Silicon substituted hydroxyapatite/VEGF scaffolds stimulate bone regeneration in osteoporotic sheep.

Silicon-substituted hydroxyapatite (SiHA) macroporous scaffolds have been prepared by robocasting. In order to optimize their bone regeneration properties, we have manufactured these scaffolds presenting different microstructures: nanocrystalline and crystalline. Moreover, their surfaces have been decorated with vascular endothelial growth factor (VEGF) to evaluate the potential coupling between vascularization and bone regeneration. In vitro cell culture tests evidence that nanocrystalline SiHA hinders pre-osteblast proliferation, whereas the presence of VEGF enhances the biological functions of both endothelial cells and pre-osteoblasts. The bone regeneration capability has been evaluated using an osteoporotic sheep model. In vivo observations strongly correlate with in vitro cell culture tests. Those scaffolds made of nanocrystalline SiHA were colonized by fibrous tissue, promoted inflammatory response and fostered osteoclast recruitment. These observations discard nanocystalline SiHA as a suitable material for bone regeneration purposes. On the contrary, those scaffolds made of crystalline SiHA and decorated with VEGF exhibited bone regeneration properties, with high ossification degree, thicker trabeculae and higher presence of osteoblasts and blood vessels. Considering these results, macroporous scaffolds made of SiHA and decorated with VEGF are suitable bone grafts for regeneration purposes, even in adverse pathological scenarios such as osteoporosis. STATEMENT OF SIGNIFICANCE: For the first time, the in vivo behavior of scaffolds made of silicon substituted hydroxyapatites (SiHA) has been evaluated under osteoporosis conditions. In order to optimize the bone regeneration properties of these bioceramics, 3D macroporous scaffolds have been manufactured by robocasting and implanted in osteoporotic sheep. Our experimental design shed light on the important issue of the biological response of nano-sized bioceramics vs highly crystalline bioceramics, as well as on the importance of coupling vascularization and bone growth processes by decorating SiHA scaffolds with vascular endothelial growth factor.

Synergistic effect of Si-hydroxyapatite coating and VEGF adsorption on Ti6Al4V-ELI scaffolds for bone regeneration in an osteoporotic bone environment.

The osteogenic and angiogenic responses to metal macroporous scaffolds coated with silicon substituted hydroxyapatite (SiHA) and decorated with vascular endothelial growth factor (VEGF) have been evaluated in vitro and in vivo. Ti6Al4V-ELI scaffolds were prepared by electron beam melting and subsequently coated with Ca10(PO4)5.6(SiO4)0.4(OH)1.6 following a dip coating method. In vitro studies demonstrated that SiHA stimulates the proliferation of MC3T3-E1 pre-osteoblastic cells, whereas the adsorption of VEGF stimulates the proliferation of EC2 mature endothelial cells. In vivo studies were carried out in an osteoporotic sheep model, evidencing that only the simultaneous presence of both components led to a significant increase of new tissue formation in osteoporotic bone. STATEMENT OF SIGNIFICANCE: Reconstruction of bones after severe trauma or tumors extirpation is one of the most challenging tasks in the field of orthopedic surgery. This scenario is even more complicated in the case of osteoporotic patients, since their bone regeneration capability is decreased. In this work we present a porous implant that promotes bone regeneration even in osteoporotic bone. By coating the implant with osteogenic bioceramics such as silicon substituted hydroxyapatite and subsequent adsorption of vascular endothelial growth factor, these implants stimulate the bone ingrowth when they are implanted in osteoporotic sheep.

Response of macrophages and neural cells in contact with reduced graphene oxide microfibers.

Graphene-based materials are revealing a great promise for biomedical applications and demonstrating attractiveness for neural repair. In the context of neural tissue damage, the dialogue between neural and immune cells appears critical for driving regeneration, thus making the understanding of their relations pivotal. Herein, the acute response of RAW-264.7 macrophages on nanostructured reduced graphene oxide (rGO) microfibers has been evaluated through the analysis of cell parameters including proliferation, viability, intracellular content of reactive oxygen species, cell cycle, apoptosis, and cell size and complexity. The influence of the direct contact of rGO microfibers on their polarization towards M1 and M2 phenotypes has been studied by analyses of both M1 (CD80) and M2 (CD163) markers and the secretion of the inflammatory cytokines TNF-α and IL-6. Finally, the capability of these rGO microfibers to regulate neural stem cell differentiation has been also evaluated. Findings reveal that rGO microfibers inhibit the proliferation of RAW-264.7 macrophages without affecting their viability and cell cycle profiles. The presence of M1 and M2 macrophages on these microfibers was confirmed after 24 and 48 h, respectively, accompanied by a decrease in TNF-α and an increase in IL-6 cytokine secretion. These rGO microfibers were also able to support the formation of a highly interconnected neural culture composed of both neurons (map2+ cells) and glial cells (vimentin+ cells). These findings encourage further investigation of these microfibers as attractive biomaterials to interact with immune and neural cells, attempting to support wound healing and tissue repair after implantation.

Pregnancy protects against antiangiogenic and fibrogenic effects of angiotensin II in rat hearts.

AIM: To investigate the difference between physiological and pathological cardiac remodelling induced, respectively, by pregnancy and angiotensin (Ang) II, and to test the hypothesis that pregnancy protects against Ang II effects. METHODS: Female Wistar rats, pregnant (n = 12) and non-pregnant (n = 12), were implanted with mini-pumps containing saline (sham) or 150 ng kg(-1) min(-1) Ang II. Ten days later echocardiography and blood pressure measurement were performed. Expression of 22 genes was assessed using RT-PCR. Microscopic sections of LV were prepared to determine collagen content (Sirius Red staining), vessel density (ß-actin immunolabelling) and myocytes diameter (Toluidine Blue). RESULTS: Heart weight (HW) was increased in Ang II treated groups compared with their controls. Furthermore, HW of Ang II treated pregnant rats (1.0 ± 0.03 g) was higher than that in non-pregnant sham (0.7 ± 0.02 g), pregnant (0.8 ± 0.01 g) and Ang II treated non-pregnant (0.8 ± 0.02 g) rats. Relative LV wall thickness showed similar pattern. Aortic pressure was significantly increased in Ang II groups. Collagen content was increased in Ang II (4.0 ± 0.5%) compared with sham (1.5 ± 0.1%) but reduced again when treated rats were pregnant (2.8 ± 0.4%). Vessel density was reduced by 47.8% after Ang II treatment in non-pregnant and by only 13.9% in pregnant rats. Gene expression analysis showed increased expression of atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), anykrin repeat domain-containing protein 1 (Ankrd-1), protein kinase C-α and -δ and tumour suppressor gene TP53 (p53) in Ang II treated groups and upregulation of ANF, BNP and Ankrd-1 remained when pregnancy was combined with Ang II. Pregnancy reduced expression of: α-myosin heavy chain, tumour necrosis factor-α, p53, endothelial nitric oxide synthase and inducible nitric oxide synthase. CONCLUSION: Pregnancy seems to counteract the detrimental effects of Ang II on fibrosis and angiogenesis in heart. In addition, pregnancy and Ang II lead to partly opposite changes in the expression of some genes important for heart function.

Multiple acute parasitization by Anisakis simplex.

Hypersensitivity to Anisakis is an increasingly prominent medical problem throughout the world, due to a better understanding of diseases induced by parasites and to modern culinary habits of eating raw or undercooked fish. We describe the case of a patient who presented epigastric pain, wheals, erythema, and pruritus 3 hours after the ingestion of fish. More than 200 larvae were obtained by endoscopy. However, the patient only developed an immune response with specific immunoglobulin E and eosinophilia peaking at day 18 and decreasing during the 17-month follow-up. Only eosinophilia reached normal limits.

[Improving the specification quality of inclusion criteria of patients in chemotherapy clinical trials]. / Mejora de la calidad de la especificación de la inclusión en ensayo clínico sobre prescripciones en quimioterapia.

OBJECTIVES: To assess the quality improvement of chemotherapy prescriptions for patients included in clinical trials (CT) analyzing whether the prescription is specified for patient participation, after having identified the major risks factors and the appropriate corrective measures are applied. METHODS: Retrospective study of omissions collected during 2006 and 2007. After collecting all the information, we analyzed the risks factors and introduced improvement measures (information to prescribers and the beginning of electronic prescription). The 2007 data were then evaluated. Variables analyzed: CT, number of prescriptions, principal investigator, prescriber, department, anti-neoplastic involved and pathology. RESULTS: Oncology and Haematology make up 57.1% of hospital trials. In 2006, 1625 chemotherapy prescriptions were received and there were 151 incorrect prescriptions (9.3%), and in 2007, 1858 prescriptions with 65 (3.5%) incorrect. In 2006, there was a higher frequency of incorrect prescriptions in Haematology (34.1%) and (Oncology (8.6%)). In 2007 Hematology had just 1.8% of omissions and in Oncology it decreased to 3.6%. As regards the pathology, the error rate has virtually disappeared in multiple myeloma prescriptions (34.1 to 2.2%) and decreased in breast cancer (10.8 to 4.4%). The non-fulfillment rate is higher when the prescriber is not the principal investigator. CONCLUSIONS: The overall frequency of prescriptions with errors decreased significantly from 9.3% to 3.5%, with an extraordinary reduction in Haematology (34.1% to 1.8%) related to the multiple myeloma trials. The corrective measures implemented have proved to be useful in CT.

Diagnosis of aspirin-induced asthma combining the bronchial and the oral challenge tests: a pilot study.

BACKGROUND: We investigated the usefulness of the bronchial challenge (BC) with lysine-acetylsalicylate (L-ASA) in the diagnosis of aspirin-exacerbated respiratory disease (AERD) using a protocol that combined both the oral challenge (OC) and the BC tests. METHODS: Adult asthmatic patients with suspected AERD who underwent BC with L-ASA were included in the study. If the BC result with L-ASA was negative, an OC was carried out to establish the diagnosis. AERD was ruled out if both the BC and the OC results were negative (nonresponders). Both responders and nonresponders were compared for age, gender, a personal or family history of atopy, underlying disease, current asthma treatment, and presence of nasal polyps. Six patients with asthma but no suggestive history of AERD were included as controls. RESULTS: Twenty-two patients completed the study. Ten patients tested positive to the BC and/or OC (responders), whereas 12 did not (nonresponders). Seven out of the 10 responders had a positive BC result and 3 a positive OC result. After BC, 4 patients had an early asthmatic response, 1 had a dual response, and 2 had isolated late responses. No significant differences were observed in the aforementioned variables between responders and nonresponders. The results of both challenges were negative in the 6 controls. CONCLUSIONS: The BC had a high positive predictive value, was safe, and when negative, the subsequent OC did not result in any severe adverse reactions. The BC elicited an isolated late asthmatic response that has not been previously described in the literature.

Glass-glass ceramic thermoseeds for hyperthermic treatment of bone tumors.

Implantable thermoseeds are synthesised from mixtures of a melt-derived glass with composition SiO(2) (40)-CaO(40)-Fe(2)O(3)(20) (mol%) and a sol-gel glass with composition SiO(2)(58)-P(2)O(5)(6)-CaO(36) (mol%). Structural, textural and magnetic properties of the samples are evaluated. In vitro bioactivity is assessed in order to determine the potential capability to bond to living bone. In spite of the low textural properties of the material, a bioactive behavior is observed as a result of the sol-gel glass content. Although the crystallization of the glass ceramic provides the magnetic phase, the presence of sol-gel glass modifies the magnetic properties, improving the heating power. For the first time, hyperthermia heating experiments as well as preliminary biocompatibility assays have been carried out for this kind of material. The ability to reach hyperthermic temperature range together with the bioactive behavior makes this biomaterial a very promising candidate for bone cancer treatment.

In vitro biocompatibility assessment of poly(epsilon-caprolactone) films using L929 mouse fibroblasts.

Biodegradable and biocompatible materials are the basis for tissue engineering. As an initial step for developing vascular grafts, the in vitro biocompatibility of poly(epsilon-caprolactone) (PCL), recently suggested for several clinical applications, was evaluated in this study using L929 mouse fibroblasts. Different cellular aspects were analyzed in order to know the cell viability during cell culture on PCL films: adhesion, proliferation, morphology, LDH release and mitochondrial function. Since topography and other surface characteristics of materials play an essential part in cell adhesion, PCL membranes with either smooth or rough surface were prepared, characterized and used to carry out cell cultures. During short culture times, PCL produced a significant stimulation of mitochondrial activity evaluated by reduction of the MTT reagent. The results provide evidences of good adhesion, growth, viability, morphology and mitochondrial activity of cells on PCL films. Therefore, it can be concluded that PCL is a suitable and biocompatible material as a scaffold for vascular graft development.

Gastroallergic anisakiasis: borderline between food allergy and parasitic disease-clinical and allergologic evaluation of 20 patients with confirmed acute parasitism by Anisakis simplex.

BACKGROUND: Human subjects can be parasitized by Anisakis simplex by eating raw or undercooked fish. Gastric anisakiasis is probably the most frequent clinical entity presenting with severe epigastric pain, vomiting, and diarrhea. In gastroallergic anisakiasis hypersensitivity symptoms predominate. OBJECTIVE: We sought to describe clinical features, laboratory data, and gastroscopic findings in gastroallergic anisakiasis. METHODS: We selected 40 patients presenting to the emergency department with an acute allergic reaction, and if we suspected acute parasitism by A simplex, a fiberoptic gastroscopy was performed. In 20 patients we could detect one or more nematodes; these patients are referred to as group A. Those in whom no worm could be found are referred to as group B (n = 20). A detailed history, clinical features, gastroscopic findings, laboratory data, and skin prick test responses were compared. RESULTS: Long-time intervals of up to 26 hours between fish intake and onset of hypersensitivity symptoms were found (group A, 5.4 +/- 6.3 hours; group B, 5.3 +/- 2.6 hours). Patients in groups A and B did not differ with respect to allergic symptoms (urticaria, angioedema, erythema, bronchospasm, and anaphylaxis) or the mainly light abdominal symptoms (upper abdominal pain, nausea, vomiting, and diarrhea). No significant differences were found with respect to age, time interval between fish intake and onset of symptoms, white cell and eosinophil counts, specific IgE levels against A simplex, or total IgE levels. CONCLUSIONS: The peculiar sometimes long-time interval between fish intake and onset of allergic symptoms render the diagnosis difficult. An early gastroscopy can confirm the diagnosis and prevent complications. We suggest that gastroallergic anisakiasis be considered a distinct clinical entity in which the predominant symptoms are hypersensitivity symptoms and in which the correct diagnosis is not only important in the management of the acute reaction but also in the prevention of further allergic episodes.

[Incidence of genitourinary infection caused by Chlamydia trachomatis in a STD center calculated by direct antigen detection]. / Incidencia de la infección genitourinaria por Chlamydia trachomatis en un centro de ETS estimada mediante detección directa de antígeno.

OBJECTIVE: Chlamydia trachomatis is one of the most common sexually transmitted agents which causes a wide spectrum of diseases including urethritis in men and endocervicitis in women. We analyzed patients with genitourinary C. trachomatis infections evaluating risk factors and the association with other sexually transmitted infections. MATERIAL AND METHODS: We processed 1,180 specimens from 913 patients (772 women and 141 men), attended at a Sexually Transmitted Diseases (STD) Center. The diagnostic of C. trachomatis infection was made by an enzyme-linked fluorescent immunoassay, Vidas Chlamydia test (bioMérieux). RESULTS: The incidence of C. trachomatis infection was 4.8% (57 cases) and was higher in women (70.1%) than in men (29.8%). The risk groups observed were: 26 prostitute, 7 contact with prostitute or risk partner, 5 homosexual, 5 promiscuous heterosexual and 14 without risk groups. Associated with this infection we observed other: 10 bacterial vaginosis, 8 Papillomavirus infection, 3 Trichomonas vaginalis infections, 2 Neisseria gonorrhoeae infections and 2 Candidiasis. The 53.4% of these patients didn't have any symptomatology at the consult moment. CONCLUSIONS: The control of patients with risk factors is important for the diagnostic of C. trachomatis and other sexually transmitted infections, because most of them were prostitutes and asymptomatic. Within men, homosexuality, contact with prostitute or risk partner were the practices with higher risk.

Usefulness of early serial measurement of specific and total immunoglobulin E in the diagnosis of gastro-allergic anisakiasis.

BACKGROUND: Sensitization to Anisakis simplex (A. simplex) has been documented to produce severe allergic reactions following ingestion of mainly raw or under-cooked parasitized fish. False positive skin prick tests (SPT) or specific IgE against this nematode and cross-reactivity restricts diagnosis. Gastric anisakiasis and gastro-allergic anisakiasis occur if fish is parasitized by live A. simplex OBJECTIVE: To investigate if serial serological analysis could be useful in the diagnosis of acute parasitation by this nematode. METHODS: We included 41 patients who experienced an allergic reaction and/or abdominal symptoms after ingestion of raw or undercooked fish and displayed specific IgE against A. simplex. Total and specific IgE were determined two times: in the 24-h period after onset of clinical symptoms and after 1 month. SPTs were performed against A. simplex and implicated fish. A fibre optic gastroscopy was performed in 22 patients. RESULTS: Median total IgE was 80.0 (Interquartile range [IQR] 41.5-186.5) kU/L in the first evaluation and 247.0 (IQR 96.5-649.5) kU/L after 1 month. Median specific IgE against A. simplex was 11.4 (IQR 7.1-33.5) kU/L in the first 24 h and 36.8 (IQR 19.5-79.5) kU/L after 1 month. A rise of total IgE was observed in 35 of 41 patients (P<0.00001) and a rise in specific IgE against A. simplex in 37 of 41 patients (P<0.00001). Mean percentage increment was 392% (215-571%; 95% confidence interval [CI]) for total IgE and 339% (177-502%; 95% CI) for specific IgE. In nine of 22 gastroscopic examinations at least one larva, identified as A. simplex, could be detected by our microbiology service. In this group (n = 9) a rise of total and specific IgE was detected in eight patients (89%) (P = 0.02). CONCLUSIONS: We consider a rise of total and specific IgE in the first month after an allergic reaction as a useful tool in the diagnosis of gastro-allergic anisakiasis (together with patient's history), even if the parasite cannot be seen with fibre optic gastroscopy. The important rise of total and specific IgE against A. simplex can be considered as a reaction induced by the live parasitizing larva in the context of a polyclonal immunological stimulation.

[Validation of the Alcohol Use Disorders Identification Test (AUDIT) in primary care]. / Validación de la prueba para la identificación de trastornos por uso de alcohol (AUDIT) en Atención Primaria.

The results of the validation for the AUDIT (Alcohol Use Disorders Identification Test) in Primary Care are here reported. A total of 326 patients who attended two primary care centers were interviewed. In a first interview they answered the AUDIT questions and later were classified on the basis of disturbances caused by alcohol use: abuse or dependency. The diagnosis of abuse or dependency was obtained with the structured interview for DSM-III-R. Reliability was determined by the test-retest correlation and internal consistency by Cronbach's alpha coefficient. Efficacy was calculated by means of the area under the curve (receiver operating characteristics). A quarter of the interviewed patients were diagnosed of some disturbance caused by alcohol use. The internal reliability of the test was acceptable (Cronbach's alpha coefficient 0.86). The test-retest correlation coefficient was 0.90. All the questions acceptably correlated with the total in the scale. A cut-off point of 8 was the most efficient for the whole sample (90% sensitivity and 90% specificity). For females, the most efficient cut-off point was 6. For patients aged over 60 years, the most efficient cut-off point was 5 for both sexes. This study confirms the usefulness of AUDIT for screening alcohol-related problems in Primary Care.

Delayed hypersensitivity to enoxaparin.

A 65-year-old woman experienced immediate itchy erythematous patches at the subcutaneous injection sites of sodium enoxaparin. An erythematous and infiltrated 40 x 20 mm lesion on the abdominal wall could be observed at the site of enoxaparin injection when she was referred to our clinic 48 h after injection. Lesions subsided spontaneously within 1 week. She had been on this treatment 1 and 3 years before without any adverse reaction. To clarify the nature of the reaction, epicutaneous tests with sodium enoxaparin, calcium nadroparin and calcium heparin were performed, all with negative results. Skin prick test with sodium enoxaparin was also negative. Biopsy of the cutaneous lesion showed spongiotic dermatitis, strongly suggesting a delayed hypersensitivity mechanism. We report here on a new case of delayed hypersensitivity to enoxaparin. Being female, overweight and having prolonged application of the drug were suggested risk factors present in our patient. Biopsy was essential for diagnosis. Although type IV hypersensitivity reactions to enoxaparin are rare, we should start to suspect this condition in order not to underdiagnose it.

Gastric anisakiasis: an underestimated cause of acute urticaria and angio-oedema?

Acute urticaria and angio-oedema are common in primary care and in the emergency unit. Food allergy is one possible cause. We describe gastric anisakiasis, in which symptoms are often not obviously related to eating raw fish. A study was made of patients presenting at the emergency department who had allergic symptoms such as urticaria or angio-oedema and had recently eaten raw or undercooked fish. They were divided into two groups. Patients in group A (n = 13) also had abdominal symptoms and were diagnosed as having gastric anisakiasis by fibre-optic gastroscopy where third-stage larvae of Anisakis simplex were visualized and extracted. Skin prick tests and specific IgE to A. simplex were positive. Patients in group B (n = 13) had only allergic symptoms after eating raw fish. Eleven of 13 patients had positive skin prick tests and specific IgE to A. simplex. Three of 15 control subjects had positive skin prick tests and specific IgE to A. simplex. Allergic symptoms appeared from 2 to 20 h (mean 5.0) after ingestion in group A and from 20 min to 23 h (mean 4.3 h) in group B. Gastric symptoms in group A disappeared rapidly after extraction of the larvae. Allergic symptoms disappeared in most cases within the first 24 h. We suggest that the allergic symptoms in group A as well as in group B were mainly due to parasitization by A. simplex in sensitized patients. Gastric anisakiasis may be a widely underdiagnosed clinical entity.