Scaffold-free, engineered porcine cartilage construct for cartilage defect repair - In vitro and in vivo study

Abstract

This study introduces an implantable scaffold-free (SF) cartilage tissue construct that is composed of chondrocytes and their self-produced extracellular matrix (ECM). Chondrocytes were isolated from the articular cartilages from knees of domestic pigs (2-week old) and monolayer-cultured for 3-4 days in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 50 mu g/mL of ascorbic acid. Briefly treated with 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA), an intact chondrocytes/ECM membrane, as a cell sheet was released from the plate bottom and subsequently centrifuged into a pellet-type construct. Each was grown in vitro for up to 5 weeks and subjected to various assays at different time points (1, 7, 14, 21, and 35 days). For in vivo implantation, full-thickness defects (n = 4) were manually created on the femoro-patellar groove of the left porcine knee and 1-week-cultured SF construct was implanted as an allograft for a month. One defect (sharp1) was an empty control and the remaining three received different recipes; construct only (sharp2) or 0.25% trypsin/EDTA-treated first and then construct and collagen gel (sharp3) or construct and collagen gel (sharp4). While the total cell numbers significantly increased by 2 weeks and then remained stable, cell viability stayed in the mid-70% range through the entire culture period. Biochemical assay found continuous glycosaminoglycan (GAG) accumulation. Histology exhibited that cell distribution was even in the construct and GAG intensity became stronger and uniform with time. Real-time reverse transcription polymerase chain reaction (RT-PCR) results showed that phenotypic stability peaked at 2 weeks, which was arable to that of freshly isolated chondrocytes. Upon analysis of the retrieved implants, some promising results were witnessed in the defects (sharp3) retaining not only their intact mass but also chondrocytic morphology with lacuna formation.