Effects Of Mesenchymal Stromal Cells Derived From Human Gingiva And Periodontal Ligament On Periodontal Regeneration

Background and ObjectiveThe ultimate goal of periodontal therapy is to achieve complete periodontal regeneration, which means the reconstitution of the soft (gingival connective tissue and periodontal ligament) and the hard tissue (cementum and alveolar bone) of the periodontium. Current therapeutics for periodontitis fails to completely and reliably reconstitute all tissues damaged through periodontal inflammation. The cell-based therapeutics has been a promising alternative for clinical trial in periodontal regeneration. Seed cells, scaffold materials, growth factors are the main contents in the traditional tissue engineering research. Looking for an alternative stem cells with abundance and ready access, and an appropriate carrier for cell transplantation is the hotspot in tissue regeneration medicine.To date, multiple mesenchymal stem cell lineages have been explored in tissue engineering. Bone marrow stem cells (BMSCs) have been extensively studied in contributing to periodontal regeneration, on account of their capacity of self-renewal and multiple differentiation potential. PDLSCs are identified as a promising alternative in periodontal regeneration. However, all of these above stem cells are not easily accessible or cultured, which hampers the utilization for clinical application.In recent years, gingiva-derived MSCs (GMSCs) have been isolated from gingival tissue and confirmed capable of forming clonogenic colonies, expressing a typical MSC surface marker profile and possessing multiple differentiatial capacity in vitro. Moreover, GMSCs also have been demonstrated to possess the ability to form new bone in vivo, and properties of anti-inflammation and immunomodulation. On account of having properties of stable morphology, uniformly homogenous and faster proliferation, GMSCs is considered being superior to BMSCs and other dental stem cells for cell therapy in regenerative medicine. Moreover, the abundance and ready access of GMSCs is the most fascinating for tissue regeneration. Thus, GMSCs might be potentially a better alternative of cell-based approaches for periodontal regeneration. However, few attempts have been done to explore ability of GMSCs to regenerate periodontal tissue and especially its possible mechanism in tissue regeneration.Cell sheet engineering is a promising strategy in tissue regeneration. Cell sheet technology may be beneficial for cell transplantation for preserving cellular junctions, endogenous extracellular matrix (ECM). Recent studies have shown that this technique is effective to form new cementum and promote periodontal attachment utilizing periodontal ligament cells and BMSCs.To estimate the potential of GMSCs and PDLSCs to reconstitute the periodontal tissue and explore their fate and differentiation in vivo, we transplanted GFP labeled GMSCs sheet into inflammed Class III furcation defects in beagle dogs to regenerate periodontium complex.Methods:1. Isolation, cultivation and identification of human GMSCs and PDLSCs1.1Isolation, cultivation of human GMSCsHuman gingival samples were harvested from healthy individuals as discarded tissues following conventional dental procedures such as the extraction of impacted third molars or crown lengthening. GMSCs were isolated, expanded, and cultured. The2rd-4th passage GMSCs were used for the following experiments. Cloning of single cells was generated by the limiting dilution method and monoclonal cells were harvested.1.2Isolation, cultivation of human PDLSCsHuman periodontal ligament samples were harvested from healthy individuals as discarded tissues following conventional dental procedures. PDLSCs were isolated, expanded, and cultured. The2rd-4th passage PDLSCs were used for the following experiments. Cloning of single cells was generated by the limiting dilution method and monoclonal cells were harvested.1.3Identification of human GMSCs and PDLSCsColony-forming unit-fibroblasts assay and Flow cytometric analysis were performed. GMSCs and PDLSCs plated in24-well plates were cultured respectively in osteogenic medium (a-MEM containing5%FBS,0.1μM dexamethasone,10mM β-glycerophosphate, and50mg/ml ascorbate-2-phosphate). Cells cultured in a-MEM containing5%FBS were as control. The mineralized nodules were characterized by2%Alizarin red S four weeks later. Cells were incubated adipogenic medium (a-MEM containing10%FBS,0.1μM dexamethasone,60μM indomethacin, and50mg/ml ascorbate-2-phosphate). Oil Red O staining was performed to identify the oil globules2weeks later.2. GMSCs and PDLSCs sheet fabrication and Alkaline phosphatase (ALP) activity assayTo create the cell sheet, GMSCs and PDLSCs were subcultured in the following medium:1) the cell sheet induction medium (a-MEM with15%FBS,0.1μM dexamethasone,10mM β-glycerophosphate, and100mg/ml ascorbate-2-phosphate);2) the osteogenic medium (a-MEM with15%FBS,0.1μM dexamethasone,10mM β-glycerophosphate, and50mg/ml ascorbate-2-phosphate);3) the normal medium (a-MEM with15%FBS). After reaching confluence (approximately at10-12d), the cells were rinsed twice with PBS, and then detached as a cell sheet. To examine the microscopic structure of the cell sheets sections were evaluated via hematoxylin and eosin (H&E) staining. Alkaline phosphatase (ALP) activity were assayed.3. In vivo transplantation of GMSC and PDLSC sheet in beagle dogs The lentiviruses encoding eGFP were used to label the3rd passage of GMSCs and PDLSC. GFP expression in transduced cells was evaluated by direct observation of cells using a fluorescence microscope. Hygromycin was used to select the stably transduced GMSCs and PDLSC. GMSC and PDLSC sheets were transplanted into the Class Ⅲ furcation defects in beagle dogs. The animals were sacrificed after8weeks. Tissue sections selected from the most central areas of the defects were used for the H&E staining, Picrosirius red and immunohistochemical staining.Results1. Isolation, identification of human GMSCs and PDLSCsPrimary cultures of the single-cell suspensions obtained from the healthy gingiva formed adherent, clonogenic cell clusters, and the cultured GMSCs and PDLSCs retained fibroblastic phenotype. Flow cytometry analysis revealed that both cells were uniformly positive for CD44, CD29, CD73, CD90, CD105markers and Stro-1, and did not express hematopoietic stem markers CD14, and CD45, and endothelial cells markers CD144, CD31and HLA-DR. To investigate the multiple differentiation potential of GMSCs and PDLSCs, the cells were cultured in the osteogenic medium for4weeks. Extensive amounts of mineralized nodules were found in the experimental groups, but the mineralized nodules formed by GMSCs were smaller and scattered than that in PDLSCs group. Moreover, after cultured in the adipogenic medium for2weeks, GMSCs and PDLSCs were found to differentiate towards adipocytes as indicated by the accumulation of lipid droplets. The form of lipid droplets in two groups has no obvious difference. These findings indicates that the single colony-derived human gingival stem cells had the basic characteristics of mesenchymal stem cells.2. GMSCs and PDLSCs sheet fabrication and Alkaline phosphatase (ALP) activity assayBoth GMSCs and PDLSCs, cultured in the cell sheet induction medium, formed a multi-layer structure with abundant ECM. After10-12d of culture, a semi-transparent cell sheet was formed which was tough enough to be completely detached from the culture dish by a scraper. In contrast, cells grown in the osteogenic medium formed a relatively fragile membrane, whereas cells grown in the normal medium without ascorbic acid could not form the multi-layer structure.In addition, both the cell sheet and osteogenic groups of each cells exhibited markedly increased ALP activity compared with the control group. No statistically significant difference in the ALP activity was detected between the cell sheet group and the osteogenic group. ALP activity under the same conditions of PDLSCs are slightly higher than the GMSCs. But the statistically significant difference between two cells is only in the osteogenic induction group.3. In vivo transplantation of GMSC and PDLSC sheet in beagle dogsThe lentiviruses encoding eGFP were transduced into the3rd passage of GMSCs and PDLSC. Bright green fluorescence was observed at48-72h using a fluorescence microscope, and infection efficiency was up to90%. The cells were purified by hygromycin and eGFP+GMSCS and eGFP+PDLSCs were harvested. Then they were expanded and cultured10to12days to fabricate the cell sheets, and then transplanted into Class Ⅲ furcation defects created in beagle dogs. The dogs were sacrificed8weeks after transplantation. The specimen slices were observed by H&E staining, immunohistochemical staining, Sirius red staining and fluorescence microscopy observation directly. Histomorphometric analysis was performed to evaluate the amount of periodontal tissue regeneration. Alveolar bone regeneration was observed in both cell sheets groups. Our results showed enhanced new bone and new cementum formation in both GMSCs and PDLSCs group when compared with that in the control group. Immunohistochemical results show the expression of GFP was strong positive in the new bone in both GMSCs and PDLSCs groups, while GFP were negative in control group. Most of the new bone are bright green under a fluorescence microscope. GFP protein was detected in the cementoblast-like cells, the periodontal ligament fibroblasts and osteoblasts. These results indicate that the transplanted GMSCs are capable of differentiating into the osteoblasts, cementoblasts and periodontal ligament fibroblasts, and therefore directly participate in the reconstitution of the periodontal tissues. To examine the periodontal ligament regeneration, Picrosirius red stained sections were observed by both light and polarizing light microscope. In both GMSCs and PDLSC groups, newly formed Sharpey’s fibers anchored into the newly regenerated cementum were arranged perpendicularly to the root surface. In contrast, the newly formed fibers were parallel to the root surface in the control group.Conclusion1. Both GMSCs and PDLSCs share the similar characteristics with other stem cells, including self-renewability, expression of specific surface marker of stem cells and multi-lineage differentiation potential.2. The osteogenic medium supplemented with100mg/ml ascorbic acid can not only induce osteogenic differentiation but also effectively fabricate cell sheets with highly biological activity.3. Both GMSCs and PDLSC sheets express similar ALP activity and have the similar potential to regenerate bone, cementum/periodontal ligament complex in Class III furcation defects created in beagle dogs.4. The mechanism of transplanted GMSCs and PDLSCs to promote periodontal regeneration might be directly differentiating into periodontal functional cells and indirectly induction effects on host cells.