Aim To comparatively evaluate changes in the proliferation and mineralization abilities of dental pulp stem cells (DPSCs) from juvenile and adult rats in a lipopolysaccharide (LPS)‐induced inflammatory microenvironment to provide a theoretical basis for the age‐related differences observed in DPSCs during repair of inflammatory injuries. Methodology DPSCs were isolated from juvenile (JDPSCs) and adult rats (ADPSCs), and senescence‐associated β‐galactosidase staining was used to compare senescence between JDPSCs and ADPSCs. Effects of LPS on JDPSCs and ADPSCs proliferation were investigated by cell counting kit‐8 assays and flow cytometry. Alizarin red staining, quantitative reverse transcription polymerase chain reaction and Western blot assay were used to examine the effects of LPS on mineralization‐related genes and proteins in JDPSCs and ADPSCs. Immunohistochemistry was used to compare interleukin‐1β (IL‐1β) and osteocalcin (OCN) expression in the pulpitis model. Unpaired Student's t‐tests and one‐way anova were used for statistical analysis. Results DPSCs were isolated from juvenile and adult rat dental pulp tissues. At low concentrations (0.1–1 μg mL−1), LPS significantly promoted the proliferation of JDPSCs (P < 0.01) and ADPSCs (P < 0.01 or P < 0.05), with the effect being stronger in JDPSCs than in ADPSCs. In addition, mineralized nodules and the expression of mineralization‐related genes (OCN, DSPP, ALP, BSP) increased significantly after stimulation with LPS (0.5 μg mL−1) in JDPSCs and ADPSCs (P < 0.01 or P < 0.05), and JDPSCs displayed a more obvious increase than ADPSCs. Western blots revealed OCN and ALP expression levels in JDPSCs treated with LPS were significantly upregulated (P < 0.05); meanwhile, ALP expression in ADPSCs increased slightly but significantly (P < 0.05), and OCN expression was not affected. Finally, IL‐1β expression was significantly higher (P < 0.05) and OCN expression was significantly lower (P < 0.05) in the inflamed dental pulp of adult rats than in juvenile rats. Conclusions A certain degree of inflammatory stimulation promoted the proliferation and mineralization of DPSCs; however, this effect declined with age. The DPSCs of adult donors in an inflammatory microenvironment have a weaker repair ability than that of juvenile donors, who are better candidates for tissues damage repair.

To apply human mesenchymal stem cells (hMSC) to regenerative medicines, it is necessary to multiply hMSC in vitro in a short period. In addition, it is desirable that the medium which is used for the hMSC multiplication is not supplemented with the serum, because the addition of the serum has risks of infection. In this study, we cultured hMSC with three kinds of medium used for multiplying hMSC (DMEM, MSCGM, STK2) and compared hMSC proliferation in each medium. As a result, it was conˆrmed that hMSC proliferation was signiˆcantly higher in STK2 medium which is a novel serum-free medium developed for hMSC multiplication. Moreover, we compared the hMSC proliferation in these media under the environment that assumed bone reproduction. When we cultured hMSC in each medium with hydroxyapatite (HAp), the proliferative inhibition by HAp depended on the additive amount, and the degree of the proliferative inhibition was diŠerent among the media but the lowest inhibitory eŠect was observed in STK2 medium.

Dental pulp cells (DPCs) are promising candidates for use as transplantable cells in regenerative medicine. However, ex vivo expansion of these cells typically requires culture media containing fetal bovine serum, which may cause infection and immunological reaction following transplantation. In addition, the proliferation and differentiation of DPCs markedly depend upon serum batches. Therefore, the present study examined whether DPCs could be expanded under serum-free conditions. DPCs obtained from four donors were identified to proliferate actively in the serum-free medium, STK2, when compared with those cells in control medium (Dulbecco's modified Eagle's medium containing 10% serum). The high proliferative potential with STK2 was maintained through multiple successive culture passages. DNA microarray analyses demonstrated that the gene expression profile of DPCs grown in STK2 was similar to that of cells grown in the control medium; however, a number of genes related to cell proliferation, including placental growth factor and inhibin-βE, were upregulated in the STK2 cultures. Following induction of osteogenesis, DPCs grown in STK2 induced alkaline phosphatase activity and calcification at higher levels compared with the control medium cultures, indicating maintenance of differentiation potential in STK2. This serum-free culture system with DPCs may have applications in further experimental studies and as a clinical strategy in regenerative medicine.