应用脱细胞气管材料建构组织工程气管的实验研究
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摘要
目的：由于缺乏有效的重建手段,广泛的气管病变仍是临床工作中遇到的棘手问题。组织工程技术为长段环形的气管缺损提供了一种新的修复途径。细胞外基质生物材料具有与正常组织相似的组成和结构,是组织工程理想的支架材料。本实验的目的是用脱细胞的方法来获取气管的细胞外基质材料,并观察脱细胞方法的脱细胞效率以及对基质完整性和材料细胞化的影响；并在体内鉴定气管材料的组织相容性和重塑的情况；最后评估体外细胞化建构的气管替代物的软骨和上皮再生的情况。
方法：我们将300g的Brown Norway (BN)大鼠和Lewis大鼠作为气管组织的供体,并用清洁剂联合酶消化的方法对取材的气管进行了脱细胞处理。在体外实验部分,我们用DAPI染色,DNA定量分析和Ⅰ、Ⅱ型主要组织相容性(抗原)复合物(MHCⅠ和Ⅱ)的免疫组织化学染色来鉴定细胞及抗原成份的脱除效率。应用组织学和生物化学定量分析法来检测气管基质成分葡萄糖胺聚糖(GAG)和胶原的保留效率,并通过组织学和扫描电子显微镜检查来观察材料的组织结构和表面显微结构的变化,同时对材料进行压缩和拉伸实验用来评估脱细胞处理之后的生物力学特性的变化。我们用接触和提取液细胞毒性实验来检测材料的生物相容性,并将软骨诱导的大鼠骨髓间充质干细胞和气管上皮细胞分别接种在材料上观察细胞与材料的相互作用。在体内实验部分,我们将软骨诱导的大鼠骨髓间充质干细胞和气管上皮细胞接种在脱细胞气管材料的管腔外内壁上来建构细胞材料复合体,之后将复合体(n=6)以及BN大鼠(n=6)和Lewis大鼠(n=6)的脱细胞气管材料植入Lewis大鼠的皮下。此外将新鲜摘取的BN(n=6)和Lewis大鼠(n=6)的气管异位移植作为对照组。经1个月的观察期后取材,对各组样本进行组织学分析来研究宿主对脱细胞材料的组织反应以及细胞材料复合体在体内的成活情况。
结果：在经过5次脱细胞循环的处理后,材料中的抗原成份被完全去除,但仍残留少量的核成份以及细胞残片。脱细胞材料的细胞外基质的胶原成分被基本保留,但GAG成份有显著的丢失。脱细胞材料的组织结构和表面显微结构未遭到明显的破坏,但脱细胞处理后气管材料的压缩和拉伸强度有所下降。材料以及其提取液对细胞没有毒性,而且材料支持骨髓间充质干细胞和气管上皮细胞的黏附和增值。在体内移植1个月后,脱细胞的BN大鼠气管材料没有引起明显的免疫排斥反应和慢性炎症反应,气管材料也未出现明显的压缩变形,但材料的管腔堵塞且软骨出现钙化。扫描电子显微镜显示复合体的细胞接种效率较低。复合体在体内的软骨基质没有细胞化的迹象,但脱细胞气管材料能够被完全上皮化及支持上皮细胞分化为纤毛细胞,有效的抑制了管腔的堵塞。
结论：清洁剂联合酶消化法可以有效的脱除抗原成份,但不能同时有效的去除细胞成份。它对基质内的GAG成份的影响较大,但保留了胶原成份和气管的组织结构。虽然材料的力学强度有所降低,但材料能够经受体内短时间的生物学压力。脱细胞处理没有对材料的再细胞化产生负面的影响,材料具有良好的生物相容性以及支持特殊组织细胞黏附和增殖的特性,是气管组织工程良好的支架材料。在体内,材料显示了良好的组织相容性,但材料无法直接用于气管的重建,需要体外细胞化的辅助。脱细胞气管材料支持接种上皮细胞的成活和分化,但是干细胞却无法进入材料的软骨基质内以产生新的软骨组织。静态的细胞种植和培养的方法影响了软骨再生和上皮内衬的效率,气管材料需要合适的生物反应器来进行体外的细胞化。
Objective:Reconstruction of long circumferential tracheal defects remains a major clinical problem owing to the lack of effective and reliable reconstructive techniques. Tissue engineering has prompted exploration into tracheal substitutes that may provide replacement options. The aims of this study were to first produce a rat tracheal matrix by decellularization, and evaluate the decellularization efficacy and the impacts of decellularization on extracellular matrix (ECM) integrity and scaffold recellularization in vitro; then to assess the host response to the decellularized tracheal matrix and matrix remodeling in vivo; finally to recellularize the matrix with syngenic cells to generate a tissue-engineered trachea in vitro and examine the cartilage and epithelial tissue regeneration of the constructs in vivo.
Methods:Tracheae were harvested from Brown Norway (BN) and Lewis rats weighing approximately 300g and were decellularized by exposing the tracheae to detergent-enzymatic treatment cycles. In vitro, the efficacies of cell and alloantigen removal were assessed by 4',6-diamidino-2-phenylindole (DAPI) staining, DNA quantification and immunohistochemical analysis of class I and II major histocompatibility complex (MHC I and II) antigens. Histological and biochemical analyses were used to evaluate desirable extracellular matrix components (glycosaminoglycans and collagen type II) retention. Matrix histoarchitecture and surface microstructure were characterized by histology and scanning electron microscope (SEM) examination. Biomechanical properties of the matrices were determined by compressive and tensile test. Contact and extract cytotoxicity tests were used to evaluate biocompatibility of the decellualrizaed tracheal matrices. Bone marrow stem cell derived chondrocytes and tracheal epithelial cells were seeded onto the matrix and SEM examination was performed to evaluate cell-matrix interaction. For the in vivo study, the bone marrow stem cell derived chondrocytes were seeded on the outer surface and freshly isolated tracheal epithelial cells were seeded to luminal surface of the acellular tracheal matrices to engineer a cell-seeded construct. The engineered tracheae (n=6) and the acellular tracheal matrices of both BN (n=6) and Lewis rats (n=6) were implanted in subcutaneous pockets in Lewis rat. In addition, fresh tracheae harvested from BN (allograft) and Lewis (isograft) rats were implanted in Lewis recipients to serve as positive (n=6) and negative (n=6) control. Host response to those different grafts and tissue remodeling were evaluated by histological analysis at 4 weeks.
Results:The major alloantigens were completely removed after 5-cycle detergent-enzymatic treatment but small amount of nuclear components and cell debris remained in the decellularized matrices. The decellularized tissue preserved the collagen type II components, but there was a significant loss of glycosaminoglycans (GAG) content. Histology and SEM showed an intact histoarchitecture with various degrees of local surface topography disturbance. Both compressive and tensile strength decreased following decellularization. Decellularized tissue and extracts were not toxic to cells. In addition, the matrices were able to support adhesion and proliferation of both stem cell derived chondrocytes and epithelial cells. Following one month in vivo implantation, acellular tracheal matrices grafts resulted in no obvious allorejection or chronic inflammation. The tubular structure of tracheal matices was well maintained. Cartilage calcification and granulation tissue growth in the lumen causing obstruction obviously due to the lack of epithelial lining were observed. The SEM examination of cell seeded matrix showed relatively low cell seeding efficacy in some engineered constructs. There was no obvious repopulation in cartilaginous portion. However, the development of ciliated cells and efficient epithelial lining were observed in tissue-engineered grafts, thus preserving luminal patency.
Conclusions:The detergent-enzymatic treatment was efficient in antigen removal but was not as efficient to eliminate cell components. The decellularization process reduced GAG components but retained collagen and major histoarchitecture of the native trachea. The biomechanical properties changed after decellularization, but the resulted matrices maintained sufficient mechanical integrity withstanding physiologic pressures in the short-term. Combined with good cell and tissue compatibility and cell adhesion properties, these decellualrized matices can be excellent scaffolds for tissue engineering of trachea. However, decellualrized matrix scaffold alone is not appropriate for tracheal reconstruction due to the lack of epithelial cells and chondrocytes. Therefore, cell seeding is required to further engineer a tracheal construct. Implantation of such cell-seeded tracheal constructs in vivo showed good epithelial cell survival and differentiation into ciliated cells, although stem cell failed to repopulate the cartilaginous matrices most likely due to static cell seeding and culture technique. A bioreactor may be needed to facilitate cell seeding and recellularization.
方法：我们将300g的Brown Norway (BN)大鼠和Lewis大鼠作为气管组织的供体,并用清洁剂联合酶消化的方法对取材的气管进行了脱细胞处理。在体外实验部分,我们用DAPI染色,DNA定量分析和Ⅰ、Ⅱ型主要组织相容性(抗原)复合物(MHCⅠ和Ⅱ)的免疫组织化学染色来鉴定细胞及抗原成份的脱除效率。应用组织学和生物化学定量分析法来检测气管基质成分葡萄糖胺聚糖(GAG)和胶原的保留效率,并通过组织学和扫描电子显微镜检查来观察材料的组织结构和表面显微结构的变化,同时对材料进行压缩和拉伸实验用来评估脱细胞处理之后的生物力学特性的变化。我们用接触和提取液细胞毒性实验来检测材料的生物相容性,并将软骨诱导的大鼠骨髓间充质干细胞和气管上皮细胞分别接种在材料上观察细胞与材料的相互作用。在体内实验部分,我们将软骨诱导的大鼠骨髓间充质干细胞和气管上皮细胞接种在脱细胞气管材料的管腔外内壁上来建构细胞材料复合体,之后将复合体(n=6)以及BN大鼠(n=6)和Lewis大鼠(n=6)的脱细胞气管材料植入Lewis大鼠的皮下。此外将新鲜摘取的BN(n=6)和Lewis大鼠(n=6)的气管异位移植作为对照组。经1个月的观察期后取材,对各组样本进行组织学分析来研究宿主对脱细胞材料的组织反应以及细胞材料复合体在体内的成活情况。
结果：在经过5次脱细胞循环的处理后,材料中的抗原成份被完全去除,但仍残留少量的核成份以及细胞残片。脱细胞材料的细胞外基质的胶原成分被基本保留,但GAG成份有显著的丢失。脱细胞材料的组织结构和表面显微结构未遭到明显的破坏,但脱细胞处理后气管材料的压缩和拉伸强度有所下降。材料以及其提取液对细胞没有毒性,而且材料支持骨髓间充质干细胞和气管上皮细胞的黏附和增值。在体内移植1个月后,脱细胞的BN大鼠气管材料没有引起明显的免疫排斥反应和慢性炎症反应,气管材料也未出现明显的压缩变形,但材料的管腔堵塞且软骨出现钙化。扫描电子显微镜显示复合体的细胞接种效率较低。复合体在体内的软骨基质没有细胞化的迹象,但脱细胞气管材料能够被完全上皮化及支持上皮细胞分化为纤毛细胞,有效的抑制了管腔的堵塞。
结论：清洁剂联合酶消化法可以有效的脱除抗原成份,但不能同时有效的去除细胞成份。它对基质内的GAG成份的影响较大,但保留了胶原成份和气管的组织结构。虽然材料的力学强度有所降低,但材料能够经受体内短时间的生物学压力。脱细胞处理没有对材料的再细胞化产生负面的影响,材料具有良好的生物相容性以及支持特殊组织细胞黏附和增殖的特性,是气管组织工程良好的支架材料。在体内,材料显示了良好的组织相容性,但材料无法直接用于气管的重建,需要体外细胞化的辅助。脱细胞气管材料支持接种上皮细胞的成活和分化,但是干细胞却无法进入材料的软骨基质内以产生新的软骨组织。静态的细胞种植和培养的方法影响了软骨再生和上皮内衬的效率,气管材料需要合适的生物反应器来进行体外的细胞化。
Objective:Reconstruction of long circumferential tracheal defects remains a major clinical problem owing to the lack of effective and reliable reconstructive techniques. Tissue engineering has prompted exploration into tracheal substitutes that may provide replacement options. The aims of this study were to first produce a rat tracheal matrix by decellularization, and evaluate the decellularization efficacy and the impacts of decellularization on extracellular matrix (ECM) integrity and scaffold recellularization in vitro; then to assess the host response to the decellularized tracheal matrix and matrix remodeling in vivo; finally to recellularize the matrix with syngenic cells to generate a tissue-engineered trachea in vitro and examine the cartilage and epithelial tissue regeneration of the constructs in vivo.
Methods:Tracheae were harvested from Brown Norway (BN) and Lewis rats weighing approximately 300g and were decellularized by exposing the tracheae to detergent-enzymatic treatment cycles. In vitro, the efficacies of cell and alloantigen removal were assessed by 4',6-diamidino-2-phenylindole (DAPI) staining, DNA quantification and immunohistochemical analysis of class I and II major histocompatibility complex (MHC I and II) antigens. Histological and biochemical analyses were used to evaluate desirable extracellular matrix components (glycosaminoglycans and collagen type II) retention. Matrix histoarchitecture and surface microstructure were characterized by histology and scanning electron microscope (SEM) examination. Biomechanical properties of the matrices were determined by compressive and tensile test. Contact and extract cytotoxicity tests were used to evaluate biocompatibility of the decellualrizaed tracheal matrices. Bone marrow stem cell derived chondrocytes and tracheal epithelial cells were seeded onto the matrix and SEM examination was performed to evaluate cell-matrix interaction. For the in vivo study, the bone marrow stem cell derived chondrocytes were seeded on the outer surface and freshly isolated tracheal epithelial cells were seeded to luminal surface of the acellular tracheal matrices to engineer a cell-seeded construct. The engineered tracheae (n=6) and the acellular tracheal matrices of both BN (n=6) and Lewis rats (n=6) were implanted in subcutaneous pockets in Lewis rat. In addition, fresh tracheae harvested from BN (allograft) and Lewis (isograft) rats were implanted in Lewis recipients to serve as positive (n=6) and negative (n=6) control. Host response to those different grafts and tissue remodeling were evaluated by histological analysis at 4 weeks.
Results:The major alloantigens were completely removed after 5-cycle detergent-enzymatic treatment but small amount of nuclear components and cell debris remained in the decellularized matrices. The decellularized tissue preserved the collagen type II components, but there was a significant loss of glycosaminoglycans (GAG) content. Histology and SEM showed an intact histoarchitecture with various degrees of local surface topography disturbance. Both compressive and tensile strength decreased following decellularization. Decellularized tissue and extracts were not toxic to cells. In addition, the matrices were able to support adhesion and proliferation of both stem cell derived chondrocytes and epithelial cells. Following one month in vivo implantation, acellular tracheal matrices grafts resulted in no obvious allorejection or chronic inflammation. The tubular structure of tracheal matices was well maintained. Cartilage calcification and granulation tissue growth in the lumen causing obstruction obviously due to the lack of epithelial lining were observed. The SEM examination of cell seeded matrix showed relatively low cell seeding efficacy in some engineered constructs. There was no obvious repopulation in cartilaginous portion. However, the development of ciliated cells and efficient epithelial lining were observed in tissue-engineered grafts, thus preserving luminal patency.
Conclusions:The detergent-enzymatic treatment was efficient in antigen removal but was not as efficient to eliminate cell components. The decellularization process reduced GAG components but retained collagen and major histoarchitecture of the native trachea. The biomechanical properties changed after decellularization, but the resulted matrices maintained sufficient mechanical integrity withstanding physiologic pressures in the short-term. Combined with good cell and tissue compatibility and cell adhesion properties, these decellualrized matices can be excellent scaffolds for tissue engineering of trachea. However, decellualrized matrix scaffold alone is not appropriate for tracheal reconstruction due to the lack of epithelial cells and chondrocytes. Therefore, cell seeding is required to further engineer a tracheal construct. Implantation of such cell-seeded tracheal constructs in vivo showed good epithelial cell survival and differentiation into ciliated cells, although stem cell failed to repopulate the cartilaginous matrices most likely due to static cell seeding and culture technique. A bioreactor may be needed to facilitate cell seeding and recellularization.
引文
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