Fund Black scientists.

Our nationwide network of BME women faculty collectively argue that racial funding disparity by the National Institutes of Health (NIH) remains the most insidious barrier to success of Black faculty in our profession. We thus refocus attention on this critical barrier and suggest solutions on how it can be dismantled.

Mechanisms of aortic carboxypeptidase-like protein secretion and identification of an intracellularly retained variant associated with Ehlers-Danlos syndrome.

Aortic carboxypeptidase-like protein (ACLP) is a collagen-binding extracellular matrix protein that has important roles in wound healing and fibrosis. ACLP contains thrombospondin repeats, a collagen-binding discoidin domain, and a catalytically inactive metallocarboxypeptidase domain. Recently, mutations in the ACLP-encoding gene, AE-binding protein 1 (AEBP1), have been discovered, leading to the identification of a new variant of Ehlers-Danlos syndrome causing connective tissue disruptions in multiple organs. Currently, little is known about the mechanisms of ACLP secretion or the role of post-translational modifications in these processes. We show here that the secreted form of ACLP contains N-linked glycosylation and that inhibition of glycosylation results in its intracellular retention. Using site-directed mutagenesis, we determined that glycosylation of Asn-471 and Asn-1030 is necessary for ACLP secretion and identified a specific N-terminal proteolytic ACLP fragment. To determine the contribution of secreted ACLP to extracellular matrix mechanical properties, we generated and mechanically tested wet-spun collagen ACLP composite fibers, finding that ACLP enhances the modulus (or stiffness), toughness, and tensile strength of the fibers. Some AEBP1 mutations were null alleles, whereas others resulted in expressed proteins. We tested the hypothesis that a recently discovered 40-amino acid mutation and insertion in the ACLP discoidin domain regulates collagen binding and assembly. Interestingly, we found that this protein variant is retained intracellularly and induces endoplasmic reticulum stress identified with an XBP1-based endoplasmic reticulum stress reporter. Our findings highlight the importance of N-linked glycosylation of ACLP for its secretion and contribute to our understanding of ACLP-dependent disease pathologies.

On Force and Form: Mechano-Biochemical Regulation of Extracellular Matrix.

The extracellular matrix is well-known for its structural role in supporting cells and tissues, and its important biochemical role in providing signals to cells has increasingly become apparent. These structural and biochemical roles are closely coupled through mechanical forces: the biochemistry of the extracellular matrix determines its mechanical properties, mechanical forces control release or display of biochemical signals from the extracellular matrix, and the mechanical properties of the matrix in turn influence the mechanical set point at which signals are sent. In this Perspective, we explain how the extracellular matrix is regulated by strain and mechanical forces. We show the impact of biochemistry and mechanical forces on in vivo assembly of extracellular matrix and illustrate how matrix can be generated in vitro using a variety of methods. We cover how the matrix can be characterized in terms of mechanics, composition, and conformation to determine its properties and to predict interactions. Finally, we explore how extracellular matrix remodeling, ligand binding, and hemostasis are regulated by mechanical forces. These recently discovered mechano-biochemical interactions have important functions in wound healing and disease progression. It is likely that mechanically altered extracellular matrix interactions are a commonly recurring theme, but due to limited tools to generate extracellular matrix fibers in vitro and lack of high-throughput methods to detect these interactions, it is hypothesized that many of these interactions have yet to be discovered.

Development of a bio-MEMS device for electrical and mechanical conditioning and characterization of cell sheets for myocardial repair.

Here we propose a bio-MEMS device designed to evaluate contractile force and conduction velocity of cell sheets in response to mechanical and electrical stimulation of the cell source as it grows to form a cellular sheet. Moreover, the design allows for the incorporation of patient-specific data and cell sources. An optimized device would allow cell sheets to be cultured, characterized, and conditioned to be compatible with a specific patient's cardiac environment in vitro, before implantation. This design draws upon existing methods in the literature but makes an important advance by combining the mechanical and electrical stimulation into a single system for optimized cell sheet growth. The device has been designed to achieve cellular alignment, electrical stimulation, mechanical stimulation, conduction velocity readout, contraction force readout, and eventually cell sheet release. The platform is a set of comb electrical contacts consisting of three-dimensional walls made of polydimethylsiloxane and coated with electrically conductive metals on the tops of the walls. Not only do the walls serve as a method for stimulating cells that are attached to the top, but their geometry is tailored such that they are flexible enough to be bent by the cells and used to measure force. The platform can be stretched via a linear actuator setup, allowing for simultaneous electrical and mechanical stimulation that can be derived from patient-specific clinical data.

Effect of Polymer Surface Modification of Superparamagnetic Iron Oxide Nanoparticle Dispersions in High Salinity Environments.

Superparamagnetic nanoparticles (SPIONs) can be used as nuclear magnetic resonance (NMR) signal enhancement agents for petroleum exploration. This enhancement effect is uniform if SPIONs are monodisperse in size and in composition; yet it is challenging to synthesize monodisperse particles that do not aggregate in high salinity petroleum brine. Here, we report a method to synthesize individual SPIONs coated with tunable surface coating densities of poly(2-acrylamido-2-methyl-1-propanesulfonic acid (pAMPS) with a catechol end-group (pAMPS*). To establish parameters under which pAMPS*-coated SPIONS do not aggregate, we compared computational predictions with experimental results for variations in pAMPS* chain length and surface coverage. Using this combined theoretical and experimental approach, we show that singly dispersed SPIONs remained stabilized in petroleum brine for up to 75 h with high surface density pAMPS*.

Fibrin-Targeted Polymerized Shell Microbubbles as Potential Theranostic Agents for Surgical Adhesions.

The development of new therapies for surgical adhesions has proven to be difficult as there is no consistently effective way to assess treatment efficacy in clinical trials without performing a second surgery, which can result in additional adhesions. We have developed lipid microbubble formulations that use a short peptide sequence, CREKA, to target fibrin, the molecule that forms nascent adhesions. These targeted polymerized shell microbubbles (PSMs) are designed to allow ultrasound imaging of early adhesions for diagnostic purposes and for evaluating the success of potential treatments in clinical trials while acting as a possible treatment. In this study, we show that CREKA-targeted microbubbles preferentially bind fibrin over fibrinogen and are stable for long periods of time (∼48 h), that these bound microbubbles can be visualized by ultrasound, and that neither these lipid-based bubbles nor their diagnostic-ultrasound-induced vibrations damage mesothelial cells in vitro. Moreover, these bubbles show the potential to identify adhesionlike fibrin formations and may hold promise in blocking or breaking up fibrin formations in vivo.

Vascular smooth muscle cell durotaxis depends on extracellular matrix composition.

Mechanical compliance has been demonstrated to be a key determinant of cell behavior, directing processes such as spreading, migration, and differentiation. Durotaxis, directional migration from softer to more stiff regions of a substrate, has been observed for a variety of cell types. Recent stiffness mapping experiments have shown that local changes in tissue stiffness in disease are often accompanied by an altered ECM composition in vivo. However, the importance of ECM composition in durotaxis has not yet been explored. To address this question, we have developed and characterized a polyacrylamide hydrogel culture platform featuring highly tunable gradients in mechanical stiffness. This feature, together with the ability to control ECM composition, allows us to isolate the effects of mechanical and biological signals on cell migratory behavior. Using this system, we have tracked vascular smooth muscle cell migration in vitro and quantitatively analyzed differences in cell migration as a function of ECM composition. Our results show that vascular smooth muscle cells undergo durotaxis on mechanical gradients coated with fibronectin but not on those coated with laminin. These findings indicate that the composition of the adhesion ligand is a critical determinant of a cell's migratory response to mechanical gradients.

Design Approaches to Myocardial and Vascular Tissue Engineering.

Engineered tissues represent an increasingly promising therapeutic approach for correcting structural defects and promoting tissue regeneration in cardiovascular diseases. One of the challenges associated with this approach has been the necessity for the replacement tissue to promote sufficient vascularization to maintain functionality after implantation. This review highlights a number of promising prevascularization design approaches for introducing vasculature into engineered tissues. Although we focus on encouraging blood vessel formation within myocardial implants, we also discuss techniques developed for other tissues that could eventually become relevant to engineered cardiac tissues. Because the ultimate solution to engineered tissue vascularization will require collaboration between wide-ranging disciplines such as developmental biology, tissue engineering, and computational modeling, we explore contributions from each field.

Synergistic Integration of Experimental and Simulation Approaches for the de Novo Design of Silk-Based Materials.

Tailored biomaterials with tunable functional properties are crucial for a variety of task-specific applications ranging from healthcare to sustainable, novel bio-nanodevices. To generate polymeric materials with predictive functional outcomes, exploiting designs from nature while morphing them toward non-natural systems offers an important strategy. Silks are Nature's building blocks and are produced by arthropods for a variety of uses that are essential for their survival. Due to the genetic control of encoded protein sequence, mechanical properties, biocompatibility, and biodegradability, silk proteins have been selected as prototype models to emulate for the tunable designs of biomaterial systems. The bottom up strategy of material design opens important opportunities to create predictive functional outcomes, following the exquisite polymeric templates inspired by silks. Recombinant DNA technology provides a systematic approach to recapitulate, vary, and evaluate the core structure peptide motifs in silks and then biosynthesize silk-based polymers by design. Post-biosynthesis processing allows for another dimension of material design by controlled or assisted assembly. Multiscale modeling, from the theoretical prospective, provides strategies to explore interactions at different length scales, leading to selective material properties. Synergy among experimental and modeling approaches can provide new and more rapid insights into the most appropriate structure-function relationships to pursue while also furthering our understanding in terms of the range of silk-based systems that can be generated. This approach utilizes nature as a blueprint for initial polymer designs with useful functions (e.g., silk fibers) but also employs modeling-guided experiments to expand the initial polymer designs into new domains of functional materials that do not exist in nature. The overall path to these new functional outcomes is greatly accelerated via the integration of modeling with experiment. In this Account, we summarize recent advances in understanding and functionalization of silk-based protein systems, with a focus on the integration of simulation and experiment for biopolymer design. Spider silk was selected as an exemplary protein to address the fundamental challenges in polymer designs, including specific insights into the role of molecular weight, hydrophobic/hydrophilic partitioning, and shear stress for silk fiber formation. To expand current silk designs toward biointerfaces and stimuli responsive materials, peptide modules from other natural proteins were added to silk designs to introduce new functions, exploiting the modular nature of silk proteins and fibrous proteins in general. The integrated approaches explored suggest that protein folding, silk volume fraction, and protein amino acid sequence changes (e.g., mutations) are critical factors for functional biomaterial designs. In summary, the integrated modeling-experimental approach described in this Account suggests a more rationally directed and more rapid method for the design of polymeric materials. It is expected that this combined use of experimental and computational approaches has a broad applicability not only for silk-based systems, but also for other polymer and composite materials.

Extracellular matrix type modulates cell migration on mechanical gradients.

Extracellular matrix composition and stiffness are known to be critical determinants of cell behavior, modulating processes including differentiation, traction generation, and migration. Recent studies have demonstrated that the ECM composition can modulate how cells migrate in response to gradients in environmental stiffness, altering a cell's ability to undergo durotaxis. These observations were limited to single varieties of extracellular matrix, but typically cells are exposed to environments containing complex mixtures of extracellular matrix proteins. Here, we investigate migration of NIH 3T3 fibroblasts on mechanical gradients coated with one or more type of extracellular matrix protein. Our results show that NIH 3T3 fibroblasts exhibit durotaxis on fibronectin-coated mechanical gradients but not on those coated with laminin, demonstrating that extracellular matrix type can act as a regulator of cell response to mechanical gradients. Interestingly, NIH 3T3 fibroblasts were also observed to migrate randomly on gradients coated with a mixture of both fibronectin and laminin, suggesting that there may be a complex interplay in the cellular response to mechanical gradients in the presence of multiple extracellular matrix signals. These findings indicate that specific composition of available adhesion ligands is a critical determinant of a cell's migratory response to mechanical gradients.

A microfluidic Transwell to study chemotaxis.

Chemotaxis is typically studied in vitro using commercially available products such as the Transwell® in which cells migrate through a porous membrane in response to one or more clearly defined chemotactic stimuli. Despite its widespread use, the Transwell assay suffers from being largely an endpoint assay, with built-in errors due to inconsistent pore size and human sampling. In this study, we report a microfluidic chemotactic chip that provides real-time monitoring, consistent paths for cell migration, and easy on-chip staining for quantifying migration. To compare its performance with that of a traditional Transwell chamber, we investigate the chemotactic response of MDA-MB-231 1833 metastatic breast cancer cells to epidermal growth factor (EGF). The results show that while both platforms were able to detect a chemotactic response, we observed a dose-dependent response of breast cancer cells towards EGF with low non-specific migration using the microfluidic platform, whereas we observed a dose-independent response of breast cancer cells towards EGF with high levels of non-specific migration using the commercially available Transwell.The microfluidic platform also allowed EGF-dependent chemotactic responses to be observed 24h, a substantially longer window than seen with the Transwell. Thus the performance of our microfluidic platform revealed phenomena that were not detected in the Transwell under the conditions tested.

Design of Multistimuli Responsive Hydrogels Using Integrated Modeling and Genetically Engineered Silk-Elastin-Like Proteins.

Elastomeric, robust, and biocompatible hydrogels are rare, while the need for these types of biomaterials in biomedical-related uses remains high. Here, a new family of genetically engineered silk-elastin copolymers (SELPs) with encoded enzymatic crosslinking sites is developed for a new generation of stimuli-responsive yet robust hydrogels. Input into the designs is guided by simulation, and realized via genetic engineering strategies. The avoidance of gamma irradiation or chemical crosslinking during gel fabrication, in lieu of an enzymatic process, expands the versatility of these new gels for the incorporation of labile proteins and cells. In the present study, the new SELP hydrogels offers sequence dependent, reversible stimuli-responsive features. Their stiffness covers almost the full range of the elasticity of soft tissues. Further, physical modification of the silk domains provided a secondary control point to fine-tune mechanical stiffness while preserving stimuli-responsive features, with implications for a variety of biomedical materials and device needs.

Sentinel lymph node biopsy is indicated for patients with thick clinically lymph node-negative melanoma.

BACKGROUND: Sentinel lymph node biopsy (SLNB) is indicated for the staging of clinically lymph node-negative melanoma of intermediate thickness, but its use is controversial in patients with thick melanoma. METHODS: From 2002 to 2012, patients with melanoma measuring ≥4 mm in thickness were evaluated at a single institution. Associations between survival and clinicopathologic characteristics were explored. RESULTS: Of 571 patients with melanomas measuring ≥4 mm in thickness and no distant metastases, the median age was 66 years and 401 patients (70.2%) were male. The median Breslow thickness was 6.2 mm; the predominant subtype was nodular (45.4%). SLNB was performed in 412 patients (72%) whereas 46 patients (8.1%) presented with clinically lymph node-positive disease and 113 patients (20%) did not undergo SLNB. A positive SLN was found in 161 of 412 patients (39.1%). For SLNB performed at the study institution, 14 patients with a negative SLNB developed disease recurrence in the mapped lymph node basin (false-negative rate, 12.3%). The median disease-specific survival (DSS), overall survival (OS), and recurrence-free survival (RFS) for the entire cohort were 62.1 months, 42.5 months, and 21.2 months, respectively. The DSS and OS for patients with a negative SLNB were 82.4 months and 53.4 months, respectively; 41.2 months and 34.7 months, respectively, for patients with positive SLNB; and 26.8 months and 22 months, respectively, for patients with clinically lymph node-positive disease (P<.0001). The median RFS was 32.4 months for patients who were SLNB negative, 14.3 months for patients who were SLNB positive, and 6.8 months for patients with clinically lymph node-positive disease (P<.0001). CONCLUSIONS: With an acceptably low false-negative rate, patients with thick melanoma and a negative SLNB appear to have significantly prolonged RFS, DSS, and OS compared with those with a positive SLNB. Therefore, SLNB should be considered as indicated for patients with thick, clinically lymph node-negative melanoma.

Monodisperse Micro-Oil Droplets Stabilized by Polymerizable Phospholipid Coatings as Potential Drug Carriers.

There is a critical need to formulate stable micron-sized oil droplets as hydrophobic drug carriers for efficient drug encapsulation, long-term storage, and sustained drug release. Microfluidic methods were developed to maximize the stability of micron-sized, oil-in-water (o/w) emulsions for potential use in drug delivery, using doxorubicin-loaded triacetin oil as a model hydrophobic drug formulation. Initial experiments examined multiple flow conditions for the dispersed (oil) and continuous (liposome aqueous) phases in a microfluidic device to establish the parameters that influenced droplet size. These data were fit to a mathematical model from the literature and indicate that the droplet sizes formed are controlled by the ratio of flow rates and the height of the device channel, rather than the orifice size. Next, we investigated effects of o/w emulsion production methods on the stability of the droplets. The stability of o/w emulsion produced by microfluidic flow-focusing techniques was found to be much greater (5 h vs 1 h) than for emulsions produced by mechanical agitation (vortexing). The increased droplet stability was attributed to the uniform size and lipid distribution of droplets generated by flow-focusing. In contrast, vortexed populations consisted of a wide size distribution that resulted in a higher prevalence of Ostwald ripening. Finally, the effects of shell polymerization on stability were investigated by comparing oil droplets encapsulated by a photopolymerizable diacetylene lipid shell to those with a nonpolymerizable lipid shell. Shell polymerization was found to significantly enhance stability against dissolution for flow-focused oil droplets but did not significantly affect the stability of vortexed droplets. Overall, results of these experiments show that flow-focusing is a promising technique for generating tunable, stable, monodisperse oil droplet emulsions, with potential applications for controlled delivery of hydrophobic drug formulations.

The effects of mechanical stimulation on controlling and maintaining marrow stromal cell differentiation into vascular smooth muscle cells.

For patients suffering from severe coronary heart disease (CHD), the development of a cell-based tissue engineered blood vessel (TEBV) has great potential to overcome current issues with synthetic graft materials. While marrow stromal cells (MSCs) are a promising source of vascular smooth muscle cells (VSMCs) for TEBV construction, they have been shown to differentiate into both the VSMC and osteoblast lineages under different rates of dynamic strain. Determining the permanence of strain-induced MSC differentiation into VSMCs is therefore a significant step toward successful TEBV development. In this study, initial experiments where a cyclic 10% strain was imposed on MSCs for 24 h at 0.1 Hz, 0.5 Hz, and 1 Hz determined that cells stretched at 1 Hz expressed significantly higher levels of VSMC-specific genetic and protein markers compared to samples stretched at 0.1 Hz. Conversely, samples stretched at 0.1 Hz expressed higher levels of osteoblast-specific genetic and protein markers compared to the samples stretched at 1 Hz. More importantly, sequential application of 24-48 h periods of 0.1 Hz and 1 Hz strain-induced genetic and protein marker expression levels similar to the VSMC profile seen with 1 Hz alone. This effect was observed regardless of whether the cells were first strained at 0.1 Hz followed by strain at 1 Hz, or vice versa. Our results suggest that the strain-induced VSMC phenotype is a more terminally differentiated state than the strain-induced osteoblast phenotype, and as result, VSMC obtained from strain-induced differentiation would have potential uses in TEBV construction.

Effect of sequence features on assembly of spider silk block copolymers.

Bioengineered spider silk block copolymers were studied to understand the effect of protein chain length and sequence chemistry on the formation of secondary structure and materials assembly. Using a combination of in vitro protein design and assembly studies, we demonstrate that silk block copolymers possessing multiple repetitive units self-assemble into lamellar microstructures. Additionally, the study provides insights into the assembly behavior of spider silk block copolymers in concentrated salt solutions.

Adsorption of superparamagnetic iron oxide nanoparticles on silica and calcium carbonate sand.

Superparamagnetic iron oxide (SPIO) nanoparticles have the potential to be used in the characterization of porous rock formations in oil fields as a contrast agent for NMR logging because they are small enough to traverse through nanopores and enhance contrast by shortening NMR T2 relaxation time. However, successful development and application require detailed knowledge of particle stability and mobility in reservoir rocks. Because nanoparticle adsorption to sand (SiO2) and rock (often CaCO3) affects their mobility, we investigated the thermodynamic equilibrium adsorption behavior of citric acid-coated SPIO nanoparticles (CA SPIO NPs) and poly(ethylene glycol)-grafted SPIO nanoparticles (PEG SPIO NPs) on SiO2 (silica) and CaCO3 (calcium carbonate). Adsorption behavior was determined at various pH and salt conditions via chemical analysis and NMR, and the results were compared with molecular theory predictions. Most of the NPs were recovered from silica, whereas far fewer NPs were recovered from calcium carbonate because of differences in the mineral surface properties. NP adsorption increased with increasing salt concentration: this trend was qualitatively explained by molecular theory, as was the role of the PEG grafting in preventing NPs adsorption. Quantitative disagreement between the theoretical predictions and the data was due to NP aggregation, especially at high salt concentration and in the presence of calcium carbonate. Upon aggregation, NP concentrations as determined by NMR T2 were initially overestimated and subsequently corrected using the relaxation rate 1/T2, which is a function of aggregate size and fractal dimension of the aggregate. Our experimental validation of the theoretical predictions of NP adsorption to minerals in the absence of aggregation at various pH and salt conditions demonstrates that molecular theory can be used to determine interactions between NPs and relevant reservoir surfaces. Importantly, this integrated experimental and theoretical approach can be used to gain insight into NP mobility in the reservoir.

Adsorption of acid and polymer coated nanoparticles: a statistical thermodynamics approach.

A molecular theoretical description is developed to describe the adsorption of nanoparticles (NPs) that are coated with polymers and functionalized with (surface) acid groups. Results are presented for the adsorption onto both negatively and positively charged surfaces as a function of pH and salt concentration, polymer coating, and NP size. An important finding is that nanoparticles that are coated with weak charge regulating acid molecules such as citric acid develop an asymmetric charge distribution close to a charged surface, due to their finite size. Depending on the sign of the surface charge of the adsorbing surface, a nanoparticle close to the surface either gains more charge or loses charge compared to its "bulk" degree of charge. This in turn influences the amount of NPs that adsorb. The effect of adsorption of negatively charged NPs onto a positively charged surface shows a nonmonotonical variation with pH. The described charging mechanism reveals that details such as size of the NP and acid distribution on the NP need to be considered to provide an accurate understanding of the adsorption process.

The impact of body mass index on esophageal cancer.

BACKGROUND: Surgeons are increasingly operating on patients who are overweight or obese. The influence of obesity on surgical and oncologic outcomes has only recently been addressed. We focus this review on obesity and its impact on esophageal cancer. METHODS: Recent literature and our own institutional experience were reviewed to determine the impact of body mass index on the perioperative and long-term outcomes of patients with esophageal cancer. RESULTS: With few exceptions, no significant differences were seen in perioperative outcomes or survival in patients treated for esophageal cancer when stratified by body mass index. CONCLUSIONS: Although obesity poses increased operative challenges to the surgeon, surgical and oncologic outcomes remain unchanged in obese patients compared with patients who are not obese.

Stability of superparamagnetic iron oxide nanoparticles at different pH values: experimental and theoretical analysis.

The detection of superparamagnetic nanoparticles using NMR logging has the potential to provide enhanced contrast in oil reservoir rock formations. The stability of the nanoparticles is critical because the NMR relaxivity (R(2) ≡ 1/T(2)) is dependent on the particle size. Here we use a molecular theory to predict and validate experimentally the stability of citric acid-coated/PEGylated iron oxide nanoparticles under different pH conditions (pH 5, 7, 9, 11). The predicted value for the critical surface coverage required to produce a steric barrier of 5k(B)T for PEGylated nanoparticles (MW 2000) was 0.078 nm(-2), which is less than the experimental value of 0.143 nm(-2), implying that the nanoparticles should be stable at all pH values. Dynamic light scattering (DLS) measurements showed that the effective diameter did not increase at pH 7 or 9 after 30 days but increased at pH 11. The shifts in NMR relaxivity (from R(2) data) at 2 MHz agreed well with the changes in hydrodynamic diameter obtained from DLS data, indicating that the aggregation behavior of the nanoparticles can be easily and quantitatively detected by NMR. The unexpected aggregation at pH 11 is due to the desorption of the surface coating (citric acid or PEG) from the nanoparticle surface not accounted for in the theory. This study shows that the stability of the nanoparticles can be predicted by the theory and detected by NMR quantitatively, which suggests the nanoparticles to be a possible oil-field nanosensor.