Stem Cell Research Center, University of Pittsburgh, Pittsburgh, PA 15219, USA; Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA 15213, USA.
Stem Cell Research Center, University of Pittsburgh, Pittsburgh, PA 15219, USA; Department of Orthopaedic Surgery, University of Pittsburgh, Pittsburgh, PA 15213, USA; Department of Orthopaedic Surgery, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.
Biomaterials. 2015 Aug;60:9-19. doi: 10.1016/j.biomaterials.2015.03.036. Epub 2015 May 14.
Ruptured human anterior cruciate ligaments (ACL) contain vascular stem cells capable of enhancing the healing of tendon grafts. In the current study we explored the role that neo-angiogenesis plays in ACL healing. ACL-derived CD34+ cells were isolated via Fluorescence Activated Cell Sorting (FACS) from the rupture sites of human ACLs. The cells were then virally transduced to express either vascular endothelial growth factor (VEGF) or soluble FLT-1 (sFLT-1), which is an antagonist of VEGF. We established five groups: CD34+VEGF(100%), where 100% of the cells were transduced with VEGF, CD34+VEGF(25%), where only 25% of the cells were transduced with VEGF, CD34+, CD34+sFLT-1, and a No cells group. The CD34+sFLT1 group had a significant reduction in biomechanical strength compared to the CD34+ group at 4 and 8 weeks; whereas the biomechanical strength of the CD34+VEGF(25%) group was significantly greater than the CD34+ group at week 4; however, no difference was observed by week 8. Immunohistochemical staining demonstrated a significantly lower number of isolectin B4 and hCD31 positive cells, markers associated with angiogenesis, in the CD34+sFLT1 group, and a higher number of isolectin B4 and hCD31 positive cells in the CD34+VEGF(100%) and CD34+VEGF(25%) groups compared to the CD34+ group. Graft maturation was significantly delayed in the CD34+sFLT1 group and accelerated in the CD34+VEGF(25%) group compared to the CD34+ group. In conclusion, blocking VEGF reduced angiogenesis, graft maturation and biomechanical strength following ACL reconstruction. Native expression of VEGF by the CD34+ cells improved tendon graft maturation and biomechanical strength; however, over-expression of VEGF impeded improvements in biomechanical strength.
断裂的人前交叉韧带（ACL）包含能够增强肌腱移植物愈合的血管干细胞。在目前的研究中，我们探讨了新血管生成在 ACL 愈合中的作用。通过荧光激活细胞分选（FACS）从人 ACL 的破裂部位分离出 ACL 衍生的 CD34+细胞。然后，通过病毒转导使这些细胞表达血管内皮生长因子（VEGF）或可溶性 FLT-1（sFLT-1），后者是 VEGF 的拮抗剂。我们建立了五个组：CD34+VEGF(100%)，其中 100%的细胞被转导了 VEGF；CD34+VEGF(25%)，其中只有 25%的细胞被转导了 VEGF；CD34+、CD34+sFLT-1 和无细胞组。与 CD34+组相比，在 4 周和 8 周时，CD34+sFLT1 组的生物力学强度显著降低；而在第 4 周时，CD34+VEGF(25%)组的生物力学强度明显大于 CD34+组；但在第 8 周时没有差异。免疫组织化学染色显示，在 CD34+sFLT1 组中，与血管生成相关的异凝集素 B4 和 hCD31 阳性细胞数量明显减少，而在 CD34+VEGF(100%)和 CD34+VEGF(25%)组中，异凝集素 B4 和 hCD31 阳性细胞数量明显增加。与 CD34+组相比，CD34+sFLT1 组的移植物成熟明显延迟，CD34+VEGF(25%)组的移植物成熟加速。结论：ACL 重建后，阻断 VEGF 会减少血管生成、移植物成熟和生物力学强度。CD34+细胞中 VEGF 的天然表达改善了肌腱移植物的成熟和生物力学强度；然而，VEGF 的过度表达会阻碍生物力学强度的改善。
