生长因子对成人关节软骨细胞的促增殖作用

目的观察不同生长因子对成人关节软骨细胞（adult human articular chondrocytes，AHAC）增殖的影响，探索AHAC体外大量扩增的方法。方法以酶消化法从成人关节软骨分离细胞，条件培养基培养；传2代细胞分别用不同浓度成纤维细胞生长因子2（fibroblast growth factor-2，FGF-2）、转化生长因子β1（transforming growth factor β1，TGF—β1）、血小板衍生因子bb（platelet derived growth factor-bb，PDGF-hb）、肝细胞生长因子（hepatocyte growth factor，HGF）或其不同组合作用。用MTT法比较细胞增殖情况，用组化和免疫组化检测观察细胞表型变化。结果FGF-2、TGF—β1、PDGF—bb、HGF均有促AHAC增殖的作用，其最大效应剂量分别是50ng/ml、1ng／ml、1ng／ml、20ng／ml。5ng／ml FGF-2＋1ng/ml TGF-β1有最强的促增殖作用，继续加用PDGF—bb和（或）HGF无进一步促进作用；用这一因子组合培养AHAC，可以传10代以上，细胞扩增2000倍以上，且传9代细胞仍弱表达Ⅱ型胶原和aggrecan。结论FGF-2、TGF-β1、PDGF—bb、HGF均对AHAC有一定的促增殖作用；5ng／ml FGF-2＋1ng/ml TGF-β1有最大的促增殖效应，细胞短期内大量扩增，且在大量扩增的同时维持了一定的软骨细胞表型，因此是合适的AHAC体外大量扩增促进剂。     

两种诱导方法对去分化软骨细胞Ⅱ型胶原的诱导

目的：用两种不同的诱导方法使去分化的永生化软骨细胞第50代重新表达软骨细胞的Ⅱ型胶原标志性表型。方法：利用NA无血清诱导培养法以及离心管聚集体诱导培养法分别诱导培养去分化的永生化人关节软骨细胞第50代，继用Ⅰ、Ⅱ、Ⅲ型胶原的免疫组化染色、Ⅱ型胶原的RT-PCR检测、胶原定量检测该永生化软骨细胞的Ⅱ型胶原表型表达情况。结果：NA无血清诱导培养法和离心管聚集体诱导培养法均能成功诱导去分化永生化人关节软骨细胞的Ⅱ型胶原表达，但二者促进Ⅱ型胶原分泌并无显著差异。结论：去分化永生化人关节软骨细胞可以经不同的诱导方法而重新表达Ⅱ型胶原表型，无血清诱导培养基与离心管聚集体培养法都能使Ⅱ型胶原重新表达。     

藻酸钠微球三维立体培养恢复去分化软骨细胞表型的实验研究

[目的] 研究在连续体外单层培养条件下不同接种密度对大鼠关节软骨细胞分化状态的影响,并探讨藻酸钠微球三维立体培养恢复去分化关节软骨细胞表型的调节机制.[方法]以正常密度(3×10<,4>/cm<'2>)或低密度(3×10<'2>/cm<'2>)分别连续体外单层传代培养大鼠膝关节软骨细胞使其去分化,RT-PCR检测Ⅰ、Ⅱ型胶原和聚集蛋白聚糖mRNA的表达以确定其分化状态.取正常密度培养时的去分化软骨细胞,藻酸钠微球包被4周以恢复其表型,在该立体培养过程中将特异的SIRT1抑制剂-EX-527加入到培养基中抑制其表达,甲苯胺兰染色微球中软骨细胞分泌的细胞外基质,Western blot检测各种条件下SIRT1和Sox-9的表达.[结果] 当以正常密度连续体外单层培养时,软骨细胞在第4代发生去分化,低密度时于第1代即明显去分化,此时SIRT1和Sox9表达明显降低.藻酸钠微球三维立体培养能显著地增强去分化软骨细胞中Ⅱ型胶原、聚集蛋白聚镛mRNA以及SIRT1、Sox9蛋白的表达,同时降低Ⅰ型胶原mRNA的表达.EX-527不仅限制藻酸钠微球中的软骨细胞周围细胞外基质的大量生成而且还抑制了SIRT1和Sox9的表达.[结论]关节软骨细胞连续体外单层培养的去分化与细胞接种密度有关,低密度培养加速去分化过程.藻酸钠微球三维立体培养恢复去分化关节软骨细胞表型的作用可能是通过细胞-细胞、细胞-细胞外基质间的相互作用,从而激活SIRT1的表达,继而增强Sox9的转录活性来实现.     

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。     

两种诱导方法对去分化软骨细胞Ⅱ型胶原的诱导

目的:用两种不同的诱导方法使去分化的永生化软骨细胞第50代重新表达软骨细胞的Ⅱ型胶原标志性表型.方法:利用NA无血清诱导培养法以及离心管聚集体诱导培养法分别诱导培养去分化的永生化人关节软骨细胞第50代,继用Ⅰ、Ⅱ、Ⅲ型胶原的免疫组化染色、Ⅱ型胶原的RT-PCR检测、胶原定量检测该永生化软骨细胞的Ⅱ型胶原表型表达情况.结果:NA无血清诱导培养法和离心管聚集体诱导培养法均能成功诱导去分化永生化人关节软骨细胞的Ⅱ型胶原表达,但二者促进Ⅱ型胶原分泌并无显著差异.结论:去分化永生化人关节软骨细胞可以经不同的诱导方法而重新表达Ⅱ型胶原表型,无血清诱导培养基与离心管聚集体培养法都能使Ⅱ型胶原重新表达.     

兔关节软骨细胞聚集培养的生物学性状观察

目的 观察聚集培养软骨细胞生物学性状的变化,为软骨细胞移植建立合适的体外培养方法。方法2001年11月至2004年6月酶消化法分离成年兔关节软骨细胞,分别低密度单层培养和高密度聚集培养,组化及免疫组化法观察细胞表型变化。结果低密度培养时,前3代细胞增殖迅速,但很快去分化,3代以后增殖缓慢,细胞表型大部丢失;聚集培养时,软骨细胞去分化速度减缓;传3代后细胞聚集培养,细胞表型部分恢复。结论聚集培养利于维持软骨细胞表型,原代细胞聚集培养或传代培养后聚集培养是较好的获取大量优良软骨细胞的培养方式。     

人脂肪来源的成体干细胞体外向软骨细胞诱导分化的研究

目的研究人脂肪来源的成体干细胞（ADSC）体外能否向软骨细胞成功分化，探讨其作为组织工程软骨种子细胞的可行性。方法自成人皮下取少量脂肪组织，经机械剪切及Ⅰ型胶原酶消化后获取成体干细胞，体外培养扩增；流式细胞仪鉴定细胞表型；第5代细胞采取离心管中微块法培养，培养液中加入转化生长因子（hTGF-β2）等诱导剂诱导其向软骨细胞分化；诱导14d的细胞团经消化后获得单细胞悬液，接种于玻片上行形态学观察，甲苯胺兰、免疫组织化学染色及逆转录-聚合酶链式反应（RT—PCR）用以鉴定软骨细胞表型。结果从人体皮下脂肪组织中可成功分离到ADSC，细胞可于体外大量扩增；第5代细胞大多数表达CD29、CD90，而CD14、CD45抗原表达阴性；RT—PCR结果提示诱导后的细胞可表达黏多糖（GAG）及Ⅱ型胶原，诱导后的细胞块分离成单个细胞后可见其形态较诱导前发生明显变化，甲苯胺兰染色和Ⅱ型胶原免疫组织化学染色为阳性。对照组未加诱导因子的细胞则未能观察到上述变化。结论成人皮下脂肪组织中可分离到大量ADSC；ADSC可在体外成功诱导向软骨细胞分化；ADSC有望作为组织工程软骨构建中的种子细胞。     

诱导骨髓间充质干细胞向软骨细胞分化的体外研究

目的 探讨转化生长因子β1（transforming growth factor β1，TGF—β1）、胰岛素样生长因子1（insulinlike growth factor1，IGF-1）在诱导骨髓间充质干细胞（marrow mesenchymal stem ceils，MSCs）向软骨细胞分化过程中的相互作用，并研究细胞密度对MSCs向软骨细胞分化的影响。方法 取健康昆明种小白鼠骨髓，用全骨髓贴壁法筛选获得MSCs，体外培养传代。采用特定的诱导培养使MSCs向软骨细胞分化，按培养基内添加生长因子的不同分成3个实验组和对照组。实验组分别为：TGF—β1＋IGF-1联合应用组（TGF—β1 10ng／ml、IGF-1 50ng／m1）；TGF—β1单独应用组（TGF—β1 10ng／m1）；IGF-1单独应用组（IGF-1 50ng／m1）；对照组不添加任何生长因子。TGF—β1＋IGF-1联合应用组于诱导14d和21d，分别进行甲苯胺蓝染色及免疫荧光双染法鉴定；于诱导7、14和21d各组分别提取诱导细胞总RNA，进行RT—PCR扩增，检测TGF—β1、IGF-1对诱导细胞Ⅱ型胶原表达量的影响；比较MSCs在平板培养及细胞团培养时，Ⅱ型胶原表达量的差异。结果TGF—β1＋IGF-1联合应用组诱导培养14d，诱导软骨细胞甲苯胺蓝染色呈阳性，免疫荧光染色可见诱导软骨细胞的细胞外基质含有Ⅱ型胶原。各组基因扩增产物的凝胶电泳可见，TGF—β1＋IGF-1联合应用组和TGF—β1单独应用组Ⅱ型胶原扩增片段呈阳性；IGF-1单独应用组和对照组，未见Ⅱ型胶原扩增条带；凝胶成像系统灰度扫描示Ⅱ型胶原表达量TGF—β1＋IGF-1联合应用组各时间点均比TGF—β1单独应用组明显增加（P〈0．05）。细胞团培养模式下，诱导细胞表达Ⅱ型胶原比平板培养模式更加显著。结论 MSCs向软骨细胞诱导分化时，IGF-1对TGF—β1有明显的促进作用；细胞培养密度提高有利于MSCs成软骨细胞表型。     

成人骨髓基质干细胞体外定向诱导分化为软骨细胞的实验研究

目的 建立临床成人骨髓基质干细胞(MSCs)体外培养、定向诱导分化为软骨细胞的途径。方法抽取成人骨髓，Percol密度梯度离心法进行体外培养，贴壁细胞传代，取第3代细胞在培养基中添加软骨分化诱导剂地塞米松、维生素C和不同剂量转化生长因子-β(TGF-β)，培养16d后,在倒置显微镜观察细胞形态，甲苯胺蓝染色蛋白多糖，逆转录一聚合酶链反应(RT-PCR)、免疫细胞化学(SABC法)检测Ⅱ型胶原表达，诱导后MSCs与新型材料聚乳酸和羟基乙醇共聚物(PL-GA)复合。结果 Percoll密度梯度离心法培养可获得均一的。MSCs；5、10ng TGF-β诱导分化的MSCs生长迅速。呈典型的软骨细胞形态，甲苯胺蓝染色阳性，Ⅱ型胶原表达阳性，MSCs对材料PL-GA黏附力强。结论 可以从成人骨髓中培养出MSCs，并可定向诱导分化为软骨细胞，5～10ng TGF-β为最佳诱导剂量，成人MSCs可用作临床自体软骨组织工程种子细胞。     

三维立体培养法诱导去分化软骨细胞Ⅱ型胶原的重新表达

目的用藻酸盐三维立体培养法及离心管聚集体诱导培养法促进去分化的永生化软骨细胞第50代(IHACs50)重新表达软骨细胞的Ⅱ型胶原标志性表型。方法利用藻酸盐三维立体培养法以及离心管聚集体诱导培养法分别诱导培养去分化的永生化人关节软骨细胞第50代,然后用Ⅰ、Ⅱ、Ⅲ型胶原的免疫组织化学染色、Ⅱ型胶原的逆转录-聚合酶链反应(RT-PCR)检测、激光共聚焦显微镜检测、胶原定量检测该永生化软骨细胞的Ⅱ型胶原表型表达情况。结果免疫组织化学染色、激光共聚焦显微镜检测、RT-PCR检测分别提示藻酸盐三维立体培养法和离心管聚集体诱导培养法使去分化永生化人关节软骨细胞的Ⅱ型胶原染色阳性以及621 bp的Ⅱ型胶原mR- NA表达,Western blot和3H-脯氨酸标记SDS-PAGEⅡ型胶原定量检测发现前者促进Ⅱ型胶原分泌较后者多。结论去分化永生化人关节软骨细胞可以经不同的诱导方法而重新表达Ⅱ型胶原表型,藻酸盐三维立体培养法因与软骨细胞的生理环境相似,故促进Ⅱ型胶原重新表达较多。     

Expansion of chondrocytes for tissue engineering in alginate beads enhances chondrocytic phenotype compared to conventional monolayer techniques

Chondrocytes are known to dedifferentiate when cultured in monolayer culture, which may compromise the efficacy of cartilage repair systems in which cells are expanded by repeat passage in monolayer prior to implantation. We tested the hypothesis that repeat passage in alginate beads can provide sufficient expansion of cells, while producing cells with enhanced chondrocytic phenotype. Bovine articular chondrocytes were seeded in 2% alginate beads or in monolayer. 4 passages at 7-day intervals were performed. Values of 9.1 days for monolayer expansion and 12.5 days for alginate expansion were estimated for a 10-fold increase in cell number. For assessment of chondrocytic and fibroblastic phenotype, expanded cells were seeded in alginate beads or on glass coverslips and cultured for 7 days. On subsequent seeding in alginate, cells which had previously been subcultured in alginate showed higher levels of both DNA and GAG synthesis than cells passaged in monolayer. Furthermore, the alginate-passaged cells retained a chondrocytic phenotype, indicated by synthesis of type II collagen and chondroitin-6-sulphate, while cells passaged inmonolayer synthesised type I collagen, indicating a fibroblastic phenotype. In conclusion, expansion of cells for autologous cartilage repair systems, using subculture within alginate beads, provides a potentially attractive alternative to monolayer expansion.     

Expression of a stable articular cartilage phenotype without evidence of hypertrophy by adult human articular chondrocytes in vitro

Chondrocytes that were isolated from adult human articular cartilage changed phenotype during monolayer tissue culture, as characterized by a fibroblastic morphology and cellular proliferation. Increased proliferation was accompanied by downregulation of the cartilage-specific extracellular matrix proteoglycan, aggrecan, by cessation of type-II collagen expression, and by upregulation of type-I collagen and versican. This phenomenon observed in monolayer was reversible after the transfer of cells to a suspension culture system. The transfer of chondrocytes to suspension culture in alginate beads resulted in the rapid upregulation of aggrecan and type-II collagen and the downregulation of expression of versican and type-I collagen. Type-X collagen and osteopontin, markers of chondrocyte hypertrophy and commitment to endochondral ossification, were not expressed by adult articular chondrocytes cultured in alginate, even after 5 months. In contrast, type-X collagen was expressed within 2 weeks in a population of cells derived from a fetal growth plate. The inability of adult articular chondrocytes to express markers of chondrocyte hypertrophy has underscored the fundamental distinction between the differentiation pathways that lead to articular cartilage or to bone. Adult articular chondrocytes expressed only hyaline articular cartilage markers without evidence of hypertrophy.     

Increased accumulation of superficial zone protein (SZP) in articular cartilage in response to bone morphogenetic protein-7 and growth factors.

The purpose of this study was to investigate the role of bone morphogenetic proteins (BMPs), such as BMP-7, growth factors, and cytokines, in the accumulation of superficial zone protein (SZP) in bovine articular cartilage. Calf superficial articular cartilage discs and chondrocytes were obtained for explant and monolayer culture systems, respectively. Dose- and time-dependent actions of BMP-7 on SZP accumulation were investigated in both explant and monolayer culture systems. In addition, actions of various morphogens and growth factors [BMP-2, BMP-4, fibroblast growth factor 2 (FGF-2), insulin-like growth factor 1 (IGF-1), platelet-derived growth factor (PDGF), and transforming growth factor beta (TGF-beta1)], and cytokines [interleukin (IL)-1alpha, IL-1beta, and tumor necrosis factor (TNF-alpha)] alone, and in combination with BMP-7, on SZP accumulation were investigated in monolayer culture systems. SZP accumulation was quantified in both the cartilage and the medium using SDS-PAGE and subsequent immunoblotting. In both explant and monolayer cultures, BMP-7 increased SZP accumulation in a dose- and time-dependent fashion (p < 0.05). Furthermore, SZP accumulation was significantly increased in monolayer cultures by FGF-2, IGF-1, PDGF, and TGF-beta1 (p < 0.05). Both IL-1alpha and TNF-alpha significantly reduced SZP accumulation (p < 0.05). The inhibition of SZP accumulation by TNF-alpha was partially alleviated by concurrent treatment with BMP-7. The results of this investigation provide novel insights into the role of morphogens, especially BMP-7, growth factors, and cytokines in the accumulation of SZP in articular cartilage. This information has clinical implications because stimulation of SZP may ameliorate the pathology of joint function in arthritis. Furthermore, tissue engineering approaches to articular cartilage may depend on the optimal synthesis and assembly of SZP in the superficial zone to ensure functional tissue architecture.     

Flow cytometric analysis of the human articular chondrocyte phenotype in vitro

OBJECTIVE: To develop flow cytometry for the study of human articular cartilage cell phenotype and to validate the method on chondrocytes cultured in different in-vitro systems. METHODS: Chondrocyte phenotype was modulated by culturing the cells under different in-vitro conditions: i.e. in monolayer and in suspension culture in gelled agarose. Monolayer cultured chondrocyte phenotype was assayed by immunohistochemical staining with monoclonal antibodies against chondrocyte-specific aggrecan, type II and I collagen. Flow cytometry was used to quantify the proportions of chondrocytes expressing these extracellular matrix molecules in both culture conditions. To exclude the effects of cell-harvesting methods on the presence of cell-bound ECM molecules, non-proteolytic isolation procedures were used to obtain the chondrocytes for flow cytometry. Subconfluent cells from monolayer cultures were detached with EDTA. Chondrocytes cultured in gelled agarose were obtained after the agarose was enzymatically digested with agarase. RESULTS: Immunohistochemical staining showed that monolayer-cultured chondrocytes, in the presence of serum, gradually lost the expression of chondrocyte-specific aggrecan and type II collagen, while type I collagen was increasingly expressed. Flow cytometry allowed monolayer cultured chondrocyte phenotype to be assessed reproducibly. Chondrocyte phenotype was characterized through the cell membrane-associated extracellular matrix antigens. EDTA, used to obtain single cells from monolayer cultures, did not affect the cell-associated matrix. Where the chondrocytes had been cultured in gelled agarose, flow cytometry allowed quantification of the percentages of chondrocytes maintaining or reexpressing their original phenotype. The agarase digestion procedure used to isolate the cells from the agarose gel did not affect the plasma membrane-associated extracellular matrix antigens. CONCLUSION: Flow cytometry allows quantification of cells expressing aggrecan, type II and I collagen in their cell-associated extracellular matrix. A continuously increasing number of specific monoclonal antibodies will broaden the range of applications offered by this method.     

Expansion of chondrocytes for tissue engineering in alginate beads enhances chondrocytic phenotype compared to conventional monolayer techniques

Chondrocytes are known to dedifferentiate when cultured in monolayer culture, which may compromise the efficacy of cartilage repair systems in which cells are expanded by repeat passage in monolayer prior to implantation. We tested the hypothesis that repeat passage in alginate beads can provide sufficient expansion of cells, while producing cells with enhanced chondrocytic phenotype. Bovine articular chondrocytes were seeded in 2% alginate beads or in monolayer. 4 passages at 7-day intervals were performed. Values of 9.1 days for monolayer expansion and 12.5 days for alginate expansion were estimated for a 10-fold increase in cell number. For assessment of chondrocytic and fibroblastic phenotype, expanded cells were seeded in alginate beads or on glass coverslips and cultured for 7 days. On subsequent seeding in alginate, cells which had previously been subcultured in alginate showed higher levels of both DNA and GAG synthesis than cells passaged in monolayer. Furthermore, the alginate-passaged cells retained a chondrocytic phenotype, indicated by synthesis of type II collagen and chondroitin-6-sulphate, while cells passaged in monolayer synthesised type I collagen, indicating a fibroblastic phenotype. In conclusion, expansion of cells for autologous cartilage repair systems, using subculture within alginate beads, provides a potentially attractive alternative to monolayer expansion.     

Expansion of chondrocytes for tissue engineering in alginate beads enhances chondrocytic phenotype compared to conventional monolayer techniques

Chondrocytes are known to dedifferentiate when cultured in monolayer culture, which may compromise the efficacy of cartilage repair systems in which cells are expanded by repeat passage in monolayer prior to implantation. We tested the hypothesis that repeat passage in alginate beads can provide sufficient expansion of cells, while producing cells with enhanced chondrocytic phenotype. Bovine articular chondrocytes were seeded in 2% alginate beads or in monolayer. 4 passages at 7-day intervals were performed. Values of 9.1 days for monolayer expansion and 12.5 days for alginate expansion were estimated for a 10-fold increase in cell number. For assessment of chondrocytic and fibroblastic phenotype, expanded cells were seeded in alginate beads or on glass coverslips and cultured for 7 days. On subsequent seeding in alginate, cells which had previously been subcultured in alginate showed higher levels of both DNA and GAG synthesis than cells passaged in monolayer. Furthermore, the alginate-passaged cells retained a chondrocytic phenotype, indicated by synthesis of type II collagen and chondroitin-6-sulphate, while cells passaged in monolayer synthesised type I collagen, indicating a fibroblastic phenotype. In conclusion, expansion of cells for autologous cartilage repair systems, using subculture within alginate beads, provides a potentially attractive alternative to monolayer expansion.