Natural biopolymers such as human fibrin are appealing to tissue engineers, because fibrin is biocompatible, bioresorbable, and essential in normal wound healing. There have been numerous studies to date to develop a fibrin-based injectable cell delivery system, albeit with varying success. We propose that the outcome of fibrin cell delivery can, in part, be attributed to the relative concentrations of fibrinogen and thrombin solutions (i.e., formulations) and the structure of the final 3D fibrin clot. Formulation-dependent proliferation of human mesenchymal stem cells (hMSCs) within 3D fibrin clots was investigated in vitro. Our results indicate that hMSCs are viable in all fibrin sealant formulations investigated, and proliferation rates vary with fibrin formulations. Furthermore, the fibrinogen solution, not thrombin, was found to have a more dominant role on hMSC proliferation, with dilute fibrinogen solutions promoting greater hMSC proliferation. Confocal and electron microscopy reveal formulation dependence on 3D fibrin clot structure, with dilute fibrinogen solutions yielding more open, homogeneous microstructures. This study suggests that the concentrations of fibrinogen and thrombin solutions must be carefully considered for cell delivery because they affect 3D fibrin clot structure and cell proliferation.

Fibrin is a substance formed through catalytic conversion of coagulation constituents: fibrinogen and thrombin. The kinetics of the two constituents determines the structural properties of the fibrin architecture. We have shown previously that changing the fibrinogen and thrombin concentrations in the final three-dimensional (3D) fibrin matrix influenced cell proliferation and differentiation. In this study, we further examined the effect of changing fibrinogen and thrombin concentrations in the absence or presence of fibroblasts on the structural modulus or stiffness of 3D fibrin matrices. We have prepared fibroblast-free and fibroblast-embedded 3D fibrin matrices of different fibrinogen and thrombin formulations, and tested the stiffness of these constructs using standard mechanical testing assays. Results showed that there was a corresponding increase in stiffness with increasing thrombin and fibrinogen concentrations; the increase was more notable with fibrinogen and to a lesser degree with thrombin. The effect of fibroblasts on the stiffness of the fibrin construct was also examined. We have observed a small increase in the stiffness of the fibroblast-incorporated fibrin construct as they proliferated and exhibited spreading morphology. To our knowledge, this is the first comprehensive report detailing the relationship between fibrinogen and thrombin concentrations, cell proliferation, and stiffness in 3D fibrin matrices. The data obtained may lead to optimally design suitable bioscaffolds where we can control both cell proliferation and structural integrity for a variety of tissue engineering applications.

Fibrin sealant products are used in hemostasis and tissue sealing, and potentially as a cell delivery vehicle. In this study, fibrin sealant was evaluated as a delivery vehicle for human dermal fibroblasts. Fibroblast proliferation and migration were assessed in various dilutions of fibrin sealant by changing the fibrinogen and thrombin concentration. Fibroblasts proliferated well within three-dimensional (3-D) fibrin clots consisting of fibrinogen (5-17 mg/mL) and thrombin (1-167 U/mL). These fibroblasts also retained good morphology and growth characteristics after migrating out of the 3-D fibrin clots. Furthermore, using Western blot and fluorescence-activated cell-sorting analysis, we found that the expression of growth factors and interleukins in the entire fibroblast-fibrin construct was dependent on the fibrin sealant formulation. For example, in a formulation in which fibroblasts showed modest proliferation and migration, interleukin 8 was secreted to a lesser extent than in a formulation that supported robust proliferation and migration. To our knowledge, this is the first time that it has been shown that modifying the concentration of fibrinogen and thrombin affects fibroblast behavior within formed 3-D fibrin clots. In addition, some of these formulations present an ideal delivery vehicle for fibroblasts that could be used for the treatment of chronic wounds.

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.

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We have shown that human mesenchymal stem cells (hMSCs) have the potential to differentiate into bone when seeded within three-dimensional (3-D) fibrin constructs. Proteins endogenous to the fibrin construct and those secreted by cells in the 3-D constructs provide cues that can promote differentiation of hMSCs along with mechanical support for cell growth and migration. In this study, we decided to take a step back and examine the effect different extracellular matrix (ECM) proteins--fibrinogen, fibronectin, and collagen type I--had on hMSC osteogenic differentiation on two-dimensional (2-D) monolayer cultures. Briefly, 24-well tissue culture plates pre-coated with either fibrinogen (10 mg/mL), fibronectin (10 μg/mL), or collagen type I (1 mg/mL) were seeded with 25,000 cells/well and cultured in normal growth medium or in osteogenic induction medium. At days 1, 7, 14, 21, and 30, cultures were assessed for cell growth using alamarBlue(®) and osteogenic indicators using alkaline phosphatase and Von Kossa staining. The results show that collagen type I stained positive for calcium deposition the greatest by day 30 in both osteogenic medium and standard culture medium. However, fibrinogen had the greatest staining in osteogenic medium at day 21 and fibronectin was the only substrate to promote calcium deposition in standard culture medium at day 21. These results suggest that the osteogenic differentiation of hMSCs is influenced by both culturing conditions and substrate and that together they have a synergistic effect. By knowing the effect ECM proteins in 3-D fibrin construct have on promoting osteogenic differentiation of hMSCs, the fabrication of complex, biomimetic models designed to manipulate hMSC differentiation toward an osteoblastic lineage will be improved.

Wound healing in healthy individuals proceeds at an optimal rate. However, in patients, with -- e.g.-- locally impaired blood flow or diabetes, chronic wounds develop and often become infected. Chronic wounds mean a low quality of life for the afflicted patients, not to mention enormous costs. Rather than using recombinant growth factors to accelerate wound healing, we employed the toll-like receptor agonist macrophage-activating lipopeptide-2 (MALP-2) to improve the healing of full-thickness excision skin wounds in an animal model with obese, diabetic mice. A gene array experiment suggested that MALP-2 stimulates the release of various mediators involved in wound healing. Further data to be presented in this study will show (i) that MALP-2 is capable of stimulating the appearance of the monocyte chemoattractant protein-1 at the wound site, (ii) that this leads to increased leucocyte and, in particular, macrophage infiltration and (iii) that MALP-2-treated wounds closed 2 weeks earlier than vehicle-treated controls. MALP-2, thus, appears to stimulate the early inflammatory process needed to set in motion the ensuing consecutive natural steps of wound healing resulting in wound closure.

Research in the last few years have focused on the use of three-dimensional (3D) fibrin construct to deliver growth factors and cells. Three-dimensional construct permeability and porosity are important aspects for proper nutrient uptake, gas exchange, and waste removal—factors that are critical for cell growth and survival. We have previously reported that the mechanical strength (stiffness) of 3D fibrin constructs is dependent on the fibrinogen and thrombin concentration. In this study, we established two new in vitro models to examine how fibrin composition affects the final 3D fibrin construct permeability and pore size; thereby, influencing the diffusivity of macromolecules throughout the network of fibrin fibrils. Flow measurements of both liquid and fluoresceinated-dextran microparticles are conducted to calculate the permeability and pore size of 3D fibrin constructs of different fibrinogen and thrombin concentrations. Similarly, the diffusivity of liquid and fluoresceinated-dextran microparticles through these 3D fibrin constructs are determined through diffusion models. Data from these studies show that the structural permeability and pore size of 3D fibrin constructs directly correlate to fibrinogen and thrombin concentration in the final 3D fibrin construct. More specifically, at a constant thrombin concentration of 2 or 5 μ/mL, pore size of the 3D fibrin constructs is dependent on fibrinogen if the concentration is 5 mg/mL and to a lesser extent if the concentration is 10–15 mg/mL. These findings suggest that fibrin's diffusive property can be manipulated to fabricate 3D constructs that are optimized for cellular growth, protein transport, and for the controlled delivery of bioactive molecules such as growth factors.