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Background: Platelet Rich Plasma (PRP) has recently emerged as a potential treatment for osteoarthritis (OA), but composition heterogeneity hampers comparison among studies, with the result that definite conclusions on its efficacy have not been reached. Objective: 1) To develop a novel methodology to prepare a series of standardized PRP releasates (PRP-Rs) with known absolute platelet concentrations, and 2) To evaluate the influence of this standardization parameter on the anti-inflammatory properties of these PRP-Rs in an in vitro and an ex vivo model of OA. Methods: A series of PRPs was prepared using the absolute platelet concentration as the standardization parameter. Doses of platelets ranged from 0% (platelet poor plasma, PPP) to 1.5·105 platelets/µl. PRPs were then activated with CaCl2 to obtain releasates (PRP-R). Chondrocytes were stimulated with 10% of each PRP-R in serum-free culture medium for 72 h to assess proliferation and viability. Cells were co-stimulated with interleukin (IL)-1β (5 ng/ml) and 10% of each PRP-R for 48 h to determine the effects on gene expression, secretion and intra-cellular content of common markers associated with inflammation, catabolism and oxidative stress in OA. OA cartilage explants were co-stimulated with IL-1β (5 ng/ml) and 10% of either PRP-R with 0.75·105 platelets/µl or PRP-R with 1.5·105 platelets/µl for 21 days to assess matrix inflammatory degradation. Results: Chondrocyte viability was not affected, and proliferation was dose-dependently increased. The gene expression of all pro-inflammatory mediators was significantly and dose-independently reduced, except for that of IL-1β and IL-8. Immunoblotting corroborated this effect for inducible NO synthase (NOS2). Secreted matrix metalloproteinase-13 (MMP-13) was reduced to almost basal levels by the PRP-R from PPP. Increasing platelet dosage led to progressive loss to this anti-catabolic ability. Safranin O and toluidine blue stains supported the beneficial effect of low platelet dosage on cartilage matrix preservation. Conclusion: We have developed a methodology to prepare PRP releasates using the absolute platelet concentration as the standardization parameter. Using this approach, the composition of the resulting PRP derived product is independent of the donor initial basal platelet count, thereby allowing the evaluation of its effects objectively and reproducibly. In our OA models, PRP-Rs showed antiinflammatory, anti-oxidant and anti-catabolic properties. Platelet enrichment could favor chondrocyte proliferation but is not necessary for the above effects and could even be counter-productive.

[Abstract] Healthy cartilage maintenance relies on an equilibrium among the anabolic and catabolic processes in chondrocytes. With the onset of osteoarthritis (OA), increased interleukin (IL)-1β levels induce an inhibition of the synthesis of extracellular matrix (ECM) proteins, as well as an increase in proteases. This eventually leads to a predominance of the catabolic phenotype and the progressive loss of articular cartilage. Hydrogen sulfide (H2S) is a small gaseous molecule recognized as the third endogenous gasotransmitter. When administered exogenously, it has shown anti-inflammatory and anti-catabolic properties in several in vitro and in vivo models. Here, OA cartilage disks were co-cultured in vitro with IL-1β (5 ng/ml) and NaSH or GYY4137 (200 or 1000 μM) for 21 days. The ability of these two H2S-producing compounds to avoid long term extracellular matrix (ECM) destruction was evaluated. We used a glycosaminoglycan (GAG) quantification kit histology and immunohistochemistry (IHC) to evaluate matrix proteins degradation and matrix metalloproteinases (MMP) abundance. Through the GAGs quantification assay, safranin O (S-O) and toluidine blue (TB) stains, and keratan/chondroitin sulfate (KS/ChS) IHCs it was shown that co-stimulation with H2S-forming reagents effectively avoided GAGs destruction. Both Masson's trichrome (MT) stain and collagen (col) type II IHC, as well as aggrecan (agg) IHC demonstrated that not only were these proteins protected but even promoted, their abundance being higher than in the basal condition. Further, stains also demonstrated that positivity in the inter-territorial and intra-cellular for the different matrix components were rescued, suggesting that NaSH and GYY4137 might also have pro-anabolic effects. In addition, a clear protective effect against the increased MMPs levels was seen, since increased MMP3 and 13 levels were subsequently reduced with the co-stimulation with sulfide compounds. In general, GYY4137 was more effective than NaSH, and increasing the dose improved the results. This study demonstrates that H2S anti-catabolic effects, which had been previously proven in short-term (24–48 h) in vitro cellular models, are maintained over time directly in OA cartilage tissue. Instituto de Salud Carlos III; PI12/00329

Osteoarthritis is the most prevalent rheumatic disease. During disease progression, differences have been described in the prevalence of chondroitin sulfate (CS) isomers. Marine derived-CS present a higher proportion of the 6S isomer, offering therapeutic potential. Accordingly, we evaluated the effect of exogenous supplementation of CS, derived from the small spotted catshark (Scyliorhinus canicula), blue shark (Prionace glauca), thornback skate (Raja clavata) and bovine CS (reference), on the proliferation of osteochondral cell lines (MG-63 and T/C-28a2) and the chondrogenic differentiation of mesenchymal stromal cells (MSCs). MG-G3 proliferation was comparable between R. clavata (CS-6 intermediate ratio) and bovine CS (CS-4 enrichment), for concentrations below 0.5 mg/mL, defined as a toxicity threshold. T/C-28a2 proliferation was significantly improved by intermediate ratios of CS-6 and -4 isomers (S. canicula and R. clavata). A dose-dependent response was observed for S. canicula (200 &micro, g/mL vs 50 and 10 &micro, g/mL) and bovine CS (200 and 100 &micro, g/mL vs 10 &micro, g/mL). CS sulfation patterns discretely affected MSCs chondrogenesis, even though S. canicula and R. clavata CS up-regulated chondrogenic markers expression (aggrecan and collagen type II) these were not statistically significant. We demonstrate that intermediate values of CS-4 and -6 isomers improve cell proliferation and offer potential for chondrogenic promotion, although more studies are needed to elucidate its mechanism of action.

[Abstract] Osteoarthritis (OA) is the most common form of arthritis and it is a leading cause of disability in the elderly. Its complete etiology is not known although there are several metabolic, genetic, epigenetic, and local contributing factors involved. At the moment, there is no cure for this pathology and treatment alternatives to retard or stop its progression are intensively being sought. Hydrogen sulfide (H2S) is a small gaseous molecule and is present in sulfurous mineral waters as its active component. Data from recent clinical trials shows that balneotherapy (immersion in mineral and/or thermal waters from natural springs) in sulfurous waters can improve OA symptoms, in particular, pain and function. Yet, the underlying mechanisms are poorly known. Hydrogen sulfide is also considered, with NO and CO, an endogenous signaling gasotransmitter. It is synthesized endogenously with the help of three enzymes, cystathionine gamma-lyase (CTH), cystathionine beta-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase (3-MPST). Here, the expression of these three enzymes was demonstrated by quantitative real-time polymerase chain reaction (qRT-PCR) and their protein abundance [by immunohistochemistry and Western blot (WB)] in human articular cartilage. No significant differences were found in CBS or CTH expression or abundance, but mRNA and protein levels of 3-MPST were significantly reduced in cartilage form OA donors. Also, the biosynthesis of H2S from OA cartilage, measured with a specific microelectrode, was significantly lower than in OA-free tissue. Yet, no differences were found in H2S concentration in serum from OA patients and OA-free donors. The current results suggest that reduced levels of the mitochondrial enzyme 3-MPST in OA cartilage might be, at least in part, responsible for a reduction in H2S biosynthesis in this tissue and that impaired H2S biosynthesis in the joint might be a contributing factor to OA. This could contribute to explain why exogenous supplementation of H2S, for instance with sulfurous thermal water, has positive effects in OA patients. Instituto de Salud carlos III; PI12/00329 Instituto de Salud Carlos III; PI16/02124 Instituto de Salud Carlos III; RETIC-RIER-RD12/0009/0018 Xunta de Galicia; IN607A 2017/11

Review [Abstract] Platelet-rich plasma (PRP) is defined as a volume of plasma with a platelet concentration higher than the average in peripheral blood. Many basic, preclinical and even clinical case studies and trials report PRP’s ability to improve musculoskeletal conditions including osteoarthritis, but paradoxically, just as many conclude it has no effect. The purpose of this narrative review is to discuss the available relevant evidence that supports the clinical use of PRP in osteoarthritis, highlighting those variables we perceive as critical. Here, recent systematic reviews and meta-analyses were used to identify the latest randomized controlled trials (RCTs) testing a PRP product as an intra-articular treatment for knee osteoarthritis, compared with an intra-articular control (mostly hyaluronic acid). Conclusions in the identified RCTs are examined and compared. In total, five recent meta-analyses and systematic reviews were found meeting the above criteria. A total of 19 individual trials were identified in the five reviews but only 9 were level of evidence I RCTs, and many had moderate or high risks of bias. At present, results from these RCTs seem to favor PRP use over other intra-articular treatments to improve pain scales in the short and medium term (6–12 months), but the overall level of evidence is low. As a result, clinical effectiveness of PRP for knee osteoarthritis treatment is still under debate. This is, prominently, the result of a lack of standardization of PRP products, scarceness of high quality RCTs not showing high risks of bias, and poor patient stratification for inclusion in the RCTs. Insituto de Salud Carlos III PI12/00329 Insituto de Salud Carlos III; PI16/02124

Poster presentation

Conference abstract

[Abstract] Background: Rheumatoid arthritis (RA) and osteoarthritis (OA) are widespread rheumatic diseases characterized by persistent inflammation and joint destruction. Hydrogen sulfide (H2S) is an endogenous gas with important physiologic functions in the brain, vasculature and other organs. Recent studies have found H2S to be a mediator in inflammatory joint diseases. Objective: This review summarizes the recent literature in this area highlighting relevant developments. Conclusions: Several authors have found that H2S exhibited anti-inflammatory, anti-catabolic and/or anti-oxidant effects in rodent models of acute arthritis and in in vitro models using human synoviocytes and articular chondrocytes from RA and OA tissues. The earliest studies used fast-dissolving salts, such as NaSH, but GYY4137, which produces H2S more physiologically, shortly appeared. More recently still, new H2S-forming compounds that target mitochondria have been synthesized. These compounds open exciting opportunities for investigating the role of H2S in cell bioenergetics, typically altered in arthritides. Positive results have also been obtained when H2S is administered as a sulphurous water bath, an option meriting further study. These findings suggest that exogenous supplementation of H2S may provide a viable therapeutic option for these diseases, particularly in OA.

The aim of this work is to evaluate the potential of cryogels to be used as scaffolds in tissue engineering. Scaffolds based on the α-tricalcium phosphate reinforced PDMAEMA (Poly(dimethyl aminoethyl methacrylate))/PHEMA (poly(hydroxyethyl methacrylate)) system were prepared and human trabecular bone-derived cells (HTBs) and bone marrow derived-mesenchymal stem cells (BM-MSCs) cultured on them. Several features, such as porosity, pore shape, molecular weight between crosslinks and mesh size, are studied. The most suitable PDMAEMA/PHEMA ratio for cell proliferation has been assessed and the viability, adhesion, proliferation and expression of osteoblastic biochemical markers are evaluated. The PDMAEMA/PHEMA ratio influences the scaffolds porosity. Values between 53% ± 5.7% for a greater content in PHEMA and 75% ± 5.5% for a greater content in PDMAEMA have been obtained. The polymer ratio also modifies the pore shape. A greater content in PDMAEMA leads also to bigger network mesh size. Each of the compositions were non-cytotoxic, the seeded cells remained viable for both BM-MSCs and HTBs. Thus, and based on the structural analysis, specimens with a greater content in PDMAEMA seem to provide a better structural environment for their use as scaffolds for tissue engineering. The α-tricalcium phosphate incorporation into the composition seems to favor the expression of the osteogenic phenotype., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

Introduccion: El sulfuro de hidrogeno (H2S), compuesto activo de las aguas mineromedicinales sulfuradas, ha demostrado ser un protector tisular en varias patologias [1,2]. Estudios previos en nuestro grupo han demostrado que dos compuestos que producen H2S presentan efectos anti-inflamatorios y anti-catabolicos en condrocitos de tejido de pacientes con artrosis (OA) estimulados con interleuquina 1β (IL1β) [3]. En el presente trabajo, hemos evaluado la capacidad de estos mismos compuestos, NaSH y GYY4137 para inhibir los procesos catabolicos inducidos por la IL1β en cartilago humano.

Background: A growing number of findings support the hypothesis that type 2 diabetes is an independent risk factor of osteoarthritis (OA). However, the mechanisms underlying the connection between both diseases remain unclear. Hydrogen sulfide (H2S) plays an important role in the pathogenesis of diabetes and its complications. In relation, we and other authors have observed a protective impact of H2S induction on activation of pathological pathways in the chondrocyte (1).

Cell-cell interactions are of crucial importance for the formation of tissues, homeostasis and regeneration processes as well as reactions on foreign bodies including implants. So far, however, the importance of heterotypic cell-cell interactions in the in vitro evaluation of implant surfaces has been largely neglected. This work aims to develop an in vitro methodology that enables the in-depth investigation of heterotypic cell-cell interactions in a mixed co-culture system, and to validate it with a primary adult human bone-derived osteoblast cells (HBCs) – abdominal fibroblasts (HAFs) system. The methodology proposed combines a simple live labelling step, semiautomated fluorescence image acquisition and analysis to characterize the interactions between different cell types (cell population dynamics) in co-culture in terms of cell proliferation and cell spatial distribution of each cell type. In this co-culture system, direct cell-cell contacts between the two cell types were permitted while the determination of cell-type specific responses could still be elucidated. We could show that HAF proliferation was reduced in a way negatively correlated with the seeding HBC/HAF ratio, i.e., a high proportion of HBC in the co-culture had an inhibitory effect on HAF proliferation. In all cultures segregation was found after 4 and 7 days of co-culture. HBCs were segregated at low ratios while HAFs were segregated at high ratios. Cell-cell distances depended on the total cell number in the co-culture but the dependence was different for each cell type.

Calcium phosphate cement (CPC) has in situ-setting ability and bioactivity, but the brittleness and low strength limit CPC to only non-load-bearing bone repairs. Human umbilical cord mesenchymal stem cells (hUCMSCs) can be harvested without an invasive procedure required for the commonly studied bone marrow MSCs. However, little has been reported on hUCMSC delivery via bioactive scaffolds for bone tissue engineering. The objectives of this study were to develop CPC scaffolds with improved resistance to fatigue and fracture, and to investigate hUCMSC delivery for bone tissue engineering. In fast fracture, CPC with 15% chitosan and 20% polyglactin fibers (CPC–chitosan–fiber scaffold) had flexural strength of 26 MPa, higher than 10 MPa for CPC control (p < 0.05). In cyclic loading, CPC–chitosan–fiber specimens that survived 2 × 106 cycles had the maximum stress of 10 MPa, compared to 5 MPa of CPC control. CPC–chitosan–fiber specimens that failed after multiple cycles had a mean stress-to-failure of 9 MPa, compared to 5.8 MPa for CPC control (p < 0.05). hUCMSCs showed excellent viability when seeded on CPC and CPC–chitosan–fiber scaffolds. The percentage of live cells reached 96–99%. Cell density was about 300 cells/mm2 at day 1; it proliferated to 700 cells/mm2 at day 4. Wst-1 assay showed that the stronger CPC–chitosan–fiber scaffold had hUCMSC viability that matched the CPC control (p > 0.1). In summary, this study showed that chitosan and polyglactin fibers substantially increased the fatigue resistance of CPC, and that hUCMSCs had excellent proliferation and viability on the scaffolds.

This work investigates the settling response of bi-disperse silicone oil-based magnetorheological fluids with varying solids loading (content) of Fe magnetic particles at three ratios of nanoparticles to micron size particles. The solid loading (content) of Fe magnetic particles in silicone oil was 50 or 60% by weight with nanoparticle fractions of 0, 10, or 15% by weight of the total solids loading. Sedimentation experiments are conducted using Z-axis translating laser light scattering at ambient temperature. Sedimentation velocities are determined from clear layer separation and functional particulate transport by creeping flow. Settling rates of 1—3 μm/s have been observed in the dispersions without nanoparticles. Viscosity testing shows lower yield stress with nanoparticle inclusion and the typical rise in shear stress with increasing shear rate and applied magnetic field using both the linear Bingham and the non-linear Hershel—Bulkley models. Adding nanoparticles also reduced sedimentation velocity by roughly two orders of magnitude from those without nanoparticles. The nanoparticles act as plugs or otherwise interact in forming soft gel particulates leading to a denser sedimentation front yielding fewer percolation holes through which the fluid can permeate. Either way, the addition of nanoparticles leads to a more effective stabilization mechanism for the dispersion.

Calcium phosphate cement (CPC) sets in situ and forms apatite with excellent osteoconductivity and bone-replacement capability. The objectives of this study were to formulate an injectable tetracalcium phosphate-dicalcium phosphate cement (CPC(D)), and investigate the powder/liquid ratio and needle-size effects. The injection force (mean +/- SD; n = 4) to extrude the paste increased from (8 +/- 2) N using a 10-gauge needle to (144 +/- 17) N using a 21-gauge needle (p0.05). With the 10-gauge needle, the mass percentage of extruded paste was (95 +/- 4)% at a powder/liquid ratio of 3; it decreased to (70 +/- 12)% at powder/liquid = 3.5 (p0.05). A relationship was established between injection force, F, and needle lumen cross-sectional area, A: F = 5.0 + 38.7/A(0.8). Flexural strength, S, (mean +/- SD; n = 5) increased from (5.3 +/- 0.8) MPa at powder/liquid= 2 to (11.0 +/- 0.8) MPa at powder/liquid = 3.5 (p0.05). Pore volume fraction, P, ranged from 62.4% to 47.9%. A relationship was established: S = 47.7 x (1 - P)(2.3). The strength of the injectable CPC(D) matched/exceeded the reported strengths of sintered porous hydroxyapatite implants that required machining. The novel injectable CPC(D) with a relatively high strength may be useful in filling defects with limited accessibility such as periodontal repair and tooth root-canal fillings, and in minimally-invasive techniques such as percutaneous vertebroplasty to fill the lesions and to strengthen the osteoporotic bone.

Six different tetracalcium phosphate (TTCP) products were synthesized by solid state reaction at high temperature by varying the overall calcium to phosphate ratio of the synthesis mixture. The objective was to evaluate the effect of the calcium to phosphate ratio on a TTCP-dicalcium phosphate dihydrate (DCPD) cement. The resulting six TTCP-DCPD cement mixtures were characterized using X-ray diffraction analysis, scanning electron microscopy, and pH measurements. Setting times and compressive strength (CS) were also measured. Using the TTCP product with a Ca/P ratio of 2.0 resulted in low strength values (25.61 MPa) when distilled water was used as the setting liquid, even though conversion to hydroxyapatite was not prevented, as confirmed by X-ray diffraction. The suspected CaO presence in this TTCP may have affected the cohesiveness of the cement mixture but not the cement setting reaction, however no direct evidence of CaO presence was found. Lower Ca/P ratio products yielded cements with CS values ranging from 46.7 MPa for Ca/P ratio of 1.90 to 38.32 MPa for Ca/P ratio of 1.85. When a dilute sodium phosphate solution was used as the setting liquid, CS values were 15.3% lower than those obtained with water as the setting liquid. Setting times ranged from 18 to 22 min when water was the cement liquid and from 7 to 8 min when sodium phosphate solution was used, and the calcium to phosphate ratio did not have a marked effect on this property.

Calcium phosphate cement (CPC) can be molded and self-hardens in vivo to form resorbable hydroxyapatite with excellent osteoconductivity. The objective of this study was to develop an injectable, macroporous and strong CPC, and to investigate the effects of porogen and absorbable fibers. Water-soluble mannitol was used as porogen and mixed with CPC at mass fractions from 0% to 50%. CPC with 0-40% mannitol was fully extruded under a syringe force of 10 N. The paste with 50% mannitol required a 100-N force which extruded only 66% of the paste. At fiber volume fraction of 0-5%, the paste was completely extruded. However, at 6% and 7.5% fibers, some fibers were left in the syringe after the paste was extruded. The injectable CPC scaffold had a flexural strength (mean+/-sd; n=5) of (3.2+/-1.0) MPa, which approached the reported strengths for sintered porous hydroxyapatite implants and cancellous bone. In summary, the injectability of a ceramic scaffold, a macroporous CPC, was studies for the first time. Processing parameters were tailored to achieve high injectability, macroporosity, and strength. The injectable and strong CPC scaffold may be useful in surgical sites that are not freely accessible by open surgery or when using minimally invasive techniques.

The progression of the setting reaction of a tetracalcium phosphate (TTCP) –dicalcium phosphate dihydrate (DCPD) rapid setting cement was investigated as a function of time. Compressive strength and extent of conversion to hydroxyapatite (HA) were obtained at different incubation times. The results indicated a rapid development of both strength and HA conversion in the early stages of the reaction, which slowed down after 4 h, presumably as a result of HA formation on the surface of the reactants. This hypothesis was supported by scanning electron microscopy examination of cement fracture surfaces.

The development of a calcium phosphate cement, comprising tetracalcium phosphate (TTCP) and dicalcium phosphate dihydrate (DCPD), that hardens in 14 min with water as the liquid or 6 min with a 0.25 mol/L sodium phosphate solution as the liquid, without using hydroxyapatite (HA) seeds as setting accelerator, is reported. It was postulated that reduction in porosity would increase cement strength. Thus, the effects of applied pressure during the initial stages of the cement setting reaction on cement strength and porosity were studied. The cement powder comprised an equimolar mixture of TTCP and DCPD (median particle sizes 17 and 1.7 μm, respectively). Compressive strengths (CS) of samples prepared with distilled water were 47.6 ± 2.4 MPa, 50.7 ± 4.2 MPa, and 52.9 ± 4.7 MPa at applied pressures of 5 MPa, 15 MPa, and 25 MPa, respectively. When phosphate solution was used, the CS values obtained were 41.5 ± 2.3 MPa, 37.9 ± 1.7 MPa, and 38.1 ± 2.3 MPa at the same pressure levels. Statistical analysis of the results showed that pressure produced an improvement in CS when water was used as liquid but not when the phosphate solution was used. Compared to previously reported TTCP-DCPD cements, the greater CS values and shorter setting times together with a simplified formulation should make the present TTCP-DCPD cement a useful material as a bone substitute for clinical applications. © 2004 Wiley Periodicals, Inc.

Las aguas minero-medicinales (AMM) representan un recurso natural de indudable valor terapeutico. Sus principales indicaciones incluyen las patologias musculo-esqueleticas, y en concreto, la artrosis. La artrosis (OA, osteoarthritis) es la patologia reumatica mas frecuente. Mas del 70% de las personas mayores de 50 anos presenta signos radiologicos de OA. Diversos ensayos clinicos han demostrado resultados positivos al evaluar los efectos de diversas AMM sobre pacientes con OA, sobre todo en indices de dolor y funcion. Por ejemplo, recientemente se obtuvieron mejoras significativas en los indices de dolor, funcion y rigidez de la escala WOMAC en pacientes con OA de cadera tratados con banos en agua sulfurada a las 12 semanas del tratamiento. Sin embargo, los estudios sobre los mecanismos fisiologicos y moleculares responsables de estos efectos son relativamente escasos. En el presente trabajo se revisaron las evidencias actuales sobre los posibles efectos anti-inflamatorios y antioxidantes de AMM o de sus componentes minerales principales (en concreto, el sulfuro de hidrogeno, H2S) en modelos in vitro de artrosis. La mayor parte de los estudios in vitro emplearon cultivos de condrocitos y/o sinoviocitos de tejido OA. En ellos se indujo un ambiente inflamatorio mediante estimulacion con interleuquina (IL) 1 y se estudiaron los efectos de la coestimulacion con compuestos sinteticos que producen H2S (NaSH y GYY4137). En particular, se evaluo la influencia en la sobreproduccion de marcadores inflamatorios y de estres oxidativo provocada por esta citoquina. De manera consistente, estos trabajos demostraron la inhibicion de la produccion de otras citoquinas tales como IL6 o IL8, de la liberacion de oxido nitrico (NO), y del aumento de prostaglandina E2 o metaloproteinasas. Respecto a las rutas de senalizacion implicadas, tanto NaSH como GYY4137 consiguieron una reduccion de la activacion de varias quinasas activadas por mitogenos (MAPKs) y de la ruta del factor de transcripcion NF-B, ambas implicados en la perpetuacion de la inflamacion cronica que caracteriza la OA. Al tiempo que indujeron la activacion de la ruta Akt de supervivencia celular. Cabe destacar otro estudio, con una metodologia novedosa, en el que las celulas articulares se estimularon directamente con AMM de alta mineralizacion (rica en sulfatos, calcio, magnesio y hierro) en diferentes proporciones. Se encontro que el aumento de NO y de su enzima inducible de produccion, y de los niveles de apoptosis celular provocados por IL1 se veian reducidos al utilizar 25% y 50% de AMM en los cultivos celulares. En conclusion, la literatura revisada sugiere que el AMM o sus componentes minerales principales, en particular H2S, presentan actividad anti-inflamatoria y antioxidante en modelos in vitro de OA. Estos estudios contribuyen a un mejor entendimiento de los mecanismos de accion que subyacen los efectos beneficiosos de las AMMs encontrados en los ensayos clinicos existentes.

La fibromialgia (FM) es un sindrome de dolor cronico que afecta al 2,5% de la poblacion espanola, preferentemente mujeres, caracterizada por dolor generalizado, fatiga, rigidez matutina, sintomas psiquicos, dificultades en las actividades de la vida diaria y mala calidad de sueno1. El tratamiento es sintomatico e incluye un amplio rango de intervenciones terapeuticas, entre ellas, la balneoterapia2-3. El objetivo de este estudio es evaluar la efectividad del tratamiento termal en el balneario de Lugo (Espana), en el manejo de pacientes con fibromialgia a corto y medio plazo. 76 pacientes con FM, con una media de edad de 53,4 anos, fueron aleatorizados en dos grupos: grupo de balneoterapia (38) y grupo control (38). Los pacientes del grupo de balneoterapia recibieron tratamiento ambulatorio en el balneario de Lugo que consistio en ejercicio dirigido en piscina de agua sulfurada a 37o C durante 20 minutos, seguido de aplicacion de peloides en zonas dolorosas a 45o durante 15 minutos, finalizando con una sesion de relajacion dirigida por un psicologo. La balneoterapia fue aplicada ambulatoriamente durante tres meses, 2 dias por semana. Todos los pacientes del grupo tratamiento continuaron con su tratamiento medico al igual que el grupo control. Un investigador que fue cegado a la intervencion valoro a los pacientes en 4 ocasiones: al inicio, al mes de comenzar, a los tres meses y 3 meses despues del finalizar el tratamiento. Las medidas fueron FIQ (Fibromyalgia Impact Questionnaire), numero de puntos dolorosos, EVA de dolor, fatiga, depresion, ansiedad, calidad del sueno, rigidez, bienestar global, cefalea, insomnio y consumo de farmacos. Los dos grupos al comienzo del estudio eran homogeneos no existiendo diferencias en ninguno de los parametros analizados. Al mes de comenzar el tratamiento la mejoria observada en el grupo tratamiento fue significativa en el numero de puntos dolorosos y en el insomnio. Al final de la intervencion de 3 meses, el grupo que recibio tratamiento termal mejoro significativamente con respecto al grupo control en todas las variables analizadas: intensidad del dolor, no de puntos dolorosos, fatiga, rigidez, insomnio, depresion, FIQ, toma de antidepresivos, de antiinflamatorios y analgesicos con respecto al inicio. Los resultados se mantuvieron o incluso mejoraron algunos parametros a los 3 meses de finalizado el tratamiento, especialmente la depresion, el dolor, la ansiedad, la rigidez y el FIQ. Podemos concluir que la inclusion de la balneoterapia en el tratamiento de los pacientes con fibromialgia constituye un tratamiento complementario con alta relevancia clinica en la fibromialgia.

El agua minero-medicinal sulfurada (AMS) es una fuente natural de sulfuro de hidrogeno (H2S). Esta molecula esta ganando reconocimiento como agente antiinflamatorio en modelos in vitro de artrosis (OA) [1]. En este trabajo se evaluaron los efectos de AMS recogida de un manantial publico en la provincia de Pontevedra, Espana, sobre condrocitos articulares de cartilago OA y la linea celular de condrocitos T/C-28a2. Los parametros evaluados fueron la viabilidad de las celulas, la produccion de oxido nitrico (NO) y la expresion genica de diversos mediadores de inflamacion, en concreto, sintasa inducible de NO (iNOS), interleuquina 6 (IL6) y metaloproteinasa 13 (MMP13). Ademas, se estudio la abundancia proteica de iNOS y los niveles de MMP13 en los sobrenadantes celulares. Las celulas se estimularon directamente con el AMS con y sin IL1 (5 ng/mL). Para ello el medio de cultivo celular se preparo disolviendo medio minimo esencial de Dulbecco (DMEM) en polvo en agua destilada (AD) o AMS, que posteriormente se mezclaron en distintas proporciones (25%, 50% y 75% de AMS). Para comprobar la citocompatibilidad de los medios preparados se utilizo un test de viabilidad con el reactivo alamarBlue® y la linea celular. En el resto de los experimentos se utilizaron condrocitos humanos primarios. Las celulas se coestimularon con IL1 (5 ng/mL) y los medios preparados con distintas proporciones de AMS. La produccion de NO se cuantifico utilizando la reaccion de Griess y la expresion genica de los marcadores de inflamacion se determino mediante qRTPCR. Para la determinar la abundancia proteica se utilizo, en el caso de iNOS una inmunocitoquimica y en el caso de la MMP13 un ELISA. El test de viabilidad permitio comprobar que ninguna de las proporciones de AMS utilizadas provocaba un efecto negativo sobre la viabilidad celular. Mediante microscopia optica y de manera cualitativa se observo como las celulas perdieron su morfologia habitual in vitro al estimularlas con IL1 y como las proporciones crecientes de AMS contrarrestaron este efecto. Con 100% AMS se habia recuperado la morfologia normal. La estimulacion con IL1 tambien provoco un aumento tanto en la produccion de NO, como de la expresion genica y la abundancia proteica de su enzima inducible. Todos estos efectos fueron parcialmente contrarrestados por el AMS, siendo 75% y 100% las proporciones con mejores resultados. La expresion de IL6 tambien se redujo debido a la presencia del AMS. Respecto a los niveles de MMP13 en los sobrenadantes celulares, se observo una tendencia descendente al aumentar la proporcion de AMS, mientras que su expresion genica unicamente se redujo al utilizar el medio preparado con 100% AMS. Los presentes resultados demuestran que el agua mineromedicinal sulfurada utilizada en este trabajo puede ejercer efectos antioxidantes y antiinflamatorios en condrocitos humanos OA cuando se utiliza como medio de cultivo. Estos resultados proporcionan evidencias adicionales sobre el potencial terapeutico del sulfuro de hidrogeno en la artrosis, que se suman a los obtenidos previamente utilizando compuestos sinteticos sulfurados [2].

Transglutaminases (TGases) are enzymes that catalyze covalent isopeptide crosslinks between reactive lysine and glutamine residues in proteins. Higher than normal local concentrations of TGase have been correlated with increased protein aggregation in vivo. These insoluble protein aggregates are the hallmark of several neurodegenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, although each aggregating protein involved is disease specific. Because TGase is implicated in protein aggregation, there is evidence that its regulation may retard disease progression. Here we report on a laser light transmission technique as an in vitro tool to gauge the efficacy of creatine, a candidate inhibitor, to regulate aggregation. Sedimentation velocities of protein-coated particles in TGase-containing water-glycerol solutions were tracked with different levels of creatine. Sedimentation velocities were converted to apparent aggregate sizes using Stoke's law of sedimentation. The results indicated that creatine promoted up to a 20% reduction in protein aggregation in vitro. This technique may prove to be useful in identifying other functional TGase inhibitors.

Calcium phosphate cement (CPC) sets in situ to form resorbable hydroxyapatite with chemical and crystallographic similarity to the apatite in human bones, hence it is highly promising for clinical applications. The objective of the present study was to develop a CPC that is fast setting and has high strength in the early stages of implantation. Two approaches were combined to impart high early strength to the cement: the use of dicalcium phosphate dihydrate with a high solubility (which formed the cement CPC(D)) instead of anhydrous dicalcium phosphate (which formed the conventional cement CPC(A)), and the incorporation of absorbable fibers. A 2 x 8 design was tested with two materials (CPC(A) and CPC(D)) and eight levels of cement reaction time: 15 min, 30 min, 1 h, 1.5 h, 2 h, 4 h, 8 h, and 24 h. An absorbable suture fiber was incorporated into cements at 25% volume fraction. The Gilmore needle method measured a hardening time of 15.8 min for CPC(D), five-fold faster than 81.5 min for CPC(A), at a powder:liquid ratio of 3:1. Scanning electron microscopy revealed the formation of nanosized rod-like hydroxyapatite crystals and platelet crystals in the cements. At 30 min, the flexural strength (mean +/- standard deviation; n = 5) was 0 MPa for CPC(A) (the paste did not set), (4.2 +/- 0.3) MPa for CPC(D), and (10.7 +/- 2.4) MPa for CPC(D)-fiber specimens, significantly different from each other (Tukey's at 0.95). The work of fracture (toughness) was increased by two orders of magnitude for the CPC(D)-fiber cement. The high early strength matched the reported strength for cancellous bone and sintered porous hydroxyapatite implants. The composite strength S(c) was correlated to the matrix strength S(m): S(c) = 2.16S(m). In summary, substantial early strength was imparted to a moldable, self-hardening and resorbable hydroxyapatite via two synergistic approaches: dicalcium phosphate dihydrate, and absorbable fibers. The new fast-setting and strong cement may help prevent catastrophic fracture or disintegration in moderate stress-bearing bone repairs.

[Abstract] Objective. Hydrogen sulfide (H2S), the third gasotransmitter together with NO and CO, is emerging as a regulator of inflammation. To test if it might offer therapeutic value in the treatment of osteoarthritis (OA) we evaluated the effects of two exogenous sources of H2S, NaSH and GYY4137, on inflammation and catabolic markers that characterize OA. Method. Human chondrocytes (CHs) were isolated from OA tissue. Cells were stimulated with a pro-inflammatory cytokine (interleukin-1β, IL1β, 5 ng/ml) and the ability of the two H2S sources to ameliorate its effects on the cells was tested. Nitric oxide (NO) production was quantified through the Griess reaction. Protein levels of inducible NO synthase (NOS2) and matrix metalloproteinase 13 (MMP13) were visualized through immunocytochemistry (ICC). Relative mRNA expression was quantified with qRT-PCR. Prostaglandin-2 (PGE-2), interleukin 6 (IL6) and MMP13 levels were measured with specific EIAs. NFκB nuclear translocation was visualized with immunofluorescence. Results. Both H2S sources led to significant reductions in NO, PGE-2, IL6 and MMP13 released by the cells and at the protein level. This was achieved by downregulation of relevant genes involved in the synthesis routes of these molecules, namely NOS2, cyclooxigenase-2 (COX2), prostaglandin E synthase (PTGES), IL6 and MMP13. NFκB nuclear translocation was also reduced. Conclusion. NaSH and GYY4137 show anti-inflammatory and anti-catabolic properties when added to IL1β activated osteoarthritic CHs. Supplementation with exogenous H2S sources can regulate the expression of relevant genes in OA pathogenesis and progression, counteracting IL1β pro-inflammatory signals that lead to cartilage destruction in part by reducing NFκB activation. Instituto de Salud Carlos III; CIBER BBN CB06/01/0040