转化人关节软骨细胞的Ⅱ型胶原表型诱导

目的 用离心管聚集体培养（Aggregate culture)诱导转化人关节细胞的Ⅱ型胶原。方法 将体外长期培养的第30，40，50代转化软骨细胞消化后进行离心管培养，比较细胞在单层培养，离心管聚集体培养，以及在普通培养基，RAI诱导培养基下[2型骨形态发生蛋白（BMP－2）＋抗坏血酸盐（Ascorbate) 胰岛素（insulin)]，Ⅰ，Ⅱ，Ⅲ型胶原的表达和胞外基质发生情况。结果 在单层培养条件下，转化软骨细胞只表达Ⅰ型胶原，而在BAI诱导培养基及离心管聚集体培养下转化软有细胞表达的Ⅱ型胶原和大量胞外基质。结论 离心管聚集体培养是诱导转化软骨细胞Ⅱ型胶原的有效方式。     

Distinct osteogenic mechanisms of bones of distinct origins

Mammalian bones have three distinct origins (paraxial mesoderm, lateral plate mesoderm, and neural crest) and undergo two different modes of formation (intra-membranous and endochondral). Bones derived from the paraxial mesoderm and lateral plate mesoderm mainly form through the endochondral process. During this process, hypertrophic chondrocytes play a vital role in inducing both osteogenesis and angiogenesis. One of the essential osteogenic factors secreted from hypertrophic chondrocytes is Indian hedgehog (Ihh). In contrast, bones derived from the neural crest mainly form through the intramembranous pro-cess and do not require Ihh. Thus, depending on their origin, bones have distinct signaling properties, which need to be considered in the research and application of bone biology.Presented at the 18th Annual Research Meeting of the Japanese Orthopaedic Association, Kitakyushu, Japan, October 17, 2003     

牵张力对体外培养兔鼻软骨细胞影响的实验研究

目的：探讨牵张力对体外培养的不同年龄兔鼻软骨细胞增殖活性的影响及牵张力大小与兔鼻软骨细胞增殖活性改变的量效关系。方法：将第4代体外培养的新生及6周龄新西兰白兔兔鼻软骨细胞置于细胞膜式牵张力施加装置上培养，流式细胞仪检测不同的牵张力(5kPa、10kPa)在0-12h内对兔鼻软骨细胞增殖活性的影响。结果：0-10h内5kPa牵张力组软骨细胞增殖指数随着牵张力作用时间的延长不断上升，增殖指数峰值位于10h处；0～8h内10kPa牵张力组软骨细胞增殖指数随着牵张力作用时间的延长不断上升，增殖指数峰值位于8h处；5kPa较10kPa牵张力对体外培养兔鼻软骨细胞具有更大的促增殖作用；牵张力对体外培养的新生兔兔鼻软骨细胞具有更大的促增殖作用。结论：牵张力可促进体外培养的新生及六周龄新西兰白兔兔鼻软骨细胞增殖活性。提示我们鼻软骨牵张方法不仅适用于临床矫治新生儿唇腭裂伴发鼻畸形，而且有可能用于矫治1岁左右婴儿甚至更大年龄患儿的唇腭裂伴发鼻畸形。     

软骨细胞老化特征及机制的研究进展

软骨细胞的老化是一个极其复杂的过程，其特征包括：细胞不可逆的生长停滞于G1期；老化相关β-半乳糖苷酶的表达；端粒长度缩短；软骨细胞分化特征的改变。目前认为其机制为基因表达的程序性或减进性改变，包括肿瘤抑制基因p53和pRb等在细胞生长停滞中的作用；端粒结合蛋白和端粒酶对端粒长度的调节；细胞骨架蛋白的重组使软骨细胞形态的变化；基质降解酶类表达增加以及各种细胞因子的变化对软骨细胞代谢的影响。     

Cyclic tensile stretch load and oxidized low density lipoprotein synergistically induce lectin-like oxidized ldl receptor-1 in cultured bovine chondrocytes, resulting in decreased cell viability and proteoglycan synthesis.

Mechanical stimulation is known to be an essential factor in the regulation of cartilage metabolism. We tested the hypothesis that expression of lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) can be modulated by cyclic tensile stretch load in chondrocytes. Cyclic loading of repeated stretch stress at 10 cycles per minute with 10 kPa of stress for 6 h induced expression of LOX-1 to 2.6 times control in cultured bovine articular chondrocytes, equivalent to the addition of 10 microg/mL oxidized low density lipoprotein (ox-LDL) (2.4 times control). Application of the cyclic load to the chondrocytes along with 10 microg/mL ox-LDL resulted in synergistically increased LOX-1 expression to 6.3 times control. Individual application of cyclic loading and 10 microg/mL ox-LDL significantly suppressed chondrocytes viability (84.6% +/- 3.4% and 80.9% +/- 3.2% of control at 24 h, respectively; n = 3; p < 0.05) and proteoglycan synthesis [81.0% +/- 7.1% and 85.7% +/- 5.2% of control at 24 h, respectively; p < 0.05 when compared with 94.6% +/- 4.6% for native-LDL (n = 3)]. Cyclic loading and 10 microg/mL ox-LDL synergistically affected cell viability and proteoglycan synthesis, which were significantly suppressed to 45.6% +/- 4.9% and 48.7% +/- 6.7% of control at 24 h, respectively (n = 3; p < 0.01 when compared with individual application of cyclic loading or 10 microg/mL ox-LDL). In this study, we demonstrated synergistic effects of cyclic tensile stretch load and ox-LDL on cell viability and proteoglycan synthesis in chondrocytes, which may be mediated through enhanced expression of LOX-1 and which has important implications in the progression of cartilage degeneration in osteoarthritis.     

鹿茸生长素对家兔关节软骨细胞代谢的影响

目的 研究鹿茸生长素对关节软骨细胞增殖的影响。方法 采用家兔关节软骨细胞体外培养和加入同位素3H—TdR标记方法，观察其对3H—TdR掺入细胞DNA合成的影响。结果 加入鹿茸生长素组的3H—TdR的掺入值与对照组有明显差异，细胞增殖作用明显。结论 鹿茸生长素能明显刺激关节软骨细胞的增殖。     

An ultrastructural study of cartilage resorption at the site of initial endochondral bone formation in the fetal mouse mandibular condyle

An ultrastructural study was undertaken on cartilage resorption at the site of initial endochondral bone formation in the mouse mandibular condyle on d 16 of pregnancy. After resorbing the bone collar, the osteoclasts extended their cell processes into the cartilage matrix and made contact with hypertrophic chondrocytes. By means of cell processes or vacuolar structures, these osteoclasts entrapped the calcified cartilage matrices, cell debris, and the degraded uncalcified cartilage matrices. In particular, since the calcified cartilage matrices were sometimes seen to be disrupted within the osteoclastic vacuolar structures, they were probably disposed of by the osteoclasts. Invading endothelial cells giving rise to capillaries also directly surrounded the degraded uncalcified cartilage matrices and small deposits of cell debris. In addition, hypertrophic chondrocytes that had attached to or were in the process of attaching to the invading osteoclasts often enclosed the small calcified cartilage matrices. Other cell types that have often been reported in other regions of cartilage resorption were not seen at the site of initial endochondral bone formation in this study. Our findings in relation to cartilage resorption may therefore represent unique features of the site of initial endochondral bone formation site. We consider that the manner of cartilage resorption is likely to vary by site, age, and species.     

兔关节软骨细胞的琼脂糖凝胶培养及其力学性质的研究

目的对兔膝关节软骨细胞进行体外琼脂糖凝胶三维培养,并对其力学性能进行检测。方法将琼脂糖凝胶、细胞、血清和培养基在正确的比例下混合,形成软骨细胞凝胶块。取培养7d、14d和21d时的软骨细胞凝胶块分别对细胞外基质进行组织学观察和免疫组化染色,同时采用Instron 5544材料试验机检测其极限应力、极限应变、抗压模量和切线模量的力学性能变化。结果在琼脂糖凝胶中培养的关节软骨细胞具有典型的软骨细胞特性,能够正常合成细胞外基质,形成有一定弹性和抗压缩能力的软骨样凝胶块。而且随着培养时间增长,基质合成增加,培养21d时的极限应力(0.0226±0.006)N/mm、抗压模量(0.608±0.061)N/mm和切线模量(0.096±0.004)N/mm,比7d时的(0.0204±0.004)MPa,(0.558±0.036)N/mm,(0.029±0.002)N/mm和14d时的(0.0213±0.008)N/mm,(0.586±0.095)N/mm,(0.049±0.005)N/mm有明显增加,但极限应变却没有明显差异(P>0.05)。结论软骨细胞-琼脂糖凝胶块的力学性能与细胞外基质的合成直接相关。     

Expansion of human nasal chondrocytes on macroporous microcarriers enhances redifferentiation

Articular cartilage has a limited capacity for self-repair. To overcome this problem, it is expected that functional cartilage replacements can be created from expanded chondrocytes seeded in biodegradable scaffolds. Expansion of chondrocytes in two-dimensional culture systems often results in dedifferentiation. This investigation focuses on the post-expansion phenotype of human nasal chondrocytes expanded on macroporous gelatin CultiSpher G microcarriers. Redifferentiation was evaluated in vitro via pellet cultures in three different culture media. Furthermore, the chondrogenic potential of expanded cells seeded in polyethylene glycol terephthalate/ polybuthylene terephthalate (PEGT/PBT) scaffolds, cultured for 14 days in vitro, and subsequently implanted subcutaneously in nude mice, was assessed.

Chondrocytes remained viable during microcarrier culture and yielded doubling times (1.07±0.14 days) comparable to T-flask expansion (1.20±0.36 days). Safranin-O staining from pellet culture in different media demonstrated that production of GAG per cell was enhanced by microcarrier expansion. Chondrocyte–polymer constructs with cells expanded on microcarriers contained significantly more proteoglycans after subcutaneous implantation (288.5±29.2 μg) than those with T-flask-expanded cells (164.0±28.7 μg). Total collagen content was similar between the two groups.

This study suggests that macroporous gelatin microcarriers are effective matrices for nasal chondrocyte expansion, while maintaining the ability of chondrocyte differentiation. Although the exact mechanism by which chondrocyte redifferentiation is induced through microcarrier expansion has not yet been elucidated, this technique shows promise for cartilage tissue engineering approaches.     

Direct protective effect of interleukin-10 on articular chondrocytes in vitro

Objective To assess whether interleukin-10 (IL-10) is chondroprotective in vitro.Methods Chondrocytes were isolated from femoral cartilage of rats (7-10 days) by digesti on with collagenase Ⅱ.The first passage cells were grown in 24- well plates with DMEM, supplemented with 10% fetal bovine serum, for 2-4 days.The ce lls were then cultured in 0.1% fetal bovine serum DMEM medium, and given respec tively interleu kin-1 (IL-1) 100 μ/ml, IL-1 100 μ/ml+recombinant murine interleukin-10 ( rmIL-10) 20 ng/ml, rmIL-10 20 ng/ml, and cultured for 48 hours.Scanning el ectron morphology and immunohistochemical study of nitric oxide synthase 2 and matric metalloproteinase 3 mRNA in situ hybridization were performe d.Cell proliferation and morphology were observed under inverted microscope fr om the beginning of cell culture for three weeks.Results IL-1 stimulated granule production in the cytoplasma of chondrocytes, and the c ells died in the second and third weeks of culture.IL-10 antagonized IL-1, p rotected the cells from death and maintained chondrocyte proliferation.Scannin g electron morphology showed that IL-1 stimulated the formation of numerous mic rovilli on the cell surface, while thin and less numerous microvilli were found in cultures with IL-10.Immunohistochemical study and in situ hybridization sh owed that IL-10 inhibited NOS2 and MMP3 expression.Conclusion IL-10 not only inhibits the synthesis of inflammatory cytokines, but also direc tly protects chondrocytes by antagonizing IL-1.