Human Mesenchymal Stem Cell Proliferation and Osteogenic Differentiation in Fibrin Gelsin Vitro

Abstract

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.

Natural polymers for the microencapsulation of cells

“…Furthermore, when fibrin is used as the matrix to encapsulate stem cells, the concentration of thrombin and fibrinogen can influence their proliferation rate and their differentiation. The results from Catelas et al [185] show that formulations containing a lower concentration of fibrinogen (5 mg ml 21 ) can support human mesenchymal stem cell growth, while a higher concentration (50 mg ml 21 ) has an increased potential for their differentiation into osteoblasts. In mammals, fibrin can degrade rapidly owing to the presence of proteolytic enzymes (fibrinolysis).…”

Influencing biophysical properties of fibrin with buffer solutions

“…Thrombin buffer solution, on the other hand, contains calcium and sodium chlorides. High fibrinogen, and to a lesser extent low thrombin, concentrations have been shown to reduce the proliferation of human MSCs [7][8][9] and dermal fibroblasts [3]. The fibrinogen concentration thresholds determined by these studies, however, varied from 18 to 50 mg/mL fibrinogen.…”

“…Scaffolds leading to better cell proliferation are not necessarily more appropriate for cell differentiation as illustrated by the study of Catelas et al [8]. They showed that high-fibrinogen content, though lowering cell proliferation rates, improved osteogenic differentiation of human BMSCs.…”

“…More recently, they also have been proposed as scaffolding for cell delivery in several tissue engineering applications [2]. Numerous in vitro studies, indeed, have demonstrated the ability of fibrin gels to support survival, proliferation, and/or differentiation of many cell types [3][4][5][6], including mesenchymal stromal cells (MSCs) [7][8][9].…”

Regenerative Dentistry: Animal Model for Regenerative Endodontology

“…We filled the canal space with fibrin gel, which acts as a scaffold for immigrating cells [37]. The selection of the scaffold in the cleaned and disinfected pulp space is also considered to be essential for the success of tissue engineering in the current context [8,11,38].…”