0702 - Age-dependence of Osteocyte Lacunar Density Is Sexually Dimorphic in Human Vertebral Cancellous Bone

INTRODUCTION Human vertebral cancellous bone is one of the primary sites for age-related bone loss. In aging females bone loss occurs due to an increased rate of resorption while in males, bone loss results from a decline in bone formation rate (1). This sexual dimorphism in the mechanism of bone loss may be a consequence of the changes in endocrine, paracrine and autocrine factors which regulate bone cell differentiation and proliferation, however, any dimorphism at the cellular level is not fully understood. Several recent studies have examined the role of osteocytes in the control of bone remodeling. It is accepted that the osteocyte response to a mechanical or biological event can be release of an anabolic or catabolic signal. Thus, sexual dimorphism in the mechanism of vertebral cancellous bone loss may be related to differences in the population or response of osteocytes. The objective of this study was to investigate the age and gender related differences in the osteocyte population and its relationship with bone volume fraction in human vertebral cancellous bone. Osteocyte lacunar density (a measure of the osteocyte population) and its relationship with age and bone volume fraction was found to be sexually dimorphic. METHODS Previously prepared histological sections from 19 white males (Ages 36-86), 16 black males (Ages 37-96), 12 white females (Ages 25-89) and 17 black females (Ages 23-91) were used. Preparation of these sections included drilling an 8 mm diameter core from each human T12 vertebral body followed by en bloc staining in 1% basic fuchsin followed by sectioning (3 sections per donor) and polishing of undecalcified sections to 100 μm thickness (3). For morphometric analyses and quantification of osteocyte lacunae, blue light epifluorescence was used as it penetrates only the first few microns and osteocyte lacunae could be identified due to the fluorescence of basic fuchsin present at the lacunar edges and canalicular processes. One section per donor was randomly chosen and quantified using a standard point counting stereological techniques at 125X magnification. Output included the total number of osteocyte lacunae per bone area (Ot.Lc.Dn.), number of osteocytes per total area (Ot.Lc.N/TA) and bone volume fraction or BV/TV (BV/TV = Bone area/Total area). For statistical analyses, non-parametric tests were used as not all variables in this study were normally distributed. The relationships between Ot.Lc.Dn. and Ot.Lc.N/TA with age and BV/TV were analyzed for gender difference using multiple regression models. RESULTS No race related differences in the osteocyte lacunar density (Ot.Lc.Dn.) (males:p=0.54; females:p=0.94) were found. Osteocyte lacunar density (Ot.Lc.Dn.) increased significantly with age in females (p=0.002). In contrast, males showed a non-significant decrease in the osteocyte lacunar density (Ot.Lc.Dn.) with age (p=0.233) (Fig. 1). Age adjusted osteocyte lacunar density (Ot.Lc.Dn.) was significantly higher in females (644+123 #/mm) than males (435+130 #/mm) (p<0.001). The number of osteocyte lacunae per total area (Ot.Lc.N/TA) showed significant negative and positive correlations with age (not shown) and BV/TV (Fig.2), respectively, in both males and females (p<0.001). Age adjusted comparisons of the number of osteocyte lacunae per total area (Ot.Lc.N/TA) indicated that females had a higher number of osteocyte lacunae per total area than males (p< 0.001). DISCUSSION Sexual dimorphism in the microanatomy of human vertebral cancellous bone is considered to be related to the different age-related mechanisms of bone loss (1,2). The results of our study have identified gender related differences in the two measures of osteocyte population (Ot.Lc.Dn. and Ot.Lc.N/TA) and the relationship of osteocyte lacunar density (Ot.Lc.Dn.) with age. In this study osteocyte lacunae were regarded to accurately represent the osteocyte population. In the spine, a constant osteocyte viability of 92 + 4% is maintained with aging (4). The gender related differences in the osteocyte lacunar density (Ot.Lc.Dn.) and its dependence on age provide a possible explanation to the sexually dimorphic mechanism of bone loss in human vertebral cancellous bone. Studies on transgenic mice (5) suggest that transforming growth factor beta (TGF β) increases differentiation of osteoblasts into osteocytes leading to an increase in the number of osteocytes. Levels of TGF β in bone’s local microenvironment are controlled and regulated by several factors including estrogen (6) and androgen (7) levels which are sexually dimorphic. Recent evidence shows that estrogen withdrawal causes an increased production of TGF β in osteoclasts (8) and compared to men, postmenopausal women have higher levels of TGF β in their bone matrix (9). A relatively greater amount of TGF β present in these situations could promote differentiation of osteoblasts into osteocytes resulting in a higher number of osteocytes in females than in males. Nevertheless, it should be noted that an increased differentiation of osteoblasts into osteocytes may not necessarily occur with a concomitant increase in bone matrix as the results of this study demonstrate that females have less bone matrix per osteocyte [Females had a higher number of osteocyte lacunae (Ot.Lc.N/TA) and a higher osteocyte lacunar density (Ot.Lc.Dn.) than males]. Thus, it would appear that the increased differentiation of osteoblasts into osteocytes in females does not compensate for postmenopausal bone loss. The relationship between the number of osteocytes per total area (Ot.Lc.N/TA) and BV/TV was also sexually dimorphic suggesting gender related differences in mechanotransduction by osteocytes and/or regulation of bone matrix. A higher density of osteocytes gives bone a greater capacity to respond to an external stimuli. This may result in the existence of a more sensitive system in females than in males. REFERENCES 1)Aaron et al. CORR 215:260-71,1985; 2)Mosekilde Bone 10:425-32,1989; 3)Wenzel etal. Bone 19:89-95,1996; 4)Dunstan et.al. Cal. Tiss. Int.53:S113-6, 1993; 5)Erlebacher et al. Mol. Bio. Cell 9:1903-18, 1998; 6)Hughes and Boyce The Endocri.. 21:55-61, 1998; 7)Gill et al. Endocin. 139:546-50, 1998; 8)Robinson et al., Endocri. 137:615-21, 1995; 9)Pfeilschifter et al. JBMR 13:716-30, 1998.