【作者】 刘莉；

【导师】 张志民；

【作者基本信息】 吉林大学 ， 口腔医学（专业学位）， 2022， 博士

【摘要】 根尖周病变会导致根尖周牙槽骨、牙周膜和牙骨质的破坏。对于大面积根尖周病变、牙周牙髓联合病变及颊舌向贯通病变,根管治疗不能完全治愈,可以通过根尖外科手术进行治疗。常规根尖手术后,病变区愈合较慢,根尖周组织多以修复方式愈合,即形成与原组织结构和功能不同的新生组织。而根尖周病变理想的愈合方式是根尖周膜新附着的形成,包括根尖区牙槽骨、牙周膜和牙骨质的再生,即根尖区牙周组织的再生。牙周膜是连接牙槽骨和牙骨质两种硬组织之间的软组织界面,解剖结构精细复杂,是根尖周膜新附着形成中的难点。虽然在临床上,大多数研究重点都放在修复牙槽骨的丢失以防止牙齿丢失,但牙周膜软组织的广泛丢失对疾病的发展至关重要。组织工程的相关技术可以重建丢失组织的形态和功能,为根尖区牙周膜的再生提供可行的方法。牙周膜间充质干细胞(periodontalligamentstemcells,PDLSCs)是目前研究应用最广泛的牙源性间充质干细胞。然而,获得PDLSCs,需要拔除健康牙齿,并且PDLSCs培养成功率低,耗费时间长,细胞表型严重受到传代次数的影响。因此,PDLSCs在牙周治疗中的转化应用受到了严重限制。基于此,研究者们试图探索其他来源的干细胞。牙龈来源的间充质干细胞(gingiva-derived mesenchymal stem cells,GMSCs)具有来源丰富、易于分离、无需拔牙等优势,并且现有研究充分证实GMSCs具有干细胞的生物特性,包括自我更新、多向分化和免疫调节等。GMSCs有望作为一种理想的干细胞来源在未来的再生医学中得到应用。尽管有一些报道涉及GMSCs移植用于组织再生,但如何构建包括细胞、生长因子及生物活性材料的组织工程复合体,发挥它们之间的相互协同及促进作用,以达到成功的组织再生,仍然是研究者们致力于解决的问题,也是本研究的主要研究内容。第二章中,我们提取并纯化PDLSCs和GMSCs两种细胞,使用克隆形成率、流式细胞术、成骨及成脂诱导后茜素红及油红O染色等方法,对提取的细胞进行鉴定。使用CCK-8、划痕实验、ALP染色及ALP活性测定、RT-qPCR的方法对两种细胞的增殖能力、迁移能力、成骨分化能力及成纤维分化能力进行对比。发现在增殖能力、迁移能力方面,GMSCs显著高于PDLSCs。而在诱导分化的能力上,PDLSCs无论是在成骨分化还是在成纤维的分化能力上,均显著优于GMSCs。表明GMSCs虽可作为组织再生的种子细胞,但如何增强GMSCs的分化能力使之替代PDLSCs,仍需要进一步研究。并且GMSCs虽可进行成纤维向分化,但是否能够分化为特异性的牙周膜组织,以及如何构建用于牙周膜组织再生的复合体,也是我们后续实验要研究的重点。第三章中,我们通过调整各成分配比,构建3种功能性水凝胶基底材料,通过扫描电镜、万能力学实验机、流变仪、CCK-8、Calcein-AM/PI染色及BCA等方法表征了水凝胶的理化特性及生物相容性。结果显示,制备的3种水凝胶均具有适合作为组织工程材料的孔隙结构、降解率、缓释能力及良好的生物相容性。进一步,通过CCK-8、RT-qPCR等检测手段着重分析了不同刚度的水凝胶对细胞生物学行为的影响。结果显示,随着水凝胶刚度的增加,细胞的伸展程度和增殖率均随之增加。而在与牙周膜生理性刚度相近的CS-GNP-2水凝胶表面培养的细胞,显著高表达牙周膜特异性标志基因PLAP-1、COL-1、SCX和POSTN。说明壳聚糖与京尼平质量比为100:2的CS-GNP-2水凝胶可促进干细胞牙周膜向的定向分化,可以作为牙周膜再生理想的支架材料。第四章中,我们将不同剂量的TGF-β1载入CS-GNP-2水凝胶,并在其上培养GMSCs,以相同条件下培养的PDLSCs作为对照。通过ELISA、CCK-8、RT-qPCR 及 Western blot 实验,检测 TGF-β1 在水凝胶内 的释放动力学,不同浓度的CS-GNP/TGF-β1对GMSCs的增殖的影响,以及对牙周膜特异性标记物PLAP-1、COL-1、SCX和POSTN的mRNA及蛋白表达的影响。评价CS-GNP/TGF-β1/GMSCs体系是否可辅助GMSCs代替PDLSCs,在体外细胞实验中有效促进牙周膜组织再生。结果显示;TGF-β1在CS-GNP水凝胶内以剂量依赖的方式释放入培养介质中,载10ng/mLTGF-β1的水凝胶可在长期诱导的过程中,显著促进牙周膜相关标记物mRNA和蛋白的表达,CS-GNP/TGF-β1/GMSCs可作为诱导牙周膜组织再生的复合体,应用于后续体内实验中进行进一步的检验。第五章,在C57BL/6小鼠体内研究CS-GNP水凝胶的体内降解率和生物相容性,并且将CS-GNP/TGF-β1/GMSCs与牙骨质片复合,植入裸鼠皮下组织,进行异位牙周膜组织再生。HE染色结果显示,CS-GNP水凝胶在小鼠体内具有良好的生物相容性,并且可随着时间延长而逐渐降解。免疫组化结果显示,实验组裸鼠皮下新生组织中牙周膜特异性标记物PLAP-1、COL-1和POSTN的表达显著高于对照组,表明CS-GNP/TGF-β1/GMSCs复合体具有优良的促进牙周膜组织再生能力,能够作为今后根尖周组织及牙周组织再生研究领域的备选方案。更多还原

【Abstract】 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.更多还原