Deep Tissue Injury: What Do Cells in the Buttocks Sense During Sitting?

Transcription

1 Deep Tissue Injury: What Do Cells in the Buttocks Sense During Sitting? Amit Gefen, PhD Dept. of Biomedical Engineering Faculty of Engineering Tel Aviv University

2 Cellular deformations and chronic wounds In our research of the aetiology of chronic wounds, we study the hypothesis that prolonged cell deformations trigger tissue damage due to changes in the mechanochemical environment of cells, which in turn enhances tissue degeneration

3 Populations at risk for pressure ulcers Bed-bound or chair-bound patients with impaired mobility or sensory capacities Patients post spinal cord injury, brain trauma or stroke Patients who undergo prolonged surgery Prosthetic users

4 Distinct mechanisms of deep versus superficial pressure ulcers

5 Multi-scale approach Organ Tissue Cell

6 Organ-scale internal tissue loads during sitting determined using subject-specific computational models Linder-Ganz et al. Journal of Biomechanics 2008

7 Internal tissue loads associated with prolonged sitting: Comparison of paraplegic data to healthy Fat tissue deformations Muscle tissue deformations Linder-Ganz et al. Journal of Biomechanics 2008

8 Intrinsic Risk Factors e.g. internal tissue loads are posture-dependent Shabshin et al. Clinical Biomechanics 2010

9 Internal tissue loads are support-dependent Shabshin et al. Journal of Rehabilitation Research and Development 2010

10 The ROHO project: Computer simulation studies of the seated buttocks on an air-cell cushion

11 The ROHO project: Computer simulation studies of the seated buttocks on an air-cell cushion (cont.) Effective Stress [MPa]

12 The ROHO project: Computer simulation studies of the seated buttocks on an air-cell cushion

13 Pressure ulcers and deep tissue injury (DTI) at a tissue-scale Histopathology data (Phosphotungstic Acid Hematoxylin staining) for muscle tissue of albino rats, which was damaged by compression of 80kPa for 2 hours. Linder-Ganz et al. Journal of Biomechanics 2006

14 Tolerance of muscle tissue to sustained deformations: TIME IS A FACTOR Muscle tissue stress [mmhg] Linder-Ganz et al. Journal of Biomechanics 2006

15 Organ Tissue Cell

16 Methods: Production of tissueengineered muscles Gefen et al. Journal of Biomechanics 2008

17 Methods: Production of BAMs from a cell/gel mold Gefen et al. Journal of Biomechanics 2008

18 The experimental setup Gefen et al. Journal of Biomechanics 2008

19 Gefen et al. Journal of Biomechanics 2008 Measurement of the lowest strain causing cell death Boundary of BAM Anchoring Velcro Contact region Indentor X2.5 Propidium iodide was used to fluorescently-stain the development of necrosis in 15 BAMs subjected to strains up to 80% The lowest strain causing cell-death was determined every 15min, during 285min-long trials, from fluorescence images of the damage region Time-series of propidium iodide fluorescence images

20 Results: raw data *Experimental strain data were fitted to the sigmoid function proposed by Linder-Ganz et al. (J. Biomech. 2006)

21 Results: Strain-time thresholds from the mean curve fit parameters Gefen et al. Journal of Biomechanics 2008

22 Summary so far The time-dependence tolerance of muscle tissue does relate to celllevel processes The decrease in tolerance does not necessarily relates to ischemia (as traditionally thought): there is no vasculature in the tissue-engineered muscles An alternative explanation would therefore be that the decrease in tolerance relates to impaired mass transport at the extracellular matrix or intracellularly, which might impair cellular homeostasis

23 Background: Deep tissue injury and diffusion Much of the metabolic transport in muscles is based on free diffusion In partially ischemic muscles there is less convection, so free diffusion is particularly important Supported postures involve large muscle tissue deformations which may affect diffusion (e.g. peak strains in glutei muscles under the ischial tuberosities are 60-80% for sitting; Linder-Ganz et al., J. Biomech. 2008) It is necessary to determine how diffusion properties of muscle tissue are affected by large strains

24 Molecular weights of muscle metabolites

25 Ischemic tissue shows mild temperature drops Linder-Ganz & Gefen, Annals of Biomedical Engineering 2007

26 Gefen et al. Journal of Biomechanics 2008 Methods: Theory of fluorescence recovery after photobleaching (FRAP) BASLINE FLUORESNCE ROI BLEACHED REGION ACTUAL RAW DATA: BLEACH RECOVERY FADE OF BLEACH

27 Gefen et al. Journal of Biomechanics 2008 Experimental setup for the FRAP studies BAM specimens were placed on a confocal microscope stage:

28 Methods: Experimental design Gefen et al. Journal of Biomechanics 2008 Baseline 50-60% strain applied Compare diffusion coefficients D 50-60% strain + 3 C temperature drop applied

29 Results: Normalized D in unloaded versus loaded BAMs- paired tests 1,0 0,9 0,8 0,7 0,68 Unloaded Loaded 1,0 0,9 0,8 0,7 0,79 Unloaded Loaded 0,6 0,5 0,4 0,3 0,2 0,35 N=5 * 0,6 0,5 0,4 0,3 0,2 0,39 N=5 * 0,1 0,0 0,1 0,0 Normailzed Diffusion 1 Coefficient 10kD Normailzed Diffusion 1 Coefficient 20kD 1,0 0,9 0,8 0,7 0,6 0,5 0,4 0,3 0,2 0,69 N=5 Unloaded 0,39 * Loaded D in BAMs were normalized with respect to medium 0,1 0,0 Normailzed Diffusion 1 Coefficient 150kD Gefen et al. Journal of Biomechanics 2008

30 Results: Normalized D in loaded BAMs without/with temperature drops (paired tests) 1,00 0,90 0,80 0,70 0,60 0,50 0,40 0,30 0,20 0,10 0,73 N=5 Loaded at 37 ºC Loaded at 34 ºC 0,63 * 0,00 **Diffusion coefficients were normalized with respect to D of the Ratio of diffusion coefficients of loaded over unloaded BAM at 37 ºC unloaded BAM at 37 C Gefen et al. Journal of Biomechanics 2008

31 Multiphysics simulations of glucose transport in muscle tissue Ruschkewitz and Gefen, Comput Methods in Biomech Biomed Eng 2011

32 HYPOTHESIS: Membrane-stretch-induced cell death in DTI Slomka et al. Cellular and Molecular Bioengineering 2009

33 Organ Tissue Cell

34 Idealized model of a compressed cell: Simulations Slomka et al. Cellular and Molecular Bioengineering 2009 Tensional strains There is a critical compression level (~25% for this model) above which large tensional strains start to develop in the plasma membrane

35 Confocal-microscopy-based 3D FE modeling

36 Confocal-microscopy-based 3D FE modeling Details of the cell structure can be included, e.g. the nucleus and cytoskeletal fibers Slomka & Gefen Journal of Biomechanics 2010

37 Maximal global cell deformation (GCD) is 65% Slomka & Gefen Journal of Biomechanics 2010 Simulations of cell distortion experiments Cell compression model

38 Slomka & Gefen Journal of Biomechanics 2010 Simulations of cell distortion experiments Cell compression model: Plasma membrane

39 Slomka et al. Cellular and Molecular Bioengineering 2009 Idealized model of cells seeded in a matrix Fluorescent ly-stained live cells in a gel Source: BBSRC bioscience for the future, To look at whether cells also deform in tension when they reside in a compressed extracellular matrix - a condition closer to the in vivo state - we test a 2D model of a microscopic construct with embedded cells

40 Idealized model of cells seeded in a matrix: Simulations Slomka et al. Cellular and Molecular Bioengineering 2009

41 Experimental approach Slomka and Gefen, Annals of Biomedical Engineering, 2012 Leopold and Gefen, Journal of Tissue Viability, 2012 Leopold and Gefen, Medical Engineering & Physics, 2012

42 Uptake of large molecules is strain-magnitude-dependent as well as molecular-weight-dependent Nuc leus (a) * p<0.01 with respect to strain of 3% for the corresponding molecular mass; ** p<0.05 (b) Confocal imaging of C2C12 cells subjected to a uniaxial tensile strain of 9% for 3 hours (a) versus control, undeformed cells (b). The left column shows combined morphological staining (actin stress fibers in red; nuclei in purple/blue) and imaging of uptake of a 4kDa Dextran dye (green fluorescence), and the right column shows just the Dextran uptake, for clarity. Leopold and Gefen, Medical Engineering & Physics 2012

43 Modeling the experiments can provide relationships between the increase in plasma membrane (PM) permeability and PM strains Slomka and Gefen, Annals of Biomedical Engineering, 2012

44 The transport property changes occur at a time-scale that could explain the loss of tissue/cell tolerance to deformation Tissue/cell deformation Time Linder-Ganz et al. Journal of Biomechanics 2006 Gefen et al. Journal of Biomechanics 2008

45 Some practical implications for the clinical setting Gefen, OWM 2008

46 Join us at the next European Pressure Ulcer Advisory Panel (EPUAP) Conferences! Upcoming annual meeting

47 Focused meetings Join us at the EPUAP Conferences!

48 Acknowledgements Students and former students: Noa Slomka, M.Sc. Ran Sopher, M.Sc. Ms. Shira Or-Tzadikario, M.Sc. Ms. Naama Shoham, M.Sc. Ms. Efrat Leopold, M.Sc. Ayelet Levy, B.Sc. Dr. Jonathan Elsner, Ph.D. Dr. Eran Linder-Ganz, Ph.D. Dr. Sigal Portnoy, Ph.D. International Collaborators: Prof. Cees Oomens (Eindhoven University of Technology, the Netherlands) Prof. Jane Nixon (University of Leeds, UK) Prof. Dan Bader (Southampton University, UK) Prof. Yohan Payan (Joseph Fourier University Grenoble) Funding: Israeli Ministry of Science, The University of Grenoble France, The Leeds Fund for International Research Collaboration, The ROHO Group No conflicts of interest exist Collaborators in Israel: Dr. Itzhak Siev-Ner, M.D. Dr. Ziva Yizhar, Ph.D. Dr. Nogah Shabshin, M.D. Ms. Anat Kristal