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

Bacterial Growth Kinetics under a Novel Flexible Methacrylate Dressing Serving as a Drug Delivery Vehicle for Antiseptics

A flexible methacrylate powder dressing (Altrazeal®) transforms into a wound contour conforming matrix once in contact with wound exudate. We hypothesised that it may also serve as a drug delivery vehicle for antiseptics. The antimicrobial efficacy and influence on bacterial growth kinetics in combination with three antiseptics was investigated in an in vitro porcine wound model. Standardized in vitro wounds were contaminated with Staphylococcus aureus (MRSA; ATCC 33591) and divided into six groups: no dressing (negative control), methacrylate dressing alone, and combinations with application of 0.02% Polyhexamethylene Biguanide (PHMB), 0.4% PHMB, 0.1% PHMB + 0.1% betaine, 7.7 mg/mL Povidone-iodine (PVP-iodine), and 0.1% Octenidine-dihydrochloride (OCT) + 2% phenoxyethanol. Bacterial load per gram tissue was measured over five days. The highest reduction was observed with PVP-iodine at 24 h to log10 1.43 cfu/g, followed by OCT at 48 h to log10 2.41 cfu/g. Whilst 0.02% PHMB resulted in a stable bacterial load over 120 h to log10 4.00 cfu/g over 120 h, 0.1% PHMB + 0.1% betaine inhibited growth during the first 48 h, with slightly increasing bacterial numbers up to log10 5.38 cfu/g at 120 h. These results indicate that this flexible methacrylate dressing can be loaded with various antiseptics serving as drug delivery system. Depending on the selected combination, an individually shaped and controlled antibacterial effect may be achieved using the same type of wound dressing

Biocomposites Based on Thermoplastic Starch Reinforced with Recycled Paper Cellulose Fibers

Biocomposites sheets were prepared by compression molding from mixtures of corn starch plasticized by glycerol as matrix and cellulose fibers, extracted from used office paper, as reinforcement filler with contents ranging from 0 to 8% wt/wt of fibers to matrix. Properties of composites were determined by mechanical tensile test, differential scanning calorimetry, thermogravimetric analysis, water absorption measurement, and scanning electron microscopy. The results showed that higher fibers content raised the tensile strength and elastic modulus up to 109% and 112%, respectively, when compared to the non-reinforced thermoplastic starch (TPS). The addition of the fibers improved the thermal resistance and decreased the water absorption up to 63.6%. Scanning electron microscopy illustrated a good adhesion between matrix and fibers

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

Preparation of Composite Electrospun Membranes Containing Strontium-Substituted Bioactive Glasses for Bone Tissue Regeneration

Barrier membranes used for the treatment of bone tissue defects caused by periodontitis lack the ability to promote new bone tissue regeneration. However, the addition of an osteogenic component to membranes may enhance their regenerative potential. Here the manufacturing of composite membranes made of poly(caprolactone) and strontium-substituted bioactive glass is described using the solution-electrospinning technique, with particles located both inside and on the surface of the fibers. All membranes are characterized using scanning electron microscopy and energy dispersive X-ray spectroscopy, and glass dissolution from within the fibers is investigated in water. In vitro material cytotoxicity is determined using a rat osteosarcoma cell line. Electrospun fibers exhibit porous surfaces and regions of increased diameter where the particles are accumulated. The glass dissolves after immersion in water, releasing dissolution products that are associated with increased pH. Further evidence suggests accelerated polymer degradation due to interactions between both components, which may provide the additional benefit of reducing the pH changes associated with glass dissolution. All compositions are biocompatible in vitro, with the exception of membranes with >50 μg of glass on their surface. In conclusion, these membranes show great potential for bone healing applications, including guided bone regeneration and scaffolds for musculoskeletal tissue engineering

Fabrication of Porous Scaffolds with a Controllable Microstructure and Mechanical Properties by Porogen Fusion Technique

Macroporous scaffolds with controllable pore structure and mechanical properties were fabricated by a porogen fusion technique. Biodegradable material poly (d, l-lactide) (PDLLA) was used as the scaffold matrix. The effects of porogen size, PDLLA concentration and hydroxyapatite (HA) content on the scaffold morphology, porosity and mechanical properties were investigated. High porosity (90% and above) and highly interconnected structures were easily obtained and the pore size could be adjusted by varying the porogen size. With the increasing porogen size and PDLLA concentration, the porosity of scaffolds decreases, while its mechanical properties increase. The introduction of HA greatly increases the impact on pore structure, mechanical properties and water absorption ability of scaffolds, while it has comparatively little influence on its porosity under low HA contents. These results show that by adjusting processing parameters, scaffolds could afford a controllable pore size, exhibit suitable pore structure and high porosity, as well as good mechanical properties, and may serve as an excellent substrate for bone tissue engineering