Aspartate 1-decarboxylase (ADC) and 3,4-dihydroxyphenylalanine decarboxylase (DDC) provide ␤-alanine and dopamine used in insect cuticle tanning. ␤-Alanine is conjugated with dopamine to yield N-␤-alanyldopamine (NBAD), a substrate for the phenol oxidase laccase that catalyzes the synthesis of cuticle protein cross-linking agents and pigment precursors. We identified ADC and DDC genes in the red flour beetle, Tribolium castaneum (Tc), and investigated their functions. TcADC mRNA was most abundant prior to the pupal-adult molt. Injection of TcADC double-stranded (ds) RNA (dsTcADC) into mature larvae resulted in depletion of NBAD in pharate adults, accumulation of dopamine, and abnormally dark pigmentation of the adult cuticle. Injection of ␤-alanine, the expected product of ADC, into dsTcADC-treated pupae rescued the pigmentation phenotype, resulting in normal rust-red color. A similar pattern of catechol content consisting of elevated dopamine and depressed NBAD was observed in the genetic black mutants of Tribolium, in which levels of TcADC mRNA were drastically reduced. Furthermore, from the Tribolium black mutant and dsTcADC-injected insects both exhibited similar changes in material properties. Dynamic mechanical analysis of elytral cuticle from beetles with depleted TcADC transcripts revealed diminished cross-linking of cuticular components, further confirming the important role of oxidation products of NBAD as cross-linking agents during cuticle tanning. Injection of dsTcDDC into larvae produced a lethal pupal phenotype, and the resulting grayish pupal cuticle exhibited many small patches of black pigmentation. When dsTcDDC was injected into young pupae, the resulting adults had abnormally dark brown body color, but there was little mortality. Injection of dsTcDDC resulted in more than a 5-fold increase in levels of DOPA, indicating that lack of TcDDC led to accumulation of its substrate, DOPA.Insect cuticle tanning (sclerotization and pigmentation) is a complex process that involves the oxidative conjugation and cross-linking of cuticular proteins by quinones, which renders the proteins insoluble and also hardens and darkens the exoskeleton (1). The quinones are derived from catecholic metabolites of the amino acid tyrosine, produced by a series of enzymatic modifications, including hydroxylation, decarboxylation, N-acyl transfer, and oxidation ( Fig. 1) (2). During tanning, cross-links form between adjacent polypeptide chains, causing progressive hardening, dehydration, and close packing of the polymers. This cross-linking occurs as a result of reactions of quinones and quinone methides derived from N-acylcatecholamines with nucleophilic side chain groups of structural proteins, leading to changes in mechanical properties and coloration (3). The major catechols used by insects as cuticle tanning agent precursors are dopamine, N-␤-alanyldopamine (NBAD), 2 and N-acetyldopamine (NADA) (1). Different types of quinones and quinone methides produced from catecholic metabolites may contribute to the variety of ...

ECM-based materials are appealing for tissue engineering strategies because they may promote stem cell recruitment, cell infiltration, and cell differentiation without the need to supplement with additional biological factors. Cartilage ECM has recently shown potential to be chondroinductive, particularly in a hydrogel-based system, which may be revolutionary in orthopedic medicine. However, hydrogels composed of natural materials are often mechanically inferior to synthetic materials, which is a major limitation for load-bearing tissue applications. The objective was therefore to create an unprecedented hydrogel derived entirely from native cartilage ECM that was both mechanically more similar to native cartilage tissue and capable of inducing chondrogenesis. Porcine cartilage was decellularized, solubilized, and then methacrylated and UV photocrosslinked to create methacrylated solubilized decellularized cartilage (MeSDCC) gels. Methacrylated gelatin (GelMA) was employed as a control for both biomechanics and bioactivity. Rat bone marrow-derived mesenchymal stem cells were encapsulated in these networks, which were cultured in vitro for 6 weeks, where chondrogenic gene expression, the compressive modulus, swelling, and histology were analyzed. One day after crosslinking, the elastic compressive modulus of the 20% MeSDCC gels was 1070 ± 150 kPa. Most notably, the stress strain profile of the 20% MeSDCC gels fell within the 95% confidence interval range of native porcine cartilage. Additionally, MeSDCC gels significantly upregulated chondrogenic genes compared to GelMA as early as day 1 and supported extensive matrix synthesis as observed histologically. Given that these gels approached the mechanics of native cartilage tissue, supported matrix synthesis, and induced chondrogenic gene expression, MeSDCC hydrogels may be promising materials for cartilage tissue engineering applications. Future efforts will focus on improving fracture mechanics as well to benefit overall biomechanical performance.

A new method for encapsulating cells in interpenetrating network (IPN) hydrogels of superior mechanical integrity was developed. In this study, two biocompatible materials-agarose and poly(ethylene glycol) (PEG) diacrylate-were combined to create a new IPN hydrogel with greatly enhanced mechanical performance. Unconfined compression of hydrogel samples revealed that the IPN displayed a fourfold increase in shear modulus relative to a pure PEG-diacrylate network (39.9 vs. 9.9 kPa) and a 4.9-fold increase relative to a pure agarose network (8.2 kPa). PEG and IPN compressive failure strains were found to be 71% ± 17% and 74% ± 17%, respectively, while pure agarose gels failed around 15% strain. Similar mechanical property improvements were seen when IPNs-encapsulated chondrocytes, and LIVE/DEAD cell viability assays demonstrated that cells survived the IPN encapsulation process. The majority of IPN-encapsulated chondrocytes remained viable 1 week postencapsulation, and chondrocytes exhibited glycosaminoglycan synthesis comparable to that of agarose-encapsulated chondrocytes at 3 weeks postencapsulation. The introduction of a new method for encapsulating cells in a hydrogel with enhanced mechanical performance is a promising step toward cartilage defect repair. This method can be applied to fabricate a broad variety of cell-based IPNs by varying monomers and polymers in type and concentration and by adding functional groups such as degradable sequences or cell adhesion groups. Further, this technology may be applicable in other cell-based applications where mechanical integrity of cell-containing hydrogels is of great importance.

SYNOPSISThe equilibrium and dynamic swelling behavior of glassy polymers immersed in solvents can be modified by controlling the history of the polymer sample, which includes prior swelling and the drying method, or by copolymerization with other monomers. In this paper, the swelling kinetics in water of ionic hydrogels of 2-hydroxyethyl methacrylate copolymerized with potassium 3-sulfopropylmethacrylate and/or ethylene glycol dimethacrylate have been studied at 23°C. The dimensional changes of a swelling polymer sheet can be controlled through incorporation of anisotropic stresses in the initially dry, glassy polymer. These anisotropic stresses do not affect the swelling kinetics as long as the sample is partially glassy. However, differences in the initial stresses cause sharply different swelling kinetics once the polymer becomes entirely rubbery, due to differences in dimensional changes. Increasing the percentage of ionic comonomer in the polymer increases the equilibrium degree of swelling and the water sorption rate without changing the time for equilibration or the swelling transport mechanism. In contrast, increasing the percentage of cross-linker in the polymer not only reduces the degree of swelling and water sorption rate, but also increases the equilibration time and shifts the water transport mechanism from Fickian diffusion to anomalous transport. I NTRO DU CTlO NThe equilibrium and dynamic swelling behavior of poly ( 2-hydroxyethyl methacrylate) (PHEMA) hydrogels has been studied by many researchers because of PHEMA's importance in biomedical applications.'-6 Control of these properties is particularly important for polymer-mediated drug delivery devices and hydrogel absorbents. The kinetic response depends both upon the history of a given gel sample and its chemical composition. Although the properties of glassy polymers are well known to be history-dependent, the effect of history on swelling kinetics has been little studied. Franson and Peppas found that repeated cycles of swelling and drying caused the degree of swelling to increase with each cycle without affecting the swelling rate or transport mechanism.2 Modification of the swelling behavior

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A tough and ductile, ultrathin film, double-network (DN), biopolymer-based hydrogel displaying the yielding phenomenon was synthesized from methacrylated chondroitin sulfate (MCS) and polyacrylamide (PAAm). The DN of MCS/PAAm exhibited a failure stress more than 20 times greater than the single network (SN) of either MCS or PAAm and exhibited yielding stresses over 1500 kPa. In addition, the stress–strain behavior with a yielding region was also seen in a hydrogel of MCS and poly(N,N-dimethyl acrylamide) (PDMAAm). By replacing PAAm with PDMAAm, interactions known to toughen networks are removed. This demonstration supports the idea that the brittle/ductile combination is key to the DN effect over specific interactions between the networks. The MCS/PAAm and MCS/PDMAAm DN hydrogels had comparable mechanical properties to the archtypal DN hydrogels of poly(2-acrylamido-2-methylpropanesulfonic acid) (PAMPS)/PAAm. In addition, these tough and ductile, biopolymer-based, double-network hydrogels demonstrated a substantial yielding region.

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Hydrogels-water swollen cross-linked networks-have demonstrated considerable promise in tissue engineering and regenerative medicine applications. However, ambiguity over which rheological properties are needed to characterize these gels before crosslinking still exists. Most hydrogel research focuses on the performance of the hydrogel construct after implantation, but for clinical practice, and for related applications such as bioinks for 3D bioprinting, the behavior of the pre-gelled state is also critical. Therefore, the goal of this review is to emphasize the need for better rheological characterization of hydrogel precursor formulations, and standardized testing for surgical placement or 3D bioprinting. In particular, we consider engineering paste or putty precursor solutions (i.e., suspensions with a yield stress), and distinguish between these differences to ease the path to clinical translation. The connection between rheology and surgical application as well as how the use of paste and putty nomenclature can help to qualitatively identify material properties are explained. Quantitative rheological properties for defining materials as either pastes or putties are proposed to enable easier adoption to current methods. Specifically, the three-parameter Herschel-Bulkley model is proposed as a suitable model to correlate experimental data and provide a basis for meaningful comparison between different materials. This model combines a yield stress, the critical parameter distinguishing solutions from pastes (100-2000 Pa) and from putties (>2000 Pa), with power law fluid behavior once the yield stress is exceeded. Overall, successful implementation of paste or putty handling properties to the hydrogel precursor may minimize the surgeon-technology learning time and ultimately ease