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.

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.

Effects of cryo-processing on the mechanical and biological properties of poly(vinyl alcohol)-gelatin theta-gels.

Poly(vinyl alcohol) (PVA), a synthetic, nontoxic polymer, is widely studied for use as a biomedical hydrogel due to its structural and physicomechanical properties. Depending on the synthesis method, PVA hydrogels can exhibit a range of selected characteristics-strength, creep resistance, energy dissipation, degree of crystallinity, and porosity. While the structural integrity and behavior of the hydrogel can be fine-tuned, common processing techniques result in a brittle, linear elastic material. In addition, PVA lacks functionality to engage and participate in cell adhesion, which can be a limitation for integrating PVA materials with tissue in situ. Thus, there is a need to further engineer PVA hydrogels to optimize its physicomechanical properties while enhancing cell adhesion and bioactivity. While the inclusion of gelatin into PVA hydrogels has been shown to impart cell-adhesive properties, the optimization of the mechanical properties of PVA-gelatin blends has not been studied in the context of traditional PVA hydrogel processing techniques. The incorporation of poly(ethylene glycol) with PVA prior to solidification forms an organized, cell instructive hydrogel with improved stiffness. The effect of cryo-processing, i.e., freeze-thaw (FT) cycling was elucidated by comparing 1 FT and 8 FT theta-cryo-gels and cryo-gels. To confirm the viability of the gels, human mesenchymal stem cell (hMSC) protein and sulfated glycosaminoglycan assays were performed to verify the nontoxicity and influence on hMSC differentiation. We have devised an elastic PVA-gelatin hydrogel utilizing the theta-gel and cryo-gel processing techniques, resulting in a stronger, more elastic material with greater potential as a scaffold for complex tissues.

PVA-gelatin hydrogels formed using combined theta-gel and cryo-gel fabrication techniques.

Poly(vinyl alcohol) (PVA) is a synthetic, biocompatible polymer that has been widely studied for use in bioengineered tissue scaffolds due to its relatively high strength, creep resistance, water retention, and porous structure. However, PVA hydrogels traditionally exhibit low percent elongation and energy dissipation. PVA material and mechanical properties can be fine-tuned by controlling the physical, non-covalent crosslinks during hydrogel formation through various techniques; PVA scaffolds were modified with gelatin, a natural collagen derivative also capable of forming reversible hydrogen bonds. Blending in gelatin and poly(ethylene glycol) (PEG) with PVA prior to solidification formed a highly organized hydrogel with improved toughness and dynamic elasticity. Theta-gels were formed from the solidification of warm solutions and the phase separation of high molecular weight gelatin and PVA from a low molecular PEG porogen upon cooling. While PVA-gelatin hydrogels can be synthesized in this manner, the hydrogels exhibited low toughness with increased elasticity. Thus, theta-gels were additionally processed using cryo-gel fabrication techniques, which involved freezing theta-gels, lyophilizing and re-hydrating. The result was a stronger, more resilient material. We hypothesized that the increased formation of physical hydrogen bonds between the PVA and gelatin allowed for the combination of a stiffer material with energy dissipation characteristics. Rheological data suggested significant changes in the storage moduli of the new PVA-gelatin theta-cryo-gels. Elastic modulus, strain to failure, hysteresis and resilience were studied through uniaxial tension and dynamic mechanical analysis in compression.

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.

Dynamic mechanical response of polyvinyl alcohol-gelatin theta-gels for nucleus pulposus tissue replacement.

Intervertebral disk degeneration is one of the most significant contributors to low back pain. Thus, there is significant interest in designing new treatments and nucleus pulposus (NP) tissue replacements. Herein, the authors propose a biosynthetic material, comprised of a polyvinyl alcohol (PVA) and gelatin theta-gel, as an acellular NP tissue replacement. Theta-gels form during the solidification of PVA and gelatin (phase I), and the phase separation of a disklike short-chain polyethylene glycol (PEG, phase II). The PVA concentration and weight ratio of PVA to PEG were optimized, in order to achieve mechanical properties resembling NP tissue. Mechanical and material properties were analyzed for the PVA-gelatin theta-gels under static and dynamic conditions. Cyclic stress-strain testing demonstrated the theta-gels' ability to relax and perform properly under dynamic loading. Altering the molecular weight and concentration of the theta-gel constituents allows for a tunable material that can match a variety of native tissue properties.

Mechanical properties and failure analysis of visible light crosslinked alginate-based tissue sealants.

Moderate to weak mechanical properties limit the use of naturally-derived tissue sealants for dynamic medical applications, e.g., sealing a lung leak. To overcome these limitations, we developed visible-light crosslinked alginate-based hydrogels, as either non-adhesive methacrylated alginate (Alg-MA) hydrogel controls, or oxidized Alg-MA (Alg-MA-Ox) tissue adhesive tissue sealants, which form covalent bonds with extracellular matrix (ECM) proteins. Our study investigated the potential for visible-light crosslinked Alg-MA-Ox hydrogels to serve as effective surgical tissue sealants for dynamic in vivo systems. The Alg-MA-Ox hydrogels were designed to be an injectable system, curable in situ. Burst pressure experiments were conducted on a custom-fabricated burst pressure device using constant air flow; burst pressure properties and adhesion characteristics correlated with the degrees of methacrylation and oxidation. In summary, visible light crosslinked Alg-MA-Ox hydrogel tissue sealants form effective seals over critically-sized defects, and maintain pressures up to 50mm Hg.

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.