异体软骨细胞诱导骨髓基质细胞成软骨分化的量效关系

目的 探讨利用异体软骨细胞作为诱导因素,与骨髓基质细胞(BMSCs)共培养体外构建软骨复合物的可行性,以及两种细胞混合比例与构建软骨质量的量效关系.方法 体外分别培养扩增BMSCs与异体软骨细胞,软骨细胞与BMSCs的混合比例分别为1:9(A组)、2:8(B组)、3:7(C组)、100%软骨细胞(D组)、100%BMSCs(E组),以5.0×107/ml的细胞终浓度接种于聚羟基乙酸 (PGA)材料支架上培养,各组标本均于体外培养6周后取材,通过大体观察、糖胺聚糖(GAG)含量测定、组织学以及免疫组织化学等方法检测组织工程化软骨形成的情况.结果 各组细胞均与材料黏附良好,C、D两组标本基本保持原有的大小和形状,外观洁白、光滑类似软骨组织;组织学显示两组均有连续的软骨陷窝样结构形成,免疫组织化学显示有大量Ⅱ型胶原沉积.定量结果显示,C组的GAG含量达到D组的70%以上.其他各组培养物体积明显缩小,组织学及免疫组织化学显示软骨样组织形成不佳.结论 异体软骨细胞与BMSC共培养可以构建出较好的软骨组织,表明软骨细胞对BMSCs发挥诱导作用,但是软骨细胞必须达到一定的数量要求.     

软骨细胞诱导骨髓基质细胞构建软骨的体内外比较

目的 应用软骨细胞诱导骨髓基质细胞(Bone Marrow Stromal Cells,BMSCs)体外构建软骨,比较其体内植入前后软骨相关生物学特性的差异,探讨共培养构建软骨临床应用的可行性.方法 体外分别培养扩增猪关节软骨细胞和BMSCs,两者按2:8比例混合(软骨细胞:BMSC),以5.0×107/ml的细胞终浓度接种于聚乳酸包埋的聚羟基乙酸支架,体外培养8周后部分标本植入裸鼠皮下,再经体内8周后取材.通过大体观察、糖胺聚糖含量(GAG)测定、生物力学测试、组织学,以及免疫组化等方法对体内植入前后标本的软骨相关生物学特性进行比较.结果 体外培养8周时,所有标本均形成了软骨样组织,但质地较软,组织结构相对较为松散.体内植入8周后,共培养构建软骨能维持良好的软骨外观,而且GAG含量及弹性模量均明显高于体外标本(p＜0.01),组织学及免疫组化显示体内标本的组织结构致密,基质及Ⅱ型胶原显色程度均明显强于体外标本.结论 软骨细胞诱导BMSCs构建的软骨在体内皮下环境中能维持良好的软骨特性,而且植入体内后能继续向成熟软骨发育.     

软骨细胞诱导骨髓基质干细胞构建带内支撑的组织工程化软骨

目的探讨以聚羟基乙酸(PGA)包裹特定形态的医用假体材料多孔高密度聚乙烯(HDPE,商品名为MEDPOR)为支架,应用软骨细胞诱导骨髓基质干细胞(BMSCs),共培养构建特定形态的带内支撑组织工程化软骨医用假体的可能性。方法以直径3mm、长5mm的圆柱形HDPE,外裹1mm厚PGA为支架,将体外分别培养的新生猪BMSCs和耳郭软骨细胞按7:3混合,以10×107/ml细胞浓度接种于支架上,同时以相同浓度的单纯软骨细胞和单纯BMSCs分别接种,作为阳性对照组(PC组)和阴性对照组(NC组)。经体外培养2周及在裸鼠皮下移植4、8周后取材,行大体观察、组织学、组织化学及免疫组化检测。结果各组细胞均与材料黏附良好。实验组和阳性对照组均形成了大体形态良好的HDPE-软骨复合体,内支撑的HDPE与外层软骨结合紧密。组织学可见成熟的软骨陷窝结构,软骨渗入HDPE孔隙内部、异染基质及Ⅱ型胶原呈强阳性表达。结论以HDPE为内支撑,外裹PGA的支架,接种混合细胞,可于皮下构建特定形态、组织学良好的HDPE-软骨复合体。     

软骨细胞诱导骨髓基质干细胞构建带内支撑的组织工程化软骨

目的 探讨以聚羟基乙酸(PGA)包裹特定形态的医用假体材料--多孔高密度聚乙烯(HDPE,商品名为MEDPOR)为支架,应用软骨细胞诱导骨髓基质干细胞(BMSCs),共培养构建特定形态的带内支撑组织工程化软骨医用假体的可能性.方法 以直径3 mm、长5 mm的圆柱形HDPE,外裹 1 mm厚PGA为支架,将体外分别培养的新生猪BMSCs和耳郭软骨细胞按7∶3混合,以10×10 7/ml细胞浓度接种于支架上,同时以相同浓度的单纯软骨细胞和单纯BMSCs分别接种,作为阳性对照组(PC组)和阴性对照组(NC组).经体外培养2周及在裸鼠皮下移植4、8周后取材 ,行大体观察、组织学、组织化学及免疫组化检测.结果 各组细胞均与材料黏附良好.实验组和阳性对照组均形成了大体形态良好的HDPE-软骨复合体,内支撑的HDPE与外层软骨结合紧密.组织学可见成熟的软骨陷窝结构,软骨渗入HDPE孔隙内部、异染基质及Ⅱ型胶原呈强阳性表达.结论 以HDPE为内支撑,外裹PGA的支架,接种混合细胞,可于皮下构建特定形态、组织学良好的HDPE-软骨复合体.     

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。     

自体骨髓间充质干细胞和软骨细胞共培养复合同种异体完全脱蛋白骨修复关节软骨全层缺损的研究

[目的]探讨自体骨髓间充质干细胞(bone m arrow-derived m esenchym al stem cells,BMSCs)与软骨细胞共培养复合同种异体完全脱蛋白骨(fu lly deprote in ized bone,FDB)修复关节软骨缺损的可行性,评价修复效果,为优化种子细胞源提供依据。[方法]取浓度为3×106/m l的第二代BMSCs和软骨细胞,按2:1比例混匀共培养作为种子细胞。FDB与共培养细胞复合接种植入修复缺损为实验A组、单纯FDB为对照B组和不处理为空白对照C组,移植8、16周后经人体观察、组织学评分和免疫组化染色评价缺损的修复。[结果]共培养的软骨细胞基质合成丰富,细胞增殖快。A组缺损修复组织呈软骨样,表面光滑平坦,与周围软骨整合的软骨细胞更为成熟。B组和C组的修复组织呈纤维组织和无修复。组织学评分表明A组优于B、C 2对照组,差异具有统计学意义(P<0.01),B组与C组差异无统计学意义(P>0.05)。免疫组化染色显示A组修复组织的细胞为透明软骨样细胞,柱状排列,Ⅱ型胶原染色阳性,与周围软骨及软骨下骨整合良好。[结论]自体BMSCs与软骨细胞共培养作为种子细胞,BMSCs能增强软骨细胞的增殖,促进软骨细胞基质合成,缩短软骨细胞培养时间和减少传代次数,节省大量的软骨细胞,与FDB复合后能有效修复关节软骨缺损。     

软骨细胞与骨髓基质细胞共培养裸鼠体内构建软骨组织的实验研究

目的：探讨软骨细胞在裸鼠体内促进骨髓基质细胞（BMSCs）向软骨分化并形成软骨组织的可行性。方法：从SD大鼠中分别分离出BMSC和软骨细胞进行体外培养。收集软骨细胞培养上清液,作为BMSCs诱导液从第2代开始进行诱导分化,7天后取出标本,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。SD大鼠BMSCs与软骨细胞按一定比例（7：3）混匀,取5.0×107个混合细胞/ml的各组细胞悬液接种至壳聚糖生物材料,体外培养一周后植入裸鼠皮下,相同数量的单纯软骨细胞或BMSCs同样方法植入,分别作为阳性对照及阴性对照,1.5×107个软骨细胞同样植入作为低浓度软骨细胞对照。各组均8周后取材检测。结果：经诱导后的大鼠BMSCs的Ⅱ型胶原免疫组化检测阳性,RT-PCR检测Ⅱ型胶原和aggrecanmRNA呈阳性表达;混合细胞组及阳性对照组均形成了成熟的软骨,组织学可见成熟软骨陷窝、异染基质及Ⅱ型胶原表达;BMSCs组仅形成了纤维性组织;低浓度软骨细胞组在局部形成了少量软骨。结论：软骨细胞能在一定程度上提供软骨形成的微环境,诱导BMSCs在裸鼠体内向软骨组织分化并形成软骨组织。 还原     

骨髓基质细胞移植修复半月板无血运区损伤的实验研究

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

力学刺激促进软骨细胞与骨髓基质干细胞共培养体外软骨分化

目的：研究不同的应力刺激对软骨细胞与骨髓基质干细胞（BMSCs）共培养体外构建组织工程化软骨的影响。方法：分离、培养、扩传兔MSCs及软骨细胞,二者按7：3比例混和,以5．0×107／ml的细胞密度接种于聚羟基乙酸（PGA）支架上,一周后根据不同的施加力分为4组：离心组、摇床组、搅拌组,静止培养作为对照组。6周后取材行相关检测。结果：三受力组形成的细胞材料复合物基本保持原来的体积与外形。HE染色结果显示大量成熟软骨陷窝形成,细胞外基质沉积均匀;Safranin-O及甲苯胺兰染色显示有大量的GAG形成,免疫组化检测II型胶原表达强阳性。三受力组标本组织湿重、体积、GAG含量等指标均优于对照组。结论：力学刺激有利于促进少量软骨细胞与BMSCs共培养体外软骨分化;并在三维支架材料上构建组织工程化软骨。     

体内外联合培养提高组织工程管状软骨构建质量

目的 探讨利用组织工程技术提高管状软骨的构建质量.方法 PLA/PGA共纺材料均匀地缠绕在硅胶模具上形成管状支架材料.从兔耳郭软骨分离软骨细胞,在体外培养扩增后接种于支架材料,软骨细胞-材料复合物先体外培养2 d.实验动物分为3组:直接植入组(DI组),自体软骨细胞材料复合物直接植入兔颈部皮下.联合培养组(CC组),复合物继续用相同的培养基在体外培养2周后再植入兔颈部皮下.单纯材料组(SO组),在颈部皮下单纯植入材料和硅胶模具.在植入后1,2,4和8周定时取材,行大体观测,组织学染色,生物化学和分子生物学检测和生物力学评定,主要评估炎症反应程度和软骨形成情况.结果 植入1周时,DI和SO组材料纤维周围有明显的炎症反应,大量白细胞浸润;第4周时,两组炎症明显减退,DI组形成的工程软骨被纤维组织分隔成散在的岛状;第8周时,材料基本降解,炎症消散,但DI组软骨无改善.体外培养2周后,CC组软骨细胞已分泌了许多细胞外基质,将部分降解的材料纤维包裹在内,此时植入体内没有引起明显的炎症反应;第4周时,材料完全降解并形成了相对均质、成熟的软骨组织,各项检测指标均优于同时间点DI组构建的软骨,接近正常气管软骨.结论 体内外联合培养可以明显减轻软骨细胞-PLA/PGA复合物植入具有免疫功能的实验动物自体内引发的炎症反应,提高组织工程管状软骨的构建质量.     

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

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.     

Immunological response against allogeneic chondrocytes transplanted into joint surface defects in rats

Rat chondrocytes isolated from the articular-epiphyseal cartilage complex were transplanted into defects prepared in articular cartilage and subchondral bone. Transplants were taken for examination after 3 and 8 wk. Cartilage formed by syngeneic chondrocytes did not evoke formation of infiltrations. Contrary to that, in the vicinity of cartilage produced by allogeneic chondrocytes numerous infiltrating cells were present and cartilage resorption could be observed. Cyclosporine-A (CsA) treatment of recipients of allogeneic chondrocytes only partially suppressed accumulation of infiltrating cells and matrix resorption. Antichondrocyte immune response of chondrocyte graft recipients was studied by evaluation of spleen mononuclear cells (SMC) stimulation in mixed splenocytechondrocyte cultures and by evaluation of antichondrocyte cytotoxic antibodies. No difference in stimulation of SMC from intact rats by syngeneic and allogeneic chondrocytes was observed. Stimulation by allogeneic chondrocytes was slightly but significantly higher in recipients of syngeneic grafts. SMC of allogenic chondrocyte recipients were strongly stimulated by allogeneic chondrocytes. This response was absent in recipients treated with CsA. Spontaneous antichondrocyte cytotoxic antibody activity was detected in intact rats and in recipients of syngeneic grafts. In recipients of allogeneic chondrocytes the antibody response against allogeneic chondrocytes was raised but was statistically not significant owing to the considerable variation in the level of spontaneously occurring antichondrocyte antibodies.     

The arthroscopic implantation of autologous chondrocytes for the treatment of full-thickness cartilage defects of the knee joint

Autologous chondrocyte implantation is an established option for the treatment of full-thickness cartilage defects of the knee. Open implantation has a high morbidity. On a resorbable polymer fleece, autologous chondrocytes can be implanted arthroscopically. Transosseous anchoring assures high initial stability of the implant. Tibial defects can be addressed. The arthroscopic technique for the implantation of autologous chondrocytes eliminates a substantial amount of the side effects known to occur after open autologous chondrocyte implantation procedures.     

膨体聚四氟乙烯（ePTFE）内支撑组织工程软骨的构建实验研究

目的：探讨骨髓间充质干细胞和软骨细胞混合培养体外构建ePTFE软骨复合体的可能性。方法：实验组：分离、获取、扩增兔骨髓间充质干细胞和软骨细胞,二者按7：3比例混和,接种到以膨体聚四氟乙烯（ePTFE）为内支撑外裹聚羟基乙酸（PGA）支架上,体外培养8周后,行大体、组织学、Ⅱ型胶原免疫组织化学和生物化学检测。对照组：利用实验组支架单纯接种骨髓间充质干细胞行体外培养。结果：实验组：体外培养8周后形成形态良好的软骨样组织复合体,组织学可见成熟软骨陷窝、异染基质、Ⅱ型胶原表达阳性。对照组：无软骨形成。结论：兔骨髓间充质干细胞和软骨细胞混合培养,可以在体外构建出特定形状、结构组织学良好的ePTFE软骨复合体。     

Mitotic activity in chondrocyte transplantation from the in vitro phase to the in vivo phase

The transplantation of devitalized allogenic matrices vehiculating autologous chondrocytes, previously isoled and seeded on them could be a solution to the problem of repairing lesions of the joint cartilage. For the matrix/cell "composite" to be "graftable" the cells must continue to duplicate and produce cartilaginous matrix even after transport in vivo. The present study analyzes the mitotic activity of chondrocytes planted on devitalized allogenic cartilage and grafted in living animals. Chondrocytes of joint cartilage of lambs were isolated enzymatically and then seeded in vitro on devitalized allogenic cartilaginous matrices for 3 weeks. At the end of the co-culture period, these matrix/chondrocyte composites were transplanted in subcutaneous pockets of athymic mice. The experimental and control samples were evaluated subsequent to explantation by histological study and incorporation of tritiated thymidine. The results obtained revealed an important decrease in the values for the incorporation of thymidine beginning from experimental time 0 (pre-implant evaluation) up to day 28 after implantation, followed by a mild increase at the experimental time of 42 days. This study demonstrated the tendency of articular chondrocytes cultivated in vitro and subsequently transplanted in vivo on a support of devitalized allogenic cartilaginous matrix to modify mitotic activity from very high values for the first experimental times, typical of the in vitro phases of cellular expansion, to very low values, more similar to the behavior of articular chondrocytes in vivo.     

Evaluation of articular cartilage with 3D-SPGR MRI after autologous chondrocyte implantation

To assess the maturation process of the cartilage after autologous chondrocyte implantation (ACI), we performed a longitudinal study with three-dimensional spoiled gradient-recalled (3D-SPGR) magnetic resonance imaging (MRI). Five knees of five patients on which ACI of the femoral condyle was performed were studied. The signal intensity of reparative tissue approached that of normal articular cartilage with time. The volume of reparative tissue remained at an almost constant level after implantation. During second-look arthroscopy, the areas on which ACI was performed were covered with hyaline-like cartilage, and the reparative tissue removed by biopsy consisted of normal chondrocytes and extracellular matrix. The increased signal intensity of the reparative tissue represents maturation of implanted autologous chondrocytes. 3D-SPGR MRI is thought to be useful for evaluating reparative tissue after autologous chondrocyte implantation.     

Repair of human articular cartilage after implantation of autologous chondrocytes

Tissue engineering is an increasingly popular method of addressing pathological disorders of cartilage. Recent studies have demonstrated its clinical efficacy, but there is little information on the structural organisation and biochemical composition of the repair tissue and its relation to the adjacent normal tissue. We therefore analysed by polarised light microscopy and immunohistochemistry biopsies of repair tissue which had been taken 12 months after implantation of autologous chondrocytes in two patients with defects of articular cartilage. Our findings showed zonal heterogeneity throughout the repair tissue. The deeper zone resembled hyaline-like articular cartilage whereas the upper zone was more fibrocartilaginous. The results indicate that within 12 months autologous chondrocyte implantation successfully produces replacement cartilage tissue, a major part of which resembles normal hyaline cartilage.     

自体富血小板血浆对自体脂肪颗粒组织移植存活率影响的研究

目的探讨混合注射富血小板血浆最佳浓度,并通过临床混合注射自体富血小板血浆及自体移植脂肪组织,观察自体富血小板血浆对移植脂肪颗粒组织存活率的影响。方法提取抽脂术中获取的脂肪颗粒组织中的脂肪来源干细胞,实验分为4组：A组（对照组）加入10％胎牛血清,B组添加5％富血小板血浆,C组添加10％富血小板血浆,D组添加15％富血小板血浆,传代培养,定时获取细胞行MTT实验,测试细胞生长曲线,并观察细胞形态变化。临床实验选取15例患者,抽取腹部脂肪组织颗粒后进行纯化;同时抽取静脉血,采用离心法提取自体富血小板血浆。纯化的脂肪颗粒与自体富血小板血浆按照10：1的质量比进行混合注射移植。术前、术后1周及术后3个月,患者在B超下行皮下组织厚度测定。结果第3代脂肪来源干细胞培养观察显示,C组有较好的细胞增殖性及活力;临床研究皮下组织厚度增加率为（158．4±83．1）％;术后3个月,皮下组织厚度增加率为（106．4±70．7）％。结论10％富血小板血浆混合浓度对细胞增殖较为适宜,且临床应用可以有效提高自体脂肪颗粒组织移植的存活率,移植后远期仍能达到较为满意的效果,可在临床上进一步推广应用。     

Response of human chondrocytes prepared for autologous implantation to growth factors

This study investigated metabolism of autologous chondrocytes after initial expansion immediately before implantation. Chondrocytes cultured in either monolayers or alginate beads were treated with insulin-like growth factor-1 (IGF-1), osteogenic protein-1 (OP-1), or a combination. Proteoglycan synthesis and DNA content were tested in both cultures. Alginate beads also were analyzed with live/dead cell assay, safranin O/fast green stain for histology, and immunohistochemistry with antibodies against collagen type II and VI, aggrecan, decorin, and fibronectin. In monolayers, autologous chondrocytes changed their morphologic appearance. In alginate, they maintained chondrocytic phenotype. Growth factors, especially combined, promoted cell survival and induced chondrocyte proliferation. OP-1 stimulated the largest cartilage-specific matrix and the most accumulation of collagen type II and fibronectin, although the overall matrix synthesized by autologous chondrocyte implantation cells was smaller than that produced by normal chondrocytes. The clinical implications of this study suggest a significant promise for anabolic growth factors in cartilage repair as a potential modifying therapy for the enhancement of chondrocytic phenotype of autologous chondrocytes.     

Stem cells and debrided waste: two alternative sources of cells for transplantation of cartilage

Implantation of autologous chondrocytes and matrix autologous chondrocytes are techniques of cartilage repair used in the young adult knee which require harvesting of healthy cartilage and which may cause iatrogenic damage to the joint. This study explores alternative sources of autologous cells. Chondrocytes obtained from autologous bone-marrow-derived cells and those from the damaged cartilage within the lesion itself are shown to be viable alternatives to harvest-derived cells. A sufficient number and quality of cells were obtained by the new techniques and may be suitable for autologous chondrocyte and matrix autologous chondrocyte implantation.