Raman microspectroscopy reveals unsaturation heterogeneity at the lipid droplet level and validates an in vitro model of bone marrow adipocyte subtypes.

Bone marrow adipocytes (BMAds) constitute the most abundant stromal component of adult human bone marrow. Two subtypes of BMAds have been described, the more labile regulated adipocytes (rBMAds) and the more stable constitutive adipocytes (cBMAds), which develop earlier in life and are more resilient to environmental and metabolic disruptions. In vivo, rBMAds are enriched in saturated fatty acids, contain smaller lipid droplets (LDs) and more readily provide hematopoietic support than their cBMAd counterparts. Mouse models have been used for BMAds research, but isolation of primary BMAds presents many challenges, and thus in vitro models remain the current standard to study nuances of adipocyte differentiation. No in vitro model has yet been described for the study of rBMAds/cBMAds. Here, we present an in vitro model of BM adipogenesis with differential rBMAd and cBMAd-like characteristics. We used OP9 BM stromal cells derived from a (C57BL/6xC3H)F2-op/op mouse, which have been extensively characterized as feeder layer for hematopoiesis research. We observed similar canonical adipogenesis transcriptional signatures for spontaneously-differentiated (sOP9) and induced (iOP9) cultures, while fatty acid composition and desaturase expression of Scd1 and Fads2 differed at the population level. To resolve differences at the single adipocyte level we tested Raman microspectroscopy and show it constitutes a high-resolution method for studying adipogenesis in vitro in a label-free manner, with resolution to individual LDs. We found sOP9 adipocytes have lower unsaturation ratios, smaller LDs and higher hematopoietic support than iOP9 adipocytes, thus functionally resembling rBMAds, while iOP9 more closely resembled cBMAds. Validation in human primary samples confirmed a higher unsaturation ratio for lipids extracted from stable cBMAd-rich sites (femoral head upon hip-replacement surgery) versus labile rBMAds (iliac crest after chemotherapy). As a result, the 16:1/16:0 fatty acid unsaturation ratio, which was already shown to discriminate BMAd subtypes in rabbit and rat marrow, was validated to discriminate cBMAds from rBMAd in both the OP9 model in vitro system and in human samples. We expect our model will be useful for cBMAd and rBMAd studies, particularly where isolation of primary BMAds is a limiting step.

Short-term high-dose zoledronic acid enhances crystallinity in mandibular alveolar bone in rats.

Owing to its antiresorptive properties, zoledronic acid (ZOL) is commonly used in the management of benign as well as malignant bone diseases. This molecule targets sites where bone is actively remodeling, and high concentrations have been reported in the jaw. The purpose of this study was to investigate whether treatment of male rats with ZOL, at a dosage equivalent to that used for antitumor treatment, impacts the short-term qualitative properties of mandibular bone independent of bone remodeling. Thirty rats were randomly assigned to treatment either with ZOL or with serum-vehicle (control) (weekly injections: 100 µg kg-1 for 6 wk, n = 15 per group). Using the tetracycline double-labeling technique, remodeled bone areas, corresponding to the preferential site of bisphosphonate binding, were found in the alveolar bone along the alveolar bone proper. The composition of bone in these areas was characterized using Raman microspectroscopy and compared with adjacent, non-remodeled, older bone. The ZOL-treated group exhibited higher crystallinity in the remodeled bone areas (+2%), reflecting an early maturation of the apatite mineral after ZOL injection. Our findings highlight a direct and rapid effect of clinically relevant anti-tumoral ZOL doses on the qualitative properties of mandibular bone, especially on mineral crystallinity in the vicinity of the teeth, namely, the alveolar bone proper.

Long Term Ovariectomy-Induced Osteoporosis is Associated with High Stearoyl-CoA Desaturase Indexes in Rat Femur.

Osteoporosis is characterized by a bone loss associated to an increased bone marrow adiposity; however, it is still unclear what kind of lipids are involved. Therefore, the main purpose of this study was to see if there is any local bone lipid changes related to osteoporosis, by using the ovariectomy-induced osteoporosis (OVX) rat model. Female SD rats (operated at 6 months of age for skeletal maturity) were divided in control SHAM and OVX groups (n = 6/group) and maintained for 9 month post-surgery. Lipids were analyzed in two compartments of femoral diaphyses: bone marrow (BM) and mineralized tissue (MT), by chromatographic methods. As expected, osteoporotic femurs had a larger BM mass associated with a two-fold increase of lipid content. The MT had a similar lipid enrichment, indicating that adiposity affected the mineral part as well. The main lipids concerned were triglycerides, sphingomyelin, phosphatidylcholine and phosphatidylserine in BM, and triglycerides and cholesterol esters in MT. The increase of both energy-storage and membrane-associated lipids in BM suggested that cell number and/or size was enhanced to allow more triglyceride storage. Interestingly, in MT of osteoporotic femurs, sphingomyelin was decreased, suggesting that its catabolism could be linked to osteoporosis. In both femoral compartments, fatty acid profiles were enriched in 14:0 and 16:1, lowered in 18:0 and 20:4 n-6, and two-fold higher stearoyl-CoA desaturase indexes (16:1/16:0 and 18:1/18:0 ratios), suggesting an increased de novo lipogenesis in osteoporotic femurs. Thus, the present study is first to report local changes of individual lipids in rat osteoporotic femurs and suggests that osteoporosis is a pathologic condition associated with an enhanced de novo lipogenesis. Further studies will be needed to better understand the consequences of these lipid changes in osteoporotic bones.

Brief Report From the 4th International Meeting on Bone Marrow Adiposity (BMA2018).

The 4th International Meeting on Bone Marrow Adiposity (BMA2018) was hosted at the premises of the Regional Government of Hauts de France in Lille, from August 29th to August 31st 2018. This congress brought together physicians and scientists working on rheumatology and bone biology, oncology, hematology, endocrinology, and metabolic diseases, all interested in bone marrow adiposity. They shared their opinions, hypothesis, and original results. Six invited keynotes were given by S. Badr, B.C.J. van der Eerden, M.J. Moreno Aliaga, O. Naveiras, C.J. Rosen, and A.V. Schwartz. Twenty-one short talks were also given. This report briefly summarizes the scientific content of the meeting and the progress of the working groups of the BMA Society (http://bma-society.org/).

Mandibular bone is protected against microarchitectural alterations and bone marrow adipose conversion in ovariectomized rats.

Osteoporosis is a disease that leads to a loss of bone mass and to alterations in the bone microarchitecture that occur in a site-specific manner; however it remains controversial in the jaw. The involvement of bone marrow adipose tissue (BMAT) in the bone metabolism has been suggested in several physiopathological contexts, such as in aging and osteoporosis. To test whether the BMAT content is related to mandibular bone loss, this study aimed to investigate the potential correlations between the trabecular bone microarchitecture on one hand and BMAT content and its spatial distribution in relation to bone surface on the other hand during aging and ovariectomy (OVX) during a long-term follow-up in a mature rat model. No age-related microarchitectural or BMAT changes were observed in the mandible. The OVX-induced bone loss was three-fold lower in the mandible than in the tibia and was observed only in the alveolar bone (not in the condyle). We also report a delayed increase in the mandibular BMAT content that remained 4-6-fold lower compared to tibia. This low BMAT content in the mandible was located at a distance from the trabecular bone surface (only 5% in contact with the bone surface versus 87% in the tibia). These findings highlight a specific mandibular response to OVX, in particular fewer microarchitectural alterations compared to that in the tibia. For the latter, the trabecular bone thickness and surface were correlated with the BMAT content. Oral functions may have a protective effect on the mandibular BMAT conversion in an OVX context.

Marrow adiposity and bone: Review of clinical implications.

There is growing interest in the relationship between bone marrow fat (BMF) and skeletal health. Progress in clinical studies of BMF and skeletal health has been greatly enhanced by recent technical advances in our ability to measure BMF non-invasively. Magnetic resonance imagery (MRI) with or without spectroscopy is currently the standard technique for evaluating BMF content and composition in humans. This review focuses on clinical studies of marrow fat and its relationship with bone. The amount of marrow fat is associated with bone mineral density (BMD). Several studies have reported a significant negative association between marrow fat content and BMD in both healthy and osteoporotic populations. There may also be a relationship between marrow fat and fracture (mostly vertebral fracture), but data are scarce and further studies are needed. Furthermore, a few studies suggest that a lower proportion of unsaturated lipids in vertebral BMF may be associated with reduced BMD and greater prevalence of fracture. Marrow fat might be influenced by metabolic diseases associated with bone loss and fractures, such as diabetes mellitus, obesity and anorexia nervosa. An intriguing aspect of bariatric (weight loss) surgery is that it induces bone loss and fractures, but with different impacts on marrow fat depending on diabetic status. In daily practice, the usefulness for clinicians of assessing marrow fat using MRI is still limited. However, the perspectives are exciting, particularly in terms of improving the diagnosis and management of osteoporosis. Further studies are needed to better understand the regulators involved in the marrow fat-bone relationship and the links between marrow fat, other fat depots and energy metabolism.

Unraveling the compromised biomechanical performance of type 2 diabetes- and Roux-en-Y gastric bypass bone by linking mechanical-structural and physico-chemical properties.

Type 2 diabetes mellitus (T2DM) is a metabolic disorder associated with obesity and hyperglycemia. Roux-en-Y gastric bypass (RYGB) surgery is a common treatment for severely obese patients and T2DM. Both RYGB and T2DM are linked to increased skeletal fragility, though the exact mechanisms are poorly understood. Our aim was to characterize the structural, mechanical and compositional properties of bones from diet-induced obese and RYGB-treated obese (bypass) mice to elucidate which the exact factors are contributing to the increased skeletal fragility. To achieve this, a combinatory approach including microfocus X-ray computed tomography, 3-point bending, finite element modeling and Raman spectroscopy, was used. Compared to aged-matched lean controls, the obese mice displayed decreased cortical thickness, trabecular bone loss, decreased stiffness and increased Young's modulus. For the bypass mice, these alterations were even more pronounced, and additionally they showed low mineral-to-matrix ratio in the cortical endosteal area. Accumulation of the advanced glycation end-product (AGE) pentosidine was found in the cortex of obese and bypass groups and this accumulation was correlated with an increased Young's modulus. In conclusion, we found that the increased fracture risk in T2DM- and post-RYGB bones is mainly driven by accumulation of AGEs and macro-structural alterations, generating biomechanical dysfunctionality.

A Novel microCT Method for Bone and Marrow Adipose Tissue Alignment Identifies Key Differences Between Mandible and Tibia in Rats.

Bone homeostasis is influenced by the bone marrow adipose tissue (BMAT). BMAT distribution varies from one anatomical location in the skeleton to another. We developed an advanced microfocus computed tomography imaging and analysis protocol that allows accurate alignment of both the BMAT distribution and bone micro-architecture as well as calculation of the distance of the BMAT adipocytes from the bone surface. Using this protocol, we detected a different spatial BMAT distribution between the rat tibia and mandible: in the proximal metaphysis of the tibia a large amount of BMAT (~ 20% of the total BMAT) was located close to the bone surface (< 20 µm), whereas in the alveolar ridge ~ 30% of the total BMAT was located between 40 and 60 µm from the bone surface. In the alveolar ridge of rats, the trabecular bone volume was 48.3% higher compared to the proximal metaphysis of the tibia (p < 0.0001) and the percentage of adiposity determined to the relative marrow volume was lower (1.5%) compared to the proximal metaphysis of the tibia (9%, p = 0.0002). Interestingly, in the tibia a negative correlation was found between the percentage of adiposity in the total volume and the trabecular thickness (r =- 0.74, p = 0.037). The present study highlights that in comparison to tibial proximal metaphysis, the mandibular bone exhibits a massive trabecular network and a low BMAT content with almost no contact with the bone surface. These findings are of great interest because of the importance of the fat-bone interaction and its potential relevance to several resorptive bone diseases.

Simultaneous three-dimensional visualization of mineralized and soft skeletal tissues by a novel microCT contrast agent with polyoxometalate structure.

Biological tissues have a complex and heterogeneous 3D structure, which is only partially revealed by standard histomorphometry in 2D. We here present a novel chemical compound for contrast-enhanced microfocus computed tomography (CE-CT), a Hafnium-based Wells-Dawson polyoxometalate (Hf-POM), which allows simultaneous 3D visualization of mineralized and non-mineralized skeletal tissues, such as mineralized bone and bone marrow vasculature and adipocytes. We validated the novel contrast agent, which has a neutral pH in solution, by detailed comparison with (immuno)histology on murine long bones as blueprint, and showed that Hf-POM-based CE-CT can be used for virtual 3D histology. Furthermore, we quantified the 3D structure of the different skeletal tissues, as well as their spatial relation to each other, during aging and diet-induced obesity. We discovered, based on a single CE-CT dataset per sample, clear differences between the groups in bone structure, vascular network organization, characteristics of the adipose tissue and proximity of the different tissues to each other. These findings highlight the complementarity and added value of Hf-POM-based CE-CT compared to standard histomorphometry. As this novel technology provides a detailed 3D simultaneous representation of the structural organization of mineralized bone and bone marrow vasculature and adipose tissue, it will enable to improve insight in the interactions between these three tissues in several bone pathologies and to evaluate the in vivo performance of biomaterials for skeletal regeneration.

Doses effects of zoledronic acid on mineral apatite and collagen quality of newly-formed bone in the rat's calvaria defect.

Due to their inhibitory effects on resorption, bisphosphonates are widely used in the treatment of diseases associated to an extensive bone loss. Yet, little is known about bisphosphonates effects on newly-formed bone quality. In the present study, adult male Sprague-Dawley rats (n=80) with a bone defect calvaria area were used and short-term effects of zoledronic acid (ZA) were studied on the healing bone area. Three ZA treatments were tested by using either: 1°) a low single dose (120µgZA/kg, n=10; equivalent to human osteoporosis treatment), 2°) a low fractionated doses (20µgZA/kg daily for 6days either a total of 120µg/kg, n=15), and 3°) a high fractionated doses, (100µgZA/kg weekly for 6weeks, n=15; equivalent to 6months of human bone metastasis treatment). For each treatment, a control "vehicle" treatment was performed (with an identical number of rats). After ZA administration, the intrinsic bone material properties were evaluated by quantitative backscattered electron imaging (qBEI) and Raman microspectroscopy. Neither single nor fractionated low ZA doses modify the intrinsic bone material properties of the newly-formed bone compared to their respective control animals. On the opposite, the high ZA treatment resulted in a significant decrease of the crystallinity (-25%, P< 0.05) and of the hydroxyproline-to-proline ratio (-30%, P<0.05) in newly-formed bones. Moreover, with the high ZA treatment, the crystallinity was positively correlated with the hydroxyproline-to-proline ratio (ρ=0.78, P<0.0001). The present data highlight new properties for ZA on bone formation in a craniofacial defect model. As such, ZA at high doses disrupted the apatite crystal organization. In addition, we report here for the first time that high ZA doses decreased the hydroxyproline-to-proline ratio suggesting that ZA may affect the early collagen organization during the bone healing.

The Role of Bone Marrow Fat in Skeletal Health: Usefulness and Perspectives for Clinicians.

CONTEXT: There is growing interest in the relationship between bone marrow fat (BMF), bone mineral density (BMD), and fractures. Moreover, BMF might be influenced by metabolic diseases associated with bone loss and fractures, such as type 2 diabetes mellitus (T2DM), anorexia nervosa (AN), and obesity. METHODS: The primary-source literature for this review was acquired using a PubMed search for articles published between January 2000 and April 2015. Search terms included BMF, BMD, fractures, T2DM, AN, and obesity. The titles and abstracts of all articles were reviewed for relevant subjects. RESULTS: Magnetic resonance imaging, with or without spectroscopy, was used to noninvasively quantify BMF in humans. A negative relationship was found between BMD and BMF in both healthy and osteopenic/osteoporotic populations. Data are lacking on the relationship between BMF and fractures. Studies in populations of individuals with metabolic diseases such as T2DM, AN, and obesity have shown BMF abnormalities. CONCLUSIONS: We conclude that most human data demonstrate an inverse relationship between BMF and BMD, but data on the relationship with fractures are inconsistent and need further study. In daily practice, the usefulness for clinicians of assessing BMF using magnetic resonance imaging is still limited. However, the perspectives are exciting, particularly in terms of improving the diagnosis and management of osteoporosis.

Dexamethasone in osteogenic medium strongly induces adipocyte differentiation of mouse bone marrow stromal cells and increases osteoblast differentiation.

BACKGROUND: Osteoblasts and adipocytes share a common mesenchymal stem cell origin. Therefore, it has been suggested that the accumulation of marrow adipocytes observed in bone loss is caused by a shift in the commitment of mesenchymal stem cells from the osteogenic pathway to the adipogenic pathway. Supporting this hypothesis the competition between adipogenic and osteogenic lineages was widely demonstrated on partially homogeneous cell populations. However, some data from mouse models showed the existence of an independent relationship between bone mineral content and bone marrow adiposity. Therefore, the combination of adipogenesis and osteogenesis in primary culture would be helpful to determine if this competition would be observed on a whole bone marrow stromal cell population in a culture medium allowing both lineages. In this aim, mouse bone marrow stromal cells were cultured in a standard osteogenic medium added with different concentrations of Dexamethasone, known to be an important regulator of mesenchymal progenitor cell differentiation. RESULTS: Gene expression of osteoblast and adipocyte markers, biochemical and physical analyses demonstrated the presence of both cell types when Dexamethasone was used at 100 nM. Overall, our data showed that in this co-differentiation medium both differentiation lineages were enhanced compared to classical adipogenic or osteogenic culture medium. This suggests that in this model, adipocyte phenotype does not seem to increase at the expense of the osteoblast lineage. CONCLUSION: This model appears to be a promising tool to study osteoblast and adipocyte differentiation capabilities and the interactions between these two processes.

Mica dust and pneumoconiosis: example of a pure occupational exposure in a muscovite milling unit.

OBJECTIVE: We present pulmonary disorders of four employees who were exposed to high concentration of pure mica dust in a muscovite milling unit. METHOD: All cases underwent traditional examinations with a dual-energy chest computed tomographic scan. An analysis of exhaled breath condensate by Raman microspectrometry and of mineralogical content of a lung biopsy was performed for one case. RESULTS: All cases showed bilateral micronodular ground glass opacities and mediastinal and hilar hyperdense lymph nodes consistent with the nodal sequestration of mineral particles. Histological analysis showed giant cell granulomas without typical silicotic nodule with high concentration of birefringent particles consistent with mica. Mica particles found in the exhaled breath condensate were identical to particles in ambient air at the company. CONCLUSION: Occupational exposure to mica dust is responsible for diffuse infiltrative lung disease by overload processes.

Reproducibility of 1H MR spectroscopy of hip bone marrow at 3 Tesla.

PURPOSE: To assess the reproducibility of fat content (FC) values in five different anatomical areas of proximal femur by the means of (1)H 3 Tesla MR spectroscopy and to evaluate if any statistical difference exists when comparing right side to left side FC values in the same individual. MATERIALS AND METHODS: Thirty-three volunteers underwent unilateral (1)H MR spectroscopy of the hip. From 1 to 4 weeks later, they repeat the MR examination of the same hip to assess the reproducibility of the technique. Fifteen other volunteers underwent a bilateral (1)H MR hip spectroscopy to compare right and left side FC values. RESULTS: The reproducibility of (1)H MR spectroscopy was high in all the anatomic areas considered, ranging from 0.90 to 0.98. No statistically significant difference was found when the fat content values on the right side were compared to those on the left side. (P > 0.16). CONCLUSION: The 3 Tesla (1)H MR spectroscopy of hip bone marrow permitted highly reproducible fat content values in all the five anatomic areas examined. No statistical significant difference existed when comparing fat content values of the right side to those of the left side in the same individual.

Laser preconditioning on cranial bone site: analysis of morphological vascular parameters.

BACKGROUND AND OBJECTIVES: Bone vascularization is a key factor in the bone healing process following X-ray irradiation. Preserving the vascular network from X-ray-induced injury is a relevant approach in the promotion of bone healing. Previously, we developed a protocol of laser preconditioning (810 nm diode laser, 36 J/cm²) prior to X-ray radiation (18.75 Gy) which protects the bone vascular network from deleterious effects of X-ray radiation. The aim of this present work is to characterize the effects of laser preconditioning on the bone through a morphological analysis of vascular parameters. MATERIALS AND METHODS: Digital images of the vascular plexus were taken through an optical bone chamber which was implanted onto the calvaria of rabbits. Bespoke software was used for the quantification of the vessels (classified in four groups according to their diameter), vessel length, and number of nodes at weeks 0, 4, and 8. Twenty rabbits were divided into four groups: control group #1 (n = 5); laser group #2 (n = 5). X-ray radiation group #3 (n = 5), laser preconditioning 24 hours prior to X-ray radiation group #4 (n = 5). RESULTS: The bone vascular network was stable for groups #1 and #2. Statistical analysis showed a significant reduction of each observed vascular parameter for groups #3 and #4. In the laser preconditioned group #4 the loss was less marked than in the X-ray group #3, especially for large vessels (diameter >50 µm). DISCUSSION AND CONCLUSION: We provide in vivo microcirculatory evidence to support the concept of laser preconditioning of bone. A computer-based semi-automatic system is described to quantify superficial bone vascular network parameters that had been treated by laser preconditioning prior to X-ray radiation. Laser preconditioning significantly attenuates the deletion of the superficial bone vascular network irradiated by X-ray, especially concerning large diameter vessels.

A preliminary investigation into the effects of X-ray radiation on superficial cranial vascularization.

Radiation therapy (RT) is an established treatment modality for malignant neoplasms. RT induces tissue damage that may lead to osteoradionecrosis in more severe cases. Suitable animal models to study RT-induced changes in membranous craniofacial bone are currently not available. The aim of this study was therefore to quantify RT-induced changes in cranial microcirculation using a newly developed calvaria chamber model and to relate these changes to RT-induced histological damage. New Zealand white rabbits received a total radiation dose of 18.75 Gy through the calvaria chamber, and the number of vessels, the vessel length density (VLD), and angiogenic sprouting were quantified on a weekly basis during a 12-week period. At the end of 12 weeks, the RT-treated (n = 5) or control (n = 5) calvarias were biopsied for histopathological analysis. RT resulted in a steep reduction in the number of vessels and the VLD during the first 3 weeks, particularly in larger-diameter vessels, followed by a flat stabilization/remodeling phase in the subsequent 9 weeks that never restored to baseline values. Histomorphometric analysis revealed a high degree of osteocytic depletion, prominent hypocellularity in the lacunae and intraosseous vasculature, enlarged and nonconcentric Haversian systems, and a severely disorganized bone matrix in the RT-treated calvarias. Despite the prevalence of some angiogenic potential, the RT-induced effects in the early phase persisted in the intermediate to late phase, which may have contributed to the poor recovery of the RT-treated bone.

[Osteonecrosis of the jaw and bisphophonates in oncology]. / Ostéonécrose des maxillaires et bisphosphonates en cancérologie.

Bisphosphonates are potent osteoclastic inhibitors that are indicated in the prevention of bone complications. They could also be of interest in the prevention of bone metastases. Several recent international publications have highlighted the onset of osteonecrosis of the jaw (ONJ) in patients treated with bisphosphonates. These osteonecroses manifest in the form of bone exposure, recent tooth mobility, swelling and inflammation and, occasionally, localised pain but they can remain asymptomatic for weeks or even months. The prevalence of these osteonecroses in cancer patients treated with bisphosphonates could range from 1 to 10%. In most cases (60 to 80%), ONJ develops after alveolo-dental surgery (e.g. tooth extraction). Length of exposure to bisphosphonate probably increases the risk. Our recommendations regarding the diagnosis, classification, prevention and treatment of cases of ONJ observed during bisphosphonate administration are based on published studies and our experience. It is obvious that the use of bisphosphonates is undoubtedly beneficial in the treatment of bone complications but the incidence of ONJ during long-term treatments and at high doses warrants preventive measures. These measures are straightforward : bucco-dental repair prior to treatment, good hygiene and regular monitoring during treatment. Current, non-invasive procedures are still permitted. In other cases, the suspension of treatment is indicated until healing is complete. The increase in the incidence of ONJs, serious adverse events, raises the issue regarding duration and administration of bisphosphonate treatment in the management of bone metastases. Studies are currently underway in an attempt to answer this issue.

Laser preconditioning of calvarial bone prior to an X-ray radiation injury: a preliminary in vivo study of the vascular response.

BACKGROUND AND OBJECTIVES: Thermal preconditioning prior to injury induces a cytoprotective effect on soft tissues and promotes their recovery. Lasers are an adequate tool to generate controlled and reproducible heat. X-ray irradiation induces a chronic antiangiogenic effect on bone, affecting its healing and remodeling processes. The aim of this study was to investigate the effect of laser preconditioning on the re-vascularization of X-ray irradiated bone. MATERIALS AND METHODS: A bone chamber was implanted onto the calvaria of rabbits to study the vascularization process. Digital pictures were taken of the vascular plexus at the target bone site using a modified digital camera. Vascular density (VD) was determined using image processing. It was defined as the ratio of blood vessel pixels to the total number of pixels to the region of interest. Laser preconditioning was performed with a diode laser (810 nm, 2 W, 3 seconds, 48 J/cm(2), 4 mm). A 12-week follow-up study was performed on 20 rabbits divided into four groups: #1: control group (n = 5); #2: laser irradiation alone (n = 5). #3: X-ray radiation (18.75 Gy) alone (n = 5), #4: laser preconditioning 24 hours prior to X-ray radiation (n = 5). RESULTS: VD remained stable during the 12-week follow up for group #1. No significant difference was observed between laser irradiation group (#2) and control group (#1) (P>0.5). The angiolytic action of X-ray radiation was confirmed in groups #3 and #4, which were statistically different from group #1 (P<0.001). However, the decrease of the vascularization was limited in group #4 highlighting a different evolution between group #3 and #4 (P<0.05). These results were confirmed by histological analysis. DISCUSSION AND CONCLUSION: The bone chamber is an effective reproducible method for the longitudinal analysis of the dynamics of vascularization. Our findings have shown that laser preconditioning is capable of preserving vascularization in an X-ray irradiated bone site, thus suggesting a novel approach for promoting the healing of bone tissue in which the vascular supply has been damaged.

Thirteen cases of jaw osteonecrosis in patients on bisphosphonate therapy.

INTRODUCTION: We report on our experience with 13 cases of jaw osteonecrosis in patients treated with amino-bisphosphonates. METHOD: Data were collected by a regional observatory for jaw osteonecrosis in northern France via letters sent to all physicians likely to manage patients with this condition. All study patients were evaluated at a multidisciplinary jaw osteonecrosis clinic between June and December 2005. RESULTS: We identified 13 cases, in 12 women and 1 man, with a mean age of 62.6 years. Intravenous amino-bisphosphonate therapy was given for metastatic bone disease from breast cancer in 7 patients and multiple myeloma in 5 patients; the remaining patient was on oral alendronate for osteoporosis. Mean treatment duration was 24 months. A history of dental extraction was found in 11 (84.6%) patients. The mandible was involved in all 13 patients and the maxillary in 3 (23%) patients. Amino-bisphosphonate therapy was discontinued in all 13 patients. We suggest a classification scheme for the clinical and computed-tomography patterns seen in our patients. CONCLUSION: Jaw osteonecrosis is a severe complication of amino-bisphosphonate therapy. In addition to the application of published guidelines, we propose discontinuing bisphosphonate therapy whenever possible. We are evaluating our classification scheme to identify early diagnostic criteria and/or clinical and computed-tomography outcome criteria that would improve the management of patients with jaw osteonecrosis.