Proliferation Of Pulp Stem Cells Research Articles

[Senolytic effects on dental pulp stem cell's proliferation and differentiation during long-term expansion].

Objective: To investigate the impact of intermittent senescent cell clearance on the proliferation and differentiation of dental pulp stem cells (DPSC) in long-term, large-scale expansion, and to explore strategies for maintaining the youthful state of DPSC in vitro. Methods: Human-derived dental pulp stem cells were isolated from healthy permanent teeth extracted for orthodontic or impeding eruption reasons, provided by the Department of Oral and Maxillofacial Surgery at West China Hospital of Stomatology, Sichuan University. Long-term, large-scale in vitro expansion of DPSC was conducted. The study compared young DPSC (passage 5) with aged DPSC (passage 25) using cellular senescence-associated β-galactosidase staining, colony formation assay, and Alizarin Red S staining for osteogenic differentiation induction. To assess the differences between the two cell populations in terms of senescence and amplification and differentiation ability. Medicine screening for the most effective senolytic was compared among 5 common senolytics [Navitoclax (ABT-263), curcumin, dasatinib, fisetin, and quercetin]. The clearance efficacy was compared using cellular senescence-associated β-galactosidase staining to reflect the changes in senescent cell ratio. The senolytic with the highest efficacy was chosen for further experiments. The passage at which the proportion of senescent cells significantly increased was identified, and the selected senolytic was administered three times at three-generation intervals from that passage to remove senescent cells. Both the control and senolytic-treated groups were estimated by fluorescence cellular senescence-associated β-galactosidase staining, real-time flurogenic quantitative PCR (qPCR), colony formation assay, wound healing assay, and Alizarin Red S staining for osteogenic differentiation induction. Subcutaneous heterotopic osteogenesis was performed in nude mice and the grafts were analyzed by HE staining and alkaline phosphatase (ALP) immunohistochemical staining. Results: The proportion of senescent cells increased as the expansion extended, leading to decreased proliferation and osteogenic differentiation ability of senescent DPSC compared to young DPSC (P<0.05). Senescent DPSC exhibited altered mRNA expression levels of senescence-related genes, including p21, p16INK4a, IL-6, and Ki67 (P<0.001). Among the five senolytics, ABT-263 demonstrated higher clearance efficiency (P<0.05). After intermittent ABT-263 treatment during expansion, the proportion of senescent cells in the senolytic-treated group [(6.72±2.34)%] was significantly lower than that in the control group [(31.82±0.57)%] (P<0.001). qPCR confirmed that compared with the control group, mRNA expressions of p21, p16INK4a, and IL-6 in the senolytic-treated group were significantly decreased (P<0.05), while mRNA expressions of Ki67 were significantly increased (P<0.01). Furthermore, the cell healing ability and osteogenic differentiation ability of the senolytic-treated group were higher than those of the control group (P<0.05). In vivo experimental results indicated that the relative new bone area [(2.36±0.48)%] after DPSC transplantation in the senolytic-treated group was greater than that in the control group [(1.00±0.46)%] (P<0.05), and the expression of ALP was higher than that in the control group (P<0.01). Conclusions: ABT-263 can effectively eliminate senescent cells in long-term large-scale DPSC expansion. Continuous: treatment with ABT-263 during cultivation can maintain the proliferation and differentiation ability of DPSC both in vivo and in vitro.

Periostin Is a Candidate Regulator of the Host Microenvironment in Regeneration of Pulp and Dentin Complex and Periodontal Ligament in Transplantation with Stem Cell-Conditioned Medium.

The microenvironment is required for tissues to maintain their properties in vivo. This microenvironment encompasses the types and three-dimensional arrangement of cells forming the tissues, and their interactions with neighboring cells and extracellular matrices, as represented by the stem cell niche. Tissue regeneration depends not on the original tissue source of the transplanted cells, but on the microenvironment in which they are transplanted. We have previously reported pulp regeneration in a heterotopic root graft model by transplantation of conditioned medium alone, which suggests that host-derived cells expressing receptors for migration factors in conditioned medium migrate into the root canal and cause pulp regeneration. Regenerative medicine is needed to restore the original function of complex tissues. To achieve this, it is necessary to reproduce the changes in the microenvironment of the host tissue that accompany the regenerative response. Therefore, it is important to reproduce the microenvironment in vivo for further development of tissue regeneration therapy. Periostin is also found in the epithelial-mesenchymal junction, with expression sites that differ depending on the mineralized matrix stage, and is involved in regulation of calcification. We investigate whether periostin contributes to microenvironmental changes in regenerated pulp tissue. Dental pulp stem cells were induced into dentin, and gene expression of DSPP, nestin, DMP1, Runx2, and periostin was analyzed by qPCR and protein expression by IHC. Similarly, gene expression was analyzed using qPCR and protein expression using IHC in regenerated dental pulp obtained by ectopic transplantation. Since these regenerated tissues were observable on the same slice, it was possible to understand changes in the microenvironment within the tissues. Periostin promoted proliferation of pulp stem cells, migration in type I collagen, and calcification in regenerated pulp, which strongly suggests that periostin is a promising candidate as a factor that contributes to the microenvironment of regenerated pulp.

Simvastatintreatment promotes proliferation of human dental pulp stem cells via modulating PI3K/AKT/miR-9/KLF5 signalling pathway.

Simvastatin serves as an effective therapeutic potential in the treatment of dental disease via alternating proliferation of dental pulp stem cells. First, western‐blot and real‐time quantitative PCR were used to detect the effect of simvastatin or LY294002 on the expression levels of AKT, miR‐9 and KLF5, or determine the effect of miR‐9. Simvastatin, KLF5 and AKT significantly enhanced the proliferation of pulp stem cells, whilst this effect induced by simvastatin was suppressed by LY294002, AKT siRNA, KLF5 siRNA and miR‐9, and simvastatin dose‐dependently upregulated the expression of PI3K. Furthermore, simvastatin upregulated PI3K and p‐AKT expression in a concentration‐dependent manner. LY294002 abrogated the upregulation of p‐AKT expression levels induced by simvastatin, and LY294002 induced the miR‐9 expression and simvastatin dose‐dependently inhibited the expression of miR‐9, by contrast, LY294002 reduced the KLF5 expression and simvastatin dose‐dependently promoted the expression of KLF5. And using computational analysis, KLF5 was found to be a candidate target gene of miR‐9, and which was further verified using luciferase assay. Finally, the level of KLF5 in cells was much lower following the transfection with miR‐9 and KLF5 siRNA, and the level of AKT mRNA in cells was significantly inhibited after transfection with AKT siRNA than control. These findings suggested simvastatin could promote the proliferation of pulp stem cells, possibly by suppressing the expression of miR‐9 via activating the PI3K/AKT signalling pathway, and the downregulation of miR‐9 upregulated the expression of its target gene, KLF5, which is directly responsible for the enhanced proliferation of pulp stem cells.

Investigation the role of Fe3O4 in the silica based bioactive polycrystalline modified with ZnO and CaF2

The aim of this article was to investigation the function of silica based polycrystalline that modified with ZnO and CaF2 and doped with 6% Fe3O4. The human pulp stem cells cultured in the presence of samples. The effects of heating-induced crystallization on the glass structure were studied by DSC and x-ray diffraction. The particle size of prepared samples was studied by TEM analysis. The bioactivity of samples were analyzed by apatite-formation ability in DMEM solution through SEM-EDX, biocompatibility was evaluated through the SHEDs cells proliferation, adhesion and spreading on surface of samples by MTT assay and ALP activity assay. Optical and SEM images displayed that co-operation of ZnO and CaF2 led to appropriate circumstance for cells proliferation, adhesion and spreading. Proliferation of pulp stem cells after 7 d of incubation in culture media containing ZC8, FC8 and ZFC4 yielded values 0.3, 1.7 and 2.8 in comparison with control, respectively.

[Bioactive glass 45S5-silk fibroin membrane supports proliferation and differentiation of human dental pulp stem cells].

To investigate the effect of bioactivity glass 45S5- silk fibroin(BG45S5- SF) membrane on growth, proliferation and differentiation of human dental pulp stem cells(hDPSC), and to provide new ideas and method for the regeneration of pulp-dentine complex. hDPSC seed on pure silk fibroin membrane (protein membrane group) and BG45S5-SF membrane with different concentrations(1 000, 5 000 mg/L, composite membrane group A and B, respectively) were prepared, and the materials were incubated in cell culture fluid for 24 h. No material membrane orifice plate was used as blank control group. Contact angle meter was used to measure surface contact angle of protein membrane and composite membrane group(each group had three repeated holes). Cell proliferation was assessed by cell counting kit- 8 on the 4, 7, 14, and 21 days. The state of adhesion and growth of hDPSC on the materials surface was evaluated by scanning electron microscopy and cytoskeleton staining; and alkaline phosphatase (ALP) activity was measured to evaluate the cell differentiation potential. The expression of odontoblastic differentiation-related genes was measured by real-time PCR. Surface contact angle of the protein membrane group and composite membrane group A and group B were 89.51° ± 0.12°, 70.32° ± 0.07° and 71.31° ± 0.09° respectively. hDPSC adhered well on each materials surface on the 7, 14, 21 days, ALP activity and differentiation genes of composite membrane group A and B rised more significantly than the blank control group and protein membrane group did (P<0.05). Dentin matrix protein1(DMP- 1), dentin sialoprotein(DSP), ALP, osteocalcin(OC) mRNA expression reached peak on the 14 days in group A, and in group B on the 21 days. Bone sialoprotein(BSP) mRNA expression in both group A and B reached peak on the 21 days. BG45S5- SF membrane is able to support the proliferation and showed the potential of odontoblastic differentiation for hDPSC. This finding suggests that BG45S5-SF membrane was a kind of tissue engineering film material with the regeneration potential for pulp-dentine complex.

Effects of mineral trioxide aggregate on proliferation and differentiation of dental pulp stem cells from young permanent teeth in vitro

Objective To investigate the effects of the different concentration of mineral trioxide aggregate (MTA) on the proliferation and differentiation of dental pulp stem cells (DPSCs) from the young permanent teeth. Methods DPSCs were isolated from the young permanent teeth and cultured by tissue explant method. The expression of STRO-1 was detected by using immunofluorescence technology. DPSCs were cultured with different concentrations of MTA (0.02, 0.20, 2.00, 20.00 g/L). Cell proliferation was detected by MTT array. Cells were cultured in the appropriate concentration of MTA for 4 weeks, and then stained by Alizarin red to detect their mineralized nodule formation capacity. The cells were cultured with the appropriate concentration of MTA and collected after 12, 24, 36, 48 h. The mRNA expression of ALP, BSP, OC and DSP after the treatment of MTA were detected by quantitative PCR. Results DPSCs were positive for STRO-1. The capacity of 0.20 g/L MTA promoting the proliferation of DPSCs was stronger than other concentrations. After 4 weeks, the mineralized nodules of DPSCs were observed after alizarin red staining. The PCR showed that with increasing induction time, the expression levels of DSP and OC were up-regulated. But that of ALP and BSP was increased first and then decreased. Conclusions In this study, MTA can promote the proliferation of DPSCs at 0.02, 0.20, 2.00 g/L concentration. It can induce odontoblast differentiation effectively by 0.20 g/L MTA. Key words: Mineral trioxide aggregate; Dental pulp; Stem cells; Permanent teeth