Obtaining of a suitable osteochondral graft for articular cartilage engineering

Abstract

Introduction. Chondral injuries are common following a knee trauma. There are numerous studies with different ways to obtain a suitable graft for articular cartilage regeneration, but without imposing results. Material and methods. From two freshly sacrificed rabbits the distal femurs were harvested and frozen at -84°C for one week. From each distal femur all tissues except cartilage and subcondral bone were removed and small pieces of normal osteochondral tissue (NOCT) were taken. The remaining osteochondral tissue has been demineralized in 0,6M HCl (Chem-Lab, Belgium) over night and again small pieces of demineralized osteochondral tissue (OCDT) were cutted with a scalpel and placed in a PBS solution for 24 hours. The remaining OCDT were separated in 4 groups. Two groups were decellularized in 0,5% and 1% SDS (Sigma, UK) and another two in 0,5% and 1% Triton X-100 (HiMedia, India). The decellularization lasted for 24 hours. At the next day the decellularized and demineralized osteochondral tissues (OCDDT) were washed with distilled water and PBS for 24 hours. All tissues were dessicated through centrifugation at 4000 rpm for 10 min (Hettich, Germany). From all types of OCT were cutted from three to nine pieces 20 mg each and quantification of DNA was performed with GeneJET Genomic DNA Purification Kit (Thermo Fisher, Lithuania). The results were read with spectrophotometer NanoDrop 2000c at wavelength of 260 nm (Thermo Fisher, USA). The best decellularized tissue and OCDT were tested for cytotoxicity with MTT test (ISO 10993-5) with mesenchymal stem cells and chondrocytes. Results. The average of DNA content in a rabbit NOCT is 36 ng/μl, in OCDT 4,23 ng/μl, OCDDT with 0,5% and 1% SDS is 3,23 ng/μl and 2,16 ng/μl respectively and in OCDDT with 0,5% and 1% TritonX-100 is 1,96 ng/μl and 0,96 ng/μl. At the MTT assay with mesenchymal stem cells and chondrocytes on the OCDT and OCDDT with 1% TritonX-100, we obtained a higher cell viability in both cases more than 80%. Conclusions. Obtaining a suitable osteochondral tissue for cartilaginous tissue engineering is very difficult because this process involves utilisation of a very toxic chemicals that harm this tissue. A shorter exposure period to chemical agents and preliminary modeling of the graft is mandatory. Also the OCDDT with 1% TritonX-100 shows the best results compared to others.