Influence Of Two Different Mineralization-Inducing Media On Osteo/Odontoblastic Differentiation Capacity Of Stem Cells From Apical Papilla

OBJECTIVEStem cells from apical papilla (SCAP) play a major role in the root dentin formation, it’s also regarded as an important source of stem cells for tooth regeneration. Mineralization-inducing media is required to induce SCAP into osteo/odontoblastic differentiation before it’s function can be fully explored. There are a variety of mineralization-inducing media, adding different supplements or different concentrations, and its mechanism is different. Recently KH2PO4has been proposed as a mineralization-inducing media supplement, however, compared to the other supplement of mineralization-inducing media, the role of KH2PO4is unclear yet. In this study, the influence of mineralization-inducing media containing KH2PO4to proliferation and differentiation of SCAP were evaluated by in vitro and in vivo assays, its superiority of mineralization induced capacity was compared to that of other mineralization-inducing media. METHODSCAPs were isolated from apical papilla tissue of human fresh extracted third molar, purified and then treated with two distinct mineralization-inducing media(50μg/mL ascorbic acid, lOnM Dexamethasone, lOmM (3-glycerophosphate), MM1and MM2, differing in KH2PO4concentrations(0mM v.s.1.8mM). Proliferation and differentiation of SCAP were evaluated both in vitro and in vivo. In vitro, proliferation of SCAP was determined by MTT and FCM assays, then the growth curve was drawn and the proliferation index was calculated. Differentiation of SCAP was determined by the following assays:Alkaline phosphatase (ALP) assay, alizarin red staining, Western blot and Real-time reverse transcriptase-polymerase chain reaction (Real-time RT-PCR). osteo/odontoblastic differentiation index was evaluated from the protein and gene level (DSP/DSPP, OCN/OCN). In vivo, dental papilla tissue was isolated from4-7days SD rats molar embryos, SCAP pellets were put into the prepared tooth fragments, incubated with two distinct mineralization-inducing media2h, tooth fragments with SCAP cell pellets were carefully seeded onto the absorbable gelatin sponges(AGS) immersed with two distinct mineralization-inducing media and transplantation procedures were performed under renal capsules of8-10week-old female SD rats. All retrieved tissues at day14post-transplantation were fixed in4%polyoxymethylene, after the process of decalcified embedded, serially paraffin sectioned were performed, the sections were stained with hemotoxylin and eosin (HE)for histological analysis, osteo/odontoblastic differentiation index was evaluated(DSP,OCN) for immunohistochemical analysis. RESULTSResults indicated that SCAP of mineralization-inducing media added KH2PO4group had a higher proliferative capacity, and upregulated expression of osteo/odontoblast-specific markers. In vitro, MTT and FCM revealed that the proliferation rate of SCAPs in both mineralization-inducing media groups were higher than that in Control group. SCAPs in MM2presented a higher growth rate than those in MM1. Both mineralization-inducing media groups presented a higher ALP activity than Control group, in which ALP levels in MM2was significantly higher than those in MM1. SCAPs in MM2produced more calcium nodules and showed higher calcium concentrations than those in other two groups by alizarin red staining. From the protein and gene level, Real-time RT-PCR and Western blot showed osteo/odontoblastic differentiation index of MM2had significant expression at the early stage (e.g. DSP/DSPP, OCN/OCN. In vivo, retrieved implants were prepared for histological examination, HE staining showed that there was no mineralized tissues formed near the canal orifice in Control group. All root segments in both mineralization-inducing media groups brought about the formation of new mineralized tissues inside the root canal space, and MM2presented larger amounts of mineralized tissues. The expression of mineralization-related proteins (OCN, DSP) were determined by immunohistochemical techniques. Weak positive staining against OCN and positive staining against DSP were detected in Control group, Positive staining against OCN and DSP were observed in both mineralization media groups, and MM2presented the stronger positive staining against DSP. Collectively, SCAPs in MM2showed greater mineralized matrix-forming capacity in vivo. CONTROLCLUSIONWithin the limitation of this study, we conclude that mineralization-inducing media supplemented with KH2PO4enhanced SCAP into osteo/odontoblastic differentiation at the early stage. Both the poliferation and differentiation of the SCAP increased induced by mineralization-inducing media containing KH2PO4. KH2PO4may be a cost-effective mineralization induction supplement.