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Scientific Unit
Efficient wound treatments to target specific events in the healing process of chronic wounds constitute a significant aim in regenerative medicine. In this sense, nanomedicine can offer new opportunities to improve the effectiveness of existing wound therapies. The aim of this study was to develop catechol bearing polymeric nanoparticles (NPs) and to evaluate their potential in the field of wound healing. Thus, NPs wound healing promoting activities, potential for drug encapsulation and controlled release, and further incorporation in a hydrogel bioink formulation to fabricate cell-laden 3D scaffolds are studied. NPs with 2 and 29 M % catechol contents (named NP2 and NP29) were obtained by nanoprecipitation and presented hydrodynamic diameters of 100 and 75 nm respectively. These nanocarriers encapsulated the hydrophobic compound coumarin-6 with 70% encapsulation efficiency values. In cell culture studies, the NPs had a protective effect in RAW 264.7 macrophages against oxidative stress damage induced by radical oxygen species (ROS). They also presented a regulatory effect on the inflammatory response of stimulated macrophages and promoted upregulation of the vascular endothelial growth factor (VEGF) in fibroblasts and endothelial cells. In particular, NP29 were used in a hydrogel bioink formulation using carboxymethyl chitosan and hyaluronic acid as polymeric matrices. Using a reactive mixing bioprinting approach, NP-loaded hydrogel scaffolds with good structural integrity, shape fidelity and homogeneous NPs dispersion, were obtained. The in vitro catechol NPs release profile of the printed scaffolds revealed a sustained delivery. The bioprinted scaffolds supported viability and proliferation of encapsulated L929 fibroblasts over 14 days. We envision that the catechol functionalized NPs and resulting bioactive bioink presented in this work offer promising advantages for wound healing applications, as they: 1) support controlled release of bioactive catechol NPs to the wound site; 2) can incorporate additional therapeutic functions by co-encapsulating drugs; 3) can be printed into 3D scaffolds with tailored geometries based on patient requirements.
Natural polymers have been widely used for biomedical applications in recent decades. They offer the advantages of resembling the extracellular matrix of native tissues and retaining biochemical cues and properties necessary to enhance their biocompatibility, so they usually improve the cellular attachment and behavior and avoid immunological reactions. Moreover, they offer a rapid degradability through natural enzymatic or chemical processes. However, natural polymers present poor mechanical strength, which frequently makes the manipulation processes difficult. Recent advances in biofabrication, 3D printing, microfluidics, and cell-electrospinning allow the manufacturing of complex natural polymer matrixes with biophysical and structural properties similar to those of the extracellular matrix. In addition, these techniques offer the possibility of incorporating different cell lines into the fabrication process, a revolutionary strategy broadly explored in recent years to produce cell-laden scaffolds that can better mimic the properties of functional tissues. In this review, the use of 3D printing, microfluidics, and electrospinning approaches has been extensively investigated for the biofabrication of naturally derived polymer scaffolds with encapsulated cells intended for biomedical applications (e.g., cell therapies, bone and dental grafts, cardiovascular or musculoskeletal tissue regeneration, and wound healing)
Hydrogel-based bio-inks have recently attracted more attention for 3D printing applications in tissue engineering due to their remarkable intrinsic properties, such as a cell supporting environment. However, their usually weak mechanical properties lead to poor printability and low stability of the obtained structures. To obtain good shape fidelity, current approaches based on extrusion printing use high viscosity solutions, which can compromise cell viability. This paper presents a novel bio-printing methodology based on a dual-syringe system with a static mixing tool that allows in situ crosslinking of a two-component hydrogel-based ink in the presence of living cells. The reactive hydrogel system consists of carboxymethyl chitosan (CMCh) and partially oxidized hyaluronic acid (HAox) that undergo fast self-covalent crosslinking via Schiff base formation. This new approach allows us to use low viscosity solutions since in situ gelation provides the appropriate structural integrity to maintain the printed shape. The proposed bio-ink formulation was optimized to match crosslinking kinetics with the printing process and multi-layered 3D bio-printed scaffolds were successfully obtained. Printed scaffolds showed moderate swelling, good biocompatibility with embedded cells, and were mechanically stable after 14 days of the cell culture. We envision that this straightforward, powerful, and generalizable printing approach can be used for a wide range of materials, growth factors, or cell types, to be employed for soft tissue regeneration.
The increasing prevalence of dry eye syndrome in aging and digital societies compromises long-term contact lens (CL) wear and forces users to regular eye drop instillation to alleviate discomfort. Here a novel approach with the potential to improve and extend the lubrication properties of CLs is presented. This is achieved by embedding lubricant-secreting biofactories within the CL material. The self-replenishable reservoirs autonomously produce and release hyaluronic acid (HA), a natural lubrication and wetting agent, long term. The hydrogel matrix regulates the growth of the biofactories and the HA production, and allows the diffusion of nutrients and HA for at least 3 weeks. The continuous release of HA sustainably reduces the friction coefficient of the CL surface. A self-lubricating CL prototype is presented, where the functional biofactories are contained in a functional ring at the lens periphery, outside of the vision area. The device is cytocompatible and fulfils physicochemical requirements of commercial CLs. The fabrication process is compatible with current manufacturing processes of CLs for vision correction. It is envisioned that the durable-by-design approach in living CL could enable long-term wear comfort for CL users and minimize the need for lubricating eye drops.
Microbial biofactories allow the upscaled production of high-value compounds in biotechnological processes. This is particularly advantageous for compounds like flavonoids that promote better health through their antioxidant, antibacterial, anticancer and other beneficial effects but are only produced in small quantities in their natural plant-based hosts. Bacteria like E. coli have been genetically modified with enzyme cascades to produce flavonoids like naringenin and pinocembrin from coumaric or cinnamic acid. Despite advancements in yield optimization, the production of these compounds still involves high costs associated with their biosynthesis, purification, storage and transport. An alternative production strategy could involve the direct delivery of the microbial biofactories to the body. In such a strategy, ensuring biocontainment of the engineered microbes in the body and controlling production rates are major challenges. In this study, these two aspects are addressed by developing engineered living materials (ELMs) consisting of probiotic microbial biofactories encapsulated in biocompatible hydrogels. Engineered probiotic E. coli Nissle 1917 able to efficiently convert cinnamic acid into pinocembrin were encapsulated in poly(vinyl alcohol)-based hydrogels. The biofactories are contained in the hydrogels for a month and remain metabolically active during this time. Control over production levels is achieved by the containment inside the material, which regulates bacteria growth, and by the amount of cinnamic acid in the medium.
