The use of commercial fibrin glue in dermal replacement material reduces angiogenic and lymphangiogenic gene and protein expression in vitro

Abstract

Background: Commercial fibrin glue is increasingly finding its way into clinical practice in surgeries to seal anastomosis, and initiate hemostasis or tissue repair. Human biological glue is also being discussed as a possible cell carrier. To date, there are only a few studies addressing the effects of fibrin glue on the cell-molecular level. This study examines the effects of fibrin glue on angiogenesis and lymphangiogenesis, as well as adipose-derived stem cells (ASCs) with a focus on gene and protein expression in scaffolds regularly used for tissue engineering approaches.

Methods: Collagen-based dermal regeneration matrices (DRM) were seeded with human umbilical vein endothelial cells (HUVEC), human dermal lymphatic endothelial cells (LECs), or adipose-derived stem cells (ASC) and fixed with or without fibrin glue according to the experimental group. Cultures were maintained for 1 and 7 days. Finally, angiogenic and lymphangiogenic gene and protein expression were measured with special regard to subtypes of vascular endothelial growth factor (VEGF) and corresponding receptors using Multiplex-qPCR and ELISA assays. In addition, the hypoxia-induced factor 1-alpha (HIF1a) mediated intracellular signaling pathways were included in assessments to analyze a hypoxic encapsulating effect of fibrin polymers.

Results: All cell types reacted to fibrin glue application with an alteration of gene and protein expression. In particular, vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor B (VEGFB), vascular endothelial growth factor C (VEGFC), vascular endothelial growth receptor 1 (VEGFR1/FLT1), vascular endothelial growth receptor 2 (VEGFR2/KDR), vascular endothelial growth receptor 3 (VEGFR3/FLT4) and Prospero Homeobox 1 (PROX1) were depressed significantly depending on fibrin glue. Especially short-term fibrin effect led to a continuous downregulation of respective gene and protein expression in HUVECs, LECs, and ASCs.

Conclusion: Our findings demonstrate the impact of fibrin glue application in dermal regeneration with special regard to angiogenesis and lymphangiogenesis. In particular, a short fibrin treatment of 24 hours led to a decrease in gene and protein levels of LECS, HUVECs, and ASCs. In contrast, the long-term application showed less effect on gene and protein expressions. Therefore, this work demonstrated the negative effects of fibrin-treated cells in tissue engineering approaches and could affect wound healing during dermal regeneration.

Keywords: Adipose-derived stem cells; Enzym-linked Immunosorbent Assay; Fibrin glue; Hypoxia-inducible factor 1-alpha; Lymphatic endothelial cells; Multiplex-RT-PCR; Vascular Endothelial Growth Factor; angiogenesis; gene and protein expression; human umbilical vein endothelial cells; lymphangiogenesis.

Figure 1.

Schematic representation of the experimental workflow: Living adipose-derived stem cells, endothelial and lymph endothelial cells were seeded into 3D collagen-based scaffolds and fixed either with or without fibrin glue. The control group was performed with the abandonment of a scaffold application to evaluate the effect of a 3D cell architecture. Seeded scaffolds were incubated under normoxic conditions (21% pO₂) for 1 and 7 days, respectively. Upon incubation, fibrin glue affects lymphangiogenic and angiogenic protein and gene expression by inducing the downregulation of corresponding growth factors or receptors.

Figure 2.

(a) The cross-sectional image of a collagen-based scaffold with fibrin application after 1 day, which is intended to be in contact with the wound ground, shows a homogeneous distribution of the fibrin glue with almost no air pockets. Scale bar represents 500 μm. (b) The stereoscopic image shows a round, fibrin-coated scaffold with a diameter of 10 mm, which can serve as a multiple-cell carrier. Scale bar represents 5 mm.

Figure 3.

Use of fibrin glue reduces angiogenic and lymphangiogenic gene expression in scaffolds after 24 hours: Gene expression of endothelial and lymph endothelial cells was evaluated by Multiplex-RT-PCR and showed a significant increase after 24 hours in absence of fibrin application compared to cells fixed with fibrin glue into scaffolds. The following angiogenic and lymphangiogenic genes were analyzed: (a) Hypoxia-inducible factor 1 alpha (b) Prospero homeobox 1 (c) Vascular endothelial growth factor C (d) Vascular endothelial growth factor receptor 3 (e) Vascular endothelial growth factor A (f) Vascular endothelial growth factor B (g) Vascular endothelial growth factor receptor 1 (h) Vascular endothelial growth factor receptor 2. All experiments were repeated at least three times (ns = not significant; *p < 0.05; **p ≤ 0.01; ***p < 0.001).

Figure 4.

Reduced angiogenic and lymphangiogenic protein expression level by fibrin glue application in scaffolds after 24 hours: The protein level of endothelial and lymph endothelial cells measured by ELISA showed a higher protein synthesis in HUVECs, LECs, and ASCs in absence of fibrin application, compared to fibrin containing conditions. Angiogenic and lymphangiogenic protein expression was significantly reduced in ACS, HUVECs, and LECs by the presence of fibrin glue. The following angiogenic and lymphangiogenic genes were analyzed: (a) Hypoxia-inducible factor 1 alpha (b) Prospero homeobox 1 (c) Vascular endothelial growth factor C (d) Vascular endothelial growth factor receptor 3 (e) Vascular endothelial growth factor A (f) Vascular endothelial growth factor B (g) Vascular endothelial growth factor receptor 1 (h) Vascular endothelial growth factor receptor 2. All experiments were repeated at least three times (ns = not significant; *p < 0.05; **p ≤ 0.01; ***p < 0.001).

Figure 5.

Long-term application of fibrin glue in scaffolds induces downregulation of angiogenic and lymphangiogenic genes: Relative angiogenic and lymphangiogenic gene expression in ASCs, LECS, and HUVECs analyzed by Multiplex-RT-qPCR after 7 days of fibrin application in scaffolds revealed a reduced expression profile compared to cells in fibrinless scaffolds. Following angiogenic and lymphangiogenic genes were analyzed: (a) Hypoxia-inducible factor 1 alpha (b) Prospero homeobox 1 (c) Vascular endothelial growth factor C (d) Vascular endothelial growth factor receptor 3 (e) Vascular endothelial growth factor A (f) Vascular endothelial growth factor B (g) Vascular endothelial growth factor receptor 1 (h) Vascular endothelial growth factor receptor 2. All experiments were repeated at least three times (ns = not significant; *p < 0.05; **p ≤ 0.01; ***p < 0.001).

Figure 6.

Long-term application of fibrin glue in scaffolds reduces angiogenic and lymphangiogenic protein expression in scaffolds: Quantification of protein levels by ELISA after continuous fibrin treatment over 7 days showed significant downregulation of VEGFA, VEGFB, VEGFC, VEGFR3 levels regarding ASCs, LECS, HUVECs in scaffolds. The lack of fibrin glue was capable to stimulate protein expression in all cell types over 7 days and thus promoting angiogenesis and lymphangiogenesis in scaffolds. The following angiogenic and lymphangiogenic genes were analyzed: (a) Hypoxia-inducible factor 1 alpha (b) Prospero homeobox 1 (c) Vascular endothelial growth factor C (d) Vascular endothelial growth factor receptor 3 (e) Vascular endothelial growth factor A (f) Vascular endothelial growth factor B (g) Vascular endothelial growth factor receptor 1 (h) Vascular endothelial growth factor receptor 2. All experiments were repeated at least three times (ns = not significant; *p < 0.05; **p ≤ 0.01; ***p < 0.001).