Porous Scaffold Based On Fish Collagen For Cartilage Tissue Engineering

Background:Scaffold materials are one of the three elements in tissue engineering,and the research on material properties is also developing rapidly with the in-depth study of tissue engineering.Collagen has many advantages,such as wide sources,easy availability,inert immunogenicity,good biosafety and biocompatibility,suitable biodegradability,more functional groups,easy to process into various shapes,etc.Therefore,it has become one of the most ideal tissue engineering cartilage scaffold materials.Mammal-derived collagen（MC）,which is mainly sourced from bovine tendon and porcine skin,is a commonly used scaffold for cartilage tissue engineering.Although bovine collagen-based scaffolds have many advantages,they also have many shortcomings.For example,bovine collagen is in short supply and the price is high.In particular,because of the large population of our country,beef is one of the food sources,and the demand is too large,leading to bovine collagen source is limited,and it is still mainly imported products.Secondly,bovine collagen also has potential transmission risks of zoonotic viruses such as swine flu,mad cow disease,and foot-and-mouth disease.At the same time,due to restrictions on religious etiquette,some countries and regions also prohibit the extraction and application of bovine collagen.Therefore,based on the above shortcomings,finding a collagen material that can replace bovine collagen has become an urgent problem.Our country has a long coastline,rich marine resources,and a wide variety of species.Since the 13th Five-Year Plan,the development and application of marine biological materials has gradually become a hot spot and an area of key national support.Fish collagen extracted from marine organisms has become the key to solving the problem.Studies have shown that collagen extracted from jellyfish,sharks,salmon and other marine organisms can not only promote the chondrogenic differentiation of stem cells,but also has a certain antioxidant effect,which is beneficial to maintain the vitality of chondrocytes and resist cell aging,At the same time,whether under physiological or pathological conditions,the degradation products of fish collagen have an effect on the metabolism of chondrocytes.All these studies suggest that fish collagen has great application prospects in the field of cartilage regeneration.At the same time,compared with bovine collagen,fish collagen has a richer source of raw materials,a lower price,and can avoid the spread of zoonotic diseases,and is expected to become an important source of scaffold materials for cartilage tissue engineering.Objective:This study mainly explores the feasibility of fabricating porous scaffolds based on fish collagen to construct tissue engineering cartilage.We prepared two different types of scaffolds by freeze-drying and electrospinning to explore whether they could be used to construct tissue-engineered cartilage.Methods:（1）Freeze drying method.The fish collagen is mixed with deionized water in different proportions（0.5%,1%and 2%）,and then freeze-dried to prepare porous scaffolds.The general view,scanning electron microscope observation,porosity,material stability,water absorption and mechanics are performed.The scaffolds were implanted subcutaneously in rats for 14 days for immunogenicity evaluation,Chondrocytes derived from rabbit ear in the second passage were seeded into the scaffolds with different volume ratios and cultured in vitro.The examination of Living&dead staining and cell proliferation assay were conducted after in vitro cultivation for 1,4 and 7 days.Moreover,Second-generation chondrocytes were prepared into a cell suspension of 1.0×10⁸ cells/ml,uniformly inoculated on the 2%FC scaffold for 4 h at 37°C/5%CO₂ and then cultured in DMEM containing 10%FBS.After 2 weeks of cultivation,the constructs were either maintained in vitro for another 8 weeks or were subcutaneously implanted into rabbits for an additional 8weeks.The chondrogenesis were observed by histological examination after in vitro culture for 4 weeks and 8 weeks or were subcutaneously implanted into rabbits for an additional 8 weeks.And the Young’s modulus,GAG content,DNA content and total collagen content were measured.Finally,through the above experiments,the 2%ratio scaffold that is most suitable for cartilage growth was selected to repair rabbit knee articular cartilage defects,and histological staining evaluation were performed at 6 and 12 weeks after surgery.（2）Electrostatic spinning method.After mixing fish collagen and polycaprolactone（PCL）at 9:1,7:3 and 5:5 respectively,the fiber membrane was prepared by electrostatic spinning technology.The gross view and scanning electron microscope observation were conducted,and the protein absorption rate,material degradation rate,water absorption and mechanical properties were tested.Chondrocytes derived from rabbit ear in the second passage were seeded into the scaffolds with different volume ratios and cultured in vitro.The examination of Living&dead staining and cell proliferation assay were conducted after in vitro cultivation for 1,4 and 7 days.Then,the chondrocytes-nanofiber membrane constructs were cultured in vitro for 4 weeks or were subcutaneously implanted into rabbits for an additional 8 weeks,and their cartilage formation was observed by histological examination,young’s modulus,GAG content,DNA content and total collagen content were detected.Results:（1）Freeze-drying method.In this study,we prepared and optimized the porous scaffolds with a 2%concentration of fish collagen,which showed satisfactory physical and chemical properties,including appropriate pore size and porosity,appropriate degradation rate,good biocompatibility,high mechanical strength and low immunogenicity.In addition,on the basis of the fish-collagen porous scaffold,we achieved mature and stable cartilage regeneration in vitro and subcutaneous transplantation in rabbits,and observed the repair of in-situ cartilage defect.These results provide experimental basis for the future clinical application of porous fish collagen scaffolds.（2）Electrostatic spinning method.This study shows that the electrostatic spinning fibrous membrane with a ratio of fish collagen to PCL of 9:1 is suitable for cartilage regeneration,which has good physical and chemical properties,including suitable pore structure and high biocompatibility.In addition,based on the scaffolds,satisfactory cartilage regeneration was obtained after subcutaneous implantation in rabbits.These results provide a theoretical basis for the future clinical application of electrospun nanofiber scaffolds.Conclusion:（1）The electrospun nanofiber scaffolds with 9:1 concentration ratio of fish Collagen to PCL and the freeze-drying scaffolds with 2%concentration of fish collagen are suitable for cartilage growth.（2）The raw material of Fish Collagen can overcome the problem of Mammal Collagen,and has the function of promoting cartilage growth,so it has great potential in clinical application.