在体构建组织工程软骨的实验研究
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摘要
第一部分利用一种新型的组织工程室在体构建组织工程软骨的实验研究
实验一兔自体耳廓软骨细胞的分离培养与鉴定的实验研究
目的:探讨兔耳廓软骨细胞分离、培养的方法及其传代过程中细胞形态、细胞外基质的变化。方法：取兔耳廓软骨采用胰蛋白酶结合Ⅱ型胶原酶消化的方法进行分离、培养及鉴定。结果：原代培养的软骨细胞呈多角形，第4代开始细胞表型发生改变。细胞培养3代以内，仍能保持表型及生物学特性的稳定。结论：采用二种或多种酶联合消化的方法可缩短消化时间，提高细胞纯度及培养效率；本实验培养的第2代新西兰大白兔耳廓软骨细胞，具备很强的增殖及分泌合成细胞外基质的能力，能提供实验所需的足量软骨细胞。适用于软骨组织工程的研究。
实验二体外构建软骨细胞-支架复合物的实验研究
观察比较胶原凝胶及PLGA胶原凝胶复合支架在体外对兔耳廓软骨细胞黏附、分化及增殖的效果。方法：构建软骨细胞/胶原凝胶和软骨细胞/PLGA胶原凝胶复合物，并进行体外培养观察。通过大体形态、组织学、扫描电镜观察两者在体外培养过程中形成软骨的能力。结果:术后14天，接种在不同支架上的软骨细胞复合物均形成了白色新生软骨样组织；扫描电镜可见软骨细胞呈圆形，在支架材料内均匀分布，黏附及生长良好，分泌大量的细胞外基质；HE切片见复合物中软骨细胞分布均匀，细胞外基质融合成片；甲苯胺蓝及番红“O”细胞外基质特异性染色呈阳性。结论：体外培养环境下，软骨细胞在胶原凝胶及PLGA凝胶支架材料上都能维持正常生长及合成细胞外基质的能力，但是其在体内及长期效果还有待于进一步实验研究。
实验三利用在体组织工程室构建组织工程软骨的实验研究
目的：初步探索我科自主研制的新型多孔硅胶组织工程室内渗液微环境是否能支撑软骨细胞-支架复合物的存活及生长，构建组织工程软骨的可行性。方法：将构建好的软骨细胞-支架复合物置入组织工程室内，再植入实验兔背部皮下，无需和血管沟通；对照组无组织工程室，直接将复合物植入同一实验兔背部皮下。术后8周进行大体形体观察、组织学及免疫组织化学染色、RT-PCR检测。结果：通过大体形体、组织学及免疫组织化学染色、RT-PCR检测结果，显示在实验兔背部皮下组织工程室内，构建的软骨细胞-支架复合物均形成了软骨样组织；而直接植入实验兔背部皮下的复合物最终消失或者形成一纤维化的结节。结论：本实验打破了目前大多数在体组织工程室需要血管化的思路，在具有免疫活性的动物体内无需和血管沟通，应用自主研制的新型多孔硅胶组织工程室内微环境成功构建了自体组织工程软骨，为软骨组织工程技术过渡到临床应用提供了理论依据。
第二部分构建无外支架组织工程软骨修复肋软骨供区缺损的实验研究
实验一兔骨髓间充质干细胞的分离培养和鉴定的实验研究
目的：探讨兔骨髓间充质干细胞（bone marrow mesenchymal stemcells，BMSCs）分离、培养及其鉴定的方法。方法：应用密度梯度离心和贴壁筛选法分离、培养兔BMSCs，观察兔BMSCs的生长特点、形态学特征，体外经成软骨、成脂肪和成骨诱导液分别诱导BMSCs向软骨、脂肪和骨细胞分化。并进行甲苯胺蓝、油红“O”及茜素红染色。结果：采用密度梯度离心和贴壁筛选相结合的方法，可以简单有效地获取大量高纯度，增殖能力强的BMSCs，可以向软骨、骨、脂肪等细胞成功分化。结论：采用密度梯度离心和贴壁筛选相结合的方法是培养兔BMSCs简便有效的方法，可以满足进一步实验的要求。
实验二BMSCs膜片的构建及其成软骨诱导的实验研究
目的：探讨应用连续培养及简单的机械方法，培养获取BMSCs膜片，并在体外向软骨细胞定向诱导。方法：将兔BMSCs在成软骨诱导条件下，连续培养14天，用细胞刮刀沿培养皿底壁外周向中心仔细刮擦，获得完整的细胞膜片。经组织学、电镜等方法检测细胞膜片的理化特性。结果：应用连续培养及细胞刮刀可获得完整的BMSCs膜片。在成软骨诱导培养条件下，BMSCs仍快速增殖，形成的细胞膜片经HE染色显示其由6-8层细胞组成，甲苯胺蓝、番红“O”染色呈阳性；电镜下可见细胞分布均匀、紧密排列，分泌细胞外基质。结论：通过连续培养及简单的机械方法可以有效获取BMSCs膜片，BMSCs膜片具有体外成软骨分化的潜能，有望用来构建组织工程软骨。
实验三成软骨诱导BMSCs膜片修复肋软骨供区缺损的实验研究
目的：采用兔胸廓损伤动物模型，观察成软骨诱导的BMSCs膜片对肋软骨供区再生修复的影响。方法：16只实验兔随机分成4组（每组4只）。切取各实验组兔双侧4-6肋一段肋软骨，缺损部位采取3种不同方法处理，①不缝合软骨膜；②BMSCs膜片折叠数层成圆筒状填塞入肋软骨缺损处缝合；③成软骨诱导的BMSCs膜片同法折叠数层成圆筒状填塞入肋软骨缺损处，缝合封闭缺损，3种方法在各实验组兔两侧肋软骨中两两配对，健康对照组不做处理。术后16周后处死动物取材进行大体观察，常规HE染色，测量各实验组兔肋软骨的平均宽度，并行生物力学检测测定所有肋软骨的抗压强度及弯曲强度。结果：各实验组家兔的胸廓整体形态均较良好，各组及各处理方法之间并无明显差别。方法1处理的修复组织平均宽度显著大于健康对照组肋软骨的平均宽度(P <0.01)，方法2、3处理的修复组织平均宽度与健康对照组肋软骨平均宽度相比无显著性差异(P>0.05)；生物力学检测显示：3种处理方法之间均存在差异(P<0.01)，方法3处理的修复组织的抗压、弯曲强度与健康对照组比较差异无统计学意义（P＞0.05），方法1，2处理的修复组织的抗压、弯曲强度明显低于健康对照组(P<0.01)，方法2处理的修复组织的抗压、弯曲强度优于方法1；组织切片HE染色病理观察，可见方法1、2处理的修复组织主要为纤维组织，方法3处理的修复组织内，可见新生的软骨细胞和大量的软骨细胞外基质。结论：成软骨诱导的BMSCs膜片可以促进肋软骨供区软骨细胞的再生，修复肋软骨供区缺损，维持胸廓的正常形态和稳定性，从而降低术后胸廓畸形的发生率。
Use of Autologous Chondrocytes and Bioinert PerforatedChambers to Tissue-Engineer Cartilage In Vivo
1. The study of isolation,culture and identification of rabbit chondrocytes
Objective: Auricular chondrocytes were isolated from the earscartilages of newzealand white rabbits with the methods of collagenase Ⅱandtrypsin enzymatic digestion. Methods: chondrocytes were harvested from rabbitears by enzymatic digestion. The morphological changes of cell and growthfeatures were observed through the phase-contrast microscope every day. Thechondrocytes were indentified with toluidine blue staining and chondrocytesgrowth curve was depicted. Results: Chondrocytes could be isolatedsuccessfully with collagenase Ⅱ and trypsindigestion.The primarily culturedchondrocytes were polygonal, and became dedifferentiated after3passages. Thechondrocytes maintained the morphology and histological staining patternwithin the first3passages. Conclusions: The study showed that the method of isolation and culture of chondrocytes was simple and feasible. The secondpassage chondrocytes grow well and are suitable for experiment.
2. Design and fabrication of the chondrocytes/Collagen andchondrocytes/PLGA-Collagen In Vitro
Objective: To compare the effect between the Collagen gel andPLGA/Collagen gel hybrid scaffolds as vehicles for chondrogenesis in vitro.Methods: P2rabbit auricular chondrocytes(RACS) were seeded into thescaffolds;(1) RACSwere seeded into Collagen gel scaffold;(2) RACSwereseeded into PLGA/Collagen gel scaffold.After culturing for14days in vitro，thesamples were evaluated by HE, toluidine blue， safranin-O staining, andelectronical microscope scanning. Results: RACS/Collagen gel andRACS/PLGA gel samples formed white cartilage-like tissue in vitro when theywere harvested14days later. HE，toluidine blue and safranin-O staining showedthe chondrocytes were surrounded by much ECM. It was observed thatchondrocytes attached, growed, and proliferated well on the scaffold by SEM.Conclusions: These results indicated that collagen gel and PLGA gel scaffoldswere suitable for use and the two kinds of scaffolds can be used fortissue-engineering cartilage construction.
3. Using bioinert perforated chambers to tissue engineer cartilage in vivo
Objective: To explore the potential applications of a chamber for invivo tissue engineering, and to establish a novel model for in vivotissue-engineered cartilage. Methods: Four experimental groups were includedin this study:(A) chambers+chondrocytes/collagen gel;(B) chambers+chondrocytes/PLGA gel;(C) chondrocytes/collagen gel alone; and (D) chondrocytes/PLGA gel alone. Groups C and D served as controls. The sampleswere implanted subcutaneously in the donor rabbit, and the contents wereharvested at8weeks after implantation. Results: Histological andimmunohistochemical staining and RT-PCR results revealed regeneratedcartilage-like tissue in group B and small, irregularly shaped islands ofopalescent tissue in group A. In contrast, the control groups displayed vascularinvasion and inflammatory reaction, which eventually led to fibrosis andabsorption. Conclusions: Reproduced cartilages were obtained in animmunocompetent animal model through the use of a bioinert perforatedchamber.
Recreating Engineering-Cartilage Without Scaffolds OnCostal Cartilage Repair and Regeneration
1. The study of isolation, culture and identification of rabbits bone marrowmesenchymal stem cells
Objective: To culture and amplify rabbit bone marrow mesenchymalstem cells (BMSCs) by using density gradient centrifugation combiningadherent cultivation method, and to identify them. Methods: The growth andmorphology of BMSCs were observed by phase-contrast microscope. TheBMSCs growth curve was depicted by MTT; chondrogenic inductor, adiposeinductor and osteogenic inductor were used to induce the BMSCs intochondrocytes, adipocytes and osteoblast in vitro. The differentiated cells wereidentified by toluidine blue, oil red O and alizarin red staining respectively.Results: The BMSCs could be obtained by the density gradient centrifugationcombining adherent cultivation method and the BMSCs could be differentiated into chondrocytes, adipocytes and osteoblasts after induction treatment.Conclusions: The density gradient centrifugation combining adherentcultivation method was a relatively simple and feasible way which could isolateand culture BMSCs. The BMSCs could proliferate and multi-differentiate verywell.This suggested that BMSCs were superior seed cells for cartilage tissueengineering.
2. Fabrication and chondrogenic differentiation of BMSCs Sheet
Objective: To obtain BMSCs sheet using continuous culturecombining simple mechanical method and to assess the feasibility ofchondrogenic differentiation. Methods: BMSCs were cultured in the culturemedium supplemented with chondrogenic inductor for14days.The harvestedcell sheets were evaluated by histological and scanning electronical microscope.Results: The intact BMSCs sheet could be gained by continuous culture andsimple mechanical method. HE staining showed that the cell sheet was made of6to8layers of cells. The safranin-O, toluidine blue staining showed positiveresults and scanning electron microscope examination revealed that the cellsarranged closely and the matrix secreted abundantly. Conclusions: The studyindicated that the BMSCs sheet could be generated by using this simpletechnique, and the sheet could differentiate into chondrocytes..
3. The Effects of chondrogenic BMSCs Sheet on Costal Cartilage Repairand Regeneration
Objective: The purpose of the study was to investigate the effects ofchondrogenic BMSCs Sheet on costal cartilage repair and regeneration. Methods: Sections of the costal cartilage of16male rabbits were removed withthe costal perichondrium and the costal cartilage junction was intactly left. Therabbits were treated with three different methods: Method1: The costalperichondrium was left unsutured; Method2: The costal perichondrium wassutured to form a tubular structure after the implantation of alone BMSCs Sheet;Method3: The costal perichondrium was sutured to form a tubular structure afterthe implantation of chondrogenic BMSCs Sheet. The specimens were harvestedafter16weeks postoperatively. Results: The average width of the costal cartilagein the method1group was significantly wider than that of the control group(P<0.01). The biomechanical strength of the costal cartilage in themethod1,2groups were significantly weaker than that of the control group (P<0.01); No obvious differences in the average widths and the biomechanicalstrength were tested between the method3group and that of the control group(P>0.05).HE-stained results showed the repaired tissues in1、2methods weremainly fibrous cartilages, and new cartilage-like tissue regneneration could befound in the3method group, The chondrocyte-like cells were surrounded bythe abundant cartilaginous matrix in the regenerated cartilage. Conclusions: Ourstudy has indicated that chondrogenic BMSCs Sheet could promote theregneneration of new cartilage, which significantly enhanced the strength of therepaired tissue, strengthened the stability of the thorax, and thus, indirectlyreduced the incidence of thoracic deformities.
实验一兔自体耳廓软骨细胞的分离培养与鉴定的实验研究
目的:探讨兔耳廓软骨细胞分离、培养的方法及其传代过程中细胞形态、细胞外基质的变化。方法：取兔耳廓软骨采用胰蛋白酶结合Ⅱ型胶原酶消化的方法进行分离、培养及鉴定。结果：原代培养的软骨细胞呈多角形，第4代开始细胞表型发生改变。细胞培养3代以内，仍能保持表型及生物学特性的稳定。结论：采用二种或多种酶联合消化的方法可缩短消化时间，提高细胞纯度及培养效率；本实验培养的第2代新西兰大白兔耳廓软骨细胞，具备很强的增殖及分泌合成细胞外基质的能力，能提供实验所需的足量软骨细胞。适用于软骨组织工程的研究。
实验二体外构建软骨细胞-支架复合物的实验研究
观察比较胶原凝胶及PLGA胶原凝胶复合支架在体外对兔耳廓软骨细胞黏附、分化及增殖的效果。方法：构建软骨细胞/胶原凝胶和软骨细胞/PLGA胶原凝胶复合物，并进行体外培养观察。通过大体形态、组织学、扫描电镜观察两者在体外培养过程中形成软骨的能力。结果:术后14天，接种在不同支架上的软骨细胞复合物均形成了白色新生软骨样组织；扫描电镜可见软骨细胞呈圆形，在支架材料内均匀分布，黏附及生长良好，分泌大量的细胞外基质；HE切片见复合物中软骨细胞分布均匀，细胞外基质融合成片；甲苯胺蓝及番红“O”细胞外基质特异性染色呈阳性。结论：体外培养环境下，软骨细胞在胶原凝胶及PLGA凝胶支架材料上都能维持正常生长及合成细胞外基质的能力，但是其在体内及长期效果还有待于进一步实验研究。
实验三利用在体组织工程室构建组织工程软骨的实验研究
目的：初步探索我科自主研制的新型多孔硅胶组织工程室内渗液微环境是否能支撑软骨细胞-支架复合物的存活及生长，构建组织工程软骨的可行性。方法：将构建好的软骨细胞-支架复合物置入组织工程室内，再植入实验兔背部皮下，无需和血管沟通；对照组无组织工程室，直接将复合物植入同一实验兔背部皮下。术后8周进行大体形体观察、组织学及免疫组织化学染色、RT-PCR检测。结果：通过大体形体、组织学及免疫组织化学染色、RT-PCR检测结果，显示在实验兔背部皮下组织工程室内，构建的软骨细胞-支架复合物均形成了软骨样组织；而直接植入实验兔背部皮下的复合物最终消失或者形成一纤维化的结节。结论：本实验打破了目前大多数在体组织工程室需要血管化的思路，在具有免疫活性的动物体内无需和血管沟通，应用自主研制的新型多孔硅胶组织工程室内微环境成功构建了自体组织工程软骨，为软骨组织工程技术过渡到临床应用提供了理论依据。
第二部分构建无外支架组织工程软骨修复肋软骨供区缺损的实验研究
实验一兔骨髓间充质干细胞的分离培养和鉴定的实验研究
目的：探讨兔骨髓间充质干细胞（bone marrow mesenchymal stemcells，BMSCs）分离、培养及其鉴定的方法。方法：应用密度梯度离心和贴壁筛选法分离、培养兔BMSCs，观察兔BMSCs的生长特点、形态学特征，体外经成软骨、成脂肪和成骨诱导液分别诱导BMSCs向软骨、脂肪和骨细胞分化。并进行甲苯胺蓝、油红“O”及茜素红染色。结果：采用密度梯度离心和贴壁筛选相结合的方法，可以简单有效地获取大量高纯度，增殖能力强的BMSCs，可以向软骨、骨、脂肪等细胞成功分化。结论：采用密度梯度离心和贴壁筛选相结合的方法是培养兔BMSCs简便有效的方法，可以满足进一步实验的要求。
实验二BMSCs膜片的构建及其成软骨诱导的实验研究
目的：探讨应用连续培养及简单的机械方法，培养获取BMSCs膜片，并在体外向软骨细胞定向诱导。方法：将兔BMSCs在成软骨诱导条件下，连续培养14天，用细胞刮刀沿培养皿底壁外周向中心仔细刮擦，获得完整的细胞膜片。经组织学、电镜等方法检测细胞膜片的理化特性。结果：应用连续培养及细胞刮刀可获得完整的BMSCs膜片。在成软骨诱导培养条件下，BMSCs仍快速增殖，形成的细胞膜片经HE染色显示其由6-8层细胞组成，甲苯胺蓝、番红“O”染色呈阳性；电镜下可见细胞分布均匀、紧密排列，分泌细胞外基质。结论：通过连续培养及简单的机械方法可以有效获取BMSCs膜片，BMSCs膜片具有体外成软骨分化的潜能，有望用来构建组织工程软骨。
实验三成软骨诱导BMSCs膜片修复肋软骨供区缺损的实验研究
目的：采用兔胸廓损伤动物模型，观察成软骨诱导的BMSCs膜片对肋软骨供区再生修复的影响。方法：16只实验兔随机分成4组（每组4只）。切取各实验组兔双侧4-6肋一段肋软骨，缺损部位采取3种不同方法处理，①不缝合软骨膜；②BMSCs膜片折叠数层成圆筒状填塞入肋软骨缺损处缝合；③成软骨诱导的BMSCs膜片同法折叠数层成圆筒状填塞入肋软骨缺损处，缝合封闭缺损，3种方法在各实验组兔两侧肋软骨中两两配对，健康对照组不做处理。术后16周后处死动物取材进行大体观察，常规HE染色，测量各实验组兔肋软骨的平均宽度，并行生物力学检测测定所有肋软骨的抗压强度及弯曲强度。结果：各实验组家兔的胸廓整体形态均较良好，各组及各处理方法之间并无明显差别。方法1处理的修复组织平均宽度显著大于健康对照组肋软骨的平均宽度(P <0.01)，方法2、3处理的修复组织平均宽度与健康对照组肋软骨平均宽度相比无显著性差异(P>0.05)；生物力学检测显示：3种处理方法之间均存在差异(P<0.01)，方法3处理的修复组织的抗压、弯曲强度与健康对照组比较差异无统计学意义（P＞0.05），方法1，2处理的修复组织的抗压、弯曲强度明显低于健康对照组(P<0.01)，方法2处理的修复组织的抗压、弯曲强度优于方法1；组织切片HE染色病理观察，可见方法1、2处理的修复组织主要为纤维组织，方法3处理的修复组织内，可见新生的软骨细胞和大量的软骨细胞外基质。结论：成软骨诱导的BMSCs膜片可以促进肋软骨供区软骨细胞的再生，修复肋软骨供区缺损，维持胸廓的正常形态和稳定性，从而降低术后胸廓畸形的发生率。
Use of Autologous Chondrocytes and Bioinert PerforatedChambers to Tissue-Engineer Cartilage In Vivo
1. The study of isolation,culture and identification of rabbit chondrocytes
Objective: Auricular chondrocytes were isolated from the earscartilages of newzealand white rabbits with the methods of collagenase Ⅱandtrypsin enzymatic digestion. Methods: chondrocytes were harvested from rabbitears by enzymatic digestion. The morphological changes of cell and growthfeatures were observed through the phase-contrast microscope every day. Thechondrocytes were indentified with toluidine blue staining and chondrocytesgrowth curve was depicted. Results: Chondrocytes could be isolatedsuccessfully with collagenase Ⅱ and trypsindigestion.The primarily culturedchondrocytes were polygonal, and became dedifferentiated after3passages. Thechondrocytes maintained the morphology and histological staining patternwithin the first3passages. Conclusions: The study showed that the method of isolation and culture of chondrocytes was simple and feasible. The secondpassage chondrocytes grow well and are suitable for experiment.
2. Design and fabrication of the chondrocytes/Collagen andchondrocytes/PLGA-Collagen In Vitro
Objective: To compare the effect between the Collagen gel andPLGA/Collagen gel hybrid scaffolds as vehicles for chondrogenesis in vitro.Methods: P2rabbit auricular chondrocytes(RACS) were seeded into thescaffolds;(1) RACSwere seeded into Collagen gel scaffold;(2) RACSwereseeded into PLGA/Collagen gel scaffold.After culturing for14days in vitro，thesamples were evaluated by HE, toluidine blue， safranin-O staining, andelectronical microscope scanning. Results: RACS/Collagen gel andRACS/PLGA gel samples formed white cartilage-like tissue in vitro when theywere harvested14days later. HE，toluidine blue and safranin-O staining showedthe chondrocytes were surrounded by much ECM. It was observed thatchondrocytes attached, growed, and proliferated well on the scaffold by SEM.Conclusions: These results indicated that collagen gel and PLGA gel scaffoldswere suitable for use and the two kinds of scaffolds can be used fortissue-engineering cartilage construction.
3. Using bioinert perforated chambers to tissue engineer cartilage in vivo
Objective: To explore the potential applications of a chamber for invivo tissue engineering, and to establish a novel model for in vivotissue-engineered cartilage. Methods: Four experimental groups were includedin this study:(A) chambers+chondrocytes/collagen gel;(B) chambers+chondrocytes/PLGA gel;(C) chondrocytes/collagen gel alone; and (D) chondrocytes/PLGA gel alone. Groups C and D served as controls. The sampleswere implanted subcutaneously in the donor rabbit, and the contents wereharvested at8weeks after implantation. Results: Histological andimmunohistochemical staining and RT-PCR results revealed regeneratedcartilage-like tissue in group B and small, irregularly shaped islands ofopalescent tissue in group A. In contrast, the control groups displayed vascularinvasion and inflammatory reaction, which eventually led to fibrosis andabsorption. Conclusions: Reproduced cartilages were obtained in animmunocompetent animal model through the use of a bioinert perforatedchamber.
Recreating Engineering-Cartilage Without Scaffolds OnCostal Cartilage Repair and Regeneration
1. The study of isolation, culture and identification of rabbits bone marrowmesenchymal stem cells
Objective: To culture and amplify rabbit bone marrow mesenchymalstem cells (BMSCs) by using density gradient centrifugation combiningadherent cultivation method, and to identify them. Methods: The growth andmorphology of BMSCs were observed by phase-contrast microscope. TheBMSCs growth curve was depicted by MTT; chondrogenic inductor, adiposeinductor and osteogenic inductor were used to induce the BMSCs intochondrocytes, adipocytes and osteoblast in vitro. The differentiated cells wereidentified by toluidine blue, oil red O and alizarin red staining respectively.Results: The BMSCs could be obtained by the density gradient centrifugationcombining adherent cultivation method and the BMSCs could be differentiated into chondrocytes, adipocytes and osteoblasts after induction treatment.Conclusions: The density gradient centrifugation combining adherentcultivation method was a relatively simple and feasible way which could isolateand culture BMSCs. The BMSCs could proliferate and multi-differentiate verywell.This suggested that BMSCs were superior seed cells for cartilage tissueengineering.
2. Fabrication and chondrogenic differentiation of BMSCs Sheet
Objective: To obtain BMSCs sheet using continuous culturecombining simple mechanical method and to assess the feasibility ofchondrogenic differentiation. Methods: BMSCs were cultured in the culturemedium supplemented with chondrogenic inductor for14days.The harvestedcell sheets were evaluated by histological and scanning electronical microscope.Results: The intact BMSCs sheet could be gained by continuous culture andsimple mechanical method. HE staining showed that the cell sheet was made of6to8layers of cells. The safranin-O, toluidine blue staining showed positiveresults and scanning electron microscope examination revealed that the cellsarranged closely and the matrix secreted abundantly. Conclusions: The studyindicated that the BMSCs sheet could be generated by using this simpletechnique, and the sheet could differentiate into chondrocytes..
3. The Effects of chondrogenic BMSCs Sheet on Costal Cartilage Repairand Regeneration
Objective: The purpose of the study was to investigate the effects ofchondrogenic BMSCs Sheet on costal cartilage repair and regeneration. Methods: Sections of the costal cartilage of16male rabbits were removed withthe costal perichondrium and the costal cartilage junction was intactly left. Therabbits were treated with three different methods: Method1: The costalperichondrium was left unsutured; Method2: The costal perichondrium wassutured to form a tubular structure after the implantation of alone BMSCs Sheet;Method3: The costal perichondrium was sutured to form a tubular structure afterthe implantation of chondrogenic BMSCs Sheet. The specimens were harvestedafter16weeks postoperatively. Results: The average width of the costal cartilagein the method1group was significantly wider than that of the control group(P<0.01). The biomechanical strength of the costal cartilage in themethod1,2groups were significantly weaker than that of the control group (P<0.01); No obvious differences in the average widths and the biomechanicalstrength were tested between the method3group and that of the control group(P>0.05).HE-stained results showed the repaired tissues in1、2methods weremainly fibrous cartilages, and new cartilage-like tissue regneneration could befound in the3method group, The chondrocyte-like cells were surrounded bythe abundant cartilaginous matrix in the regenerated cartilage. Conclusions: Ourstudy has indicated that chondrogenic BMSCs Sheet could promote theregneneration of new cartilage, which significantly enhanced the strength of therepaired tissue, strengthened the stability of the thorax, and thus, indirectlyreduced the incidence of thoracic deformities.
引文
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