Pathophysiological and therapeutic aspects of dentoalveolar resorption

Pathophysiological and therapeutic aspects of dentoalveolar resorption Angela Pierce, MDS, PhD, FRACDS*

Key words: Bone, dentine, odontoclast, osteoclast, resorption.

Abstract Both physiological and pathological forms of bone and tooth resorption are seen in the dentoalveolar complex, and much of the non-restorative component of a clinician’s work is occupied by problems related to loss of bone or dentine. Bone resorption is also a feature of many systemic diseases, some of which can affect the alveolar bone. The aim of this report is to summarize some recent work in which the dentoalveolar complex has been used as a model system to study physiological, pathological and therapeutic aspects of resorption of bone and dentine.

(Received for publication December 1987. Accepted October 1988.)

Introduction Resorption of the dentoalveolar complex is seen

in a variety of pathological situations confronting the dental surgeon. Bone resorption is found in periodontal disease, and can create prosthetic problems after tooth loss. It can also occur in associ- ation with local pathology, such as tumours, inflammatory lesions and cysts. Pathological resorption of dentine commonly follows a traumatic incident, but may also be associated with orthodontic therapy, local pathology and impacted teeth.

The cells responsible for resorption of bone and dentine, osteoclasts and dentinoclasts (collectively referred to as ‘clastic’ cells), are usually large and multinucleated, but it is not uncommon to see smaller mononuclear cells. These cells are highly

*Departments of Pathology and Dentistry, The University of Adelaide.

vacuolated and have many mitochondria, indicative of their high metabolic rate. Unique to resorbing cells are two membrane specializations: the ruffled border, a network of membranous folds which inter- digitate with the hard tissue and are thought to engineer the resorption process; and the clear zone, an area of organelle-free cytoplasm which encircles the rumed border and anchors the cell to the bone surface. The mononuclear precursor cells of osteoclasts are thought to be derived from the spleen andor bone marrow and pass through the blood to the resorption site where they fuse to form multinucleated osteoclasts.’ There is some debate as to whether this stem cell is part of the mononuclear phagocyte system2 but, irrespective of origin, osteoclasts are phagocytic cells which share many functional similarities with the mono- cytelmacrophage.

Resorption of bone is influenced by a number of humoral factors, such as parathyroid hormone (PTH), vitamin D and calcitonin. These substances appear to regulate the degree of activity of the total pool of resorbing cells. There are also local regulatory factors, such as prostaglandins, cytokines and growth factors, which have been found to have powerful effects on resorbing cell^.^-^

The dentoalveolar complex is a convenient structure for the examination of hard tissue resorption processes. Not only is it possible to examine active physiological bone resorption during eruption in aivo, but root resorption can also be induced and dentinoclasts examined either in siru or in culture. The aims of the present series of investigations were to further characterize the relationship between clastic cells and their substrata in both a physiological state and following corticosteroid and calcitonin administration and, based on these findings, to examine the therapeutic effects of such agents in experimentally-induced inflammatory root resorption.

Australian Dental Journal 1989;34(5):4374. 437

Fig. 1.-Transmission electron micrograph of part of an osteoclast resorbing alveolar bone (B) in a dernineral- ized specimen. Resorbing structure areas are outlined and were quantitated using image analysis. 1 and 2 represent

clear zone areas, and 3 the rumed border. (Bar = 5 pm.)

Materials and methods 1. Transmission electron microscopy: Morphological, immunochemical and stereological analysis of osteoclast function

In order to study the ultrastructural appearance of osteoclasts in vivo, the resorbing bone around erupting molar teeth in young rats was examined using transmission electron microscopy (TEM).6-8 The alveoli were processed according to routine techniques, and the relationship between the organic and inorganic phases of bone in osteoclast attachment and endocytosis was studied. Special attention was paid to mineral preservation in specimens used for examination of inorganic material.6 Sections of undemineralized specimens were either collected quickly from the water bath, or collected after longer intervals from a bath with alkaline adjustment; KOH was used to increase the pH of the water bath in these cases.

Some specimens of resorbing alveoli were also embedded in a low-temperature embedding resin and sections of osteoclasts were incubated using immunocytogold techniques in order to examine

receptor-mediated endocytosis more specifically.’ Sections were incubated with mouse IgM anti- clathrin at a dilution of 1 pglmL, and goat anti- mouse IgM linked to 10 nm gold particles was used to detect the reaction.

Other animals were injected with various corticosteroids (hydrocortisone 4 mg/100 g body mass and prednisolone 1 mgllOO g body mass); or 50 MRC Units/100 g body mass calcitonin (a known inhibitor of osteoclastic function) or 0.2 mL NaCl (controls), prior to sacrifice, and the morphological response of osteoclasts to these substances was quantitated using image analysis and established stereological techniques.8 Total cytoplasmic area, area of rumed border, and areas of clear zones were traced and calculated using an IBAS I image analysis unit.? Relative areas of clear zone and ruffled borders as a percentage of total cytoplasmic volume were also calculated. Results between experimental groups were compared for statistically significant differences at P*O.OS using the two- sample Student’s t-test. Figure 1 illustrates the ruffled border and clear zone areas which were measured in each osteoclast.

438 Australian Dental Journal 1989;34:5.

Fig. 2. -Electron micrograph of undemineralized section illustrating bone mineral crystals within the channels of the ruflled border and associated vacuoles. B =bone, C = clear zone. (Bar = 0.5 pm.)

Fig. 3.-High magnification view of ‘close contact’ types of adhesion between cell and mineral in the clear zone (arrowheads). (Bar = 20 nm.)

Australian Dental Journal 1989;34:5. 439

Fig. 4. -Electron micrograph illustrating the relationship between the collagenous organic bone matrix (0) and the ruffled border in demineralized specimens. No collagen is evident within the channels of the ruffled border

or its associated vacuoles. (Bar = 0.5 m.)

2. Scanning electron microscopy: Analysis of isolated dentinoclasts

Osteoclasts are difficult to examine in vitro due to technical problems in removing sufficient numbers from resorbing bone surfaces. The dentinoclast was chosen as an alternative clastic cell due to its similarity to the osteoclast (it may be iden- tical), and the ease with which these cells can be removed from resorbing root ~urfaces .~ External root resorption was established in rat molar teeth, and dentinoclasts were removed and cultured on coverslips in media to which various experimental substances had been added, prior to fixation and examination for morphological responses using scanning electron microscopy (SEM).8.t0 Using this technique, the spreading behaviour of these cells in vitro in response to various corticosteroids

mol/L hydrocortisone and mol/L prednisolone), and calcitonin (5 MRC Units), could be examined and related to the in vivo T E M results.

tZeiss, West Germany.

SLcdcrmix Paste. Lederle Pharmaceuticals, Wolfratshausen, West Germany.

Furthermore, the specific response of dentinoclasts to an antibiotickorticosteroid pastet used in the treatment of root resorption, was studied. This medicament was applied at a final concentration of

mol/L triamcinolone.

3. Histomorphometric analysis of experimentally- induced inflammatory root resorption

Studies were also undertaken to assess the effect of various therapeutic substances on experimentally- induced inflammatory root resorption in monkey teeth. I1.'' A histomorphometric technique based on that of A n d r e a ~ e n ~ ~ . ' ~ was used, whereby the responses in the periodontal membrane (PDM) at various cervico-apical levels were classified as either normal PDM, inflamed PDM without root resorption, surface resorption, inflammatory resorption, or ankylosis.

The periodontal reaction pattern was quantitated by dividing the number of observations showing one of the reactions listed above by the total number of observation points, and multiplying the result by 100. Thus, for each tooth, the 5 periodontal reactions were expressed as percentages of the total root


Fig. 5.-Lateral surface of an osteoclast facing and in intimate contact with an osteoblast (0). Coated pits and vesicles are indicated with arrowheads. M =mineral crystals within an intracellular vacuole. The mmed border

is not included within this section. (Bar = 1 pm.)

surface. The means of the periodontal reactions were calculated and the Mann-Whitney U test was used to test the level of significance of the differences between the experimental groups (P4 0.05).15

Results Many osteoclasts were evident along the surface

of the resorbing bone adjacent to the tooth follicle. The ultrastructural appearance of osteoclasts in undemineralized sections is illustrated in Figure 2. Bone mineral was apparent within the channels of the rumed border, and also within cytoplasmic vacuoles.6 The attachment area of the clear zone to mineral was characterized by areas of adhesion at regular intervals along the bone surface (Fig. 3). Two types of attachment were noted, ‘close contacts’ and ‘focal contacts’, and were found in different parts of the clear zones.6 Examination of demineralized sections revealed that collagen was only detected within the Howship’s lacuna area, and no collagen fragments were evident within the channels of the rumed border or intracellularly (Fig.

Coated pits and vesicles were not evident within 4).

the ruflled border or clear zone, but were numerous along the dorsal surface of the cells, particularly lateral to the clear zone areas (Fig. 5). Immunogold incubations revealed that these pits and vesicles were associated with the typical coating protein, clathrin.’ No clathrin was found within the ruflled border or clear zone.

Transmission electron microscopy stereological analysis of osteoclastic responses to calcitonin and corticosteroids in oivo revealed that both calcitonin at one and four hours (Fig. 6) and prednisolone at four hours resulted in a significant reduction in areas of clear zones and ruflled borders (‘resorbing structures’) compared with control specimens.8 Relative areas of resorbing structures were increased in specimens treated with hydrocortisone at one and four hours.

The numbers of dentinoclasts cultured with calcitonin, prednisolone and hydrocortisone on coverslips were all reduced compared with the control cultures.8 This was accompanied by plasma membrane changes resulting in the gradual detachment of all cells from the coverslips (Fig. 7 and 8). The hydrocortisone-treated cells appeared to swell


Fig. 6.-Osteoclast in wiw four hours after injection with calcitonin. Note lack of rumed border and clear zones which have been replaced by an 'intermediate zone' prior to detachment (arrowhead). B =bone. (Demineralized.

Bar = 5 pm.)

before detachment, whereas the others showed shrinkage. Culturing of dentinoclasts with Ledermix paste produced a similar response whereby cells assumed a spherical appearance and eventually became detached from the coverslip.

Histomorphometric analyses of inflammatory resorption induced in monkey teeth revealed that after eight weeks, the majority of the root surfaces of control teeth were covered with inflammatory root resorption (Fig. 9). Treatment with either Ledermix or calcitonin eliminated inflammatory root and the predominant response in the periodontal membrane was one of ankylosis (Fig. 10) and surface resorption (Fig. 1 la, b). The reactions in the PDM are illustrated graphically in Figure 12.

Discussion As the results of the above studies indicate, ther-

apeutic measures aimed at the treatment of pathological hard tissue resorption should be aimed at specific inhibition of the expression of resorbing structures (rumed borders and clear zones) by clastic cells. These structures develop normally as a result

of attachment of the cell to the mineral component of the s u b ~ t r a t u m ~ . ~ . ' ~ (Fig. 3), followed by a motile phase in which the cell moves to and spreads at the site of resorption." It is only during the spreading stage that the cell develops a rumed border9 which is thought to be the site of active endocytosis of hard tissue components, and exocytosis of solubilizing agents such as acids and enzymes. The development of the rumed border is intimately associated with clear zone formation. The following discussion will focus on the details of this process and its implications in the selection and development of therapeutic measures.

In the non-resorbing state, bone is covered by osteoblasts and bone-lining cells which are thought to prevent osteoclasts from reaching the bone surface by forming a cellular mechanical barrier which is only penetrated after exposure of osteoblasts to resorption stimuli such as parathyroid hormone.18 Recent evidence suggests that osteoblasts may play a strong regulatory role in res~rpt ion '~ and it has been established that several humoral and local factors exert their stimulatory effects on osteoclastic resorption via the osteo-


Fig. 7.-Scanning electron micrograph ofcontrol dentinoclast 30 minutes after in wirro culture with NaCl. Note the peripheral skirt of smooth cytoplasm. The cell is well-spread along the coverslip surface. (Bar = 5 pm.)

Fig. 8.-Dentinoclast 30 minutes after culture with prednisolone. The cell has assumed a spherical shape prior to detachment from the coverslip surface. (Bar = 5 pm.)

Australian Dental Journal 1989345 443

Fig. 9.-Light micrograph of monkey incisor infected and replanted after one hour of bench-drying showing inflammation within the PDM and extensive destruction of dentine (D) after eight weeks. P =pulp. (H&E.

Bar = 0.5 mm.)

blast.20-22 These effects include release of enzymes involved in the removal of the non-mineralized osteoid cover of bone19.23.24 which has previously been shown to resist attachment by resorbing cells.24 As indicated in the results of the present studiesY6 attachment by osteoclasts occurs directly to the mineral component of bone (Fig. 3). Furthermore, the ensuing endocytic processes principally involve dislodgement of mineral (Fig. 2). Recently, it has been proposed that receptors for the Fc part of the IgG complex on the osteoclast surface participate in both of these p r o c e s s e ~ . ~ ~

It has been suggested that the attachment between mineral and cell in the area of the clear zone forms a tight seal, but the relationship between the mineral and the clear zone depicted in Figure 3 indi- cates that adhesion is mediated by intermittent adhesion structures which project from the plasma membrane of the clear zone to the mineral phase of the bone.6,26 Furthermore, a tracer protein, horse- radish peroxidase, has been demonstrated within the ruffled border soon after injection, suggesting that the clear zone does not form a ~ e a l . * ~ . ~ ' Hence the prime hnction of the clear zone adhesion structures

may be to act as anchorage sites during cell move- ment, as the results of the present studies indicate.6 These sites have been characterized in witro on other motile cellsY2' and support microcinematographic studies of osteoclasts moving along a bone surface as they resorb it.29

Endocytosis of dislodged mineral is not the only mechanism by which clastic cells remove mineralized tissue components. It has been suggested that acid hydrolases are released into the Howship's lacunae which is acidified by a membrane-bound proton pump transporting H' ions into the resorption area.3o This low pH not only provides an optimal environment for the secreted enzymes, but it may also be responsible for early mineral dissolution. Some studies have suggested that the organic matrix is degraded by enzymes after mineral dissolution occurs e ~ t r a c e l l u l a r l y . ~ ~ However, ultrastructural studies, including those described above, do not support this sequence of events as mineral crystals and conglomerates have been observed within cytoplasmic vacuoles in undemineralized sections (Fig. 2), indicating that at least some of the mineral dissolution occurs


Fig. 10.-Monkey incisor eight weeks after replantation and five weeks treatment with Lcdcrmix. Note the attachment of bone (B) to dentine (D) in an area of ankylosis. (H&E. Bar = 50 pm.)

i n t r a ~ e l l u l a r l y . ~ ~ ~ ~ Furthermore, it would appear that mineral is phagocytosed after the initial break- down of supporting organic matrix, as collagen fibres are never observed free of mineral in undemineralized sections, and are not seen intracellularly or within the channels of the ruffled border (Fig. 4).

The ruffled border membrane has a special coating which remains to be identified but which may be associated with its endocytotic ability. Results of the present studies7 indicate that the ruffled border is not associated with the unique receptor-mediated endocytosis protein, clathrin, suggesting that endocytosis at the ruffled border may be mediated by a different and special coated membrane structure. Clathrin-associated coated pits and vesicles were evident outside the clear zone- ruffled border area on osteoclasts (Fig. 9, but the role of these endocytotic vesicles which are common to all mammalian cells is probably one of nutrient ingestion or hormonal and local factor re~ognition.~

The studies discussed above outline a functional sequence of events and emphasize the importance of adhesion structures for the proper resorbing hnction of a clastic cell. As indicated in the results of

the therapeutic studies, the primary effect of these treatments is to interfere with the ability of the cell to anchor itself to the mineralized surface. Calcitonin is a potent osteoclast inhibitor and exerts its effects through a receptor on the osteoclast

It decreases osteoclastic motility and causes loss of resorbing It has also been reported to have an effect on osteoclastic enzyme levels .35

Loss of resorbing structures after exposure to calcitonin, observed in both TEM (Fig. 6) and SEM studies, was also seen following administration of some corticosteroids in vivo and in vitro (Fig. 8). These inhibitory effects comprised a decrease in ruffled border and clear zone areas relative to total cytoplasmic volume (determined using TEM stereological techniques), accompanied by a gradual detachment of the cells in vitro observed using SEM.8 The lack of inhibition shown by hydrocortisone in vivo may be explained by the complexity of systemic effects which corticosteroids are capable of exerting. For example, corticosteroid administration also causes a compensatory increase in PTH production which stimulates osteoclastic bone res~rption.~" However, it appears as if the direct

Australian Dental Journal 1989345 445

Fig. 1 la,b. -Area of surface resorption in calcitonin-treated monkey incisor showing repair of exposed dentine by new cementum deposition (arrowhead). Polarization shows new PDM fibres inserting into the reparative

cementum (1 1 b). (H&E. Bar = 30 pm.)

100

90

80

Y 70

2 u a 60

& 50

LL 40

W? 30

20

10

0

3 ln

z 0

PER 10 DO NTAL R EACTlO N S

CONT L LED CONT C CALC EXPERIMENTAL GROUP

LEGEND

ANKYLOSIS

INFLAM R E S

SURF RES

PDM INFLAM

NORMAL PDM

Fig. 12.-Graph showing the periodontal reaction patterns for Ledermix control (Cont L), Ledermix (Led), calcitonin control (Cont C) and calcitonin (Calc) eight weeks after inflammatory resorption was induced in monkey teeth. Therapeutic substances were applied three weeks after replantation and both

control groups were left untreated. (See text for explanation of PDM reactions.)


effect of corticosteroids on osteoclasts and dentinoclasts is primarily one of inhibition. This was also seen when dentinoclasts were cultured with Ledermix, where it was demonstrated that the corticosteroid component, triamcinolone, directly inhibited these cells in vim."

In the clinical situation, the primary inhibitory effect of the corticosteroid would be more likely to occur independently of secondary compensatory mechanisms because the medicaments are placed into the confined space of the root canal with direct access via dentinal tubules to the resorbing cells on the root surface. The inhibition of inflammatory root resorption by Ledermix and calcitonin seen in the histomorphometric studies may be easily explained when viewed in the perspective of the preceding discussion of clastic cell behaviour. The corticosteroid component of Ledermix acts by inhibiting spreading and attachment of dentinoclasts along the root surface, as well as by reducing inflammation within the periodontal membrane." At the same time, the antibiotic component exerts its effects on the bacteria in the dentinal tubules which maintain the resorption via the induced inflammation in the PDM. It is likely that calcitonin directly inhibits attachment of the dentinoclast, thereby permitting reparative cementum to form at the sites of resorption (Fig. 1 la, b) and confining the bacteria and their products to the closed dentinal tubules.I2 This hormone may be a useful adjunct in the treatment of difficult cases of external root resorption.

In conclusion, these studies have shown that detailed knowledge of resorbing mechanisms is essential for the precise targeting of therapeutic measures in the treatment of localized destructive processes in the dentoalveolar complex. Future studies aimed at more detailed biochemical charac- terization of the mechanisms of resorption will provide a basis for the development of alternative and superior therapies.

Acknowledgements The Australian Dental Research Fund Inc., and

the School of Dentistry, Karolinska Institutet, Stockholm are acknowledged for their support of this work.

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Address for correspondenceheprints: Department of Dentistry,

The University of Adelaide, GPO Box 498,

Adelaide, South Australia, 5001.


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Pathophysiological and therapeutic aspects of dentoalveolar resorption