Carnes D L, De La Fontaine J, Cochran D L, Mellonig J T, Keogh B, Harris S E, Ghosh-Choudhury N, Dean D D, Boyan B D, Schwartz Z
Department of Endodontics, University of Texas Health Science Center at San Antonio, 78284-7774, USA.
J Periodontol. 1999 Apr;70(4):353-63. doi: 10.1902/jop.1999.70.4.353.
Because of the wide variation in the ability of human demineralized freeze-dried bone allograft (DFDBA) to reproducibly induce new bone formation, there is a need for a reliable measure of bone induction activity. In this study we examined an immature osteoprogenitor cell line for its potential utility in measuring the activity of DFDBA in vitro.
We characterized the response of 2T9 cells, an immature osteoprogenitor cell line derived from the calvariae of transgenic mice containing the SV40 T-antigen driven by the mouse bone morphogenetic protein (BMP)-2 promoter, to recombinant human BMP-2 by measuring alkaline phosphatase specific activity, osteocalcin production, and matrix mineralization. Responses were compared to those obtained with 1,25-(OH)2D3. In addition, 2T9 cells were cultured with active or inactive human DFDBA in the presence or absence of BMP-2. We also tested the hypothesis that radio-opacity of tissue following implantation of DFDBA in vivo correlates with the ability of human DFDBA to induce new bone. DFDBA from 9 different donors, stratified by age, were implanted subcutaneously in the thorax of 18 nude (nu/nu) mice. Tissue was harvested at 36 days postoperatively and examined histologically and biochemically for calcium and phosphorus uptake.
2T9 cells exhibited a dose- and time-dependent response to soluble BMP-2. Proliferation was decreased and alkaline phosphatase activity, osteocalcin production, and mineralized nodule formation were increased. The effects were dose- and time-dependent. Peak effects on alkaline phosphatase and osteocalcin were noted on day 8, whereas mineral deposition did not begin to occur until day 12. 1,25-(OH)2D3 did not regulate these effects unless used with BMP-2. When the cells were exposed to active or inactive DFDBA in the presence or absence of BMP-2, no effect on 2T9 cell differentiation was observed. This indicated that DFDBA released no soluble factors with bone inductive ability and that if any active factors were adsorbed to the DFDBA, they were inactivated. When DFDBA was implanted subcutaneously in the thorax of nude mice, there was no histologic evidence of new bone formation. However, there was a donor age-dependent decrease in Ca and P uptake of the implanted tissue, reflecting a donor age-dependent decrease in remineralization of DFDBA.
These data indicate that cell culture assays like the one used in this study may not be appropriate indicators of bone induction ability by DFDBA since soluble factors may not be responsible for bone induction in vivo. Nonetheless, in vitro assays are still needed. While Ca and P uptake by DFDBA-implanted tissue in the present study correlated with the age-dependent decrease in bone induction at intramuscular sites in a previously reported study, these data show that early x-rays may actually detect remineralization and not new bone formation. Thus, assessment of bone induction ability may still depend on histologic analysis of animal models.
由于人脱矿冻干骨同种异体移植（DFDBA）诱导新骨形成的能力差异很大，因此需要一种可靠的骨诱导活性测量方法。在本研究中，我们检测了一种未成熟的骨祖细胞系在体外测量DFDBA活性方面的潜在用途。
我们通过测量碱性磷酸酶比活性、骨钙素产生和基质矿化，对2T9细胞（一种从未成熟转基因小鼠颅骨中分离得到的骨祖细胞系，其含有由小鼠骨形态发生蛋白（BMP）-2启动子驱动的SV40 T抗原）对重组人BMP-2的反应进行了表征。将这些反应与用1,25-(OH)2D3获得的反应进行比较。此外，在有或没有BMP-2的情况下，将2T9细胞与活性或非活性人DFDBA一起培养。我们还测试了以下假设：DFDBA在体内植入后组织的不透射线性与人DFDBA诱导新骨的能力相关。将来自9个不同供体（按年龄分层）的DFDBA皮下植入18只裸（nu/nu）小鼠的胸部。术后36天收获组织，并进行组织学和生化检查以检测钙和磷的摄取。
2T9细胞对可溶性BMP-2表现出剂量和时间依赖性反应。增殖减少，碱性磷酸酶活性、骨钙素产生和矿化结节形成增加。这些效应具有剂量和时间依赖性。在第8天观察到碱性磷酸酶和骨钙素的峰值效应，而矿物质沉积直到第12天才开始出现。1,25-(OH)2D3除非与BMP-2一起使用，否则不会调节这些效应。当细胞在有或没有BMP-2的情况下暴露于活性或非活性DFDBA时，未观察到对2T9细胞分化的影响。这表明DFDBA没有释放具有骨诱导能力的可溶性因子，并且如果有任何活性因子吸附到DFDBA上，它们也会失活。当DFDBA皮下植入裸鼠胸部时，没有新骨形成的组织学证据。然而，植入组织的钙和磷摄取存在供体年龄依赖性下降，反映了DFDBA再矿化的供体年龄依赖性下降。
这些数据表明，像本研究中使用的细胞培养测定可能不是DFDBA骨诱导能力的合适指标，因为可溶性因子可能不是体内骨诱导的原因。尽管如此，体外测定仍然是需要的。虽然本研究中DFDBA植入组织的钙和磷摄取与先前报道的肌肉内部位骨诱导的年龄依赖性下降相关，但这些数据表明早期X射线实际上可能检测到的是再矿化而不是新骨形成。因此，骨诱导能力的评估可能仍然依赖于动物模型的组织学分析。
