Browsing by Author "Mendes, AF"
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- Aloenxertos criopreservados no tratamento de defeitos osteocartilagíneosPublication . Judas, F; Mendes, AFA reparação cirúrgica dos defeitos da cartilagem articular representa uma das situações mais difíceis de tratar em Ortopedia. Os aloenxertos osteocartilagíneos devem ser reservados para a reconstrução de defeitos significativos envolvendo cartilagem e osso (> 3 cm de diâmetro e 1 cm de profundidade), isto é, nas lesões demasiado extensas para serem corrigidas através de outras técnicas. Os aloenxertos osteocartilagíneos criopreservados apresentam vantagens em relação aos frescos, que incluem uma maior segurança microbiológica, menor capacidade imunogénica ligada ao tecido ósseo e estão disponíveis em maior número. No entanto, os aloenxertos osteocartilagíneos frescos mantêm uma maior viabilidade condrocitária e, por isso, oferecem um melhor desempenho clínico. Embora permita recuperar um maior número de condrócitos vivos, a utilização de crioprotectores está ainda longe de originar a protecção completa e eficaz de todos os condrócitos presentes na cartilagem articular, o que compromete significativamente o desempenho clínico a médio ou a longo termo dos aloenxertos osteocartilagíneos criopreservados. A combinação de um potente agente crioprotector como parece ser a arbutina, com meios mecânicos capazes de exercer uma pressão adequada poderá ser a chave para se alcançar uma percentagem significativa de condrócitos vivos após o processo de descongelação da cartilagem criopreservada e, assim, poderá assegurar a eficácia clínica, a médio e longo prazos, dos aloenxertos osteocartilagíneos criopreservados.
- Assessment of strategies to increase chondrocyte viability in cryopreserved human osteochondral allografts: evaluation of the glycosylated hydroquinone, arbutin.Publication . Rosa, SC; Gonçalves, J; Judas, F; Lopes, C; Mendes, AFOBJECTIVE: Allogeneic cartilage is used to repair damaged areas of articular cartilage, requiring the presence of living chondrocytes. So far, no preservation method can effectively meet that purpose. Identification of more effective cryoprotective agents (CPAs) can contribute to this goal. The aim of this study was to determine whether the glycosylated hydroquinone, arbutin, alone or in combination with low concentrations of other CPAs, has cryoprotective properties towards human articular cartilage. MATERIAL AND METHODS: Human tibial plateaus were procured from multi-organ donors, with the approval of the Ethics Committee of the University Hospital of Coimbra. The tibial plateaus were treated with or without arbutin (50 or 100mM), alone or in combination with various concentrations of dimethyl sulfoxide (DMSO) and glycerol, for 0.5-1.5h/37 degrees C, then frozen at -20 degrees C and 24h later transferred to a biofreezer at -80 degrees C. Two to 3 months later, thawing was achieved by immersion in cell culture medium at 37 degrees C/1h. Chondrocyte viability was assessed before and after freeze-thawing using a colorimetric assay based on the cell's metabolic activity and fluorescent dyes to evaluate cell membrane integrity. RESULTS: Before freezing, chondrocyte metabolic activity was identical in all the conditions tested. After freeze-thawing, the highest activity, corresponding to 34.2+/-2.1% of that in the Fresh Control, was achieved in tibial plateaus incubated in 50mM arbutin for 1h whereas in those left untreated it was 11.1+/-4.7. Addition of DMSO and glycerol to arbutin did not increase chondrocyte viability any further. Fluorescence microscopy confirmed these results and showed that living chondrocytes were mainly restricted to the superficial cartilage layers. CONCLUSION: Arbutin seems to be an effective cryoprotective agent for osteochondral allografts with potential benefits over DMSO and glycerol.
- Chondrocyte Viability in Fresh and Frozen Large Human Osteochondral Allografts: Effect of Cryoprotective AgentsPublication . Judas, F; Rosa, S; Teixeira, L; Lopes, C; Mendes, AFChondrocyte survival is a major goal for the effective storage and clinical performance of human osteochondral allografts. The majority of animal and human cryopreservation studies conducted so far have been performed in small osteochondral cylinders. Using human tibial plateaus as a model for large osteochondral pieces, this work sought to evaluate the cryoprotective efficiency of glycerol and dimethylsulfoxide (DMSO), and to identify cryopreservation conditions suitable for use in tissue banks. Human tibial plateaus harvested from 7 cadaveric tissue donors were incubated in the presence or absence of cryoprotective agents (CPA): 10% or 15% glycerol and 10% DMSO in a Ham F-12 nutrient mixture. Chondrocyte viability was assessed immediately after thawing, using the MTT reduction assay and a fluorescence microscopic method. The tibial plateaus frozen in the absence of CPA showed a significant decrease in chondrocyte viability. The use of CPA significantly increased chondrocyte viability compared with cartilage frozen without CPA (nearly 50% versus 80% living chondrocytes with 10% glycerol versus 10% DMSO, respectively) relative to that in fresh cartilage. In this regard, 10% DMSO was slightly more effective than either 10% or 15% glycerol, eliciting the recovery of approximately 15% relative to the living chondrocyte content in fresh cartilage. In all conditions, fluorescence microscopic studies showed that surviving chondrocytes were restricted to the superficial cartilage layer. Human tibial plateaus seemed to be a good experimental model to establish cryopreservation methods applicable to large human osteochondral pieces in tissue banks.
- Cortical strut allografting in reconstructive orthopaedic surgeryPublication . Judas, F; Saavedra, MJ; Mendes, AF; Dias, RMany approaches are used in the repair of skeletal defects in reconstructive orthopaedic surgery, and bone grafting is involved in virtually every procedure. Autografting remains the gold standard for replacing bone loss. However, the limited amount of bone that can be harvested and the morbidity associated with that procedure are major constraints to the clinical use of autografts. In contrast, bone allografts can be used in any kind of surgery, whether involving minor defects or major bone loss. Cortical strut allografts unite to host bone through callus formation, restoring bone stock and can be used as an onlay biological plate. These struts can be made from hemicylinders of tibia being fixed to host bone by circumferential metallic cables or by screws. The purpose of this study was to analyze the radiographic outcomes of twelve cryopreserved cortical onlay strut allografts, used in a group of nine patients, for revision hip arthroplasty of the femoral side, to stabilize femoral periprosthetic fractures, to reinforce poor cortical bone and to treat one atrophic femoral nonunion. The average follow-up period was 4.3 years (range, 1.6 to 9 years). No fractures, nonunions or progressive resorption of the bone allografts were observed. All struts were incorporated to the native femur with minimal resorption, within the first year after surgery. There was no failure of any of the allograft reconstructions.The results obtained show that cortical onlay strut allografts, either alone or in conjunction with metallic plate or cancellous bone allografts, are a valuable adjunct for reconstructive surgery of the hip and to treat atrophic femoral nonunion.
- Cortical strut allografting in reconstructive orthopaedic surgeryPublication . Judas, F; Saavedra, MJ; Mendes, AF; Dias, RMany approaches are used in the repair of skeletal defects in reconstructive orthopaedic surgery, and bone grafting is involved in virtually every procedure. Autografting remains the gold standard for replacing bone loss. However, the limited amount of bone that can be harvested and the morbidity associated with that procedure are major constraints to the clinical use of autografts. In contrast, bone allografts can be used in any kind of surgery, whether involving minor defects or major bone loss. Cortical strut allografts unite to host bone through callus formation, restoring bone stock and can be used as an onlay biological plate. These struts can be made from hemicylinders of tibia being fixed to host bone by circumferential metallic cables or by screws. The purpose of this study was to analyze the radiographic outcomes of twelve cryopreserved cortical onlay strut allografts, used in a group of nine patients, for revision hip arthroplasty of the femoral side, to stabilize femoral periprosthetic fractures, to reinforce poor cortical bone and to treat one atrophic femoral nonunion. The average follow-up period was 4.3 years (range, 1.6 to 9 years). No fractures, nonunions or progressive resorption of the bone allografts were observed. All struts were incorporated to the native femur with minimal resorption, within the first year after surgery. There was no failure of any of the allograft reconstructions.The results obtained show that cortical onlay strut allografts, either alone or in conjunction with metallic plate or cancellous bone allografts, are a valuable adjunct for reconstructive surgery of the hip and to treat atrophic femoral nonunion.
- Differential effects of the essential oils of Lavandula luisieri and Eryngium duriaei subsp. juresianum in cell models of two chronic inflammatory diseasesPublication . Rufino, AT; Ferreira, I; Judas, F; Salgueiro, L; Lopes, MC; Cavaleiro, C; Mendes, AFCONTEXT: Effective drugs to treat osteoarthritis (OA) and inflammatory bowel disease (IBD) are needed. OBJECTIVE: To identify essential oils (EOs) with anti-inflammatory activity in cell models of OA and IBD. MATERIALS AND METHODS: EOs from Eryngium duriaei subsp. juresianum (M. Laínz) M. Laínz (Apiaceae), Laserpitium eliasii subsp. thalictrifolium Sennen & Pau (Apiaceae), Lavandula luisieri (Rozeira) Rivas-Martínez (Lamiaceae), Othantus maritimus (L.) Hoff. & Link (Asteraceae), and Thapsia villosa L. (Apiaceae) were analyzed by GC and GC/MS. The anti-inflammatory activity of EOs (5-200 μg/mL) was evaluated by measuring inducible nitric oxide synthase (iNOS) and nuclear factor-κB (NF-κB) activation (total and phosphorylated IκB-α), in primary human chondrocytes and the intestinal cell line, C2BBe1, stimulated with interleukin-1β (IL-1β) or interferon-γ (IFN-γ), IL-1β and tumor necrosis factor-α (TNF-α), respectively. RESULTS: The EO of L. luisieri significantly reduced iNOS (by 54.9 and 81.0%, respectively) and phosphorylated IκB-α (by 87.4% and 62.3%, respectively) in both cell models. The EO of E. duriaei subsp. juresianum caused similar effects in human chondrocytes, but was inactive in intestinal cells, even at higher concentrations. The EOs of L. eliasii subsp. thalictrifolium and O. maritimus decreased iNOS expression by 45.2 ± 8.7% and 45.2 ± 6.2%, respectively, in C2BBe1 cells and were inactive in chondrocytes. The EO of T. villosa was inactive in both cell types. DISCUSSION AND CONCLUSION: This is the first study showing anti-inflammatory effects of the EOs of L. luisieri and E. duriaei subsp. juresianum. These effects are specific of the cell type and may be valuable to develop new therapies or as sources of active compounds with improved efficacy and selectivity towards OA and IBD.
- Evaluation of the anti-inflammatory, anti-catabolic and pro-anabolic effects of E-caryophyllene, myrcene and limonene in a cell model of osteoarthritisPublication . Rufino, AT; Ribeiro, M; Sousa, C; Judas, F; Salgueiro, L; Cavaleiro, C; Mendes, AFOsteoarthritis is a progressive joint disease and a major cause of disability for which no curative therapies are yet available. To identify compounds with potential anti-osteoarthritic properties, in this study, we screened one sesquiterpene, E-caryophyllene, and two monoterpenes, myrcene and limonene, hydrocarbon compounds for anti-inflammatory, anti-catabolic and pro-anabolic activities in human chondrocytes. At non-cytotoxic concentrations, myrcene and limonene inhibited IL-1β-induced nitric oxide production (IC50=37.3μg/ml and 85.3µg/ml, respectively), but E-caryophyllene was inactive. Myrcene, and limonene to a lesser extent, also decreased IL-1β-induced NF-κB, JNK and p38 activation and the expression of inflammatory (iNOS) and catabolic (MMP-1 and MMP-13) genes, while increasing the expression of anti-catabolic genes (TIMP-1 and -3 by myrcene and TIMP-1 by limonene). Limonene increased ERK1/2 activation by 30%, while myrcene decreased it by 26%, relative to IL-1β-treated cells. None of the compounds tested was able to increase the expression of cartilage matrix-specific genes (collagen II and aggrecan), but both compounds prevented the increased expression of the non-cartilage specific, collagen I, induced by IL-1β. These data show that myrcene has significant anti-inflammatory and anti-catabolic effects in human chondrocytes and, thus, its ability to halt or, at least, slow down cartilage destruction and osteoarthritis progression warrants further investigation.
- Expression and function of K(ATP) channels in normal and osteoarthritic human chondrocytes: Possible role in glucose sensingPublication . Rufino, AT; Rosa, SC; Judas, F; Mobasheri, A; Lopes, MC; Mendes, AF
- Expression and function of K(ATP) channels in normal and osteoarthritic human chondrocytes: Possible role in glucose sensing.Publication . Rufino, AT; Rosa, SC; Judas, F; Mobasheri, A; Lopes, MC; Mendes, AFATP-sensitive potassium [K(ATP)] channels sense intracellular ATP/ADP levels, being essential components of a glucose-sensing apparatus in various cells that couples glucose metabolism, intracellular ATP/ADP levels and membrane potential. These channels are present in human chondrocytes, but their subunit composition and functions are unknown. This study aimed at elucidating the subunit composition of K(ATP) channels expressed in human chondrocytes and determining whether they play a role in regulating the abundance of major glucose transporters, GLUT-1 and GLUT-3, and glucose transport capacity. The results obtained show that human chondrocytes express the pore forming subunits, Kir6.1 and Kir6.2, at the mRNA and protein levels and the regulatory sulfonylurea receptor (SUR) subunits, SUR2A and SUR2B, but not SUR1. The expression of these subunits was no affected by culture under hyperglycemia-like conditions. Functional impairment of the channel activity, using a SUR blocker (glibenclamide 10 or 20 nM), reduced the protein levels of GLUT-1 and GLUT-3 by approximately 30% in normal chondrocytes, while in cells from cartilage with increasing osteoarthritic (OA) grade no changes were observed. Glucose transport capacity, however, was not affected in normal or OA chondrocytes. These results show that K(ATP) channel activity regulates the abundance of GLUT-1 and GLUT-3, although other mechanisms are involved in regulating the overall glucose transport capacity of human chondrocytes. Therefore, K(ATP) channels are potential components of a broad glucose sensing apparatus that modulates glucose transporters and allows human chondrocytes to adjust to varying extracellular glucose concentrations. This function of K(ATP) channels seems to be impaired in OA chondrocytes.
- Expression and function of the insulin receptor in normal and osteoarthritic human chondrocytes: modulation of anabolic gene expression, glucose transport and GLUT-1 content by insulinPublication . Rosa, SC; Rufino, AT; Judas, F; Tenreiro, C; Lopes, MC; Mendes, AFOBJECTIVE: Chondrocytes respond to insulin, but the presence and role of the specific high affinity insulin receptor (InsR) has never been demonstrated. This study determined whether human chondrocytes express the InsR and compared its abundance and function in normal and osteoarthritis (OA) human chondrocytes. DESIGN: Cartilage sections were immunostained for detection of the InsR. Non-proliferating chondrocyte cultures from normal and OA human cartilage were treated with 1nM or 10nM insulin for various periods. InsR, insulin-like growth factor receptor (IGFR), aggrecan and collagen II mRNA levels were assessed by real time RT-PCR. InsR, glucose transporter (GLUT)-1, phospho-InsRbeta and phospho-Akt were evaluated by western blot and immunofluorescence. Glucose transport was measured as the uptake of [3H]-2-Deoxy-d-Glucose (2-DG). RESULTS: Chondrocytes staining positively for the InsR were scattered throughout the articular cartilage. The mRNA and protein levels of the InsR in OA chondrocytes were approximately 33% and 45%, respectively, of those found in normal chondrocytes. Insulin induced the phosphorylation of the InsRbeta subunit. Akt phosphorylation and 2-DG uptake increased more intensely in normal than OA chondrocytes. Collagen II mRNA expression increased similarly in normal and OA chondrocytes while aggrecan expression remained unchanged. The Phosphoinositol-3 Kinase (PI3K)/Akt pathway was required for both basal and insulin-induced collagen II expression. CONCLUSIONS: Human chondrocytes express functional InsR that respond to physiologic insulin concentrations. The InsR seems to be more abundant in normal than in OA chondrocytes, but these still respond to physiologic insulin concentrations, although some responses are impaired while others appear fully activated. Understanding the mechanisms that regulate the expression and function of the InsR in normal and OA chondrocytes can disclose new targets for the development of innovative therapies for OA.