Standardized, high-efficiency protocol for in vitro murine jawbone osteoclast isolation and differentiation

Osteoclasts derived from distinct skeletal sites exhibit site-specific differences in osteoclastogenic potential, reflecting variations in developmental origin, local microenvironment, and regulatory signaling pathways. Jawbones differ from long bones in their remodeling characteristics, reflecting their neural crest origin, intramembranous development, and functional integration with the periodontal ligament, which together shape distinct stromal programs, mechanotransduction cues, and bone turnover set points. These differences are especially relevant when studying diseases such as periodontitis, where alveolar bone resorption is a hallmark feature. However, due to the lack of standardized protocols for the isolation and culture of jawbone derived osteoclasts, most in vitro studies rely on bone marrow–derived osteoclasts, which incompletely recapitulate craniofacial osteoclast biology and may therefore limit translational relevance. Here, we present a comprehensive protocol for the generation of osteoclasts derived from murine jawbone tissue, followed by downstream in vitro processing and analytical evaluation. The protocol is complemented by standardized methods to assess osteoclast differentiation and functional resorptive capacity using calcium phosphate coated surfaces. By enabling the reliable generation and characterization of osteoclasts from craniofacial bone, this approach provides a physiologically relevant in vitro model for studying periodontitis and other oral bone pathologies, thereby enhancing the mechanistic understanding of site-specific osteoclast biology, and supporting the development of targeted therapeutic strategies.