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1. Supercharging Carbohydrate Binding Module Alone Enhances Endocellulase Thermostability, Binding, and Activity on Cellulosic Biomass

   https://doi.org/10.1021/acssuschemeng.3c06266  

   DeChellis, Antonio; Nemmaru, Bhargava; Sammond, Deanne; Douglass, Jenna; Patil, Nivedita; Reste, Olivia; Chundawat, Shishir_P S (March 2024, ACS Sustainable Chemistry & Engineering)

   Lignocellulosic biomass recalcitrance to enzymatic degradation necessitates high enzyme loadings, incurring large processing costs for the production of industrial-scale biofuels or biochemicals. Manipulating surface charge interactions to minimize nonproductive interactions between cellulolytic enzymes and plant cell wall components (e.g., lignin or cellulose) via protein supercharging has been hypothesized to improve biomass biodegradability but with limited demonstrated success to date. Here, we characterize the effect of introducing non-natural enzyme surface mutations and net charge on cellulosic biomass hydrolysis activity by designing a library of supercharged family-5 endoglucanase Cel5A and its native family-2a carbohydrate binding module (CBM) originally belonging to an industrially relevant thermophilic microbe, Thermobifida fusca. A combinatorial library of 33 mutant constructs containing different CBM and Cel5A designs spanning a net charge range of −52 to 37 was computationally designed using Rosetta macromolecular modeling software. Activity for all mutants was rapidly characterized as soluble cell lysates, and promising mutants (containing mutations on the CBM, Cel5A catalytic domain, or both CBM and Cel5A domains) were then purified and systematically characterized. Surprisingly, often endocellulases with mutations on the CBM domain alone resulted in improved activity on cellulosic biomass, with three top-performing supercharged CBM mutants exhibiting between 2- and 5-fold increase in activity, compared to native enzyme, on both pretreated biomass enriched in lignin (i.e., corn stover) and isolated crystalline/amorphous cellulose. Furthermore, we were able to clearly demonstrate that endocellulase net charge can be selectively fine-tuned using a protein supercharging protocol for targeting distinct substrates and maximizing biocatalytic activity. Additionally, several supercharged CBM-containing endocellulases exhibited a 5–10 °C increase in optimal hydrolysis temperature, compared to native enzyme, which enabled further increase in hydrolytic yield at higher operational reaction temperatures. This study demonstrates the first successful implementation of enzyme supercharging of cellulolytic enzymes to increase hydrolytic activity toward complex lignocellulosic biomass-derived substrates. 

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2. 3D printing of drug-eluting bioactive multifunctional coatings for orthopedic applications

   https://doi.org/10.18063/ijb.v9i2.661  

   Adarkwa, E.; Roy, A.; Ohodnicki, J.; Lee, B.; Kumta, P. N.; Desai, S. (January 2023, International journal of bioprinting)

   Three-dimensional (3D) printing is implemented for surface modification of titanium alloy substrates with multilayered biofunctional polymeric coatings. Poly(lactic-co- glycolic) acid (PLGA) and polycaprolactone (PCL) polymers were embedded with amorphous calcium phosphate (ACP) and vancomycin (VA) therapeutic agents to promote osseointegration and antibacterial activity, respectively. PCL coatings revealed a uniform deposition pattern of the ACP-laden formulation and enhanced cell adhesion on the titanium alloy substrates as compared to the PLGA coatings. Scanning electron microscopy and Fourier-transform infrared spectroscopy confirmed a nanocomposite structure of ACP particles showing strong binding with the polymers. Cell viability data showed comparable MC3T3 osteoblast proliferation on polymeric coatings as equivalent to positive controls. In vitro live/dead assessment indicated higher cell attachments for 10 layers (burst release of ACP) as compared to 20 layers (steady release) for PCL coatings. The PCL coatings loaded with the antibacterial drug VA displayed a tunable release kinetics profile based on the multilayered design and drug content of the coatings. Moreover, the concentration of active VA released from the coatings was above the minimum inhibitory concentration and minimum bactericidal concentration, demonstrating its effectiveness against Staphylococcus aureus bacterial strain. This research provides a basis for developing antibacterial biocompatible coatings to promote osseointegration of orthopedic implants. 

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3. Impact of Mixing on Content Uniformity of Thin Polymer Films Containing Drug Micro-Doses

   https://doi.org/10.3390/pharmaceutics13060812  

   Buyukgoz, Guluzar G.; Castro, Jeremiah N.; Atalla, Andrew E.; Pentangelo, John G.; Tripathi, Siddharth; Davé, Rajesh N. (June 2021, Pharmaceutics)

   null (Ed.)

   The impact of mixer type and critical process parameters (CPPs) on critical quality attributes (CQAs), including the drug content uniformity (CU) of slurry-cast polymer films loaded with micro-sized poorly water-soluble drugs were investigated. Previously untested hypothesis was that the best mixer at suitable CPPs promotes uniform drug dispersion within film precursors leading to acceptable dried-film CU at low, ~0.6 wt% drug concentrations. Taguchi design was utilized to select the best of three mixers; low-shear impeller, high-shear planetary, and high-intensity vibrational, for dried-film drug concentration of ~23 wt%. As-received fenofibrate, a model poorly water-soluble drug (~6 µm) was directly mixed with the hydroxypropyl methylcellulose (HPMC) and glycerin aqueous solution. Impeller and planetary mixers yielded desirable film relative standard deviation (RSD), while vibrational mixer could not. For the lowest dried-film drug concentration of ~0.6 wt%, only planetary mixer yielded RSD <6%. The precursor drug homogeneity was a sufficient but not a necessary condition for achieving dried-film RSD <6%. Thus, proper selection of mixer and its CPPs assured desirable film CQAs. However, minor drug particle aggregation was identified via re-dispersion testing which also led to incomplete drug release. 

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4. Microfluidic chip-based co-culture system for modeling human joint inflammation in osteoarthritis research

   https://doi.org/10.3389/fphar.2025.1579228  

   Mirazi, Hosein; Wood, Scott T (April 2025, Frontiers in Pharmacology)

   Here we present a microfluidic model that allows for co-culture of human osteoblasts, chondrocytes, fibroblasts, and macrophages of both quiescent (M0) and pro-inflammatory (M1) phenotypes, maintaining initial viability of each cell type at 24 h of co-culture. We established healthy (M0-based) and diseased (M1-based) joint models within this system. An established disease model based on supplementation of IFN-γ and lipopolysaccharide in cell culture media was used to induce an M1 phenotype in macrophages to recapitulate inflammatory conditions found in Osteoarthritis. Cell viability was assessed using NucBlue™ Live and NucGreen™ Dead fluorescent stains, with mean viability of 83.9% ± 14% and 83.3% ± 12% for healthy and diseased models, respectively, compared with 93.3% ± 4% for cell in standard monoculture conditions. Cytotoxicity was assessed via a lactate dehydrogenase (LDH) assay and showed no measurable increase in lactate dehydrogenase release into the culture medium under co-culture conditions, indicating that neither model promotes a loss of cell membrane integrity due to cytotoxic effects. Cellular metabolic activity was assessed using a PrestoBlue™ assay and indicated increased cellular metabolic activity in co-culture, with levels 5.9 ± 3.2 times mean monolayer cell metabolic activity levels in the healthy joint model and 5.3 ± 3.4 times mean monolayer levels in the diseased model. Overall, these findings indicate that the multi-tissue nature ofin vivohuman joint conditions can be recapitulated by our microfluidic co-culture system at 24 h and thus this model serves as a promising tool for studying the pathophysiology of rheumatic diseases and testing potential therapeutics. 

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5. Tuning the Topography of Dynamic 3D Scaffolds through Functional Protein Wrinkled Coatings

   https://doi.org/10.3390/polym16050609  

   Oguntade, Elizabeth; Fougnier, Daniel; Meyer, Sadie; O’Grady, Kerrin; Kudlack, Autumn; Henderson, James H (March 2024, Polymers)

   Surface wrinkling provides an approach to fabricate micron and sub-micron-level biomaterial topographies that can mimic features of the dynamic, in vivo cell environment and guide cell adhesion, alignment, and differentiation. Most wrinkling research to date has used planar, two-dimensional (2D) substrates, and wrinkling work on three-dimensional (3D) structures has been limited. To enable wrinkle formation on architecturally complex, biomimetic 3D structures, here, we report a simple, low-cost experimental wrinkling approach that combines natural silk fibroin films with a recently developed advanced manufacturing technique for programming strain in complex 3D shape–memory polymer (SMP) scaffolds. By systematically investigating the influence of SMP programmed strain magnitude, silk film thickness, and aqueous media on wrinkle morphology and stability, we reveal how to generate and tune silk wrinkles on the micron and sub-micron scale. We find that increasing SMP programmed strain magnitude increases wavelength and decreases amplitudes of silk wrinkled topographies, while increasing silk film thickness increases wavelength and amplitude. Silk wrinkles persist after 24 h in cell culture medium. Wrinkled topographies demonstrate high cell viability and attachment. These findings suggest the potential for fabricating biomimetic cellular microenvironments that can advance understanding and control of cell–material interactions in engineering tissue constructs. 

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