Das Anusuya, Fishero Brian A, Christophel J Jared, Li Ching-Ju, Kohli Nikita, Lin Yong, Dighe Abhijit S, Cui Quanjun
Orthopaedic Research Laboratories, Department of Orthopaedic Surgery, University of Virginia, Charlottesville, VA 22908, USA.
Department of Otolaryngology- Head and Neck Surgery, University of Virginia, Charlottesville, VA 22908, USA.
Cell Tissue Res. 2016 Apr;364(1):125-35. doi: 10.1007/s00441-015-2301-x. Epub 2015 Oct 16.
We have previously shown that the combined delivery of mesenchymal stem cells (MSCs), vascular endothelial growth factor (VEGF) and bone morphogenetic protein 6 (BMP-6) induces significantly more bone formation than that induced by the delivery of any single factor or a combination of any two factors. We now determine whether the exogenous addition of VEGF and BMP-6 is sufficient for bone healing when MSCs are not provided. Poly(lactic-co-glycolic acid) (PLAGA) microsphere-based three-dimensional scaffolds (P) were fabricated by thermal sintering of PLAGA microspheres. The scaffolds were chemically cross-linked with 200 ng recombinant human VEGF (P(VEGF)) or BMP-6 (P(BMP-6)) or both (P(VEGF+BMP-6)) by the EDC-NHS-MES method. Release of the proteins from the scaffolds was detected for 21 days in vitro which confirmed their comparable potential to supply the proteins in vivo. The scaffolds were delivered to a critical-sized mandibular defect created in 32 Sprague Dawley rats. Significant bone regeneration was observed only in rats with P(VEGF+BMP-6) scaffolds at weeks 2, 8 and 12 as revealed by micro-computer tomography. Vascular ingrowth was higher in the P(VEGF+BMP-6) group as seen by microfil imaging than in other groups. Trichrome staining revealed that a soft callus formed in P(VEGF), P(BMP-6) and P(VEGF+BMP-6) but not in P. MSCs isolated from rat femurs displayed expression of the bone-specific marker osteocalcin when cultured with P(VEGF), P(BMP-6), or P(VEGF+BMP-6) but not with P. Robust mineralization and increased alkaline phosphatase gene expression were seen in rat MSCs when cultured on P(VEGF+BMP-6) but not on P, P(VEGF), or P(BMP-6). Thus, unlike the delivery of VEGF or BMP-6 alone, the combined delivery of VEGF and BMP-6 to the bone defect significantly enhanced bone repair through the enhancement of angiogenesis and the differentiation of endogenously recruited MSCs into the bone repair site.
我们之前已经表明，间充质干细胞（MSCs）、血管内皮生长因子（VEGF）和骨形态发生蛋白6（BMP - 6）联合递送诱导的骨形成明显多于任何单一因子或任意两种因子组合递送所诱导的骨形成。我们现在确定，在不提供MSCs的情况下，外源性添加VEGF和BMP - 6是否足以促进骨愈合。通过对聚乳酸 - 乙醇酸共聚物（PLAGA）微球进行热烧结制备了基于PLAGA微球的三维支架（P）。通过EDC - NHS - MES方法，使支架与200 ng重组人VEGF（P(VEGF)）或BMP - 6（P(BMP - 6)）或两者（P(VEGF + BMP - 6)）进行化学交联。在体外检测了蛋白质从支架中的释放情况，持续21天，这证实了它们在体内供应蛋白质的相当潜力。将这些支架递送至32只Sprague Dawley大鼠制造的临界大小的下颌骨缺损处。如微型计算机断层扫描所示，仅在植入P(VEGF + BMP - 6)支架的大鼠中，在第2、8和12周观察到显著的骨再生。通过微丝成像观察到，P(VEGF + BMP - 6)组的血管长入比其他组更高。三色染色显示，P(VEGF)、P(BMP - 6)和P(VEGF + BMP - 6)组形成了软骨痂，而P组未形成。从大鼠股骨分离的MSCs在与P(VEGF)、P(BMP - 6)或P(VEGF + BMP - 6)共培养时显示出骨特异性标志物骨钙素的表达，而与P共培养时则未显示。当在P(VEGF + BMP - 6)上培养时，大鼠MSCs出现强烈的矿化和碱性磷酸酶基因表达增加，而在P、P(VEGF)或P(BMP - 6)上培养时则未出现。因此，与单独递送VEGF或BMP - 6不同，将VEGF和BMP - 6联合递送至骨缺损处通过增强血管生成以及将内源性募集的MSCs分化至骨修复部位，显著增强了骨修复。
