gms | German Medical Science

Objectives: Critical-sized bone defects are clinically associated with a high risk of impaired regeneration and non-union development. Thus, novel therapeutic approaches to enhance bone repair are needed. TNF-stimulated gene 6 protein (TSG-6), an anti-inflammatory and pro-regenerative protein, is secreted by various cell types including mesenchymal stem cells (MSCs) and known to improve wound healing. Because wound and bone healing share similar pathways, we here investigated if TSG-6 also stimulates bone repair.

Methods: Male C57BL/6J mice (12-weeks-old) received a femoral critical-sized defect (1.5 mm). A collagen type I gel served as a carrier to transfer 10 or 50 μg recombinant human TSG-6 (rhTSG-6) into the bone defect. An unloaded collagen gel and empty defect served as controls. Bone repair was analysed using μCT and histomorphometry after 35 d (n=8/group). The impact of 50 μg rhTSG-6 on innate and adaptive immune cells in the fracture hematoma was investigated on d3 using flow cytometry (n=8/group). The role of Tsg-6 on osteogenic differentiation (OD) was investigated in vitro in primary murine MSCs by siRNA-mediated Tsg-6 KO (n=4/group), and effects were analysed by expression analysis of the osteogenic marker genes alkaline phosphatase (Alpl), Runt-related transcriptionfactor-2 (Runx2), Bone γ-carboxyglutamate protein (Bglap) and Sp7 transcription factor (Sp7), and by quantitative measurements of alkaline phosphatase activity. Statistics: Student’s t-test or ANOVA/post hoc Fisher's LSD, p<0.05.

Results and conclusion: In vivo, 50 μg rhTSG-6 significantly improved bone repair as indicated by an increased percentage of bony bridged defects and volume of newly formed bone compared to 10 μg rhTSG-6, collagen gel and empty group (Figure 1A–C [Fig. 1]). Histomorphometric analysis of the fracture callus showed a significant increased number of osteoblasts and decreased number of osteoclasts (Figure 1D–G [Fig. 1]), suggesting that 50 µg rhTSG-6 promoted bone formation via increased osteoblastogenesis and reduced osteoclastogenesis. On d3, we found significantly increased numbers of TH-cells (CD3+/CD4+) in the fracture hematoma of the 50 μg rhTSG-6-treated group compared to collagen gel and to empty group (Figure 1H [Fig. 1]). In vitro, Tsg-6 KO significantly impaired OD of mMSCs indicated by a reduced osteogenic marker gene expression and mineralization (Figure 1I–L [Fig. 1]).

Our results showed that TSG-6 improved the healing of critical-sized bone defects in a pre-clinical model. Moreover, we identified Tsg-6 as a positive regulator of osteogenesis. Our results imply that rhTSG-6 treatment might offer a new therapeutic option to enhance bone healing in patients with large bone defects. However, further mechanistic investigations are warranted.