Platelet lysates-based hydrogels incorporating bioactive mesoporous silica nanoparticles for stem cell osteogenic differentiation.

Scaffolds for bone tissue regeneration should provide the right cues for stem cell adhesion and proliferation, but also lead to their osteogenic differentiation. Hydrogels of modified platelet lysates (PLMA) show the proper mechanical stability for cell encapsulation and contain essential bioactive molecules required for cell maintenance. We prepared a novel PLMA-based nanocomposite for bone repair and regeneration capable of releasing biofactors to induce osteogenic differentiation. Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) were encapsulated in PLMA hydrogels containing bioactive mesoporous silica nanoparticles previously loaded with dexamethasone and functionalized with calcium and phosphate ions. After 21 d of culture, hBM-MSCs remained viable, presented a stretched morphology, and showed signs of osteogenic differentiation, namely the presence of significant amounts of alkaline phosphatase, bone morphogenic protein-2 and osteopontin, hydroxyapatite, and calcium nodules. Developed for the first time, PLMA/MSNCaPDex nanocomposites were able to guide the differentiation of hBM-MSCs without any other osteogenic supplementation.

Modeling of Cell-Mediated Self-Assembled Colloidal Scaffolds.

A critical step in tissue engineering is the design and synthesis of 3D biocompatible matrices (scaffolds) to support and guide the proliferation of cells and tissue growth. The most existing techniques rely on the processing of scaffolds under controlled conditions and then implanting them in vivo, with questions related to biocompatibility and implantation that are still challenging. As an alternative, it was proposed to assemble the scaffolds in loco through the self-organization of colloidal particles mediated by cells. To overcome the difficulty to test experimentally all the relevant parameters, we propose the use of large-scale numerical simulation as a tool to reach useful predictive information and to interpret experimental results. Thus, in this study, we combine experiments, particle-based simulations, and mean-field calculations to show that, in general, the size of the self-assembled scaffold scales with the cell-to-particle ratio. However, we have found an optimal value of this ratio, for which the size of the scaffold is maximal when the cell-cell adhesion is suppressed. These results suggest that the size and structure of the self-assembled scaffolds may be designed by tuning the adhesion between cells in the colloidal suspension.

Multifunctional laminarin microparticles for cell adhesion and expansion.

Microfabrication technologies have been widely explored to produce microgels that can be assembled in functional constructs for tissue engineering and regenerative medicine applications. Here, we propose microfluidics coupled to a source of UV light to produce multifunctional methacrylated laminarin microparticles with narrow distribution of sizes using photopolymerization. The multifunctional microparticles were loaded with platelet lysates and further conjugated with an adhesive peptide. The adhesive peptides dictated cell adhesiveness to the laminarin microparticles, the incorporation of platelet lysates have resulted in improved cell expansion compared to clear microparticles. Overall, our findings demonstrate that multifunctional methacrylated laminarin microparticles provide an effective support for cell attachment and expansion. Moreover, expanded cells provide the link for microparticles aggregation resulting in robust 3D structures. This suggest the potential for using the methacrylated laminarin microplatforms capable to be assembled by the action of cells to rapidly produce large tissue engineered constructs.

Cell selective chitosan microparticles as injectable cell carriers for tissue regeneration.

The detection, isolation and sorting of cells holds an important role in cell therapy and regenerative medicine. Also, injectable systems have been explored for tissue regeneration in vivo, because it allows repairing complex shaped tissue defects through minimally invasive surgical procedures. Here we report the development of chitosan microparticles with a size of 115.8 µm able to capture and expand a specific cell type that can also be regarded as an injectable biomaterial. Monoclonal antibodies against cell surface antigens specific to endothelial cells and stem cells were immobilized on the surface of the microparticles. Experimental results showed that particles bioconjugated with specific antibodies provide suitable surfaces to capture a target cell type and subsequent expansion of the captured cells. Primarily designed for an application in tissue engineering, three main challenges are accomplished with the herein presented microparticles: separation, scale-up expansion of specific cell type and successful use as an injectable system to form small tissue constructs in situ.

Photopatterned antibodies for selective cell attachment.

We present a phototriggerable system that allows for the spatiotemporal controlled attachment of selected cell types to a biomaterial using immobilized antibodies that specifically target individual cell phenotypes. o-Nitrobenzyl caged biotin was used to functionalize chitosan membranes and mediate site-specific coupling of streptavidin and biotinylated antibodies after light activation. The ability of this system to capture and immobilize specific cells on a surface was tested using endothelial-specific biotinylated antibodies and nonspecific ones as controls. Homogeneous patterned monolayers of human umbilical vein endothelial cells were obtained on CD31-functionalized surfaces. This is a simple and generic approach that is applicable to other ligands, materials, and cell types and shows the flexibility of caged ligands to trigger and control the interaction between cells and biomaterials.

Selective cell recruitment and spatially controlled cell attachment on instructive chitosan surfaces functionalized with antibodies.

Bioactive constructs to guide cellular mobilization and function have been proposed as an approach for a new generation of biomaterials in functional tissue engineering. Adult mesenchymal stem cells have been widely used as a source for cell based therapeutic strategies, namely tissue engineering. This is a heterogeneous cell population containing many subpopulations with distinct regenerative capacity. Thus, one of the issues for the effective clinical use of stem cells in tissue engineering is the isolation of a highly purified, expandable specific subpopulation of stem cells. Antibody functionalized biomaterials could be promising candidates to isolate and recruit specific cell types. Here we propose a new concept of instructive biomaterials that are able to recruit and purify specific cell types from a mixed cell population. This biomimetic concept uses a target-specific chitosan substrate to capture specific adipose derived stem cells. Specific antibodies were covalently immobilized onto chitosan membranes using bis[sulfosuccinimidyl] suberate (BS3). Quartz crystal microbalance (QCM) was used to monitor antibody immobilization/adsorption onto the chitosan films. Specific antibodies covalently immobilized, kept their bioactivity and captured specific cell types from a mixed cell population. Microcontact printing allowed to covalently immobilize antibodies in patterns and simultaneously a spatial control in cell attachment.

Immobilization of fibronectin in chitosan substrates improves cell adhesion and proliferation.

Covalent grafting of biomolecules is a strategy to improve the biocompatibility and bioactivity of materials. However, it is critical to maintain the biological activity of the biomolecule upon its attachment to the surface. In the present study we compared the biological properties of chitosan, in which the surface was enriched with fibronectin (Fn), using two methodologies: chemical immobilization, using a water-soluble carbodiimide; and simple adsorption. X-ray photoelectron spectroscopy studies confirmed the successful immobilization of Fn onto modified membranes. SaOs-2 cells were seeded onto these surfaces to assess the biological consequences of such modifications. The presence of Fn stimulated cell adhesion on chitosan. It was found that after 7 days of culture in the presence of covalently attached Fn, the cells are confluent; significantly fewer cells were detected in unmodified film and in film with adsorbed Fn. This result is consistent with the fact that considerable desorption of Fn from chitosan takes place within 24 h in culture medium. This study showed that Fn may be easily covalently attached onto chitosan substrates, improving the biological performance of the material. The technique could find applications in tissue-engineering strategies, as the surface modification of chitosan-based substrates could be carried out in more complex geometries, such as in scaffolds or particles.