Whey Protein Isolate Composites as Potential Scaffolds for Cultivated Meat.

Modern food technology has given rise to numerous alternative protein sources in response to a growing human population and the negative environmental impacts of current food systems. To aid in achieving global food security, one such form of alternative protein being investigated is cultivated meat, which applies the principles of mechanical and tissue engineering to produce animal proteins and meat products from animal cells. Herein, nonmodified and methacrylated whey protein formed hydrogels with methacrylated alginate as potential tissue engineering scaffolds for cultivated meat. Whey protein is a byproduct of dairy processing and was selected because it is an approved food additive and cytocompatible and has shown efficacy in other biomaterial applications. Whey protein and alginate scaffolds were formed via visible light cross-linking in aqueous solutions under ambient conditions. The characteristics of the precursor solution and the physical-mechanical properties of the scaffolds were quantified; while gelation occurred within the homo- and copolymer hydrogels, the integrity of the network was significantly altered with varying components. Qualitatively, the scaffolds exhibited a three-dimensional (3D) interconnected porous network. Whey protein isolate (WPI)-based scaffolds were noncytotoxic and supported in vitro myoblast adhesion and proliferation. The data presented support the hypothesis that the composition of the hydrogel plays a significant role in the scaffold's performance.

Dual-Crosslinked Alginate-Based Hydrogels with Tunable Mechanical Properties for Cultured Meat.

Cultured meat refers to the production of animal tissue by utilizing the same techniques as tissue engineering through cell culture. Various biomaterials have been designed to serve as in vitro supports for cell viability, growth, and migration. In this study, visible light and dual-crosslinked alginate hydrogels were designed to enable control of the physical and mechanical properties needed for the fabrication of cultured meat scaffolds. We hypothesized that a difference in hydrogel stiffness would influence cell behavior, indicating the efficacy of our processing methods to benefit the cultured meat field. Herein, we synthesized and created: (1) methacrylated alginate (AlgMA) to enable covalent crosslinking via visible light exposure, (2) Methacrylated alginate and arginyl-glycyl-aspartic acid RGD conjugates (AlgMA-RGD), using carbodiimide chemistries to provide cell-binding sites on the material, and (3) designer hydrogels incorporating different crosslinking techniques. The material and mechanical properties were evaluated to determine the structural integrity of the hydrogels, and in vitro cell assays were conducted to verify cytocompatibility and cell adhesion. Gelation, swell ratio, and weight loss calculations revealed longer gelation times for the AlgMA scaffolds and similar physical properties for all hydrogel groups. We showed that by adjusting the polymer concentration and the crosslinking methodology, the scaffold's mechanical properties can be controlled and optimized within physiological ranges. Incorporating dual crosslinking significantly increased the compressive moduli of the AlgMA hydrogels, compared to visible-light crosslinking alone. Moreover, the muscle satellite cells responded favorably to the AlgMA scaffolds, with clear differences in cell density when cultured on materials with significantly different mechanical properties. Our results indicate the usefulness of the dual-crosslinking alginate hydrogel system to support in vitro meat growth.

Bio-inspired green light crosslinked alginate-heparin hydrogels support HUVEC tube formation.

Alginate is a polysaccharide which forms hydrogels via ionic and/or covalent crosslinking. The goal was to develop a material with suitable, physiologically relevant mechanical properties and biological impact for use in wound treatment. To determine if the novel material can initiate tube formation on its own, without the dependance on the addition of growth factors, heparin and/or arginyl-glycyl-aspartic acid (RGD) was covalently conjugated onto the alginate backbone. Herein, cell adhesion motifs and bioactive functional groups were incorporated covalently within alginate hydrogels to study the: 1) impact of crosslinked heparin on tubular network formation, 2) impact of RGD conjugation, and the 3) biological effect of vascular endothelial growth factor (VEGF) loading on cellular response. We investigated the structure-properties-function relationship and determined the viscoelastic and burst properties of the hydrogels most applicable for use as a healing cell and tissue adhesive material. Methacrylation of alginate and heparin hydroxyl groups respectively enabled free-radical covalent inter- and intra-molecular photo-crosslinking when exposed to visible green light in the presence of photo-initiators; the shear moduli indicate mechanical properties comparable to clinical standards. RGD was conjugated via carbodiimide chemistry at the alginate carboxyl groups. The adhesive and mechanical properties of alginate and alginate-heparin hydrogels were determined via burst pressure testing and rheology. Higher burst pressure and material failure at rupture imply physical tissue adhesion, advantageous for a tissue sealant healing material. After hydrogel formation, human umbilical vein endothelial cells (HUVECs) were seeded onto the alginate-based hydrogels; cytotoxicity, total protein content, and tubular network formation were assessed. Burst pressure results indicate that the cell responsive hydrogels adhere to collagen substrates and exhibit increased strength under high pressures. Furthermore, the results show that the green light crosslinked alginate-heparin maintained cell adhesion and promoted tubular formation.

Physico-mechanical Characterization of Liquid versus Solid Applications of Visible Light Cross-Linked Tissue Sealants.

The limitations of commercially available tissue sealants have resulted in the need for a new tissue adhesives with adequate adhesion, improved mechanical properties, and innocuous degradation products. To address current limitations, a visible light cross-linking method for the preparation of hydrogel tissue sealants, based on natural polymers (chitosan or alginate), is presented. Water-soluble chitosan was generated via modification with vinyl groups. To form hydrogels, alginate and chitosan were cross-linked by green light illumination, with or without the use of a bifunctional cross-linker. Evaluation of the mechanical properties through rheological characterization demonstrated an increased viscosity of polymer blends, and differences in shear moduli despite similar gelation points upon photo-cross-linking. A comparative study on the burst pressure properties of liquid versus solid material applications was performed to determine if the tissue sealants can perform under physiological lung pressures and beyond using different application methods. Higher burst pressure values were obtained for the sealants applied as a liquid compared to the solid application. The hydrogel tissue sealants revealed no cytotoxic effects toward primary human mesenchymal stem cells. This is the first report of a direct comparison between hydrogel tissue sealants of the same formulation applied in liquid versus solid form.

Dual-crosslinked homogeneous alginate microspheres for mesenchymal stem cell encapsulation.

A smart hydrogel material was used in combination with custom microfluidic devices (MFDs) to create microspheres for human mesenchymal stem cell (MSC) encapsulation. Methods for fabricating homogeneous stimuli-responsive microspheres for MSC encapsulation and cell delivery have gained interest to increase viability and manipulate microencapsulation within microspheres 10-1000 µm in diameter. Herein, MFDs were combined with non-toxic smart hydrogel materials to tune both the size and mechanics of the microspheres. Traditional hydrogels have a single input/stimulus for crosslinking, utilize potentially toxic ultraviolet radiation, and fail to mimic surrounding musculoskeletal tissue mechanics. Thus, it is highly beneficial to encapsulate MSCs inside a mechanically-stable microsphere made from naturally-derived materials. The objectives of this research were to optimize microsphere fabrication techniques using custom microfluidic devices (MFDs), and to encapsulate viable MSCs within visible-light crosslinked smart-alginate microspheres, with tunable mechanical properties. Microsphere production was characterized optically, and MSC viability, post-encapsulation, was verified using a standard florescence assay. Cell viability was maintained in chemically-modified alginate homogenous microspheres post encapsulation, and after subsequent crosslinking via green light exposure.

Anticancer Therapeutic Alginate-Based Tissue Sealants for Lung Repair.

Injury to the connective tissue that lines the lung, the pleura, or the lung itself can occur from many causes including trauma or surgery, as well as lung diseases or cancers. To address current limitations for patching lung injuries, to stop air or fluid leaks, an adherent hydrogel sealant patch system was developed, based on methacrylated alginate (AMA) and AMA dialdehyde (AMA-DA) blends, which is capable of sealing damaged tissues and sustaining physiological pressures. Methacrylation of alginate hydroxyl groups rendered the polysaccharide capable of photo-cross-linking when mixed with an eosin Y-based photoinitiator system and exposed to visible green light. Oxidation of alginate yields functional aldehyde groups capable of imine bond formation with proteins found in many tissues. The alginate-based patch system was rigorously tested on a custom burst pressure testing device. Blending of nonoxidized material with oxidized (aldehyde modified) alginates yielded patches with improved burst pressure performance and decreased delamination as compared with pure AMA. Human mesothelial cell (MeT-5A) viability and cytotoxicity were retained when cultured with the hydrogel patches. The release and bioactivity of doxorubicin-encapsulated submicrospheres enabled the fabrication of drug-eluting adhesive patches and were effective in decreasing human lung cancer cell (A549) viability.

Dual-Cross-Linked Methacrylated Alginate Sub-Microspheres for Intracellular Chemotherapeutic Delivery.

Intracellular delivery vehicles comprised of methacrylated alginate (Alg-MA) were developed for the internalization and release of doxorubicin hydrochloride (DOX). Alg-MA was synthesized via an anhydrous reaction, and a mixture of Alg-MA and DOX was formed into sub-microspheres using a water/oil emulsion. Covalently cross-linked sub-microspheres were formed via exposure to green light, in order to investigate effects of cross-linking on drug release and cell internalization, compared to traditional techniques, such as ultraviolet (UV) light irradiation. Cross-linking was performed using light exposure alone or in combination with ionic cross-linking using calcium chloride (CaCl2). Alg-MA sub-microsphere diameters were between 88 and 617 nm, and ζ-potentials were between -20 and -37 mV. Using human lung epithelial carcinoma cells (A549) as a model, cellular internalization was confirmed using flow cytometry; different sub-microsphere formulations varied the efficiency of internalization, with UV-cross-linked sub-microspheres achieving the highest internalization percentages. While blank (nonloaded) Alg-MA submicrospheres were noncytotoxic to A549 cells, DOX-loaded sub-microspheres significantly reduced mitochondrial activity after 5 days of culture. Photo-cross-linked Alg-MA sub-microspheres may be a potential chemotherapeutic delivery system for cancer treatment.

Self-Healing and Thermoresponsive Dual-Cross-Linked Alginate Hydrogels Based on Supramolecular Inclusion Complexes.

ß-Cyclodextrin (ß-CD), with a lipophilic inner cavity and hydrophilic outer surface, interacts with a large variety of nonpolar guest molecules to form noncovalent inclusion complexes. Conjugation of ß-CD onto biomacromolecules can form physically cross-linked hydrogel networks upon mixing with a guest molecule. Herein, the development and characterization of self-healing, thermoresponsive hydrogels, based on host-guest inclusion complexes between alginate-graft-ß-CD and Pluronic F108 (poly(ethylene glycol)-b-poly(propylene glycol)-b-poly(ethylene glycol)), are described. The mechanics, flow characteristics, and thermal response were contingent on the polymer concentration and the host-guest molar ratio. Transient and reversible physical cross-linking between host and guest polymers governed self-assembly, allowing flow to occur under shear stress and facilitating complete recovery of the material's properties within a few seconds of unloading. The mechanical properties of the dual-cross-linked, multi-stimuli-responsive hydrogels were tuned as high as 30 kPa at body temperature and are advantageous for biomedical applications such as drug delivery and cell transplantation.

Design and Synthesis of an Artificial Pulmonary Pleura for High Throughput Studies in Acellular Human Lungs.

Whole organ decellularization of complex organs, such as lungs, presents a unique opportunity for use of acellular scaffolds for ex vivo tissue engineering or for studying cell-extracellular matrix interactions ex vivo. A growing body of literature investigating decellularizing and recellularizing rodent lungs has provided important proof of concept models and rodent lungs are readily available for high throughput studies. In contrast, comparable progress in large animal and human lungs has been impeded owing to more limited availability and difficulties in handling larger tissue. While the use of smaller segments of acellular large animal or human lungs would maximize usage from a single lung, excision of small acellular segments compromises the integrity of the pleural layer, leaving the terminal ends of blood vessels and airways exposed. We have developed a novel pleural coating using non-toxic ionically crosslinked alginate or photocrosslinked methacrylated alginate which can be applied to excised acellular lung segments, permits inflation of small segments, and significantly enhances retention of cells inoculated through cannulated airways or blood vessels. Further, photocrosslinking methacrylated alginate, using eosin Y and triethanolamine at 530 nm wavelength, results in a mechanically stable pleural coating that permits effective cyclic 3-dimensional stretch, i.e., mechanical ventilation, of individual segments.

Antimicrobial effects of nanofiber poly(caprolactone) tissue scaffolds releasing rifampicin.

This study quantified the antibiotic release kinetics and subsequent bactericidal efficacy of rifampicin (RIF) against Gram-positive and Gram-negative bacteria under in vitro static conditions. Antibiotic-loaded scaffolds were fabricated by electrospinning poly(caprolactone) (PCL) with 10% or 20% (w/w) RIF. Scaffold fiber diameter and RIF loading were characterized, and RIF release kinetics were measured. RIF-releasing and RIF-free scaffolds were inoculated with Pseudomonas aeruginosa and Staphylococcus epidermidis, and the suspended concentration live and dead bacteria were determined by fluorescent microscopy. Adherent bacteria and biofilm formation were examined using scanning electron microscopy. Mean fiber diameters were 557 ± 399 nm for RIF-free, 402 ± 225 nm for 10% RIF, and 665 ± 402 nm for 20% RIF scaffolds. RIF release kinetics exhibited a short-burst release during the first hour, followed by a 7 h, zero-order release during which both RIF scaffolds released ~50% of their initial RIF mass loading. P. aeruginosa and S. epidermidis suspended cell populations proliferated in accordance with logarithmic growth models when exposed to control scaffolds; however both RIF-containing scaffolds completely inhibited bacterial growth in suspension and, subsequently, prevented biofilm formation within the scaffolds through the first 6 h.

Synthesis and characterization of a Hyaluronan-polyethylene copolymer for biomedical applications.

Hyaluronan (HA)-based biomaterials are of interest for bone and cartilage tissue engineering because HA plays an important role in orthopedic tissue development, function, and repair. The goal of this project was to develop a biomaterial that incorporated the constituents of both a hydrogel and a hydrophobic polymer for biomedical applications. A series of amphiphilic graft copolymers consisting of HA, a glycosaminoglycan, and high-density polyethylene (HDPE), that is, HA-co-HDPE, were fabricated. The chemical characteristics, physical and viscoelastic properties, and cytocompatibility of novel HA-co-HDPE materials were characterized via Fourier Transform infrared (FTIR) spectroscopy, solid state nuclear magnetic resonance (ssNMR) spectroscopy, differential scanning calorimetry (DSC), dynamic shear testing, and an in vitro human osteoblast cell study. The esterification reaction between HA and functionalized HDPE resulted in semicrystalline, insoluble powder. The dynamic shear properties of HA-co-HDPE concentrated solutions were more like natural proteoglycans than the HA control. HA-co-HDPE was successfully compression molded into disks that swelled upon hydration. Osteoblasts were viable and expressed the osteoblast phenotype after 7 days of culture on HA-co-HDPE materials. These HA-co-HDPE materials may have several biomaterial applications in saline suspension or molded form, including orthopedic tissue repair.