Experimental Study On The Construction Of Perichondrium-Wrapped Tissue Engineered Cartilage

BackgroundCongenital microtia is a common disease in plastic surgery,especially among Asians.At present,the best way to solve the congenital microtia is the reconstruction surgery of the auricle with autologous costal cartilage transplantation.However,there are still many drawbacks with this surgery,such as the deformity of the thorax at the donor site of the costal cartilage,the unsatisfactory outlook of the reconstructed auricle,etc.The development of tissue engineering has offered a new way for cartilage transplantation.The use of cartilage cells in vitro to produce cartilage framework with an auricle-shape has long been achieved,while the mechanical strength and flexibility of tissue engineering auricles are still incomparable than those of natural auricles.The mobility and toughness of natural ear cartilage are largely related to the existence of the perichondrium.Nevertheless,to which extent the perichondrium contributes to the mechanical strength of the auricular cartilage has not been confirmed by any research so far.If a layer of perichondrium-like structure can be attached to the surface of tissue engineered cartilage,the above-mentioned problems may be solved once for all.At present,there are few research on artificially making perichondrium.The only research so far islimited to swelling simulation with chemical polymer materials or using animal lyophilized perichondrium for attachment,etc.If the method of tissue engineering can be used to construct perichondrium and realize the integrated production of engineered perichondrial attached cartilage,then there will be a profound impact on the development and application of cartilage tissue engineering.Objective1.To investigate the changes in the mechanical properties of natural auricular cartilage with or without perichondrium attachment,and to study the influence of perichondrium on the biomechanical properties of auricular cartilage,and provide follow-up experiments for this topic and future research on the mechanics of auricular cartilage theoretical basis;2.Try to use different tissue engineering methods to construct tissue engineered cartilage with composite perichondrium,analyze the differences in histology,molecular biology,and biomechanics of cartilage constructed by various methods,and explore the possibility and research value of constructing composite cartilage;3.Use single-cell transcriptome sequencing technology to perform cell clustering and subpopulation analysis of perichondrial cells,explore the cell composition in the perichondrium,clarify the existence of stem/progenitor cells in the perichondrium,and study its gene expression and application potential.Method1.Research on the influence of perichondrium on the mechanics of auricular cartilageThe rabbit auricle cartilage was taken to make mechanical specimens,the experimental group retained the perichondrium,and the control group was stripped of the perichondrium.Use the Instron 5967 universal material testing machine to perform tensile and compressive stress-strain tests,tensile and compressive stress relaxation tests,and tensile creep tests on the two groups of cartilage,analyze the test data and compare the hardness and adhesion of the two groups of cartilage materials.Differences in mechanical characteristics such as elasticity.2.Construction of tissue engineered cartilage with composite perichondrium2.1.Using cell sheet technology to construct tissue engineered cartilage with composite perichondriumObtain and culture porcine auricular chondrocytes and perichondrial cells,subculture in vitro to the P2 generation,and then induce and prepare chondrocyte cell sheet and perichondrial cell sheet respectively.After the cell sheet was cultured and matured,the control group folded the chondrocyte sheet into a 1 cm × 1 cm square cartilage block.On this basis,the experimental group wrapped 2 layers of perichondrial cell sheet on the upper and lower sides of the cartilage block.The two groups of constructs were incubated and matured in vitro and then transplanted into nude mice under the skin.After 4,8,and 12 weeks,the samples were taken for gross and histological,related gene expression,and biomechanical testing.2.2.Using 3D bioprinting technology to construct tissue engineered cartilage with composite perichondriumObtain and culture porcine auricular chondrocytes and perichondrial cells,subculture to P2 generation in vitro,mix the cells with hydrogel materials to configure bioprinting ink.The experimental group used a 3D-Bioplotter bioprinter to print a three-layer composite structure of "perichondrium-cartilage-perichondrium",in which the perichondrium layer was printed with chondrocytes and 10%GelMA configuration ink,and the cartilage layer was printed with perichondrial cells and 10%GelMA+1%HAMA is configured with ink printing;the control group only prints the cartilage layer with the same ink ratio.After the two groups of constructs were printed,they were incubated overnight in cell culture medium,and then transplanted into nude mice subcutaneously the next day.After 4,8,12 weeks,they were taken for gross and histological,related gene expression,and biomechanical testing.3.Subpopulation analysis of perichondrial cells based on single-cell transcriptome sequencing technologyObtain porcine auricular perichondrial cells,prepare cell suspensions,detect the quality of perichondrial cell samples,perform single-cell transcriptome sequencing on the samples,perform bioinformatics analysis on the sequencing data,Cluster the cells into groups,and analyze each subgroup.GO enrichment and KEGG enrichment analysis of the top 50 high-expressed genes of the group are performed to determine the main function of each subgroup,determine the subgroup of cartilage stem/progenitor cells according to the function,and find out the key genes according to the database gene information.Result1.The mechanical properties of rabbit auricular cartilage will be significantly decreased in all aspects after the perichondrium is removed.In the tensile test,the Young's modulus of the cartilage decreased by about 23%after the perichondrium was removed.In the compression test,the Young's modulus of the cartilage decreased by about 37%after the perichondrium was removed.In various biomechanical tests such as stress-strain,stress relaxation,and creep,the cartilage of the membrane group is superior to the cartilage of the non-membrane group in terms of mechanical properties.2.Both cell sheet technology and 3D bio-printing technology can construct a composite tissue engineered cartilage of "perichondrium-cartilage-perichondrium",but the constructed perichondrium and natural perichondrium are significantly different in general and histology.In terms of biomechanics,there was no statistical difference between cartilage constructed with perichondrium group and cartilage without perichondrium group.However,the cartilage constructs of the perichondrium group constructed by the two methods are superior to the cartilage constructs of the non-perichondrium group in the degree of cartilage formation,and the construction of perichondrium significantly promotes the formation of cartilage.3.After sequencing and analysis of perichondrial cells using single-cell transcriptome sequencing technology,we found that perichondrial cells are mainly mesenchymal stem cells.This study divided the perichondrial cells into 7 subgroups.Among them,subgroup 6 accounts for 5%of the total number of cells.According to the results of GO enrichment and KEGG enrichment,the functions of subgroup 6 such as cartilage development,collagen fiber tissue management,proteoglycan biosynthesis process,cartilage aggregation,etc.are closely related to cartilage tissue formation.Related,this group specifically expresses cartilage formation-related genes COMP,ACAN,ELN,ECM2,etc.,and is highly consistent with cartilage stem/progenitor cells in terms of function and gene expression.ConclusionThe perichondrium plays an important role in maintaining the toughness,elasticity and overall mechanical strength of the auricular cartilage.The construction of tissue-engineered cartilage with composite perichondrium by tissue engineering methods is of great significance.After obtaining a sufficient amount of perichondrial cells,cell sheet technology and 3D bioprinting technology can be used to construct tissue engineered cartilage with composite perichondrium.The composite cartilage construct is significantly better than simple cartilage in terms of cartilage formation performance.Through single-cell transcriptome sequencing of perichondrial cells,the complexity of perichondrial cell components can be found,and a group of cartilage stem/progenitor cells can be found.The development and formation of cartilage has an obvious promotion effect.However,the perichondrium constructed based on the current level in this study has a large gap with the natural perichondrium in morphology and histology and cannot have a beneficial effect on cartilage similar to the natural perichondrium in terms of mechanical properties.