Alginate foams as biomaterials
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Development of biomaterials is crucial for progress in the field of tissue engineering and regenerative medicine. The intention of biomaterials is to interface with biological systems to evaluate, treat, augment or replace any tissue, organ or function of the body. The utility of alginate as a biomaterial in tissue engineering has recently gained increasing interest. A wide variety of uses have been identified including regeneration of various tissues, delivery vehicles for bioactive molecules such as drugs, growth factors and genes, implantable space filling agents and as artificial extracellular matrices (ECM) providing support for cells in both in vivo and in vitro systems. The present work describes the development and characterization of a novel type of macroporous alginate based foams. The alginate foam is prepared by the steps of: (i) high shear mixing for incorporation of air into a viscous solution containing alginate, water, plasticizer, foaming agent and a source of gelling ions that is insoluble at neutral conditions (calcium carbonate (CaCO3)/strontium carbonate (SrCO3)), (ii) addition of a hydrolyzing glucono-δ-lactone (GDL) that induce slow dissolution of the carbonate salt, (iii) transfer of the wet foam into trays for gelation, and (iv) air drying to receive pliable and soft sheets of foams. The gelation method is well known for preparation of homogeneous alginate hydrogels, however, it has not before been utilized for preparation of macroporous dried alginate foams. Pore structure, hydration properties, mechanical properties, degradability and biocompatibility are all highly relevant for the functionality of a biomaterial scaffold. The flexibility of the method allows tuning of these properties by process and formulation parameters. The mechanisms that were influenced in the preparation process to modify the foam properties were (a) the foam density controlled by the amount of air incorporated, (b) coalescence and gelling rate of the wet foam before drying controlled by the amount of air incorporated, amount and type of gelling ion and alginate, and gelling time, (c) gel network controlled by the amount and type of gelling ion and alginate, and gelling time, (d) weight average molecular weight (MW) controlled by technique for terminal sterilization. A rabbit study was conducted to evaluate biocompatibility and degradability of the foam in vivo. Intramuscular implantation of alginate foam discs showed degradation rates related to concentration of gelling calcium ions (Ca2+) and MW. No adverse systemic signs or mortality were noted and body weights and organ weights of the rabbits were unaffected. Implantation-related inflammation changed over time as typically seen in a healing process and a transient foreign body reaction occurred that mirrored the foam degradation. The overall results showed that the foams were well tolerated. To investigate the possibility of using alginate foams as scaffolds for 3-dimentional (3D) cell cultivation, a new technique for cell immobilization was utilized to ensure gentle and fast entrapment of cells that were evenly distributed throughout the thickness of the foam. First the cells were suspended in an alginate solution and applied to a Ca alginate foam. As the solution was absorbed by the foam, the pore walls became rehydrated and Ca2+ diffused from the foam and induced gelation of the added alginate solution in situ filling the foam pores. Thereby cells were entrapped in a transparent foam-gel composite structure. The cells were cultured in such gels for extended periods and different types of multicellular structures were formed inside. Isolation of cells and intact multicellular structures were possible due to the de-gelling properties of citrate which chelates divalent cations, hence the gel dissolves. High viability of cells was retained inside the gels and controlled modification of the cell-matrix interactions were demonstrated by inclusion of alginates with covalently attached peptide sequences. The peptide sequence used was glycine-arginine-aspartic acid-glycine-proline (GRDGSP), which is present on several ECM proteins and function as a cell adhesion motif. This facilitated cell-matrix interactions which stimulated proliferation in a dose dependent manner, although variations in sensitivity between the different types of cells were seen. Tunable elastic properties of the gels were shown over the range of 1-16 kPa, hence the elasticity can be optimized for different cell types and matches the elasticity of different soft tissues. The applicability of macroporous alginate foams as biomaterials was demonstrated here. The demonstrated mechanistic and functional relationships between operational and formulation parameters can be used as a tool to tailor physical and biological properties. Good biocompatibility was confirmed by in vivo animal studies in addition to in vitro cell cultivation studies.