Evaluation of chitosan-GP hydrogel biocompatibility in osteochondral defects: an experimental approach

Background: Articular cartilage, because of its avascular nature, has little capacity for spontaneous healing, and tissue engineering approaches, employing different biomaterials and cells, are under development. Among the investigated biomaterials are the chitosan-based hydrogels. Although thoroughly studied in other mammalian species, studies are scarce in equines. So, the aim of the present study was to investigate the biocompatibility of chitosan-GP in horse joints submitted to high mechanical loads.Results: An osteochondral defect was created by arthroscopy in the medial surface of lateral trochlea of talus of left or right leg, randomly selected, from six healthy geldings. the defect was filled up with chitosan-GP. the contralateral joint received an identical defect with no implant. the chondral fragment removed to produce the defect was collected, processed and used as the Initial sample (normal cartilage) for histology, immunohistochemistry, and metabolic labelling of PGs. After 180 days, the repair tissues were collected, and also analyzed. At the end of the experiment (180 days after lesion), the total number of cells per field in repair tissues was equal to control, and macrophages and polymorphonuclear cells were not detected, suggesting that no significant inflammation was present. These cells were able to synthesize type II collagen and proteoglycans (PGs). Nevertheless, the cell population in these tissues, both in presence of chitosan-GP and in untreated controls, were heterogeneous, with a lower proportion of type II collagen-positives cells and some with a fibroblastic aspect. Moreover, the PGs synthesized in repair tissues formed in presence or absence of chitosan-GP were similar to those of normal cartilage. However, the chitosan-GP treated tissue had an disorganized appearance, and blood vessels were present.Conclusions: Implanted chitosan-GP did not evoke an important inflammatory reaction, and permitted cell growth. These cells were able to synthesize type II collagen and PGs similar to those synthesized in normal cartilage and in healing tissue without implant, indicating its chondrocyte nature.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Univ São Paulo, Fac Med Vet & Zootecnia, Dept Cirurgia, BR-09500900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04044020 São Paulo, BrazilUniv São Paulo, Fac Med Vet & Zootecnia, Dept Clin Med, BR-09500900 São Paulo, BrazilUniv São Paulo, Fac Med Vet & Zootecnia, Dept Patol, BR-09500900 São Paulo, BrazilUniversidade Federal de São Paulo, Escola Paulista Med, Dept Bioquim, BR-04044020 São Paulo, BrazilWeb of Scienc

Impact of high glucose and AGEs on cultured kidney-derived cells. Effects on cell viability, lysosomal enzymes and effectors of cell signaling pathways

We have previously reported decreased expression and activities of lysosomal cathepsins B and L in diabetic kidney. Relevant morphological changes were observed in proximal tubules, suggesting that these cells are implicated in the early stages of the disease. The aim of the present study was to investigate the mechanisms that lead to these changes. The effects of high glucose (HG) and advanced glycation end products (AGEs) on cell viability, lysosomal enzymes and other effectors of cell signaling of cultured kidney cells were studied. HG increased viable mesangial cells (ihMC) in 48 h, while epithelial tubular cells were not affected (LLC-PK1 and MDCK). In contrast, the number of viable cells was markedly decreased, for all cell lines, by AGE-BSA. Concerning lysosomal enzymes, the main cysteine-protease expressed by these cells was cathepsin B, and its concentration was much higher in epithelial than in mesangial cells. Exposure to HG had no effect on the cathepsin B activity, but AGE-BSA caused a marked decrease in LLC-PK1, and increased the enzyme activities in the other cell lines. The levels of nitric oxide (NO) was increased by AGE-BSA in all cell lines, suggesting oxidative stress, and Western blotting has shown that, among the investigated proteins, cathepsin B, mTOR and transcription factor EB (TFEB) were the most significantly affected by exposure to AGE-BSA. As mTOR induces anabolism and inhibits autophagy, and TFEB is a master transcription factor for lysosomal enzymes, it is possible that this pathway plays a role in the inhibition of lysosomal enzymes in proximal tubule cells. Published by Elsevier B.V.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Sao Paulo, SP, BrazilConselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Brasilia, DF, BrazilFundacao Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES), Brasilia, SP, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Bioquim, Disciplina Biol Mol, Rua Tres Maio 100, BR-04044020 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Med, Disciplina Nefrol, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Bioquim, Disciplina Biol Mol, Rua Tres Maio 100, BR-04044020 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Escola Paulista Med, Dept Med, Disciplina Nefrol, Sao Paulo, SP, BrazilFAPESP: 2010/16022-5FAPESP: 2013/07109-8FAPESP: 2015/03964-6CNPq: 301326/2013-4Web of Scienc

Making of an Intelligent Tutoring System (or Methodological Issues of Artificial Intelligence Research by Example)

This paper contains some methodological reflections upon developing good-quality research in Artificial Intelligence. We consider in these reflections research programmes that aim at achieving basic scientific results together with developing techniques for use in applied systems. We focus on an empirical approach to research organisation, in which the results can be assessed through the technical robustness of the scientific theories developed, as well as through the success of applications that are built using the obtained techniques. We discuss this approach through an example, namely we present the development of an Intelligent Tutoring System, and show some issues that can arise when such approach is employed. 1 Introduction Artificial Intelligence is still a new area of research, and one clear indication of this is the fact that the precise definition of what it is about is still rather contentious. Our personal view about how Artificial Intelligence can be defined begins with..

Reliability of 1,9-dimethylmethylene blue tests in comparison to agarose gel electrophoresis for quantification of urinary glycosaminoglycans

Background: the relevance of glycosaminoglycan determination in biological fluids is gradually gaining importance in the literature. Nevertheless, the results obtained by different methods vary widely. We evaluated 1,9-dimethylmethylene blue (DMB) dye-binding assays for quantification of urinary glycosaminoglycans, in comparison to densitometry after agarose gel electrophoresis.Methods: Urinary glycosaminoglycans from different mammalian species were quantified by 3 different DMB dye-binding assays. the results were compared to those obtained by densitometry after agarose gel electrophoresis of glycosaminoglycans isolated from urine samples by ion exchange chromatography.Results: Densitometry after agarose gel electrophoresis showed glycosaminoglycan urinary concentrations of 1-20 mg/l, and glycosaminoglycan/creatinine ratios of 2-25 x 10(-3), for all the mammalian species here studied. A decrease with age was observed for humans, cats and horses. in comparison, DMB assays gave much higher results - up to 200 mg/l and 500 x 10(-3) glycosaminoglycan/creatinine ratios. These values were greatly reduced after 4-h dialysis, suggesting that low molecular weight compounds do interfere. Furthermore, urinary anions such as sulfate, phosphate and citrate, react with metachromatic dyes, such as Toluidine Blue and DMB.Conclusion: DMB assays, although rapid and simple, are not appropriate to quantify urinary glycosaminoglycans in normal mammalians, since other urinary components interfere with the reactions. (c) 2006 Elsevier B.V. All rights reserved.Universidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, SP, BrazilUniv São Paulo, Fac Med Vet & Zootecn, Dept Clin Med, São Paulo, SP, BrazilUniversidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, SP, BrazilWeb of Scienc

Optical Anisotropy Of Alcian Blue-stained Acid Glycosaminoglycans.

Optical anisotropy as dispersion of birefringence (DB) (birefringence studied for light of different wavelengths) and linear dichroism (LD) (selective absorption of polarized light) in stained substrates reflects their macromolecular orientation states. Birefringence interference colors of alcian blue (AB)-stained glycosaminoglycans (GAG) and glycoconjugates observed with polarization microscopy have been found to vary, although their staining characteristics under unpolarized light are practically the same. We investigated the optical anisotropy of GAG-AB and some glycoconjugate-AB complexes used as standards, to provide a basis for interpreting results for AB-stained materials in situ. Filamentous preparations of hyaluronic acid (HA), chondroitinsulfates, proteoglycans, and a mucus sulfoglycoconjugate were studied. Anomalous DB (birefringence sign changing with the wavelength of the incident light) was generally observed, but LD was seen only in the AB-HA complex. LD simultaneous to anomalous DB characteristics on the AB-HA complex were assumed to be caused by a maximally oriented helical conformation of the HA. For the other AB-GAG studied, the optical anisotropic characteristics were suggestive of some degree of folding of their chains into a tertiary structure. The profiles of the anomalous DB curves for the AB-stained sulfoglycoconjugate differed from those of the other materials, probably due to different organization of its dye-binding sites.10978-8

Optical anisotropy of alcian blue-stained acid glycosaminoglycans

Optical anisotropy as dispersion of birefringence (DB) (birefringence studied for light of different wavelengths) and linear dichroism (LD) (selective absorption of polarized Light) in stained substrates reflects their macromolecular orientation states. Birefringence interference colors of alcian blue (AB)-stained glycosamino-glycans (GAG) and glycoconjugates observed with polarization microscopy have been found to vary, although their staining characteristics under unpolarized Light are practically the same. We investigated the optical anisotropy of GAG-AB and some glycoconjugate-AB complexes used as standards, to provide a basis for interpreting results for AB-stained materials in situ. Filamentous preparations of hyaluronic acid (HA), chondroitinsulfates, proteoglycans, and a mucus sulfoglycoconjugate were studied. Anomalous DB (birefringence sign changing with the wavelength of the incident Light) was generally observed, but LD was seen only in the AB-HA complex. LD simultaneous to anomalous DB characteristics on the AB-HA complex were assumed to be caused by a maximally oriented helical conformation of the HA. for the other AB-GAG studied, the optical anisotropic characteristics were suggestive of some degree of folding of their chains into a tertiary structure. the profiles of the anomalous DB curves for the AB-stained sulfoglycoconjugate differed from those of the other materials, probably due to different organization of its dye-binding sites. (C) 2006 Elsevier GmbH. All rights reserved.Univ Estadual Campinas, UNICAMP, Inst Biol, Dept Cell Biol, BR-13083863 Campinas, SP, BrazilUniversidade Federal de São Paulo, Dept Biochem, BR-04044020 São Paulo, BrazilAutonomous Univ Madrid, Dept Biol, E-28049 Madrid, SpainUniversidade Federal de São Paulo, Dept Biochem, BR-04044020 São Paulo, BrazilWeb of Scienc