Standardized User-Independent Confocal Microscopy Image Acquisition and Analysis for Thickness Measurements of Microscale Collagen Scaffolds.

The ability to accurately and precisely measure the thickness of biomaterial constructs is critical for characterizing both specific dimensional features and related mechanical properties. However, in the absence of a standardized approach for thickness measurements, a variety of imaging modalities have been employed, which have been associated with varying limits of accuracy, particularly for ultrathin hydrated structures. Electron microscopy (EM), a commonly used modality, yields thickness values for extensively processed and nonhydrated constructs, potentially resulting in overestimated mechanical properties, including elastic modulus and ultimate tensile strength. Confocal laser scanning microscopy (CLSM) has often been used as a nondestructive imaging alternative. However, published CLSM-derived image analysis protocols use arbitrary signal intensity cutoffs and provide minimal information regarding thickness variability across imaged surfaces. To address the aforementioned limitations, we present a standardized, user-independent CLSM image acquisition and analysis approach developed as a custom ImageJ macro and validated with collagen-based scaffolds. In the process, we also quantify thickness discrepancies in collagen-based scaffolds between CLSM and EM techniques, further illustrating the need for improved strategies. Employing the same image acquisition protocol, we also demonstrate that this approach can be used to estimate the surface roughness of the same scaffolds without the use of specialized instrumentation.

The Surgical Program in Innovation (SPIN): A Design and Prototyping Curriculum for Surgical Trainees.

PROBLEM: Health professions education does not routinely incorporate training in innovation or creative problem solving. Although some models of innovation education within graduate medical education exist, they often require participants' full-time commitment and removal from clinical training or rely upon participants' existing expertise. There is a need for curricula that teach innovation skills that will enable trainees to identify and solve unmet clinical challenges in everyday practice. To address this gap in surgical graduate education, the authors developed the Surgical Program in Innovation (SPIN). APPROACH: SPIN, a 6-month workshop-based curriculum, was established in 2016 in the Beth Israel Deaconess Medical Center Department of Surgery to teach surgical trainees the basics of the innovation process, focusing on surgeon-driven problem identification, product design, prototype fabrication, and initial steps in the commercialization process. Participating surgical residents and graduate students attend monthly workshops taught by medical, engineering, and medical technology (MedTech) industry faculty. Participants collaborate in teams to develop a novel device, fabricate a protype, and pitch their product to a panel of judges. OUTCOMES: From academic years 2015-2016 to 2017-2018, 49 trainees, including 41 surgical residents, participated in SPIN. Across this period, 13 teams identified an unmet need, ideated a solution, and designed and pitched a novel device. Ten teams fabricated prototypes. The 22 SPIN participants who responded to both pre- and postcourse surveys reported significant increases in confidence in generating problem statements, computer-aided design, fabrication of a prototype, and initial commercialization steps (product pitching and business planning). NEXT STEPS: Incorporating innovation education and design thinking into clinical training will prove essential in preparing future physicians to be lifelong problem finders and solvers. The authors plan to expand SPIN to additional clinical specialties, as well as to assess its impact in fostering future innovation and collaboration among program participants.

Continuous Formation of Ultrathin, Strong Collagen Sheets with Tunable Anisotropy and Compaction.

The multiscale organization of protein-based fibrillar materials is a hallmark of many organs, but the recapitulation of hierarchal structures down to fibrillar scales, which is a requirement for withstanding physiological loading forces, has been challenging. We present a microfluidic strategy for the continuous, large-scale formation of strong, handleable, free-standing, multicentimeter-wide collagen sheets of unprecedented thinness through the application of hydrodynamic focusing with the simultaneous imposition of strain. Sheets as thin as 1.9 µm displayed tensile strengths of 0.5-2.7 MPa, Young's moduli of 3-36 MPa, and modulated the diffusion of molecules as a function of collagen nanoscale structure. Smooth muscle cells cultured on engineered sheets oriented in the direction of aligned collagen fibrils and generated coordinated vasomotor responses. The described biofabrication approach enables rapid formation of ultrathin collagen sheets that withstand physiologically relevant loads for applications in tissue engineering and regenerative medicine, as well as in organ-on-chip and biohybrid devices.

Collagen and Elastin Biomaterials for the Fabrication of Engineered Living Tissues.

Collagen and elastin represent the two most predominant proteins in the body and are responsible for modulating important biological and mechanical properties. Thus, the focus of this review is the use of collagen and elastin as biomaterials for the fabrication of living tissues. Considering the importance of both biomaterials, we first propose the notion that many tissues in the human body represent a reinforced composite of collagen and elastin. In the rest of the review, collagen and elastin biosynthesis and biophysics, as well as molecular sources and biomaterial fabrication methodologies, including casting, fiber spinning, and bioprinting, are discussed. Finally, we summarize the current attempts to fabricate a subset of living tissues and, based on biochemical and biomechanical considerations, suggest that future tissue-engineering efforts consider direct incorporation of collagen and elastin biomaterials.

Targeting P-selectin glycoprotein ligand-1/P-selectin interactions as a novel therapy for metabolic syndrome.

Obesity-induced insulin resistance and metabolic syndrome continue to pose an important public health challenge worldwide as they significantly increase the risk of type 2 diabetes and atherosclerotic cardiovascular disease. Advances in the pathophysiologic understanding of this process has identified that chronic inflammation plays a pivotal role. In this regard, given that both animal models and human studies have demonstrated that the interaction of P-selectin glycoprotein ligand-1 (PSGL-1) with P-selectin is not only critical for normal immune response but also is upregulated in the setting of metabolic syndrome, PSGL-1/P-selectin interactions provide a novel target for preventing and treating resultant disease. Current approaches of interfering with PSGL-1/P-selectin interactions include targeted antibodies, recombinant immunoglobulins that competitively bind P-selectin, and synthetic molecular therapies. Experimental models as well as clinical trials assessing the role of these modalities in a variety of diseases have continued to contribute to the understanding of PSGL-1/P-selectin interactions and have demonstrated the difficulty in creating clinically relevant therapeutics. Most recently, however, computational simulations have further enhanced our understanding of the structural features of PSGL-1 and related glycomimetics, which are responsible for high-affinity selectin interactions. Leveraging these insights for the design of next generation agents has thus led to development of a promising synthetic method for generating PSGL-1 glycosulfopeptide mimetics for the treatment of metabolic syndrome.

Bioinspired Nanoparticulate Medical Glues for Minimally Invasive Tissue Repair.

Delivery of tissue glues through small-bore needles or trocars is critical for sealing holes, affixing medical devices, or attaching tissues together during minimally invasive surgeries. Inspired by the granule-packaged glue delivery system of sandcastle worms, a nanoparticulate formulation of a viscous hydrophobic light-activated adhesive based on poly(glycerol sebacate)-acrylate is developed. Negatively charged alginate is used to stabilize the nanoparticulate surface to significantly reduce its viscosity and to maximize injectability through small-bore needles. The nanoparticulate glues can be concentrated to ≈30 w/v% dispersions in water that remain localized following injection. With the trigger of a positively charged polymer (e.g., protamine), the nanoparticulate glues can quickly assemble into a viscous glue that exhibits rheological, mechanical, and adhesive properties resembling the native poly(glycerol sebacate)-acrylate based glues. This platform should be useful to enable the delivery of viscous glues to augment or replace sutures and staples during minimally invasive procedures.

A portable chemotaxis platform for short and long term analysis.

Flow-based microfluidic systems have been widely utilized for cell migration studies given their ability to generate versatile and precisely defined chemical gradients and to permit direct visualization of migrating cells. Nonetheless, the general need for bulky peripherals such as mechanical pumps and tubing and the complicated setup procedures significantly limit the widespread use of these microfluidic systems for cell migration studies. Here we present a simple method to power microfluidic devices for chemotaxis assays using the commercially available ALZET® osmotic pumps. Specifically, we developed a standalone chemotaxis platform that has the same footprint as a multiwell plate and can generate well-defined, stable chemical gradients continuously for up to 7 days. Using this platform, we validated the short-term (24 hours) and long-term (72 hours) concentration dependent PDGF-BB chemotaxis response of human bone marrow derived mesenchymal stem cells.

Tracking mesenchymal stem cells with iron oxide nanoparticle loaded poly(lactide-co-glycolide) microparticles.

Monitoring the location, distribution and long-term engraftment of administered cells is critical for demonstrating the success of a cell therapy. Among available imaging-based cell tracking tools, magnetic resonance imaging (MRI) is advantageous due to its noninvasiveness, deep penetration, and high spatial resolution. While tracking cells in preclinical models via internalized MRI contrast agents (iron oxide nanoparticles, IO-NPs) is a widely used method, IO-NPs suffer from low iron content per particle, low uptake in nonphagocytotic cell types (e.g., mesenchymal stem cells, MSCs), weak negative contrast, and decreased MRI signal due to cell proliferation and cellular exocytosis. Herein, we demonstrate that internalization of IO-NP (10 nm) loaded biodegradable poly(lactide-co-glycolide) microparticles (IO/PLGA-MPs, 0.4-3 µm) in MSCs enhances MR parameters such as the r(2) relaxivity (5-fold), residence time inside the cells (3-fold) and R(2) signal (2-fold) compared to IO-NPs alone. Intriguingly, in vitro and in vivo experiments demonstrate that internalization of IO/PLGA-MPs in MSCs does not compromise inherent cell properties such as viability, proliferation, migration and their ability to home to sites of inflammation.

Nanoparticle-based monitoring of cell therapy.

Exogenous cell therapy aims to replace/repair diseased or dysfunctional cells and promises to revolutionize medicine by restoring tissue and organ function. To develop effective cell therapy, the location, distribution and long-term persistence of transplanted cells must be evaluated. Nanoparticle (NP) based imaging technologies have the potential to track transplanted cells non-invasively. Here we summarize the most recent advances in NP-based cell tracking with emphasis on (1) the design criteria for cell tracking NPs, (2) protocols for cell labeling, (3) a comparison of available imaging modalities and their corresponding contrast agents, (4) a summary of preclinical studies on NP-based cell tracking and finally (5) perspectives and future directions.