Squid Derived Decellularized Cartilage Matrix

BACKGROUND:Cartilage defect is a common disease in orthopedics,mostly caused by trauma and articular cartilage degeneration.With the serious population aging,the incidence of cartilage defect increased significantly in recent decades.The limited repair capability of defected articular cartilage could develop to osteoarthritis,which seriously affects bone joint functional movements and the quality of life.The treatment methods for defected cartilage include microfracture,drilling and autologous chondrocyte implantation（ACI）,whereas these procedures lead to several postoperative complications.To date,there is still no ideal treatment for cartilage defect.Tissue engineering is a promising technique for cartilage defect repair.Scaffold for cartilage tissue engineering is a key to the success of cartilage defect repair.Although there are numerous natural and synthetic scaffolds have been introduced for cartilage tissue engineering,it has been suggested that the ideal scaffold for cartilage tissue engineering should mimic the natural constitutions and components of cartilage extracellular matrix to provide a favorable natural microenvironmental network for cell growth.The higher the similarity of the scaffold constitutions and components to the cartilage extracellular matrix,the more favorable it is for cartilage tissue regeneration.Therefore,this study focused on the application of natural cartilage extracellular matrix as scaffold for cartilage tissue engineering.At present,Matrix-induced Autologous Chondrocyte Implantation（MACI）,the third generation of ACI,is the most well-known method for cartilage tissue engineering by using biocompatible type I or type III collagen scaffolds as a carrier of autologous chondrocytes implantation to repair cartilage defect.Many studies have proved the repair efficacy of MACI is greater than ACI,but there are some reported about the disadvantages of MACI procedure,such as the need for secondary surgical implantation and the failure of grafting.In addition,type II collagen is dominant in hyaline cartilage,whereas type I and type III collagen scaffolds applied to MACI are not natural components of cartilage matrix,leading to the difficulty of ideal repair of hyaline cartilage.In recent years,researchers have attempted to use hyaline cartilage matrix as cartilage tissue engineering scaffold to repair cartilage defects.To the best of our knowledge,previous described decellularized cartilage scaffolds were derived from the cartilage of terrestrial animals（such as chicken cartilage,porcine cartilage,etc）.It is hard to preserve an intact structure of such scaffolds derived from terrestrial animals due to the dense and compact constitution,and the components of extracellular matrix cannot be easily retained after decellularization.Moreover,the risk of virus transmission of terrestrial animal cartilage is high,and its clinical application faces higher biological risk challenges.Some studies reported the decellularization methods by sliced or pulverized to reform a spongy-like scaffold to remove antigenicity in such cartilages.However,these ECM scaffolds comprising decellularized cartilage lose their natural microstructure and biomechanics in such preparations.Marine-derived materials are recognized as natural abundant materials with high biosafety,and good biocompatibility.Some of marine-derived materials have been widely used in clinical application,including chitosan and its derivatives.Squid cranial cartilage extracellular matrix has been described in several studies that its composition is similar to that of human hyaline cartilage matrix.In addition,squids inhabit an aquatic environment and experience less gravitational effects;therefore,the cartilage structure is obviously different from that of terrestrial animal cartilage.Squid cranial cartilage could be successfully decellularized using mild manner and retain original microstructures of extracellular matrix providing a noval decellularized cartilage matrix scaffold for use in cartilage tissue engineering.OBJECTIVE:In this study,squid cranial cartilage is used to produce decellularized squid cranial cartilage scaffold（DSCS）using mild manner of decellularization technique.We firstly characterized DSCS characteristics.Secondly,the biocompatibility of DSCS was evaluated via the effect of DSCS extracts on cell viability,and the cell-ladden scaffold was constructed to observe cell viability,adhesion,proliferation,and morphology.Finally,the efficacy of DSCS in repairing full-thickness knee cartilage defect was observed in the rabbit model.METHODS:（1）Comparing histological differences between fresh squid cartilage and human hyaline cartilage using histological staining;（2）Preparation of squid cartilage decellularized scaffold:After removing adherent soft tissues,deionized water was used to clean the cartilage tissue several times at 4°C.The cartilage tissue was cut to a thickness of 3 mm,and fats and other tissues were rinsed off with normal saline solution.The cartilage pieces were placed in a simulated body fluid（SBF）solution for 4 h to maintain their microstructure.The combination of 0.02%EDTA and 0.05μg/ml trypsin solution was applied for 4 h followed by three freeze-thaw cycles to complete the decellularization process.After removing the excess solution,deionized water was used to clean and remove cellular debris.The samples were placed in SBF solution for another 2 h followed by freeze-drying to obtain the DSCS;（3）Evaluation of DSCS characteristics:gross morphology,micro-CT scanning,atomic force microscopy（AFM）and scanning electron microscopy（SEM）were assessed;histological and immunohistochemial staining was performed using Hematoxylin&Eosin staining（HE）,Saffranin o-fast green staining（SO）,Toluidine Blue staining（TB）,Alcain Blue staining（AB）,type II collagen immunohistochemical staining,and DAPI fluorescence staining;Young’s modulus of DSCS was measured by biomechanical properties;water contact angle（WCA）was used to observe the hydrophilicity of the DSCS;in vitro degradation rate of DSCS was determined by phosphate buffer saline（PBS）immersion method at different time points of 1,2 and 4w;（4）Biocompatibility evaluation of DSCS extracts on cells:DSCS extracts solution was prepared according to the national standard（GB/T16886）method to evaluate its effect on rabbit articular chondrocytes and mouse preosteoblast（MC3T3-E1）proliferation and migration.Chondrocytes and MC3T3-E1 were cultured in vitro using different extract concentrations.Cells viability were evaluated using cell count kit（cck-8）at day 1,3,7 and 14.Meanwhile,the effect of extracts on chondrocyte migration was observed using scratch assay and Transwell assay;（5）Chondrocytes were seeded into the DSCS to construct cell-laden scaffold:after one week of incubation at 37℃5%CO₂,chondrocyte adhesion,viability,and morphology were observed using SEM;after incubation for 1,2,3 w,cell viability,proliferation and morphology on the DSCS were observed using fluorescence microscope and confocal microscope using live/dead staining and cytoskeleton staining,respectively;（6）In vivo animal experiments:a total of 24 New Zealand rabbits with full-thickness knee cartilage defect models were established and randomly divided into four groups:untreated control group（Ctrl）,DSCS-treated group（DSCS）,DSCS/chondrocyte-construct group（DSCS/cell）,and type I collagen scaffold-treated group（Col-I）.The experimental animals were euthanized and sacrificed at 6 and 12weeks after the initial operation.The gross morphology of cartilage defect repair of rabbit knee joint in all groups was observed and scored.The specimens were then sectioned for histological and immunohistochemical staining to observe the repair efficacy of regenerated tissue and score.RESULTS:（1）Fresh squid cranial cartilage is translucent,tough,elastic,and similar to that of human hyaline cartilage tissue;（2）The DSCS was white,with a relatively smooth and dense surface.Micro-CT scanning revealed non-uniformity in density with small cavities within the scaffold,while high density areas were dense cartilage matrix and low density areas were relatively loose cartilage matrix.SEM exhibited that the superficial surface of the scaffold was smooth and compact,whereas numerous interconnected porous structures were observed in the cross-sectional surface.Such a structure is conducive to cell growth,adhesion,migration,and the delivery of nutrients and metabolic wastes.The AFM results revealed that the roughness values（Rq）of the superficial and cross-sectional surfaces were 115 and129 nm,respectively.HE staining and DAPI fluorescence staining showed that the DSCS had no cell residue,and possessed interconnected porous inner structure of chondrocyte lacunae and numerous tiny porous structures within cartilage extracellular matrix.The positive staining of SO,TB,and AB indicated that,using mild manner of decellularization the scaffold was rich in glycosaminoglycan.Immunohistochemical staining of type II collagen was positive,indicating the scaffold conformed to hyaline cartilage matrix.Young’s modulus of DSCS scaffolds was 23.84±0.22 MPa,reflecting DSCS scaffolds had excellent biomechanical properties.The WCA was 71.98±6.44°,indicating that the scaffold had good hydrophilicity.In vitro degradation results showed that the percentage of DSCS degradation at observation time points of 1,2 and 4w were 37.64%,52.69%and70.24%,respectively;（3）Different DSCS extracts on MC3T3-E1 and chondrocyte viability were assessed using the CCK-8 assay on day 1,3,7 and 14.We observed the naturally derived scaffold extracts were not toxic to either cell type,revealing that the scaffold behaved favorable biocompatibility.In addition,the effect of DSCS extracts on chondrocyte migration using scratch and Transwell assays in vitro indicated that DSCS extracts exerted promotive effect on chondrocyte migration;（4）SEM scanning results of the cell-scaffold construct showed good cell viability and adhesion.The chondrocytes appeared spherical with a certain amount of cartilage matrix secreted.After incubated for 7,14 and 21 days,the number of cells increased significantly and showed a dispersed distribution on DSCS scaffold.No dead cells were observed at day 7,while few cells stained red at day 14 and 21 by live/dead staining.The co-staining of cytoskeleton and nucleus revealed the number of cells was significantly increased with elliptic spreading morphology by cytoskeletal staining observed under a confocal microscopy;（5）Six weeks after initial operation,the gross morphology of cartilage defect repair in each treatment groups showed that in the Ctrl group the defect sites were poor repair with sunken and rough surface,and poor integration with surrounding cartilage tissue;Col-I group was better repaired than that of Ctrl group with a sunken but smoother surface,and poor integration with surrounding cartilage tissue;the DSCS and the DSCS/cell groups obtained more favorable tissue regeneration with thickened semitransparent tissue,and well integrated with the surrounding cartilage tissue.12 weeks after initial operation,the defect sites in all group were totally regenerated.Rough subchondral bone growth surfaces were observed in the Ctrl and Col-I groups,with minimal fibrous tissue partially covering the cartilage defect sites.Whereas the cartilage defect sites in the DSCS and DSCS/cell groups were in a better state of repair with semitransparent tissue and a smooth surface that was well-integrated with the surrounding host cartilage.CONCLUSION:（1）Squid cartilage tissue is a hyaline cartilage matrix,and the cartilage structure is looser than that of human hyaline cartilage.The natural cartilage extracellular matrix scaffold was fabricated using mild manner of decellularization technique.The chondrocytes were completely removed,while the DSCS retained the original cartilage extracellular matrix structure and components（type II collagen and glycosaminoglycan）.DSCS showed relatively smooth and dense surface,with interconnected porous inner structure of chondrocyte lacunae.DSCS possessed good hydrophilicity,biomechanical property and biodegradation,which could be considered as an ideal cartilage tissue engineering scaffold;（2）DSCS derived from natural cartilage matrix possesses hyaline cartilage matrix components.The DSCS extracts have no cytotoxicity;moreover,the extracts exerted promotive effect on cell migration.These results revealed DSCS possessed favorable biocompatibility,that reached the in vitro biosafety requirement for medical materials,and possessed the biosafety and feasibility of developing an artificial cartilage material;（3）DSCS performed a natural three-dimensional network structure and essential components of cartilage extracellular matrix,which exerted a promotive effect on implanted chondrocyte adhesion,proliferation,and maintain chondrocyte morphology and phenotype.Hence,the DSCS is an ideal decellularized cartilage scaffold for use in cartilage tissue engineering;（4）The in vivo experiments revealed both DSCS scaffold and cell-laden DSCS scaffold exerted promotive effects for the regeneration of cartilage in a full-thickness cartilage defect in the rabbit model.DSCS has an advantage over marketable col-I scaffold by mimicing the native cartilage ECM microenvironment for cell growth,migration and maintain chondrocyte morphology and phenotype.DSCS scaffold exerts promotive effect of hyaline cartilage repair in the defect area with or without cell transplantation.Hence,DSCS possesses great potential for direct application in repairing cartilage defects.