Treatment of chronic immunomediated thrombocytopenia with mesenquimal stem cells in dog: case report / Tratamento de trombocitopenia imunomediada crônica com células-tronco mesenquimais em cão: relato de caso

Immunomediated thrombocytopenia is a systemic metabolic disorder in which the platelet count falls below reference values, as the patient's immune system destroys them. The main clinical signs in thrombocytopenia are petechial, hemorrhages, ecchymoses and suffusions. Hematomas can also occur in coagulation disorders. The diagnosis is based on clinical findings and hematological examinations. The treatment consists of the use of corticosteroids and immunosuppressants, delaying cell destruction, and may last for months, not always obtaining a cure for the disease. The present work reports the use of therapy with allogeneic mesenchymal stem cells, derived from the adipose tissue of dogs, for the treatment of chronic immunomediated thrombocytopenia, with an evolution of more than one year, in a Pinscher dog. The alternative treatment showed a good evolution, keeping platelets within the reference values during the treatment, giving the patient quality of life and removing the need for continuous medication for homeostasis after treatment.

A trombocitopenia imunomediada é uma desordem metabólica sistêmica, na qual a contagem plaquetária fica abaixo dos valores de referência, pois o sistema imunológico do paciente a destrói. O principal sinal clínico na trombocitopenia são hemorragias, petequiais, equimoses e sufusões. Hematomas podem ocorrer também em alterações da coagulação. O diagnóstico baseia-se nos achados clínicos e nos exames hematológicos. O tratamento consiste na utilização de corticosteroides e imunossupressores, o que retarda a destruição celular, mas pode se prolongar por meses, nem sempre obtendo cura da doença. O presente trabalho relata a utilização da terapia com células-tronco mesenquimais alogênicas, oriundas do tecido adiposo de cães, para tratamento de trombocitopenia imunomediada crônica, com evolução de mais de um ano, em um cão da raça Pinscher. O tratamento alternativo revelou boa evolução, pois manteve as plaquetas dentro dos valores de referência durante o tratamento, o que proporcionou qualidade de vida ao paciente e tornou desnecessárias medicações de uso contínuo para a homeostase após o tratamento.

Cell therapy with adipose tissue-derived human stem cells in the urinary bladder improves detrusor contractility and reduces voiding residue / A terapia celular com células-tronco humanas derivadas do tecido adiposo na bexiga urinária melhora a contratilidade do detrusor e reduz o resíduo miccional

Detrusor hypocontractility (DH) is a disease without a gold standard treatment in traditional medicine. Therefore, there is a need to develop innovative therapies. The present report presents the case of a patient with DH who was transplanted with 2 x 106 adipose tissue-derived mesenchymal stem cells twice and achieved significant improvements in their quality of life. The results showed that cell therapy reduced the voiding residue from 1,800 mL to 800 mL, the maximum cystometric capacity from 800 to 550 mL, and bladder compliance from 77 to 36.6 mL/cmH2O. Cell therapy also increased the maximum flow from 3 to 11 mL/s, the detrusor pressure from 08 to 35 cmH2O, the urine volume from 267 to 524 mL and the bladder contractility index (BCI) value from 23 to 90. The International Continence on Incontinence Questionnaire - Short Form score decreased from 17 to 8. Given the above, it is inferred that the transplantation of adipose tissue-derived mesenchymal stem cells is an innovative and efficient therapeutic strategy for DH treatment and improves the quality of life of patients affected by this disease.

A Hipocontratilidade Detrusora (HD) é uma doença sem um tratamento padrão-ouro na medicina tradicional. Logo, há a necessidade de desenvolvimento de terapias inovadoras. O presente relato apresenta um caso de paciente com HD transplantado duas vezes com 2 x 106 células-tronco mesenquimais derivadas do tecido adiposo que obteve melhoras significativas em sua qualidade de vida. Os resultados demonstraram que a terapia celular reduziu o resíduo miccional de 1.800mL para 800mL; a Capacidade Cistométrica Máxima de 800 para 550mL; a complacência de 77 para 36,6mL/cmH2O. A terapia celular também aumentou o fluxo máximo de 3 para 11mL/s; a pressão detrusora de 08 para 35cmH2O; o volume urinado de 267 para 524mL e o índice de contratilidade vesical (BCI) de 23 para 90. O score do International Continence on Incontinence Questionare - Short Form passou de 17 para 8. Diante do exposto, infere-se que o transplante de células-tronco mesenquimais derivadas do tecido adiposo é uma estratégia terapêutica inovadora e eficiente para o tratamento da HD e para melhoria da qualidade de vida de pacientes acometidos por essa doença.

Comparison of three different strategies to treat sciatic nerve regeneration: an experimental study

Purpose: To compare the effect of vein conduit filled with adipose tissue stem cells (ASC) on peripheral nerve injury regeneration. Methods: We analyzed 30 male Wistar rats surgically submitted to a 5-mm gap on the sciatic nerve. Then, the animals were divided into three groups: nerve autografting (AG, n=10), autogenous inverted glycerol-conserved vein (VG, n=10), and autogenous inverted glycerol-conserved vein + ASC (VASCG, n=10). The study endpoints were neuromotor functional analysis, gastrocnemius muscle weight, and sciatic nerve graft histomorphometry analysis. In the histologic analysis, we added a control group (naïve nerve). Results: Regarding functional analysis (Walking tract- score), the findings at week 3 showed a difference between the AG and the VG (-96.6 vs. -59.6, p=0.01, respectively) and between the VG and the inverted vein + VASCG (-59.9 vs. -88.92, p=0.02). At week 12, this study showed a difference between the AG and the VG (-64.8 vs. -47.3, p=0.004, respectively), and also a difference between the VG and the VASCG (-47.3 vs. -57.4, p=0.02, respectively). There was no difference in the histomorphometry analysis (nerve diameter, Schwann cells counting). The gastrocnemius muscles on the intervention side were more atrophic when compared to the gastrocnemius muscles on the control side. Conclusions: Our results suggested better functional recovery in the inverted vein group when compared to control group, and inverted vein + ASC group.

Mesenchymal stem cells have ameliorative effect on the colitis model via Nrf2/HO-1 pathway

Purpose: To evaluate the ameliorative effect of mesenchymal stem cells (MSCs) on acetic acid colitis model via Nrf2/HO-1 pathway in rats. Methods: In this study, 30 rats were divided into three groups. Acute colitis was induced by rectal administration of 4% solution of acetic acid. MSCs were injected intraperitoneally in the treatment group. Results: Increased levels of tumor necrosis factor-α (TNF-α), pentraxin-3, and malondialdehyde (MDA) in colitis group were revealed biochemically. Increased level of TNF-α and decreased levels of Nrf2 and interleukin-10 (IL-10) were observed in rectum tissues. Increased fibrous tissue proliferation, vascularization and inflammatory cell infiltration were described in the colitis group. Significant improvement was observed in MSCs treated group histopathologically. Increased immunopositivity of TNF-α, vascular endothelial growth factor (VEGF) and CD68 markers was observed in the colitis group cells, and decreased level of this positivity was observed in MSCs treated group. Conclusions: Biochemical, histopathological and immunohistochemical results strongly support the ameliorative effect of MSCs against acetic induced colitis model via Nrf2/HO-1 pathway in rats.

Evaluation of mesenchymal cells and dapsone for the treatment of dermonecrotic wounds caused by Loxosceles laeta venom in rabbits / Avaliação de células mesenquimais e dapsona para o tratamento de feridas dermonecróticas causadas pelo veneno de Loxosceles laeta em coelhos

This study aimed to evaluate the efficacy of mesenchymal stem cells (MSC), alone or associated with dapsone (DAP), in treating dermonecrotic wounds caused by Loxosceles laeta venom. Twenty-five male rabbits were distributed into five groups. Negative control received ultrapure water (C-), whilst all other groups were injected with 20 μg of L. laeta venom. After 4 hours, each group received one of the following treatments: PBS (C+), DAP, MSC, and DAP+MSC. Animals were evaluated daily and photographic records made for analysis of wound area. Twelve days after, animals were euthanized and skin samples removed for histological analysis. We observed that DAP showed the best percentage of wound contraction at day 3. In the treatments using MSCs, a negative value of wound contraction was observed for the isolated MSCs, as well as a lower contraction value for the association of the MSC + DAP when compared to PBS, probably, by the increase in initial infammation after the application of stem cells, due to the fact that MSCs secrete a broad spectrum of bioactive molecules such as cytokines and growth factors that favor regeneration. Histologically, it was observed that animals of C+ showed extensive areas of necrosis, ulcers, neutrophilic infiltrate, and mineralization. Collagen deposition showed increase in MSC+DAP treatment, however vascularization remained unchanged. This is the first report using MSC and MSC+DAP as a treatment for cutaneous loxoscelism and more studies are needed to determine its use as an alternative therapy for dermonecrotic lesions caused by Loxosceles spider.

Este estudo teve como objetivo avaliar a eficácia das células-tronco mesenquimais (CTMs), isoladas ou associadas à dapsona (DAP), no tratamento de feridas dermonecróticas causadas pelo veneno de Loxosceles laeta. Vinte e cinco coelhos machos foram distribuídos em cinco grupos. O controle negativo recebeu água ultrapura (C-), enquanto todos os outros grupos foram injetados com 20 μg de veneno de L. laeta. Após 4 horas, cada grupo recebeu um dos seguintes tratamentos: PBS (C+), DAP, CTMs e DAP + CTMs. Os animais foram avaliados diariamente durante 12 dias, e feitos registros fotográficos para análise da ferida e no 12º dia, foram eutanasiados e, retiradas amostras de pele para análise histológica. Observou-se que a DAP apresentou o melhor percentual de contração da ferida no terceiro dia. Nos tratamentos com CTMs, observou-se uma contração negativa da ferida tanto para as CTMs isoladas, bem como a associação CTMs + DAP em relação ao PBS, possivelmente, pelo aumento da infamação inicial após a aplicação de células-tronco. Isso é devido ao fato de que as CTMs secretam um amplo espectro de moléculas bioativas como citocinas e fatores de crescimento que favorecem a regeneração. Histologicamente, observou-se que os animais de C+ apresentaram extensas áreas de necrose, úlceras, infiltrado neutrofílico, além de mineralização. Houve aumento de deposição de colágeno no tratamento CTMs + DAP, no entanto, a vascularização permaneceu inalterada. Este é o primeiro relato usando CTMs e CTMs + DAP como tratamento para loxoscelismo cutâneo e mais estudos são necessários para determinar seu uso como terapia alternativa para lesões demonecróticas causadas pela aranha Loxosceles.

Quantification of adhesion of mesenchymal stem cells spread on decellularized vein scaffold

ABSTRACT Purpose: To evaluate methods that improve adipose-derived stem cells (ASCs) population in decellularized biological venous scaffold for tissue engineering in blood vessels, a model in rabbits. Methods: The ASC was expanded until the third passage. Inferior vena cava (IVC) was submitted to the decellularization process using 1% sodium dodecyl sulfate (SDS) or 2% sodium deoxycholate (SD) to compose 12 study groups (G): pure SD or SDS, exposed or not to 1% TritonX-100 (TX-100) and exposed or not to poly-l'lysine and laminin (PL). Scaffolds were covered with 1 × 105 or 1 × 106 ASCs diluted in 10 μL Puramatrix™. The histological analysis was done by cell counting in hematoxylin and eosin (HE) and nuclei count in immunofluorescence (IF) with 4',6-Diamidine-2'-phenylindole dihydrochloride (DAPI). Results: The study of groups in HE and IF showed similar results. For both analyses,IVC-SD-1 × 106 ASC and IVC-SD-PL-1 × 106 ASC provided the best results. The IF technique showed better sensitivity than HE, with a weak agreement between them. Conclusions: Decellularizing agent and the number of ASC influence scaffolds cellularization response and the best protocols as those ones using SD with or without the addition of PL.

The influence of adipocyte-derived stem cells (ASCs) on the ischemic epigastric flap survival in diabetic rats

ABSTRACT Purpose: To assess the effects of adipocyte-derived stem cell (ASC)-injection on the survival of surgical flaps under ischemia in diabetic rats. Methods: Diabetes was induced in 30 male Wistar rats using streptozotocin (55 mg/kg). After eight weeks, epigastric flap (EF) surgery was performed. The animals were divided into control (CG), medium-solution (MG), and ASC groups. The outcomes were: the survival area (SA), the survival/total area rate (S/TR), and expression levels (EL) of genes: C5ar1, Icam1, Nos2, Vegf-a. Results: In the ASC group, compared to CG, we observed improved flap SA (CG-420 mm2 vs. ASC-720 mm2; p=0.003) was observed. The S/TR analysis was larger in the ASC group (78%) than the CG (45%). This study showed an increase in the Vegf-a EL in the ASC group (2.3) vs. CG (0.93, p=0.0008). The Nos2 EL increased four-fold in the ASC group compared to CG, and C5ar1 EL decreased almost two-fold in the ASC group vs. the CG (p=0.02). There was no difference among the groups regarding Icam1 EL. Compared to the MG, the ASC group had a bigger flap SA (720 mm2 vs. 301 mm2, respectively), a bigger S/TR (78% vs. 32%, p=0.06, respectively) and increased EL of Vegf-a (2.3 vs. 1.3, respectively). No difference between ASC-group and MG was seen regarding Nos2 (p=0.08) and C5ar1 (p=0.05). Conclusions: This study suggests that ASCs increase the survival of EF under IR in diabetic rats.

Pigeon as a model to study peripheral projections from the horizontal semicircular canal vestibular apparatus to a brainstem target immunoreactive for AMPA

ABSTRACT Purpose: To evaluate whether the pigeon (Columba livia) is a good model for evaluating the vestibular system involved with postural maintenance during movement. Methods: This study maps the brainstem targets of the horizontal ampullary inputs from the vestibular periphery of the pigeon. We used biotin dextran amine (BDA) injection in horizontal semicircular canal (HSCC), immunohistochemistry for GluR2/3 and GluR4 AMPA and computerized histomorphology reconstruction. Results: Our results show the same distribution pattern with ipsilateral projections to vestibular nuclear complex (VNC) from the HSCC, with the majority of labeled fibers being, long, thin, with few varicosities and many ramifications. Horizontal semicircular canal projections achieve neurons belonging to all nuclei of the VNC with exception of dorsal portion of lateral vestibular nucleus and this area express GluR2/3 and GluR4 AMPA receptors reinforcing the idea of glutamate participation in these connections. Conclusions: Pigeon is an appropriated experimental model to study of projections of HSCC and reinforcing the information that the vestibular system has strong relation with the fast responses necessary for postural control. Moreover, its phylogenetic organization apparently conservation, also seems to be a fundamental characteristic for vertebrates.

The role of Rho-associated kinase inhibitor, Y-27632 on primary culture of ovine spermatogonial stem cells

Abstract The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. Rho kinase (ROCK) belongs to a family of serine/threonine kinases and involves in a wide range of fundamental cellular functions. The aim of the present study was to study the effect of ROCK inhibitor, Y-27632 (0.1-40 µM), during the primary culture of ovine SSCs. SSCs were collected from 3-5-month-old's lamb testes. The viability of SSCs, the apoptosis assay of SSCs, the intracellular reactive oxygen species (ROS) analysis, and the SSCs markers and apoptosis-related gene expressions were detected by MTT reduction assay, Annexin V-FITC/ Propidium Iodide (PI) dual staining, flow cytometry and real-time-PCR studies, respectively. Morphological analyses indicated that the 5-10 µM Y-27632 had an optimal effect on the number of presumptive SSCs colonies and the area covered by them after a 10 days culture. The cell viability, apoptosis and necrosis of SSCs after 10 days' culture were not affected in comparison with the control group, and the 20 µM of Y-27632 resulted in significantly decreased cell viability (P<0.05) and an increased necrosis of cells. On day 10 after culture, the expression of P53 was decreased with an increase from 0 to 10 µM in the Y-27632 dose. In the 20 µM Y-27632 group, the expressions of P53 and Bax were higher and the Bcl-2 was lower than other groups and these values were significantly different from 5 and 10 µM Y-27632 groups (P<0.05). The level of intracellular ROS was decreased with an increase in the Y-27632 dose from 5 to 20 µM in comparison with the control group. In conclusion, the present study demonstrated that Y-27632 at a concentration of 5-10 µM provided optimal culture conditions for the primary culture of ovine SSCs.

The role of Rho-associated kinase inhibitor, Y-27632 on primary culture of ovine spermatogonial stem cells

The access to sufficient numbers of spermatogonial stem cells (SSCs) is a prerequisite for the study of their regulation and further biomanipulation. Rho kinase (ROCK) belongs to a family of serine/threonine kinases and involves in a wide range of fundamental cellular functions. The aim of the present study was to study the effect of ROCK inhibitor, Y-27632 (0.1-40 µM), during the primary culture of ovine SSCs. SSCs were collected from 3-5-month-olds lamb testes. The viability of SSCs, the apoptosis assay of SSCs, the intracellular reactive oxygen species (ROS) analysis, and the SSCs markers and apoptosis-related gene expressions were detected by MTT reduction assay, Annexin V–FITC/ Propidium Iodide (PI) dual staining, flow cytometry and real-time-PCR studies, respectively. Morphological analyses indicated that the 5-10 µM Y-27632 had an optimal effect on the number of presumptive SSCs colonies and the area covered by them after a 10 days culture. The cell viability, apoptosis and necrosis of SSCs after 10 days culture were not affected in comparison with the control group, and the 20 µM of Y-27632 resulted in significantly decreased cell viability (P<0.05) and an increased necrosis of cells. On day 10 after culture, the expression of P53 was decreased with an increase from 0 to 10 µM in the Y-27632 dose. In the 20 µM Y-27632 group, the expressions of P53 and Bax were higher and the Bcl-2 was lower than other groups and these values were significantly different from 5 and 10 µM Y-27632 groups (P<0.05). The level of intracellular ROS was decreased with an increase in the Y-27632 dose from 5 to 20 µM in comparison with the control group. In conclusion, the present study demonstrated that Y-27632 at a concentration of 5-10 µM provided optimal culture conditions for the primary culture of ovine SSCs.(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.

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)

Retorno de cavalos da raça Puro-Sangue Inglês às corridas após tratamento de fraturas slab do osso terceiro carpiano com PRP e células-tronco / Return of Thoroughbred racing horses to the races after treating slab fractures of the third carpal bone with PRP and stem cells

Background: Despite the fact that slab fracture of the third carpal bone is an event of great worldwide relevance in racehorses, the third carpal fracture doesn’t have data on treatments and return to racing in Brazil. The search for efficient treatments and which provide recovery providing horses return to racing is an objective of sports equine medicine. Regenerative therapies like Platelet Rich Plasma (PRP) and Stem Cells (MSc) have demonstrated a great potential in the treatment of several injuries. For the treatment of three Thoroughbred racing horses at Brazilian Jockey Club, with sagittal plane slab fractures of the third carpal bone, we used the association of PRP and Stem Cells for reduction of the rest and good return to conditions athletics of these horses. Cases: Three Thoroughbred racing horses, males, 4 and 5 years old, showed slab fracture third carpal bone in different moments of their activity at Brazilian Jockey Club. Usually, the treatment for this type of fracture is the arthroscopic repair and the conservative management. We performed after initial radiographic evaluation dorsoproximal-dorsodistal oblique (DPr-DDiO) “skyline”, intra-articular applications of PRP and MSCs. The treatments were performed according to the radiographic follow-up of the lesion evolution. No other treatment was performed concomitantly with cell therapy in the 3 treated patients. Three treatments were performed in the first patient and 2 in the second and third patients. Before each treatment for all patients, we performed radiography dorsoproximal-dorsodistal oblique (DPr-DDiO) ‘skyline’ of the carpus. After this step, the animal was sedated with 10% xylazine hydrochloride at a dose of 0.5 mg/kg intravenously. Antisepsis of the carpus was performed and applied with radiographic monitoring of the needle positioning, first the PRP in the volume of 2 mL and in sequence, 20 million cells MSCs suspended in autologous...

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)

In vitro evaluation of hydroxyapatite, chitosan, and carbon nanotube composite biomaterial to support bone healing / Avaliação in vitro do biomaterial compósito à base de hidroxiapatita, quitosana e nanotubo de carbono como adjuvante no reparo ósseo

Hydroxyapatite, chitosan, and carbon nanotube composite biomaterial were developed to improve bone healing. Previous studies suggested that a combination of biomaterials and mesenchymal stem cells (MSCs) can potentially help promote bone regeneration. In the present study, we first developed hydroxyapatite, chitosan, and carbon nanotube composite biomaterial. Then, the effect of different concentrations of the extract on the viability of Vero cells (ATCC CCL-81) and MSCs obtained from sheep bone marrow using methylthiazol tetrazolium (MTT) and propidium iodide (PI) assays were evaluated. The biomaterial group demonstrated an absence of cytotoxicity, similar to the control group. Samples with 50% and 10% biomaterial extract concentrations showed higher cell viability compared to samples from the control group (MTT assay). These results suggest that the presence of this composite biomaterial can be used with MSCs. This study also concluded that hydroxyapatite, chitosan, and carbon nanotube composite biomaterial were not cytotoxic. Therefore, these could be used for performing in vivo tests.(AU)

O compósito à base de hidroxiapatita, quitosana e nanotubo de carbono foi desenvolvido com o intuito de auxiliar na consolidação óssea. Estudos anteriores sugerem que a combinação de substitutos ósseos e células-tronco mesenquimais (CTM) podem auxiliar a potencializar e promover a regeneração óssea. No presente estudo, o biomaterial foi desenvolvido e a viabilidade e a citotoxicidade de células Vero (ATCC CCL-81) e CTM obtidas de medula óssea provenientes de ovinos utilizando ensaios metil-tiazol-tetrazólio, MTT e iodeto de propídeo (PI) foram avaliadas em diferentes concentrações de extrato desse compósito. O compósito demonstrou ausência de citotoxicidade com comportamento semelhante ao grupo controle. Amostras com 50% e 10% de concentração de extrato do compósito mostraram resultados maiores comparados ao grupo controle (ensaio MTT). Esses resultados também sugerem que a presença do biomaterial pode ser utilizada em associação a CTM. Assim, esse estudo conclui que o compósito apresentado de hidroxiapatita, quitosana e nanotubo de cabono não foi considerado citotóxico e pode ser utilizado em teste in vivo.(AU)

In vitro evaluation of hydroxyapatite, chitosan, and carbon nanotube composite biomaterial to support bone healing / Avaliação in vitro do biomaterial compósito à base de hidroxiapatita, quitosana e nanotubo de carbono como adjuvante no reparo ósseo

Hydroxyapatite, chitosan, and carbon nanotube composite biomaterial were developed to improve bone healing. Previous studies suggested that a combination of biomaterials and mesenchymal stem cells (MSCs) can potentially help promote bone regeneration. In the present study, we first developed hydroxyapatite, chitosan, and carbon nanotube composite biomaterial. Then, the effect of different concentrations of the extract on the viability of Vero cells (ATCC CCL-81) and MSCs obtained from sheep bone marrow using methylthiazol tetrazolium (MTT) and propidium iodide (PI) assays were evaluated. The biomaterial group demonstrated an absence of cytotoxicity, similar to the control group. Samples with 50% and 10% biomaterial extract concentrations showed higher cell viability compared to samples from the control group (MTT assay). These results suggest that the presence of this composite biomaterial can be used with MSCs. This study also concluded that hydroxyapatite, chitosan, and carbon nanotube composite biomaterial were not cytotoxic. Therefore, these could be used for performing in vivo tests.(AU)

O compósito à base de hidroxiapatita, quitosana e nanotubo de carbono foi desenvolvido com o intuito de auxiliar na consolidação óssea. Estudos anteriores sugerem que a combinação de substitutos ósseos e células-tronco mesenquimais (CTM) podem auxiliar a potencializar e promover a regeneração óssea. No presente estudo, o biomaterial foi desenvolvido e a viabilidade e a citotoxicidade de células Vero (ATCC CCL-81) e CTM obtidas de medula óssea provenientes de ovinos utilizando ensaios metil-tiazol-tetrazólio, MTT e iodeto de propídeo (PI) foram avaliadas em diferentes concentrações de extrato desse compósito. O compósito demonstrou ausência de citotoxicidade com comportamento semelhante ao grupo controle. Amostras com 50% e 10% de concentração de extrato do compósito mostraram resultados maiores comparados ao grupo controle (ensaio MTT). Esses resultados também sugerem que a presença do biomaterial pode ser utilizada em associação a CTM. Assim, esse estudo conclui que o compósito apresentado de hidroxiapatita, quitosana e nanotubo de cabono não foi considerado citotóxico e pode ser utilizado em teste in vivo.(AU)

Spermatogonial stem cell survival in ram lambs following busulfan treatment

To clarify the effect of busulfan on the depletion of spermatogonial stem cells (SSCs) from shal rams testis, in the first experiment, lambs were treated by intraperitoneal injection with 4 mg/kg busulfan. In the second experiment, different concentrations of busulfan (1, 2 and 4 mg/kg) were injected directly into both sides of the left testis. The testes of 8 lambs were collected by standard castration procedure for histological analysis five weeks after the treatments and the left testis of remaining lambs were collected after eight weeks and a two-time enzymatic digestion process was used to isolate SSCs. The results showed that all rams that had received intraperitoneal injections of busulfan died. But by testicular injecting of same dose of the drug, 40% of the animals died. The testicular injection of rams with 1, 2 and 4 mg/kg of busulfan resulted in a dose dependent decrease in testis size and also spermatocytes population after 5 weeks of treatments. From the results of colony formation 8 weeks after treatment with busulfan, it can be concluded that only in 1 and 2 mg/kg of busulfan, recovery of endogenous germ cells was performed. In conclusion, the results demonstrated that intra-testicular injections of busulfan (2 mg/kg) reduced spermatocytes population in ram testis within 5 weeks of treatments, and this effect was reversible within 8 weeks of injection. However, it was not recommended to inject 4 mg/kg busulfan into the peritoneal cavity or testis of lambs based on its side effects.

Pluripotent stem cells proliferation is associated with placentation in dogs

Pluripotent stem cells have been studied as source of cells for regenerative medicine and acquire or genetic diseases, as an innovative therapy. Most tissues have stem cells populations, however in few quantities or impossible to be used during adult life, which lead to scientists look for new sources. Thus, this study aimed to analyze the presence of pluripotent cells in the uterus and placenta, following up non-pregnant, pregnant (begin, middle, and final), and postpartum periods in dogs. The uteri were obtained from social castration programs for population control in Pirassununga, Sao Paulo, Brazil. It was collected 20 uteri at different stages. The samples were fixed and processed for immunohistochemical analysis of NANOG, OCT4 and SOX2 expression, knowing as pluripotent stem cells makers. Our results showed positive expression for NANOG, OCT4 and SOX2 in all stages of gestation and nonpregnant uterus; however, we highlight some quantitative different between stages. OCT4 showed more expression in non-pregnant uterus than NANOG and SOX2, and its expression increased in pregnant uterus. In pregnant uterus there was more expression of NANOG than OCT4 and SOX2. Interesting, no difference was found between these markers in the other periods. In conclusion, it was possible to identify pluripotent stem cells in all periods in dog placenta and uterus, however during the early stage of pregnancy we observed more pluripotent stem cells than in all the others periods confirming the high plasticity and regeneration capacity of the uterine tissue.

Pluripotent stem cells proliferation is associated with placentation in dogs

Pluripotent stem cells have been studied as source of cells for regenerative medicine and acquire or genetic diseases, as an innovative therapy. Most tissues have stem cells populations, however in few quantities or impossible to be used during adult life, which lead to scientists look for new sources. Thus, this study aimed to analyze the presence of pluripotent cells in the uterus and placenta, following up non-pregnant, pregnant (begin, middle, and final), and postpartum periods in dogs. The uteri were obtained from social castration programs for population control in Pirassununga, Sao Paulo, Brazil. It was collected 20 uteri at different stages. The samples were fixed and processed for immunohistochemical analysis of NANOG, OCT4 and SOX2 expression, knowing as pluripotent stem cells makers. Our results showed positive expression for NANOG, OCT4 and SOX2 in all stages of gestation and nonpregnant uterus; however, we highlight some quantitative different between stages. OCT4 showed more expression in non-pregnant uterus than NANOG and SOX2, and its expression increased in pregnant uterus. In pregnant uterus there was more expression of NANOG than OCT4 and SOX2. Interesting, no difference was found between these markers in the other periods. In conclusion, it was possible to identify pluripotent stem cells in all periods in dog placenta and uterus, however during the early stage of pregnancy we observed more pluripotent stem cells than in all the others periods confirming the high plasticity and regeneration capacity of the uterine tissue.(AU)

Spermatogonial stem cell survival in ram lambs following busulfan treatment

To clarify the effect of busulfan on the depletion of spermatogonial stem cells (SSCs) from shal rams testis, in the first experiment, lambs were treated by intraperitoneal injection with 4 mg/kg busulfan. In the second experiment, different concentrations of busulfan (1, 2 and 4 mg/kg) were injected directly into both sides of the left testis. The testes of 8 lambs were collected by standard castration procedure for histological analysis five weeks after the treatments and the left testis of remaining lambs were collected after eight weeks and a two-time enzymatic digestion process was used to isolate SSCs. The results showed that all rams that had received intraperitoneal injections of busulfan died. But by testicular injecting of same dose of the drug, 40% of the animals died. The testicular injection of rams with 1, 2 and 4 mg/kg of busulfan resulted in a dose dependent decrease in testis size and also spermatocytes population after 5 weeks of treatments. From the results of colony formation 8 weeks after treatment with busulfan, it can be concluded that only in 1 and 2 mg/kg of busulfan, recovery of endogenous germ cells was performed. In conclusion, the results demonstrated that intra-testicular injections of busulfan (2 mg/kg) reduced spermatocytes population in ram testis within 5 weeks of treatments, and this effect was reversible within 8 weeks of injection. However, it was not recommended to inject 4 mg/kg busulfan into the peritoneal cavity or testis of lambs based on its side effects.(AU)