MECHANICAL CHARACTERIZATION OF PRETERM NEONATE PIG LIVER AS A FUNCTION OF HIGH-DENSITY LIPOPROTEIN (HDL)

ABSTRACT Elastography, a non-invasive imaging modality, utilizes mechanical properties of tissue as markers for disease diagnosis or staging. In the case of liver, there have been a number of studies focusing on the relationship between elastic mechanical properties and underlying disease, i.e. fibrosis and cirrhosis. In summary, these studies indicate the feasibility of elastographic tools in detecting liver diseases such as fibrosis and steatosis. There have not been any studies looking at the mechanical properties of the preterm neonate liver to date, which is important, because preterm neonates are at a greater risk for developing liver complications due to their aggressive dietary needs that are met with total parenteral nutrition (TPN). They use of elastography may be less from the use of elastographic tools since the concerns over noise levels in measurements resulting from abdominal wall thickness may be less influential. Therefore, it is necessary to establish basic preterm neonate liver mechanical properties. In this study, we measured the nonlinear (hyperelastic) mechanical properties of livers from preterm pigs that were fed common neaonatal diets, i.e. colostrum, total parenteral nutrition (TPN). 16 neonate pigs survived the feeding regime. Mechanical evaluation of 15 of these neonatal pigs was achieved with the use of uniaxial compression experiments at 0.01 s -1 strain rate. The livers averaging a weight of 34.7±7.0 (SD), were stored in phosphate buffered saline solution at 4°C until experimentation, which occurred within 30 minutes of the animal sacrifice. A minimum of three specimens from each liver was required for the computation of averaged mechanical properties. In addition to mechanical testing samples, blood serum was also obtained from these animals and common chemical parameters for liver health were measured (bilirubin, ALT, AST, HDL, LDL, etc.) Exponential form of the hyperelastic strain energy function, , where b i are the material parameters and L is the stretch ratio, was utilized to describe the hyperelastic mechanical behavior of the preterm neonate pig livers. With the use of E=6b 1 b 2 , a small-strain regime estimate of the elastic modulus of the neonate liver tissue was also computed. The mean b 1 and b 2 parameters are determined to be 97.00±44.15(SD) Pa and 1.90±0.28(SD) (n=71). The mean elastic modulus exhibited an linear dependence on the HDL values obtained from chemical analysis of the blood serum. Moreover, although relatively weak, the ratio of the HDL over LDL also correlated with the elastic modulus. To our knowledge, this is the only study to date that has focused on the mechanical properties of preterm neonatal pigs and its correlation with liver lipid profile in neonates. Future work will focus on correlating this information with histology and then devising multi-scale material characterization approaches that link underlying neonatal liver structure to its overall mechanical properties

Regional variations in growth plate chondrocyte deformation as predicted by three-dimensional multi-scale simulations

The physis, or growth plate, is a complex disc-shaped cartilage structure that is responsible for longitudinal bone growth. In this study, a multi-scale computational approach was undertaken to better understand how physiological loads are experienced by chondrocytes embedded inside chondrons when subjected tomoderate strain under instantaneous compressive loading of the growth plate. Models of representative samples of compressed bone/growthplate/bone from a 0.67mm thick 4-month old bovine proximal tibial physis were subjected to a prescribed displacement equal to 20%of the growth plate thickness. At the macroscale level, the applied compressive deformation resulted in an overall compressive strain across the proliferative-hypertrophic zone of 17%. The microscale model predicted that chondrocytes sustained compressive height strains of 12%and 6% in the proliferative and hypertrophic zones, respectively, in the interior regions of the plate. This pattern was reversed within the outer 300 μm region at the free surface where cells were compressed by 10%in the proliferative and 26%in the hypertrophic zones, in agreement with experimental observations. This work provides a new approach to study growth plate behavior under compression and illustrates the need for combining computational and experimental methods to better understand the chondrocyte mechanics in the growth plate cartilage. While the current model is relevant to fast dynamic events, such as heel strike in walking, we believe this approach provides new insight into the mechanical factors that regulate bone growth at the cell level and provides a basis for developingmodels to help interpret experimental results at varying time scales

Multiscale modeling of growth plate cartilage mechanobiology

Growth plate chondrocytes are responsible for bone growth through proliferation and differentiation. However, the way they experience physiological loads and regulate bone formation, especially during the later developmental phase in the mature growth plate, is still under active investigation. In this study, a previously developed multiscale finite element model of the growth plate is utilized to study the stress and strain distributions within the cartilage at the cellular level when rapidly compressed to 20 %. Detailed structures of the chondron are included in the model to examine the hypothesis that the same combination of mechanoregulatory signals shown to maintain cartilage or stimulate osteogenesis or fibrogenesis in the cartilage anlage or fracture callus also performs the same function at the cell level within the chondrons of growth plate cartilage. Our cell-level results are qualitatively and quantitatively in agreement with tissue-level theories when both hydrostatic cellular stress and strain are considered simultaneously in a mechanoregulatory phase diagram similar to that proposed at the tissue level by Claes and Heigele for fracture healing. Chondrocytes near the reserve/proliferative zone border are subjected to combinations of high compressive hydrostatic stresses (- 0.4 MPa), and cell height and width strains of - 12 to +9% respectively, that maintain cartilage and keep chondrocytes from differentiating and provide conditions favorable for cell division, whereas chondrocytes closer to the hypertrophic/calcified zone undergo combinations of lower compressive hydrostatic stress (- 0.18 MPa) and cell height and width strains as low as - 4 to +4 %, respectively, that promote cell differentiation toward osteogenesis; cells near the outer periphery of the growth plate structure experience a combination of low compressive hydrostatic stress (0 to - 0.15 MPa) and high maximum principal strain (20–29 %) that stimulate cell differentiation toward fibrocartilage or fibrous tissue

TREK-1 Regulates Cytokine Secretion from Cultured Human Alveolar Epithelial Cells Independently of Cytoskeletal Rearrangements

BackgroundTREK-1 deficient alveolar epithelial cells (AECs) secrete less IL-6, more MCP-1, and contain less F-actin. Whether these alterations in cytokine secretion and F-actin content are related remains unknown. We now hypothesized that cytokine secretion from TREK-1-deficient AECs was regulated by cytoskeletal rearrangements.MethodsWe determined F-actin and α-tubulin contents of control, TREK-1-deficient and TREK-1-overexpressing human A549 cells by confocal microscopy and western blotting, and measured IL-6 and MCP-1 levels using real-time PCR and ELISA.ResultsCytochalasin D decreased the F-actin content of control cells. Jasplakinolide increased the F-actin content of TREK-1 deficient cells, similar to the effect of TREK-1 overexpression in control cells. Treatment of control and TREK-1 deficient cells with TNF-α, a strong stimulus for IL-6 and MCP-1 secretion, had no effect on F-actin structures. The combination of TNF-α+cytochalasin D or TNF-α+jasplakinolide had no additional effect on the F-actin content or architecture when compared to cytochalasin D or jasplakinolide alone. Although TREK-1 deficient AECs contained less F-actin at baseline, quantified biochemically, they contained more α-tubulin. Exposure to nocodazole disrupted α-tubulin filaments in control and TREK-1 deficient cells, but left the overall amount of α-tubulin unchanged. Although TNF-α had no effect on the F-actin or α-tubulin contents, it increased IL-6 and MCP-1 production and secretion from control and TREK-1 deficient cells. IL-6 and MCP-1 secretions from control and TREK-1 deficient cells after TNF-α+jasplakinolide or TNF-α+nocodazole treatment was similar to the effect of TNF-α alone. Interestingly, cytochalasin D decreased TNF-α-induced IL-6 but not MCP-1 secretion from control but not TREK-1 deficient cells.ConclusionAlthough cytochalasin D, jasplakinolide and nocodazole altered the F-actin and α-tubulin structures of control and TREK-1 deficient AEC, the changes in cytokine secretion from TREK-1 deficient cells cannot be explained by cytoskeletal rearrangements in these cells

TREK-1 Regulates Cytokine Secretion from Cultured Human Alveolar Epithelial Cells Independently of Cytoskeletal Rearrangements.

BackgroundTREK-1 deficient alveolar epithelial cells (AECs) secrete less IL-6, more MCP-1, and contain less F-actin. Whether these alterations in cytokine secretion and F-actin content are related remains unknown. We now hypothesized that cytokine secretion from TREK-1-deficient AECs was regulated by cytoskeletal rearrangements.MethodsWe determined F-actin and α-tubulin contents of control, TREK-1-deficient and TREK-1-overexpressing human A549 cells by confocal microscopy and western blotting, and measured IL-6 and MCP-1 levels using real-time PCR and ELISA.ResultsCytochalasin D decreased the F-actin content of control cells. Jasplakinolide increased the F-actin content of TREK-1 deficient cells, similar to the effect of TREK-1 overexpression in control cells. Treatment of control and TREK-1 deficient cells with TNF-α, a strong stimulus for IL-6 and MCP-1 secretion, had no effect on F-actin structures. The combination of TNF-α+cytochalasin D or TNF-α+jasplakinolide had no additional effect on the F-actin content or architecture when compared to cytochalasin D or jasplakinolide alone. Although TREK-1 deficient AECs contained less F-actin at baseline, quantified biochemically, they contained more α-tubulin. Exposure to nocodazole disrupted α-tubulin filaments in control and TREK-1 deficient cells, but left the overall amount of α-tubulin unchanged. Although TNF-α had no effect on the F-actin or α-tubulin contents, it increased IL-6 and MCP-1 production and secretion from control and TREK-1 deficient cells. IL-6 and MCP-1 secretions from control and TREK-1 deficient cells after TNF-α+jasplakinolide or TNF-α+nocodazole treatment was similar to the effect of TNF-α alone. Interestingly, cytochalasin D decreased TNF-α-induced IL-6 but not MCP-1 secretion from control but not TREK-1 deficient cells.ConclusionAlthough cytochalasin D, jasplakinolide and nocodazole altered the F-actin and α-tubulin structures of control and TREK-1 deficient AEC, the changes in cytokine secretion from TREK-1 deficient cells cannot be explained by cytoskeletal rearrangements in these cells

Regional Variations in Growth Plate Chondrocyte Deformation as Predicted By Three-Dimensional Multi-Scale Simulations

<div><p>The physis, or growth plate, is a complex disc-shaped cartilage structure that is responsible for longitudinal bone growth. In this study, a multi-scale computational approach was undertaken to better understand how physiological loads are experienced by chondrocytes embedded inside chondrons when subjected to moderate strain under instantaneous compressive loading of the growth plate. Models of representative samples of compressed bone/growth-plate/bone from a 0.67 mm thick 4-month old bovine proximal tibial physis were subjected to a prescribed displacement equal to 20% of the growth plate thickness. At the macroscale level, the applied compressive deformation resulted in an overall compressive strain across the proliferative-hypertrophic zone of 17%. The microscale model predicted that chondrocytes sustained compressive height strains of 12% and 6% in the proliferative and hypertrophic zones, respectively, in the interior regions of the plate. This pattern was reversed within the outer 300 μm region at the free surface where cells were compressed by 10% in the proliferative and 26% in the hypertrophic zones, in agreement with experimental observations. This work provides a new approach to study growth plate behavior under compression and illustrates the need for combining computational and experimental methods to better understand the chondrocyte mechanics in the growth plate cartilage. While the current model is relevant to fast dynamic events, such as heel strike in walking, we believe this approach provides new insight into the mechanical factors that regulate bone growth at the cell level and provides a basis for developing models to help interpret experimental results at varying time scales.</p></div