Induction Of Odontoblast-like Cells From Rat Adipose Derived Stem Cells By Mip-3α Combined With Osteogenic Factors In Vitro

Tooth loss due to caries or periodontal diseases is commonly seen in clinic,especially in elder people. World health organization (WHO) listed the dentaldiseases as one of the three human non-infectious diseases with the highestincidence. Routine clinical treatments for tooth replacement, e.g., syntheticmaterials, may elicit an immune-induced host rejection response. Toothregeneration using tissue engineering concepts is a promising biological approachthat aims to regenerate natural tooth-like mineral tissues in terms of histology,morphology and function. Tooth derived stem cells, such as DPSCs, PDLSCs,DFSCs, SCAPs, SHEDs, have demonstrated their capability to differentiate intotooth related tissues, well these cells are difficult, even impossible to harvest inclinic, restricting their clinical application. Non-tooth derived stem cells, likeBMSCs, can also be induced to form tooth-like structures in vitro, but these cellsremarkably lose their differentiation ability with the donor age increasing, and itis usually a painful and risky process to obtain bone marrow from the patients. SoBMSCs is not an ideal seeding cell type for tooth regeneration either.Adipose derived stem cells (ASCs) have many clinical advantages over BMSCs and tooth derived stem cells, and their differentiation potential can bemaintained with aging. They can be easily retrieved from either liposuctionaspirates or subcutaneous adipose tissue fragments and can easily be expanded invitro. In this study, we first isolated, cultured and identified rat adipose derivedstem cells in vitro, and then investigated the the effect of odontogenic inducingfactors on rat ASCs’ differentiation into odontoblast-like cells, hoping to providea new insight for the selection of tooth tissue engineering seed cells.Methods1. Rat adipose derived stem cells were primarily isolated, cultured inDMEM/F12supplemented with10%FBS. The morphology and growthcondition of the cells were observed by invert microscope. Growth curve ofthe P3rat ASCs were analyzed by MTT, and the surface markers weredetected by flow cytometry.2. P3~P4of rat ASCs were used for osteogenic and adipogenic differentiationaccording to the conventional protocol. NAP staining, Alizarin red stainingand Oil red staining were carried out to testify the differentiation outcomes.3. P3~P4of rat ASCs were maintained in DMEM/F12supplemented with10%FBS and ondontogenic factors. At day1,4, and7, the activity of ALPenzyme were measured, and the odontoblast-specific gene dspp and proteinDSP were analyzed by RT-PCR and Western Blotting, respectively.Results1. Rat ASCs grew well in the culture medium, MTT experiments showed an“S”-like growth curve, and flow cytometry revealed a pattern of CD29+、CD49d+、CD45-surface markers.2.2weeks after osteogenic induction, some ASCs grew centripetally andgranules of high transmittance could be observed within cytoplasm. NAP staining showed a positive result.1weeks later, red calcified nodules could beseen under microscope with Alizarin red staining.3weeks after adipogenicinduction, several red round lipid droplets with different sizes were observedscattered in the cytoplasm, while the nucleus were stain blue.3. ALP activity test showed an increased ALP activity as compared to theosteogenic groups and within the odontogenic group. dspp expression andDSP protein level of the odontogenic group were higher than that of theosteogenic group. The control group showed the lowest ALP activity and nodspp expression or DSP protein synthesized.Conclusions1. The isolation and culture of rat ASCs were successful. ASCs showed aneffective self-renewal ability. Surface makers pattern revealed that ASCs inculture are a group of homogeneous and undifferentiated stem cells.2. Rat ASCs can be induced into osteogenic and adipogenic differentiation underappropriate conditions.3. After7days of odontogenic induction, rat ASCs showed an increasedmineralization ability, and can express odontoblast-specific genes andproteins. The induction period and efficacy of the induction method adoptedby this study has been improved compared to the former ones, and hasprovided a more practical way of inducing ASCs to differentiate intoodontoblast-like cells.