔关节全层软骨缺损进行修复,并对修复结果进行组织形态学观察.方法 1.生物相容性实验:包括急性毒性实验、溶血实验、免疫原性实验及慢性毒性实验.2.关节软骨修复实验:制作全层关节软骨缺损动物模型,随机于一侧植入一体化修复体,另一侧不予处理.术后4、6、8和12周分别处死动物,取修复组织行大体、放射学、组织学观察并用Wakitani法进行组织形态学评分.结果 1.生物相容性实验:(1)急性全身毒性实验动物体质量呈上升趋势,且各组体质量增加比较无显著性差异.(2)3种浓度梯度的修复材料溶血率均未超过5%.(3)慢性毒性实验:术后12周动物肝肾功能与正常对照组及术前比较无显著性差异.2.关节软骨修复实验:术后4～8周植入侧修复组织主要为透明软骨,表面光整平滑有光泽,与周围组织整合良好,对照侧无明显修复组织.Wakitani评分各组间差异均有统计学意义,实验组明显优于对照组.结论 多级结构仿生型关节软骨-骨一体化修复体具有良好的生物相容性,并且在动物体内可诱导全层关节软骨缺损后的修复.

Abstract：

Objective To observe the biocompatibility of a biomimetic designing of a multi-grade compositions in repairing articular cartilage and subchondral bone in animal bodies and repair the fullthickness defects in articular cartilage with the compositions and to study the regenerated cartilage histomorphologically. Methods Biocompatibility study: Acute general toxicity test, Haemolysis test, subcutaneous implantation test and chronic toxicity test. Articular cartilage defects repaired experimental study :The models of defects in articular cartilage were made artificially in both condylus lateralis femoris of mature rabbits. Implanted with the biomimetic designing of a multi grade compositions randomly at one side as the experimental group and the other side were untreated as the control group. The rabbits were killed at 4, 6, 8and 12 weeks after operation, respectively, with 6 ones at each time, and the macroscopic, histological, ultrastroctural examinations and semi-quantity cartilage scoring employing Wakitanifa repaired cartilage value system were performed. Results Biocompatibility study: (1) The rabbits' weight in experimental group kept growing .(2) Haemolysis rate of rats to different concentrations of diffusion solution was＜5%.(3) In chronic toxic reaction, rabbits' liver and kidney function was not different compared with the control groups at 12weeks and the index before operation. Articular cartilage defects repaired experimental study: 4-8 weeks after operation, the defects in the experimental group were partly filled with hyaline cartilage. Twelve weeks after operation, the defects in the experimental group were completely filled with mature hyaline cartilage.However, fibrous tissues were seen in the control group all the time. At 4, 6, 8, and 12 weeks postoperatively, the Wakitanifa cartilage scores were (7.60±0.98), (5.69±0.58), (4.46±0.85) and (4.35±0.12), respectively,in the experimental group and (10.25±1.05), (9.04±0.96), (8.96±0.88) and (8.88±0.68), respectively, in the control group. Differences between the control group and the experimental group were significant. Conclu sion The biomimetic designing of a multi-grade compositions has good biocompatibility and may induce cartilage regeneration to repair the full-hickness defects of articular cartilage.     

Treatment of osteonecrosis of femoral head with BMSCs-seeded bio-derived bone materials combined with rhBMP-2 in rabbits

Objective： To evaluate the effect of autologous bone marrow mesenchymal stem cells （BMSCs） seeded bio-derived bone materials （BBM） combined with recombinant human bone morphogenetic protein-2 （rhBMP-2） in repairing defect of osteonecrosis of femoral head （ONFH）.
Methods： Early-stage osteonecrosis in the left hip was induced in 36 adult New Zealand white rabbits （provided by the Animal Center of Guangxi Medical University, Nanning, China） after core decompression and delivery of liquid nitrogen into the femoral head. Then the animals were divided into three groups according to the type of implants for bone repair： 12 rabbits with nothing （Group Ⅰ, the blank control group）, 12 with BBM combined with rhBMP-2 （Group Ⅱ）, and 12 with BMSCs-seeded BBM combined with rhBMP-2 （Group Ⅲ）. At 4, 8, and 12 weeks after surgery, X-ray of the femoral head of every 4 rabbits in each group was taken, and then they were killed and the femoral heads were collected at each time point, respectively. Gross observation was made on the femoral heads. After hematoxylin and eosin staining, Lane-sandhu scores of X-ray and bone densitometry were calculated and the histomorphometric measurements were made for the new bone trabeculae.
Results： At 12 weeks after surgery, two femoral heads collapsed in Group Ⅰ, but none in Group Ⅱ or Group Ⅲ. X-ray examination showed that the femoral heads in Group I had defect shadow or collapsed while those in Group II had a low density and those in Group III presented with a normal density. Histologically, the defects of femoral heads were primarily filled with no new bone but fibrous tissues in Group Ⅰ. In contrast, new bone regeneration and fibrous tissues occurred in Group II and only new bone regeneration occurrd in Group Ⅲ. Lane-sandhu scores of X-ray, bone mineral density and rate of new bone in trabecular area in Group Ⅲ were higher significantly than those of the other two groups. Conclusions： Our findings indicate a superior choice of     

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.     

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.     

三维培养骨软骨复合体融合研究

目的 探讨三维培养体系中构建骨软骨复合体的可行性.方法 取10周以内流产胚胎组织原代培养人胚胎干细胞,按1×1O7个/ml细胞数植入自制的微流控支架三维培养系统,分别用相应细胞因子诱导分化成骨细胞和软骨细胞.体外培养3 d后植入裸鼠体内,8周后取出标本进行组织学观察.结果 支架骨端孔隙率为45%,孔径为125～150 μm;软骨端孔隙率为85%,孔径150～250 μm.组织学检查显示再造组织形成了骨和软骨复合组织,但是两者之间仍有界限,完全融合失败.结论 三维培养骨软骨复合体,可成功再造出骨软骨复合组织,但是完全融合尚需增加支架软骨端的孔隙率和孔径,以及延长体内培养的时间.

Abstract：

Objective To explore the feasibility of construction of bone cartilage complex in the three-dimensional culture. Methods The abortive embryos within 10 weeks were obtained for primary culture of human embryonic stem cells (hESCs). The hESCs about 1 × 107/ml were implanted into microfluidic three-dimensional culture system. hESCs were induced to differentiate into osteoblasts and chondrocytes by cytokine respectively. After culture in vitrofor 3 days, the system was transplanted in nude mouse.Eight weeks after implantation, the specimens were harvested and examined histologically. Results In the microfluidic three-dimensional culture system, the porosity of bony part was 45%, and the aperture was 125-150 μm; the porosity of chondral part was 85%, and the aperture was 150-250 μm. In the newly formed tissue, it demonstrated the formation of bone cartilage complex tissue under the microscopy. However, there was still a boundary between the two tissues. The perfect fusion of bone and cartilage became failure. Conclusion In the experiment, bone cartilage complex tissue can be reconstructed in three-dimensional culture system, but the perfect fusion needs to increase the porosity and the aperture of chondral part, together with culture time elongation in vivo.     

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

目的 探讨利用软骨细胞提供的软骨微环境诱导骨髓基质细胞(BMSC)在体外构建软骨组织的可行性.方法 将分离出的猪骨髓基质细胞和软骨细胞进行体外培养,收集软骨细胞培养上清液,作为骨髓基质细胞诱导液从第2代开始进行诱导分化.7 d后取出标本,免疫组织化学检测软骨特异性Ⅱ型胶原表达,RT-PCR检测Ⅱ型胶原和aggrecan的mRNA表达.体外分离培养的骨髓基质细胞与软骨细胞,扩增后两者以8∶2比例混匀,以5.0×107/ml的终浓度接种于聚羟基乙酸/聚乳酸(PGA/PLA)支架,以相同浓度的单纯软骨细胞和单纯BMSC以及20%上述浓度(1.0×107/ml)的单纯软骨细胞作为对照组.标本于8周后取材,行大体观察、湿重、蛋白多糖(GAGs)含量测定、组织学及免疫组化等相关检测.结果 经诱导后的骨髓基质细胞的Ⅱ型胶原免疫组化检测阳性,RT-PCR检测Ⅱ型胶原和aggrecan mRNA呈阳性表达.混合细胞组及阳性对照组体外培养8周后形成了单一成熟的软骨组织,并保持了支架材料的大小和形状,两组新生软骨在外观及组织学特征上也基本相同,免疫组化结果 表明两组均大量表达软骨特异性细胞外基质Ⅱ型胶原,共培养组的平均湿重和蛋白多糖(GAGs)含量均达到阳性对照组的70%以上.而单纯骨髓基质细胞组仅在局部形成了极少量幼稚的软骨样组织,且材料支架明显皱缩变形.低软骨细胞浓度组虽新生软骨湿重量能达阳性对照组的30%,但材料支架明显皱缩变形,仅在局部形成了不连续的软骨组织,新生软骨量明显少于共培养各组及阳性对照组.结论 软骨细胞能在一定程度上提供软骨形成的微环境,有效地诱导BMSC向软骨细胞分化,并在体外形成组织工程化的软骨组织.

Abstract：

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.     

Donor’s site evaluation after restoration with autografts or synthetic plugs in rabbits

AIM: To investigate donor site's area histological and immunohistochemical knee cartilage appearances after resurfacing iatrogenic defects with biosynthetic plugs orautografts. METHODS: Thirty New Zealand White rabbits were used in this study. A full-thickness cylindrical defect of 4.5 mm(diameter) × 7 mm(depth) was created with a hand drill in the femoral groove of every animal. In Group A(n = 10) the defect of the donor site was re-paired with a biosynthetic osteochondral plug, in Group B(n = 10) with an osteochondral autograft, while in Group C(control group of 10) rabbits were left untreated. RESULTS: Twenty-four weeks postoperatively, smooth articular cartilage was found macroscopically in some trocleas' surfaces; in all others, an articular surface with discontinuities was observed. Twenty-eight out of 30 animals were found with predominantly viable chondrocytes leaving the remaining two-which were found only in the control group- with partially viable chondrocytes. However, histology revealed many statistical differences between the groups as far as the International Cartilage Repair Society(ICRS) categories are concerned. Immunofluoresence also revealed the presence of collagen Ⅱ in all specimens of Group B, whereas in Group A collagen Ⅱ was found in less specimens. In Group C collagen Ⅱwas not found. CONCLUSION: The matrix, cell distribution, subchondral bone and cartilage mineralization ICRS categories showed statistically differences between the three groups. Group A was second, while group B received the best scores; the control group got the worst ICRS scores in these categories. So, the donor site area, when repairing osteochondral lesions with autografting systems, is better amended with osteochondral autograft rather than bone graft substitute implant.     

The fabrication of biomimetic biphasic CAN-PAC hydrogel with a seamless interfacial layer applied in osteochondral defect repair

Cartilage tissue engineering based on biomimetic scaffolds has become a rapidly developing strategy for repairing cartilage defects. In this study, a biphasic CAN-PAC hydrogel for osteochondral defect(OCD)regeneration was fabricated based on the density difference between the two layers via a thermally reactive,rapid cross-linking method. The upper hydrogel was cross-linked by CSMA and NIPAm, and the lower hydrogel was composed of PECDA, AAm and PEGDA. The interface between the two layers was first grafted by the physical cross-linking of calcium gluconate and alginate, followed by the chemical cross-linking of the carbon-carbon double bonds in the other components. The pore sizes of the upper and lower hydrogels were ~ 187.4 and ~ 112.6 μm, respectively. The moduli of the upper and lower hydrogels were ~ 0.065 and~ 0.261 MPa. This prepared bilayer hydrogel exhibited the characteristics of mimetic composition, mimetic structure and mimetic stiffness, which provided a microenvironment for sustaining cell attachment and viability. Meanwhile, the biodegradability and biocompatibility of the CAN-PAC hydrogel were examined in vivo. Furthermore, an osteochondral defect model was developed in rabbits, and the bilayer hydrogels were implanted into the defect. The regenerated tissues in the bilayer hydrogel group exhibited new translucent cartilage and repaired subchondral bone, indicating that the hydrogel can enhance the repair of osteochondral defects.     

膝关节软骨人体标本解剖与磁共振成像的比较研究

目的 探讨膝关节尸体标本解剖与磁共振成像(MRI)三维序列-扰相梯度回波序列(3D-FS-SPGR)测量关节软骨厚度的差异,并分析软骨组织主要成分在关节软骨不同位置的差异.方法选用国人青壮年中等身材、无明显关节病变的成年男性尸体膝关节标本2具,首先进行3D-FS-SPGR序列矢状位扫描.复冻后按解剖部位进行矢状位解剖,分别对股骨及胫骨内、外髁负重区前、后面及髌骨面软骨厚度进行测量.关节软骨石蜡切片进行维多利亚蓝-丽春红复合染色并观察.结果 软骨尸体标本解剖与3D-FS-SPGR序列测得的膝关节软骨厚度:股骨外侧髁前负重面平均分别为2.25、2.25 mm,股骨外侧髁后负重面平均分别为2.70、2.75 mm,胫骨外侧髁前负重面平均分别为2.00、2.10 mm;胫骨外侧髁后负重而平均分别为2.35、2.25 mm,股骨内侧髁前负重面平均分别为2.20、2.20 mm,股骨内侧髁后负重面平均分别为2.15、2.30 mm,胫骨内侧髁前负重面半均分别为2.20、2.45mm,胫骨内侧髁后负重面平均分别为2.70、2.95 mm,髌骨面软骨平均分别为3.08、3.15 mm.软骨组织学染色显示:关节软骨表层胶原纤维含量相对较多,软骨细胞及其周围基质相对较少;在关节软骨深层,胶原纤维含量相对较少,而软骨及软骨周围基质相对较多.结论 3D-FS-SPGR序列能够相对真实地反映关节软骨的形态及厚度.胶原纤维主要集中在软骨表层,其分布与软骨的功能相一致.

Abstract：

Objective To compare corpse sampling and MR imaging with 3D-FS-SPGR sequences in measurement of the articular cartilage thickness and to investigate knee cartilage topography. Methods Two fresh specimens of the knee joint were obtained from 2 normal young adult male corpses of medium stature. MR1 scanning was carried on the 2 specimens in sagittal 3D-FS-SPGR MR sequences. After defrosted,the knee specimens were dissected longitudinally, and the cartilage thicknesses were measured at different locations of the knee joint. Paraffin sections of the knee cartilage were observed following compound staining with victoria blue and ponceau red. Results The average cartilage thicknesses measured by dissection and MR imaging sequence were respectively: 2. 25 mm and 2. 25 mm at the anterior weight-loading surface of the femoral lateral condyle, 2. 70 mm and 2. 75 mm at the posterior weight-loading surface of the femoral lateral condyle, 2. 00 mm and 2. 10 mm at the anterior weight-loading surface of the tibial lateral condyle,2. 35 mm and 2. 25 mm at the posterior weight-loading surface of the tibial lateral condyle, 2. 20 mm and 2. 20mm at the anterior weight-loading surface of the femoral medial condyle, 2. 15 mm and 2. 30 mm al the posterior weight-loading surface of the femoral medial condyle, 2. 20 mm and 2.45 mm at the anterior weight-loading surface of the tibial medial condyle, 2. 70 mm and 2. 95 mm at the posterior weight-loading surface of the tibial medial condyle and 3. 08 mm and 3. 15 mm at patella cartilage surface. Collagen fibers were rich at the periphery of the articular cartilage with sparse chondrocytes and matrixes, while the opposite was observed at the center of the articular cartilage. Conclusions MR imaging with 3D-FS-SPGR sequences can display the actual knee cartilage topography. Collagen fibers mainly concentrate at the periphery of the articular cartilage, which accounts for the function of the articular cartilage.     

组织工程骨软骨复合体修复犬膝关节负重区骨软骨缺损的实验研究

目的 观察以骨软骨支架复合骨髓基质干细胞(bone-fflarrow mesenchymal stem cells,BMSCs)修复犬膝关节负重区骨软骨缺损的疗效.方法 利用软骨细胞外基质作为软骨支架部分,以脱细胞骨作为骨支架部分,采用相分离技术制备骨软骨双相支架,将成软骨诱导的BMSCs种植到双相支架上构建组织工程骨软骨复合体,并以此复合体修复犬膝关节股骨髁负重区骨软骨缺损,分为细胞-双相支架组(实验组)和单纯支架组(对照组).分别在术后3和6个月时取材,根据大体、组织学、Micro-CT等检测结果进行半定量或定量评估.结果 大体及组织学评价表明:同一时间点实验组的修复效果优于对照组,且实验组在术后6个月时的修复效果优于其术后3个月时,两项差异均有统计学意义;而对照组小同时间点修复效果的差异无统计学意义.Micro-CT检测结果表明实验组与对照组软骨下骨均得到重建,两者的差异尤统计学意义.结论 骨软骨双相支架复合成软骨诱导的BMSCs能成功修复犬膝关节负重区的骨软骨缺损,其修复效果明显优于单纯支架植入组.     

脱细胞骨软骨支架复合自体骨髓间充质干细胞修复羊骨软骨缺损的研究

目的探讨脱细胞骨软骨支架接种自体骨髓间充质干细胞(BMSCs)修复羊骨软骨缺损效果,探索骨软骨缺损新的修复方式。方法制备直径为8mm骨软骨脱细胞支架,培养羊BMSCs,接种于骨软骨支架,制备羊负重区骨软骨缺损模型,分空白、空白支架及细胞支架复合物3组,每组4只羊,3个月后处死动物取标本行大体及组织学检测。结果修复羊负重区骨软骨缺损模型实验结果显示细胞支架复合修复组骨软骨有较好修复,空白支架组软骨下骨基本修复、软骨侧无明显修复,空白对照组未见明显修复,缺损边缘软骨退变。结论含骨软骨连接结构的脱细胞骨软骨支架接种种子细胞能较好的修复羊负重区骨软骨缺损。     

自体骨髓间充质干细胞复合壳聚糖/羟基磷灰石支架修复兔膝骨软骨缺损

目的 探讨骨髓间充质干细胞(bone mesenehymal stem cells,BMSCs)复合壳聚糖(chitosan,CS)/羟基磷灰石(hydmxyapatite,HA)支架修复兔膝关节局部骨软骨缺损.方法 选健康日本大耳白兔36只,2～3月龄,体重1.7～2.0 kg,每只抽取自体骨髓4～6ml,体外分离培养BMSCs后以2×107/ml密度植于CS/HA支架上体外培养10 h,制成BMSCs-CS/HA支架复合物.将36只实验动物手术制成右膝股骨外侧髁负重区骨缺损模型后,随机分成A、B、C 3组,每组12只.A组植入BMSCs-CS/HA复合物,B组植入单纯CS/HA支架;C组不作任何植入,为空白对照组.分别于术后6周、12周各处死6只动物,取材后进行大体、组织学观察6根据改良Wakitani评分标准进行评分,评估软骨组织的修复情况,并行成组设计方差分析.结果 A组术后6周即可重建关节软骨缺损;修复软骨在观察期内逐渐变厚,软骨下骨有少量骨修复;术后12周透明软骨样修复,表面光整,与周围软骨色泽相近,软骨下骨有部分修复.而B组和C组12周时缺损区仍为纤维软骨样纤维组织修复,色泽浅黄.术后6、12周各组组织学半定量评分显示:股骨髁负重区修复A组评分明显优于B、C组(F=27.26,P＜0.05).结论 自体BMSCs复合CS/HA支架在体内环境下可形成透明软骨修复兔膝关节负重区骨软骨缺损.     

双相磷酸钙组织工程骨块重建股骨头内骨缺损的实验研究

目的 观察在体外构建的具有仿生结构的双相磷酸钙（biphasic calcium phosphate，BCP）组织工程骨块植入犬股骨头缺损内的骨再生情况及预防股骨头塌陷的效果。方法 以犬股骨头的松质骨样本Micro—CT（micro—computed tomography）图像为基础，提取其中的图像信息，利用三维凝胶叠层成形法制备出具有仿骨小梁结构的陶瓷支架。在体外利用诱导分化的自体骨髓间充质细胞与仿生BCP支架复合，构建组织工程骨块，将其植入10只犬的股骨头负重区骨缺损内；另取10只火作为对照组，在股骨头骨缺损区内打球植入自体松质骨粒，对比观察仿生BCP骨块的植入效果。结果 制备出的股骨头仿生BCP支架具有良好的三维空间结构，支架小梁具有一定的方向性，呈板状模型；细胞在支架表面大量生长。植入动物体内30周后，实验组火股骨头外形基本完整，新生骨质包绕支架小梁并沿支架表面生长，而对照组犬股坩头均出现不同程度的塌陷。结论 仿生BCP组织工程骨块具有良好的生物相容性，植入犬股骨头负曩区骨缺损后，能在一定程度上防止股骨头塌陷。     

胶原/羟基磷灰石支架负载软骨细胞修复兔膝关节骨软骨缺损

目的 探讨胶原复合梯度羟基磷灰石(Col/HA)双相支架负载软骨细胞修复兔膝关节骨软骨缺损的可行性及疗效.方法 构建Col/HA双相支架,将软骨细胞种植于支架培养1周,再将软骨细胞-支架复合体移植修复兔膝关节股骨髁的骨软骨缺损,并对骨软骨缺损的修复进行检测.结果 光镜及扫描电镜观察显示软骨细胞在Col/HA支架中贴附良好,表型维持稳定,分泌胞外基质.大体观察和组织学检测显示,植入体内16周后实验组软骨层呈透明软骨样修复,软骨下骨缺损有新骨构建;对照组骨软骨缺损修复不良,组织学检测以纤维性组织或纤维软骨组织形成.Wakitani评分显示实验组修复组织优于对照组,差异有统计学意义(P＜0.05).结论 双相Col/HA复合支架可作为骨软骨组织工程支架,负载软骨细胞可修复兔膝关节骨软骨缺损,重建关节软骨的结构和功能.     

Orderly osteochondral regeneration in a sheep model using a novel nano‐composite multilayered biomaterial

The objective of this article was to investigate the safety and regenerative potential of a newly developed biomimetic scaffold when applied to osteochondral defects in an animal model. A new multilayer gradient nano‐composite scaffold was obtained by nucleating collagen fibrils with hydroxyapatite nanoparticles. In the femoral condyles of 12 sheep, 24 osteochondral lesions were created. Animals were randomized into three treatment groups: scaffold alone, scaffold colonized in vitro with autologous chondrocytes and empty defects. Six months after surgery, the animals were sacrificed and the lesions were histologically evaluated. Histologic and gross evaluation of specimens showed good integration of the chondral surface in all groups except for the control group. Significantly better bone regeneration was observed both in the group receiving the scaffold alone and in the group with scaffold loaded with autologous chondrocytes. No difference in cartilage surface reconstruction and osteochondral defect filling was noted between cell‐seeded and cell‐free groups. In the control group, no bone or cartilage defect healing occurred, and the defects were filled with fibrous tissue. Quantitative macroscopic and histological score evaluations confirmed the qualitative trends observed. The results of the present study showed that this novel osteochondral scaffold is safe and easy to use, and may represent a suitable matrix to direct and coordinate the process of bone and hyaline‐like cartilage regeneration. The comparable regeneration process observed with or without autologous chondrocytes suggests that the main mode of action of the scaffold is based on the recruitment of local cells. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:116–124, 2010     

Repair of porcine articular cartilage defect with a biphasic osteochondral composite.

Autologous chondrocyte implantation (ACI) has been recently used to treat cartilage defects. Partly because of the success of mosaicplasty, a procedure that involves the implantation of native osteochondral plugs, it is of potential significance to consider the application of ACI in the form of biphasic osteochondral composites. To test the clinical applicability of such composite construct, we repaired osteochondral defect with ACI at low cell-seeding density on a biphasic scaffold, and combined graft harvest and implantation in a single surgery. We fabricated a biphasic cylindrical porous plug of DL-poly-lactide-co-glycolide, with its lower body impregnated with beta-tricalcium phosphate as the osseous phase. Osteochondral defects were surgically created at the weight-bearing surface of femoral condyles of Lee-Sung mini-pigs. Autologous chondrocytes isolated from the cartilage were seeded into the upper, chondral phase of the plug, which was inserted by press-fitting to fill the defect. Defects treated with cell-free plugs served as control. Outcome of repair was examined 6 months after surgery. In the osseous phase, the biomaterial retained in the center and cancellous bone formed in the periphery, integrating well with native subchondral bone with extensive remodeling, as depicted on X-ray roentgenography by higher radiolucency. In the chondral phase, collagen type II immunohistochemistry and Safranin O histological staining showed hyaline cartilage regeneration in the experimental group, whereas only fibrous tissue formed in the control group. On the International Cartilage Repair Society Scale, the experimental group had higher mean scores in surface, matrix, cell distribution, and cell viability than control, but was comparable with the control group in subchondral bone and mineralization. Tensile stress-relaxation behavior determined by uni-axial indentation test revealed similar creep property between the surface of the experimental specimen and native cartilage, but not the control specimen. Implanted autologous chondrocytes could survive and could yield hyaline-like cartilage in vivo in the biphasic biomaterial construct. Pre-seeding of osteogenic cells did not appear to be necessary to regenerate subchondral bone.     

自体柱状骨软骨移植修复股骨头软骨缺损的实验研究

目的探讨用自体股骨头非负重区骨软骨移植修复股骨头负重区软骨缺损。方法选用成年杂种犬18只,36个髋关节股骨头,随机抽取2只动物4个股骨头作为正常对照。分别凿取股骨头负重区及非负重区相同柱状骨软骨换位移植,于术后4、8、12、24及48周进行大体观察、组织学检查、电镜观察、CT、ECT及MRI检查。结果实验组移植后的骨软骨存活,骨软骨色泽及组织学检测显示移植的软骨细胞、骨细胞形态与正常对照组比较无显著差异,电镜观察显示其成骨细胞、骨细胞及软骨细胞与正常对照组无明显差异。结论股骨头非负重区骨软骨修复股骨头负重区软骨缺损,能达到骨愈合及修复软骨缺损的目的。     

隔离层在组织工程骨软骨复合支架中的作用

目的研究隔离层在一种新型组织工程骨软骨复合支架中对支架生物力学的影响以及在修复关节骨软骨缺损中的作用。方法在体外,检测具有隔离层的实验组骨软骨复合支架和没有隔离层的对照组支架的最大抗拉、抗剪切强度。将向软骨方向和成骨细胞方向诱导后的兔骨髓间充质干细胞（BMSCs）,分别接种在实验组和对照组的骨软骨复合支架的软骨支架和骨支架上,然后将两组骨软骨复合支架分别回植于兔自体股骨髁间窝骨软骨全层缺损处。术后3、6个月取材,对新生骨软骨组织的Micro-CT三维重建和HE组织学染色的结果进行观察;对新生软骨下骨的骨矿物质密度（BMD）、新生软骨的压缩模量进行检测。结果具有隔离层的实验组骨软骨复合支架的最大抗拉和抗剪切强度明显高于对照组（P〈0．05）;实验组新生骨软骨的Micro—CT三维重建和HE组织学染色的形态明显优于对照组;实验组新生软骨下骨的BMD值与新生软骨的压缩模量明显高于对照组（P〈0．05）。结论隔离层能显著增强骨软骨复合支架的力学性能,并显著提高骨软骨复合支架修复骨软骨缺损的能力。