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Disturbances of tooth form and eruption in the microphthalmic (mi) mouse: A light and electron microscopic study
Archs oral Bid. Printed in Great
Vol. 34, Britain.
No. 2, pp. 71-76,
1989
0003-9969/89
$3.00 + 0.00 Press plc
Copyright 0 1989Pergamon
All rights reserved
DISTURBANCES OF TOOTH FORM AND ERUPTION IN THE MICROPHTHALMIC (mi) MOUSE: A LIGHT AND ELECTRON MICROSCOPIC STUDY A. L. SYMONS, R. N. PO~RLL, G. J. SEYMOURand D. J. HARBROW Department of Social & Preventive Dentistry and Department of Oral Biology & Oral Surgery, Dental School, University of Queensland, Turbot Street, Brisbane, Queensland 4000, Australia (Accepied 23 August 1988) Summary--Changes in the surrounding alveolar bone occur during tooth eruption. The microphthalmic (mi/mi) mouse suffers from osteopetrosis and lack of bone resorption; tooth form and eruption were examined in both affected mi/mi mice and unaffected litter-mates to determine the effect of osteopetrosis on tooth development and eruption. Paraffin sections of mandibles from 3, 7, 10, 13, 15 and 20-day-old mice were ex.amined by light microscopy after staining with haematoxylin and eosin and for stable acid phosphatase activity. Mandibles from IS- and 20-day-old mice were examined by scanning electron microscopy. ‘The ultrastructure of odontoblasts was observed in 15day-old mice..Tooth eruption was significantly reduced in the mi/mi mice; the bone of affected mice increased in area with increasing age and marrow spaces narrowed. There was little bony remodeling in the mi/mi mouse, as indicated by layers of reversal lines. This lack of bone resorption affected tooth eruption and root formation. No abnormalities: were detected in odontoblasts, suggesting functional normality, but the wide predentine layer in the mi/mi mouse may indicate an alteration in dentine mineralization.
INTRODUCTION
reduced bone resorption through an osteoclast dysfunction (Marks, 1973); this results in eruptive failure of the incisors and first molars and reduced, delayed eruption of the second and third molars. Unlike the mi/mi mouse, the ia/ia rat appears to undergo spontaneous remission (Marks, 1973) during the fourth week of postnatal life and consequently the second and third molars erupt. Our aim was now to examine tooth development and eruption in the homozygous microphthalmic mouse in comparison with litter-mates free from the bone resorption defect. We sought to establish a baseline for subsequent studies with autologous and allogeneic tooth germ transplants in these animals, and with isolated tooth germs in organ culture.
disease characterized by an accumulation of bone mass which may almost obliterate marrow cavities (Marks, 1982). Microphthalmic (mijmi) mice suffer from osteopetrosis (Loutit and Sansom, 1976) and their bony defects include sclerosis of the metaphyseal zones of long bones, increased skeletal density, marrow-space obliteration, and a decrease in the size of intra-osseous foramina (Packer, 1967; Al-Douri and Johnson, 1983; Marks, 1984). The bony defects are combined with dwarfism, pseudo-albinism and microphthalmia (Loutit and Sansom, 1976). Compared with normal litter-mates, microphthalmic mice are hypocalcaemic, hypophosphataemic, have high levels of citrate in blood and bone, and have elevated levels of alkaline phosphatase (Marks, 1977). Reduced bone resorption is considered to be the main cause of ost.eopetrosis in the mi/mi mouse (Marks, 1977), due to an osteoclast abnormality (Holtrop et al., 1,981; Marks and Walker, 1981. Resolution of the osteopetrotic condition may be achieved by the infusion of appropriate spleen or bone marrow cells into an irradiated host (Walker, 1975). It is believ’ed that the infused cells either provide a local factor or factors which stimulate osteoclast function or contain the mononuclear precursors of functional multinucleate osteoclasts (Marks, 1984; Marks and Walker, 1981). Alterations in the structure of alveolar bone are required to accommodate growth and movement of an erupting tooth. Because it exhibits reduced bone resorption and tooth eruption, the microphthalmic mouse js a potential model for investigating the relationship betwesen bone resorption and tooth eruption. The incisor-absent (iajia) rat suffers from Osteopetrosis
0 8. 34,*--A
is a metabolic
MATERIALS
AND METHODS
Microphthalmic mice were obtained from stock held at the Central Animal Breeding House, University of Queensland. The homozygous (mi/mi) affected offspring were identified by their macroscopic traits of red eye colour and absence of fur pigmentation. Unaffected homozygous (+ /+) and heterozygous (mi/+) litter-mates were used as controls. Light microscopy
Mice from each group were killed at 3, 7, 10, 13, 15 and 20 days after birth. Three animals were examined from each age group for both the affected and unaffected mice. The mandibles were removed and fixed in neutral buffered formalin, demineralized in EDTA, and embedded in paraffin. Sagittal sections, 5 pm thick, were stained with haematoxylin and eosin, and for stable acid phosphatase (SAP) activity by a simultaneous azo-dye 71
A. L.
12
SYMONS et al.
coupling technique with naphthol AS-TR as the substrate and hexazotized pararosaniline as the dye. SAP sections were incubated at pH 5.0 for 60 min at 37°C (Eggert and Germain, 1980). Scanning electron microscopy
Mandibles from 15- and 20-day-old affected and unaffected microphthalmic mice were sectioned sagittally using a diamond blade. Sections were washed thoroughly and dehydrated in 70%, 95% and absolute alcohol. The specimens were mounted, gold-coated in a Dentovac vacuum evaporator, and studied in a Philips 505 scanning electron microscope, operating at 20 kV. Transmission electron microscopy
Disturbances of tooth eruption and dental form are evident by 15 days so ultrastructural investigation of odontoblasts was done at that age. Sixteen 15-day-old mice (8 affected and 8 unaffected) were perfused with 4% glutaraldehyde and 4% paraformaldehyde in 0.067 M cacodylate buffer. After fixation their mandibles were removed and tissues were placed in a buffer solution of 0.1 M sodium cacodylate, pH 7.2, at 4°C. The first molar teeth were dissected from the mandibles, post-fixed in 1% osmium tetroxide for 2 h, washed with deionized water and stained en bloc with 5% uranyl acetate for 45 min. Specimens were washed and dehydrated through a graded series of ethanols (70%, 90%, absolute). The ethanol was cleared with propylene oxide, and the tissue was infiltrated and embedded. Sections, approx. 60-70nm thick, were cut on a LKBIII ultramicrotome and stained with lead citrate. The specimens were orientated to enable sagittal sectioning of the developing first molar and examined in an Hitachi HV 1I-E transmission electron microscope at 75 kV. RESULTS
Eruption and investing bone
Although continuity between reduced enamel epithelium and oral epithelium was observed at 10 days for the incisor and at 13 days for the first and second molars, teeth in the mi/mi mouse had not erupted by 20 days. Vertical buccolingual sections of the first molar at 20 days showed penetration of the lingua1 cusp into the mouth but failure of the entire crown to erupt (Fig. 1). Scanning electron micrographs revealed very little bone occlusal to the first and second molars, but bone was closely associated with the crowns, forming a rim about the level of the occlusal surface (Fig. 2). This bony rim was observed as early as 7 days in the mi/mi mouse. In controls, eruption of the incisor and first molar was evident at 10 days and, by 13 days, the second molar was close to eruption. Both first and second molars and incisors were fully erupted by 20 days, and the enamel-free zone on the molar cusp tips may have undergone additional wear (Fig. 3). Dental tissues Enamel. As the tissues were demineralized it was not possible at the light microscopic level to detect
differences in the enamel formed by each group. Scanning electron micrographs showed no difference in the enamel thickness and crown coverage between the affected and unaffected microphthalmic mouse. In the affected mouse, enamel did not completely cover the cusp tip of the molar teeth (Fig. 4). In the unaffected mouse the cusp tips were worn after functioning in the mouth. Dentine. Dentine abnormalities were detected in the mi/mi animals from about age 7 days and they increased in severity with age. The predentine layer, though variable in thickness, tended to be much wider in the mi/mi mouse than in the control (Figs 5 and 6). The predentineedentine border in the mi/mi mouse was irregularly scalloped and interglobular dentine was observed. Root development
Root formation in the unaffected mouse began just prior to tooth eruption; by day 20, it was well established for the first and second molars (Fig. 3). The clinical root of the incisor extended distally to the level of the third molar. Roots were straight, apical foramina had not yet formed, and normal spacing was present between the developing root and investing bone. As early as 7 days the developing roots of the mi/mi mouse were distorted, and showed extensive undulation indicative of folding. This root distortion increased with age (Fig. 5). Crowns with short, twisted roots appeared to be closely related to the alveolar bone. Ankylosis was first observed at 10 days. In a 13-day second molar, there was close approximation between the developing enamel matrix and the surrounding bone (Fig. 7). Ultrastructure. No ultrastructural difference was observed between odontoblasts from affected and unaffected microphthalmic mouse. They contained a large oval, basally orientated nucleus. There was a concentration of rough endoplasmic reticulum close to the nucleus, connected to a similar, apical concentration by strands of reticulum running parallel and close to the cell membrane. The rough endoplasmic reticulum was highly organized into widely dilated cisternae at the dentine-forming part of the cell. The centrally placed Golgi apparatus was surrounded by endoplasmic reticulum. Mitochondria and free ribosomes are scattered throughout the cytoplasm. Investing bone
At 3 days there were no apparent differences between the alveolar bone of either group. However, at 7 days, there appeared to be an increase in bone trabeculation per unit area and a reduction in the size of the marrow spaces for the mi/mi mouse. This became more pronounced with age (Figs 8 and 9). SAP activity
There was little histological difference in bone between the two groups at 3 days. The bony trabeculae were well spaced around areas of soft tissue; large multinucleated cells, closely associated with bone, were SAP positive. Areas of bone itself were also positive and thus staining may indicate a lamina limitans formed during the reversal phase of bone formation.
Tooth form and eruption in mi/mi mice At 7 days there was a marked difference between the two groups: the bone area had increased in the mi/mi mouse with numerous thin trabeculae and a narrowing of marrow spaces. There was intense SAP staining of the lamina limitans and of multinucleated cells along the bone margins. The diameters of bony trabeculae appeared greater in the unaffected mouse and they were separated by larger marrow spaces. At 20 days, in the mi/mi mouse, marrow spaces had further decreased in size and the bony trabeculae were intensely stained for SAP activity, particularly along the reversal lines. Few large cells stained positively for SAP activity (Fig. 8). The bone of the unaffected mouse appeared better organized and stained less intensely for SAP activity with fewer reversal lines. Some intensely stained multinucleated cells were observed (Fig. 9). DISCUSSION
The failure of tooth eruption in the mi/mi mouse may be related to skeletal abnormalities resulting from a deficiency in osteocfast function and a disturbed bone metabolism (Packer, 1967). We found little bone superfioaf to the unerupted teeth in a number of instances, but this is not surprising as rodent teeth develop within an oval trough formed by the alveolar bone (Park, 1973). Nevertheless, the presence of bone around the developing crown may lock the unerupted t.ooth, preventing it from entering the mouth. This oss,eous rim has also been observed in the incisor-absent (ia) rat and was reported to be an impediment to molar eruption (Marks, 1976). We now report a similar rim in mi/mi mice which, unlike the ia/ia rat, do not undergo’ remission of the osteopetrotic defect. The tooth defects and failure of tooth eruption were consistent in all our affected mice. Ankylosis and poor root formation may also contribute to the failure of tooth eruption in the mi/mi mouse. Marks (1976) claims that ankylosis and poor root development are secondary to bone resorption and lack of eruption because ankyfosis occurs after eruption fails; tooth formation continues unabated in the absence of eruption and eruption occurs without ankyfosis if bone resorption is restored at birth (Marks, 1981). However, we do not know whether poor root development results from a lack of eruption or is an intrinsic defect in the microphthalmic mouse. Further studies in vivo and in vitro are required to determine if poor root development is a result of lack of eruption. During eruption the growing tooth expands but does not move towards the mouth until root formation commences. Bone resorption usually tak.es place occlusal to the crown as it moves occfusally and, in normal circumstances, little bone resorption occurs in the root apical region (Davidovitch, 1979). Indeed, there may be bone deposition apical to the erupting tooth as the rate of eruption can exceed that of root formation (Jacobson, 1983). In the affected homozygous mouse the root portion of teeth prevented from erupting by the occfusal rim of bone grew backwards into the mandibular bone, was short and distorted, and became ankylosed. During the eruptive phase of odontogenesis, developmental changes include the
13
formation of the periodontal ligament, the dentogingivaf junction and the root (Jacobson, 1983). Although Gowgief (1961) has demonstrated the eruption of rootless molars in monkeys, the eruption rate of partially formed permanent teeth is generally retarded, and the relationship between root formation and eruption remains controversial. In the mi/mi mouse, failure of the roots to develop properly may have created an additive effect, diminishing the eruptive forces. The apparent reduction in tooth size and the reduced root development in the mi/mi mouse may be related to the failure of the surrounding bone to accommodate tooth growth and expansion. On the other hand, failure of tooth eruption and abnormality in crown shape and size in the mi/mi mouse may be independent of the iniluznce of the bone and genetically determined. As microphthalmic mice are hypocafcaemic, suffering from a disturbance in calcium and phosphorus homeostasis (Marks and Walker, 1969), their dental defects could be related to a defective calcium metabolism or the inability of osteoclasts to function and release calcium from bone tissue. Abnormalities in the teeth of the mi/mi mouse became greater with age. No differences in the odontobfasts were observed at the ultrastructural level, although disturbance in the mineralization of dentine was suggested by the increased width of the predentine layer and presence of interglobular dentine. The combination of an increase in bone area and hypocafcaemia may have resulted in reduced mineral supply to the odontobfasts. With increasing age, differences in bone structure between the affected and unaffected microphthalmic mouse were more easily detected, and this was related to the absence of bony remodelling in the affected mouse. Such remodelling was evident in the unaffected mouse, in which bone trabeculae were larger and separated by large marrow spaces; there were few reversal lines but generally the bone did not stain intensely for SAP activity. SAP identifies osteoclasts and stains reversal lines intensely (Eggert and Germain, 1980; Baron, Vignery and Horowitz, 1983), giving similar results to those of tartrate-resistant acid phosphatase (Baron et al., 1986) and tartrateresistant acid ATPase (Anderson et al., 1986). We found SAP staining of reversal lines only in mi/mi mice, and it is therefore unlikely to be an artefact. Normally bone resorption is initiated only at resting surfaces, and calcified bone matrix is required for ostecclast formation and the attachment of osteobfast precursors to the bone surface (Baron et al., 1983). The nature of the defect in the microphthafmic mouse is unknown but a number of possibilities exist. There may be an absence of resting surfaces available for resorption due to overactive osteobfasts which could exert an inhibitirig focal effect on bone resorption and a reduction in osteoclast activity. However, injection of bone marrow cells reverses the defect, implying that abnormalities in the ordered sequence of bone remodelling are not a cause of the reduced resorption. As osteocfasts with high nuclear counts are considered to be more efficient than osteoclasts with fewer nuclei, the bone resorption defect may be correlated to the increase in the ratio of uninucleated
A. L. SYMONS et al.
74
to multinucleated
osteoclasts (Thesingh and Scherft, 1985; Green, Marshall and Nisbet, 1986) which may result from a defective fusion mechanism. Alternatively, the increased ratio of uninucleate cells in mi/mi mice may be the result of a shortened life span, reducing the probability of forming osteoclasts with high nuclear counts if fusion is a time-dependent
process (Marshall Ed al., 1987).
REFERENCES
Al-Douri
S. M. J. and Johnson
D. R. (1983) Ultra-
structurally abnormal bone and dentin produced by microphthalmic mice. J. Anat. 136, 715-722. Anderson G. N.. Ek-Rvlander B.. Hammarstrom L. E., Lindskog S. and Tbverud S.’ U. (1986) Immunocytochemical localization of a tartrate-resistant and vandate-sensitive acid nucleotide tri- and diphosphatase. J. Histochem. Cytochem. 34, 293-298. Baron R., Vignery A. and Horowitz M. (1983) Lymphocytes, macrophages and the regulation of bone remodelhng. Bone Min. Res. 2, 175-243. Baron R., Tran Van P., Nefussi J. and Vignery A. (1986) Kinetic and cytochemical identification of osteoclast precursors and their differentiation into multinucleated osteoclasts. Am. J. Path. 122, 363-378. Davidovitch Z. (1979) Bone metabolism associated with tooth eruption and orthodontic tooth movement. J. Periodont. SO, 22-29. Eggert F. M. and Gennain J. P. (1980) Stable acid phosphatase: I. Demonstration and distribution. Histochemistry 66, 307-3 17. Gowgiel J. M. (1961) Eruption of irradiation-produced rootless teeth in monkeys. J. dent. Res. 40, 538-547. Green P. M., Marshall M. J. and Nisbet N. W. (1986) A study of osteoclasts on calvaria of normal and osteopetrotic (mi/mi) mice by vital staining with acridine orange. Br. J. exp. Path. 67, 85-93. Holtrop M. E., Cox K. A., Eilon G., Simmons H. A. and Raisz L. G. (1981) The ultrastructure of osteoclasts in microphthalmic mice. Metab. Bone Dis. Relat. Res. 3, 123-129. Jacobson A. (1983) The physiology of tooth eruption. Birth Defects 19, 67-82. Plate Fig.
Loutit J. F. and Sansom J. M. (1976) Osteopetrosis of microphthalmic mice. A defect of the hematopoietic stem cell? Calc. Tiss. Res. 20, 251-259. Marks S. C. Jr (1973) Pathogenesis of osteopetrosis in the ia rat: reduced bone resorption due to reduced osteoclast function. Am. J. Anat. 138, 165-189. Marks S. C. (1976) Tooth eruption and bone resorption: experimental investigation of the ia (osteopetrotic) rat as a model for studying their relationships. J. oral Path. 5, 1499163. Marks S. C. (1977) Pathogenesis of osteopetrosis in the microphthalmic mouse: reduced bone resorption. Am. J. Anat. 149. 269-276. Marks S. C. (1981) Tooth eruption depends on bone resorption: experimental evidence from osteopetrotic (ia) rats. Metab. Bone Dis, Relat. Res. 3, 107-I 13. Marks S. C. (1982) Morphological evidence of reduced bone resorption in osteopetrotic (op) mice. Am. J. Anat. 163, 157-167. Marks S. C. (1984) Congenital osteopetrotic mutations as probes of the origin, structure and function of osteoclasts. Clin. Orthop. 189, 239-263. Marks S. C. and Walker D. G. (1969) The role of the parafollicular cell of the thyroid gland in the pathogenesis of congenital osteopetrosis in mice. Am. J. Anat. 126, 299-314. Marks S. C. and Walker D. G. (1981) The hematogenous origin of osteoclasts: experimental evidence from osteopetrotic (microphthalmic) mice treated with spleen cells from beige mouse donors. Am. J. Anat. 161, I-IO. Marshall M. J., Rees J. A., Nisbet N. W. and Wiseman J. (1987) Reduced life span of the osteoclast in osteopetrotic (mi and mid’) mice. Bone 2, 115-124. Packer S. 0. (1967) The eye and skeletal effects of two mutant alleles at the microphthalmic locus of Mus musculus. J. exp. 2001. 165, 2146. Park A. W. (1973) The maxillary and mandibular osteodental fissures of the laboratory rat (Rattrcs noruegius) in relation to molar eruption. Anat. Anz. 133, 125-137. Thesingh C. W. and Scherft J. P. (1985) Fusion disability of embryonic osteoclast precursor cells and macrophages in the microphthalmic osteopetrotic mice. Bone 6, 43-52. Walker D. G. (1975) Control of bone resorption by hematopoietic tissue. The induction and reversal of congenital osteopetrosis in mice through use of bone marrow and splenic transplants. J. exp. Med. 142, 651-663. 1
1. Vertical section of the lingual cusp (Ic) of a 20-day-old lingual
cusp into the oral cavity
mi/mi mouse showing (oc). x 160
penetration
of the
Fig. 2. Scanning electron micrograph showing a first molar tooth of a 20-day-old mi/mi mouse. There is little bone (b) occlusal to the developing tooth but a bone rim (br) is present around the crown. x 65 Fig. 3. Scanning
electron
micrograph showing tooth eruption of a 20-day-old (arrowed) was evident on cusp tips. x 30
unaffected
mouse.
Fig. 4. Scanning electron micrograph of a 20-day-old mi/mi mouse showing lack of tooth eruption associated bone rim (br). Cusp tips (arrowed) were not covered with enamel. x 54
Wear and
Fig. 5. Photomicrograph of the first and second molars in a 20-day-old mi/mi mouse. Root curling (rc) is evident and teeth sit on dense bone (db). In dentine (d) there is wide predentine (‘pd) layer showing areas of interglobular dentine (arrowed). x 50 Fig. 6. Photomicrograph of the first molar in a 20-day-old unaffected mouse. The predentine layer is narrow and no interglobular dentine is observed. x 50 Fig. 7. Photomicrograph
Fig. 8. Photomicrograph (arrowed) are intensely
of a 13-day-old mi/mi mouse showing close proximity matrix (em) to bone (b). x 160
(arrowed)
of dentine (d) and enamel
Plate 2 of 20-day-old mi/mi bone stained for SAP activity. The numerous reversal lines stained and the bone comprises numerous thin trabeculae with small marrow spaces (ms). x 140
Fig. 9. Photomicrograph of 20-day-old unaffected bone stained for SAP activity. Bone is better organized with greater areas of bone mass (bm) and large marrow spaces (ms). x 140
Tooth
form and eruption
Plate
1
in mi/mi mice
75
76
Vol. 34, Britain.
No. 2, pp. 71-76,
1989
0003-9969/89
$3.00 + 0.00 Press plc
Copyright 0 1989Pergamon
All rights reserved
DISTURBANCES OF TOOTH FORM AND ERUPTION IN THE MICROPHTHALMIC (mi) MOUSE: A LIGHT AND ELECTRON MICROSCOPIC STUDY A. L. SYMONS, R. N. PO~RLL, G. J. SEYMOURand D. J. HARBROW Department of Social & Preventive Dentistry and Department of Oral Biology & Oral Surgery, Dental School, University of Queensland, Turbot Street, Brisbane, Queensland 4000, Australia (Accepied 23 August 1988) Summary--Changes in the surrounding alveolar bone occur during tooth eruption. The microphthalmic (mi/mi) mouse suffers from osteopetrosis and lack of bone resorption; tooth form and eruption were examined in both affected mi/mi mice and unaffected litter-mates to determine the effect of osteopetrosis on tooth development and eruption. Paraffin sections of mandibles from 3, 7, 10, 13, 15 and 20-day-old mice were ex.amined by light microscopy after staining with haematoxylin and eosin and for stable acid phosphatase activity. Mandibles from IS- and 20-day-old mice were examined by scanning electron microscopy. ‘The ultrastructure of odontoblasts was observed in 15day-old mice..Tooth eruption was significantly reduced in the mi/mi mice; the bone of affected mice increased in area with increasing age and marrow spaces narrowed. There was little bony remodeling in the mi/mi mouse, as indicated by layers of reversal lines. This lack of bone resorption affected tooth eruption and root formation. No abnormalities: were detected in odontoblasts, suggesting functional normality, but the wide predentine layer in the mi/mi mouse may indicate an alteration in dentine mineralization.
INTRODUCTION
reduced bone resorption through an osteoclast dysfunction (Marks, 1973); this results in eruptive failure of the incisors and first molars and reduced, delayed eruption of the second and third molars. Unlike the mi/mi mouse, the ia/ia rat appears to undergo spontaneous remission (Marks, 1973) during the fourth week of postnatal life and consequently the second and third molars erupt. Our aim was now to examine tooth development and eruption in the homozygous microphthalmic mouse in comparison with litter-mates free from the bone resorption defect. We sought to establish a baseline for subsequent studies with autologous and allogeneic tooth germ transplants in these animals, and with isolated tooth germs in organ culture.
disease characterized by an accumulation of bone mass which may almost obliterate marrow cavities (Marks, 1982). Microphthalmic (mijmi) mice suffer from osteopetrosis (Loutit and Sansom, 1976) and their bony defects include sclerosis of the metaphyseal zones of long bones, increased skeletal density, marrow-space obliteration, and a decrease in the size of intra-osseous foramina (Packer, 1967; Al-Douri and Johnson, 1983; Marks, 1984). The bony defects are combined with dwarfism, pseudo-albinism and microphthalmia (Loutit and Sansom, 1976). Compared with normal litter-mates, microphthalmic mice are hypocalcaemic, hypophosphataemic, have high levels of citrate in blood and bone, and have elevated levels of alkaline phosphatase (Marks, 1977). Reduced bone resorption is considered to be the main cause of ost.eopetrosis in the mi/mi mouse (Marks, 1977), due to an osteoclast abnormality (Holtrop et al., 1,981; Marks and Walker, 1981. Resolution of the osteopetrotic condition may be achieved by the infusion of appropriate spleen or bone marrow cells into an irradiated host (Walker, 1975). It is believ’ed that the infused cells either provide a local factor or factors which stimulate osteoclast function or contain the mononuclear precursors of functional multinucleate osteoclasts (Marks, 1984; Marks and Walker, 1981). Alterations in the structure of alveolar bone are required to accommodate growth and movement of an erupting tooth. Because it exhibits reduced bone resorption and tooth eruption, the microphthalmic mouse js a potential model for investigating the relationship betwesen bone resorption and tooth eruption. The incisor-absent (iajia) rat suffers from Osteopetrosis
0 8. 34,*--A
is a metabolic
MATERIALS
AND METHODS
Microphthalmic mice were obtained from stock held at the Central Animal Breeding House, University of Queensland. The homozygous (mi/mi) affected offspring were identified by their macroscopic traits of red eye colour and absence of fur pigmentation. Unaffected homozygous (+ /+) and heterozygous (mi/+) litter-mates were used as controls. Light microscopy
Mice from each group were killed at 3, 7, 10, 13, 15 and 20 days after birth. Three animals were examined from each age group for both the affected and unaffected mice. The mandibles were removed and fixed in neutral buffered formalin, demineralized in EDTA, and embedded in paraffin. Sagittal sections, 5 pm thick, were stained with haematoxylin and eosin, and for stable acid phosphatase (SAP) activity by a simultaneous azo-dye 71
A. L.
12
SYMONS et al.
coupling technique with naphthol AS-TR as the substrate and hexazotized pararosaniline as the dye. SAP sections were incubated at pH 5.0 for 60 min at 37°C (Eggert and Germain, 1980). Scanning electron microscopy
Mandibles from 15- and 20-day-old affected and unaffected microphthalmic mice were sectioned sagittally using a diamond blade. Sections were washed thoroughly and dehydrated in 70%, 95% and absolute alcohol. The specimens were mounted, gold-coated in a Dentovac vacuum evaporator, and studied in a Philips 505 scanning electron microscope, operating at 20 kV. Transmission electron microscopy
Disturbances of tooth eruption and dental form are evident by 15 days so ultrastructural investigation of odontoblasts was done at that age. Sixteen 15-day-old mice (8 affected and 8 unaffected) were perfused with 4% glutaraldehyde and 4% paraformaldehyde in 0.067 M cacodylate buffer. After fixation their mandibles were removed and tissues were placed in a buffer solution of 0.1 M sodium cacodylate, pH 7.2, at 4°C. The first molar teeth were dissected from the mandibles, post-fixed in 1% osmium tetroxide for 2 h, washed with deionized water and stained en bloc with 5% uranyl acetate for 45 min. Specimens were washed and dehydrated through a graded series of ethanols (70%, 90%, absolute). The ethanol was cleared with propylene oxide, and the tissue was infiltrated and embedded. Sections, approx. 60-70nm thick, were cut on a LKBIII ultramicrotome and stained with lead citrate. The specimens were orientated to enable sagittal sectioning of the developing first molar and examined in an Hitachi HV 1I-E transmission electron microscope at 75 kV. RESULTS
Eruption and investing bone
Although continuity between reduced enamel epithelium and oral epithelium was observed at 10 days for the incisor and at 13 days for the first and second molars, teeth in the mi/mi mouse had not erupted by 20 days. Vertical buccolingual sections of the first molar at 20 days showed penetration of the lingua1 cusp into the mouth but failure of the entire crown to erupt (Fig. 1). Scanning electron micrographs revealed very little bone occlusal to the first and second molars, but bone was closely associated with the crowns, forming a rim about the level of the occlusal surface (Fig. 2). This bony rim was observed as early as 7 days in the mi/mi mouse. In controls, eruption of the incisor and first molar was evident at 10 days and, by 13 days, the second molar was close to eruption. Both first and second molars and incisors were fully erupted by 20 days, and the enamel-free zone on the molar cusp tips may have undergone additional wear (Fig. 3). Dental tissues Enamel. As the tissues were demineralized it was not possible at the light microscopic level to detect
differences in the enamel formed by each group. Scanning electron micrographs showed no difference in the enamel thickness and crown coverage between the affected and unaffected microphthalmic mouse. In the affected mouse, enamel did not completely cover the cusp tip of the molar teeth (Fig. 4). In the unaffected mouse the cusp tips were worn after functioning in the mouth. Dentine. Dentine abnormalities were detected in the mi/mi animals from about age 7 days and they increased in severity with age. The predentine layer, though variable in thickness, tended to be much wider in the mi/mi mouse than in the control (Figs 5 and 6). The predentineedentine border in the mi/mi mouse was irregularly scalloped and interglobular dentine was observed. Root development
Root formation in the unaffected mouse began just prior to tooth eruption; by day 20, it was well established for the first and second molars (Fig. 3). The clinical root of the incisor extended distally to the level of the third molar. Roots were straight, apical foramina had not yet formed, and normal spacing was present between the developing root and investing bone. As early as 7 days the developing roots of the mi/mi mouse were distorted, and showed extensive undulation indicative of folding. This root distortion increased with age (Fig. 5). Crowns with short, twisted roots appeared to be closely related to the alveolar bone. Ankylosis was first observed at 10 days. In a 13-day second molar, there was close approximation between the developing enamel matrix and the surrounding bone (Fig. 7). Ultrastructure. No ultrastructural difference was observed between odontoblasts from affected and unaffected microphthalmic mouse. They contained a large oval, basally orientated nucleus. There was a concentration of rough endoplasmic reticulum close to the nucleus, connected to a similar, apical concentration by strands of reticulum running parallel and close to the cell membrane. The rough endoplasmic reticulum was highly organized into widely dilated cisternae at the dentine-forming part of the cell. The centrally placed Golgi apparatus was surrounded by endoplasmic reticulum. Mitochondria and free ribosomes are scattered throughout the cytoplasm. Investing bone
At 3 days there were no apparent differences between the alveolar bone of either group. However, at 7 days, there appeared to be an increase in bone trabeculation per unit area and a reduction in the size of the marrow spaces for the mi/mi mouse. This became more pronounced with age (Figs 8 and 9). SAP activity
There was little histological difference in bone between the two groups at 3 days. The bony trabeculae were well spaced around areas of soft tissue; large multinucleated cells, closely associated with bone, were SAP positive. Areas of bone itself were also positive and thus staining may indicate a lamina limitans formed during the reversal phase of bone formation.
Tooth form and eruption in mi/mi mice At 7 days there was a marked difference between the two groups: the bone area had increased in the mi/mi mouse with numerous thin trabeculae and a narrowing of marrow spaces. There was intense SAP staining of the lamina limitans and of multinucleated cells along the bone margins. The diameters of bony trabeculae appeared greater in the unaffected mouse and they were separated by larger marrow spaces. At 20 days, in the mi/mi mouse, marrow spaces had further decreased in size and the bony trabeculae were intensely stained for SAP activity, particularly along the reversal lines. Few large cells stained positively for SAP activity (Fig. 8). The bone of the unaffected mouse appeared better organized and stained less intensely for SAP activity with fewer reversal lines. Some intensely stained multinucleated cells were observed (Fig. 9). DISCUSSION
The failure of tooth eruption in the mi/mi mouse may be related to skeletal abnormalities resulting from a deficiency in osteocfast function and a disturbed bone metabolism (Packer, 1967). We found little bone superfioaf to the unerupted teeth in a number of instances, but this is not surprising as rodent teeth develop within an oval trough formed by the alveolar bone (Park, 1973). Nevertheless, the presence of bone around the developing crown may lock the unerupted t.ooth, preventing it from entering the mouth. This oss,eous rim has also been observed in the incisor-absent (ia) rat and was reported to be an impediment to molar eruption (Marks, 1976). We now report a similar rim in mi/mi mice which, unlike the ia/ia rat, do not undergo’ remission of the osteopetrotic defect. The tooth defects and failure of tooth eruption were consistent in all our affected mice. Ankylosis and poor root formation may also contribute to the failure of tooth eruption in the mi/mi mouse. Marks (1976) claims that ankylosis and poor root development are secondary to bone resorption and lack of eruption because ankyfosis occurs after eruption fails; tooth formation continues unabated in the absence of eruption and eruption occurs without ankyfosis if bone resorption is restored at birth (Marks, 1981). However, we do not know whether poor root development results from a lack of eruption or is an intrinsic defect in the microphthalmic mouse. Further studies in vivo and in vitro are required to determine if poor root development is a result of lack of eruption. During eruption the growing tooth expands but does not move towards the mouth until root formation commences. Bone resorption usually tak.es place occlusal to the crown as it moves occfusally and, in normal circumstances, little bone resorption occurs in the root apical region (Davidovitch, 1979). Indeed, there may be bone deposition apical to the erupting tooth as the rate of eruption can exceed that of root formation (Jacobson, 1983). In the affected homozygous mouse the root portion of teeth prevented from erupting by the occfusal rim of bone grew backwards into the mandibular bone, was short and distorted, and became ankylosed. During the eruptive phase of odontogenesis, developmental changes include the
13
formation of the periodontal ligament, the dentogingivaf junction and the root (Jacobson, 1983). Although Gowgief (1961) has demonstrated the eruption of rootless molars in monkeys, the eruption rate of partially formed permanent teeth is generally retarded, and the relationship between root formation and eruption remains controversial. In the mi/mi mouse, failure of the roots to develop properly may have created an additive effect, diminishing the eruptive forces. The apparent reduction in tooth size and the reduced root development in the mi/mi mouse may be related to the failure of the surrounding bone to accommodate tooth growth and expansion. On the other hand, failure of tooth eruption and abnormality in crown shape and size in the mi/mi mouse may be independent of the iniluznce of the bone and genetically determined. As microphthalmic mice are hypocafcaemic, suffering from a disturbance in calcium and phosphorus homeostasis (Marks and Walker, 1969), their dental defects could be related to a defective calcium metabolism or the inability of osteoclasts to function and release calcium from bone tissue. Abnormalities in the teeth of the mi/mi mouse became greater with age. No differences in the odontobfasts were observed at the ultrastructural level, although disturbance in the mineralization of dentine was suggested by the increased width of the predentine layer and presence of interglobular dentine. The combination of an increase in bone area and hypocafcaemia may have resulted in reduced mineral supply to the odontobfasts. With increasing age, differences in bone structure between the affected and unaffected microphthalmic mouse were more easily detected, and this was related to the absence of bony remodelling in the affected mouse. Such remodelling was evident in the unaffected mouse, in which bone trabeculae were larger and separated by large marrow spaces; there were few reversal lines but generally the bone did not stain intensely for SAP activity. SAP identifies osteoclasts and stains reversal lines intensely (Eggert and Germain, 1980; Baron, Vignery and Horowitz, 1983), giving similar results to those of tartrate-resistant acid phosphatase (Baron et al., 1986) and tartrateresistant acid ATPase (Anderson et al., 1986). We found SAP staining of reversal lines only in mi/mi mice, and it is therefore unlikely to be an artefact. Normally bone resorption is initiated only at resting surfaces, and calcified bone matrix is required for ostecclast formation and the attachment of osteobfast precursors to the bone surface (Baron et al., 1983). The nature of the defect in the microphthafmic mouse is unknown but a number of possibilities exist. There may be an absence of resting surfaces available for resorption due to overactive osteobfasts which could exert an inhibitirig focal effect on bone resorption and a reduction in osteoclast activity. However, injection of bone marrow cells reverses the defect, implying that abnormalities in the ordered sequence of bone remodelling are not a cause of the reduced resorption. As osteocfasts with high nuclear counts are considered to be more efficient than osteoclasts with fewer nuclei, the bone resorption defect may be correlated to the increase in the ratio of uninucleated
A. L. SYMONS et al.
74
to multinucleated
osteoclasts (Thesingh and Scherft, 1985; Green, Marshall and Nisbet, 1986) which may result from a defective fusion mechanism. Alternatively, the increased ratio of uninucleate cells in mi/mi mice may be the result of a shortened life span, reducing the probability of forming osteoclasts with high nuclear counts if fusion is a time-dependent
process (Marshall Ed al., 1987).
REFERENCES
Al-Douri
S. M. J. and Johnson
D. R. (1983) Ultra-
structurally abnormal bone and dentin produced by microphthalmic mice. J. Anat. 136, 715-722. Anderson G. N.. Ek-Rvlander B.. Hammarstrom L. E., Lindskog S. and Tbverud S.’ U. (1986) Immunocytochemical localization of a tartrate-resistant and vandate-sensitive acid nucleotide tri- and diphosphatase. J. Histochem. Cytochem. 34, 293-298. Baron R., Vignery A. and Horowitz M. (1983) Lymphocytes, macrophages and the regulation of bone remodelhng. Bone Min. Res. 2, 175-243. Baron R., Tran Van P., Nefussi J. and Vignery A. (1986) Kinetic and cytochemical identification of osteoclast precursors and their differentiation into multinucleated osteoclasts. Am. J. Path. 122, 363-378. Davidovitch Z. (1979) Bone metabolism associated with tooth eruption and orthodontic tooth movement. J. Periodont. SO, 22-29. Eggert F. M. and Gennain J. P. (1980) Stable acid phosphatase: I. Demonstration and distribution. Histochemistry 66, 307-3 17. Gowgiel J. M. (1961) Eruption of irradiation-produced rootless teeth in monkeys. J. dent. Res. 40, 538-547. Green P. M., Marshall M. J. and Nisbet N. W. (1986) A study of osteoclasts on calvaria of normal and osteopetrotic (mi/mi) mice by vital staining with acridine orange. Br. J. exp. Path. 67, 85-93. Holtrop M. E., Cox K. A., Eilon G., Simmons H. A. and Raisz L. G. (1981) The ultrastructure of osteoclasts in microphthalmic mice. Metab. Bone Dis. Relat. Res. 3, 123-129. Jacobson A. (1983) The physiology of tooth eruption. Birth Defects 19, 67-82. Plate Fig.
Loutit J. F. and Sansom J. M. (1976) Osteopetrosis of microphthalmic mice. A defect of the hematopoietic stem cell? Calc. Tiss. Res. 20, 251-259. Marks S. C. Jr (1973) Pathogenesis of osteopetrosis in the ia rat: reduced bone resorption due to reduced osteoclast function. Am. J. Anat. 138, 165-189. Marks S. C. (1976) Tooth eruption and bone resorption: experimental investigation of the ia (osteopetrotic) rat as a model for studying their relationships. J. oral Path. 5, 1499163. Marks S. C. (1977) Pathogenesis of osteopetrosis in the microphthalmic mouse: reduced bone resorption. Am. J. Anat. 149. 269-276. Marks S. C. (1981) Tooth eruption depends on bone resorption: experimental evidence from osteopetrotic (ia) rats. Metab. Bone Dis, Relat. Res. 3, 107-I 13. Marks S. C. (1982) Morphological evidence of reduced bone resorption in osteopetrotic (op) mice. Am. J. Anat. 163, 157-167. Marks S. C. (1984) Congenital osteopetrotic mutations as probes of the origin, structure and function of osteoclasts. Clin. Orthop. 189, 239-263. Marks S. C. and Walker D. G. (1969) The role of the parafollicular cell of the thyroid gland in the pathogenesis of congenital osteopetrosis in mice. Am. J. Anat. 126, 299-314. Marks S. C. and Walker D. G. (1981) The hematogenous origin of osteoclasts: experimental evidence from osteopetrotic (microphthalmic) mice treated with spleen cells from beige mouse donors. Am. J. Anat. 161, I-IO. Marshall M. J., Rees J. A., Nisbet N. W. and Wiseman J. (1987) Reduced life span of the osteoclast in osteopetrotic (mi and mid’) mice. Bone 2, 115-124. Packer S. 0. (1967) The eye and skeletal effects of two mutant alleles at the microphthalmic locus of Mus musculus. J. exp. 2001. 165, 2146. Park A. W. (1973) The maxillary and mandibular osteodental fissures of the laboratory rat (Rattrcs noruegius) in relation to molar eruption. Anat. Anz. 133, 125-137. Thesingh C. W. and Scherft J. P. (1985) Fusion disability of embryonic osteoclast precursor cells and macrophages in the microphthalmic osteopetrotic mice. Bone 6, 43-52. Walker D. G. (1975) Control of bone resorption by hematopoietic tissue. The induction and reversal of congenital osteopetrosis in mice through use of bone marrow and splenic transplants. J. exp. Med. 142, 651-663. 1
1. Vertical section of the lingual cusp (Ic) of a 20-day-old lingual
cusp into the oral cavity
mi/mi mouse showing (oc). x 160
penetration
of the
Fig. 2. Scanning electron micrograph showing a first molar tooth of a 20-day-old mi/mi mouse. There is little bone (b) occlusal to the developing tooth but a bone rim (br) is present around the crown. x 65 Fig. 3. Scanning
electron
micrograph showing tooth eruption of a 20-day-old (arrowed) was evident on cusp tips. x 30
unaffected
mouse.
Fig. 4. Scanning electron micrograph of a 20-day-old mi/mi mouse showing lack of tooth eruption associated bone rim (br). Cusp tips (arrowed) were not covered with enamel. x 54
Wear and
Fig. 5. Photomicrograph of the first and second molars in a 20-day-old mi/mi mouse. Root curling (rc) is evident and teeth sit on dense bone (db). In dentine (d) there is wide predentine (‘pd) layer showing areas of interglobular dentine (arrowed). x 50 Fig. 6. Photomicrograph of the first molar in a 20-day-old unaffected mouse. The predentine layer is narrow and no interglobular dentine is observed. x 50 Fig. 7. Photomicrograph
Fig. 8. Photomicrograph (arrowed) are intensely
of a 13-day-old mi/mi mouse showing close proximity matrix (em) to bone (b). x 160
(arrowed)
of dentine (d) and enamel
Plate 2 of 20-day-old mi/mi bone stained for SAP activity. The numerous reversal lines stained and the bone comprises numerous thin trabeculae with small marrow spaces (ms). x 140
Fig. 9. Photomicrograph of 20-day-old unaffected bone stained for SAP activity. Bone is better organized with greater areas of bone mass (bm) and large marrow spaces (ms). x 140
Tooth
form and eruption
Plate
1
in mi/mi mice
75
76