BMP9-loaded collagen scaffold promotes periodontal regeneration via microenvironmental modulation of PDLSCs and macrophages.

Periodontal tissue regeneration requires coordinated cellular responses among multiple cell types, supporting matrix components, and signaling proteins, including bone morphogenetic proteins (BMPs). Despite extensive studies on BMP2, the regenerative role of BMP9 and its interplay with BMP2 in shaping bone and cementum repair remain poorly understood. Notably, BMP9 was independent of the antagonist Noggin, and molecular dynamics simulations confirmed that the Noggin-BMP9 complex exhibited greater root mean square deviation (RMSD) values and unstable interactions in periodontal ligament stem cells (PDLSCs). Functionally, BMP9 notably enhanced the osteogenic and cementoblastic differentiation of PDLSCs, elicited minimal osteoclast activation, and improved the survival of PDLSCs and macrophages by attenuating stress-induced apoptosis and preserving mitochondrial membrane potential. BMP9 also induced epithelial-mesenchymal transition (EMT) and exhibited synergy with endogenous matrix proteins to enhance mineralization. To optimize BMP localization in the application site, sponge type collagen scaffolds were attempted. Type I collagen sponges crosslinked with carbodiimide (EDC/NHS) and dehydrothermal (DHT) were compared, and the DHT-crosslinked sponge was selected for its larger pores, higher absorbency, and higher BMP9 loading efficiency. In a canine periodontal defect model, BMP9-loaded DHT-collagen sponges facilitated integrated regeneration of cementum, periodontal ligament, and alveolar bone, accompanied by localized ApoBD-like structures associated with PDLSC- and macrophage-related markers. Transcriptomic profiling revealed notable induction of gene sets related to EMT and cementogenesis, underscoring the role of BMP9 in driving periodontal tissue homeostasis. Collectively, these findings establish high-affinity BMP9 delivery via a collagen sponge as an effective and clinically translatable strategy for periodontal tissue regeneration.