动物源性骨软骨支架修复兔膝关节复合缺损

目的探讨软骨细胞-动物源性骨软骨支架复合体修复兔膝关节骨软骨复合缺损的可行性和影响因素。方法将改良贴壁离心法获取的骨髓间充质干细胞（bone marrowm esenchymal stem cells,BMSCs）/诱导分化的软骨细胞共培养后与经深低温冷冻、脱脂、脱钙、真空冷冻干燥和辐照消毒的动物源性骨软骨支架复合,构建共培养细胞＋软骨-骨一体化复合支架。27只新西兰大白兔随机分为实验组（A组）、对照组（B组）和空白组（C组）,每组9只。于兔股骨髁间窝处钻一深6mm的骨软骨复合缺损,A组植入共培养细胞＋骨软骨复合支架,B组植入骨软骨复合支架,C组不植入任何支架材料和细胞,分别于术后4周、8周和12周取材,行大体观察、苏木精—伊红染色和甲苯胺蓝染色,并对各标本的软骨切片进行组织学评分。结果随着时间的延长,A组大体观察见复合缺损区已完全修复,局部无凹陷,新生组织和周围组织融合;B组新生组织仍不能完全填充缺损;C组缺损区仍明显。苏木精—伊红染色和甲苯胺蓝染色见A组软骨缺损区由新生的透明软骨样组织修复,细胞呈柱状排列,极性好,软骨陷窝明显,骨缺损区由骨样组织修复,新生软骨和软骨下骨以及宿主骨界面耦合良好;B组新生软骨细胞无软骨陷窝,排列混乱,各界面藕合欠理想;C组可见陈旧性肉芽组织生长并突出于缺损区表面。甲苯胺蓝染色阳性率和组织学评分结果表明,A组与B、C两组之间的差异具有统计学意义（P〈0.05）。结论 BMSCs/诱导分化的软骨细胞共培养细胞复合动物源性骨软骨支架对兔膝关节软骨和软骨下骨的复合缺损具有修复作用。     

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。     

人真皮成纤维细胞体外构建组织工程化软骨的初步探索

目的 利用软骨细胞提供的体外软骨诱导微环境,探讨人真皮成纤维细胞在体外构建软骨的可行性.方法 分别培养猪的软骨细胞与人真皮成纤维细胞,将2种细胞按1:1(软骨细胞:成纤维细胞)比例混匀,以5.0×10~7/ml的终浓度接种于聚羟基乙酸支架(PGA,直径9 mm,高2mm)作为共培养组,相同终浓度的单纯软骨细胞和单纯成纤维细胞分别接种于相同支架作为阳性对照及阴性对照.每组各接种3个标本,每个接种细胞悬液200 μl.全部标本均于体外培养8周后取材,通过大体观察、湿重测定、组织学及免疫组化等相关检测对构建软骨进行评价.结果 软骨细胞组(阳性对照组)基本保持了复合物初始的大小和形状,组织周边和中央均有较均质的软骨陷窝样结构形成,表达软骨特异性细胞外基质;共培养组的组织稍有缩小,组织周边也有软骨陷窝样结构形成,表达软骨特异性细胞外基质,但组织内大部分区域形成了纤维样组织,特别是通过人核抗原免疫组化和对应的Safranin O染色结果,可以看到少量人核抗原阳性的细胞形成了较成熟的陷窝样结构,表达软骨特异性基质.单纯成纤维细胞组(阴性对照组)在体外培养过程中逐渐皱缩变形,未形成软骨样组织.结论 软骨细胞共培养体系可以有效地诱导人真皮成纤维细胞中一定比例的细胞向软骨分化,并能在体外构建软骨样组织.     

软骨细胞与成纤维细胞共培养体外构建软骨的初步研究

目的 软骨微环境对软骨形成具有重要作用.本实验探讨软骨细胞与成纤维细胞共培养体外构建软骨的可行性.方法 分别培养猪软骨细胞与人成纤维细胞,将两种细胞按3:7(软骨细胞:成纤维细胞)比例混匀,以5.0×107/ml终浓度接种于聚羟基乙酸支架(PGA,直径9mm,高2mm)作为共培养组,相同终浓度单纯软骨细胞和单纯成纤维细胞分别接种于相同支架作为阳性对照及阴性对照.每组各接种3例标本,每例接种细胞悬液200μl.全部标本均体外培养12周后取材,通过大体观察、组织学及免疫组化等检测对构建软骨进行评价.结果 各组细胞均与材料支架粘附良好.体外培养12周后,阳性对照组(软骨细胞组)及共培养组均形成成熟的软骨样组织,组织学及免疫组化显示有成熟的软骨陷窝样结构及Ⅱ型胶原表达.软骨细胞组在体外培养过程中能基本保持复合物初始的大小和形状,形成的软骨在组织学上也教为均匀一致.共培养组在培养过程中稍有缩小,在复合物的周边区域形成了均质的软骨样组织,但在近中心区域存在一定量的纤维性组织.阴性对照组(单纯成纤维细胞组)在体外培养过程中逐渐皱缩变形,未形成软骨样组织.结论 软骨微环境在体外软骨分化及软骨形成中具有重要作用,软骨细胞与成纤维细胞体外共培养能形成软骨样组织.     

膝关节滑膜间质干细胞分离、培养及其多向分化潜能特性的研究

目的 探讨晚期骨关节炎患者膝关节滑膜间质干细胞(synovium-derived mesenchymalstem cells,SMSCs)体外分离、培养的可行性及其在体外向脂肪细胞、成骨细胞和软骨细胞定向分化的特性.方法 取膝关节滑膜组织,胶原酶消化获得有核细胞.挑选单细胞克隆,筛选获得SMSCs.流式细胞技术检测细胞表面特异性抗原标志.培养至第三代,分别向脂肪细胞、成骨细胞和软骨细胞诱导分化.油红O染色鉴定向脂肪细胞分化;碱性磷酸酶染色、茜素红染色鉴定向成骨细胞分化;甲苯胺蓝染色鉴定向软骨细胞分化.RT-PCR检测脂肪细胞、成骨细胞标志基因.Ⅱ型胶原免疫组化染色检测软骨细胞Ⅱ型胶原的表达.结果 原代SMSCs体外培养呈葵花样细胞集落,传代后可见圆形巨噬样细胞和纺锤形成纤维样细胞,融合后呈成纤维细胞样生长.CD44、CD90呈阳性,CD34、CD71和CD45呈阴性.向脂肪细胞诱导21d,油红O染色阳性;RT-PCR检测有脂蛋白酶、乙二腈及PPARγ2表达;向成骨细胞诱导7、28 d,ALP,茜素红染色阳性,有ALP、Osteopontin及Osteocalcin表达;向软骨细胞诱导21d,甲苯胺蓝染色阳性,Ⅱ型胶原免疫组化染色阳性.结论 晚期骨关节炎患者膝关节滑膜组织可以分离、培养获得SMSCs. SMSCs具有向脂肪细胞、成骨细胞和软骨细胞发生定向分化的潜能.     

兔关节软骨块和骨髓间充质干细胞共培养

[目的]探讨与兔关节软骨块共培养对兔骨髓间充质干细胞(RBMSCs)分化的影响。[方法]全骨髓贴壁培养法分离培养RBMSCs,对P3代细胞进行氯甲基苯甲酰氨荧光染料(CM)标记。用4周龄雄性新西兰大白兔股骨头关节面制备软骨块。分为3组,分别为单纯细胞组、细胞+软骨组和单纯软骨组,于第7、14、21 d细胞爬片,行甲苯胺蓝染色法检测和II型胶原免疫组化染色。[结果]经过3周培养,单纯细胞组细胞仍保持骨髓间充质干细胞的特点;细胞+软骨组RBMSCs与兔关节软骨块混合共培养后,RBMSCs细胞形态由长梭形逐渐变为短小的三角形或不规则形,表现出典型的"铺路石"样改变;甲苯胺蓝染色阳性和II型胶原免疫组化阳性,符合软骨细胞特点;单纯软骨组软骨块在培养液中成活,无细胞生长。[结论]兔关节软骨块所营造的微环境能够促进RBMSCs向软骨细胞方向分化,此种RBMSCs能够在兔关节软骨块表面贴附生长。     

可注射性藻酸钙凝胶载体组织工程性软骨的实验研究

目的 对经体外培养的软骨细胞与藻酸钙凝胶载体复合形成的组织工程性软骨进行组织形态学观察。方法 取 2周龄新西兰白兔的膝关节软骨细胞,经体外培养两次传代后与藻酸钠溶液复合,使细胞密度为 5× 10 6/ml、 NaCl 0.135 mol/L、 K 2HPO 4 0.1 mol/L、藻酸钠的质量分数为 1％,然后注入葡萄糖酸钙使终浓度为 40 mmol/L。在实验组 (A、 B组 )兔的背部皮下注入复合藻酸钙载体的软骨细胞悬液,对照组 (C、 D组 )动物或注入无载体的软骨细胞悬液或注入无细胞的藻酸钙载体,分别于注射后第 2周、第 4周取材,进行 HE和甲苯胺蓝染色。结果 实验组,于注射复合软骨细胞的藻酸钙凝胶载体第 2周时, HE染色结果显示皮下注射点出现大量软骨细胞增殖,第 4周时有软骨组织形成;甲苯胺蓝染色见有软骨细胞和软骨基质形成。在注入无载体的软骨细胞悬液组 (C组 ),部分动物的皮下注射点有少量软骨组织形成,而在注入无细胞的藻酸钙载体组 (D组 )则未见软骨形成。结论 应用可注射性藻酸钙凝胶载体复合软骨细胞可在同种异体动物体内形成新生软骨组织。     

成人骨髓间充质干细胞体外诱导分化为软骨细胞的实验研究

目的研究体外三维培养条件下,成人骨髓间充质干细胞(BMSCs)定向分化软骨细胞可行性。方法用密度梯度离心法分离纯化BMSCs,并传代扩增后,用离心三维培养法在软骨诱导剂下进行诱导培养,以常规培养液培养的BMSCs作为阴性对照,于诱导培养第21天取出标本行苏木精-伊红染色、甲苯胺蓝染色、s-100蛋白免疫组织化学检测。结果BMSCs经21d的离心三维诱导培养后,培养管出现软骨外观组织块,组织切片甲苯胺蓝染色呈明显异染,s-100蛋白免疫细胞化学检测阳性。对照组阴性。结论成人BMSCs在三维培养条件下可成功诱导分化为软骨细胞。     

犬骨髓基质干细胞体外定向分化为软骨细胞

目的 体外诱导犬骨髓基质干细胞(BMSCs)定向分化为软骨细胞，探讨体外诱导成软骨的方法和条件。方法 自犬肋骨取骨髓2～3ml，体外行原代和传代培养扩增，顺序加入碱性成纤维细胞生长因子(bFGF)和转化生长因子β1(TGF-β1)，以培养瓶内较高细胞浓度培养，诱导BMSCs分化为软骨细胞。甲苯胺蓝、阿新蓝染色检测软骨基质的分泌，免疫组织化学染色检测软骨特异性Ⅱ型胶原表达。结果 诱导的软骨样细胞甲苯氨蓝异染性、阿新蓝染色阳性；Ⅱ型胶原免疫组织化学检测阳性。结论 应用bFGF和TGF-β1体外可以诱导犬BMSCs分化为软骨细胞，诱导的软骨细胞可作为软骨组织工程较理想的种子细胞。     

软骨细胞与脂肪基质细胞共培养体外构建软骨的初步研究

目的 探讨软骨细胞与脂肪基质细胞(adipose-derived stromal cells,ADSCs)共培养体外构建软骨的可行性,并阐明软骨细胞提供的软骨微环境能否诱导ADSCs向软骨细胞分化并形成软骨组织.方法 分别培养扩增人ADSCs与猪耳软骨细胞,将2种细胞按7:3(ADSCs:软骨细胞)比例混匀,以5.0×107/ml的细胞终浓度接种于聚羟基乙酸/聚乳酸(PGA/PLA,直径8 mm,高2 mm)支架作为共培养组,以相同终浓度的单纯软骨细胞和单纯ADSCs分别接种相同支架作为阳性对照组及阴性对照组,以30％上述浓度(1.5×107/ml)的单纯软骨细胞接种作为低浓度软骨细胞对照组.每组各接种6例标本,每例接种细胞悬液200μl.全部标本均于体外培养8周时取材,通过大体观察、组织学、免疫组化及湿重、蛋白多糖定量检测等方法对新生软骨进行初步评价.多样本t检验统计分析各组湿重及蛋白多糖含量差异.结果 各组细胞均与材料粘附良好.共培养组及阳性对照组体外培养8周时基本保持了复合物初始大小和形状,大体观察2组均形成了较成熟软骨组织,组织学显示大量软骨基质和软骨陷窝形成,免疫组化显示软骨特异性细胞外基质Ⅱ型胶原分泌.定量测定结果表明,共培养组的平均湿重为(174±12) mg,平均蛋白多糖含量为(7.6±0.4) mg,两者分别达到阳性对照组的75％ (P＜ 0.01)和79％ (P＜ 0.01).阴性对照组(单纯ADSCs组)明显皱缩变形,组织学未见成熟软骨陷窝.低浓度软骨细胞组明显变薄,新生软骨平均湿重为(85 ±5) mg,是阳性对照组的37％ (P＜ 0.01),只在局部形成了不连续的软骨组织.结论 软骨细胞与ADSCs共培养能够在体外构建较成熟的软骨组织,软骨细胞能够诱导ADSCs成软骨分化及体外形成软骨组织.     

Teratoma formation and hepatocyte differentiation in mouse liver transplanted with mouse embryonic stem cell-derived embryoid bodies

We previously reported that mouse embryonic stem (ES) cells are capable of differentiating into hepatocytes in cultured embryoid bodies (EBs) and that hepatocytes generate in the recipient liver injected with cultured day-9 EB cells via spleen without the formation of a teratoma. Because ES cells frequently form teratomas in recipient mice, we investigated incidence of teratoma formation when day-9 EBs derived from ES cells were transplanted directly into the subcapsule of mouse liver. In contrast to injection of day-9 EB cells through the portal vein via the spleen, direct subcapsular injection of cultured day-9 EB cells into liver, and even of cultured day-15 EBs, resulted in an high incidence of teratoma in the liver. In teratomas of livers injected directly with day-15 EBs, hepatocytes were detected singly and in clusters. These results imply that undifferentiated cells capable of developing into teratomas exist in cultured EBs, and even in cultured day-15 EBs containing differentiated hepatocytes.     

Isolation of hepatocyte-like cells from mouse embryoid body cells

We previously reported that embryoid body (EB) cells derived from embryonic stem (ES) cells are capable of differentiating into functional hepatocyte-like cells both in vitro and in vivo. Because transplantation of EB-derived cells into the liver via the spleen resulted in a low incidence of teratoma formation, purification of hepatocyte-like cells is required to prevent teratoma formation. The aim of this study was to purify hepatocyte-like cells from cultured EBs. For the isolation of hepatocyte-like cells, EBs cultured for 15 days were treated with trypsin-EDTA. The disaggregated cells were plated on a gelatin-coated dish as a monolayer. These cells were separated by Percoll gradient centrifugation, enriched by magnetic cell sorting, and purified by FACS. The purified hepatocyte-like cells in monolayer cultures were positive for immunostaining for albumin and expressed albumin mRNA, but not Oct3/4 mRNA. Transplantation of the purified hepatocyte-like cells derived from mouse ES cells might be an effective treatment for liver failure.     

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。     

Stem cell-derived chondrocytes for regenerative medicine

The regenerative capacity of cartilage is limited. Transplantation methods used to treat cartilage lesions are based mainly on primary cultures of chondrocytes, which dedifferentiate during cultivation in vitro and lose their functional properties. Stem cells are considered as an alternative source to generate cells for two reasons: first, they can almost indefinitely divide in culture, and second, they are able to differentiate into various mature cell types. Herein, we asked the question whether chondrocytes could be differentiated from mouse embryonic stem (ES) cells to a state suitable for regenerative use. When cultivated as embryoid bodies (EBs), murine ES cells differentiate into mesenchymal progenitor cells, which progressively develop into mature, hypertrophic chondrocytes and transdifferentiate into calcifying cells recapitulating all of the cellular processes of chondrogenesis. Chondrocytes isolated from EBs exhibit a high regenerative capacity. They dedifferentiate initially in culture, but later reexpress stable characteristics of mature chondrocytes. However, in cultures of chondrocytes isolated from EBs, additional mesenchymal cell types can be observed. Mesenchymal stem (MS) cells from bone marrow have already been used in tissue engineering settings. We compared the chondrogenic differentiation of MS and ES cells.     

Enrichment of hepatocytes differentiated from mouse embryonic stem cells as a transplantable source

BACKGROUND: We previously reported that hepatocytes can be differentiated from embryonic stem (ES) cells by way of embryoid body (EB) formation and are transplantable into the mouse liver. However, the transplantation of EB-derived cells frequently resulted in teratoma formation in the recipient liver. In the present study, we eliminated the tumorigenic cells from EB outgrowths and examined the effects of enriched ES-cell-derived hepatocyte transplantation into an injured liver. METHODS: On day 15 in culture, the EBs were partially disaggregated and subcultured. Hepatocytes in the subcultured cells were examined by the expression of hepatocyte markers. Undifferentiated cells contaminating in the EB-derived cells were eliminated by Percoll discontinuous gradient centrifugation. Furthermore, undifferentiated cells, endothelial cells, and macrophages were eliminated by magnetic cell sorting using platelet/endothelial cell adhesion molecule (PECAM)-1 and Mac-1 antibodies. These enriched ES-cell-derived hepatocytes were then transplanted into the injured mouse liver. RESULTS: Percoll centrifugation and PECAM-1 antibodies eliminated the undifferentiated cells expressing Oct-3/4 from the EB-derived cells. ES-cell-derived hepatocytes showed expression of liver-related genes, synthesis of urea and glycogen, and structural characteristics during subculture. A transplantation study showed that the enriched ES-cell-derived hepatocytes integrated into the injured mouse liver and produced no teratomas. When the ES-cell-derived hepatocytes were transplanted into a CCl4-injured liver, the liver function was subsequently improved. CONCLUSIONS: Functional hepatocytes can be differentiated from mouse ES cells by way of EB formation. The elimination of undifferentiated cells from the EBs provides transplantable cells for liver failure without tumorigenicity.     

软骨细胞和骨髓间质干细胞混合培养构建组织工程软骨的实验研究

目的 探讨软骨细胞和骨髓间质干细胞(bone mesenchymal stem cells,BMSCs)混合培养对构建组织工程软骨的影响,并确定两者的最佳比例.方法 体外分离培养兔(1月龄)关节软骨细胞和BMSCs,按不同比例(软骨细胞和BMSCs的浓度比为:4:1、2:1、1:1、1:2、1:4及单纯软骨细胞)混合培养一代.以4×107/ml的细胞终浓度接种40μl于聚乳酸-羟基乙酸共聚物[poly(lactic-co-glycolic acid),PLGA:4mm×4mm×2mm),静态培养2 d,然后移至周期性压力场(压力0～200 kPa,频率:0.1 Hz,时间:8 h/d)培养3周.收获组织工程软骨行大体观察,组织切片,定量检测糖胺聚糖(glyeosaminoglycans,GAGs)含量、DNA含量及Ⅱ型胶原染色面积.结果 软骨细胞和BMSCs混合培养组与单纯软骨细胞组相比,体积较大,表面光滑,有弹性,有光泽.组织学检查显示混合培养组结构致密,细胞外基质分布更均匀,其中软骨细胞和BMSCs的浓度比为2:1组可见软骨陷窝.混合培养组的Ⅱ型胶原染色面积、GAGs含量、DNA含量高于单纯软骨细胞组,其中软骨细胞和BMSCs的浓度比为2:1组含量最高,与其他比例混合组比较,差异有统计学意义.结论 软骨细胞和BMSCs混合培养能提高组织工程软骨的质量,其中以软骨细胞和BMSCs的浓度比为2:1最佳.     

同种异体软骨细胞与自体骨髓基质干细胞混合共培养构建软骨皮下移植的可行性研究

背景：软骨组织工程的种子细胞问题是目前研究的热点和难点,如何找到一种既能够避免对自体软骨进行取材又能够达到稳定软骨构建目的的方法呢？本研究尝试利用少量同种异体羊软骨细胞作为软骨诱导微环境提供者,与扩增后的羊自体BMSC混合共培养并植入皮下环境,探讨利用同种异体软骨细胞共培养构建软骨皮下移植的可行性。方法：本实验对山羊软骨细胞和BMSC分别进行取材和分离培养扩增,并将以上细胞分为以下四组进行混合并接种在PGA支架材料上：A组：100%自体软骨细胞;B组：30%自体软骨细胞＋70%自体BMSCs;C组：30%同种异体软骨细胞＋70%自体BMSCs;D组：100%同种异体软骨细胞。经过体外构建6周后植入羊皮下进行体内构建12周,对所形成的组织块进行大体观察和组织学染色等评价。结果：自体软骨细胞组和自体软骨细胞混合自体BMSC组皮下移植后可见成熟软骨组织形成,但同种异体软骨细胞参与的两组（包括同种异体软骨细胞混合自体BMSC的实验组和单纯异体软骨细胞组）在皮下环境中都因为较强的免疫反应未能形成软骨组织。结论：同种异体软骨细胞以及PGA支架材料的存在对于组织工程软骨在羊皮下环境的构建有负面影响。