Experimental Study Of Chitosan Hydrogel Loaded With GMSCs And TGF-β1 To Promote The Regeneration Of Periodontal Ligament

Periapical lesions can lead to the destruction of alveolar bone,periodontal ligament,and cementum.For large areas of periapical lesions,combined periodontal and endodontic lesions,and buccal-lingual penetrating lesions,root canal treatment can not be completely cured and can be treated by apical surgery.After conventional apical surgery,the healing of the lesion area is slow,and the periapical tissue is mostly healed by repair,that is,the formation of new tissue with a different structure and function from the original tissue.The ideal healing mode of periapical lesions is the formation of new attachment of periapical ligament,that is,the regeneration of periodontal complex in the apical region,including the regeneration of periodontal ligament,cementum,and alveolar bone in the apical region.The periodontal ligament is a soft tissue interface connecting the alveolar bone and cementum.It has a sophisticated and complex anatomical structure,which is challenging to form a new attachment of the periapical ligament.Although clinically,most studies have focused on repairing the loss of alveolar bone to prevent tooth loss,extensive loss of periodontal ligament soft tissue is essential for developing the disease.The periodontal tissue engineering techniques can reconstruct the lost tissue’s morphology and function and provide a feasible method for regenerating periodontal ligament in the apical region.Periodontal ligament stem cells(PDLSCs)are the most widely used Periodontal tissue regeneration cells.However,to obtain PDLSCs,tooth extraction is required,and the success ratio of PDLSC culture is low and it takes a long time.Therefore,the translational application of PDLSC in periodontal therapy is severely limited.Based on this,the researchers sought to explore the possibility of other sources of stem cells.Gingival-derived mesenchymal stem cells(GMSCs)have the advantages of abundant sources,easy separation,and no tooth extraction,and existing studies have fully confirmed that GMSCs have the biological characteristics of stem cells.They include self-renewal,multidirectional differentiation,and immune regulation.GMSCs are expected to be an ideal source of stem cells for future use in regenerative medicine.Although there are some reports about GMSC transplantation for tissue regeneration,how to construct tissue engineering complexes for tissue regeneration,including cells,growth factors,and scaffold materials,and exert their synergies and promoting effects to achieve successful tissue regeneration is still a problem that researchers are committed to solving.In Chapter 2,PDLSCs and GMSCs were extracted and purified,and the extracted cells were identified by clonal formation ratio,flow cytometry,alizarin red,and oil-red O staining after osteogenesis and lipid induction.CCK-8,scratch test,ALP staining,and RT-qPCR were used to compare the proliferation ability,migration ability,osteogenic differentiation ability,and fibroblast differentiation ability of the two kinds of cells.It was found that GMSCs were significantly higher than PDLSCs in proliferation and migration ability.In terms of the ability to induce differentiation,PDLSCs were significantly superior to GMSCs in both osteogenic and fibrogenic differentiation.These results indicate that GMSCs can be used as seed cells for tissue regeneration,but how to enhance the differentiation ability of GMSCs to replace PDLSCs still needs further study.In addition,although GMSCs can undergo fibroblast differentiation,whether they can be differentiated into specific periodontal ligament tissue and how to construct a complex for periodontal ligament regeneration are also the focus of our subsequent experiments.In Chapter 3,we constructed three functional hydrogel substrates by adjusting the ratio of each component and characterized the physicochemical properties and biocompatibility of hydrogels by scanning electron microscopy,universal testing machine,rheometer,CCK-8,Calcein-AM/PI staining and BCA.The results showed that the three hydrogels had the pore structure,degradation ratio,sustained release ability,and good biocompatibility as tissue engineering scaffolds.Furthermore,the effects of hydrogels with different stiffness on cell biological behavior were analyzed by CCK-8 and RT-qPCR.The results showed that with the increase of hydrogel stiffness,the extension degree and proliferation ratio of cells increased.However,cells cultured on CS-GNP-2 hydrogel surface with similar physiological stiffness to periodontal ligament showed significantly high expression of periodontal ligament specific marker genes PLAP-1,COL-1,SCX,and POSTN.These results suggest that CS-GNP-2 hydrogel with the mass ratio of chitosan to genipin 100:2 can promote the directional differentiation of periodontal ligament of stem cells and be used as an ideal scaffold material for periodontal ligament regeneration.In Chapter 4,we loaded different doses of TGF-β1 into CS-GNP-2 hydrogel and cultured GMSCs on it.PDLSC cultured under the same conditions were used as control.The release kinetics of TGF-β1 in the hydrogel was detected by ELISA,the effects of different concentrations of CS-GNP/TGF-β1 on the proliferation of GMSCs was detected by CCK-8,and the mRNA and protein expression of PLAP-1,COL-1,SCX and POSTN,which are specific markers of periodontal ligament were detected by RT-qPCR and Western blot.To evaluate whether the CS-GNP-2/TGF-β1/GMSCs system can assist GMSCs to replace PDLSCs and effectively promote periodontal tissue regeneration in vitro.Results show that;TGF-β1 was released into the culture medium in a dose-dependent manner in the hydrogel.The hydrogel loaded with 10 ng/mL TGF-β1 could significantly promote the expression of mRNA and protein of periodontal ligament related markers during long-term induction.CS-GNP/TGF-β1/GMSCs could be used as a complex to induce periodontal ligament tissue regeneration.It can be used for further examination in subsequent in vivo experiments.In chapter 5,in vivo degradation ratio and biocompatibility of CS-GNP-2 hydrogels were studied in C57BL/6 mice,and CS-GNP/TGF-β1/GMSCs were compounded with cementum slices and implanted into the subcutaneous tissue of nude mice for ectopic periodontal ligament tissue regeneration.HE staining showed that CSGNP-2 hydrogel had good biocompatibility in mice and could be degraded gradually with time.Immunohistochemical results showed that the expression of periodontal ligament specific markers PLAP-1,COL-1,and POSTN in the experimental group was significantly higher than in the control group,indicating that CS-GNP/TGF-β1/GMSCs complex had excellent ability to promote periodontal ligament tissue regeneration.It can be used as an alternative in the field of periapical and periodontal tissue regeneration in the future.