Patterns of distribution of phosphomono-esterases on surfaces of demineralized bone

Summary Decalcification over short periods (5 days) with MnNa₂ EDTA, MgNa₂ EDTA and EGTA according to a method described in the present paper, creates sections of high quality with simultaneous good preservation of phosphomonoesterases on bone surfaces. In fact, the enzyme distribution seems to be comparable to that obtained by using undecalcified sections.Na₂ EDTA creates, on the other hand, poor preservation of alkaline phosphatase probably due to the fact that this chelate contrary to the other chelates removes the essential metal from the protein, leaving an unstable enzyme molecule which undergoes denaturation.Decalcification over longer periods (15 days) does not influence the pattern of distribution of acid phosphatase, whereas the alkaline phosphatase reaction becomes depressed in certain surface areas. The significance of this differential distribution is discussed. It might be an indication of differential processes of bone transformations in such a way that bone surfaces corresponding to areas of enzyme reactions are depository whereas bone surfaces corresponding to areas of lack of enzyme reaction are resorptive. New experimental designs are, however, necessary before the phenomenon is fully perceived. Two different coupling agents were used in connexion with the demonstration of acid phosphatase reaction. When HPR was used as the coupler the final enzyme distribution coincided with that usually described in the literature, i.e., strong reaction of cells adjacent to resorptive surfaces and weak reaction of cells adjacent to depository surfaces. When, however, Fast dark blue R was used all surface cells reacted markedly. This method also revealed certain cell types with nuclear reaction.     

The significance of a differential distribution of phosphomonoesterases on bone surfaces after prolonged demineralization

Summary An observed differential distribution of alkaline and acid phosphatase on the surfaces of growing bones may serve to describe transformative processes of bone growth. This conclusion has been reached by comparing the distribution of the two enzymes on the surfaces of fibulae from young rats with the patterns of apposition and resorption on the periosteal surfaces of this bone, revealed by in vivo staining with alizarin red S. Presence of reaction to acid phosphatase is, as shown before, an indication of resorptive surfaces, while the presence of reaction to alkaline phosphatase is an indication of depository surfaces.     

The significance of a differential distribution of phosphomonoesterases on bone surfaces after prolonged demineralization.

An observed differential distribution of alkaline and acid phosphatase on the surfaces of growing bones may serve to describe transformative processes of bone growth. This conclusion has been reached by comparing the distribution of the two enzymes on the surfaces of fibulae from young rats with the patterns of apposition and resorption on the periosteal surfaces of this bone, revealed by in vivo staining with alizarin red S. Presence of reaction to acid phosphatase is, as shown before, an indication of resorptive surfaces, while the presence of reaction to alkaline phosphatase is an indication of depository surfaces.     

Wolff's law and the problem of muscle attachment on resorptive surfaces of bone

During the growth of a bone, outer (periosteal) surfaces in many areas undergo normal remodeling processes involving resorptive removal. Attachments of muscles commonly occur on such outer resorptive surfaces. The cortex in these regions grows in an inward direction by bone deposition on endosteal surfaces. In some areas of a bone, a portion of a muscle can be inserted onto a depository surface, but other parts of the same muscle may be attached onto an adjacent resorptive surface. It has been generally assumed that the pull of a muscle acts to directly stimulate deposition of new bone, and that attachments of muscle are thereby responsible for determining the gross morphology of a whole bone. In view of the foregoing considerations, a re-evaluation and an expansion of this concept is now needed. Muscle pull, in many regions of a bone, can be associated with normal cortical recession (involving surface resorption) as well as with outward bone deposition.     

Histochemical studies on the localization and activity of acid phosphatase in calcifying tissues

Summary Acid phosphatase activity has been studied in cold microtome sections and using simultaneous azo coupling method in developing teeth and bone, and serial sections were made for the demonstrations of alkaline phosphatase.1. In developing teeth, strongest activity of acid phosphatase was found in the distal portion of high columnar ameloblasts associated with heavy calcification in the rodent incisor, and ameloblasts and odontoblasts in adjacent occlusal surface in molar teeth. However, the activity of immatured ameloblast and crevicular aspects of molar were weaker.2. In the epiphyseal bone trabeculae a striking acid phosphatase reaction was found.3. As regards to the effects of decalcifying solutions to the enzymatic activity, the use of EDTA decalcifying agent (10% and pH 7 to 4) showed the best results. That is, a decrease of decalcifying time and a greater preservation of acid phosphatase activity.With 11 Figures in the Text     

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.     

Ethylenediaminetetraacetic Acid in Ammonium Hydroxide for Reducing Decalcification Time

Ethylenediaminetetraacetic acid (EDTA) solution is used to decalcify bone specimens for histological examination. Sodium hydroxide (NaOH) has been used to dissolve EDTA and to bring EDTA solutions to neutral pH. This solution, however, requires several weeks to decalcify bone specimens. We investigated a new de-calcification fluid using concentrated ammonium hydroxide (NH₄OH) to dissolve EDTA and to adjust the pH to neutral. Decalcification was performed using a magnetic stirrer with and without vacuum, or with a sonic cleaner. Decalcification end point was confirmed using both the weight loss and X-ray methods. After decalcification, specimens were processed through paraffin and sections were stained with hematoxylin and eosin. Decalcification employing NH₄OH required an average of six days. Light microscopy indicated good retention of cellular detail.     

A comparative study of facial growth in Homo and Macaca

Sections were prepared throughout all areas of the various facial bones in young, growing Rhesus monkeys. The detailed distribution of resorptive and depository surfaces and the distribution of endosteal and periosteal bone tissue types were determined. From this information, the sequence of remodeling changes associated with the growth of the facial skeleton was then interpreted. This study is a sequal to previous reports in which growth and remodeling processes in the human face were described using similar procedures. In the present report, growth changes in the monkey and human facial skeleton are compared and contrasted. The general plan of facial growth is similar in both species, but major differences exist in the area of the muzzle. The maxillary arch in the monkey is entirely depository in nature, and it grows in a forward and downward direction as the maxillary tuberosity simultaneously grows backward. In the human, the forward part of the maxillary arch is resorptive in character. This contrasting growth factor results in a downward but not forward movement of this area. The result is decreased prognathism. Other differences in growth pattern exist in the forehead, malar, chin, and orbit. The developmental and phylogenetic basis for the upright human face is discussed and evaluated.     

Stain Solubilities Part III

Ethylenediaminetetraacetic acid (EDTA) solution is used to decalcify bone specimens for histological examination. Sodium hydroxide (NaOH) has been used to dissolve EDTA and to bring EDTA solutions to neutral pH. This solution, however, requires several weeks to decalcify bone specimens. We investigated a new de-calcification fluid using concentrated ammonium hydroxide (NH₄OH) to dissolve EDTA and to adjust the pH to neutral. Decalcification was performed using a magnetic stirrer with and without vacuum, or with a sonic cleaner. Decalcification end point was confirmed using both the weight loss and X-ray methods. After decalcification, specimens were processed through paraffin and sections were stained with hematoxylin and eosin. Decalcification employing NH₄OH required an average of six days. Light microscopy indicated good retention of cellular detail.     

Histochemical studies of acid and alkaline phosphatases in rat tooth germs with undecalcified resin-embedded specimens.

A novel technique for the histochemical demonstration of acid phosphatase (AcPase) and alkaline phosphatase (AkPase) in hard tissues has been proposed. Fresh, unfixed, undecalcified samples of rat tooth germs and surrounding structures were embedded in LR Gold resin at -20 degrees C. Sections of 2 microns were taken and subsequently processed for enzyme histochemistry. AkPase reaction product appeared as strong linear staining outlining cell boundaries and was present in the enamel organ, dental pulp, and osteoblast cells. Tartrate-resistant AcPase staining was seen exclusively in the osteoclasts of developing alveolar bone. Our results demonstrated that the use of unfixed, undecalcified LR Gold resin-embedded specimens for histochemistry is a novel technique which may be of value for certain studies when decalcification of specimens is undesirable. The technique appears to give good preservation of enzyme activity combined with the ability to prepare sections with excellent morphological detail.     

Ultrastructural cytochemistry of phosphatases in the ductal component of pleomorphic adenoma of human parotid and submandibular salivary glands

Summary Some ductal cells of pleomorphic adenomas showed evidence of secretory activity, with apical secretory granules, thiamine pyrophosphatase activity in the Golgi apparatus, and acid phosphatase activity in GERL-like structures and in immature secretory granules. Alkaline phosphatase activity was demonstrated rarely at the luminal plasma membrane and in intracellular vesicles, suggesting resorptive activity. ATPase reaction product was associated with contiguous surfaces of tumour cells, particularly of those cells adjacent to the basement membrane, these latter cells apparently differentiating in a different manner to the luminal cells. A comparison of luminal ductal cells of the tumours with normal salivary glands revealed most similarity with intercalary ductal cells.     

An in vitro production of bone specific alkaline phosphatase

Induced alkaline phosphatase has been extracted from osteosarcoma cells grown in tissue culture medium. The extracted enzyme has been purified. Using electrophoresis, inhibition studies, and thermolability, the enzyme was categorized as alkaline phosphatase of osseous origin. Antibodies to this enzyme were reacted against alkaline phosphatase extracted from cadaveric bone, liver, intestine, kidney and fresh placenta. The antibodies were specific against alkaline phosphatase of osseous origin only. No cross-reaction occurred with the enzyme extracted from other sources. The data derived from these studies indicate that alkaline phosphatase of bone is a specific enzyme of osseous tissue. Furthermore, the enzyme has specific antigenic and other properties which distinguish it from alkaline phosphatases from other sources. A model for in vitro production of a specific alkaline phosphatase of bone is presented.     

The enzyme histochemistry of developing odontoblasts in cattle, pigs and horses

Synopsis The histochemical distribution of some hydrolytic and oxidative enzymes in developing odontoblasts and subodontoblasts in cattle, pigs and horses has been observed in cryostat sections of teeth that have been decalcified with neutral EDTA.Undifferentiated dental epithelium and immature odontoblasts of the bell stage tooth germ showed lower levels of enzymatic activity as compared with the well-developed tooth germ.When the dentine matrix began to form, the young odontoblasts appeared to have a significantly positive reaction for acid phosphatase, and gradually other enzymes developed an activity at the top of the cusp.Odontoblasts as well as subodontoblastic-rich cells showed strong enzymatic activities for hydrolytic and oxidative enzymes, that is, they were strongly reactive for alkaline and acid phosphatase and lactate and malate dehydrogenases, and moderately reactive for other oxidative enzyme systems.It is suggested that the subodontoblastic layer is concerned with the biosynthesis of dentinal matrix as well as with the odontoblasts themselves.     

Immunocytological investigation of protein synthesis in Escherichia coli.

Ferritin-conjugated specific antibodies have been used to localize beta-galactosidase and both the monomer and active dimer of alkaline phosphatase in frozen thin sections of cells of Escherichia coli O8 strain F515. The even distribution of the ferritin marker throughout cells that had been induced for beta-galactosidase synthesis, frozen, sectioned, and exposed to ferritin-anti-beta-galactosidase conjugate showed that this enzyme was present throughout the cytoplasm of these cells. Frozen thin sections of cells that had been derepressed for the synthesis of alkaline phosphatase were exposed to both ferritin-anti-alkaline phosphatase monomer and ferritin-anti-alkaline phosphatase dimer conjugates, and the ferritin markers showed a peripheral distribution of both the monomer and the dimer of this enzyme. This indicates that alkaline phosphatase is present only in the peripheral regions of the cell and argues against the existence of a cytoplasmic pool of inactive monomers of this enzyme. This peripheral location of both the monomers and dimers of alkaline phosphatase supports the developing concensus that this enzyme is, like other wall-associated enzymes, synthesized in association with the cytoplasmic membrane and vectorially transported to the periplasmic area, where it assumes its tertiary and quaternary structure and acquires its enzymatic activity.     

Light microscopic localization of alkaline phosphatase in fetal bovine bone using immunoperoxidase and immunogold-silver staining procedures

We localized alkaline phosphatase in the metaphyses of fetal bovine tibial bone by use of avidin-biotin-immunoperoxidase and immunogold-silver staining procedures. Low melting-point, paraffin-embedded sections of periodate lysine-paraformaldehyde-fixed undecalcified bone were used for immunostaining. We suggest that the combination of intact embryonic bone with this fixative and the immunohistochemical procedures used in this study may have helped to preserve antigenicity and thus to improve the efficiency of immunolabeling. Similar patterns of alkaline phosphatase localization were produced by the immunoperoxidase and immunogold-silver staining methods. The latter, although free of immunoreagents such as diaminobenzidine, must be monitored closely to avoid nonspecific staining during the silver enhancement procedure. Both methods revealed a concentration of the enzyme in osteoblasts and in areas of osteoid that lined the bone trabeculae. The results support the findings of earlier enzyme cytochemical studies in which osteoblasts were shown to have significant alkaline phosphatase activity.     

The effects of different decalcification protocols on TUNEL and general cartilage staining

Apoptosis is characterized by DNA strand breaks with a 3'-OH terminus, which are analyzed by terminal deoxy(d)-UTP nick end labeling (TUNEL). Proteinase K digestion is thought to be an essential step in the TUNEL procedure. The effects of decalcifying reagents on general staining and the TUNEL assay for cartilage sections are largely unknown. The effects of these reagents on retention and integrity of DNA in chondrocytes have not been described until now. We evaluated the effects of various decalcifying solutions, including 10% EDTA, 10% citric acid, 5% trichloroacetic acid, 5% acetic acid and a commercial hydrochloric acid-based reagent, on general cartilage staining and the TUNEL assay for cartilage. The effects of proteinase K on nucleus preservation were also examined. Decalcification with 10% EDTA gave the best result for general cartilage staining. Chondrocyte DNA was retained and intact after using this reagent. Decalcification with 10% EDTA is also the safest method of decalcification if the TUNEL assay is applied to cartilage. Proteinase K digestion may have adverse effects on nucleus preservation in cartilage. Awareness of these effects is important whenever the TUNEL assay is applied.     

Freeze-drying of bone tissue: immunocytochemistry and enzyme histochemistry on paraffin embedded and low-temperature resin embedded specimens.

A simple protocol of tissue preparation was sought, which would enable marker enzymes of bone cells and extracellular matrix antigens to be localized in the same tissue section with high optical resolution. For this purpose, snap-frozen samples of rat fetal skeletal tissues were dried in a FDU 010 freeze-drying unit (Balzers) for 8-12 h at -50 to -40 degrees C and 0.02 bar. Freeze-dried tissues were either vacuum-infiltrated at 45 degrees C and embedded undemineralized in Paraplast, or vacuum-infiltrated overnight at 4 degrees C and embedded undemineralized in glycol methacrylate. These procedures enabled enzyme cytochemistry for alkaline phosphatase and tartrate-resistant acid phosphatase, and immunocytochemical staining for collagen types I, III, and laminin to be performed on the same sections. No pretreatment of the sections was necessary to reveal collagen antigenicity. This study reveals the possibility of complementing immunocytochemical studies of extracellular matrix with enzyme cytochemistry and, above all, with the excellent tissue preservation and high resolution afforded by plastic embedding.     

Fine structural localization of alkaline phosphatase in the granular pneumonocytes of hamster lung.

The fine structural localization of nonspecific alkaline phosphatase was studied in the granular pneumonocytes (type II alveolar epithelial cells) of hamster lung by incubating sections of glutaraldehyde-fixed tissues in a medium containing lead ions and sodium beta-glycerophosphate or alpha-naphthyl acid phosphate. The specificity of the reaction was tested by exposing the sections to inhibitors of alkaline phosphatase. The results showed that alkaline phosphatase activity was present in the inclusion bodies of granular pneumonocytes. The enzyme reaction was strong in the membrane lining the inclusion bodies and a weaker reaction was generally detectable in the inclusion contents. Although only a proportion of the inclusion bodies showed enzyme activity, there was no obvious correlation between the reactivity of the inclusions and their intracellular position or size. The other organelles were unreactive. The finding of alkaline phosphatase activity within the inclusion bodies of granular pneumonocytes is an enigma as these organelles are generally considered to be lyosomes.     

The effects of different decalcification protocols on TUNEL and general cartilage staining.

Apoptosis is characterized by DNA strand breaks with a 3'-OH terminus, which are analyzed by terminal deoxy(d)-UTP nick end labeling (TUNEL). Proteinase K digestion is thought to be an essential step in the TUNEL procedure. The effects of decalcifying reagents on general staining and the TUNEL assay for cartilage sections are largely unknown. The effects of these reagents on retention and integrity of DNA in chondrocytes have not been described until now. We evaluated the effects of various decalcifying solutions, including 10% EDTA, 10% citric acid, 5% trichloroacetic acid, 5% acetic acid and a commercial hydrochloric acid-based reagent, on general cartilage staining and the TUNEL assay for cartilage. The effects of proteinase K on nucleus preservation were also examined. Decalcification with 10% EDTA gave the best result for general cartilage staining. Chondrocyte DNA was retained and intact after using this reagent. Decalcification with 10% EDTA is also the safest method of decalcification if the TUNEL assay is applied to cartilage. Proteinase K digestion may have adverse effects on nucleus preservation in cartilage. Awareness of these effects is important whenever the TUNEL assay is applied.     

Identification of non-specific alkaline phosphatases in hyphal cells of the fungus Neurospora crassa by in situ histochemistry

The present study was designed to identify alkaline phosphatases in non-permeabilized hyphal cells of the fungus Neurospora crassa by staining these enzymatic activities with a modified azo dye coupling method. Our strategy allowed the identification of three non-specific alkaline phosphatase activities, one of them possibly being a novel putative enzyme, which is not responsive to either Mg(2+) or EDTA. Another alkaline phosphatase activity, whose location in the hyphal cell is regulated by phosphate, is stimulated by Mg(2+), inhibited by EDTA, and somehow dependent on the expression of the pho-2(+) -encoded Pi-repressible alkaline phosphatase.