中国组织工程研究 ›› 2025, Vol. 29 ›› Issue (22): 4748-4760.doi: 10.12307/2025.432

• 细胞外基质材料 extracellular matrix materials • 上一篇    下一篇

组织工程化口腔黏膜等效物的血管化特征

石丽娟1,2,3,韦  建1,2,3,张  旋1,2,3,何凌霄1,2,3,江小茜1,2,3,聂敏海1,2,3,陈佳娜1,2,3,刘旭倩1,2,3   

  1. 1西南医科大学附属口腔医院牙周黏膜病科,四川省泸州市   646000;2口颌面修复重建和再生泸州市重点实验室,四川省泸州市   646000;3西南医科大学口腔医学研究所,四川省泸州市   646000
  • 收稿日期:2024-03-21 接受日期:2024-05-06 出版日期:2025-08-08 发布日期:2024-12-06
  • 通讯作者: 刘旭倩,博士,副教授,硕士生导师,西南医科大学附属口腔医院牙周黏膜病科,四川省泸州市 646000;口颌面修复重建和再生泸州市重点实验室,四川省泸州市 646000;西南医科大学口腔医学研究所,四川省泸州市 646000
  • 作者简介:石丽娟,女,1996年生,江西省丰城市人,汉族,西南医科大学在读硕士,主要从事牙周黏膜病学研究。
  • 基金资助:
    口腔疾病防治全国重点实验室开放课题项目(SKLOD2024OF04),项目负责人:刘旭倩;泸州市科知局科技创新人才计划项目(2023RCX171),项目负责人:刘旭倩;四川省医学科研课题计划项目(S23043),项目负责人:刘旭倩;彭州市-西南医科大学联合项目(2023PZXNYD12),项目负责人:刘旭倩

Vascularization characteristics of tissue-engineered oral mucosa equivalents

Shi Lijuan1, 2, 3, Wei Jian1, 2, 3, Zhang Xuan1, 2, 3, He Lingxiao1, 2, 3, Jiang Xiaoxi1, 2, 3, Nie Minhai1, 2, 3, Chen Jiana1, 2, 3, Liu Xuqian1, 2, 3   

  1. 1Department of Periodontal Mucosal Diseases, Affiliated Stomatological Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China; 2Luzhou Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Luzhou 646000, Sichuan Province, China; 3Institute of Stomatology, Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • Received:2024-03-21 Accepted:2024-05-06 Online:2025-08-08 Published:2024-12-06
  • Contact: Liu Xuqian, MD, Associate professor, Master’s supervisor, Department of Periodontal Mucosal Diseases, Affiliated Stomatological Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China; Luzhou Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Luzhou 646000, Sichuan Province, China; Institute of Stomatology, Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • About author:Shi Lijuan, Master candidate, Department of Periodontal Mucosal Diseases, Affiliated Stomatological Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China; Luzhou Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Luzhou 646000, Sichuan Province, China; Institute of Stomatology, Southwest Medical University, Luzhou 646000, Sichuan Province, China
  • Supported by:
    1Department of Periodontal Mucosal Diseases, Affiliated Stomatological Hospital of Southwest Medical University, Luzhou 646000, Sichuan Province, China; 2Luzhou Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Luzhou 646000, Sichuan Province, China; 3Institute of Stomatology, Southwest Medical University, Luzhou 646000, Sichuan Province, China

摘要:


文题释义:

口腔黏膜等效物:是一种通过组织工程技术在体外构建的三维口腔黏膜模型,上层为上皮细胞,以模拟口腔黏膜上皮;下层为镶嵌有成纤维细胞的3D支架,以模拟口腔黏膜固有层。
激光捕获显微切割技术:是一种将同种细胞群从其自身生态位中分离出来进行下游分子检测的方法,能够精确获取组织中的目标细胞群,高质量提取组织中的DNA和RNA。


背景:前期研究中三维细胞重建组织工程化口腔黏膜等效物结构类似于正常口腔黏膜,即存在类上皮样结构、类固有层样结构、类血管腔样结构,并已初步实现了等效物的血管化建立,但其血管化特征尚不十分明确。

目的:采用血管内皮细胞特异性标志物表达谱关联激光捕获显微切割系统靶向获取血管化口腔黏膜等效物的血管样结构,评价其成血管能力,揭示其血管化特征。
方法:分别从人牙龈上皮组织和固有层组织原代培养人牙龈上皮细胞、人牙龈成纤维细胞、人牙龈间充质干细胞,人牙龈间充质干细胞经单克隆扩增培养后诱导分化形成血管内皮样细胞。将人牙龈上皮细胞、人牙龈成纤维细胞、血管内皮样细胞分层负载于脱细胞血管基质-0.25%类人Ⅰ型胶原支架上,构建血管化口腔黏膜等效物。将血管化口腔黏膜等效物(实验组)与脱细胞血管基质-0.25%类人Ⅰ型胶原支架(对照组)分别植入裸鼠背部皮下,14  d后两组切口表面涂布生物胶,实验组生物胶表面接种人牙龈上皮细胞,对照组不接种细胞,继续饲养14 d后取材,利用形态学观察口腔黏膜等效物分层结构;采用较为全面的血管内皮细胞特异性标志物表达谱对口腔黏膜等效物中的新生血管样结构进行免疫组化、免疫荧光标记,进行血管化特征分析;采用激光捕获显微切割系统靶向捕获免疫组化特异性标记的口腔黏膜等效物中新生血管样结构,靶向分析其血管化特征。

结果与结论:①形态学观察显示口腔黏膜等效物细胞层次清晰,结构类似于正常口腔黏膜,即存在类上皮样结构、类固有层样结构、类血管腔样结构,类血管腔样结构内存在散在红细胞;②口腔黏膜等效物组中EdU Apollo示踪种子细胞结果显示:EdU Apollo 488标记的人牙龈上皮细胞呈绿色荧光表达;DAPI标记的人牙龈成纤维细胞呈蓝色荧光表达,体内形成类固有层样结构;EdU Apollo 567标记的血管内皮样细胞呈红色荧光表达,体内形成类血管样结构;③血管内皮细胞特异性标志物表达谱免疫荧光标记血管结构显示,与正常口腔黏膜相比,口腔黏膜等效物中CD31、CD51、CD54、CD105、Tie-2、VWF、血管内皮生长因子受体1、血管内皮生长因子受体2表达升高(P < 0.000 1),CD34表达无明显变化(P > 0.05);④与特异性标记的口腔黏膜血管结构相比,激光捕获显微切割系统靶向捕获的口腔黏膜等效物血管样结构中CD51、CD54、CD105、Tie-2、VWF、血管内皮生长因子受体1、血管内皮生长因子受体2表达升高(P < 0.000 1),CD31、CD34表达无明显变化(P > 0.05);⑤结果表明,通过三维细胞分层重建的口腔黏膜等效物能够实现良好的血管化,其血管化特征符合新生血管生成的免疫学功能及特点;血管化助力三维细胞分层重建的口腔黏膜等效物再生。

https://orcid.org/0009-0009-6026-9697 (石丽娟) 

中国组织工程研究杂志出版内容重点:生物材料;骨生物材料;口腔生物材料;纳米材料;缓释材料;材料相容性;组织工程

关键词: 脱细胞血管基质, 血管内皮样细胞, 三维细胞重建, 血管化, 口腔黏膜等效物, 激光捕获显微切割系统

Abstract: BACKGROUND: In previous studies, the equivalent structure of three-dimensional cell reconstruction of tissue engineering oral mucosa is similar to normal oral mucosa, including epithelial-like structure, lamina propria-like structure, and vascular lumen-like structure, and has initially achieved the establishment of vascular equivalent, but its vascularization characteristics are not very clear. 
OBJECTIVE: Vascular-like structures of vascularized oral mucosa equivalent were obtained by targeting vascular endothelial cells specific marker expression profiles correlated with laser capture microdissection system, and their vascularization ability was evaluated to reveal their vascularization characteristics.
METHODS: Human gingival epithelial cells were cultured from human gingival epithelium and human gingival fibroblasts, human gingival mesenchymal stem cells were cultured from human gingival lamina propria. Human gingival mesenchymal stem cells were induced to differentiate into vascular endothelial-like cells after monoclonal expansion culture. Human gingival epithelial cells, human gingival fibroblasts, and vascular endothelial-like cells were loaded with acellular vascular matrix-0.25% human-like collagen type I scaffold to construct the vascularized oral mucosa equivalent. The layered structure of oral mucosa equivalent (experimental group) and the acellular vascular matrix-0.25% human-like collagen type I scaffold (control group) were implanted subcutaneously into the back of nude mice, respectively. 14 days later, the incision surface of the two groups was coated with biogel. The biogel surface of the experimental group was inoculated with human gingival epithelial cells, while the control group was not inoculated with cells. The samples were collected after 14 days of feeding. The layered structure of oral mucosa equivalent was observed by morphology. The neovascular-like structures in oral mucosa equivalents were labeled by immunohistochemistry and immunofluorescence with a more comprehensive expression profile of vascular endothelial cells, and the vascularization characteristics were analyzed. A laser capture microdissection system was used to capture the neovascularization structures in the oral mucosa equivalents specifically labeled by immunohistochemistry and analyze their vascularization characteristics.
RESULTS AND CONCLUSION: (1) The morphology showed that the cell level of oral mucosa equivalent was clear, and the structure was similar to that of normal oral mucosa, that is, there were epithelioid structures, lamina-like structures, and vascular cavelike structures, and there were scattered erythrocytes in the vascular cavelike structures. (2) The results of EdU Apollo tracer seed cells in the oral mucosa equivalent group showed that human gingival epithelial cells labeled with EdU Apollo 488 showed green fluorescence expression. DAPI labeled human gingival fibroblasts showed blue fluorescence expression and formed lamina-like structures in vivo. EdU Apollo 567 labeled vascular endothelial-like cells showed red fluorescence expression and formed a vascular-like structure in vivo. (3) Vascular endothelial cell specific marker expression profile immunofluorescence labeling of vascular structure showed that compared with normal oral mucosa, the expressions of CD31, CD51, CD54, CD105, Tie-2, VWF, vascular endothelial growth factor receptor 1, and vascular endothelial growth factor receptor 2 in oral mucosa equivalents were increased (P < 0.000 1). There were no significant changes in CD34 expression (P > 0.05). (4) Compared with the specifically labeled oral mucosal vascular structures, the expression levels of CD51, CD54, CD105, Tie-2, VWF, vascular endothelial growth factor receptor 1, and vascular endothelial growth factor receptor 2 of the oral mucosa equivalents targeted by the laser capture microdissection system were increased (P < 0.000 1). There were no significant changes in expression of CD31 and CD34 (P > 0.05). (5) The results showed that the oral mucosa equivalent reconstructed by three-dimensional cell stratification could achieve good vascularization, and its vascularization characteristics were consistent with the immunological function and characteristics of neovascularization. Vascularization helps three-dimensional cell layer reconstruction of oral mucosa equivalent regeneration.

Key words: acellular vascular matrix, vascular endothelial-like cell, three-dimensional cell reconstruction, vascularization, oral mucosa equivalent, laser capture microdissection system

中图分类号: