骨愈合是多个功能事件组合在一起的精细调整的过程，涉及早期的炎症免疫调节和后续血管生成、成骨分化以及生物矿化等多个事件的重合与交替。浙江大学医学院范顺武与林贤丰课题组受“骨膜成骨”概念启发，制备一种可注射的骨膜细胞外基质水凝胶并评估其在骨愈合过程中对免疫炎症、血管生成、成骨分化等修复事件的调节作用。采用酶消化法制备脱细胞骨膜细胞外基质(PEM)水凝胶。经组织学染色观察脱细胞组织结构与成分保留，定量测定 DNA 和胶原含量。以 I 型胶原水凝胶作为通用基材对照，通过扫描电镜观察两类水凝胶的微观结构。通过质谱分析推测PEM 水凝胶的潜在生物功能。与 Raw264.7 细胞共培养 3 天后通过 qPCR 和免疫荧光法评估 PEM 水凝胶体外对巨噬细胞极化标志物的表达调节。与 HUVECs 细胞共培养 6 小时后，通过内皮细胞管样结构形成实验评估 PEM 水凝胶体外促血管生成活性。与成骨细胞共培养 14 天后，通过 qPCR 和免疫荧光法评估 PEM 水凝胶对成骨分化相关基因表达的影响。PEM 水凝胶在体植入大鼠颅骨临界缺损模型后，通过常规和特殊组织学染色评估血管增生和新骨形成与成熟，免疫荧光法检测在体巨噬细胞极化，Micro-CT 扫描检测评估矿化骨形成。相比较于单一成分的 I 型胶原水凝胶，PEM 水凝胶以独特的生化组成，在早期免疫调节及后期的血管生成、成骨分化等修复事件方面均显现更优越的调节作用，这些生物效应与骨再生的自然过程相配合，最终加速骨修复。因此，PEM水凝胶在骨组织工程中具有广阔的应用前景。
Bone healing is a process of fine-tuning of multiple functional events, which involves the coincidence and alternation of early inflammatory immune regulation and subsequent angiogenesis, osteogenic differentiation and biomineralization. Professor Fan Shunwu and Professor Lin Xianfeng from Zhejiang University of Medical inspired by the concept of periosteal osteogenesis, they prepared an injectable periosteal extracellular matrix hydrogel which its regulatory effect on immune inflammation, angiogenesis, osteogenic differentiation and other repair events during bone healing was evaluated.Decellularized periosteum extracellular matrix (PEM) hydrogel was prepared by enzyme digestion method. Histological staining was used to observe the decellularized tissue structure and component retention, and then quantitatively determine the DNA and collagen content. Using type I collagen hydrogel as a universal substrate control, the microstructures of the hydrogels were observed by SEM. The potential biological functions of PEM hydrogels were inferred by mass spectrometry analysis. After co-culture with Raw264.7 cells for 3 days, the expression regulation of macrophage markers in vitro by PEM hydrogels was evaluated by qPCR and immunofluorescence. After co-cultivation with HUVECs for 6 h, the angiogenic activity of PEM hydrogels in vitro was evaluated by endothelial cell tube-like formation assay.Compared with the single component I collagen hydrogel, PEM hydrogel has a unique biochemical composition, which shows superior regulatory effects in early immune regulation and later angiogenesis, osteogenic differentiation and other repair events. These biological effects cooperate with the natural process of bone regeneration, and ultimately accelerate bone repair. Therefore, PEM hydrogel has broad application prospects in bone tissue engineering.