Bone Biomechanics Image Analysis Back to Research

Cellular and Molecular Mechanisms of In Vivo Trabecular Bone Response to Mechanical and Hormonal Stimulation

Trabecular bone adaptation plays a significant role in the etiology of many metabolic bone diseases such as osteoporosis, osteopetrosis, bone loss in microgravity and the long term success or failure of porous implants in total joint arthroplasty. Parathyroid hormone (PTH) is an important anabolic agent when administrated intermittently. The long term goal of this research program is to use an in vivo model to study the biophysical signal transduction pathways mediating bone remodeling. In this project, the trabecular bone response to combined mechanical loading and PTH stimulation is quantified utilizing a novel in vivo rat tail vertebra model coupled with µCT image based finite element technique. Specifically, a controlled mechanical load is applied to a rat tail vertebra and detailed 3D stress/strain environment in the trabecular bone tissue is determined using an advanced finite element microstructural model of the same vertebra. The cellular and molecular responses are quantified using histomorphometric, RT-PCR and in situ hybridization techniques in the presence or absence of intermittent PTH treatment. This approach begins to elucidate the quantitative relationship between local cellular bone response and local mechanical environment.

In Vivo Rat Tail Model and µCT based Finite Element Modeling

Wolff's Law of Trabecular Adaptation

Related Publications

  1. Guo, X. E., Eichler, M. J., Takai, E. and Kim, C. H. (2002) A Rat Tail Vertebrae Model for Trabecular Bone Adaptation Studies, J. Biomech. 35, pp. 363-368. Link
  2. Kim, C. H., Takai, E., Zhou, H., von Stechow, D., Müller, R., Dempster, D. W. and Guo, X. E. (2003) Trabecular Bone Response to Mechanical and Parathyroid Hormone Stimulation: The Role of Mechanical Microenvironments, J. Bone and Miner. Res., 18(12), p2116-2125. Link
  3. Kim, C. H., Takai, E., Culella, N., and Guo X. E., Measurements of In Vivo Strains in the Rat Tail Vertebra, Proc. 2002 ASME IMECE2002, BED, New Orleans, LA, November 17–22, 2002. Download

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Cellular and Molecular Mechanisms of In Vivo Intervertebral Disc Response to Mechanical Loads 

Excessive or abnormal compressive forces on human IVDs are associated with accelerated degeneration and altered composition. In vivo animal models offer the ability to gain temporal development of disc degenerative disease under investigator imposed mechanical interventions. We have applied the rat tail dynamic loading model for in vivo studies of the IVD response to loading using the in vivo rat-tail compressive loading device described above. Detailed measurements of IVD biochemical and mechanical properties are taken and to employed in constitutive models used to describe the material behavior of the IVD as a soft hydrated tissue, as demonstrated in our studies of articular cartilage ( e.g. multiphasic nature and anisotropy).

Control IVD
IVD Loaded at 10Hz

Histology Sections of IVD (20X). The H & E Staining

 
Histology Sections of IVD (20X). The Safranin-O Staining of the IVD

Related Publications

  1. Kim, C. H., Han, S. H., Chen, F. H., Chan, M. L., Ateshian, G. A., Hung, C. T., Guo, X. E. Intervertebral Disc Response to In Vivo Dynamic Loading in a Rat-Tail Model, 49th Orthopaedic Research Society Annual Meeting, 2003. Download
  2. Han, S. H., Ho, M. M., Kim, C. H., Chen, F. H., Weidenbaum, M., Ateshian, G. A., Hung, C. T., and Guo, X . E., In Vivo Hyperphysiologic Load at High Frequencies is Detrimental to Intervertebral Disc Composition in Rat Tails, 50th Orthopaedic Research Society Annual Meeting, 29, San Francisco, Ca, March 7-10, 2004. Download

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3D Trabecular Bone Explant Culture Model

Osteocytes are mature bone cells, which are encased in three-dimensional (3D) mineralized extracellular bone matrix, and are interconnected through numerous intercellular processes that extend through the canaliculi channel system. Osteocytes form gap junctions with osteoblasts, and are thought to play an important role in the coordination of osteoclast and osteoblast activities in bone adaptation. However, few studies have demonstrated direct interactions between osteoblasts and osteocytes in vitro, and no studies have examined these interactions with osteocytes in their native 3D environment under mechanical loading. We have developed a trabecular bone explant culture that allows osteocytes to be cultured in their native 3D bone matrix environment, and also allows controlled cell seeding on the surface of the trabeculae, such as osteoblasts. This explant culture can be subjected to well-defined mechano-chemical stimuli such as hydrostatic pressure loading or hormonal stimulation to study mechano-signal transduction in bone cells in situ.

Bone Core with Live Osteocytes

Bone Core with Live Osteocytes and Seeded Osteoblasts

Related Publications

  1. Takai, E., Mauck, R.L., Hung, C.T., and Guo, X.E. (2004) Osteocyte Viability and Regulation of Osteoblast Function in a 3D Trabecular Bone Explant Under Dynamic Hydrostatic Pressure, J. Bone and Miner. Res.,19(9):1403-10. Link
  2. Takai, E. , Huang, M., Mauck, R.L., Hung, C.T., and Guo, X.E. Osteocytes Regulate Osteoblast Function in a 3D Trabecular Bone Explant Under Dynamic Hydrostatic Pressure, 50th Orthopaedic Research Society Annual Meeting, 29, San Francisco, Ca, March 7-10, 2004. Download
  3. Takai, E., Kinnebrew, G. H., Hung, C. T. and Guo, X. E. Co-culture of Osteocytes and Osteoblasts in a 3D Trabecular Bone Explant, 49th Orthopaedic Research Society Annual Meeting, 2003. Download

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Tissue Engineering of Osteochondral Constructs

Since partial defects in articular cartilage do not heal spontaneously, whereas injuries that penetrate the subchondral bone undergo some repair, we in collaboration with the Tissue Engineering Laboratory at Columbia University, are developing osteochondral tissue engineered constructs in order to enhance graft-host tissue interactions after implantation in vivo . Using a custom-made cell seeder, osteoblast-like cells obtained from trabecular bone chips are seeded onto live or devitalized trabecular bone cores. Then a custom template is used to create osteochondral constructs of bone cores with agarose hydrogels containing chondrocytes. Both osteoblasts and chondrocytes maintain their phenotypes in this co-culture. Since this co-culture approach maintains the 3D extracellular matrix environment of the osteocytes, and osteoblasts there is a potential to utilize this system for mechano-signal transduction studies between different bone cells in situ , in addition to the development of new bone tissue engineering using live bone scaffolds.

Osteochondral Construct: Bone Core with Agarose Hydrogel Containing Chondrocytes

Live-Dead Staining of Bone-Gel Interface Region (Green=Live Cells; Red=Dead Cells)

Related Publications

  1. Hung, C.T., Lima, E.G., Mauck, R.L., Takai, E., LeRoux, M.A., Lu, H.H., Stark, R.G., Guo, X.E., and Ateshian, G.A. (2003) Anatomically shaped osteochondral constructs for articular cartilage repair, J. Biomech. 36(12), p1853-1864. Link
  2. Takai, E., Lima, E., Lu, H. H., Ateshian, G. A., Guo, X. E. and Hung, C. T. Natural Trabecular Bone as a Mineralized Substrate for osteochondral Tissue Engineered Hydrogel Constructs, 49th Orthopaedic Research Society Annual Meeting, 2003. Download

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Osteocyte Mechanosignal Transduction

During the past three decades, there have been extensive in vitro studies addressing mechano-signal transduction mechanisms in bone cells. Osteocytes, mature bone cells comprising the bone cell population that exist in the greatest number in vivo, are encased in three-dimensional mineralized extracellular bone matrix (in lacunae), and are interconnected through numerous intercellular processes that extend through the canaliculi channel system. In this respect, osteocytes form a cell network, linked by gap junctions that are ideally situated to sense and respond to mechanical events that arise from normal physiological loading, such as fluid shear. Accordingly, there has been a great deal of focus on the response of osteocytes to a variety of mechanical stimuli including fluid shear. To gain the greatest insights into bone mechanotransduction, it is critical to develop a system that can both establish normal osteocyte communication in an environment that closely resembles that in situ and that can permit application of physiologic levels of pressure-driven fluid flow in vitro. Our approach to this problem is to incorporate microfabrication techniques and self-assembled monolayers (SAMs) to develop novel 2D osteocyte network cultures as well as a 3D microfluidics system to investigate bone cell mechanotransduction.
Fibronectin Patterned Glass Coverslip; Circles are 20µm in Diameter and the Lines are 50µm Long x 2µm Wide
Osteocytes Cultured in a Network Pattern
 
Calcium Resoponse of Bone Cells in Network Pattern . Center Cell is Stimulated by Indentation Using an Atomic Force Microscope

Related Publications

  1. Tang, Z., Chao, G., Tucay, A., Takai, E., Djukic, D., Lind, M.L., Hung, C.T., Guo. X.E., West, A., Osgood, R., and Yardley, J.T. (2003) XYZ on a Chip: Nanoscale Fabrication, Fluidics, and Optics Directed Toward Applications Within Biology and Medicine, in Organic Nanophotonics, Edited by F. Charra, V.M. Agranovich, and F. Kajzar, Kluwer Academic Publishers, Dordrecht, Netherlands, pp127-138. Link
  2. Takai, E. , Hung, C.T., Tucay, A., Djukic, D., Linde, M. L., Costa, K.D., Yardley, J.T., and Guo, X.E., Design of a Microfluidics System for 3D Culture of Osteocytes In Vitro, Proc. 2002 ASME IMECE2002, BED, New Orleans, LA, November 17–22, 2002. Download
  3. Takai, E. , Hung, C.T., Costa, K.D., Yardley, J.T., and Guo, X.E. Controlled Culture of Bone Cellular Networks in 2D and 3D, 50th Orthopaedic Research Society Annual Meeting, 29, San Francisco, Ca, March 7-10, 2004. Download

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Osteoblast Extracellular Matrix Protein Interactions

The nature and adhesion strength of osteoblasts bound to various extracellular matrix (ECM) proteins as well as the subsequent cellular events and cytoskeletal reorganization have not been fully elucidated nor quantified. Various ECM proteins such as type I collagen (COLI), fibronectin (FN), and vitronectin (VN) are known to modulate osteoblast adhesion, signaling responses, morphology, and phenotypic expression. These ECM proteins are also believed to promote cell adhesion via integrins. Integrin mediated adhesion has been shown to activate focal adhesion kinase (FAK), which plays a role in cytoskeletal reorganization. FAK mediated cytoskeletal reorganization may change the mechanical properties of the cell and influence adhesion strength. Furthermore, these changes in the cell are likely to affect biological responses of the cell under mechanical stimuli such as fluid shear or stretch. Our goal is to determine how adhesion of osteoblasts to various ECM protein alter their cytoskeleton and morphology, and to examine the subsequent changes in cell stiffness and adhesion strength. In collaboration with the Cardiac Biomechanics Laboratory at Columbia University, we have been incorporating Atomic Force Microscopy (AFM) to determine the apparent Young’s modulus of osteoblasts adherent to various ECM proteins.

Bioscope AFM Mounted on an Olympus IX 70 Inverted Microscope
AFM Probe Tip Indenting an Osteoblast
   
AFM Imaging Mode: Osteoblast Adhered to Fibronectin Coated Surface
AFM Imaging Mode: Osteoblast Adhered to Glass Surface

Related Publications

  1. Takai, E., Katz, R. W., Landesberg, R., Hung, C. T., and Guo, X. E. Adhesion Strength, Focal Adhesion Kinase Activation, and Cytoskeletal Organization of Osteoblasts on Various Substrates, Trans. 48th Orthopaedic Research Society Annual Meeting, 27:535, Dallas, TX, Feb. 10-13, 2002. Download
  2. Guo, X. E., Shyu, J., Takai, E., Hung, C. T. and Costa, K. D. Substrates Influence Osteoblast Elastic Modulus Measured by Atomic Force Microscopy, Trans. 48th Orthopaedic Research Society Annual Meeting, 27:521, Dallas, TX, Feb. 10-13, 2002. Download

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Research Grants

1. Biomedical Engineering Research Grant: Quantification of In Vivo Cellular Adaptation of Trabecular Bone by Mechanical Stimulation, The Whitaker Foundation, 1/1/98-12/31/2000.

2. 1RO3 AR45832: Bone Response to Combined PTH and Mechanical Stimulation, The National Institute of Musculoskeletal, Arthritis and Skin Diseases and American Society for Bone and Mineral Research, 10/1/98-9/30/2001.

3. Whitaker Foundation TF 97-0086: Gene Expression of Bone Cells in Response to Combined Mechanical and PTH Stimulation, 12/15/2001-12/14/2002

4. NIH/NIAMS RO1 AR48287: Bone Response to Combined Mechanical and PTH Stimulation, 5/1/2002-4/30/2006

5. NIH/NIAMS R21 AR4879 (PI: Clark T. Hung): Novel Determination of Chondrocyte Material Properties, 3/1/2002-2/28/2005

6. NIH/NIAMS RO1 AR049922 (PI: Clark T. Hung): Intervertebral Disc Response to Cyclic Loading In Vivo, 9/15/2002-9/14/2006

Bone Biomechanics Image Analysis Back to Research