Focus on the Physics of Cancer

Despite the spectacular achievements of molecular biology in the second half of the twentieth century and the crucial advances it permitted in cancer research, the fight against cancer has brought some disillusions. It is nowadays more and more apparent that getting a global picture of the very diverse and interlinked aspects of cancer development necessitates, in synergy with these achievements, other perspectives and investigating tools. In this undertaking, multidisciplinary approaches that include quantitative sciences in general and physics in particular play a crucial role. This `focus on' collection contains 19 articles representative of the diversity and state-of-the-art of the contributions that physics can bring to the field of cancer research.Comment: Invited editorial review for the `Focus on the Physics of Cancer' published by the New journal of Physics in 2011--201

Polarized cortical tension drives zebrafish epiboly movements

The principles underlying the biomechanics of morphogenesis are largely unknown. Epiboly is an essential embryonic event in which three tissues coordinate to direct the expansion of the blastoderm. How and where forces are generated during epiboly, and how these are globally coupled remains elusive. Here we developed a method, hydrodynamic regression (HR), to infer 3D pressure fields, mechanical power, and cortical surface tension profiles. HR is based on velocity measurements retrieved from 2D+T microscopy and their hydrodynamic modeling. We applied HR to identify biomechanically active structures and changes in cortex local tension during epiboly in zebrafish. Based on our results, we propose a novel physical description for epiboly, where tissue movements are directed by a polarized gradient of cortical tension. We found that this gradient relies on local contractile forces at the cortex, differences in elastic properties between cortex components and the passive transmission of forces within the yolk cell. All in all, our work identifies a novel way to physically regulate concerted cellular movements that might be instrumental for the mechanical control of many morphogenetic processes.Peer ReviewedPostprint (author's final draft

Direct cell seeding on collagen-coated silicone mandrels to generate cell-derived tissue tubes

The large number of patients suffering from cardiovascular diseases has led to a high demand for functional arterial replacements. A variety of approaches to vascular graft tissue engineering have shown promise, including seeding cells onto natural and synthetic scaffolds or by culturing cell sheets which are subsequently rolled into a tube without exogenous scaffolds. The goal of this project is to develop and characterize cell-derived, fully biological small diameter tissue engineered tubes by seeding and culturing cells directly on tubular supports. Rat aortic smooth muscle cells were seeded onto collagen-coated silicone mandrels and cultured for 14 days. Cells proliferated on the mandrels to form tubes (1.19 mm inner diameter, 1.68 +/- 0.1 mm outer diameter and 230 +/- 63 microns thick; n=72). Histological analysis of the developed tissue tubes demonstrated circumferential alignment of smooth muscle cells, abundant glycosaminoglycan production and some amount of collagen production. On inflating at a constant rate, it was observed that the tissue tubes dilated to an average burst pressure of 256 +/- 76 mmHg; (n=11). In order to observe the effects of addition of soluble factors on extracellular matrix synthesis and mechanical properties, tissue tubes were grown in culture medium supplemented with 50 microgram/ml sodium ascorbate. A significant decrease in outer diameter and wall thickness (1.57 +/- 0.02 mm and 189 +/- 10 microns; n=6 respectively) in the treated groups was observed as compared to (1.66 +/- 0.06 mm and 234 +/- 32 microns; n=6; p\u3c0.05) for the untreated control groups. A slight increase in collagen production was observed by visual assessment of histological images of the ascorbate-treated tissue tubes. This suggests that by using a direct cell seeding approach, it is possible to develop completely biologic small diameter cell-derived tissue tubes that can withstand handling, and it may also possible to modulate matrix synthesis by optimizing cell culture conditions

A DIC based technique to measure the contraction of a skeletal muscle engineered tissue

Tissue engineering is a multidisciplinary science based on the application of engineering approaches to biologic tissue formation. Engineered tissue internal organization represents a key aspect to increase biofunctionality before transplant and, as regarding skeletal muscles, the potential of generating contractile forces is dependent on the internal fiber organization and is reflected by some macroscopic parameters, such as the spontaneous contraction. Here we propose the application of digital image correlation (DIC) as an independent tool for an accurate and noninvasive measurement of engineered muscle tissue spontaneous contraction. To validate the proposed technique we referred to the X-MET, a promising 3-dimensional model of skeletal muscle. The images acquired through a high speed camera were correlated with a custom-made algorithm and the longitudinal strain predictions were employed for measuring the spontaneous contraction. The spontaneous contraction reference values were obtained by studying the force response.The relative error between the spontaneous contraction frequencies computed in both ways was always lower than 0.15%. In conclusion, the use of a DIC based systemallows for an accurate and noninvasive measurement of biological tissues’ spontaneous contraction, in addition to the measurement of tissue strain field on any desired region of interest during electrical stimulation

Neutrophil biomechanical properties and immune function in health and inflammatory disease

Low density granulocytes (LDGs) are a poorly understood class of immune cells found in patients with chronic inflammatory diseases including psoriasis and systemic lupus erythematosus (SLE). Research completed at the National Institutes of Health (NIH) revealed that in the context of SLE, LDGs release higher levels of type 1 interferons, undergo increased NETosis, and accordingly drive inflammation. Meanwhile, advances in mechanical phenotyping at the University of Cambridge have driven hypotheses of neutrophil trafficking and immune function being intimately linked to cellular biomechanical properties (e.g. density, stiffness, morphology). This thesis analyses the intersection of immune cellular biomechanical phenotypes and their function. Specifically, it focuses on the role of neutrophils and LDGs in inflammatory diseases. In this thesis, real-time deformability cytometry (RT-DC) was optimised as a high-throughput mechanical phenotyping technique for the analysis of neutrophils. This enabled development of a protocol to recover purified neutrophils to their whole blood mechanical phenotype. Neutrophil biomechanical properties were analysed by RT-DC, lattice light-sheet microscopy, confocal microscopy and scanning electron microscopy. Neutrophil immunologic functions (e.g. NETosis, macropinocytosis) were imaged using florescence microscopy. To analyse the contribution of biomechanical properties to neutrophil trafficking, a novel microfluidic microvasculature mimetic was developed. An endothelial flow assay was used to image neutrophils interacting with endothelial cells. Finally, the complete proteomes and phosphoproteomes of LDGs and normal dense neutrophils (NDNs) were obtained from five healthy donors and five SLE patients. Several key insights were gained. Firstly, hypotonic lysis and magnetic column-based isolation techniques are damaging to neutrophil biomechanical properties, but purification of neutrophils retaining their biomechanical properties can be achieved by using gradients and column-free magnetic systems followed by recovery at 37 degrees Celsius. Secondly, the biphasic biomechanical kinetics of neutrophil priming were described; cells contract briefly before immediately expanding. The expansion phase was determined to be macropinocytosis dependent. Thirdly, SLE LDGs are phenotypically rougher than autologous SLE NDNs or healthy LDGs. This appears to impact their microvasculature trafficking abilities, as SLE LDGs were increasingly trapped in the narrow channels of a three- dimensional microvasculature mimetic. These results suggest a role for biomechanical properties in modulation of neutrophil trafficking, indicating that SLE LDGs may be increasingly retained in microvasculature networks, similar to what has been described for primed neutrophils. Finally, unbiased proteomics quantified 4109 proteins and 875 phosphoproteins in four neutrophil subsets (healthy unstimulated NDNs, healthy primed NDNs, SLE NDNs, and SLE LDG). This shed new light into neutrophil heterogeneity at the protein level and to my knowledge, is the first proteomic profile of the SLE LDG. In addition to findings pertaining to SLE LDG biology and function, differential phosphorylation of proteins associated with cytoskeletal organisation were identified in SLE LDGs relative to SLE NDNs, suggesting a phosphoproteomic explanation for the SLE LDGs’ distinct biomechanical phenotype. When taken together, this work could have important pathogenic implications in the context of SLE manifestations in various organs and the development of small vessel vasculopathy.Kathleen Bashant was funded by the NIH-Cambridge Scholars Program This research was supported by the NIHR Cambridge Biomedical Research Centre (BRC-1215-20014*). The views expressed are those of the author and not necessarily those of the NIHR or the Department of Health and Social Care

Enzymatically degradable versatile hydrogel platform for cell sheet engineering

The structural organization of cells and their associated extracellular matrix (ECM) is critical to overall tissue function. Recapitulating the complex, highly organized structure of a target tissue is a key to achieve the unique functional characteristics of native tissue. However, achieving this goal requires a system in which substrate physicochemical properties such as modulus, topology and surface chemistry can be modulated. Here, we developed a cell sheet-based harvest & transfer system that can rapidly produce patterned 2D cell sheets in any physiologically relevant size and shape for various cell types. We further show that these cell sheets can be stacked one on top of the other with high cell viability while preserving the patterns, and that they remain sufficiently intact in vivo to allow neovascularization. We can thus use this system to mimic both the 2D and 3D structure of native tissue structure. A further advantage of our system is its substrate modulus tuning capability, which allows us to provide an optimal biomechanical environment for the differentiation and phenotypic stabilization of specific cell types. Because hydrogels theoretically have no limit in 2D shape and size, this system is scalable for producing quality controlled multiple cell sheets in a short period of time. Our model should also aid in understanding the mechanisms that underlie cell-cell and cell-ECM communication in 3D environments, which will be imperative to improving engineered tissue design. We thus ultimately envision that our system could allow the rapid fabrication of functionalized three dimensional thick tissues from multiple stacks of cell sheets derived from autologous cells, which would be an important step forward in both tissue modeling and regenerative medicine in general. Finally, this system can also potentially serve as a powerful model to study in vivo tissue formation and growth as well as cancer cell behavior

Physical limits to sensing material properties

Constitutive relations describe how materials respond to external stimuli such as forces. All materials respond heterogeneously at small scales, which limits what a localized sensor can discern about the global constitution of a material. In this paper, we quantify the limits of such constitutional sensing by determining the optimal measurement protocols for sensors embedded in disordered media. For an elastic medium, we find that the least fractional uncertainty with which a sensor can determine a material constant $\lambda_0$ is approximately \begin{equation*} \frac{\delta \lambda_0}{\lambda_0 } \sim \left( \frac{\Delta_{\lambda} }{ \lambda_0^2} \right)^{1/2} \left( \frac{ d }{ a } \right)^{D/2} \left( \frac{ \xi }{ a } \right)^{D/2} \end{equation*} for $a \gg d \gg \xi$, $\lambda_0 \gg \Delta_{\lambda}^{1/2}$, and $D>1$, where $a$ is the size of the sensor, $d$ is its spatial resolution, $\xi$ is the correlation length of fluctuations in the material constant, $\Delta_{\lambda}$ is the local variability of the material constant, and $D$ is the dimension of the medium. Our results reveal how one can construct microscopic devices capable of sensing near these physical limits, e.g. for medical diagnostics. We show how our theoretical framework can be applied to an experimental system by estimating a bound on the precision of cellular mechanosensing in a biopolymer network.Comment: 33 pages, 3 figure

A Review on Pressure Ulcer: Aetiology, Cost, Detection and Prevention Systems

Pressure ulcer (also known as pressure sore, bedsore, ischemia, decubitus ulcer) is a global challenge for today’s healthcare society. Found in several locations in the human body such as the sacrum, heel, back of the head, shoulder, knee caps, it occurs when soft tissues are under continuous loading and a subject’s mobility is restricted (bedbound/chair bound). Blood flow in soft tissues becomes insufficient leading to tissue necrosis (cell death) and pressure ulcer. The subject’s physiological parameters (age, body mass index) and types of body support surface materials (mattress) are also factors in the formation of pressure ulcer. The economic impacts of these are huge, and the subject’s quality of life is reduced in many ways. There are several methods of detecting and preventing ulceration in human body. Detection depends on assessing local pressure on tissue and prevention on scales of risk used to assess a subject prior to admission. There are also various types of mattresses (air cushioned/liquid filled/foam) available to prevent ulceration. But, despite this work, pressure ulcers remain common.This article reviews the aetiology, cost, detection and prevention of these ulcers

Self-Assembling Peptides for Cartilage Regeneration

Loss of glycosaminoglycans (GAGs) in osteoarthritic (OA) cartilage contributes to a decrease in mechanical properties and function in vitro, and is considered to be a major contributor to disease progression. The aims of this investigation were to test the hypothesis that a combination of self-assembling peptides (SAPs) and chondroitin sulfate (glycosaminoglycan; GAG) would restore the biomechanical properties of GAG depleted porcine condylar cartilage, ideally to a level intrinsic to native porcine condylar cartilage. The SAPs investigated were members of the P11 series which have been designed to spontaneously self-assemble into three-dimensional fibrilar hydrogels, in response to physiological conditions. Initial studies were carried out to determine which of three peptides (P11-4, P11-8 and P11-12) demonstrated high β-sheet percentage, long-woven fibrilar networks and high stiffness; when mixed with chondroitin sulfate at two different GAG molar ratios (1:16 and 1:64) in physiological conditions, using FTIR analysis, transmission electron microscopy and rheology. The β-sheet percentage, dimensions of fibrils and stiffness were dependent upon the peptide, GAG molar ratio and Na2+ salt concentration. P11-4 and P11-8: GAG mixtures had high β-sheet percentage ranging from 50.6-91 % and 81.7-92 %, respectively. Fibril lengths of the P11-4 and P11-8: GAG mixtures were in the range 498- 3518 nm and the elastic shear modulus (G’) ranged from 4,479-10,720 Pa and 7,722-26,854 Pa, respectively. P11-4 and P11-8: GAG mixtures were selected for further investigation. In order to produce a GAG depleted cartilage model, porcine femoral condylar cartilage was subjected to three different methods of GAG depletion (1) coating the surface with chondroitinase ABC (2) injecting chondroitinase ABC into the cartilage (3) washing the condyles in sodium dodecyl sulfate (SDS). GAG depletion was successfully achieved following two 24 hour washes in 0.1 % (w/v) SDS and buffer washes. Histological analysis of safranin O stained sections revealed an absence of GAGs. Quantification of GAGs using the dimethylemethylene blue assay revealed that 75 % of GAGs had been removed. In order to assess the effects of peptide: GAG mixtures on the biomechanical properties of the GAG depleted porcine condylar cartilage a biomechanical test method was developed. A series of indentation tests using different loads, followed by finite element analysis of the data were performed on native and GAG depleted porcine condylar cartilage; to identify a suitable load for detection of a significant difference in the deformation, equilibrium elastic modulus and permeability between the native and GAG depleted porcine condylar cartilages. A load of 0.31 N was identified as the most appropriate. GAG depleted porcine condylar cartilage was injected with P11-4 and P11-8 alone, P11-4 and P11-8 : GAG mixtures at a molar ratio of 1:64 and chondroitin sulfate alone. The average percentage deformation of the medial condylar cartilage samples injected with P11-4 alone and P11-4: GAG mixture was 15.5 % and 8.7 % and for P11-8 alone and P11-8: GAG mixture was 11.4 % and 9.1 % respectively; compared to 6.3 % for the native cartilage and 12.6 % for the GAG depleted cartilage. The average equilibrium elastic modulus of the medial cartilage samples injected with P11-4 alone and P11-4: GAG mixture was 0.16 MPa and 0.43 MPa and for P11-8 alone and P11-8: GAG, 0.23 MPa and 0.35 MPa, respectively; compared to 0.49 MPa for the native cartilage and 0.21 MPa for the GAG depleted cartilage. Statistical analysis (ANOVA) showed that a mixture of P11-4: GAG, but not P11-8: GAG restored both the percentage deformation and equilibrium elastic modulus of the GAG depleted cartilage to levels that were not significantly different to the native cartilage. This study has shown that the use of P11-4 in combination with chondroitin sulfate has future potential for development as a minimally invasive treatment for early stage osteoarthritis

The impact of biomechanics on corneal endothelium tissue engineering

The integrity of innermost layer of the cornea, the corneal endothelium, is key to sustaining corneal transparency. Therefore, disease or injury causing loss or damage to the corneal endothelial cell population may threaten vision. Transplantation of corneal tissue is the standard treatment used to replace malfunctioning corneal endothelial cells. However, this surgery is dependent upon donor tissue, which is limited in supply. Hence, tissue engineers have attempted to construct alternative transplantable tissues or cell therapies to alleviate this problem. Nevertheless, the intrinsic non-dividing nature of corneal endothelial cells continues to foil scientists in their attempts to yield large numbers of cells in the laboratory for use in such novel therapies. Interestingly, the contribution of the biomechanical properties of the underlying extracellular matrix (ECM) on cell division, tissue development and maintenance has been extensively investigated in other many cell types. However, the impact of biomechanics on corneal endothelial cell behaviour is relatively unexplored. Here, we describe contemporary tissue engineering solutions aimed at circumventing donor tissue scarcity. We review the ECM structure and biomechanical features of corneal endothelial cells. We discuss the alterations of ECM in endothelial disease development and progression and point out the role of ECM in developing a tissue-engineered corneal endothelium. We highlight the main biomechanical cues, including topographical and mechanical features, that impact cellular behaviors. Finally, we discuss the influence of biomechanical cues on cell and tissue development, and how corneal endothelial cells response to individual biomechanical stimuli in tissue engineering, which have implications for designing an engineered endothelium and maintaining cell function