Our first efforts in this area focused on the formation of calcium phosphate minerals using thermally responsive liposomes, in which mineral formation was activated when the liposome suspension was heated from ambient to physiologic temperature (Chemistry of Materials, vol. 10, 1998, pp. 117-124). Separately, calcium- and phosphate-loaded liposomes were prepared and mixed together to form a suspension highly supersaturated with respect to calcium phosphate solids such as hydroxyapatite. The suspension could be stored for several weeks at room temperature without detectable mineral formation, reflecting the low bilayer permeability of ions in the gel state. However, when the liposome suspension was heated to the Tm of the lipid mixture, calcium and phosphate ions were released into the extravesicular fluid, where they rapidly reacted to form apatite and other calcium phosphate minerals. In subsequent in-vitro experiments we demonstrated how this approach can be used to deposit apatite on human enamel and dentin surfaces by exploiting the increased temperature of the oral cavity (Polyhedron, vol. 13, 2000, pp. 357-363). A more recent paper described the use of liposomal mineralization in conjunction with thermally triggered collagen gel assembly for in-situ formation of a mineralized collagen composite biomaterial (Biomaterials, vol. 24, 2003, pp. 4881-4890). The results of these studies suggest that this approach to mineral formation could be used to remineralize tooth surfaces or even possibly for in-situ formation of bone repair materials.

We have also used this liposome strategy to elicit rapid in-situ formation of peptide and polymer hydrogel biomaterials. By utilizing calcium-reactive macromolecules such as alginates and calcium-dependent enzymes that crosslink proteins, we have developed liposomal suspensions that are stable and fluid at room temperature, but rapidly form polymer hydrogels at physiological temperature. For example, in the alginate system (Biomaterials, vol. 22, 2001, pp. 453-462), a precursor fluid consisting of Ca-loaded liposomes dispersed in a low viscosity Na-alginate solution remained fluid for several days at ambient temperature. Heating of the precursor fluid to Tm triggered Ca2+ release from the liposomes; the Ca2+ in turn crosslinked Na-alginate to form a Ca-alginate hydrogel. Characterization of the gelation kinetics revealed that gelation is very rapid in this system (on the order of tens of seconds) when the temperature of the precursor fluid reaches Tm. This system is suitable for injectable hydrogels for drug delivery and cartilage tissue engineering (ACS Symposium Series, vol. 709, 1998, pp. 203-211).

Other Transglutaminase Crosslinked Hydrogels.
Recently we reported the design and characterization of short peptide substrates of TG enzymes, which when coupled to branched PEG polymers are capable of being crosslinked into hydrogels rapidly under physiologic conditions (Journal of the American Chemical Society, vol. 125, 2003, pp. 14298-14299). Ongoing aspects of this project include the use of combinatorial chemistry to study the effect of peptide length and amino acid sequence on peptide substrate reactivity and specificity. This information will be used to design new conjugates of short TG substrate peptides and biocompatible polymers designed to be rapidly crosslinked by TG enzymes. Additionally, these new polymers are being developed for an injectable cartilage repair strategy, in which an aqueous liposome/polymer/cell suspension will be injected into a cartilage defect, after which the fluid solidifies to entrap chondrocyte or stem cells for regeneration of the defect. Although other groups have developed injectable cartilage repair materials, a unique aspect of our approach is the design of the TG crosslinkable polymers, which are designed to provide direct covalent bonding of the biomaterial to the tissue at the margins of the defect, since macromolecules in cartilage have been shown to be substrates of TG.