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Available online at www.sciencedirect.com

journal homepage: www.elsevier.com/locate/yexcr

Review Article

Cell culture in autologous fibrin scaffolds for applications in tissue engineering Pilar de la Puentea,n, Dolores Ludeñab a

Department of Radiation Oncology, Cancer Biology Division, Washington University in St Louis School of Medicine, St. Louis, MO 63108, USA b Pathology Service, University Hospital of Salamanca, Salamanca, Spain

article information

abstract

Article Chronology:

In tissue engineering techniques, three-dimensional scaffolds are needed to adjust and guide cell

Received 16 October 2013

growth and to allow tissue regeneration. The scaffold must be biocompatible, biodegradable and

Received in revised form

must benefit the interactions between cells and biomaterial. Some natural biomaterials such as fibrin

11 December 2013

provide a structure similar to the native extracellular matrix containing the cells. Fibrin was first used

Accepted 18 December 2013

as a sealant based on pools of commercial fibrinogen. However, the high risk of viral transmission of

Available online 28 December 2013

these pools led to the development of techniques of viral inactivation and elimination and the use of

Keywords:

autologous fibrins. In recent decades, fibrin has been used as a release system and three-dimensional

Autologous

scaffold for cell culture. Fibrin scaffolds have been widely used for the culture of different types of

Fibrin scaffolds

cells, and have found several applications in tissue engineering. The structure and development of

Tissue engineering Fibrinogen Cell culture

scaffolds is a key point for cell culture because scaffolds of autologous fibrin offer an important alternative due to their low fibrinogen concentrations, which are more suitable for cell growth. With this review our aim is to follow methods of development, analyze the commercial and autologous fibrins available and assess the possible applications of cell culture in tissue engineering in these three-dimensional structures. & 2013 Elsevier Inc. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biomaterials as scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Fibrin: Concept, nature and clinical use . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . From sealants to three-dimensional scaffolds for cell culture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Adipose tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bone engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cardiac tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cartilage engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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n Correspondence to. Department of Radiation Oncology, Cancer Biology Division, Washington University in Saint Louis School of Medicine, 4511 Forest Park Ave., Room 3103, St. Louis, MO 63108, USA. Fax: þ34 314 362 9790. E-mail address: [email protected] (P. de la Puente).

0014-4827/$ - see front matter & 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.yexcr.2013.12.017

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Delivery systems. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Applications in differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Muscle tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Neural tissue engineering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Ocular tissue engineering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Respiratory tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Skin engineering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tendon and ligament engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Vascular tissue engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Methods for the building of fibrin scaffolds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Autologous fibrin vs. commercial fibrin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction Tissue engineering is an interdisciplinary scientific area that attempts to restore or improve the biological functions of damaged tissues or of tissues that are no longer able to carry out their function. Biomaterials are used as three-dimensional structures that contain cells and biologically active molecules [1]. The scaffold adjusts and guides cell growth and also allows tissue regeneration in a three-dimensional structure [2]. To be feasible, a tissue graft must accomplish two requirements: first, it must allow the development of neovascularization in the area where the avascular matrix has been implanted. This helps to prevent the erosion, infection and necrosis of the graft in which the coordinated and sequential action of certain growth factors will play an important role. Second, grafts must ensure the scarring of the area, thanks to the proliferation and differentiation of the cell component, which helps to regenerate the epithelium [1]. Some authors have considered the possibility of using avascular grafting without the need to add any cell type. In other studies, the existence of epithelial and vascular invasion from the organ towards the graft has been observed [3]. Nevertheless, most studies tend towards the use of different cell types to produce the regeneration of epithelium, ensure the preservation of the capillary network and/or promote graft vascularization [4–7]. In tissue engineering techniques, the cellular component is usually autologous (i.e., from the patient). This is preserved in in vitro culture until it can be introduced into a biocompatible three-dimensional scaffold, together with new biologically active substances that will promote angiogenesis and cell proliferation. The resulting tissue graft is preserved under adequate culture conditions until in vivo implantation is carried out (Fig. 1). In short, thanks to the techniques of tissue engineering three important components have become available for obtaining a feasible graft: the biomaterial that will act as scaffold, the cell component and inducer substances. Below we describe and review the biomaterials most widely used for the development of scaffolds in cell culture.

Biomaterials as scaffolds The ideal scaffold must be developed using biocompatible materials with surface properties that will benefit cell adhesion,

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proliferation and differentiation [6] and will not produce inflammatory reactions after implantation [2]. Depending on their origin, biomaterials can be classified as natural and synthetic. The first group generally includes proteins and polysaccharides (collagen, fibrin, alginate, hyaluronic acid, etc.), whereas the second one is composed of metallic, ceramic or polymeric materials, such as polyglycolic acid (PGA) and polylactic acid (PLA). All of them have been used to manufacture scaffolds in tissue engineering [6–8]. A comparison of natural and synthethic materials is given in Table 1 [6–18]. Natural biomaterials mimic the structure and composition of the native extracellular matrix. Their stimulating effects allow the inclusion of growth factors and other proteins able to boost cellular functions. However, they deteriorate easily and can transfer pathogens, and their variability depends on the structure of the original natural polymer [10]. Certain natural polymers such as collagen have been used for different purposes in soft tissue engineering, for example in skin.

A. Patientbiopsy

B. In vitro cellculture

C. Cell component is introduced in 3D biomaterial D. Inducer substances are introduced in 3D biomaterial

F. Implantation

E. In vitro culture of 3D biomaterial

Fig. 1 – Outline of the process of techniques of autologous tissue engineering. A patient biopsy is obtained (A) to extract the cell component. This is seeded in an in vitro culture (B), after which the cell component (C) is introduced with inducer substances (D) in a biomaterial or scaffold and is cultured (E) until it is implanted in vivo (F).

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Table 1 – Pros and cons of natural and synthetic materials to manufacture scaffolds for tissue engineering. Pros Natural materials (Collagen, fibrin, alginate, etc.)

Synthetic materials (PGA, PLA, PLGA, etc.)

Contras

– Usually biocompatible, biodegradable, non toxic or immunogenic – Mimic the native extracellular matrix (structure, composition and stimulatory effects) – Good transport of nutrients to cells and product from cells

– – – – –

Relative biocompatible Easy to handle Controlled degradation rates Architectural and mechanical properties can be controlled No risk of pathogens transmission

Nevertheless, the use of collagen has raised some concern because it is difficult to handle and also due to its antigenicity and immunogenicity [11]. Moreover, the biodegradability and the low mechanical strength of untreated collagen limit the use of this material [12]. Thus, cross-linking and remodeling of collagen are effective ways to slow down the biodegradation rate [13,14]. Some natural materials such as collagen and fibrin share good cell adhesion, proliferation and distribution properties [8,15],. In the case of fibrin scaffolds, these have high levels of cell proliferation, a uniform distribution of cells in a three-dimensional structure, and good adhesion [1,8,16]. Unlike xenogenic gelatin and collagen, which may induce intense inflammatory responses, fibrin can prevent the potential risk of a infection or of foreignbody reaction when produced from the patients’ own blood [17]. The most significant disadvantages lie in their low mechanical strength and their rapid deterioration [16,18]. Conversely, synthetic biomaterials are easy to handle. They have controlled degradation rates and do not transfer pathogens. However, they usually need surface molecules to help the interactions between cells and the material, and, moreover, their degradation may produce unwanted subproducts [10]. In tissue engineering, the synthetic biodegradable materials most commonly used are PGA, PLA, and the copolymer poly DL-lactic co-glycolic acid (PLGA). When these polymers are implanted in an in vivo model, there is a massive release of acid products, which triggers an inflammatory response [19]. An interesting alternative can be found in the use of natural biomaterials, such as fibrin. They are similar in composition and structure to the native extracellular matrix and also have the ability to exert certain stimuli on cells that are equivalent to those of the original tissue, releasing proteins and growth factors that enhance cell functions.

Fibrin: Concept, nature and clinical use In physiological conditions, fibrin plays an important role in tissue injuries [1] since it acts as a support for the haemostatic plug, forming a protein mesh that results from the polymerization of fibrinogen. Fibrinogen is a very large (340 kD) and long (45 nm long and 9 nm in diameter) glycoprotein consisting of three pairs of polypeptide chains (Aα, Bβ, and γ) bound by disulphide bridges. It is divided into: (i) the central node, the E domain, composed of six amino-terminal ends of the protein chains and bound by

– Possible transfer of pathogens – Usually require modifications and processing techniques – Mechanical and degradation properties depend on application

– Variability due to natural variability – Require surface modification to improve cell interactions – Mechanical and degradation properties are affected by processing techniques

– Degradation may produce products that cause reactions or inflammatory response

Fibrinopeptides A & B

Fibrinogen Thrombin Ca2+

A) Proteolysis of fibrinogen Fibrinopeptides A & B

Unstable Fibrin Thrombin

Factor XIII

Ca2+

B) Fibrin stabilization

Factor XIIIa

Stable Fibrin

Fig. 2 – Formation of the fibrin clot. (A) The action of thrombin on fibrinogen produces a molecule of unstable fibrin (after the release of fibrinopeptides (A and (B)) in a reaction that depends on calcium ions. (B) Fibrin is able to adhere to itself through the action of factor XIIIa (plasma factor XIII is activated by the action of thrombin in a reaction that depends on calcium ions) and produces a stable fibrin clot. disulphide bridges, (ii) two external D nodes composed of carboxy-terminus ends, (iii) the carboxy-terminus end of the Aα chain, which is a flexible arm that may change in position to bind to other molecules of fibrinogen [20–22]. Fibrinogen has binding sites related to fibrin assembly and also binding sites for proteins of the clotting system. In the E domain, thrombin binds to fibrinogen through the substrate site and a low-affinity non-substrate site; i.e., where thrombin cannot perform its biological activity. Fibrinogen also has another highaffinity site at the C-terminal end of the γ´ chain and a binding site for the B subunit of Factor XIII [23,24]. In the formation of the fibrin network, fibrinogen is converted into fibrin through the action of thrombin and activated Factor XIII (Factor XIIIa). This process depends to a large extent on calcium ions [23] and consists of two different stages: (i) proteolysis of fibrinogen by thrombin, followed by the polymerization of fibrin monomers, and (ii) fibrin stabilization through the action of Factor XIIIa (Fig. 2).

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The concentration of fibrinogen in plasma, which is released through the liver, varies across a narrow range (2 to 4 g/L) [25,26]. However, these small variations change the structure of fibrin [21,27]. Cells adhere to fibrin either directly, via integrin receptors (mainly integrin ανβ3) on tripeptide amino acid sequences such as arginin-glycine-aspartic acid (RGD), or indirectly, based on the capacity of fibrin to bind blood-borne extracellular matrix (ECM) proteins, for example fibronectin and vitronectin [28,29]. Moreover, fibrin can bind several growth factors, both directly and indirectly, through heparin. The cellular response is further regulated by a number of bioactive molecules bound by fibrin, such as growth factors (basic fibroblast growth factor and vascular endothelial growth factor) and enzymes and proenzymes, (plasminogen activactor and plasminogen) [30]. Enzymes (thrombin), proenzymes (plasminogen) and enzyme inhibitors also have the capacity to bind to fibrin and control the balance between clotting and fibrinolysis [25]. The first clinical use of fibrin emulsion dates from 1909 and it was used to improve wound healing [31]. However, purified thrombin and fibrinogen were first combined in 1944 for the adhesion of skin grafts in soldiers with large burns [32]. Despite this, the concept of fibrin as currently held would not be used until the beginning of the seventies, thanks to the significant development in isolation techniques and the concentration of clotting factors. Tissucol/Tisseel™ and Beriplast HS/Beriplast P™ were the first fibrin sealants marketed in Europe, but today there is a huge variety of products available on the market; they all have different compositions [33] and, consequently, different adhesive properties [34]. In the nineties, the licence for the clinical use of commercial fibrinogen pools was suspended by the FDA (Food and Drug Administration), thus preventing the import of commercial fibrins from Europe. This was due to the associated high risk of viral transmission [35]. Several techniques of viral inactivation and elimination were developed to ensure a reduction in such viral transmission. The procedures for the selection, testing, and screening of plasma donors incorporate highly sensitive techniques [31], which reduce the possibility of viral transmission significantly [36,37]. Fibrin has been used as a sealant in several clinical applications: for example, in cardiovascular and thoracic surgery [33], in neurosurgery [38] and in plastic and reconstructive surgery [39].

From sealants to three-dimensional scaffolds for cell culture When fibrin is used as a sealant or adhesive, high tensile strength and adhesion strength are needed and their mechanical properties require a high concentration of fibrinogen [1,40]. As the concentration of fibrinogen increases, fibrin becomes more compact and rigid and less permeable [21]. An ideal threedimensional structure for cell culture requires low concentrations of fibrinogen in order to contribute to improved cell proliferation. Accordingly, the optimal composition of fibrinogen in a fibrin scaffold would be approximately 3–5 mg/ml [1,48,49]. In recent decades, fibrin has not only been used as a sealant system, because owing to its chemical and mechanical properties it

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can also serve: (i) as a drug delivery system [42] (drugs, antibiotics or chemotherapy agents), (ii) to release cells and growth factors [43,44], and (iii) as a three-dimensional scaffold able to host cell growth and differentiation [1,46,47]. A three-dimensional scaffold is necessary for cell adhesion, proliferation and differentiation [6]. The scaffolds used in tissue engineering must be biocompatible and biodegradable, must have a porous structure, and must not be toxic or immunogenic. Some natural and synthetic scaffolds fulfill all these properties, one of them being fibrin scaffolds. Fibrin is an excellent scaffold for tissue engineering because of its biocompatibility and biodegradability in soft tissue tests [51], and the initial stability of the grafted stem cells [52]. Fibrin has been used not only for tissue-engineering techniques; other strategies include delivery systems, and it is also used as a culture differentiation tool. Thus, the following review strategy focuses on successful studies of cultured fibrin matrices for tissue engineering applications. Table 2 shows some of the several clinical applications related to the tissue engineeringspecific techniques of cell culture in fibrin scaffolds.

Adipose tissue engineering Borges et al. [53] proposed the first patented connection of tissueengineered microvessels in adipose tissue to a host vessel system without applying exogenous angiogenic growth factors or temporary transfection. The co-transplantation of endothelial cell spheroids together with angiogenic mesenchymal cells in fibrin scaffolds permits the engineering of complex three-dimensional implants. Two years later, Cho et al. [54] demonstrated that volume-stable adipose tissues can be engineered in vivo using mechanical support structures of PGA fiber-based matrices with PLA suspended in a fibrin matrix. This technique offers the potential for the augmentation of adipose tissues with volume conservation. Zhang et al. [55] showed that the injection of a fibrin glue scaffold with adiposederived stem cells can create adipose tissue-like new-born tissues at the injection sites. Accordingly, cell-fibrin matrices may help to explore the possibility of building tissue-engineered adipose tissue and find a new approach for repairing soft tissue defects.

Bone engineering Perka et al. [52] elaborated a tissue-graft of periosteal cells seeded into a biodegradable PGLA polymer or fibrin beads for the reconstruction of critical-size bone defects, observing that such beads may serve especially well for the repair of circumscribed contained defects. Yamada et al. [56] recreated three-dimensional templates for bone growth, resulting in new bone formation at heterotopic sites by a combination of fibrin glue plus β-tricalcium phosphate (β-TCP) and mesenchymal stem cells. Recently, Zhou and Xu. [57] evaluated the efficiency of stem cell-encapsulating fibrin microbeads for injection and bone regeneration. Overall, cell-fibrin combinations may be useful as bone graft substitutes due to their capacity to induce bone formation.

Cardiac tissue engineering Jockenhoevel et al. [16] reported that scaffolds play a central role in the creation of 3-D structures in cardiovascular tissue

Table 2 – Clinical applications related to tissue engineering-specific techniques of cell culture in fibrin scaffolds. Tissue engineering techniques of cell culture in fibrin scaffolds Study Adipose tissue engineering

Borges et al. [53] Cho et al. [54]

Zhou and Xu [57] Cardiac tissue engineering

Cells

Aim

Findings

Fibrin on chorioallantoic membrane PGA fiber-based matrices with PLA suspended in a fibrin matrix Fibrin glue

Human preadipocytes and dermal endothelial cells Human preadipocytes

Formation of a capillary-like system

Formation of adipose tissues with capillary networks connected to the host vessel system in a cell fibrin graft

In vivo engineered volume-stable adipose tissues

Fibrin matrix served as a space-filling matrix for preadipocyte implantation

Human adiposederived stem cells Rabbit periosteal cells

Built tissue-engineered adipose tissue

Adipose tissue-like new-born tissues were found in the injection sites

Reconstruction of critical size bone defects

Fibrin structures as carrier materials showed intense bone formation

Rat mesenchymal stem cells

Provide 3D templates for bone growth resulting in new bone formation at heterotopic sites Deliver stem cells inside injectable scaffolds to promote bone regeneration

Successful bone formation eight weeks after implantation

PGLA polymer fleece / fibrin beads Fibrin glue plus β-tricalcium phosphate (β-TCP) Alginate-fibrin microbeads Fibrin gel Silicone chambers and fibrin gel

Neonatal cardiac myocytes

Weber et al. [59]

Fibrin and textile coscaffold

Ovine umbilical artery cells

Tissue engineering-specific techniques of cell culture in fibrin scaffolds Cartilage Eyrich et al. Fibrin Primary bovine engineering [60] chondrocytes Li et al. [61] Fibrin gel inside a Mesenchymal stem PLGA sponge cells Muscle tissue engineering

Nervous tissue

Huang et al. [70] Page et al. [71]

Fibrin gel

Rat myoblasts

Fibrin microthreads

Human muscle cells

Liu et al. [72]

Fibrin hydrogel

Sakiyama et al. [73]

Fibrin gel

Human umbilical cord mesenchymal stem cells Dorsal root ganglia cells

Stem cell-encapsulating fibrin microbeads are suitable for injection and the synthesis of bone mineral

Creation of cardiovascular tissue structures based on patient cells Formation and characterization of contractile, vascularized three-dimensonal cardiac tissue Development of the first tissueengineered heart valve based on a tubular leaflet design

Autologous fibrin scaffold can recreate 3D cardiovascular structures

Develop a long-term stable fibrin gel for in vitro cartilage engineering Develop a construct for simultaneous regeneration of cartilage and subchondral bone Develop three-dimensional engineered muscles Provide an efficient delivery system for cell-based therapies and improve regeneration of large musculoskeletal wounds Investigate the encapsulated hUCMSC proliferation and myogenic differentiation for muscle tissue engineering Improve the potential of fibrin to promote nerve regeneration

Cultivation of primary bovine chondrocytes in fibrin gels resulted in the development of an adequate cartilaginous tissue PLGA/fibrin gel restores functional cartilage with the expression of chondrogenesis-marker genes

Newly formed constructs resemble normal myocardial tissue in terms of their contractile and morphological characteristics A fibrin-based tubular heart-valve showed simple construction and the advantages of a living structure

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Jockenhoevel et al. [16] Birla et al. [58]

Human umbilical cord mesenchymal stem cells Myofibroblast cells

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Bone engineering

Zhang et al. [55] Perka et al. [52] Yamada et al. [56]

Scaffold

Fibrin gels provide a method to engineer three-dimensional functional muscle tissue Development of a platform for microthread-based delivery of autologous cells, which has the potential to improve healing outcomes in large skeletal muscle wounds Successful myogenic differentiation with the formation of multinucleated myotubes. The fibrin construct seemed promising for muscle tissue engineering applications Incorporation of heparin-binding peptides into fibrin gels enhanced peripheral nerve regeneration through nerve guide tubes

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Table 2 (continued ) Tissue engineering techniques of cell culture in fibrin scaffolds Study

engineering

Respiratory tissue engineering

Skin engineering

Vascular tissue engineering

Aim

Findings

Willerth et al. [74]

Fibrin scaffold

Murine embryonic stem (ES) cells

Evaluation of ES cell differentiation and proliferation in fibrin scaffolds

Zurita el al. [65]

Matrices of fibrin and blood plasma

Human stromal stem cells

Analyze survival and neural differentiation of human stromal stem cells supported in biological matrices of fibrin

Development of an optimal fibrin scaffold for evaluating ES cell differentiation and proliferation in culture, and for use as a platform for neural tissue engineering Fibrin is an excellent biological scaffold for bone marrow stromal cells in cell therapy strategies in the nervous system

Han et al. [75]

Fibrin gel

Alaminos et al. [76]

Fibrin-agarose scaffold

Develop a bioengineered ocular surface tissue replacement in fibrin gel Construct a full-thickness biological substitute of the rabbit cornea

Autologous bioengineered ocular surface with a combination of presumed corneal epithelial stem cells in a cross-linked fibrin gel In vitro development of a full-thickness rabbit corneal model

Kim et al. [77]

Fibrin/hyaluronic acid (HA) gel

Human corneal epithelial stem cells Rabbit epithelial, stromal, and endothelial cells Rabbit chondrocytes

Manufacture tissue-engineered trachea by using fibrin/hyaluronic acid gel

Cornelissen et al. [78]

Fibrin gel

Respiratory epithelial cells

Test the suitability of fibrin for respiratory tissue engineering

Geer et al. [79]

Fibrin

Human keratinocytes

Carriel et al. [80]

Fibrin-agarose scaffold

Hankemeier et al. [81]

Fibrin

Human dermal fibroblasts and epithelial keratinocytes Human Bone Marrow Stromal Cells

Paxton et al. [82]

Fibrin gel

Embryonic chick tendon fibroblasts

Develop a matrix for keratinocyte migration and support reepithelialization of skin Develop a scaffold that supports the generation of a proper human skin construct Improve the healing process in a standardized patellar tendon window defect Production of tissue-engineered constructs with suitable replacement tissue characteristics

Functional epithelial regeneration without graft rejection and inflammation were observed after repair of a tracheal resection using fibrin/HA implants Cells grown on fibrin gel revealed adequate differentiation. Thus, fibrin gel might prove to be a suitable scaffold for respiratory tissue engineering Fibrin can be used as a substrate for keratinocyte growth and for wound reepithelialization

Cummings et al. [83]

Fibrin and collagen

Rat aortic smooth muscle cells

Lesman et al. [84]

Fibrin and/or PLA/ PLGA

Endothelial cells, fibroblasts and tissue specific skeletal myoblast cells

Evaluate the use of fibrin as an alternative, or additional, matrix in vascular tissue engineering Generate vascular networks by applying the multicellular culturing technique within 3D fibrin-based constructs

Fibrin-agarose artificial skin showed adequate biocompatibility and proper biomechanical properties. Injection of human BMSC in a fibrin glue matrix appeared to lead to more mature tissue formation and regular patterns of cell distribution Fibrin helped to analyze the morphological changes occurring during the early-stage formation and maturation of tissue-engineered boneto-bone ligament-like constructs The addition of fibrin to collagen-based blood vessel constructs containing vascular smooth muscle cells produced desirable changes in the mechanical and morphological properties of the tissue A fibrin and PLA/PLGA synthetic scaffold provided a way to enhance vascularization in vitro and in vivo

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Tendons & ligaments engineering

Cells

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Ocular tissue engineering

Scaffold

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engineering, in which fibrin gel could combine the whole array of the important properties of an ideal scaffold (autologous, moldable and with controllable degradation). Birla et al. [58] reported that engineering cardiac tissue in three dimensions is limited by the ability to supply nourishment to the cells at the center of the construct. Their study demonstrated the in vivo survival, vascularization, organization, and functionality of transplanted myocardial cells in silicone chambers and fibrin gel. Recently, Weber et al. [59] proposed a fibrin-based tubular heart valve as an attractive new system, which combines the simplicity of construction and the reliability of implantation with the advantages of a living structure (self-repair, remodelling and physiological haemodynamics). In this sense, cell-seeded fibrin scaffolds have shown good results as cardiac grafts.

Cartilage engineering The goal of articular cartilage tissue engineering is to provide cartilaginous constructs to replace abnormal cartilage. Eyrich et al. [60] proposed fibrin gels for cartilage engineering because it is essential that hydrogel scaffold systems maintain long-term shape stability and mechanical integrity for applications in cartilage tissue engineering. Li et al. [61] proposed PLGA/fibrin gel/MSCs complexes to restore functional cartilage. The regenerated tissues had good integration with the host tissues, expressed chondrogenesismarker genes, and restored functional cartilage. Interestingly, Ahmed et al. [62] characterized fibrin hydrogel-degrading enzymes during the development of tissue engineering scaffolds. Their data suggest that plasmin and MMPs contribute independently to fibrin hydrogel breakdown, but that either enzyme can achieve extracellular matrix breakdown. Thus, cell-seeded fibrin may be an accessible and useful tool for cartilage engineering.

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Muscle tissue engineering Huang et al. [70] reported fibrin-based gels as a novel method to engineer three-dimensional functional muscle tissue by culturing myoblasts. This model allows long-term cultures of skeletal muscle cells to be obtained and may be used to model the development of skeletal muscle in vitro. Page et al. [71] developed a platform for a microthread-based delivery of autologous cells, which has the potential to improve healing outcomes in large skeletal muscle wounds by using a scaffold delivery system for cell-based therapies with fibrin microthreads. Recently, Liu et al. [72] described the encapsulation of a human umbilical cord stem cells in a new macroporous and injectable fibrin for muscle tissue engineering, with successful myogenic differentiation and the formation of multinucleated myotubes. In sum, cell-based fibrin has been used successfully for the proliferation and encapsulation of muscle cells for muscle tissue engineering applications.

Neural tissue engineering As an example of matrix design in tissue engineering, Sakiyama et al. [73] showed that the incorporation of heparin-binding peptides into fibrin gels enhanced peripheral nerve regeneration through nerve guide tubes. Furthermore, Willerth et al. [74] optimized fibrin scaffolds for the differentiation of murine embryonic stem cells into neural lineage cells. Their fibrin scaffolds could be used as a platform for neural tissue engineering applications, such as the treatment for spinal cord injury. Accordingly, Zurita al. [65] studied the usefulness of platelet-rich plasma as a scaffold for stromal cells in regenerative therapies of the nervous system. Thus, fibrin can be considered a good biological scaffold for cell survival and neural differentiation.

Ocular tissue engineering Delivery systems Montana et al. [63] reviewed fibrin adhesives as drug delivery systems (owing to their slow lysis) for several molecules such as growth factors, anesthetic drugs, antibiotics, chemotherapeutic agents, and also cells. There are several studies that have addressed the use of fibrin as a delivery system.

Applications in differentiation Cell differentiation to adipogenic, osteogenic and chondrogenic lineages [40] has been demonstrated in autologous fibrin scaffolds. Fibrin scaffolds had already been used for the differentiation of embryonic stem cells [64] and stromal human cells [65] but little is known about the differentiation potential inside fibrin scaffolds. Adipogenic differentiation is easily obtained in monolayer cultures and only a few researchers have attempted to accomplish this in three-dimensional structures [66]. The use of scaffolds in osteogenic differentiation is more frequent because a firm structure is needed as a support for bone formation [67,68]. Finally, the pellet culture system is used in chondrogenic differentiation because a threedimensional structure it required to help to cartilage formation [69]. Thus, fibrin scaffolds have been used as a niche for cells and the fibrin matrix provides the three-dimensional structure required for cell adhesion, proliferation and differentiation. Such scaffolds are completely autologous, biocompatible, easy to develop and can be used for implantation in several therapeutic applications [47].

Han et al. [75] investigated a bioengineered ocular surface tissue consisting of human corneal epithelial stem cells in a cross-linked fibrin gel for potential transplants, which represents a potential improvement in current attempts to create a transportable, pliable, and stable tissue replacement. Alaminos et al. [76] created a complete rabbit corneal substitute using a fibrin-agarose scaffold. Their findings suggest that it is possible to develop a fullthickness rabbit corneal model in the laboratory by culturing three types of corneal cells in different positions. Thus, cellseeded fibrin has been used efficiently for the development of stable ocular tissue substitutes.

Respiratory tissue engineering Kim et al. [77] reported a strategy to manufacture a tissueengineered trachea by using fibrin/hyaluronic acid gel cultured with chondrocytes, and they evaluated the feasibility of creating tracheal cartilage. Their data showed functional epithelial regeneration without graft rejection or inflammation after repair of a tracheal resection. Cornelissen et al. [78] cultured respiratory epithelial cells on fibrin gel. Their data suggested that fibrin gel might be a suitable scaffold for respiratory tissue engineering due to the good proliferation, functionality, and differentiation of cells. In sum, cell-seeded fibrin might be used in respiratory tissue engineering due to its ability to promote efficient proliferation of cells and functional epithelial regeneration.

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Skin engineering Geer et al. [79] developed an in vitro model of wound re-epithelialization, based on engineered composite skin equivalents of human keratinocytes, in which fibrin can be used as a substrate for keratinocyte growth and migration. Accordingly, this model could be used to study molecular mechanisms of re-epithelialization. Carriel et al. [80] developed an equivalent model of fibrin-agarose skin able to reproduce the structure and histological architecture of native human skin, especially after long-term in vivo implantation, suggesting that these tissues could reproduce native skin. Thus, applied in skin engineering fibrin might be a useful structure for cell growth.

Tendon and ligament engineering Hankemeier et al. [81] studied the tissue engineering of tendons and ligaments using human bone marrow stromal cells in a liquid fibrin matrix. They suggested that ideal matrices for the tissue engineering of ligaments and tendons should allow homogenous cell seeding and offer sufficient stability. In this regard, fibrin could offer an excellent structure for cell culture. Paxton et al. [82] investigated the morphological changes that occur during the early-stage formation and maturation of tissue-engineered bone-to-bone ligament-like constructs by using a cell-seeded fibrin gel. Accordingly, further studies with fibrin might be necessary to verify the application of cell-seeded fibrin for tendon and ligament engineering.

Vascular tissue engineering Cummings et al. [83] demonstrated that the properties of engineered vascular tissues can be modulated by a combination of selected extracellular matrix components, such as collagen, fibrin, and collagen-fibrin mixtures. The addition of fibrin to collagen-based blood vessel constructs containing vascular smooth muscle cells produced desirable changes in the mechanical and morphological properties of the tissue. Lesman et al. [84] used 3D fibrin gels alone or in combination with synthetic PLA/PLGA sponges to support in-vitro construct vascularization and to enhance neovascularization upon implantation. The combination of PLLA/PLGA sponges with fibrin matrices provided added mechanical strength and featured highly mature vessel-like networks. Their findings revealed that a complex biomaterial platform involving fibrin and a PLA/PLGA synthetic scaffold provided a way to enhance vascularization in vitro and in vivo. Accordingly, cell-seeded fibrin might be used for vascular tissue engineering. To conclude, it is clear that in recent decades fibrin scaffoldsupported cell cultures, either alone or combined with other materials, have been used in several different therapeutic applications and provide a relevant support for tissue engineering.

Methods for the building of fibrin scaffolds The methods used to develop both commercial and autologous fibrins isolate and concentrate blood fibrinogen by means of a first centrifugation combined with cryoprecipitation methods and other chemical methods [85–87]. Sometimes, the methods for fibrinogen concentration demand expensive and difficult techniques that alter

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the nature of the fibrin. However, the fibrin products thus obtained may be useful as sealants or adhesives. The 3D scaffold structure of fibrin for cell culture is composed of a network of interconnected pores, through which (i) cells migrate, multiply and spread along the scaffold, (ii) the diffusion of nutrients and waste products is easier and (iii) vascularization is promoted and developed [2]. Fibrinogen is the most important factor because it defines the pore structure [88]. A low concentration of fibrinogen (physiological concentrations) provides an appropriate matrix for cell growth, migration and differentiation, and is characterized by a network of thin fibers forming a dense structure [48,50,89]. Commercial fibrins contain high concentrations of fibrinogen (60–115 mg/ ml), their use is expensive [1], and they also entail the risk of viral transmission. Although the manufacturing procedures for fibrin sealants include processing steps designed to reduce the risk of viral transmission [36], autologous fibrins are a good choice because they are developed from autologous blood from only one patient, extensively tested for markers of disease, and not from pools. In addition, if no fibrinogen concentration methods are used, it is easier and cheaper to produce them. There is a wide range of useful components to produce fibrins: fibrinogen, thrombin, Factor XIIIa, calcium ions and antifibrinolytic agents (such as aprotinin and tranexamic acid). As stated above, thrombin (generally dissolved in a calcium chloride solution) converts fibrinogen into fibrin monomers, to form unstable fibrin clots. Factor XIIIa catalyzes the cross-linking of adjacent fibrin fibers, after which the clot becomes mechanically stable. The use of calcium chloride in the elaboration of fibrin scaffolds is because calcium ions facilitate the conversion of fibrinogen molecules to fibrin and enhance the activity of factor XIIIa, which helps stabilize the 3D structure of fibrin networks. The interaction of calcium ions with coagulation proteins is probably the reason why the bonding strength of fibrin adhesive is increased and the gelling time is decreased [90,91]. One of the disadvantages of fibrin is that it degrades rapidly, so the use of antifibrinolytic agents (such as aprotinin or tranexamic acid) is useful to prevent clot lysis. Tranexamic acid is a plasminogen inhibitor and an antifibrinolytic agent. It is an alternative to aprotinin for controlling the in vitro degradation rate [92]. Tranexamic acid reduces fibrin degradation, maintains integrity, and does not affect the viability of cells or calcium deposits [93]. It has been described in several studies that thrombin values are unrelated to better cell proliferation [94,95]. This is why thrombin is not needed in the development of 3D scaffolds. The techniques for developing 3D scaffolds using plasma, fibrinogen and purified and isolated thrombin components could be useful for cell culture and hence for their use in tissue engineering. The large amounts of molecules in blood plasma could benefit the cell adhesion of molecules such as fibronectin [96] or fibrinogen [97] itself or could promote fibroblast proliferation. Tissue repair can also be stimulated and accelerated by growth factors (TGF-β, bFGF, EGF VEGF, etc.), platelets, cytokines and enzymes [25,48,79,92].

Autologous fibrin vs. commercial fibrin The best known and most widely used commercial fibrins available are Tissucol™, Beriplast™ and Quixil™. The composition of these fibrin sealants differs in the quantities of plasma proteins, fibrinogen, thrombin, Factor XIIIa, fibronectin, plasminogen and antifibrinolytic

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agents [63]. However, Hale et al. [98] have recently published a study in which they assessed the effect of scaffold dilution on the migration of mesenchymal stem cells (MSCs) from fibrin hydrogels. They observed that MSC migration from fibrin hydrogels increased with the dilution of the fibrinogen component for the both autologous and commercial sources. For instance, in stromal cells cultured in vitro it was noted that autologous fibrin scaffolds represent an excellent biological scaffold. By contrast, commercial fibrins afforded a poor cell survival [65]. Thus, commercial fibrins are designed for use as sealants and this complicates their use as three-dimensional scaffolds for cell culture. As regards autologous fibrins, there are two commercial versions used as sealants: Vivostat™ and CryoSeal™. These sealants are marketed as medical devices, but the manufacturing system fails in reproducibility with regard to their mechanical properties and clinical outcomes [68]. By contrast, autologous fibrins can be produced in a faster and cheaper way. Fairly recently, a new autologous fibrin scaffold [1,47] was introduced; it is cheap, easy to use, natural (with physiological concentrations of fibrinogen), implantable, highly available and has low fibrinogen concentrations. The key point to this model is that no fibrinogen concentration techniques, such as cryoprecipitation or other chemical methods, are used. This system is adequate both for cell culture and cell differentiation [1,47]. Autologous blood is obtained, centrifuged to separate it from the plasma (physiological concentrations in plasma are 2–4 mg/ml [21]) and frozen at  40 1C until used [1]. Accordingly, more natural and less modified fibrins are used and the nature of the fibrin (with its physiological concentrations) is preserved. It should not be forgotten that if both the cells and the plasma components of the fibrin gel are of human origin, this technique provides the potential for a totally autologous bioengineered replacement tissue. In sum, scaffolds made of autologous fibrin are excellent candidates for tissue engineering experiments because they promote regeneration and angiogenesis [41]. They also offer a suitable matrix for cell growth and differentiation [1,46,47] and, owing to their porous morphology, they are suitable for both cell culture systems [45] and as release systems of growth factors, such as VEGF [43,44] and bFGF [99].

Conclusions The composition and methods used to develop fibrin scaffolds are crucial for cell culture and possible therapeutic applications in tissue engineering. Autologous fibrin scaffolds have low fibrinogen concentrations, which means that they are suitable for cell culture. An autologous carrier could prevent the complications of other techniques or those deriving from the use of commercial fibrins. Thus, autologous fibrin has proved to be a valuable scaffold for tissue engineering.

Conflict of interest None.

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Acknowledgments This research was supported by the Diputación Provincial of León, the Tissue Bank San Francisco Clinic Foundation of León and the University of León thanks to a fellowship grant from the Manuel Elkin Patarroyo Foundation.

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