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While matrix-assisted autologous chondrocyte implantation has emerged as a promising therapy to treat focal chondral defects, matrices that support regeneration of hyaline cartilage remain challenging. The goal of this work was to investigate the potential of a matrix metalloproteinase (MMP)-sensitive poly(ethylene glycol) (PEG) hydrogel containing the tethered growth factor, transforming growth factor β3 (TGF-β3), and compare cartilage regeneration in vitro and in vivo. The in vitro environment comprised chemically-defined medium while the in vivo environment utilized the subcutaneous implant model in athymic mice. Porcine chondrocytes were isolated and expanded in 2D culture for 10 days prior to encapsulation. The presence of tethered TGF-β3 reduced cell spreading. Chondrocyte-laden hydrogels were analyzed for total sulfated glycosaminoglycan and collagen contents, MMP activity, and spatial deposition of aggrecan, decorin, biglycan, and collagens type II and I. The total amount of extracellular matrix (ECM) deposited in the hydrogel constructs was similar in vitro and in vivo. However, the in vitro environment was not able to support long-term culture up to 64 days of the engineered cartilage leading to the eventual breakdown of aggrecan. The in vivo environment, on the other hand, led to more elaborate ECM, which correlated with higher MMP activity, and an overall higher quality of engineered tissue that was rich in aggrecan, decorin, biglycan and collagen type II with minimal collagen type I. Overall, the MMP-sensitive PEG hydrogel containing tethered TGF-β3 is a promising matrix for hyaline cartilage regeneration in vivo. Statement of Significance Regenerating hyaline cartilage remains a significant clinical challenge. The resultant repair tissue is often fibrocartilage, which long-term cannot be sustained. The goal of this study was to investigate the potential of a synthetic hydrogel matrix containing peptide crosslinks that can be degraded by enzymes secreted by encapsulated cartilage cells (i.e., chondrocytes) and tethered growth factors, specifically TGF-β3, to provide localized chondrogenic cues to the cells. This hydrogel led to hyaline cartilage-like tissue growth in vitro and in vivo, with minimal formation of fibrocartilage. However, the tissue formed in vitro, could not be maintained long-term. In vivo this hydrogel shows great promise as a potential matrix for use in regenerating hyaline cartilage.

Organophosphorus compounds (OPs), developed as pesticides and chemical warfare agents, are extremely toxic chemicals that pose a public health risk. Of the different detoxification strategies, organophosphate-hydrolyzing enzymes have attracted much attention, providing a potential route for detoxifying those exposed to OPs. Phosphotriesterase (PTE), also known as organophosphate hydrolase (OPH), is one such enzyme that has been extensively studied as a catalytic bioscavenger. In this review, we will discuss the protein engineering of PTE aimed towards improving the activity and stability of the enzyme. In order to make enzyme utilization in OP detoxification more favorable, enzyme immobilization provides an effective means to increase enzyme activity and stability. Here we present several such strategies that enhance the storage and operational stability of PTE/OPH. Organophosphorus compounds (OPs) are extremely toxic chemicals that pose a public health risk. Organophosphate-hydrolyzing enzymes are a potential route for detoxifying those exposed to OPs, and phosphotriesterase (PTE) is one such enzyme. In this review, we will discuss the protein engineering of PTE aimed towards improving the activity and stability of the enzyme. We also present several enzyme immobilization strategies that enhance the storage and operational stability of PTE.

Photopolymerizable poly(ethylene glycol) (PEG) hydrogels are a promising platform for chondrocyte encapsulation and cartilage tissue engineering. This study demonstrates that during the process of encapsulation, chondrocytes alter the formation of PEG hydrogels leading to a reduction in the bulk and local hydrogel crosslink density. Freshly isolated chondrocytes were shown to interact with hydrogel precursors, in part through thiol-mediated events between dithiol crosslinkers and cell surface free thiols, depleting crosslinker concentration and causing a reduction in the bulk hydrogel crosslink density. This effect was more pronounced with increasing cell density at the time of encapsulation. Encapsulation of chondrocytes in fluorescently labeled hydrogels exhibited a gradient in hydrogel density around the cell, which was abrogated by treatment of the cells with the antioxidant estradiol prior to encapsulation. This gradient led to spatial variations in the degradation behavior of a hydrolytically degradable PEG hydrogel, creating regions devoid of hydrogel surrounding cells. Collectively, findings from this study indicate that the antioxidant defense mechanisms in chondrocytes alter the resultant properties of PEG hydrogels formed by free-radical polymerizations. These interactions will have a significant impact on tissue engineering, affecting the local microenvironment around cells and how tissue grows within the hydrogels. STATEMENT OF SIGNIFICANCE: Cell encapsulations in synthetic hydrogels formed by free-radical polymerizations offer numerous benefits for tissue engineering. Herein, we studied cartilage cells and identified that during encapsulation, cells interfered with hydrogel formation through two distinct mechanisms. Thiol-mediated events between monomers led to monomer depletion and a lower crosslinked hydrogel. Cells' antioxidant defense mechanisms interfered with free-radicals and inhibited hydrogel formation near the cell. These cell-mediated effects led to softer hydrogels and created unique hydrogel degradations patterns causing rapid degradation around the cells. The latter has benefits for tissue engineering, where these regions provide space for tissue growth. Overall, this study demonstrates that cells play a key role in how the hydrogel structure forms when cells are present.

Proteins are some of the most versatile and studied macromolecules with extensive biomedical applications. The natural and biological origin of proteins offer such materials several advantages over their synthetic counterparts, such as innate bioactivity, recognition by cells and reduced immunogenic potential. Furthermore, proteins can be easily functionalized by altering their primary amino acid sequence and can often be further self-assembled into higher order structures either spontaneously or under specific environmental conditions. This review will feature the recent advances in protein-based biomaterials in the delivery of therapeutic cargo such as small molecules, genetic material, proteins, and cells. First, we will discuss the ways in which secondary structural motifs, the building blocks of more complex proteins, have unique properties that enable them to be useful for therapeutic delivery. Next, supramolecular assemblies, such as fibers, nanoparticles, and hydrogels, made from these building blocks that are engineered to behave in a cohesive manner, are discussed. Finally, we will cover additional modifications to protein materials that impart environmental responsiveness to materials. This includes the emerging field of protein molecular robots, and relatedly, protein-based theranostic materials that combine therapeutic potential with modern imaging modalities, including near-infrared fluorescence spectroscopy (NIRF), single-photo emission computed tomography/computed tomography (SPECT/CT), positron emission tomography (PET), magnetic resonance imaging (MRI), and ultrasound/photoacoustic imaging (US/PAI).

Enzyme-sensitive hydrogels are promising cell delivery vehicles for cartilage tissue engineering. However, a better understanding of their spatiotemporal degradation behavior and its impact on tissue growth is needed. The goal of this study was to combine experimental and computational approaches to provide new insights into spatiotemporal changes in hydrogel crosslink density and extracellular matrix (ECM) growth and how these changes influence the evolving macroscopic properties as a function of time. Hydrogels were designed from aggrecanase-sensitive peptide crosslinks using a simple and robust thiol–norbornene photoclick reaction. To study the influence of variations in cellular activity of different donors, chondrocytes were isolated from either juvenile or adult bovine donors. Initial studies were performed to validate and calibrate the model against experiments. Through this process, two key features were identified. These included spatial variations in the hydrogel crosslink density in the immediate vicinity of the cell and the presence of cell clustering within the construct. When these spatial heterogeneities were incorporated into the computational model along with model inputs of initial hydrogel properties and cellular activity (i.e., enzyme and ECM production rates), the model was able to capture the spatial and temporal evolution of ECM growth that was observed experimentally for both donors. In this study, the juvenile chondrocytes produced an interconnected matrix within the cell clusters leading to overall improved ECM growth, while the adult chondrocytes resulted in poor ECM growth. Overall, the computational model was able to capture the spatiotemporal ECM growth of two different donors and provided new insights into the importance of spatial heterogeneities in facilitating ECM growth. Our long-term goal is to use this model to predict optimal hydrogel designs for a wide range of donors and improve cartilage tissue engineering.

Concentrating on the case of poly(ethylene glycol) hydrogels, this paper introduces a methodology that enables a natural integration between the development of a so-called mechanistic model and experimental data relating material's processing to response. In a nutshell, we develop a data-driven modeling component that is able to learn and indirectly infer its own parameters and structure by observing experimental data. Using this method, we investigate the relationship between processing conditions, microstructure and chemistry (cross-link density and polymer-solvent interactions) and response (swelling and elasticity) of non-degradable and degradable PEG hydrogels. We show that the method not only enables the determination of the polymer-solvent interaction parameter, but also it predicts that this parameter, among others, varies with processing conditions and degradation. The proposed methodology therefore offers a new approach that accounts for subtle changes in the hydrogel processing.

Degradable hydrogels have been developed to provide initial mechanical support to encapsulated cells while facilitating growth of neo-tissues. When cells are encapsulated within degradable hydrogels, the process of neo-tissue growth is complicated by the coupled phenomena of transport of large extracellular matrix macromolecules and the rate of hydrogel degradation. If hydrogel degradation is too slow, neo-tissue growth is hindered, whereas if it is too fast, complete loss of mechanical integrity can occur. Therefore, there is a need for effective modelling techniques to predict hydrogel designs based on the growth parameters of the neo-tissue. In this article, hydrolytically degradable hydrogels are investigated due to their promise in tissue engineering. A key output of the model focuses on the ability of the construct to maintain overall structural integrity as the construct transitions from pure hydrogel to an engineered neo-tissue. We show that heterogeneity in cross-link density and cell distribution are key elements for this successful transition and ultimately to achieve tissue growth. Specifically, we find that optimally large regions of weak cross-linking around cells in the hydrogel and well-connected and dense cell clusters create the optimum conditions needed for neo-tissue growth while maintaining structural integrity. Experimental observations using cartilage cells encapsulated in a hydrolytically degradable hydrogel are compared with model predictions to show the potential of the proposed model.

The use of enzymes as therapeutics is very promising because of their catalytic activity and specificity. However, intracellular delivery of active enzymes is challenging due to their low stability and large size. The production of protein-enzyme nanoparticles was investigated with the goal of developing a protein carrier for active enzyme delivery. β-Galactosidase (β-gal), an enzyme whose deficiency is the cause of some lysosomal storage disorders, was incorporated into enhanced green fluorescent protein nanoparticles prepared via desolvation. Particle size was found to be sensitive to the type of cross-linker, cross-linking time, and the presence of imidazole. The results indicate that β-gal activity is highly retained (>70%) after particle fabrication and >85% of protein is incorporated in the particles. Protein-enzyme nanoparticles exhibited higher internalization in multiple cell lines in vitro, compared with the soluble enzyme. Importantly, β-gal retained its activity following intracellular delivery. These data demonstrate that protein nanoparticles are a biocompatible, high-efficiency alternative for intracellular delivery of active enzyme therapeutics.

Female broiler-type chickens were fed diets containing different concentrations of protein and fat in two experiments. The first experiment was for 4 weeks. The mean weights of the abdominal fat pads were inversely related to dietary concentrations of protein between 20 and 35%. Adipocyte size and cellularity of the fat pads were lower with 30 than 20% protein. Fat deposition in the abdominal pad was increased in 3-week-old chicks by isocaloric substitution of oil for starch in the diets. At 4 weeks of age the effect of oil was not significant with 30 or 20% dietary protein (no measurements were made in chicks fed 35 or 25% protein). In Experiment 2, the development of the abdominal fat pad in birds fed 18 to 33% protein was monitored for 22 weeks. By 7 weeks the distribution of adipocyte size was bimodal. The initial adipocyte population showed comparatively little increase in numbers after 12 weeks, but cell size continued to increase. The numbers of adippcytes in the second population of adipocytes was still increasing rapidly at 22 weeks, although the size of the cells in this population remained small. At 22 weeks the small cells constituted 62% of the total adipocyte population but contained only 2.4% of the lipid in the fat pad. Adipocyte cell size was significantly affected by dietary protein and energy until 9 weeks of age, but the effects subsequently declined.

One thousand broiler-type pullets were reared with feed restriction from 5 weeks of age to attain a target average body weight of 1.64 kg at 18 weeks of age. Thereafter feed was supplied at the rate of 130 g per bird per day until 47 weeks at which time half the population was fed ad libitum. An additional treatment was imposed at 86 weeks when 12 birds were gradually but severely restricted in feed intake until, by 97 weeks, they were receiving only 40 g feed per day. Birds restricted in feed intake until 47 weeks and then fed ad libitum increased consumption sharply and in proportion to body weight for approximately 2 weeks. Thereafter, intake declined to about 20% in excess of the restricted level for the next 16 weeks. Body weight increased with the increased feed intake. Average adipocyte diameter increased with the age of the birds even when feed was restricted. There was no increase in the number of adipocytes present in the retroperitoneal fat pad after 27 weeks of age. The size distribution of adipocytes was consistently bimodal except following severe feed restriction when the weight of the fat pad regressed and the secondary peak of small adipocytes disappeared.