Introduction: Treatment of cavitary defects within healthy tissues requires an injectable matrix to provide a framework and restore the functions of the lost extracellular matrix. Gelatin-hydroxyphenyl propionic acid (Gtn-HPA)[1] and hyaluronic acid-tyramine (HA-Tyr)[2],[4] are injectable natural biopolymer matrices that are capable of undergoing horseradish peroxidase (HRP)-catalyzed covalent cross-linking in vivo. Gtn-HPA provides the necessary cell adhesion ligands to promote cell attachment, proliferation[3], and migration, and to facilitate osteogenesis. HA-Tyr offers relative mechanical robustness and the biological benefits of HA[4].
Material and Methods: A range of 2wt% Gtn-HPA/HA-Tyr composites were tested with the goal of identifying a suitable ratio for an osteogenic application: pure Gtn-HPA; Gtn-HPA:HA-Tyr=3:1, 1:1 and 1:3 (v:v); and pure HA-Tyr—all cross-linked by 0.1U/ml HRP and 1.2mM of H2O2. Modulus was evaluated with unconfined compression. Cell viability was calculated by counting live/dead cell numbers, and cell proliferation was monitored at days 1, 4 and 7. 3-D migration assay was designed for continuous observation using an annulus-core construct. Goat bone marrow mesenchymal stem cells (bMSCs) were seeded in collagen I annulus gels (simulating healthy tissue). Growth factors were incorporated into core gels containing different ratios of Gtn-HPA and HA-Tyr. Migrated cells were counted and distances measured on days 4 and 7. For osteogenic differentiation, bMSC-seeded gels were cultured in osteogenic medium for 28 days. Smooth muscle actin was stained to show the formation of nodules whereas osteopontin and osteocalcin were marked to demonstrate the osteogenic differentiation and calcium deposition. All the markers were imaged by confocal microscopy in bulk samples of gels.
Results and Discussion: The compressive modulus for pure HA-Tyr was about 7-fold higher than Gtn-HPA, resulting in higher moduli for the HA-Tyr rich (>50%) gels. A greater number of cells adhered to and spread on the Gtn-HPA rich gels, compared to the HA-Tyr on which the cells remained rounded and cell number was low. Gtn-HPA rich-gels also showed greater cell viability and proliferation rate. In the migration assay employing PDGF-BB as a chemoattractant, a substantially greater number of cells migrated into the high Gtn-HPA ratio gels, while no cells entered the pure HA-Tyr matrix. When 10ng/ml FGF-2 was incorporated into gels together with PDGF-BB, the migrated cell numbers were elevated in Gtn-HPA rich groups, and doubled in the 3:1 group. After 28 days of osteogenic induction, a larger size and greater number of cell nodules and greater calcium deposition were observed within the Gtn-HPA rich gels. In contrast, individually arranged rounded bMSCs with minimal calcium deposition were observed in HA-Tyr high ratio gels.
Conclusion: Gtn-HPA is highly permissive of MSC adhesion, proliferation, migration and osteogenic differentiation. The addition of up to 25% HA-Tyr does not significantly decrease this cell behavior, but contributes to the mechanical behavior and decreases degradation rate. For cell migration, PDGF-BB and FGF-2 in combination are more effective in chemoattraction compared to PDGF-BB alone. Large cell nodules with Ca deposition are found in MSC-seeded Gtn-HPA-rich hydrogels grown in osteogenic medium.
Department of Veteran Affairs, U.S.; A-STAR, Singapore
References:
[1] Wang LS. Biomaterials 2010;31:8608-8616.
[2] Toh WS. Biomaterials 2012;33:3835-3845.
[3] Lim TC. FASEB.J 2013;27:1023-1033.
[4] Kurisawa M. Journal of Materials Chemistry 2010; 20:5371–5375