Decellularized Amniotic Membrane Scaffold as a Pericardial Substitute: An In Vivo Study.

BACKGROUND: In the development of new biomaterials for pericardium substitute, acellular amniotic membrane (AAM) presents potential for new applications in regenerative medicine. We studied an AAM as a pericardial substitute to achieve a suitable, cost effective, abundant matrix for the purpose of using it as graft for tissue repair. METHODS: Twenty Wistar rats were randomly divided into 2 groups (n = 10/group) and had their pericardiums excised. In the experimental group, the excised pericardium segment was substituted by a 7-mm-diameter patch of decellularized AAM sutured to the lesion area. After 4 weeks, the heart's outer layer of both groups was evaluated. The structure and component characteristics of the scaffold were determined with the use of hematoxylin and eosi, Alizarin Red S, and immumohistochemical staining and scanning electron microscopy. RESULTS: Histopathologic examination of the AAM patches revealed that the integrity of the AAM was preserved, and no calcification was observed on the surface of the myocardium. We also observed thicker pericardium repair tissue in the AAM group compared with the control group. AAM patches, by virtue of their low immunogenicity, evoked minimal host-versus-graft reaction. CONCLUSIONS: We conclude that AAM appears to be an ideal substitute for pericardium lesions, because it is integrated into the biologic tissue owing to its low immunogenicity and its ability to diminish the occurrence of adhesions and scarring, increasing the pericardium thickness.

Effect of platelet-rich plasma therapy associated with exercise training in musculoskeletal healing in rats.

BACKGROUND: Muscle healing is a time-dependent process associated with an increase in the total amount of local collagen fibers. Platelet-rich plasma therapy (PRPT) associated with exercise may improve this healing process. The aim of this study was to demonstrate the regenerative effect of PRPT in association with exercise training on musculoskeletal healing. METHODS: Male Wistar rats were submitted to an injury in the vastus lateralis muscle and randomly divided into 4 groups (n = 5/group): sedentary sham-operated (SSO); sedentary group submitted to PRPT (SPR); swim-trained (SWT); and swim-trained group submitted to PRPT (SWP). Serum lactate level was used to confirm the training protocol effectiveness to increase aerobic fitness. The collagen fiber concentration was measured by the polarization colors in picrosirius red-stained tissue sections. RESULTS: Lactate levels decreased in both training groups (SWT and SWP; P < .05) after training (SWT: from 6.2 ± 0.44 to 4.7 ± 0.22 mmol/L; SWP: from 5.5 ± 0.99 to 4.0 ± 0.78 mmol/L). There were less type 1 collagen fibers in SWP group compared with other groups (SSO = 31.8 ± 10.3, SSP = 32.3 ± 13.5, SWT = 14.6 ± 13.4, SWP = 5.7 ± 4.7, P < .05), while there were more type 3 collagen fibers on SWP (SSO = 68.7 ± 9.8, SSP = 71.2 ± 12.2, SWT = 85.4 ± 13.4, SWP = 94.4 ± 4.6, P < .05) in the injured region. CONCLUSION: Exercise in association with PRPT enhances the skeletal muscle-healing process.

Regeneration of skin tissue promoted by mesenchymal stem cells seeded in nanostructured membrane.

BACKGROUND: The mesenchymal stem cell therapy has proven to be an effective option in the treatment of skin injuries. The combination of these cells with nanostructured membranes seems to be the future for tissues recovery. The aim of this project was to use biomolecules of polysaccharides to be incorporated on regenerated cellulose membranes and to prospect the improvement as bioactive wound dressings with mesenchymal stem cells. METHODS: The biocomposites were obtained after defibrillation with the use of never-dried bacterial cellulose to form a pulp, and, after the films were regenerated, in the presence of gellan gum with or without fluconazole. Membrane atomic force microscopy was performed for comparison of their structures. RESULTS: Adipose-derived mesenchymal stem cells were obtained from human adipose tissue liposuction in accordance with Zuk et al. The flow cytometric analysis and induction tests for adipocytes and osteocytes were performed. In vitro assays were performed on different membranes to evaluate the ability of these cells to adhere at 2 hours and proliferate at 7 days; the results were obtained by use of the MTT cell counting technique. In vivo testing allowed us to observe cell migration and participation in wound-healing by fluorescence labeling of the cells with BrdU. The bioactive curative, seeded with cells, was tested in skin burned in a murine model. CONCLUSIONS: The bacterial cellulose with gelan gum membrane incorporated with fluconazole presented the best performance in adhesion and proliferation tests. The cells can be identified in burned host tissue after occurrence of the wound.

Angiogenesis without functional outcome after mononuclear stem cell transplant in a doxorubicin-induced dilated myocardiopathy murine model.

OBJECTIVES: Cell transplantation is considered a novel approach in the treatment of myocardiopathy. The objective of this study was to evaluate the effects of autologous mononuclear stem cell therapy in doxorubicin-induced dilated myocardiopathy by conducting both functional and histopathologic analysis. METHODS: Seventy male rats were doxorubicin injected intraperitoneally for 2 weeks. At 1 month, the animals that had demonstrated left ventricular ejection fractions less than 40% were randomly divided into a mononuclear stem cell group and controls. Mononuclear stem cells were isolated. All animals underwent echocardiographic study: baseline, pre-cell therapy, and at 1 month post-cell therapy, and analyzed by the nonparametric Mann-Whitney test. Transplants were performed by subepicardial injections. Standard staining was performed. RESULTS: Twenty-three animals were randomly treated: mononuclear stem cell and control groups, with 11 rats completing the study. Cell viability was 85%. Mononuclear stem cells (n=5; 5x106 cells /300 microL medium) and control (n=6; 300 microL medium) were used. The resulting left ventricular ejection fraction in the cell therapy group was not significantly different compared with controls (p=0.54). New vessels were demonstrated in the subepicardial region. CONCLUSIONS: Autologous mononuclear stem cell therapy was not functionally effective in doxorubicin-induced dilated myocardiopathy in the animal model under study with the experimental conditions, despite occurrence of angiogenic activity.

Evaluation of bone marrow mesenchymal stem cell standard cryopreservation procedure efficiency.

INTRODUCTION: Mesenchymal stem cells are obtained from a variety of sources, particularly bone marrow. These cells have great potential for clinical research due to their potential to regenerate tissue. As is well known, the cryopreservation process can store any cell type, particularly blood cells, for an indeterminate time. OBJECTIVE: The aim of this study was to analyze the efficiency of standard cryopreservation procedures for adult mesenchymal stem cells from bone marrow. METHODS: Mononuclear stem cells isolated from 10 Wistar male rats were cultivated for 4 weeks to obtain mesenchymal stem cells. The parameters considered in this study were trypan blue exclusion test and annexin V conjugated with 7-amino-actinomycin for flow cytometry before cryopreservation in liquid nitrogen vapor phase for 1 month and after thawing. RESULTS: The viabilities determined by the trypan blue exclusion test were 94.76% and 90.58%, and the flow cytometry assay (annexin V conjugated with 7-amino-actinomycin) were 85.52% and 66.25%, before cryopreservation and after thawing, respectively. CONCLUSIONS: Standard procedures for cryopreservation were not efficient for those cells. The flow cytometry assay was more sensitive than the trypan blue exclusion test to demonstrate nonviability.

Functional outcome of bone marrow stem cells (CD45(+)/CD34(-)) after cell therapy in chronic spinal cord injury in Wistar rats.

BACKGROUND: Therapy with diverse cell types has been proposed to regenerate spinal cord injuries seeking to minimize the consequences for the lives of chronic patients. The types considered are: mononuclear and mesenchymal adult stem cells, embryonic stem cells, and Schwann cells. MATERIALS AND METHODS: Ninety male Wistar rats that underwent spinal cord contusion injury (NYU Impactor) were followed with the Basso, Beattie, and Bresnahan (BBB) locomotor rating scale for 14 days. Animals with scores < or = 16 were randomly divided into 2 groups: control (vehicle) versus cell therapy group. The mononuclear fraction (CD45(+)/CD34(-)) obtained by puncture-aspiration of the bone marrow was isolated by a density gradient (d = 1.077). The parenchymal cell infusion was performed using a syringe (100 U/1 mL) with a 30G1/2 needle. The animals were followed for 10 days before euthanasia. Statistical analyses comparing groups were performed by the Mann-Whitney test and group comparisons by the Wilcoxon test. RESULTS: Among 90 injured rats, 65 (72.2%) survived, including 44 whose scores were < or = 16. Eleven animals finished the study in the control group (64.7%) and 17 in the therapy group (80.9%). The statistical analyses did not demonstrate significance (P > .05) for either test. CONCLUSION: Mononuclear adult stem cell therapy was not demonstrated to be functionally effective for chronic spinal cord injury.

Immunophenotypic expression by flow cytometric analysis of cocultured skeletal muscle and bone marrow mesenchymal stem cells for therapy into myocardium.

The product generated by skeletal muscle and bone marrow mesenchymal stem cell cocultures has been demonstrated to improve the functional outcomes after cell therapy in postinfarction or Chagas myocardiopathy. This coculture method allows cell interactions in vitro, diminishing the operational costs of the culture/expansion as well as leading to angiogenesis and myogenesis for regeneration of the injured heart. Flow cytometric analysis may better characterize the cellular types in this model. Our objective was to use flow cytometry to analyze the immunophenotype expressed in this coculture model. The coculture was performed in accordance with Carvalho for 21 days. Flow cytometry was performed before and after coculture to characterize the immunophenotypic profile of cellular subsets, namely, the surface markers CD31, CD34, CD44H, CD45, CD49d, CD54, CD73, CD90, CD105, CD106, Myo-D, M-cadherin, and Connexin-43. Statistics were performed by the nonparametric Friedman test (P < .05) with post-hoc analysis by the nonparametric Wilcoxon test (P < or = .017, Bonferroni correction). The results demonstrated statistical significance for CD45(+) in 89.49% of mononuclear cells, 3.58% in skeletal muscle cells, and 4.74% among cocultured cells (P = .0094); and CD90(+) in 36.18% of mononuclear cells, 6.01% in skeletal muscle cells, and 48.94% among cocultured cells (P = .0420). The cocultured cells expressed the markers CD73(++), CD90(+++), CD45(-), CD34(+), CD105(-/+), CD106(-/+), M-cadherin(-/+), and Connexin-43(-/+). In conclusion, flow cytometric analysis showed a heterogeneous adherent cell population in this coculture model.

Functional outcome of bone marrow stem cells (CD45(+)/CD34(-)) after cell therapy in acute spinal cord injury: in exercise training and in sedentary rats.

BACKGROUND: Cell therapy and exercise training may be options for spinal cord regeneration. Our objective was to evaluate the functional effects of autologous bone marrow stem cell (CD45(+)/CD34(-)) transplantation in acute spinal cord injury in exercise training and in sedentary rats. MATERIALS AND METHODS: Fifty-five adult male Wistar rats underwent spinal cord contusion by Impactor (NYU). Locomotor rating scale was performed every 48 hours for 48 days. Animals with scores < or = 12 were randomly divided into 4 groups: sedentary without parenchymal cell infusion; sedentary with parenchymal cell infusion; swimming training without parenchymal cell infusion; and swimming training with parenchymal cell infusion. Bone marrow stem cells were isolated by puncture-aspiration of the bone marrow and density gradient (d = 1.077). The animals underwent a 60-minute swimming session 6 times/week supporting an overload of 3% of body weight for 6 consecutive weeks. Comparisons between the groups in relation to differences between the beginning to the end of scores used the nonparametric Bonferroni test and post-hoc Mann-Whitney U test to identify significance. RESULTS: Forty-two rats that obtained scores < or = 12 underwent therapy with 9 animals in each of the 4 groups as completors (n = 36). There was significance (P < or = .008) for sedentary without parenchymal cell infusion vs swimming training with parenchymal cell infusion. CONCLUSION: The combination of bone marrow stem cell therapy (CD45(+)/CD34(-)) and exercise training resulted in significant functional improvement in acute spinal cord injury.

Functional outcome of bone marrow stem cells: mononuclear versus mesenchymal stem cells after cellular therapy in myocardial scar in Wistar rats.

Experimental studies have suggested that a bone marrow stem cell transplant into the heart produces a favorable impact on tissue perfusion, yielding a new perspective on myocardial regeneration. Studies in human beings have demonstrated an improved clinical and functional cardiac state, which has been explained mainly by the angiogenic potential of the stem cells. Our objective was to compare the functional outcome of mononuclear stem (MoSC) and mesenchymal stem (MeSC) cell therapy after myocardium infarction in rats. Forty-two rats with myocardial infarctions underwent autologous transplantation of MoSC and MeSC in animals with ejection fractions lower than 40%. The functional analysis was performed using echocardiography at baseline and at 1 month after direct injection into the ventricular wall using: 5 x 10(6) MoSC (n = 08) or 2.5 x 10(6) MeSC (n = 13) or medium controls (n = 21). Statistical significance was accepted when P < .05. Intragroup comparisons of baseline versus 1-month follow-up were performed with paired t tests. Kruskal-Wallis was used as appropriate. There was a difference in baseline left ventricular ejection fraction (LVEF) and left ventricular-end dyastolic volume between all groups. After 1 month, LVEF decreased in the control group but remained unchanged in MoSC and MeSC groups. In all groups we observed myocardial remodeling. In conclusion, we have not demonstrated functional effectiveness with either MoSC or MeSC cell type, but potentially improved myocardial perfusion needs to be analyzed.

Proliferation of bone marrow mesenchymal stem cells, skeletal muscle cells and co-culture of both for cell myocardium therapy in Wistar rats.

The best results of cell therapy are achieved by a greater quantity of cells, delivery to the correct place, and cell conditions of viability with proliferation and without apoptosis. The quantification of cellular growth, including proliferation and viability, has become an essential tool. The objective of this study was to analyze cell proliferation in 14-day cultures of bone marrow mesenchymal stem cells (BMMSC), skeletal muscle cells (SMC), and co-culture of both types of cells (CO). Forty-four adult Wistar male rats (250-300g) received cultured cells CO (n = 22), BMMSC (n = 10), and SMC (n = 12). All cultured cells were started with the same concentration: 5 x 10(5)/mL, under similar conditions and maintained in an incubator with 5% CO(2) at 37 degrees C, which was changed every 48 hours for 14 days. The cell count was performed in Neubauer's chamber to calculate the proliferation index (IP). Statistical analysis was performed by the nonparametric Kruskal-Wallis and Wilcoxon tests. P values <.05 were considered statistically significant. The results showed that IP was positive in all groups. In conclusion, proliferation capacity was demonstrated in all groups. SMC IP was greater than the others, although it was the most heterogeneous.

Simultaneous autologous transplantation of cocultured mesenchymal stem cells and skeletal myoblasts improves ventricular function in a murine model of Chagas disease.

BACKGROUND: Cellular transplantation is emerging as a promising strategy for the treatment of postinfarction ventricular dysfunction. Whether its beneficial effects can be extended to other cardiomyopathies remains an unexplored question. We evaluated the histological and functional effects of simultaneous autologous transplantation of co-cultured stem cells and skeletal myoblasts in an experimental model of dilated cardiomyopathy caused by Chagas disease, characterized by diffuse fibrosis and impairment of microcirculation. METHODS AND RESULTS: Wistar rats weighing 200 grams were infected intraperitoneally with 15 x 10(4) trypomastigotes. After 8 months, 2-dimensional echocardiographic study was performed for baseline assessment of left ventricle (LV) ejection fraction (EF) (%), left ventricle end-diastolic volume (LVEDV) (mL), and left ventricle end-systolic volume (LVESV) (mL). Animals with LV dysfunction (EF <37%) were selected for the study. Autologous skeletal myoblasts were isolated from muscle biopsy and mesenchymal stem cells from bone marrow aspirates were co-cultured in vitro for 14 days, yielding a cell viability of >90%. Eleven animals received autologous transplant of 5.4 x 10(6)+/-8.0 x 10(6) cells (300 microL) into the LV wall. The control group (n=10) received culture medium (300 microL). Cell types were identified with vimentin and fast myosin. After 4 weeks, ventricular function was reassessed by echo. For histological analysis, heart tissue was stained with hematoxylin and eosin and immunostained for fast myosin. After 4 weeks, cell transplantation significantly improved EF and reduced LVEDV and LVESV. No change was observed in the control group. CONCLUSIONS: The co-transplant of stem cells and skeletal myoblasts is functionally effective in the Chagas disease ventricular dysfunction.

Cell transplantation after the coculture of skeletal myoblasts and mesenchymal stem cells in the regeneration of the myocardium scar: an experimental study in rats.

UNLABELLED: In myocardial infarction and Chagas's disease, some physiopathological aspects are common: cardiomyocyte loss due to ischemia leads to a reduction of contractility and heart function. Different cells have been proposed for cellular cardiomioplasty. OBJECTIVE: Our goal was to evaluate the method of co-culture of skeletal muscle (SM) and mesenchymal stem cells (MSC) for cell therapy of heart failure in Chagas's disease (CD) and myocardium postinfarction (MI). METHODS: For MI, 39 rats completed the study at 1 month. Seventeen rats received cell therapy into the scar and 22 rats only medium. For CD, 15 rats completed the study at 1 month including 7 that received cell therapy and eight followed the natural evolution. All animals underwent ecocardiographic analysis at baseline and 1 month. Left ventricular, ejection fraction, end systolic, and end dyastolic volume were registered and analyzed by ANOVA. The co-culture method of SM and MSC was performed at 14 days (DMEM, with 15% FCS, 1% antibiotic, IGF-I, dexamethasone). Standard stain analysis was performed. RESULTS: For MI ejection fraction in the animals that received the co-cultured cells increased from 23.52+/-8.67 to 31.45+/-8.87 (P=.006) versus the results in the control group: 26.68+/-6.92 to 22.32+/-6.94 (P=.004). For CD, ejection fraction in animals that received the co-cultured cells increased from 31.10+/-5.78 to 53.37+/-5.84 (P<.001) versus the control group values of 36.21+/-3.70 to 38.19+/-7.03 (P=0.426). Histopathological analysis of the animals receiving co-cultured cells demonstrated the presence of myogenesis and angiogenesis. CONCLUSION: The results validated the product of SM and MSC co-cultures for treatment of diseases.

Cell transplantation: differential effects of myoblasts and mesenchymal stem cells.

BACKGROUND: Cellular transplantation has emerged as a novel therapeutic option for treatment of ventricular dysfunction. Both skeletal myoblasts (SM) and mesenchymal stem cells (MSC) have been proposed as ideal cell for this aim. The aim of this study is to compare the efficacy of these cells in improving ventricular function and to evaluate the different histological findings in a rat model of severe post-infarct ventricular dysfunction. METHODS: Myocardial infarction was induced in Wistar rats by left coronary occlusion. Animals with resulting ejection fraction (EF) lower than 40% were included. Heterologous SM were obtained by lower limb muscle biopsy and MSC by bone marrow aspiration. Nine days after infarction, rats received intramyocardial injection of SM (n=8), MSC (n=8) or culture medium, as control (n=11). Echocardiographic evaluation was performed at baseline and after 1 month. Histological evaluation was performed after HE and Gomori's trichrome staining and immunostainig against desmin, fast myosin and factor VIII. RESULTS: There was no difference in baseline EF and left ventricular end diastolic (LVEDV) and systolic volume (LVESV) between all groups. After 1 month a decrease was observed in the EF in the control group (27.0+/-7.10% to 21.46+/-5.96%, p=0.005) while the EF markedly improved in SM group (22.66+/-7.29% to 29.40+/-7.01%, p=0.04) and remained unchanged in the MSC group (23.88+/-8.44% to 23.63+/-10.28%, p=0.94). Histopathology identified new muscular fibers in the group that received SM and new vessels and endothelial cells in the MSC. CONCLUSION: Skeletal myoblasts transplantation resulted in myogenesis and improvement of ventricular function. In contrast, treatment with mesenchymal stem cells resulted in neoangiogenesis and no functional effect.

Could the coculture of skeletal myoblasts and mesenchymal stem cells be a solution for postinfarction myocardial scar?

Currently two lines of research have been proposed for treatment of heart failure in an attempt to address its main cause: skeletal myoblast (SM) transplants, which increase the contractile muscular mass, and mesenchymal stem cell (MSC) transplants, which increase neoangiogenesis. The objective of this study was to establish methods whereby cocultures of SM and MSC proliferate and expand, making possible the interaction of these cell types prior to their transplantation to the myocardium. Seeking to support the survival of these cells after myocardial transplantation and achieve subsequent functional improvement, SM and MSC from 10 rats were isolated and cultivated in DMEM medium supplemented with 15% fetal calf serum, 1% ATB, and growth factors. Following plating in variable proportions of satellite cells/mononuclear cells namely 2:1, 1:1, 1:2, morphological observations were made regarding cell survival, adhesion to substrate, and confluence. After 48 hours nonadherent cells were aspirated from the flasks, leaving the adherent cells, SM, and MSC. The better level of cell proliferation was observed with the proportion 2:1 cocultivated at a concentration of 5 x 10(5)/mL for 14 days. The results were satisfactory; the cell production was up to 10(8), increasing the chances of transplant success after myocardial infarction. Transplants with this model are ongoing.

Aneural culture of rat myoblasts for myocardial transplant.

Due to the peculiar characteristics of skeletal muscle, myoblast transplants have emerged as a therapy for cardiomyopathy, particularly after myocardial infarction. The objectives of this study were to define the mean time of cultivation necessary to obtain a cellular concentration of 10(6) to expand the mass for transplant, and to identify the proliferation phase of myoblasts. Ten myoblast cultures were performed using newborn Wistar rats. The isolation method used enzymatic dissociation in culture medium (HAM-F12 and 199) supplement with basic-fibroblast growth factor (b-FGF) and insulin growth factor (IGF-I). The mean cultivation time to obtain the desired concentration of 10(6) was 7 days, with expansion of up to 10(8)/g. When b-FGF was used, the cellular yield was approximately 10(7), with IGF-I the cellular yield was approximately 10(8), independent of the medium. We concluded that IGF-I is the better option for mass cellular expansion of myoblasts for application in myocardial transplants.