Effect Of Mesenchymal Stem Cells On Regeneration Of Periodontal Tissue/Bone Defect

Background and ObjectiveThe most important goal of periodontitis treatment is to restore the architecture and function of periodontium lost because of periodontitis and to achieve the regeneration of periodontium. The periodontal tissue regeneration includes the hard tissue (alveolar bone and cementum) regeneration and soft tissue (gingiva and periodontal ligament membrane) regeneration. GTR is a classical method of periodontal tissue repair. Barrier membrane can separate epithelium and gingival connective tissue from the root surface, which not only preventes the epithelial migration along the root surface but aggregates the periodontal precursor cells. These aggregated cells migrate, proliferate, differentiate, and then form new attachment. But this method cannot ensure the periodontal precursor cells can preferentially attach to the root in technic and it is hard to manipulate in clinical practice. And GTR cannot make sure complete regeneration of periodontium. The development of tissue engineering offers a new thread for periodontal tissue regeneration. Three basic biological factors are involved in tissue regeneration, which are seed cells, differentiation inducing factors and matrical materials. The seed cells can differentiate into osteoblasts, cementoblasts and periodontal ligament cells under suitable conditions. Embryonic stem cells and mesenchymal stem cells both can be the seed cells of the periodontal tissue engineering.MSCs come from the inchoate mesodermal cells and have capacity of self-renew. MSCs can differentiate into different cell lineages, including nerve cells, osteoblasts, muscle cells and adiopocytes. MSCs play a significant role in cell-based therapy and tissue engineering. MSCs originally isolated from bone marrow. However, the drawback in high viral pollution, cell collection, aging and limited proliferative property restricts the utility of BMSCs. Therefor, seeking for a candidate is a problem. PDLSCs can be used in periodontal tissue regeneration. However, the drawback in cell collection, hard isolation and amplification hinders the clinical application of PDLSCs.GMSCs described as previous reports were easily isolated and expanded in vitro and uniformly homogenous. GMSCs own more advantages over PDLSCs. Therefore it is proposed that GMSCs may be more significant in clinical practice than PDLSCs as candidate seed cells for periodontal tissue engineering.UCMSCs own more advantages over BMSCs. Aside from ease of isolation, no viral infection; UCMSCs possess higher proliferative capacity than BMSCs, and without drawbacks of embryonic stem cells, including source deficiency, xenoma rejection and ethical concerns. The utility of UCMSCs support the clinical application of MSCs.Up to now, the efficacy of utilizing the GMSCs and UCMSCs in periodontal tissue regeneration remains obscure. Our studies are focus on investigating the ability of GMSCs and UCMSCs in periodontal fenestrated model and critical-sized calvarial defects model. We firstly demonstrated that GMSCs and UCMSCs could repair bony defects and periodontal tissue defects, and provide proofs for their clinical application in periodontal regeneration.Methods:1. Isolation and cultivation and differentiation of human GMSCs and UCMSCs1.1 Isolation and cultivation and differentiation of human GMSCsGigiva was collected from artificial eruption and tooth (without dental caries and periodontal disease) extraction because of teeth orthopedic. Primary cultured gingival cells were gained by tissue adherent method. After passaged, cells which belong to P3 or P4 were prepared for further experiments. GMSCs were got from single cell clones which were generated by limiting dilution method. After GMSCs were cultured under osteogenic differentiation conditions with 10-8mol/L dexamethasone?10mmol/L?-sodium glycerophosphate?50mg/L vitamin C for 2 weeks and 4 weeks, calcified tubercles were determined by ALP staining and Von Kossa staining. After GMSCs were cultured under adipogenic differentiation conditions with 10-8mol/L dexamethasone,200?M indomethacin, 10?M insulin and 0.5mM isobtityl-methylxanthine (IBMX) for 2 weeks, adipogenic differentiation was determined by Oil Red O staining. After 7 weeks under chondrogenic induction medium supplemented with 50nM ascorbate-2-phosphate, 10ng/ml TGF-?1 and 6.25?g/ml insulin and 1% antibiotic/antimycotic, chondrogenic differentiation of GMSCs was detected by alcian blue staining. The specific genes of oseoblasts, adipocytes and chondrocytes were evaluated by RT-PCR. The GMSCs cultured under normal conditions were control groups.1.2 Isolation and cultivation and differentiation of human UCMSCsHuman umbilical cords were processed with enzyme digestion after harvest under asepsis. After passaged, cells which belong to P3 or P4 were prepared for further experiments. UCMSCs were obtained from single cell clones which were generated by limiting dilution method. After UCMSCs were cultured under the same osteogenic differentiation conditions, adipogenic differentiation conditions and chondrogenic induction medium as GMSCs, the osteoblasts, adipocytes, chondrocytes and the specific genes were evaluated by ALP staining, Von Kossa staining, Red Oil O staining, alcian blue staining and RT-PCR. The UCMSCs cultured under normal conditions were control groups.2.In vitro amplification of GFP+GMSCs, GFP+UCMSCs and transplatationTo trace directly the distribution and differentiation of GMSCs and UCMSCs in vivo, the lentiviral vector with enhanced green fluorescent protein (pBPLV-eGFP) was used to label the third-passage GMSCs and UCMSCs. Lentiviral expression vector was firstly transfected into the packaging cells 293FT, and then 293FT cells produced high-level lentiviruses. GMSCs and UCMSCs transfected by lentiviruses were sorted by flow cytometry, then GFP+GMSCs and GFP+UCMSCs were collected and amplified for further experiments. GFP+GMSCs and GFP+UCMSCs cultured under osteogenic differentiation conditions for 10 days were transplanted into periodontal fenestration model and critical-sized calvarial defects model with collagen gel matrix. To investigate repair ability of GMSCs and UCMSCs, the samples were treated by H&E staining and immunohistochemical staining. The control groups of animal experiments were transplantation of collagen gel matrix alone.Result1. Isolation and cultivation and differentiation of human GMSCs and UCMSCs1.1 Isolation and cultivation and differentiation of human GMSCsAfter 5 days, there were lots of cells around the adherent histic debris. Cells were subcultured at 70%-80% confluency with trypsin digestion. GMSCs were obtained by limiting dilution method. According to flow cytometric analysis,GMSCs could express the CD29, CD 105, CD90, STRO-1, but could not express the CD34 and CD45. Cultured under osteogenic conditions for 2 weeks and 4 weeks, GMSCs could be induced into osteoblasts, which was positive of alkaline phosphatase by ALP staining and form calcified tubercles stained dark brown by Von Kossa staining. The specific genes of osteoblast, ALP and OCN, could be detected by RT-PCR. After incubated in adipogenic induction medium for 2 weeks, GMSCs could differentiate into Oil Red O-positive adipocytes with accumulated lipid, and expressed two adipocyte specific transcripts Adipsin and PPARy2, as detected by RT-PCR. After GMSCs were cultured under chondroenic induction, chondrogenic differentiation was confirmed by alcian blue staining with the presence of sulfated proteoglycans within the extracellular matrix. Likewise, chondrogenic specific genes, CoL?and AGC were confirmed by RT-PCR analysis.1.2 Isolation and cultivation and differentiation of human UCMSCsAfter 3 days, short fusiform cells were observed under inverted microscope. Cells were subcultured at 70%-80% confluency with trypsin digestion. UCMSCs were obtained from the single cell clones which were generated by limiting dilution method. According to flow cytometric analysis,UCMSCs could express CD29, CD 105, CD90 and CD44, but could not express CD34 and CD45. Cultured under osteogenic conditions for 2 weeks and 4 weeks, UCMSCs could be induced into osteoblasts, which was positive of alkaline phosphatase by ALP staining and form calcified tubercles stained dark brown by Von Kossa staining. The specific genes of osteoblast, ALP and OCN, could be detected by RT-PCR. After incubated in adipogenic induction medium for 2 weeks, UCMSCs could differentiate into Oil Red O-positive adipocytes with accumulated lipid, and expressed Adipsin and PPARy2, as detected by RT-PCR. After UCMSCs were cultured under chondroenic induction, chondrogenic differentiation was confirmed by alcian blue staining with the presence of sulfated proteoglycans within the extracellular matrix. Likewise, chondrogenic specific genes, CoL?and AGC were confirmed by RT-PCR analysis.2. In vitro amplification of GFP+GMSCs, GFP+UCMSCs and transplatationGMSCs and UCMSCs transfected by lentiviruses with eGFP were sorted by flow cytometry, then GFP+GMSCs and GFP+UCMSCs were collected and amplified in vitro. After GFP+GMSCs and GFP+UCMSCs cultured under osteogenic differentiation conditions for 10 days, GFP+GMSCs and GFP+UCMSCs were transplanted into periodontal fenestration model and critical-sized calvarial defects model with collagen gel matrix. After 8 weeks, in GFP+GMSCs transplantation groups, the defects in mandible and calvaria were sealing and newly formed bone were detected by H&E staining. The immunohistochemical analysis of OPN, CoL I and GFP were positive in experimental groups. In GFP+UCMSCs transplantation groups, there were newly formed bone, cementum and periodontal ligament in the periodontal fenestration model, and new bone in calvarial defects. The immunohistochemical analysis of OPN, CoL I and GFP were positive in experimental groups.Conclusion1. GMSCs and UCMSCs share the similar characteristics with other stem cells, including self-renewal ability, expression of specific surface marker of stem cells and tri-lineage differentiation potential.2. GMSCs can repair boney defect and can be ideal candidate of seed cells for bone regeneration; UCMSCs can repair bony defects and periodontal tissue defect, could be a novel source for stem cell-based therapy in periodontal tissue reconstruction in clinical applications.