A comparison of methods to assess cell mechanical properties.

The mechanical properties of cells influence their cellular and subcellular functions, including cell adhesion, migration, polarization, and differentiation, as well as organelle organization and trafficking inside the cytoplasm. Yet reported values of cell stiffness and viscosity vary substantially, which suggests differences in how the results of different methods are obtained or analyzed by different groups. To address this issue and illustrate the complementarity of certain approaches, here we present, analyze, and critically compare measurements obtained by means of some of the most widely used methods for cell mechanics: atomic force microscopy, magnetic twisting cytometry, particle-tracking microrheology, parallel-plate rheometry, cell monolayer rheology, and optical stretching. These measurements highlight how elastic and viscous moduli of MCF-7 breast cancer cells can vary 1,000-fold and 100-fold, respectively. We discuss the sources of these variations, including the level of applied mechanical stress, the rate of deformation, the geometry of the probe, the location probed in the cell, and the extracellular microenvironment.

Respiratory Therapy Faculty Knowledge of and Attitudes Toward Interprofessional Education.

BACKGROUND: Interprofessional education (IPE) improves collaboration and patient care through joint education between health professions. Respiratory therapy (RT) faculty were surveyed to evaluate their knowledge and attitudes toward IPE. We report current opportunities for IPE from faculty and compare responses from associate's, bachelor's, and master's degree programs and profit versus nonprofit institutions. METHODS: We developed an online survey based on IPE literature and questions modified for the RT discipline. The survey was distributed by email to 874 faculty from the Commission on Accreditation for Respiratory Care accredited programs. RESULTS: The response rate was 33%. Faculty identified IPE as an important component of RT education (n = 207, 80%) but reported challenges in integrating IPE into current curriculum. Overall, communication was ranked as the most important IPE competency (n = 104, 39%) and ethics least important (n = 131, 49%). When asked how many credit hours are required to teach IPE, 48% of respondents reported that they were unsure of an appropriate time requirement. Significant differences between associate's and bachelor's/master's degree program faculty were found on the following topics: institutional resources needed for IPE (P < .001), faculty availability (P < .001), curriculum availability for IPE (P = .02), and importance of including IPE at academic health center campuses (P < .001). CONCLUSIONS: IPE is recognized as an important component of RT education by all faculty respondents. However, significant differences in knowledge and attitudes toward IPE exist between faculty in associate's versus bachelor's/master's degree programs. Revisiting the current accreditation standards program may allow IPE to take a more prominent role in RT curricula.

Biomimetic tissue-engineered systems for advancing cancer research: NCI Strategic Workshop report.

Advanced technologies and biomaterials developed for tissue engineering and regenerative medicine present tractable biomimetic systems with potential applications for cancer research. Recently, the National Cancer Institute convened a Strategic Workshop to explore the use of tissue biomanufacturing for development of dynamic, physiologically relevant in vitro and ex vivo biomimetic systems to study cancer biology and drug efficacy. The workshop provided a forum to identify current progress, research gaps, and necessary steps to advance the field. Opportunities discussed included development of tumor biomimetic systems with an emphasis on reproducibility and validation of new biomimetic tumor models, as described in this report.

Physical biology in cancer. 1. Cellular physics of cancer metastasis.

One of the major challenges in cancer research today is developing new therapeutic strategies to control metastatic disease, the spread of cancer cells from a primary tumor to seed in a distant site. Advances in diagnosis and treatment options have increased the survival rate for most patients with local tumors; however, less progress has been made in treatment of disseminated disease. According to the SEER Cancer Statistics Review, 1975-2010, in the case of breast and prostate cancers, only one in four patients diagnosed with distant metastatic disease will survive more than five years. Current research efforts largely focus on identifying biological targets, such as specific genes and signaling pathways that drive two key steps of metastasis, invasion from the primary tumor and growth in the secondary site. On the other hand, there are phenotypic traits and dynamics in the metastatic process that are not encoded by single genes or signaling pathways but, rather, a larger system of events and biophysical characteristics. Connecting genomic and pathway investigations with quantitative physical phenotypic characteristics of cells, the physical microenvironment, and the physical spatiotemporal interactions of the metastatic process provides a stronger complementary understanding of the disease.

Measuring the evolution and output of cross-disciplinary collaborations within the NCI Physical Sciences-Oncology Centers Network.

Development of effective quantitative indicators and methodologies to assess the outcomes of cross-disciplinary collaborative initiatives has the potential to improve scientific program management and scientific output. This article highlights an example of a prospective evaluation that has been developed to monitor and improve progress of the National Cancer Institute Physical Sciences-Oncology Centers (PS-OC) program. Study data, including collaboration information, was captured through progress reports and compiled using the web-based analytic database: Interdisciplinary Team Reporting, Analysis, and Query Resource. Analysis of collaborations was further supported by data from the Thomson Reuters Web of Science database, MEDLINE database, and a web-based survey. Integration of novel and standard data sources was augmented by the development of automated methods to mine investigator pre-award publications, assign investigator disciplines, and distinguish cross-disciplinary publication content. The results highlight increases in cross-disciplinary authorship collaborations from pre- to post-award years among the primary investigators and confirm that a majority of cross-disciplinary collaborations have resulted in publications with cross-disciplinary content that rank in the top third of their field. With these evaluation data, PS-OC Program officials have provided ongoing feedback to participating investigators to improve center productivity and thereby facilitate a more successful initiative. Future analysis will continue to expand these methods and metrics to adapt to new advances in research evaluation and changes in the program.

Analysis of cell binding and internalization of multivalent PEG-based gene delivery vehicles.

RGD peptides have been incorporated into several gene delivery vehicles to enhance specific interactions of nonviral vehicles with the cell surface. However, there are contradictory results regarding the effect of linear RGD peptides on specific cell surface binding of polyethylene glycol (PEG)-conjugated gene delivery vehicles. This study sought to understand how coupling RGD peptides to PEG vehicles affects cell binding and internalization using a novel four arm PEG backbone. Coupling multiple RGD peptides to the PEG backbone increased the affinity of the vehicle for the cell surface, and that the PEG backbone did not reduce the affinity of RGD peptides for integrin receptors in both kinetic and equilibrium studies. Kinetic studies suggest that cellular internalization of PEG-based vehicles is not regulated by the RGD peptides on the vehicle, but rather by nonspecific interactions with heparan sulfate proteoglycans either alone or in combination with integrins. These results suggest that while increasing the number of RGD peptides per vehicle increases cell binding, but it does not contribute to increased internalization or transfection efficiency.

Synergistic enhancement of human bone marrow stromal cell proliferation and osteogenic differentiation on BMP-2-derived and RGD peptide concentration gradients.

Rational design of bioactive tissue engineered scaffolds for directing bone regeneration in vivo requires a comprehensive understanding of cell interactions with the immobilized bioactive molecules. In the current study, substrates possessing gradient concentrations of immobilized peptides were used to measure the concentration-dependent proliferation and osteogenic differentiation of human bone marrow stromal cells. Two bioactive peptides, one derived from extracellular matrix protein (ECM), GRGDS, and one from bone morphogenic protein-2 (BMP-2), KIPKASSVPTELSAISTLYL, were found to synergistically enhance cell proliferation, up-regulate osteogenic mRNA markers bone sialoprotein (BSP) and Runt-related transcription factor 2, and produce mineralization at densities greater than 130 pmol cm(-2) (65 pmol cm(-2) for each peptide). In addition, COOH-terminated self-assembled monolayers alone led to up-regulated BSP mRNA levels at densities above 200 pmol cm(-2) and increased cell proliferation from day 3 to day 14. Taking further advantage of both the synergistic potentials and the concentration-dependent activities of ECM and growth-factor-derived peptides on proliferative activity and osteogenic differentiation, without the need for additional osteogenic supplements, will enable the successful incorporation of the bioactive species into biorelevant tissue engineering scaffolds.

Modulus-driven differentiation of marrow stromal cells in 3D scaffolds that is independent of myosin-based cytoskeletal tension.

Proliferation and differentiation of cells are known to be influenced by the physical properties of the extracellular environment. Previous studies examining biophysics underlying cell response to matrix stiffness utilized a two-dimensional (2D) culture format, which is not representative of the three-dimensional (3D) tissue environment in vivo. We report on the effect of 3D matrix modulus on human bone marrow stromal cell (hBMSC) differentiation. hBMSCs underwent osteogenic differentiation in poly(ethylene glycol) hydrogels of all modulus (300-fold modulus range, from 0.2 kPa to 59 kPa) in the absence of osteogenic differentiation supplements. This osteogenic differentiation was modulus-dependent and was enhanced in stiffer gels. Osteogenesis in these matrices required integrin-protein ligation since osteogenesis was inhibited by soluble Arginine-Glycine-Aspartate-Serine peptide, which blocks integrin receptors. Immunostained images revealed lack of well-defined actin filaments and microtubules in the encapsulated cells. Disruption of mechanosensing elements downstream of integrin binding that have been identified from 2D culture such as actin filaments, myosin II contraction, and RhoA kinase did not abrogate hBMSC material-driven osteogenic differentiation in 3D. These data show that increased hydrogel modulus enhanced osteogenic differentiation of hBMSCs in 3D scaffolds but that hBMSCs did not use the same mechanosensing pathways that have been identified in 2D culture.

The modulation of dendritic cell integrin binding and activation by RGD-peptide density gradient substrates.

Dendritic cells (DCs) are central regulators of the immune system that operate in both innate and adaptive branches of immunity. Activation of DC by numerous factors, such as danger signals, has been well established. However, modulation of DC functions through adhesion-based cues has only begun to be characterized. In this work, DCs were cultured on surfaces presenting a uniform gradient of the integrin-targeting RGD peptide generated using the recently established "universal gradient substrate for click biofunctionalization" methodology. Surface expression of activation markers (costimulatory molecule CD86 and stimulatory molecule MHC-II) and production of cytokines IL-10 and IL-12p40 of adherent DCs was quantified in situ. Additionally, bound alpha(V) integrin was quantified in situ using a biochemical crosslinking/extraction method. Our findings demonstrate that DCs upregulated CD86, MHC-II, IL-10, IL-12p40 and alpha(V) integrin binding as a function of RGD surface density, with production of IL-12p40 being the marker most sensitive to RGD surface density. Surface expression of activation markers demonstrated moderate correlation with alpha(V) integrin binding, while cytokine production was highly correlated with alpha(V) integrin binding. This work demonstrates the utility of the surface density gradient platform as a high-throughput method to investigate RGD density-dependent DC adhesive responses. Furthermore, this quantitative analysis of DC integrin-based activation represents a first of its type, helping to establish the field of adhesion-based modulation of DCs as a general mechanism that has previously not been defined, and informs the rational design of biomimetic biomaterials for immunomodulation.

The use of immobilized osteogenic growth peptide on gradient substrates synthesized via click chemistry to enhance MC3T3-E1 osteoblast proliferation.

In this study, we report the use of surface immobilized peptide concentration gradient technology to characterize MC3T3-E1 osteoblast cell response to osteogenic growth peptide (OGP), a small peptide found naturally in human serum at mumol/L concentrations. OGP was coupled to oxidized self assembled monolayer (SAM) gradients by a polyethylene oxide (PEO) linker using click chemistry. After 4h incubation with MC3T3-E1 cells, OGP functionalized surfaces had higher cell attachment at low peptide concentrations compared to control gradients. By day 3, OGP gradient substrates had higher cell densities compared to control gradients at all concentrations. MC3T3-E1 cell doubling time was 35% faster on OGP substrates relative to SAM gradients alone, signifying an appreciable increase in cell proliferation. This increase in cell proliferation, or decrease in doubling time, due to OGP peptide was reduced by day 7. Hence, immobilized OGP increased cell proliferation from 0 days to 3 days at all densities indicating it may be useful as a proliferative peptide that can be used in tissue engineering substrates.

Characterization of a multifunctional PEG-based gene delivery system containing nuclear localization signals and endosomal escape peptides.

Endosomal escape and nuclear localization are two barriers to gene delivery that need to be addressed in the design of new nonviral gene delivery vehicles. We have previously synthesized low-toxicity polyethylene glycol (PEG)-based vehicles with endosomal escape functionalities, but it was determined that the transfection efficiency of PEG-based vehicles that escaped the endosome was still limited by poor nuclear localization. Two different nuclear localization signal (NLS) peptides, SV40 and TAT, were coupled to PEG-based vehicles with DNA-binding peptides (DBPs) to determine the effect of NLS peptides on the transfection efficiency of PEG-based gene delivery vehicles. Coupling one SV40 peptide, a classical NLS, or two TAT peptides, a nonclassical NLS, to PEG-DBP vehicles increased the transfection efficiency of PEG-DBP/DNA particles 15-fold and resulted in similar efficiency to that of a common cationic polymer vehicle, polyethylenimine (PEI). The transfection efficiency of both types of PEG-DBP-NLS particles was further increased 7-fold in the presence of chloroquine, suggesting that the transfection efficiency of PEG-DBP-NLS particles is limited by their ability to escape the endosome. To develop particles that could escape the endosome and target the nucleus, a mixture of PEG-DBP-NLS vehicles and PEG-based vehicles with DBPs and endosomal escape peptides were complexed with plasmid DNA to form multifunctional particles that had a transfection efficiency 2-3 times higher than that of PEI. Additionally, the PEG-based vehicles were less toxic and more resistant to nonspecific protein adsorption than PEI, making them an attractive alternative for nonviral gene delivery.

The effect of endosomal escape peptides on in vitro gene delivery of polyethylene glycol-based vehicles.

BACKGROUND: With recent progress in gene therapy clinical trials, there is an even greater demand to advance the development of nonviral gene delivery vehicles. We have previously developed poly(ethylene glycol) (PEG)-based vehicles with transfection efficiency similar to polyethyleneimine and low cytotoxicity. It was hypothesized that conjugating endosomal escape peptides (EEPs) to PEG-based vehicles would further increase their transfection efficiency. The present study aimed to determine how two different EEPs, INF7 and H5WYG, which destabilize the endosomal membrane at different pHs, affect the efficiency of PEG-based vehicles. METHODS: INF7 and H5WYG were conjugated to PEG-tetraacrylate (PEG-TA) via a Michael-type addition at the desired molar ratios. The pH-dependent membrane lytic activity, transfection efficiency, particle size, zeta potential, and endosomal escape kinetic rate constants were determined. RESULTS: Fusogenic peptides, INF7 and H5WYG, showed pH-dependent membrane lytic activity when conjugated to PEG-TA. The highest membrane lytic activity of PEG-INF7 and PEG-H5WYG conjugates occurred at pH 5 and 5.5, respectively. Coupling one INF7 peptide to PEG-DNA binding peptide (DBP) vehicles increased the transfection efficiency ten-fold and showed greater transfection efficiency than PEG-DBP vehicles coupled with H5WYG peptide. Fitting a first-order kinetic model to the average intracellular pH of the vehicle/DNA particles over time determined that coupling EEPs to PEG-DBP vehicles increased the endosomal escape rate constant by two orders of magnitude. CONCLUSIONS: Endosomal escape is a key step in nonviral cellular trafficking and thus the transfection efficiency of nonviral vehicles can be increased by targeting release of DNA from the endosome with EEPs.

Synthesis and characterization of four-arm poly(ethylene glycol)-based gene delivery vehicles coupled to integrin and DNA-binding peptides.

The goal of this study was to develop a gene delivery vehicle that can specifically target cell surface receptors with low nonspecific protein adsorption and low cytotoxicity. Toward this goal, four-arm poly(ethylene glycol) vehicles were functionalized with DNA-binding peptides (DBPs) and integrin-binding (RGD) peptides. We have previously described a novel PEG-based gene delivery vehicle functionalized with DBPs that successfully transfected Chinese hamster ovary (CHO) cells with low toxicity and low protein adsorption. This work investigated whether incorporating RGD peptides onto PEG-DBP vehicles could target specific cell surface receptors and increase transfection efficiency of HEPG2 cells. DBP and RGD peptides were coupled onto PEG-tetraacrylate (PEG-TA) in three combinations (molar ratios of DBP:RGD of 1:3, 2:2, and 3:1) and characterized by measuring particle size, zeta potential, and transfection efficiency as a function of charge ratio (peptide amine groups:DNA phosphate). Nonspecific protein adsorption and cytotoxicity of PEG-DBP-RGD vehicles were also measured. Dynamic light scattering showed that PEG-DBP-RGD vehicles condensed DNA into particles having mean diameters of 250-300 nm and zeta potentials ranging from -10 to 7 mV. It was found that coupling two RGD peptides to the PEG-DBP 2 vehicle increased the transfection efficiency at a polymer/DNA charge ratio of 5:1 (+/-) and 6:1 (+/-) and that these vehicles had transfection efficiencies similar to those of polyethylenimine (PEI)/DNA particles. However, coupling one or three RGD peptides to PEG-DBP vehicles did not increase the transfection efficiency. Additionally, the PEG-DBP-RGD/DNA particles adsorbed less protein than PEI particles and were less toxic to HEPG2 cells.

Short bowel syndrome: parenteral nutrition versus intestinal transplantation. Where are we today?

Current management of short bowel syndrome (SBS) revolves around the use of home TPN (HPN). Complications include liver disease, catheter-related infections or occlusions, venous thrombosis, and bone disease. Patient survival with SBS on TPN is 86% and 75% at 2 and 5 years, respectively. Surgical management of SBS includes nontransplant surgeries such as serial transverse enteroplasty and reanastomosis. Small bowel transplant has become increasingly popular for management of SBS and is usually indicated when TPN cannot be continued. Posttransplant complications include graft-versus-host reaction, infections in an immunocompromised patient, vascular and biliary diseases, and recurrence of the original disease. Following intestinal-only transplants, patient and graft survival rate is 77% and 66% after 1 year. After 5 years the survival figures are 49% and 34%, respectively. Future improvements in survival and quality of life will enhance small bowel transplant as a viable treatment option for patients with SBS.

Development of novel poly(ethylene glycol)-based vehicles for gene delivery.

The purpose of this research was to develop and characterize a gene delivery vehicle with a poly(ethylene glycol) (PEG) backbone with the aim of overcoming limitations, such as cytotoxicity and rapid clearance, associated with current commonly used non-viral carriers. PEG was functionalized with DNA-binding peptides (DBPs) to make a vehicle (DBP-PEG) capable of condensing DNA. Complexes of plasmid DNA and DBP-PEG were formed and characterized by measuring particle size, zeta potential, and transfection efficiency as a function of N:P charge ratios (DBP-PEG amino groups:DNA phosphate). Dynamic light scattering showed that DBP-PEG was able to condense DNA efficiently resulting in a population of particles in the range of 250-300 nm. Neutral or slightly positive zeta potentials were measured for charge ratios of 3.5:1 and greater. DBP-PEG/DNA complexes, made with plasmids encoding the green fluorescent protein (GFP) and beta-Galactosidase (beta-Gal) genes, were used to transfect Chinese hamster ovary (CHO) cells. DBP-PEG/DNA was capable of transfecting cells and maximum transfection efficiency was observed for N:P ratios from 4:1 to 5:1, corresponding to zeta potentials from -4 to +1.6 mV. The effect of the DBP-PEG vehicle on cell viability was assayed. DBP-PEG was associated with a higher percentage of viable cells ( approximately 95%) than either polyethylenimine (PEI) or poly-L-lysine (PLL), and with transfection efficiency greater than PLL, but with somewhat lower than PEI. The results of this work demonstrate that PEG can be used as the backbone for gene delivery vehicles.