Organoids as host models for infection biology - a review of methods.

Infectious diseases are a major threat worldwide. With the alarming rise of antimicrobial resistance and emergence of new potential pathogens, a better understanding of the infection process is urgently needed. Over the last century, the development of in vitro and in vivo models has led to remarkable contributions to the current knowledge in the field of infection biology. However, applying recent advances in organoid culture technology to research infectious diseases is now taking the field to a higher level of complexity. Here, we describe the current methods available for the study of infectious diseases using organoid cultures.

Biofunctional Nanofibrous Substrate for Local TNF-Capturing as a Strategy to Control Inflammation in Arthritic Joints.

Rheumatoid arthritis (RA) is an autoimmune disease that affects the synovial cavity of joints, and its pathogenesis is associated with an increased expression of pro-inflammatory cytokines, namely tumour necrosis factor-alpha (TNF-α). It has been clinically shown to have an adequate response to systemic administration of TNF-α inhibitors, although with many shortcomings. To overcome such limitations, the immobilization of a TNF-α antibody on a nanofibrous substrate to promote a localized action is herein proposed. By using this approach, the antibody has its maximum therapeutic efficacy and a prolonged therapeutic benefit, avoiding the systemic side-effects associated with conventional biological agents' therapies. To technically achieve such a purpose, the surface of electrospun nanofibers is initially activated and functionalized, allowing TNF-α antibody immobilization at a maximum concentration of 6 µg/mL. Experimental results evidence that the biofunctionalized nanofibrous substrate is effective in achieving a sustained capture of soluble TNF-α over time. Moreover, cell biology assays demonstrate that this system has no deleterious effect over human articular chondrocytes metabolism and activity. Therefore, the developed TNF-capturing system may represent a potential therapeutic approach for the local management of severely affected joints.

Chondrogenesis-inductive nanofibrous substrate using both biological fluids and mesenchymal stem cells from an autologous source.

During the last decade, many cartilage tissue engineering strategies have been developed, being the stem cell-based approach one of the most promising. Transforming Growth Factor-ß3 (TGF-ß3) and Insulin-like Growth Factor-I (IGF-I) are key proteins involved in the regulation of chondrogenic differentiation. Therefore, these two growth factors (GFs) were immobilized at the surface of a single electrospun nanofibrous mesh (NFM) aiming to differentiate human Bone Marrow-derived Mesenchymal Stem Cells (hBM-MSCs). The immobilization of defined antibodies (i.e. anti-TGF-ß3 and anti-IGF-I) allows the selective retrieval of the abovementioned GFs from human platelet lysates (PL). Biochemical assays, involving hBM-MSCs cultured on biofunctional nanofibrous substrates under basal culture medium during 28 days, confirm the biological activity of bound TGF-ß3 and IGF-I. Specifically, the typical spherical morphology of chondrocytes and the immunolocalization of collagen type II confirmed the formation of a cartilaginous ECM. Therefore, the proposed biofunctional nanofibrous substrate is able to promote chondrogenesis.

The functionalization of natural polymer-coated gold nanoparticles to carry bFGF to promote tissue regeneration.

Gold nanoparticles (AuNPs) enable the treatment and real-time monitoring of several diseases, providing an exciting and advantageous nanomedicine strategy. These NPs have therefore been adequately functionalized to enable them to carry growth factors (GF), namely basic fibroblastic (bF) GF, which play an essential role in different and important cellular processes including cellular proliferation, survival, migration and differentiation. The AuNPs were coated with natural polymers, chitosan and heparin, to enhance their physicochemical properties such as suspension stability. The polyelectrolyte coating was monitored using a quartz crystal microbalance with dissipation, size and zeta-potential analysis. The natural polymer-coated AuNPs have a spherical shape and a positive surface charge due to chitosan amino groups, enabling their biofunctionalization with monoclonal antibodies to target specific biomolecules. Additionally, cellular assays with the chondrocyte cell line ATDC5 show that the NPs are cytocompatible at relevant concentrations. As a proof of concept of their potential application in tissue regeneration, the natural polymer-coated AuNPs were further functionalized with an antibody to selectively bind the desired GF. The bFGF concentration reached in the NPs without compromising the cytocompatibility demonstrates the potential of this carrier for tissue regeneration.

Electrospun Nanofibrous Meshes Cultured With Wharton's Jelly Stem Cell: An Alternative for Cartilage Regeneration, Without the Need of Growth Factors.

Many efforts are being directed worldwide to the treatment of OA-focal lesions. The majority of those efforts comprise either the refinement of surgical techniques or combinations of biomaterials with various autologous cells. Herein, we tested electrospun polycaprolactone (PCL) nanofibrous meshes for cartilage tissue engineering. For that, articular chondrocytes (hACs) isolated from human osteoarthritic joints and Wharton's Jelly Stem Cells (hWJSCs) are cultured on electrospun nanofiber meshes, without adding external growth factors. We observed higher glycosaminoglycans production and higher over-expression of cartilage-related genes from hWJSCs cultured with basal medium, when compared to hACs isolated from osteoarthritic joints. Moreover, the presence of sulfated proteoglycans and collagen type II is observed on both types of cell cultures. We believe that this effect is due to either the electrospun nanofibers topography or the intrinsic chondrogenic differentiation potential of hWJSCs. Therefore, we propose the electrospun nanofibrous scaffolds in combination with hWJSCs as a viable alternative to the commercial membranes used in autologous chondrogenic regeneration approaches.

The influence of patterned nanofiber meshes on human mesenchymal stem cell osteogenesis.

A specially designed electroconductive collector enables the electrospinning of P-NFM composed of areas of parallel/uniaxially aligned fibers and areas of random/orthogonal nanofiber distribution. The biological relevance of P-NFM is demonstrated using hBMSCs as an autologous cell source. The structures induce cell orientation along the uniaxially aligned fibers, mainly during earlier culturing periods under basal and osteogenic differentiation conditions. The microtopography of the P-NFM also controls the deposition of mineralized extracellular matrix along the pre-defined fiber direction. Genotypic characterization confirms the successful differentiation into the osteogenic lineage.