MiR-10a-5p Regulates Osteogenic/Cementogenic Differentiation Of Human Peridontal Ligament Stem Cells

ObjectivePeridontal ligament is a layer of soft connective tissue, contains rich blood vessels and cells, located between the root and the alveolar bone. It anchors teeth, disperses the bite forces and shoulders many other important functions. Periodontal ligament cell is a group of heterogeneous cells. Some of these cells can differentiate into osteoblasts and cementoblasts. Cementoblast can form cementum. Osteoblast is able to differentiate into bone tissues.Micro RNAs(mi RNAs) are short, noncoding RNAs, they act as key regulators in diverse biological processes of their target genes by m RNA degradation or translational repression. Growing evidence shows that mi RNA has played an important role in proliferation and differentiation of stem cells. Our group has used mi RNAs array and quantitative RT-PCR(q-PCR) to compare the differences of mi RNAs expression in human periodontal ligament stem cells(h PDLSCs) before and after been induced by apical tooth germ cell conditioned medium(APTG-CM). We revealed that mi R-10a-5p was down-regulated during osteoblast/cementoblast differentiation of h PDLSCs. This study verifies the biological functions of mi R-10a-5p in h PDLSCs by in vitro and in vivo experiments. It provides the theoretical bases to improve regenerative capacity of hPDLSCs.MethodsWe used Magnetic-activated cell separation(MACS) to acquire homogeneous populations of h PDLSCs. Immunocytochemical staining and osteogenic/adipogenic differentiation potential were evaluated. We cultured rats’ apical tooth germ cells from8 days Sprauge-Dawley rats to construct APTG-CM. We changed mi R-10a-5p expression of h PDLSCs by using transient transfection of mi R-10a-5p mimics or inhibitor, then q-PCR and Western Blot were used to compare the difference of osteoblastic/cementoblastic differentiation capacity after changing the endogenous mi R-10a-5p expression. HA-TCP scaffolds were co-culutured with h PDLSCs after transfection, and implanted into the dorsal surfaces of BALB/C nude mice. Wesacrificed the mice to make tissue sections to verify the mineral characteristics in vivo by HE staining after 8 weeks.ResulsThe positive immunomagnetic separated cells had several characteristics of h PDLSCs,as indicated by osteogenic/adipogenic stainings and positive immunocytochemical stainings of mesenchymal stem cell markers STRO-1 and CD146. After increasing the expression level of mi R-10a-5p, q-PCR and Western blot results showed that the osteoblastic/cementoblastic differentiation of h PDSLCs were reduced. Meanwhile, it was enhanced after decreasing the expression of endogenous mi R-10a-5p. No major changes were observed by HE staining of the implants.ConclusionsMi R-10a-5p acted as a negative regulator of h PDLSCs osteogenesis/cementogenesis.