Integrity of the osteocyte bone cell network in osteoporotic fracture: Implications for mechanical load adaptation

Transcription

1 IOP Conference Series: Materials Science and Engineering Integrity of the osteocyte bone cell network in osteoporotic fracture: Implications for mechanical load adaptation To cite this article: J S Kuliwaba et al 2010 IOP Conf. Ser.: Mater. Sci. Eng View the article online for updates and enhancements. Related content - The characterization of human cortical bone microdamage by nuclear magnetic resonance Qingwen Ni and Daniel P Nicolella - A comparison between the patella and the calcaneus using ultrasound velocity and attenuation as predictors of bone mineral density S M Han and J Davis - Comparison of speed of sound and ultrasound attenuation in the os calcis to bone density of the radius, femur and lumbar spine P Rossman, J Zagzebski, C Mesina et al. This content was downloaded from IP address on 15/05/2018 at 09:38

2 Integrity of the osteocyte bone cell network in osteoporotic fracture: implications for mechanical load adaptation J S Kuliwaba 1,2, L Truong 1, J D Codrington 1,3 and N L Fazzalari 1,2 1 and Joint Research Laboratory, Directorate of Surgical Pathology, SA Pathology and Hanson Institute, Adelaide, South Australia, 5000, Australia 2 Discipline of Anatomy and Pathology, School of Medical Sciences, The University of Adelaide, Adelaide, South Australia, 5005, Australia 3 School of Mechanical Engineering, The University of Adelaide, Adelaide, South Australia, 5005, Australia Julia.Kuliwaba@health.sa.gov.au Abstract. The human skeleton has the ability to modify its material composition and structure to accommodate loads through adaptive modelling and remodelling. The osteocyte cell network is now considered to be central to the regulation of skeletal homeostasis; however, very little is known of the integrity of the osteocyte cell network in osteoporotic fragility fracture. This study was designed to characterise osteocyte morphology, the extent of osteocyte cell apoptosis and expression of sclerostin protein (a negative regulator of bone formation) in trabecular bone from the intertrochanteric region of the proximal femur, for postmenopausal women with fragility hip fracture compared to age-matched women who had not sustained fragility fracture. Osteocyte morphology (osteocyte, empty lacunar, and total lacunar densities) and the degree of osteocyte apoptosis (percent caspase-3 positive osteocyte lacunae) were similar between the fracture patients and non-fracture women. The fragility hip fracture patients had a lower proportion of sclerostin-positive osteocyte lacunae in comparison to sclerostin-negative osteocyte lacunae, in contrast to similar percent sclerostinpositive/sclerostin-negative lacunae for non-fracture women. The unexpected finding of decreased sclerostin expression in trabecular bone osteocytes from fracture cases may be indicative of elevated bone turnover and under-mineralisation, characteristic of postmenopausal osteoporosis. Further, altered osteocytic expression of sclerostin may be involved in the mechano-responsiveness of bone. Optimal function of the osteocyte cell network is likely to be a critical determinant of bone strength, acting via mechanical load adaptation, and thus contributing to osteoporotic fracture risk. 1. Introduction Osteoporosis is a progressive bone disease characterised by loss of bone strength, resulting in an increased risk of fragility fractures that occur with minimal or no trauma [1]. strength is determined by a number of inter-related variables which, in addition to bone mineral density, include bone structural properties such as whole bone geometry and microarchitecture, and bone material properties such as the degree of bone matrix mineralisation, collagen characteristics, microdamage c 2010 Published under licence by Ltd 1

3 accumulation, and the integrity of the osteocyte-canalicular cell network. The skeleton has the ability to modify its material composition and structure to accommodate mechanical loads through adaptive modelling and remodelling [1]. Osteocytes, the most abundant cell type in bone, are ideally located to influence bone remodelling through their syncytial relationship with surface bone cells and cells of the bone marrow. The osteocyte-canalicular cell network is now considered to play a central and multi-functional role in regulating skeletal homeostasis via mechanical load adaptation [2]. As such, its optimal function may be a critical determinant of bone strength, which thus contributes to fragility fracture risk. Sclerostin, a secreted protein produced almost exclusively by mature osteocytes [3,4], potently regulates osteoblast development and bone formation by binding to low-density lipoprotein receptorrelated protein (LRP)5/LRP6 and preventing canonical wingless-type (Wnt) signalling [5]. Sclerostin is a key bone formation inhibitor [6] that plays a central role in determining the normal extent of bone formation and consequently protects against the deleterious effects of uncontrolled bone growth (sclerosteosis). The role of sclerostin in osteoporotic fragility fracture is unknown. This study was designed to characterise osteocyte morphology, the extent of osteocyte cell apoptosis and expression of sclerostin protein in trabecular bone from the proximal femur, for postmenopausal women with fragility hip fracture compared to age-matched women who had not sustained fragility fracture. 2. Methods 2.1. Human bone specimens Trabecular bone cores (10 mm diameter) from the intertrochanteric region of the proximal femur were obtained from 10 postmenopausal women undergoing hemi-arthroplasty surgery for a non-traumatic subcapital femoral fracture, and from 10 postmenopausal women at total hip arthroplasty surgery for primary hip osteoarthritis. The non-fracture group comprised patients with hip osteoarthritis as there is an inverse association between osteoarthritis and osteoporosis. Individuals with osteoarthritis rarely experience osteoporosis (especially for fractures of the hip) and osteoporotic patients have a very low incidence of osteoarthritis [7]. The mean (standard deviation [SD]) age of the fracture patients was 75.8 (5.1) years (age range years) and the non-fracture women 75.5 (6.4) years (age range years). Informed consent and Royal Adelaide Hospital Research Ethics Committee approval were obtained for the collection of these bone specimens Immunohistochemistry specimens were fixed in 4% paraformaldehyde, decalcified in 15% ethylene-diamine-tetra acetic acid (EDTA), dehydrated and embedded in paraffin wax. 5 µm-thick sections were subjected to immunohistochemical localisation of activated caspase-3 (Casp3) and sclerostin (Sost) protein using the Labelled Streptavidin Biotin Method with diaminobenzidine tetrahydrochloride (DAB) as the chromogen. Sections were counterstained with haematoxylin. The optimal concentrations for anticaspase-3 (rabbit polyclonal anti-human Caspase 3 active, R&D Systems) and anti-sclerostin (mouse monoclonal anti-human SOST, R&D Systems) were determined at 6.67 and 0.40 µg/ml, respectively. Positive control tissues used were human lymph node (for Casp3) and normal iliac crest bone biopsy and postmortem femoral trabecular bone (for Sost). Negative control tissues used for Sost were human appendix and spleen. For each study case, negative controls (without primary antibody) were included Histoquantitation Osteocyte morphology and osteocyte immunostaining patterns for Casp3 and Sost were determined by counting cell and lacunar numbers using the Quantimet 550IW Image Analyser (Leica DM6000B; 20 objective). The number of osteocyte-occupied lacunae and empty (osteocyte-devoid) lacunae were quantitated; the sum of both is the number of total lacunae. The number of osteocytes (Ot), empty lacunae (EL), and total lacunae (TL) were expressed per bone area (#/mm 2 ), and percent empty 2

4 lacunae calculated as EL/TL (%). The proportion of osteocyte lacunae immuno-negative and immunopositive for Casp3 and Sost were quantified Statistical analysis The osteocyte morphometric and immunostaining data were normally distributed (Shapiro-Wilk statistic). Data were expressed as mean (SD). Differences between women with/without fragility fracture and between immuno-negative and immuno-positive data were evaluated by Student s t-test. Relationships between morphometric parameters and age were determined by regression analysis. The critical value for statistical significance was chosen as p = Results The osteocyte morphometric parameters, osteocyte density (Ot.Dn), total lacunar density (TL.Dn), empty lacunar density (EL.Dn), and percent empty lacunae (EL/TL), were not statistically different between the postmenopausal fragility hip fracture patients and the age-matched non-fracture women (Table 1). Table 1. Comparison of mean values for osteocyte and lacunar density measured in trabecular bone from the intertrochanteric region of the proximal femur between non-fracture postmenopausal women and women with fragility hip fracture a. Variable Non-fracture women Fracture patients (n = 10) (n = 10) % Difference p Ot.Dn (#/mm 2 ) 486 (98.8) 446 (79.0) TL.Dn (#/mm 2 ) 588 (121) 530 (112) EL.Dn (#/mm 2 ) (54.2) 84.9 (55.7) EL/TL (%) 17.3 (6.2) 14.4 (7.1) a Values are mean (SD). Trabecular bone tissue from the proximal femur for postmenopausal women with/without fragility fracture was immunostained for activated Casp3 to identify apoptotic cells. Positive Casp3 immunostaining was observed in the cytosol of osteocytes, lacunar walls and canaliculi in trabecular bone from both fracture and non-fracture women (Figure 1). Casp3-positive immunoreactivity was also detected in marrow cells (Figure 1a) [8]. a b c Figure 1. Caspase-3 immunostaining in osteocytes, lacunar walls and canaliculi (arrows) in trabecular bone from the intertrochanteric region of the proximal femur for (a) 84-year-old woman with a subcapital femoral neck fracture, and (b) 79-year-old woman without fragility fracture (hip osteoarthritis). Caspase-3 immunoreactivity also present in bone marrow cells (arrowhead, a). (c) Absence of staining without primary antibody in serial section to (b). Original magnification: 20 (a) and 40 (b and c). 3

5 Sost immunostaining was osteocyte specific; present in osteocytes, lacunar walls, and canaliculi in femoral trabecular bone from postmenopausal women with/without fragility fracture (Figure 2). Sostnegative osteocytes were spatially associated with trabecular bone forming surfaces (+osteoblasts; Figure 2b), suggesting that these osteocytes have been recently embedded. Sost-positive osteocytes were numerous and found in interstitial bone. Osteoblasts, bone lining cells, osteoclasts, and marrow cells were Sost-negative, as expected (Figure 2). a b c Figure 2. Sclerostin immunostaining in osteocytes, lacunar walls and canaliculi (arrows) in trabecular bone from the intertrochanteric region of the proximal femur for (a) 79-year-old woman without fragility fracture (hip osteoarthritis), and (b) 78-year-old woman with a subcapital femoral neck fracture. Sclerostin-negative osteocytes (arrowheads) spatially associated with bone forming surfaces (area between dashed lines representative of bone forming surface with osteoblasts present, b). (c) Absence of staining without primary antibody in serial section to (b). Original magnification: 40. The extent of osteocyte apoptosis, measured as percent Casp3-positive osteocyte lacunae (Casp3.PosL/TL), was not different between the fracture and non-fracture women. For both groups, the percentage of Casp3-negative lacunae (Casp3.NegL/TL) was significantly higher than percent Casp3-positive lacunae (Figure 3a). Casp3 Neg/Pos Lacunae per Total Lacunae (%) a Non-fracture women Fracture patients Sost Neg/Pos Lacunae per Total Lacunae (%) Non-fracture women Fracture patients b Casp3.NegL/TL (%) Casp3.PosL/TL (%) Sost.NegL/TL (%) Sost.PosL/TL (%) Figure 3. The percentage of osteocyte lacunae immuno-negative and immuno-positive for (a) caspase-3 (Casp3) and (b) sclerostin (Sost) in trabecular bone from the intertrochanteric region of the proximal femur for non-fracture postmenopausal women and women with fragility hip fracture. Values are mean (SD), n = 10 per group. p < , p < The percentage of Sost-positive (Sost.PosL/TL) and Sost-negative (Sost.NegL/TL) lacunae were similar between the fracture and non-fracture women (Figure 3b). However, the fracture patients had a 4

6 lower percentage of Sost-positive lacunae in comparison to Sost-negative lacunae, in contrast to similar percent Sost-positive/Sost-negative lacunae for non-fracture women (Figure 3b). Further, Sost.PosL/TL declined with age in the fracture group, whereas no age-related change was observed for the non-fracture group (Figure 4). Sost.PosL/TL (%) Non-fracture women Fracture patients Age (years) Figure 4. Relationship between percentage of sclerostin (Sost) immuno-positive osteocyte lacunae and age in trabecular bone from the intertrochanteric region of the proximal femur for non-fracture postmenopausal women and women with fragility hip fracture. The regression line shows a significant reduction in Sost-positive lacunae with age for fracture patients; r = -0.85, p < Discussion and Conclusions Femoral trabecular bone from postmenopausal women with fragility hip fracture has similar osteocyte morphology (Table 1), and a similar degree of osteocyte apoptosis (Figure 3a), in comparison to agematched women without fragility fracture. In human femoral trabecular bone positive sclerostin immunostaining was specific to osteocytes. Sclerostin-negative osteocytes were spatially associated with trabecular bone forming surfaces and sclerostin-positive osteocytes were predominantly located in interstitial bone (Figure 2), consistent with data reported for iliac crest biopsies [4] and femoral neck cortices [9]. Given the known role of sclerostin as an inhibitor of bone formation and subsequent bone mineralisation [3,6], the unexpected finding of decreased sclerostin expression in trabecular bone osteocytes from fragility fracture cases (Figure 3b), may be indicative of elevated bone turnover and under-mineralisation, characteristic of postmenopausal osteoporosis [10,11]. Our data contrast to Power et al [9], who recently reported an increased proportion of sclerostin-positive osteonal osteocytes in femoral neck cortical bone from hip fracture patients (women and men). Specifically, osteocytes increased their tendency to express sclerostin faster with osteonal maturation for fracture patients, compared with non-fracture individuals [9]. The opposing finding of our study to that of Power et al [9] may be explained by the structural and material heterogeneity both within and between the cortex and trabecular bone [12]. Furthermore, there may be fundamental differences in the molecular cell regulation of bone turnover between these bone compartments. There is evidence for varied regulation of sclerostin expression according to the skeletal localisation [13,14]. Recent data reported from rodent studies of mechanical loading and unloading [15,16], suggest that osteocytic control of mechano-transduction may be mediated by sclerostin. In humans, it is unknown whether altered osteocytic expression of sclerostin is involved in mechanical load adaptation. The relationship between osteocyte morphology, bone architecture, mechano-responsiveness, and apoptosis is clearly complex and requires further investigation. 5

7 An association between sclerostin expression and local stress/strain distribution was shown by Robling et al [15] through in vivo mechanical compression testing of rodent ulnae. The regions within the bone experiencing the highest stresses (in this case the outer medial and lateral cortices due to the induced bending load) corresponded with the greatest reduction in sclerostin expression levels and the formation of new bone. The observation in the current study of sclerostin-negative osteocytes generally located towards the outer surface of trabeculae (Figure 2b), where bending stresses within an individual trabecula would be maximum, is consistent with the findings of Robling et al [15]. The distribution of stresses and strains at a whole bone level is dependent on the global loading of the bone as well as the bone macro and microstructure, such as the trabecular architecture. In regards to the present work, the stresses experienced by the individual trabeculae, and potentially the sclerostin expression level, will be affected by the trabecula orientation and location within the proximal femur. Relationships between the local trabecular and global structural mechanical response and sclerostin expression could therefore be further understood via computational modelling of the proximal femur such as by finite element analysis. Optimal function of the highly active mechano-sensing osteocyte-canalicular cell network may be a critical determinant of bone strength contributing to fragility fracture risk. The central role of the osteocyte cell network in regulating skeletal homeostasis presents the osteocyte cell as an ideal therapeutic target for novel preventative treatments for bone loss associated with osteoporosis and other conditions. Acknowledgments We kindly thank the Orthopaedic Surgeons and Nursing Staff of The Department of Orthopaedics and Trauma, Royal Adelaide Hospital, and the Mortuary Staff of Surgical Pathology, SA Pathology, for support and cooperation in the collection of bone tissue specimens. This study was financially supported by the National Health and Medical Research Council of Australia. References [1] Seeman E 2008 quality: the material and structural basis of bone strength. J. Miner. Metab [2] wald L F 2007 Osteocytes as dynamic multifunctional cells. Ann. N. Y. Acad. Sci [3] Winkler D G et al Osteocyte control of bone formation via sclerostin, a novel BMP antagonist. EMBO J [4] Poole K E, van Bezooijen R L, Loveridge N, Hamersma H, Papapoulos S E, Lowik C W and Reeve J 2005 Sclerostin is a delayed secreted product of osteocytes that inhibits bone formation. FASEB J [5] Semenov M, Tamai K and He X 2005 SOST is a ligand for LRP5/LRP6 and a Wnt signaling inhibitor. J. Biol. Chem [6] Li X et al Targeted deletion of the sclerostin gene in mice results in increased bone formation and bone strength. J. Miner. Res [7] Dequeker J, Boonen S, Aerssens J and Westhovens R 1996 Inverse relationship osteoarthritisosteoporosis: what is the evidence? What are the consequences? Br. J. Rheumatol [8] Krajewska M, Wang H G, Krajewski S, Zapata J M, Shabaik A, Gascoyne R and Reed J C 1997 Immunohistochemical analysis of in vivo patterns of expression of CPP32 (Caspase-3), a cell death protease. Cancer Res [9] Power J, Poole K E, van Bezooijen R, Doube M, Caballero-Alias A M, Lowik C, Papapoulos S, Reeve J and Loveridge N 2010 Sclerostin and the regulation of bone formation: Effects in hip osteoarthritis and femoral neck fracture. J. Miner. Res. Feb 23 (epub ahead of print) [10] Tsangari H, Findlay D M, Zannettino A C, Pan B, Kuliwaba J S and Fazzalari N L 2006 Evidence for reduced bone formation surface relative to bone resorption surface in female femoral fragility fracture patients

8 [11] Sutton-Smith P, Beard H and Fazzalari N L 2008 Quantitative backscattered electron imaging of bone in proximal femur fragility fracture and medical illness. J. Microsc. 229(Pt 1) 60 6 [12] Parfitt A M 1996 Skeletal heterogeneity and the purposes of bone remodelling: Implications for the understanding of osteoporosis, eds R Marcus R, et al. (San Diego: Academic) pp [13] Silvestrini G, Ballanti P, Sebastiani M, Leopizzi M, Di Vito M and Bonucci E 2008 OPG and RANKL mrna and protein expressions in the primary and secondary metaphyseal trabecular bone of PTH-treated rats are independent of that of SOST. J. Mol. Histol [14] Silvestrini G, Ballanti P, Leopizzi M, Sebastiani M, Berni S, Di Vito M and Bonucci E 2007 Effects of intermittent parathyroid hormone (PTH) administration on SOST mrna and protein in rat bone. J. Mol. Histol [15] Robling A G et al Mechanical stimulation of bone in vivo reduces osteocyte expression of Sost/sclerostin. J. Biol. Chem [16] Lin C, Jiang X, Dai Z, Guo X, Weng T, Wang J, Li Y, Feng G, Gao X and He L 2009 Sclerostin mediates bone response to mechanical unloading through antagonizing Wnt/beta-catenin signaling. J. Miner. Res