Pancreatic cancer orthotopic graft in a murine model

Purpose: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with increasing incidence. Even if progress have been made, the five-year overall survival remains lower than 10%. There is a desperate need in therapeutic improvements. In the last two decades, new in-vitro models have been developed and improved, including tridimensional-culture spheroids and organoids. However, animal studies remain mandatory in the upscaling before clinical studies. Orthotopic and syngeneic grafting is a robust model to test a drug efficiency in a tumor and its microenvironment. Methods: We described a method for orthotopic and syngeneic graft of KRAS mutated, p53 wildtype, 8305 cells in a C57BL/6J mouse model. Results: With this microsurgical method, 30 mice were grafted, 24 by a junior and six by a senior, resulting in 95,8 and 100% of (partial and total) successful tumoral implantation, respectively. Twenty mice underwent ultrasound follow-up. It was an efficient method for the tumoral growth evaluation. At day 16 after grafting, 85% of the tumors were detectable by ultrasound, and at day 22 all tumors were detected. Conclusions: The presented method appears to be a robust and reliable method for pre-clinical studies. A junior master student can provide positive results using this technique, which can be improved with training.

Short-term outcomes of trochlear recession surgery combined with "Watermelon" autograft for treatment of canine medial patellar luxation: a case series / Resultados a curto prazo da cirurgia de recessão trocolear combinada com o auto-enxerto "Melancia" para tratamento da luxação patelar medial canina: uma série de casos

The aim of this study was to present a novel surgical method for correction of medial patellar luxation in small-breed dogs with trochlear dysplasia and marked hypoplasia of the medial femoral condyle. The "Watermelon" approach was applied together with trochlear wedge recession or trochlear block recession and consisted in increasing the height of the medial condyle with an osteochondral autograft resembling a watermelon slice, placed into an additional slot in the medial femoral condyle to prevent the postoperative reluxation of the patella. The study cohort included 19 dogs (25 joints) from small breeds (Pinscher, Pomeranian, and Chihuahua) with second-grade medial patellar luxation. Fourteen joints were submitted to wedge recession surgery combined with "Watermelon" grafting, and 11 joints: to block recession surgery with "Watermelon" grafting. The sulcus-deepening trochleoplasty combined with "Watermelon" grafting was clinically successful and with low percentage of minor postoperative complications. The mean duration of anesthesia was significantly longer for block recession combined with "Watermelon" (P<0.001), but the recovery period was shorter (P<0.05).

O objetivo deste estudo foi apresentar um novo método cirúrgico para correção da luxação patelar medial em cães de raça pequena com displasia troquelar e hipoplasia marcada do côndilo femoral medial. A abordagem "Melancia" foi aplicada juntamente com a recessão da cunha trocolear ou recessão do bloco trocolear e consistiu em aumentar a altura do côndilo medial com um auto-enxerto osteocondral parecido com uma fatia de melancia, colocado em uma fenda adicional no côndilo femoral medial para evitar o relaxamento pós-operatório da patela. A coorte do estudo incluiu 19 cães (25 juntas) de raças pequenas (Pinscher, Pomeranian, e Chihuahua) com luxação patelar medial de segundo grau. Catorze juntas foram submetidas à cirurgia de recessão em cunha combinada com enxerto de "melancia", e 11 juntas: para bloquear a cirurgia de recessão com enxerto de "melancia". A trocleoplastia de "melancia" combinada com o enxerto de "melancia" foi clinicamente bem sucedida e com baixo percentual de pequenas complicações pós-operatórias. A duração média da anestesia foi significativamente maior para a recessão em bloco combinada com "Melancia" (P<0,001), mas o período de recuperação foi mais curto (P<0,05).

Maturation and quality of 'BRS Magna' grapes influenced by rootstocks in rainy season

Seasonal variations and production factors can influence the quality of grapes. Considering the Sub-middle region of the São Francisco Valley, Brazil, differentiated by production in rainy and warmer periods of the year, the climatic conditions in each season affect the duration of the phenological phases and quality of the grapes. The objective of this study was to characterize the changes in the quality components during maturation of 'BRS Magna' grapes on different rootstocks, in rainy seasons under tropical conditions, as a support to define the harvest time. The experiment was conducted in Petrolina, Pernambuco, Brazil, from Jan to Apr 2017 (cycle 1) and Dec 2017 to Apr 2018 (cycle 2) evaluating rootstocks and age of the fruits. Bunches were picked from the start of maturation to the harvest: 49, 56, 61, 64 and 68 days after fruit set (DAF) for cycle 1, and 35, 41, 48, 55, 61, and 66 DAF for cycle 2. In cycle 1 'IAC 572' and 'Paulsen 1103' rootstocks provided higher sugar contents in grapes, which was only repeated in cycle 2 for 'Paulsen 1103'. The accumulation of anthocyanins and yellow flavonoids was favored by 'IAC 313' and 'IAC 572' rootstocks in cycle 1, and by 'IAC 313' and 'IAC 766' in cycle 2. A higher content of polyphenols was conferred on 'IAC 572' in cycle 1 and on 'IAC 766' in cycle 2, which also had a greater antioxidant capacity. Although there were differences between rootstocks, harvesting at approximately 64 DAF maximized grape quality for all treatments.(AU)

Fluorescence spectroscopy for clinical transplantation liver grafts monitoring: possibilities offered by 408 nm excitation

Purpose: Fluorescence spectroscopy techniques have been investigated aiming to reduce the invasiveness of methods for investigation of tissue. In transplantation procedures, it may offer the possibility of a complementary technique for the monitoring of liver grafts' conditions prior to and during the transplantation procedure stages involving cold perfusion. The objective of this study was to evaluate fluorescence spectroscopy under violet light excitation (408 nm) for the monitoring of clinical hypothermic liver transplantation procedures. Methods: Organ grafts were monitored from before the removal of the donor's body to 1 h after the implant into the receptor's body. Fluorescence spectroscopy was assessed over five stages within these transplant stages. Results: The study provided evidence of a correlation between fluorescence information collected during liver grafts transplantation and the survival of patients. Conclusions: Fluorescence spectroscopy can become a tool to monitor transplantation grafts, providing objective information for the final decision of surgeons to use organs.

Compatibility and initial development of grapevines BRS Magna grafted on different rootstocks / Compatibilidade e desenvolvimento inicial de videiras ‘BRS Magna enxertada em diferentes porta-enxertos

Rootstocks are widely used in viticulture due to their resistance to biotic and abiotic stress. Additionally, rootstocks can affect vine growth and fruit quality. This study evaluated the compatibility and initial developmental of the BRS Magna grafted on different rootstocks The wedge graft technique on woody cuttings was utilized. The percentage of survival ranged from 0% (VR043-43) to 98.33% (101-14 MGT), and the rootstock were grouped into three distinct groups. IAC 313 Tropical and SO4 rootstocks were those ones with the highest vigor in relation to initial shoot growth. However, IAC 572 Jales, Harmony, 3309 Couderc and Gravesac had the best balance between initial shoot growth and root development. The BRS Magna when grafted on rootstocks IAC 313 Tropical, SO4 and 101-14 MGT showed the highest initial development rates, while when grafted on R99, R110 and 420A, it showed the lowest initial development.(AU)

Os porta-enxertos são amplamente utilizados na viticultura devido à sua resistência aos estresses biótico e abiótico. Além disso, os porta-enxertos podem afetar o crescimento da videira e a qualidade dos frutos. O objetivo deste estudo foi avaliar a compatibilidade e o desenvolvimento inicial de videiras BRS Magna enxertada em diferentes porta-enxertos. A técnica de enxertia em fenda foi utilizada. A porcentagem de sobrevivência variou de 0% (VR043-43) a 98,33% (101-14 MGT) e os porta-enxertos foram agrupados em três grupos distintos. Os porta-enxertos IAC 313 Tropical e SO4 foram os de maior vigor em relação ao crescimento inicial da parte aérea. Já IAC 572 Jales, Harmony, 3309 Couderc e Gravesac tiveram o melhor equilíbrio entre o crescimento inicial da parte aérea e o desenvolvimento das raízes. BRS Magna quando enxertada nos porta-enxertos IAC 313 Tropical, SO4 e 101-14 MGT apresentou as maiores taxas de desenvolvimento inicial, enquanto que, quando enxertada em R99, R110 e 420A, apresentou os menores valores iniciais de desenvolvimento.(AU)

Techniques of orthotopic renal transplantation. II. Size-matched porcine grafts in monkey recipients

ABSTRACT Purpose As a classical xenotransplantation model, porcine kidneys have been transplanted into the lower abdomen of non-human primates. However, we have improved upon this model by using size-matched grafting in the orthotopic position. The beneficial aspects and surgical details of our method are reported herein. Methods Donors were two newborn pigs (weighting 5 to 6 kg) and recipients were two cynomolgus monkeys (weighting, approximately, 7 kg). After bilateral nephrectomy, kidneys were cold-transported in Euro-Collins solution. The porcine kidney was transplanted to the site of a left nephrectomy and fixed to the peritoneum. Results Kidneys transplanted to the lower abdomen by the conventional method were more susceptible to torsion of the renal vein (two cases). In contrast, early-stage blood flow insufficiency did not occur in orthotopic transplants of theleft kidney. Conclusions Size-matched porcine-primate renal grafting using our method of transplanting tothe natural position of the kidneys contributes to stable post-transplant blood flow to the kidney.

Enxerto cutâneo em semeadura associado à malha não aderente em membro pélvico de um cão / Island skin graft associated with non-adherent mesh in a dog's pelvic limb

Background: Large skin defects are caused by tumor excision, making appropriate reconstruction and complete healingof the lesion a challenge for surgeons. There are some difficulties in reaching these goals, especially in cases of surgicalwound in the limbs, due to the scarce amount of skin and its reduced elasticity, which limit the possibility of flaps whencompared to the head, neck, and trunk. This study reports a case of wound closure on the lateral skin in the femoral regionof a dog’s pelvic limb via island skin graft associated with the implantation of a non-adherent cellulose acetate mesh andintensive postoperative care.Case: An 8-year-old Rottweiler female dog was attended at Pontifical Catholic University of Paraná’s Veterinary Clinic(CVE) in Curitiba, Paraná, presenting a tumor located laterocaudally to the right stifle joint. After preoperative examinations the patient underwent tumor surgery; however, two more surgical procedures were required due to suture dehiscence in the region, which resulted in increased wound size. At first, the wound was treated for granulation tissue to beformed. Subsequently, the island skin grafting technique was chosen to close the wound, associated with the implantationof a non-adherent cellulose acetate mesh imbibed with petrolatum emulsion to keep the grafted fragments in place. Themesh was fixed in a simple interrupted suture pattern using 2-0 nylon thread. The lateral regions of the chest and abdomen were chosen as donor skin beds due to their large dimensions, skin elasticity, and ease in defect reconstruction. Thefragments were obtained using a 10-mm biopsy punch and scalpel, and the defects were sutured in a simple interruptedpattern using 2-0 nylon thread. The patient remained hospitalized for movement restriction and postoperative monitoringfor 72 h, and the bandage remained untouched during this time interval. Thereafter, the patient...

Enxerto cutâneo em semeadura associado à malha não aderente em membro pélvico de um cão / Island skin graft associated with non-adherent mesh in a dog's pelvic limb

Background: Large skin defects are caused by tumor excision, making appropriate reconstruction and complete healingof the lesion a challenge for surgeons. There are some difficulties in reaching these goals, especially in cases of surgicalwound in the limbs, due to the scarce amount of skin and its reduced elasticity, which limit the possibility of flaps whencompared to the head, neck, and trunk. This study reports a case of wound closure on the lateral skin in the femoral regionof a dogs pelvic limb via island skin graft associated with the implantation of a non-adherent cellulose acetate mesh andintensive postoperative care.Case: An 8-year-old Rottweiler female dog was attended at Pontifical Catholic University of Paranás Veterinary Clinic(CVE) in Curitiba, Paraná, presenting a tumor located laterocaudally to the right stifle joint. After preoperative examinations the patient underwent tumor surgery; however, two more surgical procedures were required due to suture dehiscence in the region, which resulted in increased wound size. At first, the wound was treated for granulation tissue to beformed. Subsequently, the island skin grafting technique was chosen to close the wound, associated with the implantationof a non-adherent cellulose acetate mesh imbibed with petrolatum emulsion to keep the grafted fragments in place. Themesh was fixed in a simple interrupted suture pattern using 2-0 nylon thread. The lateral regions of the chest and abdomen were chosen as donor skin beds due to their large dimensions, skin elasticity, and ease in defect reconstruction. Thefragments were obtained using a 10-mm biopsy punch and scalpel, and the defects were sutured in a simple interruptedpattern using 2-0 nylon thread. The patient remained hospitalized for movement restriction and postoperative monitoringfor 72 h, and the bandage remained untouched during this time interval. Thereafter, the patient...(AU)

Diminuição do índice de necrose em enxertos cutâneos em coelhos mediante a aplicação de células tronco mesenquimais xenógenas / Reducing of the necrosis index in skin grafts in rabbits through application of xenogenous mesenchymal stem cells

O presente estudo teve como objetivo avaliar a inflamação em auto-enxertos cutâneos obtidos no terceiro, sétimo e décimo quarto dia de pós-operatório, além disso, buscou-se determinar diferenças no processo de cicatrização no grupo tratado com células tronco mesenquimais xenógenas em relação ao grupo controle utilizando a avaliação microscópica e imuno-histoquímico. A avaliação microscópica foi realizada utilizando cortes histológicos corados pela técnica de histoquímica com hematoxilina-eosina (HE), e a imuno-histoquímica, com cortes submetidos a anticorpos específicos. As variáveis analisadas foram quantidade de vasos, células inflamatórias, COX-2, Macrófagos e presença de necrose. Os dados foram analisados estatisticamente pelo software R. A quantidade de vasos foi maior (p<0,0001) no grupo tratamento (GT) durante o dia 3, enquanto no grupo controle (GC) foi maior no dia 7. No dia 3 houve menor porcentagem de necrose no grupo tratamento (GT) (p = 0,038). Nos demais dias avaliados não houve diferença entre a porcentagem de necrose observada nos dois tratamentos (p = 0,98), sendo de 53% para o grupo controle (GC) e 47% para o grupo tratamento (GT). Em relação ao número de macrófagos não houve diferença entre os grupos (p = 0,5637). Entretanto, entre os dias houve diferença significativa (p = 0,0223), sendo menor número de macrófagos no terceiro dia. A imunomarcação de COX-2 foi similar entre os grupos (p = 0,5637) e entre os dias (p = 0,9843). Portanto, o emprego das células tronco mesenquimais xenógenas em enxertos cutâneos promoveu menor ocorrência de necrose, favorecendo sua cicatrização, e não induziu o processo inflamatório, sendo assim factível seu uso em cirurgias reconstrutivas.

The present study aimed to assess inflammation in skin autografts obtained on the third, seventh and fourteenth postoperative day, in addition, it sought to determine differences in the healing process in the group treated with xenogenous mesenchymal stem cells in relation to to the control group using microscopic and immunohistochemical evaluation. Microscopic evaluation was performed using histological sections, stained by the hematoxylin-eosin (HE) histochemistry technique, and immunohistochemistry with sections were subjected to specific antibodies. The variables analyzed were the number of vessels, inflammatory cells (COX-2 and Macrophages) and the presence of necrosis. The data were analyzed statistically by software R. The number of vessels was higher (p< 0.0001) ) in the treatment group (GT) during day 3, while in the control group (CG) it was higher on day 7. On day 3 there was a lower percentage of necrosis in the treatment group (GT) (p = 0.038). On the other evaluated days, there was no difference between the percentage of necrosis observed in the two treatments (p = 0.98), being 53% for the control group (CG) and 47% for the treatment group (GT). Regarding the number of macrophages, there was no difference between groups (p = 0.5637). However, between days there was a significant difference (p = 0.0223), with a lower number of macrophages on the third day. The immunostaining of COX-2 was similar between groups (p = 0.5637) and between days (p = 0.9843). Therefore, the use of xenogenous mesenchymal stem cells in skin grafts promoted a lower occurrence of necrosis, favoring its healing, and did not induce the inflammatory process, thus making its use in reconstructive surgery feasible.

Diminuição do índice de necrose em enxertos cutâneos em coelhos mediante a aplicação de células tronco mesenquimais xenógenas / Reducing of the necrosis index in skin grafts in rabbits through application of xenogenous mesenchymal stem cells

O presente estudo teve como objetivo avaliar a inflamação em auto-enxertos cutâneos obtidos no terceiro, sétimo e décimo quarto dia de pós-operatório, além disso, buscou-se determinar diferenças no processo de cicatrização no grupo tratado com células tronco mesenquimais xenógenas em relação ao grupo controle utilizando a avaliação microscópica e imuno-histoquímico. A avaliação microscópica foi realizada utilizando cortes histológicos corados pela técnica de histoquímica com hematoxilina-eosina (HE), e a imuno-histoquímica, com cortes submetidos a anticorpos específicos. As variáveis analisadas foram quantidade de vasos, células inflamatórias, COX-2, Macrófagos e presença de necrose. Os dados foram analisados estatisticamente pelo software R. A quantidade de vasos foi maior (p<0,0001) no grupo tratamento (GT) durante o dia 3, enquanto no grupo controle (GC) foi maior no dia 7. No dia 3 houve menor porcentagem de necrose no grupo tratamento (GT) (p = 0,038). Nos demais dias avaliados não houve diferença entre a porcentagem de necrose observada nos dois tratamentos (p = 0,98), sendo de 53% para o grupo controle (GC) e 47% para o grupo tratamento (GT). Em relação ao número de macrófagos não houve diferença entre os grupos (p = 0,5637). Entretanto, entre os dias houve diferença significativa (p = 0,0223), sendo menor número de macrófagos no terceiro dia. A imunomarcação de COX-2 foi similar entre os grupos (p = 0,5637) e entre os dias (p = 0,9843). Portanto, o emprego das células tronco mesenquimais xenógenas em enxertos cutâneos promoveu menor ocorrência de necrose, favorecendo sua cicatrização, e não induziu o processo inflamatório, sendo assim factível seu uso em cirurgias reconstrutivas.(AU)

The present study aimed to assess inflammation in skin autografts obtained on the third, seventh and fourteenth postoperative day, in addition, it sought to determine differences in the healing process in the group treated with xenogenous mesenchymal stem cells in relation to to the control group using microscopic and immunohistochemical evaluation. Microscopic evaluation was performed using histological sections, stained by the hematoxylin-eosin (HE) histochemistry technique, and immunohistochemistry with sections were subjected to specific antibodies. The variables analyzed were the number of vessels, inflammatory cells (COX-2 and Macrophages) and the presence of necrosis. The data were analyzed statistically by software R. The number of vessels was higher (p< 0.0001) ) in the treatment group (GT) during day 3, while in the control group (CG) it was higher on day 7. On day 3 there was a lower percentage of necrosis in the treatment group (GT) (p = 0.038). On the other evaluated days, there was no difference between the percentage of necrosis observed in the two treatments (p = 0.98), being 53% for the control group (CG) and 47% for the treatment group (GT). Regarding the number of macrophages, there was no difference between groups (p = 0.5637). However, between days there was a significant difference (p = 0.0223), with a lower number of macrophages on the third day. The immunostaining of COX-2 was similar between groups (p = 0.5637) and between days (p = 0.9843). Therefore, the use of xenogenous mesenchymal stem cells in skin grafts promoted a lower occurrence of necrosis, favoring its healing, and did not induce the inflammatory process, thus making its use in reconstructive surgery feasible.(AU)

Platelet-concentrated and platelet poor-plasma promote different pattern on immunohistochemical expression of TGF-β1, however they impairs the osteoneogensis in calvarial defects treated with autograft due suppression of osteocalcin

ABSTRACT Purpose Herein we evaluated the effects of platelet concentrate (PC) and platelet-poor plasma (PPP) on bone repair using noncritical defects in the calvaria of rabbits and compared them to the presence of TGF-β1 and osteocalcin on reparative sites. Methods Five noncritical defects of 8.7 mm in diameter were created on the calvaria of 15 animals. Each defect was treated differently, using autograft (ABG), ABG associated with PC (ABG + PC), ABG with PPP (ABG + PPP), isolated PPP, and blood clot (control). The animals were submitted to euthanasia on the second, fourth and sixth week post-surgery. Results The defects that received ABG+PC or PPP demonstrated lower bone formation when compared to specimens that received ABG in the same period. These results coincided to significant higher immunopositivity for TGF-β1 for specimens that received PC, and lower presence of cytokine in the group PPP. However, either higher or lower presence of TGF-β1 were also correlated to lower presence of osteocalcin. Likewise, these results were similar to findings in specimens treated only with PPP when compared to control. Conclusions PC and PPP were not effective when applied in association with ABG. Similarly, isolated use of PPP was not beneficial in optimizing the bone repair.

Fecal microbiota transplantation via colonoscopy in a dog with Clostridioides (Clostridium) difficile infection / Transplante de microbiota fecal via colonoscopia em um cão com infecção por Clostridioides (Clostridium) difficile

In dogs, antimicrobial therapy for Clostridioides (Clostridium) difficile infection (CDI) is based solely on metronidazole, leaving limited treatment options in case of recurrent disease. Fecal microbiota transplantation (FMT) has been successfully used in humans with recurrent CDI, whereas the usefulness of this approach is largely unknown in dogs. In the present study, a dog with a chronic-recurring diarrhea was treated with FMT via colonoscopy. CDI was confirmed by A/B toxin detection and isolation of toxigenic C. difficile from ribotype 106, a strain also commonly associated with nosocomial infection in humans. The dog recovered well after the procedure and C. difficile was no longer isolated from its stool sample. The present research suggested that FMT could be a useful tool to treat recurrent CDI in dogs, corroborating the actual protocol in humans.(AU)

Em cães, a terapia antimicrobiana para infecções por Clostridioides (Clostridium) difficile é baseada apenas no uso de metronidazol, limitando as opções de tratamento nos casos de recorrência. O transplante de microbiota fecal (FMT) tem sido utilizado com sucesso em seres humanos com infecções recorrentes por C. difficile, porém a utilidade desse método é ainda amplamente desconhecida em cães. O presente trabalho relata a utilização de FMT para o tratamento de um cão com diarreia crônica-recorrente por C. difficile. A infecção foi confirmada por detecção das toxinas A/B e isolamento de uma estirpe toxigênica do ribotipo 106, linhagem comumente associada a infecção em seres humanos. Após o transplante via colonoscopia, o animal se recuperou do quadro e C. difficile não mais foi encontrado em novas amostras fecais. O presente trabalho sugere que o FMT possa ser útil para o tratamento de quadros de C. difficile em cães, corroborando protocolo atual de tratamento em seres humanos.(AU)

Future strategies to improve short- and long-term outcomes of renal transplantation in dogs / Estratégias futuras para melhorar os resultados de transplante renal em cães a curto e longo prazo

Transplants for cats with naturally occurring renal disease have been introduced into clinical practice, but canine renal transplantation represents a greater challenge because of the lack of a balanced immunosuppressive protocol, difficulty in selecting compatible canine kidney donors, and absence of transplantation monitoring protocols. This and other important factors will be discussed in this review to help improve short- and long-term outcomes for renal transplantation in dogs.(AU)

O transplante renal em gatos com doença renal naturalmente adquirida está cada vez mais sendo introduzido na prática clínica. O transplante renal em cães, por sua vez, representa um desafio maior devido a falta da definição de um protocolo imunossupressor equilibrado, dificuldade na seleção de doadores compatíveis e ausência de protocolos de monitoramento de transplantes. Esses e outros fatores serão abordados nesta revisão afim de melhorar os resultados, tanto a curto quanto a longo prazo do transplante renal em cães.(AU)

Avaliação da eficácia do sistema rigeneracon no tratamento de lesões de calvária em ratos / [Evaluation of the efficacy of the rigeneracon system in the treatment of calvary injuries in rats]

Defeitos ósseos constituem um problema de saúde global. O sistema Rigenera permite a extração de microenxertos ricos em células-tronco mesenquimais (CTMs). Objetivou-se avaliar o processo de regeneração óssea por enxertos obtidos pelo sistema Rigenera em defeitos críticos na calvária de ratos. Foram utilizados 18 ratos Wistar, machos, pesando 285±29g, distribuídos em três grupos (n=6), sendo cada animal controle de si mesmo, denominados G15-Controle e G15-Tratado (15 dias); G30-Controle e G30-Tratado (30 dias) e G60-Controle e G60-Tratado (60 dias). Foram realizadas duas lesões de 5mm de diâmetro em cada antímero da calvária. Nos grupos tratados, foram utilizados microenxertos autólogos de cartilagem xifoide, obtidos pelo sistema Rigenera. O defeito contralateral serviu como controle em todos os animais. Os animais foram eutanasiados aos 15, 30 e 60 dias após a cirurgia, e as amostras foram processadas para a histoquímica. Nos grupos controle, não foram observados sinais de regeneração óssea, enquanto nos grupos tratamento foram verificadas áreas de formação óssea e tecido mesenquimal ativado. O sistema Rigenera foi eficiente na obtenção de microenxertos autólogos, para terapia celular em defeito crítico de calvária de ratos. Com o aprimoramento do protocolo, o sistema Rigenera poderá ser amplamente utilizado no tratamento de lesões ósseas.(AU)

Bone defects are a global health problem. The Rigenera system allows the extraction of micro grafts rich in mesenchymal stem cells (MSCs). The objective of this study was to evaluate the bone regeneration process by grafts obtained by the Rigenera system in defects in the rats calvarian. Eighteen male Wistar rats were used, weighing 285 ± 29g, distributed in three groups (n = 6), where each animal was treatment and control, called G15-Control and G15-Treated (15 days); G30-Control and G30-Treated (30 days) and G60-Control and G60-Treated (60 days). Two 5mm diameter lesions were performed on each calvaria side. In the treated groups, autologous micrograft from xiphoid cartilage, obtained by the Rigenera system, were used. The other defect served as a control in all animals. The animals were euthanized at 15, 30 and 60 days after the surgery and the samples were processed for histochemistry. In the control groups, no signs of bone regeneration were observed, while in the treatment groups, areas of bone formation and activated mesenchymal tissue were verified. The Rigenera system was efficient in obtaining autologous micrograft for cell therapy in a critical calvaria defect in rats. Rigenera system can be widely used in the treatment of bone injuries.(AU)

P-I metalloproteinases and L-amino acid oxidases from Bothrops species inhibit angiogenesis

Snake venoms are composed of pharmacologically active proteins that are evolutionarily diverse, stable and specific to targets. Hence, venoms have been explored as a source of bioactive molecules in treating numerous diseases. Recent evidences suggest that snake venom proteins may affect the formation of new blood vessels. Excessive angiogenesis has been implicated in several pathologies including tumours, diabetic retinopathy, arthritis, inter alia. In the present study, we have examined the effects of P-I metalloproteinases isolated from Bothrops moojeni (BmMP-1) and Bothrops atrox (BaMP-1) and L-amino acid oxidases (LAAO) isolated from B. moojeni (BmLAAO) and B. atrox (BaLAAO) on biochemical and functional aspects of angiogenesis. Methods: P-I metalloproteinases and LAAO were purified from venom by molecular size exclusion and ion-exchange chromatography and subsequently confirmed using mass spectrometry. The P-I metalloproteinases were characterized by azocaseinolytic, fibrinogenolytic and gelatinase activity and LAAO activity was assessed by enzyme activity on L-amino acids. Influence of these proteins on apoptosis and cell cycle in endothelial cells was analysed by flow cytometry. The angiogenic activity was determined by in vitro 3D spheroid assay, Matrigel tube forming assay, and in vivo agarose plug transformation in mice. Results: P-I metalloproteinases exhibited azocaseinolytic activity, cleaved α and partially β chain of fibrinogen, and displayed catalytic activity on gelatin. LAAO showed differential activity on L-amino acids. Flow cytometry analysis indicated that both P-I metalloproteinases and LAAO arrested the cells in G0/G1 phase and further induced both necrosis and apoptosis in endothelial cells. In vitro, P-I metalloproteinases and LAAO exhibited significant anti-angiogenic properties in 3D spheroid and Matrigel models by reducing sprout outgrowth and tube formation. Using agarose plug transplants in mice harbouring P-I metalloproteinases and LAAO we demonstrated a marked disruption of vasculature at the periphery. Conclusion: Our research suggests that P-I metalloproteinases and LAAO exhibit anti-angiogenic properties in vitro and in vivo.(AU)

P-I metalloproteinases and L-amino acid oxidases from Bothrops species inhibit angiogenesis

Snake venoms are composed of pharmacologically active proteins that are evolutionarily diverse, stable and specific to targets. Hence, venoms have been explored as a source of bioactive molecules in treating numerous diseases. Recent evidences suggest that snake venom proteins may affect the formation of new blood vessels. Excessive angiogenesis has been implicated in several pathologies including tumours, diabetic retinopathy, arthritis, inter alia. In the present study, we have examined the effects of P-I metalloproteinases isolated from Bothrops moojeni (BmMP-1) and Bothrops atrox (BaMP-1) and L-amino acid oxidases (LAAO) isolated from B. moojeni (BmLAAO) and B. atrox (BaLAAO) on biochemical and functional aspects of angiogenesis. Methods: P-I metalloproteinases and LAAO were purified from venom by molecular size exclusion and ion-exchange chromatography and subsequently confirmed using mass spectrometry. The P-I metalloproteinases were characterized by azocaseinolytic, fibrinogenolytic and gelatinase activity and LAAO activity was assessed by enzyme activity on L-amino acids. Influence of these proteins on apoptosis and cell cycle in endothelial cells was analysed by flow cytometry. The angiogenic activity was determined by in vitro 3D spheroid assay, Matrigel tube forming assay, and in vivo agarose plug transformation in mice. Results: P-I metalloproteinases exhibited azocaseinolytic activity, cleaved α and partially β chain of fibrinogen, and displayed catalytic activity on gelatin. LAAO showed differential activity on L-amino acids. Flow cytometry analysis indicated that both P-I metalloproteinases and LAAO arrested the cells in G0/G1 phase and further induced both necrosis and apoptosis in endothelial cells. In vitro, P-I metalloproteinases and LAAO exhibited significant anti-angiogenic properties in 3D spheroid and Matrigel models by reducing sprout outgrowth and tube formation. Using agarose plug transplants in mice harbouring P-I metalloproteinases and LAAO we demonstrated a marked disruption of vasculature at the periphery. Conclusion: Our research suggests that P-I metalloproteinases and LAAO exhibit anti-angiogenic properties in vitro and in vivo.(AU)

Behavior of free omental grafts without vascular anastomosis implanted in cats experimental wounds / Comportamento do enxerto omental livre sem anastomose vascular implantado em feridas experimentais de gatos

It is known that skin healing in cats is slower and has a higher complication rate than in dogs, what leads to search for new resources to enhance this process. Omentum properties in the aid of tissue repair are known and, among the forms of extraperitoneal omentalization, the use of free omental grafts without vascular anastomosis is advantageous, but little studied; there is no knowledge about its effect on skin healing, nor if its still viable when implanted in the subcutaneous space. Thus, the present study aimed to describe possible alterations produced by free omental graft without anastomosis inserted in the subcutaneous space, by means of macroscopic clinical analysis of the implantation region in cats. Twenty intact female cats were divided into two groups (Group 1: treated animals; Group 2: controls) of similar size. All animals were subjected to ovariosalpingohysterectomy (OSH) prior to the induction of an experimental wound for the implantation of the omental graft. Wounds were evaluated on days 1, 2, 4, 8, and 15 postsurgery and then weekly until no lesions were noted. Data regarding color, consistency, presence or absence of crusts, wound resistance, and volume measurements were recorded. There was activation of the omentum on the 4th day of evaluation with reduction of hemorrhage and an increase in the resistance to traction of the experimental wound. We concluded that the omentum without vascular anastomosis is capable of maintaining its viability and exert positive influence on the repair process without showing deleterious signs on the implantation site.(AU)

Sabe-se que a cicatrização de pele em gatos é mais lenta e apresenta índice maior de complicações que nos cães, dai a importância da busca de novos recursos para estimular o processo cicatricial. São conhecidas as propriedades do omento no auxílio ao reparo tecidual e, dentre as formas de omentalização extraperitoneal, o uso do omento livre sem anastomose vascular é vantajoso, porém pouco estudado; não há conhecimento sobre seu efeito na cicatrização cutânea, tampouco se permanece viável quando implantado no espaço subcutâneo. Sendo assim, o presente trabalho tem como objetivo avaliar possíveis alterações produzidas pelo enxerto omental livre sem anastomose inserido no espaço subcutâneo, por meio da avaliação clínica macroscópica da região de implantação em gatos. Foram utilizadas 20 gatas, de idade entre um e sete anos, sem alterações em exame de sangue, FIV/FELV negativas. Os animais foram divididos em dois grupos (Tratado e Controle) de igual tamanho, diferindo quanto à utilização do enxerto de omento livre. Foi realizada cirurgia de ovariossalpingohisterectomia (OSH) em todos os animais, previamente à criação da ferida experimental para implantação ou não do enxerto omental. As feridas foram avaliadas nos dias 1, 2, 4, 8 e 15 do pós-operatório e a partir de então semanalmente, até nenhuma alteração ser observada. Foram anotados dados referentes à coloração, consistência, presença de crosta, resistência da ferida e medidas do volume. Nos resultados destacou-se a ativação do omento no quarto dia de avaliação, observada pelo aumento de volume, com redução de hemorragia e aumento na resistência da ferida experimental à tração. Conclui-se que o omento em sua forma livre sem anastomose é capaz de manter sua viabilidade e exercer influência positiva sobre o processo de reparo, sem demonstrar sinais deletérios sobre a região implantada.(AU)

Mechanical properties of the sciatic nerve following combined transplantation of analytically extracted acellular allogeneic nerve and adipose-derived mesenchymal stem cells

Purpose To investigate the effects of Chemically Extracted Acellular Nerves (CEANs) when combined with Adipose-Derived mesenchymal Stem Cell (ADSC) transplantation on the repair of sciatic nerve defects in rabbits. Methods A total of 71 six-month-old Japanese rabbit were used in this study. Twenty rabbits served as sciatic nerve donors, while the other 51 rabbits were randomly divided into Autologous Nerve Transplantation Group (ANT, n=17), CEAN group (n=17) and CEAN-ADSCs group (n=17). In all these groups, the rabbits left sciatic nerves were injured before the experiment, and the uninjured sciatic nerves on their right side were used as the control (CON). Electrophysiological tests were carried out and sciatic nerves were prepared for histomorphology and stretch testing at 24 weeks post-transplant. Results There were significant differences between ANT and Con groups in amplitude (AMP): P=0.031; motor nerve conduction velocity (MNCV): P=0.029; Maximum stress: P=0.029; and Maximum strain P=0.027. There were also differences between the CEAN and CEAN+ADSCs groups in AMP: P=0.026, MNCV: P=0.024; Maximum stress: P=0.025 and Maximum strain: P=0.030. No significant differences in these parameters were observed when comparing the ANT and CEAN+SACN groups (MNCV: P=0.071) or the CEAN and ANT groups (Maximum stress: P=0.069; Maximum strain P=0.077). Conclusion Addition of ADSCs has a significant impact on the recovery of nerve function, morphology, and tensile mechanical properties following sciatic nerve injury.(AU)

Effects of canine adipose-derived mesenchymal stem cells on the epithelialization of rabbits' skin autograft (Oryctolagus cuniculus) / Efeitos das células-tronco mesenquimais derivadas do tecido adiposo de cães na epitelização de autoenxertos de pele em coelhos (Oryctolagus cuniculus)

The present study aimed to evaluate the effects of mesenchymal stem cells derived from canine adipose tissue in the healing process of full-thickness mesh skin grafts in rabbits. The stem cells were collected from young dogs; and, after characterization, remained in cryopreservation, in independent doses containing 2 x 106 cells. The mesh distal limb graft technique was performed in 60 rabbits, divided into three groups, CG (Control Group), GT1 (Intralesional Stem Cell Treated Group), and GT2 (Intravenous Stem Cell Treated Group), containing 20 animals each. After grafting, each group was randomly divided into four subgroups according to euthanasia time 3, 7, 14, and 30 days, containing five animals in each group. Animals of GT1_14, GT1_30, and GT2_14, GT2_30 subgroups received a second dose of xenogeneic cells on the seventh day. Meanwhile, animals from GT1_30 and GT2_30 received the third dose of xenogeneic cells on day 14. The groups treated with xenogeneic stem cells positively affected type III collagen re-epithelialization and deposition, and possibly GT1 had a controlled inflammatory response. However, no effect on angiogenesis. Thus, it was possible to demonstrate tolerance and therapeutic action of mesenchymal stem cells from canine adipose tissue in skin grafts in rabbits.(AU)

O presente estudo teve como principal objetivo avaliar os efeitos das células-tronco mesenquimais derivadas do tecido adiposo de cães no processo de cicatrização de autoenxertos de pele de espessura total em malha em coelhos. As células-tronco foram coletadas de cães jovens, após a caracterização estas permaneceram em criopreservação, em doses individuais contendo 2 x 106 células. A técnica de enxerto em malha na região distal do membro foi realizada em 60 coelhos, divididos em três grupos, GC (Grupo Controle), GT1 (Grupo tratado com células-tronco intralesional) e GT2 (Grupo tratado com células-tronco via endovenosa), contendo 20 animais cada. Imediatamente após a enxertia, cada grupo foi dividido aleatoriamente em quatro subgrupos, de acordo com o tempo de eutanásia 3, 7, 14 e 30 dias contendo cinco animais cada. Animais dos subgrupos GT1_14, GT1_30 e GT2_14, GT2_30 receberam uma segunda dose de células xenógenas no sétimo dia. Ademais, animais do GT1_30 e do GT2_30 receberam a terceira dose de células xenógenas no dia 14. Os grupos tratados com células-tronco xenógenas tiveram um efeito positivo na reepitelização e deposição de colágeno tipo III, e possivelmente, o GT1 teve uma resposta inflamatória controlada, entretanto o efeito na angiogênese não foi observado. Dessa forma, foi possível demonstrar que houve tolerância e ação terapêutica das células-tronco mesenquimais derivadas do tecido adiposo de cães em enxertos de pele em coelhos.(AU)

Effects of canine adipose-derived mesenchymal stem cells on the epithelialization of rabbits' skin autograft (Oryctolagus cuniculus) / Efeitos das células-tronco mesenquimais derivadas do tecido adiposo de cães na epitelização de autoenxertos de pele em coelhos (Oryctolagus cuniculus)

The present study aimed to evaluate the effects of mesenchymal stem cells derived from canine adipose tissue in the healing process of full-thickness mesh skin grafts in rabbits. The stem cells were collected from young dogs; and, after characterization, remained in cryopreservation, in independent doses containing 2 x 106 cells. The mesh distal limb graft technique was performed in 60 rabbits, divided into three groups, CG (Control Group), GT1 (Intralesional Stem Cell Treated Group), and GT2 (Intravenous Stem Cell Treated Group), containing 20 animals each. After grafting, each group was randomly divided into four subgroups according to euthanasia time 3, 7, 14, and 30 days, containing five animals in each group. Animals of GT1_14, GT1_30, and GT2_14, GT2_30 subgroups received a second dose of xenogeneic cells on the seventh day. Meanwhile, animals from GT1_30 and GT2_30 received the third dose of xenogeneic cells on day 14. The groups treated with xenogeneic stem cells positively affected type III collagen re-epithelialization and deposition, and possibly GT1 had a controlled inflammatory response. However, no effect on angiogenesis. Thus, it was possible to demonstrate tolerance and therapeutic action of mesenchymal stem cells from canine adipose tissue in skin grafts in rabbits.(AU)

O presente estudo teve como principal objetivo avaliar os efeitos das células-tronco mesenquimais derivadas do tecido adiposo de cães no processo de cicatrização de autoenxertos de pele de espessura total em malha em coelhos. As células-tronco foram coletadas de cães jovens, após a caracterização estas permaneceram em criopreservação, em doses individuais contendo 2 x 106 células. A técnica de enxerto em malha na região distal do membro foi realizada em 60 coelhos, divididos em três grupos, GC (Grupo Controle), GT1 (Grupo tratado com células-tronco intralesional) e GT2 (Grupo tratado com células-tronco via endovenosa), contendo 20 animais cada. Imediatamente após a enxertia, cada grupo foi dividido aleatoriamente em quatro subgrupos, de acordo com o tempo de eutanásia 3, 7, 14 e 30 dias contendo cinco animais cada. Animais dos subgrupos GT1_14, GT1_30 e GT2_14, GT2_30 receberam uma segunda dose de células xenógenas no sétimo dia. Ademais, animais do GT1_30 e do GT2_30 receberam a terceira dose de células xenógenas no dia 14. Os grupos tratados com células-tronco xenógenas tiveram um efeito positivo na reepitelização e deposição de colágeno tipo III, e possivelmente, o GT1 teve uma resposta inflamatória controlada, entretanto o efeito na angiogênese não foi observado. Dessa forma, foi possível demonstrar que houve tolerância e ação terapêutica das células-tronco mesenquimais derivadas do tecido adiposo de cães em enxertos de pele em coelhos.(AU)