物理性刺激对骨膜软骨生成的影响

目的探讨物理性刺激对骨膜软骨生成方面的影响,以期培养出一种与正常关节软骨更相似的软骨组织。方法从新西兰大白兔胫骨近端内侧取下骨膜,将骨膜固定在支架上,然后将细胞支架悬吊在旋转瓶内,用水流产生的剪切应力去刺激骨膜。通过宏观观察、体积大小测量、组织切片染色与细胞外基质(ECM)成分的比较及生物力学测试分析软骨体外生长的最佳环境。结果宏观观察发现软骨生长的方向与水流的方向相同。组织切片染色可见有两层不同形态的软骨细胞和不同密度的ECM,免疫组织化学染色见在剪切应力的刺激下,软骨表面可分泌浅层蛋白质及润滑剂,且在不同大小的剪切应力刺激时,软骨表面还会产生不同厚度的表层。结论剪切应力刺激能使骨膜上的干细胞分化形成软骨,同时证明力学环境不仅影响细胞的分化与生长,而且影响细胞的形态与ECM的分泌。     

Matrix Vesicles Promote Mineralization in a Gelatin Gel

Extracellular matrix vesicles (MVs) are associated with initial calcification in a variety of tissues, but the mechanisms by which they promote mineralization are not certain. In this study, MVs isolated from fourth passage rat growth plate chondrocyte cultures were included within a gelatin gel into which calcium and phosphate ions diffused from opposite ends. In this gel, apatite formation occurs by 3.5 days in the absence of mineralization promoters, allowing measurement of the ability of different factors to ``nucleate' apatite before this time or to assess the effects of molecules which modulate the rate and extent of mineral deposition. Mineral ion accumulation and crystal type are assayed at 5 days. In this study, MV protein content in the central band of a 10% gelatin gel was varied by including 100 μl of a Tris-buffered solution containing 0–300 μg/ml MV protein. There was a concentration-dependent increase in mineral accretion. Whereas 10 μg MV protein in the gel did not significantly promote apatite formation as compared with vesicle-free gels, 20 and 30 μg MV protein in the gel did promote apatite deposition. Inclusion of 10 mM β-glycerophosphate in the gels, along with MVs, did not significantly increase apatite formation despite the demonstrable alkaline phosphatase activity of the MVs. In contrast, MVs at all concentrations significantly increased apatite accumulation when proteoglycan aggregates or ATP, inhibitors of apatite formation and proliferation, were included in the gel. Slight increases in calcium, but not phosphate accumulation, were also noted when an ionophore was included with the MVs to facilitate Ca ion transport into the vesicles. FT-IR analysis of the mineral formed in the vesicle-containing gels revealed the presence of a bone-like apatite. These data suggest that MVs facilitate mineralization by providing enzymes that modify inhibitory factors in the extracellular matrix, as well as by providing a protected environment in which mineral ions can accumulate. Received: 28 January 1996 / Accepted: 9 August 1996     

Matrix vesicles are enriched in metalloproteinases that degrade proteoglycans

Summary This study examined the presence of extracellular matrix processing enzymes in matrix vesicles produced by rat costochondral resting zone and growth zone chondrocytes in culture. Optimum procedures for the extraction of each enzyme activity were determined. Enzyme activity associated with chondrocyte plasma membrane microsomes was used for comparison. There was a differential distribution of the enzyme activities related to the cartilage zone from which the cells were isolated. Acid and neutral metalloproteinase (TIMP), plasminogen activator, and betaglucuronidase were highest in the growth zone chondrocyte (GC) membrane fractions when compared with matrix vesicles and plasma membranes isolated from resting zone chondrocyte (RC) cultures. There was a threefold enrichment of total and active acid metalloproteinase in GC matrix vesicles, whereas no enrichment in enzyme activity was observed in RC matrix vesicles. Total and active neutral metalloproteinase were similarly enriched twofold in GC matrix vesicles. TIMP, plasminogen activator, and betaglucuronidase activities were highest in the plasma membranes of both cell types. No collagenase, lysozyme, or hyaluronidase activity was found in any of the membrane fractions. The data indicate that matrix vesicles are selectively enriched in enzymes which degrade proteoglycans. The highest concentrations of these enzymes are found in matrix vesicles produced by growth zone chondrocytes, suggesting that this may be a mechanism by which the more differentiated cell modulates the matrix for calcification.     

Morphological characteristics of the life cycle of resting cartilage cells in mouse rib investigated in intrasplenic isografts

Summary Resting cartilages taken from 2-day-old mouse ribs were transplanted into spleens in order to carry out morphological investigations of the life cycles of their chondrocytes. The explants were isografted for periods of up to 60 days and examined at light and electron microscopic levels, using von Kossa's reaction or osmium-potassium ferrocyanide (OPF) fixation. By day 3 after transplantation, resting cartilage containing immature chondrocytes was well adapted to splenic tissue and by 7 days after transplantation these chondrocytes had changed into early hypertrophic chondrocytes containing large vacuoles, glycogen aggregates and abundant secretory organelles. It was also demonstrated by von Kossa's reaction that the initial calcification occurred in the territorial matrix during this period. In spite of the hypertrophic chondrocytes in the central zone being surrounded by an extensively calcified matrix during days 14–21 after transplantation, these cells had well-preserved organized organelles, except that Golgi-associated elements and endoplasmic reticulum revealed a tendency toward degenerative changes. With increased duration of the grafting period, from 30–60 days, the calcification zone progressed gradually, and the number of hypertrophic chondrocytes embedded in the calcified matrix decreased considerably. By day 60, degenerating hypertrophic chondrocytes of two types were distinguished: flattened cells containing large vacuoles, poorly developed Golgi apparatuses, and rough endoplasmic reticulum; and shrunken dark cells displaying terminal hypertrophy. During the present study, we observed no vascular invasion into the calcified matrix, or appearance of bone-related cells, and the morphological changes from the resting chondrocytes to cellular hypertrophy accompanied by the formation of a calcified matrix were observed at day 60. These findings indicate that resting cartilage cells of the mouse have the capacity for terminal differentiation when transplanted into the spleen.     

The Effects of Extracellular Matrix on Tissue Engineering Construction of Cartilage in Vitro

In the study of bone and cartilage diseases in our laboratory, chondrocyte stereo-differentiation mod-el was cultured into cartilage tissue. The cultured cartilage tissue was closely similar to natural tissue inboth histological condition and biochemical metabolism. The model mimicked the chondrocyte differenti-ation and metabolism in vivo completely〔1〕. We studied some essential extracellular matrices, includingcollagen, proteoglycan(PG) and hyaluronic acid(HA) on the construction of grow…     

mTOR signaling contributes to chondrocyte differentiation.

The mammalian Target Of Rapamycin (mTOR) is a nutrient-sensing protein kinase that regulates numerous cellular processes. Fetal rat metatarsal explants were used as a physiological model to study the effect of mTOR inhibition on chondrogenesis. Insulin significantly enhanced their growth. Rapamycin significantly diminished this response to insulin through a selective effect on the hypertrophic zone. Cell proliferation (bromodeoxyuridine incorporation) was unaffected by rapamycin. Similar observations were made when rapamycin was injected to embryonic day (E) 19 fetal rats in situ. In the ATDC5 chondrogenic cell line, rapamycin inhibited proteoglycan accumulation and collagen X expression. Rapamycin decreased content of Indian Hedgehog (Ihh), a regulator of chondrocyte differentiation. Addition of Ihh to culture medium reversed the effect of rapamycin. We conclude that modulation of mTOR signaling contributes to chondrocyte differentiation, perhaps through its ability to regulate Ihh. Our findings support the hypothesis that nutrients, acting through mTOR, directly influence chondrocyte differentiation and long bone growth.     

体外传代培养对关节软骨细胞成软骨能力的影响

目的　研究不同代体外培养关节软骨细胞成软骨能力 ,选择软骨组织工程最佳种子细胞。方法　将兔膝关节软骨消化分离出软骨细胞 ,传代培养至第 6代 ,每代细胞与模板支架材料Pluron icF 12 7混匀形成复合物 ,使细胞浓度达 5× 10 7/ml,将复合物注射于自体兔背部皮下 ,9周后取材 ,进行大体观察、新生软骨湿重与体积测量、组织学观察。结果　体外培养关节软骨细胞随传代次数的增加 ,形态从圆形逐渐向梭形转变。 1～ 3代软骨细胞成软骨能力最强 ,新生软骨的湿重与体积最大 ,组织结构与正常软骨相同 ;第 4代成软骨能力减弱 ,软骨基质分泌减少 ,新生软骨湿重和体积减小 ,与前 3代比较差异有显著性 ;第 5代仅形成分散于纤维组织内的软骨细胞团 ;第 6代则未发现有软骨样组织形成。结论　①在体外单层培养条件下 ,随传代次数的增加关节软骨细胞逐渐去分化 ,成软骨能力逐渐降低直到完全丧失。②前 3代软骨细胞均具备形成正常软骨的能力 ,都可用作组织工程的种子细胞 ,但传代培养的目的是使细胞得以最大限度扩增 ,因此第 3代关节软骨细胞是可被应用的最佳种子细胞     

Temporal changes in cytoskeletal organisation within isolated chondrocytes quantified using a novel image analysis technique

This paper examines temporal changes in the organisation of the cytoskeleton within isolated articular chondrocytes cultured for up to 7 days in agarose constructs. Fluorescent labelling and confocal microscopy were employed to visualise microtubules (MT), vimentin intermediate filaments (VIF) and actin microfilaments (AMF). To quantify the degree of cytoskeletal organisation within populations of cells, a novel image analysis technique has been developed, and fully characterised. Organisation was quantified in terms of an Edge Index, which reflects the density of ‘edges’ present within the confocal images as defined by a Sobel digital filter. This parameter was shown to be independent of image intensity and, for all three cytoskeletal components, was validated statistically against a visual assessment of organisation. Both MT and VIF exhibited fibrous networks extending throughout the cytoplasm, while AMF appeared as punctate units associated with the cell membrane. The use of the Edge Index parameter revealed statistical significant temporal variation, in particular associated with VIF and AMF. These findings indicate the possibility of cytoskeletal mediated temporal variation in many aspects of cell behaviour following isolation from the intact tissue. Furthermore, the image analysis techniques are likely to be useful for future studies aiming to quantify changes in cytoskeletal organisation.     

猪关节软骨细胞体外培养及其形成糖胺多糖能力的实验研究

目的:观察软骨细胞体外培养的情况及其合成糖胺多糖的特点。方法:通过Ⅱ型胶原酶消化法获得高活性的软骨细胞,定量接种6×106个细胞于培养皿,加入培养基,每周传代一次,连续5周,留取所有培养液用阿利新蓝法(AlcianBlue)测定软骨细胞外分泌基质糖胺多糖(GAG)的变化;利用倒置显微镜观察原代及传代软骨细胞的生长情况。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,体外培养的软骨细胞从传代后合成糖胺多糖类基质,第二代传代细胞分泌GAG的能力最强。结论:软骨细胞体外培养生长良好,合成大量的糖胺多糖类基质。传代第二代是最佳接种时间。