Fibrin is a provisional matrix during tissue repair and is an attractive biomaterial for cell delivery. Fibrin hydrogel properties can be tuned using a variety of techniques including altering protein concentrations, pH, calcium content, and overall ionic strength. As an alternative to increasing the concentration of clotting proteins, we demonstrated that tailoring the NaCl content in the pre-gel solution alters gel stiffness, pore size, fiber diameter, and permeability. The supplementation of fibrin gels with increasing NaCl concentrations slowed gelation time from 4 minutes to 10 minutes, retaining gelation time within in a clinically acceptable period without risking osmotic shock.

We hypothesized that fibrin gels with increased bulk stiffness would provide substrate-mediated cues to entrapped cells, and these cues would instruct cell function. We examined this hypothesis in two distinct applications: neurogenesis and osteogenesis. Neurite extension from dorsal root ganglia (DRGs) entrapped in fibrin gels correlated with substrate stiffness. We observed significant decreases in neurite length as NaCl content increased from 0.8% (w/v) to 3.5% (w/v). Furthermore, we observed significant reductions in degradation area surrounding the entrapped DRG with increasing salt concentration.

We determined that neurite extension within fibrin gels is dependent on fibrinolysis and is mediated by the secretion of serine proteases and MMPs by entrapped DRGs, as confirmed by culturing cells in the presence of inhibitors against these enzymes and real-time-polymerase chain reaction. We also explored the contribution of fibrin gels with varying NaCl content toward the dual potential of mesenchymal stromal cells (MSC) in bone repair: osteogenic differentiation and trophic factor secretion.

Gel volume was better preserved in fibrin hydrogels containing greater NaCl concentrations (which also exhibited higher compressive moduli), while human MSC entrapped in fibrin gels with lower NaCl content rapidly contracted the material. Early MSC proliferation was markedly accelerated (over 200% increase in DNA) in gels containing less NaCl compared to gels with the highest NaCl content. Alkaline phosphatase activity and calcium deposition, early and late indicators of osteogenic differentiation, respectively, also were highest in fibrin gels with more NaCl, yet secretion of vascular endothelial growth factor (VEGF) was lowest. Fibrin gel osteoconductivity was dramatically improved by adding polymeric substrata coated with bone-like mineral, exhibiting near linear increases in both calcium and phosphate entrapment over 21 days.

Collectively, these data demonstrate that the biophysical properties of fibrin gels can be tailored by the simple addition of NaCl. Salt-mediated control of fibrin gel properties is sufficient to significantly direct cell function as it relates to tissue engineering and regenerative medicine.

Kent Leach

Univ. of California, Davis, UC Davis School of Medicine

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