Self-assembled Hydrogel Fiber Bundles from Oppositely Charged Polyelectrolytes Mimic Micro-/nanoscale Hierarchy of Collagen.

Fiber bundles are present in many tissues throughout the body. In most cases, collagen subunits spontaneously self-assemble into a fibrilar structure that provides ductility to bone and constitutes the basis of muscle contraction. Translating these natural architectural features into a biomimetic scaffold still remains a great challenge. Here, we propose a simple strategy to engineer biomimetic fiber bundles that replicate the self-assembly and hierarchy of natural collagen fibers. The electrostatic interaction of methacrylated gellan gum (MeGG) with a countercharged chitosan (CHT) polymer led to the complexation of the polyelectrolytes. When directed through a polydimethylsiloxane (PDMS) channel, the polyelectrolytes formed a hierarchical fibrous hydrogel demonstrating nano-scale periodic light/dark bands similar to D-periodic bands in native collagen and aligned parallel fibrils at micro-scale. Importantly, collagen-mimicking hydrogel fibers exhibited robust mechanical properties (MPa scale) at a single fiber bundle level and enabled encapsulation of cells inside the fibers under cell-friendly mild conditions. Presence of carboxyl- (in gellan gum) or amino- (in chitosan) functionalities further enabled controlled peptide functionalization such as RGD for biochemical mimicry (cell adhesion sites) of native collagen. This biomimetic aligned fibrous hydrogel system can potentially be used as a scaffold for tissue engineering as well as a drug/gene delivery vehicle.

Engineering Enriched Microenvironments with Gradients of Platelet Lysate in Hydrogel Fibers.

Gradients of physical and chemical cues are characteristic of specific tissue microenvironments and contribute toward morphogenesis and tissue regeneration upon injury. Recent advances on microfluidics and hydrogel manipulation raised the possibility of generating biomimetic biomaterials enriched with bioactive factors and encapsulating cells following designs specifically tailored for a target application. The novelty of this work relies on the combination of methacrylated gellan gum (MeGG) with platelet lysate (PL), aiming to generate novel advanced 3D PL-enriched photo-cross-linkable hydrogels and overcoming the lack of adhesion sites provided by the native MeGG hydrogels. This combination takes advantage of the availability, enriched growth factor composition, and potential autologous application of PL while simultaneously preserving the ability provided by MeGG to tailor mechanical properties, protein release kinetics, and shape of the construct according to the desired goal. Incorporation of PL in the hydrogels significantly improved cellular adhesion and viability in the constructs. The use of microfluidic tools allowed the design of a fiber-like hydrogel incorporating a gradient of PL along the length of the fiber. These spatial protein gradients led to the viability and cell number gradients caused by maintenance of human umbilical vein endothelial cells (HUVECs) survival in the fibers toward the PL-enriched sections in comparison with the nonloaded MeGG sections of the fibers. Altogether, we propose a proof of concept strategy to design a PL gradient biomaterial with potential in tissue engineering approaches and analysis of cell-microenvironment interactions.

The effect of chitosan on the in vitro biological performance of chitosan-poly(butylene succinate) blends.

Chitosan blends with synthetic biodegradable polymers have been proposed for various biomedical applications due to their versatile mechanical properties and easier processing. However, details regarding the main surface characteristics that may benefit from the blending of these two types of materials are still missing. Hence, this work aims at investigating the surface properties of chitosan-based blends, illustrating the way these properties determine the material-proteins interactions and ultimately the behavior of osteoblast-like cells. The surface characteristics of modified and nonmodified blends were assessed using complimentary techniques such as optical microscopy, scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR-ATR), X-ray photoelectron spectroscopy (XPS), contact angle measurements and surface energy calculations. The adsorption of human serum albumin (HSA) and human plasma fibronectin (HFN) onto the different surfaces was quantified by association of an indirect method with a colorimetric assay. It was found that the presence of chitosan on the surface promoted the adsorption of proteins. Moreover, a preferential adsorption of albumin over fibronectin was registered. The in vitro biological performance of the studied materials was further investigated by a direct contact assay with an osteoblastic-like cell line (SaOs-2). A synergistic effect of the two components of the blend was observed. While the synthetic polyester promoted the adhesion of SaOs-2, the presence of chitosan significantly enhanced the osteoblastic activity of these cells. This work further confirmed the interest in designing polymeric blends with natural polymers as a successful strategy to enhance the biological performance of a biomaterial.

Development of micropatterned surfaces of poly(butylene succinate) by micromolding for guided tissue engineering.

Native tissues present complex architectures at the micro- and nanoscale that dictate their biological function. Several microfabrication techniques have been employed for engineering polymeric surfaces that could replicate in vitro these micro- and nanofeatures. In this study, biomimetic surfaces of poly(butylene succinate) (PBS) were engineered by a micromolding technique. After the optimization of the system parameters, 20 surfaces with different combinations of groove and ridge sizes were developed and characterized by scanning electron microscopy (SEM). The influence of the engineered microfeatures over the viability and attachment of human adipose derived adult stem cells (hASCs) was evaluated. hASCs cultured onto the engineered surfaces were demonstrated to remain viable for all tested patterns. SEM and immunostaining showed adequate attachment and spreading of the stem cells for all the patterned groove/ridge combinations. This study indicated that it is possible to engineer micropatterned surfaces of PBS and that the developed structures could have great potential for tissue engineering where cell alignment is an essential requisite.

Microfabricated photocrosslinkable polyelectrolyte-complex of chitosan and methacrylated gellan gum.

Chitosan (CHT) based polyelectrolyte complexes (PECs) have been receiving great attention for tissue engineering approaches. These hydrogels are held together by ionic forces and can be disrupted by changes in physiological conditions. In this study, we present a new class of CHT-based PEC hydrogels amenable to stabilization by chemical crosslinking. The photocrosslinkable anionic methacrylated gellan gum (MeGG) was complexed with cationic CHT and exposed to light, forming a PEC hydrogel. The chemical characterization of the photocrosslinkable PEC hydrogel by Fourier transform infrared spectroscopy (FTIR) revealed absorption peaks specific to the raw polymers. A significantly higher swelling ratio was observed for the PEC hydrogel with higher CHT content. The molecular interactions between both polysaccharides were evaluated chemically and microscopically, indicating the diffusion of CHT to the interior of the hydrogel. We hypothesized that the addition of MeGG to CHT solution first leads to a membrane formation around MeGG. Then, migration of CHT inside the MeGG hydrogel occurs to balance the electrostatic charges. The photocrosslinkable feature of MeGG further allowed the formation of cell-laden microscale hydrogel units with different shapes and sizes. Overall, this system is potentially useful for a variety of applications including the replication of microscale features of tissues for modular tissue engineering.

An automated two-phase system for hydrogel microbead production.

Polymeric beads have been used for protection and delivery of bioactive materials, such as drugs and cells, for different biomedical applications. Here, we present a generic two-phase system for the production of polymeric microbeads of gellan gum or alginate, based on a combination of in situ polymerization and phase separation. Polymer droplets, dispensed using a syringe pump, formed polymeric microbeads while passing through a hydrophobic phase. These were then crosslinked, and thus stabilized, in a hydrophilic phase as they crossed through the hydrophobic-hydrophilic interface. The system can be adapted to different applications by replacing the bioactive material and the hydrophobic and/or the hydrophilic phases. The size of the microbeads was dependent on the system parameters, such as needle size and solution flow rate. The size and morphology of the microbeads produced by the proposed system were uniform, when parameters were kept constant. This system was successfully used for generating polymeric microbeads with encapsulated fluorescent beads, cell suspensions and cell aggregates proving its ability for generating bioactive carriers that can potentially be used for drug delivery and cell therapy.

Modified Gellan Gum hydrogels with tunable physical and mechanical properties.

Gellan Gum (GG) has been recently proposed for tissue engineering applications. GG hydrogels are produced by physical crosslinking methods induced by temperature variation or by the presence of divalent cations. However, physical crosslinking methods may yield hydrogels that become weaker in physiological conditions due to the exchange of divalent cations by monovalent ones. Hence, this work presents a new class of GG hydrogels crosslinkable by both physical and chemical mechanisms. Methacrylate groups were incorporated in the GG chain, leading to the production of a methacrylated Gellan Gum (MeGG) hydrogel with highly tunable physical and mechanical properties. The chemical modification was confirmed by proton nuclear magnetic resonance (1H NMR) and Fourier transform infrared spectroscopy (FTIR-ATR). The mechanical properties of the developed hydrogel networks, with Young's modulus values between 0.15 and 148 kPa, showed to be tuned by the different crosslinking mechanisms used. The in vitro swelling kinetics and hydrolytic degradation rate were dependent on the crosslinking mechanisms used to form the hydrogels. Three-dimensional (3D) encapsulation of NIH-3T3 fibroblast cells in MeGG networks demonstrated in vitro biocompatibility confirmed by high cell survival. Given the highly tunable mechanical and degradation properties of MeGG, it may be applicable for a wide range of tissue engineering approaches.

Blending polysaccharides with biodegradable polymers. II. Structure and biological response of chitosan/polycaprolactone blends.

Blends of polycaprolactone (PCL) and chitosan (CHT) were prepared by casting from the mixture of solutions of both components in suitable solvents. PCL, and CHT, form phase separated blends with improved mechanical properties and increased water sorption ability with respect to pure PCL. The morphology of the system was investigated by scanning electron microscopy (SEM), atomic force microscopy (AFM) and confocal microscopy. Dispersed domains of CHT in the semicrystalline PCL matrix were found in samples with less than 20% CHT but cocontinuous phase morphologies are found in blends with 20% or more CHT. This feature was corroborated by the temperature dependence of the elastic modulus measured by dynamic mechanical properties as a function of temperature. It was observed that for those blends above 20 wt% CHT, the mechanical stability of the system was kept even after melting of the PCL phase. Primary human chondrocytes were cultured on the different substrates. Cell morphology was studied by SEM and the viability and proliferation was investigated by the colorimetric MTT assay. Different protein conformations were found by AFM on CHT and PCL samples which were related to the biological performance of the substrates. Hydrophilicty of the material is not directly related to the biological response and the sample with 20 wt% CHT shows better results than the other blends with respect to chondrocyte viability and proliferation. However, the results obtained in the blends are worse than in pure PCL. It seems to be correlated with the surface energy of the different blends rather than hydrophilicity.