Decline in Osteocyte Lacunar Density in Human Cortical Bone Is Associated With Accumulation of Microcracks With Age

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1 Bone Vol. 26, No. 4 April 2000: Decline in Osteocyte Lacunar Density in Human Cortical Bone Is Associated With Accumulation of Microcracks With Age D. VASHISHTH, 1 O. VERBORGT, 2 G. DIVINE, 3 M. B. SCHAFFLER, 4 and D. P. FYHRIE 1 1 Bone and Joint Center, Henry Ford Hospital, Detroit, MI, USA 2 Department of Orthopaedics & Traumatology, University of Antwerp, Wilrijk, Belgium 3 Biostatistics and Research Epidemiology, Henry Ford Health Sciences Center, Detroit, MI, USA 4 Department of Orthopaedics, The Mount Sinai School of Medicine, New York, NY, USA Despite osteocytes ideal position to sense the local environment and thereby influence bone remodeling, the function of osteocytes in bone remains controversial. In this study, histomorphometric examination of male and female femoral middiaphyseal cortical bone was conducted to determine if bone s remodeling response, indicated by tissue porosity and accumulation of damage, is associated with osteocyte lacunar density (number of osteocyte lacunae per bone area). The results support the sensory role of the osteocyte network as the decline in osteocyte lacunar density in human cortical bone is associated with the accumulation of microcracks and increase in porosity with age. Porosity and microcrack density increased exponentially with a decline in osteocyte lacunar density indicating that a certain minimum number of osteocytes is essential for an operational network. No gender-related differences were found in the relationship of osteocyte lacunar density to age, porosity, or microcrack density. The coefficient of variation of osteocyte lacunar density increased linearly with age, indicating that aging bone tissue is characterized by increased heterogeneity in the spatial organization of osteocytes. Osteocyte lacunar density, porosity, and microcrack density exhibited the same exponential probability density distribution in the donor population, indicating their regulation by similar biological phenomena. (Bone 26: ; 2000) 2000 by Elsevier Science Inc. All rights reserved. Key Words: Cortical bone; Osteocyte; Lacunae; Microcracks; Porosity; Aging. Introduction Osteocytes are by far the most abundant cells in bone. In skeletally mature human bones, osteocytes are located in ellipsoidal cavities (lacunae) and are extensively connected to one another as well as to the bone forming cells or osteoblasts by cytoplasmic processes inside the canaliculi. Despite osteocytes ideal location to sense the local environment and Address for correspondence and reprints: Deepak Vashishth, Ph.D., Department of Biomedical Engineering, Jonnson Engineering Center, Room 7046, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY vashid@rpi.edu thereby influence bone remodeling, the function of osteocytes in bone remain controversial. Previous investigators noted that osteocytes are capable of bone resorption 6,7,29 and formation. 5 However, the current trend is to regard osteocytes as sensory cells that are capable of: (i) detecting the local mechanical and biochemical environments, and (ii) conveying the signal to one another and to osteoblasts, thereby influencing bone s remodeling response. 1,9,17,19,23 25,31 Osteocytes apparently can utilize multiple pathways to communicate with one another and with osteoblasts. Several studies have theorized that osteocytes execute their mechanosensory function by the transduction of mechanical loads into biochemical signals capable of influencing osteoblasts and osteoclasts. 8,31 Recent work from our laboratory has shown that osteocyte cell death by DNA fragmentation or apoptosis is induced by fatigue loading and that osteocyte apoptosis coincides with osteoclastic bone resorption. 33 In either case, a cell-to-cell communication network is apparently used by bone to sense its local environment, adapt to mechanical loading, and to control the repair of microdamage. Mori et al. 18 observed that, in human trabecular bone, an increase in microdamage coincides with a decrease in osteocyte lacunar density. This suggests that a cause-and-effect relationship exists between osteocyte lacunar density and microdamage. The aim of the present study was to extend the aforementioned concept to cortical bone and to determine if bone s remodeling response, indicated by tissue porosity and accumulation of damage, is reflected by osteocyte lacunar density. Based on Mori et al. s study, and a previous study on the age-related accumulation of microdamage in femoral middiaphyseal compact bone, 26 we hypothesized that osteocyte lacunar density would decrease with age and correlate with age-related accumulation of microdamage and increase in bone porosity. Materials and Methods Specimen Preparation Previously prepared histological sections from femoral middiaphyseal cortical bone were used for this study. The sections were obtained from 16 men (age range years, mean age years) and 9 women (age range years, mean age years) who died suddenly and had no prior history of bone diseases. The preparation of these specimens included bulk 2000 by Elsevier Science Inc /00/$20.00 All rights reserved. PII S (00)

2 376 D. Vashishth et al. Bone Vol. 26, No. 4 Decline in osteocyte lacunar density April 2000: Figure 1. Photomicrographs (original magnification 125) of cross sections of femoral middiaphyseal cortical bone (posterior region) from: (a) a 17-year-old man; (b) a 73-year-old man; (c) a 28-year-old woman; and (d) a 52-year-old woman. The difference between the osteocyte lacunar density can be clearly seen between (a) and (b) and between (c) and (d). staining of a 3-cm-long femoral middiaphyseal cylinder in 1% basic fuchsin in an ascending series of ethanol (70%, 80%, 90%, and 100%) followed by sectioning (three sections per individual) and polishing of undecalcified cross sections to approximately 100 m thickness. 26 This method of staining and sectioning allows accurate assessment of preexisting damage in the bone at the time of donor death. 10 Morphometric Analyses and Quantification of Osteocyte Lacunar Density Based on a preliminary study conducted on sections from three male and three female donors under blue-violet epifluorescent light ( nm excitation and 470 nm barrier filter) and bright light, it was decided to use blue-violet light for illumination because it penetrates only the first few microns of the bone section, allowing osteocyte lacunae to be quantified without projection due to section thickness (100 m). Osteocyte lacunae can be readily identified under blue-violet light due to the fluorescence of the lacunar edges and canalicular processes. Preliminary examination determined that: (1) measuring five fields adequately quantified the average osteocyte lacunar density of a section (as the moving average of the osteocyte lacunar density stabilized after three fields); and (2) the posterior cortex of the specimens was highly osteonal. Quantification of osteocyte lacunae was, therefore, conducted on the posterior cortex under blue-violet epifluorescent light. The osteocyte lacunae were counted using a standard point-counting stereological technique at a magnification of 125. The output included the total number of osteocyte lacunae per bone area (osteocyte lacunar density) and percent porosity. Porosity included haversian canals and resorption spaces. Three sections were counted per specimen with all sections from all donors randomized (after blinding) using a random number generator (Microsoft EXCEL, version 5.0). All data were collected by a single observer (D.V.) who was blinded to age, gender, or section identification. Statistical Analyses As the osteocyte lacunar density was not normally distributed in both genders (men: not normally distributed [Kolgomorov Smirnov distribution 0.257, p 0.006]; women: normally distributed [Kolgomorov-Smirnov distribution 0.194, p 0.20]) comparison between men and women was done using the nonparametric Mann Whitney signed-rank test (SIGMASTAT 2.0). The relationship of osteocyte lacunar density and porosity to age and between osteocyte lacunar density and previously published microcrack data 26 were analyzed for gender differences by using multiple regression models parameterized to include a term for the difference in slope (SAS system). The coefficient of variation, calculated by dividing the standard deviation by the mean,

3 Bone Vol. 26, No. 4 April 2000: D. Vashishth et al. Decline in osteocyte lacunar density 377 Figure 2. The decline in the osteocyte lacunar density (number of osteocyte lacunae per bone area) vs. age in men (a) and women (b). The decline in osteocyte lacunar density vs. age was best described by an exponential relationship in both men (p 0.001) and women (p 0.036). The osteocyte lacunar density (p 0.308) and its relationship with age (p 0.534) was not significant for men and women. Figure 3. Coefficient of variation (defined as standard deviation divided by mean [COV] vs. age for (a) men and (b) women. The relationship of COV to age was significant in men (p 0.008) but not in women (p 0.46). was also plotted with age and tested for correlation using the Spearman rank order correlation test (SIGMASTAT 2.0). Cumulative probability of occurrence vs. density/size was plotted for osteocyte lacunae, porosity, and previously published microcrack data 25 to establish how these variables were distributed in the donor population (men and women). The resulting data were curve fit using TABLECURVE 2D software (Jandel Scientific) to obtain an estimated cumulative distribution function. The cumulative distribution function was then differentiated to obtain the probability density function. In the context of this study, the probability density function describes the probability of the occurrence of a continuous variable associated with a physical process. Results Influence of Age and Gender The osteocyte lacunar density decreased with age in both men and women. Figure 1a d shows cross sections of the femoral mid-diaphysis posterior cortex of young and old male and female specimens. The decline of osteocyte lacunar density with age could be best described by an exponential relationship in both men (p 0.001) (Figure 2a) and women (p 0.036) (Figure 2b). The osteocyte lacunar density was not different between men and women (p 0.308; Mann Whitney rank sum test). Decline of osteocyte lacunar density vs. age was also not significantly different between men and women (p 0.534). The coefficient of variation of osteocyte lacunar density increased significantly with age in men (Figure 3a) (p 0.008; Spearman rank order correlation test), but not in women (Figure 3b) (p 0.46; Spearman test). Relationship of Osteocyte Lacunar Density to Microcracks and Porosity The relationships of osteocyte lacunar density to microcracks and porosity could be best described by an inverse exponential relationship in both men and women (Figures 4 and 5), and were not significantly different between men and women (p for porosity vs. osteocyte lacunar density; p for osteocyte lacunar density vs. microcracks). The inverse exponential relationships with a sharp inflexion point, shown in Figures 4 and 5, are indicative of the two distinct phases in the relationships of osteocyte lacunar density to microcracks and porosity. The first phase, characterized by higher osteocyte lacunar density ( 600 osteocyte lacunae/mm 2 ), is associated with little or no change in the microcrack density or porosity due to a decrease in the osteocyte lacunar density. In contrast, the second phase, characterized by lower osteocyte density ( 600 osteocyte lacunae/

4 378 D. Vashishth et al. Bone Vol. 26, No. 4 Decline in osteocyte lacunar density April 2000: Figure 4. Porosity vs. osteocyte lacunar density in (a) men and (b) women. The relationship was significant for both men (p 0.001) and women (p 0.025), but there was no gender-related difference (p 0.773). Figure 5. Microcrack density 26 vs. osteocyte lacunar density in (a) men and (b) women. The relationship was significant for both men (p 0.002) and women (p ), but there was no gender-related difference (p 0.299). mm 2 ), shows sharp increases in the microcrack density and porosity in response to a decrease in osteocyte lacunar density. Probability Density Distribution An exponential function of the general form: y 1 a exp[ ((x b)/c)] was consistent with the distribution of microcracks (Figure 6a), porosity (Figure 6b), and osteocyte lacunae (Figure 6c) in the donor population. The probability density functions obtained by differentiating the distribution functions for microcracks, porosity, and osteocyte lacunae are given in Table 1. Discussion The osteocyte network is believed to sense the local environment in bone and affect bone remodeling by modulating the response of the bone forming (osteoblasts) and resorbing (osteoclasts) cells. 8,31 The results of this investigation support the sensory role of the osteocyte network and suggest that it may play an important role in mechanical homeostasis of the adult skeleton. In this study no direct measurement of the osteocyte population was done using the markers of cellular integrity, and the osteocyte lacunar density was considered to be a quantitative measure of the osteocyte network. Previous studies have shown that not all the lacunae contain osteocytes, and that the percentage of empty lacunae in bone depends on the anatomical location. 11 More significantly, for purposes of the present investigation, no previous study has demonstrated a decline in the percent of empty lacunae with age. The percentage of empty lacunae has been reported to increase significantly, 12,35,36 or increase nonsignificantly, 4,20 with age. It is, therefore, reasonable to believe that the age-related decline in osteocyte density is more pronounced than the age-related decline in lacunar density. To our knowledge, this is the first time an age-related exponential decline in osteocyte lacunar density has been demonstrated for human femoral cortical bone. Previous studies on the relationship between lacunar density and age have been restricted to trabecular bone. Mullender et al. 20 found a linear decline in lacunar density with age in the iliac crest. In contrast, Mori et al. 18 reported no age-related change in lacunar density of femoral head trabecular bone until the age of 70 years, followed by a sudden sharp decline. Taken together, the results of Mullender s, Mori s, and our study suggest that the relationship between osteocyte lacunar density and age varies with anatomical location and bone type (cortical, trabecular). One may speculate on the reasons for the age-related decline in osteocyte lacunar density in cortical bone. Studies by Frost 12 have demonstrated that, with age, lacunae fill with mineralized

5 Bone Vol. 26, No. 4 April 2000: D. Vashishth et al. Decline in osteocyte lacunar density 379 Figure 6. Cumulative probability of occurrence vs. density/size for (a) osteocyte lacunar density; (b) porosity; and (c) previously published microcrack data in the donor population (men and women). tissue (micropetrosis) and, therefore, micropetrosis can cause an age-related decline in osteocyte lacunar density without any change in bone porosity. Alternatively, evidence from in vitro studies on bone resorption suggests that bone with osteocytes is resorbed more readily than bone devoid of osteocytes. 27 Thus, with increasing age, osteocyte-rich bone may be selectively resorbed. Furthermore, as bone formation activity declines and/or bone resorption activity increases with advancing age, selective resorption of osteocyte-rich bone will gradually lead to bone tissue with low osteocyte density and high porosity. Results of our study support this latter scenario, because, with advancing age, both a decline in osteocyte density (Figure 2) and an increase in porosity were observed. The coefficient of variation for osteocyte lacunar density, indicative of heterogeneity or increased variation, also increased linearly with age (Figure 3). Again, this age-related increase in the coefficient of variation is consistent with the notion that, with advancing age, certain areas of bone are preferentially resorbed and selectively repopulated with osteocytes. Such a remodeling process will lead to the clustering of osteocytes, which, in turn, will result in an increased heterogeneity in the spatial organization of osteocytes. A similar phenomenon has been observed to occur with osteons (which contain osteocytes) in equine third metacarpal bone. 16 A noteworthy finding of this study is the accumulation of microdamage in concert with the decline in osteocyte lacunar density. These results are consistent with a previous study conducted in our laboratory 22 where accumulation of microdamage was found in the areas of compromised osteocyte integrity. More recently, Mori et al. 18 also found an inverse relationship between osteocyte lacunar density and microcrack density. Our results can be best described by an exponential relationship, which indicates that, above a certain density threshold (approximately 600 #/mm 2 ) (Figure 5), an age-related decline in cortical bone s osteocyte lacunar density is associated with little or no change in microcrack density. However, once that threshold is reached, even a small change is associated with large increases in microcrack density. The relationship between osteocyte lacunar density and porosity (Figure 6) was similar to that between microcrack density and osteocyte lacunar density. Based on the aforementioned results, it is difficult to say whether the accumulation of microdamage and increased porosity cause an impaired osteocyte network or vice versa. In either case, however, the damaged and porous bone do accumulate with age, which suggests that an impaired osteocyte network is indeed associated with the failure of bone to repair itself. More significantly, from the results of this study, it appears that a minimum number of osteocytes is essential for an operational network. There are many reasons why a minimum number of osteocytes may be essential. The well-defined spatial organization of osteocytes relies on the fine network of canaliculi for the transport of metabolites. 14,15,21 Furthermore, based on the evidence from other animal cells, osteocytes can be expected to be under a social control where they rely on signals from other cells to survive. 28 The observation that, in in vitro cultures, osteocytes reestablish their network 34 supports the concept of interreliance and social control in the osteocyte population. It is believed that isolated or limited cell death by necrosis 30,32 or apoptosis 33 is unlikely to affect the efficiency of the osteocyte network. Osteocytes may function as a neural network system, communicating using protein molecules, organized in series (i.e., in a hierarchical order) or parallel or both. Such networks are common in biological systems 13 and recent studies have shown that the adaptation property of these networks is mainly a consequence of connectivity. 2,3 Therefore, it is believed that, once the network s connectivity falls below a certain minimum value, its capability for signal transduction is seriously compromised. In this study, no gender-related differences were found in osteocyte lacunar density nor in its relationships with microcracks and porosity. Osteocyte lacunar density, porosity, and microcrack density exhibited the same exponential probability

6 380 D. Vashishth et al. Bone Vol. 26, No. 4 Decline in osteocyte lacunar density April 2000: Table 1. Probability density function for the osteocyte lacunar density, porosity, and previously published microcrack data in human middiaphyseal femoral cortical bone Variable Probability density function Limits Osteocyte lacunar density 0.24e [(#Ot.Lc/mm )/140] Ot.Lc.Dn 448/mm 2, Ot.Lc.Dn 888/mm 2 Porosity 0.13e [((porosity (%)) 2.09)/8.69] Porosity 3.7%, porosity 37.9% Microcrack density 2.28e [(# microcracks/mm2 1.06)/1.07] MCD 0.001/mm 2, MCD 5.6/mm 2 density distribution in the donor population. This suggests that the occurrence of osteocyte lacunar density, microcracking, and porosity may be regulated by similar biological phenomena, such as bone turnover and aging. Acknowledgments: This study was supported by NIH Grants AR and AR Tissues were provided by the Musculoskeletal Transplant Foundation. References 1. Aarden, E. M., Burger, E. H., and Nijweide, P. J. 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