Fabrication Of Bilaver Collagen/Microporous Nanofiber Scaffolds And Its Application To Articular Osteochondral Tissue Engineering

Background:With the increase of global aging population and the growth of the sports injury, the incidence of osteoarthritis is raising, which influences thousands of people's life. There are three types of cartilage defects:partial cartilage defect, full-thickness cartilage defect, and osteochondral lesion which accounts for 5% of all cartilage defects. Articular cartilage is an organ without vascular, lymphatic and nerve tissues thus resulting in poor regeneration capacity after injury. This leads to a great challenge for people both in clinical orthopedics and sports medicine. Though little attention was paid in the past few decades, the regeneration of subchondral bone is drawing more and more interests because of its importance in maintaining the function of cartilage. In our research, we designed two kinds of subchondral bone scaffolds and analyzed their effect and possible mechanism in the subchondral bone repair and osteochondral regeneration. Our study includes three parts:(1) Constructing the bilayer collagen/microporous nanofiber scaffolds (2) Examining the biological performance of the bilayer collagen/microporous nanofibers scaffolds (3) investigating the effect of bilayer scaffolds on in situ osteochondral regeneration and its mechanism. Furthermore, we discussed the effects of different bilayer scaffolds on subchondral bone regeneration and its relation to the design of subchondral bone scaffolds.Part I Constructing the bilayer collagen/microporous nanofiber scaffolds for subchondral bone repairOBJECTIVE:To construct bilayer subchondral bone scaffold for in vitro biological performance examination and in situ subchondral bone regeneration.METHODS:Collagen-â… was separated from pig tendon and purified. The bilayer collagen/collagen (COL-COL) scaffolds were constructed with a porous upper layer and a dense lower layer through lypohilization. Poly-L-lactic acid nanofibers were made by electrospinning and sliced into micron stripes by frozen section. Meanwhile, the bilayer collagen/PLA nanofibers(COL-PLA) scaffolds were constructed with collagen upper layer and PLA porous lower layer. The microstructure of the scaffolds was evaluated by SEM.RESULTS:In our study, we constructed two kinds of bilayer scaffolds:COL-COL scaffold and COL-PLA scaffold. The size of each scaffold was different according to its application for in vitro and in situ regeneration. For in vitro experiments, the scaffolds were 15mm in diameter and 2mm in height (both upper and lower layers was lmm). The scaffolds for in situ regeneration were 4mm in diameter and 4mm in height (both upper and lower layers are 2mm). The pore size of the upper layer in COL-COL scaffold was 100-300um while the lower layer was 50-150um. And it was 50-150um and 100-300um respectively in COL-PLA scaffold.CONCLUSIONS:COL-COL and COL-PLA scaffolds were successfully fabricated according to the requirements of in vitro biological test and in situ subchondral bone regeneration.Part II Examining the biological performance of the bilayer collagen/microporous nanofibers scaffoldsOBJECTIVE:To investigate the cell adhesion and osteogenic differentiation of mesenchymal stem cells on the subchondral bilayer scaffolds.METHODS:Human bone mesenchymal stem cells(hBMSC) and rabbit bone mesenchymal stem cells(rBMSC) were isolated and seeded on the scaffolds. Cell adhesion was evaluated by SEM. Meanwhile, cell proliferation was detected by MTT assay, and the enrichment of calcium nodules on the bilayer scaffolds was examined by von Kossa assay. Moreover, expression of osteogenic genes on the bilayer scaffolds was analyzed using real time-PCR.RESULTS:SEM images indicate that cells adhere to collagen and PLA bilayer scaffolds well. MTT results showed that cells exhibited good proliferation on both scaffolds. Von Kossa staining on COL-PLA scaffold was deeper black than COL-COL scaffold, suggesting that COL-PLA scaffold concentrate more calcium nodules. Real time-PCR result revealed that the expression of osteogenic differentiation genes both OCN and Runx2 were higher in COL-PLA scaffold.CONCLUSIONS:Cells adhered to both scaffolds well and COL-PLA scaffold exhibited greater potential of osteogenic differentiation induction than COL-COL scaffold.Partâ…¢In vivo Subchondral bone regeneration in situ using COL-COL and COL-PLA bilayer scaffoldsOBJECTIVE:In this study, we investigated the effects of the bilayer scaffolds on subchondral bone regeneration and the possible mechanism.METHODS:23 New Zealand Rabbits were divided into 3 groups:.Non-treated group, COL-COL group and COL-PLA group. The osteochondral defect was made in the bilateral patellofemoral knee femoral condyle position (cylinder defect with 4mm in diameter and 3.5mm-4mm in depth). Then COL-COL scaffold or COL-PLA scaffold were transplanted into the defect respectively.6 and 12 weeks after operation, the rabbits were anaesthetized and sacrificed for the general observation, histological staining, mechanical evaluation and micro-CT examination. Statistical analysis was also done to identify the significance of differences among groups.RESULTS:In general, the COL-PLA group obtained the highest score, and the non-treated group was the worst. Regarding the subchondral bone regeneration, COL-PLA group formed a thicker cancellous bone and a thicker hyaline cartilage layer.CONCLUSIONS:COL-PLA scaffold was more suitable for the subchondral bone regeneration, showing its potential in clinical application.