Randee L. Hunter, Amanda M. Agnew Skeletal Biology Research Laboratory, The Ohio State University
INTRODUCTION
MATERIALS AND METHODS
o Increasing age has been shown to increase fracture risk independently from measured bone mass or mineral density (Seeman 2007). Determining factors of bone quality include mass, microarchitecture, material properties of the extracellular matrix, microdamage accumulation, osteocyte density, and remodeling rate (Burr 2014, Burr and Akkus 2014).
o Cortical bone samples from the distal third of the radius, the midshaft 6th rib, and the midshaft femur were obtained from 30 cadavers (15 males and 15 females) ranging in age from 49 to 100 years old. Undecalcified transverse thin (80 µm) sections were prepared using standard histological procedures and subsequently imaged under bright field light.
o Osteocytes and their extensive lacunocanalicular network form a functional syncytium capable of controlling their local microenvironment which in turn governs the bone as a whole (Bonewald 2011). The density of apoptotic osteocytes has been significantly correlated with indices of remodeling (Bellido 2014, Hedgecock et al. 2007). Increased osteocyte apoptosis is caused by a multitude of systemic and mechanical factors including increased reactive oxygen species (Almeida 2010), microdamage accumulation, endogenous glucocorticoids, and nutrient deprivation (Bellido and Hill 2014, Bonewald 2011). By influencing the components of the basic multicellular unit (BMU), osteocyte density has a direct impact on intracortical porosity.
o The influence of age and sex on regional osteocyte lacunar density (Ot.Lc.N/B.Ar) and intracortical porosity (%Po.Ar) were investigated separately using Pearson correlations. Regional relationships between Ot.Lc.N/Ct.Ar and %Po.Ar were quantified for males, females and the total sample. Lastly, correlations were performed to investigate the intraindividual or systemic relationship of osteocyte lacunar density changes with age for each anatomical site
o All variables were collected using ImageJ and/or ArcGIS® software (see Table 1 and Figure 1).
Variable
Abbreviation
Description
Total Subperiosteal Area
Tt.Ar
Total cross sectional area (mm2)
Total Cortical Area
Ct.Ar
Area between periosteal and endosteal borders (mm2)
Po.Ar
Total area of all pores located within the cortex excluding lacunar area (mm2)
Porosity Area
Method Manual Manual Semi-automatic B
o This study is the first to analyze human cortical bone variation in osteocyte lacunar density from multiple skeletal sites to establish regional and systemic age and sex related trends. Using their lacunae as a proxy, the objective of this project is to explore the effects of increasing age and sex related trends on osteocyte viability at these anatomical sites. Additionally, the regional and systemic relationships between osteocyte lacunar density and intracortical porosity are investigated.
Amount of cortical bone present (mm2)
Ct.Ar-Po.Ar
%Po.Ar
Area of intracortical porosity relative to cortical area
Po.Ar/Ct.Ar
Ot.Lc.N
Number of osteocyte lacunae within the cortex
Bone Area
B.Ar
Percent Porosity Osteocyte Lacunar Number
Osteocyte Lacunar Density
Automatic
Ot.Lc.N/B.Ar
Number of lacunae present relative to amount of cortical bone (#/mm2)
Ot.Lc.N/B.Ar
Ot.Lc.N/Ct.Ar
Number of lacunae present relative to cortical area (#/mm2)
Ot.Lc.N/Ct.Ar
A
C
o Intraskeletal variation in changes of osteocyte density is important to quantify in order to understand the effects of age and sex on differential skeletal fragility.
Table 1: Bone size and quality variables measured or calculated are defined here. All variables for the rib and radius were collected using ImageJ. Due to the size of the femoral cross-sections, Tt.Ar and Ct.Ar were measured using ArcGIS software.
Figure 1: Example of Ot.Lc.N and Po.Ar quantification demonstrated on PMHS 6882 distal radius (A). B) ImageJ automatically detects and counts osteocyte lacunae outlined in red overlay masks. C) Semi-automated porosity area (Po.Ar)
RESULTS AND DISCUSSION
CONCLUSIONS
o Mean Ot.Lc.N/B.Ar values per anatomical site are demonstrated in Figure 2. There were no significant sex differences in osteocyte lacunar density or intracortical porosity (p-value ranges from 0.302 to 0.954) allowing for pooling of the sample to investigate systemic trends.
o Osteocyte lacunar density decreases in both males and females with increasing age due to systemic and mechanical influences on the viability of the cells.
o Osteocyte lacunar density (Ot.Lc.N/B.Ar) decreased with age in the radius, rib and femur for males and females (see Figure 3). The femur displayed the largest range in Ot.Lc.N/B.Ar (515.17 mm-2 to 1222.95 mm-2) compared to the radius (738.26 mm-2 to1301.56 mm-2) or the rib (620.91 mm-2 to1128.81 mm-2 ). o Ct.Ar, rather than B.Ar, was used to calculate lacunar density for correlations with %Po.Ar. Regionally, Ot.Lc.N/Ct.Ar was significantly negatively correlated with intracortical porosity in all elements further supporting the relationship between apoptotic osteocytes and BMU initiation and regulation (see Figure 4). o For the total sample, Ot.Lc.N/B.Ar demonstrate no clear systemic relationship between elements (see Table 2). This suggests that despite being under the same systemic changes occurring with age, osteocyte viability is differentially affected by these factors potentially mitigated by the local mechanical environment and the sensitivity of the cellular population. Males: r= -0.604 (p=0.009) Females: r= -0.335 (p= 0.112) All: r=-0.456 (p=0.006) R2= 0.208
Males: r= -0.290 (p=0.147) Females: r= -0.405 (p=0.067) All: r=-0.337 (p=0.034) R2= 0.114
Males: r= -0.519 (p=0.029) Females: r= -0.039 (p=0.446) All: r=-0.241 (p=0.104) R2= 0.058
Femur- Rib Femur-Radius Rib-Radius Variable r (p-value) r (p-value) r (p-value) Ot.Lc.N/B.Ar -0.125 (0.519) 0.312 (0.093) -0.050 (0.798) %Po.Ar 0.566 (0.001) 0.643 (0.000) 0.425 (0.022) Table 2: Pearson correlation coefficients (r) for intraskeletal changes in Ot.Lc.N/B.Ar and %Po.Ar
Interval Plot Osteocyte Lacunar Density 95% CI for the Mean
A
Males: r=-0.638 (p=0.003) Females: r=-0.560 (p=0.015) All: r=-0.626 (p=0.000) R2= 0.392
Males: r=-0.685 (p=0.003) Females: r=-0.790 (p=0.000) All: r=-0.746 (p=0.000) R2= 0.556
Males: r=-0.506 (p=0.033) Females: r=-0.476 (p=0.037) All: r=-0.486 (p=0.004) R2= 0.236
Density (Ot.Lc.N/B.Ar)
Figure 3: Regional trends in osteocyte lacunar density with age. Pearson correlation coefficients (r) reported separately for males and females in investigate the influence of sex on Ot.Lc.N/B.Ar decrease with age. With no significant differences between males and females, the sample was pooled and R2 is reported.
B
1200
1100
o In determining bone quality to assess skeletal fragility, the amount of intracortical porosity is crucial to the prevention of fracture. Osteocyte apoptosis, despite its cause, regulates and controls the initiation of bone resorption. Intracortical porosity increases with age and is moderately to strongly (r=-0.486 to -0.790) correlated with decreases in osteocyte lacunar density at each anatomical site. o This study demonstrates the variation in osteocyte lacunar density with age and sex both regionally and systemically. The lack of intraskeletal correlations suggests a mitigating effect of mechanical influences on osteocyte viability. Decreases in osteocyte cellular populations have a measurable effect on bone quality manifested as intracortical porosity. CONTACT INFORMATION
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[email protected]
REFERENCES CITED
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800 Sex
Figure 4: Regional osteocyte lacunar density correlations with intracortical porosity. Pearson correlation coefficients reported for males and females separately. With no significant differences between males and females in either Ot.Lc.N/Ct.Ar or %Po.Ar, the sample was pooled and R2 is reported.
o The femur demonstrates the largest range of Ot.Lc.N/B.Ar (followed by the radius then the rib). With increasing age and less mobility, the femur may experience the greatest variation in mechanical environment between individuals whereas the rib remains consistently loaded during pulmonary ventilation. The radius may experience an intermediate amount of variation.
Males
Females Rib
Males Females Radius
Males Females Femur
Figure 2: Regional osteocyte lacunar density (Ot.Lc.N/B.Ar). Mean values for the total sample -2 -2 (n=30) for each element are B as follows: Rib: 939.23 mm ± 132.33. Femur: 939.80 mm ± 178.22. Radius: 1074.50 mm-2 ± 137.088. There are no significant differences between the femur and rib (p=1.00). Radius density values are significantly higher than the femur and rib (p= 0.003).
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ACKNOWLEDGEMENTS Thanks to the donors for their gifts. Thank you to everyone in the Skeletal Biology Research Laboratory and Injury Biomechanics Research Center who have provided great insight, and invaluable aide.
