Gronowicz G, Woodiel F N, McCarthy M B, Raisz L G
Department of Orthopaedics, University of Connecticut Health Center, Farmington.
J Bone Miner Res. 1989 Jun;4(3):313-24. doi: 10.1002/jbmr.5650040305.
We have developed a bone organ culture system that mineralizes in vitro. Fetal rat parietal bones (20 days old) were cultured in a chemically defined serum-free medium containing physiological 3 mM phosphate. During 5 days in culture, calcium content increased from 26 to 55 micrograms and dry weight increased from 137 to 194 micrograms. After 2 days in vivo, the calcium content of the parietal bone showed a comparable increase to 49 micrograms and dry weight increased to 183 micrograms. During culture, the mineralized bone area in thick sections increased from 11 to 23%, which paralleled the doubling in calcium content. Fluorescent calcein labeling during the 5 day culture period demonstrated that calcification occurs in an ordered pattern. Protein synthesis was assessed by measuring incorporation of [3H]proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP). The percentage collagen synthesis decreased from 17.5% at 0 time to 5.0% at 2 days and then increased to 9.4% at 5 days of culture. Varying the inorganic phosphate concentration in the medium or adding beta-glycerol phosphate was found to affect mineralization. After 5 days in culture, bones treated with 1 mM phosphate exhibited a large region of unmineralized osteoid with only a 23% increase in calcium content compared with 112% in control (3 mM phosphate) bones and a 28% increase in dry weight compared with a 40% increase in control. Treatment for 5 days with 6 mM phosphate or 1, 3, or 10 mM beta-glycerol phosphate had no significant effect on dry weight compared to control bones. However, bone calcium content increased significantly from 55 +/- 5 micrograms in control cultures to 105 +/- 7 with 6 mM phosphate, 74 +/- 6 with 3 mM beta-glycerol phosphate, and 75 +/- 5 micrograms with 10 mM beta-glycerol phosphate. Calcified area measured by histomorphometry was also significantly greater than in control bones, but this was mainly due to ectopic calcification in the periosteum, representing from 23 to 74% of the total increase in calcified matrix in bones cultured with 6 mM phosphate or 1-10 mM beta-glycerol phosphate. Ultrastructural analysis demonstrated that ectopic calcification was associated with cell death and debris. Therefore, calcification with beta-glycerol phosphate and high concentrations of inorganic phosphate differed from mineralization in vivo or in bones cultured with a physiologically concentration of phosphate.
我们开发了一种能在体外矿化的骨器官培养系统。将20日龄的胎鼠顶骨在含有生理浓度3 mM磷酸盐的化学成分明确的无血清培养基中培养。在培养的5天中，钙含量从26微克增加到55微克，干重从137微克增加到194微克。在体内培养2天后，顶骨的钙含量可比地增加到49微克，干重增加到183微克。在培养过程中，厚切片中的矿化骨面积从11%增加到23%，这与钙含量的翻倍相平行。在5天的培养期内进行荧光钙黄绿素标记表明钙化以有序模式发生。通过测量[3H]脯氨酸掺入胶原酶可消化蛋白（CDP）和非胶原蛋白（NCP）来评估蛋白质合成。胶原蛋白合成的百分比从0天时的17.5%降至2天时的5.0%，然后在培养5天时增加到9.4%。发现改变培养基中的无机磷酸盐浓度或添加β-甘油磷酸会影响矿化。培养5天后，用1 mM磷酸盐处理的骨骼表现出大片未矿化的类骨质，钙含量仅增加23%，而对照（3 mM磷酸盐）骨骼增加112%，干重增加28%，对照增加40%。与对照骨骼相比，用6 mM磷酸盐或1、3或10 mMβ-甘油磷酸处理5天对干重没有显著影响。然而，骨钙含量从对照培养物中的55±5微克显著增加到6 mM磷酸盐时的105±7微克、3 mMβ-甘油磷酸时的74±6微克和10 mMβ-甘油磷酸时的75±5微克。通过组织形态计量学测量的钙化面积也显著大于对照骨骼，但这主要是由于骨膜中的异位钙化，在用6 mM磷酸盐或1 - 10 mMβ-甘油磷酸培养的骨骼中，异位钙化占钙化基质总增加量的23%至74%。超微结构分析表明异位钙化与细胞死亡和碎片有关。因此，用β-甘油磷酸和高浓度无机磷酸盐进行的钙化与体内或用生理浓度磷酸盐培养的骨骼中的矿化不同。
