Background: There are few studies on stem cell isolation in wild animals that provide isolation and culture protocols of these cells in vitro. Among the wild species studied, we present the collared peccary (Tayassu tajacu) as a model with potential to obtain and use MSC in preclinical studies. These animals are phylogenetically close to the domestic pig, popularly known as peccaries and found naturally in South America, Central America and the South of the United States. The aim of the present study was to establish a protocol for the isolation, in vitro cell expansion, differentiation and assessment of the stromal MSC growth curve before and after thawing.Materials, Methods & Results: Mesenchymal stem cells (MSC) from collared peccary bone marrow (Tayassu tajacu) were isolated and expanded by centrifuge in Ficoll® solution and cultured in DMEM® High Glucose medium. The culture was assessed by assays of colony forming units CFU-F and growth curve by saturation (GCS). Cultures in the third passage, with 70% confluence, were replicated at 105 cells/mL concentration in the culture media to induce osteogenic cell differentiation and adipogenic cell differentiation, respectively. The MSC were frozen in nitrogen for 40 days, thawed and re-assessed for cell viability and GCS.Discussion: The bone marrow collected presented high mononuclear cellularity, with a mean variability of 94.5% and 60.83 ± 4.27 UFC were identified in the samples and cells with fibroblast-like-cell morphology were observed. When they were expanded, the mean cell viability was 95%, the mean cell concentration obtained was 233.31 ± 20.04 cells per 25cm2 bottle and the culture reached the growth plateau in GCS between the 13th and 16th day. The osteoblastic cell differentiation assay showed after 18 days, morphology similar to osteoblasts, with irregular cytoplasm limits, cell prolongation formation and flattened appearance. After staining with Alizarin Red, the nucleus presented a wine red coloring and the cytoplasm, more basophilic and well-defined, with calcium deposits inside the cells. The cultures submitted to adipogenic differentiation were large, hexagonal, irregular and presented birrefringent cytoplasm granules after the third week of culture. When stained with Oil Red it was observed that the cytoplasm granules were scattered small fat vacuoles and stained maroon. The viability after thawing was 78% and the mean cell concentration obtained in GCS was 199.71 ± 14.72 cells per 25 cm2 bottle. The curves reached the saturation plateau early, on the eighth day of observation. From then onwards the cultures entered became exhausted and the cell concentration of the samples decreased progressively until minimum values. These results showed the presence of a well-defined MSC population in the collared peccary bone marrow with a high rate of replication in vitro and potential for differentiation confirmed by the adipogenic and osteogenic lines. The cryopreservation technique adopted presented satisfactory results, but indicated a significant cell stress after thawing that justifies investigation of the apoptosis rates induced post thawing in the species. Furthermore, the bone marrow collection did not harm the animals and the facility of stromal MSC isolation and culture qualifies the collared peccary as a viable alternative model to obtain MSC and for studies in the area of cell therapy.
Allogeneic mesenchymal stem cells and xenogenic platelet rich plasma, associated or not, in the repair of bone failures in rabbits with secondary osteoporosis¹ 9-Experimental SurgeryActa Cir Bras. 2017;32(9):767-780 AbstractPurpose: To assess the efficacy of allogeneic mesenchymal stem cells and xenogenic platelet rich plasma in the treatment of bone failure of osteoporotic rabbits secondary to estrogenic deprivation and iatrogenic hypercortisolism. Methods: Eight female rabbits underwent ovarian resection and corticoid therapy to induce clinical status of osteoporosis. Four failures were produced in the tibiae, with each failure being treated with hemostatic sponge, allogenic mesenchymal stem cells, xenogenic plateletrich plasma and the association between both. The animals were divided into two groups, evaluated radiographically and histopathologically at 30 and 60 days post treatment. Results: A radiographically confirmed consolidation of bone failures treated with allogeneic mesenchymal stem cells, associated with the histopathological image of mature and immature bone tissue, without evidence of osteopenia, was compared with the other groups, in which radiolucent failures with osteopenia and fibrosis were still present, denoting the satisfactory effect of the first treatment in detriment to the others. Conclusion:The treatment of bone failures of rabbits with secondary osteoporosis with allogeneic mesenchymal stem cells induced greater bone consolidation with mature and immature bone tissue production (p<0.01), when compared to the other treatments.
One herein reports a successful case of celiotomy by plastrotomy for removal of foreign bodies in yellow-footed tortoise (Geochelone denticulata). The animal was treated at the Veterinary Hospital of the Federal University of Piaui, with appetite loss, regurgitation, constipation, lethargy, reluctance to walk and slightly reddish ocular mucous membranes. Radiographic examination was performed, confirming the presence of foreign bodies in the stomach. The tortoise underwent celiotomy by plastrotomy for the removal of the foreign bodies. The opening of the plastron was performed through the abdominal shields, with the aid of a circular mini grinding saw. One performed an incision in the midline between the two abdominal veins to access the abdominal cavity. A gastrotomy for removal of the foreign bodies (nails, toothpicks, stones, pieces of plastic, glass and crockery pieces) was performed after the location of the stomach. The surgery was successful and confirmed with radiographic evaluation in the immediate postoperative period. The celiotomy by plastrotomy for removal of foreign bodies in that animal proved to be a viable, very important and safe technique to the survival of chelonians.
Stem cells derived from adipose tissue (ADSC) have been used in cell therapy as an alternative to treat chronic and degenerative diseases. Using biomedical and image trials to track the cells when infused in the target tissue is essential to control cell migration and adhesion. The objective of the present study was to label and assess the adhesion of goat adipose tissue-derived stem cells (g-ADSC) after cell infusion in animal models by tracking luminescent intracytoplasmatic nanocrystals. The cells were labeled by using Qdots. The g-ADSCs infused with nanocrystal were prepared either fresh or fixed and further visualized under a fluorescence microscope. The labeled cells were infused in the goat mammary glands and mouse testicles and kidneys via tail vein injection. Thirty days after cell infusion, biopsy was carried out for analyses. The g-ADSC cultures were presented with high cellularity and fibroblast morphology, even after infusion of the nanocrystals. It was possible, by processing in paraffin and under fluorescence microscopy, demonstrating the success of the labeling in the long term. Freezing mammary gland biopsies in liquid NO did not alter the quality of labeling with Qdots. Therefore, g-ADSCs can be labeled with intracytoplasmatic nanocrystals (Qdots) enabling their in vitro and ex vivo tracking.
RESUMO.-[Papel das células-tronco mesenquimais autólogas em comparação com plasma rico em plaquetas na cicatrização de feridas cutâneas em camundongos diabéticos.] Lesões cutâneas crônicas afetam 15% dos pacientes diabéticos e humanos representam um risco 15 a 46 vezes maior de amputações de membros em comparação com as pessoas com a glicemia normal. Supõe-se que a metade destas amputações poderia ser evitada por meio do tratamento precoce das feridas cutâneas com, por exemplo, uma adequada terapia celular. Objetivos: Neste estudo, a ação do transplante autólogo de células estaminais mesenquimais (MSC) foi avaliada em comparação com o tratamento com plasma rico em plaquetas autólogo (PRP) na cicatrização de lesões cutâneas induzidas em camundongos diabéticos. Estes animais foram previamente tratados com estreptozotocina para induzir diabetes mellitus e feridas redondas de 1,5 cm de diâmetro foram criadas na região posterior. Os diâmetros dos ferimentos e tempo de cicatrização foram avaliados durante 30 dias e os resultados fo- (7):617-624. Departamento de Clínica e Cirurgia Veterinária, Universidade Federal do Piauí, Campus Socopo, Teresina, PI 64049-550, Brazil. E-mail: firstname.lastname@example.org Chronic cutaneous lesions affect 15% of diabetic human patients and represent a risk 15 to 46 times larger of limb amputations compared to people with normal glycemia. It is assumed that half of these amputations could be prevented by early treatment of wounds, for example, with proper cell therapy. Objectives: In this study, the action of the autologous transplant of mesenchymal stem-cells (MSC) was evaluated compared to the treatment with autologous platelet rich plasma (PRP) in the cicatrization of cutaneous lesions induced in diabetic mice. These animals were previously treated with streptozootocin to induce diabetes mellitus and round wounds of 1.5cm in diameter were created in the posterior region. Diameters of the wounds and healing time were evaluated during 30 days and the results were submitted to variance analysis and Tukey's test average. It was noticed that the animals treated with MSC presented a more accelerated cicatrization of the cutaneous lesion than the animals treated with PRP. However, the treatment with PRP presented better results than just the daily asepsis of the lesions with saline or covering them with semi-permeable bandage. Besides, the use of semi-permeable bandage kept the cutaneous lesions of diabetic mice did not interfere negatively with cicatrization, proved to be harmless to use, but kept the cutaneous lesions more hydrated than the ones exposed to the environment.
Background: Osteochondral knee failures are among the most common causes of disability among the elderly human population and animal athletes. The xenogeneic transplantation of mesenchymal stem cells is a questionable therapeutic alternative that, despite the low expression of Major Histocompatibility Complex type II by these cells, still has relevantuncertainties about the safety and clinical efficacy. The main objective of the present study was to investigate whether the xenogeneic transplantation of mesenchymal stem cells induces hyaline cartilage formation, without histopathological evidence of rejection, in osteochondralfailures of goats.Materials, Methods & Results: Five female goats were used, submitted to three surgical osteocondral failures in the right knee, treated with xenogenic mesenchymal stem cells of dental pulp, xenogenic platelet-rich plasma and hemostatic sponge of hydrolyzed collagen, respectively. The lesions were evaluated after 60 days of treatment, aiming to identify thepresence of hyaline cartilage or fibrocartilage and the subchondral bone pattern (regenerated or disorganized). Transplantation of xenogenic mesenchymal stem cells induced predominant formation of hyaline cartilage (P < 0.05), with no histopathological evidence of inflammationwhen compared to the other treatments. Therapies with xenogeneic platelet-rich plasma and hemostatic sponge of hydrolyzed collagen induced greater formation of fibrocartilaginous cartilage, with no significant difference between them (P > 0.05). Macroscopically, the lesions of the stem cell treated group showed formation of firm repair tissue, opaque staining, integrated with adjacent cartilage and with the failure filling almost completely. The groups treated with PRP and hemostatic sponge of hydrolyzed collagen presented, on average, partial filling of the lesion, with irregular shape and darkened coloration.Discussion. The absence of macroscopic and histopathological evidences of an inflammatory process on the surface and in the internal portion of the osteochondral lesions treated with xenogeneic stem cells, probably due to the low expression of Major Histocompatibility Complex type II by these cells, which would theoretically induce low rejection response. Such observations are of great importance, since graft-versus- host disease syndrome is a serious condition, responsible for the low therapeutic efficacy with transplantation of cells or grafts in humans. The formation of fibrocartilage, although without macro and microscopic evidence of degeneration or necrosis, in the osteochondral failures treated with PRP and hemostatic collagen sponge suggest that paracrine factors of the local microenvironment of the osteochondral failure are possibly responsible for the formation of fibrocartilaginous tissue or by inhibition of normal cartilage formation. The fibrocartilage formed in the Plasmaand Control groups, contributed to the commitment in the filling of the lesion, contrasting with the almost complete fill of the lesions treated with stem cells. The xenotransplantation of mesenchymal stem cells induced formation of hyaline cartilage and did not promote histopathological evidence of rejection in osteochondral lesions of goat knees. The treatments with PRP and hemostatic sponge of hydrolyzed collagen induced greater formation of fibrocartilaginous cartilaginous surface in the osteochondral failures.
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