Faborication Of Oriented Scaffold Derived From Native Cartilage Extracellular Matrix And Its Related Cartilage Tissue Engineering Research

BACKGROUND Degenerative and rheumatic diseases,traumatic events and excision of tumors are the main reasons for articular cartilage impairs.By itself,cartilage has very limited capabilities of regeneration,if not treated,they extend to be osteoarthritis.Nowadays,numerous strategys have been made to promote reparation of the cartilage defect such as drilling,microfracture,autograft of periosteum,perichondrium and osteochondral autogratt.However all of these generally leads to a mechanical and biological inferior repair tissue based on fibrocartilage.Moreover there is a shortage of autogenous donor sources for grafting.However,the development of the ACI technique and the progress in the tissue-engineering field in the early nineties,have suggested that the repair of cartilage actually might be feasible,leading to the development of numerous strategies to repair or replace the damaged surfaces.In many approaches,cells are grown on biomaterial scaffolds and then implanted into the defect,where new functional tissue is formed,remodelled and integrated into the body.In a typical cartilage tissue engineering approach,to control cartilage repair tissue formation in three dimensions(3D),a highly porous scaffold is critical.And the biological, biochemical,mechanical,and structural properties of scaffolds for cartilage tissue engineering are known to be of great importance.However most materials currently investigated lack a scaffold-structure similar to that of native cartilage and they strongly differ in their biological and biochemical composition.In addition to defining the 3D geometry for the tissue to be engineered,the scaffold provides the microenvironment(synthetic temporary extracellular matrix) for regenerative cells,supporting cell attachment,proliferation,differentiation,and neo tissue genesis.Therefore,the chemical composition,physical structure,and biologically functional are all important attributes to biomaterials for tissue engineering.To serve as the temporary extracellular matrix(ECM) for regenerative cells,it may be beneficial for the scaffold to emulate certain advantageous features of the natural cartilage ECM.However,it is likely unnecessary and impractical for a scaffold to entirely duplicate the cartilage ECM. A natural cartilage ECM may actually not be the ideal scaffold for cartilage tissue engineering applications because mature cartilage tissue matrix often does not possess the highly interconnected macro-or micro-pore structures to allow for quick and uniform cell population throughout,which is essential for a tissue engineering/repair process.Therefore optimal cartilage tissue engineering scaffold should have certain artificially designed scaffold features(such as porosity,pore size,interpore connectivity,etc.) Based on the above discussion,we previously shattered the natural cartilage and collected the nano-material derived cartilage extracellular matrix which biochemical composition mimic the native cartilage ECM,and by freeze-dry technique,we fabricated a porous scaffold,and the scaffold were proved to be sure for supporting the cartilage regenerating in vitro and in vivo.Base on our previous research,considered the structured feature of the native cartilage tissue,we attempt to fabricate the oriented scaffolds that characterized by a perpendicular pore channel structure and biomechanical properties similar to native cartilage tissue.And on the other hand,our previously cartilage materials were human cartilage,which were shortage of donor sources, so in this study we use the porcine cartiage to fabricate the scaffolds,because the porcine cartilage is economic and its homology is similar to human.OBJECTIVE This study aims to further promote the technology and procedure of collecting the native cartilage ECM material.To fabricate oriented scaffold and investigate the charactarization of the scaffolds.To investigate the effect of the oriented scaffold on the the attachment,proliferation,distribution and alignment of chondrocytes seeded in the scaffolds cultured in vitro.And to test the clinical applicability of the scaffolds,we repaired rabbit osteochondral defect using OS and NOS combined the autologous BMSCs.METHOD①Cartilage slices were shattered in sterile phosphate-buffered saline(PBS),and the suspension were differentially centrifugated untill the micro-fiber of the acellular articular cartilage extracellular matrix were divided from the residue cartilage fragments.At last the supernatant were centrifugated, the precipitation were collected and were made into 2～3%suspension.Using unidirectional solidification as a freezing process and freeze-dried method the articular cartilage extracellular matrix derived oriented scaffolds were fabricated. The scaffolds were then cross-linked by exposure to ultraviolet radiation and immersion in a carbodiimide solution.And the cartilage articular cartilage extracellular matrix scaffolds with random and spherical pores were prepared as a control at the same time.②Oriented cartilage extracellular matrix derived scaffolds were fabricated by using unidirectional solidification as a freezing process and freeze-dried method.After being labeled with PKH26,rabbits chondrocytes were seeded onto the scaffolds and incubated for 1,3,5 and 7 days. Attachment,proliferation and viability of the cells were measured by mitochondrial dehydrogenase activity(CCK-8 assay),the morphology, distribution and alignment of the chondrocytes in the scaffolds was analyzed by invert microscopy,fluorescent microscopy,scanning electron microscopy and H&E staining.Chondrocytes seeded into nonoriented cartilage extracellular matrix derived scaffolds were used as controls.③The chondrocytes/scaffold compounds were incubated for 1 week at 37℃,5%CO2 incubator,divided into two groups:chondrocytes/OS,chondrocytes/NOS.And then implanted respectively into subcutaneous pockets on the backs of 6 athymic nude mouse. The composites were harvested and examined with histology at 4weeks after implantation.④A full-thickness articular-cartilage defect(4ram in diameter) was created in the patellar grove of distal femur of rabbits.The rabbits were divided into five groups:(A) the defects were left empty.(B) filled with OS.(C) filled with NOS.(D) filled with BMSCs/OS.(E) filled with BMSCs/NOS.Specimens were harvested at 3,6 and 12 months postoperatively,and assessing the reconstituted defects grossly,histologically,biochemically and biomechanically.RESULTS:There were no cells in the cartilage extracellular matrix fabricated from the shattered cartilage slices;and the fiber size of the acellular articular cartilage extracellular matrix was nano scale.The histological staining showed that toluidine blue,safranin O,alcian blue and anti-collagenⅡimmunohistochemistry staining of acellular articular cartilage extracellular matrix fabricated from the shattered cartilage slices were positive.By SEM analysis a perpendicular pore-channel structures in the scaffolds were verified with channel diameters about 100μm.The porosity and the water uptake had no difference between the oriented scaffolds and the nonoriented scaffolds(p＞0.05).Mechanical testing showed that compression modulus and tension modulus of the oriented scaffolds were much higher than those of the nonoriented scaffolds(p＜0.05). Chondrocytes seeded in both scaffolds showed good attachment and viability from day 1 to day 7,most of the cells in both scaffolds were spheric morphology. However,proliferation of chondrocytes seeded in oriented scaffolds(OS) was higher than that of chondrocytes seeded in nonoriented scaffolds(NOS).Moreover, chondrocytes seeded in the OS widely distributed within the scaffolds and aligned themselves along the oriented pore mimic a columnar arrangement whereas chondrocytes seeded in the NOS randomly distributed within the scaffolds and its amount was less.In nude mice model,histological examination showed that both of OS and NOS support the chondrogenisis in that toluidine blue,safranin O, alcian blue and anti-collagenⅡimmunohistochemistry staining of the chondrocytes/scaffolds constructs were positive and there were large mount extracellular matrix around the cells.The difference of the two kinds of scaffolds was that chondrocytes seeded in OS and the collagen aligned mimic a columnar arrangement whereas chondrocytes seeded in the NOS and collagen randomly distributed within the scaffolds.In animal experimental,results indicated that every group have been repaired by fibrocartilage or hyaline cartilage in different degree.By histological and histochemical grading scale evaluation of area of defect,the group filled with BMSCs/OS or BMSCs/NOS was better to repair full-thicks cartilage defect than the others.And the score of BMSCs/OS groups were higher than that of the BMSCs/NOS groups.There was significance between the BMSCs/OS and BMSCs/NOS groups,especially in early stage after the implantation.CONCLUSION The ACECM derived oriented scaffolds have promising composition,and structural,mechanical properties similar to that of nature cartilage,can support the chondrocytes to accommodate and guide their growth into cartilagous tissue,and can repair articular cartilage defects when combined with BMSCs.This optimized scaffold might be a ideal cartilage tissue engineering scaffolds for cartilage defect regeneration.