This study analyzed human mesenchymal stem cell (hMSC) behavior in a fibrin sealant. hMSC morphology, proliferation, and osteogenic differentiation were analyzed after up to 28 days of incubation in eight different formulations of fibrin gels (Tisseel) prepared with various concentrations of fibrinogen complex (FC) and thrombin. Cell morphology and distribution within the gels were observed by fluorescence microscopy after cell staining with calcein dye. Cell proliferation was assessed by measuring the fluorescence intensity of the cell suspension stained with calcein dye after dissolution of the gels. A standard alkaline phosphatase (ALP) assay, von Kossa staining, and real-time reverse transcriptase-polymerase chain reaction (RT-PCR) were used to analyze hMSC osteogenic differentiation. Cell behavior varied depending on the gel formulation. Proliferation was higher in the formulations containing a low FC concentration, but ALP activity was higher in the formulations containing a high FC concentration. Variations in thrombin concentration had a lesser effect. Small nodules of mineralization were observed at days 21 and 28 in a formulation containing a high FC concentration, in addition to a marked increase in bone sialoprotein (BSP) gene expression level as well as a lower increase in ALP and osteopontin (OPN) levels. However, there was no significant increase in osteocalcin (OCN) expression, a late marker of osteogenic differentiation, up to day 28. In conclusion, this study demonstrated that hMSC morphology, proliferation, and osteogenic differentiation in fibrin gels depended on the FC/thrombin ratio. hMSCs appeared to undergo osteogenic differentiation when seeded in Tisseel fibrin sealant containing a high FC concentration, but they did not fully differentiate into mature osteoblasts.
Over the last several years, our in vitro and in vivo studies have focused on optimizing the use of fibrin to deliver cells. We have shown that some three-dimensional (3D) fibrin constructs with specific fibrinogen and thrombin concentration support robust proliferation of normal human dermal fibroblasts, whereas different fibrinogen and thrombin concentrations support high mesenchymal stem cell proliferation in 3D fibrin constructs. In this article, we found that normal human epithelial keratinocytes proliferate well in 3D fibrin constructs consist of fibrinogen concentration ranging from 17 to 33 mg/mL and thrombin concentration of 1 U/mL. Further, using a new proliferation assay, we studied the proliferation of fibroblasts and keratinocytes cocultured in various 3D fibrin constructs of different fibrinogen and thrombin concentrations. We found that 3D fibrin constructs with a range of fibrinogen concentration (5-34 mg/mL) and a thrombin concentration of 1 U/mL produce an optimal cell proliferation for both cell types when cocultured. This profile of proliferation is different from that seen when keratinocytes or fibroblasts are incorporated separately in 3D fibrin constructs. In conclusion, we found that one needs to choose the fibrinogen and thrombin concentration carefully depending on the cell type to deliver; that is, different fibrin constructs with different fibrinogen and thrombin concentration are required to deliver fibroblasts or keratinocytes alone or to codeliver both cell types. Moreover, there seems to be a cross-talk between keratinocytes and fibroblasts when they are cointroduced in 3D fibrin constructs. This feedback could be due to the effects of growth factors produced by the two cell types in the 3D fibrin constructs.
Fibrin Sealant products such as TisseelR have been used over the last 25 years to reach hemostasis during surgery or to seal tissues. Moreover, there were many studies on the use of fibrin sealant in cell and bioactive substance delivery. Our In Vitro and In Vivo studies, over the last 2 years, have focused on the use of fibrin sealant to deliver human fibroblasts or keratinocytes to overcome the healing deficiency in chronic wounds. We have shown that some fibrin formulations supported a high fibroblast proliferation and other fibrin formulations supported a high proliferation of human keratinocytes. In this study, we examined the use of fibrin sealant in the co‐delivery of both human – derived keratinocytes and fibroblasts. The study report examined the cell proliferation of these two cell types in various formulations of fibrin sealant. Fibroblasts and keratinocytes were mixed with various dilutions of the Sealer Protein solution and added to culture plates before adding the Thrombin solution to form fibrin clots containing both cell types. We found that a low to medium sealer protein concentration (1–34 mG/mL) and a very low thrombin concentration (1 U/mL) in the final fibrin clots provided for an optimal cell proliferation for both cell types within these fibrin clots. This profile of proliferation was different from that seen when keratinocytes alone or fibroblasts alone were incorporated in the fibrin clots. Morphologically, it was difficult to determine the cell type from examining cell morphology, thus, it was difficult to determine the cell distribution. In conclusion, we found that various modified formulations of fibrin sealant may be chosen when co‐ delivering fibroblasts and keratinocytes. Moreover, there seems to be a positive feedback between keratinocytes and fibroblasts when they were co – introduced in the fibrin clots. This feedback could be carried by growth factors. Future studies will determine his signaling process.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.