Damage associated molecular patterns in necrotic femoral head inhibit osteogenesis and promote fibrogenesis of mesenchymal stem cells

In Legg-Calve-Perthes disease (LCPD), a loss of blood supply to the juvenile femoral head leads to extensive cell death and release of damage-associated molecular patterns (DAMPs). Over time chronic inflammatory repair process is observed with impaired bone regeneration. Increased fibrous tissue and adipose tissue are seen in the marrow space with decreased osteogenesis in a piglet model of LCPD, suggesting inhibition of osteoblastic differentiation and stimulation of fibroblastic and adipogenic differentiation of mesenchymal stem cell (MSC) during the healing process. Little is known about the DAMPs present in the necrotic femoral head and their effects on MSC differentiation. The purpose of this study was to characterize the DAMPs present in the femoral head following ischemic osteonecrosis and to determine their effects on MSC differentiation. Necrotic femoral heads were flushed with saline at 48 h, 2 weeks and 4 weeks following the induction of ischemic osteonecrosis in piglets to obtain necrotic bone fluid (NBF). Western blot analysis of the NBF revealed the presence of prototypic DAMP, high mobility group box 1 (HMGB1), and other previously described DAMPs: biglycan, 4-hydroxynonenal (4-HNE), and receptor activator of NF-kappa B ligand (RANKL). ELISA of the NBF revealed increasing levels of inflammatory cytokines IL1 beta, IL6 and TNF alpha with the temporal progression of osteonecrosis. To determine the effects of NBF on MSC differentiation, we cultured primary porcine MSCs with NBF obtained by in vivo necrotic bone flushing method. NBF inhibited osteoblastic differentiation of MSCs with significantly decreased OSX expression (p = 0.008) and Von Kossa/Alizarin Red staining for mineralization. NBF also significantly increased the expression of proliferation markers Ki67 (p = 0.03) and PCNA (p < 0.0001), and fibrogenic markers Vimentin (p = 0.02) and Fibronectin (p = 0.04). Additionally, NBF treated MSC cells showed significantly elevated RANKL/OPG secretion ratio (p = 0.003) and increased expression of inflammatory cytokines IL1 beta (p = 0.006) and IL6 (p < 0.0001). To specifically assess the role of DAMPs in promoting the fibrogenesis, we treated porcine fibroblasts with artificial NBF produced by bone freeze-thaw method. We found increased fibroblastic cell proliferation in an NBF dose-dependent manner. Lastly, we studied the effect of HMGB1, a prototypic DAMP, and found that HMGB1 partially contributes to MSC proliferation and fibrogenesis. In summary, our findings show that DAMPs and the inflammatory cytokines present in the necrotic femoral head inhibit osteogenesis and promote fibrogenesis of MSCs, potentially contributing to impaired bone regeneration following ischemic osteonecrosis as observed in LCPD.