Production and characterization of cryogel-nanofiber bi-layered scaffolds for use in the repair of osteochondral defects

Osteochondral defects refer to conditions in which damage to both articular cartilage and subchondral bone is seen simultaneously. Self-healing of osteochondral defects caused by degenerative diseases is difficult. There is a need for approaches that take into account the physical and chemical properties of cartilage, bone and the cartilage-bone interface for the treatment of these diseases. For this reason, intensive studies are carried out on alternative treatment methods. Among these methods, tissue scaffolds produced with the bone tissue engineering approach come to the fore in the treatment of osteochondral injuries. The most important advantage of bi-layer scaffolds is that they contain physical or chemical components specific to cartilage and bone development. In tissue engineering, cryogels and nanofibers are often used as tissue scaffolds. In this study, Poly(ɛ-caprolactone) (PCL) nanofiber scaffolds were obtained by electrospinning method. Solutions containing PCL were prepared at a concentration of 15% (w/v). Chloroform and dimethylformamide (30:70, v:v) system was used as solvent. The voltage applied to the solution was 13 kV and the distance between the syringe tip containing the polymer solution and the collector was kept constant as 10 cm and the feed rate was adjusted as 1 ml/h. By keeping these parameters constant, nanofiber scaffolds were produced by electrospinning method. Chitosan:gelatin-based cryogel scaffolds were produced using the cryogelation method. Chitosan and gelatin were prepared by dissolving acetic acid in 6% aqueous solution with a total polymer concentration of 2% (w/v) and a chitosan:gelatin ratio of 50:50 (w:w). 6% (v/v) glutaraldehyde solution was used as crosslinker. Integration of nanofiber and cryogel layers was carried out by crosslinking at -16 °C cryogelation temperature in a cooled circulator. PCL nanofibers, which have a structure that will support cartilage regeneration, have been successfully integrated with chitosan:gelatin cryogels reinforced with biodegradable, hydroxyapatite (HA) at different rates (10, 30, 50, 100%) with properties that can mimic bone tissue. The surface morphology of the fabricated scaffolds was examined by SEM analysis. The mean pore diameter of the cryogel layer varied between 36.87±6.79 µm and 20.30±5.8 µm. The increase in the amount of HA caused a decrease in the pore diameter. The morphology of PCL nanofibers was observed as bead-free and smooth network structure. The mean fiber diameter was calculated as 258±35 nm. The integration of the two layers has been demonstrated by SEM images. The chemical structure of the scaffolds was investigated by FTIR. When the spectra were examined, peaks of PCL nanofiber and chitosan:gelatin cryogel were found in the bi-layer tissue scaffold. In addition, peaks of hydroxyapatite were observed in the cryogel layer. When the swelling behavior was examined, it was observed that the high rate of degradation of chitosan:gelatin cryogel and poly(ɛ-caprolactone) nanofiber bilayer scaffold (CS:Gel Cr/PCL Nf) with the highest swelling ratio occurred. The scaffold with the lowest swelling and degradation rate was determined as 100% hydroxyapatite added chitosan:gelatin cryogel and poly(ɛ-caprolactone nanofiber bi-layer scaffold (100HA-CS:Gel Cr/PCL Nf). The increase in the amount of HA decreased the swelling rate and the degradation rate. When the mechanical performance of the bi-layer scaffolds was examined, the average mechanical strength value of the samples was between 2 ±11 and 60±25 kPa, and Young's modulus values were between 24±11 and 60±25 kPa/%. Addition of HA to bi-layer scaffolds increased the mechanical strength properties. As a result of the study, it has been revealed that the developed cryogel/nanofiber bi-layer scaffolds are a potential product that can be used for the repair of osteochondral defects in bone tissue engineering applications.