Krause A, Cowles E A, Gronowicz G
Department of Orthopaedic Surgery, MC 1110, University of Connecticut Health Center, Farmington, Connecticut 06032, USA.
J Biomed Mater Res. 2000 Dec 15;52(4):738-47. doi: 10.1002/1097-4636(20001215)52:4<738::aid-jbm19>3.0.co;2-f.
The intracellular signaling pathway for osteoblast adhesion to the orthopedic implant material Ti6Al4V (TIV) was investigated and compared to integrin-mediated adhesion to extracellular matrix proteins. Primary osteoblasts from fetal rat calvaria were plated onto TIV, fibronectin (FN), and poly-L-lysine (PLL) and the levels of focal adhesion kinase (FAK), mitogen-activated protein kinase (MAPK), and AP-1 transcription factors, c-fos and c-jun, were compared by Western and Northern blots. Cells on all substrates showed maximum FAK phosphorylation within 60 min and then a decrease at 2 and 24 h. However, the subsequent signal transduction pathway differed on PLL compared to TIV and FN. MAPK was phosphorylated similarly in osteoblasts attached to FN and TIV, whereas cells on PLL demonstrated no MAPK phosphorylation. On TIV and FN, c-fos and c-jun mRNA levels were maximal within 1 h and then plateaued or declined by 2 h. On PLL, they increased at 2 h. Within 1 h, c-fos protein was stimulated in cells attached to TIV and FN and decreased in cells on PLL. c-jun protein increased on all substrates compared to unplated cells. Cytoskeletal changes visualized by phalloidin fluorescence microscopy at 4 h of culture were delayed on TIV compared to FN. In addition, approximately 50% fewer cells adhered to TIV compared to FN or PLL. By 24 h, a well-spread cytoskeleton with focal adhesion sites was apparent on TIV and FN, but cells on PLL were rounded with minimal cell spreading. During 6 days of culture, cells on FN and TIV proliferated, whereas the number of cells on PLL remained the same or decreased, depending on the initial plating density. We conclude that osteoblast adhesion to TIV implants is similar to osteoblast adhesion to FN and leads to osteoblast proliferation. These data provide evidence for the biocompatibility of TIV at a molecular level.
研究了成骨细胞与骨科植入材料Ti6Al4V（TIV）黏附的细胞内信号通路，并将其与整合素介导的细胞与细胞外基质蛋白的黏附进行了比较。将来自胎鼠颅骨的原代成骨细胞接种到TIV、纤连蛋白（FN）和聚-L-赖氨酸（PLL）上，通过蛋白质免疫印迹法和Northern印迹法比较黏着斑激酶（FAK）、丝裂原活化蛋白激酶（MAPK）以及AP-1转录因子c-fos和c-jun的水平。所有底物上的细胞在60分钟内FAK磷酸化达到最大值，然后在2小时和24小时时下降。然而，与TIV和FN相比，PLL上随后的信号转导途径有所不同。附着于FN和TIV的成骨细胞中MAPK的磷酸化情况相似，而PLL上的细胞未显示MAPK磷酸化。在TIV和FN上，c-fos和c-jun mRNA水平在1小时内达到最大值，然后在2小时时趋于平稳或下降。在PLL上，它们在2小时时升高。在1小时内，附着于TIV和FN的细胞中c-fos蛋白受到刺激，而PLL上的细胞中c-fos蛋白减少。与未接种的细胞相比，所有底物上的c-jun蛋白均增加。培养4小时时，通过鬼笔环肽荧光显微镜观察到的细胞骨架变化在TIV上比在FN上延迟。此外，与FN或PLL相比，附着于TIV的细胞数量减少了约50%。到24小时时，TIV和FN上出现了具有黏着斑位点的铺展良好的细胞骨架，但PLL上的细胞呈圆形，细胞铺展极少。在6天的培养过程中，FN和TIV上的细胞增殖，而PLL上的细胞数量保持不变或减少，这取决于初始接种密度。我们得出结论，成骨细胞与TIV植入物的黏附类似于成骨细胞与FN的黏附，并导致成骨细胞增殖。这些数据为TIV在分子水平上的生物相容性提供了证据。
