Lung Tissue Engineering Based On Collagen And Matrigel

Currently, chronic obstructive pulmonary disease (COPD), Asthma and pulmonary fibrosis (PF) have become lung disease with high mortality. Accompanied by changes in climate and environment, human lung diseases were now facing significant growth trend. Nearly half a century, as people's basic theory for the study of lung deepening, as well as the launch mechanism of lung disease, oxygen therapy, surgery and rehabilitation treatment of lung disease has progressed. Nevertheless, lung transplantation remains the treatment of advanced lung disease. However, the lack of lung organ sources, many patients simply can not currently be effectively treated. Find new ways and means to treat lung diseases have become a top priority. The continuous development of tissue engineering has been a new hope for the treatment of lung disease.Tissue engineering to reconstruct human tissue and organ is an interdisciplinary that integrates life science, materials science and engineering principles. In recent years, tissue engineering has made breakthrough on the skin, cartilage, nerves, blood vessels and heart tissue. In the meantime, some products of tissue engineering have been put into the clinical application.Pulmonary parenchyma consists of the lung bronchial and its terminal branches at all levels of alveolar. Lung tissue engineering has a rapid development in recent years. Researchers have successfully reconstructed tissue engineered alveolar and bronchial mucosa in vitro. Nevertheless, for the reconstruction of lung in vitro, there are still many problems, such as how to choose cell scaffold material, how to optimize the cell-scaffold nurturing environment in vitro and to further improve the quality of the reconstructed lung tissue or other issues and so on.This paper carried out reconstruction of the alveolar and bronchial mucosa in vitro using tissue engineering. About reconstructing alveolar in vitro, we used mouse fetal pulmonary cells (FPCs) as seeding cells and collagen/Matrigel provided three dimensional growth environments for cells. And for now there is no effective means to control the reconstruction of alveolar structures in vitro situation. We present the first application of alginate-poly-L-lysine-alginate (APA) microcapsules to play the role of space occupying and control the formation of alveolus-like structures. In the meantime, we successful reconstructed alveolus-like structures in vitro, which achieved expected results. About engineered bronchial mucosa, we used collagen/Matrigel as embryonic lung fibroblasts and bronchial epithelial cells as scaffolds. We successful reconstructed the bronchial mucosa-like structure in vitro and studied regeneration of human bronchial epithelial cells with Matrigel. At present, there is no report on regeneration of the bronchial epithelial cells with Matrigel.This paper based on scaffold of reconstructed lung tissue in vitro (APA microcapsules), and different structures of reconstructed lung tissue (alveolar and bronchial mucosa-like structures), the main research contents contained three parts, outlined as follows:Part I: Preparation of APA microcapsules and evaluation of physical & biological propertiesMicrocapsules can be formed as a semipermeable membrane immunoisolation barrier structure; its physical and biological characteristics of the preparation process by controlling the physical parameters can be changed. The purpose of our research was to prepare APA microcapsules used to reconstruct alveolus-like structure, and assess their physical and biological characteristics. We used electrostatic field was 6kV/1.5cm, the speed of the pump was 15mL/h, needle inner diameter was 0.09mm, the concentration of sodium alginate was 2%, concentration of poly-lysine was 0.05%, the reaction time was 6min, diameter of APA microcapsules were 200-300μm. The average diameter of human alveolar about 250μm. Therefore, under the controlling of these parameters is more suitable for preparation of microcapsules for reconstructing alveolus-like structure.In the physical performance evaluation, the results show that APA microcapsules had good mechanical strength. About microcapsule permeability, we prepared APA microcapsules can 4kD free diffusion of small molecules into the microcapsules, 70kD middle molecular substances can be some passed, and 150kD macromolecular material can not be or most can not be encapsulated membrane. Performance in biology observation of APA microcapsules form after abdominal transplantation showed good biocompatibility.In this part of the study, we prepared the APA microcapsules, mastered the key technologies of developing APA microcapsules, and groped a series of conditions developing APA microcapsules, and its physical and biological properties were evaluated. We found that the size of the prepared APA microcapsules was suitable for construction of alveolar structure, and had good biocompatibility. Part II: Study of reconstructing tissue engineered alveolus-like structures in vitro used collagen/Matrigel-APA microcapsules as scaffoldsConstruction of the distal lung tissue, alveolar-like structure to achieve is important. The purpose of this study is to use FPCs as seeding cells, using microcapsules play a space occupying effect and prepare biological materials and then by changing the preparation process of the physical parameters to control the size and shape of the alveolar to achieve the construction of tissue-engineered structures well.Generally observed, FPCs and collagen/Matrigel-APA microcapsules can be formed tissue sheets, and then the sheets were gradually shrinking. Observed 14 day-cultured significantly shrink and continued to train to 21 days. We could observe the final formation of butterfly-like appearance. Under inverted microscope, we found that cells in three-dimensional scaffold materials formed vesicle-like structure. APA microcapsules maintained good integrity, the seed cells grew around the microcapsules. H.E. staining showed that the vesicle-like structures of 7 day-cultured and 14 day-cultured sheets were very similar to the structure of normal lung tissue in mice. By immunohistochemical staining pan-CK, Vimentin, and SpC-positive cells could be seen in 7 day-cultured and 14 day-cultured sheets. Observed by transmission electron microscopy lamellar bodies gained in 7 day-cultured and 14 day-cultured sheets, indicating typeⅡalveolar cells in alveolus-like structures maintained the undifferentiated state. Scanning electron microscope found that the longitudinal section of lung tissue sheets had cystic structure.This part of the study, successfully constructed the alveolar structures in vitro, microcapsules played space occupying effect, and typeⅡalveolar cells could maintain the undifferentiated state.Part III: Study of reconstructing tissue engineered bronchial mucosa in vitro used collagen/Matrigel as scaffolds Bronchial mucosa in the lungs plays an important role in the department. Weintend to use human embryonic lung fibroblast cells (MRC-5) and human bronchial epithelial cells (Beas-2B) as seed cells as well as employed collagen/Matrigel as scaffolds, meanwhile applied air-liquid interface culture method to reconstruct airway mucosa in vitro. We also want to study the effect of Matrigel on differentiation of epithelial cells. Among them, the epithelial cells inoculated on the fibroblasts after collagen/Matrigel mixture gelation. In the study, also by Matrigel was added before epithelial cells inoculation way to explore the effect of Matrigel on epithelial cell regeneration.Under inverted microscope, we found that fibroblasts in collagen/Matrigel scaffolds formed a network like structure in collagen/Matrigel scaffold and built bronchial mucosal surface of airway epithelial cell fusion. H.E. staining showed that in Beas-2B cells formed intact epithelial cell layer under the surface of Matrigel was added. On the contrary, not dropping Matrigel, airway epithelial cell layer was incomplete. Immunohistochemical staining showed that Matrigel under the dropping of bronchial mucosa pan-CK positive cells were more shows that Matrigel promoted the growth of epithelial cells; without dropping Matrigel, pan-CK positive cells were few. Dropping Matrigel under the Beas-2B cells had no effect on the growth of fibroblasts. Scanning electron microscopy of bronchial mucosa in vitro found that dropping Matrigel under the Beas-2B cells had ciliated-like structure formation, which indicated Matrigel can promote epithelial cell differentiation.This part of the study, reconstructed the bronchial mucosa in vitro successfully, and confirmed that the Matrigel can promote epithelial cell regeneration.In summary, this study, about scaffolds development, we mastered key technologies developed APA microcapsules, and explore a series of development conditions for microcapsules, successfully prepared APA microcapsules for the alveolar structure, and with good physical properties and biocompatibility. Construction of the alveolar, we used FPCs as seeding cells, collagen/Matrigel as scaffolds. And APA microcapsules played full role of space occupying in vitro to construct the alveolar structure. By regulating the size and shape of microcapsules can effectively control size and shape of alveoli in alveolus-like structure. We achieved initial control of alveolar structure. About bronchial mucosa, we took Beas-2B and MRC-5 as seeding cells, collagen/Matrigel as scaffolds and successfully reconstructed the bronchial mucosa in vitro, and confirmed that Matrigel can influence bronchial epithelial cell regeneration.Construction of the engineered lung tissue supply new technologies and means for treatment of lung diseases. In addition, above mentioned engineered lung tissue also can serve as a model of lung disease or the treatment of lung disease research, drug screening and toxicity testing.