Deferoxamine Protects Stromal/Stem Cells of "Lull pgm System"-Processed Lipoaspirates Against Damages Induced by Mitochondrial Respiration Inhibition.

BACKGROUND: The ischemic environment of the receiving area compromises the outcome of autologous fat grafts. The aim of this study was to isolate and expand the stromal vascular fraction from patient lipoaspirates and investigate the gain in cell viability exerted by some protective agents against the blockage of mitochondrial respiration. METHODS: The aspirates were (1) washed, using the "Lull pgm system," (2) centrifuged and (3) decanted. The corresponding stromal vascular fractions were isolated, and after cell adherence selection, the stromal/stem cell subpopulations were exposed to Antimycin A for 1 h. Then, the protection induced by cell pretreatment with deferoxamine, diazoxide and IGF-1 was evaluated. RESULTS: The residual cell viability of the "Lull pgm system"-washed samples was greater than that of the centrifuged samples (p < 0.05), and this advantage was maintained during the following 12 days of culture. The administration of 400 µM deferoxamine before Antimycin A treatment increased the number of viable cells from 56.5 to 80.8% (p < 0.05). On the contrary, the pretreatment with 250 µM diazoxide or 0.1 µg/ml IGF-1 did not exert any significant pro-survival action. Echinomycin abolished the positive effect of deferoxamine, suggesting that its protection involved HIF-1α. CONCLUSIONS: Adipose-derived stromal-stem cells survive the inhibition of mitochondrial respiration better if the lipoaspirate is washed using the "Lull pgm system" rather than centrifuged. Moreover, a significant contribution to cell survival can be obtained by preconditioning stromal-stem cells with deferoxamine. In a clinical perspective, this drug could be safely administered before surgery to patients undergoing autologous fat transfer. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266.

Complete Disaggregation of MCF-7-derived Breast Tumour Spheroids with Very Low Concentrations of α-Mangostin Loaded in CD44 Thioaptamer-tagged Nanoparticles.

Background: α-Mangostin (αMG) is a natural substance that exerts a wide range of antitumor effects. Recently, we described that free αMG was able to dissociate multicellular tumour spheroids (MCTSs) generated from breast carcinoma cells and to reduce their cellular viability and motility. Here, αMG was encapsulated into lipidic nanoparticles (NPs), conjugated or not to a CD44 thioaptamer, and the anticancer action evaluated against MCF-7 breast MCTSs. Methods: NPs containing αMG were formulated with a core of polylactic-co-glycolyc acid. Some of them were decorated with a CD44 thioaptamer using as catalysts 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide. Both size and density of MCF-7-derived MCTSs were monitored during 72 h of treatment with NPs carrying 0.1, 0.5 and 1.0 µg/ml final concentrations of αMG. MCTSs were cultured on Matrigel or gelatine to better simulate the extracellular environment. Results: The NPs without thioaptamer and conveying 0.1 µg/ml αMG caused a significant dissociation of the MCTSs grown in gelatine after 24 h of treatment (p < 0.01). The most significant disaggregation of MCTSs was obtained using NPs carrying 0.5 µg/ml αMG (p < 0.01). A similar dissociating effect was observed when MCTSs were cultured in Matrigel under the same conditions for 48 - 72 h. By contrast, only concentrations over 1.0 µg/ml of free αMG were able to provoke a damage to MCTSs, consisting in a substantial reduction in their size (p < 0.05). Since the MCTS dissociation induced by αMG-loaded NPs occurred only in the presence of Matrigel or gelatine, an impairment of cell contacts to collagen fibres was likely responsible of this effect. Finally, the treatment of MCTSs with αMG-loaded NPs that were conjugated to the CD44 thioaptamer caused a similar decrease in density but a lower expansion of the spheroid, suggesting that a significant number of cells were died or arrested in cycle. Conclusion: Very low concentrations of αMG delivered by lipidic NPs are sufficient to provoke a substantial disaggregation of MCF-7 MCTSs that involves cell-to-collagen contacts. Similarly, the treatment of MCTSs with NPs conjugated to a CD44 thioaptamer leads to MCTS dissociation but through a more damaging action that causes also a reduction in cell number.

Effects of α-Mangostin on Viability, Growth and Cohesion of Multicellular Spheroids Derived from Human Breast Cancer Cell Lines.

Background: α-Mangostin (αMG) is extracted from Garcinia mangostana Linn and exerts antiproliferative activities. Although several researches on αMG were performed using cell monolayers, the in vitro pharmacological effects on 3D cancer models have never been investigated. Aim of the present study was to find new anticancer properties of αMG by evaluating the changes that this compound provokes in multicellular tumour spheroids (MCTSs). Methods: MCTSs were generated from MDA-MB-231 and MCF-7 breast tumour cell lines and then treated with 0.1÷30 µg/ml αMG for 24 and 48 h. MCTS size, density, and cell migration were determined by software elaboration of phase contrast images captured by a digital camera. Cell viability was evaluated by resazurin and acid phosphatase assays, while cell apoptosis was assessed by a fluorescent assay of caspase activity. The distribution of living cells inside MCTSs was shown by live/dead fluorescence staining. Results: A dose-dependent decrease in cell viability was obtained by treating MDA-MB-231 spheroids with αMG for 48 h (IC50 = 0.70-1.25 µg/ml). A significant reduction in spheroid volume, paralleled by its increased compactness, was observed only at concentration of 30 µg/ml, but not with lower doses of αMG. By contrast, αMG in the range of 5-15 µg/ml increased the size of MCTSs due to a parallel reduction in cell aggregation. The same window of concentrations was also able to stimulate cell apoptosis in a dose-dependent manner. Bimodal volumetric effects were also obtained by treating the spheroids generated from the MCF-7 cells with 0.1÷30 µg/ml αMG for 48 h. Finally, doses higher than 5 µg/ml caused a progressive impairment in cell migration from the edge of MDA-MB-231 MCTSs. Conclusion: After exposure at doses of αMG just above IC50, MDA-MB-231 spheroids showed a significant reduction in cell adhesion that did not stimulate cell migration but, on the contrary, blunted cell motility. These findings suggest a novel anticancer feature of αMG that could be taken into consideration to improve conventional drug penetration into the tumour bulk.

Lull pgm system: A suitable technique to improve the regenerative potential of autologous fat grafting.

Autologous fat grafting and methods of purification of harvested tissue have become one of the most current themes in regenerative medicine. The aim of this study was to evaluate the in vitro regenerative potential of abdomen lipoaspirates subjected to a combined washing-decantation purifying procedure, the Lull pgm System (Lull). Blood cells and stromal-vascular fraction (SVF) cells contained in the aspirates were investigated and compared with those obtained through more conventional fat-processing methods, that is, the decantation and Coleman's centrifugation techniques. The lowest number of erythrocytes, which are proinflammatory cells, was observed in the Lull samples, corresponding to about 50% of those isolated by decantation and centrifugation. The highest amount of SVF cells were isolated from the Lull samples whose number of colony forming units, representative of the amount of adipose-derived stem cells (ADSCs), was about fourfold and sixfold higher than in the decantation and centrifugation samples, respectively. Adipocyte and osteoblast commitment of SVF cells obtained from all the three procedures also confirmed that the subpopulation of ADSCs was actively represented in the processed aspirates. Moreover, the growth rate of the SVF cells was more accentuated in the samples obtained from decantation and Lull than centrifugation. In conclusion, Lull seems to be the best processing technique for adipose tissue graft with respect to decantation and centrifugation, because it clears more efficiently the fat from proinflammatory blood cells and provides the greatest number of proliferating SFV cells and ADSCs.

Hyaluronan and cardiac regeneration.

Hyaluronan (HA) is abundantly expressed in several human tissues and a variety of roles for HA has been highlighted. Particularly relevant for tissue repair, HA is actively produced during tissue injury, as widely evidenced in wound healing investigations. In the heart HA is involved in physiological functions, such as cardiac development during embryogenesis, and in pathological conditions including atherosclerosis and myocardial infarction. Moreover, owing to its relevant biological properties, HA has been widely used as a biomaterial for heart regeneration after a myocardial infarction. Indeed, HA and its derivatives are biodegradable and biocompatible, promote faster healing of injured tissues, and support cells in relevant processes including survival, proliferation, and differentiation. Injectable HA-based therapies for cardiovascular disease are gaining growing attention because of the benefits obtained in preclinical models of myocardial infarction. HA-based hydrogels, especially as a vehicle for stem cells, have been demonstrated to improve the process of cardiac repair by stimulating angiogenesis, reducing inflammation, and supporting local and grafted cells in their reparative functions. Solid-state HA-based scaffolds have been also investigated to produce constructs hosting mesenchymal stem cells or endothelial progenitor cells to be transplanted onto the infarcted surface of the heart. Finally, applying an ex-vivo mechanical stretching, stem cells grown in HA-based 3D scaffolds can further increase extracellular matrix production and proneness to differentiate into muscle phenotypes, thus suggesting a potential strategy to create a suitable engineered myocardial tissue for cardiac regeneration.

Pharmacologically active microcarriers influence VEGF-A effects on mesenchymal stem cell survival.

Resistance of transplanted mesenchymal stem cells (MSCs) in post-ischemic heart is limited by their poor vitality. Vascular-endothelial-growth-factor-A (VEGF-A) as such or slowly released by fibronectin-coated pharmacologically-active-microcarriers (FN-PAM-VEGF) could differently affect survival kinases and anti-apoptotic mediator (e.g. Bcl-2). Therefore VEGF-A or FN-PAM-VEGF could differently enhance cell proliferation, and/or resistance to hypoxia/reoxygenation (H/R) of MSCs. To test these hypotheses MSCs were incubated for 6-days with VEGF-A alone or with FN-PAM-VEGF. In addition, MSCs pre-treated for 24-hrs with VEGF-A or FN-PAM-VEGF were subsequently exposed to H/R (72-hrs 3% O(2) and 3-hrs of reoxygenation). Cell-proliferation and post-hypoxic vitality were determined. Kinases were studied at 30-min., 1- and 3-days of treatment. Cell-proliferation increased about twofold (P < 0.01) 6-days after VEGF-A treatment, but by a lesser extent (55% increase) with FN-PAM-VEGF (P < 0.05). While MSC pre-treatment with VEGF-A confirmed cell-proliferation, pre-treatment with FN-PAM-VEGF protected MSCs against H/R. In the early phase of treatments, VEGF-A increased phospho-Akt, phospho-ERK-1/2 and phospho-PKCε compared to the untreated cells or FN-PAM-VEGF. Afterword, kinase phosphorylations were higher with VGEF, except for ERK-1/2, which was similarly increased by both treatments at 3 days. Only FN-PAM-VEGF significantly increased Bcl-2 levels. After H/R, lactate dehydrogenase release and cleaved Caspase-3 levels were mainly reduced by FN-PAM-VEGF. While VEGF-A enhances MSC proliferation in normoxia, FN-PAM-VEGF mainly hampers post-hypoxic MSC death. These different effects underscore the necessity of approaches suited to the various conditions. The use of FN-PAM-VEGF could be considered as a novel approach for enhancing MSC survival and regeneration in hostile environment of post-ischemic tissues.

Restored perfusion and reduced inflammation in the infarcted heart after grafting stem cells with a hyaluronan-based scaffold.

The aim of this study is to investigate the blood perfusion and the inflammatory response of the myocardial infarct area after transplanting a hyaluronan-based scaffold (HYAFF(®) 11) with bone marrow mesenchymal stem cells (MSCs). Nine-week-old female pigs were subjected to a permanent left anterior descending coronary artery ligation for 4 weeks. According to the kind of the graft, the swine subjected to myocardial infarction were divided into the HYAFF(®) 11, MSCs, HYAFF(®) 11/MSCs and untreated groups. The animals were killed 8 weeks after coronary ligation. Scar perfusion, evaluated by Contrast Enhanced Ultrasound echography, was doubled in the HYAFF(®) 11/MSCs group and was comparable with the perfusion of the healthy, non-infarcted hearts. The inflammation score of the MSCs and HYAFF(®) 11/MSCs groups was near null, revealing the role of the grafted MSCs in attenuating the cell infiltration, but not the foreign reaction strictly localized around the fibres of the scaffold. Apart from the inflammatory response, the native tissue positively interacted with the HYAFF(®) 11/MSCs construct modifying the extracellular matrix with a reduced presence of collagene and increased amount of proteoglycans. The border-zone cardiomyocytes also reacted favourably to the graft as a lower degree of cellular damage was found. This study demonstrates that the transplantation in the myocardial infarct area of autologous MSCs supported by a hyaluronan-based scaffold restores blood perfusion and almost completely abolishes the inflammatory process following an infarction. These beneficial effects are superior to those obtained after grafting only the scaffold or MSCs, suggesting that a synergic action was achieved using the cell-integrated polymer construct.

Priming adult stem cells by hypoxic pretreatments for applications in regenerative medicine.

The efficiency of regenerative medicine can be ameliorated by improving the biological performances of stem cells before their transplantation. Several ex-vivo protocols of non-damaging cell hypoxia have been demonstrated to significantly increase survival, proliferation and post-engraftment differentiation potential of stem cells. The best results for priming cultured stem cells against a following, otherwise lethal, ischemic stress have been obtained with brief intermittent episodes of hypoxia, or anoxia, and reoxygenation in accordance with the extraordinary protection afforded by the conventional maneuver of ischemic preconditioning in severely ischemic organs. These protocols of hypoxic preconditioning can be rather easily reproduced in a laboratory; however, more suitable pharmacological interventions inducing stem cell responses similar to those activated in hypoxia are considered among the most promising solutions for future applications in cell therapy. Here we want to offer an up-to-date review of the molecular mechanisms translating hypoxia into beneficial events for regenerative medicine. To this aim the involvement of epigenetic modifications, microRNAs, and oxidative stress, mainly activated by hypoxia inducible factors, will be discussed. Stem cell adaptation to their natural hypoxic microenvironments (niche) in healthy and neoplastic tissues will be also considered.

Comparison between stem cells harvested from wet and dry lipoaspirates.

Adipose-derived stem cells (ASC) are usually isolated from lipoaspirates, but it is not known if the anesthetic solution injected into adipose tissue affects cell yield and functions. Two different samples were drawn from the abdominal region of female subjects. In the first, a physiological solution containing lidocaine/adrenaline was injected (wet liposuction, WL), while in the contralateral area, the sample was collected without injecting any solution (dry liposuction, DL). The aspirates were processed to investigate the yield of the stromal-vascular fraction (SVF) cells and ASC frequency, growth rate, apoptosis, and differentiation potential. The solid dried mass of fresh WL isolates was lower than that of DL isolates (p < 0.01) due to the presence, in the former, of a liquid solution. As a consequence, the amount of WL-SVF cells was 18.7% lower than those obtained from DL (p < 0.01); this difference was also observed under culture conditions. In addition, the number of colony-forming unit-fibroblasts (CFU-Fs) obtained from 1 × 10(3) SVF cells was 25.5% lower in WL-aspirates than DL-aspirates (p < 0.05) owing, at least in part, to the observed presence of ASC [corrected] in the liquid solution of the WL isolates. After WL and DL, no differences were observed in ASC growth rate, apoptosis, or differentiation potential toward adipogenic, osteogenic, and endothelial cell lineages. In conclusion, WL yields about 40% fewer ASC than DL due to the combined effect of tissue dilution and the reduced frequency of ASC in the SVF. The main biological features of ASC are suitable for cell-based therapies.

Localization of mesenchymal stem cells grafted with a hyaluronan-based scaffold in the infarcted heart.

BACKGROUND: Permanence of grafted stem cells in the infarcted myocardial area has been suggested to be favored by tissue engineering strategies, including the application of a scaffold as a cell support. However, an estimation of how many cells remain localized in the site of transplantation has never been done. The aim of this work was to investigate the localization of mesenchymal stem cells (MSCs) grafted with a well cell-adhesive polymer in the scar region of the infarcted heart. MATERIALS AND METHODS: Rat MSCs were engineered in a hyaluronan-based scaffold (HYAFF(®)11) for 3 wk. The hearts of donor rats were also explanted, subjected to coronary artery ligation, and grafted into the abdomen of syngeneic rats. Two wk after coronary ligation a small dish of the HYAFF(®)11/MSC construct was introduced into a pouch created in the ventricular wall of the infarct area and left for 2 wk. RESULTS: Under ex vivo conditions, MSCs tightly adhered to the hyaluronan fibers and secreted abundant extracellular matrix. In contrast, HYAFF(®)11 was not more surrounded by the engrafted MSCs 2 wk after construct transplantation. Most MSCs migrated near the border zone of the infarcted area close to the coronary vessels. Moreover, the infarcted region of the heart was enriched in capillaries and the degree of fibrosis was attenuated. CONCLUSIONS: Two wk after transplantation most MSCs grafted in the infarcted myocardium with HYAFF(®)11 had left the scaffold and moved to the border zone. Nevertheless, this treatment increased the myocardial vascularization and reduced the degree of fibrosis in the scar area.

A brief very-low oxygen tension regimen is sufficient for the early chondrogenic commitment of human adipose-derived mesenchymal stem cells.

PURPOSE: The aim of this study was to evaluate the effects exerted over chondrogenic commitment of human adipose-derived mesenchymal stem cells (ADSCs) by a very low oxygen tension (<1% pO2). MATERIALS/METHODS: Cell morphology, mRNA levels of chondrocyte-specific marker genes and the involvement of p38 MAPK signalling were monitored in human ADSCs under a very low oxygen tension. RESULTS: Cell morphology was significantly changed after two days of hypoxic preconditioning when they featured as elongated spindle-shaped cells. SRY-box containing gene 9, aggrecan and collagen type II mRNA levels were enhanced under severe hypoxic culture conditions. Moreover, the inhibition of p38 MAPK resulted in a substantial reduction in transcription of the above-mentioned specific genes, proving the pivotal role of this pathway in the transcriptional regulation of chondrogenesis. CONCLUSIONS: Here, we propose a protocol showing the early commitment of stem cells towards the chondrogenic phenotype in only 2 days of culture via a very low hypoxic environment, in the absence of growth factors added in the culture medium.

Comparison between Culture Conditions Improving Growth and Differentiation of Blood and Bone Marrow Cells Committed to the Endothelial Cell Lineage.

The aim of this study was to compare different cell sources and culture conditions to obtain endothelial progenitor cells (EPCs) with predictable antigen pattern, proliferation potential and in vitro vasculogenesis. Pig mononuclear cells were isolated from blood (PBMCs) and bone marrow (BMMCs). Mesenchymal stem cells (MSCs) were also derived from pig bone marrow. Cells were cultured on fibronectin in the presence of a high concentration of VEGF and low IGF-1 and FGF-2 levels, or on gelatin with a lower amount of VEGF and higher IGF-1 and FGF-2 concentrations. Endothelial commitment was relieved in almost all PBMCs and BMMCs irrespective of the protocol used, whilst MSCs did not express a reliable pattern of EPC markers under these conditions. BMMCs were more prone to expand on gelatin and showed a better viability than PBMCs. Moreover, about 90% of the BMMCs pre-cultured on gelatin could adhere to a hyaluronan-based scaffold and proliferate on it up to 3 days. Pre-treatment of BMMCs on fibronectin generated well-shaped tubular structures on Matrigel, whilst BMMCs exposed to the gelatin culture condition were less prone to form vessel-like structures. MSCs formed rough tubule-like structures, irrespective of the differentiating condition used. In a relative short time, pig BMMCs could be expanded on gelatin better than PBMCs, in the presence of a low amount of VEGF. BMMCs could better specialize for capillary formation in the presence of fibronectin and an elevated concentration of VEGF, whilst pig MSCs anyway showed a limited capability to differentiate into the endothelial cell lineage.

Leupeptin preserves cardiac nitric oxide synthase 3 during reperfusion following long-term cardioplegia.

The objective of this study was to investigate how long-term cardioplegia/reperfusion affects cardiac nitric oxide synthase 3 (NOS3). To this aim, rat hearts were mounted in a perfusion apparatus and equilibrated with a modified Krebs-Henseleit solution (KH). The hearts were then arrested by soaking them in cold St. Thomas Hospital II solution (STH) for 5, 7, and 15 h. Reperfusion was performed by low-flow cold STH delivering for 1 h followed by 15-min aerobic normothermic KH perfusion. Cardioplegia preserved the amount of NOS3 irrespective of the duration of the cardiac arrest. NOS3 content was also unaffected by reperfusion following 5 and 7 h of cardioplegia. On the contrary, reperfusion performed after 15 h of cardioplegia caused a marked reduction in the amount of NOS3 protein, in both endothelial and cardiac muscle cells, and NOS activity. The involvement of intracellular proteolysis as a cause of reduction in NOS3 cardiac level was then investigated by delivering 0.1 mmol/L of either calpain I and II inhibitors or 0.05 mmol/L leupeptin during heart reperfusion. Only the treatment with leupeptin preserved NOS3, indicating that lysosomal proteases rather then cytoplasmic calpains were mainly responsible for the cleavage of this enzyme. The observed decrease in GSH/GSSG ratio and activation of JNK in the reperfused heart suggested that proteolysis could be triggered by reactive oxygen species.

Doxorubicin and α-Mangostin oppositely affect luminal breast cancer cell stemness evaluated by a new retinaldehyde-dependent ALDH assay in MCF-7 tumor spheroids.

According to cancer stem cell theory, only a limited number of self-renewing and cloning cells are responsible for tumor relapse after a period of remittance. The aim of the present study was to investigate the effects of Doxorubicin and α-Mangostin, two antiproliferative drugs, on both tumor bulk and stem cells in multicellular tumor spheroids originated from the luminal MCF-7 breast cancer cell line. A new and original fluorimetric assay was used to selectively measure the activity of the retinaldehyde-dependent isoenzymes of aldehyde dehydrogenase (RALDH), which are markers of a subpopulation of breast cancer stem cells. The administration of 5 µg/ml (12.2 µM) α-Mangostin for 48 h provoked: i) a marked disaggregation of the spheroids, leading to a doubling of their volume (p < 0.01), ii) a 40 % decrease in cell viability (p < 0.01), evaluated by the acid phosphatase assay, and iii) a reduction by more than 90 % of RALDH activity. By contrast, Doxorubicin given for 48 h in the range of 0.1-40 µM did not significantly reduce cell viability and caused only a modest modification of the spheroid morphology. Moreover, 40 µM Doxorubicin increased RALDH activity 2.5-fold compared to the untreated sample. When the two drugs were administered together using 5 µg/ml α-Mangostin, the IC50 of Doxorubicin referred to cell viability decreased six-fold and the RALDH activity was further reduced. In conclusion, the combined administration of Doxorubicin and α-Mangostin provoked a significant cytotoxicity and a remarkable inhibition of RALDH activity in MCF-7 tumor spheroids, suggesting that these drugs could be effective in reducing cell stemness in luminal breast cancer.

Difluoromethylornithine stimulates early cardiac commitment of mesenchymal stem cells in a model of mixed culture with cardiomyocytes.

The efficiency of in vitro mesenchymal stem cell (MSC) differentiation into the myocardial lineage is generally poor. In order to improve cardiac commitment, bone marrow GFP+MSCs obtained from transgenic rats were cultured with adult wild type rat cardiomyocytes for 5 days in the presence of difluoromethylornithine (DFMO), an inhibitor of polyamine synthesis and cell proliferation. The percentage of GFP+MSCs showing cardiac myofibril proteins (cMLC2, cTnI) was about threefold higher after DFMO addition (3%) relative to the untreated control (1%). Another set of experiments was performed with cardiomyocytes incubated for 1 day in the absence of glucose and serum and under hypoxic conditions (pO2 < 1%), in order to simulate severe ischemia. The percentage of cardiac committed GFP+MSCs was about 5% when cultured with the hypoxic/starved cardiomyocytes and further increased to 7% after DFMO addition. The contemporary presence of putrescine in DFMO-treated cells markedly blunted differentiation, while the cytostatic mitomycin C was not able to induce cardiac commitment. The involvement of histone acetylation in DFMO-induced differentiation was evidenced by the strong attenuation of cardiac commitment exerted by anacardic acid, an inhibitor of histone acetylase. Moreover, the percentage of acetylated histone H3 significantly increased in bone marrow MSCs obtained from wild type rats and treated with DFMO. These results suggest that polyamine depletion can represent a useful strategy to improve MSC differentiation into the cardiac lineage, especially in the presence of cardiomyocytes damaged by an ischemic environment.

* An Engineered Multiphase Three-Dimensional Microenvironment to Ensure the Controlled Delivery of Cyclic Strain and Human Growth Differentiation Factor 5 for the Tenogenic Commitment of Human Bone Marrow Mesenchymal Stem Cells.

At present, injuries or rupture of tendons are treated by surgical repair or conservative approaches with unpredictable clinical outcome. Alternative strategies to repair tendon defects without the undesirable side effects associated with the current options are needed. With this in mind, a tissue engineering approach has gained considerable attention as a promising strategy. Here we investigated a synthetic three-dimensional (3D) microenvironment able to interact with stem cells and inducing, via coupled biochemical and physical signals, their early commitment toward the tenogenic lineage. This multiphase 3D construct consisted of a braided hyaluronate elastic band merged with human bone marrow mesenchymal stem cells (hBMSCs) and poly-lactic-co-glycolic acid microcarriers loaded with human growth differentiation factor 5 (hGDF-5) by means of fibrin hydrogel. The multiphase structure allowed hBMSC culture under cyclic strain within a microenvironment where a controlled amount of hGDF-5 was regularly delivered. The cooperative biochemical and physical stimuli induced significantly increased expression of tenogenic markers, such as collagen type I and III, decorin, scleraxis, and tenascin-C, within only 3 days of dynamic hBMSC culture. This approach opens exciting perspectives for future development of engineered tendon tissue substitutes.

Long-term treatment with N-acetylcysteine, but not caloric restriction, protects mesenchymal stem cells of aged rats against tumor necrosis factor-induced death.

The survival of mesenchymal stem cells (MSCs) to tumor necrosis factor alpha (TNFalpha) stimulation was evaluated after a long-term antioxidant treatment, or caloric restriction, in aged rats. MSCs were isolated from bone marrow of 30-month-old rats which orally received N-acetylcysteine in the last 18 months. The necrotic cell death-induced in vitro by TNFalpha, determined by trypan blue exclusion, was markedly attenuated in MSCs obtained from treated vs. control aged rats (percent mean+/-SEM: 10.9+/-2.17 vs. 17.8+/-0.53; p<0.05). Also, the proliferation rate of MSCs from control, but not N-acetylcysteine-treated, aged rats evaluated up to 2 weeks was significantly higher than that of MSCs from younger (4-month-old) rats. No significant effect was observed relative to the parameters investigated when the aged rats were previously subjected to a hypocaloric diet for 18 months. In conclusion, a prolonged supplementation with N-acetylcysteine in rats can increase resistance to necrotic death of MSCs and may also counteract an excessive rate of MSC proliferation.

Mechanical Actuation Systems for the Phenotype Commitment of Stem Cell-Based Tendon and Ligament Tissue Substitutes.

High tensile forces transmitted by tendons and ligaments make them susceptible to tearing or complete rupture. The present standard reparative technique is the surgical implantation of auto- or allografts, which often undergo failure.Currently, different cell types and biomaterials are used to design tissue engineered substitutes. Mechanical stimulation driven by dedicated devices can precondition these constructs to a remarkable degree, mimicking the local in vivo environment. A large number of dynamic culture instruments have been developed and many appealing results collected. Of the cells that have been used, tendon stem cells are the most promising for a reliable stretch-induced tenogenesis, but their reduced availability represents a serious limitation to upscaled production. Biomaterials used for scaffold fabrication include both biological molecules and synthetic polymers, the latter being improved by nanotechnologies which reproduce the architecture of native tendons. In addition to cell type and scaffold material, other variables which must be defined in mechanostimulation protocols are the amplitude, frequency, duration and direction of the applied strain. The ideal conditions seem to be those producing intermittent tension rather than continuous loading. In any case, all physical parameters must be adapted to the specific response of the cells used and the tensile properties of the scaffold. Tendon/ligament grafts in animals usually have the advantage of mechanical preconditioning, especially when uniaxial cyclic forces are applied to cells engineered into natural or decellularized scaffolds. However, due to the scarcity of in vivo research, standard protocols still need to be defined for clinical applications.

Activation of glucose transport during simulated ischemia in H9c2 cardiac myoblasts is mediated by protein kinase C isoforms.

Glucose transport into cells may be regulated by a variety of conditions, including ischemia. We investigated whether some enzymes frequently involved in the metabolic adaptation to ischemia are also required for glucose transport activation. Ischemia was simulated by incubating during 3 h H9c2 cardiomyoblasts in a serum- and glucose-free medium in hypoxia. Under these conditions 2-deoxy-d-[2,6-(3)H]-glucose uptake was increased (57% above control levels, p<0.0001) consistently with GLUT1 and GLUT4 translocation to sarcolemma. Tyrosine kinases inhibition via tyrphostin had no effect on glucose transport up-regulation induced by simulated ischemia. On the other hand, chelerythrine, a broad range inhibitor of protein kinase C isoforms, and rottlerin, an inhibitor of protein kinase C delta, completely prevented the stimulation of the transport rate. A lower activation of hexose uptake (19%, p<0.001) followed also treatment with Gö6976, an inhibitor of conventional protein kinases C. Finally, PD98059-mediated inhibition of the phosphorylation of ERK 1/2, a downstream mitogen-activated protein kinase (MAPK), only partially reduced the activation of glucose transport induced by simulated ischemia (31%, p<0.01), while SB203580, an inhibitor of p38 MAPK, did not exert any effect. These results indicate that stimulation of protein kinase C delta is strongly related to the up-regulation of glucose transport induced by simulated ischemia in cultured cardiomyoblasts and that conventional protein kinases C and ERK 1/2 are partially involved in the signalling pathways mediating this process.

Nitric oxide regulates multiple functions and fate of adult progenitor and stem cells.

Nitric oxide is an endogenous gas which exerts autocrine/paracrine actions by different signaling pathways and/or direct interactions with intracellular compounds and structures. Several processes are regulated by nitric oxide in stem cells including self-renewal, viability, migration, proliferation, and differentiation. The modulation of cell functions depends on its concentrations because opposite effects can be observed when low and high levels of nitric oxide are compared. Here, the responses to nitric oxide of adult stem/progenitor cells which are often used in regenerative medicine, including mesenchymal stem cells, hematopoietic stem cells, neural stem cells, endothelial progenitor cells, satellite cells, and fibro-adipogenic precursor cells, are reviewed. Therapeutic strategies which employ drugs releasing nitric oxide or modulating nitric oxide intracellular pathways are suggested to perform new ex vivo preconditioning or in vivo treatments suitable for stem/progenitor cell therapy and tissue engineering applications.