pH and Thrombin Concentration Are Decisive in Synthesizing Stiff, Stable, and Open‐Porous Fibrin‐Collagen Hydrogel Blends without Chemical Cross‐Linker

Abstract

Fibrin‐collagen hydrogel blends exhibit high potential for tissue engineering applications. However, it is still unclear whether the underlying cross‐linking mechanisms are of chemical or physical nature. It is here hypothesized that chemical cross‐linkers play a negligible role and that instead pH and thrombin concentration are decisive for synthetizing blends with high stiffness and hydrolytic stability. Different fibrin‐collagen formulations (pure and with additional transglutaminase) are used and the blends’ compaction rate, hydrolytic stability, compressive strength, and hydrogel microstructure are investigated. The effect of thrombin concentration on gel compaction is examined and the importance of pH control during synthesis observed. It is revealed that transglutaminase impairs gel stability and it is deduced that fibrin‐collagen blends mainly cross‐link by mechanical interactions due to physical fibril entanglement as opposed to covalent bonds from chemical cross‐linking. High thrombin concentrations and basic pH during synthesis reduce gel compaction and enhance stiffness and long‐term stability. Scanning electron microscopy reveals a highly interpenetrating fibrous network with unique, interconnected open‐porous microstructures. Endothelial cells proliferate on the blends and form a confluent monolayer. This study reveals the underlying cross‐linking mechanisms and presents enhanced fibrin‐collagen blends with high stiffness, hydrolytic stability, and large, interconnected pores; findings that offer high potential for advanced tissue engineering applications.

A Fibrin‐Based Human Multicellular Gingival 3D Model Provides Biomimicry and Enables Long‐Term In Vitro Studies

“…Hydrogel Blends Preparation: Fibrin 2.5% (w/v): For the fibrin hydrogel, which was previously described and comprehensively analyzed for the me-chanical properties, [23] a fibrinogen stock solution was prepared by mixing 50 mg mL −1 fibrinogen (from bovine plasma, type I-S, 65-85% protein, Sigma-Aldrich) in PBS at 37 °C for 24 h in a water bath with stirring. The solution was sterilized by filtering through a 0.2 μm sterile syringe filter (Filtropur S, Sarstedt) and stored at −20 °C until use.…”

“…Hydrogel blends composed of fibrin and gelatin are proven to promote physiological cellular activity while providing suitable mechanical properties that offer a higher stability during in vitro cultivation. [23,24] Fibrinogen and gelatin are enzymatically cross-linked using microbial transglutaminase at physiological temperature and neutral pH, before the blend is crosslinked with thrombin. [24,25] We hypothesized that fibrin-based hydrogel blends can function as an alternative to collagen I hydrogel in gingival 3D in vitro models due to their amended mechanical properties and improved hydrolytic stability suitable for long-term in vitro cultivation.…”

“…Hydrogel blends composed of fibrin and gelatin are proven to promote physiological cellular activity while providing suitable mechanical properties that offer a higher stability during in vitro cultivation. [ 23,24 ] Fibrinogen and gelatin are enzymatically cross‐linked using microbial transglutaminase at physiological temperature and neutral pH, before the blend is crosslinked with thrombin. [ 24,25 ]…”

Engineering Mesoscopic 3D Tumor Models with a Self‐Organizing Vascularized Matrix

“…[35] However, since collagen lacks mechanical strength, it is often combined with fibrin gel to produce biofunctional blends with optimal mechanical properties. [27,[34][35][36] A polymerized fibrin gel similar to a physiological clot can be easily synthesized in vitro by mixing fibrinogen with thrombin and calcium chloride (CaCl 2 ). [35,36] For the development of our model, a naturally-derived fibrin-collagen hydrogel blend was used as ECM substitute.…”

“…[27,[34][35][36] A polymerized fibrin gel similar to a physiological clot can be easily synthesized in vitro by mixing fibrinogen with thrombin and calcium chloride (CaCl 2 ). [35,36] For the development of our model, a naturally-derived fibrin-collagen hydrogel blend was used as ECM substitute. Transglutaminase, antifibrinolytics tranexamic acid, and aprotinin were added to improve the mechanical stability of the hydrogel for long-time cultivation under fluid flow and to reduce degradation by resident cells [25,27,37] (Figure S2, Supporting Information).…”