Development of thermoplastic ternary composites and hydrogels for additive manufacturing of scaffolds for regeneration of osteochondral defect

摘要

Introduction: Regeneration of osteochondral defect requires application of biomaterials which are able to simultaneously support development of bone and cartilage. Materials envisioned for reconstruction of the bony part should exhibit good initial mechanical properties, sustain them during initial stages of regeneration and subsequently undergo degradation. Materials designed as cartilage substitute should be softer and enable encapsulation of cells to provide them with a 3D environment. In both cases, the materials should be compatible with fabrication techniques yielding 3D scaffolds with well-defined architectures, ideally, in one fabrication process. Additive manufacturing meets those criteria. The aim of this study was to develop materials for regeneration of bone and cartilage compatible with Fused Deposition Modeling (FDM) and bioplotting techniques and to fabricate scaffolds suitable for reconstruction of osteochondral defect. Materials and Methods: Bony part: Ternary composite scaffolds were fabricated using FDM. The scaffolds were composed of 70wt% of polycaprolactone (PCL), 5wt% Tricalcium Phosphate (TCP) and 25wt% poly(lactide-co-glycolide) (PLGA) with various lactide (LA) to glycolide (GA) ratios. In vitro degradation was carried out in Phosphate Buffered Saline (PBS) and Simulated Body Fluid (SBF). Changes of mass, molecular weight (MW) and mechanical stability were monitored during this experiment. Chondral part: Bioink contained 4wt% of alginate, 6wt% of gelatin-methacrylamide (GELMA) and 4wt% of chondroitin sulfate-methacrylate (CSMA) in HEPES buffer supplemented with 10% FBS. The bioink was laden with 15×10~6/ml of human mesenchymal stem cells (hMSC) and bioplotted using calcium chloride and UV-photopolymerization for gelation. Cell viability was studied using live-dead fluorescence assay. The cell-laden hydrogels were cultured in chondrogenic medium and expression of chondrogenic markers was studied by means of RT-PCR and immunofluochemistry (IF). Results and Discussion: Young's modulus of the PCL-based scaffolds was in the range of 59±3+73±2 MPa, which corresponds to stiffness of some cancellous bonesl. Decrease of the modulus followed mass loss exceeding 5 wt%. However, we observed an increase of the modulus to its initial value after 16 weeks of degradation in SBF in the case of scaffolds containing PLGA with 50 mol% of LA. The highest decrease of MW of PCL (by 60% after 24 weeks) occurred in the case of scaffolds containing PLGA with 75 mol% of LA. Prolonged retention of PLGA could accelerate hydrolysis of PCL due to catalytic effect of carboxylic groups released from the PLGA. Cell viability after bioplotting exceeded 80%. This shows the advantage of combination of quick gelation of alginate caused by Ca~(2+) and short exposure to UV-light for photopolymerization of GELMA and CSMA. The cell-laden scaffolds were stable after 28 days of culture, additionally proving the effectiveness of the applied cross-linking method. Expression of type 2 collagen and aggrecan by hMSC was confirmed by RT-PCR and IF. Conclusions: Degradation profile of PCL-based ternary composite scaffolds could be tailored by varying the LA:GA ratio. The developed bioink supported chondrogenic differentiation of hMSC. Future work will focus on combining the two materials in one fabrication process.