P53 negatively regulates the osteogenic differentiation in jaw bone marrow MSCs derived from diabetic osteoporosis

Abstract

Patients with diabetic osteoporosis (DOP) often suffer from poor osseointegration of artificial implants, which is a challenge that affects implant outcomes. The osteogenic differentiation ability of human jaw bone marrow mesenchymal stem cells (JBMMSCs) is the key to implant osseointegration. Studies have shown that the microenvironment of hyperglycemia affects the osteogenic differentiation of mesenchymal stem cells (MSC), but the mechanism is still unclear. Therefore, the aim of this study was to isolate and culture JBMMSCs from surgically derived bone fragments from DOP patients and control patients to investigate the differences in their osteogenic differentiation ability and to elucidate its mechanisms. The results showed that the osteogenic ability of hJBMMSCs was significantly decreased in the DOP environment. Mechanism study showed that the expression of senescence marker gene P53 was significantly increased in DOP hJBMMSCs compared to control hJBMMSCs according to RNA-sequencing result. Further, DOP hJBMMSCs were found to display significant senescence using β-galactosidase staining, mitochondrial membrane potential and ROS assay, qRT-PCR and WB analysis. Overexpression of P53 in hJBMMSCs, knockdown of P53 in DOP hJBMMSCs, and knockdown followed by overexpression of P53 significantly affected the osteogenic differentiation ability of hJBMMSCs. These results suggest that MSC senescence is an important reason for decreasing osteogenic capacity in DOP patients. P53 is a key target in regulating hJBMMSCs aging, and knocking down P53 can effectively restore the osteogenic differentiation ability of DOP hJBMMSCs and promote osteosynthesis in DOP dental implants. It provided a new idea to elucidate the pathogenesis and treatment of diabetic bone metabolic diseases.

Keywords: Bone regeneration; Diabetic osteoporosis; Osteogenesis; Senescence; hJBMMSCs.

Fig. 1

Isolation, culture, and identification of hJBMMSCs(human jaw bone marrow mesenchymal stem cells). A. Collection of clinical samples; B. Microscopically, Primary (P0) hJBMMSCs and the third generation (P3) hJBMMSCs were spindle-shaped. C. HJBMMSCs positively expressed CD73, CD90, CD105, and negatively expressed CD14, CD34, CD45. D. The ALP (alkaline phosphatase) staining area of osteogenic induced group was larger and darker than control group. E. RUNX2, ALP, OCN, SP7 and DLX5 were significantly up-regulated in the osteogenic induction group (Mean ± SD, n = 3). F. A large number of lipid droplets appeared in the adipogenic induced group. G. PPARG, ADI and CEPBA were significantly up-regulated in adipogenic induction group (Mean ± SD, n = 3). H. Chondrogenic differentiation was induced and then clumped. Microscopically, chondrocytes and proteins were cross-linked into a network. ***P < 0.001.

Fig. 2

Comparison of osteogenic differentiation ability between NC and DOP(diabetic osteoporosis) hJBMMSCs. A. The ALP staining area was smaller and the color was lighter in DOP group than NC group on the 10th day (Mean ± SD, n = 6). B. The ALP activity was lower in DOP group than NC group after 7 days and 14 days of osteogenic induction (Mean ± SD, n = 6). C. After osteogenic induction, ARS (alizarin red S) staining on the 21st day showed that the number of mineralized nodules in DOP group was significantly less than that in NC group (Mean ± SD, n = 6). D. semi-quantitative analysis showed that the OD (optical density) value of DOP group was significantly lower than that of NC group (Mean ± SD, n = 6). E. After osteogenic induction, the mRNA expressions of RUNX2, BMP2,ALP, OCN, SP7 and DLX5 in DOP group were significantly lower than those in NC group (Mean ± SD, n = 3). F. The protein expressions of RUNX2, BMP2, ALP and DLX5 in DOP group were significantly lower than those in NC group (Mean ± SD, n = 3). **P < 0.01,***P < 0.001.

Fig. 3

DOP model and bone defect model were established. A. Micro-CT images showed dense trabecular bone of normal rat femur, while sparse trabecular bone in the femur of GK rats in coronal plane, sagittal plane and cross section. B. A surgical procedure to create a defect in the skull. C. Micro-CT three-dimensional reconstruction showed that the area of new bone formation in DOP rats was significantly less than that in NC rats. D. The percentage of new bone formation was significantly different between the two group (Mean ± SD, n = 5). E. The coverage area of new bone was relatively small with DOP hJBMMSCs in both DOP rats and NC rats at 10 week. F. Statistical analysis showed significant differences between the two groups in both DOP rats and NC rats (Mean ± SD, n = 5). ***P < 0.001.

Fig. 4

Biological characteristics of NC and DOP hJBMMSCs. A. From the third day, the proliferation ability of DOP hJBMMSCs was significantly lower than that of NC group (1-12 d, n = 5). B. On day 10, the clone formation rate of DOP hJBMMSCs was significantly lower than that of NC group (Mean ± SD, n = 6). C. The wound healing area of DOP group was significantly smaller than that of NC group at 12 h, 24 h and 48 h (Mean ± SD, n = 6). D. The G0/G1 phase, S phase and G2/M phase of the cell cycles were different in the two groups (Mean ± SD, n = 5). *P < 0.05,**P < 0.01, ***P < 0.001.

Fig. 5

RNA-seq analysis and validation of DOP hJBMMSCs gene. A. The volcano plot illustrated the differentially expression genes between DOP and NC group. Red and green denote the upregulation and downregulation, respectively. B. Representative top 40 genes after the hierarchical clustering analysis. C. The most significantly affected categories in biological process, cellular component and molecular function by Gene ontology functional clustering on differentially expressed genes. D. The top 20 of most signifificantly affected pathway by KEGG pathway analysis of differentially expressed genes. E. Multiple samples were used to verify that P53 gene expression was significantly different between DOP group and NC group. F. The expression of P53 protein in DOP group and NC group was detected by WB, and statistical analysis showed significant differences (Mean ± SD, n = 3). ***P < 0.001.

Fig. 6

Senescence characteristics of NC and DOP hJBMMSCs. A. The SA-β-Gal (senescence-associated β-galactosidase) expression in DOP group was significantly higher than that in NC group (Mean ± SD, n = 6). B. The mitochondrial green fluorescence in DOP group was significantly stronger than that in NC group, and the ratio of red/green fluorescence was significantly lower than that of NC group (Mean ± SD, n = 6). C. In the detection of reactive oxygen species, the fluorescence intensity of DOP group was significantly stronger than that of NC group (Mean ± SD, n = 6). D. The expression of senescence related secretory phenotype genes was increased in DOP group (Mean ± SD, n = 3). E. The expression of senescence related proteins in DOP group was increased (Mean ± SD, n = 3). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 7

Changes of senescence and osteogenesis related indexes after P53 overexpression in NC group. A,B. After overexpressing P53 in NC group, the expressions of senescence-related genes and proteins were increased (Mean ± SD, n = 3). C,D. After overexpressing P53 in NC group, the expression of osteogenesis related genes and proteins decreased (Mean ± SD, n = 3). E. After overexpressing P53 in the NC group, ARS staining was performed on the 14th day after osteogenic induction, and the number of mineralized nodules was significantly less than that in the NC group. **P < 0.01, ***P < 0.001.

Fig. 8

P53 regulated the osteogenic ability of DOP hJBMMSCs. A,B. After knockdown of P53 in DOP group, the expressions of senescence related genes and proteins were significantly decreased (Mean ± SD, n = 3). C,D. After knockdown of P53 in DOP group, the expression of osteogenic related genes and proteins significantly increased (Mean ± SD, n = 3). E. In DOP + siRNA (small interfering RNA) group, ARS staining was performed after14 days of osteogenic induction, and the number of mineralized nodules was significantly higher than that in DOP group. F,G,H. After overexpression of P53 in DOP + siRNA group, the mRNA and protein of P53 were increased and the mRNA and protein of RUNX2 were decreased, significantly (Mean ± SD, n = 3). I. After overexpression of P53 in DOP + siRNA group, mineralized nodules were significantly reduced after 14 days of osteogenic induction. **P < 0.01, ***P < 0.001.