Multilayered hierarchical capsules providing cell adhesion Sites

Liquified capsules featuring (i) an external shell by layer-by-layer assembly of poly(L-lysine), alginate, and chitosan, and encapsulating (ii) surface functionalized poly(L-lactic acid) (PLLA) microparticles were developed. We hypothesize that, while the liquifi ed environment enhances the diffusion of essential molecules for cell survival, microparticles dispersed in the liquified core of capsules provide the physical support required for cellular functions of anchorage-dependent cells. The influence of the incorporation of PLL on the regime growth, thickness, and stability was analyzed. Results show a more resistant and thicker film with an exponential build-up growth regime. Moreover, capsules ability to support cell survival was assessed. Capsules containing microparticles revealed an enhanced biological outcome in cell metabolic activity and proliferation, suggesting their potential to boost the development of innovative biomaterial designs for bioencapsulation systems and tissue engineering products.The present work was financial supported by the Portuguese Foundation for Science and Technology (FCT) through the Ph.D. Grant with the Reference No. SFRH/BD/69529/2010, cofinanced by the Operational Human Potential Program (POPH), developed under the scope of the National Strategic Reference Framework (QREN) from the European Social Fund (FSE). The authors also acknowledge the financial support from the MP-2008-Small-2 project (Find&Bind) developed under the scope of the EU 7th Framework Programme

Multiphasic, multistructured and hierarchical strategies for cartilage regeneration

Cartilage tissue is a complex nonlinear, viscoelastic, anisotropic, and multiphasic material with a very low coefficient of friction, which allows to withstand millions of cycles of joint loading over decades of wear. Upon damage, cartilage tissue has a low self-reparative capacity due to the lack of neural connections, vascularization, and a latent pool of stem/chondroprogenitor cells. Therefore, the healing of articular cartilage defects remains a significant clinical challenge, affecting millions of people worldwide. A plethora of biomaterials have been proposed to fabricate devices for cartilage regeneration, assuming a wide range of forms and structures, such as sponges, hydrogels, capsules, fibers, and microparticles. In common, the fabricated devices were designed taking in consideration that to fully achieve the regeneration of functional cartilage it is mandatory a well-orchestrated interplay of biomechanical properties, unique hierarchical structures, extracellular matrix (ECM), and bioactive factors. In fact, the main challenge in cartilage tissue engineering is to design an engineered device able to mimic the highly organized zonal architecture of articular cartilage, specifically its spatiomechanical properties and ECM composition, while inducing chondrogenesis, either by the proliferation of chondrocytes or by stimulating the chondrogenic differentiation  of stem/chondro-progenitor cells. In this chapter we present the recent advances in the development of innovative and complex biomaterials that fulfill the required structural key elements for cartilage regeneration. In particular, multiphasic, multiscale, multilayered, and hierarchical strategies composed by single or multiple biomaterials combined in a welldefined structure will be addressed. Those strategies include biomimetic scaffolds mimicking the structure of articular cartilage or engineered scaffolds as models of research to fully understand the biological mechanisms that influence the regeneration of cartilage tissue

An immunomodulatory miniaturized 3D screening platform using liquefied capsules

A critical determinant of successful clinical outcomes is the host's response to the biomaterial. Therefore, the prediction of the immunomodulatory bioperformance of biomedical devices following implantation is of utmost importance. Herein, liquefied capsules are proposed as immunomodulatory miniaturized 3D platforms for the high-content combinatorial screening of different polymers that could be used generically in scaffolds. Additionally, the confined and liquefied core of capsules affords a cell-mediated 3D assembly with bioinstructive microplatforms, allowing to study the potential synergistic effect that cells in tissue engineering therapies have on the immunological environment before implantation. As a proof-of-concept, three different polyelectrolytes, ranging in charge density and source, are used. Poly(L-lysine)-, alginate-, and chitosan-ending capsules with or without encapsulated mesenchymal stem/stromal cells (MSCs) are placed on top of a 2D culture of macrophages. Results show that chitosan-ending capsules, as well as the presence of MSCs, favor the balance of macrophage polarization toward a more regenerative profile, through the up-regulation of anti-inflammatory markers, and the release of pro-regenerative cytokines. Overall, the developed system enables the study of the immunomodulatory bioperformance of several polymers in a cost-effective and scalable fashion, while the paracrine signaling between encapsulated cells and the immunological environment can be simultaneously evaluated.publishe

Engineering immunomodulatory hydrogels and cell-laden systems towards bone regeneration

The well-known synergetic interplay between the skeletal and immune systems has changed the design of advanced bone tissue engineering strategies. The immune system is essential during the bone lifetime, with macrophages playing multiple roles in bone healing and biomaterial integration. If in the past, the most valuable aspect of implants was to avoid immune responses of the host, nowadays, it is well-established how important are the crosstalks between immune cells and bone-engineered niches for an efficient regenerative process to occur. For that, it is essential to recapitulate the multiphenotypic cellular environment of bone tissue when designing new approaches. Indeed, the lack of osteoimmunomodulatory knowledge may be the explanation for the poor translation of biomaterials into clinical practice. Thus, smarter hydrogels incorporating immunomodulatory bioactive factors, stem cells, and immune cells are being proposed to develop a new generation of bone tissue engineering strategies. This review highlights the power of immune cells to upgrade the development of innovative engineered strategies, mainly focusing on orthopaedic and dental applications.publishe

Cell encapsulation in liquified compartments: Protocol optimization and challenges

Cell encapsulation is a widely used technique in the field of Tissue Engineering and Regenerative Medicine (TERM). However, for the particular case of liquefied compartmentalised systems, only a limited number of studies have been reported in the literature. We have been exploring a unique cell encapsulation system composed by liquefied and multilayered capsules. This system transfigured the concept of 3D scaffolds for TERM, and was already successfully applied for bone and cartilage regeneration. Due to a number of appealing features, we envisage that it can be applied in many other fields, including in advanced therapies or as disease models for drug discovery. In this review, we intend to highlight the advantages of this new system, while discussing the methodology, and sharing the protocol optimization and results. The different liquefied systems for cell encapsulation reported in the literature will be also discussed, considering the different encapsulation matrixes as core templates, the types of membranes, and the core liquefaction treatments.publishe

Close-to-native bone repair via tissue-engineered endochondral ossification approaches

In order to solve the clinical challenges related to bone grafting, several tissue engineering (TE) strategies have been proposed to repair critical-sized defects. Generally, the classical TE approaches are designed to promote bone repair via intramembranous ossification. Although promising, strategies that direct the osteogenic differentiation of mesenchymal stem/stromal cells are usually characterized by a lack of functional vascular supply, often resulting in necrotic cores. A less explored alternative is engineering bone constructs through a cartilage-mediated approach, resembling the embryological process of endochondral ossification. The remodeling of an intermediary hypertrophic cartilaginous template triggers vascular invasion and bone tissue deposition. Thus, employing this knowledge can be a promising direction for the next generation of bone TE constructs. This review highlights the most recent biomimetic strategies for applying endochondral ossification in bone TE while discussing the plethora of cell types, culture conditions, and biomaterials essential to promote a successful bone regeneration process.publishe

Viscous microcapsules as microbioreactors to study mesenchymal stem/stromal cells osteolineage commitment

It is essential to design a multifunctional well-controlled platform to transfermechanical cues to the cells in different magnitudes. This study introduces aplatform, a miniaturized bioreactor, which enables to study the effect of shearstress in microsized compartmentalized structures. In this system, thewell-established cell encapsulation system of liquefied capsules (LCs) is usedas microbioreactors in which the encapsulated cells are exposed to variablecore viscosities to experience different mechanical forces under a 3D dynamicculture. The LC technology is joined with electrospraying to produce suchmicrobioreactors at high rates, thus allowing the application of microcapsulesfor high-throughput screening. Using this platform for osteogenicdifferentiation as an example, shows that microbioreactors with higher coreviscosity which produce higher shear stress lead to significantly higherosteogenic characteristics. Moreover, in this system the forces experienced bycells in each LC are simulated by computational modeling. The maximum wallshear stress applied to the cells inside the bioreactor with low, and high coreviscosity environment is estimated to be 297 and 1367 mPa, respectively, forthe experimental setup employed. This work outlines the potential of LCmicrobioreactors as a reliable in vitro customizable platform with a wide rangeof applications.publishe

Liquefied microcapsules as dual-Mmcrocarriers for 3D+3D bottom-up tissue engineering

Cell encapsulation systems must ensure the diffusion of molecules to avoid the formation of necrotic cores. The architectural design of hydrogels, the gold standard tissue engineering strategy, is thus limited to a microsize range. To overcome such a limitation, liquefied microcapsules encapsulating cells and microparticles are proposed. Microcapsules with controlled sizes with average diameters of 608.5 ± 122.3 µm are produced at high rates by electrohydrodynamic atomization, and arginyl-glycyl-aspartic acid (RGD) domains are introduced in the multilayered membrane. While cells and microparticles interact toward the production of confined microaggregates, on the outside cell-mediated macroaggregates are formed due to the aggregation of microcapsules. The concept of simultaneous aggregation is herein termed as 3D+3D bottom-up tissue engineering. Microcapsules are cultured alone (microcapsule1 ) or on top of 2D cell beds composed of human umbilical vein endothelial cells (HUVECs) alone (microcapsule2 ) or cocultured with fibroblasts (microcapsule3 ). Microcapsules are able to support cell encapsulation shown by LiveDead, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulphofenyl)-2H-tetrazolium (MTS), and dsDNA assays. Only microcapsule3 are able to form macroaggregates, as shown by F-actin immunofluorescence. The bioactive 3D system also presented alkaline phosphatase activity, thus allowing osteogenic differentiation. Upon implantation using the chick chorioallontoic membrane (CAM) model, microcapsules recruit a similar number of vessels with alike geometric parameters in comparison with CAMs supplemented with basic fibroblast growth factor (bFGF).publishe

Geometrically controlled liquefied capsules for modular tissue engineering strategies

A plethora of bioinspired cell-laden hydrogels are being explored as building blocks that once assembled are able to create complex and highly hierarchical structures recapitulating the heterogeneity of living tissues. Yet, the resulting 3D bioengineered systems still present key limitations, mainly related with limited diffusion of essential molecules for cell survival, which dictates the failure of most strategies upon implantation. To maximize the hierarchical complexity of bioengineered systems, while simultaneously fully addressing the exchange efficiency of biomolecules, the high-throughput fabrication of liquefied capsules is proposed using superhydrophobic-superhydrophilic microarrays as platforms to produce the initial structures with high fidelity of geometry and size. The liquefied capsules are composed by i) a permselective multilayered membrane; ii) surface-functionalized poly(ε-caprolactone) microparticles loaded into the liquefied core acting as cell adhesion sites; and iii) cells. It is demonstrated that besides the typical spherical liquefied capsules, it is also possible to obtain multi-shaped blocks with high geometrical precision and efficiency. Importantly, the internal gelation approach used to produce such blocks does not jeopardize cell viability, evidencing the mild conditions of the proposed cell encapsulation technique. The proposed system is intended to be used as hybrid devices implantable using minimally invasive procedures for multiple tissue engineering applications.publishe