Local stress-strain distribution and load transfer across cartilage matrix at micro-scale using combined microscopy-based finite element method

Articular cartilage is the load-bearing tissue that consists of proteoglycan macromolecules entrapped between collagen fibrils in a three-dimensional architecture. To date, the drudgery of searching for mathematical models to represent the biomechanics of such a system continues without providing a fitting description of its functional response to load at micro-scale level. We believe that the major complication arose when cartilage was first envisaged as a multiphasic model with distinguishable components and that quantifying those and searching for the laws that govern their interaction is inadequate. To the thesis of this paper, cartilage as a bulk is as much continuum as is the response of its components to the external stimuli. For this reason, we framed the fundamental question as to what would be the mechano-structural functionality of such a system in the total absence of one of its key constituents-proteoglycans. To answer this, hydrated normal and proteoglycan depleted samples were tested under confined compression while finite element models were reproduced, for the first time, based on the structural microarchitecture of the cross-sectional profile of the matrices. These micro-porous in silico models served as virtual transducers to produce an internal noninvasive probing mechanism beyond experimental capabilities to render the matrices micromechanics and several others properties like permeability, orientation etc. The results demonstrated that load transfer was closely related to the microarchitecture of the hyperelastic models that represent solid skeleton stress and fluid response based on the state of the collagen network with and without the swollen proteoglycans. In other words, the stress gradient during deformation was a function of the structural pattern of the network and acted in concert with the position-dependent compositional state of the matrix. This reveals that the interaction between indistinguishable components in real cartilage is superimposed by its microarchitectural state which directly influences macromechanical behavior.

Effect of cell wall properties of plant tissue on porosity and shrinkage of dried apple

In this study cell wall properties; moisture distribution, stiffness, thickness and cell dimension have been taken into consideration. Cell wall stiffness dependent on complex combination of plant cell microstructures, composition and water holding capacity of the cell. In this work, some preliminary steps taken by investing cell wall properties of apple in order to predict change of porosity and shrinkage during drying. Two different types of apple cell wall characteristic were investigated to correlate with porosity and shrinkage after convective drying. A scanning electron microscope (SEM), 2N Intron, a pyncometer and image J software were used in order to measure and analyze cell characteristics, water dynamics, porosity and shrinkage. Cell stiffness of red delicious apple was found higher than granny smith apples. A significant relationship has found between cell wall characteristics and both heat and mass transfer. Consequently, evolution of porosity and shrinkage noticeably influenced during convective drying by the nature of cell wall. This study has brought better understanding of porosity and shrinkage of dried food stuff in microscopic (cell) level and would provide better insight to attain energy effective drying process and quality food stuff.

Differential effects of tissue culture coating substrates on prostate cancer cell adherence, morphology and behavior

Weak cell-surface adhesion of cell lines to tissue culture surfaces is a common problem and presents technical limitations to the design of experiments. To overcome this problem, various surface coating protocols have been developed. However, a comparative and precise real-time measurement of their impact on cell behavior has not been conducted. The prostate cancer cell line LNCaP, derived from a patient lymph node metastasis, is a commonly used model system in prostate cancer research. However, the cells’ characteristically weak attachment to the surface of tissue culture vessels and cover slips has impeded their manipulation and analysis and use in high throughput screening. To improve the adherence of LNCaP cells to the culture surface, we compared different coating reagents (poly-L-lysine, poly-L-ornithine, collagen type IV, fibronectin, and laminin) and culturing conditions and analyzed their impact on cell proliferation, adhesion, morphology, mobility and gene expression using real-time technologies. The results showed that fibronectin, poly-L-lysine and poly-L-ornithine improved LNCaP cells adherence and provoked cell morphology alterations, such as increase of nuclear and cellular area. These coating reagents also induced a higher expression of F-actin and reduced cell mobility. In contrast, laminin and collagen type IV did not improve adherence but promoted cell aggregation and affected cell morphology. Cells cultured in the presence of laminin displayed higher mobility than control cells. All the coating conditions significantly affected cell viability; however, they did not affect the expression of androgen receptor-regulated genes. Our comparative findings provide important insight for the selection of the ideal coating reagent and culture conditions for the cancer cell lines with respect to their effect on proliferation rate, attachment, morphology, migration, transcriptional response and cellular cytoskeleton arrangement.

PAGAT gel dosimeters for dose distribution measurements in the vicinity of high-density implants : a preliminary study

This work examined the suitability of the PAGAT gel dosimeter for use in dose distribution measurements around high-density implants. An assessment of the gels reactivity with various metals was performed and no corrosive effects were observed. An artefact reduction technique was also investigated in order to minimise scattering of the laser light in the optical CT scans. The potential for attenuation and backscatter measurements using this gel dosimeter were examined for a temporary tissue expander's internal magnetic port.

Photon beam dose distributions for patients with implanted temporary tissue expanders

This study examines the effects of temporary tissue expanders (TTEs) on the dose distributions of photon beams in breast cancer radiotherapy treatments. EBT2 radiochromic film and ion chamber measurements were taken to quantify the attenuation and backscatter effects of the inhomogeneity. Results illustrate that the internal magnetic port present in a tissue expander causes a dose reduction of approximately 25% in photon tangent fields immediately downstream of the implant. It was also shown that the silicone elastomer shell of the tissue expander reduced the dose to the target volume by as much as 8%. This work demonstrates the importance for an accurately modelled high-density implant in the treatment planning system for post-mastectomy breast cancer patients.

Establishing the impact of temporary tissue expanders on electron and photon beam dose distributions

Purpose: This study investigates the effects of temporary tissue expanders (TTEs) on the dose distributions in breast cancer radiotherapy treatments under a variety of conditions. Methods: Using EBT2 radiochromic film, both electron and photon beam dose distribution measurements were made for different phantoms, and beam geometries. This was done to establish a more comprehensive understanding of the implant’s perturbation effects under a wider variety of conditions. Results: The magnetic disk present in a tissue expander causes a dose reduction of approximately 20% in a photon tangent treatment and 56% in electron boost fields immediately downstream of the implant. The effects of the silicon elastomer are also much more apparent in an electron beam than a photon beam. Conclusions: Evidently, each component of the TTE attenuates the radiation beam to different degrees. This study has demonstrated that the accuracy of photon and electron treatments of post-mastectomy patients is influenced by the presence of a tissue expander for various beam orientations. The impact of TTEs on dose distributions establishes the importance of an accurately modelled high-density implant in the treatment planning system for post-mastectomy patients.

An analysis of DNA methylation in human adipose tissue reveals differential modification of obesity genes before and after gastric bypass and weight loss

Background Environmental factors can influence obesity by epigenetic mechanisms. Adipose tissue plays a key role in obesity-related metabolic dysfunction, and gastric bypass provides a model to investigate obesity and weight loss in humans. Results Here, we investigate DNA methylation in adipose tissue from obese women before and after gastric bypass and significant weight loss. In total, 485,577 CpG sites were profiled in matched, before and after weight loss, subcutaneous and omental adipose tissue. A paired analysis revealed significant differential methylation in omental and subcutaneous adipose tissue. A greater proportion of CpGs are hypermethylated before weight loss and increased methylation is observed in the 3′ untranslated region and gene bodies relative to promoter regions. Differential methylation is found within genes associated with obesity, epigenetic regulation and development, such as CETP, FOXP2, HDAC4, DNMT3B, KCNQ1 and HOX clusters. We identify robust correlations between changes in methylation and clinical trait, including associations between fasting glucose and HDAC4, SLC37A3 and DENND1C in subcutaneous adipose. Genes investigated with differential promoter methylation all show significantly different levels of mRNA before and after gastric bypass. Conclusions This is the first study reporting global DNA methylation profiling of adipose tissue before and after gastric bypass and associated weight loss. It provides a strong basis for future work and offers additional evidence for the role of DNA methylation of adipose tissue in obesity.

Improved fabrication of melt electrospun tissue engineering scaffolds using direct writing and advanced electric field control

Direct writing melt electrospinning is an additive manufacturing technique capable of the layer-by-layer fabrication of highly ordered 3d tissue engineering scaffolds from micron-diameter fibres. The utility of these scaffolds, however, is limited by the maximum achievable height of controlled fibre deposition, beyond which the structure becomes increasingly disordered. A source of this disorder is charge build-up on the deposited polymer producing unwanted coulombic forces. In this study we introduce a novel melt electrospinning platform with dual voltage power supplies to reduce undesirable charge effects and improve fibre deposition control. We produced and characterised several 90° cross-hatched fibre scaffolds using a range of needle/collector plate voltages. Fibre thickness was found to be sensitive only to overall potential and invariant to specific tip/collector voltage. We also produced ordered scaffolds up to 200 layers thick (fibre spacing 1 mm, diameter 40 μm) and characterised structure in terms of three distinct zones; ordered, semi-ordered and disordered. Our in vitro analysis indicates successful cell attachment and distribution throughout the scaffolds, with little evidence of cell death after seven days. This study demonstrates the importance of electrostatic control for reducing destabilising polymer charge effects and enabling the fabrication of morphologically suitable scaffolds for tissue engineering.

Probabilistic multi-tensor estimation using the tensor distribution function

Diffusion weighted magnetic resonance (MR) imaging is a powerful tool that can be employed to study white matter microstructure by examining the 3D displacement profile of water molecules in brain tissue. By applying diffusion-sensitized gradients along a minimum of 6 directions, second-order tensors can be computed to model dominant diffusion processes. However, conventional DTI is not sufficient to resolve crossing fiber tracts. Recently, a number of high-angular resolution schemes with greater than 6 gradient directions have been employed to address this issue. In this paper, we introduce the Tensor Distribution Function (TDF), a probability function defined on the space of symmetric positive definite matrices. Here, fiber crossing is modeled as an ensemble of Gaussian diffusion processes with weights specified by the TDF. Once this optimal TDF is determined, the diffusion orientation distribution function (ODF) can easily be computed by analytic integration of the resulting displacement probability function.

The rapid manufacture of uniform composite multicellular-biomaterial micropellets, their assembly into macroscopic organized tissues, and potential applications in cartilage tissue engineering

We and others have published on the rapid manufacture of micropellet tissues, typically formed from 100-500 cells each. The micropellet geometry enhances cellular biological properties, and in many cases the micropellets can subsequently be utilized as building blocks to assemble complex macrotissues. Generally, micropellets are formed from cells alone, however when replicating matrix-rich tissues such as cartilage it would be ideal if matrix or biomaterials supplements could be incorporated directly into the micropellet during the manufacturing process. Herein we describe a method to efficiently incorporate donor cartilage matrix into tissue engineered cartilage micropellets. We lyophilized bovine cartilage matrix, and then shattered it into microscopic pieces having average dimensions < 10 μm diameter; we termed this microscopic donor matrix "cartilage dust (CD)". Using a microwell platform, we show that ~0.83 μg CD can be rapidly and efficiently incorporated into single multicellular aggregates formed from 180 bone marrow mesenchymal stem/stromal cells (MSC) each. The microwell platform enabled the rapid manufacture of thousands of replica composite micropellets, with each micropellet having a material/CD core and a cellular surface. This micropellet organization enabled the rapid bulking up of the micropellet core matrix content, and left an adhesive cellular outer surface. This morphological organization enabled the ready assembly of the composite micropellets into macroscopic tissues. Generically, this is a versatile method that enables the rapid and uniform integration of biomaterials into multicellular micropellets that can then be used as tissue building blocks. In this study, the addition of CD resulted in an approximate 8-fold volume increase in the micropellets, with the donor matrix functioning to contribute to an increase in total cartilage matrix content. Composite micropellets were readily assembled into macroscopic cartilage tissues; the incorporation of CD enhanced tissue size and matrix content, but did not enhance chondrogenic gene expression.

The tensor distribution function

Diffusion weighted magnetic resonance imaging is a powerful tool that can be employed to study white matter microstructure by examining the 3D displacement profile of water molecules in brain tissue. By applying diffusion-sensitized gradients along a minimum of six directions, second-order tensors (represented by three-by-three positive definite matrices) can be computed to model dominant diffusion processes. However, conventional DTI is not sufficient to resolve more complicated white matter configurations, e.g., crossing fiber tracts. Recently, a number of high-angular resolution schemes with more than six gradient directions have been employed to address this issue. In this article, we introduce the tensor distribution function (TDF), a probability function defined on the space of symmetric positive definite matrices. Using the calculus of variations, we solve the TDF that optimally describes the observed data. Here, fiber crossing is modeled as an ensemble of Gaussian diffusion processes with weights specified by the TDF. Once this optimal TDF is determined, the orientation distribution function (ODF) can easily be computed by analytic integration of the resulting displacement probability function. Moreover, a tensor orientation distribution function (TOD) may also be derived from the TDF, allowing for the estimation of principal fiber directions and their corresponding eigenvalues.

Expression profiling in spondyloarthropathy synovial biopsies highlights changes in expression of inflammatory genes in conjunction with tissue remodelling genes

Background: In the spondyloarthropathies, the underlying molecular and cellular pathways driving disease are poorly understood. By undertaking a study in knee synovial biopsies from spondyloarthropathy (SpA) and ankylosing spondylitis (AS) patients we aimed to elucidate dysregulated genes and pathways. Methods RNA was extracted from six SpA, two AS, three osteoarthritis (OA) and four normal control knee synovial biopsies. Whole genome expression profiling was undertaken using the Illumina DASL system, which assays 24000 cDNA probes. Differentially expressed candidate genes were then validated using quantitative PCR and immunohistochemistry. Results: Four hundred and sixteen differentially expressed genes were identified that clearly delineated between AS/SpA and control groups. Pathway analysis showed altered gene-expression in oxidoreductase activity, B-cell associated, matrix catabolic, and metabolic pathways. Altered «myogene» profiling was also identified. The inflammatory mediator, MMP3, was strongly upregulated (5-fold) in AS/SpA samples and the Wnt pathway inhibitors DKK3 (2.7-fold) and Kremen1 (1.5-fold) were downregulated. Conclusions: Altered expression profiling in SpA and AS samples demonstrates that disease pathogenesis is associated with both systemic inflammation as well as local tissue alterations that may underlie tissue damaging modelling and remodelling outcomes. This supports the hypothesis that initial systemic inflammation in spondyloarthropathies transfers to and persists in the local joint environment, and might subsequently mediate changes in genes directly involved in the destructive tissue remodelling.

Hyaluronic acid enhances the mechanical properties of tissue-engineered cartilage constructs

There is a need for materials that are well suited for cartilage tissue engineering. Hydrogels have emerged as promising biomaterials for cartilage repair, since, like cartilage, they have high water content, and they allow cells to be encapsulated within the material in a genuinely three-dimensional microenvironment. In this study, we investigated the mechanical properties of tissue-engineered cartilage constructs using in vitro culture models incorporating human chondrocytes from osteoarthritis patients. We evaluated hydrogels formed from mixtures of photocrosslinkable gelatin-methacrylamide (Gel-MA) and varying concentrations (0–2%) of hyaluronic acid methacrylate (HA-MA). Initially, only small differences in the stiffness of each hydrogel existed. After 4 weeks of culture, and to a greater extent 8 weeks of culture, HA-MA had striking and concentration dependent impact on the changes in mechanical properties. For example, the initial compressive moduli of cell-laden constructs with 0 and 1% HA-MA were 29 and 41 kPa, respectively. After 8 weeks of culture, the moduli of these constructs had increased to 66 and 147 kPa respectively, representing a net improvement of 69 kPa for gels with 1% HA-MA. Similarly the equilibrium modulus, dynamic modulus, failure strength and failure strain were all improved in constructs containing HA-MA. Differences in mechanical properties did not correlate with glycosaminoglycan content, which did not vary greatly between groups, yet there were clear differences in aggrecan intensity and distribution as assessed using immunostaining. Based on the functional development with time in culture using human chondrocytes, mixtures of Gel-MA and HA-MA are promising candidates for cartilage tissue-engineering applications.

In vitro pre-vascularisation of tissue-engineered constructs: A co-culture perspective

In vitro pre-vascularization is one of the main vascularization strategies in the tissue engineering field. Culturing cells within a tissue-engineered construct (TEC) prior to implantation provides researchers with a greater degree of control over the fate of the cells. However, balancing the diverse range of different cell culture parameters in vitro is seldom easy and in most cases, especially in highly vascularized tissues, more than one cell type will reside within the cell culture system. Culturing multiple cell types in the same construct presents its own unique challenges and pitfalls. The following review examines endothelial-driven vascularization and evaluates the direct and indirect role other cell types have in vessel and capillary formation. The article then analyses the different parameters researchers can modulate in a co-culture system in order to design optimal tissue-engineered constructs to match desired clinical applications.