Studies Of Outcomes And Immunoreaction Of Engineered Cartilage Tissue In Large Animals

Background:Repair and reconstruction of auricle,nose and upper eyelid defects are essential tasks in plastic surgery practice.In order to maintain a satisfactory and durable morphology post operation,artificial protheses or autologous cartilage are frequently applied as support structures.However,commonly used protheses have the risk of rejection,infection and even extrusion,while autologous cartilage may lead to extra incisions,scarring and even malformation of the rib cage.Tissue engineering,constructing cartilage-like tissue with a small amount of chondrocytes,emerges as an alternative solution to the problem.Nevertheless,tissue-engineered cartilage,which could be successfully constructed in vitro and in immunodeficient animals such as nude mice,would evitably result in Foreign body reaction(FBR)and construct failure after implanted subcutaneously in immunocompetent mammals such as dogs,sheep and pigs.The unexpected FBR,hinders the development and wide application of cartilage tissue engineering.Therefore,it is of significance to figure out the FBR related physiological processes and participants,which may provide clues for better tissue construction in further researches.Although there have been a number of researches concerning FBR,most of them were based on unabsorbable devices like artificial joint prostheses and cardiac pacemaker.While engineered cartilage,or cell-laden scaffolds,are a mixure of materials,chondrocytes,deposited ECM and exogenous active proteins in serum.Each component of the mixure may contribute to FBS more or less and have different effects on its development.On the other hand,previous researches about FBR in immunocompetent animals were mostly conducted in mice or rats,of which immune systems are quite different from human compare to larger animals like pigs and sheep.In this research,the influence of scaffold materials and seeding cells on the FBR and inflammatory microenvironment of subcutaneously transplanted implants in pig models are to be exploredObjectives:1.To evaluate whether SGS and SGS-Mps scaffolds are suitable for cartilage tissue engineering.2.To explore the contribution of natural derived and synthetic materials to FBR development and their influence on the secretion of cytokines in local inflammatory microenvironment.3.To explore the effects of seeding cells and ECM on the development and outcomes of FBR,as well as the release of cytokines in local inflammatory microenvironment.Methods and Results:1.The evaluation of SGS and SGS-Mps as engineering scaffolds in vitro and in vivo of nude mice:Methods:Physical properties of both scaffolds like microstructure,porosity and compression modulus were estimated via scanning electron microscope,fluid replacement method and universal material testing instrument,respectively.Porcine auricular chondrocytes were harvested,proliferated to P2 and seeded on both scaffolds.Live/dead staining and CCK-8 assays were conducted to detect cell viability and growth on scaffolds during the first 21 days post seeding while quantative measurement of DNA,GAG and total collagen content,as well as relative expression of chondrogenesis related genes were committed after in vitro cultivation for 28 days.Cell-laden SGS and SGS-Mps scaffolds were then implanted subcutaneously in immunodeficiency nude mice.After cultivation in vivo for another 28 days and 84 days,implants were explanted.Appearance and histological changes were observed by HE staining and ECM staining including toluidine blue and type ?&? collagen IHC staining to evaluation chondrogenesis in vivo.Results:Both SGS and SGS-Mps scaffolds are equipped with a highly interconnected porous structure and porosity around 80%,while the mechanical strength of SGS-Mps scaffolds were in the range of natural cartilage.SGS and SGS-Mps scaffolds show a similar capability in promoting chondrocytes adhesion,proliferation and secretion of ECM during 28 days of in vitro cultivation but the advantage of SGS-Mps scaffolds over SGS ones emerges after subcutaneous implantation and became even more remarkable as time processes.2.The effects of scaffold materials on outcomes and local cytokine expression of implants:2.1 The detection of different outcomes between cell-landen scaffolds and cell sheet post subcutaneous implantation in immunocompetent pigs:Methods:Cell-laden SGS and SGS-Mps scaffolds,accompanied with cartilage cell sheets,were implanted subcutaneously in pigs from which seeding chondrocytes had been harvested,after a 28-days in vitro cultivation.Implants were then explanted at intervals from 3 days to 84 days post implantaion.Except for appearance observation,histological analyses including HE staining,toluidine blue staining and IHC staining of type ? and ? collagen were conducted to detect the development of FBR and outcomes of explants.Relative expression of chondrogenesis related genes were also tested via qPCR assays to help evaluating ECM and material hydrolysis during FBR.Furthermore,CD68/CD86 and CD68/CD206 immunofluorescence staining was applied to detect M1 and M2 polarized macrophage infiltration,respectively.Results:A classical FBR process of leukocytes infiltration,material hydrolysis and fibrous envelope formation was observed in both cell-landen scaffold groups within 21 days post implantation.The hydrolysis of ECM and material occurred during the first 14 days.In contrary,implanted cell sheets only cause mild immune reactions and ended up with mature cartilage tissue after in vivo cultivation for 84 days.Besides,SGS and SGS-Mps scaffolds differed in the FBR development tendency and long-term outcomes the former shared a drastic and rapidly developing FBR process within the first 14 days and caused large area of tissue necrosis in the long run while the latter experienced much more steady and alleviated inflammation reaction and mild hydrolysis of material until 21 days post implantation.As for macrophage phenotype,SGS scaffolds demonstrated a balanced and synchronously varying population of M1 and M2 subtypes,while SGS-Mps ones presented similar trend in the beginning but turned to a M2 polarization and anti-inflammation dominated phenotype in the later stage.2.2 The influence of scaffold materials on the release of cytokines in implant local microenvironment:Methods:Cell-laden SGS and SGS-Mps scaffolds,accompanied with cartilage cell sheets,were implanted subcutaneously in pigs from which seeding chondrocytes had been harvested,after a 28-days in vitro cultivation.Implants were then explanted 7 days and 14 days post implantation.Expression of cytokines were measured in a relatively quantative way via protein arrays among implants from three groups,and the average expression of each cytokine in three groups was compared.Cytokines that expressed significantly differently among three groups were selected and noted.The ELISA assays with larger sample amount of three selected cytokines and one other cytokines were conducted to verify the screening results.The differently expressing cytokines were then underwent an enrichment analysis based on GO and KEGG database,to figure out their involvement in biological process and diseases.Results:A group of cytokines,including IL-1 family members like IL-1a,IL-1ra,IL-18 as well as chemokines like IL-8,MIP-la and MIP-lb showed significantly higher expression in the cell-laden scaffolds compared to cell sheets,indicating their involvement in the scaffold material related FBR.Cytokine expression differences between the two cell-laden groups proved to be much less remarkable than the ones between cell sheets and each cell-laden group.The primary difference laid in the expression of IL-1 and IL-8,presenting a SGS-Mps advantage,indicating a correlation between PLLA and persist expression of these two inflammatory cytokines.In previous literature,above mentioned cytokines were frequently reported to be involved in the development of rheumatoid arthritis(RA),which was characterized with cartilage defects and infiltration of neutrophils and macrophages.Thus,treatment and medicine of RA target in antagonizing or blocking the above mentioned cytokines can provide clues for the blocking of FBR.3.The effects of seeding cells and deposited ECM on the outcomes and local cytokine expression of implants:Methods:Cell-laden SGS/SGS-Mps scaffolds were implanted with corresponding cell-free scaffolds subcutaneously in pigs from which seeding chondrocytes had been harvested.After cultivation in vivo for another 3-84 days,implants were explanted.Appearance changes were observed and histological analysis by HE staining and CD68/CD86 and CD68/CD206 immunofluorescence staining were conducted.Differently expression cytokines were screened and verified via protein arrays and ELISA among cell-laden,cell-free scaffolds and cell sheets.Results:Cell-free SGS and SGS-Mps scaffolds both showed remarkably higher expression of IL-1a,IL-8,MIP-1a and MIP-1b compared to cell sheets,which were similar to the results of the corresponding cell-laden scaffolds.Meanwhile,there was little difference in the expression of the above cytokines between cell-free scaffolds and their cell-laden counterparts.Therefore,it can be speculated that scaffold materials are the main inducer of FBR for subcutaneously implanted cell-laden scaffolds.Furthermore,cell-laden scaffolds presented with more intensive leukocytes infiltration and earlier scaffolds collapse compared to their cell-free counterparts,while they also shared a more M2 polarization tendency and better outcomes in the long run.In addition,leukocytes infiltration and scaffolds collapse were observed less productive in SGS-Mps scaffolds implants,regardless of cell-laden and cell-free one's,indicating the potential ability of PLLA microspheres in hindering leukocytes infiltration.Conclusions:1.The evaluation of SGS and SGS-Mps as engineering scaffolds in vitro and in vivo of nude mice:Both scaffolds proved to be efficient and appropriate as scaffolds for cartilage tissue engineering,and SGS-Mps ones performed even better in the long run.2.1 The detection of different outcomes between cell-landen scaffolds and cell sheet post subcutaneous implantation in immunocompetent pigs:Cell-laden SGS and SGS-Mps scaffolds both showed remarkable FBR within 21 days after implantation while deposited ECM and materials were phagocytosed and hydrolyzed within 14 days post implantation.SGS-Mps underwent milder FBR than SGS ones,with less infiltration density of macrophages,significant M2 polarization in the later stage of inflammation,and less long-term tissue necrosis.Scaffolds-free cell sheets result in less intensive FBR and mature cartilage tissue formation.2.2 The influence of scaffold materials on the cytokine release in implant local microenvironment:IL-1 family members including IL-1a,IL-1ra and IL-18,as well as inflammatory chemokines including IL-8,MIP-1a and MIP-1b are correlated with material induced FBR.PLLA microspheres are related to the continuous and high level expression of IL-1 and IL-8.Subcutaneously implanted cell-laden scaffolds shared similar involved cytokines and leukocytes with RA microenvironment,thus RA treatments can provide clues to FBR blockage.4.The effects of seeding cells and deposited ECM on outcomes and local cytokine expression of implants:Scaffold materials are primary inducer of cell-laden scaffolds FBR.Seeding cells motivate leukocytes infiltration and scaffolds collapse but also promote M2 polarization and improve long-run outcomes.PLLA microspheres,embedded in porous structure can hinder leukocytes infiltration.