关键词: 骨缺损修复, 仿生骨膜, 静电纺丝, 血管内皮生长因子, 骨髓间充质干细胞, 巨噬细胞, 工程化干细胞

Abstract: BACKGROUND: Autologous bone, allogeneic bone or artificial bone has been used to promote bone defect repair in the clinic, but the rate of non-healing is still high. The key is to ignore the importance of periosteum in the bone healing process. In the early stage of the project, the project team constructed an electrospinning membrane loaded with vascular endothelial growth factor to highly simulate the intramembranous osteogenesis of natural periosteum at the bone defect site, which promoted bone regeneration to a certain extent. However, the injured area often faces the dilemma of severe inflammatory response mediated by macrophages and lack of seed cells, resulting in the risk of inactivation or diffusion of delivered biological factors. Therefore, it is necessary to further optimize and coordinate the immune regulation and angiogenesis functions of biomimetic periosteum to promote bone repair. 
OBJECTIVE: To investigate the physicochemical properties of stem cell-engineered bionic periosteum and its role in regulating the inflammatory microenvironment to promote bone repair. 
METHODS: By combining L-polylactic acid-based microsol electrospinning, type I collagen self-assembly and gel stem cell transplantation technology, a bionic periosteum (M@C-B) was constructed, in which the core layer loaded with vascular endothelial growth factor and the shell layer delivered bone marrow mesenchymal stem cells to regulate the immune microenvironment of bone defects. The physicochemical properties of the periosteum were characterized by scanning electron microscopy, transmission electron microscopy, and Fourier transform infrared spectroscopy. A co-culture system was established between the bionic periosteum and macrophages, bone marrow mesenchymal stem cells and human umbilical vein endothelial cells to explore immune regulation and in vitro osteogenic and angiogenic abilities. Finally, the osteogenic properties of the stem cell engineered bionic periosteum were further verified in a rat femoral condyle defect model.
RESULTS AND CONCLUSION: (1) Transmission electron microscopy results showed that the micro-sol electrospinning (MS) formed a distinct core-shell structure. Scanning electron microscopy indicated that after the assembly of the collagen-I artificial periosteum (M@C) on the surface of the vascular endothelial growth factor-loaded micro-sol, a distinct “spider web-like” fibrous structure was deposited. Infrared spectroscopy further confirmed the successful self-assembly of collagen-I. Release experiments demonstrated that the M@C group mitigated the burst release phenomenon compared to the MS group, maintaining internal vascular endothelial growth factor activity and sustained release. (2) Live/dead cell staining and CCK-8 assay showed that bone marrow mesenchymal stem cells proliferated well and survived on three types of artificial periosteum: MS, purely aligned poly(L-lactic acid) (PLLA) surface self-assembled collagen-I artificial periosteum (PLLA@C), and vascular endothelial growth factor-loaded micro-sol fiber surface self-assembled collagen-I-bone marrow mesenchymal stem cells artificial periosteum (M@C-B). Among them, the M@C-B group had the highest number of live cells and the fastest proliferation rate. (3) Alkaline phosphatase staining, alizarin red staining, and osteopontin immunofluorescence staining showed that the PLLA@C and M@C-B groups significantly promoted osteogenic differentiation of bone marrow mesenchymal stem cells. Angiogenesis experiments demonstrated that the vascular endothelial growth factor-loaded groups (MS and M@C-B) had longer blood vessel lengths and more reticular vascular-like structures with more cross-linked nodes, with the M@C-B group being the most prominent. (4) Immunofluorescence and flow cytometry showed that artificial periosteum in the M@C-B group significantly inhibited the pro-inflammatory macrophage phenotype and promoted the polarization of macrophages towards the anti-inflammatory M2 phenotype. (5) In vivo studies further confirmed that the M@C-B group showed superior bone mineral density, trabecular thickness, relative bone volume, and trabecular spacing compared to other groups. (6) These results indicate that bone marrow mesenchymal stem cell-engineered artificial periosteum, through the rapid regulation of the bone defect immune microenvironment by the collagen-I-bone marrow mesenchymal stem cells outer phase and the sustained release of vascular endothelial growth factor by the micro-sol electrospinning core-shell structure of the inner phase, synergistically promotes bone healing.

Key words: bone defect repair, bionic periosteum, electrospinning, vascular endothelial growth factor, bone marrow mesenchymal stem cell, macrophage, engineered stem cell