Controlling fibroblast fibrinolytic activity allows for the bio-engineering of stable connective tissue equivalents (original) (raw)
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Frontiers in Bioengineering and Biotechnology, 2022
In tissue engineering, cell origin is important to ensure outcome quality. However, the impact of the cell type chosen for seeding in a biocompatible matrix has been less investigated. Here, we investigated the capacity of primary and immortalized fibroblasts of distinct origins to degrade a gelatin/alginate/ fibrin (GAF)-based biomaterial. We further established that fibrin was targeted by degradative fibroblasts through the secretion of fibrinolytic matrixmetalloproteinases (MMPs) and urokinase, two types of serine protease. Finally, we demonstrated that besides aprotinin, specific targeting of fibrinolytic MMPs and urokinase led to cell-laden GAF stability for at least forty-eight hours. These results support the use of specific strategies to tune fibrin-based biomaterials degradation over time. It emphasizes the need to choose the right cell type and further bring targeted solutions to avoid the degradation of fibrin-containing hydrogels or bioinks.
Preparation of a pure autologous biodegradable fibrin matrix for tissue engineering
Medical & Biological Engineering & Computing, 2000
Parallel to the growing role of tissue engineering, the need for cell embedding materials, which allow ceils to stabi/ise in a three-dimensional distribution, has increased. Although several substances have been tested, fibrin is thus far the only one that permits the clinical application of cultured tissue. To date, autologous fibrinogen has usually been polymerised with bovine thrombin, which can cause severe immunological side effects. The objective of this study was to explore the practicability of obtaining autologous thrombin from a single patient in an adequate concentration and amount. Fibrinogen was cryoprecipitated from 200 ml of freshly-frozen plasma. Thrombin was isolated from the supernatant through ionexchange chromatography. The thrombin was first bound to Sephadex A-50 and then eluated using 2ml of a salt buffer (2.0M NaCI in 0.015M trisodiumcitrate, pH 7.0).The activity of the thrombin (51NIHml 1 to 414NIHml 1) reached levels comparable to those in commercially available fibrin glues (4-500NIHml 1). The study has shown that it is possible to obtain a sufficient amount of autologous thrombin from a single donor to create a fibrin matrix of high efficiency without the risk of immunological and infectious side effects.
Enzymatic formation of modular cell-instructive fibrin analogs for tissue engineering
Biomaterials, 2007
The molecular engineering of cell-instructive artificial extracellular matrices is a powerful means to control cell behavior and enable complex processes of tissue formation and regeneration. This work reports on a novel method to produce such smart biomaterials by recapitulating the crosslinking chemistry and the biomolecular characteristics of the biopolymer fibrin in a synthetic analog. We use activated coagulation transglutaminase factor XIIIa for site-specific coupling of cell adhesion ligands and engineered growth factor proteins to multiarm poly(ethylene glycol) macromers that simultaneously form proteolytically sensitive hydrogel networks in the same enzyme-catalyzed reaction. Growth factor proteins are quantitatively incorporated and released upon cell-derived proteolytic degradation of the gels. Primary stromal cells can invade and proteolytically remodel these networks both in an in vitro and in vivo setting. The synthetic ease and potential to engineer their physicochemical and bioactive characteristics makes these hybrid networks true alternatives for fibrin as provisional drug delivery platforms in tissue engineering. r
Materials, 2019
Platelet-rich fibrin (PRF) is a blood concentrate derived from venous blood that is processed without anticoagulants by a one-step centrifugation process. This three-dimensional scaffold contains inflammatory cells and plasma proteins entrapped in a fibrin matrix. Liquid-PRF was developed based on the previously described low-speed centrifuge concept (LSCC), which allowed the introduction of a liquid-PRF formulation of fibrinogen and thrombin prior to its conversion to fibrin. Liquid-PRF was introduced to meet the clinical demand for combination with biomaterials in a clinically applicable and easy-to-use way. The aim of the present study was to evaluate, ex vivo, the interaction of the liquid-PRF constituents with five different collagen biomaterials by histological analyses. The results first demonstrated that large variability existed between the biomaterials investigated. Liquid-PRF was able to completely invade Mucograft® (MG; Geistlich Biomaterials, Wolhusen, Switzerland) and ...
Aspects of In Vitro Biodegradation of Hybrid Fibrin–Collagen Scaffolds
Polymers, 2021
The success of the regenerative process resulting from the implantation of a scaffold or a tissue-engineered structure into damaged tissues depends on a series of factors, including, crucially, the biodegradability of the implanted materials. The selection of a scaffold with appropriate biodegradation characteristics allows for synchronization of the degradation of the construct with the processes involved in new tissue formation. Thus, it is extremely important to characterize the biodegradation properties of potential scaffold materials at the stage of in vitro studies. We have analyzed the biodegradation of hybrid fibrin–collagen scaffolds in both PBS solution and in trypsin solution and this has enabled us to describe the processes of both their passive and enzymatic degradation. It was found that the specific origin of the collagen used to form part of the hybrid scaffolds could have a significant effect on the nature of the biodegradation process. It was also established, duri...
Fibrin-Based Tissue Engineering: Comparison of Different Methods of Autologous Fibrinogen Isolation
Tissue Engineering Part C: Methods, 2013
Objective: This study is focussed on the optimal method of autologous fibrinogen isolation with regard to the yield and the use as a scaffold material. This is particularly relevant for paediatric patients with strictly limited volumes of blood. Materials and Methods: The following isolation methods were evaluated: cryoprecipitation, ethanol-precipitation, ammonium sulphate-precipitation, ammonium sulphate-precipitation combined with cryoprecipitation, and polyethylene glycol-precipitation combined with cryoprecipitation. Fibrinogen yields were quantified spectrophotometrically and by electrophoretic analyses. To test the influence of the different isolation methods on the microstructure of the fibrin gels, scanning electron microscopy was used and the mechanical strength of the cell-free and cellseeded fibrin gels was tested by burst strength measurements. Cytotoxicity assays were performed to analyse the effect of various fibrinogen isolation methods on proliferation, apoptosis and necrosis. Tissue development and cell migration were analysed in all samples using immunohistochemical techniques. The synthesis of collagen as an extracellular matrix component by HUASMCs in fibrin gels was measured using hydroxyproline assay. Results: Compared to cryoprecipitation, all other considered methods were superior in quantitative analyses, with maximum fibrinogen yields of ~80% of total plasma fibrinogen concentration using ethanol-precipitation. SEM imaging demonstrated minor differences in the gel microstructure. Ethanol-precipitated fibrin gels exhibited the best mechanical properties. None of the isolation methods had a cytotoxic effect on the cells. Collagen production was similar in all gels except those from ammonium sulphate-precipitation. Histological analysis showed good cell compatibility for ethanol-precipitated gels. Conclusion: The results of the present study demonstrated that ethanol-precipitation is a simple and effective method for isolation of fibrinogen and a suitable alternative to cryoprecipitation. This technique allows minimization of the necessary blood volume for fibrinogen isolation, particularly important
2017
Interpenetrating polymer networks (IPNs) have gained great attention for a number of biomedical applications due to their improved properties compared to individual components alone. In this study, we investigated the capacity of newly-developed naturally-derived IPNs as potential biomaterials for tissue engineering. These IPNs combine the biologic properties of a fibrous fibrin network polymerized at the nanoscale and the mechanical stability of polyethylene oxide (PEO). First, we assessed their cytotoxicity in vitro on L929 fibroblasts. We further evaluated their biocompatibility ex vivo with a chick embryo organotypic culture model. Subcutaneous implantations of the matrices were subsequently conducted on nude mice to investigate their biocompatibility in vivo. Our preliminary data highlighted that our biomaterials were non-cytotoxic (viability above 90%). The organotypic culture showed that the IPN matrices induced higher cell adhesion (across all the explanted organ tissues) an...
Fibrin-filled scaffolds for bone-tissue engineering: Anin vivo study
Journal of Biomedical Materials Research, 2004
Recently, fibrin sealants that typically contain supra physiological concentrations of fibrinogen and thrombin have been investigated as matrices to facilitate the delivery of cells within biodegradable scaffolds for tissue engineering applications. It is well known from in vitro experiments that the thrombin concentration present during fibrin polymerization influences the structural properties of fibrin, and these can affect cell invasion. This study was conducted to determine whether the structural properties of fibrin can affect bony wound healing in vivo. Drill hole defects were created in the distal femurs of 20 rats. Four experimental groups were used: nontreated defects, scaffolds alone, and scaffolds filled with fibrin polymerized with either a low thrombin concentration [fibrin(low T)] or a high thrombin concentration [fibrin(high T)]. The area of bone formed at 2, 5, and 11 days after implantation was determined histomorphometrically. After 5 days, scaffolds filled with fibrin(high T) were infiltrated with less bone than empty scaffolds (p Ͻ 0.05), but no statistical difference was found between the empty scaffolds and the scaffolds filled with fibrin(low T). After 11 days, both fibrin-filled scaffolds significantly delayed bony wound healing (p Ͻ 0.004). Reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the two fibrin formulations showed no difference in ␥-␥ crosslink formation. This work demonstrates that fibrin sealants in their present state are not ideal for enhancing bone-tissue invasion into scaffolds, and that the structural properties of fibrin matrices may be an important design parameter for maximizing host tissue invasion during wound healing.