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.

Skull fractures in forensic putrefied/skeletonised cases: The challenge of estimating the post-traumatic interval.

The objective of our study was to assess the reliability of the estimation of posttraumatic survival time (PTST) in forensic cases based on microCT and histology of putrefied/dry bone samples with comparison of initial macroscopic fracture classification performed during autopsy. Macroscopic morphological patterns of bone fracture are routinely used in forensic pathology and anthropology to distinguish between antemortem, perimortem and postmortem injuries. Based on macroscopic and microscopic analysis of six craniofacial fractures, our study results illustrate the need to complete macroscopical findings and initial fracture classification with microscopic analysis to avoid any inaccuracy. MicroCT has become a powerful technique to identify early bone healing signs but histology remains the gold standard to estimate the PTST and determine vital fracture based on hemorrhage marker. Raman microspectroscopy can identify a blood clot in the fracture line.

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.

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.

Measurement by vertical scanning profilometry of resorption volume and lacunae depth caused by osteoclasts on dentine slices.

The resorption pit assay is classically used to evaluate osteoclast activity on bone or dentine slices that can be eroded by these cells. Two different types of cells were generated from peripheral blood mononuclear cells cultured in the presence of M-CSF + sRANKL or with M-CSF + LPS. At the end of the culture period (21 days), cells were discarded and the dentine slices stained with toluidine blue and examined with an NT9100 Wyco vertical scanning profilometer. The images of the dentine surface were corrected for tilt and the eroded volume was calculated on the whole images. The depth of the eroded pits was determined. The data files were used to reconstruct the surface of the slices by standardizing the ground level to compare both conditions. Osteoclasts generated with M-CSF + sRANKL were capable of resorbing a more important volume than those generated with M-CSF + LPS. In addition, the formers were able to resorb the dentine matrix more deeply. Data provided by the microscope were used to reconstruct three-dimensional images of the dentine slices with pseudo colours varying with the depth of erosion. Vertical scanning profilometry, a technique used to measure the roughness of polished or etched surfaces in metallurgic industry, can be used to accurately measure the eroded volume and the mean erosion depth done by osteoclasts in the resorption pit assay.

Does milling one-piece titanium dental implants induce osteocyte and osteoclast changes?

One-piece dental implants avoid adverse effects sometimes associated with the traditional implant-abutment interface and may provide a suitable alternative to two-piece implants; however, one-piece implants often need in situ milling, which may exacerbate cell apoptosis from excessive heat at the bone-implant interface and induce secondary crestal bone loss. Twelve implants were placed in the metaphyses of two sheep under general anesthesia. Six implants were milled with a diamond bur while the other six implants remained intact. Animals were euthanized after four days, and bone blocks were harvested. Bone samples were studied without decalcification. Osteocytes were stained with Hoechst 33342 and osteoclasts by the TRAcP reaction. Both cell types, in the cortical and trabecular bone around the implant's cervical region, were counted utilizing morphometric methods. Values were compared to areas at a distance from the cervical region. No difference was observed between milled and unmilled implants, which suggested that the amount of generated heat did not provoke osteocyte loss or induce osteoclastogenesis. Intraoral abutment preparations did not increase cellular apoptosis at the bone-implant interface after four days in the ovine model.

Osteopontin is an argentophilic protein in the bone matrix and in cells of kidney convoluted tubules.

Nucleolar organising regions (NOR) are part of the nucleolus, containing argyrophilic proteins (nucleoclin/C23, nucleophosmin/B23). They are identified by silver staining at low pH. The method also reveals osteocyte canaliculi and cement lines and granules in the cytoplasm of kidney cells in locations that mimic osteopontin distribution. Human bone and kidney sections, benign and lymphomatous pleural effusions were processed for silver staining to identify AgNOR. Sections were processed in parallel for immunohistochemistry with an antibody direct against osteopontin. In pleural effusions, AgNORs were found increased in the nuclei of lymphoma cells. In bone, Ag staining identified AgNOR in cell nuclei, as well as in osteocyte canaliculi, cement and resting lines. In the distal convoluted tubules of the kidney, silver deposits were also observed in cytoplasmic granules on the apical side of the cells. Immunolocalization of osteopontin closely matched with all these locations in bone and kidney. NOR proteins and osteopontin are proteins containing aspartic acid rich repeats that can bind Ag. Staining protocols using silver nitrate at low pH can identify these proteins on histological sections. AgNOR is a useful histochemical method to identify osteopontin in bone sections.

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.

Osteopontin is histochemically detected by the AgNOR acid-silver staining.

Silver nitrate staining of decalcified bone sections is known to reveal osteocyte canaliculi and cement lines. Nucleolar Organising Regions (NOR) are part of the nucleolus, containing argyrophilic proteins (nucleoclin/C23, nucleophosmin/B23) that can be identified by silver staining at low pH. The aim of this study was to clarify the mechanism explaining why AgNOR staining also reveals osteocyte canaliculi. Human bone and kidney sections were processed for silver staining at light and electron microscopy with a modified method used to identify AgNOR. Sections were processed in parallel for immunohistochemistry with an antibody direct against osteopontin. Protein extraction was done in the renal cortex and decalcified bone and the proteins were separated by western blotting. Purified hOPN was also used as a control. Proteins were electro-transferred on polyvinylidene difluoride membranes and stained for AgNOR proteins. In bone, Ag staining identified AgNOR in cell nuclei, as well as in osteocyte canaliculi, cement and resting lines. In the distal convoluted tubules of the kidney, silver deposits were also observed in cytoplasmic granules on the apical side of the cells. Immunolocalization of osteopontin closely matched with all these locations in bone and kidney. Ag staining of membranes at low pH revealed bands for NOR proteins and 56 KDa (kidney), 60KDa (purified hOPN) and 75 KDa (bone) bands that corresponded to osteopontin. NOR proteins and osteopontin are proteins containing aspartic acid rich regions that can bind Ag. Staining protocols using silver nitrate at low pH can identify these proteins on histological sections or membranes.