A permeable on-chip microvasculature for assessing the transport of macromolecules and polymeric nanoconstructs.

HYPOTHESIS: The selective permeation of molecules and nanomedicines across the diseased vasculature dictates the success of a therapeutic intervention. Yet, in vitro assays cannot recapitulate relevant differences between the physiological and pathological microvasculature. Here, a double-channel microfluidic device was engineered to comprise vascular and extravascular compartments connected through a micropillar membrane with tunable permeability. EXPERIMENTS: The vascular compartment was coated by endothelial cells to achieve permeability values ranging from ~0.1 µm/sec, following a cyclic adenosine monophosphate (cAMP) pre-treatment (25 µg/mL), up to ~2 µm/sec, upon exposure to Mannitol, Lexiscan or in the absence of cells. Fluorescent microscopy was used to monitor the vascular behavior of 250 kDa Dextran molecules, 200 nm polystyrene nanoparticles (PB), and 1,000 × 400 nm discoidal polymeric nanoconstructs (DPN), under different permeability and flow conditions. FINDINGS: In the proposed on-chip microvasculature, it was confirmed that permeation enhancers could favor the perivascular accumulation of ~200 nm, in a dose and time dependent fashion, while have no effect on larger particles. Moreover, the microfluidic device was used to interrogate the role of particle deformability in vascular dynamics. In the presence of a continuous endothelium, soft DPN attached to the vasculature more avidly at sub-physiological flows (100 µm/sec) than rigid DPN, whose deposition was larger at higher flow rates (1 mm/sec). The proposed double-channel microfluidic device can be efficiently used to systematically analyze the vascular behavior of drug delivery systems to enhance their tissue specific accumulation.

Beta-tricalcium phosphate ceramic triggers fast and robust bone formation by human mesenchymal stem cells.

Due to their osteoconductive and inductive properties, a variety of calcium phosphate (CaP) scaffolds are commonly used in orthopaedics as graft material to heal bone defects. In this study, we have used two CaP scaffolds with different hydroxyapatite (HA) and ß-tricalcium phosphate (ß-TCP) ratios (MBCP®; 60/40 and MBCP+ ®; 20/80) to investigate their intrinsic capacity to favour human bone marrow stem cells (hBMSCs) osteogenic differentiation capacity. We report that MBCP+ ® showed in in vitro culture model a higher rate of calcium ion release in comparison with MBCP®. In two defined coculture systems, the hBMSC seeded onto MBCP+ ® presented an increased amount of VEGF secretion, resulting in an enhanced endothelial cell proliferation and capillary formation compared with hBMSC seeded onto MBCP®. When both ceramics combined with hBMSC were implanted in a nude mouse model, we observed a faster osteogenic differentiation and enhancement mature bone deposition sustained by the presence of a vast host vasculature within the MBCP+ ® ceramics. Bone formation was observed in samples highly positive to the activation of calcium sensing receptor protein (CaSr) on the surface of seeded hBMSC that also shown higher BMP-2 protein expression. With these data we provide valuable insights in the possible mechanisms of ossification and angiogenesis by hBMSC that we believe to be primed by calcium ions released from CaP scaffolds. Evidences could lead to an optimization of ceramic scaffolds to prime bone repair.

Deciphering the relative contribution of vascular inflammation and blood rheology in metastatic spreading.

Vascular adhesion of circulating tumor cells (CTCs) is a key step in cancer spreading. If inflammation is recognized to favor the formation of vascular "metastatic niches," little is known about the contribution of blood rheology to CTC deposition. Herein, a microfluidic chip, covered by a confluent monolayer of endothelial cells, is used for analyzing the adhesion and rolling of colorectal (HCT-15) and breast (MDA-MB-231) cancer cells under different biophysical conditions. These include the analysis of cell transport in a physiological solution and whole blood over a healthy and a TNF-α inflamed endothelium with a flow rate of 50 and 100 nl/min. Upon stimulation of the endothelial monolayer with TNF-α (25 ng/ml), CTC adhesion increases from 2 to 4 times whilst cell rolling velocity only slightly reduces. Notably, whole blood also enhances cancer cell deposition from 2 to 3 times, but only on the unstimulated vasculature. For all tested conditions, no statistically significant difference is observed between the two cancer cell types. Finally, a computational model for CTC transport demonstrates that a rigid cell approximation reasonably predicts rolling velocities while cell deformability is needed to model adhesion. These results would suggest that, within microvascular networks, blood rheology and inflammation contribute similarly to CTC deposition, thereby facilitating the formation of metastatic niches along the entire network, including the healthy endothelium. In microfluidic-based assays, neglecting blood rheology would significantly underestimate the metastatic potential of cancer cells.

Hierarchical Microplates as Drug Depots with Controlled Geometry, Rigidity, and Therapeutic Efficacy.

A variety of microparticles have been proposed for the sustained and localized delivery of drugs with the objective of increasing therapeutic indexes by circumventing filtering organs and biological barriers. Yet, the geometrical, mechanical, and therapeutic properties of such microparticles cannot be simultaneously and independently tailored during the fabrication process to optimize their performance. In this work, a top-down approach is employed to realize micron-sized polymeric particles, called microplates (µPLs), for the sustained release of therapeutic agents. µPLs are square hydrogel particles, with an edge length of 20 µm and a height of 5 µm, made out of poly(lactic- co-glycolic acid) (PLGA). During the synthesis process, the µPL Young's modulus can be varied from 0.6 to 5 MPa by changing the PLGA amounts from 1 to 7.5 mg, without affecting the µPL geometry while matching the properties of the surrounding tissue. Within the porous µPL matrix, different classes of therapeutic payloads can be incorporated including molecular agents, such as anti-inflammatory dexamethasone (DEX), and nanoparticles containing imaging and therapeutic molecules themselves, thus originating a truly hierarchical platform. As a proof of principle, µPLs are loaded with free DEX and 200 nm spherical polymeric nanoparticles, carrying DEX molecules (DEX-SPNs). Electron and fluorescent confocal microscopy analyses document the uniform distribution and stability of molecular and nanoagents within the µPL matrix. This multiscale, hierarchical microparticle releases DEX for at least 10 days. The inclusion of DEX-SPNs serves to minimize the initial burst release and modulate the diffusion of DEX molecules out of the µPL matrix. The biopharmacological and therapeutic properties together with the fine tuning of geometry and mechanical stiffness make µPLs a unique polymeric depot for the potential treatment of cancer, cardiovascular, and chronic, inflammatory diseases.

Predicting the role of microstructural and biomechanical cues in tumor growth and spreading.

A continuum porous media model is developed for elucidating the role of the mechanical cues in regulating tumor growth and spreading. It is shown that stiffer matrices and higher cell-matrix adhesion limit tumor growth and spreading toward the surrounding tissue. Higher matrix porosities, conversely, favor the growth and the dissemination of tumor cells. This model could be used for predicting the response of malignant masses to novel therapeutic agents affecting directly the tumor microenvironment and its micromechanical cues.

Human Articular Chondrocytes Regulate Immune Response by Affecting Directly T Cell Proliferation and Indirectly Inhibiting Monocyte Differentiation to Professional Antigen-Presenting Cells.

Autologous chondrocyte implantation is the current gold standard cell therapy for cartilage lesions. However, in some instances, the heavily compromised health of the patient can either impair or limit the recovery of the autologous chondrocytes and a satisfactory outcome of the implant. Allogeneic human articular chondrocytes (hAC) could be a good alternative, but the possible immunological incompatibility between recipient and hAC donor should be considered. Herein, we report that allogeneic hAC inhibited T lymphocyte response to antigen-dependent and -independent proliferative stimuli. This effect was maximal when T cells and hAC were in contact and it was not relieved by the addition of exogenous lymphocyte growth factor interleukin (IL)-2. More important, hAC impaired the differentiation of peripheral blood monocytes induced with granulocyte monocyte colony-stimulating factor and IL-4 (Mo) to professional antigen-presenting cells, such as dendritic cells (DC). Indeed, a marked inhibition of the onset of the CD1a expression and an ineffective downregulation of CD14 antigens was observed in Mo-hAC co-cultures. Furthermore, compared to immature or mature DC, Mo from Mo-hAC co-cultures did not trigger an efficacious allo-response. The prostaglandin (PG) E2 present in the Mo-hAC co-culture conditioned media is a putative candidate of the hAC-mediated inhibition of Mo maturation. Altogether, these findings indicate that allogeneic hAC inhibit, rather than trigger, immune response and strongly suggest that an efficient chondrocyte implantation could be possible also in an allogeneic setting.

Encapsulation and survival of a chondrocyte cell line within xanthan gum derivative.

A chemical derivative of xanthan gum polysaccharide is investigated as a new artificial matrix for the encapsulation of chondrocytic cells. Toward this goal, a novel micro-droplet generator is developed to produce microcapsules. Microcapsules with an average diameter of 500 µm, smooth surface, and homogeneous size distribution are obtained. ATDC5 cells encapsulated in carboxymethyl xanthan (CMX) microcapsules remain viable and are observed to proliferate for prolonged culture periods with enhanced metabolic activity. Furthermore, retention of the chondrogenic phenotype is exhibited by the cells within CMX, suggesting the ability of this material to be applied in cell-delivery therapies.

Encapsulation of human articular chondrocytes into 3D hydrogel: phenotype and genotype characterization.

This chapter is intended to provide a summary of the current materials used in cell encapsulation technology as well as methods for evaluating the performance of cells encapsulated in a polymeric matrix. In particular, it describes the experimental procedure to prepare a hydrogel matrix based on natural polymers for encapsulating and culturing human articular chondrocytes with the interest in cartilage regeneration. Protocols to evaluate the viability, proliferation, differentiation, and matrix production of embedded cells are also described and include standard protocols such as the MTT and [3H] Thymidine assays, reverse transcription polymerase chain reaction (RT-PCR) technique, histology, and immunohistochemistry analysis. The assessment of cell distribution within the 3D hydrogel construct is also described using APoTome analysis.

Chitosan scaffolds incorporating lysozyme into CaP coatings produced by a biomimetic route: a novel concept for tissue engineering combining a self-regulated degradation system with in situ pore formation.

This study describes an innovative self-regulated degrading material with gradual in situ pore formation ability for bone tissue engineering applications. This approach is based on the incorporation of the lysozyme enzyme into calcium phosphate (CaP) coatings, prepared on the surface of chitosan scaffolds by means of a biomimetic coating technique with the aim of controlling their degradation rate and subsequent formation of pores. However, because lysozyme has antibacterial properties, these coatings may act as a carrier for its sustained release, preventing infection upon implantation. In order to prove the concept of in situ pore formation, the coated scaffolds (with and without lysozyme) were incubated in two different solutions at different pH to simulate normal physiological conditions (pH 7.4) and inflammatory response (pH 5). The weight loss and morphology of the scaffolds was monitored over time. At pH 7.4, the scaffolds remained more stable than at pH 5. The scaffolds incubated at pH 5 showed a rapid decrease in their initial weight, and scanning electron microscopy imaging revealed the formation of a highly porous structure. Furthermore, evaluation of the activity of the incorporated lysozyme revealed that the enzyme was able to hydrolyse the peptidoglycan of the bacteria cell walls (as detected by the decrease in optical density with time), indicating that the enzyme remained active after being incorporated into the CaP coating.