Efecto de la suplementación con vitamina D en los efectos secundarios musculoesqueléticos relacionados con los inhibidores de la aromatasa para el cáncer de mama: cohorte B-ABLE / Effect of vitamin D supplementation on aromatase inhibitor-related musculoskeletal side effects for breast cancer: B-ABLE cohort

Objetivo: Evaluar el efecto de la suplementación con vitamina D en las complicaciones musculoesqueléticas relacionadas con el tratamiento con inhibidores de la aromatasa (IA) en pacientes con cáncer de mama. Material y métodos: Estudio observacional prospectivo de mujeres en tratamiento con IA, reclutadas en la cohorte B-ABLE. Las pacientes con niveles séricos iniciales de 25(OH)D (25-hidroxivitamina D) <30 ng/ml recibieron una dosis de 16.000 UI de calcifediol oral cada 2 semanas. La artralgia y la pérdida ósea relacionadas con los IA se evaluaron a los 3 meses y al año de seguimiento, respectivamente. Los análisis de asociación del status de vitamina D a los 3 meses con eventos musculoesqueléticos se realizaron mediante modelos de regresión lineal multivariante ajustados. Además, se evaluó la asociación del dolor incidente, definido como pacientes sin dolor articular inicial, pero con una escala visual analógica (EVA) >0 a los 3 meses, mediante regresión logística. Resultados: La suplementación con vitamina D al inicio del tratamiento con IA disminuyó el riesgo tanto de artralgia incidente como de su empeoramiento. El umbral efectivo de 25(OH)D en suero para reducir el dolor articular se estableció en 40 ng/ml. Sin embargo, este umbral no se relacionó significativamente con los cambios óseos al año de seguimiento. No obstante, los niveles de vitamina D se correlacionaron inversamente con la pérdida ósea de la columna lumbar (CL) (β=0,177% [IC 95%: 0,014 a 0,340]). Conclusiones: La administración de suplementos de vitamina D con el objetivo de alcanzar niveles séricos de 25OHD de al menos 40 ng/ml es protectora para la artralgia. Los niveles de vitamina D a los tres meses podrían predecir el riesgo de pérdida ósea en CL al año de tratamiento con IA. Por lo tanto, se recomiendan dosis altas de vitamina D en estas pacientes, que son más propensas a sufrir afecciones musculoesqueléticas. (AU)

Objetive: To assess the effect of vitamin D supplementation on musculoskeletal complications related to aromatase inhibitor (AI) treatment in patients with breast cancer. Material and methods: Prospective observational study of women undergoing AI treatment, recruited in the B-ABLE cohort. Patients with baseline serum 25 (OH) D (25-hydroxyvitamin D) levels <30 ng/ml received a 16,000 IU dose of oral calcifediol every 2 weeks. Arthralgia and bone loss related to AIs were assessed at 3 months and 1 year of followup, respectively. The association analyzes of vitamin D status at 3 months with musculoskeletal events were carried out using adjusted multivariate linear regression models. In addition, the association of incident pain, defined as patients without initial joint pain, but with a visual analog scale (VAS) >0 at 3 months, was evaluated using logistic regression. Results: Vitamin D supplementation at the start of AI treatment decreased the risk of both incident arthralgia and its worsening. The effective threshold of 25 (OH) D in serum to reduce joint pain was established at 40 ng/ml. However, this threshold was not significantly related to bone changes at one year of follow-up. However, vitamin D levels were inversely correlated with lumbar spine bone loss (LS) (β=0.177% [95% CI: 0.014 to 0.340]). Conclusions: Vitamin D supplementation aimed at achieving serum 25(OH)D levels of at least 40 ng/ml is protective for arthralgia. Vitamin D levels at three months could predict the risk of bone loss in LS at one year of AI treatment. Therefore, high doses of vitamin D are recommended in these patients, who are more prone to musculoskeletal conditions. (AU)

Persistencia a los inhibidores de la aromatasa en la cohorte SIDIAP: mortalidad e influencia de los bifosfonatos / Persistence to aromatase inhibitors in the SIDIAP cohort: mortality and influence of bisphosphonates

OBJETIVO: Evaluar la persistencia a la terapia con inhibidores de la aromatasa (IA), la mortalidad asociada a la discontinuidad al tratamiento y la influencia de los bifosfonatos (BF) orales, en la práctica clínica habitual. MATERIAL Y MÉTODOS: Estudio prospectivo observacional de mujeres con cáncer de mama en tratamiento con IA entre enero de 2006 y diciembre de 2015, registradas en la base de datos SIDIAP. Se excluyeron aquellas tratadas previamente con tamoxifeno. Se estudió la persistencia al tratamiento con IA con un análisis de supervivencia: se calculó el estimador de Kaplan-Meier, y se realizó un modelo de los riesgos proporcionales (regresión de Cox) entre usuarias y no usuarias de BF ajustando por edad. Se llevó a cabo un análisis de sensibilidad teniendo en cuenta la mortalidad como riesgo competitivo (modelos de Fine y Gray). Se comparó la diferencia de mortalidad entre grupos mediante una prueba Chi cuadrado. RESULTADOS: Se observó una persistencia a los IA del 87% a 5 años de tratamiento, con una mortalidad global del 19,75%. Se registró un 7,7% menos de mortalidad en aquellas pacientes que completaron los 5 años de tratamiento respecto a las que no. Las pacientes con BF mostraron una disminución de la mortalidad (6,6%) y una disminución del riesgo de abandono de la terapia (SHR ajustado: 0,62 [IC 95%: 0,55 a 0,70]) respecto a las no usuarias. CONCLUSIONES: La permanencia a los IA y el uso de BF está asociada a una disminución de la mortalidad global. Además, el uso de BF resulta en un aumento de la adherencia al tratamiento con IA

OBJETIVE: To assess the persistence of aromatase inhibitor (AI) therapy, mortality associated with treatment discontinuation and the influence of oral bisphosphonates (BP) in routine clinical practice. MATERIAL AND METHODS: Prospective observational study of women with breast cancer undergoing AI treatment between January 2006 and December 2015, registered in the SIDIAP database. Those previously treated with tamoxifen were excluded. AI persistence was studied with a survival analysis: the Kaplan-Meier estimator was calculated, and a proportional hazards model (Cox regression) was performed between users and non-users of BP adjusting for age. A sensitivity analysis was carried out taking into account mortality as a competitive risk (Fine and Gray models). The difference in mortality between groups was compared using a Chi square test. RESULTS: A persistence to AI of 87% was observed after 5 years of treatment, with an overall mortality of 19.75%. There was 7.7% less mortality in those patients who completed the 5 years of treatment compared to those who did not. Patients with BP showed a decrease in mortality (6.6%) and a decrease in the risk of discontinuing therapy (adjusted SHR: 0.62 [95% CI: 0.55 to 0.70]) compared to non-users. CONCLUSIONS: Persistence to AI and BP use are associated with a decrease in overall mortality. Furthermore, the use of BP increases adherence to AI treatment

Factores de riesgo de fractura incidente en pacientes con cáncer de mama tratadas con inhibidores de la aromatasa: cohorte B-ABLE / Risk factors for incident fracture in patients with breast cancertreated with aromatase inhibitors: B-ABLE cohort

OBJETIVO: Los inhibidores de la aromatasa (IA) se han asociado con una pérdida de masa ósea acelerada y un mayor riesgo de fracturas osteoporóticas. Con este trabajo se pretendió evaluar los factores de riesgo de fractura incidente en pacientes con cáncer de mama que reciben IA. MATERIAL Y MÉTODOS: Estudio prospectivo-observacional de cohorte de mujeres con cáncer de mama que inician tratamiento con IA (cohorte B-ABLE). Las pacientes realizaron tratamiento durante 5 años o bien 2 ó 3 años si habían recibido previamente tamoxifeno. Se les evaluó la salud ósea desde el inicio del tratamiento hasta un año después de finalizar dicho tratamiento mediante densitometría ósea, marcadores de remodelado óseo, niveles de vitamina D y una radiografía antero-posterior y otra lateral de columna. Se realizó el cálculo de riesgo de fractura mediante la herramienta FRAX® antes de iniciar IA. Se utilizaron modelos de Cox para calcular los ratios de riesgo (HR [IC 95%]) de fractura. RESULTADOS: Un total de 943 pacientes fueron incluidas en el estudio. El 5,4% sufrieron una fractura incidente, la mayoría durante el tratamiento con IA, aunque un 21,5% ocurrieron durante el primer año después de finalizar la terapia. La mayoría de las fracturas incidentes fueron vertebrales clínicas (29,4%) y de Colles (31,4%). El 86,3% de las pacientes tenían un diagnóstico de osteopenia u osteoporosis en el momento de la fractura y el 33% tenían los niveles de β-CTX (isómero β del telopéptido carboxiterminal del colágeno tipo I) por encima de la normalidad. Las pacientes diagnosticadas de osteoporosis o con riesgo de fractura al inicio del estudio fueron tratadas con antirresortivos óseos. No se encontraron diferencias significativas en el riesgo de fractura entre pacientes con y sin tratamiento antirresortivo: HR=1,75 [IC 95%: 0,88 a 3,46]. Tampoco se encontraron diferencias entre las pacientes que habían hecho tratamiento previo con tamoxifeno respecto a las que no (HR=1,00 [IC 95%: 0,39 a 2,56]). La herramienta FRAX® dio valores de media dentro del rango de riesgo intermedio, con 13 pacientes con valores de alto riesgo de fractura principal. CONCLUSIONES: El principal factor de riesgo detectado para fractura incidente en pacientes tratadas con IA es el diagnóstico de osteopenia u osteoporosis. El cálculo de la herramienta FRAX® y la determinación de los niveles de β-CTX son herramientas útiles para identificar a pacientes de alto riesgo

OBJETIVO:Aromatase inhibitors (AI) have been associated with an accelerated loss of bone mass and an increased risk ofosteoporosis fractures. This study assesses the risk factors for incident fracture in breast cancer patients receiving AI. MATERIAL AND METHODS:Prospective‐observational cohort study of women with breast cancer who begin treatment withAI (B‐ABLE cohort). Patients were treated for 5 years or 2 or 3 years if they had previously received tamoxifen. Bone healthwas assessed from the beginning of the treatment until one year post treatment by bone densitometry, bone remodelingmarkers, vitamin D levels and an anteroposterior and lateral spine radiography. The fracture risk calculation was performedusing the FRAX® tool before starting AI. Cox models were used to calculate the risk ratios (HR [95% CI]) of fracture. RESULTS: A total of 943 patients were included in the study.5.4% suffered an incident fracture, most during AI treatment,although 21.5% occurred during the first year after the end of therapy. Most of the incident fractures were clinical vertebral (29.4%) and Colles (31.4%).86.3% of the patients had a diagnosis of osteopenia or osteoporosis at the time of the fractureand 33% had the levels of β‐CTX (β isomer of the carboxyterminal telopeptide of type I collagen) above normal. Patients diagnosed with osteoporosis or at risk of fracture at the start of the study were treated with bone antiresorptives. No significant differences in fracture risk were found between patients with and without antiresorptive therapy: HR=1.75[95% CI: 0.88 to 3.46]. Nor were differences found among patients who had previously treated with tamoxifen comparedto those who did not (HR=1.00 [95% CI 0.39 to 2.56]). The FRAX®tool gave average values within the intermediate riskrange, with 13 patients with high risk of major fracture values. CONCLUSIONS:The main risk factor detected for incident fracture in patients treated with AI is the diagnosis of osteopeniaor osteoporosis. The calculation of the FRAX® tool and the determination of β‐CTX levels are useful tools to identifyhigh‐risk patients

In-vitro comparison of hydroxyapatite coatings obtained by cold spray and conventional thermal spray technologies.

Hydroxyapatite (HA) coatings onto Ti6Al4V alloy substrates were obtained by several thermal spray technologies: atmospheric plasma spray (APS) and high velocity oxy fuel (HVOF), together with the cold spray (CS) technique. A characterization study has been performed by means of surface and microstructure analyses, as well as biological performance. In-vitro tests were performed with primary human osteoblasts at 1, 7 and 14 days of cell culture on substrates. Cell viability was tested by MTS and LIVE/DEAD assays, cell differentiation by alkaline phosphatase (ALP) quantification, and cell morphology was analyzed by scanning electron microscopy. The HA coatings showed an increase of HA crystallinity from 62,4% to 89%, but also an increase of hydrophilicity from ∼32° to 0°, with the decrease of the operating temperature of the thermal spray techniques (APS > HVOF > CS). Additionally, APS HA coatings showed more surface micro-features than HVOF and CS HA coatings; cells onto APS HA coatings showed faster attachment by acquiring osteoblastic morphology in comparison with the rounded cell morphology observed onto CS HA coatings at 1 day of cell culture. HVOF HA coatings also showed proper cell adherence but did not show extended filopodia as cells onto APS HA coatings. However, at 14 days of cell culture, higher cell proliferation and differentiation was detected on HA coatings with higher crystallinity (HVOF and CS techniques). Cell attachment is suggested to be favoured by surface micro-features but also moderate surface wettability whereas cell proliferation and differentiation is suggested to be highly influenced by HA crystallinity and crystal size.

Inflammation status in HIV-positive individuals correlates with changes in bone tissue quality after initiation of ART.

BACKGROUND: The mechanisms behind ART-induced bone changes in HIV-infected patients are poorly known. We aimed to analyse changes in inflammatory and bone markers in HIV after tenofovir disoproxil fumarate initiation, and the associations with changes in the bone strength parameters. METHODS: HIV-positive participants starting tenofovir disoproxil fumarate-based ART underwent dual-energy X-ray absorptiometry (QDR 4500 SL®, Hologic, Waltham, MA, USA) for bone mineral density (BMD), a microindentation test (OsteoProbe®, Active Life Scientific, Santa Barbara, CA, USA) for bone quality [bone material strength index (BMSi)] and phlebotomy at baseline and 48 weeks after ART. A panel of inflammatory biomarkers and bone turnover markers were measured by ELISA. HIV-negative controls underwent identical procedures once. Values are expressed as medians and IQRs, and non-parametric tests were used to perform the analysis. RESULTS: Twenty HIV-infected individuals and 20 HIV-negative control individuals were matched in terms of age and gender. HIV individuals showed higher levels of inflammatory markers. We found no differences in bone turnover markers. HIV-positive individuals presented lower BMSi values at baseline compared with controls [86 (83-90) versus 89 (88-93), respectively; P = 0.034]. We found no difference in BMD (at either of the sites evaluated). BMSi tended to increase with treatment. IL-1ß at baseline was positively correlated with changes in BMSi after ART (rho = 0.564, P = 0.014). Baseline levels of sclerostin tended to be negatively correlated with changes in BMSi (rho = -0.402, P = 0.097). We found a negative correlation between time since HIV diagnosis and changes in BMSi (rho = -0.466, P = 0.04). CONCLUSIONS: We observed a correlation between changes in bone quality and the inflammatory environment in HIV-positive individuals. Moreover, among the underlying mechanisms we highlight the Wnt pathway as having a potentially significant role in ART bone quality recovery.

Estudio genético de la fractura femoral atípica mediante la secuenciación del exoma en tres hermanas afectas y tres pacientes no relacionadas / Genetic study of atypical femoral fractures using exome sequencing in three affected sisters and three unrelated patients

Objetivos: Las fracturas atípicas de fémur (FAF) son un tipo de fracturas poco frecuentes, a menudo relacionadas con un tratamiento prolongado con bisfosfonatos (BPs). Actualmente no se conocen con exactitud sus mecanismos patogénicos y no hay pruebas para identificar aquellos pacientes con un alto riesgo de sufrir una FAF. El objetivo de este trabajo es investigar las bases genéticas de las FAFs. Material y métodos: Se secuenció el exoma completo de 3 hermanas y de 3 pacientes adicionales no relacionadas, todas tratadas con BPs durante más de 5 años. Se seleccionaron variantes compartidas por las hermanas, de baja frecuencia y potencialmente patogénicas, y se construyó una red de interacciones de genes y proteínas con los datos hallados. Resultados: Identificamos 37 variantes raras (en 34 genes) compartidas por las 3 hermanas, algunas de ellas no descritas anteriormente. La variante más llamativa fue la mutación p.Asp188Tyr en el enzima geranilgeranil pirofosfato sintasa (codificada por el gen GGPS1), de la vía del mevalonato y esencial para la función del osteoclasto. Otro hallazgo interesante fueron dos mutaciones (una en las 3 hermanas y una en una paciente no relacionada) en el gen CYP1A1, implicado en el metabolismo de los esteroides. Identificamos otras variantes que también podrían estar involucradas en la susceptibilidad a las FAFs o en el fenotipo osteoporótico subyacente, tales como las presentes en los genes SYDE2, NGEF, COG4 y la FN1. Conclusiones: Nuestros datos son compatibles con un modelo donde la acumulación de variantes de susceptibilidad podría participar en la base genética de las FAF

Objectives: Atypical femoral fractures (AFF) are rare, often related to long-term bisphosphonate (BPs) treatment. Their pathogenic mechanisms are not precisely known and there is no evidence to identify patients with a high risk of AFF. The aim of this work is to study the genetic bases of AFFs. Material and methods: Whole-exome sequencing was carried out on 3 sisters and 3 unrelated additional patients, all treated with BPs for more than 5 years. Low frequency, potentially pathogenic variants shared by the 3 sisters, were selected, were selected and a network of gene and protein interactions was constructed with the data found. Results: We identified 37 rare variants (in 34 genes) shared by the 3 sisters, some not previously described. The most striking variant was the p.Asp188Tyr mutation in the enzyme geranylgeranyl pyrophosphate synthase (encoded by the GGPS1 gene), from the mevalonate pathway and essential for osteoclast function. Another noteworthy finding was two mutations (one in the 3 sisters and one in an unrelated patient) in the CYP1A1 gene, involved in the metabolism of steroids. We identified other variants that could also be involved in the susceptibility to AFFs or in the underlying osteoporotic phenotype, such as those present in the SYDE2, NGEF, COG4 and FN1 genes. Conclusions: Our data are compatible with a model where the accumulation of susceptibility variants could participate in the genetic basis of AFFs

Estudios funcionales de variantes de DKK1 presentes en la población general / Functional studies of DKK1 variants present in the general population

Objetivo: En las últimas décadas se han identificado genes asociados a la masa ósea y al riesgo de fractura osteoporótica, varios de los cuales pertenecen a la vía de Wnt. En este proyecto se estudió la funcionalidad de 7 mutaciones de cambio de sentido del gen DKK1 -un inhibidor de la vía de Wnt- presentes en la población general. Material y métodos: Se realizaron estudios in vitro del gen reportero luciferasa para medir la actividad de la vía de Wnt en presencia o ausencia de DKK1 silvestre o mutada, y estudios de western blot, para evaluar si las distintas mutaciones afectan a su síntesis y/o a su estabilidad. Resultados: La proteína DKK1 con la variante p.Ala41Thr presenta menor actividad inhibidora de la vía en comparación con la proteína silvestre. También se observaron diferencias significativas entre los experimentos realizados en ausencia de DKK1 y los que incluyen DKK1 con la mutación p.Ala41Thr. Los western blots mostraron que la cantidad de proteína era similar para todas las variantes, tanto las mutadas como la silvestre, por lo que la pérdida de actividad de p.Ala41Thr no parecía deberse a falta de proteína. El resto de las mutaciones no presentaron un comportamiento diferente al de la proteína DKK1 silvestre. Conclusiones: La variante de cambio de sentido p.Ala41Thr de la proteína DKK1, con una frecuencia poblacional de 0,013%, presenta una pérdida parcial de su función inhibidora, que no es debida a la falta de expresión de ésta. Esta variante génica podría conllevar un aumento de la densidad mineral ósea en las personas de la población general portadoras de esta mutación

Objective: In recent decades, genes associated with bone mass and osteoporotic fracture risk have been identified, several of which belong to the Wnt pathway. In this project, the functionality of 7 missense mutations of the gene DKK1-an inhibitor of the Wnt pathway- present in the general population was studied. Material and methods: In vitro studies of the luciferase reporter gene were carried out to measure Wnt pathway activity in the presence or absence of wild-type or mutated DKK1, and western blot studies, to evaluate if the different mutations affect its synthesis and/or stability. Results: The DKK1 protein with the p.Ala41Thr variant shows lower pathway inhibitory activity compared to the wild-type protein. Significant differences were also observed between the experiments performed in the absence of DKK1 and those that include DKK1 with the p.Ala41Thr mutation. Western blots showed that the amount of protein was similar for all variants, both mutated and "wild-type, so the loss of p.Ala41Thr activity did not seem to be due to a lack of protein. The rest of the mutations did not show different behavior from that of the wild DKK1 protein. Conclusions: The missense variant p.Ala41Thr of the DKK1 protein, with a population frequency of 0.013%, shows a partial loss of its inhibitory function, which is not due to the lack of expression. This gene variant could lead to an increase in bone mineral density in those people in the general population who carry this mutation

La resistencia mecánica tisular ósea es independiente de la edad en individuos sanos / Bone tissue mechanical strength is in dependent of age in healthy individuals

Objetivo: La microindentación de impacto (MII) es una técnica que permite medir in vivo la resistencia tisular mecánica ósea. Se ha demostrado que la MII proporciona información útil sobre la evaluación de enfermedades esqueléticas, pero se desconoce el efecto que la edad puede ejercer sobre la propiedad ósea medida. El objetivo del estudio es analizar la relación entre la edad y la MII. Material y métodos: El índice de Resistencia Mineral Ósea (BMSi), la variable de medición de MII, se midió en 69 mujeres (mediana de edad: 49 años; rango: 30-81 años) y 19 varones (mediana de edad: 34 años; rango: 24-98 años) sanos. La correlación entre BMSi y la edad se analizó mediante regresión lineal. La asociación entre BMSi y edad se evaluó mediante ANOVA tras ajustar por el índice de masa corporal. El posible efecto de depleción estrogénica postmenopáusica sobre el BMSi se estudió comparando el subgrupo de mujeres más jóvenes con las más mayores mediante la prueba t de Student. Resultados: Los análisis de regresión lineal mostraron que la BMSi no se correlaciona con la edad en varones (R2=0,0016, p=0,74) ni en mujeres (R2=0,076, p=0,25). Asimismo, el análisis ajustado de ANOVA no demostró asociación entre la BMSi y la edad ni en varones (p=0,78) ni en mujeres (p=0,73). Finalmente, no se encontraron diferencias entre la BMSi entre las mujeres más jóvenes y las mayores (p=0,8). Conclusiones: La resistencia tisular mecánica ósea en individuos sanos es independiente a la edad y a la depleción estrogénica postmenopáusica

Objective: Impact microindentation (IMI) is a technique that allows the measurement of mechanicalbone tissue resistance in vivo. IMI has proven to provide useful information on the evaluation of skeletal diseases, but the effect of age on the bone property that is measured by this technique is unknown. This study aims to analyzethe relationship between age and MIH. Material and methods: Bone Material Strength index (BMSi), IMI’s output variable, was measured in 69 healthy women (median age: 49 years, range: 30-81 years) and 19 healthy men median age: 34 years, range: 24-98 years). The correlation between BMSi and age was analyzed by linear regression. The association between BMSi and age was evaluated by ANOVA after adjusting for body mass index. The potential effect of postmenopausal estrogenic depletion on BMSi was studied by comparing the younger vs the older subset of women through a t-student test. Results: Linear regression analysis showed that BMSi was not correlated with age in either men (R2=0.0016, p=0.74) or women (R2=0.076, p=0.25). Similarly, the BMI-adjusted ANOVA model revealed a lack of asso-ciation of BMSi with age in men (p=0.78) and women (p=0.73). Finally, there were not significant differences on BMSi detected between the younger and the older subset of women (p=0.8). Conclusions: Bone tissue mechanical resistance in healthy individuals is independent of age and postmenopausal estrogenic depletion

In-vitro study of hierarchical structures: Anodic oxidation and alkaline treatments onto highly rough titanium cold gas spray coatings for biomedical applications.

Hierarchical structures were obtained applying two different nanotexturing surface treatments onto highly rough commercial pure titanium coatings by cold spray: (i) anodic oxidation and (ii) alkaline treatments. An extended surface characterization in terms of topography, composition, and wettability has been performed to understand how those parameters affect to cell response. Primary human osteoblasts extracted from knee were seeded onto the as-sprayed titanium surface before and after the nanotexturing treatments. Cell viability was tested by using MTS and LIVE/DEAD assays, as well as osteoblasts differentiation by alkaline phosphatase (ALP) quantification at 3 and 10 days of cell culture. The combination of micro-/nano-roughness results in a significantly increase of cell proliferation, as well as cell differentiation after 10 days of cell culture in comparison with the non-treated coatings.

Estudio de la base genética de la reducción del Trabecular Bone Score relacionada con los inhibidores de la aromatasa / Study of the genetic basis of Trabecular Bone Score reduction related to aromatase inhibitors

Objetivos: Los inhibidores de la aromatasa (AI) son terapias endocrinas adyuvantes eficaces para pacientes con cáncer de mama, aunque se han asociado a un mayor riesgo de fractura osteoporótica. Previamente se ha demostrado una pérdida en el Trabecular Bone Score (TBS) que puede variar entre las pacientes tratadas con IA. El estudio pretendió identificar la base genética asociada al cambio en el TBS mediante el estudio de genes de la vía esteroidogénica. Material y métodos: La cohorte B-ABLE estudia de forma prospectiva a mujeres postmenopáusicas con cáncer de mama en tratamiento con IA. Se calculó el TBS a partir de los datos adquiridos en la densitometría mediante absorciometría radiológica dual (DXA) realizada al inicio y al final del tratamiento con IA. El cambio relativo del TBS se calculó como la variación porcentual del valor de TBS al final de tratamiento respecto al TBS basal. Para estudiar la posible asociación genética se genotiparon los polimorfismos de cambio de un nucleótido (SNPs) en los genes CYP11A1, CYP17A1, HDE3B2, HDE17B3, CYP19A1, CYP2C19, CYP2C9, ESR1, GC, CYP27B1, VDR y CYP24A1. Se estudió mediante regresión lineal múltiple la posible relación entre genes y cambios en TBS contemplando los modelos de herencia genética dominante, recesivo y aditivo. Resultados: Se incluyeron en el estudio un total de 212 mujeres no tratadas con bisfosfonatos en las que pudo calcularse el TBS. La mitad de las pacientes habían recibido tratamiento previo con tamoxifeno. El porcentaje de cambio intra-individual del TBS fue del -0,04% [IC del 95%: -0,05 a -0,03; p<0,001] al final de tratamiento con IA. El SNP rs6013897 en el gen CYP24A1 mostró una asociación significativa con la reducción del TBS [p=0,03565; coeficiente Beta (IC del 95%) = -1,55 (-2,98 a -0,11)]. Conclusiones: El gen CYP24A1 podría estar implicado en la variabilidad fenotípica encontrada en el deterioro de la microarquitectura ósea durante el tratamiento con IA

Objectives: Aromatase inhibitors (AI) are effective adjuvant endocrine therapies for breast cancer patients, although they have been associated with an increased risk of osteoporotic fracture. Trabecular Bone Score (TBS) loss has been previously demonstrated, although it may vary among AI-treated patients. This study aims to identify the genetic basis associated with TBS change by studying steroidogenic pathway genes. Material and methods: The B-ABLE cohort studies prospectively postmenopausal women with breast cancer under treatment with AI. TBS is calculated from the raw data acquired in dual-energy x-ray absorptiometry (DXA) scan at the outset of the study and at the end of AI-treatment. The relative TBS change was calculated as the percentage variation of the TBS value at the end of treatment from baseline. To study the possible genetic association, nucleotide polymorphisms (SNPs) were genotyped in genes CYP11A1, CYP17A1, HDE3B2, HDE17B3, CYP19A1, CYP2C19, CYP2C9, ESR1, GC, CYP27B1, VDR and CYP24A1. The possible relationship between genes and TBS changes was studied by multiple linear regression, considering models of dominant, recessive and additive genetic inheritance. Results: The study included 212 women that had not been treated with bisphosphonates and had available TBS data. Half of the patients had been treated previously with tamoxifen. The percentage of intra-individual TBS change was -0.04% [95% CI: -0.05 to -0.03; p<0.001] at the end of AI treatment. The SNP rs6013897 in the gene CYP24A1 showed a significant association with TBS reduction [p=0.03565; coefficient Beta (95% CI) = -1.55 (-2.98 to -0.11)]. Conclusions: The CYP24A1 gene could be involved in the phenotypic variability found in bone microarchitecture deterioration during AI treatment

Functionalized coatings by cold spray: An in vitro study of micro- and nanocrystalline hydroxyapatite compared to porous titanium.

Three different surface treatments on a Ti6Al4V alloy have been in vitro tested for possible application in cementless joint prosthesis. All of them involve the novelty of using the Cold Spray technology for their deposition: (i) an as-sprayed highly rough titanium and, followed by the deposition of a thin hydroxyapatite layer with (ii) microcrystalline or (iii) nanocrystalline structure. Primary human osteoblasts were extracted from knee and seeded onto the three different surfaces. Cell viability was tested by MTS and LIVE/DEAD assays, cell differentiation by alkaline phosphatase (ALP) quantification and cell morphology by Phalloidin staining. All tests were carried out at 1, 7 and 14 days of cell culture. Different cell morphologies between titanium and hydroxyapatite surfaces were exhibited. At 1 day of cell culture, cells on the titanium coating were spread and flattened, expanding the filopodia actin filaments in all directions, while cells on the hydroxyapatite coatings showed round like-shape morphology due to slower attachment. Higher cell viability was detected at all times of cell culture on titanium coating due to a better attachment at 1 day. However, from 7 days of cell culture, cells on hydroxyapatite showed good attachment onto surfaces and highly increased their proliferation, mostly on nanocrystalline, achieving similar cell viability levels than titanium coatings. ALP levels were significantly higher in titanium, in part, because of greatest cell number. Overall, the best cell functional results were obtained on titanium coatings whereas microcrystalline hydroxyapatite presented the worst cellular parameters. However, results indicate that nanocrystalline hydroxyapatite coatings may achieve promising results for the faster cell proliferation once cells are attached on the surface.

Osteoblastic cell response on high-rough titanium coatings by cold spray.

Highly rough and porous commercially pure titanium coatings have been directly produced for first time by the cold spray technology, which is a promising technology in front of the vacuum plasma spray for oxygen sensitive materials. The wettability properties as well as the biocompatibility evaluation have been compared to a simply sand blasted Ti6Al4V alloy substrate. Surface topographies were analysed using confocal microscopy. Next, osteoblast morphology (Phalloidin staining), proliferation (MTS assay), and differentiation (alkaline phosphatase activity) were examined along 1, 7 and 14 days of cell culture on the different surfaces. Finally, mineralization by alizarin red staining was quantified at 28 days of cell culture. The contact angle values showed an increased hydrophilic behaviour on the as-sprayed surface with a good correlation to the biological response. A higher cell viability, proliferation and differentiation were obtained for highly rough commercial pure titanium coatings in comparison with sand blasted substrates. Cell morphology was similar in all coatings tested; at 14 days both samples showed extended filopodia. A higher amount of calcium-rich deposits was detected on highly rough surfaces. In summary, in-vitro results showed an increase of biological properties when surface roughness increases.

Discrepancy between bone density and bone material strength index in three siblings with Camurati-Engelmann disease.

Camurati-Engelmann (CE) is a very rare disease affecting one in every million persons worldwide. It is characterized by an enlargement of long bones. We aimed to assess bone characteristics in three siblings with different tools. Even if there was an excess of bone density, quality seemed to be deteriorated. INTRODUCTION: CE disease is a rare monogenic disorder affecting approximately one in every million persons worldwide. It is mainly characterized by a progressive hyperostosis of the periosteum and endosteum of the diaphysis of long bones. Limited data are available about bone characteristics in these patients. In three siblings with CE disease, we aimed to assess bone mineral density (BMD) and trabecular bone score (TBS) by dual-energy X-ray absorptiometry (DXA) and material characteristics at tissue level using bone impact reference point indentation. METHODS: Clinical data were collected and a general laboratory workup was performed. At the lumbar spine and hip, BMD and TBS were measured using DXA imaging. Bone material strength index (BMSi) was measured by bone impact microindentation using an Osteoprobe instrument. RESULTS: All three cases had densitometric values consistent with high bone mass (sum of Z-score at the lumbar spine and hip > 4). Hip BMD was extremely high in all three siblings at both total hip and femoral neck, while at the lumbar spine, two of them had normal values but the third again had very high BMD. TBS values were in the normal range. In contrast, BMSi measurements were at low or very low levels, compared with normal controls. CONCLUSION: Despite strikingly increased BMD and normal microarchitecture, BMSi is affected in patients with CE. Microindentation could be an appropriate tool for assessing bone fragility in these patients. Bone disease in this group of patients requires further study to better understand the underlying regulatory mechanisms and their alterations.

Análisis genético de enzimas de la vía esteroidal asociadas a efectos adversos musculoesqueléticos de los inhibidores de la aromatasa / Genetic analysis of steroid pathway enzymes associated with adverse musculoskeletal effects of aromatase inhibitors

Objetivos: Identificar putativas variantes funcionales en los genes CYP11A1 y CYP17A1 asociadas a efectos musculoesqueléticos (pérdida acelerada de la masa ósea y artralgias) derivados del tratamiento con inhibidores de la aromatasa (IA). Material y métodos: La cohorte B-ABLE es un estudio prospectivo de mujeres postmenopáusicas con cáncer de mama en tratamiento con IA. La densidad mineral ósea en columna lumbar y cuello femoral se midió mediante densitometría, y el dolor articular mediante escala analógica visual. A partir de polimorfismos de cambio de un nucleótido (SNPs) en los genes CYP11A1 (rs4077581, rs11632698 y rs900798) y CYP17A1 (rs4919686, rs4919683, rs4919687, rs3781287, rs10786712, rs6163, rs743572), asociados previamente con eventos musculoesqueléticos, se construyeron los haplotipos para cada paciente de la cohorte, y se seleccionaron aquellos que mostraron mayor diferencia fenotípica (p<0,05). Dentro de cada haplotipo, se eligieron aquellas pacientes con fenotipos extremos para la secuenciación de los respectivos genes y la identificación de variantes genéticas funcionales. Finalmente, se realizó un análisis de regresión lineal múltiple contemplando los modelos de herencia genética dominante, recesivo y aditivo. Resultados: No se encontró ninguna mutación en las regiones codificantes. En la región del promotor basal del gen CYP11A1 se encontró una variante genética (D15S520) asociada a la pérdida de masa ósea del cuello de fémur a los 24 meses de tratamiento con IA. Conclusiones: Variantes en regiones reguladoras del gen CYP11A1 podrían modular la expresión de este gen, explicando así parte de la variabilidad fenotípica encontrada en la pérdida de hueso de las pacientes en tratamiento con IA (AU)

Objetives: Identify putative functional variants in the CYP11A1 and CYP17A1 genes associated with musculoskeletal effects (accelerated bone mass loss and arthralgia) derived from treatment with aromatase inhibitors (AI). Material and methods: The B-ABLE cohort is a prospective study of postmenopausal women with breast cancer undergoing AI treatment. Bone mineral density in the lumbar spine and femoral neck was measured by densitometry and joint pain using visual analogue scale. From single-nucleotide polymorphisms (SNPs) in genes CYP11A1 (rs4077581, rs11632698 and rs900798) and CYP17A1 (rs4919686, rs4919683, rs4919687, rs3781287, rs10786712, rs6163, rs743572), previously associated with musculoskeletal events, haplotypes were constructed for each pacient from the cohort, and those haplotypes that showed greatest phenotypic differences were chosen (p<0.05). Within each haplotype, patients with extreme phenotypes were chosen for the sequencing of respective genes and identifying functional genetic variants. Finally, a multiple linear regression analysis was carried out considering the models of dominant, recessive and additive genetic inheritance. Results: No mutation was found in coding regions. A genetic variant (D15S520), in the basal promoter region of gene CYP11A1, was found associated with femoral neck bone loss at 24 month of AI treatment. Conclusions: Variants in regulatory regions of the CYP11A1 gene could modulate the expression of this gene, thus explaining part of the phenotypic variability found in bone loss of patients undergoing AI treatment (AU)

Identificación de variantes genéticas asociadas con la densidad mineral ósea (DMO) en el gen FLJ42280 / Identification of genetic variants associated with bone mineral density (BMD) in the FLJ42280 gene

FLJ42280 es un posible gen de susceptibilidad a la osteoporosis. Distintos estudios de GWAs han identificado 4 SNPs no-codificantes en este gen que se asocian a la densidad mineral ósea (DMO) y el riesgo de fractura. Para descubrir la causa de la asociación entre estos SNPs y la osteoporosis, se realizó una búsqueda de variantes genéticas mediante resecuenciación de 28 kb que contienen el gen, en una selección truncada de mujeres con DMO muy baja (n=50) o muy alta (n=50) de la cohorte BARCOS (Barcelona Cohorte Osteoporosis, cohorte de mujeres postmenopáusicas de Barcelona). Las variantes encontradas se filtraron y se analizó su frecuencia en cada grupo. Se analizó el solapamiento de las variantes con elementos funcionales del proyecto ENCODE y también se calculó el desequilibrio de ligamiento entre los SNPs de la región. Finalmente, se hizo un análisis de eQTL de los 4 SNPs no-codificantes respecto a los niveles de expresión de genes cercanos a FLJ42280 en linfoblastos. Se seleccionaron 110 variantes. Las diferencias de sus frecuencias entre los dos grupos estuvieron por debajo del poder estadístico del diseño experimental. Sin embargo, 3 variantes solaparon con posibles enhancers y una solapó con un enhancer activo en osteoblastos (rs4613908). Se observó un fuerte desequilibrio de ligamiento entre los 4 SNPs no-codificantes y el SNP rs4613908, que pertenecen a un bloque que abarca el gen casi por completo. Ninguno de los SNPs no-codificantes mostró asociación con los niveles de expresión de genes cercanos a FLJ42280. En conclusión, el SNP rs4613908 podría estar implicado funcionalmente en la determinación de la DMO. Serán necesarios experimentos concretos para confirmarlo (AU)

FLJ42280 is a possible gene for susceptibility to osteoporosis. Different studies of GWAs have identified 4 non-coding SNPs in this gene associated with bone mineral density (BMD) and fracture risk. In order to ascertain the cause of the association between these SNPs and osteoporosis, we searched for genetic variants by resequencing the 28-kb gene, in a truncated selection of women with very low (n=50) or very high BMD (N=50) of the BARCOS cohort (Barcelona Cohort Osteoporosis, cohort of postmenopausal women in Barcelona). The variants found were filtered and their frequency analyzed in each group. The overlap of the variants with functional elements of the ENCODE project was calculated. Finally, an eQTL analysis of the 4 SNPs-coding was performed on the expression levels of FLJ42280 neighbor genes in lymphoblasts. In all, 110 variants were selected. The differences in their frequencies between the two groups were below the statistical power of the experimental design. However, three variants overlapped with possible enhancers and one overlapped with an active enhancer in osteoblasts (rs4613908). A strong linkage disequilibrium was observed between the 4 non-coding SNPs and the SNP rs4613908, which belong to a block spanning the gene almost completely. None of the non-coding SNPs showed association with the expression levels of FLJ42280 neighbor genes. In conclusion, the SNP rs4613908 could be involved functionally in determining BMD. Tangible experiments will be required to confirm this (AU)

Real-Time Protein and Cell Binding Measurements on Hydroxyapatite Coatings.

Although a lot of in vitro and in vivo assays have been performed during the last few decades years for hydroxyapatite bioactive coatings, there is a lack of exploitation of real-time in vitro interaction measurements. In the present work, real-time interactions for a plasma sprayed hydroxyapatite coating were measured by a Multi-Parametric Surface Plasmon Resonance (MP-SPR), and the results were compared with standard traditional cell viability in vitro assays. MP-SPR is proven to be suitable not only for measurement of molecule-molecule interactions but also molecule-material interaction measurements and cell interaction. Although SPR is extensively utilized in interaction studies, recent research of protein or cell adsorption on hydroxyapatite coatings for prostheses applications was not found. The as-sprayed hydroxyapatite coating resulted in 62.4% of crystalline phase and an average thickness of 24 ± 6 µm. The MP-SPR was used to measure lysozyme protein and human mesenchymal stem cells interaction to the hydroxyapatite coating. A comparison between the standard gold sensor and Hydroxyapatite (HA)-plasma coated sensor denoted a clearly favourable cell attachment on HA coated sensor as a significantly higher signal of cell binding was detected. Moreover, traditional cell viability and proliferation tests showed increased activity with culture time indicating that cells were proliferating on HA coating. Cells show homogeneous distribution and proliferation along the HA surface between one and seven days with no significant mortality. Cells were flattened and spread on rough surfaces from the first day, with increasing cytoplasmatic extensions during the culture time.

Estudio del patrón de expresión de microRNAs en el hueso osteoporótico / Study of miRNAs expression patterns in osteoporotic bone

Objetivos: Identificar microRNAs (miRNAs) diferencialmente expresados en muestras óseas con fractura osteoporótica respecto a huesos sanos. Material y métodos: Se extrajo RNA total a partir de hueso trabecular fresco del cuello femoral de mujeres sometidas a reemplazo de cadera, ya sea debido a fractura osteoporótica (n=6) o por artrosis en ausencia de osteoporosis (según la DMO) (n=6). Las muestras se hibridaron en un array de miRNAs y se realizaron diagramas de PCA y de mapa de calor. Para la comparación de los niveles de expresión, se fijó como significativo un umbral de cambio de >1,5 veces y un valor p <0,05 en la t de Student (corregido para múltiples pruebas). Resultados: Tanto los análisis de PCA como el mapa de calor mostraron una agrupación de las muestras según si eran de fractura o no. Se detectaron 790 miRNAs en las muestras de hueso, 82 de los cuales estaban alterados en las muestras osteoporóticas. Tras la validación en otro panel de 6 muestras osteoporóticas y 6 no osteoporóticas mediante PCR a tiempo real de los miRNAs más significativos, y para los que existía un ensayo disponible, se confirmaron los miRNAs miR-320a y miR-22-3p. Estos dos miRNAs se detectaron en cultivos de osteoblastos primarios, aunque no mantenían el mismo patrón de expresión que en las muestras de hueso total. Conclusiones: Hemos demostrado que existen diferencias en la expresión de miRNAs en muestras con fractura osteoporótica, lo que abre nuevas perspectivas para la investigación y diseño de nuevas terapias (AU)

Objectives: To identify microRNAs (miRNAs) differentially expressed in bone samples with osteoporotic fracture compared with healthy bones. Methods: Total RNA was extracted from fresh trabecular bone of the femoral neck of women undergoing hip replacement surgery, either because to osteoporotic fracture (n=6) or in the absence of osteoarthritis osteoporosis (based on BMD) (n=6). The samples were hybridized on an array of miRNAs and PCA diagrams and heat map were made. To compare expression levels, >1.5 times and a value p<0.05 Student's T test (corrected for multiple testing) was set as a threshold of significant change. Results: Both PCA analysis and the heat map showed a samples grouping whether there was fracture or not. 790 were detected miRNAs in bone samples, 82 of which were altered in the osteoporotic samples. After validation in another panel of 6 samples 6 osteoporotic and non-osteoporotic by PCR real time of the most significant miRNAs, and for which there was a test available, the miRNAs, miR-320a and miR-22-3p were confirmed. These two miRNAs were detected in cultures of primary osteoblasts, although they did not maintain the same pattern of expression in total bone samples. Conclusions: We have shown that there are differences in the expression of miRNAs in samples with osteoporotic fracture. This opens prospects for research and design of new therapies (AU)

Evolución de la DMO durante el tratamiento con inhibidores de aromatasa y su relación con el gen CYP11A1: estudio prospectivo de la cohorte B-ABLE / BMD evolution during treatment with aromatase inhibitors and its relation to the CYP11A1 gene: prospective study in the B-ABLE cohort

Objetivos: El objetivo del estudio fue analizar los cambios en la densidad mineral ósea (DMO) a lo largo del tratamiento con inhibidores de aromatasa (IA) en la práctica clínica y evaluar la asociación entre el gen CYP11A1 y la variación de DMO al final del tratamiento. Material y métodos: La cohorte B-ABLE es un estudio prospectivo de mujeres postmenopáusicas con cáncer de mama, en tratamiento con IA. Se analizó la variación de DMO durante todo el tratamiento con IA, así como las diferencias entre las pacientes tratadas y no-tratadas previamente con tamoxifeno (TMX). Tres polimorfismos (rs4077581, rs11632698 y rs900798) del gen CYP11A1, fueron genotipados para su asociación con la variación de DMO. Resultados: Las pacientes tratadas con TMX mostraron pérdidas más aceleradas de DMO que las no tratadas previamente con TMX (60% menos en columna y 46% en fémur a los 2 años y 70% menos en columna y 63% en fémur a los 3 años). No obstante, al final del tratamiento no se detectaron diferencias significativas en la pérdida de DMO entre ambos grupos de pacientes. Los 3 polimorfismos del gen CYP11A1 resultaron significativamente asociados a la variación de DMO en fémur al final del tratamiento. Conclusiones: La DMO disminuyó de forma más acelerada en las pacientes con tratamiento previo con TMX que en las que solo recibieron AI, a pesar de que no se detectaron diferencias significativas al final de tratamiento. Polimorfismos en el gen CYP11A1 están relacionados con la variación de la DMO en respuesta al tratamiento con IA (AU)

Objectives: The aim of this study was to analyze bone mineral density (BMD) changes throughout aromatase inhibitor (AI) treatment in clinical cases and also consider its association with the CYP11A1 gene and the BMD variation after treatment. Material and methods: The B-ABLE cohort is a prospective study of postmenopausal women with breast cancer, in AI treatment. BMD variation was analyzed during AI treatment, as well as the differences those patients who were treated and not treated previously with tamoxifen (TMX). Three polymorphisms (rs4077581, rs11632698 and rs900798) of the CYP11A1 gene were genotyped for their association with BMD variation. Results: TMX-treated patients presented more rapid BMD loss than those who did not undergo prior TMX treatment (60% less in spine and 46% in femur at 2 years and 70% less in the spine and 63% in the femur at 3 years). However, no significant BMD loss was detected after treatment in either group. The 3 CYP11A1 gene polymorphisms were significantly associated with BMD variation in the femur at the end of the treatment. Conclusions: BMD was reduced more rapidly in patients with prior TMX treatment than in those who only received AI, although no significant differences were detected after treatment. The 3 CYP11A1 gene polymorphisms were associated with BMD variation in response to AI treatment (AU)

CYP11A1 expression in bone is associated with aromatase inhibitor-related bone loss.

Aromatase inhibitors (AIs) used as adjuvant therapy in postmenopausal women with hormone receptor-positive breast cancer cause diverse musculoskeletal side effects that include bone loss and its associated fracture. About half of the 391 patients treated with AIs in the Barcelona-Aromatase induced bone loss in early breast cancer cohort suffered a significant bone loss at lumbar spine (LS) and/or femoral neck (FN) after 2 years on AI-treatment. In contrast, up to one-third (19.6% LS, 38.6% FN) showed no decline or even increased bone density. The present study aimed to determine the genetic basis for this variability. SNPs in candidate genes involved in vitamin D and estrogen hormone-response pathways (CYP11A1, CYP17A1, HSD3B2, HSD17B3, CYP19A1, CYP2C19, CYP2C9, ESR1, DHCR7, GC, CYP2R1, CYP27B1, VDR and CYP24A1) were genotyped for association analysis with AI-related bone loss (AIBL). After multiple testing correction, 3 tag-SNPs (rs4077581, s11632698 and rs900798) located in the CYP11A1 gene were significantly associated (P<0.005) with FN AIBL at 2 years of treatment. Next, CYP11A1 expression in human fresh bone tissue and primary osteoblasts was demonstrated by RT-PCR. Both common isoforms of human cholesterol side-chain cleavage enzyme (encoded by CYP11A1 gene) were detected in osteoblasts by western blot. In conclusion, the genetic association of CYP11A1 gene with AIBL and its expression in bone tissue reveals a potential local function of this enzyme in bone metabolism regulation, offering a new vision of the steroidogenic ability of this tissue and new understanding of AI-induced bone loss.

In vitro performance of ceramic coatings obtained by high velocity oxy-fuel spray.

Hydroxyapatite coatings obtained by plasma-spraying have been used for many years to improve biological performance of bone implants, but several studies have drawn attention to the problems arising from high temperatures and the lack of mechanical properties. In this study, plasma-spraying is substituted by high velocity oxy-fuel (HVOF) spray, with lower temperatures reached, and TiO2 is added in low amounts to hydroxyapatite in order to improve the mechanical properties. Four conditions have been tested to evaluate which are those with better biological properties. Viability and proliferation tests, as well as differentiation assays and morphology observation, are performed with human osteoblast cultures onto the studied coatings. The hydroxyapatite-TiO2 coatings maintain good cell viability and proliferation, especially the cases with higher amorphous phase amount and specific surface, and promote excellent differentiation, with a higher ALP amount for these cases than for polystyrene controls. Observation by SEM corroborates this excellent behaviour. In conclusion, these coatings are a good alternative to those used industrially, and an interesting issue would be improving biological behaviour of the worst cases, which in turn show the better mechanical properties.