Bone characteristics in condylar hyperplasia of the temporomandibular joint: a microcomputed tomography, histology, and Raman microspectrometry study.

Unilateral condylar hyperplasia (UCH) of the temporomandibular joint is a progressive deformation of the mandibular condyle of unknown origin. UCH is characterized by excessive growth of the condylar head and neck, leading to an increase in size and volume. The aim of this study was to investigate the characteristics of the bone in patients with UCH using microcomputed tomography (micro-CT), histology, and Raman microspectroscopy. The mandibular condyles of six patients with UCH were analysed using micro-CT, histology, and Raman microspectrometry and imaging, and the results were compared with those obtained for a normal control subject. Three-dimensional micro-CT models revealed focal abnormalities of the bone microarchitecture, with foci of osteosclerosis. Histological sections showed that these foci included islands of calcified cartilage matrix with live chondrocytes. Raman analysis revealed that the cartilage matrix was more heavily calcified than the bone matrix and that the cartilage could be identified by the phenylalanine (PHE) band of its matrix, as well as by its glycosaminoglycan (GAG) content. The persistence of foci of live and active chondrocytes within the bone matrix is intriguing and appears to be pathognomonic of UCH. These new findings on UCH could help to determine its pathophysiology and thus prevent this disease, which can lead to major facial deformity.

Osseointegration of two types of titanium cylinders with geometric or trabecular microarchitecture: A nanotomographic and histomorphometric study.

Porous biomaterials promote osseointegration. We have prepared porous titanium cylinders by additive manufacturing from titanium beads. Two types of morphology were tested: cylinders with geometric pores or mimicking trabecular microarchitecture. Cylinders were decontaminated and cleaned by HF/HNO3 to remove unmelted balls. Surgical implantation in ewes was performed under general anesthesia and the animals were housed for 90 and 270days. The femoral condyles were collected and analyzed by nanoCT, embedded in pMMA and analyzed by histomorphometry. No significant difference was found in terms of bone volume or bone/titanium interface between the two types of cylinders. There was no evolution over time except for the mineralization rates which decreased, reflecting the effect of the aging of the animals. The influence of the pores (geometrical or "natural") did not influence osseointegration. HF/HNO3 etching treatments are effective on the outermost surfaces but do not seem to reach the central cavities of the samples. Finally, osseointegration seems to occur only in the few millimeters around the periphery of the implants and does not extend in the center. This is explained by the absence of stress transmission within the very rigid metal cylinders, preventing bone modeling and remodeling.

Necrosis of the femoral head, X-ray microtomography (microCT) and histology of retrieved human femoral heads.

Aseptic osteonecrosis of the hip (AON) is a rare, but well-known pathology in rheumatology and orthopedic surgery that is a necrosis of the articular cartilage secondary to a necrosis of the subchondral bone. The microscopic aspect is well known, but the microCT aspect has never been reported or correlated with histopathological findings. The objective of this study was to improve the knowledge of the pathophysiology of AON using histochemistry and microCT. One hundred and sixty femoral heads with stage 3 or 4 AON were analyzed: one half of the head was sent for microCT analysis after impregnation with phosphotungstic acid (PTA) and the other half was used for histological analysis without decalcification. The microCT analysis provides relevant information on the cracked articular cartilage and the relationship with the necrotic subchondral trabecular bone well illustrated on three videos. In histology, Goldner's trichrome showed that the articular cartilage remains well preserved for a long time. In addition, toluidine blue staining reveals a modeling process, i.e. the apposition of new bone without prior resorption by osteoclasts. Rhodamine B staining (fluorescence analysis) reveals that the osteonecrotic trabeculae and subchondral bone were devoid of osteocytes. Areas of peri-necrotic osteosclerosis are due to direct bone formation on the surface of pre-existing necrotic trabeculae.

Wear debris released by hip prosthesis analysed by microcomputed tomography.

Total hip arthroplasty uses commercial devices that combine different types of biomaterials. Among them, metals, ceramics and metal oxides can be used either in the prosthesis itself or in the cement used to anchor them in the bone. Over time, all of these materials can wear out and release particles that accumulate in the periprosthetic tissues or can migrate away. We used histology blocks from 15 patients (5 titanium metallosis, 5 alumina prostheses, 5 with altered methacrylic cement) to perform a microCT study and compare it with conventional histology data. An EDS-SEM analysis was done to characterise the atomic nature of the materials involved. A morphometric analysis was also performed in 3D to count the particles and assess their density and size. The metallic particles appeared to be the largest and the ceramic particles the finest. However, microCT could not reveal the wear particles of radiolucent biomaterials such as polyethylene and the very fine zirconia particles from cement fragmentation. MicroCT analysis can reveal the extent of the accumulation of these debris in the periprosthetic tissues. LAYOUT DESCRIPTION: Hip prostheses progressively degrade in the body by releasing wear debris. They accumulate in the periprosthetic tissues. Microcomputed tomography was used to image three types of radio-opaque wear debris: metal, ceramic and zirconia used in the bone cements.

Microvascularization of the human central and peripheral nervous system: A new microcomputed tomography method.

Microcomputed X-ray tomography (microCT), developed since the late 1990s, is a miniaturized version of the tomographs used daily in medical imaging. It produces vascular images that are different from those obtained by microradiography, in particular by facilitating the vision in space, thus understanding microvascularisation. The anatomical specimens, once treated with formalin, are injected with a mixture made of gelatin containing a contrast product (barium) and then analyzed by microCT. The acquisition times that can exceed 24hours and metal sheets used for X-ray filtering vary according to the sample. The projection images are reconstructed to produce 2D sections. These are combined for the reconstruction of 3D models using a volume rendering software. Four examples will allow the imaging of microvascularization: the inferior alveolar nerve, the cerebral cortex and pia-mother, brain stem, central gray nuclei (ganglia at the base of the brain). Small capillaries are highlighted using high-end software for reconstruction. Conventional software or freeware cause a considerable loss of information on small vessels that are not visualized. The VGStudio max high-end software allows the production of videos that are particularly useful for 3D exploration and teaching (four videos are provided with this article).

Bone lesions in systemic mastocytosis: Bone histomorphometry and histopathological mechanisms.

Osteoporosis is considered the most frequent skeletal manifestation of systemic mastocytosis (SM). We performed a retrospective analysis of sixty patients (37 males and 23 females) who underwent a bone biopsy in the assessment of SM or in the assessment of unexplained bone fragility. Thirty-three had simultaneously a bone marrow biopsy with a Jamshidi's needle; this sample was used for immunohistochemical analysis (tryptase, c-KIT. CD20, VCAM-1). Bone biopsy was realized in 42 cases in the assessment of SM to provide histologic proof of the disease and in 18 cases in the assessment of unexplained bone fragility and surprisingly revealed a SM. An increased bone turnover was observed in patients with SM with elevated eroded surfaces, osteoclast number and bone formation rate. In addition to nodules of mast cells (MC), a high number of MC was directly apposed on the trabeculae, affixed on the osteoblasts or the lining cells. The VCAM-1 adhesion protein recognizing α4ß7 and α4ß1 integrins may be a candidate to explain this particular adherence. One third of the bone marrow biopsies did not exhibit MC nodules or MC infiltration and led to a false negative diagnosis for SM. SM can be discovered in the assessment of fracture or osteoporosis. Transiliac bone biopsy allows for the diagnosis of the disease more accurately than bone marrow biopsy; it also provides a histomorphometric analysis of bone remodeling.

GLP-2 administration in ovariectomized mice enhances collagen maturity but did not improve bone strength.

Osteoporosis and bone fragility are progressing worldwide. Previous published literature reported a possible beneficial role of gut hormones, and especially glucagon-like peptide-2 (GLP-2), in modulating bone remodeling. As now (Gly2)GLP-2 is approved in the treatment of short bowel syndrome, we thought to investigate whether such molecule could be beneficial in bone fragility. MC3T3 and Raw 264.7 were cultured in presence of ascending concentrations of (Gly2)GLP-2. Collagen crosslinks, maturity, lysyl oxidase activity and osteoclastogenesis were then analyzed. Furthermore, (Gly2)GLP-2, at the clinical approved dose of 50 µg/kg/day, was also administered to ovariectomized Balb/c mice for 8 weeks. Hundred µg/kg zoledronic acid (once iv) was also used as a positive comparator. Bone strength, microarchitectures and bone tissue composition were analyzed by 3-point bending, compression test, microCT and Fourier transform infrared imaging, respectively. In vitro, (Gly2)GLP-2 was potent in enhancing bone matrix gene expression but also to dose-dependently enhanced collagen maturation and post-processing. (Gly2)GLP-2 was also capable of reducing dose-dependently the number of newly generated osteoclasts. However, in vivo, (Gly2)GLP-2 was not capable of improving neither bone strength, at the femur diaphysis or lumbar vertebrae, nor bone microarchitecture. On the other hand, at the tissue material level, (Gly2)GLP-2 significantly enhances collagen maturity and reduce phosphate/amide ratio. Overall, this study highlights that despite modification of bone tissue composition, (Gly2)GLP-2, at the clinical approved dose of 50 µg/kg/day, did not provide real beneficial effects in improving bone strength in a mouse model of bone fragility. Further studies are recommended to validate the best dose and regimen of administration to significantly enhance bone strength.

Hyaluronic Acid Stimulates Osseointegration of ß-TCP in Young and Old Ewes.

Cross-linked hyaluronic acid (HyAR) increases the local concentration of growth factors. We compared ß-TCP osseointegration in old and young ewes with/without HyAR addition. A blind tunnel was drilled on the medial femoral condyle of each knee in nine young and nine old ewes and was filled with ß-TCP, ß-TCP + HyAR or left unfilled. Double labeling with calcein allowed histodynamic analysis. Ewes were sacrificed at 84 days and the knees were harvested. MicroCT provided histomorphometric parameters: trabecular bone volume, residual volume of biomaterial. Histodynamic parameters were: mineralization rate, mineralized surfaces, bone formation rate. A non-parametric ANOVA and post hoc test analyzed differences between subgroups. Osseointegration of ß-TCP was similar in the aged/young grafted groups. Trabecular bone volume was significantly increased versus ungrafted animals (p < 0.001). There were no significant difference for bone volume, residual volume of biomaterial and histodynamic parameters when a single parameter was considered but additional effects of ß-TCP and HyAR were evidenced by 3D analysis. Addition of HyAR to ß-TCP does not significantly increase bone volume but tends to increase histodynamic parameters. However, considering the reduction of osteoblastic activity in aged animals, ß-TCP, and HyAR boosts osteoblastic activity. HyAR leads to an equivalent response between young and old animals.

Polyhydroxyalkanoate (PHBV) fibers obtained by a wet spinning method: Good in vitro cytocompatibility but absence of in vivo biocompatibility when used as a bone graft.

Polyhydroxyalkanoates (PHAs) are biomaterials widely investigated for tissue-engineering applications. In this regard, we describe a method to prepare fibers of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) by a wet-spinning technique. Polymer fibers were used to test the cytocompatibility of the material in vitro. We have investigated their behavior in vitro in presence of the osteoblast-like (SaOs2) and macrophage (J774.2) cell lines. The PHBV fibers used were 100-200µm in diameter and offered a large surface for cell adhesion, similar to that they encounter when apposed onto a bone trabeculae. The fiber surface possessed a suitable roughness, a factor known to favor the adherence of cells, particularly osteoblasts. PHBV fibers were degraded in vitro by J774.2 cells as erosion pits were observable by transmission electron microscopy. The fibers were also colonisable by SaOs2 cells, which can spread and develop onto their surface. However, despite this good cytocompatibility observed in vitro, implantation in a bone defect drilled in rabbit femoral condyles showed that the material was only biotolerated without any sign of osteoconduction or degradation in vivo. We can conclude that PHBV is cytocompatible but is not suitable to be used as a bone graft as it does not favor osteoconduction and is not resorbed by bone marrow macrophages.

Microcomputed tomography (microCT) and histology of the mandibular canal in human and laboratory animals.

The inferior alveolar nerve (IAN) is a sensitive branch of the trigeminal nerve. It has an intra-bone path in the mandible, inside the mandibular canal, where it is accompanied by lymph, venous and arterial vessels. We have studied the mandibular canal in human mandibles and in some laboratory animals (mice, rats, rabbits and cats). Microcomputed tomography evidenced that the walls of the canal are made with thin plates of trabecular bone with numerous fenestrations. This aspect is evidenced in dentate subjects and become more evident in edentulous subjects with atrophy of the alveolar bone. In rats and mice, the wall of the canal is also clearly composed of trabecular plates coming from the surrounding alveolar bone of the mandible. In the rabbit, similar findings are also observed but the trajectory of the canal is more difficult to identify. In the cat, the floor of the canal is composed of the cortical bone from the basilar cortex of the mandible and the roof has a trabecular nature. Vascular injections of gelatin-barium evidenced the arterial trajectories inside the bone in rats and humans. Undecalcified bone sections in human evidenced the histological aspect of the IAN and its connective sheets. Some nervous bundles can be observed outside the epineurium. Bone remodeling is observed on the wall of the mandibular canal. These descriptive findings have a clinical relevance in dental implantology or mandibular surgery.

Metaplastic woven bone in bone metastases: A Fourier-transform infrared analysis and imaging of bone quality (FTIR).

Most osteolytic tumors are in fact mixed and contain an osteoblastic component associated with the predominant osteolytic areas. This metaplastic woven bone is always evidenced by histological analysis even in the absence of radiological expression. Metaplastic bone formation reflects the activation of new osteoblasts coming from the stimulation of the dormant lining cells. Twelve patients with secondary metastases of the iliac crest evidenced by hot spots on a 99Tc-MBP san were diagnosed by histomorphometry on bone biopsies. Fourier Transformed InfraRed analysis and Imaging (FTIRI) was used on 4µm thick sections of undecalcified bone. The mineralization degree, carbonate substitution, crystallinity and the cross-links ratio of collagen (1660/1690cm-1 bands) were determined. The matrix characteristics were analyzed and imaged in the pre-existing residual bone and in the metaplastic woven bone in the vicinity of the tumor cells. FTIRI provided images of the phosphate, amide and combination of peak ratio after having selected the peaks of interest. In addition, the matrix properties can be measured and compared between the old and newly-formed bones. Woven bone appeared poorly calcified with a low phosphate/amide ratio (P=0.03) crystallinity (P<0.0001) and carbonate substitution (P=0.003). Collagen was less mature as evidenced by lower cross-links (P=0.01). Woven bone associated with bone metastasis appears poorly mineralized and rapidly elaborated by osteoblasts. The collagenous phase of the bone matrix has a low level of reticulation. FTIRI is a powerful tool to measure and visualize the various components of the bone matrix in human diseases.

Strontium ranelate stimulates trabecular bone formation in a rat tibial bone defect healing process.

Strontium ranelate treatment is known to prevent fractures. Here, we showed that strontium ranelate treatment enhances bone healing and affects bone cellular activities differently in intact and healing bone compartments: Bone formation was increased only in healing compartment, while resorption was reduced in healing and normal bone compartments. INTRODUCTION: Systemic administration of strontium ranelate (SrRan) accelerates the healing of bone defects; however, controversy about its action on bone formation remains. We hypothesize that SrRan could affect bone formation differently in normal mature bone or in the bone healing process. METHODS: Proximal tibia bone defects were created in 6-month-old female rats, which orally received SrRan (625 mg/kg/day, 5/7 days) or vehicle (control groups) for 4, 8, or 12 weeks. Bone samples were analyzed by micro-computed tomography and histomorphometry in various regions, i.e., metaphyseal 2nd spongiosa, a region close to the defect, within the healing defect and in cortical defect bridging region. Additionally, we evaluated the quality of the new bone formed by quantitative backscattered electron imaging and by red picosirius histology. RESULTS: Healing of the bone defect was characterized by a rapid onset of bone formation without cartilage formation. Cortical defect bridging was detected earlier compared with healing of trabecular defect. In the healing zone, SrRan stimulated bone formation early and laterly decreased bone resorption improving the healing of the cortical and trabecular compartment without deleterious effects on bone quality. By contrast, in the metaphyseal compartment, SrRan only decreased bone resorption from week 8 without any change in bone formation, leading to little progressive increase of the metaphyseal trabecular bone volume. CONCLUSIONS: SrRan affects bone formation differently in normal mature bone or in the bone healing process. Despite this selective action, this led to similar increased bone volume in both compartments without deleterious effects on the newly bone-formed quality.

In vivo erosion of orthopedic screws prepared from nacre (mother of pearl).

BACKGROUND: Biodegradable biomaterials have been proposed to prepare orthopedic devices. Nacre is a natural aragonitic material made of calcium carbonate and is bioerodible. WORKING HYPOTHESIS: We postulated that nacre is biodegradable without provoking bone erosion and favors bone apposition. MATERIAL AND METHODS: We prepared orthopedic screws from nacre of the giant oyster Pinctada maxima. Threaded screws (3.5mm diameter) were implanted in 6 ewes in the upper tibial metaphysis (3 to 4 screws per animal). Their trajectory was transcortical and intramedullary to the opposite cortex. Animals were kept for 3months (n=2) and 6 months (n=4). They did not develop local inflammation. Before euthanasia, they received a double calcein labeling. Bone samples were analyzed by X-ray nanotomography and histology after embedding in poly(methyl methacrylate). The fractal dimension of the screw profiles (measured by the box-counting method) was used to quantify surface erosion. RESULTS: 3D nanotomography showed a gradual erosion of the threads, which was confirmed by a decreased fractal dimension. Histologically, multinucleated cells (non-osteoclastic appearance) were visible at the surface of the screws. No ruffled border was seen in these cells but they had extensions creeping in the organic matter between the aragonite tablets. Bone apposition was noted in the transcortical path of the screws with limited osteoconduction at the endosteum. Mineralization rate was increased in these zones composed of woven bone in contact with the nacre. DISCUSSION AND CONCLUSION: Screws prepared from nacre have the advantage of an in vivo resorbability by macrophage-derived cells and an osteoconductive apposition in contact with the material without triggering a local inflammatory reaction.

Aluminum and bone: Review of new clinical circumstances associated with Al(3+) deposition in the calcified matrix of bone.

Several decades ago, aluminum encephalopathy associated with osteomalacia has been recognized as the major complication of chronic renal failure in dialyzed patients. Removal of aluminum from the dialysate has led to a disappearance of the disease. However, aluminum deposit occurs in the hydroxyapatite of the bone matrix in some clinical circumstances that are presented in this review. We have encountered aluminum in bone in patients with an increased intestinal permeability (coeliac disease), or in the case of prolonged administration of aluminum anti-acid drugs. A colocalisation of aluminum with iron was also noted in cases of hemochromatosis and sickle cell anemia. Aluminium was also identified in a series of patients with exostosis, a frequent benign bone tumor. Corrosion of prosthetic implants composed of grade V titanium (TA6V is an alloy containing 6% aluminum and 4% vanadium) was also observed in a series of hip or knee revisions. Aluminum can be identified in undecalcified bone matrix stained by solochrome azurine, a highly specific stain allowing the detection of 0.03 atomic %. Colocalization of aluminum and iron does not seem to be the fruit of chance but the cellular and molecular mechanisms are still poorly understood. Histochemistry is superior to spectroscopic analyses (EDS and WDS in scanning electron microscopy).