Management of Airway Remodeling in a Mouse Model of Allergic Airways Inflammation Using Extracellular Vesicles from Human Bone Marrow-Derived Mesenchymal Stromal Cells

Abstract: Asthma is a chronic respiratory disease affecting 300 million people worldwide. It results in several structural changes in the airways, which are minimally accessible in clinical practice. Cell therapy using mesenchymal stromal cells (MSCs) is a promising strategy for treating asthma due to the paracrine activity of MSCs, which influences tissue regeneration and modulates the immune response. Studies using extracellular vesicles (EV) released by MSCs have demonstrated their regenerative properties in animal models. The aim of this study was to evaluate the potential of EVs isolated from human bone marrow MSCs (hBM-MSCs) to control lung tissue remodeling in ovalbumin-induced allergic asthma in Balb/c mice. We isolated hBM-MSCs from a single donor, expanded and characterized them, and then isolated EVs. Asthma was induced in 43 male Balb/c mice, divided into four groups: control, asthmatic (AS), asthmatic plus systemic EVs (EV-S), and asthmatic plus intratracheal EVs (EV-IT). Upon completion of asthma induction, animals were treated with EVs either locally (EV-IT) or intravenously (EV-S). Seven days after, we performed bronchoalveolar lavage (BAL) and the total nuclear cells were counted. The animals were euthanized, and the lungs were collected for histopathological analysis of the airways. The EV-S group showed improvement in only the total BAL cell count compared with the AS group, while the EV-IT group showed significant improvement in almost all evaluated criteria. Therefore, we demonstrate that the local application of EVs derived from hBM-MSCs may be a potential treatment in controlling asthma.

Effect of Hydroxyapatite Microspheres, Amoxicillin-Hydroxyapatite and Collagen-Hydroxyapatite Composites on Human Dental Pulp-Derived Mesenchymal Stem Cells.

In this study, the preparation and characterization of three hydroxyapatite-based bioactive scaffolds, including hydroxyapatite microspheres (HAps), amoxicillin-hydroxyapatite composite (Amx-HAp), and collagen-hydroxyapatite composite (Col-HAp) were performed. In addition, their behavior in human dental pulp mesenchymal stem cell (hDPSC) culture was investigated. HAps were synthesized through the following methods: microwave hydrothermal, hydrothermal reactor, and precipitation, respectively. hDPSCs were obtained from samples of third molars and characterized by immunophenotypic analysis. Cells were cultured on scaffolds with osteogenic differentiation medium and maintained for 21 days. Cytotoxicity analysis and migration assay of hDPSCs were evaluated. After 21 days of induction, no differences in genes expression were observed. hDPSCs highly expressed the collagen IA and the osteonectin at the mRNA. The cytotoxicity assay using hDPSCs demonstrated that the Col-HAp group presented non-viable cells statistically lower than the control group (p = 0.03). In the migration assay, after 24 h HAps revealed the same migration behavior for hDPSCs observed compared to the positive control. Col-HAp also provided a statistically significant higher migration of hDPSCs than HAps (p = 0.02). Migration results after 48 h for HAps was intermediate from those achieved by the control groups. There was no statistical difference between the positive control and Col-HAp. Specifically, this study demonstrated that hydroxyapatite-based bioactive scaffolds, especially Col-Hap, enhanced the dynamic parameters of cell viability and cell migration capacities for hDPSCs, resulting in suitable adhesion, proliferation, and differentiation of this osteogenic lineage. These data presented are of high clinical importance and hold promise for application in therapeutic areas, because Col-HAp can be used in ridge preservation, minor bone augmentation, and periodontal regeneration. The development of novel hydroxyapatite-based bioactive scaffolds with clinical safety for bone formation from hDPSCs is an important yet challenging task both in biomaterials and cell biology.

Quality control and immunomodulatory potential for clinical-grade equine bone marrow-derived mesenchymal stromal cells and conditioned medium.

This study aimed to assess the safety and reproducibility of cell therapy for its use in clinical practice. We performed immunophenotypic characterization of equine bone marrow-derived mesenchymal stromal cells (BMMSCs) by flow cytometry using CD90, CD19, CD14, CD105, CD45, and HLA-DR markers (n = 4); GTG banding cytogenetic analysis (n = 3); and microbiological quality control (n = 4). The immunomodulatory potentials of BMMSCs (n = 4) and its conditioned medium (CM, n = 3) were investigated by in vitro lymphocyte inhibition assay using phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMCs). BMMSCs populations isolated from all animals showed high expression of CD90 and CD105, and low expression of CD19, CD4, CD45, and HLA-DR. Of the 60 metaphases analyzed, 5% presented aneuploidy on random chromosomes and no contamination was found based on microbiological analyses. Both treatments significantly inhibited lymphocyte proliferation (> 50%), compared with PHA-stimulated PBMCs (p < 0.0001). These promising results for BMMSCs and CM justify their potential as a therapeutic approach for inflammatory diseases. The techniques used in this study were effective in assessing the quality and determining the minimum criteria for the clinical use of BMMSCs in veterinary medicine.

Influence of Adipose Tissue-Derived Stem Cells on the Burn Wound Healing Process.

Burns are lesions in which the thermal energy of the causative agent transfers heat to the surface of the body, causing superficial or deep damage to the skin with protein denaturation in cells and biochemical maladjustments, which delay and disrupt the cicatricial process, increasing the chances of functional and aesthetic sequelae. This study evaluates the influence of adipose tissue-derived stem cells (ADSCs) on burn healing in terms of the size of the cicatricial space and quantified measures of collagen deposition, inflammatory infiltrate, blood vessels, and lymphatic vessels. Initially, intra-abdominal adipose tissue was resected from a single donor Wistar rat that was not part of any of the subsequent groups to obtain ADSCs by isolation and cell culture. Burns were made in the left lateral abdominal region of Wistar rats by contact with a square ceramic paper with a 484 mm2 area heated to 100°C for 30 seconds. Intradermal ADSC transplantation was performed in two stages. The first was on the same day of the burn, when 3.2 × 106 ADSCs were transplanted shortly after the burned region cooled, while the second stage occurred four days later with the same number of ADSCs. The progress was evaluated by immunohistochemical methods and H&E, Masson's trichrome, Picrosirius red, and Lyve-1 immunofluorescence staining. Despite the quantitative similarity of blood vessels and the inflammatory infiltrate observed by H&E, there were statistically significant differences between the groups on the fourteenth day of evolution. The group that received ADSCs showed a reduction in the scar tissue area, increased collagen type III deposition, and a quantifiable reduction in lymphatic vessels, so we conclude that ADSCs influence the healing of total thickness burns in rats.

Expanded CD133+ Cells from Human Umbilical Cord Blood Improved Heart Function in Rats after Severe Myocardial Infarction.

Pharmacological approaches are partially effective in limiting infarct size. Cell therapies using a cell population enriched with endothelial progenitor cells (EPCs) CD133+ have opened new perspectives for the treatment of ischemic areas after infarction. This preclinical study evaluated the effect of intramyocardial transplantation of purified or expanded human umbilical cord blood-derived CD133+ cells on the recovery of rats following acute myocardial infarction (AMI). Histology studies, electrocardiogram, and fluorescence in situ hybridization (FISH) were used to evaluate heart recovery. Purified CD133+ cells, enriched in endothelial progenitor cells, when expanded in vitro acquired an endothelial-like cell phenotype expressing CD31 and von Willebrand factor (vWF). The group of infarcted rats that received expanded CD133+ cells had a more significant recovery of contraction performance and less heart remodeling than the group that received purified CD133+ cells. Either purified or expanded CD133+ cells were able to induce neovascularization in the infarcted myocardium in an equivalent manner. Few human cells were detected in the infarcted myocardium of the rats 28 days after transplantation suggesting that the effects observed might be related primarily to paracrine activity. Although both cell populations ameliorated the infarcted heart and are suitable for regeneration of the vascular system, expanded CD133+ cells are more beneficial and promising candidates for vascular regeneration.

Padronização de queimaduras térmicas em ratos por contato direto com a superfície cutânea / Standardization of thermal burns in rats by direct contact with the skin surface / Estandarización de quemaduras térmicas en ratas por contacto directo con la superficie cutánea

Introdução: Queimaduras cutâneas são lesões geralmente causadas pela energia térmica da transferência de calor, que determina um processo cicatricial desordenado. Para entender o processo e para avaliar possibilidades terapêuticas, são necessários modelos animais, assim como a padronização da elaboração da queimadura experimental. Objetivo: Detalhar uma técnica que promove queimaduras padronizadas de espessura total da pele em ratos Wistar. Método: Realizaram-se queimaduras padronizadas em 22 ratos Wistar divididos aleatoriamente em GI e GII. Acoplou-se à extremidade de um ferro de solda uma peça cerâmica de 484mm2 de superfície, que em contato com a pele durante 30 segundos, aquecida a 100° Celsius com uma pressão de 54 gramas, exercida pela própria massa do ferro da solda, determinou as queimaduras. No quarto dia, avaliou-se a coloração, umidade e elasticidade para caracterizar macroscopicamente as queimaduras e mensurou-se a área das queimaduras dos grupos, nos quais aplicou-se o teste t de Student para comparar os resultados. Resultados: Evidenciaram-se, em todos os animais, queimaduras com coloração branca cerosa, seca e inelástica. Constatou-se uma área média das queimaduras 403,5±71,48mm2 no GI e 403,7±71,19mm2 no GII (p=0,995). Conclusão: O contato da peça cerâmica de 484mm2 com pressão de massa de 54 gramas a 100° Celsius durante 30 segundos determinou queimaduras de espessura total de pele com área média semelhante entre os grupos, ao quarto dia de evolução, permitindo comparações seguras entre GI e GII ao longo do tempo cicatricial.

Introduction: Skin burns are lesions usually caused by energy of heat transfer that determine a disordered cicatricial process, to understand it and to evaluate therapeutic possibilities are needed animal models, as well as the standardization of experimental burn. Objective: To describe a technique that promotes standardized burns of total skin thickness for experimental study in Wistar rats. Methods: Standardized burns were performed on 22 Wistar rats randomly divided into GI and GII. A ceramic piece of 484 mm2 was attached to the end of a soldering iron, which in contact with the skin for 30 seconds at 100° Celsius at a pressure of 54 grams. On the fourth day, the staining, moisture and elasticity were evaluated to characterize the burns and the burn area of the groups was measured, in which Students t-test. Results: Burns characterized by waxy, dry and inelastic white color were observed in all the animals. It was found an average area of burns to 403.5±71.48mm2 in GI and 403.7±71.19mm2 in GII (p=0.995). Conclusion: The contact of the ceramic piece of 484mm2 with mass pressure of 54 grams at 100° Celsius for 30 seconds, determined total skin thickness burns with similar mean area groups, on the fourth day, allowing the safe comparison between the groups over the cicatricial time

Introducción: Las quemaduras cutáneas son lesiones en general causadas por la transferencia de calor que determinan una cicatrización desordenado, para entenderlo y evaluar posibilidades terapéuticas, se necesitan modelos animales, así como la estandarización de la quemadura experimental. Objetivo: Describir una técnica que promueve quemaduras estandarizadas del grosor total de la piel para el estudio experimental en ratas Wistar. Métodos: Se realizaron quemaduras en veintidós ratas Wistar divididas aleatoriamente en GI y GII. Se colocó una pieza cerámica de 484mm2 en un soldador en contacto con la piel durante 30 segundos a 100° Celsius a una presión de 54 gramos determinó la quemaduras. En el cuarto día, la tinción, la humedad y la elasticidad se evaluaron para caracterizar las quemaduras y se midió el área de las quemadura y se aplicó la prueba t de Student. Resultados: Se observaron quemaduras caracterizadas por un color blanco ceroso, seco e inelástico en todos los animales. Se encontró un área media de quemaduras 403.5±71.48mm2 en GI y 403.7±71.19 mm2 en GII (p=0.995). Conclusión: El contacto de la pieza de cerámica de 484 mm2 con una presión de masa de 54 gramos a 100° Celsius durante 30 segundos, determinó quemaduras de espesor total de la piel con área media similares entre los grupos en el cuarto día, lo que permite la comparación segura entre GI y el GII a lo largo del tiempo cicatricial

Human adipose-derived stem cells (ADSC) and human periodontal ligament stem cells (PDLSC) as cellular substrates of a toxicity prediction assay.

With the increasing need to develop in vitro assays to replace animal use, human stem cell-derived methods are emerging and showing outstanding contributions to the toxicological screening of substances. Adult human stem cells such as adipose-derived stem cells (ADSC) and periodontal ligament stem cells (PDLSC) were used as cell substrates for a cytotoxicity assay and toxicity prediction using the neutral red uptake (NRU) assay. First, primary cell cultures from three independent donors, from each tissue source, were characterized as mesenchymal stem cells (MSC) by plastic adherence and appropriate immunophenotype for MSC markers (positive for CD90, CD73, and CD105 and negative for CD11b, CD34, CD45, HLADR, and CD19). Furthermore, ADSC and PDLSC were able to differentiate into adipocytes and osteoblasts when maintained under the same culture conditions previously established for the NRU assay. NRU assays for three reference test substances were performed. R2 was higher than 0.85 for all conditions, showing the feasibility to calculate IC50 values. The IC50 values were then used to predict the LD50 of the test substances, which were comparable to previous results and the ICCVAM standard test report. Primary ADSC and PDLSC showed the potential to be considered as additional models for use in cytotoxicity assays.

A complexidade cicatricial em queimaduras e a possibilidade da terapia com células-tronco derivadas do tecido adiposo: revisão / The complexity of burn healing and the possibility of adipose tissue-derived stem cell therapy: review / La complejidad de la cicatrización de las quemaduras y la posibilidad de la terapia con células madre derivadas del tejido adiposo: revisión

OBJETIVO: Verificar as especificidades do processo cicatricial em queimaduras e identificar se as células-tronco derivadas do tecido adiposo (CTDA) podem se tornar aliadas em sua reparação. MÉTODO: Realizou-se uma revisão da literatura com consultas a Biblioteca Virtual de Saúde, Google Acadêmico, PubMed, Scientific Electronic Library Online, Mendeley catalog of academic literature, artigos e bibliografia especilizada em cicatrização, queimaduras, células-tronco derivadas do tecido adiposo publicados entre 2012 e 2016. RESULTADOS: As queimaduras se diferenciam de outras lesões pela intensidade da inflamação sistêmica e pela sobreposição das fases cicatriciais desorganizando-as. As CTDA são encontradas no estroma vascular do tecido adiposo e podem ser obtidas por lipoaspiração. Estas células podem interferir nas fases cicatriciais pela secreção de citocinas de crescimento, substâncias imunomoduladoras e anti-inflamatórias, assim como pela capacidade de diferenciação celular e homing. CONCLUSÃO: As queimaduras ocorrem na população mundial de forma preocupante, com características de morbidade e mortalidade. Os problemas sistêmicos e locais da reparação tecidual parecem ser atenuados pelas CTDA, sua abundante disponibilidade para o cultivo celular e suas habilidades as apontam como aliadas na recuperação das queimaduras.(AU)

OBJECTIVE: To evaluate the distinctions of the healing process in burns and identify how the adipose-tissue derived stem cells (ATSC) may become allies in this repair process. METHODS: Review of the literature was carried out with consultations at Virtual Health Library, Google Scholar, PubMed, Scientific Electronic Library Online and Mendeley catalog of academic literature and specialized bibliography in healing, burns and ATSC published between 2012 and 2016. RESULTS: Burns differentiate themselves from other lesions by the intensity of systemic inflammation and by the overlapping of the healing wound phases disorganizing them. The ATSC are found in the vascular stroma of adipose tissue and can be obtained by liposuction. These cells can interfere in the healing wound phases by the secretion of growth cytokines, immunomodulatory and anti-inflammatory substances and the ability of cell differentiation and homing. CONCLUSION: Burns occur in the world population in a worrisome way with characteristics of morbimortality. The problems of tissue repair seem to be attenuated by ATSC, their abundant availability for cell culture and their abilities point them as promising allies in healing of burn wound.(AU)

Objetivo: Verificar las especificidades del proceso cicatricial en quemaduras y identificar si las células-tronco derivadas del tejido adiposo (CTDA) pueden tornarse aliadas en su reparación. Método: Se realizó una revisión del literatura con consultas en Biblioteca Virtual de Salud, Google Académico, PubMed, Scientific Electronic Library online online, Mendeley catalog of academic literature, artículos y bibliografía especilizada, quemaduras, CTDA, publicados entre 2012 y 2016. Resultados: Las quemaduras se diferencian de otras lesiones por la intesidad de la inflamación sistémica y por la sobreposición de las fases cicatriciales desorganizándolas. Las CTDA son encontradas en el estroma vascular del tejido adiposo y pueden ser obtenidas por lipoaspiracion. Estas celulas pueden interferir en las fases cicatriciales por secrecion de citoquinas del crecimiento, sustancias inmunomoduladoras, anti inflamatorias y por la capacidad de diferenciacion celular e homing. Conclusión: Las quemaduras ocurren en la población mundial de forma preocupante con caracteristicas de morbimortalidad. Los problemas de la reparacion tecidual parecen ser atenuados por las CTDA, su abundante disponibilidad para el cultivo celular y sus habilidades las apuntan como aliadas en la recuperacion de las quemaduras.(AU)