Role of mononuclear stem cells and decellularized amniotic membrane in the treatment of skin wounds in rats.

Stem cells (SC) and amniotic membrane (AM) are recognized for their beneficial impacts on the healing of cutaneous wounds. Thus, this study evaluated the capacity of tissue repair in a skin lesion rat model. Forty Wistar rats were randomized into four groups: group I - control, with full-thickness lesions on the back, without SC or AM; group II-injected SC; group III - covered by AM; group IV-injected SC and covered by AM. Lesion closure was assessed using contraction rate (Cr). Histochemical and immunohistochemical analyses were performed, and collagen, elastic fibers, fibroblast differentiation factor (TGF-ß), collagen remodeling (MMP-8), and the number of myofibroblasts and blood vessels (α-SMA) were evaluated. On the 7th postoperative day, Cr 1st-7th day levels were higher in groups III and IV. However, on the 28th day, Cr 1st-28th day were higher in the control group. Picrosirius staining showed that type I collagen was predominant in all groups; however, the SC + AM group obtained a higher average when compared to the control group. Elastic fiber analysis showed a predominance in groups that received treatment. Groups II and IV showed the lowest expression levels of TGF-ß and MMP-8, and α-SMA was significantly lower in group IV. The application of SC and AM accelerated the initial healing phase, probably owing to their anti-inflammatory effect that favored early formation of collagen and elastic fibers.

Beneficial Roles of Cellulose Patch-Mediated Cell Therapy in Myocardial Infarction: A Preclinical Study.

Biological scaffolds have become an attractive approach for repairing the infarcted myocardium and have been shown to facilitate constructive remodeling in injured tissues. This study aimed to investigate the possible utilization of bacterial cellulose (BC) membrane patches containing cocultured cells to limit myocardial postinfarction pathology. Myocardial infarction (MI) was induced by ligating the left anterior descending coronary artery in 45 Wistar rats, and patches with or without cells were attached to the hearts. After one week, the animals underwent echocardiography to assess for ejection fraction and left ventricular end-diastolic and end-systolic volumes. Following patch formation, the cocultured cells retained viability of >90% over 14 days in culture. The patch was applied to the myocardial surface of the infarcted area after staying 14 days in culture. Interestingly, the BC membrane without cellular treatment showed higher preservation of cardiac dimensions; however, we did not observe improvement in the left ventricular ejection fraction of this group compared to coculture-treated membranes. Our results demonstrated an important role for BC in supporting cells known to produce cardioprotective soluble factors and may thus provide effective future therapeutic outcomes for patients suffering from ischemic heart disease.

Regeneration of Tracheal Tissue in Partial Defects Using Porcine Small Intestinal Submucosa.

BACKGROUND: Surgical correction of tracheal defects is a complex procedure when the gold standard treatment with primary end-to-end anastomosis is not possible. An alternative treatment may be the use of porcine small intestinal submucosa (SIS). It has been used as graft material for bioengineering applications and to promote tissue regeneration. The aim of this study was to evaluate whether SIS grafts improved tracheal tissue regeneration in a rabbit model of experimental tracheostomy. METHODS: Sixteen rabbits were randomized into two groups. Animals in the control group underwent only surgical tracheostomy, while animals in the SIS group underwent surgical tracheostomy with an SIS graft covering the defect. We examined tissues at the site of tracheostomy 60 days after surgery using histological analysis with hematoxylin and eosin (H&E) staining and analyzed the perimeter and area of the defect with Image-Pro® PLUS 4.5 (Media Cybernetics). RESULTS: The average perimeter and area of the defects were smaller by 15.3% (p = 0.034) and 21.8% (p = 0.151), respectively, in the SIS group than in the control group. Histological analysis revealed immature cartilage, pseudostratified ciliated epithelium, and connective tissue in 54.5% (p = 0.018) of the SIS group, while no cartilaginous regeneration was observed in the control group. CONCLUSIONS: Although tracheal SIS engraftment could not prevent stenosis in a rabbit model of tracheal injury, it produced some remarkable changes, efficiently facilitating neovascularization, reepithelialization, and neoformation of immature cartilage.

Tracheal repair with acellular human amniotic membrane in a rabbit model.

Surgical correction of tracheal stenosis is still a complex and challenging procedure. Acellular human amniotic membranes (AHAM) represent a promising biomaterial source for tissue regeneration. The aim of this study was to evaluate whether AHAM grafts improve tissue regeneration of the trachea in a rabbit model of tracheostomy. Twenty rabbits were randomized into 2 groups. Animals in the control group underwent surgical tracheostomy only, and animals in the AHAM group underwent surgical tracheostomy and received an AHAM graft that covered the defect site. We examined tissues at the site of tracheostomy 60 days after surgery by histological analysis with haematoxylin and eosin, Movat's pentachrome stain and immunohistochemistry by analysis with antiaggrecan antibodies. The average perimeter and area of the defect 60 days after surgery were smaller in animals in the control group than in the AHAM group (p = .011 and p = .011, respectively). Histological analysis of AHAM group revealed neovascularization, islands of immature cartilage, pseudostratified ciliated epithelium. and connective tissue at the site of AHAM engraftment, whereas only pseudostratified ciliated epithelium and connective tissue were observed at the defect site in tissues of animals in the control group. Regeneration of islands of immature cartilage tissue with hyaline pattern and pseudostratified ciliated epithelium were confirmed by immunohistochemistry analysis. These results indicate that AHAM engraftment could facilitate neovascularization and regeneration of immature cartilage in a model of tracheal injury. Its use may lower the risk of post-operative complications including stenosis of trachea.

Bone Marrow-Derived Stem Cell Populations Are Differentially Regulated by Thyroid or/and Ovarian Hormone Loss.

Bone marrow-derived stem cells (BMDSCs) play an essential role in organ repair and regeneration. The molecular mechanisms by which hormones control BMDSCs proliferation and differentiation are unclear. Our aim in this study was to investigate how a lack of ovarian or/and thyroid hormones affects stem cell number in bone marrow lineage. To examine the effect of thyroid or/and ovarian hormones on the proliferative activity of BMDSCs, we removed the thyroid or/and the ovaries of adult female rats. An absence of ovarian and thyroid hormones was confirmed by Pap staining and Thyroid Stimulating Hormone (TSH) measurement, respectively. To obtain the stem cells from the bone marrow, we punctured the iliac crest, and aspirated and isolated cells by using a density gradient. Specific markers were used by cytometry to identify the different BMDSCs types: endothelial progenitor cells (EPCs), precursor B cells/pro-B cells, and mesenchymal stem cells (MSCs). Interestingly, our results showed that hypothyroidism caused a significant increase in the percentage of EPCs, whereas a lack of ovarian hormones significantly increased the precursor B cells/pro-B cells. Moreover, the removal of both glands led to increased MSCs. In conclusion, both ovarian and thyroid hormones appear to have key and diverse roles in regulating the proliferation of cells populations of the bone marrow.

The functional effect of soybean extract and isolated isoflavone on myocardial infarction and ventricular dysfunction: the soybean extract on myocardial infarction.

BACKGROUND: Myocardial infarction is a public health problem. Functional food is an alternative treatment for cardiovascular diseases. OBJECTIVE: The objective was to analyze the functional and anatomopathological post-myocardial-infarction effects of soybean extract (SE) and isoflavone (IF). METHODS: Myocardial infarction was induced in adult Wistar rats. After 5 days, an echocardiogram was performed to determine heart rate (HR), ejection fraction (EF), systolic volume (LVESV) and diastolic volume (LVEDV). Animals with ventricular dysfunction (EF<45%) were selected for study. The animals were divided into three groups: control (n=14), SE (n=15) and IF (n=12). The IF group received 120 mg/kg/day isolated IF, and the SE group received 12.52 g/day. After 30 days, a new echocardiogram was performed. A histological exam was carried out to determine the collagen. Activity of biochemical markers [arginase, lactate dehydrogenase (LDH) and malate dehydrogenase] was measured. RESULTS: The animals of the control, IF and SE groups showed a reduction in EF after the infarction (P=.432, P=.017 and P=.320, respectively). An increase of LVESV and LVEDV was observed in all groups (P=.009, P=.001 and P=.140; and P=.003, P=.008 and P=.205, respectively). A reduction of HR was found in the SE group (P=.020). There was a greater activity of LDH in the SE group. A smaller quantity of mature collagen was found in the region proximal to the myocardial infarction in the SE group. CONCLUSION: A protective effect in the SE group was observed 30 days after the myocardial infarction.

Transplantation of SNAP-treated adipose tissue-derived stem cells improves cardiac function and induces neovascularization after myocardium infarct in rats.

Stem cell therapy has been considered a promise for damaged myocardial tissue. We have previously shown that S-nitroso-N-acetyl-D,L-penicillamine (SNAP) increases the expression of several muscular markers and VEGF in mesenchymal stem cells, indicating that transplantation of SNAP-treated cells could provide better functional outcomes. Here, we transplanted SNAP-treated adipose tissue-derived stem cells (ADSCs) in rat infarcted myocardium. After 30days, we observed a significant improvement of the ejection fraction in rats that received SNAP-treated ADSCs, compared with those that received untreated cells (p=0.008). Immunohistochemical reactions showed an increased expression of troponin T-C and von Willebrand factor, and organized vascular units in the infarcted area of tissue transplanted with treated ADSCs. SNAP exposure induced intracellular S-nitrosation, a decreased GSH/GSSG ratio, but did not increase cGMP levels. Collectively, these results indicate that SNAP alters the redox environment of ADSCs, possibly associated with a pre-differentiation state, which may improve cardiac function after transplantation.

Combined transplantation of skeletal myoblasts and mesenchymal cells (cocultivation) in ventricular dysfunction after myocardial infarction.

OBJECTIVE: Cell therapy in the myocardium has been mainly performed with satisfactory results using 2 cell types: skeletal myoblasts (myogenic) and mesenchymal cells (angiogenic). This study assessed the combined transplantation of those 2 cell types (SMM) into infarcted rats. METHODS: Myocardial infarction was induced by ligature of the left coronary artery in 26 Wistar rats. After one week, the animals underwent echocardiography for assessing ejection fraction (EF%) and left ventricular end-diastolic and systolic volumes (EDV, ESV, mL). After 2 days, the animals were reoperated on and divided into 2 groups: 1) control (n = 10), which received 0.15 mL of culture medium; and 2) SMM (n = 16), which received 7.5x10(6) heterologous skeletal myoblasts and mesenchymal cells in the infarcted region. The cells were obtained from puncture of the iliac crest and biopsy of skeletal muscle, and were cultured in vitro. After one month, the animals underwent a new echocardiography. RESULTS: No significant difference in EF, EDV, and ESV was observed between the 2 groups on baseline echocardiographic values. One month after transplantation, the following was observed: a reduction in EF in the control group (29.31 +/- 5.6% to 23.54 +/- 6.51%; P = 0.048); and an increase in EF in the SMM group (24.03 +/- 8.68% to 31.77 +/- 9.06%; P = 0.011). The presence of neovascularization and muscle fibers was identified in the regions of myocardial fibrosis in the SMM group. CONCLUSION: Cocultivation of skeletal myoblasts and mesenchymal cells is functionally effective.

O transplante em conjunto de células mioblásticas esqueléticas e mesenquimais (cocultivadas) na disfunção ventricular pós-infarto do miocárdio / Combined transplantation of skeletal myoblasts and mesenchymal cells (cocultivation) in ventricular dysfunction after myocardial infarction

OBJETIVO: A terapia celular no miocárdio tem sido realizada fundamentalmente com dois tipos celulares: as células mioblásticas esqueléticas (miogênicas) e as mesenquimais (angiogênicas) com resultados satisfatórios. Foi analisado o resultado do transplante em conjunto destas células (CEM) em ratos infartados. MÉTODOS: Foram induzidos ao infarto do miocárdio, por meio de ligadura da coronária esquerda 26 ratos Wistar. Após uma semana, os animais foram submetidos à ecocardiografia para avaliação da fração de ejeção (FE, por cento) e dos volumes diastólico e sistólico finais do ventrículo esquerdo (VDF, VSF,ml). Após dois dias os animais foram reoperados e divididos em dois grupos: 1) controle (n=10) que recebeu 0,15 ml de meio de cultura e 2) CEM (n=16) que recebeu 7.5x106 células mioblásticas esqueléticas e mesenquimais, heterólogas, na região do infarto. As células foram obtidas a partir da punção da crista ilíaca e da biópsia do músculo esquelético, ambas submetidas à cultura celular in vitro. Após um mês, os animais foram submetidos a nova ecocardiografia. RESULTADOS: Não houve diferença significativa entre os dois grupos quanto a FE, VDF e VSF nos valores ecocardiográficos de base. Um mês após o transplante, foram observados diminuição da FE no grupo controle (29.31 ± 5.6 por cento para 23.54 ± 6.51 por cento p=0.048) e acréscimo da FE no grupo CEM (24.03 ± 8.68 por cento para 31.77 ± 9.06 por cento, p=0.011). Identificou-se a presença de neovasos e fibras musculares, nas regiões de fibrose miocárdica no grupo CEM. CONCLUSAO: O cocultivo das células mioblásticas esqueléticas e das células mesenquimais é funcionalmente efetivo.

Transplante celular: análise funcional, imunocitoquímica e histopatológica em modelo experimental de miocardiopatia isquêmica utilizando diferentes células / Cellular transplant: functional, immunocytochemical and histopathologic analysis in an experimental model of ischemic heat disease using different cells

OBJETIVO: Apresentar os resultados funcionais, imunocitoquímicos e histopatológicos, in vitro ou em espécimes cardíacas após isolamento, cultura e co-cultura de células tronco mesenquimais, células mioblásticas esqueléticas e transplantadas e co-transplantadas em animais de laboratório com miocardiopatia isquêmica e fração de ejeção do ventrículo esquerdo menor de 40 por cento. MÉTODO: Foram empregados 72 ratos Wistar, divididos em quatro grupos de acordo com o meio de cultura ou das células injetáveis: Grupo controle em que foi injetado apenas o meio de cultura (22 ratos); Grupo de células tronco mesenquimais (17 ratos); Grupo de células mioblásticas esqueléticas (16 ratos) e grupo co-cultura (17 ratos). Nos estudos imunocitoquímicos, as células foram marcadas com anti-vimentina, anti-desmina e anti-miosina. Nos estudos histopatológicos, as lâminas foram coradas com Tricômio de Gomori e identificados neovasos e tecido muscular. Na análise funcional, foi medida a fração de ejeção do ventrículo esquerdo em dois momentos do seguimento, uma semana após o infarto do miocárdio e um mês após a injeção. RESULTADOS: A fração de ejeção do ventrículo esquerdo entre os quatro grupos não apresentou diferença estatística significante (P=0,276), o ecocardiograma de seguimento demonstrou diferença estatística significante (P=0,001). Essa diferença ocorreu entre o grupo controle e o grupo de células mioblásticas esqueléticas (P=0,037), entre o grupo controle e o grupo co-cultura (P<0,001) e o grupo de células tronco mesenquimais e o grupo co-cultura (P=0,025). Quando se compararam as medidas obtidas dos dois ecocardiogramas em cada grupo, encontrou-se diferença no grupo controle (P=0,005) para pior e no grupo co-cultura (P=0,006) para melhor. No estudo imunocitoquímico in vitro, identificou-se células tronco mesenquimais quando marcou-se com anti-vimentina e células musculares, com anti-desmina. Nas espécimes cardíacas, identificou-se tecido muscular marcada com anti-desmina e células mioblásticas esqueléticas marcadas com anti-miosina rápida. No estudo histopatológico, observaram-se novos vasos no grupo de células tronco mesenquimais, no grupo de células mioblásticas esqueléticas, tecido muscular e angiogênese e miogênese no grupo co-cultura. CONCLUSAO: A fração de ejeção do ventrículo esquerdo melhorou no grupo em que foram injetadas células musculares, mais acentuadamente no grupo co-cultura.