Populated collagen hydrogel and polyhydroxyalkanoate composites: novel matrices for cartilage repair and regeneration?

Abstract

Purpose \nArticular cartilage is an avascular connective tissue present in joints which covers the ends of opposing bones providing a smooth, shock absorbing surface to promote normal joint function. Normal cartilage structure is maintained by chondrocytes, the only cell type present. Abnormal function/death of these cells, due to long- term “wear and tear” and other risk factors results in cartilage loss and osteoarthritis (OA). There is currently no cure for OA, the most common treatments simply involve the management of joint pain, or surgical treatments, which are not able to restore cartilage function. \nIn recent years, cartilage repair has been approached with engineered scaffolds, able to support the growth of chondrocytes or progenitor cells to generate a neo-tissue, but the neo tissue generated is often fibrocartilage, mechanically inferior and less–durable than the hyaline cartilage normally found in healthy articular joints, indicating that further research is required to develop a more suitable scaffolds. \nThe aim of this study has been to assess the properties of collagen I hydrogel 3D scaffolds, populated with the phenotypically stable chondrocytes cell line C20/A4, strengthened with solvent cast and 3D printed Polyhydroxyalkanoate (PHA) polymer. \nPHAs are a family of biopolyesters synthesized by bacteria with monomers of varying chain lengths (C4-C16) which can be blended to produce products with different mechanical properties. PHAs are biodegradable, non-toxic and biocompatible. \nIt has been already established that PHA scaffolds are able to support chondrocyte growth and activity but despite that, they do not represent the natural environment for chondrocyte metabolism, collagen production and cartilage regeneration. A combination collagen hydrogel and PHA scaffold that addresses this issue would be highly desirable. \nMethods \nPolymer production and preparation \nPHAs were produced by Pseudomonas mendocina CH50 in batch fermentation conditions using a 15 L bioreactor with an operating fermenter volume of 10 L. with glucose as the sole carbon source under nitrogen limiting conditions. PHA film and scaffolds were either prepared using the solvent casting method incorporating large NaCl crystals (which were later dissolved out) to form a porous structure or with a 3D melt printing method. \nCollagen gel preparation and seeding: \nType I collagen constructs were prepared with chondrocytes by mixing type I collagen polymer suspension with 10\uf0b4modified Eagle’s medium (MEM) and 1M NaOH solution was added dropwise, to neutralise the acidic gel mixture. This gel solution was then mixed with a cell suspension of C-20/A4 cells in Dulbecco’s MEM (DMEM)-based medium containing 1% foetal calf serum (FCS) . 1.2 ml constructs were cast in 24 well plates either on their own or with the addition of solvent cast PHA polymers or 3D printed PHA polymers. Gels were then allowed to solidify by incubation at 37 \uf0b0C for 5-10 minutes, overlaid with DMEM medium (1% FCS) and cultured for 1 or 7 days, at 37 \uf0b0C and 5% CO2. \nCompression Studies: \nGels were subjected to compression, at 0,6KN/m2 for 5 minutes in a custom built apparatus. The displacement of gels was measured over time. \n \nResults \n \nThe compression testing data demonstrates that type I collagen gels, seeded with C-20/A4 chondrocytes (control gels), exhibit changes in their mechanical properties over time, with an increased resistance and smaller displacement at Day 7 compare to Day 1. \nThe addition of solvent cast and 3D printed scaffolds increase the mechanical resistance of the construct when compared to the populated control gels (collagen matix only) at DAY 1 and is even more evident at DAY 7. 3D printed scaffolds also show higher stiffness of the construct compared to the control and solvent cast polymer samples. \n \nCytotoxicity testing performed with Alamar blue staining, on chondrocyte populations grown on PHA films, indicates that there are no cytotoxic effects of the polymer on cell viability at any of time points analysed over 7 days. Trypan Blue staining, after 5 minutes compression test, reveals no significant difference in viability in any of the samples containing PHA polymer (solvent cast or 3D printed) compare to control samples. \n \nConclusions \nIn conclusion, these studies suggest that a composite collagen gel-PHA construct provides a supportive environment for the maintenance of chondrocyte activity increasing the initial resistance of the structure to compression, whilst the embedded chondrocytes remodel the Type I collagen gel to form a more cartilage like structure. This may offer a potential application of collagen hydrogel-PHA scaffolds for clinical use in support of cartilage regeneration in small and contained osteoarthritic lesions.