Stem Cell Research Center, Children's Hospital of Pittsburgh, Pittsburgh, PA 15219, USA.
Cell Transplant. 2012;21(8):1651-65. doi: 10.3727/096368912X647234. Epub 2012 Jun 20.
We recently reported that the ruptured regions of the human anterior cruciate ligament (ACL) contained vascular-derived stem cells, which showed the potential for high expansion and multilineage differentiation. In this study, we performed experiments to test the hypothesis that ACL-derived CD34(+) cells could contribute to tendon-bone healing. ACL-derived cells were isolated from the rupture site of human ACL by fluorescence-activated cell sorting. Following ACL reconstruction, immunodeficient rats received intracapsular administration of either ACL-derived CD34(+) cells, nonsorted (NS) cells, CD34(+) cells, or phosphate-buffered saline (PBS). We also performed in vitro cell proliferation assays and enzyme-linked immunosorbent assays for vascular endothelial growth factor (VEGF) secretion. We confirmed the recruitment of the transplanted cells into the perigraft site after intracapuslar injection by immunohistochemical staining at week 1. Histological evaluation showed a greater area of collagen fiber formation and more collagen type II expression in the CD34(+) group than the other groups at the week 2 time point. Immunostaining with isolectin B4 and rat osteocalcin demonstrated enhanced angiogenesis and osteogenesis in the CD34(+) group at week 2. Moreover, double immunohistochemical staining for human-specific endothelial cell (EC) and osteoblast (OB) markers at week 2 demonstrated a greater ability of differentiation into ECs and OBs in the CD34(+) group. Microcomputerized tomography showed the greatest healing of perigraft bone at week 4 in the CD34(+) cell group, and the failure load of tensile test at week 8 demonstrated the greatest biomechanical strength in the CD34(+) group. Furthermore, the in vitro studies indicated that the CD34(+) group was superior to the other groups in their cell proliferation and VEGF secretion capacities. We demonstrated that ACL-derived CD34(+) cells contributed to the tendon-bone healing after ACL reconstruction via the enhancement of angiogenesis and osteogenesis, which also contributed to an increase in biomechanical strength.
我们最近报道，人类前交叉韧带（ACL）的破裂区域含有血管源性干细胞，这些干细胞具有高扩增和多谱系分化的潜力。在这项研究中，我们进行了实验来验证假设，即 ACL 来源的 CD34+细胞可能有助于腱骨愈合。通过荧光激活细胞分选从 ACL 破裂部位分离 ACL 来源的细胞。ACL 重建后，免疫缺陷大鼠接受囊内注射 ACL 来源的 CD34+细胞、未分选（NS）细胞、CD34+细胞或磷酸盐缓冲盐水（PBS）。我们还进行了体外细胞增殖实验和酶联免疫吸附试验以检测血管内皮生长因子（VEGF）的分泌。我们通过第 1 周囊内注射后免疫组织化学染色证实了移植细胞被募集到移植部位周围。组织学评估显示，在第 2 周时，CD34+组的胶原纤维形成面积更大，胶原 II 型表达更多。第 2 周时，异硫氰酸荧光素 B4 和大鼠骨钙素免疫染色显示 CD34+组的血管生成和成骨增强。此外，第 2 周时人特异性内皮细胞（EC）和成骨细胞（OB）标志物的双重免疫组织化学染色显示 CD34+组向 EC 和 OB 的分化能力更强。第 4 周时微计算机断层扫描显示 CD34+细胞组周围骨的愈合效果最好，第 8 周时拉伸试验的失效负荷表明 CD34+细胞组的生物力学强度最大。此外，体外研究表明，与其他组相比，CD34+组在细胞增殖和 VEGF 分泌能力方面具有优势。我们证明 ACL 来源的 CD34+细胞通过增强血管生成和成骨作用有助于 ACL 重建后的腱骨愈合，这也有助于增加生物力学强度。
