ArchsoralBiol.Vol 22. pp.383 LO391 Pergamon Press 1977. Printed in Great Bntam.
CORTICAL STRUCTURE OF THE PIG MANDIBLE AFTER THE INSERTION OF METALLIC IMPLANTS INTO ALVEOLAR BONE P. J. Departments
ATKINSON,
KATHLEEN POWELL
and C.
W~~DHEZAD
of Oral Biology and Restorative Dentistry, University of Leeds, Leeds, Yorkshire, England
Summary-The effect of tooth extraction and endosseous metallic implants on the bone structure of adult pigs was studied using both densitometric and microradiographic techniques. Similar methods were used to study the bone structure of both growing and adult pig mandibles. The use of tetracycline and 32P-labelled phosphate in uivo indicated that the porous bone deposited periosteally is the most metabolically active bone during growth. This formation occurs over almost the entire surface of the body of the mandible throughout life but, despite an overall density increase with age, the internal structural pattern established during development, is maintained. Both tooth extraction and the introduction of metallic endosseous implants :stimulated further remodelling. Despite the different responses to tooth extraction and implantation, the internal structural pattern remained constant.
INTRODUCTION
The influence of mechanical stress on cortical bone structure is well known. However, the precise mechanisms which maintain shape and internal structure, despite mechanical and metabolic demands, are not clearly unden,tood. Mandibular growth is closely coordinated with craniofacial growth (Enlow, 1966; Moss and Salentijn, 1970). Shape and internal structure are established at an early stage of development and are maintained throughout life by surface remodelling (Atkinson, 1976). To gain more information about growth and the effects of stress, we studied the effect on bone structure of endosseous implants. MATERIAL
AND
METHODS
The animals used were large White pigs of the same herd. (1) Measurement
x Landrace
of growth
Mandibular growth was recorded in tiiuo in two litters of piglets, cone consisting of eleven l-day-old piglets and another of twelve 16-day-old piglets. Using calipers, mandibular length was measured from the most prominent part of the posterior border of the ramus to the most anterior projection of the body of the mandible. Measurements were taken at 7-day intervals three times. Similar measurements were made post-mortem on IO mandibles from animals aged from 2 days to 2.5 yr. (2) Metabolic mandible
activity
and structure
the soft tissues removed by dissection and the mandible divided at the symphysis. The entire tooth-bearing area of each half-mandible was then cut into segments. Left and right sides were considered to be equivalent. Segments from the left half were cleaned of blood and medullary soft tissue, dehydrated in alcohol and defatted using ether. As the intention was to study mandibular cortex only, teeth and cancellous bone were removed using a dental bur. The segments were then divided into approximately equal pieces on both buccal and lingual sides. Using the method described by Powell et al. (1973), the density of each sample was calculated by dividing its dry weight by its external volume. This gave an indication of the compactness of the cortical bone. The density values were colour-coded and transferred to plastic replicas of each bone so that the density distribution of the cortex of the tooth-bearing mandible could be visualised and compared with the other data. Following the mass density estimation, the samples were ashed in a muffle furnace at 580°C and the value of ash weight per unit volume obtained. This provided the information about the mineral density of each bone sample. When this was colour-coded and displayed on plastic replicas, the patterns of mineral density could be compared with the patterns of mass density or compactness. Using the left halves of 10 mandibles obtained postmortem from pigs aged 2 days to 2.5 yr, additional information was obtained of the mass density distribution in the cortex of growing and adult mandibles. (3) Histological
in the growing
examination
The right halves of each mandible were used for microradiography and histology. A thin section was taken from each segment for subsequent microradiography and the remainder used for histological study. The undemineralised sections were embedded in plastic (Crystic resin, Trylon Ltd.) and ground to a thickness of 100pm. Contact microradiographs of these sections were made using High Resolution Plate
Using a 15-day..old piglet from one of the litters, tetracycline and 32P labelled phosphate, metabolic activity was studied IVIuivo. An intra-peritoneal injection of tetracycline (15 mgfig) followed 48 hr later and by the same route by an injection of 32P-labelled phosphate (200 pCi/kg). After 4 hr, the animal was killed, 383
P. J. Atkinson, Kathleen Powell and C. Woodhead
3x4
(Kodak) and a Raymax-AEI Radiographic Unit at 15 cm film distance (20 kV 4 mA). The remaining segments from the right half-jaw were demineralised in 1.5 per cent nitric acid, embedded in wax and sectioned. The structural organisation of the section observed after staining with haematoxylin and eosin was compared with that seen in microradiographs and correlated with information obtained from the density patterns. (4) The effect of' tooth extraction plantation
and endosseous im-
Nine adult breeding sows aged 18 months to 2 yr were premeditated with phencyclidine HCl (Semylan; Parke, Davis & Co., London) 1 mg/kg body weight by intramuscular injection and anaesthetised using phenobarbitone Na (Nembutal) 12-15 mg/kg body weight by intravenous injection. The first and second premolar teeth were extracted on the left side. This ensured that the diastema and premolar regions were completely free of teeth and roots. In two animals, tooth extraction alone was carried out. In the other seven, cobalt-chromium blade implants were inserted partly into a slot of appropriate size previously prepared in the diastema using a dental bur and partly into the socket of the second premolar. The blade implants were specially cast in the shape of a trident consisting of a single post and three tapered legs. The overall length of each implant was 25mm, their broadest dimension 25 mm, and their width 2mm. Each implant was placed so that the legs were below the level of the alveolar crest, leaving the single post projecting 1Omm into the oral cavity. Implants remained in situ for various periods, re-implantation being carried out whenever an implant was rejected. Thus, two mandibles were available for examination after tooth extraction, two after one implantation, two after one re-implantation and three after a second reimplantation. The animals were subsequently killed and the mandibles removed, divided at the symphysis and segments cut as before. In this case, however, both left and right halves of each mandible were treated in the same way to permit a comparison of the treated and control side. A thin section was taken from each segment for microradiography and the remaining segments used for density estimation as before. This enabled microradiographs to be compared with density distribution at similar sites on left and right sides. By taking very small samples from selected areas for cytological study, disruption of the density pattern was avoided.
Although mandibular growth occurs predominantly at the condylar cartilage and in the ramus, remodelling on the surface of the body produces increases in width, length and depth. Remodelling during growth resulted in deposition of bone over almost the entire surface of the body of the mandible. This affected bone density and the differences in compactness of structure could be recognised on microradiographs, because the newly deposited bone consisted of porous plexiform lamellae. Nearer to the growing surface, these lamellae were loosely arranged while below this porous layer they were more compact (Plate Fig. 2a and Text Fig. 4). In older jaws, the rate of growth was less and the lamellae of the porous layer became thicker and the structure was generally more compact. However, even in the more mature animals, there was always relatively more plexiform bone in the anterior region of the growing mandible than elsewhere. This bone deposited on both buccal and lingual sides was previously observed in adult animals (Powell et al., 1973). The distribution of Howship’s lacunae suggested that some surfaces, especially those in the more compact lamellar regions, were being resorbed. The presence of these isolated areas of resorption probably accounted for the remodelling necessary to preserve minor surface depressions during growth. Mandibular bone density was less in young growing pigs than in the older animals, ranging from 0.6 to 1.3 g/ml in the 2-day-old; 0.7 to 1.7 g/ml at 42 days and 1.1 to 1.8 g/ml at 84 days. The lower density was due to the much greater porosity of the newly-formed bone and was therefore greatest in regions of most rapid growth. Overall, bone density increased with age; although density values covered a wide range in each jaw, the number of samples with a density of less than l.Og/ml diminished with age. For instance, although almost the entire jaw of the 2-dayold piglet consisted of low density bone (< 1.0 g/ml) this low density was not present in the jaw of the 84-day-old pig. Despite this general increase of density with age, more rapidly growing areas, for instance the buccal bone anteriorly and to a lesser extent the lingual bone of the same region, were always of relatively lower density. A general increase in density occurred towards the ramus and in the basal part of the man-
RESULTS
The growing mandible In viuo measurements of mandibular growth in the two groups of piglets showed that growth was extremely rapid from birth to 30 days. The length of the mandible, determined post-mortem in 10 animals of different ages, showed that growth rate was greatest during the first 80 days, after which it slowed down (Fig. 1). Both in vioo and post-mortem studies indicated that the length of the jaw increased by approximately 1.4mm/day during this period of rapid growth.
I
/
/
,
/
i
260
400
600
800
1000
Age
*
days
Fig. 1. Graph showing the growth in length of the pig jaw. The diagram shows the points from which measurements were taken.
Cortical structure of the pig mandible Ash
Density
16 doy
& Density [
g/ml
I.1 and less
Fig. 3. Diagrams showing distribution of density (dry weight per volume) and ash density (ash weight per volume) in the cortical bone of 4 pig jaws aged 16. 28, 42 and 49 days respectively.
Fig. 4. A 3-dimensional representation
of the porous bone shown in Fig. 2(a).
385
386
P. J. Atkinson,
Kathleen
dible; this applied to both buccal and lingual aspects and was observed in the youngest, 2-day-old, animal as well as in the older pigs. This pattern was similar to that previously observed in adult animals of other species (Powell et al., 1973). Despite the rapid growth that had occurred in some of the jaws, mineralisation of the newly-deposited bone did not appear to be any less than in more slowly growing parts. No variation in the mineral density of bone tissue was observed between microradiographs of regions with different rates of growth. In general, the distribution of ash density conformed to the same pattern as mass density in all mandibles, which suggested that density differences were more likely to be structural rather than the result of variation in mineralisation (Text Fig. 3). The most porous regions in the jaw of the piglet which had been labelled in uiuo showed the greatest uptake of tetracycline. The uptake of 32POim as visualised on autoradiographs also was greatest in these highly porous areas (Plate Figs. 2a and b) and corresponded with the more marked cellular activity. The 32P specific activity assessed quantitatively in each sample (c.p.m. per pg phosphate) also showed greater values in the most porous regions (Text Fig. 5). The two most actively growing regions in the body of the mandible, i.e. the anterior and the rameal ends were both demarcated by the greater specific activity and porosity. The adult pig mandible after tooth extraction plantation
and im-
The overall pattern of density distribution remained constant whether trauma was caused by tooth extraction or endosseous implantation (Text Fig. 6). Although the trauma had been localised to one part of the alveolar region, the response extended both anteriorly and posteriorly through the body of the mandible. Low density in the jaw anteriorly was due to an increase in the amount of porous bone but, as this was produced in sites which were porous during growth and as these continued to follow the growth pattern, density increased progressively along the mandible towards the ramus and from alveolar crest to inferior border. Implantation led to the depo-
Powell
and C. Woodhead
sition of porous plexiform bone and accounted for the lower density even 1 month after implantation (Text Fig. 6b). This effect of implantation on bone density persisted for a considerable time and was sometimes detectable 26 months after implantation (Text Fig. 6~). However. this overall density pattern was not due solely to the production of porous new bone. for in some instances there was increased resorption. Tooth extraction alone led exclusively to increased resorption with no stimulation of bone formation. This resorption. like bone formation, maintained a similar pattern, being more extensive on the buccal than on the lingual side but less marked in the basal part of the mandible (Text Fig. 6a). Endosseous implantation stimulated considerable periosteal reaction leading to the formation of a wide layer of porous plexiform bone (Plate Fig. 7a) in contrast with that on the unoperated side (Plate Fig. 7b). There was also an obvious proliferation of osteoblasts in the newly formed bone. It was the presence of this porous piextform bone over a wide area of the surface of the bone which led to a reduction of bone density. Resorption of the alveolar ridge occurred locally both after tooth extraction and implantation but was more marked buccally than lingually. This lowered the crest height but did not appear to influence the remodelling which modified the internal cortical structure. The porosity of the plexiform bone gradually decreased. The plexiform lamellae progressively enlarged and the bone consequently became more compact during the longer time interval (Plate Fig. 8a). The greater overall porosity of both left and right sides in the jaw one month after implantation in contrast with that after 26 months is an indication of the age difference between the two animals and emphasises the importance of comparing the treated with the non-treated side (Text Fig. 6). After the original implant had been rejected, reimplantation at the same site produced a similar but reduced bone response (Plate Fig. 8b). Repeated implantation led to an even greater reduction in response, the amount of periosteally formed bone becoming less after each re-implantation. Resorption also reduced the height of the alveolar crest after each implantation. Apparently, by repeating the surgical procedure, not only was bone formation less able to preserve the shape of the alveolar ridge by remodelling but capacity to form bone was also reduced. Whether this reduction of bone-forming ability was contributed to by a rejection phenomenon or by a reaction to metal is not clear. However, the internal structural pattern, as envisaged from the density distribution, was still preserved:
DISCUSSION
snout Fig. 5. Graph 32P (c.p.m. per border to the The change in
1
I
I
Segments
I
_ Ramus
showing the change in specific activity of pg P) in the buccal cortex from the anterior posterior part of the body 6f the mandible. porosity ml/g is also shown. Specific activity A, porosity 0.
As is well known, the body of the growing mandible in all mammals undergoes increase in length as a result of growth of the condyle and ramus. At the same time, remodelling over the entire surface of the mandible. although influenced by different mechanisms, preserves its shape and maintains dental occlusion. The present study shows how an overall pattern of mandibular structure is also maintained by remodelling from early stages of growth.
Cortical
structure
of the pig mandible
Left
Right
Density g/ml
C
Fig. 6. Diagrams showing the density (g/ml) distribution in the cortical bone of 3 pig jaws after different surgical
procedures: (a) 12 months after tooth extraction; (b) 1 month after tooth implantation; (c) 26 months after tooth extraction and implantation.
In the pig, surface remodellidg of the body of the mandible consists almost entirely of bone deposition but some areas grow more rapidly than others, as indicated by the greater metabolic activity demonstrated by in uiuo tetracycline labelling and 32PO:incorporation. Areas of greatest activity were invariably the most porous and so the degree of porosity itself might be considered as an indication of the state of growth. That growth was greater in the buccal cortex anteriorly 1:han elsewhere was shown by the greater width of porous plexiform bone. In the more slowly growing areas, increasing width of lamellae keeps pace with the deposition of new plexiform bone. Thus the width of the region consisting of more porous lamellae remains constant. By studying variations in the amount of porous plexiform bone in in each section, it was possible to indicate the main directions of growth of the body of the mandible (Text Fig. 9)
Fig. 9. Diagram
showing
extraction
main directions
and
of growth
at the The plexiform
periosteal surface of the body of the pig mandible. size of the arrows
indicate the amount bone formed.
of porous
388
P. J. Atkinson, Kathleen Powell and C. Woodhead
The density (mass per unit volume) of blocks of cortical bone from different regions showed variations which also reflected these growth patterns, rapidly growing porous regions being less dense than the more slowly growing parts. In adult pigs, as the growth rate diminished, surface remodelling continued but at a slower rate which diminished proportionally over the whole region. Thus, both mandibular shape and the internal structural pattern were preserved, despite the fact that overall, the density of the older bone was much greater than that of younger bone. The removal of the endosteal cancellous bone ensured that only changes in the cortex were being measured. The distribution of cancellous bone as judged from microradiographs was quite variable and did not appear to be related to the distribution of plexiform bone. However, in some situations, the removal of trabeculae from the endosteal surface would inevitably lead to the removal of cortical bone which had been trabeculated by resorption, as for instance that following tooth extraction. Any recorded low density in this instance would enhance the pattern and thus be even more significant. There is considerable experimental evidence to show that stresses induced in the mandible, e.g. by muscle attachments and other functional mechanisms, influence the growth of bone. When muscles are resected, for instance, bone growth is reduced (Moore, 1973) so that when muscles are unilaterally resected both the pattern and direction of growth can be altered (Schumaker and Dokladal, 1968). The eruption and the presence of teeth as well as the stresses on teeth during function all affect bone remodelling and hence bone structure (Reitan, 1951; Zengo et al., 1973; Grenoble and Knoell, 1973). In our study, although trauma influenced remodelling, the pattern of density distribution was unaltered. Even when different types of trauma led to different types of remodelling, the density pattern remained the same. For instance, although tooth extraction was followed by a loss of alveolar crest height and although internal resorption in the remaining cortical bone was increased, this increased resorption still maintained the established pattern and was in keeping with the shape changes observed by Pietrokowski and Massler (1961). The density pattern was preserved because this resorption was greatest in areas where, as judged by the data from younger bones, the overlying periosteum had been most active during growth. Even when bone formation increased following endosseous implantation, it was still most active in areas which previously had shown most activity during growth. These responses were not confined to the region of implantation but involved the periosteum of a large part of the body of the mandible. They were always more marked buccally than lingually and always more marked in the alveolar region than in basal bone. Thus, although trauma had been localised to the diastema, the response followed the path established during growth and ensured that the pattern of bone structure was preserved. It appears therefore that some regions of the mandible are always capable of a greater response than others. The coincidence that resorption and formation both lead to the production of low density bone may have implications for implantation. Any procedure
which stimulates bone formation may reverse the resorption associated with tooth loss and still maintain the structural pattern of the jaw. However, the present observation on pig does not mean necessarily that the same is true for man. One of the features of the human mandible is that resorption and not formation predominates over the bucco-labial surface (Enlow, 1966). This establishes the more prominent chin of man. If implantation in the alveolar bone above this region enhanced resorption rather than bone formation, it is conceivable that the long-term effects might not achieve the required stability of the implant. During growth, areas of greater metabolic activity in pigs such as those observed in the anterior region of the mandible will always be associated with a greater turnover of bone tissue. That such areas of different activity existed was shown by Weidmann and Rogers (1958) who found that 3zPO:m activity was always greater in alveolar bone than in the rest of the mandible. Bone with a high turnover presumably consists of large amounts of relatively immature collagen and small mineral crystals and is more labile than bone with a lower turnover presumably consisting of more mature collagen and larger crystals. Areas with a more active collagen and mineral turnover might well constitute an essential part of growth control in actively developing areas. They apparently retain this lability because, when stimulated by trauma, they also show this tendency for more rapid turnover and remodelling than other areas. That some regions are more susceptible to remodelling is perhaps also aided by the increased vascularity associated with the greater fibrinolytic activity of bone marrow (Bjorkmann and Nilsson, 1961) and of alveolar bone (Birn, 1971). A maximal response to trauma would be expected to occur during the early more active growing period and diminish with age. This is clearly demonstrated when comparing the density distribution shown in Figs. 6b and c. The younger animal (18 months, 6b) shows a much lower overall density than animal 6c (48 months). We also observed that the repeated trauma of re-implantation led to a diminished response. Thus, the age at implantation. the time between operation and inspection and the frequency of implantation are all factors influencing the structural changes. The precise nature of the mechanism controlling remodelling to produce such changes therefore still remains uncertain. The potency of cells in actively growing areas will diminish with time and it seems that such responses might depend initially on some genetic mechanism. On the other hand, the mineralised tissue itself also matures with age. The extent of stable cross-linking in collagen increases (Bailey et al., 1974) and overall, collagen will be less soluble. Crystals become larger (Robinson and Watson, 1955) and by this increase in size are also relatively more stable. Yet, as a result of increased resorption in ageing bone, the production of free mineral is often so excessive as to be precipitated leading to blocked lacunae and canaliculi (Jowsey, 1960). From our study, which shows such a regular pattern of remodelling, it is not possible to establish whether the degenerating cells or the maturing mineralised tissue leads to the diminished response
Cortical structure of the pig mandible seen with ageing or after repeated trauma. However stimulated, remodelling at any time follows a predictable course and presumably is still directed by genetically inbuilt factors. On the other hand, such a response could be following a path of least resistance, established during early growth by the same genetic mechanism. This remodelling potential, although to some extent obscured during active growth, is still discernible from the density patterns. This suggests that genetically inbuilt factors control the response of the bone tissue throughout growth. However, with ageing and also fol.lowing trauma in the adult animal, it seems more likely that trauma stimulates a bone response which follows the path of least resistance which is probably determined more by the conditioned environment of the metabolic process rather than the potential activity within the cells. Further studies ton the nature of the collagen and crystal components in the different regions of growth and remodelling could be expected to reveal information about such a mechanism. Acknowledgements~It gives pleasure to thank the staff of the Department of Animal Physiology, University of Leeds for providing and assisting with the pigs and especially to Mr. K. G. Towers, Veterinary Officer, for his advice and assistance. Th~7ks are due to Dr. C. R. Hiller for carrying out the specific activity measurements, Mr. V. Hawkins for technical assistance and Mr. F. H. Lumby for the drawing of ligure 4. The work was supported by a grant from the Mlzdical Research Council.
REFERENCES
Atkinson P. J. 1976. Patterns of bone growth. In: The Eruption and Occlusion of Teeth (Edited by Poole D. F. G. and Stack M. V.). Colston Papers No 27. Butterworths, London. Bailey A. J., Robins S. P. and Balian G. 1974. Biological significance of the intermolecular cross-links of collagen. Nature, Lond. 251, 105-109.
389
Birn H. 1971. Fibrinolytic activity of normal alveolar bone. Acta. odont. stand. 29, 141-153. Bjorkmann S. E. and Nilsson I. M. 1961. Demonstration of a fibrinolytic activator in red bone marrow. Acta haemat. 26, 273280. Chen P. S., Toribara T. Y. and Warner H. 1956. Microdetermination of phosphorus. An&t. Chem. 28, 17561758. Enlow D. H. 1966. k morphogenetic analysis of facial growth. Am. J. Orthod. 52. 283-299. Grenoble D. E. and Knoell k. C. 1973. Unified approach to the biomechanics of dental implantology. Jet Propul. Lab. q. Tech. Rev. 2, 7-17. Horowitz S. L. and Shapiro H. H. 1955. Modification of skull and jaw architecture following removal of the masseter muscle in the rat. Am. J. phys. Anthrop. 13, 301-308.
Jowsey J. 1960. Age changes in human bone. C/in. Orthop. Rel. Res. 17, 21@218. Moore W. J. 1973. An experimental study of the functional components of growth in the rat mandible. Acta anat. 85, 378-385. Moss M. L. and Salentijn L. 1970. The logarithmic growth of the human mandible. Acta anat. 77, 341-360. Pietrokowski J. and Massler M. 1967. Alveolar ridge resorption following tooth extraction. J. prosth. Dent.17, 21-27. Powell K., Atkinson P. J. and Woodhead C. 1973. Cortical bone structure of the pig mandible. Archs oral Biol. 18, 171-180. Reitan K. 1951. The initial tissue reaction incident to orthodontic tooth movement as related to the influence of function. Acta odont. stand. Suppl. 6. Robinson R. A. and Watson M. L. 1955. Crystal collagen relationships in bone as observed in the electron microscope III drystal and collagen morphology as a function of age. Ann. N.Y. Acad. Sci. 60. 596-628. Schumaker G. H. and Dokladal M. 1968. uber unterschiedliche Sekundlvergnderungen am Schldel als Folge von Kammuskelnesktianen A& anat. 69, 378-392. _ Weidmann S. M. and Rogers H. J. 1958. Studies of the skeletal tissue. 5. The in%uence of age upon the degree of calcification and incorporation of 32PO:- in bone. Biochem.
J. 69. 338-343.
Zengo A. N., Pawluk R. J. and Bassett C. A. L. 1973. Stress induced bioelectric potentials in the dento-alveolar complex. Am. J. Orthod. 64, 17-27.
390
P. J. Atkinson,
Kathleen
Powell and C. Woodhead
Plate
1
Fig. 2. Microradiograph (a) and autoradiograph (b) of a section from a mandible of a 16-day-old pig which had been given an intra-peritoneal injection of 32P in ciao. Porous areas of bone on the microradiograph show the greatest activity in the autoradiograph (b). Fig. 7. (a) Microradiograph
showing bone response to implantation after the bone on the unoperated side (b) x 20.
1 month
Fig. 8. (a) Microradiograph showing the bone response 26 months after implantation. radiograph showing the bone response 25 months after re-implantation.
compared
with
x 20 (b) Microx 20.
Cortical
structure
of the pig mandible
2b
391
CORTICAL STRUCTURE OF THE PIG MANDIBLE AFTER THE INSERTION OF METALLIC IMPLANTS INTO ALVEOLAR BONE P. J. Departments
ATKINSON,
KATHLEEN POWELL
and C.
W~~DHEZAD
of Oral Biology and Restorative Dentistry, University of Leeds, Leeds, Yorkshire, England
Summary-The effect of tooth extraction and endosseous metallic implants on the bone structure of adult pigs was studied using both densitometric and microradiographic techniques. Similar methods were used to study the bone structure of both growing and adult pig mandibles. The use of tetracycline and 32P-labelled phosphate in uivo indicated that the porous bone deposited periosteally is the most metabolically active bone during growth. This formation occurs over almost the entire surface of the body of the mandible throughout life but, despite an overall density increase with age, the internal structural pattern established during development, is maintained. Both tooth extraction and the introduction of metallic endosseous implants :stimulated further remodelling. Despite the different responses to tooth extraction and implantation, the internal structural pattern remained constant.
INTRODUCTION
The influence of mechanical stress on cortical bone structure is well known. However, the precise mechanisms which maintain shape and internal structure, despite mechanical and metabolic demands, are not clearly unden,tood. Mandibular growth is closely coordinated with craniofacial growth (Enlow, 1966; Moss and Salentijn, 1970). Shape and internal structure are established at an early stage of development and are maintained throughout life by surface remodelling (Atkinson, 1976). To gain more information about growth and the effects of stress, we studied the effect on bone structure of endosseous implants. MATERIAL
AND
METHODS
The animals used were large White pigs of the same herd. (1) Measurement
x Landrace
of growth
Mandibular growth was recorded in tiiuo in two litters of piglets, cone consisting of eleven l-day-old piglets and another of twelve 16-day-old piglets. Using calipers, mandibular length was measured from the most prominent part of the posterior border of the ramus to the most anterior projection of the body of the mandible. Measurements were taken at 7-day intervals three times. Similar measurements were made post-mortem on IO mandibles from animals aged from 2 days to 2.5 yr. (2) Metabolic mandible
activity
and structure
the soft tissues removed by dissection and the mandible divided at the symphysis. The entire tooth-bearing area of each half-mandible was then cut into segments. Left and right sides were considered to be equivalent. Segments from the left half were cleaned of blood and medullary soft tissue, dehydrated in alcohol and defatted using ether. As the intention was to study mandibular cortex only, teeth and cancellous bone were removed using a dental bur. The segments were then divided into approximately equal pieces on both buccal and lingual sides. Using the method described by Powell et al. (1973), the density of each sample was calculated by dividing its dry weight by its external volume. This gave an indication of the compactness of the cortical bone. The density values were colour-coded and transferred to plastic replicas of each bone so that the density distribution of the cortex of the tooth-bearing mandible could be visualised and compared with the other data. Following the mass density estimation, the samples were ashed in a muffle furnace at 580°C and the value of ash weight per unit volume obtained. This provided the information about the mineral density of each bone sample. When this was colour-coded and displayed on plastic replicas, the patterns of mineral density could be compared with the patterns of mass density or compactness. Using the left halves of 10 mandibles obtained postmortem from pigs aged 2 days to 2.5 yr, additional information was obtained of the mass density distribution in the cortex of growing and adult mandibles. (3) Histological
in the growing
examination
The right halves of each mandible were used for microradiography and histology. A thin section was taken from each segment for subsequent microradiography and the remainder used for histological study. The undemineralised sections were embedded in plastic (Crystic resin, Trylon Ltd.) and ground to a thickness of 100pm. Contact microradiographs of these sections were made using High Resolution Plate
Using a 15-day..old piglet from one of the litters, tetracycline and 32P labelled phosphate, metabolic activity was studied IVIuivo. An intra-peritoneal injection of tetracycline (15 mgfig) followed 48 hr later and by the same route by an injection of 32P-labelled phosphate (200 pCi/kg). After 4 hr, the animal was killed, 383
P. J. Atkinson, Kathleen Powell and C. Woodhead
3x4
(Kodak) and a Raymax-AEI Radiographic Unit at 15 cm film distance (20 kV 4 mA). The remaining segments from the right half-jaw were demineralised in 1.5 per cent nitric acid, embedded in wax and sectioned. The structural organisation of the section observed after staining with haematoxylin and eosin was compared with that seen in microradiographs and correlated with information obtained from the density patterns. (4) The effect of' tooth extraction plantation
and endosseous im-
Nine adult breeding sows aged 18 months to 2 yr were premeditated with phencyclidine HCl (Semylan; Parke, Davis & Co., London) 1 mg/kg body weight by intramuscular injection and anaesthetised using phenobarbitone Na (Nembutal) 12-15 mg/kg body weight by intravenous injection. The first and second premolar teeth were extracted on the left side. This ensured that the diastema and premolar regions were completely free of teeth and roots. In two animals, tooth extraction alone was carried out. In the other seven, cobalt-chromium blade implants were inserted partly into a slot of appropriate size previously prepared in the diastema using a dental bur and partly into the socket of the second premolar. The blade implants were specially cast in the shape of a trident consisting of a single post and three tapered legs. The overall length of each implant was 25mm, their broadest dimension 25 mm, and their width 2mm. Each implant was placed so that the legs were below the level of the alveolar crest, leaving the single post projecting 1Omm into the oral cavity. Implants remained in situ for various periods, re-implantation being carried out whenever an implant was rejected. Thus, two mandibles were available for examination after tooth extraction, two after one implantation, two after one re-implantation and three after a second reimplantation. The animals were subsequently killed and the mandibles removed, divided at the symphysis and segments cut as before. In this case, however, both left and right halves of each mandible were treated in the same way to permit a comparison of the treated and control side. A thin section was taken from each segment for microradiography and the remaining segments used for density estimation as before. This enabled microradiographs to be compared with density distribution at similar sites on left and right sides. By taking very small samples from selected areas for cytological study, disruption of the density pattern was avoided.
Although mandibular growth occurs predominantly at the condylar cartilage and in the ramus, remodelling on the surface of the body produces increases in width, length and depth. Remodelling during growth resulted in deposition of bone over almost the entire surface of the body of the mandible. This affected bone density and the differences in compactness of structure could be recognised on microradiographs, because the newly deposited bone consisted of porous plexiform lamellae. Nearer to the growing surface, these lamellae were loosely arranged while below this porous layer they were more compact (Plate Fig. 2a and Text Fig. 4). In older jaws, the rate of growth was less and the lamellae of the porous layer became thicker and the structure was generally more compact. However, even in the more mature animals, there was always relatively more plexiform bone in the anterior region of the growing mandible than elsewhere. This bone deposited on both buccal and lingual sides was previously observed in adult animals (Powell et al., 1973). The distribution of Howship’s lacunae suggested that some surfaces, especially those in the more compact lamellar regions, were being resorbed. The presence of these isolated areas of resorption probably accounted for the remodelling necessary to preserve minor surface depressions during growth. Mandibular bone density was less in young growing pigs than in the older animals, ranging from 0.6 to 1.3 g/ml in the 2-day-old; 0.7 to 1.7 g/ml at 42 days and 1.1 to 1.8 g/ml at 84 days. The lower density was due to the much greater porosity of the newly-formed bone and was therefore greatest in regions of most rapid growth. Overall, bone density increased with age; although density values covered a wide range in each jaw, the number of samples with a density of less than l.Og/ml diminished with age. For instance, although almost the entire jaw of the 2-dayold piglet consisted of low density bone (< 1.0 g/ml) this low density was not present in the jaw of the 84-day-old pig. Despite this general increase of density with age, more rapidly growing areas, for instance the buccal bone anteriorly and to a lesser extent the lingual bone of the same region, were always of relatively lower density. A general increase in density occurred towards the ramus and in the basal part of the man-
RESULTS
The growing mandible In viuo measurements of mandibular growth in the two groups of piglets showed that growth was extremely rapid from birth to 30 days. The length of the mandible, determined post-mortem in 10 animals of different ages, showed that growth rate was greatest during the first 80 days, after which it slowed down (Fig. 1). Both in vioo and post-mortem studies indicated that the length of the jaw increased by approximately 1.4mm/day during this period of rapid growth.
I
/
/
,
/
i
260
400
600
800
1000
Age
*
days
Fig. 1. Graph showing the growth in length of the pig jaw. The diagram shows the points from which measurements were taken.
Cortical structure of the pig mandible Ash
Density
16 doy
& Density [
g/ml
I.1 and less
Fig. 3. Diagrams showing distribution of density (dry weight per volume) and ash density (ash weight per volume) in the cortical bone of 4 pig jaws aged 16. 28, 42 and 49 days respectively.
Fig. 4. A 3-dimensional representation
of the porous bone shown in Fig. 2(a).
385
386
P. J. Atkinson,
Kathleen
dible; this applied to both buccal and lingual aspects and was observed in the youngest, 2-day-old, animal as well as in the older pigs. This pattern was similar to that previously observed in adult animals of other species (Powell et al., 1973). Despite the rapid growth that had occurred in some of the jaws, mineralisation of the newly-deposited bone did not appear to be any less than in more slowly growing parts. No variation in the mineral density of bone tissue was observed between microradiographs of regions with different rates of growth. In general, the distribution of ash density conformed to the same pattern as mass density in all mandibles, which suggested that density differences were more likely to be structural rather than the result of variation in mineralisation (Text Fig. 3). The most porous regions in the jaw of the piglet which had been labelled in uiuo showed the greatest uptake of tetracycline. The uptake of 32POim as visualised on autoradiographs also was greatest in these highly porous areas (Plate Figs. 2a and b) and corresponded with the more marked cellular activity. The 32P specific activity assessed quantitatively in each sample (c.p.m. per pg phosphate) also showed greater values in the most porous regions (Text Fig. 5). The two most actively growing regions in the body of the mandible, i.e. the anterior and the rameal ends were both demarcated by the greater specific activity and porosity. The adult pig mandible after tooth extraction plantation
and im-
The overall pattern of density distribution remained constant whether trauma was caused by tooth extraction or endosseous implantation (Text Fig. 6). Although the trauma had been localised to one part of the alveolar region, the response extended both anteriorly and posteriorly through the body of the mandible. Low density in the jaw anteriorly was due to an increase in the amount of porous bone but, as this was produced in sites which were porous during growth and as these continued to follow the growth pattern, density increased progressively along the mandible towards the ramus and from alveolar crest to inferior border. Implantation led to the depo-
Powell
and C. Woodhead
sition of porous plexiform bone and accounted for the lower density even 1 month after implantation (Text Fig. 6b). This effect of implantation on bone density persisted for a considerable time and was sometimes detectable 26 months after implantation (Text Fig. 6~). However. this overall density pattern was not due solely to the production of porous new bone. for in some instances there was increased resorption. Tooth extraction alone led exclusively to increased resorption with no stimulation of bone formation. This resorption. like bone formation, maintained a similar pattern, being more extensive on the buccal than on the lingual side but less marked in the basal part of the mandible (Text Fig. 6a). Endosseous implantation stimulated considerable periosteal reaction leading to the formation of a wide layer of porous plexiform bone (Plate Fig. 7a) in contrast with that on the unoperated side (Plate Fig. 7b). There was also an obvious proliferation of osteoblasts in the newly formed bone. It was the presence of this porous piextform bone over a wide area of the surface of the bone which led to a reduction of bone density. Resorption of the alveolar ridge occurred locally both after tooth extraction and implantation but was more marked buccally than lingually. This lowered the crest height but did not appear to influence the remodelling which modified the internal cortical structure. The porosity of the plexiform bone gradually decreased. The plexiform lamellae progressively enlarged and the bone consequently became more compact during the longer time interval (Plate Fig. 8a). The greater overall porosity of both left and right sides in the jaw one month after implantation in contrast with that after 26 months is an indication of the age difference between the two animals and emphasises the importance of comparing the treated with the non-treated side (Text Fig. 6). After the original implant had been rejected, reimplantation at the same site produced a similar but reduced bone response (Plate Fig. 8b). Repeated implantation led to an even greater reduction in response, the amount of periosteally formed bone becoming less after each re-implantation. Resorption also reduced the height of the alveolar crest after each implantation. Apparently, by repeating the surgical procedure, not only was bone formation less able to preserve the shape of the alveolar ridge by remodelling but capacity to form bone was also reduced. Whether this reduction of bone-forming ability was contributed to by a rejection phenomenon or by a reaction to metal is not clear. However, the internal structural pattern, as envisaged from the density distribution, was still preserved:
DISCUSSION
snout Fig. 5. Graph 32P (c.p.m. per border to the The change in
1
I
I
Segments
I
_ Ramus
showing the change in specific activity of pg P) in the buccal cortex from the anterior posterior part of the body 6f the mandible. porosity ml/g is also shown. Specific activity A, porosity 0.
As is well known, the body of the growing mandible in all mammals undergoes increase in length as a result of growth of the condyle and ramus. At the same time, remodelling over the entire surface of the mandible. although influenced by different mechanisms, preserves its shape and maintains dental occlusion. The present study shows how an overall pattern of mandibular structure is also maintained by remodelling from early stages of growth.
Cortical
structure
of the pig mandible
Left
Right
Density g/ml
C
Fig. 6. Diagrams showing the density (g/ml) distribution in the cortical bone of 3 pig jaws after different surgical
procedures: (a) 12 months after tooth extraction; (b) 1 month after tooth implantation; (c) 26 months after tooth extraction and implantation.
In the pig, surface remodellidg of the body of the mandible consists almost entirely of bone deposition but some areas grow more rapidly than others, as indicated by the greater metabolic activity demonstrated by in uiuo tetracycline labelling and 32PO:incorporation. Areas of greatest activity were invariably the most porous and so the degree of porosity itself might be considered as an indication of the state of growth. That growth was greater in the buccal cortex anteriorly 1:han elsewhere was shown by the greater width of porous plexiform bone. In the more slowly growing areas, increasing width of lamellae keeps pace with the deposition of new plexiform bone. Thus the width of the region consisting of more porous lamellae remains constant. By studying variations in the amount of porous plexiform bone in in each section, it was possible to indicate the main directions of growth of the body of the mandible (Text Fig. 9)
Fig. 9. Diagram
showing
extraction
main directions
and
of growth
at the The plexiform
periosteal surface of the body of the pig mandible. size of the arrows
indicate the amount bone formed.
of porous
388
P. J. Atkinson, Kathleen Powell and C. Woodhead
The density (mass per unit volume) of blocks of cortical bone from different regions showed variations which also reflected these growth patterns, rapidly growing porous regions being less dense than the more slowly growing parts. In adult pigs, as the growth rate diminished, surface remodelling continued but at a slower rate which diminished proportionally over the whole region. Thus, both mandibular shape and the internal structural pattern were preserved, despite the fact that overall, the density of the older bone was much greater than that of younger bone. The removal of the endosteal cancellous bone ensured that only changes in the cortex were being measured. The distribution of cancellous bone as judged from microradiographs was quite variable and did not appear to be related to the distribution of plexiform bone. However, in some situations, the removal of trabeculae from the endosteal surface would inevitably lead to the removal of cortical bone which had been trabeculated by resorption, as for instance that following tooth extraction. Any recorded low density in this instance would enhance the pattern and thus be even more significant. There is considerable experimental evidence to show that stresses induced in the mandible, e.g. by muscle attachments and other functional mechanisms, influence the growth of bone. When muscles are resected, for instance, bone growth is reduced (Moore, 1973) so that when muscles are unilaterally resected both the pattern and direction of growth can be altered (Schumaker and Dokladal, 1968). The eruption and the presence of teeth as well as the stresses on teeth during function all affect bone remodelling and hence bone structure (Reitan, 1951; Zengo et al., 1973; Grenoble and Knoell, 1973). In our study, although trauma influenced remodelling, the pattern of density distribution was unaltered. Even when different types of trauma led to different types of remodelling, the density pattern remained the same. For instance, although tooth extraction was followed by a loss of alveolar crest height and although internal resorption in the remaining cortical bone was increased, this increased resorption still maintained the established pattern and was in keeping with the shape changes observed by Pietrokowski and Massler (1961). The density pattern was preserved because this resorption was greatest in areas where, as judged by the data from younger bones, the overlying periosteum had been most active during growth. Even when bone formation increased following endosseous implantation, it was still most active in areas which previously had shown most activity during growth. These responses were not confined to the region of implantation but involved the periosteum of a large part of the body of the mandible. They were always more marked buccally than lingually and always more marked in the alveolar region than in basal bone. Thus, although trauma had been localised to the diastema, the response followed the path established during growth and ensured that the pattern of bone structure was preserved. It appears therefore that some regions of the mandible are always capable of a greater response than others. The coincidence that resorption and formation both lead to the production of low density bone may have implications for implantation. Any procedure
which stimulates bone formation may reverse the resorption associated with tooth loss and still maintain the structural pattern of the jaw. However, the present observation on pig does not mean necessarily that the same is true for man. One of the features of the human mandible is that resorption and not formation predominates over the bucco-labial surface (Enlow, 1966). This establishes the more prominent chin of man. If implantation in the alveolar bone above this region enhanced resorption rather than bone formation, it is conceivable that the long-term effects might not achieve the required stability of the implant. During growth, areas of greater metabolic activity in pigs such as those observed in the anterior region of the mandible will always be associated with a greater turnover of bone tissue. That such areas of different activity existed was shown by Weidmann and Rogers (1958) who found that 3zPO:m activity was always greater in alveolar bone than in the rest of the mandible. Bone with a high turnover presumably consists of large amounts of relatively immature collagen and small mineral crystals and is more labile than bone with a lower turnover presumably consisting of more mature collagen and larger crystals. Areas with a more active collagen and mineral turnover might well constitute an essential part of growth control in actively developing areas. They apparently retain this lability because, when stimulated by trauma, they also show this tendency for more rapid turnover and remodelling than other areas. That some regions are more susceptible to remodelling is perhaps also aided by the increased vascularity associated with the greater fibrinolytic activity of bone marrow (Bjorkmann and Nilsson, 1961) and of alveolar bone (Birn, 1971). A maximal response to trauma would be expected to occur during the early more active growing period and diminish with age. This is clearly demonstrated when comparing the density distribution shown in Figs. 6b and c. The younger animal (18 months, 6b) shows a much lower overall density than animal 6c (48 months). We also observed that the repeated trauma of re-implantation led to a diminished response. Thus, the age at implantation. the time between operation and inspection and the frequency of implantation are all factors influencing the structural changes. The precise nature of the mechanism controlling remodelling to produce such changes therefore still remains uncertain. The potency of cells in actively growing areas will diminish with time and it seems that such responses might depend initially on some genetic mechanism. On the other hand, the mineralised tissue itself also matures with age. The extent of stable cross-linking in collagen increases (Bailey et al., 1974) and overall, collagen will be less soluble. Crystals become larger (Robinson and Watson, 1955) and by this increase in size are also relatively more stable. Yet, as a result of increased resorption in ageing bone, the production of free mineral is often so excessive as to be precipitated leading to blocked lacunae and canaliculi (Jowsey, 1960). From our study, which shows such a regular pattern of remodelling, it is not possible to establish whether the degenerating cells or the maturing mineralised tissue leads to the diminished response
Cortical structure of the pig mandible seen with ageing or after repeated trauma. However stimulated, remodelling at any time follows a predictable course and presumably is still directed by genetically inbuilt factors. On the other hand, such a response could be following a path of least resistance, established during early growth by the same genetic mechanism. This remodelling potential, although to some extent obscured during active growth, is still discernible from the density patterns. This suggests that genetically inbuilt factors control the response of the bone tissue throughout growth. However, with ageing and also fol.lowing trauma in the adult animal, it seems more likely that trauma stimulates a bone response which follows the path of least resistance which is probably determined more by the conditioned environment of the metabolic process rather than the potential activity within the cells. Further studies ton the nature of the collagen and crystal components in the different regions of growth and remodelling could be expected to reveal information about such a mechanism. Acknowledgements~It gives pleasure to thank the staff of the Department of Animal Physiology, University of Leeds for providing and assisting with the pigs and especially to Mr. K. G. Towers, Veterinary Officer, for his advice and assistance. Th~7ks are due to Dr. C. R. Hiller for carrying out the specific activity measurements, Mr. V. Hawkins for technical assistance and Mr. F. H. Lumby for the drawing of ligure 4. The work was supported by a grant from the Mlzdical Research Council.
REFERENCES
Atkinson P. J. 1976. Patterns of bone growth. In: The Eruption and Occlusion of Teeth (Edited by Poole D. F. G. and Stack M. V.). Colston Papers No 27. Butterworths, London. Bailey A. J., Robins S. P. and Balian G. 1974. Biological significance of the intermolecular cross-links of collagen. Nature, Lond. 251, 105-109.
389
Birn H. 1971. Fibrinolytic activity of normal alveolar bone. Acta. odont. stand. 29, 141-153. Bjorkmann S. E. and Nilsson I. M. 1961. Demonstration of a fibrinolytic activator in red bone marrow. Acta haemat. 26, 273280. Chen P. S., Toribara T. Y. and Warner H. 1956. Microdetermination of phosphorus. An&t. Chem. 28, 17561758. Enlow D. H. 1966. k morphogenetic analysis of facial growth. Am. J. Orthod. 52. 283-299. Grenoble D. E. and Knoell k. C. 1973. Unified approach to the biomechanics of dental implantology. Jet Propul. Lab. q. Tech. Rev. 2, 7-17. Horowitz S. L. and Shapiro H. H. 1955. Modification of skull and jaw architecture following removal of the masseter muscle in the rat. Am. J. phys. Anthrop. 13, 301-308.
Jowsey J. 1960. Age changes in human bone. C/in. Orthop. Rel. Res. 17, 21@218. Moore W. J. 1973. An experimental study of the functional components of growth in the rat mandible. Acta anat. 85, 378-385. Moss M. L. and Salentijn L. 1970. The logarithmic growth of the human mandible. Acta anat. 77, 341-360. Pietrokowski J. and Massler M. 1967. Alveolar ridge resorption following tooth extraction. J. prosth. Dent.17, 21-27. Powell K., Atkinson P. J. and Woodhead C. 1973. Cortical bone structure of the pig mandible. Archs oral Biol. 18, 171-180. Reitan K. 1951. The initial tissue reaction incident to orthodontic tooth movement as related to the influence of function. Acta odont. stand. Suppl. 6. Robinson R. A. and Watson M. L. 1955. Crystal collagen relationships in bone as observed in the electron microscope III drystal and collagen morphology as a function of age. Ann. N.Y. Acad. Sci. 60. 596-628. Schumaker G. H. and Dokladal M. 1968. uber unterschiedliche Sekundlvergnderungen am Schldel als Folge von Kammuskelnesktianen A& anat. 69, 378-392. _ Weidmann S. M. and Rogers H. J. 1958. Studies of the skeletal tissue. 5. The in%uence of age upon the degree of calcification and incorporation of 32PO:- in bone. Biochem.
J. 69. 338-343.
Zengo A. N., Pawluk R. J. and Bassett C. A. L. 1973. Stress induced bioelectric potentials in the dento-alveolar complex. Am. J. Orthod. 64, 17-27.
390
P. J. Atkinson,
Kathleen
Powell and C. Woodhead
Plate
1
Fig. 2. Microradiograph (a) and autoradiograph (b) of a section from a mandible of a 16-day-old pig which had been given an intra-peritoneal injection of 32P in ciao. Porous areas of bone on the microradiograph show the greatest activity in the autoradiograph (b). Fig. 7. (a) Microradiograph
showing bone response to implantation after the bone on the unoperated side (b) x 20.
1 month
Fig. 8. (a) Microradiograph showing the bone response 26 months after implantation. radiograph showing the bone response 25 months after re-implantation.
compared
with
x 20 (b) Microx 20.
Cortical
structure
of the pig mandible
2b
391
