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PURPOSE: To compare the number and properties of bone marrow stromal cells (BMSCs) collected from bone marrow aspirate concentrate (BMAC) obtained from different harvest sites and from patients of different ages. METHODS: BMAC was obtained from two groups of patients based on age (n = 10 per group): 19.0 ± 2.7 years for the younger and 56.8 ± 12.5 for the older group. In the latter, BMAC was obtained from both iliac crest and proximal tibia for a donor-matched analysis. Mononucleated cell count and CFU-F assay were performed, together with phenotype characterization of BMSCs from iliac crest and proximal tibia, the study of chondrogenic and osteogenic differentiation capacity, histological staining and spectrophotometric quantification, and the analysis of mRNAs expression. RESULTS: Cells derived from iliac crest and proximal tibia showed the same phenotypic pattern at flow cytometry, as well as similar chondrogenic and osteogenic potential. However, a significantly higher number of mononuclear cells per ml was observed in younger patients (3.8 ± 1.8 × 107) compared to older patients (1.2 ± 0.8 × 107) (p < 0.0005). The latter yield, obtained from the iliac crest, was significantly higher than resulting from the BMAC harvested from the proximal tibia in the same group of patients (0.3 ± 0.2 × 107, p < 0.0005). This result was confirmed by the CFU-F analysis at day 10 (15.9 ± 19.4 vs 0.6 ± 1.0, p = 0.001) and day-20 (21.7 ± 23.0 vs 2.9 ± 4.2, p = 0.006). CONCLUSION: Harvest site and age can affect the quality of BMAC. BMSCs obtained from iliac crest and proximal tibia present comparable mesenchymal markers expression as well as osteogenic and chondrogenic differentiation potential, but iliac crest BMAC presents a four times higher number of mononucleated cells with significantly higher clonogenic capacity compared to the tibia. BMAC of younger patients also had a three-time higher number of mononucleated cells. The identification of BMAC characteristics could help to optimize its preparation and to identify the most suitable indications for this orthobiologic treatment in the clinical practice.
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Medula Óssea , Células-Tronco Mesenquimais , Osteogênese , Células-Tronco/metabolismo , Diferenciação CelularRESUMO
Adipose tissue-derived cell-based injectable therapies have been demonstrated to have disease-modifying effects on joint tissues in preclinical studies on animal osteoarthritis (OA) models, but clinical results are heterogeneous and not always satisfactory. The aim of this study was to investigate the influence of adipose tissue properties on the therapeutic effects of the adipose-derived product in an in vitro OA setting. Micro-fragmented adipose tissue (MF-AT) samples were obtained from 21 OA patients (mean age 51.7 ± 11.8 years, mean BMI 25.7 ± 4.1 kg/m2). The analysis of the MF-AT supernatant was performed to analyze the release of inflammatory factors. The effects of MF-AT inflammatory factors were investigated on chondrocytes and synoviocytes gene expression levels. Patients' characteristics were analyzed to explore their influence on MF-AT inflammatory molecules and on the MF-AT effects on the gene expression of chondrocytes and synoviocytes. The study results demonstrated that adipose tissue-derived products may present inflammatory properties that influence the therapeutic potential for OA treatment, with products with a higher pro-inflammatory profile stimulating a higher expression of genes related to a more inflamed and catabolic phenotype. A higher pro-inflammatory cytokine pattern and a higher pro-inflammatory effect were found in adipose tissue-derived products obtained from OA patients with higher BMI.
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Osteoartrite do Joelho , Sinoviócitos , Animais , Osteoartrite do Joelho/metabolismo , Sinoviócitos/metabolismo , Condrócitos/metabolismo , Células Cultivadas , Tecido Adiposo/metabolismoRESUMO
PURPOSE: The mechanisms of action and disease-modifying potential of platelet-rich plasma (PRP) injection for osteoarthritis (OA) treatment are still not fully established. The aim of this systematic review of preclinical evidence was to determine if PRP injections can induce disease-modifying effects in OA joints. METHODS: A systematic review was performed on animal studies evaluating intra-articular PRP injections as treatment for OA joints. A synthesis of the results was performed investigating the disease-modifying effects of PRP by evaluating studies that compared PRP with OA controls or other injectable products, different PRP formulations or injection intervals, and the combination of PRP with other products. The risk of bias was assessed according to the SYRCLE's tool. RESULTS: Forty-four articles were included, for a total of 1251 animals. The publication trend remarkably increased over time. PRP injections showed clinical effects in 80% and disease-modifying effects in 68% of the studies, attenuating cartilage damage progression and reducing synovial inflammation, coupled with changes in biomarker levels. Evidence is limited on the best PRP formulation, injection intervals, and synergistic effect with other injectables. The risk of bias was low in 40%, unclear in 56%, and high in 4% of items. CONCLUSION: Intra-articular PRP injections showed disease-modifying effects in most studies, both at the cartilage and synovial level. These findings in animal OA models can play a crucial role in understanding mechanism of action and structural effects of this biological approach. Nevertheless, the overall low quality of the published studies warrants further preclinical studies to confirm the positive findings, as well as high-level human trials to demonstrate if these results translate into disease-modifying effects when PRP is used in the clinical practice to treat OA. LEVEL OF EVIDENCE: Level II.
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Osteoartrite do Joelho , Plasma Rico em Plaquetas , Animais , Humanos , Ácido Hialurônico/uso terapêutico , Inflamação , Injeções Intra-Articulares , Modelos Animais , Osteoartrite do Joelho/tratamento farmacológico , Resultado do TratamentoRESUMO
PURPOSE: To compare the results of two groups of patients affected by osteochondritis dissecans (OCD) of the knee and treated with either osteochondral autologous transplantation (OAT) or bone-cartilage paste grafting (PG). METHODS: A total of 27 patients affected by OCD lesions of the femoral condyles were included: 15 treated with OAT, 12 with PG, with comparable baseline characteristics (mean age 22.4 ± 7.2 vs. 24.2 ± 8.5 p = n.s., mean defect size 2.2 ± 1 cm2 vs 2.6 ± 1 cm2 p = n.s.). Patients were evaluated pre-operatively and at 24 and 84 months post-operatively with the International Knee Documentation Committee (IKDC) subjective and objective scores. Sport activity level was evaluated with the Tegner activity score. Adverse events and failures were also recorded. RESULTS: The IKDC subjective score improved significantly in both groups. At 24 months, a significant improvement from 53.4 ± 9.1 to 80.8 ± 12.9 (p = 0.005) was obtained in the OAT group and from 44.6 ± 11.0 to 71.4 ± 25.3 in the PG group (p = 0.008). A further statistically significant increase was observed at 84 months in both groups. No significant differences were found between OAT and PG at both follow-ups. One OAT patient required post-operative knee mobilization under narcosis and two complained of donor site symptoms. More failures were documented in the PG vs OAT group (25% vs 0%; p = 0.043). CONCLUSION: Both PG and OAT provided overall satisfactory results up to 84 months follow-up. However, while PG presents the advantages of a less invasive approach with lower adverse events, the higher failure rate of PG should be considered when choosing between these two surgical treatment options for restoration of the articular surface in patients affected by knee OCD.
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Cartilagem Articular , Osteocondrite Dissecante , Adolescente , Adulto , Autoenxertos , Transplante Ósseo , Seguimentos , Humanos , Articulação do Joelho/diagnóstico por imagem , Articulação do Joelho/cirurgia , Osteocondrite Dissecante/cirurgia , Transplante Autólogo , Resultado do Tratamento , Adulto JovemRESUMO
There is an error in the original publication. Affiliation of corresponding author (Giulia Merli) was incorrectly published. Correct presentation is Nano-Biotechnology Laboratory-NaBi IRCCS- Istituto Ortopedico, Rizzoli, Italy.
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PURPOSE: Platelet-rich plasma (PRP) is currently the most exploited strategy in the clinical practice to provide a regenerative stimulus for tendon healing. The aim of the present study was to systematically review the available evidence on the treatment of the main tendon disorders where PRP is currently applied. METHODS: A systematic review of the literature was performed on the use of PRP as a treatment for tendinopathies focusing on the following sites: Achilles tendon, patellar tendon, rotator cuff tendons, and lateral elbow tendons. The following inclusion criteria for relevant articles were used: clinical trials written in English language up to 21 June 2016 on the use of PRP in the conservative or surgical treatment of the aforementioned tendinopathies. RESULTS: The research identified the following clinical trials dealing with the application of PRP in the selected tendons: 19 papers on patellar tendon (6 being RCTs: 4 dealing with PRP conservative application and 2 surgical), 24 papers on Achilles tendon (4 RCTs: 3 conservative and 1 surgical), 29 on lateral elbow tendons (17 RCTs, all conservative), and 32 on rotator cuff (22 RCTs: 18 surgical and 3 conservative). CONCLUSION: Patellar tendons seem to benefit from PRP injections, whereas in the Achilles tendon, PRP application is not indicated neither as a conservative approach nor as a surgical augmentation. Lateral elbow tendinopathy showed an improvement in most of the high-level studies, but the lack of proven superiority with respect to the more simple whole-blood injections still questions its use in the clinical practice. With regard to rotator cuff pathology, the vast majority of surgical RCTs documented a lack of beneficial effects, whereas there is still inconclusive evidence concerning its conservative application in rotator cuff disorders. LEVEL OF EVIDENCE: Systematic review of level I-IV trials, Level IV.
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Plasma Rico em Plaquetas , Lesões do Manguito Rotador/terapia , Tendinopatia/terapia , Tendão do Calcâneo/lesões , Tendinopatia do Cotovelo/terapia , Humanos , Injeções , Ligamento Patelar/lesões , Regeneração , Manguito Rotador , Resultado do TratamentoRESUMO
PURPOSE: The aim of this study was to document the available evidence on the use of regenerative techniques for the treatment of femoral head osteonecrosis (or avascular necrosis of femoral head, AVN) and to understand their benefit compared to core decompression (CD) alone in avoiding failure and the need for total hip replacement (THR). METHODS: The search was conducted on three medical electronic databases according to PRISMA guidelines. The studies reporting number and timing of failures were included in a meta-analysis calculating cumulative survivorship with a Kaplan-Mayer curve. Moreover, the results on failures in treatment groups reported in RCT were compared with those documented in control groups, in order to understand the benefit of biological therapies compared to CD for the treatment of AVN. RESULTS: Forty-eight studies were included in this systematic review, reporting results of different types of regenerative techniques: mesenchymal stem cell implantation in the osteonecrotic area, intra-arterial infiltration with mesenchymal stem cells, implantation of bioactive molecules, or platelet-rich plasma. Overall, reported results were good, with a cumulative survivorship of 80% after ten year follow-up, and better results when regenerative treatments were combined to CD compared to CD alone (89.9% vs 70.6%, p < 0.0001). CONCLUSION: Regenerative therapies offer good clinical results for the treatment of AVN. The combination of CD with regenerative techniques provides a significant improvement in terms of survivorship over time compared with CD alone. Further studies are needed to identify the best procedure and the most suitable patients to benefit from regenerative treatments for AVN.
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Terapia Biológica/métodos , Descompressão Cirúrgica/métodos , Necrose da Cabeça do Fêmur/terapia , Artroplastia de Quadril/estatística & dados numéricos , Necrose da Cabeça do Fêmur/mortalidade , Articulação do Quadril/cirurgia , Humanos , Análise de Sobrevida , Sobrevivência , Resultado do TratamentoRESUMO
Mitochondrial calcium uniporter (MCU) channel is responsible for Ruthenium Red-sensitive mitochondrial calcium uptake. Here, we demonstrate MCU oligomerization by immunoprecipitation and Förster resonance energy transfer (FRET) and characterize a novel protein (MCUb) with two predicted transmembrane domains, 50% sequence similarity and a different expression profile from MCU. Based on computational modelling, MCUb includes critical amino-acid substitutions in the pore region and indeed MCUb does not form a calcium-permeable channel in planar lipid bilayers. In HeLa cells, MCUb is inserted into the oligomer and exerts a dominant-negative effect, reducing the [Ca(2+)]mt increases evoked by agonist stimulation. Accordingly, in vitro co-expression of MCUb with MCU drastically reduces the probability of observing channel activity in planar lipid bilayer experiments. These data unveil the structural complexity of MCU and demonstrate a novel regulatory mechanism, based on the inclusion of dominant-negative subunits in a multimeric channel, that underlies the fine control of the physiologically and pathologically relevant process of mitochondrial calcium homeostasis.
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Canais de Cálcio/química , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Canais de Cálcio/genética , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Homeostase , Humanos , Bicamadas Lipídicas , Potencial da Membrana Mitocondrial , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Terciária de Proteína , Subunidades ProteicasRESUMO
The direct measurement of mitochondrial [Ca(2+)] with highly specific probes demonstrated that major swings in organellar [Ca(2+)] parallel the changes occurring in the cytosol and regulate processes as diverse as aerobic metabolism and cell death by necrosis and apoptosis. Despite great biological relevance, insight was limited by the complete lack of molecular understanding. The situation has changed, and new perspectives have emerged following the very recent identification of the mitochondrial Ca(2+) uniporter, the channel allowing rapid Ca(2+) accumulation across the inner mitochondrial membrane.
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Apoptose/fisiologia , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Animais , Canais de Cálcio/genética , Humanos , Mitocôndrias/genética , NecroseRESUMO
Tendinopathies are common overuse disorders that arise both in athletes and the general population. Available tendon treatments are used both for women and men without distinction. However, the existence of a sex-based difference in tendon biology is widely demonstrated. Since basic research represents the foundation for treatment development, an equal female-male representation should be pursued in preclinical studies. This systematic review quantified the current evidence by analyzing 150 studies on 8231 animals. Preclinical studies largely neglected the importance of sex, none analyzed sex-based differences, and only 4% of the studies reported disaggregated data suitable for the analysis of treatment results in males and females. There is an alarming female under-representation, in particular in the field of injective therapies. Despite the growing awareness on the importance of investigating treatments in both males and females, the investigated field proved resistant from properly designing studies including both sexes, and the lack of sex-representation remains critical.
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Sexismo , Tendinopatia , Animais , Feminino , Humanos , Masculino , Tendinopatia/epidemiologia , Tendinopatia/terapia , TendõesRESUMO
The last decade has seen exponentially growing efforts to exploit the effects of adipose derived stromal cells (ADSC) in the treatment of a wide range of chronic degenerative diseases, including osteoarthritis (OA), the most prevalent joint disorder. In the perspective of developing a cell-free advanced therapy medicinal product, a focus has been recently addressed to the ADSC secretome that lends itself to an allogeneic use and can be further dissected for the selective purification of small extracellular vesicles (sEVs). sEVs can act as "biological drug carriers" to transfer information that mirror the pathophysiology of the providing cells. This is important in the clinical perspective where many OA patients are also affected by the metabolic syndrome (MetS). ADSC from MetS OA patients are dysfunctional and "inflammatory" primed within the adipose tissue. To mimic this condition, we exposed ADSC to IL-1ß, and then we investigated the effects of the isolated sEVs on chondrocytes and synoviocytes, either cultured separately or in co-culture, to tease out the effects of these "IL-1ß primed sEVs" on gene and protein expression of major inflammatory and catabolic OA markers. In comparison with sEVs isolated from unstimulated ADSC, the IL-1ß primed sEVs were able to propagate NF-κB activation in bystander joint cells. The effects were more prominent on synoviocytes, possibly because of a higher expression of binding molecules such as CD44. These findings call upon a careful characterization of the "inflammatory fingerprint" of ADSC to avoid the transfer of an unwanted message as well as the development of in vitro "preconditioning" strategies able to rescue the antiinflammatory/anticatabolic potential of ADSC-derived sEVs.
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Acetic acid is an organic acid of great importance globally and the demand of this product is currently increasing. The production of this acid has consequently aroused more and more interest over the years, especially for more sustainable processes. From a biological point of view, acetic acid can be produced by acetogenesis using inorganic substrates like CO2 or CO (with acetogenic bacteria) and aerobic fermentation (with acetic acid bacteria or fungi). With the aim of investigating the progress of technological innovation, the methodology applied by this review was an analysis of the international patents with the Espacenet platform, which ensured a worldwide invention overview. Another criterion was the selection of a precise period of time, from 1990 to 2020. A patent review is able to create an overview of the inventions designed for the real scale implementation, providing a whole picture of the state of the art of the technological innovation change. In addition, the most representative works of literature, that consider the influence of operating conditions (T, pH, oxygenation), have been analysed for each process. The present review, with an innovative approach focused on the technological innovation change, highlighted the ongoing interest for acetic acid bioproduction by acetogenic and acetic acid bacteria. The number of patents related to acetic acid bacteria was consistent also in the past years, but recently the interest is moving forward the utilization of genetic engineering (36% of the patents) and new substrates, like agriculture waste (26% of the patens), responding to circular economy principles. On the other hand, the acetic acid production by acetogenic bacteria is most recent, with over the 90% of the patents developed in the last 10 years. In this case the interest is mainly focused on the use of synthesis gas as substrate, that could increase the process sustainability.
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Ácido Acético , Invenções , Fermentação , PublicaçõesRESUMO
OBJECTIVE: The aim of this systematic review was to analyze the evidence about the efficacy of the several synovial fluid (SF) biomarkers proposed for knee osteoarthritis (OA), categorizing them by both molecular characteristics and clinical use according to the BIPEDs criteria, to provide a comprehensive and structured overview of the current literature. DESIGN: A systematic review was performed in May 2020 on PubMed, Cochrane Library, and Embase databases about SF biomarkers in patients with knee OA. The search was limited to articles in the last 20 years on human studies, involving patients with knee OA, reporting SF biomarkers. The evidence for each selected SF biomarker was quantified according to the 6 categories of BIPEDs classification. RESULTS: A total of 159 articles were included in the qualitative data synthesis and 201 different SF biomarkers were identified. Among these, several were investigated multiple times in different articles, for a total of 373 analyses. The studies included 13,557 patients with knee OA. The most promising SF biomarkers were C4S, IL-6, IL-8, Leptin, MMP-1/3, TIMP-1, TNF-α, and VEGF. The "burden of disease" and "diagnostic" categories were the most represented with 132 and 106 different biomarkers, respectively. CONCLUSIONS: The systematic review identified numerous SF biomarkers. However, despite the high number of studies on the plethora of identified molecules, the evidence about the efficacy of each biomarker is supported by limited and often conflicting findings. Further research efforts are needed to improve the understanding of SF biomarkers for a better management of patients with knee OA.
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Biomarcadores/análise , Citocinas/metabolismo , Inflamação/metabolismo , Traumatismos do Joelho/metabolismo , Osteoartrite do Joelho , Líquido Sinovial , Humanos , Osteoartrite do Joelho/diagnóstico , Osteoartrite do Joelho/cirurgia , Líquido Sinovial/químicaRESUMO
OBJECTIVES: To quantify the placebo effect of intraarticular injections for knee osteoarthritis in terms of pain, function, and objective outcomes. Factors influencing placebo effect were investigated. DESIGN: Meta-analysis of randomized controlled trials; Level of evidence, 2. PubMed, Web of Science, Cochrane Library, and grey literature databases were searched on January 8, 2020, using the string: (knee) AND (osteoarthritis OR OA) AND (injections OR intra-articular) AND (saline OR placebo). The following inclusion criteria were used: double-blind, randomized controlled trials on knee osteoarthritis, including a placebo arm on saline injections. The primary outcome was pain variation. Risk of bias was assessed using the RoB 2.0 tool, and quality of evidence was graded following the GRADE (Grading of Recommendations Assessment, Development and Evaluation) guidelines. RESULTS: Out of 2,363 records, 50 articles on 4,076 patients were included. The meta-analysis showed significant improvements up to the 6-month follow-up: Visual Analogue Scale (VAS)-pain -13.4 mean difference (MD) (95% confidence interval [CI]: -21.7/-5.1; P < 0.001), Western Ontario and McMaster Osteoarthritis Index (WOMAC)-pain -3.3 MD (95% CI: -3.9/-2.7; P < 0.001). Other significant improvements were WOMAC-stiffness -1.1 MD (95% CI: -1.6/-0.6; P < 0.001), WOMAC-function -10.1 MD (95% CI: -12.2/-8.0; P < 0.001), and Evaluator Global Assessment -21.4 MD (95% CI: -29.2/-13.6; P < 0.001). The responder rate was 52% (95% CI: 40% to 63%). Improvements were greater than the "minimal clinically important difference" for all outcomes (except 6-month VAS-pain). The level of evidence was moderate for almost all outcomes. CONCLUSIONS: The placebo effect of knee injections is significant, with functional improvements lasting even longer than those reported for pain perception. The high, long-lasting, and heterogeneous effects on the scales commonly used in clinical trials further highlight that the impact of placebo should not be overlooked in the research on and management of knee osteoarthritis.
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Osteoartrite do Joelho/tratamento farmacológico , Manejo da Dor , Humanos , Injeções Intra-Articulares , Articulação do Joelho , Dor , Medição da Dor , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
The therapeutic ability of Mesenchymal Stem/Stromal Cells to address osteoarthritis (OA) is mainly related to the secretion of biologically active factors, which can be found within their secreted Extracellular Vesicles including small Extracellular Vesicles (sEV). Aim of this study was to investigate the effects of sEV from adipose derived stromal cells (ADSC) on both chondrocytes and synoviocytes, in order to gain insights into the mechanisms modulating the inflammatory/catabolic OA environment. sEV, obtained by a combined precipitation and size exclusion chromatography method, were quantified and characterized, and administered to chondrocytes and synoviocytes stimulated with IL-1ß. Cellular uptake of sEV was evaluated from 1 to 12 h. Gene expression and protein release of cytokines/chemokines, catabolic and inflammatory molecules were analyzed at 4 and 15 h, when p65 nuclear translocation was investigated to study NF-κB pathway. This study underlined the potential of ADSC derived sEV to affect gene expression and protein release of both chondrocytes and synoviocytes, counteracting IL-1ß induced inflammatory effects, and provided insights into their mechanisms of action. sEV uptake was faster in synoviocytes, where it also elicited stronger effects, especially in terms of cytokine and chemokine modulation. The inflammatory/catabolic environment mediated by NF-κB pathway was significantly attenuated by sEV, which hold promise as new therapeutic strategy to address OA.
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Vesículas Extracelulares/transplante , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , NF-kappa B/metabolismo , Osteoartrite/terapia , Idoso , Western Blotting , Condrócitos/metabolismo , Feminino , Humanos , Inflamação/metabolismo , Inflamação/terapia , Masculino , Transplante de Células-Tronco Mesenquimais/métodos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/ultraestrutura , Microscopia Eletrônica de Transmissão , Pessoa de Meia-Idade , Osteoartrite/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Sinoviócitos/metabolismoRESUMO
Long-term results of autologous chondrocyte implantation and matrix-assisted autologous chondrocyte transplantation in the knee are satisfying, but not enough attention has been paid to the evaluation of failures. Thus, a systematic review of the literature was performed, underlining a failure rate in the 58 included articles of 14.9% among 4294 patients, most of them occurring in the first 5 years after surgery, and with no difference between autologous chondrocyte implantation and matrix-assisted autologous chondrocyte transplantation. Failures are very heterogenously defined in the current literature. A widely accepted definition is needed, and a comprehensive definition taking into consideration the patient's perception of the outcome, not just the surgeon's or researcher's point of view, would be advisable. Finally, there is no agreement on the most appropriate treatment of failures, and further studies are needed to give better indications to properly manage patients failed after cartilage procedures. LEVEL OF EVIDENCE: Level IV.
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Cartilagem Articular/lesões , Condrócitos/transplante , Transtornos Traumáticos Cumulativos/terapia , Traumatismos do Joelho/terapia , Procedimentos Ortopédicos/métodos , Humanos , Reoperação , Transplante Autólogo/métodos , Falha de TratamentoRESUMO
Platelet-Rich Plasma (PRP) is a low-cost procedure to deliver high concentrations of autologous growth factors (GFs). Platelet activation is a crucial step that might influence the availability of bioactive molecules and therefore tissue healing. Activation of PRP from ten voluntary healthy males was performed by adding 10% of CaCl2, 10% of autologous thrombin, 10% of a mixture of CaCl2 + thrombin, and 10% of collagen type I. Blood derivatives were incubated for 15 and 30 minutes and 1, 2, and 24 hours and samples were evaluated for the release of VEGF, TGF-ß1, PDGF-AB, IL-1ß, and TNF-α. PRP activated with CaCl2, thrombin, and CaCl2/thrombin formed clots detected from the 15-minute evaluation, whereas in collagen-type-I-activated samples no clot formation was noticed. Collagen type I produced an overall lower GF release. Thrombin, CaCl2/thrombin, and collagen type I activated PRPs showed an immediate release of PDGF and TGF-ß1 that remained stable over time, whereas VEGF showed an increasing trend from 15 minutes up to 24 hours. CaCl2 induced a progressive release of GFs from 15 minutes and increasing up to 24 hours. The method chosen to activate PRP influences both its physical form and the releasate in terms of GF amount and release kinetic.
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Ca(2+)-sensitive photoproteins are ideal agents for measuring the Ca(2+) concentration ([Ca(2+)]) in intracellular organelles because they can be modified to include specific targeting sequences. Aequorin was the first Ca(2+)-sensitive photoprotein probe used to measure the [Ca(2+)] inside specific intracellular organelles in intact cells. Aequorin is a 22-kDa protein produced by the jellyfish Aequorea victoria. On the binding of Ca(2+) to three high-affinity sites in aequorin, an irreversible reaction occurs in which the prosthetic group is released and a photon is emitted. Aequorin has become widely used for intracellular Ca(2+) measurements because it offers many advantages: For example, it can be targeted with precision, functions over a wide range of [Ca(2+)], and shows low buffering capacity. In this article we describe the main characteristics of the aequorin probe and review the reasons why it is widely used to measure intracellular [Ca(2+)].
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Equorina , Cálcio/análise , Equorina/isolamento & purificação , Animais , Citosol/química , Hidrozoários , Organelas/química , Fótons , Ligação ProteicaRESUMO
Aequorin is a Ca(2+)-sensitive photoprotein isolated from the jellyfish Aequorea victoria. It is an ideal probe for measuring Ca(2+) concentration ([Ca(2+)]) in intracellular organelles because it can be modified to include specific targeting sequences. On the binding of Ca(2+) to three high-affinity sites in aequorin, an irreversible reaction occurs in which the prosthetic group coelenterazine is released and a photon is emitted. This protocol presents procedures for expressing, targeting, and reconstituting aequorin in intact and permeabilized mammalian cells and describes how to use this photoprotein to measure intracellular [Ca(2+)] in various subcellular compartments.