This study aimed to evaluate the inflammatory effect and bone formation in sterile surgical failures after implantation of a collagen sponge with mesenchymal stem cells from human dental pulp (hDPSCs) and
Material and Methods
(n=75) were divided into five experimental groups according to treatment: G1) control (blood clot); G2) Hemospon
; G3) Hemospon
in a culture medium enriched with 8%
; G4) Hemospon
in a culture medium containing hDPSCs and G5) Hemospon
in a culture medium enriched with 8%
and hDPSCs. On days 7, 15 and 30, the animals were euthanized, and the tibia was dissected for histological, immunohistochemistry and immunofluorescence analyses. The results were analyzed using nonparametric Kruskal-Wallis test and Dunn’s post-test.
On days 7 and 15, the groups with
had less average acute inflammatory infiltrate compared to the control group and the group with Hemospon
(p<0.05). No statistically significant difference was found between the groups regarding bone formation at the three experimental points in time. Osteopontin expression corroborated the intensity of bone formation. Fluorescence microscopy revealed positive labeling with Q-Tracker
in hDPSCs before transplantation and tissue repair.
The results suggest that the combination of Hemospon
and hDPSCs is a form of clinical treatment for the repair of non-critical bone defects that reduces the inflammatory cascade’s effects.
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.
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.
Stem cells are present in the adult tissues of most diverse species. Bone marrow is recognized to be the most exploited site to obtain stem cells and cell progenitors. The objective of the present study was to characterize hematopoietic progenitor (HP) morphology and analyze the performance of adherent cell progenitors (ACPs) cultivated in vitro from black-rumped agouti bone marrow (Dasyprocta prymnolopha). Bone marrow aspirates were obtained from tibia crest and used to prepare histological slides and identify cell morphology. Cells were also scattered on culture plates for later isolation, expansion, and quantification. Smears obtained from bone marrow demonstrated HPs at different stages of maturity. In culture, these cells showed fibroblastoid morphology and a strong tendency to form colonies, demonstrated by the presence of cell aggregates, cytoplasmic elongations lying side by side. An 80% cell confluence was observed at 18 days in culture and progressive reduction in the percentage of nonadherent mononuclear cells. After eight passes, a mean cell viability of 96.07% was observed, from a pool of 1.6 × 10(7) cells (ACP). Thirteen 25-cm(2) culture bottles were trypsinized, resuspended in freezing medium, stored in 14 criotubes at a concentration of 1 × 10(6) cells per milliliter, and placed in liquid nitrogen at -196°C. Agouti bone marrow demonstrated high plasticity, moreover different HP lines, and a population of adherent cells demonstrated morphology similar to mesenchymal stem cells in culture.
Resumo: As biotécnicas da reprodução são importantes ferramentas para conservação de espécies domésticas e silvestres, pois permitem a recuperação e uso futuro de material reprodutivo. O objetivo deste estudo foi avaliar parâmetros morfológicos de CCOs de cutias, obtidos pela técnica de fatiamento do ovário para utilização em protocolos de maturação e fecundação na produção in vitro de embriões. Foram utilizadas dezessete cutias fêmeas do NEPAS, CCA- UFPI, com idade e peso médios de 3,9 anos e 2,16Kg, respectivamente, que foram submetidas à ovariossalpingohisterectomia. Os ovários após dissecados e pesados em balança de precisão, foram fatiados individualmente. Procedeu-se a busca e seleção dos CCOs em estereomicroscópio, os quais foram identificados e quantificados por cada ovário, além de classificados quanto a sua morfologia segundo a quantidade de camadas de células do cumulus e ao citoplasma em quatro graus. Verificou-se que a técnica de fatiamento do ovário possibilitou a obtenção de CCOs de cutias, com recuperação de grande quantidade e de variados graus de qualidade. Não houve correlações entre a idade dos animais e o peso dos ovários; a idade e o número de CCOs obtidos; entre o peso das cutias e o peso dos ovários e o número de CCOs obtidos.
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