Isolamento e caracterização de células-tronco mesenquimais de filtros reutilizáveis e descartáveis de medula óssea

OBJETIVO: Comparar as células-tronco mesenquimais humanas obtidas de filtros de coleta reutilizáveis àquelas coletadas em filtros descartáveis e caracterizá-las utilizando os critérios da International Society for Cellular Therapy. MÉTODOS: Foram isoladas células-tronco mesenquimais humanas de kits de coleta de medula óssea reutilizáveis e descartáveis, pela lavagem dos filtros com meio de cultura. As células isoladas foram caracterizadas de acordo com os critérios estabelecidos pela International Society for Cellular Therapy, por meio das técnicas de citometria de fluxo, diferenciação in vitro e citoquímica. RESULTADOS: As amostras foram obtidas de filtro descartável (n=3) e reutilizável (n=3). Todas as amostras obtidas de filtros descartáveis produziram células-tronco mesenquimais, e todas as células-tronco mesenquimais humanas derivadas de medula óssea preencheram os critérios estabelecidos pela International Society for Cellular Therapy. CONCLUSÃO: Este estudo mostrou que as células-tronco mesenquimais também podem ser obtidas de kits de coleta reutilizáveis (que permanecem em uso em vários centros, no mundo inteiro), para serem empregadas em pesquisa como uma fonte alternativa e ética.

Effect of low-level laser therapy after rapid maxillary expansion on proliferation and differentiation of osteoblastic cells

The aim of this study was to investigate the osteoblastic activity of cells derived from the midpalatal suture upon treatment with low-level laser therapy (LLLT) after rapid maxillary expansion (RME). A total of 30 rats were divided into two groups: experimental I (15 rats with RME without LLLT) and experimental II (15 rats with RME + LLLT). The rats were euthanized at 24 h, 48 h, and 7 days after RME, when the osteoblastic cells derived from the rats' midpalatal suture were explanted. These cells were cultured for periods up to 17 days, and then in vitro osteogenesis parameters and gene expression markers were evaluated. The cellular doubling time in the proliferative stage (3-7 days) was decreased in cultured cells harvested from the midpalatal suture at 24 and 48 h after RME + LLLT, as indicated by the increased growth of the cells in a culture. Alkaline phosphatase activity at days 7 and 14 of the culture was increased by LLLT in cells explanted from the midpalatal suture at 24 and 48 h and 7 days after RME. The mineralization at day 17 was increased by LLLT after RME in all periods. Results from the real-time PCR demonstrated that cells harvested from the LLLT after RME group showed higher levels of ALP, Runx2, osteocalcin, type I collagen, and bone sialoprotein mRNA than control cells. More pronounced effects on ALP activity, mineralization, and gene expression of bone markers were observed at 48 h after RME and LLLT. These results indicate that the LLLT applied after RME is able to increase the proliferation and the expression of an osteoblastic phenotype in cells derived from the midpalatal suture.

Fibronectin expression is decreased in metastatic renal cell carcinoma following endostatin gene therapy

Tumor cells induce the disruption of homeostasis between cellular and extracellular compartments to favor tumor progression. The expression of fibronectin (FN), a matrix glycoprotein, is increased in several carcinoma cell types, including renal cell carcinoma (RCC). RCC are highly vascularized tumors and are often amenable to antiangiogenic therapy. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we examined the modulation of FN gene expression by ES gene therapy in a murine metastatic renal cell carcinoma (mRCC) model. Balb/C mice bearing Renca cells were treated with NIH/3T3-LXSN cells or NIH/3T3-LendSN cells. At the end of the experiment, the ES serum levels were measured, and the FN gene expression was assessed using real-time PCR. The tissue FN was evaluated by western blotting and by immunofluorescence analysis. The ES serum levels in treated mice were higher than those in the control group (P < 0.05). ES treatment led to significant decreases at the FN mRNA (P < 0.001) and protein levels (P < 0.01). Here, we demonstrate the ES antitumor effect that is mediated by down-regulation of FN expression in mRCC. (C) 2012 Elsevier Masson SAS. All rights reserved.

Infrared (810-nm) low-level laser therapy on rat experimental knee inflammation

Arthritis of the knee is the most common type of joint inflammatory disorder and it is associated with pain and inflammation of the joint capsule. Few studies address the effects of the 810-nm laser in such conditions. Here we investigated the effects of low-level laser therapy (LLLT; infrared, 810-nm) in experimentally induced rat knee inflammation. Thirty male Wistar rats (230-250 g) were anesthetized and injected with carrageenan by an intra-articular route. After 6 and 12 h, all animals were killed by CO(2) inhalation and the articular cavity was washed for cellular and biochemical analysis. Articular tissue was carefully removed for real-time PCR analysis in order to evaluate COX-1 and COX-2 expression. LLLT was able to significantly inhibit the total number of leukocytes, as well as the myeloperoxidase activity with 1, 3, and 6 J (Joules) of energy. This result was corroborated by cell counting showing the reduction of polymorphonuclear cells at the inflammatory site. Vascular extravasation was significantly inhibited at the higher dose of energy of 10 J. Both COX-1 and 2 gene expression were significantly enhanced by laser irradiation while PGE(2) production was inhibited. Low-level laser therapy operating at 810 nm markedly reduced inflammatory signs of inflammation but increased COX-1 and 2 gene expression. Further studies are necessary to investigate the possible production of antiinflammatory mediators by COX enzymes induced by laser irradiation in knee inflammation.

Low-level laser therapy (LLLT) in human progressive-intensity running: effects on exercise performance, skeletal muscle status, and oxidative stress

The aim of this work was to evaluate the effects of low-level laser therapy (LLLT) on exercise performance, oxidative stress, and muscle status in humans. A randomized double-blind placebo-controlled crossover trial was performed with 22 untrained male volunteers. LLLT (810 nm, 200 mW, 30 J in each site, 30 s of irradiation in each site) using a multi-diode cluster (with five spots - 6 J from each spot) at 12 sites of each lower limb (six in quadriceps, four in hamstrings, and two in gastrocnemius) was performed 5 min before a standardized progressive-intensity running protocol on a motor-drive treadmill until exhaustion. We analyzed exercise performance (VO(2 max), time to exhaustion, aerobic threshold and anaerobic threshold), levels of oxidative damage to lipids and proteins, the activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), and the markers of muscle damage creatine kinase (CK) and lactate dehydrogenase (LDH). Compared to placebo, active LLLT significantly increased exercise performance (VO(2 max) p = 0.01; time to exhaustion, p = 0.04) without changing the aerobic and anaerobic thresholds. LLLT also decreased post-exercise lipid (p = 0.0001) and protein (p = 0.0230) damages, as well as the activities of SOD (p = 0.0034), CK (p = 0.0001) and LDH (p = 0.0001) enzymes. LLLT application was not able to modulate CAT activity. The use of LLLT before progressive-intensity running exercise increases exercise performance, decreases exercise-induced oxidative stress and muscle damage, suggesting that the modulation of the redox system by LLLT could be related to the delay in skeletal muscle fatigue observed after the use of LLLT.

In vivo study of laser irradiation of fractionated drug administration based mechanism for effective photodynamic therapy in rat liver

Up-regulation of stress-activated proteins in cancer cells plays a protective role against photodynamic induced apoptosis. Post photodynamic therapy extracted normal rat liver tissue usually shows a fraction of surviving cells, the photodynamic resistant cells, residing in the necrotic region. To treat these photo-dynamic resistant cells a technique has been proposed based on fractionated drug administration of diluted photosensitizer, keeping the net concentration (5 mg/kg) constant, and subsequently varying drug light interval (DLI). Flourescence measurements were made for the presence of photosensitizer in a tissue. For qualitative analysis both histological and morphological studies were made. Although preliminary aim of this approach was not achieved but there were some interesting observation made i.e. for higher dilution of photosensitizer there was a sharp boundary between necrotic and normal portion of tissue. An increase in the absorption coefficient (alpha) from 2.7 -> 2.9 was observed as photosensitizer was diluted while the corresponding threshold dose (D (th)) persistently decreases from (0.10 -> 0.02) J/cm(2) when irradiated with a 635 nm laser fluence of 150 J/cm(2).

Low-level laser therapy (808 nm) reduces inflammatory response and oxidative stress in rat tibialis anterior muscle after cryolesion

Background and Objective Muscle regeneration is a complex phenomenon, involving coordinated activation of several cellular responses. During this process, oxidative stress and consequent tissue damage occur with a severity that may depend on the intensity and duration of the inflammatory response. Among the therapeutic approaches to attenuate inflammation and increase tissue repair, low-level laser therapy (LLLT) may be a safe and effective clinical procedure. The aim of this study was to evaluate the effects of LLLT on oxidative/nitrative stress and inflammatory mediators produced during a cryolesion of the tibialis anterior (TA) muscle in rats. Material and Methods Sixty Wistar rats were randomly divided into three groups (n?=?20): control (BC), injured TA muscle without LLLT (IC), injured TA muscle submitted to LLLT (IRI). The injured region was irradiated daily for 4 consecutive days, starting immediately after the lesion using a AlGaAs laser (continuous wave, 808?nm, tip area of 0.00785?cm2, power 30?mW, application time 47?seconds, fluence 180?J/cm2; 3.8?mW/cm2; and total energy 1.4?J). The animals were sacrificed on the fourth day after injury. Results LLLT reduced oxidative and nitrative stress in injured muscle, decreased lipid peroxidation, nitrotyrosine formation and NO production, probably due to reduction in iNOS protein expression. Moreover, LLLT increased SOD gene expression, and decreased the inflammatory response as measured by gene expression of NF-k beta and COX-2 and by TNF-a and IL-1 beta concentration. Conclusion These results suggest that LLLT could be an effective therapeutic approach to modulate oxidative and nitrative stress and to reduce inflammation in injured muscle. Lasers Surg. Med. 44: 726735, 2012. (c) 2012 Wiley Periodicals, Inc.

In vitro cultivation of canine multipotent mesenchymal stromal cells on collagen membranes treated with hyaluronic acid for cell therapy and tissue regeneration

Support structures for dermal regeneration are composed of biodegradable and bioresorbable polymers, animal skin or tendons, or are bacteria products. The use of such materials is controversial due to their low efficiency. An important area within tissue engineering is the application of multipotent mesenchymal stromal cells (MSCs) to reparative surgery. The combined use of biodegradable membranes with stem cell therapy may lead to promising results for patients undergoing unsuccessful conventional treatments. Thus, the aim of this study was to test the efficacy of using membranes composed of anionic collagen with or without the addition of hyaluronic acid (HA) as a substrate for adhesion and in vitro differentiation of bone marrow-derived canine MSCs. The benefit of basic fibroblast growth factor (bFGF) on the differentiation of cells in culture was also tested. MSCs were collected from dog bone marrow, isolated and grown on collagen scaffolds with or without HA. Cell viability, proliferation rate, and cellular toxicity were analyzed after 7 days. The cultured cells showed uniform growth and morphological characteristics of undifferentiated MSCs, which demonstrated that MSCs successfully adapted to the culture conditions established by collagen scaffolds with or without HA. This demonstrates that such scaffolds are promising for applications to tissue regeneration. bFGF significantly increased the proliferative rate of MSCs by 63% when compared to groups without the addition of the growth factor. However, the addition of bFGF becomes limiting, since it has an inhibitory effect at high concentrations in culture medium.