Establishing an osteosarcoma organoid model reflecting the bone microenvironment

Osteosarcoma is the most common primary malignant bone tumor in children and young adults and usually arises in the metaphyses of long bones during periods of rapid skeletal growth. Survival for metastatic or relapsed disease remains poor, and most in vitro studies still rely on two-dimensional plastic cultures that do not reproduce the mineralized matrix, stromal cells, and matrix-bound growth factors that shape osteosarcoma behavior in bone.To address this gap, we established a stepwise bone-mimetic system using Saos-2 osteoblastic osteosarcoma cells. Demineralized bone matrix (DBM) serves as a defined source of bone-matrix-derived growth factors (DBM-DGF), including growth factors such as TGF-beta, BMPs, and IGF-1, which act together in a combined effect. Fresh human bone chips provide a more complete bone surrogate with native mineralized matrix, matrix-derived factors, and resident bone cells. In tier 1, Saos-2 cells were treated with matrix-derived growth factor preparations that had been prescreened and grouped by osteoinductive activity. Proliferation was measured by MTT assay. Saos-2 cells showed a graded increase in proliferation across DBM-DGF groups, with the highest-activity preparations producing the strongest proliferative response. These findings indicate that DBM potency directly influences the growth-promoting strength of bone-matrix-derived signals and can be used to approximate “young,” more proliferative, growth-factor-rich bone versus more “aged,” growth-factor-poor bone.In tier 2, conditioned medium from bone-matrix-derived stromal cells is applied to Saos-2 cultures to model paracrine co-culture and test how stromal secreted factors regulate proliferation, differentiation, and migration. In tier 3, fresh human bone chips are combined with Saos-2 cells in three dimensions so that tumor cells experience intact mineralized matrix, matrix-bound growth factors, and resident bone cells at the same time, creating a bone-like microenvironment. This tiered platform directly compares isolated matrix-derived signals with a structurally and cellularly complex bone surrogate and provides a practical, more physiologically relevant model for studying tumor-bone interactions, age-related changes in the bone environment, and for preclinical testing of candidate therapies in osteosarcoma.