Fabrication Of Silk Fibroin Scaffolds And Its Application To Osteochondral Tissue Engineering

Background:Articular cartilage is a special tissue with the absence of vascular, lymphatic and nerve tissues, and low metabolic rate. The special anatomical structure of the articular cartilage determines its limited ability to repair itself. There are three types of cartilage defects:partial cartilage defect, full-thickness cartilage defect, and osteochondral lesions. The repair of articular cartilage defects has been a hot area of the orthopaedic research. Although traditional treatment methods, such as conservative treatment, arthroscopic repair treatment, tissue transplantation therapy, produce a certain beneficial effect, they only temporarily alleviate the pain and other symptoms and cartilage repair parts will eventually degrade by necrosis. The development of cartilage tissue engineering holds promise for the repair of cartilage damage.In the present study, we employed different thickness silk fibroin scaffolds to repair articular osteochondral defects, and its effect and mechanism in the subchondral bone repair and osteochondral regeneration were explored. Our study consists of three parts:(1) Construction the silk fibroin scaffolds for subchondral bone repair;(2) Evaluation of biological performance of silk fibroin scaffolds;(3) Investigation of the effect of silk fibroin scaffolds on in situ osteochondral regeneration and its mechanism. The study would pave the way for the application of silk fibroin in clinic.Part I:Constructing the silk fibroin scaffolds for subchondral bone repairOBJECTIVE:To construct silk fibroin scaffold for in vitro biological performance detection and in situ subchondral bone regeneration.METHODS:The silk fibroin aqueous solution was prepared by degumming, dissolving, dialyzing sequencely from silk fiber of a Bombyx mori silkworm cocoon. Porous structure of silk fibroin scaffolds was generated by small salt as porogen and cross-linking in a mold. Scaffold morphology was examined with scanning electron microscopy (SEM). Scaffold porosity was determined using a liquid displacement method.RESULTS:For in situ regeneration,we constructed two depth silk fibroin scaffolds that are4.0mm in diameter and4.0mm or2.0mm in height. Those scaffolds for in vitro experiment are15mm in diameter and2.0mm in height. The aperture of the silk fibroin scaffold is100-300um which is calculated from SEM images. Scaffold porosity method is88%.CONCLUSIONS:Silk fibroin scaffolds meet the requirement of in vitro biological test and in situ subchondral bone regeneration.Part II:Detecting the biological performance of the silk fibroin scaffoldsOBJECTIVE:To investigate the performance of chondrocyte adhesion and its specific gene profile on the silk fibroin scaffolds.METHODS:Human fetal chondrocyte were isolated, cultured and CFDA stainded, and employed to test cell adhesion on the silk fibroin scaffolds by inverted fluorescence microscope. Moreover, chondrocyte specific genes were examined on cells cultured in the silk fibroin scaffolds using semi-quantitative PCR. RESULTS:Inverted fluorescence microscope observation indicates that cells adhere to the silk fibroin scaffolds very well. Semi-quantitative PCR demonstrates the presence of chondrocyte marker genes.CONCLUSIONS:Cells adhere to the silk fibroin scaffolds well and the scaffolds can maintain the chondrocyte phenotype.Part III:In vivo Subchondral bone regeneration in situ using different thickness Silk Fibroin scaffoldsOBJECTIVE:We investigate the effects of the different depth silk fibroin scaffolds on subchondral bone regeneration and its possible mechanism.METHODS:11New Zealand Rabbits were randomly divided into2groups, including long scaffold group(Long, equal altitude with osteochondral defect) and short scaffold group(short, under the subcartilaginous osseous lamella). The osteochondral defect was made in the bilateral patellofemoral knee femoral condyle position (3.5-4.0mm in thickness,4.0mm in diameter). Then long scaffold or short scaffold was transplanted into the defect.6weeks and12weeks after operation, the rabbits were anaesthetized and sacrificed for the general observation, histological sections and mechanical testing. RESULTS:Compared with the long scaffold group, the short scaffold group in general showed better results, wherein the cartilage surface grew more smoothly and thicker cancellous bone formed.CONCLUSIONS:Grafting silk fibroin scaffold lower than the subcartilaginous osseous lamella promotes the regeneration of osteochondral defect, implicating its potential in clinical application.