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1.
Int J Mol Sci ; 22(12)2021 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-34203791

RESUMO

For in vitro modeling of human joints, osteochondral explants represent an acceptable compromise between conventional cell culture and animal models. However, the scarcity of native human joint tissue poses a challenge for experiments requiring high numbers of samples and makes the method rather unsuitable for toxicity analyses and dosing studies. To scale their application, we developed a novel method that allows the preparation of up to 100 explant cultures from a single human sample with a simple setup. Explants were cultured for 21 days, stimulated with TNF-α or TGF-ß3, and analyzed for cell viability, gene expression and histological changes. Tissue cell viability remained stable at >90% for three weeks. Proteoglycan levels and gene expression of COL2A1, ACAN and COMP were maintained for 14 days before decreasing. TNF-α and TGF-ß3 caused dose-dependent changes in cartilage marker gene expression as early as 7 days. Histologically, cultures under TNF-α stimulation showed a 32% reduction in proteoglycans, detachment of collagen fibers and cell swelling after 7 days. In conclusion, thin osteochondral slice cultures behaved analogously to conventional punch explants despite cell stress exerted during fabrication. In pharmacological testing, both the shorter diffusion distance and the lack of need for serum in the culture suggest a positive effect on sensitivity. The ease of fabrication and the scalability of the sample number make this manufacturing method a promising platform for large-scale preclinical testing in joint research.


Assuntos
Osso e Ossos/fisiologia , Custos e Análise de Custo , Técnicas de Cultura de Tecidos/economia , Técnicas de Cultura de Tecidos/métodos , Idoso , Idoso de 80 Anos ou mais , Agrecanas/genética , Agrecanas/metabolismo , Biomarcadores/metabolismo , Cartilagem Articular/metabolismo , Proliferação de Células , Sobrevivência Celular , Condrócitos/citologia , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Matriz Extracelular/metabolismo , Feminino , Humanos , Antígeno Ki-67/metabolismo , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Esclerose , Sobrevivência de Tecidos , Transcrição Genética , Fator de Necrose Tumoral alfa/metabolismo
2.
Aging (Albany NY) ; 13(8): 10891-10919, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33864446

RESUMO

Alzheimer's disease (AD) is frequently accompanied by progressing weight loss, correlating with mortality. Counter-intuitively, weight loss in old age might predict AD onset but obesity in midlife increases AD risk. Furthermore, AD is associated with diabetes-like alterations in glucose metabolism. Here, we investigated metabolic features of amyloid precursor protein overexpressing APP23 female mice modeling AD upon long-term challenge with high-sucrose (HSD) or high-fat diet (HFD). Compared to wild type littermates (WT), APP23 females were less prone to mild HSD-induced and considerable HFD-induced glucose tolerance deterioration, despite unaltered glucose tolerance during normal-control diet. Indirect calorimetry revealed increased energy expenditure and hyperactivity in APP23 females. Dietary interventions, especially HFD, had weaker effects on lean and fat mass gain, steatosis and adipocyte hypertrophy of APP23 than WT mice, as shown by 1H-magnetic-resonance-spectroscopy, histological and biochemical analyses. Proteome analysis revealed differentially regulated expression of mitochondrial proteins in APP23 livers and brains. In conclusion, hyperactivity, increased metabolic rate, and global mitochondrial dysfunction potentially add up to the development of AD-related body weight changes in APP23 females, becoming especially evident during diet-induced metabolic challenge. These findings emphasize the importance of translating this metabolic phenotyping into human research to decode the metabolic component in AD pathogenesis.


Assuntos
Adipócitos/patologia , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Fígado Gorduroso/diagnóstico , Intolerância à Glucose/diagnóstico , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Encéfalo/patologia , Dieta Hiperlipídica/efeitos adversos , Sacarose na Dieta/administração & dosagem , Sacarose na Dieta/efeitos adversos , Modelos Animais de Doenças , Metabolismo Energético/genética , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Feminino , Intolerância à Glucose/etiologia , Intolerância à Glucose/metabolismo , Intolerância à Glucose/patologia , Humanos , Hipertrofia/diagnóstico , Hipertrofia/etiologia , Hipertrofia/metabolismo , Hipertrofia/patologia , Fígado/patologia , Camundongos , Camundongos Transgênicos , Índice de Gravidade de Doença
3.
Sci Rep ; 10(1): 18215, 2020 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-33106576

RESUMO

Glucose hypometabolism potentially contributes to Alzheimer's disease (AD) and might even represent an underlying mechanism. Here, we investigate the relationship of diet-induced metabolic stress and AD as well as the therapeutic potential of chia seeds as a modulator of glucose metabolism in the APP23 mouse model. 4-6 (pre-plaque stage, PRE) and 28-32 (advanced-plaque stage, ADV) weeks old APP23 and wild type mice received pretreatment for 12 weeks with either sucrose-rich (SRD) or control diet, followed by 8 weeks of chia seed supplementation. Although ADV APP23 mice generally showed functioning glucose homeostasis, they were more prone to SRD-induced glucose intolerance. This was accompanied by elevated corticosterone levels and mild insulin insensitivity. Chia seeds improved spatial learning deficits but not impaired cognitive flexibility, potentially mediated by amelioration of glucose tolerance, attenuation of corticosterone levels and reversal of SRD-induced elevation of pro-inflammatory cytokine levels. Since cognitive symptoms and plaque load were not aggravated by SRD-induced metabolic stress, despite enhanced neuroinflammation in the PRE group, we conclude that impairments of glucose metabolism do not represent an underlying mechanism of AD in this mouse model. Nevertheless, chia seeds might provide therapeutic potential in AD as shown by the amelioration of cognitive symptoms.


Assuntos
Doença de Alzheimer/dietoterapia , Precursor de Proteína beta-Amiloide/genética , Cognição/efeitos dos fármacos , Modelos Animais de Doenças , Glucose/metabolismo , Resistência à Insulina , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Ração Animal , Animais , Dieta , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Salvia/química , Sementes/química
4.
J Orthop Res ; 37(8): 1723-1729, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30977553

RESUMO

There is evidence that the application of mesenchymal stromal cells (MSCs) counteracts osteoarthritis (OA) progression. However, the prospect of extracting and expanding these cells might be limited. The aim of this study was to investigate whether hyaluronic acid (HA) supplemented with MSC-recruiting chemokine C-C motif ligand 25 (CCL25) can influence the natural course of spontaneous OA in the guinea pig. CCL25 concentration in synovial fluid (SF) was quantified with enzyme-linked immunosorbent assay. Boyden chamber cell migration assay was used to test CCL25-mediated migration of guinea pig MSC. Forty-nine 11-month-old male guinea pigs were divided into seven groups. The main treatments consisted of five intra-articular injections of HA in pure form and in combination with three doses of CCL25 (63, 693, and 6,993 pg) given at a weekly interval. The severity of cartilage damage was assessed by using a modified Mankin score. The measured average physiological concentration of CCL25 in SF of animals is 85 ± 39 pg/ml. MSC showed a 3.2-fold increase in cell migration at 1,000 nM CCL25 in vitro demonstrating the biological migratory activity of CCL25 on these cells. In vivo, treatment with HA alone did not reduce OA progression. Similarly, OA scores were not found significantly reduced after treatment with 63 pg CCL25 + HA. However, when compared to pure HA, treatment with 693 pg CCL25 + HA and 6,993 pg CCL25 + HA significantly reduced the OA score from 10.1 to 7.4 (-28%) and 8.4 (-20%), respectively. These data suggest that intra-articular injections of HA supplemented with CCL25 attenuates OA. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1723-1729, 2019.


Assuntos
Artrite Experimental/tratamento farmacológico , Quimiocinas CC/uso terapêutico , Ácido Hialurônico/uso terapêutico , Osteoartrite do Joelho/tratamento farmacológico , Viscossuplementos/uso terapêutico , Animais , Cartilagem Articular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Quimiocinas CC/metabolismo , Quimiocinas CC/farmacologia , Avaliação Pré-Clínica de Medicamentos , Cobaias , Ácido Hialurônico/farmacologia , Injeções Intra-Articulares , Masculino , Células-Tronco Mesenquimais/efeitos dos fármacos , Líquido Sinovial/metabolismo , Viscossuplementos/farmacologia
5.
J Biomed Mater Res B Appl Biomater ; 107(3): 490-500, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-29663644

RESUMO

This study aimed to evaluate the potential of an anti-inflammatory polyethylene glycol (PEG) hydrogel for osteoarthritis (OA) management in an OA in vitro model. Freshly isolated porcine chondrocytes were maintained in high-density cultures to form cartilage-like three-dimensional micromasses. Recombinant porcine tumor necrosis factor-alpha (TNF-α) was used to induce OA-like changes. Normal and OA-like micromasses were treated with dendritic polyglycerol sulfate-based PEG hydrogel. Live/dead staining showed that all micromasses remained vital and presented similar morphological characteristics. Safranin-O staining demonstrated a typical depletion of glycosaminoglycans in TNF-α-treated micromasses but not in the presence of the hydrogel. There was no distinct difference in immunohistochemical detection of type II collagen. Microarray data showed that rheumatoid arthritis and TNF signaling pathways were down regulated in hydrogel-treated OA-like micromasses compared to nontreated OA-like micromasses. The hydrogel alone did not affect genes related to OA such as ANPEP, COMP, CXCL12, PTGS2, and TNFSF10, but it prevented their regulation caused by TNF-α. This study provides valuable insights toward a fully synthetic hydrogel for the intra-articular treatment of OA. The findings proved the potential of this hydrogel to prevent the development of TNF-α-induced OA with regard to proteoglycan loss and TNF-α-induced expression pattern without additional signs of differentiation and inflammation. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 490-500, 2019.


Assuntos
Materiais Biomiméticos , Regulação da Expressão Gênica/efeitos dos fármacos , Hidrogéis , Osteoartrite , Polietilenoglicóis , Proteoglicanas/metabolismo , Fator de Necrose Tumoral alfa/toxicidade , Animais , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Modelos Animais de Doenças , Hidrogéis/química , Hidrogéis/farmacologia , Osteoartrite/induzido quimicamente , Osteoartrite/tratamento farmacológico , Osteoartrite/metabolismo , Osteoartrite/patologia , Polietilenoglicóis/química , Polietilenoglicóis/farmacologia , Suínos
6.
Int J Mol Sci ; 19(5)2018 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-29783732

RESUMO

The aim of this study is to identify gene expression profiles associated with hyaluronic acid (HA) treatment of normal and osteoarthritis (OA)-like tissue-engineered cartilage. 3D cartilage micromasses were treated with tumour necrosis factor-α (TNF-α) (OA-inducer) and/or HA for 7 days. Viability was examined by PI/FDA staining. To document extracellular matrix (ECM) formation, glycosaminoglycans (GAG) were stained with Safranin-O and cartilage-specific type II collagen was detected immunohistochemically. Genome-wide gene expression was determined using microarray analysis. Normal and OA-like micromasses remained vital and showed a spherical morphology and homogenous cell distribution regardless of the treatment. There was no distinct difference in immunolabeling for type II collagen. Safranin-O staining demonstrated a typical depletion of GAG in TNF-α-treated micromasses (-73%), although the extent was limited in the presence of HA (-39%). The microarray data showed that HA can influence the cartilage metabolism via upregulation of TIMP3 in OA-like condition. The upregulation of VEGFA and ANKRD37 genes implies a supportive role of HA in cartilage maturation and survival. The results of this study validate the feasibility of the in vitro OA model for the investigation of HA. On the cellular level, no inhibiting or activating effect of HA was shown. Microarray data demonstrated a minor impact of HA on gene expression level.


Assuntos
Cartilagem/efeitos dos fármacos , Ácido Hialurônico/farmacologia , Osteoartrite/metabolismo , Transcriptoma , Animais , Cartilagem/metabolismo , Células Cultivadas , Matriz Extracelular , Osteoartrite/genética , Suínos , Engenharia Tecidual , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo
7.
Colloids Surf B Biointerfaces ; 159: 477-483, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28841497

RESUMO

Osteoarthritis (OA) is a disabling condition especially in the elderly population. The current therapeutic approaches do not halt the OA progression or reverse joint damage. In order to overcome the problem of rapid clearance of hyaluronic acid (HA), a standard viscosupplement for OA, we investigated the rheological properties of a relatively non-degradable dendritic polyglycerol sulfate (dPGS) hydrogel to determine a suitable concentration for intra articular injections that mimics HA in terms of its viscoelastic and mechanical properties. To do so, the concentration range from 3.6 to 4.8wt% of dPGS and, as a reference, blends of commercially available HAs (Ostenil®, GO-ON®, Synocrom® Forte and Synvisc®), were investigated by means of oscillating and flow rheology, thereby yielding storage (G') and loss modulus (G"), as well as yield stress and shear viscosity. In our rheological experiments we observe a pronounced coupling of the molecular weight and the rheological properties for the HAs. Furthermore, we find the dPGS hydrogel to form more compact networks with increasing concentration. From a broader comparison the current findings suggest that an overall polymer concentration of 4.0wt% dPGS has viscoelastic properties that are comparable to hyaluronic acid in the medically relevant frequency range, where for medical application the dPGS hydrogel has the advantage of being much less easily displaced from its injection place than HA.


Assuntos
Hidrogéis/química , Osteoartrite/terapia , Reologia/métodos , Ácido Hialurônico/química , Hidrogéis/uso terapêutico , Resistência ao Cisalhamento , Viscosidade
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