Characterisation of the biochemical and biophysical properties of biomimetic cartilage models

Purpose: At present, osteoarthritis (OA) treatment options are limited to pain relief until the disease reaches end-stage when patients undergo joint replacement surgeries. In recent years, tissue engineering, regenerative medicine and cell therapy have emerged as major research areas for orthopaedic surgery, with the aim being to repair damaged cartilage tissue and promote tissue regeneration through the use of biomimetic models (1). However, future success of these approaches requires the development of biomimetic models with both the desired biochemical and biophysical properties. The aim of this study was therefore to characterise a novel alginate biomimetic model of cartilage repair with regards to chondrocyte viability and cartilage matrix production and to use Raman spectroscopy to examine the model’s compositional distribution. Methods: Cartilage tissue was collected from patients with knee OA undergoing joint replacement surgery. Primary chondrocytes were isolated by collagenase digestion and then suspended (at 5.5x105 cells/ml) in a 3D alginate hydrogel matrix either with or without a cartilage explant present and cultured in either normoxic (20% O¬2) or hypoxic (5% O2) conditions. Cell viability was determined using a Cell Titre Glo assay. qRT-PCR and Western blotting measured expression of type II collagen in chondrocytes. Production of proteoglycan matrix was determined by measuring the release of sGAG. Compressive modulus of alginate was measured following culture to determine any effect of increased collagen type II expression on the mechanical properties. Raman spectroscopy was used to map regions of GAG, collagen type II and alginate in the 3D alginate samples. Results: Primary chondrocytes cultured in the 3D alginate matrix maintained viability up to 2 weeks. Primary chondrocytes cultured in 2D monolayer rapidly lost Type II collagen expression. However, when cultured in 3D alginate matrix, Type II collagen expression was recovered and was closer to freshly isolated cells than the 2D monolayer cells. Expression of Type II collagen was greater in chondrocytes cultured in 3D alginate in the presence of cartilage explant, compared to the absence of cartilage explant. Following 2 weeks of culture, sGAG was detectable in supernatants from cultured chondrocyte/alginate biomimetics. Using Raman spectroscopy to map collagen, GAG and alginate regions in the gels, the highest collagen expression was identified in regions where the alginate was most dense (n=3). There was a significant decrease (P=<0.05) in the compressive modulus of alginate following 2 weeks of chondrocyte culture in aqueous media. Conclusions: Alginate scaffolds maintain both primary human chondrocyte viability and Type II Collagen phenotype. Reduced compressive properties of alginate, following 2 weeks of chondrocyte culture, suggests that further optimisation of alginate is required before it could be considered a suitable scaffold for the development of cartilage with structural integrity.