胶原凝胶包埋软骨细胞接种CPPf/PLLA支架体外构建组织工程软骨

目的：探讨软骨细胞均匀、高效种植于三维支架的细胞接种方法。方法：将胶原凝胶包埋的软骨细胞整合入CPPf／PLLA三维支架并进行体外培养，细胞计数检测细胞粘附情况，倒置显微镜观察细胞在支架内分布的均一性，组织形态学检测细胞-胶原凝胶-支架复合物形成软骨组织的情况。结果：超过90％的种植细胞能有效、均匀种植于CPPf／PLLA支架，体外培养3周的复合物能形成较成熟的工程化软骨组织。结论：胶原凝胶包埋软骨细胞三维接种能有效提高组织工程软骨的体外构建质量，同时结合了两种材料的优势。     

胶原水凝胶支架用于软骨组织工程的实验研究

目的探讨Ⅰ型胶原浓度对胶原水凝胶理化性能的影响,以及在体外组织工程模型中不同浓度的Ⅰ型胶原水凝胶对软骨细胞表型和基因表达的影响。方法制备Ⅰ型胶原浓度为12、8、6 mg/mL的3种胶原水凝胶,记为C12、C8、C6。扫描电镜观察形貌,并通过溶胀性能和抗压性能测试表征其理化性能。取2只新生7日龄新西兰大白兔的关节软骨,酶消化法分离、培养软骨细胞。取第2代软骨细胞与3种胶原水凝胶复合体外培养(C12组、C8组及C6组),1 d后采用二乙酸荧光素(fluorescein diacetate,FDA)/碘化丙啶(propidium iodide,PI)染色,激光共聚焦显微镜观察细胞活性,体外培养14 d后行HE和甲苯胺蓝染色观察组织学形态,实时荧光定量PCR测定软骨细胞相关基因,即Ⅱ型胶原、蛋白聚糖(Aggrecan)、Ⅰ型胶原、Ⅹ型胶原、Sox9 mRNA表达。结果随着Ⅰ型胶原浓度从6 mg/mL提高到12 mg/mL,胶原水凝胶的理化性质出现明显变化,扫描电镜下纤维网络变得致密;192 h时C6、C8、C12溶胀率依次增大,分别为0.260±0.055、0.358±0.072、0.539±0.033,比较差异均有统计学意义(P<0.05);压缩模量逐渐增加,分别为(4.86±0.96)、(7.09±2.33)、(11.08±3.18)kPa,比较差异均有统计学意义(P<0.05)。体外培养1 d,3组凝胶中软骨细胞经FDA/PI染色后,绝大部分被染成绿色,仅有少数被染成红色。14 d后组织学观察示:C12组软骨细胞在组织中聚集明显,并出现明显的甲苯胺蓝异染为紫红色的现象;C8组中也有部分增殖聚集的区域,细胞周围有较明显的甲苯胺蓝异染现象;C6组细胞分布较为均匀,也有较明显的甲苯胺蓝异染现象。实时荧光定量PCR检测显示,3组Ⅱ型胶原mRNA和Aggrecan mRNA表达水平相近;Ⅰ型胶原、Sox9和X型胶原mRNA表达水平C12组最高,C6组最低,C8组介于二者之间,组间差异均有统计学意义(P<0.05)。结论提高Ⅰ型胶原浓度至12 mg/mL后,胶原水凝胶具有更好的理化性质,但软骨细胞纤维化和肥大相关基因表达也有所上调。     

骺板软骨细胞复合三维支架体外构建组织工程软骨的研究

目的探讨将骺板软骨细胞复合三维支架经体外培养,构建组织工程软骨的效果及其生物学特点. 方法将3周龄幼兔第1代骺板软骨细胞与液态的生物凝胶混合,接种于聚磷酸钙纤维/L-聚乳酸(CPPF/PLLA)三维支架材料,构建组织工程软骨组织块,连续培养4周.行大体、倒置显微镜及组织学、Ⅰ型和Ⅱ型胶原免疫组织化学光镜观察,定量检测硫酸糖胺多糖(GAG)含量. 结果构建的组织工程软骨块在培养过程中能保持其初始外形,种子细胞呈稳定的三维均相分布,外观逐渐呈乳白色、半透明,硬度亦不断增加.培养1周有软骨细胞陷窝形成,2周后形成富含Ⅱ型胶原和蛋白聚糖、具有典型软骨组织结构的工程化软骨,且Ⅰ型胶原逐渐转为阴性.4周时构建软骨的组织结构与天然骺板软骨相类似,硫酸GAG含量平均为天然骺板软骨的34%以上. 结论骺板软骨细胞复合三维支架体外培养可生成典型软骨,且可形成类似天然骺板软骨的组织结构,能满足修复骺板缺损的基本要求.体外培养1～2周可能是植入体内修复骺板缺损的较佳时机.     

Ⅰ型胶原半月板支架负载纤维软骨细胞体外培养

[目的]观察Ⅰ型胶原半月板支架对纤维软骨细胞的吸附作用及对细胞生物学性状的影响，评价其作为半月板组织工程支架的可行性及价值。[方法]构建Ⅰ型胶原半月板支架，将体外培养的兔半月板纤维软骨细胞吸附于该支架上三维立体培养，通过相差倒置显微镜、组织学、扫描电镜及免疫组织化学检测支架对半月板纤维软骨细胞的表型、增殖及功能的影响。[结果]Ⅰ型胶原能制成理想大小、形状的三维立体多孔半月板支架，纤维软骨细胞在支架孔壁贴附良好，维持表型稳定，分泌胞外基质。[结论]Ⅰ型胶原半月板支架细胞相容性良好，但力学性能相对较差，通过与其它生物材料复合以提高其机械力学强度，可望制得理想的半月板组织工程支架。     

软骨细胞三维培养扩增与液态凝胶复合构建软骨的实验研究

目的通过在微载体上进行三维培养扩增软骨细胞,并结合液态胶原构建组织工程软骨。方法比较兔软骨细胞在单层培养与微载体上进行三维培养扩增软骨细胞的保持表型能力。幼兔软骨细胞分别进行单层和微载体三维培养扩增,并进行体外球型培养评价软骨细胞保持表型能力和糖胺多糖的定量生化分析。三维培养扩增软骨细胞与液态鼠尾胶原复合构建组织工程软骨,分别以低细胞密度（2×10^6个／mL）和高细胞密度（1．2×10^7个／mL）两种细胞密度接种,培养14d后通过组织学特种染色鉴定构建组织特性。结果微载体培养的软骨细胞可以保持良好活力和保持表型能力,与单层培养体系相比较,细胞糖胺多糖的定量生化分析的差异具有统计学意义（P〈0．05）。三维培养扩增软骨细胞复合液态鼠尾胶原构建组织工程软骨,体外14d后发现高细胞密度接种时可形成形态稳定的软骨组织。组织学染色显示为透明软骨样组织。结论在微载体上进行三维培养扩增软骨细胞可以加强细胞保持表型能力。软骨细胞与液态胶原合成后,以高细胞密度（1．2×10^7个／mL）可以在体外形成形态稳定的组织工程软骨。     

软骨微粒脱细胞基质和纤维蛋白凝胶支架皮下构建组织工程软骨

目的观察皮下植入异体软骨细胞复合异种软骨微粒脱细胞基质(Cartilage microparticle acellular matrix,CMACM)和纤维蛋白胶(Fibrin glue,FG)为支架形成组织工程软骨的可能性。方法制备猪耳廓CMACM,体外培养成年兔的耳软骨细胞,将不同的混合物植人5只成年兔背部皮下。A组:异体软骨细胞复合CMACM和FG;B组:自体软骨细胞复合CMACM和FG;C组:CMACM和FG。将每只兔子背部皮肤均分为6个区,分别植入不同混合物各两个点,以备两次取材。观察并记录皮下植入体的形态变化,分别于植入后8周和12周取材,行组织学检测。结果A组和C组未能形成软骨样组织。B组8周可以形成软骨样组织,周围炎症细胞数量较多;12周时形成的软骨组织成分单一,周围没有炎症反应,类似于正常软骨,且新生的软骨组织中均长有许多小血管,新生软骨的厚度不超过1 mm。结论将同种异体软骨细胞复合CMACM和FG植入皮下,不能形成软骨样组织:而以自体软骨细胞为种子细胞则可以得到软骨样组织,但新生软骨的体积和厚度有限,并且新生的软骨组织中长有许多小血管,可能更有利于新生软骨组织的长期存活。     

软骨细胞外基质/壳聚糖复合多孔支架和骨髓间充质干细胞构建组织工程软骨

[目的]探讨软骨细胞外基质和壳聚糖制备复合多孔支架,同时并对小鼠骨髓间充质干细胞构建组织工程软骨的可行性进行观察.[方法]以猪关节软骨细胞外基质和壳聚糖为原料,采用冷冻干燥法制备软骨细胞外基质/壳聚糖复合多孔支架.通过扣描电镜观察材料内部结构及孔径大小,液体位移法测定材料的孔隙率,MTT方法检测支架浸提液毒性.将小鼠的骨髓间充质干细胞(BMSCs)分离培养并用TGF-β1成软骨诱导后,与材料复合培养,扫描电镜观察细胞在材料上的生长粘附情况.[结果]软骨细胞外基质/壳聚精复合支架具有疏松多孔结构,孔径大小(159±36)μm,孔隙率为90.5%±2.3%,复合支架中的软骨细胞外基质成分甲苯胺蓝染色、番红O染色均呈阳性,MTT结果显示支架无细胞毒性.诱导的骨髓间充质干细胞在支架表面生长良好.[结论]软骨细胞外基质/壳聚糖复合材料具有合适的孔径和孔隙率,生物相容性良好,是组织工程软骨的良好支架载体.     

不同支架材料体外构建组织工程软骨的研究

目的:体外构建组织工程软骨,筛选更为适合组织工程软骨构建的支架材料。方法:体外获取SD大鼠肋软骨细胞。采用第一代软骨细胞作为种子细胞,接种于壳聚糖/明胶和BMG/生物蛋白胶支架,体外培养的不同时间对其进行HE、甲苯胺蓝染色、Mas s on染色、免疫学检测、扫描电镜观察。结果:在培养2周时,BMG/生物蛋白胶各种染色结果显示软骨细胞在其表面以及内部分布均匀,蛋白多糖和Ⅱ型胶原染色阳性;壳聚糖/明胶表面细胞稍多于前者,但内部细胞数量极少且分布不均,染色结果不如前者明显。随着培养时间的延长各种检测均显示有大量的软骨细胞特异性的蛋白多糖和Ⅱ型胶原的表达,壳聚糖/明胶凸显出明显的优势。结论:体外成功构建组织工程软骨,软骨细胞在BMG/生物蛋白胶上的生长、增殖和分泌基质情况优于壳聚糖/明胶支架。     

离心力在体外构建组织工程软骨中的作用

目的探讨离心力对软骨细胞功能表达和组织工程软骨结构的影响。方法采用组织化学和免疫组织化学观察组织工程软骨结构以及Ⅱ型胶原表达情况,应用DMMB分光法测定组织工程软骨硫酸化糖胺多糖(GAG)的含量。结果体外培养2、4、8周时,静态培养的组织较离心培养的组织Ⅱ胶原免疫组织化学染色弱;并且其GAG含量低于离心培养组织GAG含量,各组差异均有统计学意义(F分别为12.3、10.2、9.1,P<0.05)。离心培养的组织工程软骨GAG含量于第4周达到高峰,均值为(7.60±0.79)%。结论离心力刺激软骨细胞分泌GAG和Ⅱ型胶原,并且影响组织工程软骨结构的排列。     

胶原-透明质酸-硫酸软骨素复合三维支架体外构建组织工程软骨的实验研究

目的探讨以胶原(collagen,Col)透明质酸(hyaluronic acid,HA)硫酸软骨素(chondroitin sulfate,CS)为支架材料构建组织工程软骨的可行性.方法以乙基-(3-二甲基氨基丙基)碳化二亚胺盐酸盐为交联剂通过冷冻干燥的方法制备Col-HA-CS复合支架及单纯Col支架.通过扫描电镜、HE染色对Col-HA-CS复合支架材料形态进行观察.分离培养幼兔关节软骨细胞,将体外扩增的软骨细胞接种在两种支架上,通过组织学、扫描电镜观察软骨细胞在支架上的生长形态;通过生物化学功能检测细胞-支架复合物中DNA、糖胺聚糖(glycosaminoglycan,GAG)含量;RTPCR方法检测在Col HA CS复合支架上的软骨细胞ColⅡ的表达情况.结果软骨细胞在Col-HA-CS复合支架材料上增殖分化良好,并保持软骨细胞特异的分化ColⅡ表型,培养21 d后已有软骨样组织形成,出现软骨陷窝.DNA和GAG含量测定显示软骨细胞在复合支架上随时间增加逐渐扩增并分泌大量的GAG,含量明显高于单纯Col支架材料,差异有统计学意义(P＜0.05).结论 Col-HA-CS复合支架材料可为软骨细胞生长分化及组织形成提供一个良好的环境,在软骨组织工程的支架材料领域有较广泛的应用前景.     

组织工程软骨体外构建的相关实验研究

目的 探索组织工程软骨体外构建技术体系可行性.方法 种子细胞选用胎儿软骨细胞(口服药物流产胎儿,胎龄3～6个月).酶消化法获得第1代细胞,以50×106/ml浓度均匀接种于经聚乳酸(PLA)包埋聚乙醇酸(PGA)高分子聚合物支架,形成细胞-支架复合体,在体外静态培养.分别于2周、4周、8周进行大体观察、扫描电镜及组织学检测.结果 体外构建的组织工程软骨,随培养时间延长,色泽由2周时的乳白色逐渐呈现半透明,8周时接近正常软骨外观.扫描电镜显示软骨细胞与材料具有良好相容性,培养7天PGA纤维之间有基质沉积.HE染色示2周有大量软骨陷窝形成和均匀嗜碱性基质分泌,Safranin'O染色示基质有酸性蛋白多糖分布,Massons's trichome染色示基质有胶原成分,但含量较少,经免疫组织化学检测为特异Ⅱ型胶原.培养4周胶原成分开始明显增多,软骨陷窝形态接近成熟,8周细胞外基质蛋白多糖和Ⅱ型胶原含量丰富且分布均匀.结论 以成熟软骨细胞为种子细胞,运用组织工程技术在体外能构建出具有正常软骨组织结构特征的人组织工程软骨.     

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.     

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.     

脱细胞软骨细胞外基质取向支架复合软骨细胞构建组织工程软骨的实验研究北大核心CSCD

目的探讨脱细胞软骨细胞外基质(acellular cartilage extracellular matrix,ACECM)取向支架复合软骨细胞构建组织工程软骨的可行性。方法取市售猪关节软骨组织,分离培养关节软骨细胞并传代。取第3代软骨细胞行PKH26荧光标记,MTT检测标记对细胞增殖无影响后,分别取标记及未标记的软骨细胞复合ACECM取向支架并体外培养后,大体观察支架形态,倒置显微镜、荧光显微镜观察软骨细胞在支架中的黏附、生长和分布情况,扫描电镜观察支架中细胞形态,Ⅱ型胶原免疫荧光染色观察软骨细胞外基质分泌情况。将PKH26标记的软骨细胞-支架复合物植入裸鼠背部皮下腔隙,术后观察裸鼠一般情况,4周后分子荧光活体成像系统无创伤性评估细胞-支架复合物生长情况,取材行大体观察以及番红O、甲苯胺蓝、Ⅱ型胶原免疫组织化学染色观察,评价形成软骨组织的能力。结果细胞-支架复合物体外培养7 d,大体观察呈半透明并具有一定硬度;倒置显微镜和荧光显微镜观察软骨细胞在支架上能良好黏附生长,并沿支架管道方向生长,分泌Ⅱ型胶原。细胞-支架复合物植入裸鼠皮下后,分子荧光活体成像系统观察示细胞均存活;术后4周,大体观察见复合物呈类软骨样组织,组织学染色及Ⅱ型胶原免疫组织化学染色示细胞周围软骨细胞外基质分泌,可见"陷窝"样结构形成。结论 ACECM取向支架有利于软骨细胞的黏附、增殖及取向性分布类似于正常软骨结构,并在裸鼠皮下成功异位构建组织工程软骨。     

胶原在软骨组织工程中的应用

软骨组织工程的出现,为解决软骨修复这个临床难题提供了新的方法.然而寻找一种合适的生物载体材料是目前软骨组织工程的热点.胶原是一种天然支架材料,具有良好的生物相容性、可降解性和生物活性,可作为细胞三维生长支架,并且有维持种子细胞增殖、黏附和分化的能力.本文就胶原及其复合材料在软骨组织工程方面的应用状况与前景作一综述.     

利用胶原构建皮肤组织工程支架的研究

目的利用胶原构建皮肤组织工程支架。方法用Na2S、弹性蛋白酶预处理胎牛皮得到胶原纤维；经蛋白酶M降解胶原纤维后，0．5mol／L醋酸溶液溶解得到酸溶性胶原；再用蛋白酶N处理上述酸溶性胶原，最终得到生物相容性良好的胶原溶液；将胶原溶液构建皮肤组织工程支架。通过SDS-PAGE试验，分析酸溶性胶原分子量及基本结构；用皮肤组织工程支架材料包覆大鼠创面，观察创面愈合情况，研究支架材料对大鼠创伤愈合的影响；在支架材料上种植成纤维细胞，观察细胞扩增情况，以及支架材料对细胞移植影响。结果通过特异性蛋白酶M处理得到的酸溶性胶原，显示典型的1型胶原SDS-PAGE图谱；构建的皮肤组织工程支架呈多孔海绵状，孔径为50～200μm；在支架材料上种植成纤维细胞扩增情况良好；用于修复大鼠创面，具有促进创面愈合作用。结论酸溶性胶原经蛋白酶N处理后，生物相容性良好，适合于皮肤组织工程支架的构建，并具有良好生物活性。     

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.     

软骨细胞与静电纺丝聚已内酯支架灌流培养构建组织工程软骨的实验研究

目的 采用静电纺丝聚已内酯(polycaprolactone,PCL)支架与软骨细胞复合培养,比较静态和灌流生物反应器培养条件下对细胞增殖及基质分泌的影响.方法 构建PCL支架,自制灌流生物反应器,分离兔软骨细胞,培养后接种于PCL支架,分为灌流培养组和静态培养组.在培养第3、7、14天对支架-细胞复合体行扫描电镜观察,DNA、糖胺聚糖和总胶原定量检测;在培养第14天分析软骨特异性基因表达并观察软骨基质分泌情况.结果 电镜观察PCL支架纤维直径(1.67±0.76) μm,孔径(17.65土7.11)μm,可见支架中软骨细胞黏附生长良好,灌流培养条件下细胞增殖快,且较好地保持了软骨细胞特征形态.在培养第7天,灌流培养组DNA定量高于静态培养组;在培养第3、7和14天,灌流培养组糖胺聚糖定量均高于静态培养组,灌流培养组糖胺聚糖/DNA比值均高于静态培养组.在培养第14天,灌流培养组Ⅱ型胶原、蛋白聚糖基因表达增加;软骨分化指数高于静态培养组.在培养第14天,组织学染色可见灌流培养促进细胞的增殖和渗透生长,提高了软骨基质的分泌,并见软骨陷窝样结构.结论 在灌流生物反应器培养条件下,静电纺丝PCL支架与软骨细胞复合培养可促进软骨细胞的增殖和基质的分泌,提高了组织工程软骨的质量.     

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.     

兔髂骨骺板软骨细胞体外构建骺板样软骨组织

目的　利用组织工程学技术体外构建骺板样软骨组织。方法　从 4～ 5周龄兔髂骨骺板软骨处获取软骨细胞 ,在离心管内轻微离心后 ,体外培养。行组织学观察。结果　培养至第 7天时 ,细胞呈现定向分化 ,形态与体内骺板软骨细胞相类似 :肥大软骨细胞体积较大、呈圆形或椭圆形 ;增殖、成熟软骨细胞体积小 ,呈圆形或扁圆形 ;细胞周围充满大量的细胞外基质。这些不同分化阶段的细胞形成了分化区带 ,肥大软骨细胞位于上侧 ,增殖、成熟细胞位于中间 ,其次是散在静止软骨细胞。培养第 14天 ,分化区带更加明显 ,增殖、成熟细胞和肥大软骨细胞呈现纵向定向排列。培养第 2 1天 ,组织表面出现膜样的结构。结论　体外构建的骺板样软骨组织与天然骺板的组织学形态极为相似。从髂骨处骺板处获取肥大软骨细胞进行体外构建骺板软骨材料 ,更具有临床实用性