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.

Fibrin sealants can be used to support tissue regeneration or as vehicles for delivery of cells in tissue engineering. Differences in the composition of fibrin sealants, however, could determine the success of such applications. The results presented in this article show clear differences between Fibrin sealant A (FS A) clots and Fibrin sealant B (FS B) clots with respect to their compatibility with primary human cells involved in soft tissue repair. FS A clots, which are characterized by a physiological coarse fibrin structure, promoted attachment, spreading, and proliferation of keratinocytes, fibroblasts, and endothelial cells. In contrast, FS B clots displaying a fine to medium clot structure failed to support spreading of all three cell types. Adhesion of keratinocytes was decreased on FS B clots compared to FS A clots after 3 h incubation, whereas number of attached fibroblasts and endothelial cells was initially comparable between the two fibrin sealants. However, all three cell types proliferated on FS A clots but no sustained proliferation was detected on FS B clots. We further demonstrate that the observed differences between FS A and B clots are partly based upon 1 M sodium chloride extractable constituents, like thrombin, and partly on nonextractable constituents or the fibrin structure. In conclusion, our in vitro results demonstrate that FS A clots serve as a provisional matrix that encourages adhesion and growth of keratinocytes, fibroblasts, and endothelial cells. Therefore, FS A seems to be well suited for applications in tissue engineering.

The fibroblast growth factor-binding protein (FGF-BP) binds and activates FGF-1 and FGF-2, thereby contributing to tumor angiogenesis. In this study, we identified novel binding partners of FGF-BP, and we provide evidence for a role of this protein in epithelial repair processes. We show that expression of FGF-BP increases after injury to murine and human skin, in particular in keratinocytes. This upregulation is most likely achieved by major keratinocyte mitogens present at the wound site. Most importantly, we demonstrate that FGF-BP interacts with FGF-7, FGF-10, and with the recently identified FGF-22, and enhances the activity of low concentrations of ligand. Due to the important functions of FGF-7 and FGF-10 for repair of injured epithelia, our findings suggest that upregulation of FGF-BP expression after injury stimulates FGF activity at the wound site, thus enhancing the process of epithelial repair.

Fibroblast growth factor – binding protein (FGF‐BP) is a secreted protein that appears to function as a low affinity heparin –binding protein. FGF‐BP binds to FGF‐1 and ‐2 in a non‐covalent, reversible manner to mobilize and solubilize these growth factors from their storage sites in the extracellular matrix. FGF‐BP is involved in both developmental and adult tissue homeostasis as well as in angiogenesis and tumorogenesis involving FGF‐1/2. FGF‐BP is overexpressed in several tumor types: head and neck, skin, cervical, and lung cancer, squamous cell carcinoma, and colon and breast adenocarcinoma. To establish the effect of FGF‐BP on wound healing, several forms of FGF‐BP cDNA were administered by particle‐mediated gene transfer into various animal wound models using the gene gun (Bio‐Rad). In a rat incisional wound model, gene gun cDNA delivery of full length FGF‐BP at the time of surgery produced a 117% increase of wound strength in diabetic rats at 10d, although the relative increase did not reach statistical significance (P < 0.08). Two truncated variants of FGF‐BP (pFGFbp10 and 17) were also administered in the rat incisional wound model by gene gun technique. pFGFbp17 increased the wound strength in diabetic rat 129%(p < 0.03), and the relative increase reach statistical significance (P < 0.008). In the rabbit ear ulcer model, particle‐mediated transduction of full length FGF‐BP increased collagen content by 195% and wound closure rate 38% at 10d post‐surgery. These findings show that FGF‐BP gene overexpression has a greater relative effect on wound healing in the diabetic rat model. The cDNA also had a significant effect in a rabbit excisional wound model that depended on granulation tissue formation. Truncated forms of the molecule may have higher therapeutic potency. FGF‐BP has an important role in FGF‐1/2 mobilization and macrophage functions, and FGF‐BP gene therapy for wound healing can improve the process by stimulating angiogenesis, epithelization and collagen synthesis in target tissue.

Photoautotrophic suspension-culture cells of Chenopodium rubrum that were shifted to mixotrophic growth by adding glucose were used as model system to investigate the influence of the source-sink transition in higher plants on the expression and en- zyme activities of intracellular and extracellular invertases. The complete cDNA coding for an extracellular invertase was cloned and sequenced from C. rubrum, and its identity has been proven by heterologous expression in Saccbaromyces cerevisiae. The higher activity of extracellular invertase after preincubation in the pres- ente of glucose was paralleled by an increased expression of the corresponding gene. The induction by glucose could be mimicked by the nonmetabolizable glucose analog 6-deoxyglucose. Both en- zyme activity and mRNA leve1 of extracellular invertase showed a sink-tissue-specific distribution in plants. The activity of neutra1 and acidic intracellular invertases were not affected by preincubation of autotrophic tissue cultures with sugars, nor did they show a tissue- specific distribution in plants. The data suggest that apoplastic invertase not only has an important function in phloem unloading and carbohydrate partitioning between source and sink tissues but may also have a role in establishing metabolic sinks.