Engineering active lysostaphin variants that incorporate noncanonical amino acids and characterizing the effects of site-specific PEGylation.

We describe a facile strategy to identify sites for the incorporation of noncanonical amino acids into lysostaphin-an enzyme that degrades the cell wall of Staphylococcus aureus-while retaining stapholytic activity. We used this strategy to generate active variants of lysostaphin incorporating para-azidophenylalanine. The incorporation of this "reactive handle" enabled the orthogonal site-specific modification of the enzyme variants with polyethylene glycol (PEG) using copper-free click cycloaddition. PEGylated lysostaphin variants could retain their stapholytic activity, with the extent of retention depending on the site of modification and the PEG molecular weight. The site-specific modification of lysostaphin could be useful not only for PEGylation to improve biocompatibility but also for the incorporation of the enzyme into hydrogels and other biomaterials and for studies of protein structure and dynamics. Moreover, the approach described herein could be readily applied to identify suitable sites for the incorporation of reactive handles into other proteins of interest.

Hydrolytic hydrogels tune mesenchymal stem cell persistence and immunomodulation for enhanced diabetic cutaneous wound healing.

Diabetes is associated with an altered global inflammatory state with impaired wound healing. Mesenchymal stem/stromal cells (MSC) are being explored for treatment of diabetic cutaneous wounds due to their regenerative properties. These cells are commonly delivered by injection, but the need to prolong the retention of MSC at sites of injury has spurred the development of biomaterial-based MSC delivery vehicles. However, controlling biomaterial degradation rates in vivo remains a therapeutic-limiting challenge. Here, we utilize hydrolytically degradable ester linkages to engineer synthetic hydrogels with tunable in vivo degradation kinetics for temporally controlled delivery of MSC. In vivo hydrogel degradation rate can be controlled by altering the ratio of ester to amide linkages in the hydrogel macromers. These hydrolytic hydrogels degrade at rates that enable unencumbered cutaneous wound healing, while enhancing the local persistence MSC compared to widely used protease-degradable hydrogels. Furthermore, hydrogel-based delivery of MSC modulates local immune responses and enhances cutaneous wound repair in diabetic mice. This study introduces a simple strategy for engineering tunable degradation modalities into synthetic biomaterials, overcoming a key barrier to their use as cell delivery vehicles.

Implantable antimicrobial biomaterials for local drug delivery in bone infection models.

Increased use of implantable biomedical devices demonstrates their potential in treating a wide variety of ailments and disorders in bone trauma and orthopaedic, reconstructive, and craniofacial applications. However, the number of cases involving implant failure or malfunction due to bacterial infection have also increased in recent years. Implanted devices can facilitate the growth of bacteria as these micro-organisms have the potential to adhere to the implant and grow and develop to form biofilms. In an effort to better understand and mitigate these occurrences, biomaterials containing antimicrobial agents that can be released or presented within the local microenvironment have become an important area of research. In this review, we discuss critical factors that regulate antimicrobial therapy to sites of bone infection, such as key biomolecular considerations and platforms for delivery, as well as current in vivo models and current advances in the field. STATEMENT OF SIGNIFICANCE: This review outlines the important factors that are taken into consideration for the development of biomaterials for local delivery of therapeutics to the site of bone infections. An overview of important criteria for development of this model (such as type of bone defect, antimicrobial therapeutic, and delivery vehicle) are provided, along with current research that utilizes these considerations. Additionally, this review highlights recent clinical trials that have utilized antimicrobial therapeutics for treatment of osteomyelitis.

Placenta-on-a-Chip: In Vitro Study of Caffeine Transport across Placental Barrier Using Liquid Chromatography Mass Spectrometry.

Due to the particular structure and functionality of the placenta, most current human placenta drug testing methods are limited to animal models, conventional cell testing, and cohort/controlled testing. Previous studies have produced inconsistent results due to physiological differences between humans and animals and limited availability of human and/or animal models for controlled testing. To overcome these challenges, a placenta-on-a-chip system is developed for studying the exchange of substances to and from the placenta. Caffeine transport across the placental barrier is studied because caffeine is a xenobiotic widely consumed on a daily basis. Since a fetus does not carry the enzymes that inactivate caffeine, when it crosses a placental barrier, high caffeine intake may harm the fetus, so it is important to quantify the rate of caffeine transport across the placenta. In this study, a caffeine concentration of 0.25 mg mL-1 is introduced into the maternal channel, and the resulting changes are observed over a span of 7.5 h. A steady caffeine concentration of 0.1513 mg mL-1 is reached on the maternal side after 6.5 h, and a 0.0033 mg mL-1 concentration on the fetal side is achieved after 5 h.

Lysostaphin and BMP-2 co-delivery reduces S. aureus infection and regenerates critical-sized segmental bone defects.

Staphylococcus aureus is the most common pathogen associated with bacterial infections in orthopedic procedures. Infections often lead to implant failure and subsequent removal, motivating the development of bifunctional materials that both promote repair and prevent failure due to infection. Lysostaphin is an anti-staphylococcal enzyme resulting in bacterial lysis and biofilm reduction. Lysostaphin use is limited by the lack of effective delivery methods to provide sustained, high doses of enzyme to infection sites. We engineered a BMP-2-loaded lysostaphin-delivering hydrogel that simultaneously prevents S. aureus infection and repairs nonhealing segmental bone defects in the murine radius. Lysostaphin-delivering hydrogels eradicated S. aureus infection and resulted in mechanically competent bone. Cytokine and immune cell profiling demonstrated that lysostaphin-delivering hydrogels restored the local inflammatory environment to that of a sterile injury. These results show that BMP-2-loaded lysostaphin-delivering hydrogel therapy effectively eliminates S. aureus infection while simultaneously regenerating functional bone resulting in defect healing.

Microfluidic Organ-on-a-Chip Technology for Advancement of Drug Development and Toxicology.

In recent years, the exploitation of phenomena surrounding microfluidics has seen an increase in popularity, as researchers have found a way to use their unique properties to create superior design alternatives. One such application is representing the properties and functions of different organs on a microscale chip for the purpose of drug testing or tissue engineering. With the introduction of "organ-on-a-chip" systems, researchers have proposed various methods on various organ-on-a-chip systems to mimic their in vivo counterparts. In this article, a systematic approach is taken to review current technologies pertaining to organ-on-a-chip systems. Design processes with attention to the particular instruments, cells, and materials used are presented.