Cellular turnover at the chondro-osseous junction of growth plate cartilage: analysis by serial sections at the light microscopical level

In the distal hypertrophic cell zone of growth plate cartilage, the penetration of metaphyseal vascular endothelial cells is into the noncalcified territorial and pericellular matrices. Cellular mechanisms that promote metaphyseal vascularization are understood poorly, partly because no study has addressed the question of the time sequence of cellular interactions at the chondro-osseous junction. The purpose of the present study is to make predictions about the relative and the real time duration of cellular events during vascular invasion, including an analysis of the time sequence of death of the terminal hypertrophic chondrocyte. The data from serial section analysis at the light microscopical level of tetracycline-labeled growth plates indicate that death of the terminal hypertrophic chondrocyte occurs in discrete morphological stages characterized by rapid cellular condensation followed, within minutes, by endothelial cell penetration into the vacated lacuna. Cellular condensation lasts approximately 45 min or 18% of the time a cell spends as a terminal chondrocyte. The data also demonstrate that chondrocytic death occurs prior to invasion by vascular endothelial cells and that the chondrocytic lacuna remains empty for as long as 15 min before an endothelial cell or blood vascular cell fills the space.     

胶原海绵与兔软骨细胞体外三维培养的实验研究

目的 观察用人胎盘Ⅰ型胶原海绵作为兔软骨细胞体外三维培养支架时，软骨细胞形态学特征和功能及该支架的吸附性和组织相容性。方法 将新生兔原代和传代软骨细胞接种于人胎盘Ⅰ型胶原海绵进行体外培养，用光镜和扫描电镜观察细胞形态和结构及支架的吸附性和组织相容性，用免疫组化观察细胞Ⅱ型胶原合成。结果 以胶原海绵作为支架的传代软骨细胞能维持其形态学特征及细胞功能，该支架有较好的吸附性和组织相容性。结论 人胎盘Ⅰ型胶原海绵可作为软骨细胞体外三维培养较好的支架材料。     

The influence of pulsed low-intensity ultrasound on matrix production of chondrocytes at different stages of differentiation: an explant study

The proximal and distal parts of sterna of chick embryos represent cartilage undergoing endochondral ossification and hyaline cartilage, respectively. Cartilage explants from both regions were exposed for 20 min to pulsed low-intensity ultrasound (PLIUS) with an intensity of 30 mW. cm(-2) (spatial average-temporal average) at a frequency of 1.5 MHz, with a pulse burst frequency of 1 kHz and burst duration of 200 micros. Histological and immunohistochemical analysis was performed on days 1, 3, 5 and 7 after treatment. An anabolic effect of PLIUS on matrix production was shown by an increase of up to 10% to 20% in quantitative immunohistochemical staining for type II collagen and aggrecan in the two parts of the sternum. PLIUS also increased type X collagen staining by up to 10% in certain regions of the proximal part of the sternum. Staining for type X collagen was negative in the distal part of the sternum in both PLIUS and control groups. These results suggest that PLIUS may stimulate bone formation by increasing hypertrophy of chondrocytes directed to terminal differentiation. However, PLIUS did not induce hypertrophy in hyaline cartilage; moreover, increased matrix synthesis indicates a potential role in cartilage repair.     

白细胞介素-1β对人软骨细胞Bax mRNA表达的作用

目的 探讨人透明软骨细胞中白细胞介素-1β(IL-1β)对凋亡基因Bax mRNA表达的作用。方法 应用反转录-聚合酶链反应(RT-PCR)和荧光定量PCR的方法检测IL-1β作用下传代培养的人透明软骨细胞Bax mRNA的含量。结果 对于传代培养人的软骨细胞，IL-1β浓度为1、10、100ng／ml作用12h，对Bax mRNA的调节作用存在剂量依赖关系，各组之间差异有显著性。结论 IL-1β可以按照剂量依赖方式正向调节软骨细胞Bax mRNA基因的表达，并通过对细胞凋亡的调控参与骨关节炎的发生、发展。     

Effect of adiponectin on ATDC5 proliferation,differentiation and signaling pathways

Adiponectin, an adipose-secreted adipocytokine, exhibits various metabolic functions but has no known effect on bone development through the growth plate and specifically, in chondrocytes. Using the mouse ATDC5 cell line, a widely used in vitro model of chondrogenesis, we demonstrated the expression of adiponectin and its receptors during chondrogenic differentiation. Adiponectin at 0.5 μg/ml increased chondrocyte proliferation, proteoglycan synthesis and matrix mineralization, as reflected by upregulation of the expression of type II collagen, aggrecan, Runx2 and type X collagen, and of alkaline phosphatase activity. Quantitative RT-PCR and gelatin zymography showed a significant increase in the matrix metalloproteinase MMP9's expression and activity following adiponectin treatment. We therefore concluded that adiponectin can directly stimulate chondrocyte proliferation and differentiation. To evaluate the underlying mechanisms, we examined the effect of adiponectin on the expression of chondrogenic signaling molecules: Ihh, PTHrP, Ptc1, FGF18, BMP7, IGF1 and p21 were all upregulated while FGF9 was downregulated. This study reveals novel and direct activity of adiponectin in chondrocytes, suggesting its positive effects on bone development.     

大鼠膝关节软骨细胞体外培养去分化时间的实验研究

目的:观察大鼠膝关节软骨细胞体外培养后自然退变时间,为骨关节炎研究提供合适靶细胞。方法:大鼠膝关节软骨细胞体外培养,传代,行甲苯胺蓝及Ⅱ型胶原染色鉴定软骨细胞,通过Western Blot检测其特征性Ⅱ型胶原的表达情况对传代软骨细胞表型进行鉴定。结果:大鼠膝关节第5代软骨细胞和前4代比较,Ⅱ型胶原表达明显减少(P<0.05),结论:大鼠膝关节软骨细胞体外培养前4代能维持正常的软骨细胞表型,第5代开始发生退变,前4代可以做为软骨细胞研究的靶细胞。     

透骨消痛胶囊含药血清对退变关节软骨细胞表达TNF-α、IL-1β的影响

目的观察透骨消痛胶囊含药血清对体外培养的大鼠退变软骨细胞表达肿瘤坏死因子-α（TNF-α）、白介素-1β（IL-1β）的影响,探讨透骨消痛胶囊防治骨性关节炎的作用机制。方法 4周龄SD大鼠膝关节软骨建立体外培养的软骨细胞,采用Ⅱ型胶原原位杂交法对第3代软骨细胞进行鉴定。用10 ng.mL-1IL-1β干预第3代软骨细胞复制退变软骨细胞模型。模型复制成功后,随机分为模型组和透骨消痛胶囊含药血清组,分别在培养24、48、72 h后用TaqMan real-time PCR法检测各组mRNA表达。结果透骨消痛胶囊含药血清组TNF-αmRNA、IL-1βmRNA相对表达量随着干预时间的延长逐渐下降,差异均有统计学意义（P〈0.05）。结论透骨消痛胶囊治疗骨性关节炎的部分作用机制是有效抑制退变软骨细胞TNF-α、IL-1β的表达。     

动态力学刺激对三维培养软骨细胞作用的初步研究

目的探讨在三维多孔支架环境里动态循环压力刺激对软骨细胞的作用。方法原代培养兔软骨细胞,传代扩增后接种于1.5 cm×0.5 cm×0.5 cm聚乳酸多孔（polylactic acid,PLA）支架,培养5 d后,施加0.5、1、2 N的正弦动态循环压力,频率0.017 Hz,加压8 h,以无压力组（0 N）作为对照。标本做扫描电镜及组织学切片染色,观察细胞数量、形态及排列的变化。结果在三维PLA多孔支架上,扫描电镜观察到,动态压力组细胞的增殖比无压力组更多,排列也更加有序;动态压力为2 N的时候,细胞增殖、形态比1、0.5 N及无压力组更好,贴附细胞计数与1、0.5 N及无压力组相比差异有统计学意义（P〈0.05）。结论动态循环压力对于三维培养条件下的软骨细胞有促进增殖和有序排列的作用,但何种强度的压力更适合体外构建组织工程软骨尚需进一步研究。