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- gelation kinetics (2)
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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.
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.
Fabrication of intricate and long-term stable 3D polymeric scaffolds by 3D printing technique is still a challenge. The currently used polymeric materials need long post-printing processes and washing steps. In addition, highly concentrated solutions are necessary for maintaining shape fidelity after 3D deposition. This paper reports the fabrication of dual crosslinked 3D scaffolds using a low concentrated (<10 wt-%) ink of Gelatin Methacryloyl (GelMA)/Chitosan and a novel crosslinking agent, a glycerylphytate (G1Phy) to overcome the current limitations in the 3D printing field using hydrogels. The applied methodology consisted of a first ultraviolet light (UV) photopolymerization followed by a post-printing ionic crosslinking treatment with G1Phy. This crosslinker provides a robust framework and avoids the necessity of neutralization with strong bases. The blend ink showed shear-thinning behavior and excellent printability in the form of straight and homogeneous filament. UV curing was undertaken simultaneously to 3D deposition, which enhanced precision, shape fidelity (resolution ≈ 150 µm), and prevented from collapse of the subsequent printed layers (up to 28 layers). In the second step, the novel G1Phy ionic crosslinker agent provided swelling and long term stability properties to the 3D scaffolds. The multi-layered printed scaffolds were mechanically stable at physiological conditions for at least one month. Preliminary in vitro assays using L929 Fibroblasts showed very promising results in terms of adhesion, spreading, and proliferation in comparison to other phosphate-based traditional crosslinkers (i.e. TPP). We envision that the proposed combination of the blend ink and 3D printing approach can have widespread applications in the regeneration of soft tissues.
In this paper we explore the printability of reversible networks formed by catechol functionalized PEG solutions and metal cations (Al3+, Fe3+ or V3+). The printability and shape fidelity were dependent on the ink composition (metal ion type, pH, PEG molecular weight) and printing parameters (extrusion pressure and printing speed). The relaxation time, recovery rate and viscosity of the inks were analyzed in rheology studies and correlated with thermodynamic and ligand exchange kinetic constants of the dynamic bonds and the printing performance (i.e. shape fidelity of the printed structures). The relevance of the relaxation time and ligand exchange kinetics for printability was demonstrated. Cells seeded on the materials crosslinked with Al3+, Fe3+ ions were viable and revealed well-spread morphologies during 7 day culture, indicating the potential of the formulations to be used as inks for cell encapsulation. The proposed dynamic ink design offers significant flexibility for 3D bioprinting, and enables straightforward adjustment of the printable formulation to meet application-specific needs.
Thiol-maleimide and thiol-vinylsulfone cross-linked hydrogels are widely used systems in 3D culture models, in spite of presenting uncomfortable reaction kinetics for cell encapsulation: too fast (seconds for thiol-maleimide) or too slow (minutes-hours for thiol-vinylsulfone). Here, we introduce the thiol-methylsulfone reaction as alternative cross-linking chemistry for cell encapsulation, particularized for PEG-hydrogels. The thiol-methylsulfone reaction occurs at high conversion and at intermediate reaction speed (seconds-minutes) under physiological pH range. These properties allow easy mixing of hydrogel precursors and cells to render homogeneous cell-laden gels at comfortable experimental time scales. The resulting hydrogels are cytocompatible and show comparable hydrolytic stability to thiol-vinylsulfone gels. They allow direct bioconjugation of thiol-derivatized ligands and tunable degradation kinetics by cross-linking with degradable peptide sequences. 3D cell culture of two cell types, fibroblasts and human umbilical vein endothelial cells (HUVECs), is demonstrated.
Osteoporosis is a silent bone disease and a growing health issue. Despite recent progress in diagnosis and treatment, effective therapeutic strategies are still needed. One of the possible solutions is the implantation of engineered drug-releasing scaffolds at the disease site. To boost this approach further, we aimed to develop printable materials (the inks) for the construction of patient-specific 3D scaffolds with drug-release capability. The inks were composed of chitosan – a natural osteoinductive polysaccharide, nanohydroxyapatite – a natural bone matrix ingredient improving mechanical properties, sodium alendronate – a bioactive drug, and hydroxyethyl-cellulose – a filler improving the printability. Printed scaffolds were crosslinked with citric acid or KOH. After coating with collagen and gelatin, they demonstrated biocompatibility with the adipose-derived mesenchymal stem cells and MG-63 cell line. They also showed a sustained release of alendronate for 50 days, causing a significant reduction in the expression of Cathepsin K, an osteoclast-specific gene marker, which indicates the osteoclast-inhibiting capacity of the coated scaffolds. This work demonstrates the potential of developed printable materials to find applications as cell and drug carriers for the treatment of osteoporosis.
The permeability of the human trabecular meshwork (HTM) regulates eye pressure via a porosity gradient across its thickness modulated by stacked layers of matrix fibrils and cells. Changes in HTM porosity are associated with increases in intraocular pressure and the progress of diseases such as glaucoma. Engineered HTMs could help to understand the structure–function relation in natural tissues and lead to new regenerative solutions. Here, melt electrowriting (MEW) is explored as a biofabrication technique to produce fibrillar, porous scaffolds that mimic the multilayer, gradient structure of native HTM. Poly(caprolactone) constructs with a height of 125–500 μm and fiber diameters of 10 –12 μm are printed. Scaffolds with a tensile modulus between 5.6 and 13 MPa and a static compression modulus in the range of 6 –360 kPa are obtained by varying the scaffold design, that is, the density and orientation of the fibers and number of stacked layers. Primary HTM cells attach to the scaffolds, proliferate, and form a confluent layer within 8–14 days, depending on the scaffold design. High cell viability and cell morphology close to that in the native tissue are observed. The present work demonstrates the utility of MEW for reconstructing complex morphological features of natural tissues.
Thiol-Methylsulfone Based Hydrogels: Enhanced Control on Gelation Kinetics for 3D Cell Encapsulation
(2019)
Hydrogels are useful temporal matrices for cell culture technologies. The successful mixing and encapsulation of cells within the gel requires the selection of efficient and cytocompatible gelation reactions occurring in the minute timescale under physiological conditions. The thiol-methylsulfonyl (MS) chemical reaction is introduced here as a novel chemistry to encapsulate cells in polymeric matrices. Thiol-MS crosslinking does not require a light activation step and can occur within the seconds-to-minutes timescale by adjusting the pH in the physiological range 8.0-6.6. This reaction is cytocompatible and the reaction product is hydrolytically stable in cell culture media up to 4 weeks. Cell encapsulation protocols enabling comfortable handling and yielding homogenous distribution of the embedded cells are described. All these features are relevant for the application of this crosslinking reaction to biomedical scenarios. Finally, this manuscript also compares the performance of thiol-MS hydrogels with the established thiol-maleimide and thiol-vinylsulfone hydrogels. The benefit of thiol-MS crosslinking in terms of control over hydrogelation kinetics is demonstrated.
Multifunctional wound dressings, enriched with biologically active agents for preventing or treating infections and promoting wound healing, along with cell delivery capability, are highly needed. To address this issue, composite scaffolds with potential in wound dressing applications were fabricated in this study. The poly-lactic acid/nanodiamonds (PLA/ND) scaffolds were first printed using melt electrowriting (MEW) and then coated with quaternized β-chitin (QβC). The NDs were well-dispersed in the printed filaments and worked as fillers and bioactive additions to PLA material. Additionally, they improved coating effectiveness due to the interaction between their negative charges (from NDs) and positive charges (from QβC). NDs not only increased the thermal stability of PLA but also benefitted cellular behavior and inhibited the growth of bacteria. Scaffolds coated with QβC increased the effect of bacteria growth inhibition and facilitated the proliferation of human dermal fibroblasts. Additionally, we have observed rapid extracellular matrix (ECM) remodeling on QβC-coated PLA/NDs scaffolds. The scaffolds provided support for cell adhesion and could serve as a valuable tool for delivering cells to chronic wound sites. The proposed PLA/ND scaffold coated with QβC holds great potential for achieving fast healing in various types of wounds.
The permeability of the Human Trabecular Meshwork (HTM) regulates eye pressure via a porosity gradient across its thickness modulated by stacked layers of matrix fibrils and cells. Changes in HTM porosity are associated with increases in intraocular pressure and the progress of diseases like glaucoma. Engineered HTMs could help to understand the structure-function relation in natural tissues, and lead to new regenerative solutions. Here, melt electrowriting (MEW) is explored as a biofabrication technique to produce fibrillar, porous scaffolds that mimic the multilayer, gradient structure of native HTM. Poly(caprolactone) constructs with a height of 125-500 μm and fiber diameters of 10-12 μm are printed. Scaffolds with a tensile modulus between 5.6 and 13 MPa, and a static compression modulus in the range of 6-360 kPa are obtained by varying the scaffolds design, i.e., density and orientation of the fibers and number of stacked layers. Primary HTM cells attach to the scaffolds, proliferate, and form a confluent layer within 8-14 days, depending on the scaffold design. High cell viability and cell morphology close to that in the native tissue are observed. The present work demonstrates the utility of MEW to reconstruct complex morphological features of natural tissues.Competing Interest StatementThe authors have declared no competing interest.
Advances in optogenetics and the increasing use of implantable devices for therapies and health monitoring are driving demand for compliant, biocompatible optical waveguides and scalable methods for their manufacture. Molding, thermal drawing, and dip-coating are the most prevalent approaches in recent literature. Here the authors demonstrate that extrusion printing at room temperature can be used for continuous fabrication of compliant optical waveguides with polydimethylsiloxane (PDMS) core and crosslinked Pluronic F127-diacrylate (Pluronic-DA) cladding. The optical fibers are printed from fluid precursor inks and stabilized by physical interactions and photoinitiated crosslinking in the Pluronic-DA. The printed fibers show optical loss values of 0.13–0.34 dB cm–1 in air and tissue within the wavelength range of 405–520 nm. The fibers have a Young's Modulus (Pluronic cladding) of 150 kPa and can be stretched to more than 5 times their length. The optical loss of the fibers shows little variation with extension. This work demonstrates how printing can simplify the fabrication of compliant and stretchable devices from materials approved for clinical use. These can be of interest for optogenetic or photopharmacology applications in extensible tissues, like muscles or heart.