Investigation of nano-mechanical properties of annulus fibrosus using atomic force microscopy.

We describe the use of atomic force microscopy (AFM) to investigate the nanomechanical properties of annulus fibrosus (AF)-the outer fibrous layer of an intervertebral disc (IVD) encapsulating the inner jelly-like mass known as the nucleus pulposus (NP). Disk disease, degenerated discs, slipped discs, and herniated discs are common terms often linked to back pain and are caused due to degeneration of IVD. Due to the variations in the structure and biochemical composition of the IVD, studies of macromechanical properties in the motion segment or AF may lack all significant nanomechanical responses or behaviors. Existing studies do not report the micro or nano level of mechanics of IVD components and whether the nanomechanics of this tissue mimic its macromechanical behavior is not known. Our studies used AFM to investigate the regional micromechanical properties of the AF that have been otherwise difficult due to small sample size of the tissue. Five different zones including peripheral and central were tested mechanically as well as biochemically. Qualitative biochemical staining and quantitative values of nanomechanical properties of different zones are compared and discussed in detail. The results of nanomechanical investigations described in this study not only reveal its mimic at macroscopic level, they represent an important step towards establishing a framework for testing and comparing tissue engineered IVD replacements with native tissues.

[1]  S. Bruehlmann,et al.  Regional variations in the cellular matrix of the annulus fibrosus of the intervertebral disc , 2002, Journal of anatomy.

[2]  S. Klisch,et al.  A special theory of biphasic mixtures and experimental results for human annulus fibrosus tested in confined compression. , 2000, Journal of biomechanical engineering.

[3]  J. D. Janssen,et al.  Nonhomogeneous Permeability of Canine Anulus Fibrosus , 1997, Spine.

[4]  L. Nicolais,et al.  Composite hydrogels for implants , 1998, Proceedings of the Institution of Mechanical Engineers. Part H, Journal of engineering in medicine.

[5]  Rod Balhorn,et al.  Combining constitutive materials modeling with atomic force microscopy to understand the mechanical properties of living cells , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[6]  K. Kaneda,et al.  Effects of Degeneration on the Elastic Modulus Distribution in the Lumbar Intervertebral Disc , 1996, Spine.

[7]  Ferenc Horkay,et al.  Determination of elastic moduli of thin layers of soft material using the atomic force microscope. , 2002, Biophysical journal.

[8]  V C Mow,et al.  Shear mechanical properties of human lumbar annulus fibrosus , 1999, Journal of orthopaedic research : official publication of the Orthopaedic Research Society.

[9]  M C Hay,et al.  Anatomy of the lumbar spine. , 1976, The Medical journal of Australia.

[10]  Delphine Périé,et al.  Confined compression experiments on bovine nucleus pulposus and annulus fibrosus: sensitivity of the experiment in the determination of compressive modulus and hydraulic permeability. , 2005, Journal of biomechanics.

[11]  V C Mow,et al.  Tensile Properties of Nondegenerate Human Lumbar Anulus Fibrosus , 1996, Spine.

[12]  V. Moy,et al.  Mechanical properties of L929 cells measured by atomic force microscopy: effects of anticytoskeletal drugs and membrane crosslinking. , 2006, Scanning.

[13]  V. C. Mow,et al.  Regional Variation in Tensile Properties and Biochemical Composition of the Human Lumbar Anulus Fibrosus , 1994, Spine.

[14]  P. Zysset,et al.  Nanoindentation discriminates the elastic properties of individual human bone lamellae under dry and physiological conditions. , 2002, Bone.

[15]  Peter Niederer,et al.  Investigation of the Morphology of the Lacunocanalicular System of Cortical Bone Using Atomic Force Microscopy , 2001, Annals of Biomedical Engineering.

[16]  Wilson C. Hayes,et al.  Basic Orthopaedic Biomechanics , 1995 .

[17]  Rupal V Patel,et al.  Regional structural and viscoelastic properties of fibrocartilage upon dynamic nanoindentation of the articular condyle. , 2001, Journal of structural biology.

[18]  V C Mow,et al.  The viscoelastic behavior of the non-degenerate human lumbar nucleus pulposus in shear. , 1997, Journal of biomechanics.

[19]  M. Pope,et al.  Water Content in Human Intervertebral Discs: Part II. Viscoelastic Behavior , 1987, Spine.

[20]  T. Xu,et al.  A new insight into the adsorption of bovine serum albumin onto porous polyethylene membrane by zeta potential measurements, FTIR analyses, and AFM observations. , 2003, Journal of colloid and interface science.

[21]  J. Kinney,et al.  The Importance of Intrafibrillar Mineralization of Collagen on the Mechanical Properties of Dentin , 2003, Journal of dental research.

[22]  M H Pope,et al.  Biomechanics of the lumbar spine. , 1989, Annals of medicine.

[23]  P. Gatenholm,et al.  Preparation and properties of hydrogels based on hemicellulose , 1998 .

[24]  M. Radmacher,et al.  Measuring the Elastic Properties of Thin Polymer Films with the Atomic Force Microscope , 1998 .

[25]  H. Tsuji,et al.  Structural Variation of the Anterior and Posterior Anulus Fibrosus in the Development of Human Lumbar Intervertebral Disc|A Risk Factor for Intervertebral Disc Rupture , 1993, Spine.

[26]  V. Moy,et al.  Atomic force microscopy measurements of protein-ligand interactions on living cells. , 2005, Methods in molecular biology.

[27]  Rupal V Patel,et al.  Microstructural and elastic properties of the extracellular matrices of the superficial zone of neonatal articular cartilage by atomic force microscopy. , 2003, Frontiers in bioscience : a journal and virtual library.

[28]  J. Melrose,et al.  A comparative analysis of the differential spatial and temporal distributions of the large (aggrecan, versican) and small (decorin, biglycan, fibromodulin) proteoglycans of the intervertebral disc , 2001, Journal of anatomy.

[29]  C. Rotsch,et al.  AFM IMAGING AND ELASTICITY MEASUREMENTS ON LIVING RAT LIVER MACROPHAGES , 1997, Cell biology international.

[30]  A. Engel,et al.  Surface and subsurface morphology of bovine humeral articular cartilage as assessed by atomic force and transmission electron microscopy. , 1996, Journal of structural biology.

[31]  Midhat H Abdulreda,et al.  Force spectroscopy of LFA-1 and its ligands, ICAM-1 and ICAM-2. , 2006, Biomacromolecules.

[32]  Jeremy J Mao,et al.  Cytoskeletal Changes of Mesenchymal Stem Cells During Differentiation , 2007, ASAIO journal.

[33]  M Radmacher,et al.  Measuring the elastic properties of biological samples with the AFM. , 1997, IEEE engineering in medicine and biology magazine : the quarterly magazine of the Engineering in Medicine & Biology Society.

[34]  A. M. Ahmed,et al.  Stress analysis of the lumbar disc-body unit in compression. A three-dimensional nonlinear finite element study. , 1984, Spine.

[35]  M. Radmacher,et al.  From molecules to cells: imaging soft samples with the atomic force microscope. , 1992, Science.

[36]  Mj Jackman,et al.  Biomechanics of the human spine , 1978 .

[37]  Van C. Mow,et al.  Structure and function of articular cartilage and meniscus , 2005 .

[38]  A Ratcliffe,et al.  Compressive mechanical properties of the human anulus fibrosus and their relationship to biochemical composition. , 1994, Spine.

[39]  A. Race,et al.  Effect of loading rate and hydration on the mechanical properties of the disc. , 2000, Spine.