Preparación de mallas mediante electrohilado para la inhibición de la angiogénesis

La preparación de nanofibras por electrohilado está ganando mucho interés en la actualidad debido a su posible aplicación como sistemas de liberación controlada de fármaco. La incorporación del compuesto activo en la matriz polimérica de las fibras permite su protección frente a los procesos degradativos del organismo y su liberación controlada y localizada. Numerosas enfermedades (psoriasis, retinopatías diabéticas, crecimiento de tumores) están relacionadas con alteraciones en la angiogénesis (formación de nuevos vasos sanguíneos a partir de otros ya formados), por tanto el desarrollo de nuevos compuestos que regulen adecuadamente los procesos angiogénicos se está estudiando como una posible vía en el tratamiento de estas enfermedades. En el presente trabajo se han preparado y caracterizado nanofibras de ácido poli(D,L-láctico) cargadas con ácido 5-amino-2-naftalensulfónico o el homopolímero del ácido 2-acrilamido-2-metilpropanosulfónico, compuestos inhibidores de la acción de algunos factores de crecimiento proangiogénicos dependientes de heparina. La distinta naturaleza de los compuestos activos utilizados permitieron la obtención de diferentes perfiles de liberación, dependientes de la hidrofilia del compuesto activo, su peso molecular y la biodegradabilidad del ácido poli(D,L-láctico). Ambos sistemas mostraron una alta capacidad para inhibir la mitogénesis de fibroblastos inducida por aFGF.Preparation of nanofibers by electrospinning is gaining much attention due to their potential application as drug delivery systems. The incorporation of the active compound in the polymeric matrix of the fibers prevents degradation and allows a controlled and localized delivery of the drug. Many diseases (psoriasis, diabetic retinopathy, tumor growth) have been related to alterations in the angiogenic processes, therefore the control of angiogenesis is being investigated as a possible treatment for these diseases. In this work poly(D, L-lactic acid) nanofibers loaded with sulfonated active compounds that inhibit heparin-dependent proangiogenic growth factors (5-amino acid-2-naphthalene sulfonic acid and poly(2-acrylamido-2-methylpropane sulfonic acid) have been prepared and characterized. The different nature of the active compounds gave rise different release profiles that depended on the hydrophilic nature of the drug, molecular weight, and the biodegradation of poly (D, L-lactic acid). Both systems showed a high inhibition of fibroblasts aFGF- induced mitogenesis.Peer Reviewe

Novel genipin cross-linked chitosan-silk fibroin sponges for cartilage engineering strategies

The positive interaction of materials with tissues is an important step in regenerative medicine strategies. Hydrogels that are obtained from polysaccharides and proteins are expected to mimic the natural cartilage environment and thus provide an optimum milleu for tissue growth and regeneration. In this work, novel hydrogels composed of blends of chitosan and Bombyx mori silk fibroin were cross-linked with genipin (G) and were freeze dried to obtain chitosan/silk (CSG) sponges. CSG sponges possess stable and ordered structures because of protein conformational changes from R-helix/random-coil to -sheet structure, distinct surface morphologies, and pH/ swelling dependence at pH 3, 7.4, and 9. We investigated the cytotoxicity of CSG sponge extracts by using L929 fibroblast-like cells. Furthermore, we cultured ATDC5 cells onto the sponges to evaluate the CSG sponges’ potential in cartilage repair strategies. These novel sponges promoted adhesion, proliferation, and matrix production of chondrocyte-like cells. Sponges’ intrinsic properties and biological results suggest that CSG sponges may be potential candidates for cartilage tissue engineering (TE) strategies.S.S.S. and M.T.R. thank the Portuguese Foundation for Science and Technology (FCT) for Ph.D. scholarships (SFRH/BD/8658/2002 and SFRH/BD/30745/2006, respectively). A.F.M.P. thanks the FCT and FEDER for a grant (POCI/FIS/61621/2004). This work was partially supported by the European-Union-funded STREP project HIPPOCRATES (NMP3-CT-2003-505758) and was carried out under the scope of the European NoE EXPERTISSUES (NMP3-CT-2004-500283). We also acknowledge Adriano Pedro for his contribution to the micro-CT analysis

Microfluidic-assisted electrospinning, an alternative to coaxial, as a controlled dual drug release system to treat inflammatory arthritic diseases

Inflammatory arthritic diseases are characterized by a persistent inflammation of the synovial tissues where tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) pro-inflammatory cytokines are over-expressed, leading to progressive musculoskeletal disability. Methotrexate (MTX), a disease-modifying-anti-rheumatic drug (DMARD) commonly applied in their treatment, can be used in combination with biological-DMARDs as anti-TNFα antibody to improve the treatments efficacy. However, their systemic administration comes with severe side-effects and limited therapeutic efficacy due to their off-target distribution and short half-life. To overcome such limitations, encapsulation of clinically relevant concentrations of MTX and anti-TNFα antibody into polycaprolactone (PCL) or poly(vinyl-alcohol) (PVA) microfluidic-assisted or coaxial electrospun fibrous meshes is proposed as local controlled dual drug release systems. Release studies show that microfluidic-assisted electrospinning meshes encapsulating both drugs achieved higher concentrations than coaxials. Biological assays using human articular chondrocytes (hACs) and monocytic cells (THP-1 cell line) demonstrate that fibrous meshes encapsulating the drugs are non-toxic. The systems' efficacy is proved by a significant decrease of TNFα and IL-6 concentrations in conditioned medium of lipopolysaccharide (LPS)-stimulated THP-1 cells, especially in the presence of microfluidic-assisted electrospun meshes, when compared with THP-1 conditioned medium (59.5% and 83.9% less, respectively). Therefore, microfluidic-assisted electrospinning fibrous meshes with encapsulating drugs represent an alternative to coaxial, as a local therapy for inflammatory arthritis diseases.This work was supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, for the Ph.D grant of Catarina Silva (UMINHO/BD/33/2016; NORTE-08-5369-FSE-000012), and by the Portuguese Science and Technology Foundation (FCT) for the cells project Cells4_ID (PTDC/BTM-SAL/28882/2017)

Cell sheet engineering for reproducing the bone marrow hematopoietic stem cell niche

Hematopoietic stem and progenitor cells (HSPC) are multipotent cells responsible for the maintenance and renewal of the hematopoietic lineage in the adult body. The fate of these stem cells is closely regulated by their surrounding microenvironment, or niche. The importance of the microenvironment for HSPC function has been long recognized by researchers that more than 30 years ago attempted to emulate it in 2D using a layer of bone marrow stromal cells to culture hematopoietic cells for long time periods (Dexter-type cultures). However, all the models based on feeder layers are less than perfect in recreating the hematopoietic microenvironment. The use of growth factor cocktails provided some promising results concerning the maintenance and proliferation of some cell populations but still struggle to deliver the correct microenvironment for the maintenance of suitable HSPC populations. Part of the problem of the current systems lies on the lack of the third dimension. At the same time, the proposed three-dimensional methodologies using scaffolds to engineer the bone marrow (BM) microenvironment present very limited results probably due to the scaffolding matrices’ intrinsic limitations. Therefore, an engineered BM microenvironment capable of acting as a functional HSPC niche would provide a tremendous tool for the study of hematopoiesis as well as for obtaining and maintaining HSPC. Using osteogenic cell sheets, we have previously demonstrated that it was possible to induce the ectopic formation of mature bone tissue with a clear bone marrow, avoiding the use of scaffolds. In the present work, we studied the potential of using osteogenic cell sheets to build in vitro, a 3D microenvironment capable of providing HSPC a suitable niche for their survival and proliferation. For this, we used bone marrow stromal cells and adipose-derived stem cells to produce the osteogenic cell sheets and human umbilical cord blood as a source of hematopoietic stem cells

Silk fibroin scaffolds enhance cell commitment of adult rat cardiac progenitor cells.

The use of three-dimensional (3D) cultures may induce cardiac progenitor cells to synthesize their own extracellular matrix (ECM) and sarcomeric proteins to initiate cardiac differentiation. 3D cultures grown on synthetic scaffolds may favour the implantation and survival of stem cells for cell therapy when pharmacological therapies are not efficient in curing cardiovascular diseases and when organ transplantation remains the only treatment able to rescue the patient’s life. Silk fibroin-based scaffolds may be used to increase cell affinity to biomaterials and may be chemically modified to improve cell adhesion. In the present study, porous, partially orientated and electrospun nanometric nets were used. Cardiac progenitor cells isolated from adult rats were seeded by capillarity in the 3D structures and cultured inside inserts for 21 days. Under this condition, the cells expressed a high level of sarcomeric and cardiac proteins and synthesized a great quantity of ECM. In particular, partially orientated scaffolds induced the synthesis of titin, which is a fundamental protein in sarcomere assembly

Novel genipin cross-linked chitosan-silk based sponges for the regeneration and repair of cartilage using a tissue engineering approach

Development of materials that can interactpositively with tissues is important to regenerative medicine strategies success. Cartilage tissue engineering (TE) scaffolding is a field of continuous evolution, and sponges derived from the combination of polysaccharides and proteins are expected to mimic the naturally occurring environment in the articular cartilage matrix, providing an optimum environment for tissue growth and regeneration. Chitosan (Cht) and Bombyx mori silk fibroin (SF) are excellent candidates for sponges design due to their intrinsic characteristics. The present work aimed to improve the chitosan biocompatibility through blending with Cht-SF and genipin-cross-linking. Hydrogels, produced by cross-linking of Cht-SF, were freeze-dryed to obtain the cross-linked chitosan/silk (CSG) sponges. Rheological and mechanical properties, structural aspects and morphological features of CSG sponges were evaluated. CSG sponges possess stable and ordered structures due to protein conformation changes from alpha-helix/randomcoil to beta-sheet structure, porous and globular-like surface morphologies, and pH/swelling dependence at pH 3, 7.4 and 9. To evaluate sponges â suitability for cell studies, ATDC5 chondrocyte-like cells were seeded onto CSG sponges and ATDC5 viability (MTS assay), proliferation (DNA test), morphology (SEM analysis) and matrix production (GAGs quantification) were assessed after 14, 21 and 28 days of culture. ATDC5-sponge constructs showed a significant higher adhesion, proliferation and matrix production with the time of culture when compared to Cht, suggesting CSG sponges as potential candidates for cartilage TE strategies. Acknowledgements. Thanks to Portuguese Foundation for Science and Technology, STREP Project HIPPOCRATES (NMP3-CT-2003-505758) and European NoE EXPERTISSUES (NMP3-CT-2004-500283).info:eu-repo/semantics/publishedVersio

Association of Systemic Lupus Erythematosus Clinical Features with European Population Genetic Substructure

Systemic Lupus Erythematosus (SLE) is an autoimmune disease with a very varied spectrum of clinical manifestations that could be partly determined by genetic factors. We aimed to determine the relationship between prevalence of 11 clinical features and age of disease onset with European population genetic substructure. Data from 1413 patients of European ancestry recruited in nine countries was tested for association with genotypes of top ancestry informative markers. This analysis was done with logistic regression between phenotypes and genotypes or principal components extracted from them. We used a genetic additive model and adjusted for gender and disease duration. Three clinical features showed association with ancestry informative markers: autoantibody production defined as immunologic disorder (P = 6.8×10(-4)), oral ulcers (P = 6.9×10(-4)) and photosensitivity (P = 0.002). Immunologic disorder was associated with genotypes more common in Southern European ancestries, whereas the opposite trend was observed for photosensitivity. Oral ulcers were specifically more common in patients of Spanish and Portuguese self-reported ancestry. These results should be taken into account in future research and suggest new hypotheses and possible underlying mechanisms to be investigated. A first hypothesis linking photosensitivity with variation in skin pigmentation is suggested

Lack of replication of higher genetic risk load in men than in women with systemic lupus erythematosus

Introduction: We aimed to replicate a recent study which showed higher genetic risk load at 15 loci in men than in women with systemic lupus erythematosus (SLE). This difference was very significant, and it was interpreted as indicating that men require more genetic susceptibility than women to develop SLE. Methods: Nineteen SLE-associated loci (thirteen of which are shared with the previous study) were analyzed in 1,457 SLE patients and 1,728 healthy controls of European ancestry. Genetic risk load was calculated as sex-specific sum genetic risk scores (GRS(s)). Results: Our results did not replicate those of the previous study at either the level of individual loci or the global level of GRS(s). GRS(s) were larger in women than in men (4.20 ± 1.07 in women vs. 3.27 ± 0.98 in men). This very significant difference (P < 10(-16)) was more dependent on the six new loci not included in the previous study (59% of the difference) than on the thirteen loci that are shared (the remaining 41%). However, the 13 shared loci also showed a higher genetic risk load in women than in men in our study (P = 6.6 × 10(-7)), suggesting that heterogeneity of participants, in addition to different loci, contributed to the opposite results. Conclusion: Our results show the lack of a clear trend toward higher genetic risk in one of the sexes for the analyzed SLE loci. They also highlight several limitations of assessments of genetic risk load, including the possibility of ascertainment bias with loci discovered in studies that have included mainly women

Mixed cryoglobulinemia

Mixed cryoglobulinemia (MC), type II and type III, refers to the presence of circulating cryoprecipitable immune complexes in the serum and manifests clinically by a classical triad of purpura, weakness and arthralgias. It is considered to be a rare disorder, but its true prevalence remains unknown. The disease is more common in Southern Europe than in Northern Europe or Northern America. The prevalence of 'essential' MC is reported as approximately 1:100,000 (with a female-to-male ratio 3:1), but this term is now used to refer to a minority of MC patients only. MC is characterized by variable organ involvement including skin lesions (orthostatic purpura, ulcers), chronic hepatitis, membranoproliferative glomerulonephritis, peripheral neuropathy, diffuse vasculitis, and, less frequently, interstitial lung involvement and endocrine disorders. Some patients may develop lymphatic and hepatic malignancies, usually as a late complication. MC may be associated with numerous infectious or immunological diseases. When isolated, MC may represent a distinct disease, the so-called 'essential' MC. The etiopathogenesis of MC is not completely understood. Hepatitis C virus (HCV) infection is suggested to play a causative role, with the contribution of genetic and/or environmental factors. Moreover, MC may be associated with other infectious agents or immunological disorders, such as human immunodeficiency virus (HIV) infection or primary Sjögren's syndrome. Diagnosis is based on clinical and laboratory findings. Circulating mixed cryoglobulins, low C4 levels and orthostatic skin purpura are the hallmarks of the disease. Leukocytoclastic vasculitis involving medium- and, more often, small-sized blood vessels is the typical pathological finding, easily detectable by means of skin biopsy of recent vasculitic lesions. Differential diagnoses include a wide range of systemic, infectious and neoplastic disorders, mainly autoimmune hepatitis, Sjögren's syndrome, polyarthritis, and B-cell lymphomas. The first-line treatment of MC should focus on eradication of HCV by combined interferon-ribavirin treatment. Pathogenetic treatments (immunosuppressors, corticosteroids, and/or plasmapheresis) should be tailored to each patient according to the progression and severity of the clinical manifestations. Long-term monitoring is recommended in all MC patients to assure timely diagnosis and treatment of the life-threatening complications. The overall prognosis is poorer in patients with renal disease, liver failure, lymphoproliferative disease and malignancies