软骨细胞培养的影响因素研究进展

体外培养软骨细胞的优点是可以大量扩增及研究其生命活动的状况,但要维持正常表型则受到众多复杂因素的调控。这些因素的作用不是单一的,而是存在一个复杂的网络调节作用,它们之间复杂的相互作用需要去了解。本文就这些复杂的影响因素的研究进展作一综述。     

软骨细胞植入胶原蛋白重建类软骨组织的体外培养研究

本实验采用鸡胚胎第３８期胸软骨，经酶消化示分离软骨细胞，均匀弥散植入外源性胶原蛋白交联形成的凝胶体内。经过体外培养８天，软骨细胞数目增加约３倍。组织学观察显示：软骨细胞均匀分布于胶原蛋白三维网络之间，细胞分化良好，细胞周围可见类似正常软骨组织之陷窝（Ｌａｃｕｎａ）。胶原网络间有蛋白多糖蓄积现象。组织化学分析提示软骨细胞合成分泌Ⅱ型胶原蛋白。实验结果提示：三维胶原蛋白凝胶构成类似软骨组织的细胞外生存     

软骨细胞培养及其调控

自从 196 5年ｃｈｅｓｔｍａｎ和Ｓｍｉｔｈ首先开始软骨细胞体外培养用于软骨缺损修复的研究以来[1] ,人们开始对关节软骨损伤不能通过自身的软骨增殖修复的概念有了新的认识。实验证明不仅年幼且年老的关节软骨标本仍可在体外培养出新生透明软骨[2 ] 。虽然软骨细胞增殖能力有限 ,其质与量直接影响体外培养扩增效果 ,软骨细胞生长环境不同所表现出的生物学特性也有差别。软骨细胞在悬浮培养或半固体琼脂培养基中生长良好并保持表型稳定 ,在培养瓶底水凝胶覆盖的四维培养环境中长期培养时软骨细胞可形成结节结构其细胞形态胞外基质分泌和软…     

改良猪耳郭软骨细胞体外培养的实验研究

目的：为组织工程化软骨修复机体软骨缺损提供实验基础。方法：用消化组织块培养法体外培养猪耳郭软骨细胞,并观察其在Polyglycolicacid（简称PGA）支架上的生长情况。结果：软骨细胞在pH值为7.0,含10%胎牛血清的DMEM培养液中生长良好,可传代10代,细胞数目扩增为原来的400～500倍;软骨细胞在PGA支架上生长良好,分泌基质旺盛。结论：改良消化组织块方法培养软骨细胞是一种可以大量扩增软骨细胞数目的确实可行的方法,PGA是软骨细胞良好的生长支架。     

人胎关节软骨细胞体外培养的生物学特性

目的 研究软骨组织工程中传代软骨细胞与原代的差异。方法 用５个月人胎关节软骨分离培养细胞,观察细胞存活率、贴壁率、生长曲线和组织形态学的改变。结果 ⑴软骨块在４℃下,３ｄ内细胞存活率可达９３．４％～９７．６％。⑵原代细胞为圆形或三角形;第４代有一半转变成梭形,到第６代全部变为长梭形。⑶传代细胞贴壁时间（２～３ｈ）短于原代（４～７ｈ）。玻璃瓶内贴壁率传代细胞为７８．７％～８５．５％,原代８．８％。⑷     

包埋后的几丁质与软骨细胞体外培养的实验研究

目的 探讨几丁质作为组织工程技术中细胞培养支架的可行性。方法 采用聚乳酸、卵磷脂及多聚赖氨酸分别或工同包埋几丁质与软骨细胞体外培养,观察其产亲水性的改变、对细胞吸附力和细胞功能的影响。结果 以聚乳酸包埋的几丁质对细胞的生长有抵制作用;以卵磷脂包埋的几柄质亲水性增强;以多聚赖氨酸包埋的几丁质对细胞吸附力增强;以卵磷脂和多聚赖氨酸共同包埋的几丁质具有良好的亲水性和对细胞吸附力,并可使细胞更好地发挥功能     

兔胚胎关节软骨细胞体外培养的研究

目的 探讨兔胚胎软骨细胞体外培养的生物学特性。方法 对孕4周兔胚胎关节软骨用酶消化法分离培养细胞。观察细胞存活率、贴壁率、生长曲线和组织形态学改变。结果 兔胚胎软骨细胞可从胚胎软骨组织中消化分离出来，经鉴定具有软骨细胞的特性。原代兔胚软骨细胞存活率达97％以上，细胞贴壁率达80％以上，从原代到第4代都有高增殖力，到第8代时增殖力降低。到第12代时几乎丧失细胞增殖。结论 体外培养的胚胎软骨细胞前4代适合于作修复关节软骨缺损的组织工程细胞。     

卵磷脂,多聚赖氨酸和PLA包埋PGA与软骨细胞体外培养的实验研究

组织工程技术中细胞培养支架的选择是研究的焦点之一，以ＰＬＡ包埋的ＰＧＡ无纺网是应用较为广泛的支架之一。但其亲水性差，对细胞吸附力弱，是其不足之处，此实验选择了卵巢脂和多聚赖氨酸来分别和共同包埋ＰＧＡ＋ＰＬＡ，来观察其亲水性和对细胞吸附力的改变，及对细胞功能的影响，本实验证明卵磷脂具有增强支架亲水性作用，而多聚氨酸除具有增加支架对细胞的吸附力强，还具有促进细胞功能的作用。以卵磷脂和多聚赖氨酸共同包埋     

松质骨骨基质明胶负载软骨细胞体外培养的实验研究

目的：探索松质骨骨基质明胶（BMG）作为软骨细胞培养支架的可行性。方法：分离幼兔关节软骨细胞，体外单层培养扩增，并负载于松质骨BMG上体外培养，于不同时间取材进行解剖显微镜，组织学，秀射电镜观察及免疫组化检测。结果：培养6d软骨细胞在松质骨BMG上分裂增殖。12d形成10－12层细胞的软骨组织，Safranin-O染色阳性，18d软骨组织厚度增加，胶原染色阳性，培养24－42d网眼中细胞数量增多，周围形成25－20层细胞的软骨组织，培养42d，解剖显微镜观察显示形成直径4mm的“圆盘状软骨”，电镜观察显示软骨细胞外有胶原纤维，免疫组化检测显示形成的软骨组织基质中有II型胶原，结论：松质骨BMG可作为软骨细胞培养的较好支架。     

应用碱性成纤维细胞生长因子体外扩增猪耳软骨细胞老化的规律

目的探讨碱性成纤维细胞生长因子(bFGF)体外扩增软骨细胞的老化规律。方法细胞取自猪耳弹性软骨,实验分两组:培养液含10μg/L b-FCF组(简称bFGF组),培养液无bF- GF组(简称对照组),分别收集两组多代细胞,按5×107个/ml浓度与30%Pluronic F-127/Ham’s F-12混合,按每一样本0.5 ml注射到猪自体皮下,8周后取材,从大体观察、称重及组织学染色,对再生的软骨组织进行评价。结果bFGF组和对照组从第3代开始均未能形成软骨组织,bFGF组第2代软骨细胞和原代比较,在两周内扩增70倍,是对照组的12.7倍,且体内能形成很好的软骨。结论应用bFGF体外扩增的软骨细胞在第3代开始老化,失去成软骨能力,种子细胞应取第2代,可兼顾适量细胞数与具备成软骨能力。     

破骨细胞体外培养研究进展

破骨细胞是骨吸收的主要功能细胞,体外培养破骨细胞是骨吸收研究和抑制骨吸收药物开发研究的基础.该文综述破骨细胞体外培养常用的5种方法及近年研究进展,并对各种方法进行比较.成熟破骨细胞分离培养法仍是目前获得成熟破骨细胞的最佳途径;骨髓诱导破骨细胞培养法是目前最常用的破骨细胞培养法;脾干细胞诱导培养法和外周血单核细胞诱导培养法是近年新发展起来的培养方法,能排除骨髓基质细胞及其他造血干细胞干扰,更具有应用价值;骨巨细胞瘤破骨细胞样细胞分离培养法是其他破骨细胞培养方法的一个重要补充.     

Effects of a chitosan scaffold containing TGF-beta1 encapsulated chitosan microspheres on in vitro chondrocyte culture

The objectives of this study were (1) to develop a three-dimensional chitosan scaffold in combination with transforming growth factor-beta1 (TGF-beta1)-loaded chitosan microspheres and (2) to evaluate the effect of the TGF-beta1 release on the chondrogenic potential of rabbit chondrocytes in the scaffolds. TGF-beta1 was loaded into chitosan microspheres using an emulsion-crosslinking method, resulting in spherical shapes with a size ranging from 0.3 to 1.5 microm. Controlled release of TGF-beta1, as measured by enzyme-linked immunosorbent assay (ELISA), was observed with chitosan microspheres over 7 days. Chitosan solutions (2% and 3%) were fabricated into two types of scaffolds with different pore morphologies and mechanical properties using a freeze-drying technique, with the result that scaffold with higher concentrations showed smaller pores and lower porosity, leading to a much stronger scaffold. The TGF-beta1 microspheres were incorporated into the scaffolds at a concentration of 10 ng TGF-beta1/scaffold and then chondrocytes seeded into each scaffold and incubated in vitro for 2 weeks. The 2% chitosan scaffolds showed higher cell attachment levels than the 3% chitosan scaffolds (P < 0.01), regardless of the TGF-beta1 microspheres. Both the proliferation rate and glycosaminoglycan (GAG) production were significantly higher for scaffolds incorporating TGF-beta1 microspheres than for the control scaffolds without microspheres 10 days after incubation. Extracellular matrix staining by Safranin O and immunohistochemistry for type II collagen both significantly increased in scaffolds containing TGF-beta1 microspheres. These results suggest that the TGF-beta1 microsphere incorporated in scaffolds have the potential to enhance cartilage formation.     

Low calcium levels in serum-free media maintain chondrocyte phenotype in monolayer culture and reduce chondrocyte aggregation in suspension culture

OBJECTIVE: Extracellular calcium influences chondrocyte differentiation and synthesis of extracellular matrix. Previously, calcium concentrations ranging from 0.1 mM to 2 mM have been used in vitro and these studies indicated that low calcium concentrations were generally favorable for chondrocyte culture. Our objective was to extend these findings to yet lower calcium concentrations and to comprehensively examine effects on morphology and phenotype in two culture systems. METHODS: Serum-free media containing 1 mM, 50 microM or 15 microM of calcium and a serum-containing medium were used to culture chondrocytes in suspension and in monolayer, at high and low inoculation density. RESULTS: In monolayer, at low and high density, removing serum and decreasing calcium concentration decreased cell spreading and lowered collagen type I expression whereas collagen type II expression remained stable. In suspension, cells aggregated for all media tested; however, aggregates were smaller and looser in the absence of serum. CONCLUSION: The serum-free 50 microM and 1 mM calcium media provide good alternatives to classical media for monolayer culture since both growth and chondrocyte phenotype were maintained. In suspension culture, the serum-free 1mM calcium medium also possesses the beneficial properties of limiting aggregate size while maintaining growth and phenotype.     

The in vitro effects of dehydroepiandrosterone on chondrocyte metabolism

OBJECTIVE: To investigate the in vitro effects of dehydroepiandrosterone (DHEA) on neonatal rat chondrocytes. DESIGN: Chondrocytes isolated from neonatal rat cartilage were cultured in three-dimensionally agarose beads and were treated with DHEA. METHODS: Primary culture of chondrocytes was harvested from newborn Wistar rats. The DHEA effects on chondrocyte activities were evaluated by analyzing chondrocyte proliferation, matrix protein synthesis, gene expressions of collagen, matrix metalloproteinase-1, -3 and -13 (MMP-1, -3 and -13), and cyclooxygenase-2 (COX-II), and protein synthesis of interleukin-6 (IL-6), prostaglandin E2 (PGE2) and tissue inhibitor of metalloproteinase-1 (TIMP-1). RESULTS: The DHEA treatment did affect chondrocyte proliferation and glycosaminoglycan (GAG) synthesis. DHEA suppressed the expression of MMP-1, -3 and -13 genes and PGE2 protein synthesis enhanced by lipopolysaccharide (LPS) while the COX-II and inducible nitric oxide synthase (iNOS) gene expressions were down-regulated by DHEA. CONCLUSIONS: Our study demonstrates that DHEA has an ability to modulate the imbalance between MMPs and PGE2 in the neonatal chondrocytes which suggest that it has a potential protective role against articular cartilage damage.     

Influences of buffer systems on chondrocyte growth during long-term culture in alginate

OBJECTIVE: Chondrocyte behavior is very sensitive to culture environment such as physical and biochemical conditions. As extracellular pH (pHo) and the existence of bicarbonate could affect the chondrocyte fate, hence, the purpose of this study is to investigate the buffer system effect on chondrocyte fate during relatively long-term culture. METHODS: In order to examine whether effects seen were due to bicarbonate or to pHo, we had to devise a system which could differentiate between the two effects. Culture media buffered by N-2-hydroxyethyl piperazine-N'-2-ethanesulfonic acid (HEPES) only and the combination of HEPES and bicarbonate were used. Bovine articular chondrocytes were cultured in alginate beads for up to 12 days. pHo was kept constant by culture of 3 beads in 2 ml culture medium. Cell density, intracellular pH (pHi) and glycosaminoglycan (GAG) were measured at day 5 and day 12. Cell morphology, distribution and viability in alginate beads were monitored over 12 days of culture. RESULTS: Compared to culture in the absence of bicarbonate, a higher proliferation rate of chondrocytes was observed in the presence of bicarbonate. pHi was more alkaline, about 0.2 pH unit, in the presence of bicarbonate than that in the absence of bicarbonate. About 50% more GAG was deposited in alginate beads when chondrocytes were cultured in the combination of HEPES and bicarbonate, compared to chondrocytes cultured in the absence of NaHCO3 at the end of 12 days of culture. CONCLUSION: The presence of bicarbonate results in more alkaline in the pHi of bovine chondrocytes after long-term culture. The combination of bicarbonate and HEPES in culture medium improves cell growth, matrix production in three-dimensional alginate beads.     

低氧环境对小鼠未成熟关节透明软骨细胞培养的影响

目的研究低氧和低氧模拟化合物氯化钴对小鼠未成熟关节透明软骨细胞氧感应基因和细胞表型的影响。方法经酶消化分离出小鼠未成熟关节透明软骨细胞，分别在21％氧、2％氧和150μmol/L氯化钴条件下培养一定时间。通过倒置显微镜、透射电镜和扫描电镜观察细胞形态学变化。应用定量PCR和Western Blot检测葡萄糖转运体-1（GLUT-1）、葡萄糖转运体-3（GLUT-3）、磷酸果糖激酶-1（PGK-1）和低氧诱导因子-1α（HIF—1α）的表达。应用定量PCR观察软骨细胞表型改变。应用四甲基偶氮唑盐法（MTT）检测低氧及氯化钴对软骨细胞活性的影响。用葡萄糖检测试剂盒测葡萄糖摄取量。结果不同培养条件下软骨细胞形态无明显差异。2％氧和氯化钴可增加GLUT-1、GLUT-3及PGK-1mRNA表达。2％氧和氯化钴可促进GLUT-1、GLUT-3和HIF—1α蛋白表达。低氧和氯化钴促进细胞活性，增加葡萄糖摄取并促进细胞外基质合成。结论软骨细胞能通过调节氧感应基因适应低氧环境，HIF—1α可能起关键作用。低氧能在一定程度上增加软骨细胞活性和细胞外基质合成。模拟体内氧环境培养细胞能更好地了解软骨细胞特性。     

模拟微重力环境下层状修复体与软骨细胞复合培养的实验研究

[目的]探讨模拟微重力作为软骨组织工程培养方法的作用和胶原/壳聚糖/β-磷酸三钙(trical ciumphosphate,TCP)层状梯度修复体作为关节软骨组织工程支架的可行性.[方法]体外培养新西兰大白兔关节软骨细胞并扩增,吸附于多孔胶原/壳聚糖/β-磷酸三钙层状梯度修复体上,模拟微重力和普通环境下三维立体分别培养3周,通过生长曲线、倒置相差显微镜、组织学、扫描电镜及免疫组织化学检测微重力对软骨细胞培养的影响和支架在三维立体培养对软骨细胞的表型、增殖及功能的影响.[结果]软骨细胞/修复体体外培养3周,软骨细胞模拟微重力培养组明显比普通培养组在层状修复体上分布均匀,修复体中心软骨细胞数量明显较多,并分泌细胞基质,包裹在软骨细胞周围,Ⅱ型胶原免疫组织化学染色阳性.[结论]模拟微重力环境有利于软骨细胞在三维支架上的均匀增殖,有望成为软骨组织工程中的一种重要培养方法;胶原/壳聚糖/β-磷酸三钙层状梯度修复体,细胞相容性良好,有望成为一种比较理想的关节软骨组织工程支架材料.     

Effects of fluid-induced shear on articular chondrocyte morphology and metabolism in vitro

This study tested the effects of fluid-induced shear on high density monolayer cultures of adult articular chondrocytes. Fluid-induced shear (1.6 Pa) was applied by cone viscometer to normal human and bovine articular chondrocytes for periods of 24, 48, and 72 hours. At 48 and 72 hours, fluid-induced shear caused individual chondrocytes to elongate and align tangential to the direction of cone rotation. Fluid-induced shear stimulated glycosaminoglycan synthesis by 2-fold (p < 0.05) and increased the length of newly synthesized chains in human and bovine chondrocytes. In human chondrocytes, the hydrodynamic size of newly synthesized proteoglycans also was increased. After 48 hours of fluid-induced shear, the release of prostaglandin E₂ from the chondrocytes was increased 10 to 20-fold. In human chondrocytes, mRNA signal levels for tissue inhibitor of metalloproteinase increased 9-fold in response to shear compared with the controls. In contrast, mRNA signal levels for the neutral metalloproteinases, collagenase, stromelysin, and 72 kD gelatinase, did not show such major changes. This study demonstrated that articular chondrocyte metabolism responds directly to physical stimulation in vitro and suggests that mechanical loading may directly influence cartilage homeostasis in vivo.     

Effects of individual control of pH and hypoxia in chondrocyte culture

Effects of oxygen tension (pO₂) and pH on gene and protein expression and metabolic activity of human chondrocytes were independently assessed. Chondrocytes were cultured under a range of pH (6.4–7.4) and different pO₂ (5 and 20%) during 5 days in a bioreactor. Effects on gene expression, DNA content, protein expression, and metabolic activity were determined. Linear regression analysis showed that gene expression of type I collagen (COL1), SOX9, and VEGF is significantly lower at acidic pH, while expression of aggrecan, type II collagen, and HIF1A is pH‐independent. Higher protein levels of VEGF were found under low pO₂. Acidic pH severely lowered VEGF release into medium, glucose consumption, and lactate production. Extracellular pH proved to more potently influence cell function than oxygen tension, the latter showing down‐regulation of COL1 gene expression and up‐regulation of VEGF protein under hypoxia. Hypoxic culture inhibits COL1 mRNA expression pH‐dependently, while expression of SOX9 is largely hypoxia independent, but pH dependent. Expression of HIF1A and VEGF revealed divergent pH dependencies. Subtle fluctuations in extracellular pH and oxygen tension clearly influence chondrocyte metabolism and marker expression. Sophisticated pH and oxygen control not only allows study of (patho)physiological changes, but also opens new venues in cartilage tissue engineering. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:537–545, 2010