Klein-Nulend J, Veldhuijzen J P, van de Stadt R J, van Kampen G P, Kuijer R, Burger E H
Department of Oral Cell Biology, Akademisch Centrum Tandheelhunde Amsterdam-Vrije Universiteit, The Netherlands.
J Biol Chem. 1987 Nov 15;262(32):15490-5.
We investigated the effect of mechanical stimulation by an intermittent compressive force (ICF) on proteoglycan (PG) synthesis and PG structure in calcified and noncalcified cartilage of fetal mouse long bone rudiments. Uncalcified cartilaginous long bone rudiments were cultured for 5 days in the presence of [35S]sulfate and [3H]glucosamine under control conditions (atmospheric pressure) or under the influence of ICF. ICF was generated by intermittently compressing the gas phase above the culture medium (130 mbar, 0.3 Hz). During culture, the center of the rudiments started to calcify. ICF stimulated calcification such that, after 5 days, the diaphysis of calcified cartilage was about two times as long as in the control cultures. At the end of the experiment, the rudiments were divided in a central calcified diaphysis and two noncalcified epiphyses. Diaphysis and epiphyses were pooled separately. PGs were extracted with 4 M guanidinium chloride and isolated by cesium chloride density gradient centrifugation. PGs (predigested with proteinase K or chondroitinase ABC) were characterized for hydrodynamic size of aggregates, monomers, and chondroitin sulfate chains by gel permeation chromatography and for degree of sulfation by ion exchange chromatography on high pressure liquid chromatography columns. ICF increased the amount of incorporated sulfate per tissue volume unit in the noncalcified epiphyses, but decreased this parameter in the calcified diaphysis. However, in both calcified and noncalcified cartilage, ICF increased the degree of sulfation of the chondroitin sulfate chains. No effects were found on the hydrodynamic size of the PG aggregates or monomers, but in the epiphyses ICF increased the size of the chondroitin sulfate chains. No other changes of structural characteristics of the macromolecules were observed. This study demonstrates that ICF generally stimulated the incorporation of [35S]sulfate into chondroitin sulfate chains. We conclude from the lowered [35S]sulfate content in calcified cartilage that ICF reduced the number of chondroitin sulfate chains and probably PGs while accelerating matrix calcification. It seems likely that the two effects are linked, indicating that a reduction of the number of chondroitin sulfate chains is part of the complicated process of cartilage calcification.
我们研究了间歇性压缩力（ICF）产生的机械刺激对胎鼠长骨原基钙化和未钙化软骨中蛋白聚糖（PG）合成及PG结构的影响。将未钙化的软骨性长骨原基在[35S]硫酸盐和[3H]葡糖胺存在的条件下，于对照条件（大气压）或ICF影响下培养5天。ICF通过间歇性压缩培养基上方的气相（130毫巴，0.3赫兹）产生。培养期间，原基中心开始钙化。ICF刺激了钙化过程，使得5天后，钙化软骨的骨干长度约为对照培养物中的两倍。实验结束时，将原基分为中央钙化骨干和两个未钙化骨骺。骨干和骨骺分别合并。用4M氯化胍提取PG，并通过氯化铯密度梯度离心进行分离。通过凝胶渗透色谱法对PG（用蛋白酶K或软骨素酶ABC预先消化）的聚集体、单体和硫酸软骨素链的流体力学尺寸进行表征，并通过高压液相色谱柱上的离子交换色谱法对硫酸化程度进行表征。ICF增加了未钙化骨骺中每单位组织体积掺入的硫酸盐量，但降低了钙化骨干中的该参数。然而，在钙化和未钙化软骨中，ICF均增加了硫酸软骨素链的硫酸化程度。未发现对PG聚集体或单体的流体力学尺寸有影响，但在骨骺中，ICF增加了硫酸软骨素链的尺寸。未观察到大分子结构特征的其他变化。本研究表明，ICF通常刺激[35S]硫酸盐掺入硫酸软骨素链中。我们从钙化软骨中[35S]硫酸盐含量降低得出结论，ICF减少了硫酸软骨素链以及可能的PG数量，同时加速了基质钙化。这两种效应似乎是相关的，表明硫酸软骨素链数量的减少是软骨钙化复杂过程的一部分。
