Isolation, Identification Of Rat Adipose-derived Stem Cells And Their Differentiation To Odontoblasts

BackgroundStem cells are self-renewing, undifferentiated cells that can give rise to multiple types of all specialized cells of the body. According to their origins, these cells can be divided into embryonic stem cells which constitute the foundation of individual development and adult stem cells which differentiation is the foundation of adult repair and regeneration. Recently, much attention has been paid on the post-natal adult stem cells with their advantages of reliable source, plasticity and lack of immunologic rejection for clinical application.Adipose-derived stem cell, which is confirmed as one of adult stem cells have multi-directional differentiated ability. They can differentiate to osteoblasts, cartilage cells, adipose cells, vascular endothelial cells, myocytes and cadiocytes, etc. Because of their advantages of easy to gain and little damage to tissue, adipose-derived stem cells hold the important position on the regenerative medical science, provide redundant cell resource for tissue engineering and establish substantial foundation for clinical application.ObjectiveIn this study, first of all, to isolate and culture adipose-derived stem cells, then differentiate them to odontoblasts by co-cultured with tooth germ cell conditioned medium, eventually establish pulp-dentin complex by implanted adipose-derived stem cells and epithelial cells of enamel organ into renal capsule. All of those confirmed the odontogenetic ability of adipose-derived stem cells, offered theory and experimental basis for the abundant seed cells in tooth tissue engineering, construct new research model for the micro-environment of the odontogenesis in vitro and vivo.Materials and Methods1. Cultivation, verification and orient differentiation.Adipose-derived adult stem cells were collected by enzyme digestion from inguinal fat pads of 4 days post natal mice. Immunocytochemistry was performed to detect the expression of Vimentin,CD29,CD44,CK. To detect the osteoblast differentiation, ADSC were induced in mineralized promoting medium containingβ- glycerophosphate, ascorbic acid and dexamethasone, then detected Alkaline phosphatase activity, the expression of osteocalcin and collagen I by immunocytochemistry and mineralized nodules by Von Kossa staining.2. Differentiation of adipose-derived stem cells into odontoblast-like cells in vitro ADSCs were co-cultured with tooth germ cell conditioned medium (TGC-CM) which would be changed every 24h. Cells in conditioned medium were routinely observed and photographed to evaluate their appearance. Immunocytochemical analyses were performed to detect the expression of dentin sialoprotein and reverse transcription-PCR (RT-PCR) to dentin sialophosphoprotein (DSPP) and dentin maitrix protein (DMP 1).3. Establishment and regeneration of dentin-pulp complex using adipose-derived stem cells (ADSCs) in vivo.The cell pellets containing labeled ADSCs with Hochest 33342 and epithelial cells of enamel organ were seeded on the PTSG scaffolds. The cell-scaffold compounds were cultured for 2 hours and transplanted into the renal capsule. After 12 weeks the transplantations were dislodged and evaluated by histological assay and immunocytochemical analyse.Results1. ADSCs displayed a fibroblast-like appearance, the cell population doubling time is 42.2 hours. It was also found that the cells positively expressed Vimentin, CD29, CD44 and negatively expressed CK. After the co-culture with mineralization solution, the initially fibroblast-like ADSCs displayed a circle or cube-like appearance. Together, our study indicates that mineralization solution can up-regulate the ALP activity and promote the secretion of extracellular matrix of ADSCs to form the mineralized nodules. Meanwhile, the treated cells expressed osteocalcin and collagen I by immunocytochemical technology.2. After the co-culture, the initially fibroblast-like ADSCs displayed a columnar, elongated appearance, long cellular processes and a tendency to align themselves in straight parallel lines. The treated cells were stained positively for cytoplasmic DSP by using immunocytochemical staining. As demonstrated by RT-PCR, the DMP and DSPP genes were expressed in co-cultured ADAS cells. 3. Based on the in vitro experiments, an in vivo evaluation of induced ADSCs was performed to verify whether the ADSCs form the dental tissues. Tooth-like structure was formed, and in this structure, ameloblasts, enamel, dentin, odontoblasts arranged regularly from external to interior. Odontoblast-like cells collocated parallel under pre-dentin were in the polarization and externalization state. Dentin sialophosphoprotein (DSP) was detected in the tubular dentin and ondontoblast-like cells.Conclusion1. Adipose-derived stem cells are multipotential stem cell and can be induced to osteoblast in vitro.2. TGC-CM contained multiple cytokine can create the odontogenic microenvironment and induce the adipose-derived stem cells into odontoblast-like cells in vitro.3. Dentine-pulp complex can be established using adipose-derived stem cells, epithelial cells of enamel organ and scaffold materials by tissue engineering method in vivo.