Contribution of Type H Blood Vessels to Pathologic Osteogenesis and Inflammation in an Experimental Spondyloarthritis Model.

OBJECTIVE: Spondyloarthritis (SpA) is characterized by pathologic osteogenesis, inflammation, and extensive angiogenesis in axial and peripheral tissues. Current therapies effectively target inflammation, but these therapies lack efficacy in preventing pathologic osteogenesis. Transgenic mice overexpressing transmembrane tumor necrosis factor (tmTNF-Tg mice) exhibit SpA-like features. We hypothesized that type H blood vessels, which are implicated in osteogenesis, are increased and contribute to pathology in this experimental SpA model. METHODS: We analyzed ankles, femora, and vertebrae of tmTNF-Tg mice and nontransgenic littermates and tmTNF-Tg mice on either a TNF receptor type I (TNFRI)-deficient or TNF receptor type II (TNFRII)-deficient background for osteogenesis, angiogenesis, and inflammation using advanced imaging technologies at various stages of disease. RESULTS: Compared to nontransgenic littermates, tmTNF-Tg mice exhibited an increase in vertebral type H vessels and osteoprogenitor cells in subchondral bone. These features of increased angiogenesis and osteogenesis were already present before onset of clinical disease symptoms. Type H vessels and osteoprogenitor cells were in close proximity to inflammatory lesions and ectopic lymphoid structures. The tmTNF-Tg mice also showed perivertebral ectopic type H vessels and osteogenesis, an increased number of vertebral transcortical vessels, and enhanced entheseal angiogenesis. In tmTNF-Tg mice crossed on a TNFRI- or TNFRII-deficient background, no clear reduction in type H vessels was shown, suggesting that type H vessel formation is not exclusively mediated via TNFRI or TNFRII. CONCLUSION: The contribution of type H vessels to pathologic osteogenesis in experimental SpA advances our knowledge of the pathophysiology of this disease and may also provide a novel opportunity for targeted intervention.

Osteocyte Apoptosis, Bone Marrow Adiposity, and Fibrosis in the Irradiated Human Mandible.

Purpose: To assess the effect of radiation therapy on osteocyte apoptosis, osteocyte death, and bone marrow adipocytes in the human mandible and its contribution to the pathophysiology of radiation damage to the mandibular bone. Methods and Materials: Mandibular cancellous bone biopsies were taken from irradiated patients and nonirradiated controls. Immunohistochemical detection of cleaved caspase-3 was performed to visualize apoptotic osteocytes. The number of apoptotic osteocytes per bone area and per total amount of osteocytes, osteocytes per bone area, and empty lacunae per bone area were counted manually. The percentage fibrotic tissue and adipose tissue per bone marrow area, the percentage bone marrow of total area, and the mean adipocyte diameter (µm) was determined digitally from adjacent Goldner stained sections. Results: Biopsies of 15 irradiated patients (12 men and 3 women) and 7 nonirradiated controls (5 men and 2 women) were assessed. In the study group a significant increase was seen in the number of empty lacunae, the percentage of adipose tissue of bone marrow area, and the adipocyte diameter. There was no significant difference in bone marrow fibrosis nor apoptotic osteocytes between the irradiated group and the controls. Conclusions: Irradiation alone does not seem to induce excessive bone marrow fibrosis. The damage to bone mesenchymal stem cells leads to increased marrow adipogenesis and decreased osteoblastogenic potential. Early osteocyte death resulting in avital persisting bone matrix with severely impaired regenerative potential may contribute to the vulnerability of irradiated bone to infection and necrosis.

Regional differences in microarchitecture and mineralization of the atrophic edentulous mandible: A microcomputed tomography study.

OBJECTIVE: The aim of the present study was to assess mineralization and trabecular microarchitecture in atrophic edentulous mandibles and to identify regional differences and relations with the extent of resorption. METHODS: Cortical and trabecular bone volumes in anterior, premolar and molar regions of 10 edentulous cadaveric mandibles (5 males and 5 females; mean age ± SD: 85.4 ± 8.3 years) were assessed by microcomputed tomography. Mandibular height and Cawood & Howell classes were recorded. Concerning trabecular volumes, bone mineral density (BMD), bone volume fraction, trabecular tissue volume fraction, connectivity density, trabecular number, trabecular thickness, trabecular separation, degree of anisotropy, and structural model index were measured; concerning cortical volumes porosity, BMD and cortical thickness were measured. RESULTS: In molar regions, the bone volume fraction and trabecular number were lower, whereas trabecular separation, degree of anisotropy and cortical BMD were higher compared to anterior regions. In premolar regions, mandibular height correlated negatively with trabecular number (Spearman's correlation r = 0.73, p = 0.017) and connectivity density (Spearman's correlation r = 0.82, p = 0.004), and correlated positively with trabecular separation (Spearman's correlation r = - 0.65, p = 0.04). Cortical BMD was higher at bucco-inferior cortex of molar and inferior border of premolar region and lower at anterior cranial buccal and lingual surface. CONCLUSIONS: In the premolar region, increased resorption coincides with local impairment of trabecular bone quality. Cortical bone BMD is higher in areas with highest strains and lower in areas with most mandibular resorption. Trabecular bone volume and quality is superior in the anterior region of the edentulous mandible, which might explain improved primary stability of dental implants in this region.

Loss of glucocorticoid rhythm induces an osteoporotic phenotype in female mice.

Glucocorticoid (GC)-induced osteoporosis is a widespread health problem that is accompanied with increased fracture risk. Detrimental effects of anti-inflammatory GC therapy on bone have been ascribed to the excess in GC exposure, but it is unknown whether there is also a role for disruption of the endogenous GC rhythm that is inherent to GC therapy. To investigate this, we implanted female C57Bl/6J mice with slow-release corticosterone (CORT) pellets to blunt the rhythm in CORT levels without inducing hypercortisolism. Flattening of CORT rhythm reduced cortical and trabecular bone volume and thickness, whilst bone structure was maintained in mice injected with supraphysiologic CORT at the time of their endogenous GC peak. Mechanistically, mice with a flattened CORT rhythm showed disrupted circadian gene expression patterns in bone, along with changes in circulating bone turnover markers indicative of a negative balance in bone remodelling. Indeed, double calcein labelling of bone in vivo revealed a reduced bone formation in mice with a flattened CORT rhythm. Collectively, these perturbations in bone turnover and structure decreased bone strength and stiffness, as determined by mechanical testing. In conclusion, we demonstrate for the first time that flattening of the GC rhythm disrupts the circadian clock in bone and results in an osteoporotic phenotype in mice. Our findings indicate that at least part of the fracture risk associated with GC therapy may be the consequence of a disturbed GC rhythm, rather than excess GC exposure alone, and that a dampened GC rhythm may contribute to the age-related risk of osteoporosis.

Incorporation of anterior iliac crest or calvarial bone grafts in reconstructed atrophied maxillae: A randomized clinical trial with histomorphometric and micro-CT analyses.

BACKGROUND: Autologous bone grafts have been applied successfully to severely atrophied maxilla via a preimplant procedure. Differences in graft incorporation at the microscopic level can be the decisive factor in the choice between anterior iliac crest and calvarial bone. PURPOSE: To compare conversion of anterior iliac crest bone and calvarial bone 4 months after grafting of the edentulous maxilla. MATERIALS AND METHODS: Twenty consecutive patients were randomly assigned to either anterior iliac crest (n = 10) or calvarial (n = 10) bone harvesting to reconstruct their atrophied maxillae. Biopsies were taken from both fresh bone grafts and reconstructed maxillae after 4 months healing, at time of implant placement. Micro-CT, histomorphometric and histological analyses were performed. RESULTS: Micro-CT analysis revealed that both the anterior iliac crest and calvarial bone grafts retained their volume and bone mass after being incorporated in the maxilla, but with a favor for calvarial bone grafts: calvarial bone grafts had a higher mineral density before and after incorporation. Both bone grafts types were well incorporated after 4 months of healing with preservation of bone volume and mineral density. Although the fresh bone biopsies were similar histomorphometrically, after 4 months of graft incorporation, the osteoid percentage and osteocyte count remained higher in the anterior iliac crest bone whereas the percentage of bone was higher in the calvarial bone grafts compared to the anterior iliac crest bone grafts. CONCLUSIONS: Both donor sites, that is, anterior iliac crest and calvarial bone, are well suited to provide a reliable and stable basis for implant placement 4 months after grafting with mineral density, porosity, and resorption rate in favor of calvarial bone grafts.

A Single Axial Impact Load Causes Articular Damage That Is Not Visible with Micro-Computed Tomography: An Ex Vivo Study on Caprine Tibiotalar Joints.

OBJECTIVE: Excessive articular loading, for example, an ankle sprain, may result in focal osteochondral damage, initiating a vicious degenerative process resulting in posttraumatic osteoarthritis (PTOA). Better understanding of this degenerative process would allow improving posttraumatic care with the aim to prevent PTOA. The primary objective of this study was to establish a drop-weight impact testing model with controllable, reproducible and quantitative axial impact loads to induce osteochondral damage in caprine tibiotalar joints. We aimed to induce osteochondral damage on microscale level of the tibiotalar joint without gross intra-articular fractures of the tibial plafond. DESIGN: Fresh-frozen tibiotalar joints of mature goats were used as ex vivo articulating joint models. Specimens were axially impacted by a mass of 10.5 kg dropped from a height of 0.3 m, resulting in a speed of 2.4 m/s, an impact energy of 31.1 J and an impact impulse of 25.6 N·s. Potential osteochondral damage of the caprine tibiotalar joints was assessed using contrast-enhanced high-resolution micro-computed tomography (micro-CT). Subsequently, we performed quasi-static loading experiments to determine postimpact mechanical behavior of the tibiotalar joints. RESULTS: Single axial impact loads with a mass of 15.5 kg dropped from 0.3 m, resulted in intra-articular fractures of the tibial plafond, where a mass of 10.55 kg dropped from 0.3 m did not result in any macroscopic damage. In addition, contrast-enhanced high-resolution micro-CT imaging neither reveal any acute microdamage (i.e., microcracks) of the subchondral bone nor any (micro)structural changes in articular cartilage. The Hexabrix content or voxel density (i.e., proteoglycan content of the articular cartilage) on micro-CT did not show any differences between intact and impacted specimens. However, quasi-static whole-tibiotalar-joint loading showed an altered biomechanical behavior after application of a single axial impact (i.e., increased hysteresis when compared with the intact or nonimpacted specimens). CONCLUSIONS: Single axial impact loads did not induce osteochondral damage visible with high-resolution contrast-enhanced micro-CT. However, despite the lack of damage on macro- and even microscale, the single axial impact loads resulted in "invisible injuries" because of the observed changes in the whole-joint biomechanics of the caprine tibiotalar joints. Future research must focus on diagnostic tools for the detection of early changes in articular cartilage after a traumatic impact (i.e., ankle sprains or ankle fractures), as it is well known that this could result in PTOA.

Histomorphometric and micro-CT analyses of calvarial bone grafts used to reconstruct the extremely atrophied maxilla.

BACKGROUND: Calvarial bone grafts are successful in the reconstruction of the severely atrophied maxilla as a pre-implant procedure. However, not much is known about graft incorporation at the microscopic level. PURPOSE: This study aimed to assess calvarial bone conversion 4 months after being grafted in the edentulous maxillary bone. MATERIALS AND METHODS: In 13 patients (age:65.3 ± 8.7 years) the atrophic maxilla was reconstructed with autologous calvarial bone. Biopsies were taken from fresh calvarial bone grafts and from the reconstructed maxillae after 4 months of healing. Micro-CT, histomorphometric, and histological analysis were performed. From three patients biopsies were obtained after 9, 11, or 45 months. RESULTS: The micro-CT analysis revealed that in the maxilla the calvarial bone was well preserved even after 45 months. Histology showed progressive incorporation of grafted bone within a maxillary bone. Osteoid and osteocytes were present in all biopsies indicating new bone formation and vital bone. Histomorphometrically, the percentage of grafted bone volume over total volume decreased from 79.8% (IQR78.7-83.3) in fresh calvarial grafts to 59.3% (IQR44.8-64.6) in healed grafts. The biopsies were taken after 9, 11, and 45 months showed similar values. CONCLUSIONS: Calvarial bone grafts result in stable and viable bone, good incorporation into native maxillary bone, and a minor decrease in bone volume after healing. Consequently, they provide a solid base for implant placement in severely atrophied edentulous maxillary bone.

Circadian disruption by shifting the light-dark cycle negatively affects bone health in mice.

The past decade, it has become evident that circadian rhythms within metabolically active tissues are very important for physical health. However, although shift work has also been associated with an increased risk of fractures, circadian rhythmicity has not yet been extensively studied in bone. Here, we investigated which genes are rhythmically expressed in bone, and whether circadian disruption by shifts in light-dark cycle affects bone turnover and structure in mice. Our results demonstrate diurnal expression patterns of clock genes (Rev-erbα, Bmal1, Per1, Per2, Cry1, Clock), as well as genes involved in osteoclastogenesis, osteoclast proliferation and function (Rankl, Opg, Ctsk), and osteocyte function (c-Fos) in bone. Weekly alternating light-dark cycles disrupted rhythmic clock gene expression in bone and caused a reduction in plasma levels of procollagen type 1 amino-terminal propeptide (P1NP) and tartrate-resistant acidic phosphatase (TRAP), suggestive of a reduced bone turnover. These effects coincided with an altered trabecular bone structure and increased cortical mineralization after 15 weeks of light-dark cycles, which may negatively affect bone strength in the long term. Collectively, these results show that a physiological circadian rhythm is important to maintain bone health, which stresses the importance of further investigating the association between shift work and skeletal disorders.

Resorption of the mandibular residual ridge: A micro-CT and histomorphometrical analysis.

OBJECTIVES: The aim of this study was to investigate whether the extent of mandibular resorption and gender is related to the bone turnover and microarchitecture of the edentulous mandible. PARTICIPANTS AND METHODS: A mandibular bone sample was obtained at canine position from 36 edentulous participants (50% women; mean age: 65 years) during dental implant surgery. All female participants were postmenopausal. Mandibular height, duration of edentulous state and resorption pattern (Cawood classification) were recorded. Microcomputed tomography was used to determine bone mineral density, bone volume fraction, trabecular connectivity density, trabecular number, trabecular thickness and trabecular separation. Histomorphometric analysis was used to assess bone turnover: osteoid area and surface were measured as a parameter for bone formation and osteoclast numbers were determined as a parameter for bone resorption. Correlations between micro-CT, histomorphometrical parameters and clinical data were analysed with correlation coefficients and parametric and non-parametric tests. RESULTS: Lower mandibular height was strongly associated with higher bone mineral density in trabecular bone. Women showed higher osteoclast numbers in trabecular bone than men. In trabecular bone of women, bone volume was significantly related to osteoclast numbers, osteoid surface and osteoid area. CONCLUSIONS: The higher trabecular bone mineral density found in the edentulous mandible could either indicate a restructuring process of the resorbed mandible or suggests that the inferior region of the mandible is more highly mineralised. In women, higher bone turnover is associated with lower bone volume, suggesting an effect of postmenopausal oestrogen deficiency on bone turnover in the edentulous mandible.

Mineralization-defects are comparable in fluorotic impacted human teeth and fluorotic mouse incisors.

OBJECTIVE: Fluoride excess of 0.05-0.07mgF/kgbw/day in water or food additives like salt is the principal cause of endemic dental fluorosis. How fluoride causes these defects is not clear yet. Recent studies in rodents suggest that development of enamel fluorosis is associated with insufficient neutralization of protons released during the formation of hypermineralized lines. DESIGN: Here we examined whether hypermineralization could also be assessed by MicroCT in developing molar enamel of humans exposed to fluoride. RESULT: Micro-CT analysis of hypomineralized enamel from human fluorotic molars graded by the Thylstrup-Fejerskov (TF) Index as III-IV showed weak hypermineralized lines and hypermineralized patches not seen in TF-I/II grade enamel. The mesio-distal sides of these molar teeth were significantly smaller (∼18%, p=0.02) than in TF-I/II teeth. CONCLUSION: The patterns of changes observed in human fluorotic teeth were similar to those in fluorotic rodent incisors. The data are consistent with the hypothesis that also in developing human teeth fluoride-stimulated local acidification of enamel could be a mechanism for developing fluorotic enamel.

Histological and micro Computed Tomography analysis of a femoral stress fracture associated with prolonged bisphosphonate use.

BACKGROUND: The origin of atypical femoral fractures (AFF) associated with bisphosphonate therapy remains to be elucidated. In this study, a biopsy of an AFF site is analyzed to determine whether microdamage and/or morphological changes are present in the area of the AFF. MATERIAL AND METHODS: Cortical bone from an AFF region was obtained during a preventive stabilization in a patient with a symptomatic AFF. This bone was scanned using microCT (resolution=0.01 mm), stained with basic fuchsin and analyzed histologically. RESULTS: The diameter of the Haversian canals was higher in the vicinity of the AFF compared to the bone further away from the AFF. The bone mineral density within the cortex ranged from 1020 to 1080 mg HA/cm3. We observed penetration of basic fuchsin into the matrix, which is a tell-tale sign of diffuse damage. DISCUSSION: The higher diameter of haversian canals is likely to result in higher local stresses and consequently increased microdamage. The diffuse microdamage in the biopsy may furthermore be directly related to bisphosphonate use, preventing repair of microdamage, and consequently the development of the AFF. CONCLUSION: Increased porosity of the cortex and accumulation of microdamage might have lead to a stress fracture and ultimately a complete AFF.

Effects of Decreased Occlusal Loading during Growth on the Mandibular Bone Characteristics.

BACKGROUND: Bone mass and mineralization are largely influenced by loading. The purpose of this study was to evaluate the reaction of the entire mandibular bone in response to decreased load during growth. It is hypothesized that decreased muscular loading will lead to bone changes as seen during disuse, i.e. loss of bone mass. METHODS AND FINDINGS: Ten 21-day-old Wistar strain male rats were divided into two groups (each n=5) and fed on either a hard- or soft-diet for 11 weeks. Micro-computed tomography was used for the investigation of bone mineralization, bone volume, bone volume fraction (BV/TV) and morphological analysis. Mandibular mineralization patterns were very consistent, showing a lower degree of mineralization in the ramus than in the corpus. In the soft-diet group, mineralization below the molars was significantly increased (p<0.05) compared to the hard diet group. Also, bone volume and BV/TV of the condyle and the masseter attachment were decreased in the soft-diet group (p<0.05). Morphological analysis showed inhibited growth of the ramus in the soft-diet group (p<0.05). CONCLUSION: Decreased loading by a soft diet causes significant changes in the mandible. However, these changes are very region-specific, probably depending on the alterations in the local loading regime. The results suggest that muscle activity during growth is very important for bone quality and morphology.

Osteochondral defects of the talus: a novel animal model in the goat.

Osteochondral defects of the talus pose a difficult therapeutic challenge. An experimental animal model of the ankle joint is not available. The aim of this study was to test a newly developed animal model for osteochondral defects of the ankle in vivo. Osteochondral defects were created in the talus of goat hind legs using a posterolateral surgical approach. The defects were filled with either autologous cancellous bone or donor demineralized bone matrix or left empty as control. After 12 weeks of healing, the specimens were analyzed with radiography, macroscopy, microcomputed tomography, histology, histomorphometry, and fluorescence microscopy. It was possible to create a standardized defect in each talus. The implanted material remained in place. The analyses showed that most bony tissue was generated in the defects filled with autologous bone and least in the control defects. Our findings show that a standard osteochondral defect can be created in the talus by a relatively simple procedure in a large animal that allows qualitative and quantitative evaluation. The model can be used in future experiments to investigate alternative treatment methods before they are introduced into clinical practice.

Degree of mineralization at the attachment of lateral pterygoid.

The degree of mineralization of bone (DMB) in the human mandibular condyle is heterogeneous, and differences in DMB have been related to variations in bone turnover caused by local strains. The lateral pterygoid muscle inserts at the anterior surface of the condyle. The aim of this study is to analyze the DMB at the attachment of this muscle as compared with a control region. It was hypothesized that, DMB at the attachment sites of lateral pterygoid muscles was lower than at the control regions, because of the larger number of loadings and subsequently higher remodeling rates. Also, as the human lateral pterygoid muscle is heterogeneous in its internal architecture, variations in DMB within the attachment sites were expected. 10 human mandibular condyles were scanned in a micro CT system. Within each condyle, two regions, that is, the pterygoid fovea and a posterior (control) region where no muscle was inserted, were selected to analyze regional differences in DMB. The attachment site was further divided into eight subregions to analyze subregional differences. At the pterygoid fovea the DMB of cortical bone was significantly lower than at the control region (p = 0.003) and increased in medio-lateral direction. The results of this study could suggest an influence of the lateral pterygoid muscle on bone turnover at this site.

The influence of mineralization on intratrabecular stress and strain distribution in developing trabecular bone.

The load-transfer pathway in trabecular bone is largely determined by its architecture. However, the influence of variations in mineralization is not known. The goal of this study was to examine the influence of inhomogeneously distributed degrees of mineralization (DMB) on intratrabecular stresses and strains. Cubic mandibular condylar bone specimens from fetal and newborn pigs were used. Finite element models were constructed, in which the element tissue moduli were scaled to the local DMB. Disregarding the observed distribution of mineralization was associated with an overestimation of average equivalent strain and underestimation of von Mises equivalent stress. From the surface of trabecular elements towards their core the strain decreased irrespective of tissue stiffness distribution. This indicates that the trabecular elements were bent during the compression experiment. Inhomogeneously distributed tissue stiffness resulted in a low stress at the surface that increased towards the core. In contrast, disregarding this tissue stiffness distribution resulted in high stress at the surface which decreased towards the core. It was concluded that the increased DMB, together with concurring alterations in architecture, during development leads to a structure which is able to resist increasing loads without an increase in average deformation, which may lead to damage.

Biomechanical consequences of developmental changes in trabecular architecture and mineralization of the pig mandibular condyle.

The purpose of the present study was to examine the changes in apparent mechanical properties of trabecular bone in the mandibular condyle during fetal development and to investigate the contributions of altering architecture, and degree and distribution of mineralization to this change. Three-dimensional, high-resolution micro-computed tomography (microCT) reconstructions were utilized to assess the altering architecture and mineralization during development. From the reconstructions, inhomogeneous finite element models were constructed, in which the tissue moduli were scaled to the local degree of mineralization of bone (DMB). In addition, homogeneous models were devised to study the separate influence of architectural and DMB changes on apparent mechanical properties. It was found that the bone structure became stiffer with age. Both the mechanical and structural anisotropies pointed to a rod-like structure that was predominantly oriented from anteroinferior to posterosuperior. Resistance against shear, also increasing with age, was highest in the sagittal plane. The reorganization of trabecular elements, which occurred without a change in bone volume fraction, contributed to the increase in apparent stiffness. The increase in DMB, however, contributed more dominantly. Incorporating the observed inhomogeneous distribution of mineralization decreased the apparent stiffness, but increased the mechanical anisotropy. This denotes that there might be a directional dependency of the DMB of trabecular elements, i.e. differently orientated trabecular elements might have different DMBs. In conclusion, the changes in DMB and its distribution are important to consider when studying mechanical properties during development and should be considered in other situations where differences in DMB are expected.

Daily number and lengths of activity bursts in rabbit jaw muscles.

Muscle activity has predominantly been studied for specific motor tasks not necessarily representative of normal daily behaviour. The few studies that have examined daily muscle use have quantified this by duty time, merging all levels of muscle activity. Muscle activity can also be characterized by the number, duration and level of bursts. The purpose of this study was to characterize, for various levels of muscle activity, the daily masseter and digastric actions in the rabbit. Characterization was realized by quantification of duty time (summed length of all bursts as a percentage of total time), number of bursts and distribution of burst lengths. A telemetric device was implanted in the two muscles of six rabbits, ensuring the recording of their jaw muscle activities while they moved freely. The continuously transmitted signals over 1 day were analysed. The results showed that (i) more than 100,000 bursts per day exceeded the 2% level of the maximum muscle activity in both muscles, whereas fewer than 100 bursts per day exceeded the 90% level; and (ii) the digastric muscle exhibited a significantly higher duty time than the masseter (respectively, 23% and 14% for activities exceeding the 2% level), which was mainly caused by the on average longer burst lengths at the lowest levels. The characterization of muscle activity in daily burst number and distribution of burst lengths exceeding various activity levels provides valuable information on motor control and enables further investigation of the adaptive capacity of muscles.