Cellular enzyme activity associated with tissue degradation following orthodontic tooth movement in man

Enzyme histochemical techniques were used as markers of macrophage activity and differentiation in the periodontal tissues following orthodontic tooth movement in man. The enzymes studied included lactate dehydrogenase, glucose-6-phosphate dehydrogenase, succinic dehydrogenase, acid phosphatase and its tartrate resistant isoenzyme, arylsulfatase, aminopeptidase M and prostaglandin synthetase. Chloroacetyl esterase activity was studied in order to detect possible neutrophilic degrading activity. Intense activities of arylsulfatase and prostaglandin synthetase and a moderate activity of aminopeptidase M were found in cells degrading the hyaline zone. However, no activity of tartrate resistant acid phosphatase was found in these cells. Giant cells in contact with bone surfaces adjacent to the hyaline zone exhibited an intense activity of succinic dehydrogenase, tartrate resistant acid phosphatase and aminopeptidase M. Chloroacetyl esterase activity did not change following orthodontic treatment. The results indicate that macrophages in various stages of differentiation were responsible for the degradation of the hyaline zone and alveolar bone during orthodontic tooth movement. The enzymatic differences were probably due to the influence of the immediate cellular environment. Prostaglandin synthetase activity, which may be interpreted as a sign of prostaglandin secretion, was associated with the degradation of the hyaline zone in man.     

Fibrinogen and IgG in the hyaline zone in man after orthodontic movement

Abstract – Fibrinogen and IgG were demonstrated in the hyaline zone with direct immunofluorescent staining after orthodontic tooth movement in man. It was suggested that presence of plasma proteins in the hyaline zone contributed to its hyaline appearance. Fibrin and IgG may, furthermore, attract phagocytes and form a network for the reorganization of the necrotic tissue in the hyaline zone.     

Reduction of orthodontic tooth movement by experimentally induced periodontal inflammation in mice

Orthodontic therapy is known to have an aggravating effect on the progression of destructive periodontitis if oral hygiene is not maintained. However, it is largely unknown how active periodontitis affects the velocity of orthodontic tooth movement. In this study, we examined the effect of periodontal inflammation on orthodontic tooth movement using a mouse model. Orthodontic force was applied on the maxillary first molar of mice, with or without ligature wire to induce experimental periodontitis. The distance moved by the first molar was significantly reduced by the ligature-induced experimental periodontitis. Tartrate-resistant acid phosphatase staining revealed that the number of osteoclasts present during orthodontic treatment was lower in the pressure zone of alveolar bone in the presence of periodontal inflammation. Consistently, the expression level of receptor activator of nuclear factor-κB ligand (RANKL) in the pressure zone was decreased in the ligature group. By contrast, experimental periodontitis increased the expression of cyclooxygenase-2 mRNA in the periodontal tissues, while in vitro treatment with prostaglandin E₂ decreased extracellular signal-regulated kinase phosphorylation and RANKL expression induced by mechanical stress in osteoblasts. Taken together, these results suggest that the orthodontic force-induced osteoclastogenesis in alveolar bone was inhibited by the accompanying periodontal inflammation, at least partly through prostaglandin E₂, resulting in reduced orthodontic tooth movement.     

Fibrinogen and IgG in the hyaline zone in man after orthodontic movement

Fibrinogen and IgG were demonstrated in the hyaline zone with direct immunofluorescent staining after orthodontic tooth movement in man. It was suggested that presence of plasma proteins in the hyaline zone contributed to its hyaline appearance. Fibrin and IgG may, furthermore, attract phagocytes and form a network for the reorganization of the necrotic tissue in the hyaline zone.     

Quantitative studies on acid phosphatase in developing rat bones and teeth during hypervitaminosis D

The following differences were found between bone and tooth buds with regard to the acid p-nitrophenyl phosphatase activity in whole homogenates and extracts: (a) most of the enzyme in a KCl (0.15 M) homogenate of bone was in a 6000 g min sediment but was extracted with urea, while most of the activity in tooth buds was extracted with KCl and was not affected by urea; (b) the activity in bone was stimulated by both sodium tartrate and sodium chloride in vitro, while the activity in tooth bud was markedly inhibited by sodium tartrate and unaffected by sodium chloride; (c) the activity in bone, but not in tooth buds, was completely inhibited by fluoride in vitro and relatively resistant to heat; and (d) the enzyme activity in bone, but not in teeth, was increased markedly during hypervitaminosis D. The tissue-specific effects of inhibitors in vitro and of vitamin D in vivo are in agreement with previous histochemical studies with α-naphthyl phosphate as substrate.It is concluded that most of the p-nitrophenyl phosphatase activity in bone is due to a tartrate-resistant and heat-resistant enzyme which is different from the tartrate-sensitive and heat-labile enzyme responsible for most of the activity in the odontogenic cells.     

Focal hyalinization during experimental tooth movement in beagle dogs.

The aim was to study morphological differences between the periodontal structures of beagle dogs showing different rates of tooth movement under identical experimental conditions. An orthodontic appliance was placed on the mandibular second premolar and the first molar to exert a continuous and constant reciprocal force of 25 cN. Tooth movement was recorded weekly. The dogs were killed after 1, 4, 20, 40, and 80 days for histological evaluation. Haematoxylin and eosin staining was used for tissue survey, alkaline phosphatase staining was used as a marker for active osteoblasts, and tartrate resistant acid phosphatase staining was used for osteoclasts. After 24 hours, osteoclastic and osteoblastic activity had already increased at the pressure and tension sides, respectively, and, in some samples, hyalinization was found. In case of fast-moving teeth, areas of direct bone resorption at the pressure side and deposition of trabecular bone at the tension side were found throughout the experimental period. In the periodontal ligaments of teeth showing little movement, small patches of hyalinization were found at the pressure side, mostly located buccally or lingually of the mesiodistal plane. These phenomena were found in both molars and premolars and at all time points. It is concluded that small focal hyalinizations might be a factor that could explain individual differences in the rate of tooth movement.     

Effect of a static magnetic field on orthodontic tooth movement in the rat

Orthodontic tooth movement may be enhanced by the application of a magnetic field. Bone remodelling necessary for orthodontic tooth movement involves clastic cells, which are tartrate-resistant acid phosphatase (TRAP) positive and which may also be regulated by growth hormone (GH) via its receptor (GHR). The aim of this study was to determine the effect of a static magnetic field (SMF) on orthodontic tooth movement in the rat. Thirty-two male Wistar rats, 9 weeks old, were fitted with an orthodontic appliance directing a mesial force of 30 g on the left maxillary first molar. The appliance incorporated a weight (NM) or a magnet (M). The animals were killed at 1, 3, 7, or 14 days post-appliance insertion, and the maxillae processed to paraffin. Sagittal sections of the first molar were stained with haematoxylin and eosin (H&E), for TRAP activity or immunohistochemically for GHR. The percentage body weight loss/gain, magnetic flux density, tooth movement, width of the periodontal ligament (PDL), length of root resorption lacunae, and hyalinized zone were measured. TRAP and GHR-positive cells along the alveolar bone, root surface, and in the PDL space were counted. The incorporation of a SMF (100-170 Gauss) into an orthodontic appliance did not enhance tooth movement, nor greatly alter the histological appearance of the PDL during tooth movement. However significantly greater root resorption (P = 0.016), increased width of the PDL (P = 0.017) and greater TRAP activity (P = 0.001) were observed for group M at day 7 on the compression side. At day 14 no differences were observed between the appliance groups.     

Acid phosphatase in developing teeth and bone of man and Macaque monkey

abstract — The activity of acid phosphatase in jaws and teeth from human fetuses and young monkeys has been studied by means of histochemistry. The enzyme was found in all cells of the hard tissues. The highest activities were found in osteoclasts and presecretory ameloblasts. In the monkeys, where the development of the teeth had reached the stage of enamel maturation, an equally high activity was also found in postsecretory ameloblasts. Studies with specific inhibitors suggested that there may be at least three different acid phosphatases present in the cells associated with hard tissue formation and resorption: (1) fluoride and copper resistant, molybdate and tartrate sensitive acid phosphatase was found in secretory and postsecretory ameloblasts, (2) fluoride, copper and molybdate sensitive, tartrate resistant acid phosphatase was found in osteoclasts. and (3) acid phosphatase rather resistant to all the applied inhibitors was found in outer enamel epithelium, stellate reticulum, stratum intermedium, odontoblasts and osteoblasts. The acid phosphatase activity in the enamel organ prior to enamel matrix formation was inhibited similarly to the activity in the oral epithelium and may represent a fourth type.     

Degenerative changes in masseter muscle following injection of lidocaine: a histochemical study.

Changes in succinic dehydrogenase, adenosine triphosphatase, and phosphorylase activities occurred in masseter muscle by 15 minutes following injection of 2% lidocaine. Abolishment of phosphorylase activity suggested an effect on the sarcoplasmic reticulum. Increased staining for succinic dehydrogenase and adenosine triphosphatase activities suggested damage to mitochondria and myofibrils, respectively. Leucine aminopeptidase and glucose-6-phosphate dehydrogenase activities appeared in macrophages.     

In vitro toxicity tests of zinc phosphate cement

BHK-21 (C-13) cells were exposed to freshly mixed zinc phosphate cement for 0.5, 1 and 2 h in a simulated cavity apparatus. The effect on the cells was evaluated by quantitative measurement of acid phosphatase and succinic dehydrogenase reaction product in order to determine organelle membrane permeability. After 0.5 h cell-material contact there was no significant effect. After 1 h cell-material contact, there was slight labilization of the lysosomal membranes, and a significant decrease in succinic dehydrogenase activity. The mean overall stain density in the experimental cells was 109% and 57% of the control values for acid phosphotase and succinic dehydrogenase respectively. After 2 h cell-material contact, there was significant labilization of the lysosomal membranes, and also labilization of the mitochondrial membranes. The mean overall stain densities were 114% and 75% of the control values for acid phosphatase and succinic dehydrogenase respectively. The relevance of these findings to the in vivo situation is discussed.     

Root resorption associated with orthodontic force in inbred mice: genetic contributions

Root resorption (RR) is an unwanted sequela of orthodontic treatment. Despite rigorous investigation, no single factor or group of factors that directly causes RR has been identified. The purpose of this study was to examine the effect of the genotype on susceptibility or resistance to develop RR secondary to orthodontic force. Nine-week-old male mice from eight inbred strains were used and randomly distributed into control (C) or treatment (T) groups as follows: A/J (C = 9,T = 9), C57BL/6J (C = 7,T = 8), C3H/HeJ (C = 8,T = 6), BALB/cJ (C = 8,T = 6), 129P3/J (C = 6,T = 8), DBA/2J (C = 8,T = 9), SJL/J (C = 8,T = 10), and AKR/J (C = 9,T = 8). Each of the treated mice received an orthodontic appliance to tip the maxillary left first molar mesially for 9 days. Histological sections of the tooth were used to determine RR and tartrate resistant acid phosphatase (TRAP) activity. The Wilcoxon ranked-sum non-parametric test was used to evaluate differences between the groups. The results showed that the DBA/2J, BALB/cJ, and 129P3/J inbred mouse strains are highly susceptible to RR, whereas A/J, C57BL/6J and SJL/J mice are much more resistant. The variation in the severity of RR associated with orthodontic force among different inbred strains of mice when age, gender, food, housing, and orthodontic force magnitude/duration are controlled support the hypothesis that susceptibility or resistance to RR associated with orthodontic force is a genetically influenced trait.     

The effects of low doses of fluoride on enzyme activity in rabbit serum.

Rabbits receiving water fluoridated at 10 ppm showed small changes in serum enzyme activity. No significant change in activity occurred with glutamic pyruvic transaminase, acid or alkaline phosphatase, isocitrate dehydrogenase, leucine aminopeptidase or aldolase. The decreases in activity of glutamic oxalacetic transaminase, malate dehydrogenase and lactate dehydrogenase were statistically significant at the p < 0.05 level. Such changes are the reverse of those observed in pathological conditions.     

Appliance‐induced osteopenia of dentoalveolar bone in the rat: effect of reduced bone strains on serum bone markers and the multifunctional hormone leptin

To understand, in greater detail, the molecular mechanisms regulating the complex relationship between mechanical strain and alveolar bone metabolism during orthodontic treatment, passive cross‐arch palatal springs were bonded to the maxillary molars of 6‐wk‐old rats, which were killed after 4 and 8 d. Outcome measures included serum assays for markers of bone formation and resorption and for the multifunctional hormone leptin, and histomorphometry of the inter‐radicular bone. The concentration of the bone‐formation marker alkaline phosphatase (ALP) was significantly reduced at both time points in the appliance group, accompanied by a 50% reduction in inter‐radicular bone volume; however, osteocalcin (bone Gla protein) levels remained unaffected. Bone collagen deoxypyridinoline (DPD) crosslinks increased 2.3‐fold at 4 d only, indicating a transient increase in bone resorption; in contrast, the level of the osteoclast‐specific marker, tartrate‐resistant acid phosphatase 5b (TRACP 5b), was unchanged. Leptin levels closely paralleled ALP reductions at both time points, suggesting an important role in the mechanostat negative‐feedback loop required to normalize bone mass. These data suggest that an orthodontic appliance, in addition to remodeling the periodontal ligament (PDL)–bone interface, may exert unexpected side‐effects on the tooth‐supporting alveolar bone, and highlights the importance of recognizing that bone strains can have negative, as well as positive, effects on bone mass.     

Histochemistry of fibres of masseter and temporalis muscles of edentulous monkeys Macaca mulatta

Muscle fibres of the masseter and temporalis muscles of female rhesus monkeys which had been edentulous for 4.5 yr were analysed histochemically. Fibres were classified on the basis of activities of succinic acid dehydrogenase and myofibrillar adenosine triphosphatase. Capillaries were visualized by phosphatase activity. Succinate oxidase activity was determined by differential respirometry of aliquots of whole muscle homogenates. These data were compared to data obtained from the muscles of control adult female animals. Relative to control values, the muscles of edentulous animals had lower oxidative capacity, more fast fatigable (FF) and fewer fast fatigue resistant (FR) and slow fatigue resistant (S) fibres, smaller S fibres and slightly reduced capillarity. These data indicate relative disuse of elevator muscle following removal of teeth.     

Histochemical studies of enzymatic patterns during experimental carcinogenesis in the mouse parotid gland

Histochemical studies of the distribution patterns of the following enzymes during experimental carcinogenesis induced by 9,10-dimethyl-l,2-benzanthracene in the mouse parotid gland were made; alkaline phosphatase, acid phosphatase, esterase, β-glucuronidase, aminopeptidase, aconitase, aldolase, monoamine oxidase and succinate-, lactate-, malate-, glutamate-, -glycerophosphate-, β-hydroxybutyrate-, isocitrate-, and glucose-6-phosphate dehydrogenases.

The carcinogen produced a relatively constant sequence of histological changes consisting of degenerative, proliferative, squamous metaplastic and malignant neoplastic changes. Most of the neoplasms induced were epidermoid carcinomata and fibrosarcomata, and occasionally cystadenomata and adenocarcinomata. There was one malignant pleomorphic adenoma. The carcinomata were considered from the histological and histochemical appearances to originate from duct cells and not from acinar or myoepithelial cells. Enzymatic behaviour in the epithelial cells during carcinogenesis showed a marked decrease in alkaline phosphatase, esterase and succinate dehydrogenase, but a marked increase in lactate, and glucose-6-phosphate dehydrogenase. The distribution patterns of the enzymes in induced carcinomata were similar to those in naturally-occurring epidermoid carcinomata. Mesodermal tumours showed no clear-cut patterns of enzyme reactivity.

The study suggests that the pentose cycle may play a role in the metabolism of carcinogenesis.     

Odontogenic keratocysts: a clinical and histological study with special reference to enzyme histochemistry

Abstract. Of a total of 1,420 odontogenic cysts, 52 (3.3%) were diagnosed as odontogenic keratocysts. Clinical and histological findings in these 52 cysts are reported. Frozen sections of 26 of the keratocysts were incubated to show the following enzyme activities: NADH₂- and NADPH₂-diaphorase, glucose-6-phosphate dehydrogenase, glutamate dehydrogenase, acid phosphatase, leucine aminopeptidase and ATPase. Furthermore, keratinization was studied with the rhodamine B method and lipids with the oil red O, the OTAN and the acid hematein methods. Sections from epidermis, oral mucosa, radicular cysts, residual cysts and follicular cysts served as reference material. The oxidative enzymes showed strong activity in the keratocyst epithelium which contrasted with weak activity in the reference cysts. Acid phosphatase activity was weak in all epithelia except that in keratocysts, which displayed a marked activity. In the fibrous capsule of the keratocyst a high activity of leucine aminopeptidase was recorded. This high activity contrasted with a weak activity in the reference material. The significance of the histochemical results in relation to the aggressive behavior of the keratocyst is discussed.     

The effect of silicate cement on the mitochondria and lysosomes of cultured cells assessed by quantitative enzyme histochemistry

The cytotoxic effect of the silicate cement "Silicap" was evaluated in an in vitro system which simulates the clinical usage of the material. BHK-21 (C-13) cells on cover slips were exposed to the freshly mixed material for 1h, and stained for the demonstration of succinic dehydrogenase and acid phosphatase. The mean stain density for succinic dehydrogenase and acid phosphatase in the experimental cells was 77% and 153% of the control values respectively. The mechanism of cytotoxicity of "Silicap" is discussed.     

Histochemical studies of three kinds of enzymes in dentinal caries

Aminopeptidase, acid phosphatase and nonspecific esterase in dentinal caries were investigated by histochemical method in this study. It was found that in contrast with Gram's stain, the several hydrolytic enzymes were all located within the region where bacteria invaded the dentine. This suggested that the enzymes were produced by the bacteria invading dentinal tubules. According to previous study of the functions of these enzymes, it was speculated that in the process of dentinal caries, aminopeptidase and acid phosphatase may be involved in the decomposition of structural protein of dentinal organic matrix and non-specific esterase in the decomposition of structural lipids of dentine.     

The effect of acetylsalicylic acid on orthodontic tooth movement in the guinea pig.

This study examined the influence of acetylsalicylic acid an inhibitor of prostaglandin synthesis, on orthodontic tooth movement induced with light spring forces in the guinea pig. The animal model was shown to permit reliable and accurate recording of tooth movement up to 28 days. Tooth movement was found to be highly correlated with spring forces, indicating that the model provided a sensitive test of the effect of aspirin on tooth movement. Aspirin was administered orally at the rate of 65 mg/kg per day in three divided doses and was found to effectively inhibit prostaglandin synthesis at the level of the bronchioles. However, aspirin did not appear to significantly affect tooth movement. Thus prostaglandins may not be the only mediators of the bone resorption associated with tooth movement induced by light orthodontic forces under these experimental conditions.     

An enzyme histochemical study of human salivary duct calculi

An investigation was undertaken to study the distribution of enzymes associated with submandibular gland salivary calculi. Ten calculi were freeze-sectioned and incubated for acid and alkaline phosphatases and for lactate, succinate and maleate dehydrogenases. All calculi were partly covered by a 50–210 μm wide zone of organic material consisting of connective tissue and metaplastic squamous epithelium facing the mineralized calculus, or of a structureless substance attached to the mineralized calculus. The epithelium showed an intense staining reaction for acid phosphatase and lactate dehydrogenase and a moderate reaction for succinate dehydrogenase throughout all levels of the epithelium. The structureless peripheral zone exhibited a moderate activity of acid phosphatase and succinate dehydrogenase located to an area close to the mineralized matrix. Also alkaline phosphatase and lactate dehydrogenase were found in a special pattern in the structureless zone. Sodium fluoride and sodium vanadate added to the incubation medium inhibited acid phosphatase activity whereas cupric chloride only lowered the staining reaction. Enzyme activity was found only within the peripheral zone of organic material with one exception. The results suggest that the calcification process of salivary calculi is not a passive calcification of necrotic material or mucin but rather an active process promoted by enzymes in the surrounding organic substances.