An Ion-exchange Bone Demineralization Method for Improved Time,Expense, and Tissue Preservation

Here, we describe an ethylenediaminetetraacetic acid (EDTA)-based bone demineralization procedure that uses cation-exchange resin and dialysis tubing. This method does not require solution changes or special equipment, is faster than EDTA alone, is cost-effective, and is environmentally friendly. Like other EDTA-based methods, this procedure yields superior tissue preservation than formic acid demineralization. Greater protein antigenicity using EDTA as opposed to formic acid has been described, but we also find significant improvements in carbohydrate-based histological staining. Histological staining using this method reveals cartilage layers that are not distinguishable with formic acid demineralization. Carbohydrate preservation is relevant to many applications of bone demineralization, including the assessment of osteoarthritis from bone biopsies and the use of demineralized bone powder for tissue culture and surgical implants. The improvements in time, expense, and tissue quality indicate this method is a practical and often superior alternative to formic acid demineralization:     

The rate of calcium extraction during EDTA decalcification from thin bone slices as assessed with atomic absorption spectrophotometry

Summary The rate of calcium extraction with EDTA (ethylenediamine tetraacetic acid) from thin bone slices (300 m-2mm thick) was determined by aid of an atomic absorption spectrophotometer. A 0.5 mm thick bone slice was completely decalcified with 15% (0.40 M), 8% (0.22 M), and 4% (0.11 M) EDTA in 24 h, 3 days, and 5 days, respectively (vol. 15 ml, temp. 4° C, pH 7.4). At 37 and 60° C the speed of demineralization was slightly increased as compared with that at 20° C, while no difference was observed between 4 and 20° C. Bone slices with a thickness of 0.3, 0.5, 1 and 2 mm were decalcified-in the same order-in 24 h, 2, 3, and 5 days (8% EDTA, 4° C, pH 7.4). At pH 7.4, the decalcification rate was a little slower than at pH 5.0 and 8.5. Agitation did not affect the decalcifying velocity, nor did the volume of the agent, except when the volume was very small. The demineralization of ordinary bone, containing both compact and spongy bone, was found to be more rapid than that of homogeneous bone reported earlier. The acidic buffers and New Decalc^®, which served as reference substances, exerted a more vigorous decalcifying effect than EDTA. K formate/formic acid buffer, pH 3.15, demineralized a 1 mm thick bone slice in 24 h, and 2 days was needed with Na lactate/lactic acid buffer, pH 3.70. With New Decalc^®, pH 0.9, the corresponding demineralization was accomplished in 1.5 h. Atomic absorption spectrophotometer proved to be a useful tool in the evaluation of calcium extraction velocity from bone slices.     

In situ hybridization of bone matrix proteins in undecalcified adult rat bone sections.

We have developed a method for in situ hybridization of adult bone tissue utilizing undecalcified sections and have used it to histologically examine the mRNA expression of non-collagenous bone matrix proteins such as osteocalcin (bone Gla protein, BGP), matrix Gla protein (MGP), and osteopontin in adult rats. Expression was compared with that in bone tissues of newborn rats. In the adult bone tissue, osteocalcin mRNA was strongly expressed in periosteal and endosteal cuboidal osteoblasts but not in primary spongiosa near the growth plate. Osteopontin mRNA was strongly expressed in cells present on the bone resorption surface, osteocytes, and hypertrophic chondrocytes, but not in cuboidal osteoblasts on the formation surface. Osteopontin and osteocalcin mRNAs were expressed independently and the distribution of cells expressing osteopontin mRNA corresponded with acid phosphatase-positive mononuclear cells and osteoclasts. Expression of MGP mRNA was noted only in hypertrophic chondrocytes. In newborn rat bone tissues, expression of osteocalcin mRNA was much weaker than in adult rat bone tissues. These results clearly indicate the differential expression of mRNAs of non-collagenous bone matrix proteins in adult rat bone tissues.     

Rapid demineralization in acidic buffers.

The demineralization of routine histological specimens in buffers of weakly ionized organic acids, unbuffered formic acid, and EDTA was investigated. The rate of demineralization was measured by a chemical method and from radiographs. Lactate-containing buffers and buffers of formic acid with its potassium salt were more rapid in effect than any other agent. Acidic buffers and unbuffered formic acid produced rapid diffuse demineralization with secondary precipitation of calcium salts. Preservation of dental enamel in such buffers resulted from the significantly slower rate of enamel demineralization than that for bone and dentine. In rapid demineralizing agents the secondary salts were quickly redissolved while in slow buffers these salts persisted. Multivalent ions such as citrate and maleate slowed the rate of demineralization, and a citrate-containing buffer was the slowest of all the agents tested. Demineralization in EDTA exhibited a different pattern with the establishment of a well-defined front of demineralization without apparent reprecipitation. EDTA attacked enamel, bone and dentine at the same rate. An attempt was made to relate the observed rates of demineralization to current theories of the demineralization process.     

Rapid demineralization in acidic buffers

Summary The demineralization of routine histological specimens in buffers of weakly ionized organic acids, unbuffered formic acid, and EDTA was investigated. The rate of demineralization was measured by a chemical method and from radiographs. Lactate-containing buffers and buffers of formic acid with its potassium salt were more rapid in effect than any other agent. Acidic buffers and unbuffered formic acid produced rapid diffuse demineralization with secondary precipitation of calcium salts. Preservation of dental enamel in such buffers resulted from the significantly slower rate of enamel demineralization than that for bone and dentine. In rapid demineralizing agents the secondary salts were quickly redissolved while in slow buffers these salts persisted. Multivalent ions such as citrate and maleate slowed the rate of demineralization, and a citrate-containing buffer was the slowest of all the agents tested. Demineralization in EDTA exhibited a different pattern with the establishment of a well-defined front of demineralization without apparent reprecipitation. EDTA attacked enamel, bone and dentine at the same rate. An attempt was made to relate the observed rates of demineralization to current theories of the demineralization process.Supported by the British Medical Research Council     

Localization of osteopontin in resorption lacunae formed by osteoclast-like cells: a study by a novel monoclonal antibody which recognizes rat osteopontin

The characteristics of a monoclonal antibody produced against osteoclast-like multinucleated cells (MNCs) formed in rat bone marrow cultures were examined immunohistochemically and biochemically. The in vitro immunization was performed using as immunogen the MNCs from rat bone marrow cell culture, which revealed many characteristics of osteoclasts. After screening and cloning of hybridomas, the monoclonal antibody HOK 1 was obtained. This antibody reacted weakly with stromal cells and intensely with both MNCs and their putative migratory traces on culture dishes. Immunofluorescent examination of paraffin sections revealed intense reactivity on the epithelium of the choroid plexus, the ileum and the proximal-convoluted tubules of the kidney, and also on bone cells such as osteocytes, osteoblasts, and osteoclasts. Western blotting using purified rat osteopontin verified that the antigen recognized by HOK 1 was osteopontin. Positive HOK 1 immunoreactivity was further observed in the resorption lacunae formed by a culture of MNCs on human tooth slices and on the surface of osteoclasts. The present data suggested that osteopontin is preferentially present on the resorption lacunae in resorbing calcified matrices and that osteoclasts under a specific state might trap this protein on their cell surface.     

Participation of transfused bone marrow cells in reparative osteohistogenesis

The participation of skeletal tissue cell precursors in the repairing regeneration of bone tissue was studied. Bone marrow was taken from donor animals--mice of C57Bl/6-TgN(ACTbGFP) 1 Osb line (The Jackson Laboratory Bar Harbor ME USA line). Nucleated cell fraction was isolated by centrifugation on a density percoll gradient. Recipient mice C57Bl/6 line were irradiated by 7.0-7.5 Gr dose. Intravenous infusion of donor cells and osteoclasts of tibia was done after irradiation of recipient mice. Histological preparations of bone regenerate tissues were studied on 15, 30, and 60 days by confocal microscopy. Donor cells were found as skeletal tissue precursors into periost, endost, bone marrow, and as differentiated cells of newborn tissue of regenerate--osteoblasts, osteocytes, chondrocytes. The data obtained indicate that part of donor bone marrow cells are able to progressive differentiation under recipient bone fractures.     

Acid phosphatase activity is detected preferentially in the osteoclastic lineage by pre-treatment with cyanuric chloride

We previously reported a simple method to detect osteoid matrices in decalcified bone sections by pre-treatment with cyanuric chloride. We have applied this technique to identify osteoclasts and their precursors in rats. In JB-4 sections prepared from untreated bone tissues with cyanuric chloride, both acid phosphatase (ACP) and tartrate-resistant acid phosphatase (TRAP) were found not only in osteoclasts and bone marrow mononuclear cells but also in osteoblasts. In contrast, treatment of bones with cyanuric chloride resulted in staining ACP preferentially in osteoclasts and mononuclear cells adjacent to the bone surface. In the osteoclasts and most of the ACP-positive mononuclear cells, autoradiography showed calcitonin binding. Decalcification with EDTA did not affect the staining for ACP activity in bones treated with cyanuric chloride. It was possible to simultaneously identify ACP and osteoid matrix in a decalcified section. In soft tissues without treatment with cyanuric chloride, both ACP and TRAP were detected in splenic macrophages, alveolar macrophages, and proximal convoluted ducts in kidney. Neither ACP nor TRAP was found in these cell types in the tissues treated with cyanuric chloride. This procedure provides a new, simple method to identify a more restricted population in the osteoclastic lineage than that detected by TRAP staining.     

Expression of mRNA of murine bone-related proteins in ectopic bone induced by murine bone morphogenetic protein-4

To determine whether a system of ectopic bone formation induced by osteosarcoma-derived bone-inducing substance (bone morphogenetic protein-4) can be used as a model of developing bone at the molecular level, we studied the expression of bone-related protein mRNAs in the process of ectopic bone formation using non-radioisotopic in situ hybridization. Osteonectin mRNA was detected in fibroblast-like cells, which are similar to periosteal cells from the early to middle stages of bone development. The proportion of osteonectin mRNA-expressing cells was greater than that of osteopontin mRNA-expressing cells in hypertrophic chondrocytes and osteoblast-like cells. In contrast, osteopontin mRNA was localized in a limited population of hypertrophic chondrocytes, a single layer of osteoblast-like cells adjacent to the bone trabeculae in the middle stage of bone formation, and in a limited subset of osteocytes in the late stage. A strong osteocalcin mRNA signal was detected in osteoblast-like cells from the middle to late stages and in a limited subset of osteocytes in the late stage of bone development. Since the sequential gene expression pattern of bone-related proteins in the present system is comparable to that in embryonic osteogenesis, this system may be useful as a model for studying gene expression in osteogenesis.     

Monogene Ionenkanalerkrankungen des Knochens

Although ion channels are intuitively more related to the generation of action potentials, they can influence skeletal cells, development, and homeostasis in different ways as demonstrated in this review. All major skeletal cell types, chondrocytes, osteoblasts, osteocytes, and osteoclasts, can be involved in the pathogenesis and often the interaction of these different defects is incompletely understood. Connexin 43 and TRPV4, two of the mentioned membrane proteins, predominantly conduct calcium ions and are the basis of a whole spectrum of skeletal phenotypes. While connexin 43 seems to regulate the function of osteoblasts and osteocytes, TRPV4 is crucial for chondrocytes. The other two examples are chloride-transporting membrane proteins ANO5 and ClC-7, which can cause gnathodiaphyseal dysplasia and osteopetrosis, respectively. Whereas the function of ANO5 is largely unknown, the role of ClC-7 in bone resorbing osteoclasts has been investigated in great detail.     

Methods for the Demonstration of Lipid Applied to Compact Bone

Sections of compact bone were cut from the diaphysis of the femur, tibia, and humerus from dogs and monkeys. These sections were either ground thin and decalcified, or decalcified and subjected to frozen sectioning. Decalcification of the sections was effected by immersion in either Decal, 10% formic acid, 10% formic acid-sodium citrate (pH 4.5) or 20% aqueous EDTA. Sections were routinely stained with oil red O, Sudan black B, or Fettrot 7B. In addition, Nile blue A and phosphine 3R were also employed. Sections stained with phosphine were viewed with a fluorescence microscope. Control sections were extracted with lipid solvents prior to application of the staining procedures. The results indicate that lipid is present in compact bone within the osteocytes, lacunae, canaliculi, and organic matrix. The significance of the lipid in these sites, particularly extracellularly, is unknown.     

Effects of various decalcification protocols on detection of DNA strand breaks by terminal dUTP nick end labelling

To analyse DNA strand breaks by terminal deoxy(d)-UTP nick-end labelling (TUNEL) in calcified tissues including bones and teeth, it is important to decalcify the tissues first. However, the effects of decalcifying reagents on the integrity of DNA are largely unknown. In the present study, we evaluated the usefulness of various decalcifying reagents including 10% EDTA (pH 7.4), 5% trichloroacetic acid (TCA), 5% formic acid, 5% HCl, 10% nitric acid, Plank–Rychlo's solution, Morse's solution and K-CX solution in TUNEL staining. Mouse maxilla was selected as the experimental system. Apoptotic cells naturally occurring in the epithelium were analysed. Tissues were assessed by soft X-ray imaging to confirm complete decalcification. The time required for decalcification of the tissue was 7 days with 10% EDTA and 2 days with other decalcifiers. Decalcified tissues were stained with Methyl/Green–Pyronine Y or 4, 6-diamidino-2-phenylindole for assessment of DNA integrity. Nuclei of epithelial cells were strongly positive for both dyes after decalcification with 10% EDTA, 5% TCA, Morse's solution and 5% formic acid. The other reagents failed to retain DNA. Our results demonstrated good TUNEL staining of the maxilla treated with 10% EDTA or 5% TCA . Based on the required time for processing and the signal-noise ratio, we recommend 5% TCA as the decalcifying reagent to analyse for DNA strand breaks.     

Decalcification for histochemical demonstration of hydrolytic and oxidative enzymes

Summary A method for histochemical demonstration of hydrolytic and oxidative enzymes following decalcification was described in mature bone and tooth by neutral EDTA decalcifying solution.The decalcifying solution, 0.5 M EDTA tetrasodium salt was adjusted to neutral pH with 5 M citric acid, obtained the most sufficient results for demonstration of enzymes in decalcified tissue. The hard tissue decalcified in 30 to 40 days at 4° C exhibited a good preservation of hydrolytic and oxidative enzymes histochemically, but a few structural destruction in a long term decalcification was found in soft tissues and certain organs.     

Cellular and subcellular distribution of galectin-3 in the epiphyseal cartilage and bone of fetal and neonatal mice.

Galectin-3 is a 30 kDa beta-galactoside binding protein that belongs to the galectin family of animal lectins. By immunocytochemistry we show the presence of galectin-3 protein in the differentiated chondrocytes of the epiphyseal plate cartilage of long bones of both fetal and neonatal mice. The highest concentrations of galectin-3 are found in the cytoplasm of mature and early hypertrophic chondrocytes. Very little protein is detected in the late hypertrophic chondrocytes undergoing terminal maturation and cell death. Galectin-3 has also been found in osteoblasts and osteocytes of the woven bone of the metaphysis and the cortical bone of the diaphysis, as well as in osteoclasts and mononuclear cells within bone marrow cavities. Galectin-3 is never detected extracellularly, the protein seems restricted to the cytoplasm of chondrocytes and bone cells, although it is occasionally detected in the nuclei of dense non-hypertrophic chondrocytes in the zone of calcification and in young osteoblasts. The results indicate that galectin-3 is a marker of both chondrogenic and osteogenic cell lineages. They also suggest that galectin-3 could be involved in the process of endochondral bone formation, possibly as a regulator of chondrocyte survival.     

Biochemical and immuno- and lectin-histochemical studies of solubility and retention of bone matrix proteins during EDTA demineralization

Summary The present study utilized biochemical and immuno-and lectin-histochemical methods to demonstrate solubility and retention of mineral-binding non-collagenous proteins in rat midshaft subperiosteal bone during EDTA demineralization. A monoclonal antibody (9-A-2) specific for chondroitin 4-sulphate and dermatan sulphate and wheat germ agglutinin (WGA) specific forN-acetyl-d-glucosamine,N-acetylneuraminic acid, andN-acetyl-d-galactosamine were used. Bone proteins were extracted from fresh unfixed or aldehyde-fixed specimens with a three step extraction procedure, 4 M guanidine HCl (GdnCl), aqueous EDTA without GdnCl, followed by GdnCl. For comparison with the second extraction step, ethanolic trimethylammonium EDTA (ethanolic EDTA) was substituted for aqueous EDTA. Based on protein staining and Western blot analysis of SDS-polyacrylamide gel electrophoresis of each extract using 9-A-2 and WGA, retention of mineral-binding proteins extractable from fresh specimens with aqueous EDTA was greatly increased in tissue when ethanolic EDTA was used. Their retention was even greater with prior aldehyde fixation. Maximum retention with no detectable solubility of 9-A-2 and WGA reactive proteins was obtained after ethanolic EDTA extraction of aldehyde-fixed specimens, which concomitantly provided the strongest immuno- and lectin staining. These results indicate that this combined method dramatically improves retention of PGs and glycoproteins during demineralization of bone tissues and provides the best method for localizing these glycoconjugates.     

The Use of a Cation Exchange Resin in Decalcification

Decalcification of 2-3 mm. sections of human ribs by 5-40% concentrations of both formic and nitric acid was subjected to a controlled study. The effect of adding 1 g. of phloroglucinol and of 2.5, 5 and 10 g. of the exchange resin (Win-3000) per 100 ml. of decalcifying solution was observed after staining celloidin sections with hematoxylin and Triosin or with Giemsa stain. Electrolytically decalcified material was included for comparison also. The following conclusions were drawn: (1) The addition of resin did not appreciably shorten the decalcification time nor enhance tissue preservation and staining qualities. (2) Formic acid, 20% or less, gave results superior to nitric acid in any concentration and also superior to those obtained after the electrolytic method. (3) The addition of 1% phloroglucinol to formic acid solution improved both preservation and staining. (4) Helly's fluid fixation with all combinations gave uniformly better results than formalin fixation.     

High mobility group box 1 protein regulates osteoclastogenesis through direct actions on osteocytes and osteoclasts in vitro

Old age and Cx43 deletion in osteocytes are associated with increased osteocyte apoptosis and osteoclastogenesis. We previously demonstrated that apoptotic osteocytes release elevated concentrations of the proinflammatory cytokine, high mobility group box 1 protein (HMGB1) and apoptotic osteocyte conditioned media (CM) promotes osteoclast differentiation. Further, prevention of osteocyte apoptosis blocks osteoclast differentiation and attenuates the extracellular release of HMGB1 and RANKL. Moreover, sequestration of HMGB1, in turn, reduces RANKL production/release by MLO-Y4 osteocytic cells silenced for Cx43 (Cx43^def), highlighting the possibility that HMGB1 promotes apoptotic osteocyte-induced osteoclastogenesis. However, the role of HMGB1 signaling in osteocytes has not been well studied. Further, the mechanisms underlying its release and the receptor(s) responsible for its actions is not clear. We now report that a neutralizing HMGB1 antibody reduces osteoclast formation in RANKL/M-CSF treated bone marrow cells. In bone marrow macrophages (BMMs), toll-like receptor 4 (TLR4) inhibition with LPS-RS, but not receptor for advanced glycation end products (RAGE) inhibition with Azeliragon attenuated osteoclast differentiation. Further, inhibition of RAGE but not of TLR4 in osteoclast precursors reduced osteoclast number, suggesting that HGMB1 produced by osteoclasts directly affects differentiation by activating TLR4 in BMMs and RAGE in preosteoclasts. Our findings also suggest that increased osteoclastogenesis induced by apoptotic osteocytes CM is not mediated through HMGB1/RAGE activation and that direct HMGB1 actions in osteocytes stimulate pro-osteoclastogenic signal release from Cx43^def osteocytes. Based on these findings, we propose that HMGB1 exerts dual effects on osteoclasts, directly by inducing differentiation through TLR4 and RAGE activation and indirectly by increasing pro-osteoclastogenic cytokine secretion from osteocytes.     

Simultaneous demonstration of bone alkaline and acid phosphatase activities in plastic-embedded sections and differential inhibition of the activities

Summary Bone alkaline (AlP) and acid phosphatase (AcP) activities were simultaneusly demonstrated in tissue sections obtained from mice, rats, and humans. The method involved tissue fixation in ethanol, embedding in glycol methacrylate (GMA), and demonstration of AlP and AcP activities employing a simultaneous coupling azo dye technique using substituted naphthol phosphate as a substrate. AlP activity was demonstrated first followed by AcP activity. Both enzyme activities were demonstrated in tissue sections from bones fixed and/or stored in acetone or 70% ethanol for up to 14 days or stored in GMA for 2 months. AlP activity in tissue sections from bones fixed in 10% formalin, 2% glutaraldehyde, or formal-calcium, however, was markedly inhibited after 3–7 days and was no longer detectable after 14 days of fixation. Moreover, AlP activity was diminished in tissue sections from bones fixed in 70% ethanol or 10% formalin and subsequently demineralized in 10% EDTA (pH7) for 2 days, and the activity was completely abolished in tissue sections from bones subsequently demineralized in 5% formic acid: 20% sodium citrate (1:1, pH 4.2) for 2 days. Methyl methacrylate (MMA) embedding at concentrations above 66% completely inhibited AlP activity. AcP activity, however, was only partially inhibited by formalin, glutaraldehyde, or formal-calcium after 7 or 14 days of fixation or by MMA embedding and was unaffected by the demineralizing agent formic acid-citrate for 2 days. While AcP activity was preserved in bones fixed in formalin and subsequently demineralized in EDTA, the activity was completely abolished when EDTA demineralization was carried out on bones previously fixed in 70% ethanol. These results indicate that bone AlP and AcP activities can be demonstrated simultaneously in the same section using a simple tissue preparation technique and that the activities are retained in tissues fixed and/or stored in acetone, 70% ethanol or GMA, but are differentially inactivated by other fixatives studied, and by EDTA, formic acid-citrate, and MMA embedding.Abbreviations AcP acid phosphatase - AlP alkaline phosphatase - GMA glycol methacrylate - MMA methyl methacrylate - EDTA ethylenediaminetetraacetic acid     

In vivo incidence of apoptosis evaluated with the TdT FragEL DNA fragmentation detection kit in cartilage and bone cells of the rat tibia

Previous studies have shown the occurrence of cell death by apoptosis in cartilage and bone cells, and have suggested a functional relationship between bone growth and remodelling on one hand, and numbers of apoptotic cells on the other. At present, no in vivo studies are available on the frequency of the apoptotic process measured at one time and in one place using the cartilage and bone cells of single specimens. The aim of the present investigation was to measure the in vivo incidence of apoptosis in cartilage and bone cells of the upper epiphysis and secondary ossification metaphyseal bone of the tibia in normal young adult rats. Apoptotic cells were visualized with the terminal deoxynucleotidyl transferase (TdT) FragEL DNA fragmentation detection kit, which is analogous to the TdT-mediated nick end-labelling (TUNEL) method. In the growth cartilage, only a few TUNEL-positive terminal hypertrophic chondrocytes were found; they were 1.32 +/- 0.70% of the total hypertrophic chondrocytes counted along the chondro-osseous junction. There were only a few apoptotic osteoblastic cells and osteocytes (0.22 +/- 0.22% and 0.15 +/- 0.16% of total osteoblasts and osteocytes respectively). TUNEL-positive osteoclasts were 1.03 +/- 0.57% of the total of osteoclastic cells; they usually showed only one or two apoptotic nuclei. The total number of TUNEL-positive bone marrow cells were also counted (56.78 +/- 10.29/mm2 of bone marrow spaces). Our results confirm that apoptosis does occur in hypertrophic chondrocytes and bone cells, and show that its frequency is very low. However, chiefly because of its short lifespan, the frequency of apoptosis in cartilage and bone may be higher than that shown by the TUNEL method. The static estimate that can be obtained with this method might lead to misleading conclusions on the physiological significance of such a dynamic, rapid and asynchronous process, whose precise importance in bone growth and remodelling remains to be determined.