New Horizon Workshop: Functional Properties of Chondrocytes in Articular Cartilage using Optical Imaging to Scanning Probe Microscopy

Transcription

1 New Horizon Workshop: Functional Properties of Chondrocytes in Articular Cartilage using Optical Imaging to Scanning Probe Microscopy Organizers: Yang Xia, PhD Eric Darling, PhD Speakers: Yang Xia, PhD Eric Darling, PhD Walter Herzog, PhD

2 Functional imaging of chondrocytes in cartilage by light microscopies Yang Xia, PhD Department of Physics and Center for Biomedical Research, Oakland University, Rochester, Michigan (Web: The functional (biomechanical) properties of cartilage and chondrocytes can be studied by the application of external loading to cartilage or isolated cells. In addition to image the characteristics of the chondrocytes individually at high resolution, these cells can also be used as the localizers in cartilage, which enables the study of the depth-dependent properties of articular cartilage. This talk summarizes the available approaches and new technologies in the optical imaging of chondrocytes and cartilage. Optical visualization of the functional properties of cartilage and chondrocytes can easily be traced back to the seminal work of Arnold Benninghoff in 1925 [1]. In more recent literature, the work of Broom and Myers contained the illustration of a mechanical compression device to image the cross sections of articular cartilage [2]. Although these devices may seem unsophisticated in the 21 st century, these devices, coupling with digital camera and image analysis, can provide new insights into the functional properties of tissue and cells [3]. In addition to the ordinary (unpolarized) light, the use of polarization in light microscopy can utilize the birefringent properties of the collagen fibers in cartilage to illustrate the [4, 5] organizational structures of cartilage and cellular matrices A different approach in optical visualization is to use the immunohistochemical approach to study the cells and cartilage [6, 7]. Based on the high concentration of type-vi collagen in the pericellular matrix, this approach visualizes only the cellular clusters in cartilage in aqueous solution, hence offering new information with regarding to the deformation of individual cells. Beyond the visible light spectrum, infrared light in Fourier-transform infrared imaging (FTIRI) has been used in the last two decades to study cartilage and cells. Since FTIRI is uniquely sensitive to the chemical bond vibration in cartilage, the ability to combine both spatial and chemical information in FTIRI makes it exceptionally suitable to the functional studies of cartilage and cells, where the chemical contents and bond orientations in the tissue can be quantified [8, 9]. The most recent development of light microscopy utilizes the non-linear optics principles to achieve molecular-specific visualization without the use of any staining. The most wellknown technology in this category is the second harmonics generation (SHG) microscopy. Since only the non-centrosymmetric and dense structures are capable of emitting SHG light, the collagen in cartilage and cells can enable very specific visualization of the specimen structure [10]. With further advancement in both the light source (e.g., high intensity light, monochromatic light, and light at specific wavelength) and imaging device (e.g., highsensitivity and fast camera, fine mechanical stages with external control, novel algorithms), optical imaging can provide new information that are illustrative to the MSK processes. SPONSOR: R01 grants from National Institutes of Health (AR , AR ). References:

3 1. Benninghoff A. Z Zellforsch Mikrosk Anat. 1925;2(5): Broom ND, Myers DB. Connect Tissue Res. 1980;7(4): Kahn D, Les C, Xia Y. J Biomech Eng. 2015;137(5): Xia Y, Moody J, Burton-Wurster N, Lust G. Osteoarthritis Cartilage. 2001;9(5): Alhadlaq HA, Xia Y, Hansen FM, Les CM, Lust G. Connect Tissue Res. 2007;48(2): Chen SS, Falcovitz YH, Schneiderman R, et al, Osteoarthritis and Cartilage. 2001;9(6): Choi JB, Youn I, Cao L, Leddy HA, Gilchrist CL, Setton LA, et al. J Biomech. 2007;40(12): Xia Y, Ramakrishnan N, Bidthanapally A. Osteoarthritis Cartilage. 2007;15(7): Ramakrishnan N, Xia Y, Bidthanapally Phys Med Biol. 2007;52(15): He B, Wu JP, Kirk TB, Carrino JA, Xiang C, Xu J. Arthritis Res Ther. 2014;16(2):205.

4 Physical probing of chondrocytes and their peri- and extra-cellular matrices Eric M. Darling, PhD Department of Molecular, Pharmacology, Physiology, and Biotechnology Center for Biomedical Engineering, Brown University, Providence, RI (Web: The mechanical characteristics of cartilage are central to its role in the body as a robust, near-frictionless bearing surface in articulating joints. While substantial research has focused on the macroscale properties of the tissue, investigations at the microscale hold just as much interest. Initial approaches in this area provided valuable insight that has since been leveraged with the use of modern scanning probe microscopy techniques to gain a better understanding of how tissues and cells mechanically interface in normal and diseased states. This talk will briefly describe some of the past approaches for cartilage mechanical characterization at the level of the cell, with emphasis on recent techniques and findings related to chondrocytes and their surrounding peri- and extra-cellular matrices. A variety of techniques exist for direct, physical measurement of the mechanical properties of individual cells, spanning approaches as diverse as micropipette aspiration, atomic force microscopy (AFM), optical tweezers, and microbead rheometry. 2 Micropipette aspiration was the earliest means to describe the viscoelastic properties of chondrocytes. 4 Indentation-based procedures, which included cytoindenters and cytocompressors, soon followed and allowed for characterization of cells attached to a surface (whereas micropipette aspiration tested cells in suspension). 1, 5 Today, AFM is the most common means to probe the local and whole-cell mechanical properties of chondrocytes. The integration of chondrocytes to their surrounding matrix is a key factor in understanding how they sense and respond to mechanical forces. The pericellular matrix in particular has posed an interesting structure to study since morphologically it is distinct in many ways from the extracellular matrix but forms a bridge to the chondrocyte. 6 Study of the peri- and extra-cellular matrices can be accomplished in situ using AFM by mechanically mapping regions immediately abutting a chondrocyte, providing unprecedented resolution in the mechanical properties of the tissue. 3, 7, 8 The scale of AFM tests also allows for use of small animal models, opening up many possibilities for studying how genetic manipulations influence cartilage mechanical properties. Advances in the area of scanning probe microscopy will provide faster and more accurate measurement of mechanical characteristics at the micro- and nano-scales. Application to the study of healthy vs. diseased cartilage can potentially provide insight into how changes in matrix structure influence what chondrocytes sense and how they respond. These data, in combination with the mechanical characteristics of the cells themselves, will provide a more complete picture of cartilage function. SPONSOR: National Institutes of Health (R01 AR063642, P20 GM104937), National Science Foundation (CAREER CBET ). References: 1. Athanasiou K. A., et al Biomaterials. 20 (23-24): Darling E. M. and D. Di Carlo Annu Rev Biomed Eng Darling E. M., et al Biophys J. 98 (12): Jones W. R., et al J Biomech. 32 (2):

5 5. Leipzig N. D., et al Osteoarthritis Cartilage. 14 (12): Poole C. A., et al J Orthop Res. 5 (4): Stolz M., et al Nat Nanotechnol. 4 (3): Xu X., et al Dev Biol. 418 (2):

6 Musculoskeletal Imaging using Confocal and Multi-photon Excitation Microscopy Walter Herzog, PhD, Ziad Abusara PhD, Eng Kuan Moo, PhD, and Sang-Kuy Han, PhD Human Performance Laboratory, Faculty of Kinesiology, Engineering, Medicine and Veterinary Medicine, University of Calgary, Calgary, Canada T2N 1N4 (Web: ; walter@kin.ucalgary.ca) We have been interested in the mechanical properties of articular cartilage, the relationship between mechanical loading and cell signaling in chondrocytes, and the use of microscopic imaging techniques in the visualization of individual sarcomere mechanics in contracting muscles. Rather than working with isolated cells in vitro, or cartilage explants (which has many advantages), we have focused our attention on in situ and in vivo properties of cartilage, muscles and cells. Aside from the appropriate techniques for microscopic imaging, this required the development of associated infrastructure that is typically not commercially available. These approaches will be discussed below. Our initial goal was to measure the dynamic deformations of cells in cartilage attached to its native bone. We felt that cartilage explant and isolated cell preparations might not capture the actual in situ chondrocyte mechanics adequately, and only steady-state results were available at the time when we started this work. We developed a cartilage indentation system in which the indenter was light transmissible and was located directly in front of the objective. Forces and displacement measurements were also incorporated into this design, therefore, deformations of chondrocytes in cartilage attached to its native bone were obtained as a function of indentation depth and surface pressure 4-6. Using appropriate staining, we could also measure calcium signaling of chondrocytes as a function of mechanical loading and cell deformations 7. This in situ chondrocyte loading and signaling system has been expanded from its original design and has been patented in the meantime. We then extended this approach to measuring chondrocyte mechanics across the entire cartilage depth in and in vivo model of the mouse knee 2. In these experiments, the knee joint was intact and the loading was provided by muscular stimulation that allowed for precise control of the duration and amount of loading of the knee articular surfaces. As a byproduct, this system/approach also allowed for quantification of cartilage deformation as a function of muscular force 3, and furthermore, permitted quantification of changes in synovial fluid composition as a function of acute joint loading 1. Finally, using laser-based grid markings, we can now quantify the three-dimensional cartilage strains and can visualize changes in collagen orientation with cartilage loading (unpublished). Finally, we have used second harmonic generation microscopy (SHG) for a variety of purposes. SHG is based on the interaction of a strong laser beam with highly polarizable matter with molecular organization that is without rotational symmetry (noncentrosymmetric). Such interaction results in emission of photons with twice the energy, and therefore half the wavelength, of the incident laser. Therefore, biological tissues with the aforementioned molecular organization can be visualized in a label-free manner, including the determination of single sarcomere lengths for steady-state, passive and active contractions of entire muscles in vivo, an essential ingredient for understanding the mechanics and properties of skeletal muscles during normal animal movement 8. SPONSOR: NSERC of Canada, The Canadian Institutes for Health Research (through Foundation Scheme Grants and Canada Research Chair Programme) The Killam Foundation. References 1 Z. Abusara, et al. J Biomech 46(7), 1225 (2013); 2 Z. Abusara, et al. J Biomech 44, 930 (2011); 3 Z. Abusara, et al. PloS One 11, 1 (2016); 4 S. K. Han, et al. Med Eng Phys 31, 1038 (2009);

7 5 S. K. Han, et al. J Biomech 45(14), 2450 (2012); 6 S. K. Han, et al. J R Soc Interface 7(47), (2010); S. K. Han, et al. J Orthop Res 30(3), 475 (2012); 8 Eng Kuan Moo, et al. Frontiers Physiol 7, 1 (2016).