Decellularisation and characterisation of porcine bone-medial meniscus-bone

The meniscus in the knee functions to absorb shock and transmit load within the joint. Problems arise when the meniscus is injured or damaged as the meniscus’ healing potential is limited by the limited vascular supply extending only ~20% of the way through the mature meniscus. Current treatment options are only effective at abating further degeneration and osteoarthritis due to altered joint mechanics. Replacement of the meniscus has the potential to prevent the onset of osteoarthritis by restoring native joint mechanics.
Here we aim to develop a decellularisation protocol for porcine medial meniscus with added bone blocks for easy fixation for use as a meniscal replacement. Menisci were decellularised using a low sodium dodecyl sulphate (SDS) method including freeze-thaw cycles, hypertonic, hypotonic and nuclease wash steps, as well as mechanical removal of cellular material. Decellularised menisci were then characterised histologically, immunohistochemically, biochemically and biomechanically for successful decellularisation and compared to native meniscus.
Histology revealed the absence of whole cells and nuclei from meniscus and bone, while quantification of DNA revealed ~96% and ~91% removal of DNA from meniscus and bone, respectively. Immunohistochemical analysis showed retention of major structural collagens I, II and II; however there was complete removal of collagen IV and some loss of collagen VI. Hydroxyproline assay showed retention of collagen and the collagen content of native meniscus was 802.90 (± 52.95, 95% CL) mg.g-1 dry weight and for decellularised meniscus was 935.35 (± 18.03, 95% CL) mg.g-1 dry weight, however there was a complete loss of GAGs from the decellularised matrices as revealed by Safranin O staining and quantified using dimethyl methylene blue assay (23.49 ± 8.63 μg.mg-1 vs. 0.30 ± 0.47 μg.mg-1, 95% C.I.). There was also a 51% reduction in calcium content of the decellularised bone blocks when compared to native bone blocks.
Material properties of meniscus were obtained using uniaxial tensile and indentation testing with no significant differences observed between native and decellularised groups under tension although greater deformation was seen for decellularised samples under compression. Unconfined compression of bone however revealed a significant decrease in the compressive modulus and strength of decellularised bone blocks. Differential scanning calorimetry revealed no significant differences in thermal stability between native and decellularised meniscus except for in the attachment region where collagen was mechanically manipulated prior to decellularisation. Magnetic resonance imaging (MRI) also revealed no gross differences between native and decellularised bone and meniscus.
Extract and contact cytotoxicity assays were used to determine biocompatibility of decellularised bone-meniscus-bone and residual SDS content determined using radio-labelled 14C SDS. There was 0.289 μg.mg-1 of residual SDS in decellularised bone-meniscus-bone and assays showed no cytotoxicity to 3T3 and BHK cells. The immunogenic galactose-α-1,3-galactose epitope could not be detected in decellularised meniscus by immunohistochemistry.
In conclusion, a protocol for the successful decellularisation of porcine bone-medial meniscus-bone has been developed which retains the structure and tensile properties of native meniscus and is non-cytotoxic. Further investigation is required to determine whether loss of mechanical strength in the bone will prevent effective fixation in vivo.