Açil Yahya, Springer Ingo N G, Broek Vanessa, Terheyden Hendrik, Jepsen Søren
Department for Oral and Maxillofacial Surgery, Christian-Albrechts-University of Kiel, Germany.
J Cell Biochem. 2002;86(1):90-8. doi: 10.1002/jcb.10197.
The objective of the present study was to investigate the effects of an in vitro stimulation of human osteoblasts by recombinant human bone morphogenetic protein-7 (rhBMP-7) on the collagen types and the quantity of the collagen cross-links synthesized in a three-dimensional culture on various biomaterials for bone replacement. Trabecular bone chips were harvested from human iliac crests, and cell cultures were established at standard conditions. One hundred and fifty nanograms per milliliter of rhBMP-7 was added. For the second passage a cell scraper was used to bring the cells into suspension, and 100 microl osteoblasts (at a density of 3.3 x 10(5)) were transferred onto nine blocks of either Bio-Oss, Tutoplast, or PepGen p-15. Blocks incubated with cells that were not treated with rhBMP-7 served as controls. Cell colonization of the biomaterials was observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM) after a period of 2, 4, and 6 weeks. Throughout the experiment medium, supernatants were collected and collagen was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Finally, the collagen cross-link residues hydroxylysylpyridinoline (HP) and lysylpyridinoline (LP) were quantified by HPLC. Within 4 weeks the cells became confluent on all of the studied biomaterials. All samples synthesized bone specific LP and collagen type I. However, in rhBMP-7-stimulated samples, the amount of HP and LP found was increased by 45% compared to non-stimulated samples. Cell proliferation and collagen synthesis was similar on the different biomaterials, but was consistently reduced in specimen not stimulated with rhBMP-7. In vitro stimulation of osteoblasts on Bio-Oss, Tutoplast, or PepGen p-15 with rhBMP-7 and subsequent transplantation of the constructs might lead to an enhanced osseointegration of the biomaterials in vivo.
本研究的目的是调查重组人骨形态发生蛋白-7（rhBMP-7）对人成骨细胞的体外刺激，在用于骨替代的各种生物材料上进行三维培养时，对合成的胶原类型和胶原交联数量的影响。从人髂嵴获取松质骨碎片，并在标准条件下建立细胞培养物。添加每毫升150纳克的rhBMP-7。在第二代时，使用细胞刮棒使细胞悬浮，并将100微升成骨细胞（密度为3.3×10⁵）转移到九个Bio-Oss块、Tutoplast块或PepGen p-15块上。用未用rhBMP-7处理的细胞孵育的块作为对照。在2周、4周和6周后，通过扫描电子显微镜（SEM）和透射电子显微镜（TEM）观察生物材料上的细胞定植情况。在整个实验过程中收集培养基上清液，并用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）对胶原进行表征。最后，通过高效液相色谱法（HPLC）对胶原交联残基羟赖氨酰吡啶啉（HP）和赖氨酰吡啶啉（LP）进行定量。在4周内，细胞在所有研究的生物材料上汇合。所有样品均合成了骨特异性LP和I型胶原。然而，在rhBMP-7刺激的样品中，发现的HP和LP量比未刺激的样品增加了45%。不同生物材料上的细胞增殖和胶原合成相似，但在未用rhBMP-7刺激的标本中持续减少。用rhBMP-7体外刺激Bio-Oss、Tutoplast或PepGen p-15上的成骨细胞，随后移植构建体可能会导致生物材料在体内的骨整合增强。
