Nicola L. Fazzalari

Osteal tissue macrophages are intercalated throughout human and mouse bone lining tissues and regulate osteoblast function in vitro and in vivo

Resident macrophages are an integral component of many tissues and are important in homeostasis and repair. This study examines the contribution of resident tissue macrophages to bone physiology. Using immunohistochemistry, we showed that a discrete population of resident macrophages, OsteoMacs, was intercalated throughout murine and human osteal tissues. OsteoMacs were distributed among other bone lining cells within both endosteum and periosteum. Furthermore, OsteoMacs were coisolated with osteoblasts in murine bone explant and calvarial preparations. OsteoMacs made up 15.9% of calvarial preparations and persisted throughout standard osteoblast differentiation cultures. Contrary to previous studies, we showed that it was OsteoMacs and not osteoblasts within these preparations that responded to pathophysiological concentrations of LPS by secreting TNF. Removal of OsteoMacs from calvarial cultures significantly decreased osteocalcin mRNA induction and osteoblast mineralization in vitro. In a Transwell coculture system of enriched osteoblasts and macrophages, we demonstrated that macrophages were required for efficient osteoblast mineralization in response to the physiological remodeling stimulus, elevated extracellular calcium. Notably, OsteoMacs were closely associated with areas of bone modeling in situ, forming a distinctive canopy structure covering >75% of mature osteoblasts on diaphyseal endosteal surfaces in young growing mice. Depletion of OsteoMacs in vivo using the macrophage-Fas-induced apoptosis (MAFIA) mouse caused complete loss of osteoblast bone-forming surface at this modeling site. Overall, we have demonstrated that OsteoMacs are an integral component of bone tissues and play a novel role in bone homeostasis through regulating osteoblast function. These observations implicate OsteoMacs, in addition to osteoclasts and osteoblasts, as principal participants in bone dynamics.

Assessment of Cancellous Bone Quality in Severe Osteoarthrosis: Bone Mineral Density, Mechanics, and Microdamage

The role of bone microdamage (microscopic cracks or microcracks and ultrastructural collagen matrix and bone mineral damage) in diseases such as osteoarthrosis and osteoporosis is poorly understood. Microdamage accumulation in vivo is influenced by age and cyclic loading, therefore, it would be useful if the burden of microdamage in bone could be assessed by noninvasive measures such as the radiological measurement of bone mineral density (BMD). The aim of this study was to investigate the relationship between BMD, compressive strength and stiffness, and microdamage in the cancellous bone of the proximal femur in patients with severe osteoarthrosis. Trabecular bone core samples, from the intertrochanteric region of the femur, were obtained from 34 patients, with a mean age of 70.3 +/- 11.1 years, undergoing total hip arthroplasty for osteoarthrosis. Cores selected from contact X-ray images were used for BMD measurement, compressive mechanical testing or left untested (uncrushed), en bloc staining for microdamage, and bone histomorphometry. The study shows a strong dependence of both the elastic modulus and ultimate failure stress of the bone samples on BMD and a significant relationship between the elastic modulus and trabecular anisotropy (Tr. An). In multiple linear regression, BMD and Tr. An together account for about 70% of the variance in the elastic modulus. Then including microcrack crack density (Cr.Dn) and damage volume fraction (DxV/BV) variables, Tr. An alone accounts for a relatively small amount of the variation (8.5%) in ultimate failure stress and elastic modulus. The Cr.Dn accounts for more of the variation in the ultimate failure stress than in the elastic modulus (50% vs. 7%). In this experiment, data for Cr.Dn provide a measure of damage associated with the ultimate failure of cancellous bone. In specimens that were not mechanically tested, in vivo microcrack accumulation increases exponentially with age. In conclusion, data from this study suggest that BMD and Cr.Dn are the major determinants of cancellous bone strength, whereas BMD and Tr. An are major determinants of cancellous bone stiffness. In bone specimens subjected to compressive testing there was no relationship between microdamage and BMD, suggesting that BMD cannot be used to monitor changes in the mechanical properties of bone due to microdamage accumulation.

Pro-Inflammatory Cytokines TNF-Related Weak Inducer of Apoptosis (TWEAK) and TNFα Induce the Mitogen-Activated Protein Kinase (MAPK)-Dependent Expression of Sclerostin in Human Osteoblasts †‡

We have recently shown that TNF‐related weak inducer of apoptosis (TWEAK) is a mediator of inflammatory bone remodeling. The aim of this study was to investigate the role of TWEAK in modulating human osteoblast activity, and how TWEAK and TNFα might interact in this context. Recombinant TWEAK and TNF were both mitogenic for human primary osteoblasts (NHBC). TWEAK dose‐ and time‐dependently regulated the expression of the osteoblast transcription factors RUNX2 and osterix. TWEAK inhibited in vitro mineralization and downregulated the expression of osteogenesis‐associated genes. Significantly, TWEAK and TWEAK/TNF induced the expression of the osteoblast differentiation inhibitor and SOST gene product, sclerostin. Sclerostin induction was mitogen‐activated protein kinase (MAPK) dependent. The SOST mRNA levels induced by TWEAK were equivalent to or exceeded those seen in steady‐state human bone, and the TWEAK/TNF induction of SOST mRNA was recapitulated in fresh cancellous bone explants. TWEAK‐induced sclerostin expression was observed in immature osteoblastic cells, both in cycling (Ki67+) primary NHBC and in the cell lines MC3T3‐E1 and MG‐63, as well as in human osteocyte‐like cells and in the osteocyte cell line, MLO‐Y4. Treatment of NHBC with recombinant human sclerostin mimicked the effects of TWEAK to suppress RUNX2 and osteocalcin (OCN). TWEAK, TNF, and sclerostin treatment of NHBC similarly altered levels of phosphorylated and total GSK3β and active and total levels of β‐catenin, implying that the Wnt signaling pathway was affected by all three stimuli. Sclerostin also rapidly activated ERK‐1/2 MAPK signaling, indicating the involvement of additional signaling pathways. Together, our findings suggest that TWEAK, alone and with TNF, can regulate osteoblast function, at least in part by inducing sclerostin expression. Our results also suggest new roles and modes of action for sclerostin.

The Ratio of Messenger RNA Levels of Receptor Activator of Nuclear Factor κB Ligand to Osteoprotegerin Correlates with Bone Remodeling Indices in Normal Human Cancellous Bone but Not in Osteoarthritis

The determinants of cancellous bone turnover and trabecular structure are not understood in normal bone or skeletal disease. Bone remodeling is initiated by osteoclastic resorption followed by osteoblastic formation of new bone. Receptor activator of nuclear factor κB ligand (RANKL) is a newly described regulator of osteoclast formation and function, the activity of which appears to be a balance between interaction with its receptor RANK and with an antagonist binding protein osteoprotegerin (OPG). Therefore, we have examined the relationship between the expression of RANKL, RANK, and OPG and indices of bone structure and turnover in human cancellous bone from the proximal femur. Bone samples were obtained from individuals with osteoarthritis (OA) at joint replacement surgery and from autopsy controls. Histomorphometric analysis of these samples showed that eroded surface (ES/BS) and osteoid surface (OS/BS) were positively associated in both control (p < 0.001) and OA (p < 0.02), indicating that the processes of bone resorption and bone formation remain coupled in OA, as they are in controls. RANKL, OPG, and RANK messenger RNA (mRNA) were abundant in human cancellous bone, with significant differences between control and OA individuals. In coplotting the molecular and histomorphometric data, strong associations were found between the ratio of RANKL/OPG mRNA and the indices of bone turnover (RANKL/OPG vs. ES/BS: r = 0.93, p < 0.001; RANKL/OPG vs. OS/BS: r = 0.80, p < 0.001). These relationships were not evident in trabecular bone from severe OA, suggesting that bone turnover may be regulated differently in this disease. We propose that the effective concentration of RANKL is related causally to bone turnover.

Three-Dimensional Confocal Images of Microdamage in Cancellous Bone

The accumulation of microdamage in bone may contribute to loss of bone quality in osteoporosis, and the role of microdamage in the etiology of fatigue fractures is unknown. Microdamage created during testing, ex vivo, can increase the fragility of bone by decreasing the load necessary to cause fracture. Microdamage can also accumulate in vivo, but its influence on bone fragility is unknown. To date, stained microcracks are the only criteria to have been correlated with bone mechanics, leaving the influence of ultrastructural damage on bone fragility open for scrutiny. Staining en bloc has identified three morphological features in the tissue, discrete microcracks, cross-hatch staining, and diffuse staining. The relationship between these features and their identification as microdamage remains equivocal. The purpose of this study was to investigate the three-dimensional nature of microdamage in cancellous bone and also to describe stained microcracks, cross-hatch staining, and diffuse staining and to determine whether they all relate to microdamage in bone. Laser scanning confocal microscopy that provides improved spatial resolution over bright-field microscopy was used to visualize bone damage. It was found that crack surface density was highly correlated with crack density (r = 0.95, p < 0.0001), suggesting that the crack surface of preexisting cracks increases as new cracks are formed or submicroscopic cracks become visible under bright-field microscopy. Cross-hatch staining and diffuse staining included ultra-microcracks about 10 microm in length. The ultra-microcracks in cross-hatch staining were organized in bands and surrounded by diffuse staining. This study demonstrates that damage in bone occurs over a wide range and that discrete microcracks, cross-hatch staining, and diffuse staining are all indicative of bone damage. The diffuse staining still evident in association with the ultra-microcracks seen in cross-hatch staining and diffuse staining is probably due to damage at a still smaller scale than we have been able to investigate.

Intervertebral Disc Disorganization Is Related to Trabecular Bone Architecture in the Lumbar Spine

Cancellous bone morphometry was investigated in the sagittal plane of lumbar vertebrae using histoquantitation. The aim of this study was to identify variations in cancellous bone architecture at increasing states of intervertebral disc (IVD) disorganization after age adjustment and to investigate regional variations within the whole vertebral body. Measurements were taken of the ratio of bone volume (BV) to total volume (TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N). Lumbar spines (T12‐L5) of 19 men and 8 women were removed at autopsy from an adult sample with no clinical history of bone‐related disease or histologically identifiable bone disease. It was found that degeneration of the IVD becomes more common with increasing age. After age‐adjustment, significant increases in the proportion of BV/TV were observed in the presence of advancing IVD disorganization. Significant architectural changes were observed in the anterior regions of the vertebral body with increases in Tb.Th and Tb.N and decreases in Tb.Sp. Minimal alterations were found at posterior regions. Bone loss was observed in central regions (most distant from the cortex) as IVD disorganization increased through reduction in both Tb.N and Tb.Th. The BV/TV increase in anterior areas of the centrum may be a response to a redistribution of load to the vertebral body periphery as a result of IVD disorganization. It appears that trabecular morphology is related to the condition of the associated IVD, rather than being the sole consequence of a loss of BV/TV with age. This relationship could influence the occurrence of vertebral body crush fracture.

The effect of hydration on the stiffness of intervertebral discs in an ovine model.

OBJECTIVE To determine the hydration-over-time behaviour of ovine intervertebral discs and intact joints in a saline bath at body temperature and the effect this has on their stiffness compared to air at ambient temperature. DESIGN The hydration-over-time behaviour and stiffness of the ovine functional spinal unit and disc were quantified. BACKGROUND The fluid content of an intervertebral disc is not constant but varies with external load and load history. The stiffness of ovine functional spinal units in a hydrated environment and how this compares to testing in air have not been quantified. METHODS Intervertebral discs and functional spinal units were weighed and soaked in a saline water bath at 37 degrees C and reweighed each hour for 6 h. They were then allowed to stand in air at room temperature while the time to return to initial weight was recorded. Functional spinal units were randomly assigned to two groups. Axial compression, flexion, extension, lateral bending and axial torsion tests were performed on both the intact functional spinal unit and isolated disc. Group 1 was tested in air then in a saline water bath at 37 degrees C with the testing order reversed for Group 2. RESULTS Hydration of the disc reached a plateau after an average 3-4 h of soaking with the largest increase seen in the first hour. Four hours, standing in air at room temperature, was required to return specimens to their initial weight. The functional spinal unit stiffness was significantly lower for those specimens tested in the bath compared to air. CONCLUSIONS Ovine intervertebral discs show similar hydration-over-time behaviour when compared to human discs. Stiffnesses in different modes of loading were significantly different when tested in a hydrated environment compared with the standard method of testing in air. RELEVANCE It has been shown that there are biomechanical and biochemical similarities between sheep and human intervertebral discs. Despite these similarities, no studies have looked at how ovine intervertebral discs behave over time in a hydrated environment. In humans, hydration levels are an important aspect of intervertebral disc degeneration. There is also a relationship between decreased hydration levels and increased stiffness. This study demonstrates the similarities between human and ovine hydration-over-time behaviour. The importance of intervertebral disc hydration and its effects on stiffness under different modes of loading were also demonstrated and have not been previously shown using the ovine model. In this context, the results from this study provide further support for the use of the ovine model.

Mechanical and pathologic consequences of induced concentric anular tears in an ovine model

Study Design. Relations between induced concentric tears in the sheep disc and the mechanics of the intervertebral joint and vertebral body bone were analyzed. Objective. To examine the effect of concentric disc tears on the mechanics of the spine. Summary of Background Data. Degeneration of the intervertebral disc results in changes to the mechanics and morphology of the spine, but the effect of concentric disc tears is unknown. Methods. In this study, 48 merino wethers were subjected to surgery, and discs were randomly selected for either a needlestick injury or induction of a concentric tear in the anterior and left anterolateral anulus. Sheep were randomly assigned to groups for killing at 0, 1, 3, 6, 12, and 18 months. From each sheep, two spine segments were mechanically tested: one with a needlestick injury and one with a concentric tear. Macroscopic disc morphology was assessed by three axial slices of the disc. Sagittal bone slices were taken from cranial and caudal vertebral bodies for histologic analysis. Results. Induced concentric tears decrease the stiffness of intact spine segments in left bending and the disc alone in flexion. In all other mechanical tests, the needlestick injury had the same effect as the induced concentric tear. In the isolated disc, the disc stiffness at 6 months was increased for right bending, as compared with the response at 1 month. This was associated with increased anterior lamellar thickening and increased vertebral body bone volume fraction. Conclusions. Concentric tears and needlestick injury in the anterior anulus lead to mechanical changes in the disc and both anular lamellar thickness and vertebral body bone volume fraction. A needlestick injury through the anulus parallel to the lamellae produces progressive damage.

Temporal pattern of gene expression and histology of stress fracture healing

Loading of the rat ulna is an ideal model to examine stress fracture healing. The aim of this study was to undertake a detailed examination of the histology, histomorphometry and gene expression of the healing and remodelling process initiated by fatigue loading of the rat ulna. Ulnae were harvested 1, 2, 4, 6, 8, and 10 weeks following creation of a stress fracture. Stress fracture healing involved direct remodelling that progressed along the fracture line as well as woven bone proliferation at the site of the fracture. Histomorphometry demonstrated rapid progression of basic multicellular units from 1 to 4 weeks with significant slowing down of healing by 10 weeks after loading. Quantitative PCR was performed at 4 hours, 24 hours, 4 days, 7 days, and 14 days after loading. Gene expression was compared to an unloaded control group. At 4 hours after fracture, there was a marked 220-fold increase (P<0.0001) in expression of IL-6. There were also prominent peak increases in mRNA expression for OPG, COX-2, and VEGF (all P<0.0001). At 24 hours, there was a peak increase in mRNA expression for IL-11 (73-fold increase, P<0.0001). At 4 days, there was a significant increase in mRNA expression for Bcl-2, COX-1, IGF-1, OPN, and SDF-1. At 7 days, there was significantly increased mRNA expression of RANKL and OPN. Prominent, upregulation of COX-2, VEGF, OPG, SDF-1, BMP-2, and SOST prior to peak expression of RANKL indicates the importance of these factors in mediating directed remodelling of the fracture line. Dramatic, early upregulation of IL-6 and IL-11 demonstrate their central role in initiating signalling events for remodelling and stress fracture healing.

Quantitative histomorphometric analysis of the human growth plate from birth to adolescence.

Longitudinal bone growth occurs via the transformation of growth plate cartilage into bone through a series of cell and matrix changes, termed endochondral ossification. In this study, we characterize the development of trabecular bone from growth plate cartilage in the human rib from birth to adolescence. The height of the proliferative and hypertrophic zones within the growth plate and the primary bone spongiosa decreased with increasing age, with the greatest change observed in the first year of postnatal life. Within these zones, an internal rearrangement of tissue structure occurred. The matrix volume fraction (either cartilage or bone) increased with age in each of the zones. A concomitant increase in cartilage septae thickness and bone trabecular thickness was observed. A decrease in cartilage septae number was seen in the proliferative zone and a decrease in bone trabeculae number was also observed in the primary spongiosa. However, no difference in cartilage septae number was noted in the hypertrophic zone, the region at which cartilage is transformed into bone. Together the proliferative and hypertrophic regions of the growth plate and the bone primary spongiosa appear to constitute the active growth region, with concomitant changes observed that result in longitudinal growth. In contrast, bone mineral volume in the secondary spongiosa was stable over the ages examined; however, trabecular architecture underwent consolidation as trabecular number decreased and trabecular thickness increased. The integration of the structural transformation from cartilage to bone is crucial in achieving the dual purposes of longitudinal growth and peak bone mass. The structure developed during childhood will have an important bearing on the response to bone-altering disease in later life.