Cell-Laden 3D Hydrogels of Type I Collagen Incorporating Bacterial Nanocellulose Fibers.

There is a growing interest in developing natural hydrogel-based scaffolds to culture cells in a three-dimensional (3D) millieu that better mimics the in vivo cells' microenvironment. A promising approach is to use hydrogels from animal tissues, such as decellularized extracellular matrices; however, they usually exhibit suboptimal mechanical properties compared to native tissue and their composition with hundreds of different protein complicates to elucidate which stimulus triggers cell's responses. As simpler scaffolds, type I collagen hydrogels are used to study cell behavior in mechanobiology even though they are also softer than native tissues. In this work, type I collagen is mixed with bacterial nanocellulose fibers (BCf) to develop reinforced scaffolds with mechanical properties suitable for 3D cell culture. BCf were produced from blended pellicles biosynthesized from Komagataeibacter xylinus. Then, BCf were mixed with concentrated collagen from rat-tail tendons to form composite hydrogels. Confocal laser scanning microscopy and scanning electron microscopy images confirmed the homogeneous macro- and microdistribution of both natural polymers. Porosity analysis confirmed that BCf do not disrupt the scaffold structure. Tensile strength and rheology measurements demonstrated the reinforcement action of BCf (43% increased stiffness) compared to the collagen hydrogel while maintaining the same viscoelastic response. Additionally, this reinforcement of collagen hydrogels with BCf offers the possibility to mix cells before gelation and then proceed to the culture of the 3D cell scaffolds. We obtained scaffolds with human bone marrow-derived mesenchymal stromal cells or human fibroblasts within the composite hydrogels, allowing a homogeneous 3D viable culture for at least 7 days. A smaller surface shrinkage in the reinforced hydrogels compared to type I collagen hydrogels confirmed the strengthening of the composite hydrogels. These collagen hydrogels reinforced with BCf might emerge as a promising platform for 3D in vitro organ modeling, tissue-engineering applications, and suitable to conduct fundamental mechanobiology studies.

One-step double network hydrogels of photocurable monomers and bacterial cellulose fibers.

Soft-tissue replacements are challenging due to the stringent compliance requirements for the implanted materials in terms of biocompatibility, durability, high wear resistance, low friction, and water content. Acrylate hydrogels are worth considering as soft tissue implants as they can be photocurable and sustain customized shapes through 3D bioprinting. However, acrylate-based hydrogels present weak mechanical properties and significant dimensional changes when immersed in liquids. To address these obstacles, we fabricated double network (DN) hydrogels composed of polyacrylic acid (PAA) and bacterial cellulose nanofibers (BCNFs) by one fast UV photopolymerization step. BCNFs/PAA hydrogels with a 0.5 wt% BCNFs content present an increased stiffness and a lower, non-pH-dependent swelling than PAA hydrogels or PAA hydrogels with cellulose nanocrystals. Besides, BCNFs/PAA hydrogels are biocompatible and can be frozen/thawed. Those characteristics endorse these hybrid hydrogels as potential candidates for vascular and cartilage tissue implants.

Highly Aligned Bacterial Nanocellulose Films Obtained During Static Biosynthesis in a Reproducible and Straightforward Approach.

Bacterial nanocellulose (BNC) is usually produced as randomly-organized highly pure cellulose nanofibers films. Its high water-holding capacity, porosity, mechanical strength, and biocompatibility make it unique. Ordered structures are found in nature and the properties appearing upon aligning polymers fibers inspire everyone to achieve highly aligned BNC (A-BNC) films. This work takes advantage of natural bacteria biosynthesis in a reproducible and straightforward approach. Bacteria confined and statically incubated biosynthesized BNC nanofibers in a single direction without entanglement. The obtained film is highly oriented within the total volume confirmed by polarization-resolved second-harmonic generation signal and Small Angle X-ray Scattering. The biosynthesis approach is improved by reusing the bacterial substrates to obtain A-BNC reproducibly and repeatedly. The suitability of A-BNC as cell carriers is confirmed by adhering to and growing fibroblasts in the substrate. Finally, the thermal conductivity is evaluated by two independent approaches, i.e., using the well-known 3ω-method and a recently developed contactless thermoreflectance approach, confirming a thermal conductivity of 1.63 W mK-1 in the direction of the aligned fibers versus 0.3 W mK-1 perpendicularly. The fivefold increase in thermal conductivity of BNC in the alignment direction forecasts the potential of BNC-based devices outperforming some other natural polymer and synthetic materials.

Bioprintable Lung Extracellular Matrix Hydrogel Scaffolds for 3D Culture of Mesenchymal Stromal Cells.

Mesenchymal stromal cell (MSC)-based cell therapy in acute respiratory diseases is based on MSC secretion of paracrine factors. Several strategies have proposed to improve this are being explored including pre-conditioning the MSCs prior to administration. We here propose a strategy for improving the therapeutic efficacy of MSCs based on cell preconditioning by growing them in native extracellular matrix (ECM) derived from the lung. To this end, a bioink with tunable stiffness based on decellularized porcine lung ECM hydrogels was developed and characterized. The bioink was suitable for 3D culturing of lung-resident MSCs without the need for additional chemical or physical crosslinking. MSCs showed good viability, and contraction assays showed the existence of cell-matrix interactions in the bioprinted scaffolds. Adhesion capacity and length of the focal adhesions formed were increased for the cells cultured within the lung hydrogel scaffolds. Also, there was more than a 20-fold increase of the expression of the CXCR4 receptor in the 3D-cultured cells compared to the cells cultured in plastic. Secretion of cytokines when cultured in an in vitro model of lung injury showed a decreased secretion of pro-inflammatory mediators for the cells cultured in the 3D scaffolds. Moreover, the morphology of the harvested cells was markedly different with respect to conventionally (2D) cultured MSCs. In conclusion, the developed bioink can be used to bioprint structures aimed to improve preconditioning MSCs for therapeutic purposes.