Regenerative potential of the cartilaginous tissue in mesenchymal stem cells: update, limitations, and challenges.

Advances in the studies with adult mesenchymal stem cells (MSCs) have turned tissue regenerative therapy into a promising tool in many areas of medicine. In orthopedics, one of the main challenges has been the regeneration of cartilage tissue, mainly in diarthroses. In the induction of the MSCs, in addition to cytodifferentiation, the microenvironmental context of the tissue to be regenerated and an appropriate spatial arrangement are extremely important factors. Furthermore, it is known that MSC differentiation is fundamentally determined by mechanisms such as cell proliferation (mitosis), biochemical-molecular interactions, movement, cell adhesion, and apoptosis. Although the use of MSCs for cartilage regeneration remains at a research level, there are important questions to be resolved in order to make this therapy efficient and safe. It is known, for instance, that the expansion of chondrocytes in cultivation, needed to increase the number of cells, could end up producing fibrocartilage instead of hyaline cartilage. However, the latest results are promising. In 2014, the first stage I/II clinical trial to evaluate the efficacy and safety of the intra-articular injection of MSCs in femorotibial cartilage regeneration was published, indicating a decrease in injured areas. One issue to be explored is how many modifications in the articulate inflammatory environment could induce differentiation of MSCs already allocated in that region. Such issue arose from studies that suggested that the suppression of the inflammation may increase the efficiency of tissue regeneration. Considering the complexity of the events related to the chondrogenesis and cartilage repair, it can be concluded that the road ahead is still long, and that further studies are needed.

Os avanços nos estudos com células-tronco mesenquimais (CTMs) adultas tornou a terapia regenerativa tecidual uma ferramenta promissora em diversas áreas da medicina. Na ortopedia, um dos principais desafios tem sido a regeneração do tecido cartilaginoso, sobretudo em diartroses. Na indução de CTMs, além da citodiferenciação, o contexto microambiental do tecido a ser regenerado, bem como uma disposição espacial adequada, são fatores de extrema importância. Além disso, sabe-se que a diferenciação das CTMs é basicamente determinada por mecanismos como proliferação celular (mitose), interações bioquímico-moleculares, movimento, adesão celular e apoptose. Apesar de o uso de CTMs para a regeneração da cartilagem estar ainda em âmbito de pesquisa, existem questões importantes a serem resolvidas para tornar essa terapêutica eficaz e segura. Sabe-se, por exemplo, que a expansão de condrócitos em cultura, necessária para aumentar o número de células, pode produzir fibrocartilagem, e não cartilagem hialina. No entanto, os últimos resultados são promissores. Em 2014, foi publicado o primeiro ensaio clínico fase I/II para avaliar a eficácia e a segurança da injeção intra-articular de CTMs na regeneração de cartilagem femorotibial e houve uma diminuição das áreas lesadas. Uma questão a ser explorada é o quanto modificações no próprio ambiente inflamatório articular poderiam induzir a diferenciação de CTMs já alocadas naquela região. Tal incógnita parte do princípio de estudos que sugerem que a supressão da inflamação articular aumentaria, potencialmente, a eficiência da regeneração tecidual. Considerando a complexidade dos eventos relacionados à condrogênese e ao reparo da cartilagem, conclui-se que o caminho ainda é longo, são necessárias pesquisas complementares.

Regenerative potential of the cartilaginous tissue in mesenchymal stem cells: update, limitations, and challenges / Potencial regenerativo do tecido cartilaginoso por células-tronco mesenquimais: atualização, limitações e desafios

ABSTRACT Advances in the studies with adult mesenchymal stem cells (MSCs) have turned tissue regenerative therapy into a promising tool in many areas of medicine. In orthopedics, one of the main challenges has been the regeneration of cartilage tissue, mainly in diarthroses. In the induction of the MSCs, in addition to cytodifferentiation, the microenvironmental context of the tissue to be regenerated and an appropriate spatial arrangement are extremely important factors. Furthermore, it is known that MSC differentiation is fundamentally determined by mechanisms such as cell proliferation (mitosis), biochemical-molecular interactions, movement, cell adhesion, and apoptosis. Although the use of MSCs for cartilage regeneration remains at a research level, there are important questions to be resolved in order to make this therapy efficient and safe. It is known, for instance, that the expansion of chondrocytes in cultivation, needed to increase the number of cells, could end up producing fibrocartilage instead of hyaline cartilage. However, the latest results are promising. In 2014, the first stage I/II clinical trial to evaluate the efficacy and safety of the intra-articular injection of MSCs in femorotibial cartilage regeneration was published, indicating a decrease in injured areas. One issue to be explored is how many modifications in the articulate inflammatory environment could induce differentiation of MSCs already allocated in that region. Such issue arose from studies that suggested that the suppression of the inflammation may increase the efficiency of tissue regeneration. Considering the complexity of the events related to the chondrogenesis and cartilage repair, it can be concluded that the road ahead is still long, and that further studies are needed.

RESUMO Os avanços nos estudos com células-tronco mesenquimais (CTMs) adultas tornou a terapia regenerativa tecidual uma ferramenta promissora em diversas áreas da medicina. Na ortopedia, um dos principais desafios tem sido a regeneração do tecido cartilaginoso, sobretudo em diartroses. Na indução de CTMs, além da citodiferenciação, o contexto microambiental do tecido a ser regenerado, bem como uma disposição espacial adequada, são fatores de extrema importância. Além disso, sabe-se que a diferenciação das CTMs é basicamente determinada por mecanismos como proliferação celular (mitose), interações bioquímico-moleculares, movimento, adesão celular e apoptose. Apesar de o uso de CTMs para a regeneração da cartilagem estar ainda em âmbito de pesquisa, existem questões importantes a serem resolvidas para tornar essa terapêutica eficaz e segura. Sabe-se, por exemplo, que a expansão de condrócitos em cultura, necessária para aumentar o número de células, pode produzir fibrocartilagem, e não cartilagem hialina. No entanto, os últimos resultados são promissores. Em 2014, foi publicado o primeiro ensaio clínico fase I/II para avaliar a eficácia e a segurança da injeção intra-articular de CTMs na regeneração de cartilagem femorotibial e houve uma diminuição das áreas lesadas. Uma questão a ser explorada é o quanto modificações no próprio ambiente inflamatório articular poderiam induzir a diferenciação de CTMs já alocadas naquela região. Tal incógnita parte do princípio de estudos que sugerem que a supressão da inflamação articular aumentaria, potencialmente, a eficiência da regeneração tecidual. Considerando a complexidade dos eventos relacionados à condrogênese e ao reparo da cartilagem, conclui-se que o caminho ainda é longo, são necessárias pesquisas complementares.

Tucumã fruit extracts (Astrocaryum aculeatum Meyer) decrease cytotoxic effects of hydrogen peroxide on human lymphocytes.

This study quantifies the bioactive molecules in and determines the in vitro protective effect of ethanolic extracts isolated from the peel and pulp of tucumã (Astrocaryum aculeatum, Mart.), an Amazonian fruit rich in carotenoids. The cytoprotective effect of tucumã was evaluated in lymphocyte cultures exposed to H2O2 using spectrophotometric, fluorimetric, and immunoassay assays. The results confirmed that tucumã pulp extract is rich in ß-carotene and quercetin, as previously described in the literature. However, high levels of these compounds were also found in tucumã peel extract. The extracts also contained significant amounts rutin, gallic acid, caffeic acid, and chlorogenic acid. Despite quantitative differences in the concentration of these bioactive molecules, both extracts increased the viability of cells exposed to H2O2 in concentrations ranging from 300 to 900 µg/mL. Caspases 1, 3, and 8 decreased significantly in cells concomitantly exposed to H2O2 and these extracts, indicating that tucumã cryoprotection involves apoptosis modulation.

Antimicrobial activity of Amazon Astrocaryum aculeatum extracts and its association to oxidative metabolism.

Several compounds present in fruits as polyphenols are able to kill or inhibit the growth of microorganisms. These proprieties are relevant mainly in tropical areas, as Amazonian region where infectious are highly prevalent. Therefore, this study investigated the antimicrobial activity of tucumã Amazonian fruit against 37 microorganisms. The potential role of oxidative metabolism imbalance was also studied as causal mechanism of antimicrobial activity. The results showed antibacterial effect of pulp and peel tucumã hydro-alcoholic extracts on three Gram-positive bacteria (Enterococcus faecalis, Bacillus cereus, Listeria monocytogenes) and antifungal effect against Candida albicans. The antimicrobial contribution of main chemical compounds (quercetin, rutin, ß-carotene and gallic, caffeic and chlorogenic acids) found in tucumã extracts was also investigated showing an inhibitory effect depending of the organism mainly by quercetin in bacteria and rutin in C. albicans. Analysis of kinetic of DNA releasing in extracellular medium by fluorescence using DNA Pico Green assay(®) and reactive oxygen species production (ROS) showed potential oxidative imbalance contribution on tucumã inhibitory effect. In B. cereus and C. albicans this effect was clear since after 24h the ROS levels were higher when compared to negative control group. In conclusion, tucumã extracts present antimicrobial activity to four microorganisms that have large problems of drug resistance, and the possible mechanism of action of this Amazon fruit is related to REDOX imbalance.

The in vitro genotoxic effect of Tucuma (Astrocaryum aculeatum), an Amazonian fruit rich in carotenoids.

Tucuma (Astrocaryum aculeatum) is an Amazonian fruit that presents high levels of carotenoids and other bioactive compounds such as quercetin. The extracts of tucuma peel and pulp present strong antioxidant activity which illustrate an elevated concentration that causes cytotoxic effects in human peripheral blood mononuclear cells (PBMCs). This study performed additional investigations to analyze the potential genotoxic effects of the tucuma extracts on PBMCs. The genotoxicity was evaluated by DNA fragmentation, Comet assay, and chromosomal instability G-band assays. The acute tucuma extract treatment showed genoprotective effects against DNA denaturation when compared with untreated PBMC cells. However, in the experiments with 24 and 72 h treatments to tucuma treatments, we observed low genotoxicity through a concentration of 100 µg/mL, some genotoxic effects related to intermediary concentrations (100-500 µg/mL), and more pronounced genotoxic effects on higher tucuma extract concentrations. After 24 h of treatment, the reactive oxygen species were similar among treatments and PBMC control groups. However, the caspase-1 activity related to the apoptosis and pyroptosis process increased significantly in higher tucuma concentrations. In summary, tucuma extracts, despite their higher antioxidant content and antioxidant activity, would present PBMCs genotoxic effects that are dependent on concentration and time exposition. These results need to be considered in future in vitro and in vivo studies of tucuma effects.

Isatin-3-N4-benzilthiosemicarbazone, a non-toxic thiosemicarbazone derivative, protects and reactivates rat and human cholinesterases inhibited by methamidophos in vitro and in silico.

Organophosphates (OPs), which are widely used as pesticides, are acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibitors. The inactivation of AChE results in the accumulation of acetylcholine at cholinergic receptor sites, causing a cholinergic crisis that can lead to death. The classical treatment for OP poisoning is administration of oximes, but these compounds are ineffective in some cases. Here we determined whether the new compound isatin-3-N(4)-benzilthiosemicarbazone (IBTC), which in our previous study proved to be an antioxidant and antiatherogenic molecule, could protect and reactivate AChE and BChE. Toxicity of IBTC after subcutaneous injection in mice was measured using assays for oxidized diclorofluoresceine (DCF), thiobarbituric acid reactive substances (TBARS), non-protein thiol (NPSH) levels, and catalase (CAT), sodium potassium (Na(+)/K(+)) ATPase, delta-aminolevulinic acid dehydratase (ALA-D), and glutathione peroxidases (GPx) enzyme activities. The cytotoxicity was evaluated and the enzymatic activity of cholinesterase was measured in human blood samples. Molecular docking was used to predict the mechanism of IBTC interactions with the AChE active site. We found that IBTC did not increase the amount of DCF-RS or TBARS, did not reduce NPSH levels, and did not increase CAT, (Na(+)/K(+)) ATPase, ALA-D, or GPx activities. IBTC protected and reactivated both AChE and BChE activities. Molecular docking predicted that IBTC is positioned at the peripheral anionic site and in the acyl binding pocket of AChE and can interact with methamidophos, releasing the enzyme's active site. Our results suggest that IBTC, besides being an antioxidant and a promising antiatherogenic agent, is a non-toxic molecule for methamidophos poisoning treatment.

Influence of Val16Ala SOD2 polymorphism on the in-vitro effect of clomiphene citrate in oxidative metabolism.

This study investigated the in-vitro antioxidant properties of the ovulation induction drug, clomiphene citrate, and assessed whether its effects are influenced by the Val16Ala polymorphism in the SOD2 gene, which encodes manganese superoxide dismutase enzyme. The investigation involved an in-vitro experimental protocol testing the effect of different concentrations of clomiphene citrate on antioxidant capacity, reactive oxygen species (ROS) production and peripheral blood mononuclear cell (PBMC) culture viability. A total of 58 healthy adult women were genotyped for the Val16Ala SOD2 polymorphism, and blood samples were collected to perform in-vitro experiments. ROS production and cytotoxicity assays were performed on blood and PBMC from carriers of different Val16Ala SOD2 genotypes. Clomiphene citrate exhibited antioxidant capacity and effects and decreased ROS production. The AA genotype displayed a more responsive antioxidant effect with clomiphene citrate treatment than other genotypes. AA and AV PBMC showed an increase in viability following treatment with 10 µmol/l clomiphene citrate when compared with control groups. The results suggest that clomiphene citrate exhibits antioxidant activity similar to that observed with other selective oestrogen receptor modulators, and the intensity of the effect appears to be SOD2 polymorphism dependent. This study was performed to investigate whether clomiphene citrate, a drug broadly used to evaluate reproductive function in women, presents antioxidant effects and if these effects could be influenced by genetic variation in the women. We found evidence that clomiphene citrate has some antioxidant properties similar to those observed with other selective oestrogen receptor modulators such as tamoxifen. As the antioxidant enzyme manganese superoxide dismutase (SOD2) is considered a key molecule involved in female reproductive metabolism, we also tested if a functional SOD2 gene polymorphism (Val16Ala) could influence the in-vitro antioxidant clomiphene citrate response. Significant differences of the clomiphene citrate antioxidant effect on PBMC with different Val16Ala SOD genotypes were observed in this study. Based on these results, we could speculate that alterations in SOD2 activity caused by the Val16Ala polymorphism can result in differential responses to drugs such as clomiphene citrate. In assisted reproduction clinics, clomiphene citrate is commonly used to induce ovulation, especially in patients with polycystic ovary syndrome. However, some women have clomiphene citrate resistance and either ovulation is not triggered by the drug or ovulation is induced but the pregnancy still fails. The causes of no effect of clomiphene citrate remain unclear and we cannot discard the influence of genetic effects including the Val16Ala SOD2 polymorphism. Therefore, it is important to perform complementary investigations considering the potential pharmacogenetic influence of Val16Ala SOD2 polymorphism on the treatment of polycystic ovary syndrome or in ovulation to elucidate this question.