EFFECTS OF ANATOMICAL LOCATION, AGE, GENDER, BODY MASS, HEIGHT AND BODY MASS INDEX ON ELASTIC MODULUS

OF HUMAN BONE TISSUE LAMELLAE

Participants: C. E. Hoffler, K. M. Kozloff, K. E. Moore, M. T. Dillon, M. Brown, W. Pan, P. K. Zysset, S. A. Goldstein

Keywords: bone mechanics, microstructure, nanoindentation

Introduction

Bone adapts to changing mechanical stimuli by adjusting its architecture and material properties through cell mediated processes in response to local environmental conditions. Similarly, dynamic metabolic demands locally influence bone remodeling and maintenance. Coupled together, these mechanisms prescribe the morphology, architecture and mechanical properties of bone tissue. By characterizing the mechanical properties and structure of bone tissue at increasing levels of magnification, we continue to refine our understanding of bone as the medium through which cells receive mechanical stimulation. A clearer understanding of these mechanotransduction mechanisms will lead to improved preventative and therapeutic strategies for skeletal diseases such as osteoporosis, osteogenesis imperfecta, osteomalacia or traumatic fractures.

In support of this structure-function paradigm, material characterization using nanoindentation has allowed us to approach the local mechanical environment of the cell by providing measures of lamellar level elastic properties. We have specifically quantified the influence of (1) anatomical location, (2) age and gender and (3) body mass, height and body mass index on the lamellar elastic modulus of bone.

Materials and Methods

Tissue samples for three studies were prepared using the same protocol. Specimens were sectioned along a transverse plane embedded in a weakly exothermic epoxy, surface polished and cleansed in an ultrasonic water bath. Samples were secured on platens in a custom irrigation system which maintained moisture with a gentamicin solution. The Nanoindenter II system was then used to measure the lamellar elastic modulus. Using a light microscope, indent locations were selected based on microstructure, and each location was sampled with an array of four indents.

For the anatomical location study, cortical and trabecular bone specimens were obtained from the distal radius, lateral neck and mid-diaphysis of the femur, and the fifth lumbar vertebra of 10 male cadavers (ages 41 - 85). In the distal radius, microstructures were classified as osteonal, interstitial, trabecular and primary lamellar. Femoral neck tissue was separated into osteonal, interstitial and trabecular microstructures. In the femoral diaphysis, only osteonal and interstitial tissues were present. Osteonal and interstitial tissue regions were selected in spatially adjacent pairs. Trabecular, cortical lamellar and enthesophytic microstructures were observed in vertebral specimens. All data were log normalized to correct for positive skew. A mixed model ANOVA was performed. Bone and microstructure were treated as fixed effects while region and indent number (within the array of four) were treated as random. Subject is also considered a random effect. ANOVA was followed by a Tukey's multiple range test.

For the age and gender study, lateral femoral neck bone specimens were obtained from 16 male subjects ages 40 to 85 and 11 female subjects ages 27 to 93. Age and gender effects were evaluated in the complete sample of 27 subjects and in an age-matched postmenopausal subset of 19 subjects (11 males, 8 females) beyond the age of 60. Microstructures were separated as detailed above for the anatomical location study. A general linear model was created to determine the ability of age and gender to explain lamellar level variations in elastic modulus and hardness. A similar model was developed for the beyond 60 subject group.

Finally, we examined the ability of body mass, height and body mass index to predict the elastic modulus and hardness of bone lamellae. From the original sample of 27 cadavers, three subsets were isolated based on weight and height availability. A subset of 21 subjects (12 male, 9 female) was used for the mass study while 19 subjects (10 male, 9 female) were used for the height study. Another subset of 18 subjects (9 male, 9 female) with available weight and height data was used for the height and body mass index study. Body mass index (BMI) is defined as (mass in kg)2/ (height in meters). General linear models containing weight and sex and containing height and BMI were developed.

All statistical analyses employed SAS 6.11 (SAS Institute, Inc., Cary, NC). Repeated measures analyses were performed to compare values between bone microstructures.

Results

As demonstrated in Figure 1a, significant variations between anatomical locations within the same microstructure were found. Differences were also found between microstructures within the same anatomical locations (not shown). Neither age, gender, body mass, height or body mass index were found to correlate with lamellar elastic modulus. This independence was consistent for all microstructures and is illustrated for age and trabecular lamellae in Figure 1b.

Discussion

The principal finding of the anatomical location study is that there are distinct differences in the extracellular matrix elastic properties of bone that vary consistently with anatomic location. This implies that extrapolations of bone matrix mechanical behavior from one organ to another are likely erroneous. The independence of lamellar elastic modulus and hardness from age and gender suggests that age and gender related fragility increases involve the regulation of tissue mass and organization, and not the inherent quality of the extracellular matrix. The present study also suggests that increased bone mass maintenance known to occur in heavier individuals is not accompanied by increases in the inherent mechanical quality of the extracellular matrix.

Figure 1. a. Variations in elastic modulus between corresponding tissue microstructures in different anatomic locations. Significant differences within microstructures are indicated by *, # (p=0.0001). b. Independence of trabecular tissue elastic modulus from age. All properties were measured on room temperature moist bone, loading at 10 nm/s to a maximum depth of 500 nm.