Diels-Alder Photoclick Patterning of Extracellular Matrix for Spatially Controlled Cell Behaviors.

Strategies that mimic the spatial complexity of natural tissues can provide cellular scaffolds to probe fundamental questions in cell biology and offer new materials for regenerative medicine. Here, the authors demonstrate a light-guided patterning platform that uses natural engineered extracellular matrix (ECM) proteins as a substrate to program cellular behaviors. A photocaged diene which undergoes Diels-Alder-based click chemistry upon uncaging with 365 nm light is utilized. By interfacing with commercially available maleimide dienophiles, patterning of common ECM proteins (collagen, fibronectin Matrigel, laminin) with readily purchased functional small molecules and growth factors is achieved. Finally, the use of this platform to spatially control ERK activity and migration in mammalian cells is highlighted, demonstrating programmable cell behavior through patterned chemical modification of natural ECM.

Design, Synthesis, and Application of a Water-soluble Photocage for Aqueous Cyclopentadiene-based Diels-Alder Photoclick Chemistry in Hydrogels.

Spatiotemporally functionalized hydrogels have exciting applications in tissue engineering, but their preparation often relies on radical-based strategies that can be deleterious in biological settings. Herein, the computationally guided design, synthesis, and application of a water-soluble cyclopentadienone-norbornadiene (CPD-NBD) adduct is disclosed as a diene photocage for radical-free Diels-Alder photopatterning. We show that this scalable CPD-NBD derivative is readily incorporated into hydrogel formulations, providing gels that can be patterned with dienophiles upon 365 nm uncaging of cyclopentadiene. Patterning is first visualized through conjugation of cyanine dyes, then biological utility is highlighted by patterning peptides to direct cellular adhesion. Finally, the ease of use and versatility of this CPD-NBD derivative is demonstrated by direct incorporation into a commercial 3D printing resin to enable the photopatterning of structurally complex, printed hydrogels.

Implementation, results and face validity of the Consultation and Relational Empathy measure in a Canadian department of surgery.

BACKGROUND: The Consultation and Relational Empathy (CARE) Measure, a validated questionnaire designed to assess patients' perceptions of their physician's communication skills and empathy, has been used to assess empathy in medical specialties but has seldom been applied to surgery. We assessed empathy and communication skills among a group of surgeons within a single academic institution. METHODS: All surgeons within our department of surgery were invited to participate. Patients seen in clinics of participating surgeons were recruited prospectively from July 2018 to February 2019. At the end of each clinical encounter, they were asked to complete a CARE survey. Surveys were analyzed according to previously validated inclusion and exclusion criteria. We calculated mean scores for each surgeon and surgical division. About 6 months after study completion, surgeons were provided with their individual score and de-identified division scores, and were asked to complete a follow-up survey assessing their attitudes toward the CARE Measure. RESULTS: Of the 82 surgeons invited, 51 (62%) agreed to participate; 7 had fewer than 25 completed surveys and were excluded from analysis. A total of 1801 surveys for 44 surgeons (33 male and 11 female) were included in the final analysis. The average CARE score across the department was 46.9 (95% confidence interval [CI] 46.6-47.1). Female surgeons received significantly higher scores than male surgeons (mean 47.6 [95% CI 47.1-48.0] v. 46.7 [95% CI 46.4-48.0]). Of the 35 surgeons who responded to the follow-up survey, 31 (89%) felt that the questions in the CARE Measure applied to their practice, and half of these reported that they intended to make changes in response to the feedback. CONCLUSION: We found high communication and empathy scores among surgeons in the outpatient setting, with enough variability to encourage continued improvement. The CARE Measure appears to have face validity among surgeons, and the vast majority found it relevant to their practice. Further study is needed to formally assess the relevance, performance, reliability and construct validity of this measure.

Shining Light on Cyclopentadienone-Norbornadiene Diels-Alder Adducts to Enable Photoinduced Click Chemistry with Cyclopentadiene.

A new Diels-Alder (DA)-based photopatterning platform is presented, which exploits the irreversible, light-induced decarbonylation and subsequent cleavage of cyclopentadienone-norbornadiene (CPD-NBD) adducts. A series of CPD-NBD adducts have been prepared and systematically studied toward the use in a polymeric material photopatterning platform. By incorporating an optimized CPD-NBD adduct into polymer networks, it is demonstrated that cyclopentadiene may be unveiled upon 365 nm irradiation and subsequently clicked to a variety of maleimides with spatial control under mild reaction conditions and with fast kinetics. Unlike currently available photoinduced Diels-Alder reactions that rely on trapping transient, photocaged dienes, this platform introduces a persistent, yet highly reactive diene after irradiation, enabling the use of photosensitive species such as cyanine dyes to be patterned. To highlight the potential use of this platform in a variety of material applications, we demonstrate two proof-of-concepts: patterned conjugation of multiple dyes into a polyacrylate network and preprogrammed ligation of streptavidin into poly(ethylene glycol) hydrogels.

An Optogenetic Platform to Dynamically Control the Stiffness of Collagen Hydrogels.

The extracellular matrix (ECM) comprises a meshwork of biomacromolecules whose composition, architecture, and macroscopic properties, such as mechanics, instruct cell fate decisions during development and disease progression. Current methods implemented in mechanotransduction studies either fail to capture real-time mechanical dynamics or utilize synthetic polymers that lack the fibrillar nature of their natural counterparts. Here we present an optogenetic-inspired tool to construct light-responsive ECM mimetic hydrogels comprised exclusively of natural ECM proteins. Optogenetic tools offer seconds temporal resolution and submicron spatial resolution, permitting researchers to probe cell signaling dynamics with unprecedented precision. Here we demonstrated our approach of using SNAP-tag and its thiol-targeted substrate, benzylguanine-maleimide, to covalently attach blue-light-responsive proteins to collagen hydrogels. The resulting material (OptoGel), in addition to encompassing the native biological activity of collagen, stiffens upon exposure to blue light and softens in the dark. Optogels have immediate use in dissecting the cellular response to acute mechanical inputs and may also have applications in next-generation biointerfacing prosthetics.