Unique heterologous fibrin biopolymer with hemostatic, adhesive, sealant, scaffold and drug delivery properties: a systematic review

Abstract Fibrin biopolymers, previously referred as “fibrin glue” or “fibrin sealants”, are natural biomaterials with diverse applications on health. They have hemostatic, adhesive, sealant, scaffold and drug delivery properties and have become widely used in medical and dental procedures. Historically, these biomaterials are produced from human fibrinogen and human or animal thrombin, and the possibility of transmission of infectious diseases by human blood is not ruled out. In the 1990s, to overcome this problem, a new heterologous biomaterial composed of a thrombin-like enzyme purified from Crotalus durissus terrificus venom and a cryoprecipitate rich in fibrinogen extracted from buffaloes Bubalus bubalis blood has been proposed. Therefore, a systematic review of studies on exclusively heterologous fibrin sealants published between 1989 and 2018 was carried out using the following databases: PubMed, SciELO and Google Scholar. The keyword used was “heterologous fibrin sealant”. The search resulted in 35 scientific papers in PubMed, four in SciELO and 674 in Google Scholar. After applying the inclusion/exclusion criteria and complete reading of the articles, 30 studies were selected, which formed the basis of this systematic review. It has been observed that the only completely heterologous sealant is the one produced by CEVAP/UNESP. This heterologous biopolymer is proven effective by several studies published in refereed scientific journals. In addition, clinical trials phase I/II for the treatment of chronic venous ulcers authorized by the Brazilian Health Regulatory Agency (ANVISA) were completed. Preliminary results have indicated a safe and promising effective product. Phase III clinical trials will be proposed and required to validate these preliminary findings.


Background
Fibrin sealants are biological materials composed of fibrinogen and thrombin. In the presence of calcium and factor XIII, thrombin converts fibrinogen to soluble fibrin, forming a stable clot and mimicking the final step of the coagulation cascade. Due to its property, this product has indications based mainly on its hemostatic and tissue adhesive properties. Thus, its recent clinical and experimental uses include drug administration and tissue engineering applicability [1].
The use of fibrin compounds as a hemostatic agent occurred for the first time in Germany in 1909 [2]. In the 1940s, it was used as an adhesive in the recovery of injured peripheral nerves through the association of autologous fibrinogen and thrombin [3], and also for skin graft fixation [4,5].
Sutures are the conventional technique in surgical procedures; however, due to the formation of fistulas, granulomas, tissue ischemia, and lacerations in some cases, tissue engineering has been stimulating the use of fibrin-based sealants [6]. Thus, these products have been used in current clinical and surgical applications, mainly to approach the edges of the skin, to produce adherence to other tissues and to provide hemostasis [6][7][8][9].
Traditionally, fibrin sealants are produced in two ways: using autologous or homologous blood derivatives [10,11]. Autologous sealants use the patient's own blood. Although biocompatible and presenting no risk of infectious diseases transmission, they are not feasible in emergency surgeries [12]. As an alternative, the homologous fibrin sealants produced by a pool of human blood has been used [13]. However, in these cases the literature suggests risks of infectious diseases transmission such as hepatitis, HIV and human parvovirus [14][15][16].
Therefore, a group of Brazilian researchers from the Center for the Study of Venoms and Venomous Animals (CEVAP) of São Paulo State University (UNESP), Brazil, proposed a new heterologous fibrin sealant (HFS) using a snake venom fraction extracted from Crotalus durissus terrificus and a cryoprecipitate rich in fibrinogen obtained from Bubalus bubalis buffaloes blood [11,[17][18][19].
Snake venoms are composed of several proteolytic enzymes, mainly serine proteases [20][21][22]. These enzymes act by activating or inhibiting the specific blood factor involved in platelet aggregation, coagulation and/or fibrinolysis [20,23,24]. In in vitro experiments, a thrombin-like enzyme from the South American rattlesnake acts similarly to human or animal thrombins transforming fibrinogen into fibrin and forming a robust insoluble fibrin-net [20,22].
The first experimental studies using the HFS produced by CEVAP were performed by Juan et al. in 1995 [25], who presented the preparation procedures and observed good adhesive and hemostatic properties in the repair of nerve injuries in rats. In 1998, Thomazini-Santos et al. [26] showed that the cryoprecipitate extracted from bubaline blood had higher concentrations of fibrinogen and better performance when compared with blood from humans, bovines, ovine or equines. Additionally, they concluded that antifibrinolytic agents were not required to this sealant to achieve successful coaptation of skin surgical edges [27]. Stolf et al. [28] applied for the first time this new sealant in humans as an alternative to conventional suture in the nasolabial region, and described a good adhesive capacity and healing of this material. After these conclusions, the researchers standardized the unique HFS using gyroxin -a serinoprotease obtained from the South American rattlesnake -and a cryoprecipitate extracted from Bubalus bubalis buffaloes [9,11].
Thereby, due to its clinical importance and increasing use in several health areas, the aim of this systematic review was to evaluate and compare studies on exclusively heterologous fibrin sealants produced worldwide.

Methods
A systematic review of the literature on HFS was carried out in November 2018 from PubMed, SciELO and Google Scholar databases taking into account work conducted between 1989 and 2018. The used keywords were "heterologous fibrin sealant". The inclusion criteria were the analysis of the title and abstract, in order to identify studies that used only heterologous components in the fibrin sealant. In vitro studies and reviews were not selected for detailed analysis, only experimental studies.

Results
The search resulted in 35 scientific papers in PubMed, four in SciELO and 674 in Google Scholar. After applying the inclusion/ exclusion and reading criteria, a total of thirty studies were selected to form the basis of this systematic review ( Figure 1). Table 1 summarizes the main articles included.  [25] To evaluate the efficacy of fibrin sealant in sciatic nerve repair in rats.
Application of sealant fractions in sciatic nerves of rats. Initially fibrinogen was obtained from human plasma.
The results obtained were similar to those of conventional glue.  [42] To evaluate fibrin sealant derived from snake venom as an alternative to conventional uterine suture after ovine cesarean surgery.
Twenty-eight pregnant sheep divided into four groups of seven animals: six animals were submitted to surgery with fibrin sealant and one animal underwent the conventional hysterorrhaphy.
The healing of the wound showed good macroscopic appearance 30 days after surgery with the application of the sealant. However, microscopically, the uterus did not return to pre-pregnancy conditions 30 days after the experimental cesarean section.
Animals submitted to conventional cesarean section showed a better process of wound healing.
Further studies on the use of this new sealant in hysterorrhaphy after cesarean section in several animal species are required. [43] To assess the effect of snake venom derived from fibrin glue on the viability of split-thickness skin graft in dogs.

Rahal et al. (2004)
One group of nine dogs: a skin graft was collected from the left lateral thoracic area and fixed using several single sutures. On the right side the fibrin sealant + suture was applied.
Compared to the sutured graft, the graft fixed with the sealant showed more cells and a greater number of collagen fibers in the papillary layer of the dermis.
Fibrin sealant increased the survival of the autogenous skin graft, but has moderate adhesive power.
The fibrin sealant did not have sufficient adhesive strength to fix the graft on its own, especially at the site where the wound was induced. [44] Evaluation of the healing process in canine uterus hysterorrhaphy using fibrin sealant derived from snake venom.
All groups presented total regeneration of the epithelium, regardless of the treatment. The thickness of healing tissue was higher in G2 than G1 and G3.
The experimental model was appropriate to achieve the proposed objectives. Fibrin sealant produced less inflammation in the exudative phase and eased the evolution of proliferation and maturation phases. Limited number of patients, sample and statistical analyzes. The histological results were not sufficient to achieve a conclusion. [38] To assess the effect of the new fibrin sealant in the repair process of venous ulcers in 24 adult patients.

Gatti et al. (2011)
Two groups: G1 (control, n = 11) treated with essential fatty acids and Unna's boot and, G2 (n = 13) treated with essential fatty acids, fibrin sealant and Unna's boot. Fibrin sealant was applied in the first and fourth week of treatment.
Fibrin sealant was effective in the healing of chronic venous ulcers, presenting ease of application, preparation of the wound bed, reduction of pain and greater number of discharges after eight weeks.
The application of fibrin sealant can improve wound healing processes and increase healing levels.
Preliminary phase I study and major studies with a larger number of patients should be conducted to evaluate the best way to apply the new sealant. [47] Evaluation of the osteogenic capacity of the new fibrin sealant (FS) combined with bone graft and laser irradiation.
The analyses showed absence of inflammatory infiltrate in the bone defect. Bone neoformation occurred in all groups, more intensely in G6.
The osteogenic effect of the 7 J/cm 2 laser has proved to be very efficient, and its combination with fibrin sealant derived from snake venom can accelerate the regeneration process.
The combination of laser and sealant becomes a therapeutic resource to be further investigated in bone regeneration research. [48] To assess the effects of fibrin sealant on functional recovery, neuronal survival, synaptic plasticity and glial reaction of the motor neuron after ventral root reimplantation.
G2 showed no significant changes in the microglial response compared to G1. However, the astroglial reaction was significantly reduced in this group.
The root reimplantation performed with fibrin sealant increased neuronal survival and improved recovery of motor function, improving the regeneration process.
It is not clear whether it is the reimplantation site or the sealant that may be responsible for neuronal survival.  [35] To analyze the combination between fibrin sealant (FS) and rhBMP-2 or P1 in the repair of bone defects in rats.
There was a statistically significant difference (p < 0.05) in all groups after six weeks in relation to the volume of newly formed bones in the surgical area.
The new fibrin sealant proved to be biocompatible and the combination with rhBMP-2 showed greater osteogenic and osteoconductive capacity for bone healing.
The role of fibrin sealants in healing and osteogenesis remains not fully understood.  [50] To investigate the effectiveness of mesenchymal stem cells associated with fibrin sealant in the peripheral regenerative process after sciatic nerve tubulization.
Fifteen Lewis rats divided into three groups (n = 5). G1: unilateral sciatic nerve transection followed by implantation of polycaprolactone tubular prosthesis; G2: tube filled with fibrin sealant, and G3: tube filled with fibrin sealant and mesenchymal stem cells.
Sixty days after tubulization, the group with mesenchymal stem cells had a higher myelinated axon counting, more compact fibers and a tendency to increase the thickness of the myelin sheath. Cell treated animals also had better motor function.
The study confirms the efficiency of mesenchymal stem cell treatments after nerve tubulization. In addition, the use of fibrin sealant increases cell reactivity, leading to better compaction of myelinated axons and improving motor recovery.
Although sensory and motor recovery could be detected by stimulating the toes, recovery through gait recovery could not be recorded. It was not possible to observe the total bone repair, since a period of six weeks was not sufficient for the complete recovery of the critical defect in the femur of rats. [60] To determine the conditions that improve functional recovery after sciatic nerve neurorrhaphy using human embryonic stem cells (hESC) and heterologous fibrin sealant.

Mozafari et al. (2018)
A 5-mm segment of the sciatic nerve of mice was removed and rotated 180 degrees to simulate an injury, and the stumps were sutured. Then, the heterologous fibrin sealant and/or hESC was applied at the lesion site.
Sensory function improved when hESCs was used.
The new heterologous fibrin sealant can facilitate nerve repair.
For enhanced functional recovery and better motor neuron reinnervation, fibrin sealant and cell therapy should be used in combination with neurotrophic factors.

Spejo et al. (2018) [61]
Evaluation of the use of fibrin sealant as a scaffold to fill the gap formed during induced nerve injury and to retain the stem cells applied at the lesion site.
The groups AI and AI + DMEM, suffered a huge degeneration of the injured motor neurons. The groups treated with fibrin sealant, stem cells and fibrin sealant + stem cells, obtained high levels of motor neurons surviving the induced lesion.
The study demonstrates that mesenchymal stem cell therapy has a neuroprotective activity and, when associated with fibrin sealant, provides a better scaffold to retain cells at the lesion site.
Further studies, including clinical cases, are needed to understand and enhance the recovery of injured complexes.

Discussion
The unique heterologous fibrin sealant (HFS) produced by CEVAP was the only completely heterologous material validated and included in this review. It possesses several advantages such as: fast production process; low cost; potential to act as a scaffold for stem cells and biomaterials, and as a new drug delivery system [9,62]. Moreover, another positive point is its wide applicability in medical, veterinary and dental practice due to the possibility of custom formulation and replacement of conventional sutures. Considering all the properties described for this bioproduct, which go beyond the adhesive capacity, the name "sealant" was reconsidered and it has recently been called "fibrin biopolymer". In Figure 2, the evolution since the 1990s of the name fibrin glue is summarized.
Standardized for more than 20 years by Brazilian researchers, the heterologous fibrin biopolymer (HFB) is composed of natural products extracted from animals, without humanblood derivatives (Fig. 3). Therefore, it does not offer risk of transmission of infectious diseases, in addition to possessing low production costs, abundant raw material and possibility of custom formulation to the procedure type [9,11]. The economic impact of this HFB is due to the reduction of surgical time with similar performance or even better when compared to traditional sutures [6,11]. Thus, this systematic review aimed to gather, analyze and evaluate the available evidence on the use of HFB in the period of 30 years in in vivo experimental studies. At the end of this, it was verified by the selected studies in worldwide literature research its effectiveness in different clinical applications.  The experimental studies regarding this bioproduct began in 1995 by Juan et al. [25], as previously mentioned, and are being continued to date. Experiments were performed on different organs and animals such as rats, rabbits, dogs, sheep and horses. In this pioneer study, the fibrin sealant was applied in the sciatic nerve of rats, and showed similar efficacy to conventional glue. Also in an animal model, this sealant allowed the coaptation of intestinal segments and proved efficient in obtaining the anastomosis of the colon of rats [41]. However, its use in anastomosis requires additional studies in larger animals to allow analysis in larger organs.
In 1998, Sartori Filho et al. [40] studied its adhesive properties in ovine, using the fibrin sealant and observing their effects on testicular biopsy. The sealant was easily applied, presenting faster repair properties. Its efficacy in hemorrhage control was important to prevent bruising that causes adhesion formation. The advantage over the traditional method (needles and suture lines, for example) was also highlighted, since these was no longer needed.
Chalhoub et al. [42] performed hysterorrhaphy after cesarean sections of sheep. The mortality rate of the animals was 12.5% , which was considered high. As expected, all the operated animals maintained the placenta, which compromised the healing process, having a strong influence on the results of animals in which the sealant was used. Therefore, further studies using this biomaterial on hysterorrhaphy after cesarean section in different animal species are required.
A third study in ovine was carried out. This time, the implantation of the sealant in chondral defects of cartilage was analyzed [51]. The applicability of the biomaterial was excellent, showed stability and did not trigger undesirable effects such as inflammation, allowing a normal repair process.
In dog models, Moraes et al. [44] assessed the healing process in canine uterus hysterorrhaphy. The results indicate that fibrin sealant may have contributed to moderating the exudative phase, facilitating fibroplasia. In addition, the presence of fibrin in the sealant helps in the formation of a connective base, where the cells can proliferate and form a scar. A complementary study by the same team validated, through biomechanical tests, higher stiffness of the tissue treated with this sealant [63].
Moreover, the study by Rahal et al. [43] sought to analyze the effect of the fibrin sealant derived from snake venom on skin grafts in dogs. The graft adhered to the fibrin sealant showed histological characteristics similar to the sutured graft, but tissue repair of the former was more pronounced, besides having more cells and a greater number of collagen fibers in the papillary layer of the dermis. Its use also had an important effect in reducing the time of surgery, since it minimized the use of suture materials, which is an advantage especially for patients with high surgical risk.
Ferraro et al. [45,64], evaluated the healing strength of the tendon of the thoracic limbs of dogs and their clinical evolution, using fibrin sealant as a substitute for tenorrhaphy. The assessment of four biomechanical properties (resilience, rigidity, maximum limit and stability limit) allowed observing that tendon healing achieved progressive resistance with maximum tensile strength time. Thus, fibrin sealant derived from snake venom obtained positive results, promoting healing in the flexor tendon of dogs [64].
Sampaio et al. [46], proposed a model to repair corneal ulcers in dogs. Although fibrinolysis occurred within the first 48 hours, the healing of the eyes treated with fibrin sealant did not present complications. The sealant helped in the local repair, being evident the ease of application and the low cost. In the assessment of peripheral nerve recovery, rat models were adopted and fibrin sealant was used for the reimplantation of dorsal spinal roots [49] and ventral [32,54,58,61,65] after its avulsion, contributing in the process of regeneration. Benitez et al. [49] and Barbizan et al. [32,48] also used bone marrow mononuclear cells associated with and homogenized to the fibrin sealant, respectively, which provided more lasting results for neuroprotection, significantly improving motor function. In addition, Cartarozzi et al. [50] found that the application of the association of mesenchymal stem cells and sealant improved the axonal regeneration and reactivity of Schwann cells.
Moreover, Vidigal de Castro et al. [54] compared the treatment of axonal injury between two sealants: one composed of animal blood and snake venom (CEVAP), and another derived of human blood (fibrin sealant homologue). Both sealants were equally efficient, but the heterologous was highlighted as a safer alternative, being a biological and biodegradable product, and without human blood derivatives.
Biscola et al. [53] also compared these two sealants, with the objective of evaluating the end-to-end coaptation of the sciatic nerve in newborn rats. The results were favorable for both sealants, promoting neuroprotection and regeneration of motor and sensory axons. However, the CEVAP fibrin sealant presented greater ease in manipulation at the time of surgery. This corroborates the study by Mozafari et al. [60], in which the fibrin sealant facilitated the repair of the sciatic nerve, allowing adhesion and nerve regeneration.
Two studies [52,56] evaluated the use of fibrin sealant in the repair of facial nerve lesions with the addition of photobiomodulation therapy. The sealant allowed less manipulation of the nerve stumps compared to the suture, and when combined with the low-level laser, showed better morphofunctional results and nerve regeneration. Buchaim et al. [33] demonstrated that the fibrin sealant was viable for nerve repair. When associated with laser therapy, the sealant showed an even more satisfactory result.
In the study by Buchaim et al. [57], concerning qualitative, quantitative and functional aspects in the facial nerve regeneration process, the results showed that LLLT increased axonal regeneration and accelerated the functional recovery of vibrissae. Thus, both the suture and the fibrin sealant allowed the growth of axons, and the latter presented good manipulation properties, in addition to the shorter surgery time for nerve repair (Fig. 4).
The most recent study on nerve reconstruction by Leite et al. [66] validated the use of the fibrin sealant as improvement to suture in sciatic repair, where a protective effect at the lesion site due to the use of HFS was observed.
In the studies that sought to evaluate the ability of the sealant in relation to bone repair in rats [34][35][36]47], the osteogenic potential was highlighted. When combined with low-level laser therapy [36,47], or with other biomaterials such as hydroxyapatite [34] and rhBMP-2 [35], the regeneration process was accelerated, proving the biocompatibility of the sealant with this tissue (Fig. 5).
In order to evaluate the osteogenic potential of the natural rubber membranes of highly bioactive clones of Hevea brasiliensis (RRIM 600 and IAN 873) and Hancornia speciosa, Floriano et al. [55] used fibrin sealant to correct membranes in bone defects in rabbit calvaria, replacing cyanoacrylate. The sealant proved to be efficient for this type of application, in which the membranes showed good adhesion to the bone surface, and there were no negative responses in the regions where the sealant was applied.
Gasparotto et al. [62] suggested for the first time the use of the new fibrin sealant in vitro as a scaffold for mesenchymal stem cells, being able to maintain cell survival without interfering with differentiation. In addition to the known advantages of its low cost of production and non-transmission of infectious diseases, Orsi et al. [59] evaluated for the first time the performance of the sealant as an in vivo scaffold for the transplantation of mesenchymal stem cells in the repair of critical defects of the femur in osteoporotic rats. In that present study, histological analysis revealed that at 14 days after surgery, the animals treated with HFS + MSCs and HFS + MSCs D showed a higher formation of bone cells at the lesion site than the control, suggesting that the sealant served as scaffold. Microscopy electron transmission (MET) analysis showed that fibrin sealant was non-toxic to cells, since the nucleus and cell morphology were very similar to the control group. Therefore, mesenchymal stem cells were proven to promote bone repair when associated with a biological scaffold, showing for the first time non-toxicity to cells. Recently, Cassaro et al. [67] validated the osteogienic potencial of HFS+MSCs on repair of bone defect in femur of rats.
When fibrin is formed after the addition of the cryoprecipitate rich in fibrinogen extracted from buffalo blood, the fibers are randomly formed with spaces between them of different diameters. Figure 6 shows the electron microscopy of the heterologous fibrin sealant at different magnifications and a captured mesenchymal stem cell.
In addition to the application in animal models, clinical trials with the HFB started in 1999 carried out by Stolf [28]. The HFS was for the first time applied in humans as an alternative to conventional suture. Twenty-one Caucasian patients with basal cell carcinoma tumor in the nasolabial region received the product. Skin grafting of the right nasolabial fold was made using HFS, while the left nasolabial fold was sutured. The comparative study of both areas in the same patient showed erythema and edemas on the sutured areas, while dehiscence and serumhemorrhagic exudation were seen on the glued areas 48 hours after surgery. The cosmetic evaluation of the scar formation was excellent for the glued area and good for the sutured area. The patients did not show any local or systemic adverse events, which makes the new product a valuable alternative method for skin surgery.
In 2007, Barbosa et al. [29] used HFS in the dental area for the first time. They studied 15 non-smoking patients who needed bilateral gingival grafts around the bicuspid mandibular area. They concluded that the HFS is an alternative to the traditional fibrin adhesive and may represent an alternative to sutures in periodontal surgery [29,30]. In addition, Chiquito et al. [8] compared HFS and conventional suture on gingival defects and considered the first as efficient as the traditional treatment.
Gatti et al. [37,38] used the HFB for the first time to treat chronic venous ulcers assessing its effects on the repair process. They concluded that the application of fibrin sealant may contribute to the wound healing process. Control trials phase I/II will be necessary to evaluate the best way to apply the HFB. In 2015, Abbade et al. [39] used the HFB, previously standardized by Ferreira Jr et al. [9], in a clinical trial phase I/II treating 31 patients. They concluded that HFB was safe for the treatment of chronic venous ulcers according to the proposed dosages. Multicenter clinical trial phase III will be required to establish the definitely efficacy of the product.
Clinical trials conducted so far show that HFB is a versatile, easy to apply, low-cost preparation, which reduces pain, does not transmit infectious diseases by human blood, and does not present adverse events. Therefore, at this moment the CEVAP team is looking for opportunities and financial support to start the clinical trials phase III and registration by the Brazilian Health Regulatory Agency.

Conclusion
In conclusion, the studies on different tissue types showed that the fibrin biopolymer is a promising material that achieved the proposed objectives due to the ease of application (reduces surgical time), regenerative properties and biocompatibility with other materials. It is also a safer alternative, since it is a biological and biodegradable product, without human blood derivatives.
As previously mentioned, due to the properties demonstrated in studies that used the heterologous sealant produced by CEVAP, the current denomination was modified to fibrin biopolymer in accordance with other sealants and the literature. In addition to the satisfactory results presented in this review, several studies continue to be conducted to a greater understanding of its performance in different tissues and potential of clinical use.

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