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Guided bone regeneration (GBR) is a promising strategy for repairing bone defects using bioactive membranes. In this study, a new type of GBR membrane based on the small intestinal submucosa (SIS) was created, and its surface structure, cytological characteristics, and bone defect repair ability were compared with commonly used membranes. Our results show that compared to the Heal-all and Dentium membranes, the SIS membrane has an asymmetric structure that does not affect the proliferation of bone marrow mesenchymal stem cells (BMSCs). Instead, it increased their formation of calcium nodules and expression of bone morphogenetic protein-2 (BMP-2), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteopontin (OPN). Six weeks after their insertion into a rat calvarial defect model, increased bone growth was observed in the SIS membrane group. Our results indicate that the SIS membrane has good biocompatibility and is more effective in promoting early bone formation than existing membranes. Given the wide range of source materials and simple preparation processes available, SIS membrane is a promising candidate for guided bone regeneration. [ABSTRACT FROM AUTHOR]