Technological advances in fibrin for tissue engineering

Abstract

Fibrin is a promising natural polymer that is widely used for diverse applications, such as hemostatic glue, carrier for drug and cell delivery, and matrix for tissue engineering. Despite the significant advances in the use of fibrin for bioengineering and biomedical applications, some of its characteristics must be improved for suitability for general use. For example, fibrin hydrogels tend to shrink and degrade quickly after polymerization, particularly when they contain embedded cells. In addition, their poor mechanical properties and batch-to-batch variability affect their handling, long-term stability, standardization, and reliability. One of the most widely used approaches to improve their properties has been modification of the structure and composition of fibrin hydrogels. In this review, recent advances in composite fibrin scaffolds, chemically modified fibrin hydrogels, interpenetrated polymer network (IPN) hydrogels composed of fibrin and other synthetic or natural polymers are critically reviewed, focusing on their use for tissue engineering.

Keywords: Fibrin hydrogels in tissue engineering; PEGylated fibrin hydrogels; fibrin-polymer composite scaffolds; natural polymer-fibrin hydrogels; particles encapsulated in fibrin hydrogels.

Figure 1.

Diagram of the fibrin structure and its polymerization process: (a) Structure of the fibrinogen macromolecule with D-domains and the central E-domain. The domains are linked by three pairs of different polypeptide chains: α, β and γ. (b) Fibrinogen is converted to fibrin monomer by thrombin. It cleaves fibrinopeptides A and B, exposing knobs A and B. (c) Then, the cleaved fibrinopeptides bind to holes a and b, respectively, known as the specific unions A:a and B:b, and form the protofibril molecule. (d) Protofibril bundling is formed by lateral aggregation of protofibrils, owing to interactions of the AC regions, and leading up to thick fibrin fibers. (e) Then, the branching and lateral aggregation of fibers form the fibrin network. (f) Image of a fiber network forming a gel obtained with a light sheet microscope. Reprinted from Belcher et al.

Figure 2.

Scheme of fibrin-based hybrid scaffold formation in two steps: the fabrication of different solid scaffolds using different strategies (a, b, and c). (a) fibrin precursor solution containing cells is injected into the solid scaffold, and subsequent fibrin gelation occurs. A freeze-drying scaffold processing by phase separation: A polymer with a solvent is frozen to form a freeze-dried scaffold because of solvent sublimation. The resulting scaffold exhibits a porous morphology. (b) Electrospinning scaffold: A polymer with a solvent is dosed using a syringe on a collector drum with a determined electric current entailing solvent evaporation and the formation of a solid polymer fiber mesh. The formed scaffold exhibits a fibrous morphology. (c) 3D printed scaffold: a scaffold is designed in software, and the 3D printer reproduces the design layer by layer using needles loaded with a polymer that acts as bio-ink. The resulting scaffold exhibits a filamentous morphology. The left part of the image was adapted from Roacho-Pérez et al.

Figure 3.

Applications of fibrin-polymer solid composite scaffolds in different tissues engineering. (a) Analysis of in vitro constructs using immunohistochemistry of fibrin/PLGA, which demonstrated strong immunopositivity of collagen type II, and PLGA, with minimal collagen type II expression, after 2 weeks. The immunopositivity of both constructs for collagen type I was moderate. Reprinted from Sha’ban et al. (b) Micropillar array of poly (dimethylsiloxane) (PDMS) at square mesh (5 × 5 cm) (top images) with poured fibrin gel, which guided organization of the cells and produced angiogenic sprouts (white arrows) during 2 weeks of in vitro culture (middle images), and demonstrated a high degree of vascularization after 2 weeks of subcutaneous implantation in SCID-Beige mice (bottom images). Reprinted from Song et al. (c) Research of scaffolds of polyvinyl alcohol (PVA) with serum albumin (SA), as a degradation agent by enzymes, at different concentrations. PVA and SA were previously methacrylated before being copolymerized into PVA-SA scaffolds via free radical copolymerization. Finally, fibrin gel with fibroblasts was infiltrated and polymerized by thrombin, resulting in enzymatically degradable IPNs. It promotes cell growth and mimics the physiological microenvironment of tissues. Reprinted from Bidault et al. (d) Design for PLCL-fibrin scaffold fabrication in cartilage using stromal cells from rabbit bone marrow. Clusters of cells were aggregated using a hanging drop method and added inside the scaffolds using fibrin-gel infiltration. Reprinted from Lee et al.

Figure 4.

Modification of fibrin hydrogels by synthetic polymers. (a) Process of the formation of a PEGylated hydrogel. First, PEG is functionalized to react in the presence of the amine groups of a protein. Then, the di-functional PEG is added to the fibrinogen leading to a new type of fibrin hydrogel (reprinted from Roberts et al.). (b) Scheme of an IPN formation of fibrinogen with Pluronic^® F-127 (F127) and poly(methy)methacrylate (PMMA) polymers, thanks to thrombin. The hydrogel is formed by a dual-syringe system which dispenses a Fb/F127/PMMA solution and thrombin and fills a meniscal region, (reprinted from An et al.). (c) The top part shows a polyethylene glycol-platelet free plasma hydrogel treated 4 mm ex vivo explant that was cultured for 14 days. It is stained with wheat germ (red) to visualize the plasma membrane and counterstained with DAPI for nuclei (blue). At the bottom, an enlarged epidermis formed over the PEG-PFP hydrogel is shown (reprinted from Stone et al.).

Figure 5.

Modification of fibrin hydrogels with natural polymers. (a) Elastic modulus graph of collagen-fibrin mixed hydrogels at different collagen concentrations, adapted from Coradin et al. (b) Proliferation assay of fibroblasts seeded inside plasma and 10% of oxidized alginate hydrogels after (left) 48 h and (right) 7 days, reprinted from Sanz-Horta et al. (c) Hematoxylin & eosin staining of cross-section, of an implantation after 12 days in a mouse, of an hydrogel of fibrin-hyaluronic acid containing red blood cells (represented by red arrows), adapted from Hinsenkamp et al. The presence of fibrin enhances the formation of blood vessels, and the infiltration of cells and extracellular. The use of fibrin was also found to support the biological process of matrix remodeling. (d) Elastin-fibrin hydrogel preparation: Elastin-N3 with plasma and AmchaFibrin is mixed to Elastin-Cyclo with NaCl and CaCl₂ at 37°C, adapted from Stojic et al.

Figure 6.

Modification of fibrin hydrogels with tri-natural-component hydrogels: (a) scheme of incorporation of collagen-HA-fibrin hydrogels with articular chondrocyte cells into intervertebral disks to regenerate damaged disk tissues, reprinted from Gansau et al. and (b) proliferation assay thanks to Live/dead staining of murine fibroblasts inside of collagen, alginate and fibrin (CAF) hydrogels at different collagen concentrations, adapted from Montalbano et al.

Figure 7.

(A) Scheme and characteristics of interspersion of alginate nanobeads into fibrin hydrogel for soft tissue engineering and (B) Stained mesenchymal stem cells (red): with alginate nanobeads alone at 6 h (a) and 48 h (d), into fibrin composite with alginate nanobeads at 6 h (b) and 48 h (e), and into fibrin network at 6 h (c) and 48 h (f). Scale bar: 5 μm. Reprinted from Deepthi et al.