A In Vitro Study Of Adipose Tissue-deprived Stem Cells Induced To Differentiate Into Cementoblasts

BackgroundCementum, a thin mineralized tissue produced by cementoblasts, covers the tooth root surface and anchors teeth to surrounding alveolar bone, which plays a crucial role in maintenance of tooth attachment. However, cementum regeneration is generally difficult for the reason that cementoblasts are scarce in root cementum and not easy to isolate. Despite considerable research, sources of cementoblasts for regeneration are largely restricted to dental stem cells, such as dental follicle cells and Hertwig's epithelial root sheath (HERS) cells from tooth germs, stem cells from periodontal ligament, and ectomesenchymal cells from the first branchial arch, etc, which are not readily available clinically. This shortage of cementoblasts has significantly limited the development of periodontal tissue engineeringAdipose tissue-derived stem cells (ADSCs) have recently been widely studied in regenerative medicine because of their ease of harvesting and high proliferation. ADSCs are capable of differentiating to osteoblasts in an osteogenic microenvironment. Cementoblasts show many similarities in phenotypes to osteoblast and are considered a subpopulation of osteoblasts by some. Therefore, it may be feasible to induce ADSCs to differentiate along the cementoblast lineage if an optimal cementogenic microenvironment is provided.To establish this microenvironment, it is necessary to recapitulate the process of embryogenesis and morphogenesis involved in the developmental formation of cementum. Although the origin of cementoblasts remains a matter of debate, it has been well established that dental follicle cells penetrate disintegrating HERS and contact with unmineralized dentin matrix of the root surface prior to any cementum formation. Thus, dental follicle and dentine matrix may contain some biological mediators which are necessary to differentiation of cementoblasts. Dentin non-collagenous proteins (dNCPs), major component of dentine matrix, include glycoproteins/sialoproteins, phosphoproteins, proteoglycans and growth factors, which are considered to play an important role in differentiation of cementoblasts and cementogenesis.Therefore, we collected dental follicle cell conditioned medium and combined it with dNCPs in this study, to create a cementogenic microenvironment and induce ADSCs to differentiate into cementoblasts. This strategy may provide an alternative stem cell resource for periodontal tissue engineering and a better understanding of regulatory mechanisms for cementogenesis in periodontal development.Materials and Methods1.Culture and identification of ADSCs Adipose tissues were isolated from the inguinal region of 7-day-old Sprague–Dawley(SD) rats and finely minced into small pieces, followed by digestion with type I collagenase solution (0.1 mg/ml, Sigma, USA) for 1 h at 37°C. The cell suspension was centrifuged at 1000 r/min for 5 minutes. The supernatant was discarded and the cell pellet was suspended in Dulbecco Modified Eagles Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100 IU/ml penicillin and 100?g/ml streptomycin), then cultured at 37°C in 5% CO2. Cells were used at the third passage for the following experiments.Before used in the induction experiments, ADSCs isolated and cultured in this study were identified by morphological analysis, immunohistochemical staining, flow cytometry and multi-lineage differentiation capacity assays.2.Primary selection of conditions for cementogenic differentiation of ADSCsCell culture and conditioned media preparation: 5-day-old SD rats were selected and killed. Dental follicle and apical papilla tissue was dissected respectively from the first molars under a dissecting microscope. Culture and identification of these two cells were the same as above. Cells at the third passage were cultured. Once the cells reached confluence, culture medium was changed every day and collected for 3 days. The collected media were filtered through a 0.2?m Millipore strainer and mixed with an equal volume of fresh DMEM supplemented with 10% FBS, then stored at ?80°C before used as dental follicle cell conditioned media (DFCCM) and apical papilla cell conditioned media (APCCM).Primary selection of induction conditions: ADSCs at the third passage were cultured in DFCCM, APCCM, 10?g/ml dNCPs+ DMEM/F12 with5%FBS, 10?g/ml dNCPs +DFCCM, 10?g/ml dNCPs +APCCM, DMEM/F12 with 5%FBS(control) respectively for six days. Culture medium was changed every other day. Treated ADSCs were assayed by RT-PCR and Western blotting.3.In vitro experiment of cementogenic differentiation of ADSCs treated with dNCPs/DFCCMBased on primary selection of induction conditions above, the effects of dNCPs/DFCCM on cementogenic differentiation of ADSCs were further studied in this experiment. The changes on morphology, proliferative capacity, alkaline phosphatase activity, in vitro mineralization behaveiors, protein and gene expression for cementum attachment protein (CAP) and mineralization-related markers were assayed in ADSCs treated with dNCPs/DFCCM.Results1.ADSCs isolated from 7-day-old SD rats for in vitro primary culture and the 4–6th passages showed high proliferative capacity. Positive expression of vimentin was detected in ADSCs by immunohistochemical staining but no expression of CK was detected. Flow cytometry assay showed that ratios of CD29, CD44, CD90, CD105, STRO-1 positive cells in ADSCs were 93.09%, 75.11%, 90.20%, 95.86%, 30.09% respectively. In adipogenic, osteogenic, neuron-like differentiation experiments, ADSCs isolated in this study showed the ability of multipotential differentiation.2.Dental follicle cells and apical papilla cells for in vitro primary culture and the 4-6th passages showed good proliferative capacity and characteristics of mesenchymal stem cells. In immunohistochemical stainingand flow cytometry assay, vimentin, CD29, CD44, CD90, CD105 and STRO-1 were detected to positively express in both cell populations. In the experiment to optimize culture conditions for cementogenic differentiation of ADSCs, relative high expressions of CAP, bone sialoprotein(BSP), osteocalcinOCN, type I collagen (Col I),osteopontin(OPN) and osteonectin(ON) were detected at mRNA or protein levels in dNCPs/DFCCM treatment group.3.To further confirm the effects of dNCPs/DFCCM on cementogenic differentiation of ADSCs, more detailed experiments were performed. After treratment with dNCPs/DFCCM, ADSCs changed from a fibroblast-like to cementoblast-like morphology and significantly lost their proliferative capacity. Alkaline phosphatase activity and an in vitro mineralization assay indicated that dNCPs/DFCCM greatly enhanced the mineralization behaviour of differentiated ADSCs, in which mineralization-related markers including cementum attachment protein, bone sialoprotein, osteocalcin, osteopontin and osteonectin were detected at mRNA or protein levels, whereas dentin sialophosphoprotein and dentin sialoprotein were not detected, implying a cementoblast-like phenotype.Conclusions1.ADSCs isolated and cultured in this study show high proliferative capacity and characteristics of mesenchymal stem cells. They could differentiate into multi-lineage cells in corresponding media, such as adipocyte, osteoblast lineage, neuron-like cell lineage.2.Dental follicle cells and apical papilla cells were successly isolated and cultured in vitro. They were identified by immunohistochemical stainingand flow cytometry assay. The results and tissue extraction sites confirmed that dental follicle cells and apical papilla cells were originated from mesenchymal progenitors and showed some characteristics of mesenchymal stem cells.3.The effects of DFCCM, APCCM and dNCPs on cementogenic differentiation of ADSCs were investingated in a primary study. DFCCM and dNCPs showed promotion effect on cementogenic differentiation of ADSCs, especially combination of them, while APCCM showed promotion effect more of odontogenic differentiation.4.After treatment with dNCPs/DFCCM, ADSCs underwent morphological change from a fibroblast-like to cementoblast-like morphology and significantly lost their proliferative capacity. Alkaline phosphatase activity and an in vitro mineralization assay indicated that dNCPs/DFCCM greatly enhanced the mineralization behaviour of differentiated ADSCs, in which mineralization-related markers including CAP, BSP, OCN, OPN and ON were detected at mRNA or protein levels, whereas dentin sialophosphoprotein and dentin sialoprotein were not detected, implying a cementoblast-like phenotype.In summary, the lines of evidence presented here suggest that ADSCs are capable of differentiating to the cementoblast lineage following treatment with dNCPs/DFCCM, which includes multiple growth factors and other molecules necessary for signaling cementogenic differentiation. ADSCs, easily harvested clinically, could be an optimal source for cementogenic cells and very feasible for periodontal regeneration. However, further studies are necessary to address the underlying mechanisms involved in dNCPs/DFCCM–mediated cementogenesis.