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PURPOSE: This study assessed the regeneration potential of mesenchymal stem cells (MSC) from adipose tissue associated with platelet-rich plasma (PRP) in bone regeneration. METHODS: Thirty Wistar rats (Rattus norvegicus albinos) were divided into five groups (according to the grafting material and time to euthanasia): (1) autograft - 14 days (control), (2) autograft - 28 days (control), (3) MSC + PRP - 14 days, (4) MSC + PRP + papaverine - 14 days and (5) MSC + PRP + papaverine - 28 days. After euthanasia, the graft was removed and histological slides were prepared. They were assessed by a blinded pathologist using a previously published histological scale as parameter. RESULTS: There was some degree of neoformed bone trabeculae (NBT) in 93.3% of the samples, as well as osteoblastic activity (OA). The autograft groups (14 and 28 days) had higher levels in the formation of bone trabeculae. Nonparametric data were analyzed using the Wilcoxon-Mann-Whitney test and proved not to be statistically significant at p < 0.05. CONCLUSIONS: Experimental parietal bone reconstruction, combining MSC, PRP and papaverine presented regeneration in all groups with no significant difference among them.

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Abstract Purpose: This study assessed the regeneration potential of mesenchymal stem cells (MSC) from adipose tissue associated with platelet-rich plasma (PRP) in bone regeneration. Methods: Thirty Wistar rats (Rattus norvegicus albinos) were divided into five groups (according to the grafting material and time to euthanasia): (1) autograft - 14 days (control), (2) autograft - 28 days (control), (3) MSC + PRP - 14 days, (4) MSC + PRP + papaverine - 14 days and (5) MSC + PRP + papaverine - 28 days. After euthanasia, the graft was removed and histological slides were prepared. They were assessed by a blinded pathologist using a previously published histological scale as parameter. Results: There was some degree of neoformed bone trabeculae (NBT) in 93.3% of the samples, as well as osteoblastic activity (OA). The autograft groups (14 and 28 days) had higher levels in the formation of bone trabeculae. Nonparametric data were analyzed using the Wilcoxon-Mann-Whitney test and proved not to be statistically significant at p < 0.05. Conclusions: Experimental parietal bone reconstruction, combining MSC, PRP and papaverine presented regeneration in all groups with no significant difference among them.

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Purpose: This study assessed the regeneration potential of mesenchymal stem cells (MSC) from adipose tissue associated with platelet-rich plasma (PRP) in bone regeneration. Methods: Thirty Wistar rats (Rattus norvegicus albinos) were divided into five groups (according to the grafting material and time to euthanasia): (1) autograft - 14 days (control), (2) autograft - 28 days (control), (3) MSC + PRP - 14 days, (4) MSC + PRP + papaverine - 14 days and (5) MSC + PRP + papaverine - 28 days. After euthanasia, the graft was removed and histological slides were prepared. They were assessed by a blinded pathologist using a previously published histological scale as parameter. Results: There was some degree of neoformed bone trabeculae (NBT) in 93.3% of the samples, as well as osteoblastic activity (OA). The autograft groups (14 and 28 days) had higher levels in the formation of bone trabeculae. Nonparametric data were analyzed using the Wilcoxon-Mann-Whitney test and proved not to be statistically significant at p 0.05. Conclusions: Experimental parietal bone reconstruction, combining MSC, PRP and papaverine presented regeneration in all groups with no significant difference among them.(AU)

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Background: Colitis murine models have become essential tools to investigate the molecular and cellular mechanisms that lead to infl ammatory bowel disease (IBD), such as ulcerative colitis (UC). DSS-induced colitis model faithfully reproduces many of the clinical presentation and immunological disturbances observed in UC. Notwithstanding mice can show differential susceptibilities and responsiveness to dextran sodium sulfate (DSS), and varying DSS concentration and molecular weights appear to be associated with the severity of inflammation. The aim of this study was to analyze the features of mice induced colitis using different DSS concentrations and molecular weights. Materials, Methods and Results: C57BL/6 mice received 2% of high molecular weight DSS (36 000 - 50 000) in drinking water (HDSS2%) or 5% of the same molecular weight (HDSS5%); other group received 5% of low molecular weight DSS (10 000) (LDSS5%). During the 7 days of DSS administration, animals were observed for weight loss, stool consistency and presence of blood feces to determine the disease activity index (DAI). On day 8, colons were removed, measured and weighed for indirect assessment of infl ammation. The tissue samples were processed for histological analysis and blood samples were collected for hematological analysis. Our results demonstrated that HDSS5% group began to show significant clinical signs starting from day 1, HDSS2% on day 2 and LDSS5% on day 3 (P < 0.05). However, from day 3, HDSS5% group presented DAI significantly higher than other groups (P < 0.001). In addition, DSS administration for 7 days was associated with significant (P < 0.05) changes in mice body weight compared to control animals. Group HDSS2% showed a weight loss of 23.8%±3.0, and HDSS5% and LDSS5% groups, presented weight loss of 32.65%±0.0 and 8.7%±1.7, respectively. From day 6, HDSS5% group presented weight loss significantly greater than HDSS2% and LDSS5% groups (P < 0.05). In colon macroscopic analysis, high molecular weight DSS groups showed a significantly macroscopic colon changes (P = 0.001) and hematological parameters alteration (P < 0.005) compared to control group. In histological features of colitis, these groups presented a higher histological score compared to normal colon (P < 0.001), with crypt damage, mucosal ulceration and cell inflammatory infiltration. Mice from group LDSS5% did not present significant macroscopic colon changes, hematological parameters alteration, and histological score compared to control group. Discussion: Results of the present study evidenced that acute colonic mucosal injury induced by DSS is dependent on the concentration and molecular weight of DSS administered in drinking water, and these findings are important consideration for reproducible induction of experimental colitis with this model. Moreover, DSS with high molecular weight and high concentration can initiate a severe colitis, which may not be an appropriate model for studies of therapeutic regeneration of the colonic mucosa. Thus, identification of differences in mice response to DSS could provide the basis for investigations of susceptibility or resistance to colitis. DSS-induced colitis model study contributes to the understanding of IBD and in the finding for new therapies targeting the reduction of inflammation.

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

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AIM: To investigate the interaction between mesenchymal stem cells (MSCs) and bone grafts using two different cultivation methods: static and dynamic. METHODS: MSCs were isolated from rat bone marrow. MSC culture was analyzed according to the morphology, cell differentiation potential, and surface molecular markers. Before cell culture, freeze-dried bone (FDB) was maintained in culture for 3 d in order to verify culture medium pH. MSCs were co-cultured with FDB using two different cultivation methods: static co-culture (two-dimensional) and dynamic co-culture (three-dimensional). After 24 h of cultivation by dynamic or static methods, histological analysis of Cell adhesion on FDB was performed. Cell viability was assessed by the Trypan Blue exclusion method on days 0, 3 and 6 after dynamic or static culture. Adherent cells were detached from FDB surface, stained with Trypan Blue, and quantified to determine whether the cells remained on the graft surface in prolonged non-dynamic culture. Statistical analyses were performed with SPSS and a P < 0.05 was considered significant. RESULTS: The results showed a clear potential for adipogenic and osteogenic differentiation of MSC cultures. Rat MSCs were positive for CD44, CD90 and CD29 and negative for CD34, CD45 and CD11bc. FDBs were maintained in culture for 3 d and the results showed there was no significant variation in the culture medium pH with FDB compared to pure medium pH (P > 0.05). In histological analysis, there was a significant difference in the amount of adhered cells on FDB between the two cultivation methods (P < 0.05). The MSCs in the dynamic co-culture method demonstrated greater adhesion on the bone surface than in static co-culture method. On day 0, the cell viability in the dynamic system was significantly higher than in the static system (P < 0.05). There was a statistical difference in cell viability between days 0, 3 and 6 after dynamic culture (P < 0.05). In static culture, cell viability on day 6 was significantly lower than on day 3 and 0 (P < 0.05). CONCLUSION: An alternative cultivation method was developed to improve the MSCs adhesion on FDB, demonstrating that dynamic co-culture provides a superior environment over static conditions.

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Embryonic stem cells are pluripotent cell lines with the capacity of self-renewal and a broad differentiation plasticity. They are isolated from preimplantation embryos and can be cultured in vitro for long time without losing their pluripotency. Embryonic stem cells can also differentiate in vitro with the proper combination of growth and differentiation factors, cells will differentiate into more advanced stages of embryogenesis generating different adult cell type. In the present study, we induced the in vitro differentiation of mouse embryonic stem cells (line R1) into cardiomyocytes and neuronal cells. These differentiations were evaluated by reverse transcription-polymerase chain reaction to verify presence of tissue-specific markers.

Células-tronco embrionárias são linhagens celulares pluripotentes capazes de se multiplicar indefinidamente e com grande capacidade de diferenciação celular. São isoladas de embriões em estágio pré-implantacional e podem ser cultivadas por longo tempo em laboratório sem perder sua pluripotencialidade. Células-tronco embrionárias podem, ainda, se diferenciar in vitro através da adição de fatores de crescimento e diferenciação ao meio de cultivo. As células se diferenciarão em estágios mais avançados de embriogênese, gerando tipos diferentes de células adultas. No presente estudo, induzimos a diferenciação in vitro de células-tronco embrionárias de camundongos (linhagem R1) em células de tecido cardíaco e nervoso. A diferenciação foi avaliada pela reação em cadeia da polimerase precedida de transcrição reversa para verificar a presença de marcadores tecido-específicos.

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