Proteoglycans of alveolar bone of diabetic and non-diabetic mice: a histochemical study

The effect of diabetes mellitus on the interdental alveolar bone has been long debated. The present study reported the distribution of glycosaminoglycans (GAG) in normal and diabetic alveolar bone using histochemical techniques. Animals were rendered diabetic and killed at 2, 4, 6 and 9 weeks after injections. Tissues were stained with Alcian blue 8GX dye (pH 2.5) to demonstrate GAG and the intensity of the staining reactions compared with age-matched controls. During the experiment, weights of control animals did not change significantly; weights of diabetic animals were significantly less than initial weights from 0-6 weeks (p less than 0.001), but became nearly equal by 9 weeks. Staining intensity of diabetic bone demonstrated initial decrease (0-4 weeks) followed by a marked increase (4-9 weeks) suggesting an early decline in bone GAG levels followed by increased bone GAG levels as compared to age-matched control and initial levels. Bone GAG levels were significantly different between diabetics and age-matched controls at 2 (p less than 0.005) 4 (p less than 0.001), 6 (p less than 0.001) and 9 (p less than 0.001) weeks after streptozotocin injections. Digestion with chondroitinase AC, ABC and streptomyces hyaluronidase suggested significant differences between control and diabetic bone matrix in the levels of chondroitin 4 and 6 sulfates (p less than 0.05) and hyaluronic acid (p less than 0.001) but not dermatan sulfate. In control and diabetic bone, chondroitin sulfates were located within the bone matrix, dermatan sulfate within bone matrix and Sharpey fiber bundles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alveolar bone of BB/W rats: a morphometric and histochemical study

The present study reported histochemical changes in alveolar bone glycosaminoglycans (GAG) (using Safranin O) and in interdental bone height in three groups of BB/W rats: diabetic, diabetes prone, and diabetes resistant. Safranin O staining intensity suggested that total GAG levels were highest in diabetic bone (p less than 0.05 compared to diabetes resistant, p less than 0.005 compared to diabetes prone) but not significantly different between diabetes prone and resistant groups. Following chondroitinase AC and ABC digestion, staining reactions suggested that the highest levels of dermatan sulfate were in the diabetes resistant group (p less than 0.001 compared to diabetic, p less than 0.001 compared to diabetes prone) and the highest levels of chondroitin sulfates were in the diabetes prone group (p less than 0.001). Coincidently the mean height of diabetes prone interdental septum was significantly less than that of diabetes resistant or diabetic groups (p less than 0.05). The study suggested that 1) diabetes and "prediabetes" produce significant changes in levels of chondroitin 4, 6, and dermatan sulfates within alveolar bone, 2) in "prediabetic" animals, interdental bone loss occurs prior to the onset of clinical symptoms and in the absence of local irritating factors, the bone height appears to return to normal levels, and 3) there may be a correlation between alveolar bone height and relative levels of dermatan sulfate.     

Whole-mount bone and cartilage staining of chick embryos with minimal decalcification

Abstract

Whole-mount staining with Alcian blue for cartilage and alizarin red for bone has been widely used for visualizing the skeletal patterns of embryos and small adult vertebrates. The possibility of decalcification by the acidic Alcian blue solution is known, but standard staining protocols do not always avoid this issue. We investigated the effects of acidity on the stainability of developing bones in stage 36 chick embryos and developed an optimal procedure for obtaining reliable results with minimal decalcification. The diaphyses of long bone rudiments and the maxillofacial membranous bones progressively lost their stainability with alizarin red when the chick embryos were soaked for long periods in the preceding acidic Alcian blue staining solution for cartilage. Unless the acidity was neutralized with an alkaline solution, the remaining acidity in the specimens rendered the pH sufficiently low to prevent the subsequent alizarin red staining of the bones. These findings indicate that the mineralizing bones at the early stages of development are labile to acidity and become decalcified more substantially during the staining process than previously appreciated. The following points are important for visualizing such labile mineralizing bones in chick embryos: 1) fixing with formaldehyde followed by soaking in 70% ethanol, 2) minimizing the time that the specimens are exposed to the acidic Alcian blue solution, and 3) neutralizing and dehydrating the specimens by an alkaline-alcohol solution immediately after the cartilage staining. When the exact onset and/or an early phase of ossification are of interest, the current double-staining procedure should be accompanied by a control single-staining procedure directed only toward bone.     

Differential use of Alcian blue and toluidine blue dyes for the quantification and isolation of anionic glycoconjugates from cell cultures: application to proteoglycans and a high-molecular-weight glycoprotein synthesized by articular chondrocytes

Alcian blue and toluidine blue dyes form complexes with anionic glycoconjugates (AG) such as proteoglycans (PG) and glycosaminoglycans (GAG). However, the Alcian blue-AG complexes do not readily dissociate, while the toluidine blue-AG complexes do so in salt solutions. This differential dissociation of the dye-AG complexes has been utilized in the analysis and isolation of radiolabeled AG elaborated by articular chondrocyte cultures incubated with the radiolabeled precursors of AG. For the rapid quantification of newly synthesized (35)S-labeled PG, small replicate aliquots of the radiolabeled culture media were applied directly to cellulose acetate strips, stained with Alcian blue and the stained immobilized radiolabeled PG was quantified by liquid scintillation counting. Comparison of anionic glycoconjugates quantified in the culture media employing toluidine blue and Alcian blue staining on cellulose acetate trips gave similar results. Staining on cellulose acetate strips using these two dyes is particularly suited for the simultaneous processing of large numbers of samples, as illustrated by the screening of the effects of biological materials and drugs on AG synthesis, in cultures labeled with [(35)S]-sulfate and [(3)H]-glucosamine. The Alcian blue and toluidine blue precipitation methods yielded similar results for the total AG recovered from the media of TGF-beta-stimulated chondrocytes. Electrophoretic analysis of toluidine blue- and Alcian blue-precipitated AG followed by autoradiography and Alcian blue staining in combination with silver nitrate demonstrated that both dyes yielded similar pattern of bands on gels. However, some AG from Alcian blue precipitate did not enter the gel, suggesting incomplete dissociation of Alcian blue-AG complex. The application of the toluidine blue precipitation method, in combination with enzymatic digestion of the GAG chains of the PGs, is illustrated by the isolation of a non-PG high-molecular-weight AG, as well as the PGs from the media of chondrocyte cultures stimulated by TGF-beta.     

Chondroitin sulfate proteoglycan in the extracellular matrix of the canine superior olivary nuclei

The perineuronal extracellular matrix of the canine superior olivary nuclei was examined by the histochemical method. The extracellular matrix was stained with Alcian blue (pH 1.0 and 2.5), high iron diamine and ruthenium red. The staining intensity of Alcian blue in the extracellular matrix was remarkably reduced after chondroitinase ABC digestion but not after that of heparitinase or hyaluronidase. These results indicate that the extracellular matrix consists of proteoglycans and contains the chondroitin sulfate proteoglycan.     

OPN deficiency suppresses appearance of odontoclastic cells and resorption of the tooth root induced by experimental force application

Osteopontin (OPN) is a major non-collagenous bone matrix protein implicated in the regulation of cell function. Although OPN is rich in the cementum of the tooth, the significance of OPN in this tissue is not understood. Tooth root resorption is the most frequent complication of orthodontic tooth movement (TM). The objective of this study was to examine the pathophysiological role of OPN in cementum of the tooth root. For this purpose, the upper right first molar (M1) in OPN-deficient and wild-type (WT) mice was subjected to mechanical force via 10 gf NiTi coil spring while the left side molar was kept intact to serve as an internal control. Micro-CT section and the level of tartrate resistant acid phosphatase (TRAP)-positive cells on the tooth root surface defined as odontoclasts were quantified at the end of the force application. In WT mice, force application to the tooth caused appearance of odontoclasts around the mesial surface of the tooth root resulting in tooth root resorption. In contrast, OPN deficiency significantly suppressed the force-induced increase in the number of odontoclasts and suppressed root resorption. This force application also induced increase in the number of TRAP-positive cells in the alveolar bone on the pressure side defined as osteoclasts, while the levels of the increase in osteoclastic cell number in such alveolar bone were similar between the OPN-deficient and WT mice. These observations indicate that OPN deficiency suppresses specifically tooth root resorption in case of experimental force application.     

Alkali burns of the rabbit cornea. II. A histochemical study of glycosaminoglycans.

In alkali burned rabbit cornea the stainability of glycosaminoglycans in cold microtome setions was investigated. Staining by Alcian blue in 3% acetic acid, Alcian blue in various MgCl2 concentration and toluidine blue (pH 4.5) was employed. From the 1st to the 4th experimental day the intensity of reactions was decreased. This is most probably due to an increased hydration of the corneal stroma. On the 7th day hydration was markedly suppressed and reached nearly the normal level. In this time interval a decreased stainability of glycosaminoglycans was seen accompanied by a complete loss of staining in the marginal zone. On the 14th day the stainability in the traumatized area began to restore and in the marginal zone appeared. On the 32nd day the staining intensity of both areas was normalised, however when lower concentrations of MgCl2 were used; in the presence of higher concentrations of MgCl2 the decreased staining intensity persisted and points to a lower sulfatation of glycosaminoglycans. This was particularly remarkable in the area bordering the injured zone. This decrease runs parallel to the increased activities of acid glycosidases (especially of acid beta-galactosidase) which were reported previously.     

Staining of interphase nuclei and mitotic figures in cultured cells with alcian blue 8GX

Cultured endothelial cells derived from bovine calf pulmonary artery were subjected to a variety of fixatives and stained with 1% Alcian blue 8GX at pH 2.59 to 3.26. Within this range of pH, interphase nuclei and especially mitotic figures were (a) strongly stained in cells fixed with 10% formalin (phosphate buffered or unbuffered) or 2.5% buffered glutaraldehyde, (b) weakly stained or unstained in cells fixed in formaldehyde containing divalent cations, and (c) unstained in cells fixed in acetic acid-containing fluids. However, optimal nuclear staining with Alcian blue under the conditions of this study was judged to be achieved after fixation with neutral phosphate buffered 10% formalin. Endothelial cell cytoplasm exhibited a similar fixative-dependent staining. At pH 2.59 the cytoplasm of interphase cells fixed in formaldehyde (containing no divalent cations) or glutaraldehyde remained unstained; however, at higher pH cytoplasmic staining did occur and it increased as pH increased. In contrast, when these latter fixatives were employed the cytoplasm of mitotic cells stained at all pH levels tested. In cultured endothelial cells after appropriate fixation, 1% Alcian blue 8GX (pH 2.59) was found to possess the ability to stain nuclei with a selectivity and intensity that compared favorably to those of the Feulgen reaction of Heidenhain iron hematoxylin but without the latters' length and complexity. Therefore, this procedure may provide a rapid, simple, and selective method for visualizing interphase nuclei or mitotic figures, or both in the majority of cultured cells.     

Alveolar bone response distal to applied orthodontic forces in ovariectomized rats

Objectives:The aim of this study was to investigate the effect of the application of orthodontic tooth forces on the alveolar bone distal to the loaded teeth, in ovariectomized female rats.Methods:Twenty-four eight-month-old Wistar rats were divided into one group ovariectomized at the age of six months and one control. An orthodontic appliance delivering a mesial traction force of 60 gr* was placed on the right maxillary 1^st molar of all animals for 14 days. Histology of the alveolar bone, of the adjacent and distal teeth to the loaded molar and the contralateral side, was performed following euthanasia.Results:In the non-ovariectomized rats, extensive resorption was noticed in the direction of the orthodontic movement in the 2^nd and 3^rd molar interdental space, whereas the respective contralateral interdental space did not show any remodeling activity. Ovariectomized rats displayed reduced osseous tissue in the interdental space of both sides. The alveolar bone in the interradicular area of the 2^nd loaded molar revealed frontal resorption, whereas, the alveolar interradicular bone of the contralateral 2^nd molar showed internal resorption.Conclusions:In conclusion, orthodontic forces applied to the dentoalveolar complex of ovariectomized rats affect bone remodeling, even in areas distal to the site of force application. This finding should be taken into account during orthodontic treatment of women during menopause.     

Staining of interphase nuclei and mitotic figures in cultured cells with Alcian blue 8GX

Summary Cultured endothelial cells derived from bovine calf pulmonary artery were subjected to a variety of fixatives and stained with 1% Alcian blue 8GX at pH 2.59 to 3.26. Within this range of pH, interphase nuclei and especially mitotic figures were (a) strongly stained in cells fixed with 10% formalin (phosphate buffered or unbuffered) or 2.5% buffered glutaraldehyde, (b) weakly stained or unstained in cells fixed in formaldehyde containing divalent cations, and (c) unstained in cells fixed in acetic acid-containing fluids. However, optimal nuclear staining with Alcian blue under the conditions of this study was judged to be achieved after fixation with neutral phosphate buffered 10% formalin. Endothelial cell cytoplasm exhibited a similar fixative-dependent staining. At pH 2.59 the cytoplasm of interphase cells fixed in formaldehyde (containing no divalent cations) or glutaraldehyde remained unstained; however, at higher pH cytoplasmic staining did occur and it increased as pH increased. In contrast, when these latter fixatives were employed the cytoplasm of mitotic cells stained at all pH levels tested. In cultured endothelial cells after appropriate fixation, 1% Alcian blue 8GX (pH 2.59) was found to possess the ability to stain nuclei with a selectivity and intensity that compared favorably to those of the Feulgen reaction or Heidenhain iron hematoxylin but without the latters’ length and complexity. Therefore, this procedure may provide a rapid, simple, and selective method for visualizing interphase nuclei or mitotic figures, or both in the majority of cultured cells.     

Keratan sulfate epitopes exhibit a conserved distribution during joint development that remains undisclosed on the basis of glycosaminoglycan charge density.

Changes in glycosaminoglycan (GAG) content and distribution are vital for joint development. However, their precise character has not been established. We have used immunohistochemistry (IHC) and "critical electrolyte" Alcian blue staining to assess such changes in developing chick and rabbit joints. IHC showed chondroitin sulfate labeling in chick epiphyseal cartilage but not in interzones. In contrast, prominent labeling for keratan sulfate (KS) was restricted to chick cartilage-interzone interfaces. In rabbit knees, KS labeling was also prominent at presumptive cavity borders, but weak in interzone and cartilage. Selective pre-digestion produced appropriate loss of label and undersulfated KS was undetectable. Quantification of Alcian blue staining by scanning and integrating microdensitometry showed prominent hyaluronan-like (HA-like) interzone staining, with chondroitin sulfate and weaker KS staining restricted to epiphyseal cartilage. Hyaluronidase decreased HA-like staining in the interzone. Surprisingly, keratanases also reduced HA-like but not sulfated GAG (sGAG-like) staining in the interzone. Chondroitinase ABC had little effect on HA-like staining but decreased sGAG staining in all regions. Rabbit joints also showed HA-like but not KS staining in the interzone and strong chondroitin sulfate-like staining in epiphyseal cartilage. Our findings show restricted KS distribution in the region close to the presumptive joint cavity of developing chick and rabbit joints. Alcian blue staining does not detect this moiety. Therefore, it appears that although histochemistry allows relatively insensitive quantitative assessment of GAGs, IHC increases these detection limits. This is particularly evident for KS, which exhibits immunolabeling patterns in joints from different species that is consistent with a conserved functional role in chondrogenesis.     

Evaluation of improved zirconyl hematoxylin compared to the classical pH 2.5 Alcian blue method for demonstrating acid mucins

Acid mucins have diagnostic significance for many pathological conditions, especially in certain tumors. We compared the classical pH 2.5 Alcian blue method to a new, improved zirconyl hematoxylin (IZH) method for demonstrating acid mucins using two fixatives: Bouin`s solution and 10% neutral buffered formalin (NBF). We used rabbit small intestine, large intestine and trachea. Specimens were fixed in Bouin`s solution and NBF. A total of 160 paraffin sections were prepared and stained with pH 2.5 Alcian blue and IZH. The stained acid mucins were assessed using digital image analysis software. Stained mucins were quantified for each staining procedure and fixative. No important differences were observed in acid mucin staining by either method after either fixative. The IZH method provides results as good as pH 2.5 Alcian blue and can be used to obtain reliable staining for acid mucins.     

Evaluation of improved zirconyl hematoxylin compared to the classical pH 2.5 Alcian blue method for demonstrating acid mucins

Abstract

Acid mucins have diagnostic significance for many pathological conditions, especially in certain tumors. We compared the classical pH 2.5 Alcian blue method to a new, improved zirconyl hematoxylin (IZH) method for demonstrating acid mucins using two fixatives: Bouin`s solution and 10% neutral buffered formalin (NBF). We used rabbit small intestine, large intestine and trachea. Specimens were fixed in Bouin`s solution and NBF. A total of 160 paraffin sections were prepared and stained with pH 2.5 Alcian blue and IZH. The stained acid mucins were assessed using digital image analysis software. Stained mucins were quantified for each staining procedure and fixative. No important differences were observed in acid mucin staining by either method after either fixative. The IZH method provides results as good as pH 2.5 Alcian blue and can be used to obtain reliable staining for acid mucins.     

Type, location and role of glycosaminoglycans in cloned differentiated chick retinal pigmented epithelium

In clonal culture, colonies of 3–4 week old chick retinal pigmented epithelial cells exhibit Alcian Blue positive extracellular matrix (ECM) material on the surface of the cells. Alcian blue positive ECM is located between undifferentiated cells at the edges of the disc-shaped colonies and beneath the differentiated cells in the colony center. The latter material is associated with the basement membrane. The staining properties suggest that glycosaminoglycans (GAG) are present in these regions. Extraction of GAG from homogenates of colonies, followed by electrophoresis on cellulose acetate strips, results in three bands with mobilities similar to those of hyaluronic acid, heparan sulfate, and chondroitin sulfate, respectively. All three bands label with [³H]glucosamine, and the last two also label with [³⁵S]sulfate. The composition appeared to differ when colonies were grown in different media. Digestion of the GAG preparations with various enzymes suggests that bands II and III represent heparan sulfate and chondroitin sulfate, respectively, in colonies grown in Ham's F10g medium. The composition of band I is as yet undetermined. In minimal Eagle's medium (MEM), bands I and III consisted of hyaluronic acid and chondroitin sulfate, respectively, while band II had properties suggestive of a copolymer of heparan sulfate and an unidentified GAG. Cells release only one [³H]glucosamine-labelled GAG into the medium. This material has a mobility similar to hyaluronic acid and is digested by Streptomyces hyaluronidase, suggesting that it is hyaluronic acid. Staining with Alcian Blue at different pH suggests that it may represent the material associated with the upper surface of the cells. Some of the ECM located between the undifferentiated cells and associated with the basement membrane in the differentiated regions of the colonies stains with Alcian Blue at pH 1.0 and 0.2 suggesting that it may contain GAGs found in bands I and II. Colonies treated with medium containing 6-diazo-5-oxo-L-norleucine (DON), an inhibitor of GAG synthesis, for 48 hr showed a reduced Alcian Blue staining of the ECM in the undifferentiated regions. After 72 hr of treatment with DON, the undifferentiated cells had detached from the plate, whereas the differentiated cells remained intact. The results suggest that the GAG may be involved in cellular adhesion, particularly of the undifferentiated cells.     

Localization and characterization of acharan sulfate in the body of the giant African snail Achatina fulica

Acharan sulfate is a glycosaminoglycan (GAG), having the structure →4)-2-acetamido-2-deoxy-α- -glucopyranose(1→4)-2-sulfo-α- -idopyranosyluronic acid (1→, isolated from the body of the giant African snail Achatina fulica. This GAG represents 3–5% of the dry weight of this snail's soft body tissues. Frozen sections and polyester wax sections of the snail's body were stained by Alcian blue-periodic acid-Schiff's reagent (PAS) to localize acharan sulfate. Alcian blue staining indicated that GAG was mainly secreted into the outer surface of the body from internal granules. A highly mucous material was collected and treated and the acharan sulfate was recovered by ethanol and cetyl pyridinium chloride precipitation. Crude acharan sulfate was purified by DEAE-Sephacel ion-exchange chromatography. Depolymerization of intact mucus and purified acharan sulfate fractions by heparin lyase II (heparitinase I) from Flavobacterium heparinum produced an unsaturated disaccharide as a major product, establishing the repeating unit of acharan sulfate. These results demonstrate that mucus in the granule and secreted to the outside of the body is composed entirely of acharan sulfate.     

Microdamage in porcine alveolar bone due to functional and orthodontic loading

Bone remodelling has been associated with microdamage. The aim of this study was to investigate the presence of microdamage in the alveolar bone and its potential role in the initiation of bone remodelling following the application of an orthodontic load. The three-dimensional morphology of the alveolar bone was investigated by means of high resolution micro-CT scanning. In 25, 3-month-old, male Danish land-race pigs, the alveolar bone around the lower right and left first molars was analysed. The right first molar was moved buccally with a force of 130 cN by means of a custom-made cantilever made of a TMA 0.017 x 0.025 inch wire. The left molar was left untreated. After 1, 2, 4, 7 and 15 days of treatment the regions containing the right and left molars were excised and en bloc stained in basic fuchsin and the presence of microdamage detected. Diffuse damage was present in the alveolar bone of both the treated and the untreated teeth on both sides. On the lingual sides, diffuse damage showed the same orientation as the periodontal fibres. Bone microcracks were also detected on both the treated and untreated teeth. On the buccal surfaces they where often observed in close proximity to scalloped resorption surfaces. After 1 day of treatment, the presence of microcracks on the buccal-treated side was particularly marked. To conclude, bone microdamage is present in porcine alveolar bone in form of both microcracks and diffuse damage, suggesting that microdamage-driven remodelling also occurs in the alveolar bone. The presence of bone microcracks in the direction of the orthodontic force at day 1 suggests that they could represent the first damage induced by the orthodontic load that has to be repaired.     

Comparsion of staining characteristics of Toto bodies

Toto bodies are eosinophilic structures that resemble the cells of the superficial cell layer of the oral epithelium. Toto bodies commonly are associated with inflammatory gingival and other mucosal lesions including pyogenic granuloma, irritational fibroma, epulis fissuratum, peripheral giant cell granuloma and inflammatory hyperplastic gingivitis. We evaluated staining characteristics of Toto bodies to establish their origin and to identify their significance in lesions. We investigated pyogenic granuloma, fibroma and leukoplakia with epithelium that exhibited Toto bodies after hematoxylin and eosin (staining. Sections were stained with Alcian blue, periodic acid-Schiff and Ayoub-Shklar stains to evaluate staining intensity and distribution. More Toto bodies were found in pyogenic granuloma than in fibroma and leukoplakia. PAS and Alcian blue staining exhibited mild intensity and did not establish the origin of Toto bodies. High staining intensity and diffuse distribution of stain was observed using Ayoub-Shklar staining, which indicated that Toto bodies originate from keratin.     

Identification of superficial zone articular chondrocyte stem/progenitor cells

Identification of progenitor/stem cell populations that differentiate specifically towards superficial zone articular chondrocytes is an unmet challenge for cartilage tissue engineering. Using fluorescence activated cell sorting (FACS) analysis we found a characteristic pattern of "side population" (SP) stem cells identified by the Hoechst 33342 dye. We established micromass cultures from this population of cells and tested their chondrogeneic potential. Control (untreated) cultures were minimally stained for Alcian blue - a marker of chondrogenesis. However, with BMP-7 treatment, Alcian blue staining was increased. Superficial zone protein - a specific marker for articular cartilage superficial zone chondrocytes - increased with BMP-7 and/or TGF-beta1 treatment in SP micromass cultures. Our results demonstrate the presence of stem/progenitor cells in the SP fraction isolated from the surface zone of bovine cartilage and have the ability to specifically differentiate towards the superficial zone articular chondrocyte.     

Histochemical method for demonstrating quail mast cell types simultaneously

Abstract

The development and application of selective staining methods for routine detection of mast cells are of considerable interest, because these cells play an important role in health and disease. The composition of cytoplasmic mast cell granules depends on the species and type of mast cell. The study reported here was conducted to investigate the combined use of aldehyde fuchsin (AF) and the Alcian blue-critical electrolyte concentration (AB-CEC) (pH 5.8, 0.3 M MgCl₂) techniques for differentiating avian mast cell subtypes. Tissue samples from skin, intestines, and lungs of six healthy adult quail and two control rats were fixed in Carnoy's solution and 10% formolin for routine histological processing. To determine the staining properties of sulfated glycosaminoglycans (GAGs), a three-step staining technique was applied using berberine sulfate, AF, and AB-CEC. In quail, AF positivity following application of the AB-CEC technique was found only in the lungs, mostly in cells that gave a berberine sulfate-positive reaction, and this positivity was determined to be localized particularly in the nucleus and perinuclear cytoplasm. In other regions, the pale AF staining of cells that did not emit fluorescence when stained with berberine sulfate was determined to be replaced by a blue color after application of AB-CEC. The AF/AB-CEC (pH 5.8, 0.3 M MgCl₂) technique demonstrated that rat and quail mast cells varied in both GAG types and their distribution within the cell. Especially in avian species, this technique can be applied to distinguish mast cells according to their GAG content. It can be used as an alternative to the AB/safranin O staining procedure for differentiating mast cells that contain and lack heparin.     

Distribution of 3H-proline within transseptal fibers of the rat following release of orthodontic forces

Maxillary right first molar teeth of rats were tipped mesially with an orthodontic appliance for 2 weeks (experimental group), 3H-proline was injected, and orthodontic forces were removed 6 hr later (time 0). The contralateral molar teeth of treated (internal control group) and age- and weight-matched untreated animals (external control group) were also studied. Diastemata were created between the molar teeth by the orthodontic appliance, and transseptal fibers between first and second (P less than 0.001) and second and third molars (P less than 0.005) were significantly lengthened as compared to external and internal controls at time 0. Diastemata between molar teeth were closed 5 days after removal of orthodontic force. Transseptal fibers adjacent to the source of the orthodontic force (mesial region) had the highest mean number of 3H-proline-labeled proteins at time 0 and at all times following removal of the force (P less than 0.001), and had the highest rate of labeled protein removal (P less than 0.001). Half-lives for removal of 3H-proline-labeled transseptal fiber proteins were significantly greater in mesial and distal regions and significantly less in middle regions of experimentals than in corresponding regions of external controls (P less than 0.001). These data suggest the following: 1) transseptal fibers adjust their length by rapid remodeling in regions experiencing a tensile force; 2) collagenous protein turnover within the middle third of the transseptal fibers is more rapid subsequent to release of orthodontic force than during normal physiologic drift, suggesting that this region adapts rapidly to changes in adjacent tooth position and that these fibers do not play a significant role in relapse of orthodontically relocated teeth; and 3) significant differences in turnover rates of 3H-proline-labeled transseptal ligament proteins of external and internal control quadrants suggest that tooth movement produces both local and systemic effects on collagenous protein metabolism.