Chronic venous ulcers: a review on treatment with fibrin sealant and prognostic advances using proteomic strategies

Venous ulcers are the main causes of chronic lower-limb ulcers. The healing difficulties encourage the research and development of new products in order to achieve better therapeutic results. Fibrin sealant is one of these alternatives. Besides being a validated scaffold and drug delivery system, it possesses excellent healing properties. This review covered the last 25 years of the literature and showed that the fibrin sealant is used in various clinical situations to promote the healing of different types of ulcers, especially chronic ones. These are mostly venous in origin and usually does not respond to conventional treatment. Commercially, only the homologous fibrin sealants obtained from human blood are available, which are highly efficient but very expensive. The heterologous fibrin sealant is a non-commercial experimental low-cost product and easily produced due to the abundance of raw material. The phase I/II clinical trial is already completed and showed that the product is safe and promisingly efficacious for the treatment of chronic venous ulcers. In addition, clinical proteomic strategies to assess disease prognosis have been increasingly used. By analyzing liquid samples from the wounds through proteomic strategies, it is possible to predict before treatment which ulcers will evolve favorably and which ones will be difficult to heal. This prognosis is only possible by evaluating the expression of isolated proteins in exudates and analysis using label-free strategies for shotgun. Multicentric clinical trials will be required to evaluate the efficacy of fibrin sealant to treat chronic ulcers, as well as to validate the proteomic strategies to assess prognosis.(AU)

Neuroprotection and immunomodulation by dimethyl fumarate and a heterologous fibrin biopolymer after ventral root avulsion and reimplantation

Background: Ventral root avulsion (VRA) is an experimental approach in which there is an abrupt separation of the motor roots from the surface of the spinal cord. As a result, most of the axotomized motoneurons degenerate by the second week after injury, and the significant loss of synapses and increased glial reaction triggers a chronic inflammatory state. Pharmacological treatment associated with root reimplantation is thought to overcome the degenerative effects of VRA. Therefore, treatment with dimethyl fumarate (DMF), a drug with neuroprotective and immunomodulatory effects, in combination with a heterologous fibrin sealant/biopolymer (FS), a biological glue, may improve the regenerative response. Methods: Adult female Lewis rats were subjected to VRA of L4-L6 roots followed by reimplantation and daily treatment with DMF for four weeks. Survival times were evaluated 1, 4 or 12 weeks after surgery. Neuronal survival assessed by Nissl staining, glial reactivity (anti-GFAP for astrocytes and anti-Iba-1 for microglia) and synapse preservation (anti-VGLUT1 for glutamatergic inputs and anti-GAD65 for GABAergic inputs) evaluated by immunofluorescence, gene expression (pro- and anti-inflammatory molecules) and motor function recovery were measured. Results: Treatment with DMF at a dose of 15 mg/kg was found to be neuroprotective and immunomodulatory because it preserved motoneurons and synapses and decreased astrogliosis and microglial reactions, as well as downregulated the expression of pro-inflammatory gene transcripts. Conclusion: The pharmacological benefit was further enhanced when associated with root reimplantation with FS, in which animals recovered at least 50% of motor function, showing the efficacy of employing multiple regenerative approaches following spinal cord root injury.(AU)

Neuroprotection and immunomodulation by dimethyl fumarate and a heterologous fibrin biopolymer after ventral root avulsion and reimplantation

Ventral root avulsion (VRA) is an experimental approach in which there is an abrupt separation of the motor roots from the surface of the spinal cord. As a result, most of the axotomized motoneurons degenerate by the second week after injury, and the significant loss of synapses and increased glial reaction triggers a chronic inflammatory state. Pharmacological treatment associated with root reimplantation is thought to overcome the degenerative effects of VRA. Therefore, treatment with dimethyl fumarate (DMF), a drug with neuroprotective and immunomodulatory effects, in combination with a heterologous fibrin sealant/biopolymer (FS), a biological glue, may improve the regenerative response. Methods: Adult female Lewis rats were subjected to VRA of L4-L6 roots followed by reimplantation and daily treatment with DMF for four weeks. Survival times were evaluated 1, 4 or 12 weeks after surgery. Neuronal survival assessed by Nissl staining, glial reactivity (anti-GFAP for astrocytes and anti-Iba-1 for microglia) and synapse preservation (anti-VGLUT1 for glutamatergic inputs and anti-GAD65 for GABAergic inputs) evaluated by immunofluorescence, gene expression (pro- and anti-inflammatory molecules) and motor function recovery were measured. Results: Treatment with DMF at a dose of 15 mg/kg was found to be neuroprotective and immunomodulatory because it preserved motoneurons and synapses and decreased astrogliosis and microglial reactions, as well as downregulated the expression of pro-inflammatory gene transcripts. Conclusion: The pharmacological benefit was further enhanced when associated with root reimplantation with FS, in which animals recovered at least 50% of motor function, showing the efficacy of employing multiple regenerative approaches following spinal cord root injury.(AU)

The effect of fibrin glue in preventing staple-line leak after sleeve gastrectomy. An experimental study in rats

Abstract Purpose To evaluate the effect of fibrin glue on staple-line leak after sleeve gastrectomy. Methods Fourteen adult wistar rats 300 gr were randomized into two groups: Control group (n=7) and study group (n=7). All the rats underwent sleeve gastrectomy using lineer stapler. In the study group, fibrin glue was used to reinforce the staple-line. The rats were sacrificed 7 days after surgery. The stomach was resected, submerged in saline and exposed to excess pressure to obtain a burst pressure value. The gastric staple line was evaluated histopathologically according to the Ehrlich Hunt scale. The results of the two groups were compared. Results The mean Ehrlich-Hunt scores for inflammation, fibroblastic activity and neo-angiogenesis were similar between the groups (p>0.05). Collagen deposition was significantly higher in study group (3.42±0.53) when compared with control group (2.57±0.78) (p=0.035). The mean burst pressure was 137.8±8.5 mmHg for control group and 135.0±8.1 mmHg for study group (p=0.536). Conclusion Reinforcement of the staple-line with fibrin glue has no effect on the burst pressure after sleeve gastrectomy. More studies are needed to evaluate the precautions against leak after sleeve gastrectomy.(AU)

Fibrin biopolymer as scaffold candidate to treat bone defects in rats

Background:Bone tissue repair remains a challenge in tissue engineering. Currently, new materials are being applied and often integrated with live cells and biological scaffolds. The fibrin biopolymer (FBP) proposed in this study has hemostatic, sealant, adhesive, scaffolding and drug-delivery properties. The regenerative potential of an association of FBP, biphasic calcium phosphate (BCP) and mesenchymal stem cells (MSCs) was evaluated in defects of rat femurs.Methods:Adult male Wistar rats were submitted to a 5-mm defect in the femur. This was filled with the following materials and/or associations: BPC; FBP and BCP; FBP and MSCs; and BCP, FBP and MSCs. Bone defect without filling was defined as the control group. Thirty and sixty days after the procedure, animals were euthanatized and subjected to computed tomography, scanning electron microscopy and qualitative and quantitative histological analysis.Results:It was shown that FBP is a suitable scaffold for bone defects due to the formation of a stable clot that facilitates the handling and optimizes the surgical procedures, allowing also cell adhesion and proliferation. The association between the materials was biocompatible. Progressive deposition of bone matrix was higher in the group treated with FBP and MSCs. Differentiation of mesenchymal stem cells into osteogenic lineage was not necessary to stimulate bone formation.Conclusions:FBP proved to be an excellent scaffold candidate for bone repair therapies due to application ease and biocompatibility with synthetic calcium-based materials. The satisfactory results obtained by the association of FBP with MSCs may provide a more effective and less costly new approach for bone tissue engineering.(AU)

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

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.(AU)

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

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.(AU)

Comparison of platelet rich plasma versus fibrin glue on colonic anastomoses in rats

Purpose: To compare platelet rich plasma (PRP) and fibrin glue about the effect of anastomotic healing. Methods: Thirty six Wistar-Albino male rats diveded into 3 groups according to control (Group1), PRP (Group 2) and fibrin glue (Tisseel VH) (Group 3). The colon was transected with scissor and subsequently an end to end anastomosis was performed using continuous one layer 6/0 vicryl sutures. Postoperative 7th day effect of anastomotic healing measuring with tissue hydroxyproline (TH) level and anastomotic bursting pressure (ABP); moreover comparison of cytokine (IL-6 and IL-10) and procalcitonin levels on 1st,3rd and 7th days. Results: There was no statistically significant difference of the ABP and hydroxyproline levels between PRP and fibrin glue on the 7th day. There was no statistically significant difference between levels of proinflammatory cytokine (IL-6) (P=0.41), anti-inflammatory cytokine (IL-10) (P=0.35), and procalcitonin levels (P=0.63) on 1, 3 and 7 days. Conclusion: Fibrin glue and platelet rich plasma are shown to be effective in healing intestinal anastomoses without superior to each other. (AU)

Combination of heterologous fibrin sealant and bioengineered human embryonic stem cells to improve regeneration following autogenous sciatic nerve grafting repair

Background Peripheral nerve injury is a worldwide clinical problem, and the preferred surgical method for treating it is the end-to-end neurorrhaphy. When it is not possible due to a large nerve gap, autologous nerve grafting is used. However, these surgical techniques result in nerve regeneration at highly variable degrees. It is thus very important to seek complementary techniques to improve motor and sensory recovery. One promising approach could be cell therapy. Transplantation therapy with human embryonic stem cells (hESCs) is appealing because these cells are pluripotent and can differentiate into specialized cell types and have self-renewal ability. Therefore, the main objective of this study was to find conditions under which functional recovery is improved after sciatic nerve neurorrhaphy. We assumed that hESC, either alone or in combination with heterologous fibrin sealant scaffold, could be used to support regeneration in a mouse model of sciatic nerve injury and repair via autografting with end-to-end neurorrhaphy. Methods Five millimeters of the sciatic nerve of C57BL/6 J mice were transected off and rotated 180 degrees to simulate an injury, and then stumps were sutured. Next, we applied heterologous fibrin sealant and/or human embryonic stem cells genetically altered to overexpress fibroblast growth factor 2 (FGF2) at the site of the injury. The study was designed to include six experimental groups comprising neurorrhaphy (N), neurorrhaphy + heterologous fibrin sealant (N + F), neurorrhaphy + heterologous fibrin sealant + doxycycline (N + F + D), neurorrhaphy + heterologous fibrin sealant + wild-type hESC (N + F + W), neurorrhaphy + heterologous fibrin sealant + hESC off (N + F +T), and neurorrhaphy + heterologous fibrin sealant + hESC on via doxycycline (N + F + D + T). We evaluated the recovery rate using Catwalk and von Frey functional recovery tests, as well as immunohistochemistry analysis. Results The experiments indicated...

Combination of heterologous fibrin sealant and bioengineered human embryonic stem cells to improve regeneration following autogenous sciatic nerve grafting repair

Background Peripheral nerve injury is a worldwide clinical problem, and the preferred surgical method for treating it is the end-to-end neurorrhaphy. When it is not possible due to a large nerve gap, autologous nerve grafting is used. However, these surgical techniques result in nerve regeneration at highly variable degrees. It is thus very important to seek complementary techniques to improve motor and sensory recovery. One promising approach could be cell therapy. Transplantation therapy with human embryonic stem cells (hESCs) is appealing because these cells are pluripotent and can differentiate into specialized cell types and have self-renewal ability. Therefore, the main objective of this study was to find conditions under which functional recovery is improved after sciatic nerve neurorrhaphy. We assumed that hESC, either alone or in combination with heterologous fibrin sealant scaffold, could be used to support regeneration in a mouse model of sciatic nerve injury and repair via autografting with end-to-end neurorrhaphy. Methods Five millimeters of the sciatic nerve of C57BL/6 J mice were transected off and rotated 180 degrees to simulate an injury, and then stumps were sutured. Next, we applied heterologous fibrin sealant and/or human embryonic stem cells genetically altered to overexpress fibroblast growth factor 2 (FGF2) at the site of the injury. The study was designed to include six experimental groups comprising neurorrhaphy (N), neurorrhaphy + heterologous fibrin sealant (N + F), neurorrhaphy + heterologous fibrin sealant + doxycycline (N + F + D), neurorrhaphy + heterologous fibrin sealant + wild-type hESC (N + F + W), neurorrhaphy + heterologous fibrin sealant + hESC off (N + F +T), and neurorrhaphy + heterologous fibrin sealant + hESC on via doxycycline (N + F + D + T). We evaluated the recovery rate using Catwalk and von Frey functional recovery tests, as well as immunohistochemistry analysis. Results The experiments indicated...(AU)

Multiple uses of fibrin sealant for nervous system treatment following injury and disease

Lesions to the nervous system often produce hemorrhage and tissue loss that are difficult, if not impossible, to repair. Therefore, scar formation, inflammation and cavitation take place, expanding the lesion epicenter. This significantly worsens the patient conditions and impairment, increasing neuronal loss and glial reaction, which in turn further decreases the chances of a positive outcome. The possibility of using hemostatic substances that also function as a scaffold, such as the fibrin sealant, reduces surgical time and improve postoperative recovery. To date, several studies have demonstrated that human blood derived fibrin sealant produces positive effects in different interventions, becoming an efficient alternative to suturing. To provide an alternative to homologous fibrin sealants, the Center for the Study of Venoms and Venomous Animals (CEVAP, Brazil) has proposed a new bioproduct composed of certified animal components, including a thrombin-like enzyme obtained from snake venom and bubaline fibrinogen. Thus, the present review brings up to date literature assessment on the use of fibrin sealant for nervous system repair and positions the new heterologous bioproduct from CEVAP as an alternative to the commercial counterparts. In this way, clinical and pre-clinical data are discussed in different topics, ranging from central nervous system to peripheral nervous system applications, specifying positive results as well as future enhancements that are necessary for improving the use of fibrin sealant therapy.(AU)

Multiple uses of fibrin sealant for nervous system treatment following injury and disease

Lesions to the nervous system often produce hemorrhage and tissue loss that are difficult, if not impossible, to repair. Therefore, scar formation, inflammation and cavitation take place, expanding the lesion epicenter. This significantly worsens the patient conditions and impairment, increasing neuronal loss and glial reaction, which in turn further decreases the chances of a positive outcome. The possibility of using hemostatic substances that also function as a scaffold, such as the fibrin sealant, reduces surgical time and improve postoperative recovery. To date, several studies have demonstrated that human blood derived fibrin sealant produces positive effects in different interventions, becoming an efficient alternative to suturing. To provide an alternative to homologous fibrin sealants, the Center for the Study of Venoms and Venomous Animals (CEVAP, Brazil) has proposed a new bioproduct composed of certified animal components, including a thrombin-like enzyme obtained from snake venom and bubaline fibrinogen. Thus, the present review brings up to date literature assessment on the use of fibrin sealant for nervous system repair and positions the new heterologous bioproduct from CEVAP as an alternative to the commercial counterparts. In this way, clinical and pre-clinical data are discussed in different topics, ranging from central nervous system to peripheral nervous system applications, specifying positive results as well as future enhancements that are necessary for improving the use of fibrin sealant therapy.(AU)

Heterologous fibrin sealant derived from snake venom: from bench to bedside - an overview

Hemostatic and adhesive agents date back to World War II, when homologous fibrin sealant came onto scene. Considering that infectious diseases can be transmitted via human blood, a new heterologous fibrin sealant was standardized in the 1990s. Its components were a serine protease (a thrombin-like enzyme) extracted from the venom of Crotalus durissus terrificus snakes and a fibrinogen-rich cryoprecipitate extracted from the blood of Bubalus bubalis buffaloes. This new bioproduct has been used as a coagulant, sealant, adhesive and recently as a candidate scaffold for mesenchymal stem cells and bone and cartilage repair. This review discusses the composition of a new heterologous fibrin sealant, and cites published articles related to its preclinical applications aiming at repairing nervous system traumas and regenerating bone marrow. Finally, we present an innovative safety trial I/II that found the product to be a safe and clinically promising candidate for treating chronic venous ulcers. A multicenter clinical trial, phase II/III, with a larger number of participants will be performed to prove the efficacy of an innovative biopharmaceutical product derived from animal venom.(AU)

Heterologous fibrin sealant derived from snake venom: from bench to bedside - an overview

Hemostatic and adhesive agents date back to World War II, when homologous fibrin sealant came onto scene. Considering that infectious diseases can be transmitted via human blood, a new heterologous fibrin sealant was standardized in the 1990s. Its components were a serine protease (a thrombin-like enzyme) extracted from the venom of Crotalus durissus terrificus snakes and a fibrinogen-rich cryoprecipitate extracted from the blood of Bubalus bubalis buffaloes. This new bioproduct has been used as a coagulant, sealant, adhesive and recently as a candidate scaffold for mesenchymal stem cells and bone and cartilage repair. This review discusses the composition of a new heterologous fibrin sealant, and cites published articles related to its preclinical applications aiming at repairing nervous system traumas and regenerating bone marrow. Finally, we present an innovative safety trial I/II that found the product to be a safe and clinically promising candidate for treating chronic venous ulcers. A multicenter clinical trial, phase II/III, with a larger number of participants will be performed to prove the efficacy of an innovative biopharmaceutical product derived from animal venom.(AU)

Nerve growth factor with fibrin glue in end-to-side nerve repair in rats / Fator de crescimento nervoso em cola de fibrina no reparo término-lateral de nervos em ratos

PURPOSE: To determine the effects of end-to-side nerve repair performed only with fibrin glue containing nerve growth in rats. METHODS: Seventy two Wistar rats were divided into six equal groups: group A was not submitted to nerve section; group B was submitted to nerve fibular section only. The others groups had the nerve fibular sectioned and then repaired in the lateral surface of an intact tibial nerve, with different procedures: group C: ETS with sutures; group D: ETS with sutures and NGF; group E: ETS with FG only; group F: ETS with FG containing NGF. The motor function was accompanied and the tibial muscle mass, the number and diameter of muscular fibers and regenerated axons were measured. RESULTS: All the analyzed variables did not show any differences among the four operated groups (p>0.05), which were statistically superior to group B (p<0.05), but inferior to group A (p>0.05). CONCLUSION: The end-to-side nerve repair presented the same recovery pattern, independent from the repair used, showing that the addition of nerve growth factor in fibrin glue was not enough for the results potentiating.(AU)

OBJETIVO: Determinar os efeitos do reparo nervoso término-lateral realizado apenas com cola de fibrina contendo fator de crescimento nervoso em ratos. MÉTODOS: Setenta e dois ratos Wistar foram distribuídos em seis grupos: A - não submetido à secção nervosa; B - secção do nervo fibular (sem reparo); Os outros grupos tiveram o nervo fibular seccionado e então reparado na superfície lateral do nervo tibial intacto, com diferentes procedimentos: C - RNTL com suturas; D - RNTL com suturas e FCN; E - RNTL apenas com CF; F - RNTL com CF contendo FCN. A função motora foi acompanhada e a massa do músculo tibial, o número e o diâmetro das fibras musculares e axônios regenerados foram medidos. RESULTADOS: Não houve diferença entre as variáveis avaliadas nos quatro grupos operados (p>0,05), os quais foram superiores ao grupo B (p<0,05), mas inferiores ao grupo A (p>0,05). CONCLUSÕES: O reparo nervoso término-lateral mostrou o mesmo padrão de recuperação, independente do tipo de reparo utilizado, evidenciando que a adição de fator de crescimento nervoso na cola de fibrina não foi suficiente para a potencialização dos resultados.(AU)

Comparative study between fibrin glue and platelet rich plasma in dogs skin grafts / Estudo comparativo entre a cola de fibrina e o plasma rico em plaquetas em enxertos cutâneos em cães

PURPOSE: Compare fibrin glue (Tissucol®) and platelet-rich plasma in full-thickness mesh skin grafts in dogs. METHODS: Eighteen dogs were used, divided into two groups: fibrin glue (FG) and platelet-rich plasma (PRP). In all the animals, a full-thickness 3x3 cm mesh skin graft was implanted. In the left limb, the biomaterial was place between the graft and the receptor bed, according to the group, while the right limb served as the control group. All the animals were evaluated clinically every 48 hours until the 14th day, using the variables of exudation, coloration, edema and cosmetic appearance. Three animals were evaluated histologically, on the third, seventh and tenth postoperative days, using the variables of fibroblasts, collagen, granulation tissue, microscopic integration-adherence and acute inflammation. RESULTS: Clinical evaluations showed that the group CF showed better scores for all variables compared to PRP group. On the histological evaluations PRP group had a higher presence of fibroblasts in the seventh and fourteenth days. CONCLUSION: The fibrin glue group was clinically superior to the platelet-rich group when used on full-thickness skin grafts in dogs.(AU)

OBJETIVO: Comparar a cola de fibrina (Tissucol®) e o plasma rico em plaquetas em enxertos cutâneos de espessura completa em malha em cães. MÉTODOS: Foram utilizados 18 cães, distribuídos em dois grupos, cola de fibrina (CF) e plasma rico em plaquetas (PRP). Em todos os animais foi realizado um enxerto cutâneo de 3x3 cm, em malha de espessura completa. No membro esquerdo foi colocado o biomaterial entre o enxerto e o leito receptor, cada qual em seu grupo, o membro direito serviu como grupo controle. Todos os animais foram avaliados clinicamente a cada 48 horas até o décimo quarto dia, através das variáveis: exsudação, coloração, edema e aspecto cosmético; histologicamente em três animais, no terceiro, sétimo e décimo quarto dia de pós-operatório através das variáveis: fibroblastos, colágeno, tecido de granulação, integração-aderência microscópica e inflamação aguda. RESULTADOS: Avaliações clínicas demonstraram que o grupo CF apresentou melhor escores em todas variáveis quando comparado com o grupo PRP. Nas avaliações histológicas o grupo PRP apresentou maior presença de fibroblastos ao sétimo e décimo quarto dia. CONCLUSÃO: A cola de fibrina foi clinicamente superior ao grupo plasma rico em plaquetas quando usados em enxertos cutâneos de espessura completa em cães.(AU)

The laparoscopy splenic injury repair: the use of fibrin glue in a heparinized porcine model / O reparo de lesão esplênica por laparoscopia: o uso da cola de fibrina em porcos heparinizados

PURPOSE: To investigate the effectiveness of fibrin glue (laparoscopic via) into promote the hemostasis of a spleen injury on a heparinized porcine model. METHODS: Eighteen Landrace porcine were submitted to laparoscopic spleen injury and randomly distributed: GHA (heparin plus adhesive), GH (heparin without adhesive) and GS (Sham - without heparin or adhesive). Ten minutes before the surgical procedures a single IV dose (200UI/kg) of heparin sodium was administrated only to groups GHA and GH. In the GHA, adhesive was applied after the mechanical injury and recorded the time until the polymerization and clot formation. RESULTS: No significant differences occurred among the groups (Fisher test) considering the weight and surgery time. The blood amount in the abdominal cavity on GH was significantly higher in comparison to the sham group and especially with the GHA (p<0.004). No significant differences were observed in the body temperature, heart rate, cardiac output, means arterial pressure, pulmonary artery pressure during the experiment. The activated partial thromboplastin time (APTT) was lower in the GHA in comparison to GH (p<0.003). CONCLUSION: The fibrin biological adhesive applied by laparoscopy is effective for hemostasis of minor spleen injury in a porcine model under the effect of anticoagulant drug.(AU)

OBJETIVO: Investigar a eficácia da cola de fibrina (via laparoscópica) na hemostasia de uma lesão no baço de porco heparinizado. MÉTODOS: Dezoito suínos Landrace foram submetidos a lesão do baço e distribuídos aleatoriamente: GHA (heparina adesivo), GH (heparina sem adesivo) e GS (Sham - sem heparina ou adesivo). Dez minutos antes dos procedimentos uma dose única (200UI/kg) de heparina sódica (EV) foi administrada nos grupos GHA e GH. A fibrina (GHA) foi aplicada após a lesão e registrado o tempo até a polimerização e formação do coágulo. RESULTADOS: Não houve diferenças significativas entre os grupos (teste de Fisher), considerando o peso e o tempo de cirurgia. A quantidade de sangue na cavidade abdominal de GH foi significativamente maior em comparação ao GS e, especialmente, com o GHA (p<0,004). Não foram observadas diferenças significativas na temperatura corporal, frequência cardíaca, débito cardíaco, pressão arterial ou pressão da artéria pulmonar durante o experimento (20 minutos). O tempo de tromboplastina parcial ativada (TTPA) foi menor no GHA em relação ao GH (p<0,003). CONCLUSÃO: A cola de fibrina biológica aplicada por laparoscopia é eficaz para a hemostasia de lesões no baço menor em um modelo suíno sob o efeito de drogas anticoagulantes.(AU)

End-to-side nerve repair using fibrin glue in rats / Anastomose nervosa término-lateral usando cola de fibrina em ratos

Purpose: To evaluate axonal regeneration after end-to-side nerve repair with fibrin glue in rats. Methods: Forty-five Wistar rats were divided into three groups: group A (n=15), were not submitted to surgery (control group); group B (n=15) were submitted to fibular transection without repair; and group C (n=15), were submitted to fibular transection with end-to-side nerve anastomosis using fibrin glue, in the lateral surface of an intact tibial nerve. The three groups were submitted to walking track (30 and 90 days) and posterior morphometrical analysis (90 days). Results: The functional tests demonstrated that there was no difference in the walking track during the study in group A (p>0.05). The group B had walking pattern impairment in the two tests (p>0.05). The group C had walking pattern impairment in the first test, with important recovery in the second test (p<0.05). The morphometrical assessment revealed significantly higher number of regenerated mielinates axons in group C, compared to group B (p<0.05). Conclusion: The end-to-side nerve repair with fibrin glue shows axonal recovery, demonstrated through functional and morphometrical ways in rats.(AU)

Objetivo: Avaliar a regeneração axonal após anastomose nervosa término-lateral (ATL) usando cola de fibrina em ratos. Métodos: Quarenta e cinco ratos Wistar distribuídos em três grupos: os animais do grupo A (n=15) não foram submetidos à secção nervosa (grupo controle); os animais do grupo B (n=15) foram submetidos apenas à secção do nervo fibular, sem posterior anastomose; e os animais do grupo C (n=15) foram submetidos à secção do nervo fibular e à ATL com cola de fibrina no nervo tibial. Posteriormente, os animais foram submetidos a dois testes de marcha (30 e 90 dias) e à análise morfométrica (90 dias). Resultados: A análise estatística dos testes de marcha demonstrou que o grupo A não apresentou alteração no padrão de caminhada durante o estudo (p>0,05). O grupo B apresentou prejuízo motor no primeiro e no segundo teste (p>0,05). O grupo C apresentou um padrão de atrofia no primeiro teste, com recuperação da marcha no segundo teste (p<0,05). Na análise morfométrica, o grupo C apresentou regeneração axonal significativamente superior ao grupo B (p<0,05). Conclusão: A ATL realizada com cola de fibrina resultou em regeneração axonal no rato, demonstrada tanto histologicamente quanto funcionalmente.(AU)

Ultrastructural evaluation of human keratinocyte growth and differentiation on a fibrin substrate / Avaliação ultraestrutural do crescimento e da diferenciação de queratinócitos sobre um substrato de fibrina

PURPOSE: In order to circumvent several difficulties that have been met in the routine use of the in vitro keratinocyte cultures using the standard procedure described by Rheinwald and Green, and obtain a more resilient and the least possible immunogeneic skin substitute for a future clinical application, this work studied a new keratinocyte culture system, which envisages the utilization of a fibrin substrate in association with high densities of human keratinocytes. METHODS: Through light and transmission electron microscopy and immunohistochemical assays, long-term proliferative and differentiative characteristics of keratinocytes cultured onto a fibrin gel under immerse and air-liquid interface culture conditions were evaluated. RESULTS: Despite the absence of a dermal substitute, the results demonstrated that the proposed composite was constituted of a transparent and elastic fibrin film covered by a well-attached, multistratified epithelium with morphological characteristics that resemble human epidermis, including the neoformation, albeit incomplete, of the basement membrane. CONCLUSIONS: Increased mechanical resistance due to the presence of an easy handling substrate, the delivery of nonclonfluent keratinocytes as well as the removal of animal-derived cells from the culture system suggest its potential use for future transplantation purposes.(AU)

OBJETIVO: Com o intuito de contornar diversas dificuldades encontradas no uso rotineiro de queratinócitos cultivados in vitro pela técnica descrita por Rheinwald e Green, e obter um substituto cutâneo mais resistente e o menos imunogênico possível para futuras aplicações clínicas, este trabalho avaliou um novo sistema de cultura de queratinócitos que prevê a utilização de um substrato de fibrina em associação com queratinócitos humanos em alta densidade. MÉTODOS: Através de microscopia óptica e eletrônica e análise imunohistoquímica, foram avaliadas as características proliferativas e de diferenciação em longo prazo de queratinócitos cultivados em condição imersa e na interface ar-líquido. RESULTADOS: Apesar da ausência de um substituto dérmico, foi demonstrado que o composto proposto constituiu-se de um substrato de fibrina transparente e elástico coberto por epitélio multi-estratificado, bem aderido, com características morfológicas semelhantes à epiderme humana, incluindo a neo-formação, embora incompleta, da membrana basal. CONCLUSÕES: A maior resistência mecânica com a presença de um substrato de fácil manuseio, a possível liberação de queratinócitos não-confluentes, e a remoção de células com origem animal dos sistemas de cultura sugerem que o composto proposto neste estudo apresenta grande potencial para uso clínico futuro.(AU)

COLA DE FIBRINA CANINA PRODUZIDA COM FIBRINOGÊNIO OBTIDO POR CRIOPRECIPITAÇÃO E PRECIPITAÇÃO COM PROTAMINA A PARTIR DE DIFERENTES CATEGORIAS DE PLASMA POBRE EM PLAQUETAS

A cola de fibrina tem sido utilizada em diferentes procedimentos cirúrgicos como agente hemostático, selante e de suporte adesivo. No entanto, seu emprego na Veterinária ainda é limitado devido à falta de formulações não dependentes dos componentes de origem humana e de validação baseada em necessidades e condições cirúrgicas de animais. Objetivou-se avaliar a viabilidade da produção de cola de fibrina canina com fibrinogênio obtido por crioprecipitação (crio) e precipitação por protamina a partir de fontes plasmáticas mais disponíveis em bancos de sangue e centros hospitalares. Quatro categorias de plasma pobre em plaquetas foram utilizadas: plasma fresco (FR), congelado dentro de oito horas da colheita e processado em menos de uma semana após congelamento; plasma fresco congelado (FFP), com armazenamento inferior a um ano; plasma fresco congelado que ultrapassou um ano de armazenamento (eFFP), e plasma congelado com período entre colheita e congelamento maior que oito horas e de armazenamento superior a um ano (FP). No estudo in vitro de cada técnica, avaliou-se a concentração de fibrinogênio precipitado por meio do método de Clauss, as propriedades reológicas do gel por tromboelastografia (TEG) e as características estruturais do coágulo por microscopia eletrônica de varredura (SEM). O estudo in vivo consistiu da avaliação da praticidade de aplicação e das propriedades hemostáticas e adesivas das colas de fibrina resultantes em fígado e intestino de coelho (Oryctolagus cuniculus). Em avaliação prévia do protocolo de crio quanto ao uso de bolsas ou tubos, o aproveitamento do material inicial não diferiu, mas os tubos se mostraram mais simples, rápidos e homogêneos para o processamento, além de permitirem aumento da concentração final. O protocolo crio em comparação ao de protamina foi superior na precipitação de fibrinogênio coagulável nas avaliações de Clauss e TEG. Os coágulos formados se mostraram semelhantes entre os dois protocolos na SEM, no modelo de hemostasia hepática e na adesão à serosa intestinal. O uso de aprotinina com o protocolo de protamina não prejudicou a aplicação da cola sobre o intestino. Na crio, plasmas com maior tempo de armazenamento (eFFP, FP) se mostraram significativamente superiores aos mais frescos (FFP, FR) nas análises por Clauss e TEG. Não foi possível identificar diferenças estatísticas entre os tipos de plasma no protocolo protamina em nenhum dos parâmetros avaliados. Estudos adicionais e ajustes nos testes para avaliação de soluções concentradas são necessários para determinação do efeito dos protocolos e tempo de armazenamento do plasma congelado sobre o fibrinogênio precipitado e demais componentes plasmáticos na cola de fibrina. Adequações e pesquisas ainda são necessárias para aproveitamento da precipitação de fibrinogênio por protamina e a partir de plasma fresco com a finalidade de obtenção rápida de cola de fibrina. Bolsas de plasma menos requisitadas em bancos de sangue veterinários representam uma fonte importante de fibrinogênio para a produção de cola de fibrina canina em centros hospitalares apropriadamente equipados, viabilizando seu uso em diferentes aplicações cirúrgicas e pesquisas relacionadas.

Canine Fibrin Glue Produced with Fibrinogen Concentrated from Cryo- and Protamine Precipitation Using Different Platelet Poor Plasma Categories Fibrin glue (FG) has been widely used in surgery for hemostatic, adhesive, sealant, and would healing support. In veterinary surgery, however, its use has been hindered by lack of specie-specific formulations and validation of its properties and biological characteristics. This study evaluated methods of fibrinogen precipitation from canine plasma envisioning autologous and allogeneic FG production for surgical use. The efficacy of cryo and protamine fibrinogen precipitation methods in producing canine FG was assessed by analysis on feasibility of each protocol with most available canine plasma sources, rheological and structural characteristics of the resultant FG clot and the hemostatic and adhesive properties of FG during in vivo application. The plasma categories studied included fresh plasma (FR), obtained and frozen within 8 hours from blood collection and processed within a week; fresh frozen plasma (FFP), frozen within 8 hours from blood collection and stored for up to a year; expired fresh frozen plasma (eFFP), plasma frozen within 8 hours from blood collection but stored for more than a year; and, frozen plasma (FP), which was frozen after 8 hours from collection and stored for more than a year. Comparison of cryoprecipitation among plasma types was previously performed in both 120-mL bags and 50-mL tubes and analyzed by Clauss. Total precipitation capacity did not differ significantly between bags and tubes. Nevertheless, the processing was more easily and homogeneously performed in tubes and allowed tailoring the final concentration. Cryoprecipitation generated better results in Clauss and TEG in comparison to protamine protocol. The resultant fibrin glue clots of cryo- and protamine-precipitation showed similar ultrastructure in scanning electron microscopy (SEM) and performance in the in vivo evaluations with the rabbit hepatic and intestinal incision models. The use of aprotinin in the protamine clot seemed beneficial in the intestinal evaluation. With cryoprecipitation, eFFP and FP were superior to FFP in the assessments performed by Clauss and TEG. Fresh plasma performed poorly with cryoprecipitation. Significant differences were not detected among plasma categories processed with protamine precipitation in any of the assays performed. While cryoprecipitation was more reliable regarding homogeneity and capacity to increase final fibrinogen concentration, protamine protocol was faster and simpler considering the equipment required. Although, older plasma units generated significantly more cryo-precipitated and/or clottable fibrinogen, further studies are needed to validate the assays with such high concentrated solutions and to elucidate the effect of freezing storage on precipitation and clottability of fibrinogen intended for FG production. Adjustments on protamine protocol and improvements on fibrinogen precipitation from fresher plasma sources would support the use of autologous or allogeneic plasma for on-site production of canine FG. Veterinary hospitals, blood banks, and patients can benefit from usage of surplus plasma units for FG production aiming surgical and scientific needs.