176 Formulational Dependence of Human Mesenchymal Stem Cell Behavior in Fibrin Sealant

Abstract

Objective: The use of fibrin as an injectable cell carrier to repair osseous defects has been reported with varying degrees of success. This study investigates fibrin formulational dependence on human mesenchymal stem cell (hMSC) proliferation and differentiation. Methods: 2 × 104 hMSCs (Bio Whittaker) per fibrin gel were suspended in fibrin formulations with varied fibrinogen (5, 17, 50 mg/ml) and thrombin (250, 167, and 1 U/ml); and cultured in 24-well plates at 37 °C, 5% CO2 for up to 2 weeks. Formulational influences on clot structure were evaluated by confocal microscopy and electron microscopy. hMSC proliferation and morphology were monitored by calcein staining. hMSC-conditioned medium was assayed using zymography (MMP-2, MMP-9), fibrin overlay (uPA), and reverse overlay (PAI-1) to examine the effect of fibrin formulation on protease activity and fibrinolysis. Results/Discussions: Confocal and electron microscopy revealed formulation dependence on three-dimensional fibrin structure, with lower fibrinogen concentrations yielding more open, homogeneous microstructures. Although hMSCs are viable in all fibrin formulations investigated, proliferation rates varied with fibrinogen concentration, with lower fibrinogen concentrations (i.e., 5 mg/ml) promoting greater hMSC proliferation. Protease levels and activities were sensitive to culture time and fibrin formulation. PAI-1 expression was elevated in hMSCs inside fibrin gels, while uPA activity was low. Both the pro and active form of MMP2 was detected by gelatin zymography, but no MMP9 was detected. This study suggests that fibrinogen:thrombin ratio can significantly influence hMSCs proliferation and interactions, and should be carefully considered when developing formulations for optimal delivery of hMSCs. Acknowledgments: UCLA Bioengineering Departmental Fellowship (WH); NIH Grant R01GM055081 (TLT)