Abstract
Introduction
Articular hyaline cartilage has a unique structural composition that allows it to endure high load, distribute load to bone and enables low friction movement in joints. A novel acellular xenogenic graft is proposed as a biological cartilage replacement, for repair of osteochondral defects. Acellular porcine cartilage has been produced using repeated freeze thaw cycles and washing using hypotonic buffers and sodium dodecyl sulphate solution (SDS; Keir, 2008). DNA content of the acellular matrix was reduced by 93.3% compared to native cartilage as measured by nanodrop spectrophotometry of extracted DNA, with a corresponding reduction in glycosaminoglycan (GAG) content.
Hypothesis
It was hypothesised that penetration of decellularisation solutions into the native tissue could be improved through deformation of the cartilage under confined compression and then allowing the osteochondral pin to recover in solution, allowing removal of cellular DNA and greater retention of the GAGs.
Methods
A stainless steel rig was manufactured to apply a ramped load through a perforated indenter, using a tensile testing machine (Instron). Osteochondral pins (9.8 mm diameter; n = 3) were loaded to a contact stress of 5 MPa for 20 minutes, and then placed in SDS 0.1% (v/v) in hypotonic Tris buffer; pH 8.0 and then treated according to the established protocol. Porcine osteochondral pins (n = 3) were treated using the standard protocol without compression for comparison. Histological and biochemical analyses were performed and analysis of variance on the results.
Results
There was no significant difference in the reduction in DNA content of loading modified versus standard protocol decellularisation cartilage (0.017 mcg.mg-1 +/− 0.006 mcg.mg-1 (95% CI), loaded, 0.018 mcg.mg-1 +/− 0.011 mcg.mg-1 (95 % CI), standard protocol). There was no significant difference in the GAG content (19.769 mcg.mg-1 +/− 33.581 mcg.mg-1 (95% CI), loaded, and 13.530 mcg.mg-1 +/− 21.207 mcg.mg-1 (95 % CI), standard protocol). DAPI fluorescence microscopy demonstrated qualitatively that there was residual DNA in deep zones and at the cartilage bone interface in the standard decellularisation protocol cartilage, whilst this was to a lesser degree with loading modified cartilage.
Discussion
Loading modification at 5 MPa contact stress did not improve the level of DNA reduction or GAG retention in acellular porcine cartilage. Whilst decellularisation of a xenogenic graft is possible to yield an acellular scaffold a significant reduction in GAG content occurs, with a consequent deleterious effect on the material properties. Further work is required on GAG replenishment in the scaffold.
