Neel S. Joshi

Versatile click alginate hydrogels crosslinked via tetrazine–norbornene chemistry

Alginate hydrogels are well-characterized, biologically inert materials that are used in many biomedical applications for the delivery of drugs, proteins, and cells. Unfortunately, canonical covalently crosslinked alginate hydrogels are formed using chemical strategies that can be biologically harmful due to their lack of chemoselectivity. In this work we introduce tetrazine and norbornene groups to alginate polymer chains and subsequently form covalently crosslinked click alginate hydrogels capable of encapsulating cells without damaging them. The rapid, bioorthogonal, and specific click reaction is irreversible and allows for easy incorporation of cells with high post-encapsulation viability. The swelling and mechanical properties of the click alginate hydrogel can be tuned via the total polymer concentration and the stoichiometric ratio of the complementary click functional groups. The click alginate hydrogel can be modified after gelation to display cell adhesion peptides for 2D cell culture using thiol-ene chemistry. Furthermore, click alginate hydrogels are minimally inflammatory, maintain structural integrity over several months, and reject cell infiltration when injected subcutaneously in mice. Click alginate hydrogels combine the numerous benefits of alginate hydrogels with powerful bioorthogonal click chemistry for use in tissue engineering applications involving the stable encapsulation or delivery of cells or bioactive molecules.

Contrast Enhanced Computed Tomography can predict the glycosaminoglycan content and biomechanical properties of articular cartilage

OBJECTIVE An early hallmark of osteoarthritis (OA) is the progressive loss of glycosaminoglycans (GAGs), the extracellular matrix (ECM) component of articular cartilage that confers it with compressive stiffness. Our aim in this work is to establish the feasibility of using Contrast Enhanced Computed Tomography (CECT) with an anionic iodinated contrast agent - Cysto Conray II - as a minimally invasive tool to measure the changes in the GAG content as well as the compressive stiffness of articular cartilage. METHODS The GAG content of mated osteochondral plugs excised from bovine patello-femoral joints was progressively degraded using chondroitinase ABC. The mated plugs were then immersed in an anionic, tri-iodinated contrast agent, imaged using peripheral quantitative computed tomography (pQCT), subjected to an unconfined compressive stress relaxation test and the GAG content measured using 1,9-dimethylmethylene blue (DMMB) assay. Partial correlation analysis was performed to compare the variation in X-ray attenuation measured by pQCT to the variation in GAG content and in equilibrium compressive modulus. RESULTS The X-ray attenuation of cartilage exposed to an anionic, tri-iodinated, contrast agent measured by quantitative computed tomography (QCT) accounted for 83% of the variation in GAG content (r(2)=0.83, P<0.0001) and 93% of the variation in the equilibrium compressive modulus (r(2)=0.93, P<0.0001). CONCLUSION Using a mated osteochondral plug model to evaluate the biochemical composition and biomechanical properties of cartilage, this study demonstrates the interrelationships between X-ray attenuation, GAG content, and equilibrium compressive modulus, and that CECT can be used to monitor and quantify changes in the GAG content and biomechanical properties of articular cartilage.

Programmable biofilm-based materials from engineered curli nanofibres

The significant role of biofilms in pathogenicity has spurred research into preventing their formation and promoting their disruption, resulting in overlooked opportunities to develop biofilms as a synthetic biological platform for self-assembling functional materials. Here we present Biofilm-Integrated Nanofiber Display (BIND) as a strategy for the molecular programming of the bacterial extracellular matrix material by genetically appending peptide domains to the amyloid protein CsgA, the dominant proteinaceous component in Escherichia coli biofilms. These engineered CsgA fusion proteins are successfully secreted and extracellularly self-assemble into amyloid nanofibre networks that retain the functions of the displayed peptide domains. We show the use of BIND to confer diverse artificial functions to the biofilm matrix, such as nanoparticle biotemplating, substrate adhesion, covalent immobilization of proteins or a combination thereof. BIND is a versatile nanobiotechnological platform for developing robust materials with programmable functions, demonstrating the potential of utilizing biofilms as large-scale designable biomaterials.

Cationic contrast agents improve quantification of glycosaminoglycan (GAG) content by contrast enhanced CT imaging of cartilage

Minimally invasive and non‐destructive methods to quantify glycosaminoglycans (GAGs) in articular cartilage extracellular matrix are of significant interest for the biochemical analysis of cartilage and diagnosis and tracking of osteoarthritis in vivo. Here, we report the use of cationic iodinated contrast agents in comparison to conventional anionic contrast agents for the quantitative monitoring of GAG concentrations with peripheral quantitative computed tomography. Using an ex vivo bovine osteochondral plug model, the cationic contrast agents were evaluated for their ability to distribute into articular cartilage and generate a positive relationship with GAG content. The cationic agents resulted in much higher equilibrium X‐ray attenuations in cartilage extracellular matrix (ECM) than anionic agents. Experiments with samples subjected to enzymatic GAG degradation demonstrated that the cationic agents were up to five times more sensitive (p = 0.0001) to changes in GAG content and had a 24% higher correlation (p = 0.002) compared to the anionic agent (R2 = 0.86, p < 0.0001 compared with R2 = 0.62, p = 0.004). The natural inhomogeneous distribution of GAGs in the ECM could clearly be identified in undegraded samples.

Applications of dendrimers in tissue engineering.

Despite the wide-spread use of dendrimers in biomedical applications, their use in the fabrication of tissue engineering scaffolds has been limited. The highly branched, multivalent nature of dendrimers makes them ideal candidates for a variety of tissue engineering applications, including as crosslinking agents, modulators of surface charge and surface chemistry, and as primary components in scaffolds that mimic natural extracellular matrices. Compared to linear polymers, the multiple end groups of dendrimers may potentially offer more control over factors such as cell proliferation rates and biodegradation profiles through systematic variation of generation size, concentration, and end group chemistry. The combination of dendrimers and other traditional scaffold polymers, such as proteins, carbohydrates, and linear synthetic polymers has led to the creation of hybrid scaffolds with new physical, mechanical, and biochemical properties. This review describes examples where dendritic macromolecules have been incorporated into scaffolds for the regeneration of a variety of tissues and cell types and highlights areas where dendrimers have yet to be utilized.

Hydrogels for Osteochondral Repair Based on Photocrosslinkable Carbamate Dendrimers

First generation, photocrosslinkable dendrimers consisting of natural metabolites (i.e., succinic acid, glycerol, and beta-alanine) and nonimmunogenic poly(ethylene glycol) (PEG) were synthesized divergently in high yields using ester and carbamate forming reactions. Aqueous solutions of these dendrimers were photocrosslinked with an eosin-based photoinitiator to afford hydrogels. The hydrogels displayed a range of mechanical properties based on their structure, generation size, and concentration in solution. All of the hydrogels showed minimal swelling characteristics. The dendrimer solutions were then photocrosslinked in situ in an ex vivo rabbit osteochondral defect (3 mm diameter and 10 mm depth), and the resulting hydrogels were subjected to physiologically relevant dynamic loads. Magnetic resonance imaging (MRI) showed the hydrogels to be fixated in the defect site after the repetitive loading regimen. The ([G1]-PGLBA-MA) 2-PEG hydrogel was chosen for the 6 month pilot in vivo rabbit study because this hydrogel scaffold could be prepared at low polymer weight (10 wt %) and possessed the largest compressive modulus of the 10% formulations, a low swelling ratio, and contained carbamate linkages, which are more hydrolytically stable than the ester linkages. The hydrogel-treated osteochondral defects showed good attachment in the defect site and histological analysis showed the presence of collagen II and glycosaminoglycans (GAGs) in the treated defects. By contrast, the contralateral unfilled defects showed poor healing and negligible GAG or collagen II production. Good mechanical properties, low swelling, good attachment to the defect site, and positive in vivo results illustrate the potential of these dendrimer-based hydrogels as scaffolds for osteochondral defect repair.

Effect of Contrast Agent Charge on Visualization of Articular Cartilage Using Computed Tomography: Exploiting Electrostatic Interactions for Improved Sensitivity

The synthesis and evaluation of a new class of cationic iodinated contrast agents for the imaging of cartilage using computed tomography (CT) are described. In direct comparisons with anionic contrast agents, the cationic contrast agents afforded higher equilibrium concentrations in the articular cartilage of ex vivo rabbit femurs and thus greater imaging sensitivity. Variations in CT intensity across the sample reflected the inhomogeneous distribution of glycosaminoglycans in the tissue as confirmed by histological analysis. We anticipate that this work represents the first step in the development of sensitive, nondestructive CT-based methods to characterize the biochemical properties of cartilage using cationic contrast agents.

Portable, On-Demand Biomolecular Manufacturing

Synthetic biology uses living cells as molecular foundries for the biosynthesis of drugs, therapeutic proteins, and other commodities. However, the need for specialized equipment and refrigeration for production and distribution poses a challenge for the delivery of these technologies to the field and to low-resource areas. Here, we present a portable platform that provides the means for on-site, on-demand manufacturing of therapeutics and biomolecules. This flexible system is based on reaction pellets composed of freeze-dried, cell-free transcription and translation machinery, which can be easily hydrated and utilized for biosynthesis through the addition of DNA encoding the desired output. We demonstrate this approach with the manufacture and functional validation of antimicrobial peptides and vaccines and present combinatorial methods for the production of antibody conjugates and small molecules. This synthetic biology platform resolves important practical limitations in the production and distribution of therapeutics and molecular tools, both to the developed and developing world.

Characterization of a Three-Component Coupling Reaction on Proteins by Isotopic Labeling and Nuclear Magnetic Resonance Spectroscopy

A three-component Mannich-type electrophilic aromatic substitution reaction was previously developed to target the phenolic side chain of tyrosine residues on proteins. This reaction proceeds under mild conditions and provides a convenient alternative to lysine-targeting strategies. However, the use of reactive aldehydes, such as formaldehyde, warrants careful inspection of the reaction products to ensure that other modifications have not occurred. Through the use of isotopically enriched reagents, nuclear magnetic resonance (NMR)-based studies were used to obtain structural confirmation of the tyrosine-modification products. These experiments also revealed the formation of a reaction byproduct arising from the indole ring of tryptophan residues. Cysteine residues were shown to not participate in the reaction, except in the case of a reduced disulfide, which formed a dithioacetal. We anticipate that this analysis method will prove useful for the detailed study of a number of bioconjugation reactions.

Engineered catalytic biofilms: Site-specific enzyme immobilization onto E. coli curli nanofibers

Biocatalytic transformations generally rely on purified enzymes or whole cells to perform complex transformations that are used on industrial scale for chemical, drug, and biofuel synthesis, pesticide decontamination, and water purification. However, both of these systems have inherent disadvantages related to the costs associated with enzyme purification, the long‐term stability of immobilized enzymes, catalyst recovery, and compatibility with harsh reaction conditions. We developed a novel strategy for producing rationally designed biocatalytic surfaces based on Biofilm Integrated Nanofiber Display (BIND), which exploits the curli system of E. coli to create a functional nanofiber network capable of covalent immobilization of enzymes. This approach is attractive because it is scalable, represents a modular strategy for site‐specific enzyme immobilization, and has the potential to stabilize enzymes under denaturing environmental conditions. We site‐specifically immobilized a recombinant α‐amylase, fused to the SpyCatcher attachment domain, onto E. coli curli fibers displaying complementary SpyTag capture domains. We characterized the effectiveness of this immobilization technique on the biofilms and tested the stability of immobilized α‐amylase in unfavorable conditions. This enzyme‐modified biofilm maintained its activity when exposed to a wide range of pH and organic solvent conditions. In contrast to other biofilm‐based catalysts, which rely on high cellular metabolism, the modified curli‐based biofilm remained active even after cell death due to organic solvent exposure. This work lays the foundation for a new and versatile method of using the extracellular polymeric matrix of E. coli for creating novel biocatalytic surfaces. Biotechnol. Bioeng. 2015;112: 2016–2024.