Jankerle N. Boeloni

Osteogenic differentiation of mesenchymal stem cells from osteopenic rats subjected to physical activity with and without nitric oxide synthase inhibition

Physical activity has potent and complex effects on bones. We hypothesized that physical activity has a positive effect upon osteopenic rat bones because it stimulates osteogenic differentiation of bone marrow mesenchymal stem cells (MSCs). We also postulated that local nitric oxide concentrations mediate the effects of physical activity on bones. The objective of this study was to investigate the osteogenic differentiation in vitro of MSCs from osteopenic female rats subjected to physical activity with and without nitric oxide synthase inhibition. We used MSCs from the femurs of Wistar female rats divided into six groups: Group 1, sham-operated (control); Group 2, sedentary osteopenic; Group 3, active osteopenic; Group 4, sham-operated with L-NAME; Group 5, sedentary osteopenic with L-NAME; and Group 6, active osteopenic with L-NAME. The cells were cultured at 37 degrees C and 5% CO2. Cells were phenotypically characterized with anti-CD45, anti-CD90, anti-CD73, and anti-CD54 using a FACScan cytometer. MSCs were cultured in osteogenic medium for 7, 14 and 21 days. Alkaline phosphatase activity, the capacity of dimethylthiazol conversion in formazan crystals, collagen synthesis and the number of mineralized nodules were analyzed. The means of all of the variables were compared using the SNK test. MSCs did not express CD45 in 96.94% of the cells, but there was expression of CD73, CD54 and CD90 in 93.99%, 95.10% and 86.77% of the cells, respectively. MSCs from osteopenic rats showed less osteogenic differentiation. Surprisingly, physical activity increased the osteogenic differentiation of MSCs in osteopenic rats. Inhibition of nitric oxide synthase in vivo had a negative effect upon the osteogenic potential of MSCs from normal rats and from osteopenic rats subjected to physical activity. Our results suggest that nitric oxide stimulates MSCs osteogenic differentiation and that nitric oxide mediates the beneficial effects of physical activity upon MSCs osteogenic differentiation.

Intra-bone marrow injection of mesenchymal stem cells improves the femur bone mass of osteoporotic female rats

The effect of intra-bone injection of differentiated rat bone marrow mesenchymal stem cells (BMMSCs) into the femur of osteoporotic female rats was studied. Osteoporosis was induced in Wistar female rats by bilateral ovariectomy. Then, 0.75 million BMMSCs isolated from healthy rats were injected into the femurs of osteoporotic rats. Histomorphometric analysis and histology clearly revealed improvements in the treated group as compared to untreated group. In 2 months, the femurs of treated rats, unlike untreated rats, showed trabecular bone percentage almost similar to the femurs from control healthy rats. To confirm the origin of newly formed bone, the experiment was repeated with BMMSCs isolated from green fluorescent protein transgenic rats. Confocal microscopy demonstrated green fluorescent protein-positive cells at the surface of trabecular bone of the treated rats. We investigated in vitro osteogenic differentiation of BMMSCs isolated from osteoporotic rats by studying alkaline phosphatase activity, collagen synthesis, and the ability to form mineralized nodules. Osteoporotic BMMSCs showed less differentiation capabilities as compared to those isolated from healthy rats. The results clearly demonstrated the importance of BMMSCs in osteoporosis and that the disease can be treated by injection of BMMSCs.

Polyhydroxybutyrate-co-hydroxyvalerate structures loaded with adipose stem cells promote skin healing with reduced scarring.

Currently available skin substitutes are still associated with a range of problems including poor engraftment resulting from deficient vascularization, and excessive scar formation, among others. Trying to overcome these issues, this work proposes the combination of poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) structures with adipose-derived stem cells (ASCs) to offer biomechanical and biochemical signaling cues necessary to improve wound healing in a full-thickness model. PHBV scaffold maintained the wound moisture and demonstrated enough mechanical properties to withstand wound contraction. Also, exudate and inflammatory cell infiltration enhanced the degradation of the structure, and thus healing progression. After 28 days all the wounds were closed and the PHBV scaffold was completely degraded. The transplanted ASCs were detected in the wound area only at day 7, correlating with an up-regulation of VEGF and bFGF at this time point that consequently led to a significant higher vessel density in the group that received the PHBV loaded with ASCs. Subsequently, the dermis formed in the presence of the PHBV loaded with ASCs possesses a more complex collagen structure. Additionally, an anti-scarring effect was observed in the presence of the PHBV scaffold indicated by a down-regulation of TGF-β1 and α-SMA together with an increase of TGF-β3, when associated with ASCs. These results indicate that although PHBV scaffold was able to guide the wound healing process with reduced scarring, the presence of ASCs was crucial to enhance vascularization and provide a better quality neo-skin. Therefore, we can conclude that PHBV loaded with ASCs possesses the necessary bioactive cues to improve wound healing with reduced scarring.

Fetal growth restriction in hypothyroidism is associated with changes in proliferative activity, apoptosis and vascularisation of the placenta

The objective of this study was to evaluate fetal weight, histomorphometric changes and proliferative activity, apoptosis and angiogenesis of the placenta in rats with hypothyroidism. Thirty-six adult female rats were divided into two groups with 18 animals each: control and hypothyroidism. Hypothyroidism was induced by daily administration of propylthiouracil (1 mg/animal). The administration began five days before becoming pregnant and the animals were sacrificed at 14 or 19 days of gestation. The control group received a placebo. The number and weight of fetuses and the rate of fetal death was determined, as well as the morphometric characteristics, the immunohistochemical expression of cell division control protein 47 (CDC)-47 and vascular endothelial growth factor (VEGF) and the number of apoptotic cells in the placental disk. The data were analysed by Mann-Whitney U test. Hypothyroidism reduced the weight of fetuses and of the uterus and placenta (P<0.05), altered the thickness of the placental labyrinth and spongiotrophoblast (P<0.05), increased the population of glycogen cells in the spongiotrophoblast (P<0.05), interfered with the vascular development of the placental labyrinth and decreased VEGF expression (P<0.05), reduced the expression of CDC-47 and cellularity and increased the apoptotic rate in the placental disk (P<0.05). We conclude that hypothyroidism affects fetal weight by altering the proliferative activity, apoptosis and vascularisation of the placenta.

Physical activity improves age-related decline in the osteogenic potential of rats' bone marrow-derived mesenchymal stem cells

To examine whether physical activity increases osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs) from adult rats compared with young rats.

Comparison of the osteogenic potential of mesenchymal stem cells from the bone marrow and adipose tissue of young dogs

BackgroundThe aim of the present study was to compare the osteogenic potential of mesenchymal stem cells extracted from the bone marrow (BM-MSCs) and adipose tissue (AD-MSCs) of young dogs. The following parameters were assessed: dimethyl thiazolyl diphenyl tetrazolium (MTT) conversion, alkaline phosphatase (ALP) activity, collagen and mineralised matrix synthesis, and the expressions of osterix, bone sialoprotein (BSP), and osteocalcin (OC).ResultsMTT conversion was greater in BM-MSCs compared to AD-MSCs after 14 and 21 days of differentiation; ALP activity was greater in differentiated AD-MSCs on day 7; collagen synthesis was greater in BM-MSCs on days 14 and 21; the percentage of mineralized area per field was greater in BM-MSCs compared to AD-MSCs; osterix expression was greater in BM-MSCs in days 14 and 21, and BSP and OC expression levels were greater in BM-MSCs at all the investigation time-points.ConclusionsIt was concluded that the osteogenic potential was greater in BM-MSCs than AD-MSCs when extracted from young dogs.

Additive effect of rPb27 immunization and chemotherapy in experimental paracoccidioidomycosis.

Paracoccidioidomycosis, PCM, the major systemic mycosis in Latin America, is caused by the termally dimorphic fungus Paracoccidioides brasiliensis and requires extended periods of chemotherapy with a significant frequency of relapsing disease. The search for new alternatives of treatment is necessary. rPb27 is an antigenic protein from P. brasiliensis that already showed a significant protective activity as a vaccine for PCM in experimental models. The cDNA of rPb27 was subcloned into a pET-DEST 42 plasmid, expressed in E. coli with a his-tag and purified by affinity chromatography. Immunization with this recombinant protein and chemotherapy were used together in an attempt to improve treatment of PCM. For this, BALB/c mice were challenged with pathogenic P. brasiliensis strain and after immunized with rPb27, in the presence of Corynebacterium parvum and Al(OH)3, some groups were also treated with fluconazole. After 40 days of treatment, the combined drug/rPb27 administration controlled PCM in the liver and spleen, with long lasting protection, and largely preserved tissues structures of these organs. Additionally, in the lungs after 40 days of treatment there was a significant reduction in the fungal load and size of lesions. At the same time, the levels of TNF-α were higher than infected-only mice. Moreover, significant levels of anti-rPb27 specific IgG1, IgG2a and IgG2b isotypes were detected in the sera of mice immunized with rPb27 fluconazole treated or not. These results showed an additive protective effect of rPb27 immunization and chemotherapy, suggesting that an rPb27-based vaccine can be used to enhance PCM antifungal treatment.

The combined use of Paracoccidioides brasiliensis Pb40 and Pb27 recombinant proteins enhances chemotherapy effects in experimental paracoccidioidomycosis

Paracoccidioides brasiliensis is the etiological agent of paracoccidioidomycosis (PCM), a chronic granulomatous mycosis prevalent in Latin America, and cell-mediated immunity represents the main mode of protection against this fungal infection. The conventional treatment for this mycosis involves long periods of therapy resulting in sequels and a high frequency of relapse. The search for new alternative methods of treatment is thus necessary. With this aim, the objective of this work was to evaluate the potential of rPb27 and rPb40 immunization to reduce treatment length and the frequency of relapse when used as an adjuvant to fluconazole chemotherapy in experimental PCM. Combined treatment with the drug and the two proteins reduced CFUs in the lung, liver and spleen to undetectable levels and largely preserved the tissue structure of these organs. At the same time, IFN-γ and TNF-α levels were higher in mice treated as described above than in infected-only mice, while very low production of IL-10 and TGF-β was observed in this treated group. Thus, the combined treatment, using immunization with the two recombinant proteins in addition to fluconazole chemotherapy, showed an additive protective effect after intratracheal challenge. These results provide new prospects for immunotherapy as a treatment for PCM.

Comparative study of osteogenic differentiation potential of mesenchymal stem cells derived from bone marrow and adipose tissue of osteoporotic female rats

Abstract Osteoporosis causes reduction of osteogenic differentiation of mesenchymal stem cells (MSCs) from bone marrow and adipose tissue. This study was designed to compare the osteogenic potential of bone marrow mesenchymal stem cells (BMMSCs) and adipose-derived stem cells (ADSCs) of ovariectomized (OVX) rats. MSC were harvested from bone marrow and inguinal fat pads of six OVX rats. The limitations of this report are that cells from different animals were pooled for the purpose of the experiments that were carried out in this study. At 7, 14 and 21 d of osteogenic differentiation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion, alkaline phosphatase activity and gene expression for collagen I, osteocalcin, bone sialoprotein, osteopontin and bone morphogenetic protein-2 bone morphogenetic protein-2 (BMP-2) were analyzed. At 21 d, percentage of cells per field and percentage of mineralized nodule were analyzed. The data were subjected to analysis of variance, and the means were compared by Student–Newman–Keuls test. The cells, regardless of group, showed phenotypic characteristics consistent with stem cells. MTT conversion, alkaline phosphatase activity, percentage of mineralized nodule and expression of collagen I, osteocalcin and BMP-2 of ADSCs from OVX rats were higher when compared to BMMSCs from OVX rats in at least one of the evaluated periods (p < 0.05). However, bone sialoprotein and osteopontin expression were smaller than those observed in BMMSCs for all evaluated periods (p < 0.05). It was concluded that the ADSCs from OVX rats have higher osteogenic potential when compared to BMMSCs from OVX rats. This result suggests that the treatment of osteoporosis with autologous ADSCs may be more efficient.

Triiodotironina não aumenta a diferenciação osteogênica reduzida pela idade de células-tronco mesenquimais da medula óssea de ratas

OBJECTIVE To examine if triiodothyronine (T3) increases osteogenic differentiation in bone marrow mesenchymal stem cells (BMMSCs) of adult rats compared with young rats. MATERIALS AND METHODS BMMSCs were cultured in osteogenic medium and distributed into six groups: 1) BMMSCs of young rats; 2) BMMSCs of adult rats; 3, 4, 5 and 6) BMMSCs of adult rats with T3 (0.01, 1, 100 to 1000 nM). We analyzed alkaline phosphatase activity, dimethylthiazol (MTT) conversion, and collagen synthesis at 7, 14, and 21 days, and percentage of cells per field and number of mineralized nodules at 21 days of differentiation. RESULTS T3 reduced MTT conversion, alkaline phosphatase activity, collagen synthesis, and the synthesis of mineralizalized nodules in at least one of the doses and periods studied (p < 0.05). Values were lower when compared with young and adult rats BMMSCs (p < 0.05) without T3. CONCLUSION T3 has a negative effect on the factors involved in osteogenic differentiation of BMMSC from adult rats.