Deletion of p16 accelerates fracture healing in geriatric mice

Abstract

The biomarker p16 plays a role in aging and is upregulated in aged organs and cells, including bone marrow mesenchymal stem cells (BM-MSCs), which play a leading role in fracture healing. Several studies have reported delayed fracture healing in geriatric mice. However, the relationship between p16 expression and fracture healing in geriatric mice remains poorly understood. In this study, we found that fracture healing was accelerated in p16 deletion (p16^-/-) mice, and the number of migrated BM-MSCs from p16^-/- mice increased. The expressions of SDF-1 and CXCR4 were also upregulated in p16^-/- mice. Increased cell percentage at S phase in cell cycle, enhanced expressions of CDK4/6, pRB, and E2F1, decreased expression of RB, and elevated expressions of SOX9, PCNA, and COL2A1 were detected in p16^-/- mice. The expressions of COL10A1, MMP13, OSTERIX, and COL1A1 were also high in p16^-/- mice. Moreover, the expressions of p-AKT, p-mTOR, HIF-1α, and VEGF-A in BM-MSCs and expression of VEGF-A in callus were upregulated in p16^-/- mice. The expression of VEGF in the serum of p16^-/- mice was also higher than that of wild type mice. Thus, deletion of p16 enhances migration, division, and differentiation of BM-MSCs, promotes proliferation and maturation of chondrocytes, activates osteoblastogenesis, and facilitates vascularization to accelerate fracture healing, providing a novel strategy to treat fracture in the elderly.

Keywords: endochondral ossification; fracture healing; geriatric mice; p16; vascularization.

Figure 1

Deletion of p16 accelerated fracture healing in geriatric mice. Representative micrographs of (A) H&E staining and (B) toluidine blue staining and (C) percentages of soft and bony callus at different postoperative timepoints. (D) Representative X-ray radiographs and (E) coronal reconstruction images of micro-CT scans of callus. (F) Quantitative analysis of callus microstructural parameters, including bone mineral density (BMD) and bone volume to total volume ratio (BV/TV). n=4, *P<0.05, **P<0.01, ***P<0.001, compared with WT mice.

Figure 2

Deletion of p16 promoted BM-MSCs migration. A. Transwell assay was performed to assess the migration of BM-MSCs. B, C. Western blot analysis was performed to determine the protein expression levels of SDF-1 in clots at the injury site on postoperative day 1 and CXCR4 in BM-MSCs. β-actin was used as loading control. D, F. The quantified protein levels of SDF-1 and CXCR4 were evaluated by densitometric analysis. E, G. The mRNA levels of SDF-1 and CXCR4 were measured by qRT-PCR, calculated as ratio relative to GAPDH mRNA, and expressed relative to WT. n=4, **P<0.01, ***P<0.001, compared with WT mice.

Figure 3

Deletion of p16 encouraged BM-MSCs to proliferate and differentiate into chondrocytes. A. Cell-cycle distribution of BM-MSCs was determined by flow cytometry. B. Western blot analysis was performed to assess the protein expression levels of members of the CDK4/6-pRB-E2F pathway, including CDK4, CDK6, RB, pRB, and E2F1 in BM-MSCs. β-actin was used as loading control. C. The quantified protein levels of CDK4, CDK6, RB, pRB, and E2F1 in BM-MSCs were evaluated by densitometric analysis. D. The mRNA levels of CDK4, CDK6, RB, pRB and E2F1 in BM-MSCs were measured by qRT-PCR, calculated as ratio relative to GAPDH mRNA and expressed relative to WT. E. Representative immunohistochemical micrographs of SOX9, a key regulator of chondrocyte differentiation. F. The percentages of SOX9-positive cells in callus on postoperative days 7, 10, and 14. G. The mRNA levels of SOX9 in callus on postoperative days 7, 10, and 14 were measured by qRT-PCR, calculated as ratio relative to GAPDH mRNA and expressed relative to WT. n=4, *P<0.05, **P<0.01, ***P<0.001, compared with WT mice.

Figure 4

Deletion of p16 promoted chondrocyte proliferation. Representative immunofluorescent micrographs of (A) PCNA, an indicator of proliferation, and (C) COL2A1, a specific marker of chondrocytes in chondrocytes differentiated from BM-MSCs. The percentages of (B) PCNA-positive cells and (D) COL2A1-positive areas in chondrocytes differentiated from BM-MSCs. (E) CCK-8 assay was performed to measure the absorbance at 450 nm to assess chondrocyte proliferation in chondrocytes differentiated from BM-MSCs. Representative immunohistochemical micrographs of (F) PCNA and (I) COL2A1 in callus on postoperative days 7, 10 and 14. The percentages of (G) PCNA-positive cells and (J) COL2A1-positive areas in callus on postoperative days 7, 10, and 14. (G) The mRNA levels of (H) PCNA and (K) COL2A1 in callus on postoperative days 7, 10, and 14 were measured by qRT-PCR, calculated as ratio relative to GAPDH mRNA and expressed relative to WT. n=4, *P<0.05, **P<0.01, ***P<0.001, compared with WT mice.

Figure 5

Deletion of p16 promoted chondrocyte maturation. Representative immunohistochemical micrographs of (A) COL10A1, a specific marker of hypertrophic chondrocytes and (D) MMP13 in callus on postoperative days 10 and 14. The percentages of (B) COL10A1-positive and (E) MMP13-positive areas in callus on postoperative days 10 and 14. The mRNA levels of (C) COL10A1 and (F) MMP13 in callus on postoperative days 10 and 14 were measured by qRT-PCR, calculated as ratio relative to GAPDH mRNA and expressed relative to WT. n=4, **P<0.01, ***P<0.001, compared with WT mice.

Figure 6

Deletion of p16 facilitated osteoblastogenesis. (A) Western blot analysis was performed to determine the protein expression levels of osteoblastic differentiation markers, including OSTERIX and COL1A1 in induced BM-MSCs. β-actin was used as loading control. (B) The quantified protein levels of OSTERIX and COL1A1 in induced BM-MSCs were evaluated by densitometric analysis. (C) The mRNA levels of OSTERIX and COL1A1 in induced BM-MSCs were measured by qRT-PCR, calculated as ratio relative to GAPDH mRNA and expressed relative to WT. (D) ALP staining and alizarin red staining images of induced BM-MSCs. Representative immunohistochemical micrographs of (E) OSTERIX in callus on postoperative days 7, 10 and 14, and (H) COL1A1 in callus on postoperative days 10, 14 and 21. The percentages of (F) OSTERIX-positive cells in callus on postoperative days 7, 10, and 14, and (I) COL1A1-positive areas in callus on postoperative days 10, 14, and 21. The mRNA levels of (G) OSTERIX in callus on postoperative days 7, 10, and 14, and (J) COL1A1 in callus on postoperative days 10 and 14 were measured by qRT-PCR, calculated as ratio relative to GAPDH mRNA and expressed relative to WT. n=4, *P<0.05, **P<0.01, ***P<0.001, compared with WT mice.

Figure 7

Deletion of p16 stimulated vascularization by activating the AKT/mTOR/HIF-1α pathway. HUVEC tube formation assay was performed to determine the differentiation of HUVECs into capillary-like structures. A. Representative micrographs and quantitation of numbers of HUVEC meshes. B. Representative immunohistochemical micrographs of VEGF-A in callus on postoperative days 7, 10, and 14. C. The percentages of VEGF-A-positive cells in callus on postoperative days 7, 10, and 14. D. The serum VEGF level detected with ELISA. E. Western blot analysis was employed to assess the protein expression levels of members of the AKT/mTOR/HIF-1α pathway, including p-AKT, AKT, p-mTOR, mTOR, HIF-1α, and VEGF-A. β-actin was used as loading control. F. The quantified protein levels of CDK4, CDK6, RB, pRB, and E2F1 were evaluated by densitometric analysis. n=4, **P<0.01, ***P<0.001, compared with WT mice.