目的 探讨海螵蛸（cuttlebone，CB）/牛血清白蛋白（bovine serum albumin，BSA）光交联水凝胶干预大鼠颅骨临界尺寸骨缺损（critical-sized bone defects，CSBD）模型的作用与潜在机制。方法 SD大鼠随机分为对照组、阳性对照组、BSA组、0.5% CB/BSA组、1.5% CB/BSA组和3.0% CB/BSA组，每组6只。采用5 mm外径环钻建立大鼠CSBD模型，对照组仅造模，无植入物；阳性对照组植入Bio-Oss^® Collagen骨填充材料；BSA组、0.5% CB/BSA组、1.5% CB/BSA组和3.0% CB/BSA组分别植入CB质量分数为0、0.5%、1.5%和3%的CB/BSA光交联水凝胶。术后8周，通过微型计算机断层扫描（micro-computed tomography，Micro-CT）对骨组织微结构变化进行分析；苏木素-伊红（hematoxylin-eosin，HE）和Masson染色观察颅骨组织病理改变；免疫组化和免疫荧光法分别检测颅骨组织I型胶原α1（collagen type I alpha 1，COL1A1）、血小板内皮细胞黏附分子-1（platelet endothelial cell adhesion molecule-1，PECAM-1）及骨钙素（osteocalcin，OCN）的阳性表达及荧光强度；qRT-PCR和Western blotting分别检测颅骨组织Wnt家族成员3A（Wnt family members 3A，WNT3A）、β-连环蛋白（β-catenin）、血管内皮生长因子A（vascular endothelial growth factor A，VEGFA）、COL1A1、PECAM-1、OCN的基因及蛋白表达。为评估水凝胶的体内安全性，采集大鼠腹主动脉血分析血常规和血清肝肾功能指标变化。结果 Micro-CT结果显示，对照组大鼠骨缺损区未见新生骨形成，表明其在观察期内未能实现骨性愈合。病理组织切片结果显示，对照组缺损区以疏松的纤维组织为主；而CB/BSA光交联水凝胶组可见新骨形成。与对照组比较，CB/BSA光交联水凝胶组大鼠的骨体积分数、骨密度、骨小梁厚度显著升高（P＜0.05），骨表面积与骨体积比显著降低（P＜0.05），颅骨组织COL1A1、PECAM-1、OCN阳性表达和荧光强度显著增加（P＜0.05），COL1A1、PECAM-1、β-catenin、WNT3A、VEGFA、OCN的表达水平显著上调（P＜0.05），其中以3.0% CB/BSA光交联水凝胶组的综合效果最佳。各组大鼠血常规和血清肝肾功能指标均未见明显异常，主要脏器未见炎症、坏死或纤维化等病理改变。结论 CB/BSA光交联水凝胶展现出良好的骨修复作用与体内安全性，其促成骨效应可能是通过激活Wnt/β-catenin信号通路介导。

Objective To investigate the effect and potential mechanism of cuttlebone (CB)/bovine serum albumin (BSA) photocrosslinked hydrogel on rats with critical sized bone defects (CSBD) model in skull tissues. Methods SD rats were randomly divided into control group, positive control group, BSA group, 0.5% CB/BSA group, 1.5% CB/BSA group and 3.0% CB/BSA group, with six rats in each group. A rat CSBD model was established using a 5 mm outer diameter ring drill, while the control group only underwent modeling without implants, positive control group implanted with Bio-Oss^® collagen bone filling material, CB/BSA photocrosslinked hydrogels with CB mass fraction of 0, 0.5%, 1.5% and 3% were implanted in BSA group, 0.5% CB/BSA group, 1.5% CB/BSA group and 3.0% CB/BSA group, respectively. After eight weeks after surgery, the microstructural changes of bone tissue were analyzed using micro computed tomography (Micro CT). Hematoxylin-eosin (HE) and Masson staining were used to observe pathological changes in skull tissue. Immunohistochemistry and immunofluorescence were used to detect the positive expressions and fluorescence intensity of collagen type I alpha 1 (COL1A1), platelet endothelial cell adhesion molecule-1 (PECAM-1) and osteocalcin (OCN) in skull tissue, respectively. qRT-PCR and Western blotting were used to detect the gene and protein expressions of Wnt family members 3A (WNT3A), β-catenin, vascular endothelial growth factor A (VEGFA), COL1A1, PECAM-1 and OCN in skull tissue, respectively. In order to evaluate the safety of hydrogel in vivo, the abdominal aorta blood of rats was collected to analyze the changes of blood routine and serum liver and kidney function indexes. Results Micro-CT results showed that no new bone formation was observed in the bone defect area of rats in control group, indicating that bone healing was not achieved during the observation period. The pathological tissue section results showed that the defect area in control group was mainly composed of loose fibrous tissue. In CB/BSA photocrosslinked hydrogel group, new bone formation was observed. Compared with control group, bone volume/tissue volume (BV/TV), bone mineral density (BMD and trabecular thickness (Tb.Th) of rats in CB/BSA photocrosslinked hydrogel group were significantly increased (P < 0.05), bone surface/bone volume (BS/BV) was significantly decreased (P < 0.05), the positive expressions and fluorescence intensity of COL1A1, PECAM-1, OCN in skull tissue were significantly increased (P < 0.05), and the expression levels of COL1A1, PECAM-1, β-catenin, WNT3A, VEGFA, OCN were significantly increased (P < 0.05), among which, the 3.0% CB/BSA photocrosslinked hydrogel group had the best comprehensive effect. No significant abnormalities were found in the blood routine and serum liver and kidney function indicators of rats in each group, and no pathological changes such as inflammation, necrosis or fibrosis were observed in the main organs. Conclusion CB/BSA photocrosslinked hydrogel shows good bone repair effect and safety in vivo, and its osteogenic effect may be mediated by activation of Wnt/β-catenin signaling pathway.

R285.5

国家自然科学基金地区基金项目（8216140594）；广西壮族自治区科技厅自然科学基金项目（2022JJA140818）；广西中医药大学2025年校级博士研究生科研创新项目（YCBXJ2025026）；广西中医药大学2025年自治区级硕士研究生科研创新项目（YCSW2025452）；广西中医药大学研究生科研创新项目（YCLT2025008）；北海市科技局研发项（北科合2023258021）