Development of a gelatin methacryloyl double-layer membrane incorporated with nano-hydroxyapatite for guided bone regeneration

Abstract

Guided bone regeneration (GBR) is an effective technique for treating bone defects, with barrier membranes playing a critical role in preventing soft tissue invasion while supporting bone formation. However, conventional collagen GBR membranes have limitations, including poor mechanical strength, high swelling ratio, rapid biodegradation, and fragile structures. In this study, we developed a heterogeneous double-layer membrane with tunable physical, chemical, and biological properties, fabricated through simple photopolymerization and lyophilization of gelatin methacryloyl (GelMA) and nanohydroxyapatite (nHA). By adjusting the crosslinking time, methacrylation degree, and nHA concentration, the cryogels showed porous microstructures with different pore sizes ranging from 93 to 360 μm. Compressive mechanical testing, swelling measurements, and in vitro/in vivo biodegradation assays confirmed that the methacrylation of gelatin increased the compressive modulus to 29.02 MPa (p = 0.0002), reduced the swelling ratio to 714% (p = 0.002), and slowed the degradation rate to 41.2% after 48 hours (p = 0.002). Incorporating nHA further enhanced the mechanical properties and extended the degradation time. GelMA and nHA–GelMA cryogels exhibited excellent biocompatibility and promoted osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), particularly in the nHA–GelMA cryogel with large pore sizes. We selected a GelMA cryogel with the smallest pore size for optimal barrier function and an nHA–GelMA cryogel with the highest osteogenic potential to construct the double-layer GBR membrane. In a rat calvarial defect model, this novel membrane significantly enhanced bone regeneration, demonstrating markedly improved bone volume/tissue volume (BV/TV) and bone mineral density (BMD) compared to the control group (p = 0.0042 and p = 0.0088, respectively), with efficacy comparable to that of a commercial GBR membrane. These findings demonstrate the promising potential of this simple, tunable double-layer GelMA/nHA cryogel membrane as a superior alternative for GBR applications.