Undecalcified Bone Sections Research Articles

Microscopic Age Changes in the Human Occipital Bone

The value of histological examination of the human occipital bone for estimation of age-at-death was assessed. Undecalcified sections of occipital bone from eighteen male Caucasian subjects between the ages of 21 and 70 years were prepared for analysis using polarized light microscopy. The fractional volumes of primary osteons, secondary osteons, osteon fragments, and lamellar bone in both the outer and inner cortical tables were determined. It was found that with increasing age there is a decrease in the fractional volume of primary osteons and a significant decrease in the fractional volume of lamellar bone. The fractional volume of secondary osteons was not found to change significantly with age, while the fractional volume of osteon fragments significantly increases. The microscopic results reflect the continuous process of bone remodeling that is responsible for the variation in cortical parameters with age and is the primary basis for age predicting methods. While observable changes in the occipital bone do occur with increasing age, the amount of random variation in the parameters examined preclude their use for accurate age estimation.

Microwaveable Toluidine Blue Stain for Surface Staining of Undecalcified Bone Sections

Toluidine blue stain has been widely used for staining undecalcified bone sections, but previous techniques required removal of embedding plastic or surface etching with both alcohol and acid solutions. In this study, solutions of toluidine blue at different pHs were used to stain unetched and acidetched sections. A group of the test staining solutions were heated in an effort to enhance section staining. Acid-etching prior to staining was found to be necessary to achieve bone matrix staining, but methanol etching could be eliminated if staining solutions were heated to 55–60°C. Sections stained with the heated solutions buffered to pH 7.0 or 8.0 had optimum morphology. (The J Histotechnol 17:357, 1994)

Monoclonal antibodies with selective reactivity against osteoblasts and osteocytes in human bone.

Monoclonal antibodies (MAb) may provide valuable tools for studying osteoblast differentiation. We therefore raised a panel of MAb reactive with cells of this phenotype using 1,25(OH)2D3-treated human trabecular osteoblast-like cells (HOBS) as the immunogen. Immunohistochemical studies on various tissues, including undecalcified cryostat sections of fetal and adult human bone, identified 11 bone cell-reactive MAb. Of these, 2 demonstrated particularly selective reactivities against osteocytes (OB/M) and osteoblasts (OB/L). These reactivities were also seen in developing bone from rat, rabbit, and marmoset. OB/L and OB/M demonstrated limited reactivity against a small number of human tissues from the extensive panel of substrates tested. Both MAb exhibited reactivity against discrete populations of cells in the large and small intestine. In addition, OB/L reacted with cells in the basal epidermis of skin and OB/M with cells in blood vessel walls. Both antibodies demonstrated reactivity against a variety of cultured osteoblast-like cell lines and other cultured cell types. These MAb may therefore provide a valuable means of studying osteoblast ontogeny.

Tissue Segregation Enhances Calvarial Osteogenesis in Adult Primates

Calvarial defects of adult mammals have a limited potential for regeneration. Osteogenesis may be impaired by the intrusion within the defect of nonosteogenic tissues during healing, which inhibits centripetal bone deposition from the margins of the defects. Using a primate model, we evaluated bone regeneration in calvarial defects treated with demineralized bone matrix versus defects treated according to the strategy of guided tissue regeneration. Sixty-four defects, 25 mm in diameter, were prepared in 16 adult male baboons (Papio ursinus). In each animal, 2 contralateral defects were covered with a template of porous polymer, 35 mm in diameter. Templates were positioned over the calvarial margins to prevent penetration of the pericranium and the temporalis muscle within the defects, thus maintaining tissue segregation during healing. The third defect was implanted with allogeneic demineralized bone matrix, as positive control. The fourth defect was left untreated and was used as negative control. Undecalcified bone sections (7 microns thick) were prepared from the harvested specimens 3 and 6 months after surgery. Although untreated defects showed limited osteogenesis after fusion of the pericranium with the dura, defects covered with the polymeric template often showed extensive bone deposition extending centripetally from the margins of the craniotomies. Histomorphometry demonstrated that defects treated with demineralized bone matrix and defects covered with the polymeric template had greater amounts of bone and osteoid when compared with untreated defects (p < 0.01). At 6 months, greater amounts of bone formed in demineralized bone matrix-treated defects when compared with defects covered with the polymeric template (p < 0.01). The results of this study show that calvarial membranous bones of adult primates retain the potential to regenerate when segregation between the different tissue components participating in the healing of the wound is maintained. In the same model, however, implantation of allogeneic demineralized bone matrix, used as a positive control provided the most effective treatment for the defects.

Staining and Histomorphometry of Microcracks in the Human Femoral Head

We developed staining techniques that permit identification and histomorphometric analysis of microcracks in the human femoral head 1) from thick, ground bone sections (100 microns) by prestaining with the Villanueva mineralized bone stain (MIBS), and 2) from plastic embedded, undecalcified thin bone sections (5-15 microns) by staining in gallocyanin chrome alum-Villanueva blood stain methods. Both methods represent a significant improvement in the stainability of the microcracks, cellular and tissue elements, and the simultaneous assessment of osteoid seams and tetracycline markers by histomorphometry. Shrinkage and other artifacts were minimized, which helped to clarify some of the uncertainties arising from artifacts resulting from some bone staining methods. Histomorphometric analyses of microcracks were conducted on thick, ground sections of subchondral and trabecular bone. Microcracks were more prevalent in the subchondral bone and osteochondral junction than in the more distant trabeculae. We have consistently localized microcrack areas in bone tissues prepared in these ways.

Microwaveable Toluidine Blue Stain for Surface Staining of Undecalcified Bone Sections

Microwaveable Toluidine Blue Stain for Surface Staining of Undecalcified Bone Sections

Reconstruction of the bone--bone marrow organ by osteogenin, a bone morphogenetic protein, and demineralized bone matrix in calvarial defects of adult primates.

Information concerning the efficacy of osteogenin, a bone morphogenetic protein, and demineralized bone matrix in orthotopic sites in nonhuman primates is a prerequisite for potential clinical application in humans. After exposure of the calvaria, 84 cranial defects, 25 mm in diameter, were prepared in 26 adult male baboons (Papio ursinus). Defects were implanted with insoluble collagenous bone matrix (ICBM, the inactive collagenous residue after dissociative extraction of bone matrix with 4 M guanidine hydrochloride) reconstituted with osteogenin fractions isolated from baboon bone matrix by chromatography on heparin-Sepharose and hydroxyapatite-Ultrogel (Og Hep-HA) or osteogenin further purified using Sephacryl S-200 gel filtration chromatography (Og S-200). Baboon osteogenin with the highest biologic activity in a rodent bioassay, as determined by alkaline phosphatase activity, calcium content, and histologic analysis, was used for orthotopic implantation in baboons. Additional defects were implanted with baboon demineralized bone matrix (DBM) or ICBM without osteogenin as control. Defects also were grafted with corticocancellous bone harvested from the iliac crest or left ungrafted to monitor the spontaneous regeneration potential of the adult baboon calvaria. Undecalcified bone sections at 7 microns were prepared from the harvested specimens 30 and 90 days after surgery. Histomorphometry demonstrated that Og S-200 induced copious amounts of bone and osteoid as early as day 30 (P < 0.01 versus ICBM, autogenous grafts and untreated defects). At day 90, in implants of Og S-200, Og Hep-HA, and DBM, bone and marrow formation was extensive, culminating in complete regeneration of the craniotomies. In implants of DBM, bone formed with an intervening phase of cartilage development. This provides the phenotypic evidence of endochondral bone differentiation by induction in defects of membranous calvarial bone in adult primates. These results establish the potential therapeutic application of osteogenin and demineralized bone matrix for the architectural reconstruction of the bone-bone marrow organ in humans.

Histomorphometric changes in iliac trabecular bone during pregnancy and lactation in beagle dogs.

Changes in bone metabolism throughout pregnancy and lactation in female beagle dogs were examined by histomorphometry and measurement of serum biochemical constituents related to bone. A total of eight dogs, including one dog observed repeatedly from 1 year and 8 months to 4 years and 7 months of age, were used. For double bone labeling to obtain the bone dynamic parameters, dogs were each injected twice, at an interval of 7 days, with fluorochromes such as tetracycline or calcein at the time just after mating, just after delivery, and before weaning. At the time just after weaning, the iliac bone was biopsied. Histomorphometric analysis of trabecular bone area of undecalcified iliac bone sections was performed using a semiautomatic image analyzer. Serum biochemical parameters related to bone such as parathyroid hormone, calcitonin, calcium, phosphorus and alkaline phosphatase activity were periodically measured. Bone histomorphometric analysis revealed significant increases in the mean values of osteoid volume and mineral apposition rate as compared with those of age-matched no pregnancy dogs. The mineral apposition rate measured with the passage of time was increased after delivery and was further increased before weaning compared with that at the time of mating. There were no significant changes in mean values of bone volume, mean trabecular thickness, bone formation rate and mineralizing lag time.(ABSTRACT TRUNCATED AT 250 WORDS)

Integrin subunit expression by human osteoblasts and osteoclasts in situ and in culture.

The extracellular matrix may be considered as an insoluble local mediator which plays an important role in regulating cell function. Communication between the cell and its matrix occurs via the integrins, a family of transmembrane proteins composed of non-covalently linked alpha and beta subunits. The aim of this study was to establish which integrins are present on human bone cells in situ and in culture, using cryostat sections of undecalcified human bone, osteoclastoma tissue and cultured human osteoblasts. Integrin subunit expression was identified indirectly using alkaline phosphatase anti-alkaline phosphatase conjugates and FITC-labelled secondary antibodies. Subunits expressed by cultured human osteoblast-like cells were then quantified by FACS analysis. Staining patterns observed in situ show that osteoblasts and osteoclasts possess different integrin subunits. Osteoblasts primarily express alpha 1, alpha 3 and beta 1 and weakly express alpha 2. Osteoclasts express alpha 2, alpha V, beta 1 and beta 3. Subunits alpha 4, alpha 5, alpha 6, alpha L, alpha M and beta 2 were not expressed by either of these cell types. Expression of beta 1 by all cells of the osteoblastic lineage was constitutive, but alpha 1 and alpha 3 subunits were expressed by osteoblasts actively synthesizing bone and some of the osteoblast lining cells. All integrin subunits identified on osteoblasts in situ were maintained on culture but there was an increased expression of alpha 2 and alpha V subunits were weakly positive. Expression of alpha 2, alpha 3, alpha V and beta 1 subunits was independent of cell density but expression of alpha 1 was much greater in confluent cultures.(ABSTRACT TRUNCATED AT 250 WORDS)

LR White resin sectioning of undecalcified rabbit long bone: A protocol for crease-free preparations with enhanced polychromatic staining

LR White resin sectioning of undecalcified rabbit long bone: A protocol for crease-free preparations with enhanced polychromatic staining

Effect of calcitonin on bone histomorphometry and bone metabolism in rheumatoid arthritis

Twenty-four women (mean age +/- SD 49 +/- 13 years) with classical or definite rheumatoid arthritis (disease duration 15 +/- 8 years) were treated with synthetic salmon calcitonin (SCT) nasal spray 200 IU three times a week for 3 months. Bone biopsies from the iliac crest were taken before and after SCT treatment. Histomorphometrical quantification of undecalcified bone sections was made using the manual point-counting method. SCT decreased the resorption surface of trabecular bone (ES/BS) significantly (P less than 0.001). There was also a significant increase (P less than 0.05) in trabecular bone volume (BV/TV) after 3 months of treatment, whereas no statistically significant changes were found in osteoid parameters. There were no significant changes in biochemical analyses of bone metabolism. We conclude that SCT might be useful in the prevention of bone loss in RA.

Production and characterization of osteoclast-selective monoclonal antibodies that distinguish between multinucleated cells derived from different human tissues.

Osteoclastoma-derived giant cells were used to produce 11 mouse monoclonal antibodies (MAb) reactive against human osteoclasts on undecalcified sections of adult human bone. All exhibited unique reactivities across a wide range of human tissues. Three in particular demonstrated distinctive reactivities; C35 was highly selective for bone osteoclasts, C27 showed selective reactivity for osteoclasts, tissue macrophages and blood-borne monocytes, and C22 showed selective membrane staining of osteoclasts. Consequently, C22 was used to coat Dynabeads to affinity-purify viable human osteoclasts from osteoclastoma-derived cell suspensions. Immunocytochemical staining of inflammatory osteoarthritic synovium/granulation tissue demonstrated positivity in the majority of giant cells with MAb C22 and C27. In contrast, C35 reacted with only very occasional giant cells. Furthermore, multinucleated cells formed in long-term human bone marrow cultures demonstrated similar selective staining. C27 stained all giant cells and the majority of mononuclear cells. C22 detected only a small proportion of giant cells. In contrast to its staining on bone osteoclasts, C22 demonstrated granular cytoplasmic staining in cultured giant cells. C35 stained no cells at all in these cultures. These MAb can therefore distinguish between giant cells of various origins and authentic mature osteoclasts. Alternatively, they can recognize antigens expressed at different stages of osteoclast differentiation and therefore provide an excellent tool for the study of the human osteoclast lineage.

Modified Giemsa Stain for Polymethyl Methacrylate Embedded Undecalcified Bone and Bone Marrow Specimens

A modified Giemsa azure-eosin method for staining polymethyl methacrylate (PMMA) plastic embedded bone sections is described. The method is similar to those used to stain glycol methacrylate sections but differs in timing and addition of air drying steps. The stain yields effective differentiation of marrow cells, growth plate, calcified cartilage, osteoid, bone matrix and bone cells in undecalcified sections of bone. The stain does not interfere with the resolution of fluorochrome labeled specimens. Hence, the same slide can beused to evaluate static and dynamic parameters of bone metabolism. (The J Histotechnol 14:85, 1991)

Iron as a possible aggravating factor for osteopathy in itai-itai disease, a disease associated with chronic cadmium intoxication

Itai-itai disease is thought to be the result of chronic cadmium (Cd) intoxication. We examined 23 autopsy cases of itai-itai disease and 18 cases of sudden death as controls. Urine and blood samples from 10 patients were collected before they died and revealed the presence of severe anemia and renal tubular injuries. Undecalcified sections of iliac bone were stained with Aluminon reagent, and ammonium salt of aurintricarboxylic acid, and Prussian blue reagent in all cases of itai-itai disease. These two reagents reacted at the same mineralization fronts. X-ray microanalysis revealed the presence of iron at mineralization fronts in itai-itai disease. Five patients showed evidence of hemosiderosis in the liver, spleen, and pancreas, probably as a result of post transfusion iron overload. Renal calculi and calcified aortic walls were also stained with Prussian blue reagent in several patients. Neither ferritin nor transferrin were visualized at mineralization fronts in itai-itai disease by immunohistochemical staining. These results suggest that iron is bound to calcium or to calcium phosphate by a physicochemical reaction. A marked osteomalacia was observed in 10 cases of itai-itai disease by histomorphometry. Regression analyses of data from cases of itai-itai disease suggested that an Aluminon-positive metal inhibited mineralization and that renal tubules were injured. Since bone Cd levels were increased in itai-itai disease, it is likely that renal tubules were injured by exposure to Cd. Therefore, stainable bone iron is another possible aggravating factor for osteopathy in itai-itai disease, and a synergistic effect between iron and Cd on mineralization is proposed.

Toxicological Study of DTPA as a Drug(VI). Effects of Intravenously Injected Ca-DTPA, Ca-EDTA, CBMIDA and Orally Administered Zn-DTPA to Bone Metabolism in Beagle Dogs.

Effects of four kinds of chelating agents, Ca-DTPA (calcium diethylenetriaminepentaacetic acid), Ca-EDTA (calcium ethylenediaminetetraacetic acid), CBMIDA [catechol-3, 6-bis(methyleiminodiacetic acid)] and Zn-DTPA (zinc DTPA), on bone metabolism were examined in beagle dogs by bone histomorphometry and measurement of serum biochemical constituents related to bone metabolism. Ca-DTP, Ca-EDTA or CBMIDA (150μmol/kg) was injected intravenously to dogs for 1 month, respectively. Three doses (30, 150 and 300μmol/kg) of Zn-DTPA were administered orally to dogs for 1 month respectively. All dogs received twice tetracycline hydrochloride infections at an interval of 7 days before the beginning of administration of chelating agents and also twice calcein injections at the same time schedule prior to sacrifices for analyzing bone dynamics. Bone samples were obtained from ilium and undecalcified bone sections were made. Bone histomorphometry of cancellous bone area of ilium was performed using an image analyzer.Bone volume and mean trabecular thickness did not change in any of the groups. Osteoid volume in the CBMIDA group increased (p<0.05). Osteoid volume and mean osteoid thickness in the 150μmol/kg of Zn-DTPA group decreased (p<0.05 and p<0.01). Mineral apposition rate and bone formation rate did not change in any groups except the CBMIDA and 150μmol/kg of Zn-DTPA groups, in which fluorescent bone labeling was absent or obscure, revealing inhibition of bone mineralization.Serum total calcium levels did not change in any of the groups. Serum phosphorus level decreased significantly in the 30μmol/kg dose of Zn-DTPA group (p<0.05). Parathyroid hormone level increased in the 30μmol/kg dose of Zn-DTPA (p<0.05), while it decreased in the 150μmol/kg dose of Zn-DTPA group (p<0.05).The results suggest that the protracted therapy using the above four kinds of chelating agents may incur damages of bone such as decrease of bone volume and inhibition of mineralization.

A Method for Histological Preparation of Undecalcified Bone Sections Containing Acrylic Bone Cement

An improved and time reducing method is presented for the histological evaluation of bone containing polymethylmethacrylate (PMMA) bone cement. The undecalcified bone was embedded in epoxy resin and section of 50-100 microns thickness were produced using a commercially available cutting grinding system. The sections were stained with Stevenel's blue and van Gieson picrofuchsin or a modified hematoxylineosin. PMMA bone cement was not dissolved and remained enabling examination in situ of an intact cement bone interface and tissue reaction without decalcification.

Comparison of Methods for Staining Mast Cells in Undecalcified Sections of Bone

Staining for mast cells in undecalcified sections of bone was evaluated with a variety of toluidine blue (TB) preparations. Attention was given to color change in granules, nuclear clarity, background staining, and appearance of bone. Our buffered TB stain, which was developed to highlight bone detail, resulted in violaceous staining of the granules in mast cells, often restricting their identification. An acidified TB stain and a new stain of TB dissolved in 30% ethanol resulted in purple granules. Prestaining with Congo red prior to acidified TB allowed good bone detail and full expression of metachromasia with red appearance of the granules, giving the best view of the mast cell. (The J Histotechnol 13: 49, 1990)

An improved method for preparing cryostat sections of undecalcified bone for multiple uses.

We developed a technique that permits the use of serial sections (7-20 microns) from a single fixed piece of bone tissue for immunofluorescence, measurement of fluorescent bone labels, enzyme histochemistry, and general staining. This technique combines modifications of previously established methods with perfusion of the polymer polyvinylpyrrollidone (PVP) to improve sectioning, and produces reliable sections with good preservation of both hard and soft tissues. The combination of techniques from several workers, the use of perfusion with a polymer to increase the sectionability of the bone, and the addition of a gelatin adhesive on top of pressure-sensitive adhesives represent a significant improvement over previously described methods. The sections obtained are usable for immunocytochemistry, general staining, enzyme histochemistry, and visualization of fluorescent bone labels. We have consistently used tissues prepared in this manner for immunohistochemical demonstration of neuropeptides in skeletal tissues and for localizing tartrate-resistant acid phosphatase (TRAP). In addition, other tissues obtained from PVP-perfused rats, such as brain, spinal cord, muscle, gut, and sympathetic ganglia, are also well preserved and demonstrate immunohistochemical staining comparable to and possibly superior to that obtained with normal fixation protocols.

Cement Line Staining in Undecalcified Thin Sections of Cortical Bone

A technique for demonstrating cement lines in thin, undecalcified transverse sections of cortical bone has been developed. Cortical bone samples are processed and embedded undecalcified in methyl methacrylate plastic. After sectioning at 3-5 microns, cross-sections are transferred to a glass slide and flattened for 10 min. Sections of cortical bone are stained for 20 sec free-floating in a fresh solution of 1% toluidine blue dissolved in 0.1% formic acid. The section is dehydrated in t-butyl alcohol, cleared in xylene, and mounted with Eukitt's medium. Reversal lines appear as thin, scalloped, dark blue lines against a light blue matrix, whereas bone formation arrest lines are thicker with a smooth contour. With this technique cellular detail, osteoid differentiation, and fluorochrome labels are retained. Results demonstrate the applicability of a one-step staining method for cement lines which will facilitate the assessment of bone remodeling activity in thin sections of undecalcified cortical bone.

A light and scanning electron microscopic study of osteocyte activity in calcium-deficient rats

The material herein is an extension of an earlier study of osteocyte lacunae in calcium-deficient rats, utilizing morphometric measurements in undecalcified bone sections and scanning electron microscopy. The results confirm our earlier finding that bone resorption resulting from a low-calcium diet is not accompanied by osteocytic resorption.