Fig 2: Knockdown of CHRDL1 inhibited BMP4-induced osteoblast differentiation of hBMSCs. hBMSCs were either transfected with si-CHRDL1 or NC-siRNA, 24 h later, both groups were induced by BMP-2, BMP-4, or BMP-7. 3 days after transfection, mRNAs were harvested for real-time quantitative PCR (A-C). Western blotting was used to detect levels of CHRDL1, p-SMAD1/5/9, and total SMAD1/5/9 in hBMSCs transfected with NC-siRNA and si-CHRDL1 either with or without treatment with rhBMP-4 (D). Relative levels of p-SMAD/t-SMAD were plotted graphically in panel (E). The experiment was repeated three times. Data were reported as mean ± SE (n = 3) (*P <0.05 and #P <0.01).

Fig 3: CHRDL1 overexpression enhanced BMP-4-induced osteoblast differentiation in vitro. (A,B) CHRDL1 mRNA expression and protein levels were detected at 72 h after transfection of pLVX-CHRDL1. ***P <0.01 compared with pLVX-vector. (C) The mRNA levels of COL1A1, ALP, OCN, and OPN were detected at 72 h after pLVX-CHRDL1 transfection and 48 h after osteogenic induction. *P <0.05; ***P <0.01 vs. pLVX-vector transfected sample; and #P <0.01 vs. rhBMP-4 administrated sample. (D,E) ALP and Alizarin Red staining after transfection with pLVX-CHRDL1 and rhBMP-4 addition separately or in combination when cultured in osteogenic induction medium for 14 days. Data are presented as mean ±SD (n = 3). *P <0.05; ***P <0.01 vs. pLVX-vector transfected sample; and #P <0.001 vs. rhBMP-4 administrated sample. (F) Western blot analysis of BMPR II, p-Smad1/5/9, total Smad1/5/9, Runx2, CHRDL1, and GAPDH at 48 h after transfection with pLVX-CHRDL1 and rhBMP-4 addition separately or in combination. GAPDH was used as loading control. (G) mRNA levels of ALP, COL1A1, OCN, and OPN were detected 48 h after treating with LDN-193189 or its vehicle (DMSO). Data were presented as mean ± SD (n = 3); (*P <0.05 and #P <0.01). All experiments were repeated independently in triplicate.

Fig 4: Knockdown of CHRDL1 suppressed hBMSCs osteogenesis. Three siRNAs were generated to suppress CHRDL1 expression. (A,B) CHRDL1 mRNA and protein expression levels were detected 72 h after siRNAs transfection. Densitometric analysis of immunoblot band intensities for CHRDL1 normalized by GAPDH were also detected. (C) CHRDL1 expression was detected at 0, 3, 7, and 10 days after siRNA2 or siRNA3 transfection. (D) In vitro growth of NC-siRNA and siRNA3 transfected hBMSCs were measured by CCK8 assay at 24, 48, 72, 96 h after transfection. (E) The mRNA expression levels of OCN, COL1A1, OPN, ALP, OSX, and RUNX2 were all detected using real-time quantitative PCR at 48 h after osteogenic induction and 72 h after siRNA3 transfection. (F,G) ALP staining, ALP quantitative analysis and Alizarin Red staining were performed to detect osteogenesis of hBMSCs after siRNA3 transfection. All experiments were repeated independently in triplicate. Data were presented as mean ± SD (n = 3) (*P <0.05 and #P <0.01).

Fig 5: Knockdown and overexpression of CHRDL1 affected bone repair in a mouse model of femoral bone defect. (A) Lateral views of 3D reconstruction of defective femur (top panel) and mineralized bone formed in hole region (lower panel) by micro-CT. Representative images of each group. (B) 3D structural parameters of trabecular BV/TV, Tb.N, Tb.Sp, and Tb.Th of mineralized bone formed in hole region by micro-CT; (C) H & E staining also shows new bone accumulation in hole regions of si-CHRDL1 and pLVX-CHRDL1 treated mice. CHRDL1 expression detected by immunofluorescent assay around newly formed bone in each group were also shown. (Original magnification: 100 × ). All experiments were repeated independently in triplicate. (*P <0.05 and #P <0.01).