Structure-Function Relations in Mineralized Tissues

Damage Accumulation in Bone

During normal activity, the skeleton is subjected to repetitive loads.  Bone must resist this loading and balance the processes of damage and repair to maintain its structural integrity.  Loss of structural integrity, however, is a significant medical and economic problem.  For example, about 250,000 hip fractures occur each year in people over the age of 50.  Of particular importance is that bone fractures in the elderly can result in significant morbidity, and the costs related to fracture are believed to be over $7B/yr.  The build-up of damage (due to a true increase in damage accumulation or deficiencies in biological mechanisms of repair) may be a contributing factor to increased skeletal fragility with age.  Damage accumulation and fracture are also associated with prolonged exercise, degenerative diseases and other insults that inhibit remodeling, such as some drugs.

A more quantitative understanding of the mechanisms controlling tissue damage is dependent upon understanding the relationship between the formation and progression of damage and mechanical history.  Accordingly, an objective of this aspect of our program is to detect, analyze and predict damage accumulation in bone as a function of mechanical history.  This objective is achieved, in part, by utilizing acoustic emission (AE) (Kohn et al. J. Mater. Sci. 1992, Crit. Rev. Biomed. Engr. 1995, Handbook of Adv. Mater. Testing 1995). By detecting damage at an early stage and establishing relations between mechanical history, AE signal parameters and damage morphology, important insights into mechanisms of damage nucleation and growth have been developed (Rajachar et al. ASME 1999, ASTM 1999, J. Biomech. accepted). Based on pattern recognition analyses of AE signals, we are able to classify specific signal parameters with specific ultrastructural damage mechanisms, at a probability level of 90-95% (Chow et al. Ann. Biomed. Engr. submitted). Histological observation of microcracks, combined with associated AE waveforms, provides evidence that incipient damage manifests itself in a shearing mode. The results of these analyses may ultimately lead to non-invasive diagnostics. Toward this end, we are developing the capability to use AE during in-vivo loading.

Relationships Between Ultrastructural Deformation and Local Tissue Composition

A second important aspect of this research is to study the relationship between mechanical history and damage in bone at various levels of organizational hierarchy.  We analyze damage at the tissue level - histologically; at the ultrastructural level – using x-ray and spectroscopic methods to relate damage to crystal orientation and chemical composition; and at the molecular level - by analyzing protein and gene expression.  As a means of relating mechanical properties to chemical and ultrastructural parameters measured at the same dimensional scale, nano-scale mechanical properties have been measured via atomic force microscopy (AFM) (Tsai et al. MRS 1999, Arch. Oral Biol. submitted).  An understanding of local, sub-micron-level mechanical properties not only yields information about the relative contribution of individual tissue constituents, but may also provide a better understanding of disease and regenerative processes.  

Some of our most recent and exciting results relate to the coupling between mechanical and chemical changes in bone.  By combining mechanical testing with chemical information attained via Raman spectroscopy, molecular level changes can be observed in the mineral phases and the organic supporting matrix as bone is subjected to increasing stress or strain, and comparative spectroscopic markers have been determined for damaged and undamaged regions of bone (Timlin et al. Anal. Chem. 2000, Carden et al. SPIE 2001, 2002, Calcif. Tissue Int. 2002).  Our collective data, which includes dimensional scales of damage ranging from 1-10 mm to several mm, has demonstrated that additional significant phosphate species, as well as organic species, evolve when bone is mechanically deformed.  These observations have been made in real-time during static and dynamic axial deformation of whole bones, as well as from analyzing the same region of sub-section of bone before and after localized indentation.  During the former types of tests, changes in Raman data are correlated with increases in stress and strain, whereas during the latter tests, Raman spectra are correlated with local load and moduli.  Secondary inorganic and organic species are therefore believed to result from damage, rather than be a causative factor, and we propose that bone responds to mechanical strain by undergoing a pressure-induced phase transformation and/or spatial segregation of crystallites. The information gleaned from these studies gives valuable insight into the mechanical behavior of bone at a previously unobserved level and provides evidence that bone can resist damage and undergo microstructural adaptation via an acellular physical/chemical pathway. 

Mechanically-Mediated Tissue Adaptation 

Continuing experiments started while on sabbatical at NIH, we now also seek to study cellular and molecular aspects of mechanically-mediated damage and adaptation.  We seek to relate loading parameters to biological output (e.g. damage, histology, histomorphometric parameters, gene expression).  Utilizing these approaches, we have undertaken studies in 3 general areas: (1) analysis of in-vivo induced damage; (2) analysis of age-related alterations in skeletal response to in-vivo loading; and (3) analysis of how bones with genetic alterations respond to mechanical loading.  This third approach utilizes several transgenic models of musculoskeletal disease. 

Regarding the first area, based on advances made with AE analysis of in-vitro induced damage, we now seek to develop an in-vivo model of microdamage and, utilizing AE, test the hypothesis that the magnitude and morphology of damage accumulation in-vivo is dependent upon mechanical history and age.  The results of these studies may lead to the use of AE as a non-invasive diagnostic tool.  Regarding the second and third areas, we utilize transgenic and aged mouse models to study in-vivo response to mechanical loading.  We hypothesize that mechanical integrity of bone is related to phenotypic expression of osteocytes and/or ligands produced by other cell types.  This hypothesis is tested via mechanically loading normal and genetically altered mice (initially, with bigylcan knock outs, which result in an osteoporotic-like phenotypic), and evaluating an integrated set of hierarchical properties by analyzing mechanical, histological and histomorphometric data, along with relative patterns of protein and matrix gene expression.  Relatively short-term mechanical loading is able to partially reverse the osteoporotic phenotype, as indicated by QCT measurements, histomorphometry and mechanical testing (Kohn et al., 2002a, in preparation).  In addition, Western Blots, immunohistochemistry and in-situ hybridization indicate a compensatory up-regulation of non-collagenous proteins, both as a molecular response to the absence of the biglycan gene, as well as in response to mechanical loading (Kohn et al. 2002b, in preparation).  Additionally, harvesting and culturing stem cells from loaded bones revealed an increase in colony forming unit efficiency, even after as little as 2 days of loading.  This result is indicative of a memory effect retained through the culturing period, and may have therapeutic benefits in creating functional tissue equivalents for transplantation.  Finally, to address the hypothesis of osteocyte regulation, we developed a strategy to isolate osteocytes from intact bone and extract mRNA from this isolated cell compartment (Kohn et al. 2002c, in preparation).  This strategy us now being used to analyze alterations in gene expression induced by alterations in mechanical loading.

OVERVIEW

Common hip associated fractures

Quantitative assessment of damage

Heirarchical structure of bone

Chemical changes due to deformation

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