Sex differences in patients with systemic lupus erythematosus from Northwest Spain.

To further establish potential differences according to sex in systemic lupus erythematosus (SLE) patients from Southern Europe. We assessed clinical and epidemiological data of patients diagnosed with SLE according to the 1982 American College of Rheumatology classification criteria at the single hospital for a well-defined population of Northwest Spain, between 1987 and 2006. Prevalence in December 2006 and age-standardized incidence rates in the whole period were estimated. Kaplan-Meier method was used in order to estimate the probability of survivorship. Women outnumbered men [127 (84.7%) vs. 23 (15.3%)]. The median age at the time of disease diagnosis in men was 54 years versus 43 in women (p < 0.001). Annual incidence rates were higher in women [5.9 (95% confidence interval--CI 4.9-7.0) per 100,000 population] than in men [1.1 (95% CI 0.7-1.7) per 100,000 population; p < 0.001]. Raynaud's phenomenon was more common in women (40.9 vs. 3.0%; p = 0.01). While the frequency of secondary Sjögren's syndrome was increased in women (p = 0.02), renal disease at the time of diagnosis (39.1 vs. 15.0%; p < 0.01) and over the course of the disease was more common in men (43.5 vs. 24.4%; p = 0.06). Higher frequency of thrombocytopenia (39.1 vs. 16.5%; p = 0.01) and lower frequency of anti-SSA (13.0 vs. 31.5%; p = 0.08) and anti-SSB (0 vs. 17.7%; p = 0.03) were observed in men. The 5- and 10-year survival probabilities were nonsignificantly reduced in men (91.3 and 78.3 3% vs. 94.6 and 89.2% in women). The frequency of some clinical manifestations is different in men and women with SLE. Higher awareness of these peculiarities may help to establish appropriate diagnosis and management of SLE in men.

Mechanics Predicts Effective Critical-Size Bone Regeneration Using 3D-Printed Bioceramic Scaffolds.

BACKGROUND: 3D-printed bioceramic scaffolds have gained popularity due to their controlled microarchitecture and their proven biocompatibility. However, their high brittleness makes their surgical implementation complex for weight-bearing bone treatments. Thus, they would require difficult-to-instrument rigid internal fixations that limit a rigorous evaluation of the regeneration progress through the analysis of mechanic-structural parameters. METHODS: We investigated the compatibility of flexible fixations with fragile ceramic implants, and if mechanical monitoring techniques are applicable to bone tissue engineering applications. Tissue engineering experiments were performed on 8 ovine metatarsi. A 15 mm bone segment was directly replaced with a hydroxyapatite scaffold and stabilized by an instrumented Ilizarov-type external fixator. Several in vivo monitoring techniques were employed to assess the mechanical and structural progress of the tissue. RESULTS: The applied surgical protocol succeeded in combining external fixators and subject-specific bioceramic scaffolds without causing fatal fractures of the implant due to stress concentrator. The bearing capacity of the treated limb was initially altered, quantifying a 28-56% reduction of the ground reaction force, which gradually normalized during the consolidation phase. A faster recovery was reported in the bearing capacity, stiffening and bone mineral density of the callus. It acquired a predominant mechanical role over the fixator in the distribution of internal forces after one post-surgical month. CONCLUSION: The bioceramic scaffold significantly accelerated in vivo the bone formation compared to other traditional alternatives in the literature (e.g., distraction osteogenesis). In addition, the implemented assessment techniques allowed an accurate quantitative evaluation of the bone regeneration through mechanical and imaging parameters.

Robocasting and Laser Micromachining of Sol-Gel Derived 3D Silica/Gelatin/ß-TCP Scaffolds for Bone Tissue Regeneration.

The design and synthesis of sol-gel silica-based hybrid materials and composites offer significant benefits to obtain innovative biomaterials with controlled porosity at the nanostructure level for applications in bone tissue engineering. In this work, the combination of robocasting with sol-gel ink of suitable viscosity prepared by mixing tetraethoxysilane (TEOS), gelatin and ß-tricalcium phosphate (ß-TCP) allowed for the manufacture of 3D scaffolds consisting of a 3D square mesh of interpenetrating rods, with macropore size of 354.0 ± 17.0 µm, without the use of chemical additives at room temperature. The silica/gelatin/ß-TCP system underwent irreversible gelation, and the resulting gels were also used to fabricate different 3D structures by means of an alternative scaffolding method, involving high-resolution laser micromachining by laser ablation. By this way, 3D scaffolds made of 2 mm thick rectangular prisms presenting a parallel macropore system drilled through the whole thickness and consisting of laser micromachined holes of 350.8 ± 16.6-micrometer diameter, whose centers were spaced 1312.0 ± 23.0 µm, were created. Both sol-gel based 3D scaffold configurations combined compressive strength in the range of 2-3 MPa and the biocompatibility of the hybrid material. In addition, the observed Si, Ca and P biodegradation provided a suitable microenvironment with significant focal adhesion development, maturation and also enhanced in vitro cell growth. In conclusion, this work successfully confirmed the feasibility of both strategies for the fabrication of new sol-gel-based hybrid scaffolds with osteoconductive properties.

Structural optimization of 3D-printed patient-specific ceramic scaffolds for in vivo bone regeneration in load-bearing defects.

Tissue engineering has recently gained popularity as an alternative to autografts to stimulate bone tissue regeneration through structures called scaffolds. Most of the in vivo experiments on long-bony defects use internally-stabilized generic scaffolds. Despite the wide variety of computational methods, a standardized protocol is required to optimize ceramic scaffolds for load-bearing bony defects stabilized with flexible fixations. An optimization problem was defined for applications to sheep metatarsus defects. It covers biological parameters (porosity, pore size, and the specific surface area) and mechanical constraints based on in vivo and in vitro results reported in the literature. The optimized parameters (59.30% of porosity, 5768.91 m-1 of specific surface area, and 360.80 µm of pore size) and the compressive strength of the selected structure were validated in vitro by means of tomographic images and compression tests of six 3D-printed samples. Divergences between the design and measured values of the optimized parameters, mainly due to manufacturing defects, are consistent with the previous studies. Using the mixed experimental-mathematical scaffold-design procedure described, they could be implanted in vivo with instrumented external fixators, therefore facilitating biomechanical monitoring of the regeneration process.

Response of osteoblasts and preosteoblasts to calcium deficient and Si substituted hydroxyapatites treated at different temperatures.

Hydroxyapatite (HA) is a calcium phosphate bioceramic widely used for bone grafting and augmentation purposes. The biological response of HA can be improved through chemical and microstructural modifications, as well as by manufacturing it as macroporous implants. In the present study, calcium deficient hydroxyapatite (CDHA) and Si substituted hydroxyapatite (SiHA) macroporous scaffolds have been prepared by robocasting. In order to obtain different microstructural properties, the scaffolds have been treated at 700°C and 1250°C. The scaffolds have been characterized and tested as supports for both osteoblast growth and pre-osteoblast differentiation, as fundamental requisite for their potential use in bone tissue engineering. Morphology, viability, adhesion, proliferation, cell cycle, apoptosis, intracellular content of reactive oxygen species and interleukin-6 production were evaluated after contact of osteoblasts-like cells with CDHA and SiHA materials. An adequate interaction of osteoblasts-like cells and preosteoblasts-like cells with all these scaffolds was observed. However, the higher bone cell proliferation and differentiation on CDHA and SiHA scaffolds treated at 1250°C and the lower adsorption of albumin and fibrinogen on these materials in comparison to those treated at 700°C, suggest a better tissue response to CDHA and SiHA materials treated at high temperature.

Effect of Polymer Infiltration on the Flexural Behavior of ß-Tricalcium Phosphate Robocast Scaffolds.

The influence of polymer infiltration on the flexural strength and toughness of ß-tricalcium phosphate (ß-TCP) scaffolds fabricated by robocasting (direct-write assembly) is analyzed. Porous structures consisting of a tetragonal three-dimensional lattice of interpenetrating rods were impregnated with biodegradable polymers (poly(lactic acid) (PLA) and poly(ε-caprolactone) (PCL)) by immersion of the structure in a polymer melt. Infiltration increased the flexural strength of these model scaffolds by a factor of 5 (PCL) or 22 (PLA), an enhancement considerably greater than that reported for compression strength of analogue materials. The greater strength improvement in bending was attributed to a more effective transfer of stress to the polymer under this solicitation since the degree of strengthening associated to the sealing of precursor flaws in the ceramic rod surfaces should remain unaltered. Impregnation with either polymer also improved further than in compression the fracture energy of the scaffolds (by more than two orders of magnitude). This increase is associated to the extraordinary strengthening provided by impregnation and to a crack bridging toughening mechanism produced by polymer fibrils.

Reinforcing bioceramic scaffolds with in situ synthesized ε-polycaprolactone coatings.

In situ ring-opening polymerization of ε-caprolactone (ε-CL) was performed to coat ß-tricalcium phosphate (ß-TCP) scaffolds fabricated by robocasting in order to enhance their mechanical performance while preserving the predesigned macropore architecture. Concentrated colloidal inks prepared from ß-TCP commercial powders were used to fabricate porous structures consisting of a three-dimensional mesh of interpenetrating rods. Then, ε-CL was in situ polymerized within the ceramic structure using a lipase as catalyst and toluene as solvent, to obtain a highly homogeneous coating and full impregnation of in-rod microporosity. The strength and toughness of scaffolds coated by ε-polycaprolactone (ε-PCL) were significantly increased (twofold and fivefold increase, respectively) over those of the bare structures. Enhancement of both properties is associated to the healing of preexisting microdefects in the bioceramic rods. These enhancements are compared to results from previous work on fully impregnated structures. The implications of the results for the optimization of the mechanical and biological performance of scaffolds for bone tissue engineering applications are discussed.

Impregnation of ß-tricalcium phosphate robocast scaffolds by in situ polymerization.

Ring-opening polymerization of ε-caprolactone (ε-CL) and L-lactide (LLA) was performed to impregnate ß-tricalcium phosphate (ß-TCP) scaffolds fabricated by robocasting. Concentrated colloidal inks prepared from ß-TCP commercial powders were used to fabricate porous structures consisting of a 3D mesh of interpenetrating rods. ε-CL and LLA were in situ polymerized within the ceramic structure by using a lipase and stannous octanoate, respectively, as catalysts. The results show that both the macropores inside the ceramic mesh and the micropores within the ceramic rods are full of polymer in either case. The mechanical properties of scaffolds impregnated by in situ polymerization (ISP) are significantly increased over those of the bare structures, exhibiting similar values than those obtained by other, more aggressive, impregnation methods such as melt-immersion (MI). ISP using enzymatic catalysts requires a reduced processing temperature which could facilitate the incorporation of growth factors and other drugs into the polymer composition, thus enhancing the bioactivity of the composite scaffold. The implications of these results for the optimization of the mechanical and biological performance of scaffolds for bone tissue engineering applications are discussed.

Biological properties of solid free form designed ceramic scaffolds with BMP-2: in vitro and in vivo evaluation.

Porous ceramic scaffolds are widely studied in the tissue engineering field due to their potential in medical applications as bone substitutes or as bone-filling materials. Solid free form (SFF) fabrication methods allow fabrication of ceramic scaffolds with fully controlled pore architecture, which opens new perspectives in bone tissue regeneration materials. However, little experimentation has been performed about real biological properties and possible applications of SFF designed 3D ceramic scaffolds. Thus, here the biological properties of a specific SFF scaffold are evaluated first, both in vitro and in vivo, and later scaffolds are also implanted in pig maxillary defect, which is a model for a possible application in maxillofacial surgery. In vitro results show good biocompatibility of the scaffolds, promoting cell ingrowth. In vivo results indicate that material on its own conducts surrounding tissue and allow cell ingrowth, thanks to the designed pore size. Additional osteoinductive properties were obtained with BMP-2, which was loaded on scaffolds, and optimal bone formation was observed in pig implantation model. Collectively, data show that SFF scaffolds have real application possibilities for bone tissue engineering purposes, with the main advantage of being fully customizable 3D structures.

Systemic lupus erythematosus in northwestern Spain: a 20-year epidemiologic study.

To further investigate the epidemiology of systemic lupus erythematosus (SLE) in southern Europe, we assessed the incidence, prevalence, clinical spectrum of the disease, flares, and survival of patients diagnosed with SLE in the Lugo region of northwestern Spain. Between January 1987 and December 2006, 150 Lugo residents were diagnosed as having SLE according to the 1982 American College of Rheumatology criteria for the classification of SLE. Women outnumbered men (127 [84.7%] vs. 23 [15.3%]). The mean age at the time of disease diagnosis was 46.1 ± 19.6 years. The mean follow-up from the time of disease diagnosis was 7.8 ± 4.5 years. The age- and sex-adjusted annual incidence rate over the 20-year study period was 3.6 (95% confidence interval [CI], 3.0-4.2) per 100,000 population aged 15 years and older. The overall annual incidence rate over the 20-year study period in women (5.9/100,000 population aged ≥ 15 yr; 95% CI, 4.9-7.0) was higher than in men (1.1/100,000 population aged ≥ 15 yr; 95% CI, 0.7-1.7) (p < 0.001). By December 31, 2006, the overall age-adjusted SLE prevalence in the Lugo region for patients who fulfilled at least 4 of 1982 American College of Rheumatology criteria was 17.5 per 100,000 population aged 15 years and older (95% CI, 12.6-24.1). Prevalence in women (29.2/100,000 population aged ≥ 15 yr; 95% CI, 20.0-40.7) was higher than in men (5.8/100,000 population aged ≥ 15 yr; 95% CI, 2.0-12.0). The most frequent clinical manifestation was arthritis. As reported in population-based studies on SLE patients of European descent, renal disease was observed in only 27.3% of the patients. The rate of flares was 0.084/year. A younger age and the presence of nephritis at the time of disease diagnosis were associated with the development of flares during the follow-up of Lugo patients. Compared with the general population the probability of survival in patients with SLE was significantly reduced (p = 0.04). In conclusion, the present study establishes a baseline estimate of the incidence and clinical spectrum of SLE in northwestern Spain. According to our results, the incidence of SLE in northwestern Spain is slightly higher than that reported in most European regions. Patients with SLE from northwestern Spain have a later average age onset and a lower frequency of nephritis than in the African-American population. However, our data show a reduced probability of survival in Spanish patients with SLE.

Improving the compressive strength of bioceramic robocast scaffolds by polymer infiltration.

The effect of polymer infiltration on the compressive strength of ß-tricalcium phosphate (TCP) scaffolds fabricated by robocasting (direct write assembly) is analyzed in this work. Porous structures consisting of a tetragonal three-dimensional mesh of interpenetrating rods were fabricated from concentrated TCP inks with suitable viscoelastic properties. Biodegradable polymers (polylactic acid (PLA) and poly(ε-caprolactone) (PCL)) were infiltrated into selected scaffolds by immersion of the structure in a polymer melt. Infiltration increased the uniaxial compressive strength of these model scaffolds by a factor of three (PCL) or six (PLA). It also considerably improved the mechanical integrity of the structures after initial cracking, with the infiltrated structure retaining a significant load-bearing capacity after fracture of the ceramic rods. The strength improvement in the infiltrated scaffolds was attributed to two different contributions: the sealing of precursor flaws in the ceramic rod surfaces and the partial transfer of stress to the polymer, as confirmed by finite element analysis. The implications of these results for the mechanical optimization of scaffolds for bone tissue engineering applications are discussed.

Production of pectinases by A. niger: influence of fermentation conditions

Response surface methodology was used for optimization of polygalacturonase (PG) and pectinesterase (PE) production in submerged fermentation by A.niger. A Central Composite Experimental Design was applied, consisting of 22 experiments, including eight central points. Variables studied were: fermentation time (24 to 120 h), pH (3.5 to 6.5) and initial concentration of pectin (5 to 20 g/l). Maximum PE production was 220 U/l, after 74 h of culture, in a medium containing 20 g/l of pectin (pH 6.5). The optimal conditions for PG production were pH: 4.1, 20 g/l of pectin and 94 h of fermentation with a maximum value of 1032 U/l. Under these conditions, the PE production was low (15 U/l). A liquid extract with high PG activity and low PE activity could be suitable to be used in food processing in order to reduce the production of methanol.

A metodologia de superfície de resposta foi utilizada para a otimização da produção de poligalacturonasa (PG) e pectinesterasa (PE), por A. niger em fermentação submergida. Foi aplicado um Desenho Experimental Composto Central abrangendo 22 experiências, incluindo oito pontos centrais. As variáveis estudadas foram: tempo de fermentação (24 a 120 h), pH (3.5 a 6.5) e concentração inicial de pectina (5 a 20 g/l). A produção máxima de PE foi de 220 U/l, após 74h de cultivo, 20 g/l de pectina e pH 6.5. As condições ótimas para a produção de PG foram pH 4.1, 20 g/l de pectina e 94 h de fermentação, com um valor máximo de 1032 U/l. Sob estas condições, a produção de PE foi baixa (15 U/l). Um extrato líquido com alta atividade PG e baixa atividade PE poderia ser conveniente para ser utilizado no processamento e alimentos, visando reduzir a produção de metanol.