Extracellular Matrix Derived From Human Urine-derived Stem Cells Enhances The Expansion,Adhesion,Spreading And Differentiation Of Human Periodontal Ligament Stem Cells

Background:Human periodontal ligament stem cells(hPDLSCs)are one of the most promising types of seed cells in periodontal tissue regeneration.Suitable biomaterials are additional essential components that must cooperate with seed cells for in vivo expansion or in vitro implantation.Extracellular matrix(ECM)derived from mesenchymal stem cells(MSCs)was recently reported to be a promising substrate with which to culture MSCs that could be applied in biomaterial scaffolds.Human urine-derived stem cells(hUSCs)have several advantages;their collection is non-invasive and easy,and hUSCs are low in cost,potentially making them a suitable and efficient source of ECM.The purpose of this study was to characterise the biological properties of ECM derived from hUSCs(UECM)and evaluate the effects of UECM on hPDLSCs.Materials and Methods:In vitro studies: hUSCs were isolated from fresh midstream urine collected from healthy males volunteers(20-25 years old)while hPDLSCs were isolated from healthy premolars extracted for orthodontic treatment from donors 12 to 18 years of age.Cell surface markers in hPDLSCs and hUSCs were examined by flow cytometric analysis.Osteogenic and adipogenic differentiation of them were also assessed.Both hUSCs and hPDLSCs were seeded on tissue culture plastic(TCP)and stimulated to produce ECM.Immunofluorescent staining and scanning electronic microscopy(SEM)were used to observed the components and structures of UECM.After 8 days of stimulation,the samples were decellularized,leaving only ECM.Then,hPDLSCs were seeded onto UECM-,PECMand fibronectin-coated TCP and untreated TCP.At days 1,3,5,7,and 9after cell seeding,cell proliferation was evaluated with a CCK-8 assay.hPDLSCs adhesion on different ECM substrates was measured by both static and dynamic methods after 1-hour seeding.Spreading and morphology of cells grown on ECM were assessed after 6-hour seeding.hPDLSCs differentiation on ECM-coated surfaces was assessed after osteogenic,adipogenic and angiogenic induction.In vivo studies: UECM and PECM were thoroughly mixed with nano hydroxyapatite(HA)powder.First,hPDLSCs were respectively cultured on UECM,PECM and untreated TCP in osteogenic media for 7 days.Then hPDLSCs cultured on TCP were digested and mixed with untreated HA powder(HA group)while hPDLSCs cultured on UECM were mixed with prepared HA powder mixed with UECM(HA/UECM group)and hPDLSCs cultured on PECM were mixed with prepared HA powder mixed with PECM(HA/PECM group).Next,the mixtures were implanted subcutaneously into the bilateral dorsal surfaces of nude mice.After 6 weeks,the implants were harvested,fixed in paraformaldehyde,decalcified with EDTA at room temperature and embedded in paraffin.To track osteogenic differentiation of the implanted hPDLSCs,slides of grafted tissues were assessed by H&E staining,Masson staining,and immunohistochemical staining using antibodies against human POSTN and OCN.Results:1.Flow cytometry analysis showed that hPDLSCs and hUSCS were positive for CD90,CD146 and negative for the markers CD31,CD34 and CD45.Alizarin red and Oil Red O staining showed calcium mineral deposits and lipid-laden lobules were both observed in hUSCs and hPDLSCs.2.SEM revealed that UECM were composed of dense bundles of fibres.Immunofluorescent staining revealed that fibronectin and collagen I were more abundant than laminin 5 in UECM.3.The CCK-8,adhesion tests and morphology observation showed UECM promoted the proliferation,adhesion and spreading of hPDLSCs.4.RT-PCR revealed the expression levels of RUNX2,POSTN,VEGFA and CD31 were significantly upregulated in the UECM group.Western blot showed that the expression levels of RUNX2,POSTN were significantly upregulated in the UECM group.Alizarin red staining showed more calcium mineral deposits were found in the UECM group.5.H&E and Masson staining showed that more abundant collagen fibers were found in the HA/UECM group compared to HA group.Immunohistochemical staining showed that the expressions of POSTN and OCN were significantly higher in the HA/UECM than HA group.Conclusions:Our study suggests UECM consists of dense bundles of fibers as a suitable biomaterial in which to culture hPDLSCs as UECM enhances their proliferation,adhesion,spreading,osteogenic and angiogenic differentiation providing an original perspective on different cell-specific ECM.