Berendsen Agnes D, Pinnow Emily L, Maeda Azusa, Brown Aaron C, McCartney-Francis Nancy, Kram Vardit, Owens Rick T, Robey Pamela G, Holmbeck Kenn, de Castro Luis F, Kilts Tina M, Young Marian F
Craniofacial and Skeletal Diseases Branch, NIDCR, NIH, Bethesda, MD 20892, USA.
LifeCell Corporation, Branchburg, NJ 08876, USA.
Matrix Biol. 2014 Apr;35:223-31. doi: 10.1016/j.matbio.2013.12.004. Epub 2013 Dec 25.
Matrix proteoglycans such as biglycan (Bgn) dominate skeletal tissue and yet its exact role in regulating bone function is still unclear. In this paper we describe the potential role of (Bgn) in the fracture healing process. We hypothesized that Bgn could regulate fracture healing because of previous work showing that it can affect normal bone formation. To test this hypothesis, we created fractures in femurs of 6-week-old male wild type (WT or Bgn+/0) and Bgn-deficient (Bgn-KO or Bgn-/0) mice using a custom-made standardized fracture device, and analyzed the process of healing over time. The formation of a callus around the fracture site was observed at both 7 and 14 days post-fracture in WT and Bgn-deficient mice and immunohistochemistry revealed that Bgn was highly expressed in the fracture callus of WT mice, localizing within woven bone and cartilage. Micro-computed tomography (μCT) analysis of the region surrounding the fracture line showed that the Bgn-deficient mice had a smaller callus than WT mice. Histology of the same region also showed the presence of less cartilage and woven bone in the Bgn-deficient mice compared to WT mice. Picrosirius red staining of the callus visualized under polarized light showed that there was less fibrillar collagen in the Bgn-deficient mice, a finding confirmed by immunohistochemistry using antibodies to type I collagen. Interestingly, real time RT-PCR of the callus at 7 days post-fracture showed a significant decrease in relative vascular endothelial growth factor A (VEGF) gene expression by Bgn-deficient mice as compared to WT. Moreover, VEGF was shown to bind directly to Bgn through a solid-phase binding assay. The inability of Bgn to directly enhance VEGF-induced signaling suggests that Bgn has a unique role in regulating vessel formation, potentially related to VEGF storage or stabilization in the matrix. Taken together, these results suggest that Bgn has a regulatory role in the process of bone formation during fracture healing, and further, that reduced angiogenesis could be the molecular basis.
诸如双糖链蛋白聚糖（Bgn）之类的基质蛋白聚糖在骨骼组织中占主导地位，但其在调节骨功能的确切作用仍不清楚。在本文中，我们描述了Bgn在骨折愈合过程中的潜在作用。我们假设Bgn可以调节骨折愈合，因为先前的研究表明它可以影响正常的骨形成。为了验证这一假设，我们使用定制的标准化骨折装置在6周龄雄性野生型（WT或Bgn+/0）和Bgn基因缺陷型（Bgn-KO或Bgn-/0）小鼠的股骨上制造骨折，并随时间分析愈合过程。在骨折后7天和14天，WT和Bgn基因缺陷型小鼠的骨折部位周围均观察到骨痂形成，免疫组织化学显示Bgn在WT小鼠的骨折骨痂中高表达，定位于编织骨和软骨内。对骨折线周围区域的微计算机断层扫描（μCT）分析表明，Bgn基因缺陷型小鼠的骨痂比WT小鼠小。同一区域的组织学检查还显示，与WT小鼠相比，Bgn基因缺陷型小鼠的软骨和编织骨较少。在偏振光下观察到的骨痂的天狼星红染色显示，Bgn基因缺陷型小鼠的纤维状胶原蛋白较少，这一发现通过使用I型胶原抗体的免疫组织化学得到证实。有趣的是，骨折后7天骨痂的实时逆转录聚合酶链反应（RT-PCR）显示，与WT相比，Bgn基因缺陷型小鼠的相对血管内皮生长因子A（VEGF）基因表达显著降低。此外，通过固相结合试验表明VEGF可直接与Bgn结合。Bgn无法直接增强VEGF诱导的信号传导，这表明Bgn在调节血管形成中具有独特作用，可能与VEGF在基质中的储存或稳定有关。综上所述，这些结果表明Bgn在骨折愈合过程中的骨形成过程中具有调节作用，此外，血管生成减少可能是其分子基础。
