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.

Introduction: The clinical need for a biodegradable material with broad application is evidenced by the fact that tissue loss as a result of injury or disease provides reduced quality of life for many at significant socio-economic cost. The development of simple biodegradable materials, with broad applicability and tissue/ cell specificity has to date proved elusive. Natural biopolymers such as alginate and chitosan are structural biomaterials of increasing significance to tissue repair and regeneration due to their potential for fabrication, design and efficient, environmentally benign synthesis. We describe the development of innovative microcapsule scaffolds based on chitosan and alginate that can be tailored to a range of cell types for a variety of tissues.

Methods: Semi-permeable polysaccharide microcapsules were produced by a one-step method, in which the deposition of a semi-permeable alginate/chitosan membrane around droplets of sodium alginate was coupled with in-situ precipitation of amorphous calcium phosphate as described by Leveque et al (2002)*. A variety of human cell types including mesenchymal stem cells, osteoprogenitors selected using the STRO-1 antibody by magnetically activated cell separation (MACS), osteoprogenitors transfected with adenovirus expressing Green Fluorescent Protein (GFP) and chondrocytes were mixed with sodium alginate and encapsulated within alginate/chitosan and calcium phosphate.

Results: Hybrid spheres (750–10,000um) were generated encapsulating primary human osteoprogenitor cells, STRO-1 selected osteoprogenitors and AdGFP transfected osteoprogenitors. Encapsulated cells remain viable inside the polysaccharide microcapsules for 2 weeks as shown by positive alkaline phosphatase staining of encapsulated cells. Cells expressing GFP were observed within microspheres indicating the e ability to deliver cells/factors as well as the potential for gene therapy. Encapsulation and delivery of active BMP-2 was confirmed using the promyoblast cell line C2C12 known to be exquisitely sensitive to BMP-2. Nucleation of calcium phosphate occurred within the polysaccharide membrane and could be controlled by the phosphate concentration in the alginate droplets to produce hybrid microcapsules with enhanced mechanical strength. Thin walled capsules were shown to split and degrade in culture within 2–4 days releasing viable osteoprogenitor cells indicating the ability to manipulate the mechanical integrity and to programme degradation of the microspheres. Finally we have shown that aggregation of the microspheres into extended frameworks can be achieved using a designed droplet/vapour aerosol system resulting in foams of aggregated beads.

Discussion and Conclusion: A variety of human skeletal cells have been encapsulated within polysaccharide/ calcium phosphate microspheres and extended frameworks with specifiable dimensions. These composite scaffolds offer stable mechanical and chemical biomimetic environments conducive to normal cell function. Natural polysaccharides are also highly amenable to complexation with a range of bioactive molecules and consequently offer tremendous potential in tissue engineering and regeneration of hard and soft tissues.