Areas Of Bone Matrix Research Articles

Melatonin is more effective on bone metabolism when given at early night than during the day in ovariectomized rats

Melatonin has diverse effects, and has been reported to promote bone formation in addition to regulating the sleep–wake cycle. In the present study, we investigated the effects of melatonin on bone metabolism using ovariectomized (OVX) rats; a model of postmenopausal osteoporosis. Here, we focused on the differences in bone formation when melatonin was subcutaneous injected at day or early night. The OVX rats were injected with melatonin once daily (0.8 or 8 mg/head) between 11:00 to 14:00 or 18:00 to 19:30 for the day or early night, respectively, for six weeks. After completion of the injection, the femur and tibia in the OVX rats were dissected under general anesthesia and examined by quantitative computed tomography (pQCT) and histological analysis, respectively. Interestingly, the trabecular bone mineral density in the femur metaphysis of the OVX rats receiving 8 mg/head melatonin at early night was higher than those receiving melatonin during the day and they recovered to a similar level as the rats with sham treatment. In the diaphysis, the pQCT analysis results indicated that there was no significant difference in bone mineral density between the day and early night melatonin-injected OVX rats. Histological analysis of the secondary trabecular bone in the tibia of the OVX rats, revealed that the bone matrix area of the group receiving 8 mg/head melatonin at early night was higher compared with that of the day group and had a significant difference compared with OVX treatment rats. Taken together, the subcutaneous melatonin injection in OVX rats at early night was found to promote trabecular bone formation better than melatonin injection during the day. The timing of melatonin injection is a crucial factor when examining the influence of bone metabolism.

Dose-dependent Effect of Zoledronic Acid on the Development of Medication-Related Jaw Osteonecrosis (MRONJ)-Like Lesion in C57Bl/6 Mice: A Histological Preliminary Study

Medication-related osteonecrosis of the jaw (MRONJ) is a potentially painful condition affecting patients with previous or continuous treatment with antiresorptive or antiangiogenic agents. In this context, the cumulative use of zoledronic acid (ZL) is important duration medication-related risk factors for patients needing tooth extraction. In this preliminary study, we investigated the development of MRONJ-type lesions in C57Bl/6 male mice (ethical committee approval n°00262-2019) at the late time point of socket healing after continuous and cumulative administration of zoledronic acid (ZL) in 2 different dosages. Mice at age of 4 to 6 months were divided, receiving intraperitoneal injection of saline solution (SS, control, n = 3) or ZL at a concentration of 250ug/Kg (n = 3) and 500ug/Kg (n = 4) once a week, starting 4 weeks before extraction of the right upper incisor and continuing until the 30-day post-surgical period. At the end of 30 days, bone samples were collected for histological (H&E and Goldner Trichrome [GT] staining), histomorphometric and immunohistochemical analyzes for bone (Rankl), and inflammatory (COX-2) markers. While the control group exhibit a dental socket filled with mature bone, the ZL-250 and ZL-500 groups presented a significant and gradual increase in the area density of inflammatory infiltrate dependent of each dosage. Bone matrix area density was significantly lower in ZL-250 and ZL-500 groups, and GT staining revealed a gradual impairment in bone matrix in ZL-250 and ZL-500 groups compared to control (Figure 1). Area density of COX2+ cells, an inflammatory parameter positively related to bone neoformation, was also decreased in both treated groups compared to the control. On the other hand, area densities of fibroblasts, RANKL+ cells, and empty osteocyte lacunae were increased in ZL-250 and ZL-500 groups. Interestingly, the area density of detached osteoclasts, a typical characteristic of ZL effect, was higher in the ZL250 group compared to the SS, while ZL500 group presented a lower density of attached osteoclasts compared to the others. In summary and despite of limitations of the present study, the final outcome of alveolar bone healing was significantly affected in C57BL/6 mice using both dosages used in this study, but with increasing effects on histological MRONJ parameters at the higher dosage.

Collagen production of osteoblasts revealed by ultra-high voltage electron microscopy.

In the bone, collagen fibrils form a lamellar structure called the "twisted plywood-like model." Because of this unique structure, bone can withstand various mechanical stresses. However, the formation of this structure has not been elucidated because of the difficulty of observing the collagen fibril production of the osteoblasts via currently available methods. This is because the formation occurs in the very limited space between the osteoblast layer and bone matrix. In this study, we used ultra-high-voltage electron microscopy (UHVEM) to observe collagen fibril production three-dimensionally. UHVEM has 3-MV acceleration voltage and enables us to use thicker sections. We observed collagen fibrils that were beneath the cell membrane of osteoblasts elongated to the outside of the cell. We also observed that osteoblasts produced collagen fibrils with polarity. By using AVIZO software, we observed collagen fibrils produced by osteoblasts along the contour of the osteoblasts toward the bone matrix area. Immediately after being released from the cell, the fibrils run randomly and sparsely. But as they recede from the osteoblast, the fibrils began to run parallel to the definite direction and became thick, and we observed a periodical stripe at that area. Furthermore, we also observed membrane structures wrapped around filamentous structures inside the osteoblasts. The filamentous structures had densities similar to the collagen fibrils and a columnar form and diameter. Our results suggested that collagen fibrils run parallel and thickly, which may be related to the lateral movement of the osteoblasts. UHVEM is a powerful tool for observing collagen fibril production.

Ovariectomy-associated changes in interradicular septum and in tibia metaphysis in different observation periods in rats

In order to standardize an experimental model to study the effects of absence of ovarian hormones in maxillary bones compared with long bones, the aim of this research was to analyze the influence of ovariectomy (OVX) on rat alveolar bone and tibiae, in different observation periods. Thirty-six female rats were ovariectomized or sham operated. After 60, 90 or 120 days, the animals were sacrificed and their hemimandibles, maxillae and tibiae were removed and routinely prepared for hematoxylin and eosin staining. The percentage of bone matrix area in bone septum in the first molar furcation region, and in tibial metaphysis was calculated, and data were submitted to statistical analysis (p<0.05). As regards the histomorphometrical analysis in jaw bones, there was no statistical difference between groups, while the effects of ovariectomy on tibiae were seen as early as 60 days. According to the methods used, there was no significant influence of absence of ovarian hormones on interradicular septum of mandibular or maxillary first molars in the periods studied, despite the reduction in bone matrix area in tibia metaphysis as early as 60 days.

Lyophilized xenograft: a case series of histological analysis of biopsies.

The reconstruction of an acetabular bone defect is one of the most important aspects of revision total hip arthroplasty surgery. It can be done by the use of grafts. Therefore, many kinds of grafts may be used and lyophilized xenograft is an alternative example. The purpose of the study was to evaluate the histological findings of lyophilized bovine xenografts used in previous total hip arthroplasty revision surgery. A case series was carried out from July 2000 to April 2013 with the approval of the Hospital Ethics Committee. Fourteen subjects were analyzed. Of these, 64.3 % were female. The average age of the patients was 52.36 ± 18.55. Neoformed bone was present in 85.7 % of subjects, and constituted 61.79 % of the total bone matrix area. The diagnosis of xenograft absorption was present in 12 subjects. A strong inverse correlation between the percentage of neoformed bone and the percentage of xenograft in the total bone matrix by analysis of biopsies was found by the Pearson test (p = 0.001). No inflammatory response was found in the clinical status of the patients or in the histological analysis. Lyophilized bovine xenograft shown to be safe until the present moment, with acceptable results. Most of the cases presented new bone formation as expected (considered values greater than 30 %) and for this reason the xenograft has proven to be an osteoconductive compatible scaffold/trellis for the bone ingrowth. Therefore, it can be considered an alternative to other bone grafts in the treatment of bone loss.

Natural bone collagen scaffold combined with OP‐1 for bone formation induction in vivo

The scaffold is a key element to osteogenic tissue engineering as it provides a microenvironment for bone formation. Natural bone collagen scaffold (NBCS) is a novel biomaterial scaffold acid-extracted from organic human bone. The objective of this study was to characterize NBCS and evaluate the osteoconductivity of the scaffold, in combination with osteogenic protein-1 (OP-1), using a rabbit posteolateral lumbar fusion model. Thirty two rabbits were divided into 4 experimental groups, autograft, NBCS alone, OP-1 alone or NBCS combined with OP-1. Bone formation was evaluated by micro-CT, quantitative histological analysis, immunohistochemistry and semi-quantitative RT-PCR at 6 weeks postoperatively. By scanning electronic microscope, we showed that NBCS maintains a porous, interconnecting microarchitecture. Micro-CT analysis demonstrated that NBCS combined with OP-1 significantly induced (p < 0.01) bone formation at the fusion site as compared to control groups. This was confirmed by quantitative histological analysis which demonstrated that the NBCS combined with OP-1 significantly enhanced bone matrix area (17.7 mm(2)) (p < 0.05) and bone marrow cavity size (71.3 mm(2)) (p < 0.05) as compared to the controls. Immunohistochemical assessment and RT-PCR also demonstrated that NBCS combined with OP-1 enhanced type I collagen and osteonectin expression. Together, these results suggest that NBCS is an effective scaffold for osteogenesis, and combined with growth factors such as OP-1, possesses both osteoconductive and osteoinductive properties that are sufficient for bone regeneration.

Radiographic and histomorphometric analysis of bone healing using autogenous graft associated with platelet-rich plasma obtained by 2 different methods

The aim of this study was to conduct radiographic and histomorphometric analysis of bone healing in the calvaria of rabbits, using an autogenous graft associated with PRP obtained by 2 different methods. Thirty rabbits were divided into control and experimental groups. Lesions were produced in the calvaria and filled with autogenous graft (control) or autogenous graft and PRP obtained by the Anitua or modified Sonnleitner methods. The animals were humanely killed 15 days after surgery and the calvarias were radiographed. The radiographs were digitized to assess the radiographic density. By histologic images of the lesion, the bone matrix was quantified. There were no significant differences in the radiographic density and the bone matrix area between the groups. The association of PRP with autogenous bone did not improve the healing process, irrespective of the method used early during healing.

In vivo fatigue microcracks in human bone: Material properties of the surrounding bone matrix

Human bones sustain fatigue damage in the form of in vivo microcracks as a result of the normal everyday loading activities. These microcracks appear to preferentially accumulate in certain regions of bone and most notably in interstitial bone matrix areas. These are remnants of old bone tissue left unremodelled, which show a higher than average mineral content and consequently the occurrence of microcracks has been attributed to the possible brittleness brought about by such hypermineralisation. There is a need, therefore, for information on the in situ bone matrix properties in the vicinity of such in vivo microcracks to elucidate the possible causes of their appearance. The present study examined the elastic, strain rate (viscous) and plastic properties of bone matrix in selectively targeted areas by nanoindentation and in both quasistatic and dynamic mode. The results showed that in vivo crack areas are not as stiff as some well-known extremely mineralised and brittle bone examples (bulla, rostrum); the strain rate effects of crack regions were identical to those of other regions of human bone and agreed well with values collected for human bone in the past at the macroscale; while the plasticity index of the crack regions was also not statistically different from most bone examples (including human at random, bovine, bulla and rostrum) except antler, which showed lower plasticity and thus a greater fraction of elastic recovery in indentation energy. It is difficult, therefore, to explain the susceptibility of these interstitial regions to crack in terms of the mineral content and its after-effects on elasticity, viscosity and plasticity alone, but one need to attribute the cracks to the cumulative loading history of these areas, or raise the suggestion that these areas of bone matrix are in some measure 'aged' or material/quality defective.

Effects of Dietary Calcium Levels on the Histomorphology of Proximal Tibia in Vitamin D-deficient Chicks.

This study was conducted to clarify the effects of dietary calcium levels on tibia histomorphology in young growing chicks fed a vitamin D-deficient diet. Male day-old chicks were fed vitamin D-deficient diets either normal (1.0%) or high (2.0%) in calcium for 4 weeks. Proximal tibiae were excised and longitudinal sections were examined by light microscopy. Morphological differences were quantified by a computerlized histomorphometry. The feeding of the normal calcium diet caused significant increase in the width of epiphyseal cartilage and histomorphometric indices of bone resorption. Osteoclastic resorbing surface and all osteoclast parameters, measured at metaphysis, greatly increased in the normal calcium diet chicks compared to the controls fed the vitamin D-replete diet. The area of osteoblasts remarkably decreased in the normal calcium diet chicks. Changes in these bone cell parameters are thus associated with marked decrease in trabecular bone volume, expressed as percentage of the mineralized bone matrix area. In contrast, tibiae of the high calcium diet chicks were not significantly different from bones of the control chicks with respect to histological features and most parameters. Thus, tibia histomorphology in the high calcium diet chicks showed normalization of bone as observed in the vitamin D-replete diet chicks. These results indicate that in the growing chick continued feeding of a diet with high calcium content can maintain nearly normal epiphyseal cartilage width and metaphyseal bone histomorphology and mineralization in the absence of vitamin D.

Effects of 17.BETA.-Estradiol on the Histomorphometry of Tibial Diaphysis in Chick Embryo.

Studies were carried out to clarify the effects of 17β-estradiol on tibia histo-morphometry in chick embryos. Varying levels of 17β-estradiol (0, 20, 40, or 80μg) were injected every other day from the 10th to 18th day of incubation into the air chamber of eggs. On the 20th day of incubation, right tibiae were excised and diaphyseal cross sections were examined by light microscopy. Morphological differences were quantified using a computerlized histomorphometric technique. The administration of two higher doses of 17β-estradiol caused significant increase in histomorphometric indices of bone formation. Total bone area, measured at middiaphysis, greatly increased in the 17β-estradiol-treated embryos compared to controls receiving the vehicle alone. This increase was associated with marked increase in bone volume, expressed as percentage of the bone matrix area. The number and areas of periosteal osteoblasts were remarkably enhanced in the 17β-estradiol-treated group. 17β-Estradiol treatment did not affect the two histomorphometric indices of bone resorption, nuclei number and area of osteoclast. Exogenous 17β-estradiol would thus appear to induce periosteal osteoblast differentiation and stimulate bone formation in the chick embryo tibia.

Ultrastructural immunolocalization of a major phosphoprotein in embryonic chick bone.

Immunocytochemistry utilizing the protein A-gold technique was used to examine the ultrastructural cellular and extracellular distribution of a major phosphoprotein in chick bone. HCl-extracts of embryonic and neo-natal chick bones contain a major 66kD phosphoprotein (BPP) which was purified and used to raise polyclonal antibodies in rabbits. The mid-diaphyseal regions of 8-, 12- and 18-day embryonic chick tibiae were fixed with 1% glutaraldehyde and embedded in Epon or Lowicryl. Electron microscopy following incubation of tissue sections with the antibody and the protein A-gold complex revealed specific immunolabeling over the rER and Golgi apparatus of osteoblasts and over those areas of bone matrix containing Ca and P as determined by electron probe x-ray microanalysis. These included extracellular areas in the matrix undergoing early mineralization and electron dense patches occurring at the mineralization front and extending throughout the more mature bone regions. Biochemical analyses of bone tissue processed similarly to that used for immunocytochemistry confirmed the retention of phosphoprotein in the tissue. The spatial correlation of phosphoprotein in the extracellular matrix with Ca-P mineral deposits confirms an earlier report using 33Pi and radioautography and may indicate a role for phosphoproteins in calcification.

Estradiol-17β-Induced Morphometric Changes in Chick Embryo Femurs

The influence of exogenous estradiol-1717β (E2) on chick embryo femur morphology was assessed. Fertile chicken eggs were air cell injected with 18.75, 37.5, or 75 μg E2/.l ml distilled water: ethanol (7:3, v/v) on Days 10, 12, and 14 of incubation. Controls received vehicle only (.1 ml) on the same days. On Day 16 of incubation, right femurs were excised, and midshaft cross sections were examined by light microscopy. Structural differences were quantified using a computerized histomorphometric technique. Injection of E2 produced significant (P<.01) dose-dependent elevations in mean femur total osseous tissue area, bone matrix area, percent of matrix surfaces covered by osteoblasts, and percent of matrix area occupied by osteocytes when compared with controls. Estradiol was ineffective in inducing medullary bone formation in chick embryo femurs.We conclude that E2 is involved in bone matrix formation processes in chick embryos. The lack of medullary bone formation upon E2 treatment in chick embryos is perhaps an indication that, as in growing (immature) birds, it is likely that endogenous testosterone levels in Day 16 chick embryos are insufficient to support medullary bone growth.

Treatment of Renal Bone Disease with 1α-Hydroxylated Derivatives of Vitamin D&lt;sub&gt;3&lt;/sub&gt;&lt;subtitle&gt;CLINICAL, BIOCHEMICAL, RADIOGRAPHIC AND HISTOLOGICAL RESPONSES&lt;/subtitle&gt;

Forty patients with severe bone disease and chronic renal failure were treated with 1 alpha-hydroxycholecalciferol (1 alpha-OHD3) or 1,25-dihydroxycholecalciferol (1,25(OH)2D3) for 7--49 months (total = 738 patient months). There were symptomatic, biochemical and radiographic improvements in the majority of patients (greater than 70 per cent). Paired bone biopsies, taken before and during treatment in 26 patients, showed no change in bone matrix area, whereas matrix area decreased in a control group of 26 patients over the same period. There were small but consistent decreases in bone marrow fibrosis and in bone cell (osteoblast and osteoclast) counts in treated patients but not in controls. However, the proportion of patients who showed histological 'cure', in the sense of complete reversal of marrow fibrosis or excess osteoid was no greater in the treated than in the control group...

Osteocyte types in the developing mouse calvarium.

Tissue maps, and cell characteristics and properties were recorded in a study under the optical microscope of the development of the mouse calvarium from pre-natal to 26 days. Osteocyte populations in left and right halves of the calvarium were similar, but decreased with time for a given volume. Small isolated areas of bone matrix stained for phosphate (or carbonate) in a more readily available form from that in the surrounding matrix, which could be stained after sectioning but failed to stain in bulk. Osteocyte types were defined on the basis of histochemical methods for calcium and phosphate, which were associated inside bone cells in a complex manner, varying with time and position. The calcium and phosphate were not always present within the cell in discrete regions and were not always present in the same place in a given cell type. On the basis of a study of changes in cell types with time in selected sites a sequence of “loading” and “unloading” is proposed.