Construction Of Artificial Articular Cartilage Under Compression Load And Repair Of Cartilage Defects

Knee articular cartilage is an important part of joint activity,and its health is related to people's daily life activities.Due to the lack of its unique structure,it is difficult to repair itself once it is defective.At present,the repair methods used are generally effective,and the appearance of cartilage tissue engineering brings a new method for the repair of cartilage defects.It is extremely necessary to develop cartilage engineering materials for the study of knee articular cartilage defects.In this paper,the cartilage extracellular matrix type II collagen and the interest material silk fibroin are used as the basic materials.Since both materials have significant disadvantages as a repair material,the two are compositely modified and used as a repair material.Silk fibroin solution and type II collagen were first extracted from natural silk and fresh bovine scapula.Preparation of composite film(SF50,SF70,SF90)according to different ratios of silk fibroin and type II collagen.The most suitable ratio is tested by the examination of apparent physical properties,mechanical properties and biocompatibility.First,the three ratiometric materials were adjusted to have a common dry-to-wet ratio by adding deionized water.Different forms of the composite membrane were obtained by a freeze dryer and air-dried at room temperature for testing.The dried composite membrane was tested for mechanical properties of the three composite membranes using an Instron universal capability tester,and the mechanical experimental data was verified by establishing a constitutive model.The water absorption and degradation rate of the composite membrane after lyophilization were tested,and then the chondrocyte ADTC-5 was inoculated.The biocompatibility of the composite membranes with different ratios was detected by fluorescence staining,MTT and scanning electron microscopy.The results showed that when the ratio of silk fibroin and type II collagen was 7:3,the composite membrane showed more stable physical properties and better mechanical properties.The constitutive model and mechanical experimental data also showed relatively consistent.Through the detection of biocompatibility,the SF70 composite membrane showed the largest proliferation of chondrocytes.This indicates that the ratio of SF70 composite membrane is suitable as a ratio of cartilage repair.After the verification of different ratio experiments is completed.The SF70 mixed material was used for filtration to remove bubbles.Scaffold is prepared through a cryogenic bio 3D printer and freeze dryer.The scaffold was subjected to deacidification and co60 irradiation sterilization.The chondrocyte ADTC-5 was inoculated on a scaffold for compound culture.In this paper,the mechanical load is introduced innovatively,and the composite scaffold is dynamically cultured by verifying the gradient compressive strain(0%,5%,10%,15%,20%),and the optimal compressive strain suitable for cell proliferation is 10%.At 10%compressive strain,after 1 day,3 days,5 days and 7 days of dynamic culture,MTT,scanning electron microscopy,PCR fluorescence quantification and HE staining were performed.Scanning electron microscopy showed that the number of cells increased with the increase of dynamic culture days,and the cell's basic outer matrix was connected to cover the surface of the scaffold.The late results confirmed the increase in cell number in terms of quantity and protein secretion on gene expression.After the completion of the phase-matching experiment,the verification is completed.The SF70 is used as a mixed material for filtration to remove bubbles,which is prepared by a low temperature biological 3D printer,freeze-dried under vacuum,and then subjected to deacidification and co60 irradiation sterilization.The treated scaffold was subjected to a porosity test,and the chondrocyte ADTC-5 was inoculated to perform a composite culture.In this paper,the mechanical load is introduced in an innovative way.By verifying the compressive strain of the gradient(0%,5%,10%,15%,20%),the compressive loading of the composite scaffold is carried out,and the optimal compressive strain suitable for cell proliferation is obtained.It is 10%.At 10% compressive strain,cyclic loading was performed for 1 day,3 days,5 days,and 7 days,respectively,by MTT,scanning electron microscopy,PCR fluorescence quantification,and HE staining.At 10% compressive strain,the scaffolds and cells were cultured and observed by scanning electron microscopy.It was found that the number of cells increased with the increase of dynamic culture days,and the cells were connected to the surface of the scaffold.MTT,HE staining quantitatively,Real-time PCR from gene expression,Western Blot from protein secretion confirmed the increase in cell number.Finally,according to the previous experimental basis,the scaffold was prepared into a cylinder having a diameter of 3.5 mm by using a hole-making device.At the same time,a circular defect of 3.5 mm in diameter was prepared from the femoral end of the 3-4 month old New Zealand white rabbit articular cartilage by a hole-making device.The cylindrical scaffold was then implanted into a circular defect,and after suturing for 2 weeks,4 weeks,6weeks,and 8 weeks,the ear vein was injected with air embolization and the defect was removed.Micro-CT,HE and Immunohistochemistry was used to observe the defect repair.The results show that the naked eye can see that as the repair process progresses,the scaffold gradually degrades and new cartilage begins to form.The results of Micro-CT showed that the scaffold not only showed repair of cartilage,but also found that the subchondral bone began to repair,which provided practical experience for the subsequent repair and integration of hyaline cartilage-cartilage calcification layer-subchondral bone.Histological and immunohistochemical staining revealed the formation of new cartilage and subchondral bone.In summary,through the self-exploration and seeking to verify,the silk fibroin/type II collagen ratio of 7:3 mixed material shows great potential and possibility for the proliferation and growth of chondrocytes and the repair of cartilage defects.It has become a new composite material for repairing cartilage defects in later tissue engineering.