Cartilage is a realistic target for tissue engineering given the avascular nature and cellular composition of the tissue. Much of the work in this field has been largely empirical, indicating the need for alternative approaches to the design of cartilage formation protocols. Given the heterogeneity associated with human mesenchymal populations, continuous cell lines may offer an alternative to model and simplify cartilage generation protocols. We therefore exploited the potential of the murine chondrocytic ATDC5 cell line to, i) delineate the process of chondrocyte differentiation in monolayer culture and three-dimensional micromass pellet culture systems, and ii) model cartilage formation utilising appropriate scaffold and bioreactor (perfused and rotating) technologies. Monolayer cultures of ATDC5 cells over a 28-day period in presence of insulin demonstrated various stages of chondrocyte differentiation- proliferative, pre-hypertrophic, hypertrophic and finally, mineralisation of cartilaginous nodules. This was confirmed by gene and protein expression, by qPCR and Western blotting respectively, of chondrogenic differentiation markers- Sox-9, Bcl-2, Type II and X collagens. Pellet cultures of ATDC5 cells under chondrogenic conditions (10 ng/ml TGF-beta3, 1X ITS {insulin, transferrin, selenium}, 10 nanomolar dexamethasone, 100 micromolar ascorbate-2-phosphate) illustrated a gradual progression from an aggregation of cells at day 7, to initiation of matrix synthesis at day 14, followed by formation of well-defined cartilaginous structures at day 21. Chondrogenic differentiation at day 21 was evident by numerous proliferative/ pre-hypertrophic chondrocytes, staining for Sox-9, Aggrecan, Type II collagen and PCNA, lodged in distinct lacunae embedded in cartilaginous matrix of proteogly-cans and Type II collagen. Inclusion of TGF-beta3 in the chondrogenic medium during pellet culture beyond 21 days maintained the pre-hypertrophic phenotype, even at day 28. In contrast, removal of TGF-beta3, addition of 50 nanomolar thyroxine and reduction of dexa-methasone to 1 nanomolar in the chondrogenic medium stimulated hypertrophy at day 28, evident by down-regulation of Sox-9 expression. ATDC5 cells cultured on Polyglycolic acid fleece in the rotating bioreactor or encapsulated in chitosan /alginate and cultured in the perfused bioreactor for 21 days, formed cartilaginous explants reminiscent of hyaline cartilage. Thus, ATDC5 cells constitute an ideal cell line to elucidate the steps of chondrocyte differentiation and cartilage formation.

The ability to generate replacement human tissues on demand is a major clinical need. Indeed the paucity of techniques in reconstructive surgery and trauma emphasize the urgent requirement for alternative strategies for the formation of new tissues and organs. The idea of biomimesis is to abstract good design principles and optimizations from nature and incorporate them in the construction of synthetic materials and structures. Direct appropriation of natural inorganic skeletons is also biomimetic since their unique properties inform us on ways to generate functional, optimized scaffolds.

A number of well characterized natural skeletons were investigated as potential scaffolds for tissue regeneration using mesenchymal stem cell populations. Marine sponges, sea urchin skeletons and nacre were found to possess unique functional properties that supported human cell attachment, growth and proliferation and provided organic/ inorganic extracellular matrix analogues for guided tissue regeneration.

A good understanding of the processses involved in biomineralisation and the emergence of complex inorganic forms has inspired synthetic strategies for the formation of biological analogues (organised inorganic materials with biological form). We have developed two functional examples of biological structures generated using biomimetic materials chemistry with applications for human tissue regeneration. Mineralised biopoly-saccharide microcapsules provided enclosed micro-environments with an appropriate physical structure and physiological milieu, for the support of the initial stages of tissue regeneration combined with a capacity to deliver human cells, plasmid DNA and controlled release of biological factors such as cytokines. Calcium carbonate porous microspheres analogous to microscopic coccolithophore shells provided a template for tissue formation and a mechanism for the delivery of DNA and functional biological factors. These biomi-metic structures have considerable potential as scaffolds for skeletal repair and regeneration, particularly when combined with inductive and stimulatory biological factors (cytokines, morphogens, signal molecules) and plasmid DNA carrying with them chemical cues that modulate and direct permanent tissue formation complimentary with the host.

Polysaccharide (alginate and chitosan) capsules coated with a unique self-assembled semi-crystalline shell of calcium phosphate provide an enclosed biological system for the spatial and temporal delivery of human cells and bioactive factors. The aim of this study was to demonstrate plasmid DNA entrapment, delivery and transfection of adjacent cells inside capsules, embedded capsules and plated. Bacterial plasmid DNA and/or bone cells (SaOS) was added to solution of sodium alginate solution supplemented with phosphate ions and mixed thoroughly. Alginate droplets were fed through a syringe into a solution of chitosan supplemented with calcium ions. Guest capsules were inserted into soft, pliable host capsules soon after immersion in chitosan solution. Capsules were then immersed in 2mL DMEM 10% FCS in 6-well plastic plates for up to 7 days to enable transfection to occur. Encapsulated bone cells were stained with standard X-Gal to show transfected cells expressing beta-galactosidase. DNA delivery and transfection was demonstrated within capsules containing SaOS cells and plasmid, an admixture of SaOS bone cells and plasmid (51%) and from capsules containing DNA alone suspended in media over plated SaOS one cells. We also demonstrate capsule transfection of encapsulated cells in vivo. Transfection efficiency is highest when plasmid is entrapped and released from embedded capsules followed by plasmid/ SaOS admixture within capsules and lowest efficiency was observed with plated SaOS cells (with a transfection efficiency of 5%). The ability to regulate shell decomposition by manipulating the degree of mineralization and the strength of gelling, and release of capsule contents provides a mechanism for programmed release of gene modulated cells into the biological environment. The beta-galactosidase plasmid was found to be strongly associated with the chitosan/ calcium phosphate shell as shown by ethidium-homodimer-1 staining of encapsulated DNA and this may assist the transfer from gel to cell. Programmed non-viral delivery of genes using biomaterial constructs is an important approach to gene therapy and orchestrated tissue regeneration. These unique biomineralised polysaccharide capsules provide a facile technique, and an enclosed biomimetic micro-environments with specifiable degradation characteristics, for the safe encapsulation and delivery of functional quantities of plasmid DNA with the implicit therapeutic implications therein.