High thrombin concentrations in fibrin sealants induce apoptosis in human keratinocytes

Abstract

Over the last century many studies have been performed to assess the impact of fibrin sealant (FS) components on cells. Because of the noncovalent bonding of thrombin to fibrin during fibrin clot formation, we wanted to further evaluate the impact of fibrin bound thrombin on cell viability. Initially, we quantified the activity of thrombin in three different, commercially available FS. This information was used to prepare fibrin clots covering a range of thrombin concentrations from 4 to 820 IU mL(-1), but which were identical with respect to all other constituents. Although these fibrin clots did not differ in their three-dimensional structure, clots prepared with highly concentrated thrombin (820 IU mL(-1)) failed to support adhesion and spreading of primary human keratinocytes (NHEK). The number of attached cells was also significantly reduced on high thrombin activity clots. We hypothesized that these observations are not only the consequence of decreased proliferation but of apoptotic mechanisms, since the expression of cleaved caspase 3 and 7 was strongly enhanced on fibrin clots with high thrombin activity. This was accompanied by an induction of expression of Trail-R2 which is a receptor known to mediate apoptosis signals. Blocking of thrombin activity by hirudin led to an improvement of cell morphology and to an increase in number of attached cells. In addition, the induction of caspase 3 and 7 was also reduced. Thus, here we report for the first time that fibrin bound thrombin does not only decrease proliferation (as already published by others), it also does induce NHEK apoptosis when present at high concentrations.

Effect of Fibrin Formulation on Initial Strength of Tendon Repair and Migration of Bone Marrow Stromal Cells in Vitro

“…They concluded that the most favorable combination for survival of bone marrow stromal cells was 20 mg/mL of fibrinogen and 87 NIH units/mL of thrombin, values intermediate between those of our control and low-low groups. The ratio of dead to total cells in our study was <30%, a finding consistent with previous reports 18,27 . Gugerell et al reported that thrombin concentrations of >400 NIH units/mL induced apoptosis of human keratinocytes 27 .…”

“…The ratio of dead to total cells in our study was <30%, a finding consistent with previous reports 18,27 . Gugerell et al reported that thrombin concentrations of >400 NIH units/mL induced apoptosis of human keratinocytes 27 . Given our findings, we agree with Gugerell et al that much lower thrombin concentrations are appropriate for fibrin gels used for cell delivery.…”

“…After one, three, or seven days in culture, cells were incubated with calcein AM (acetomethoxy) and ethidium homodimer-1 fluorescent dye (EthD) solution (LIVE/DEAD Viability/Cytotoxicity Kit, for mammalian cells; Life Technologies) to stain for living and dead cells, according to the manufacturer's protocol 27 . Stained specimens were examined with a laser scanning confocal microscope (LSM780; Zeiss) for living and dead-cell counting and to examine cell morphology.…”

A novel bioreactor for the generation of highly aligned 3D skeletal muscle-like constructs through orientation of fibrin via application of static strain

“…In contrast to other approaches using rather low fibrinogen to thrombin ratios [10,11,34,39], we chose a higher ratio (20 mg/mL fibrinogen, 0.625 U/mL thrombin) in our experimental setup for several reasons: (i) the stiffness is directly proportional to the fibrinogen concentration. The obtained constructs displayed a Young's modulus of about 17 kPa, which lies within the stiffness range proposed to be most suitable for myogenic differentiation [43,47,73]; (ii) the fibrinogen to thrombin ratio directly correlates with permeability and pore size as well as the ultimate tensile strength of the scaffold [16]; (iii) lower thrombin concentrations give slower clotting times, increasing the homogeneity of the fibrin meshwork, and have less detrimental effects on cell viability [74].…”

Protein nanocoatings on synthetic polymeric nanofibrous membranes designed as carriers for skin cells

“…Gugerell et al reported that high concentrations (about 820 U/mL) of thrombin activated apoptotic mechanisms in keratinocytes, and also decreased their attachment and spreading on fibrin sealants. 45 Kubo et al showed that fibrin and fibrinogen were nonadhesive for keratinocytes, due to a lack in these cells of α v β 3 integrin receptors, which are important for binding cells to fibrin molecules. 46 In our study, the HaCaT keratinocytes were able to adhere to fibrin-coated membranes, though they were not so well flattened as on collagen-coated membranes.…”