Scaffold-Free Strategy To Construct Composite And Biomimetic Tissue-Engineering Trachea For Orthotopic Transplantation

ABSTRACT ⅠConstruction of chondrocyte sheets and the mechanism of promoting extracellular matrix productionObjective:In order to build the tissue-engineered trachea to solve the problem of airway reconstruction after long-segment tracheal defects,and to avoid the adverse effects associated with the use of scaffold materials,in this part of the study,we propose to build chondrocyte sheets with the help of pure chondrocytes in a high-density implantation,and to realize a three-dimensional culture pattern with the help of mutual accumulation of chondrocytes.The physical parameters of the chondrocyte sheets and the production of cartilage matrix within the sheets were investigated under different growing density and culture time conditions to screen the optimal construction parameters.Materials and methods:Primary chondrocytes were extracted from the auricular cartilage of experimental animals,and cultured and expanded in vitro.The cell densitometry was 1-fold density,approximately1*105/cm2,when the monolayer chondrocytes were spread all over the culture flask and in contact with each other.three sets of small-sized chondrocyte membrane sheets(24-well plates were used as the growing carriers)were grown at 5-fold,10-fold and 20-fold of the above density in a stepwise and high-density manner.The dead or alive status and metabolic activity of the chondrocytes inside the sheets were evaluated with the help of live-dead cell double staining and CCK-8 assay.The growth and development of chondrocyte sheets and matrix production were investigated with the help of histological section staining and quantification of cartilage-specific extracellular matrix components.The molecular mechanism of chondrocyte membrane secretion was investigated by polymerase chain reaction(PCR).Results:The results showed that with the help of trypsin and type Ⅱ collagenase digestion,about 1*108 primary chondrocytes could be extracted from a single auricular cartilage of experimental rabbits,and completed in vitro culture and expansion.To avoid excessive passaging leading to chondrocyte de-differentiation,P2 generation chondrocytes were used in this study for chondrocyte sheets.The results of live-dead double staining showed that no obvious cell death was observed at the early stage after chondrocyte sheets implantation,and the percentage of live cells was maintained at more than 90%.CCK-8 assay showed that the overall activity of chondrocyte sheets was good,with no significant cell death or loss.The results of tissue section staining and related quantitative assays showed that the secretion and deposition of cartilage extracellular matrix inside the sheets gradually increased with the extension of the in vitro culture time,which could form a natural cavity-like cartilage lacuna structure,and the thickness and wet weight of chondrocyte membrane gradually increased.The 10fold density group could show a relevant advantage relative to the 5-fold density group,while the 20-fold density group had no significant advantage relative to the 10-fold density group and could show delamination during the culture process,which was not conducive to the construction of tubular cartilage structures.The results of PCR suggested that the three-dimensional culture mode within the chondrocyte sheets could be highly expressed in the ACAN,ELN and COL2 genes,which were positively associated with extracellular matrix secretion,while the COL 10 gene,which was negatively associated with chondrogenesis,was significantly down-regulated by 2 weeks of culture compared with the normal two-dimensional flat culture.Conclusion:Primary chondrocytes extracted from the autologous auricular cartilage of experimental animals were cultured and expanded in vitro,and P2generation cells could be used to construct chondrocyte sheets.During the in vitro culture,no significant cell death or loss was observed within the chondrocyte sheets,and the metabolic activity was good.By comprehensively assessing the thickness,wet weight and matrix generation of chondrocyte membrane sheets in different density groups,the 10-fold density was used for chondrocyte sheets in the subsequent study.In addition,the three-dimensional culture pattern of chondrocytes inside the sheets could promote the cells to highly express ACAN,ELN and COL2 genes and down-regulate COL1,COL 10 gene,thus promoting the secretion and deposition of cartilage extracellular matrix.ABSTRACT ⅡConstruction of airway epithelial cell membrane sheets and the study of their differentiation potentialObjective:In this part of the study,the efficiency differences of different extraction methods of primary epithelial cells were explored and compared.In vitro experiments were conducted to investigate the specific phenotypic maintenance of epithelial cell patches under different seed cell densities and proliferation algebraic conditions.To explore the differentiation potential of epithelial cell sheet by creating gas-liquid plane culture conditions.At the same time,the technical details of obtaining the epithelial cell sheet completely through temperature-sensitive culture technology were explored,and the growth state of the epithelial cell membrane with the cartilage cell membrane as the base layer was explored with the help of in vitro co-culture model,so as to verify the feasibility of the application of the epithelial cell sheet in tissue engineering endotracheal wall functionalization.Materials and methods:Primary airway epithelial cells were extracted from the airways of experimental rabbits by the clamp method to compare the extraction efficiency of epithelial cells under the condition of preserving the tissue fragments of the airway lining or not.The cell densities of epithelial cells in the culture flasks were calculated as 100%,which was about 4*105/cm2.For reference,four groups of epithelial cell sheet with different initial densities were grown in a gradient of 6%,12%,25%and 50%to observe the growth status of epithelial cells,the rate of cell colony formation and fusion,and the maintenance of the characteristic phenotype of epithelial cells.The epithelial cell sheets were grown in Transwell inner chambers to create airliquid plane culture conditions to observe the differentiation potential of airway epithelial cells.A co-culture model with a composite structure of chondrocyte sheet and epithelial cell sheet was constructed to observe the changes of epithelial cell growth status during the co-culture process.Results:Primary epithelial cells could be successfully extracted from the airway of rabbits by the clamp method,and the preserve of the tissue fragments of the airway lining could effectively improve the extraction efficiency of epithelial cells.During the in vitro culture,epithelial cells proliferated and formed cell colonies,which grew and fused with each other to form layers within 7-9 days.In vitro culture up to 2 weeks,the immunofluorescence staining results indicated that the low-density group only locally low-expressed the epithelial-specific marker CK-14,while the 50%high-density implantation group could stably high-express CK-14,which showed that the highdensity implantation method could effectively avoid the process of epithelial mesenchymal transition during proliferation and facilitate the formation of epithelial cell sheets with complete function and differentiation potential.Transferring the temperature-sensitive culture dishes with epithelial cell sheets to 20℃ for 30 minutes allowed the epithelial cell sheets to separate from the bottom of the dishes.Immunofluorescence showed high expression of the epithelial-specific markers CK-14 and Pan-CK,and the molecular characterization of epithelial cells was well maintained in epithelial cell sheets.The epithelial cell sheets were intact and continuous,and the barrier function of the epithelial structure was preserved.When the cells were cultured for 4 weeks,under air-liquid plane culture conditions.Through scanning electron micrograph,epithelial cells were arranged regularly and the tight junctions between cells were well maintained,and the cilia could be seen in clusters at the cell tips.Immunofluorescence staining revealed that about half of the cells highly expressed the ciliated cell-specific marker β-Tubulin,while some cells also expressed the specific marker MUC5AC,which is associated with mucus secretion function in cupula cells.The chondrocyte sheets and epithelial cell sheets co-culture model showed that using chondrocyte membrane as a substrate layer could promote relatively high expression of the specific marker CK-14 in epithelial cells compared to simple epithelial cell cultivation.Conclusion:Primary airway epithelial cells can be extracted from autologous tissues of experimental animals and cultured and expanded in vitro.With the help of temperature-sensitive culture technology,selecting epithelial cells in early generation for high-density cultivation can more effectively maintain the phenotype and differentiation potential of epithelial cells inside the epithelial cell sheets.The epithelial cell sheets cultured by the air-liquid plane method could gradually differentiate into pseudo-complex structures in vitro,and were functioned with mucus secretion and cilia oscillation,and cilia sprouted from the cell tip under scanning electron microscopy.This chapter verifies that epithelial cell sheets are a feasible solution to provide inner wall function for tissue-engineered trachea,and are expected to increase the functions of defense and mucus of tissue-engineered trachea after transplantation in vivo.ABSTRACT ⅢCombined tubular cartilage structure and epithelial cell sheet to construct composite bionic tissue engineering tracheaObjective:In this part of the study,the feasibility of constructing tubular cartilage structures with the help of chondrocyte sheets was investigated.Meanwhile,the fusion ability and matrix regeneration of multilayered chondrocyte sheets inside the tubular cartilage structure during in vitro and in vivo culture were analyzed,and the changes of their mechanical properties were explored to evaluate the rationality of using them as support structures in tissue-engineered trachea.After that,the feasibility of constructing a bionic tissue-engineered trachea with a composite structure by transferring airway epithelial cell sheets to the inner wall of a tubular cartilage structure was investigated.Materials and methods:In order to meet the requirements for the construction of tissue-engineered trachea,large-size chondrocyte sheets were constructed at 10 times the initial cell density according to the results of the previous study.After 2 weeks of culture,the chondrocyte sheets were laminated,clipped,convoluted,and shaped into tubular cartilage structures,and were placed in a special device for 4 weeks of in vitro culture.Tubular cartilage structures were then surgically placed under the skin of nude mice and cultured in vivo for 2 weeks.During these cultures,tissue sections were stained to assess the internal fusion and matrix application of the tubular cartilage structures,and the stress-strain curves were plotted by mechanical properties tests to assess the resistance of the tubular cartilage to compressive loading.Combined with temperature-sensitive culture techniques and sticker tools,epithelial cell sheets were transferred to the inner wall of the tubular cartilage structure,placed in a sealed-bottom well plate and cultured vertically,and the growth of epithelial cells was observed by immunofluorescent staining after 1 week.Results:Large-sized chondrocyte sheets with manipulable ability could be successfully constructed,and the tubular cartilage structure could be constructed after the reprocessing process.After 4 weeks of in vitro chondrogenic differentiation culture,it could become morphologically stable tubular cartilage structure.In general,the tubular cartilage structure was white with slight transparency,elastic,and could recover its shape after squeezing the lumen to maintain the lumen patency.With the secretion and deposition of cartilage matrix,the chondrocyte sheets could fuse with each other and become a whole.In particular,under polarized light microscopy after Sirius red staining,the extracellular matrix of the tubular cartilage was rich in collagen fibers and interlaced,constituting a natural lattice-like fiber arrangement without fiber delamination or gap formation.After implantation of the tubular cartilage structure under the skin of nude mice for 2 weeks.Removal of the graft and the white tubular cartilage structure was well integrated with the surrounding tissues of nude mice origin and wrapped by vascular-rich connective tissue.Staining of the tissue sections revealed a smooth inner border of the tubular cartilage structure,with the outer area in direct contact with the host,and a mild infiltration of the nude mouse’s own tissue.Inside the tubular cartilage structure,dense rows of chondrocytes were seen to further develop and mature,with increased cell volume,widened cartilage lacuna,increased secretion of extracellular matrix,and further fusion of the multilayered chondrocyte sheets into a monolithic structure.The mechanical properties of the tubular cartilage structure cultured in vitro for 4 weeks were found to be enhanced after 2 weeks of implantation in nude mice,and the mechanical properties were found to be similar to those of natural trachea in terms of compression resistance.The epithelial cell membrane was successfully transferred to the inner wall of the tubular cartilage with the aid of a sticker tool,and airway epithelial cells with high expression of the specific marker CK-14 were visualized by immunofluorescence after 1 week of in vitro culture.Conclusion:After the reprocessing process,the flattened chondrocyte sheets constructed in the previous section can be upgraded to a three-dimensional tubular structure with spatial conformation.After 4 weeks of in vitro culture,the tubular cartilage structure was stable,the internal cartilage extracellular matrix was well secreted,and the multiple layers of superimposed chondrocyte sheets were fused with each other.When the tubular cartilage structure was implanted subcutaneously in nude mice,it was found that the tissue-engineered cartilage could further develop and mature into a natural cartilage-like structure,and could exhibit a stress-strain pattern that resisted lateral compression similar to that of the natural one.With the help of a special sticker tool,epithelial cell sheets can be transferred to the inner wall of the tubular cartilage structure to construct a composite tissue-engineered trachea.ABSTRACT ⅣIn vivo pre-vascularization of composite bionic tissue-engineered trachea and evaluation of the effect after orthotopic transplantationObjective:In this part of the study,the vascularization elements were perfected for tissue-engineered trachea by means of autologous pre-vascularization to create conditions for the survival of cartilage and epithelial components after transplantation and the in vivo function of the grafts.After that,a long-segment tracheal defect animal model was constructed,and the tissue-engineered trachea with all three elements of cartilage,epithelium and vascularization was transplanted in situ to observe the respiratory movements and survival of the experimental animals after transplantation.Materials and methods:After the rabbits were anesthetized,the skin was incised longitudinally about 6 cm directly above the trachea.Ophthalmic scissors were used to separate the subcutaneous,fascial and muscular tissues layer by layer until the trachea was exposed.The loose connective tissue adjacent to the trachea was separated,the sternomastoid muscle gap was found,and the gap was bluntly separated and enlarged,and the tissue-engineered trachea was placed in the muscle and tracheal gap,followed by layer-by-layer suturing of the muscle and fascia,and closure of the incision.After 2 weeks of pre-vascularization,the incision was reopened and the tissue-engineered trachea was dissociated.According to the length of the tissue-engineered trachea,the native trachea was detached and cut to construct a long-segment tracheal defect model.The upper anastomosis was firstly sutured,followed by the same method of anastomosis of the lower incision.Samples were taken 4 weeks after in situ transplantation,and cartilage and epithelial regeneration within the tissue-engineered trachea was assessed by conventional staining,immunofluorescence,and scanning electron microscopy.Results:After 2 weeks of in vivo pre-vascularization,the tissue-engineered trachea was seen to be wrapped by vascular-rich connective tissue of the recipient’s own origin,suggesting a good vascularization effect.At 4 weeks after orthotopic transplantation,the surface of the tissue-engineered trachea was seen to be covered with hyperplastic fibrous tissue of recipient origin and thickened by local wrapping,resulting in a thickening of the overall tracheal morphology within the surgical interval,within which the anastomotic surgical suture was visible.When the tissue-engineered trachea was cut along the membrane cloth,it was seen that the lumen was clear and the inner wall was smooth,with a small amount of connective tissue covering it,and no obvious inflammatory exudation or granulation tissue proliferation was seen.The staining of the tissue sections showed that continuous strips of cartilage structures were visible in the tissue,and there were obvious cartilage lacuna structures around the chondrocytes,suggesting that the chondrocytes were developing and depositing extracellular matrix components of cartilage,and the Alcian blue staining was obviously positive.Hematoxylin eosin staining suggested that the epithelial layer of the airway could be observed in the inner wall near the anastomosis of the tissue-engineered trachea and the native trachea,and the morphology was similar to that of the epithelial structure of the natural ones.Epithelial-like structures could be observed in the middle position of the tissue-engineered trachea,and immunofluorescence staining suggested that the epithelial cell-specific marker CK-14 was positive,and pseudo-complex columnar epithelial cells at different stages of initial maturation were observed under scanning electron microscopy,with obvious ciliated structures at the top of some cells.Conclusion:To improve the survival and functional performance of tissueengineered trachea after orthotopic transplantation,tissue-engineered trachea can be implanted into the paratracheal muscle space of experimental rabbits with good prevascularization effect after 2 weeks.By constructing a long-segment tracheal defect model,after orthotopic transplantation of tissue-engineered trachea,the experimental animals survived well,and cartilage development and extracellular matrix deposition could be seen in the tissue-engineered trachea,and the airway epithelium could be formed and cilia-like differentiated to play local immune barrier and defense functions.