Mineralized polysaccharide capsules as biomimetic microenvironments for cell, gene and growth factor delivery in tissue engineering

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1 HIGHLIGHT Soft Matter Mineralized polysaccharide capsules as biomimetic microenvironments for cell, gene and growth factor delivery in tissue engineering David W. Green, a Stephen Mann b and Richard O. C. Oreffo* a DOI: /b604786f This paper presents an overview of our recent studies on mineralized polysaccharide capsules and their potential as multifunctional scaffolds for the organisation and delivery of human cell types, DNA and growth factors. Microcapsules containing these encapsulates are readily produced at room temperature in the form of alginate beads that are stabilized by an outer shell consisting of an ultra thin chitosan calcium phosphate membrane. Modulation of the shell thickness and composition influences the rates of release and diffusion of encapsulated cells, growth factors and genes to produce microcapsules with tailored spatial and temporal properties that offer significant potential as novel biomimetic delivery systems in tissue engineering applications such as skeletal tissue repair and regeneration. We demonstrate the use of mineralized microcapsules to enable regeneration from encapsulated human mesenchymal populations, functional quantities of growth factor capture, and gene transfection. Significantly, we highlight the ability to fabricate integrated capsules consisting of spatially separated multiple components in nested combination that may provide phased temporal release of appropriate growth factors, analogous to the natural regeneration process. a Bone & Joint Group, Developmental Origins of Health & Disease, University of Southampton, UK. roco@soton.ac.uk; dwg@soton.ac.uk b School of Chemistry, University of Bristol, Bristol BS8 1TS, UK. s.mann@bris.ac.uk Introduction Matrices generated from natural polysaccharides such as starch, cellulose derivatives, heparin sulfate, hyaluronic acid, chitin, chitosan, and alginate are receiving considerable attention as biomimetic environments for cell adhesion, differentiation and proliferation, and as scaffolds in tissue engineered constructs. 1,2 These materials have a range of properties, are readily available, easily processed and bio-compatible, and are therefore of immediate clinical relevance. For example, sodium alginate, which is present in large amounts in seaweed, is David W. Green Dr David Green received his BSc in botany and zoology from the University of Reading in 1994, where he specialized in biomimetics and the structural design of organisms under the tutorship of Julian Vincent. After working at the Natural History Museum, London he completed aphdatastonuniversityto develop a biomimetic artificial cornea. His current interest is the development of biomimetic approaches to tissue engineering scaffold design and synthesis and the use of natural skeletal biomatrices. Stephen Mann Stephen Mann is Professor in the School of Chemistry and Director of the Centre for Organized Matter Chemistry at the University of Bristol. His current research interests concern the biomimetic synthesis, characterization and emergence of complex forms of organized matter across extended length scales. He has published over 300 scientific papers and was elected as Fellow of the Royal Society, UK, in Soft Matter, 2006, 2, This journal is ß The Royal Society of Chemistry 2006

2 an anionic polysaccharide that can form soft, compliant hydrogels by cross-linking with Ca 2+ ions. In contrast, chitosan is a water-soluble cationic polysaccharide derived by deacetylation of chitin, which can be readily obtained as the stiff, insoluble matrix of crustacean shells. At the supramolecular level, both alginate and chitosan form extended helical structures that are stable in water. Chitosan has been used widely as a scaffold for tissue engineered epidermis 3 and cartilage, 4 and alginate gels are specifically suited toward chondrocyte growth and differentiation particularly when coupled with a RGD (arginineglycine-aspartate) tri-peptide adhesion motif. 5 The sequences of chemical units along the alginate backbone determine the physical (swelling, de-swelling, fracture toughness, elasticity) and pharmacological (binding associations, diffusion coefficients, and release profiles) properties, as well as the bioactivity. In particular, the strength of the alginate network and associated level of biological activity is dependent on the quantity of guluronic acid residues combined with the size and frequency of guluronic blocks along the polymer backbone. For example, alginates with high guluronic acid concentrations form stiffer gels with greater porosity and less swelling, while mannuronic-enriched gels are softer and less porous but swell more readily 6 Other studies have shown that films and gels of chitosan or alginate are amenable to controlled inorganic crystallization 7,8 indicating that numerous types of inorganic organic composites can be prepared based on these biopolymers. Combinations of alginate and chitosan in the form of stabilised microcapsules Richard O. C. Oreffo are of considerable current research interest as the gel-like beads offer convenient and effective systems for numerous biotechnological, pharmacological and biomedical applications. 9,10 Bioactive components are usually entrapped within millimetre-sized gel beads of Ca-alginate and coated with polycationic polyelectrolytes, such as poly-l-lysine or chitosan, to improve the microcapsule stability and controlled permeability The coating process is readily achieved for example by immersing droplets of Ca-alginate into chitosan solutions or sodium alginate into Ca-containing solutions of chitosan. In both cases, interfacial charge matching between the anionic and cationic polysaccharides produces a semi-permeable membrane around the alginate droplet. 14 Moreover, the gel-like interior can be subsequently liquified by removing the metal ion-induced cross-links using a Ca 2+ -complexing agent such as citrate to produce hollow microcapsules. 15 Previous studies have shown that the mechanical strength and permeability of the alginate chitosan membrane surrounding the polysaccharide microcapsules are influenced by changes in the polyelectrolyte molar masses and concentrations. 12 Moreover, Livage and co-workers 15 demonstrated that a thin external coating of amorphous silica can be deposited on preformed alginate poly-l-lysine microspheres, and that the composite structure had improved mechanical strength and controlled release behaviour. Herein, we summarise some recent studies on the fabrication and use of novel alginate chitosan microbead constructs that comprise outer shells of calcium phosphate and Richard Oreffo is Professor of Musculoskeletal Science and Director of Enterprise in the School of Medicine, University of Southampton. He leads a group focused on developing strategies to regenerate bone and cartilage using stem cell technology, gene/growth factors and innovative scaffolds. He has published over 65 peer-reviewed papers and is a member of the ARC Scientific Research Committee and MRC Basic Users Stem Cell Panel. internally encapsulated bio-components such as human bone marrow stromal cells. Our approach is based on mimicking natural processes such as biomineralization, in particular, the use of diffusion-controlled processes for the deposition of inorganic minerals within porous organic polymeric matrices. 16 This method has the key advantage that changes in the mineralization process can be tailored to meet specific requirements associated with cell growth and differentiation, release of functional biomolecules, and gene delivery, for example. Such materials should have significant potential as hybrid constructs in applications such as skeletal tissue engineering, bone repair and regeneration. Preparation of mineralized polysaccharide microcapsules Mineral-polysaccharide microcapsules are produced by a one-step method in which the deposition of a porous alginate chitosan membrane around droplets of sodium alginate is coupled with the controlled precipitation of calcium phosphate arising from counter-diffusion of ions across the polysaccharide interface. Typically, millimetre-sized gel beads of Ca-alginate chitosan are prepared by adding droplets of sodium alginate to Ca 2+ -containing aqueous solutions of chitosan at near neutral ph (Fig. 1a, b). The size and shape of the beads can be controlled through the use of different types of syringe tip. In each case, a thin white membrane is observed immediately around the alginate droplet due to interfacial complexation of the oppositely charged polyelectrolytes (Fig. 1c, d). This outer membrane hardens with time due to deposition of calcium phosphate but remains permeable to Ca 2+ ions, such that within 1 h the droplets are also internally stabilized in the form of gellike beads due to cross-linking of the alginate network with Ca 2+ ions. The mineralized microspheres can be readily handled with tweezers, stored in distilled water for several months or oven-dried without loss of structure (Fig. 1e). Nucleation of calcium phosphate occurs within the membrane rather than as a discrete external coating, and therefore the thickness of the mineralized outer shell is readily controlled by the phosphate concentration present in the This journal is ß The Royal Society of Chemistry 2006 Soft Matter, 2006, 2,

3 Fig. 1 Fabrication of mineralized polysaccharide capsules and nested structures. (a) Phosphatecontaining solution of sodium alginate solution and syringe for droplet transfer. The solution also contains encapsulates such as cells, DNA and functional biomolecules. (b) Spontaneous formation of phosphate-containing alginate beads by addition of droplets into a calciumcontaining solution of chitosan. (c) Schematic showing formation of mineralized chitosan alginate outer membrane on alginate droplet containing encapsulates (black squares). The calcium phosphate shell is produced by counter diffusion of ions across the polysaccharide membrane, and the alginate interior is gelled by crosslinking with Ca 2+ ions. (d) Magnified optical image of edge of a mineralized microcapsule showing calcium phosphate outer shell (thin white line) (scale bar = 200 mm). (e) Optical micrograph of a single capsule, (scale bar = 50 mm). (f) Manual insertion of a small guest capsule with high mineral content (white circle) into a host capsule within a mineralized shell (scale bar = 3 mm). alginate droplets. In addition, the core and shell properties can be carefully modulated particularly by alteration of calcium and phosphate ratios and gelling time. 17 Initial studies indicated that the mineralized microspheres show enhanced mechanical strength and reduced permeability to encapsulated molecules such as haemoglobin and ibuprofen. 17 Moreover, as the beads have potential as biomimetic microenvironments for tissue regeneration and as delivery systems for cells, genes and biomolecules, various types of cells, DNA and growth factors were incorporated into the mineralized polysaccharide capsules under different conditions with the aim of tailoring the diffusion and mechanical properties that matched specific biological requirements. Examples of these studies are described in detail in the following sections. adipocytes, chondrocytes, bone marrow cells, and mesenchymal stem cells have been encapsulated within mineralized polysaccharide capsules (Fig. 2a, b). 18 Confocal microscopy of capsules stained with a fluorescent dye indicates that the encapsulated cell populations are viable for up to 14 days. Immunoselected and un-selected human osteoprogenitor cells stain positively for alkaline phosphatase activity at two weeks showing excellent maintenance of the osteoblast phenotype within the capsules. Similarly, histological analyses of encapsulated adipocytes or chondrocytes indicate that these cell populations remain viable after seven days of incubation within the mineralized microspheres. Alginate matrices have been shown to be suitable for the culture of chondrocytic populations as the polysaccharide is homologous to host cartilage matrix tissue, and the conformation of cells embedded in the alginate hydrogel is analogous to the conformation of cartilage cells in vivo. In vitro cell viability is high and can vary between 73 81% depending on cell type and density of cell contacts. 19 Significantly, encapsulation of admixtures of cell types within mineralized polysaccharide capsules results in improvements in the quantity and maturity of in vitro tissue formation compared with single cell types. 20 This effect is dependent on cell loading. In the absence of appropriate biomolecules the cells are relatively inactive within the microcapsules, but increased levels of cell organization should be possible by incorporating spatial and signalling cues into the alginate backbone by chemical augmentation. The above results indicate that the alginate interior of mineralized polysaccharide capsules functions as an extracellular matrix (ECM) analogue for capturing and positioning cells in spatial arrays. The outer shell comprises a semi-permeable membrane reinforced by calcium phosphate, which together provide a diffusive barrier that shields and protects encapsulated cells from potentially detrimental external factors such as neutrophils, mast cells, B cells, etc., while spatially confining desirable Human cell encapsulation within mineralized polysaccharide capsules A number of human cell types and immortalised cell lines, including Fig. 2 Mineralized polysaccharide capsules containing: (a) human bone marrow stromal cells, and (b) immuno-selected mesenchymal stem cells stained red for the phenotypic marker of bone, alkaline phosphatase, indicating the osteogenic phenotype of these cells. (Scale bars = 0.5 mm) 734 Soft Matter, 2006, 2, This journal is ß The Royal Society of Chemistry 2006

4 growth factors. Importantly, as cell-to- ECM and cell-to-cell contacts stimulate or induce cells to differentiate and organise into coherent arrays or networks through specific non-covalent macromolecule interactions that culminate in tissue formation, it seems feasible that these processes could be modulated by fine-tuning of the chemical and physical properties associated with the microcapsule interior. Human cell, growth factor and gene delivery within polysaccharide capsules in vitro and in vivo tissue formation Tissue formation is known to occur within alginate and alginate chitosan complexes. For example, Alsberg et al. demonstrated in vivo formation of complex, mature cartilage tissue, including a growth plate analogue, within an alginate matrix. 21 This occurred however after a period of 40 weeks, and tissue engineering specifications ideally require tissue formation within shorter time frames. This requires biochemical induction and further spatial and positional cues such as those provided by cell membrane peptide sequences like RGD that are involved in human cell attachment. Rowley and Mooney were particularly successful in chemical attachment of sequences to alginate polymers with concomitant improvement in cell responses and activities. 21,22 Furthermore, increased synergy between engineered tissue assembly and host tissue assembly requires physiologically responsive scaffolds that are activated and actuated by cues present during healing and host bone formation. Current strategies favour using cell populations cultured in vitro onto scaffolds possessing an osteogenic phenotype as differentiated populations appear to interact better with host bone cells and generate enhanced natural bone regeneration. 23 A key factor in tissue engineering is to provide scaffolds with osteogenic potential (for example impregnated with the bone morphogenic protein, BMP-2) combined with characterised cells displaying an osteogenic phenotype. 24 In this respect, we used mineralized polysaccharide microcapsules for cell and growth factor co-encapsulation to demonstrate that capture of functional Fig. 3 In vivo bone tissue formation within polysaccharide capsules. (a,b) Thin section showing osteoid formation by human marrow stromal cells encapsulated within a mineralized polysaccharide capsule containing rhbmp-2 (100 ng ml 21 ), and after four weeks implantation (scale bar = 100 mm). (c) X-Ray image of polysaccharide microcapsules containing human bone marrow stromal cells and rhbmp-2, and incubated within a diffusion chamber for eight weeks (scale bar = 2.5 mm); arrows indicate areas of mineralised tissue formation within the capsules. (d) Control X-ray image of diffusion chamber containing capsules with human bone marrow stromal cells but no rhbmp-2 growth factor after eight weeks (scale bar = 2.5 mm). quantities of biological growth factors together with the controlled endogenous and exogenous delivery to human cell populations can be used successfully for cell differentiation and osteoinduction. 18 Functional delivery was determined by encapsulation of the growth factors rhbmp-2 and BMP retentate (an admix of BMP-2,3,4,5,7 molecules) within polysaccharide capsules containing promyoblast C2C12 cells and human mesenchymal populations. Induction of C2C12 cells occurs along a pre-determined osteoblast lineage. Similarly, rhbmp-2-containing capsules co-cultured with plated C2C12 cells induces cell differentiation and significantly enhances bone tissue formation specifically within the polysaccharide microspheres. In an animal model enhanced bone tissue formation, as shown by the presence of osteoid, occurs inside capsules containing rhbmp-2 and human marrow stromal cells at four weeks (Fig. 3a,b). Bone mineralization occurs at eight weeks as shown by X-ray analysis (Fig. 3c,d). Other factors are able to supplant or augment rhbmp-2 such as pleiotrophin or an admixture of BMPs and TFG-beta. In addition, growth factor stimulation of encapsulated mesenchymal cell populations can occur within in vitro static culture using capsules carrying rhbmp-2 or mesenchymal stem cells (MSC) alone embedded within an rhbmp-2 host capsule. rhbmp-2 delivered using such strategies augments MSC growth and differentiation. 25 We found that within a period of 7 21 days capsules readily disintegrate due to swelling or encapsulated cell activities (Fig. 4a c). The challenge is to prescribe these processes of cell encapsulation, activation and release in a designated manner. In other studies, plasmid DNA constructs (pclacz, pcgfp) co-cultured within mineralized polysaccharide capsules were shown to display functional activity leading to cell transfection. 26 This procedure offers an innovative alternative approach to gene delivery as it provides a safe and sustainable method for implantation of the recombinant cells Fig. 4 Capsule fragmentation and release properties. (a) Initial stage of capsule fracture at seven days showing tearing of the outer shell (top arrow) and across the alginate core (scale bar = 0.5 mm). (b) Optical image of a highly mineralized capsule containing a dispersion of hydroxyapatite powder showing extensive rupture after 21 days due to extensive swelling of the alginate core (scale bar = 1 mm). (c) Remnants of a ruptured capsule showing released mesenchymal stem cells adhered to a plastic tissue plate surface and stained to show alkaline phosphatase activity (scale bar = 50 mm). This journal is ß The Royal Society of Chemistry 2006 Soft Matter, 2006, 2,

5 Fig. 5 Host guest capsule systems: (a) single polysaccharide capsule containing high density of hydroxyapatite particles (scale bar = 1 mm). (b) Cross-section through a single mineralized polysaccharide capsule containing human bone marrow stromal cells mixed with chitin nanofibrils to generate an open core texture for enhanced cellular contacts and activities. Sample is after 12 days; (red arrow: human bone marrow stromal cells; white arrow: chitin nanofibril domains; yellow arrow: chitosan) (scale bar = 20 mm), (c) single mineralized polysaccharide capsule containing nacre particles (yellow arrows) and human bone marrow stromal cells stained for alkaline phosphatase (scale bar = 0.5 mm). (d) Single guest capsule (green sphere) embedded within host capsule containing human bone marrow stromal cells stained for cell viability (green fluorescence) (scale bar = 0.5 mm). and the functional expression of their recombinant products under conditions of controlled release. Work is additionally being carried out toward intracapsular and extra-capsular plasmid transfection of primary and immortalised human bone cells as well as the delivery of functional biomolecules, ECM components and bioactive reagents such as hydroxyapatite (Fig. 5a), glycosaminoglycans, chitin nanofibrils (Fig. 5b) and seashell nacre platelets (Fig. 5c). Host guest capsule system The procedure for preparing mineralized polysaccharide microspheres can be developed for the generation of more complex architectures such as capsuleswithin-capsules (Fig. 5d). These nested structures enable multiple cell types and bio-factors to be loaded and spatially differentiated within the confinement of a single discrete entity that can exhibit modulated degradation properties. The method involves the physical insertion of gelled guest capsules into soft pre-formed microspheres. Moreover, the host and guest beads can have different mineral contents and therefore different rates of shell disintegration allowing for multitemporal release of various biological factors embedded with guest capsules. For example, encapsulated mesenchymal populations display enhanced alkaline phosphatase expression (a known osteogenic marker) following co-culture within a guest capsule embedded within an rhbmp-2-containing host capsule. The functional inter- and counter-diffusion of growth factors in these nested structures is confirmed by differentiation of C2C12 cultured within a host capsule containing a rhbmp-2 guest capsule. In other studies, double layered capsules are generated in which the encapsulated cells are embedded within a shell surrounding a highly mineralized alginate core containing a dispersion of hydroxyapatite powder. These constructs are analogous to the cellular assemblies associated with boundary organized bone formation. 16 Conclusions and future work The above studies demonstrate the potential of mineralized polysaccharide capsules for cell, growth factor and gene delivery and the implications therein for tissue engineering. The simplicity and adaptability of these capsules provides significant potential for exploitation of many human cell, gene and pharmaceutical drug-based therapies. Our work demonstrates that the capsules can be programmed to disintegrate depending on the composition of the outer shell and the strength of gelation in the alginate interior. Thus, the polysaccharide capsules provide a suitable and viable environment for a wide range of cell types, biomolecules and genes. In addition, host guest capsules enable different factors to be co-encapsulated within a spatially delineated environment to provide synergies between cells and their interactions with stimulatory/inductive factors to orchestrate regenerative processes. By providing environmental conditions that drive higher order cellular functions such as controlling mineralization (as opposed to differentiation and gene expression), these structures meet a fundamental and strategic tissue engineering aim. There is a diverse number of possible developments and novel applications for mineralized polysaccharide capsules, particularly in tissue engineering and biotechnology. Current work in our group is focused on the combination of multiple cell types and growth factors involved in musculoskeletal repair, together with release profiles in a prescribed order. In addition, we propose to chemically tailor alginates to include useful peptide sequences and functional biomolecules, and combine bio-responsive additives such as hydroxyapatite with the alginate core. Attempts are also being made to coat individual cells with alginate chitosan CaP thin films using emulsion technologies. Methods are also being developed to generate capsular nanostructures and macrostructures comprising bonded arrays of mineralized polysaccharide beads. Ultimately, in vivo work will be required to validate these strategies using clinically relevant models to demonstrate the potential for bone repair in the first instance as well as the utility of such exciting biomimetic approaches in other hard and soft tissues. Mineralized microcapsules provide facile tissue engineering scaffold technology for effective, tailored delivery of growth factors, enzymes, genes and human cells. Microcapsules can also be tailored for a range of cell and tissue 736 Soft Matter, 2006, 2, This journal is ß The Royal Society of Chemistry 2006

6 types. Development of such strategies will be essential for enhanced effectiveness of a tissue engineered approach to repair and regeneration of hard and soft tissues. References 1 S. V. Madihally and H. W. Matthew, Biomaterials, 1999, 20, K. H. Bae, J. J. Yoon and T. G. Park, Biotechnol. Prog., 2006, 22, G. Saintigny, M. Bonnard, O. Damour and C. Collombel, Acta Derm. Venereol., 1993, 73, J. K. Suh and H. W. Matthew, Biomaterials, 2000, 21, N. G. Genes, J. A. Rowley, D. J. Mooney and L. J. Bonassar, Arch. Biochem. Biophys., 2004, 422, A. Martinsen, G. Skjåk-Bræk and O. Smidsrød, Biotechnol. Bioeng., 1989, 33, C. C. Barrias, M. Lamghari, P. L. Granja, M. C. Miranda and M. A. Barbosa, J. Biomed. Mater. Res. A, 2005, 74, H. Wakayama, S. R. Hall and S. Mann, J. Mater. Chem., 2005, 15, M. F. A. Goosen, G. M. Oshea, H. M. Gharapetian, S. Chou and A. M. Sun, Biotechnol. Bioeng., 1985, 27, P. R. Hari, T. Chandy and C. P. Sharma, J. Microencapsulation, 1996, 13, A. Bartkowiak and D. Hunkeler, Chem. Mater., 1999, 11, A. Bartkowiak and D. Hunkeler, Chem. Mater., 2000, 12, O. Gåserød, O. Smidsrød and G. Skjåk- Bræk, Biomaterials, 1998, 19, O. Gåserød, A. Sannes and G. Skjåk- Bræk, Biomaterials, 1999, 20, T. Coradin, E. Mercey, L. Lisnard and J. Livage, Chem. Commun., 2001, 23, S. Mann, in Biomineralization; Principles and Concepts in Bioinorganic Materials Chemistry, Oxford University Press, Oxford, UK, I. Lévêque, K. H. Rhodes and S. Mann, J. Mater. Chem., 2002, 12, D. W. Green, I. Lévêque, D. Walsh, D. Howard, X. B. Yang, K. Partridge, S. Mann and R. O. C. Oreffo, Adv. Funct. Mater., 2005, 15, D. W. Green, K. A. Partridge, I. Lévêque, D. Walsh, R. Tare, S. Mann and R. O. C. Oreffo, J. Bone Miner. Res., 2005, 20, J. C. Pound, D. W. Green, J. B. Chaudhuri, H. I. Roach and R. O. C. Oreffo, Eur. Cells Mater., 2005, 10, E. Alsberg, K. W. Anderson, A. Albeiruti, J. A. Rowley and D. J. Mooney, Proc. Natl. Acad. Sci. U. S. A., 2002, 99, J. A. Rowley and D. J. Mooney, J. Biomed. Mater. Res., 2002, 60, A. J. Salgado, M. E. Gomes and R. L. Reis, in Learning from Nature How to Design New Implantable Biomaterials, ed. R. L. Reis and S. Weiner, Kluwer Academic, Dordrecht, 2003, ch. 4, pp R. O. C. Oreffo, C. Cooper, M. Mason and M. Clements, Stem Cell Rev., 2005, 1, D. Green, I. Lévêque, D. Walsh, D. Howard, S. X. Yang, K. Partridge, S. Mann and R. O. C. Oreffo, J. Bone Miner. Res., 2004, 19, D. Green, K. Partridge, I. Lévêque, S. Mann and R. O. C. Oreffo, Int. J. Exp. Path., 2005, 86, A6. This journal is ß The Royal Society of Chemistry 2006 Soft Matter, 2006, 2,