Plasma rico em plaquetas: estudo comparativo entre a dupla centrifugação e o filtro E-PET (Equine Platelet Enhancement Therapy) / Comparative study of double spin and use of E-PET filter (Equine Platelet Enhancement Therapy) to obtain platelet rich plasma in horses

O plasma rico em plaquetas (PRP) consiste em uma alta concentração de plaquetas em um pequeno volume de plasma, sendo, em média, quatro vezes maior que a concentração sérica. O uso de PRP é justificado pela alta concentração de fatores de crescimento presentes em grânulos no interior das plaquetas, que possuem diversas funções como proliferação celular, quimiotaxia, angiogênese e diferenciação celular, que ampliam o poder de reparação tecidual. Há diversos protocolos para obtenção do PRP em equinos descritos na literatura, dentre os quais destacam-se os de dupla centrifugação, os automatizados e os filtros. Há diferenças substanciais no conteúdo do PRP dependendo do seu método de obtenção, principalmente no que se diz respeito à quantidade de leucócitos, plaquetas e concentração de fatores de crescimento. Sendo assim, o objetivo deste estudo foi comparar a utilização do concentrado de plaquetas obtido por protocolo de dupla centrifugação e o obtido pelo filtro E-PET (Equine Platelet Enhancement Therapy), levando-se em consideração a concentração plaquetária e leucocitária final, a quantificação de fatores de crescimento (TGFß e PDGF-BB) e a facilidade de realização entre tais métodos. Utilizou-se nove animais para a obtenção de PRP por dupla centrifugação e através do filtro E-PET, não havendo diferença estatística (p>0,05) entre os métodos de obtenção em relação à concentração plaquetária e leucocitária, entretanto, houve diferença estatística (p=0,002; p=0,004, respectivamente) em relação a concentração de TGFß e PDGF-BB. Dessa forma, concluiu-se que o filtro E-PET mostrou-se um método mais efetivo, sendo possível sua utilização à campo, além de proporcionar uma maior concentração de fatores de crescimento TGFß e PDGF-BB.(AU)

The platelet-rich plasma (PRP) consists of a high concentration of platelets in a small volume of plasma, four times greater (average) than the serum concentration. The use of PRP is justified by the high concentration of growth factors present in granules in the platelets, which have several functions such as cell proliferation, chemotaxis, angiogenesis and differenciation, which extend the power of tissue repair. There are several protocols to obtain PRP in horses described in the literature, among which are highlighted the double centrifugation, automated and filters. There are substantial differences in the PRP content depending on its method of production, especially when it concerns the amount of leukocytes, platelets and concentration of growth factors. Thus, the aim of this study was to compare the use of platelet concentrates obtained by double centrifugation protocol and obtained by the filter E-PET (Equine Platelet Enhancement Therapy) taking into account the platelet and leukocyte final concentration, the quantification of growth factors (TGFß and PDGF-BB) and the facility to do those methods. Nine horses were used to obtain PRP by double centrifugation and through the E-PET filter, with no statistical difference (p>0.05) between the methods relative to the platelet and leukocyte concentration; however, there was statistical difference (p=0.002 and p=0.004 respectively) compared with the concentration of TGFß and PDGF-BB. It was concluded that the E-PET filter proved to be a more effective method possible to use in the field and to provide a higher concentration of growth factors (TGFß and PDGF-BB).(AU)

Plasma rico em plaquetas: estudo comparativo entre a dupla centrifugação e o filtro E-PET (Equine Platelet Enhancement Therapy) / Comparative study of double spin and use of E-PET filter (Equine Platelet Enhancement Therapy) to obtain platelet rich plasma in horses

O plasma rico em plaquetas (PRP) consiste em uma alta concentração de plaquetas em um pequeno volume de plasma, sendo, em média, quatro vezes maior que a concentração sérica. O uso de PRP é justificado pela alta concentração de fatores de crescimento presentes em grânulos no interior das plaquetas, que possuem diversas funções como proliferação celular, quimiotaxia, angiogênese e diferenciação celular, que ampliam o poder de reparação tecidual. Há diversos protocolos para obtenção do PRP em equinos descritos na literatura, dentre os quais destacam-se os de dupla centrifugação, os automatizados e os filtros. Há diferenças substanciais no conteúdo do PRP dependendo do seu método de obtenção, principalmente no que se diz respeito à quantidade de leucócitos, plaquetas e concentração de fatores de crescimento. Sendo assim, o objetivo deste estudo foi comparar a utilização do concentrado de plaquetas obtido por protocolo de dupla centrifugação e o obtido pelo filtro E-PET (Equine Platelet Enhancement Therapy), levando-se em consideração a concentração plaquetária e leucocitária final, a quantificação de fatores de crescimento (TGFß e PDGF-BB) e a facilidade de realização entre tais métodos. Utilizou-se nove animais para a obtenção de PRP por dupla centrifugação e através do filtro E-PET, não havendo diferença estatística (p>0,05) entre os métodos de obtenção em relação à concentração plaquetária e leucocitária, entretanto, houve diferença estatística (p=0,002; p=0,004, respectivamente) em relação a concentração de TGFß e PDGF-BB. Dessa forma, concluiu-se que o filtro E-PET mostrou-se um método mais efetivo, sendo possível sua utilização à campo, além de proporcionar uma maior concentração de fatores de crescimento TGFß e PDGF-BB.(AU)

The platelet-rich plasma (PRP) consists of a high concentration of platelets in a small volume of plasma, four times greater (average) than the serum concentration. The use of PRP is justified by the high concentration of growth factors present in granules in the platelets, which have several functions such as cell proliferation, chemotaxis, angiogenesis and differenciation, which extend the power of tissue repair. There are several protocols to obtain PRP in horses described in the literature, among which are highlighted the double centrifugation, automated and filters. There are substantial differences in the PRP content depending on its method of production, especially when it concerns the amount of leukocytes, platelets and concentration of growth factors. Thus, the aim of this study was to compare the use of platelet concentrates obtained by double centrifugation protocol and obtained by the filter E-PET (Equine Platelet Enhancement Therapy) taking into account the platelet and leukocyte final concentration, the quantification of growth factors (TGFß and PDGF-BB) and the facility to do those methods. Nine horses were used to obtain PRP by double centrifugation and through the E-PET filter, with no statistical difference (p>0.05) between the methods relative to the platelet and leukocyte concentration; however, there was statistical difference (p=0.002 and p=0.004 respectively) compared with the concentration of TGFß and PDGF-BB. It was concluded that the E-PET filter proved to be a more effective method possible to use in the field and to provide a higher concentration of growth factors (TGFß and PDGF-BB).(AU)

Avaliação clínico-radiográfica do uso de quitosana, hidroxiapatita e plasma rico em plaquetas na cicatrização óssea em coelhos / Clinical and radiographic evaluation of the use of chitosan, hydroxyapatite and platelet-rich plasma in bone healing in rabbits

The aim of the study was to evaluate the use of chitosan, hydroxyapatite and platelet-rich plasma in bone healing in rabbits. Twelve animals were submitted to surgical procedure to perform bone defect in both members, a control and other treatment, were carried out clinical and orthopedic ratings daily, and radiographic of members of animals seven and 45 days after surgical procedures, where the latter were evaluated for bone reaction. No differences were observed between groups in clinical, radiographs showed slight differences between the control and treatment. Even the radiographic examination revealed the progression of bone reaction, other evaluations are necessary to obtain more data about the biomaterials effect on bone healing.

Avaliação clínico-radiográfica do uso de quitosana, hidroxiapatita e plasma rico em plaquetas na cicatrização óssea em coelhos / Clinical and radiographic evaluation of the use of chitosan, hydroxyapatite and platelet-rich plasma in bone healing in rabbits

The aim of the study was to evaluate the use of chitosan, hydroxyapatite and platelet-rich plasma in bone healing in rabbits. Twelve animals were submitted to surgical procedure to perform bone defect in both members, a control and other treatment, were carried out clinical and orthopedic ratings daily, and radiographic of members of animals seven and 45 days after surgical procedures, where the latter were evaluated for bone reaction. No differences were observed between groups in clinical, radiographs showed slight differences between the control and treatment. Even the radiographic examination revealed the progression of bone reaction, other evaluations are necessary to obtain more data about the biomaterials effect on bone healing.(AU)

Secretoma no tratamento de lesão dermonecrótica induzida pelo veneno de Loxosceles intermedia

Objetivou-se avaliar os efeitos do secretoma, em feridas dermonecróticas em coelhos submetidos à injeção de veneno de Loxosceles intermedia. Foram utilizados 16 coelhos machos, adultos, Nova Zelândia, com peso médio de 2,0 kg, distribuídos em quatro grupos (n=4). À exceção do grupo controle (grupo I), que foi submetido apenas à aplicação de secretoma (60g de secretoma diluído em tampão fosfato-salina a 0,5%), todos os outros grupos foram submetidos à administração de 10g de veneno de Loxosceles intermedia, diluído em NaCl 0,9%, via intradérmica (ID) na região interescapular e, tratados 30 minutos após a injeção do veneno, da seguinte forma: grupo II (NaCl 0,9%, via ID); grupo III (60g de secretoma diluído em tampão fosfato-salina 0,5%, via ID) e, grupo IV (60g de secretoma diluído em tampão fosfato-salina 0,5% via endovenosa - EV). Os animais foram avaliados diariamente e realizados registros fotográficos em altura pré-definida de 30 cm para posterior análise da evolução da área da ferida por morfometria. Amostras de sangue foram coletadas imediatamente antes da aplicação do veneno (tempo 0) e 3, 9 e 15 dias após, para avaliação e monitoração de parâmetros hematológicos e bioquímicos séricos e plasmáticos. Após 15 dias os animais foram eutanasiados, submetidos a necropsia e amostras de pele ao redor da lesão foram coletadas para posterior análise histológica. Os resultados demonstraram que os animais do GI não apresentaram edema, eritema, halo ou necrose. No primeiro dia da injeção do veneno, após tratamentos com secretoma, os animais do GIII e GIV, apresentaram maior grau de edema, quando comparado aos animais do GII. Todavia, no 15ª dia de avaliação, o edema foi menor nos animais do GIII e GIV e, de forma inversa, maior no GI. O eritema foi observado nos grupos que receberam veneno de L. intermedia (GII, GIII e GIV), e comparativamente, no primeiro dia, nos grupos II e III foram similares entre si, e diferentes do GIV que apresentou menor diâmetro de eritema (p<0,05). O halo hemorrágico não foi observado no GI e nos animais que receberam veneno (GII, GIII e GIV), nos tempos 1 e 3, não houve diferença (p>0,05). Deve ser salientado que, no nono dia, somente nos animais do GIV, ainda havia halo hemorrágico. Todavia, macroscopicamente, no GIV, apenas um animal apresentou evolução para ferida dermonecrótica. Na avaliação microscópica, não foram observadas alterações na pele dos animais do GI, entretanto, apesar de todos animais que foram desafiados com o veneno, apresentarem alterações muito semelhantes, como necrose e infiltração heterofílica, no GIV, os animais apresentaram ativação fibroblástica, desenvolvimento precoce de tecido conjuntivo, neovascularização e reepitelização tecidual, conferindo alternativa comprovadamente eficaz em relação ao processo de cicatrização. Em relação a hematologia não houve alteração digna de nota e na bioquímica sérica, somente houve aumento na concentração de CK no tempo 3, nos grupos GIII e GIV e, posterior redução a partir do nono dia. Esses mesmos grupos também apresentaram aumento de LDH e ureia, porém os valores permaneceram dentro dos parâmetros fisiológicos para a espécie leporina. Conclui-se que a terapia com o secretoma pode ser utilizada na cicatrização da ferida dermonecrótica no loxoscelismo.

The objective was to evaluate the effects of the secretome, in dermonecrotic wounds in rabbits subjected to injection of Loxosceles intermedia. Sixteen male, adult, New Zealand rabbits, with a mean weight of 2.0 kg, were distributed in four groups (n=4). Except for the control group (group I), which was subjected only to the application of secretome (60g of secretome diluted in 0.5% phosphate-saline buffer), all other groups were subjected to the administration of 10g of L. intermedia venom, diluted in 0.9% NaCl, via intradermal (ID) in the interscapular region and, treated 30 minutes after the venom injection, as follows: group II (NaCl 0.9%, via ID); Group III (60g of secretome diluted in 0.5% phosphate-saline buffer, via ID) and, group IV (60g of secretome diluted in 0.5% phosphate-saline buffer intravenous - IV). Animals were evaluated daily and photographic records were taken at a predefined height of 30 cm for later analysis of the evolution of the wound area by morphometry. Blood samples were collected immediately before venom application (time 0) and 3, 9 and 15 days after, for evaluation and monitoring of hematological and serum and plasma biochemical parameters. After 15 days, the animals were euthanized, submitted to necropsy, and skin samples around the lesion were collected for subsequent histological analysis. The results showed that the animals in GI did not have edema, erythema, hemorrhagic halo or necrosis. On the first day of venom injection, after secretome treatments, the animals in GIII and GIV showed a more significant degree of edema, when compared to the animals in GII. However, on the 15th day of evaluation, the edema was lower in the animals of GIII and GIV, and conversely, higher in GI. Erythema was observed in the groups that received L. intermedia venom (GII, GIII and GIV), and comparatively, on day 1st, groups II and III were similar to each other, and different from GIV which showed smaller erythema diameter (p<0.05). The hemorrhagic halo was not observed in GI and in the animals that received venom (GII, GIII, and GIV), at times 1 and 3, there was no difference (p>0.05). It should be noted that on the 9th day, only in the animals of GIV, there was still a hemorrhagic halo. However, macroscopically, in GIV, only one animal showed evolution to a dermonecrotic wound. In the microscopic evaluation, no changes were observed in the skin of animals of GI, however, although all animals that were challenged with the venom presented very similar changes, such as necrosis and heterophilic infiltration, in GIV, the animals showed fibroblastic activation, early development of connective tissue, neovascularization, and tissue reepithelialization, conferring a proven effective alternative in relation to the healing process. Regarding hematology, there was no noteworthy change, and in biochemistry serum profile, there was only an increase in CK concentration at time 3, in groups GIII and GIV, and a subsequent reduction from day 9. These same groups also showed an increase in LDH and urea, but the values remained within the physiological parameters for the leporine species. It is concluded that secretome therapy can be used in dermonecrotic wound healing in loxoscelism.

Liquid and gel Platelet Rich Plasma as skin healing adjuvant / Plasma Rico em Plaquetas em suas frações líquida e em gel como adjuvantes da cicatrização cutânea

Background: In recent decades, many researches have been conducted on processes involved in tissue repairing, mainly in the development of resources and technology designed to improve the wound healing progress. Platelet rich plasma (PRP) derived from autologous blood is defined as a plasma volume with platelet concentration higher than physiological level. It is an autogenous and low cost source of growth factors, which are essential for tissue regeneration due to their angiogenic, mitogenic, and chemotactic properties. The aim of this study was evaluate two forms of PRP- liquid and gel - regarding their capacity to influence quality and repair time of standardized skin injuries. Materials, Methods & Results: New Zealand healthy rabbits were distributed in three groups (n = 6): control group (CG), liquid platelet rich plasma group (LIQPRP), and gel platelet rich plasma group (GELPRP). Acute skin lesions were inducted in two areas approximately 2 cm close to scapular edge and depth including epidermis, dermis, and hypodermis to external muscular fascia. Animals received treatment according to each group. Injuries were measured with digital pachymeter in two directions: longer length (l) and longer width (w), every two days. Areas and healing rates were calculated. Microscopic analysis samples were collected on days seven and 14 and evaluated through hematoxylin and eosin [...](AU)

Liquid and gel Platelet Rich Plasma as skin healing adjuvant / Plasma Rico em Plaquetas em suas frações líquida e em gel como adjuvantes da cicatrização cutânea

Background: In recent decades, many researches have been conducted on processes involved in tissue repairing, mainly in the development of resources and technology designed to improve the wound healing progress. Platelet rich plasma (PRP) derived from autologous blood is defined as a plasma volume with platelet concentration higher than physiological level. It is an autogenous and low cost source of growth factors, which are essential for tissue regeneration due to their angiogenic, mitogenic, and chemotactic properties. The aim of this study was evaluate two forms of PRP- liquid and gel - regarding their capacity to influence quality and repair time of standardized skin injuries. Materials, Methods & Results: New Zealand healthy rabbits were distributed in three groups (n = 6): control group (CG), liquid platelet rich plasma group (LIQPRP), and gel platelet rich plasma group (GELPRP). Acute skin lesions were inducted in two areas approximately 2 cm close to scapular edge and depth including epidermis, dermis, and hypodermis to external muscular fascia. Animals received treatment according to each group. Injuries were measured with digital pachymeter in two directions: longer length (l) and longer width (w), every two days. Areas and healing rates were calculated. Microscopic analysis samples were collected on days seven and 14 and evaluated through hematoxylin and eosin [...]

SINOVITE EM EQUINOS: EFEITO DAS CÉLULAS TRONCO MESENQUIMAIS SOBRE A INFLAMAÇÃO ARTICULAR

A alta exigência do sistema locomotor de equinos atletas resulta em pequenas lesões articulares que, cumulativamente, provocam inflamação sinovial e liberação de diversos mediadores inflamatórios no microambiente articular, resultando no desenvolvimento da osteoartrite (OA). A utilização de células tronco mesenquimais (CTMs) nestes casos visa modificar a progressão desta enfermidade. O objetivo do presente trabalho foi verificar o comportamento das CTMs alogênicas derivadas da membrana sinovial (CTMms) frente aos eventos inflamatórios em casos de sinovite aguda em equinos. A sinovite foi induzida utilizando 0,5ng de LPS via intra-articular (IA) e os animais foram tratados 8 horas após a indução. O grupo controle recebeu como tratamento 2 ml de PBS IA, enquanto o grupo tratado recebeu 107 CTMms IA. Foram realizados, de maneira seriada, exames físicos, exames do aparelho locomotor, análise citológica do líquido sinovial e a determinação da concentração sinovial de TGF- e PGE2. Não foram observadas diferenças entre os grupos quanto à claudicação e parâmetros do exame físico. A análise citológica revelou diminuição significativa de linfócitos e macrófagos no grupo tratado (P= 0,0059 e P= 0,0003, respectivamente) que também apresentou menores níveis de TGF- (P= 0,0467) no momento 24h. Os demais parâmetros não apresentaram diferença significativa em nenhum momento. Os dados avaliados sugerem que a inflamação aguda tenha inicialmente causado inibição da capacidade imunomoduladora das CTMs, que foi retomada após 24h, com o declínio da inflamação articular, e que a interação das CTMs com os mediadores inflamatórios presentes no líquido sinovial está diretamente relacionada à condição inflamatória articular no momento da aplicação. Para a otimização do potencial imunomodulador das CTMs, os autores sugerem a adoção de estratégias para o controle da inflamação local antes de sua administração

The high requirement of the locomotor system of equine athletes often results in small joint injuries that can cause synovial inflammation and release of inflammatory mediators, leading to the development of osteoarthritis (OA). Mesenchymal stem cells (MSCs) are used in these cases to modify OA progression. This study aimed to verify the behavior of allogeneic synovial membrane derived MSCs (MSCsm) against inflammatory events in acute equine synovitis. Synovitis was induced by intra-articular (IA) administration of 0.5 ng of LPS and the subjects were treated 8 hours post-induction. The control group received 2 ml of PBS IA as treatment, while the treated group received 107 MSCsm IA. Physical exams, lameness evaluations, cytological analysis of synovial fluid and determination of synovial concentrations of TGF- and PGE2 were performed. No differences were observed in lameness and physical exam parameters between the groups. Cytological analysis revealed a significant decrease in lymphocytes and macrophages in the treated group (P=0.0059 and P=0.0003, respectively), which also had lower levels of TGF- (P=0.0467) at 24 h. The other parameters did not present significant difference. Our data suggest that acute inflammation initially caused inhibition of the immunomodulatory capacity of MSCs, which was resumed after 24h with the decline of joint inflammation, and that the interaction of MSCs with the inflammatory mediators in the synovial fluid is directly related to the inflammatory condition at the time of application. To optimize the immunomodulatory potential of MSCs, the authors suggest the adoption of strategies to control local inflammation prior to its administration.

Comparação entre três protocolos de obtenção de plasma rico em plaquetas (PRP) utilizando o coelho como modelo experimental / Comparison of Three Protocols to Obtain Platelet Rich Plasma (PRP) with Rabbits as Experimental Model

Background: Platelet rich plasma (PRP) is a blood-derived source of growth factors and several cytokines, which are essential for tissue regeneration and important for wound healing due to their angiogenic, mitogenic, and chemotactic activities. To date no protocol has been established for PRP production. Standardization of this technique should consider fundamental factors such as experimental model used, blood collection method, anticoagulant choice, rotation and amount of centrifugations, elapsed time between sample activation and its clinical use in order to ensure quality and biological effects of the product. This study aimed to compare three protocols for PRP achievement in order to evaluate platelet enrichment ability and method reproducibility for further use in clinical investigations regarding PRP therapeutic properties. Materials, Methods & Results: New Zealand higid rabbit's whole blood was collected in tubes containing sodium citrate. Samples were obtained through exsanguination, via abdominal aortic puncture, and separated in four aliquots designed for PRP processing and basal platelet count. The count was conducted at the time blood was collected and after every concentration protocol. Methods were tested in triplicates, and three different individuals repeated each technique for three times, reaching 27 repetitions. Selected methodologies consisted in two centrifugations protocols: protocol A used 250 g for 10 min for the first separation, and another 10 min at 2000 g during the second centrifugation; protocol B proposed that first centrifugation would last 20 min at 160 g, and the second would last 15 min at 400 g, and protocol C consisted of 10 min at 400 g for the first separation, and 10 min at 800 g during second separation. Protocols were performed at the same time in three similar centrifuges, in order to standardize the variables (operator, time, environment, equipments), and also to diminish biases. Comparison objects in this study include: ability of raising platelet concentration, time required for preparing the final product, reproduction handiness, and need for equipment for proper hemoconcentrated production. Achieved platelet count in each protocol and basal value were analyzed following randomized complete. Kurskal-Wallis test was used for independent samples comparison, considering a 5% significance level. For each tested sample, elapsed time for product preparation was evaluated. Subjective analyzes comprehended execution easiness and the need for special material, and were evaluated through questionnaire after each protocol. Protocol A showed a 25-fold increase in platelet count, whereas protocols B and C had 13 and 7-fold, respectively. Results indicate all protocols were efficient in concentrating the samples at least 3 times more than basal count. Elapsed time for product preparation in each protocol was 35, 52, and 41 min for A, B, and C methods, respectively. Subjective analyzes considered protocols A and C as low complexity, and protocol B was defined as medium complexity in regards to execution. With reference to material accessibility for protocols, all were considered of easy reproducibility. Discussion: Besides analyzing experimental model and most proper way to access blood collection, this study was limited to verify in a quantitative manner the platelet concentration in specific protocols, without evaluating their biological effects. Therefore, in regards to proposed objectives - relation between platelet concentration increase, spent time, and easiness of protocol - we conclude that protocol A, formulated by Nagae et al. (2007), was the method that most fitted the work needs, and greatly suited the challenges posed.

Comparison of three protocols to obtain platelet rich plasma (PRP) with rabbits as experimental model / Comparação entre três protocolos de obtenção de plasma rico em plaquetas (PRP) utilizando o coelho como modelo experimental

Background: Platelet rich plasma (PRP) is a blood-derived source of growth factors and several cytokines, which are essential for tissue regeneration and important for wound healing due to their angiogenic, mitogenic, and chemotactic activities. To date no protocol has been established for PRP production. Standardization of this technique should consider fundamental factors such as experimental model used, blood collection method, anticoagulant choice, rotation and amount of centrifugations, elapsed time between sample activation and its clinical use in order to ensure quality and biological effects of the product. This study aimed to compare three protocols for PRP achievement in order to evaluate platelet enrichment ability and method reproducibility for further use in clinical investigations regarding PRP therapeutic properties. Materials, Methods & Results: New Zealand higid rabbits whole blood was collected in tubes containing sodium citrate. Samples were obtained through exsanguination, via abdominal aortic puncture, and separated in four aliquots designed for PRP processing and basal platelet count. The count was conducted at the time blood was collected and after every concentration protocol. Methods were tested in triplicates, and three different individuals repeated each technique for three times, reaching 27 repetitions. Selected methodologies consisted in two c

O plasma rico em plaquetas derivado de sangue autólogo é definido como um volume de plasma com uma concentração plaquetária acima dos níveis fisiológicos. É uma fonte autógena e de baixo custo de fatores de crescimento (FC). FC são moléculas bioativas fundamentais no reparo e regeneração de diversos tecidos, capazes de estimular a mitogênese, angiogênese, quimiotaxia, proliferação e diferenciação celular. Entre os fatores liberados pelas plaquetas destacam-se: Fator de Crescimento Derivado de Plaquetas (PDGF), Fator de Crescimento Transformador Beta (TGF- ), Fator de Crescimento Endotelial Vascular (VEGF) e Fator de Crescimento Epitelial (EGF). [...]

Influência do anticoagulante na obtenção e criopreservação do plasma rico em plaquetas (PCR) em equinos e muares

O plasma rico em plaquetas (PRP) é um produto biológico utilizado na promoção de melhor reparo tecidual, por meio dos fatores de crescimento presentes em sua composição. O objetivo do presente trabalho foi comparar a viabilidade da criopreservação das plaquetas de muares e equinos e a influência de dois anticoagulantes citrato dextrose (ACD) e citrato de sódio (CS). Foram utilizados oito animais de cada espécie, com duas replicações cada. O PRP foi obtido em uma centrifugação única de 131g, durante oito minutos. As amostras foram congeladas em nitrogênio líquido após serem submetidas à curva de resfriamento controlado. Foi utilizado uma solução de DMSO a 3% em glicose como criopreservante. As análises foram feitas em aparelho hematológico e na câmara de Neubauer; a avaliação morfológica foi feita em microscopia óptica de contraste de fases. A concentração plaquetária do PRP em relação ao sangue total foi de 1,42 vezes para equinos e 2,00 vezes para muares, em ACD; e 1,39 vezes para equinos e 2,69 vezes para muares utilizando-se o CS. O protocolo utilizado foi mais efetivo em concentrar as plaquetas de muares do que de equinos, embora a qualidade do PRP congelado tenha sido semelhante nas duas espécies. Constatou-se o aumento das células em estado ativado nas amostras criopreservadas, independente da espécie ou anticoagulante empregado. A contagem de plaquetas em ACD e CS, no sangue total, foi mais fidedigna do que a contagem no EDTA para a produção do PRP. Baseando-se na premissa de que a presença leucocitária é deletéria à qualidade do PRP, o anticoagulante CS mostrou-se mais eficiente na redução de leucócitos totais em relação ao ACD, em ambas as espécies. Por não haver diferenças significativas entre a qualidade do PRP criopreservado obtido entre os diferentes anticoagulantes empregados (principalmente em parâmetros morfológicos plaquetários), sugere-se o CS como melhor anticoagulante na obtenção do PRP em equinos e muares. Aplicações clínicas são necessárias para validar os efeitos biológicos do PRP, fresco e criopreservado, especialmente em muares, que mostraram particularidades hematológicas em relação aos equinos.

Platelet-Rich Plasma (PRP) is a biological product often used to promote tissue repair, due to its composition of growth factors. The aim of this study was to compare the viability of platelet cryopreservation on two species, horses and mules, and the influence of two different anticoagulants dextrose citrate (ACD) and sodium citrate (SC). Eight animals of each species were used, with two replicas each. PRP was obtained after single centrifugation at 131 g in eight minutes. Samples were frozen in liquid nitrogen after exposure to a controlled cooling rate. A solution of glucose and 3% DMSO was used as a cryoprotectant. Blood analysis were performed on hematologic analyzer, Neubauer chamber and morphology was evaluated through optical microscopy of phase inversion. Platelet concentration on PRP when compared to total blood samples were 1,42 times in horses and 2,00 in mules, using ACD; and 1,39 times in horses and 2,69 times in mules using SC. The protocol was more efficient in concentrating mules platelets when compared to horses, although both species cryopreservation had similar quality. There was a significant increase of cells in activated status after cryopreservation, despite the species or anticoagulant employed. Platelet count on ACD and SC, on total blood, was more truthful in ACD and SC when compared to EDTA, in order to establish PRP parameters. Based upon the negative effects of leukocytes on PRP, SC was more efficient in reducing total leukocyte count when compared to ACD on both species. Due to the fact that there were no significant differences between cryopreserved PRP and the anticoagulants employed (especially regarding morphology parameters of platelets), it is suggested that SC was a better anticoagulant to obtain PRP on both mules and horses. Clinical trials should be performed, especially in mules, due to its hematological distinctive features when compared to horses.

Células tronco mesenquimais de origem adiposa associadas a enxertos livres de pele de espessura total em modelo murino

Enxertos livres de pele de espessura total são indicados para cobrir grandes defeitos de pele em superfícies flexoras ou em extremidades distais e sofrem lesão por isquemia e reperfusão pela própria natureza do procedimento cirúrgico. O objetivo deste trabalho foi testar a associação de células tronco mesenquimais de origem adiposa (ADSCs) heterólogas a enxertos cutâneos autólogos de espessura total em ratos Wistar. Enxertos de 12 mm de diâmetro foram executados no dorso de 30 ratos, em dois locais: cranial e caudal. Os ratos foram distribuídos em seis grupos (n=5): grupo ADSC_G recebeu, no enxerto, 1x10 6 ADSCs em 200 µL de Solução Salina 0,9% (SS); grupo ADSC_B recebeu 1x106 ADSCs em 200 µL de SS na borda do leito receptor; grupo ADSC_GB, metade da mesma suspensão na borda e outra metade no enxerto. Os grupos controle, SS_G e SS_B, receberam apenas SS no enxerto ou nas bordas respectivamente. No grupo S, o enxerto não recebeu nenhum tratamento durante as cirurgias. Curativos do tipo tie-over permaneceram até o 5º dia. Na cirurgia (d0), aos 5 (d5) e 14 (d14) dias de pós-operatório, os desenhos dos enxertos foram digitalizados e suas áreas mensuradas (software ImageJ). As avalições clínicas consideraram peso, presença de secreções e ocorrência de epidermólise. A planimetria demonstrou a taxa de pele normal, de pele avermelhada e de ulceração, assim como a contração dos enxertos entre os intervalos d0d5, d5-d14 e d0-d14. As amostras dos enxertos foram obtidas em d14 para coloração com Hematoxilina e eosina e Tricrômico de Masson. A epiderme foi avaliada para espessamento, ceratose, acantose, degeneração hidrópica, infiltrado inflamatório. A derme foi avaliada quanto a rarefação pilosa, tecido de granulação, infiltrado inflamatório, deposição de colágeno. Os resultados foram expressos em média e desvio padrão, e a análise estatística utilizou as Equações de Estimações Generalizadas (GEE), com nível de significância de 5%. O grupo ADSC_G apresentou melhores aspectos macroscópicos, menos ocorrência de epidermólise e de rarefação pilosa e uma das menores médias de área de ulceração, juntamente com os outros grupos tratados com ADSCs. O grupo ADSC_G demonstrou as menores médias de alterações histopatológicas como: espessamento da epiderme, degeneração hidrópica, tecido de granulação. O mesmo grupo ficou entre as menores médias de acantose e infiltrado inflamatório na derme, ao mesmo tempo que apresentou as maiores médias de VEGF no subcutâneo e no tecido de granulação. Dessa forma, pode-se concluir que as ADSCs protegeram os enxertos dos efeitos deletérios da isquemia, assim como e sugerem-se novos estudos em fases iniciais da cicatrização para compreensão dos mecanismos envolvidos.

Evaluation of adipose derived stem cells (ADSC) effect on full-thickness skin graft (FTSG) as a wound healing model in ischemic conditions. Two 12 mm diameter FTSGs were harvested and placed onto dorsal recipient beds of twenty-four Wistar rats, in two anatomic regions: cranial and caudal. Rats were randomized into five groups. Before grafting, group E FTSGs received subfascial injection of 1X106 ADSCs diluted in 200 µL of physiologic saline. Group EC FTSGs received only physiologic saline. Group B received ADSCs in the recipient bed edges; group C received physiologic saline in the edges. Group ADSC_GB received the same ADSCs number and volume, half in the graft and half in the edges. Using planimetry, grafts were analyzed for graft´s contraction rate (d0, d5, d14), normal skin rate and occurrence of epidermolysis and failure rate (d14). FTSGs samples were obtained on the d14 to hematoxylin-eosin and Massons Trichrome staining for epidermal analysis (epidermal thickening, keratosis, acanthosis, hydropic degeneration, inflammatory infiltrate) and dermal analysis (hairless, granulation tissue, inflammatory infiltrate, collagen deposition). The obtained results were expressed by mean (n=5). Statistical significance (p<0,05) was calculated using Generalized Estimating Equations. The ADSC_G group had better macroscopic aspects, less occurrence of epidermolysis and hairless and one of the lowest averages of ulceration area, along with the other groups treated with ADSCs. The ADSC_G group showed the lowest mean on the histopathological changes measured such as thickening of the epidermis, hydropic degeneration, less granulation tissue. The same group was among the lowest acanthosis and inflammatory infiltrate averages in the dermis, while presented with the greatest of VEGF average in the subcutaneous and in the granulation tissue. Thus it can be concluded that the ADSCs may have protected the grafts from the deleterious effects of ischemia and suggest new studies in the early stages of healing to understand the mechanisms involved.

Células-tronco mesenquimais e plasma rico em plaquetas como adjuvantes da cicatrização de lesões cutâneas experimentais em coelhos Nova Zelândia

Muito se tem investido em pesquisa na compreensão dos processos e fenômenos envolvidos nas diversas fases da reparação tissular e, principalmente, no desenvolvimento de recursos e tecnologias com o objetivo de favorecer os avanços no tratamento de feridas. Este trabalho foi realizado com o objetivo de avaliar os efeitos biológicos da associação células-tronco mesenquimais (CTMs) e plasma rico em plaquetas (PRP) como adjuvantes da cicatrização cutânea de feridas padronizadas em coelhos. Foram utilizados 37 coelhos Nova Zelândia, dentre os quais, 36 foram distribuídos em seis grupos: grupo controle (GC), grupo células-tronco mesenquimais (GCTM), grupo plasma rico em plaquetas na forma líquida (GPRPLIQ), grupo plasma rico em plaquetas na forma de gel (GPRPGEL), grupo associação células-tronco mesenquimais e plasma rico em plaquetas na forma liquida (GCTM+PRPLIQ), grupo associação células-tronco mesenquimais e plasma rico em plaquetas na forma de gel (GCTM+PRPGEL). Após indução de lesões cutâneas agudas e padronizadas, os animais receberam o tratamento de acordo com o grupo que compunham. Macroscopicamente, a cada dois dias, foram analisadas as medidas das lesões e calculadas a área e a taxa de cicatrização das mesmas. Amostras para análise microscópica foram coletadas em sete e 14 dias e avaliadas quanto à presença de células inflamatórias, angiogênese, formação de fibrose colagênica, proliferação epitelial e fibroblástica. Com base nos resultados obtidos foi possível concluir que: 1) a associação CTM e PRP em suas frações terapêuticas (CTM+PRPGEL e CTM+PRPLIQ) não acelerou o processo de cicatrização de feridas cutâneas agudas, na avaliação morfométrica aos 14 dias de pós-operatório; 2) a aplicação local da CTM e das frações de PRP quando utilizadas de maneira isolada não conseguiram acelerar o tempo necessário ao processo cicatricial na avaliação morfométrica aos 14 dias de pósoperatório; 3) a utilização do PRP na sua forma líquida obteve índices de epitelização menores em relação ao controle em avaliações histopatológicas aos 14 dias de pósoperatório; 4) à exceção do ocorrido com o grupo PRP líquido, os demais tratamentos não alteraram significativamente as fases inflamatória, proliferativa e inicial do remodelamento, segundo dados histopatológicos; 5) a terapia com as células-tronco mesenquimais promoveu cicatrizes esteticamente melhores na avaliação clínica, especialmente aos 14 dias de observação. Sugere-se a necessidade de novos estudos e a utilização de outras ferramentas diagnósticas para melhores subsídios de interpretação sobre os resultados encontrados

Much has been invested on research regarding comprehension of the processes and phenomena that are involved in several stages of tissue repairing and mainly on resources and technologies development in order to support advances on wound healing. This study has been performed in order to evaluate the biological effects of mesenchymal stem cells (CTM) and platelet rich plasma (PRP) association as adjuvants on skin healing of standardized wounds in rabbits. Thirty-seven New Zealand rabbits have been used and, among these animals, 36 were allocated into six groups: control group (GC), mesenchymal stem cells group (GCTM), liquid platelet rich plasma group (GPRPLIQ), gel platelet rich plasma group (GPRPGEL), mesenchymal stem cells associated with liquid platelet rich plasma group (GCTM+PRPLIQ) and mesenchymal stem cells associated with gel platelet rich plasma group (GCTM+PRPGEL). After the induction of acute and standardized skin wounds, animals received the treatment according to the group they belonged to. Macroscopically, every two days, wounds measures were taken to calculate their area and healing rate. Samples for microscopically analysis were collected on day 7 and on day 14 and were evaluated for the presence of inflammatory cells, angiogenesis, collagen fibrosis formation, epithelial and fibroblastical proliferation. Basing on the results it was concluded that: 1) CTM and PRP in its two therapeutic fractions association (GCTM+PRPLIQ and GCTM+PRPGEL) did not accelerate the healing process of acute skin wounds, on morphometric assessment at 14 days after surgery; 2) local injection of CTM and PRP fractions when isolated did not accelerate the time required for wound healing on morphometric assessment at 14 days after surgery; 3) PRP application on its liquid fraction showed lower levels of epithelization related to control on histopathological evaluation at 14 days after surgery; 4) except GPRPLIQ, other treatments did not alter significantly inflammatory, proliferative nor initial remodeling phases, based on histopathological data; 5) CTM therapy promoted aesthetically better scars on clinical evaluation, especially at 14 days of observation. It is suggested the requirement of further studies and use of different diagnostic tools to obtain better interpretation of the achieved results

Comparison of three protocols to obtain platelet rich plasma (PRP) with rabbits as experimental model / Comparação entre três protocolos de obtenção de plasma rico em plaquetas (PRP) utilizando o coelho como modelo experimental

Background: Platelet rich plasma (PRP) is a blood-derived source of growth factors and several cytokines, which are essential for tissue regeneration and important for wound healing due to their angiogenic, mitogenic, and chemotactic activities. To date no protocol has been established for PRP production. Standardization of this technique should consider fundamental factors such as experimental model used, blood collection method, anticoagulant choice, rotation and amount of centrifugations, elapsed time between sample activation and its clinical use in order to ensure quality and biological effects of the product. This study aimed to compare three protocols for PRP achievement in order to evaluate platelet enrichment ability and method reproducibility for further use in clinical investigations regarding PRP therapeutic properties. Materials, Methods & Results: New Zealand higid rabbits whole blood was collected in tubes containing sodium citrate. Samples were obtained through exsanguination, via abdominal aortic puncture, and separated in four aliquots designed for PRP processing and basal platelet count. The count was conducted at the time blood was collected and after every concentration protocol. Methods were tested in triplicates, and three different individuals repeated each technique for three times, reaching 27 repetitions. Selected methodologies consisted in two c

O plasma rico em plaquetas derivado de sangue autólogo é definido como um volume de plasma com uma concentração plaquetária acima dos níveis fisiológicos. É uma fonte autógena e de baixo custo de fatores de crescimento (FC). FC são moléculas bioativas fundamentais no reparo e regeneração de diversos tecidos, capazes de estimular a mitogênese, angiogênese, quimiotaxia, proliferação e diferenciação celular. Entre os fatores liberados pelas plaquetas destacam-se: Fator de Crescimento Derivado de Plaquetas (PDGF), Fator de Crescimento Transformador Beta (TGF- ), Fator de Crescimento Endotelial Vascular (VEGF) e Fator de Crescimento Epitelial (EGF). [...]