Maintained Properties of Aged Dental Pulp Stem Cells for Superior Periodontal Tissue Regeneration

Abstract

Owing to excellent therapeutic potential, mesenchymal stem cells (MSCs) are gaining increasing popularity with researchers worldwide for applications in tissue engineering, and in treatment of inflammation-related and age-related disorders. However, the senescence of MSCs over passaging has limited their clinical application owing to adverse effect on physiological function maintenance of tissues as well as disease treatment. An inflammatory microenvironment is one of the key contributors to MSC senescence, resulting in low regeneration efficiency. Therefore, MSCs with high resistance to cellular senescence would be a benefit for tissue regeneration. Toward this end, we analyzed the senescence properties of different types of stem cells during culture and under inflammation, including dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), bone marrow mesenchymal stem cells (BMMSCs), and adipose-derived stem cells (ADSCs). Overall, the DPSCs had higher proliferation rates, lower cellular senescence, and enhanced osteogenesis maintenance compared to those of non-dental MSCs cultured from passage three to six. The expression profiles of genes related to apoptosis, cell cycle, and cellular protein metabolic process (contributing to the cell self-renewal ability and metabolic processes) significantly differed between DPSCs and BMMSCs at passage three. Moreover, DPSCs were superior to BMMSCs with regards to resistance to lipopolysaccharide-induced apoptosis and senescence, with enhanced osteogenesis in vitro, and showed improved periodontal regeneration after injection in a miniature pig periodontitis model in vivo. Overall, the present study indicates that DPSCs show superior resistance to subculture and inflammation-induced senescence and would be suitable stem cells for tissue engineering with inflammation.

Keywords: inflammation; mesenchymal stem cells; periodontitis; senescence.

Figure 1.. DPSCs have higher proliferation and osteogenic capability maintenance compared to PDLSCs, ADSCs, and BMMSCs during aging

(A) Gross view of different MSCs at passages three and six. (B) Cell count analysis showed that both passage three and six DPSCs have a higher proliferation rate than that of other stem cells. (C) Cell cycle analysis also indicated more DPSCs at G2 phase compared to other stem cells at both passage three and six. (D) Population doubling of different MSCs at passage three and passage six showed that DPSCs have a higher PD value. (E, F) Real-time RT-PCR analysis revealed that NANOG, SOX2, TERT, and OCT4 were expressed at higher levels in passage three and six DPSCs than in the other stem cells. (G) Alizarin Red staining and quantitative measurement revealed no significant difference in mineralization of DPSCs, PDLSCs, ADSCs and BMMSCs at passage three, while both ADSCs and BMMSCs of passage six exhibited declined mineralization potential and were lower compared to DPSCs and PDLSCs. (H) ADSCs and BMMSCs showed higher osteogenesis ability in vivo than DPSCs and PDLSCs of passage three, with no significant difference among groups at passage six. Values are means ± SDs. One-way ANOVA was used to determine statistical significance. Error bars represent SDs (n = 5). *P ≤ 0.05; **P ≤ 0.01; NS: no significance. Scale bar: 100 μm. B: bone-like tissue, Fib: fibrous tissue, HA: hydroxyapatite/ tricalciumphosphate.

Figure 2.. DPSCS show higher ability for senescence resistance than PDLSCs, ADSCs, and BMMSCs

(A) Senescence-associated β-galactosidase (SA β-gal) staining of DPSCs, PDLSCs, ADSCs, and BMMSCs at passage three. (B) Quantitative analysis indicated significantly more SA β-gal-positive cells in passage three BMMSCs and ADSCs compared to DPSCs and PDLSCs. (C) SA β-gal staining of DPSCs, PDLSCs, ADSCs, and BMMSCs at passage six. (D) Quantitative analysis indicated significantly more SA β-gal-positive cells in passage six PDLSCs, BMMSCs, and ADSCs compared to DPSCs. (E) Reactive oxygen species (ROS) staining of DPSCs, PDLSCs, ADSCs, and BMMSCs at passage three. (F) Quantitative analysis indicated significantly lower ROS generation in passage three DPSCs and PDLSCs compared to ADSCs and BMMSCs. (G) ROS staining of DPSCs, PDLSCs, ADSCs, and BMMSCs at passage six. (H) Quantitative analysis indicated significantly lower ROS generation in passage six DPSCs compared to PDLSCs, ADSCs, and BMMSCs. (I) Real-time RT-PCR analysis revealed that theP16, P21, P53 were downregulated in DPSCs compared to other MSCs at passage three. (J) Real-time RT-PCR analysis revealed that theP16, P21, P53 were downregulated in DPSCs compared to other MSCs at passage six. Values are means ± SDs. One-way ANOVA was used to determine statistical significance. Error bars represent SDs (n = 5). *P ≤ 0.05; **P ≤ 0.01. Scale bar: 100 μm.

Figure 3.. DPSCs have reduced activation of the MAPK signaling pathway and a lower apoptosis expression profile compared to BMMSCs

(A) Affymetrix GeneChip Human Gene 1.0 ST arrays analysis of BMMSCs and DPSCs. (B) The top 13 GO terms for the differentially expressed genes (-LgP > 28) between BMMSCs and DPSCs. (C) The top 13 pathways enriched for differentially expressed genes between BMMSCs and DPSCs. (D) Path-net analysis of the interaction network covering the significantly changed pathways between DPSCs and BMMSCs. (E) Real-time RT-PCR analysis revealed that the MAPK1 and CANT1 level were decreased, and the PIK3R5 and PKLR levels were increased in BMMSCs at passage three compared to DPSCs. GAPDH was used as an internal control. Values are means ± SDs. Student’s t-tests were used to determine statistical significance. Error bars represent SDs (n = 5). **P ≤ 0.01

Figure 4.. DPSCs show higher resistance to LPS-induced senescence and dysfunction of osteogenesis compared to BMMSCs

(A) SA β-gal staining of DPSCs and BMMSCs under TNFα and LPS stimulation. Quantitative analysis indicated significantly more SA β-gal-positive cells in BMMSCs compared to DPSCs under TNFα and LPS stimulation. (B) Annexin V/PI staining and quantitative analysis showed lower rates of cell apoptosis in DPSCs than BMMSCs under LPS and TNFα stimulation. (C) Alizarin Red staining and quantitative measurements of osteogenesis of DPSCs and BMMSCs under LPS and TNFα stimulation. (D) HE staining and quantitative measurements of the osteogenic property of DPSCs and BMMSCs in vivo under LPS and TNFα stimulation. Values are means ± SDs. Student’s t-tests were used to determine statistical significance. Error bars represent SDs (n = 5). *P ≤ 0.05; **P ≤ 0.01; NS: no significance. Scale bar: 100 μm.

Figure 5.. More hard tissue regeneration in the periodontium after DPSCs injection compared to BMMSCs injection in vivo

(A-C) Gross micro-CT view of periodontal defect regeneration in the control group (saline injection), DPSC group, and BMMSC group. (D-F) Histopathological assessment of periodontal bone regeneration by HE staining in the periodontal defects of the DPSCs group and BMMSCs group. (D’-F’) Impaired sulcular epithelia and lymphocyte infiltration were evident in the control group compared with the DPSCs and BMMSCs groups. (D’’-F’’, G, H) Alveolar bone heights in the DPSCs and BMMSCs groups were much larger than those of the control group. Values are means ± SDs. One-way ANOVA was used to determine statistical significance. Error bars represent SDs (n = 5). *P ≤ 0.05; **P ≤ 0.01. Scale bar: 100 μm.