Repair of wounds in the periodontium of the rat. Influence of periodontal ligament on osteogenesis

Archs oral Bid. Vol. 15, pp. 1183-1204, 1970. PergamonPress.Printedin Great Britain.

REPAIR OF WOUNDS IN THE PERIODONTIUM OF THE RAT. INFLUENCE OF PERIODONTAL LIGAMENT ON OSTEOGENESIS A. H.

MELCHER

Faculty of Dentistry, University of Toronto, Toronto 2, Canada

124 Edward Street,

Summary-Bilateral wounds were made in the alveolar bone of 15 Wistar rats to expose the mid l/2-2/3 of the periodontal ligament on the buccal aspect of the mesiobucal root of the mandibular first molars. On one side of the jaw the periodontal ligament was removed from the apical half of the wound, and on the other from the coronal half. Three animals were sacrificed at each of 4 days, 1 week, 2 weeks, 3 weeks and 4 weeks after operation. Eighteen hours before death, each animal was given an intraperitonesl injection of 0.6 mg Vinblastine Sulphate. No difference was seen between repair of wounds in which the periodontal ligament was removed from the coronal half and those in which it was removed from the apical half. Four days after operation the periodontal ligament that had been exposed in the wound was relatively acellular, but many of the remaining cells were dividing and were arrested in metaphase. The undisturbed periodontal ligament adjacent to the wound wasnotably cellular when compared with the rest of the ligament, and many of the cells exhibited mitotic figures arrested in metaphase. Four weeks after operation the exposed periodontal ligament was repopulated by cells which appeared to have originated from the two sources of dividing cells described above. Bone for repair of the wound was provided by endosteal and periosteal callus. In 8 of the 18 wounds observed between 2 and 4 weeks after operation bony callus had invaded the half of the periodontal space in the wound from which periodontal ligament had been extirpated, and there was ankylosis between bone and tooth. In none of these wounds was there invasion of the periodontal space in the half of the wound in which periodontal ligament was exposed but retained, or of adjacent undisturbed periodontal ligament. In the remaining wounds, and in the half of the ankylosed wounds from which periodontal ligament was not extirpated, new bone was laid down external to the periodontal space. It is postulated that cells of periodontal ligament and their progeny have the capacity to inhibit osteogenesis.

THE NEIGHBOURING mineralized connective tissues, cementum and bone, are separated throughout the life of the tooth by the soft tissue-containing periodontal space. This space is not usually invaded by bone, so that ankylosis between bone and tooth rarely occurs. New bone can be deposited on the periodontal aspect of alveolar bone, for example following orthodontic movement of the tooth, but osteogenesis in this situation is apparently inhibited except under controlled, physiological conditions. Irreversible and progressive resorption, and consequent destruction of cementum and dentine of the root of permanent teeth also does not occur normally, although this can supervene physiologically prior to shedding of primary teeth, and pathologically as a consequence of pressure from misplaced teeth or new growths. There is experimental evidence to support the belief that the connective tissue of 1183

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the periodontal ligament possesses the capacity to inhibit both osteogenesis in the periodontal space and progressive resorption of the connective tissues of the root. This evidence has been obtained from observations made on reimplanted teeth. it has been found that if the periodontal ligament is removed from the root of the tooth prior to reimplantation, much of the periodontal space is often occupied by newlyformed bone which gains attachment to the cementum of the root of the tooth. This change is frequently accompanied by progressive resorption of the root followed by replacement with bone (HAMMER,1955; LYE and WAERHAUG,1961; ANDREASEN and HJBRTINGHANSEN1966; SHERMAN,1968). Further support for the view that these occurrences might be prevented by the periodontal ligament has been provided by HUEBSCHet al. (1952) and RADDEN(1959). These investigators found that signs of osteogenesis in a given site on the wall of a healing extraction socket are evident only after the organized periodontal ligament in that situation has disappeared. The question that arises is whether the collagen fibres of the periodontal ligament provide a physical barrier to osteogenesis, or whether inhibition of osteogenesis is achieved essentially by the cells of the ligament. The collagen fibres in the periodontal ligament of necrotic implants of alveolar process and periodontal ligament disappear and do not inhibit osteogenesis (unpublished). This finding suggests that the cells are probably essential, either directly or indirectly, for the osteodepressive capacity of periodontal ligament. However, it is not known how inhibition is achieved. It is conceivable that it could be accomplished by exertion of some influence on osteogenic cells or their precursors, as discussed by MELCHER(1969), or perhaps through some substance secreted by the cells into the extracellular substance of the soft connective tissue, or both. It is also not clear to what extent the cells of wounded periodontal ligament retain their osteodepressive capacity during repair. Invasion of the periodontal space by newly-formed bone and its attachment to the root of the tooth is not wholly inhibited in reimplantation experiments in which the periodontal ligament adhering to the cementum is carefully conserved (SHERMAN,1968). Furthermore, in the implantation experiments referred to above, it is not known to what extent the cells in the periodontal ligament left adhering to the alveolar bone after extraction of the tooth continue to exert an osteodepressive effect on the adjacent bone cells. The present investigation was undertaken firstly, to explore further the short-term changes which result in repair of the wounded periodontal ligament of teeth of limited eruption and secondly, to examine the osteodepressive influence of periodontal ligament during the early part of the repair process.

MATERIALS AND METHODS Fifteen albino rats of the Wistar strain, weighing about 250 g, were used in the investigation. Experimental wounds were made over the external aspect of the mesiobuccal root of both mandibular first molars in each animal. Under Nembutal and ether anaesthesia, the external aspect of the masseter muscle and the mandible anterior to it was exposed through a skin incision along the inferior border of the jaw. The anterior insertion of the muscle was elevated to expose the underlying bone. Using an

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operating microscope and a slowly rotating number 3 round bur held in a straight handpiece, bone was removed until the periodontal ligament covering the mid l/2-2/3 of the root could be seen. The thin sliver of bone remaining over the ligament was then removed using a sharp probe and small dental excavator. The periodontal ligament and some of the underlying cementum was then removed from half of the wound using a small excavator; on one side of the jaw the ligament was removed from the apical half of the wound, and on the other side from the coronal half of the wound (Fig. 1). The soft tissues were subsequently sutured in layers with 000 gut, and the wounds allowed to heal for varying periods up to 4 weeks. Three animals were killed at each time-period of 4 days, 1 week, 2 weeks, 3 weeks, and 4 weeks.

FIG. 1. A diagrammatic

representation

of the operative

procedure.

Eighteen hours before death, each animal was given an intrapcritoneal injection of a stathmokinetic drug, 0 - 6 mg of Vinblastine sulphate in 0 *5 ml normal saline (Donated by Lilly Research Centre Limited, Windlesham, Surrey, England). All animals were killed at 10 a.m. by coal-gas inhalation. The mandibles were dissected from each animal and tied for 24 hr in 10 per cent formol-saline. Serial sections in the coronal plane were subsequently prepared by a method designed to facilitate location and orientation of small structures. After fixation the mandibles were radiographed in the bucco-lingual plane, and the wound identified on the radiograph. Using the radiograph as a guide the mandible was sliced coronally l-2 mm anterior to the wound, and parallel to it, with a saline-cooled diamond disc rotated in a conventional dental engine. The trimmed specimen was then radiographed in the bucco-lingual plane. After embedding in paraffin wax, the angle between the anterior margin of the wound and the sliced surface of the specimen as seen on the second radiograph was used to orientate the specimen on the

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microtome so that the anterior surface of the wound was parallel to the knife. The distance between the sliced surface of the specimen and the anterior margin of the wound was then measured on the second radiograph, and an amount of tissue equal to half the measured distance removed from the specimen by cutting on the microtome. One section was then cut, mounted, dewaxed, and examined under phasecontrast. From this section it was possible to determine the position of the cut-face of the specimen relative to the wound. If the section was adjacent to the wound a start was made on cutting serial sections. If not, further tissue was cut and discarded and another sample-section examined under phase-contrast. This process was continued until sufficient tissue had been removed to expose the tissues adjacent to the wound, at which time serial sections of the whole wound were prepared. These were stained with haematoxylin and eosin, and examined in the light microscope. OBSERVATIONS

Histological observations made on the healing wound showed that the process of repair was essentially the same irrespective of whether the periodontal ligament was removed from the apical or the coronal half of the wound. Consequently, in the description below, no distinction will be made between the two types of wound. Four days after wounding The bone defect was made opposite both cellular cementum and acellular cementurn; the latter was present in the coronal part of the wound. Cementocytes were present in the cementum where the periodontal ligament had been exposed but left intact. Where the periodontal ligament had been extirpated, the underlying cellular cementum exhibited empty cementocyte lacunae (Fig. 2). The junction between the half of the wound in which the periodontal ligament had been exposed but not removed, and the half of the wound from which the periodontal ligament had been extirpated always occurred opposite cellular cementum. Consequently, the junction between the part of the cementum containing occupied lacunae and the part of the cementum containing empty lacunae served to delineate the two halves of the wound throughout the experiment. When the periodontal ligament had been removed from the coronal part of the wound, the full thickness of the acellular cementum was sometimes found to have been removed, exposing the dentine. The wound in the soft tissues and bone, and in the part of the periodontal space from which the periodontal ligament had been removed, was occupied by fibrin, polymorphonuclear leucocytes and by soft and hard tissue debris resulting from the operation (Fig. 3). Very few inflammatory cells could be identified in the periodontal ligament in the vicinity of the wound. The periodontal ligament that had been left in the wound was found to be covered by occasional thin slivers of necrotic bone that had not been removed at operation (Fig. 3). The cellularity of the exposed ligament was markedly reduced (Fig. 3) when compared with the ligament in the experimental site before operation (Fig. 4). However, many of the cells of the exposed ligament that survived the operation appeared to retain their vitality and their capacity to

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reproduce, as judged by the numbers of these that exhibited mitotic figures (Fig. 5). A band of periodontal ligament containing few cells, many of which appeared to be dividing, was also often seen surrounding the part of the wound from which the periodontal ligament had been removed. This tissue had probably been traumatised at operation. The periodontal ligament adjacent to the wound, which had not been disturbed at operation, was markedly cellular when compared with the rest of the periodontal ligament surrounding the root (see Fig. 9). The cells in this part of the periodontal ligament exhibited numerous mitotic figures (Fig. 6) and this contrasted strongly with those in the rest of the ligament, few if any of which were seen to be dividing. The mitotic figures present in the undisturbed ligament adjacent to the wound were sometimes found to be situated perivascularly. New bony callus was never found to have been laid down in the wound, and there was little resorption of bone in the wall of the wound. Few osteocyte lacunae in the bone adjacent to the margin of the wound were found to be empty. Small deposits of subperiosteal callus were sometimes found to have been deposited on the inferior border of the mandible, at a site remote from the wound. These did not contain cartilage. Cells with nuclei that had been arrested in metaphase were found to be associated with these. One week after wounding The appearance of the wounds in this series is similar to those examined 4 days after operation, but the process of repair could be seen to have advanced a little (Fig. 7). The wound in the bone, the part of the wound from which the periodontal ligament had been removed, and the wound in the overlying soft tissue, were still partly occupied by fibrin and tissue debris, but were being invaded by primitive connective tissue cells, the nuclei of many of which exhibited mitotic figures (Fig. 8). The appearance of the exposed periodontal ligament that had been left in the wound and that of the undisturbed periodontal ligament adjacent to the wound was similar to that seen 4 days after operation, except that cells appeared to be migrating from the latter into the former (Fig. 9). Numerous mitotic figures were still evident in both situations. The histological appearance of the tissue was such as to suggest that it was unlikely that the exposed, relatively acellular ligament remaining in the wound was being invaded by primitive connective tissue cells lying external to it. No new bony callus was found to have been laid down in the wound, and no subperiosteal callus had been deposited adjacent to the wound. However, deposits of subperiosteal callus were again found in a situation remote from the wound, on the inferior border of the mandible. Cartilage was not found to be associated with this callus. Two weeks after wounding The most striking feature in 3 out of the 6 wounds examined in this series is that new bony callus was found to have been laid down in the periodontal space, and to have gained attachment to the cementum of the root (Figs. 10 and 11). In all of these instances, this phenomenon was found to have occurred in the half of the wound from

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which the periodontal ligament had been removed, and never to have occurred where the periodontal ligament had been exposed but left intact in the wound. This observation was confirmed by examining the cellular cementum immediately underlying the attached bone for the presence of cementocytes. The width of the attachment between bone and tooth varied in different wounds and in different parts of the same wound. The bone that had proliferated into the periodontal space was sometimes separated from the adjacent highly cellular area of periodontal ligament by a band of ligament exhibiting reduced cellularity (Figs. 10 and 12), and never was seen to have invaded the undisturbed periodontal ligament surrounding the wound (Fig. 12). Associated with invasion of the periodontal space by bone, there was occasional evidence of localized resorption of the tooth by multinucleated giant cells (Fig. 11). Examination of serial sections showed that the bone that had invaded the periodontal space had originated as subperiosteal callus, endosteal callus, or a combination of both. The subperiosteal callus had been deposited on all aspects of the wound. There was evidence that some resorption of bone from the periodontal aspect of the alveolar process had taken place during the 2 weeks after operation. The endosteal callus could have been deposited by perivascular cells exposed as a result of this resorption, or by perivascular cells exposed as a consequence of wounding. The trabeculae of bony callus that had invaded the periodontal space were usually covered by osteoblasts, and were clearly discernible from the contiguous soft connective tissue (Fig. 12). Sometimes, however, in other parts of the same wound, the bone was not covered by an easily recognizable layer of plump osteoblasts, and it and the adjacent soft connective tissue appeared to blend with one another (Figs. 15 and 18). The associated osteoblasts were often spindle-shaped or epithelioid (KNESE, 1964). Much of the bone that had been laid down in the periodontal space possessed the characteristic of woven bone, viz. incorporation of collagen fibres from adjacent soft connective tissue. When sections were examined under polarized light, fibres could be seen coursing from the adjacent periodontal ligament into the bone and from the bone into the overlying soft connective tissue. Fragments of bone debris were often seen to have been incorporated into the newly deposited bony callus (Fig. lo), but examination of serial sections provided no evidence that osteogenesis had been induced by the fragments of necrotic bone. Two weeks after wounding, subperiosteal callus was frequently found to have been deposited in the vicinity of the wound margin, and in different wounds was seen on all aspects. Unlike the subperiosteal callus deposited at a distance from the wound, the callus adjacent to the wound was frequently found to be associated with cartilage, most of the cells of which were hypertrophic. The cartilage was varyingly seen on both the coronal and apical walls of the wound (Figs. 12-14) but never in the periodontal space When deposited on the coronal wall, the subperiosteal callus was sometimes seen to have proliferated superior to the alveolar crest (Fig. 14), and to contain collagen fibres that originated in the cementum at the neck of the tooth and that formerly had passed over the alveolar crest and into the gingiva. The subperiosteal callus present adjacent to the margin of the wound was often separate from that which had been laid down more remotely at the inferior border of the mandible.

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Three weeks after wounding

Two out of the 6 wounds examined at this time period show evidence of osteogenesis in the periodontal space in the half of the wound from which the periodontal ligament had been removed, and ankylosis between bone and tooth. None of the wounds showed penetration of the periodontal space by bony callus in the half of the wound from which the periodontal ligament had not been removed. The appearance of the new bone that had formed in the periodontal space was similar to that seen 2 weeks after operation. New subperiosteal callus, endosteal callus, or both, had been laid down in all the wounds. Where the bony callus had not penetrated the periodontal space, new bone was deposited on the periphery of the space (Fig. 16). The new periodontal space thus formed appeared to be of more regular thickness in the half of the wound in which periodontal ligament had been exposed but not removed than in the half of the wound from which the periodontal ligament had been extirpated, and the contained connective tissue to be of more orderly arrangement (Figs. 16-18). In none of the wounds examined had the breach in the alveolar process been wholly repaired by new bone. Cells that had been arrested in metaphase while dividing were often seen to be present on the external aspect of the callus that had been laid down to replace the excised alveolar process, but rarely if ever on its periodontal aspect. However, where the periodontal space had been invaded by new bone, mitotic figures were frequently seen in the adjacent connective tissue in the periodontal space. Most of the periodontal ligament that had been left exposed in the wounds was found to have been repopulated by cells (Figs. 16 and 17). Occasional areas adjacent to the cementum were still seen to be relatively acellular (Fig. 19), but many of the cells contained therein exhibited mitotic figures.The soft connective tissue that had replaced the periodontal ligament removed at operation was less well organized than that in the other half of the wound (Figs. 16-18). It showed varying degrees of cellularity and varying amounts of extracellular substance, and, whereas the collagen fibres in the ligament in the other half of the wound were well orientated, those in this half of the wound ran in varying directions (Figs. 16-l 8). When examined in polarized light, fibres in all parts of the periodontal space were often seen to be continuous with those of the new bony callus. Where continuity of the alveolar process had not been restored fibres in the periodontal space were seen to be continuous with, and to intermingle with fibres in the overlying connective tissue. In this situation the external limit of the periodontal ligament was often marked by an increase in cellularity. Occasional spicules of bone debris were found in the soft connective tissue of the periodontal space, but there was no evidence that these had induced osteogenesis in their vicinity. Four weeks after wounding

In 3 of the 6 wounds examined in this series, new bone had been deposited in the periodontal space, and had fused with the tooth. In these instances, there was evidence that there had been some resorption of the root by multinucleated osteoclasts. The A.O.B. 15/12-F

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histological features of wounds exhibiting ankylosis, and of wounds in which the periodontal space was reconstituted by bony callus examined at this time period were similar to those seen in wounds examined three weeks after operation, except that repair was more advanced and the newly deposited connective tissue tended to be more mature. In this series, as in the three-week series, the newly constituted periodontal space was of more regular dimension where the periodontal ligament had been left in situ than in the half of the wound from which it had been extirpated, and was often very narrow in the latter situation (Fig. 20). No convincing evidence was obtained that new cementum had been deposited on the surface of the root from where periodontal ligament had been extirpated, nor that the new connective tissue that had been laid down in the space had become attached to the tooth. Large deposits of subperiosteal callus were often seen on all aspects of these wounds. Active osteogenesis appeared sometimes still to be evident at the alveolar crest. Large deposits of subperiosteal callus extending from the inferior border of the mandible to the margin of the wound could be seen, and these often had elevated muscle attachments away from the surface of the mandible. In three of the six wounds comprising the series, examination of serial sections showed that the continuity of the alveolar process had been re-established, and the part of the periodontal space that had been exposed was once again covered by bone. In one of these wounds, there was evidence of ankylosis. DISCUSSION

(a) Repair of the periodontium (i) Periodontal ligament. The change in the histological appearance of the part of the periodontal ligament that was exposed, but not removed from the wound, was striking. The loss of cellularity that occurred gave the collagen fibres a hyaline appearance. A similar change in the appearance of periodontal ligament that has been compressed between root and bone has been reported by SANDSTEDT (1904) and by MACAPANPAN et al. (1954). The second group of authors have inferred that this change is related to interference with blood supply, and this explanation could reasonably be given for the observations made in the present experiment. The blood supply of the periodontal ligament of rat molars has been described by KINDLOVAand MATENA (1962) and by CARRANZA et al. (1966). Arteries leave the alveolar bone at all levels to enter and supply the ligament of mandibular molars. It seems likely, therefore, that the removal of the alveolar bone in wounding interfered with the blood supply of the underlying periodontal ligament, and that this resulted in the death of most of the cells in the affected area. However, the finding of mitotic figures in the relatively acellular, exposed ligament provides evidence that some of the cells were able to obtain adequate nourishment, and it might be supposed that this was provided by branches of vessels that entered the ligament in the vicinity of the wound. There is unfortunately no direct evidence to support this supposition; this will have to be provided by studies specifically aimed at examining vascularization of the wounds. The periodontal ligament that had become relatively acellular after exposure by

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wounding was repopulated by cells during the period of this experiment. Pools of cells from which cells could have migrated into the exposed periodontal ligament were identified by the presence of a high percentage of nuclei that had been arrested in metaphase by the Vinblastine sulphate that was administered 18 hr before death. In contrast to these areas, few mitotic figures were seen in cells resident in the rest of the periodontal ligament. This observation may appear strange in that the periodontal ligament of rat molars has been shown to exhibit a relatively high turnover of cells (WEISSet al., 1968). The explanation may lie in the fact that Vinblastine, which is an alkaloid obtained from Vincu rosea, has a marked mitodepressive effect in addition to its mitoclastic effect; in this way it differs from colchicine (DEYSSON, 1968). Thus, in the rest of the ligament, where the mitotic index was probably lower than in the vicinity of the wound, most of the cells may have been prevented from dividing. Three distinct areas showed a population of actively dividing cells: the relatively acellular periodontal ligament exposed at wounding, the undisturbed periodontal ligament adjacent to the wound and the cells of the soft connective tissue external to the wound. Histological observations suggested that the exposed periodontal ligament was repopulated by cells derived from the first two pools. The half of the wound from which the periodontal ligament had been extirpated appeared to have been repopulated by cells derived from the adjacent undisturbed periodontal ligament and, in some instances possibly, by cells derived from the overlying soft connective tissue. The periodontal ligament has been recognized by a number of authors (PFEIFFER,1963 ; WILDERMAN,1963; RAMFJORD et al., 1966) as a source from which cells can be culled for repair of connective tissues. The collagen fibres newly deposited in the half of the wound from which the periodontal ligament had been extirpated were of less regular dimension and orientation than were those that had been left at operation in the other half of the wound, and the connective tissue appeared to be of varying cellularity. It is unlikely that these new fibres deposited in the peridontal space would have become regularly orientated prior to their becoming subject to the forces acting upon the tooth, as in other situations the orientation of fibres has been shown to depend on the forces exerted upon them (see, for example, BASSETT,1962). The fibres could not have been stressed until they had become attached to the root of the tooth through the medium of newly synthesized cementum. No evidence that cementum had been deposited during the experimental period was obtained, and this is consistent with the observation that deposition of new cementum after wounding can be a tardy process (LINGHORNEand O’CONNEL,1950; MORRISand THOMPSON,1963). (ii) Cementum. The finding of empty lacunae deep to sites from which the periodontal ligament had been extirpated, and of lacunae occupied by cementocytes deep to periodontal ligament that had been exposed during wounding, was not unexpected. There is evidence that cementocytes are viable, as they are active metabolically (FULLMER, 1967) and are capable of resorbing cementum (BBLANGER,1968). It is reasonable to suppose that they obtain their nutrition by diffusion of substances from the periodontal ligament, and to postulate that extirpation of the overlying ligament

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completely removes their source of nutrition and leads to their death. The fact that removal of bone overlying periodontal ligament resulted in loss of most of the cells of the ligament left in situ, but not of the underlying cementocytes, supports the belief that the nutritional requirement of the cementocytes is lower than that of the ligament cells. It may be postulated further that the surviving cementocytes were maintained on metabolites that reached the cells from undisturbed tissues adjacent to the wound. (iii) Alveolar bone. The finding that few osteocyte lacunae in the bone adjacent to the wound were empty, and the limited resorption of bone from the wall of the wound, was consistent with the slow cutting speed used in preparation of the wound. Despite this, no new endosteal or subperiosteal callus was evident in the vicinity of the wound one week postoperatively. However, small deposits of subperiosteal callus were seen on the inferior border of the mandible opposite the wound as early as four days after operation. The reason for osteogenesis in the latter situation is unknown. Perhaps it was due largely to operative trauma which resulted from making the skin incision or retracting tissue. It is also not possible to explain why the osteoblastic reaction at the inferior border of the mandible was initiated so much earlier than that in the vicinity of the wound. It was evident, however, that, although the two deposits frequently had become continuous four weeks after operation, the subperiosteal callus that contributed to wound repair was that which was deposited initially adjacent to the wound. No cartilage was seen to be associated with the subperiosteal callus deposited at the inferior border of the mandible, an observation consistent with others made on repair of wounds in the body of the rat mandible (RETIEF and DREYER, 1967). Between one and two weeks after operation, however, deposits of cartilage were laid down in the subperiosteal callus adjacent to all aspects of the wounds examined in this study. These were replaced by bone four weeks after operation. Chondrogenesis did not accompany deposition of endosteal callus. A number of reasons have been given to explain why chondrogenesis precedes osteogenesis in repair of some wounds. These include poor blood supply (HAM, 1930; PRITCHARDand RUZICKA, 1950 ; GIRGISand PRITCHARD,1958 ; BASSETT and HERRMANN,1961; CAVADIAS and TRUETA, 1965; SLEDGE,1968), age, species and regional differences, and physical stress (see, for example, MCLEAN and URIST, 1968; PRITCHARD,1964). When account is taken of the situation of the wound made in this study, it seems that here stress should be considered as the most obvious stimulus for condrogenesis. However, the role in formation of cartilage played by a poor blood supply to the tissues cannot be ignored. Bone laid down in repair of the wound appears to have been deposited by cells of the periosteum and endosteum, but not by cells of the periodontal ligament. In the rat, a scaffolding of endosteal callus appears to provide support for periosteal callus proliferating in wounds of the femur (MELCHERand IRVING,1962) and the mandible in the region of the inferior dental nerve (RETIEFand DREYER,1967). There was no evidence that periosteal callus laid down in wounds examined in this study proliferated across a previously deposited support of endosteal callus. The endosteal and periosteal bone appeared to proliferate together to effect closure or near closure of the wound four weeks after operation. They were deposited either on the external

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aspect of the periodontal ligament remaining in the wound, or on the connective tissue that had been newly deposited in the wound, or on the cementum of the tooth. Chips of bone inadvertently left in the wound at the time of operation were incorporated into the bony callus, but examination of serial sections did not provide any support of STALLARD and HIATT’S (1968) belief that these fragments may induce new bone formation. (b) The osteodepressive capacity of healing periodontal ligament As discussed in the introduction, it is possible that osteogenesis may be inhibited by periodontal ligament. The present study has provided some additional evidence to support the belief that osteogenesis may be inhibited by viable periodontal ligament: (a) The periodontal ligament that was exposed but not removed from the wound, and which subsequently became repopulated by cells, was never invaded by new bone. The bony callus was always deposited on its external aspect. (b) In 8 of 18 wounds observed between 2 and 4 weeks after operation, bony callus had invaded the part of the periodontal space from which the periodontal ligament had been extirpated, and had gained attachment to the tooth. Despite this, there was never evidence in these wounds that the bony callus had invaded adjacent undisturbed periodontal ligament. It is noteworthy that the two areas of the periodontal space into which new bone was never found to have penetrated were occupied by cells of the periodontal ligament, or by cells that were probably the progeny of cells of the periodontal ligament. In contrast to this, the area of the periodontal space into which developing callus sometimes proliferated was the part of the wound from which periodontal ligament had been extirpated. It seems possible that this part of the wound was not always colonised by connective tissue cells that had originated in periodontal ligament, and that cells from the non-specialized overlying soft connective tissue may sometimes have migrated into the area (see section a above). There is as yet no direct proof that the cells of the periodontal ligament and their progeny have the capacity to inhibit osteogenesis. But, if they are in fact capable of achieving this, it would be possible to explain the observations made in this investigation. When the whole of the area from which the periodontal ligament has been extirpated is repopulated by the progeny of periodontal ligament cells, new bone should not be able to invade it. On the other hand, when this area, or part of it, is colonized by connective tissue cells that have not originated in the periodontal ligament, bone could be laid down in it, and possibly could gain attachment to the tooth. Thus, in repair of wounds of the periodontal ligament, maintenance of the width of the periodontal space could depend on the capacity of the cells of the ligament to make good the defect. Maintenance of the width of the periodontal space after wounding could then possibly depend on cells originating in periodontal ligament being able to fill the area to the exclusion of other cells. In the wounds in which bone had invaded the periodontal space, and had ankylosed with the tooth, there was evidence, 3 weeks after operation, of the presence of mitotic figures on the periodontal aspect of the bony callus. In wounds of the same time period

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in which bony callus had not penetrated the periodontal space, there was no evidence that cells on the periodontal aspect of the callus were dividing. The possible significance of this observation in relation to continuing osteogenesis has been discussed previously (MELCHER,1969). The most striking feature in the wounds in which there was evidence of ankylosis between bone and tooth was the frequent absence of a clear-cut border between bony callus and soft connective tissue. In other situations the developing bone was covered by a layer of osteoblasts, and could be differentiated readily from the contiguous soft connective tissue. The former appearance suggests that new bone was being deposited in the soft connective tissue; perhaps, a “creeping replacement”. The absence of a well-ordered array of osteoblasts raises the question of whether cells of soft connective tissue were being induced to osteogenesis. This appears to be something that happens regularly in repair of fractures (for example, see PRITCHARD,1964), and has been seen in other wounds (R~~EDI and BASSETT, 1967). If this is the case, and the cells of the developing bone are able to induce some connective tissue cells to become osteoblastic, then it seems likely that cells of periodontal ligament exhibit another property, the capacity to resist induction to osteogenesis.

R&m&Des plaies bilat&ales ont BtCproduites dans 1’0s alvkolaire de 15 rats Wistar pour exposer le milieu l/2-2/3 du ligament ptriodontique sur l’aspect buccal de la racine mCdio-buccale de la premiere molaire mandibulaire. D’un c&6 du maxillaire le ligament pCriodontique a CtBenlevC de la moitik apicale de la plaie et de l’autre c&B on a enlevt la moitib coronaie. Trois animaux ont Bt&sacrifiCs chaque fois, 4 jours, 1 semaine, 2 semaines, 3 semaines apr& l’opkration. Dix-huit heures avant la mort on a don& g chaque animal une injection intra-p&ritonbale de 0,6 mg de sulphate de vinblastine. Aucune diff&ence n’a &tBremarqu& entre la rkparation des plaies dans lesquelles le ligament p&iodontique a CtB enlevk de la moitiC coronale et celles dans lesquelles le ligament a Btt enlevk de la moitib apicale. Quatre jours apr&s l’opbation, le ligament pCriodontique qui a &6 expose dans la plaie a CtB trouv6 rklativement acellulaire mais on a vu des nombreuses celluies restantes se divisant et s’arr&er en mktaphase. Le ligament p&iodontique intact, adjacent B la plaie, Btait notablement cellulaire, cornpark avec le reste du ligament et des nombreuses cellules exhibaient des aspects mitotiques, arr&% en mktaphase. Quatre semaines apr&s 1’opQation le ligament pCriodontique expost Ctait repeuplk par des cellules et ces-ci paraissaient avoir l’origine dans les deux sources de cellules divisantes, d&rites plus haut. L’os pour la reparation de la plaie 6tait procur par les cals endosttal et p&iostal. Dans 8 de 18 plaies observ&s entre 2 et 4 semaines apr&s l’op&ation, le cal osseux avait invadk la moitiC de l’espace p&iodontique qui avait Btk extirpQ et il y avait une ankylose entre 1’0s et la dent. Dans aucune de ces plaies, il n’y a pas eu invasion de l’space p&iodontique dans la moitie de la plaie dans laquelle le ligament pCriodontique ttait expose, mais le ligament p&iodontique adjacent resta intact. Dans les plaies restantes et dans la moiti6 des plaies ankylos&s, dans lesquelles le ligament p&iodontique n’avait pas t!t&extirp& il y a eu des d&p&s de mat&e osseuse neuve g l’ext&ieur de l’espace pCriodontique. 11est suppod que les cellules du ligament p&iodontique et leurs descendantes ont la capacitC d’inhiber l’ost&og&&e. Zusammenfassuq-Es wurden beiderseitige Einschnitte in den Alveolarknochen von 15 Wistar Ratten gemacht, um das mittlere l/2-2/3 des periodontalen Ligaments auf der buccalen Seite der mesiobucalen Wurzel des ersten Backenzahns im Unterkiefer freizulegen. Auf einer Seite des Kiefers wurde das periodontale Ligament von der apikalen Hglfte der Wunde und auf der anderen von der koronalen Hiilfte entfemt. An jedem von 4 Tagen 1 Woche, 2 Wochen, 3 Wochen und 4 Wochen nach der Operation opferte man drei Tiere. Achtzehn Stunden vor Todeseintritt wurde jedem Tier eine intraperitoneale Injektion mit 0,6 mg. Vinblastinsulfat gegeben. Es zeigte sich kein

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Unterschied in der Heihmg der Wunde, von welcher das periodontale Ligament von der koronalen Kalfte entfemt wurde im Vergleich mit der Wunde, von welcher das periodontale Ligament von der apikalen Halfte entfemt wurde. Vier Tage nach der Operation fand man, dass das in der Wunde exponierte periodontale Ligament verhlltnismbsig azellular war; es wurde aber beobachtet, dam sich viele der verbleibenden Zellen teilten und in der Metaphase zum Stillstand kamen. Das an die Wunde angrenzende, ungestijrte Ligament war im Vergleich mit dem Rest des Ligaments bemerkenswert zellular und viele der Zellen zeigten mitotische, in der Metaphase festgehaltene Formen. Vier Wochen nach der Operation ftillte sich das exponierte Ligament wieder mit Zellen, welche von den zwei Quellen der obenerwlhnten Zellteilung herzustammen schienen. Zur Verheilung der Wunde wurde Knochen aus dem endostealen und periostealen Kallus beigestellt. In acht von 18 Wunden, die beobachtet wurden, war im Laufe von 2-4 Wochen nach der Operation knochiger Kallus in die Hllfte des periodontalen Raumes in der Wunde, von welcher periodontales Ligament entfemt worden war, eingednmgen und Ankylose fand zwischen dem Knochen und dem Zahn statt. Bei keiner der Wunden fand Eindringen in den periodontalen Raum in der Halfte statt, in welcher das periodontale Ligament exponiert, aber zurtickgelassen wurde, ebenso nicht im angrenzenden, ungestorten, periodontalen Ligament. In der verbleibenden Wunde und in der Halfte der Knochenverwachsungswunden, von denen periodontales Ligament nicht entfemt worden war, wurde neuer Knochen ausserhalb des periodontalen Raumes abgesetzt. Es wird behauptet, dass Zellen aus dem periodontalen Ligament die Flhigkeit besitzen, selbst und durch ihre Abkijmmlinge die Osteogenesis aufzuhalten. REFERENCES ANDREASEN,J. 0. and HJORTING-HANSEN,E. 1966. Replantation of teeth-II. Histological study of 22 replanted anterior teeth in humans. Actu odont. scund. 24,287-306. BASSE-IT,C. A. L. 1962. Current concepts of bone formation. J. Bone Jt Surg. 44A, 1217-1244. BASSETT,C. A. L. and HERRMANN,I. 1961. Influence of oxygen concentration and mechanical factors on differentiation of connective tissue in vitro. Nature, Land. 190,460-461. B~LANGER,L. F. 1968. Resorption of cementum by cementocyte activity (“cementolysis”). Cult. Tim Res. 2, 229-236. CARRANZA,F. A. JR., ITOIZ, M. E., CABRINI,R. L. and Dorro, C. A. 1966. A study of periodontal vascularization in different laboratory animals. J.periodont. Res. 1,120-128. CAVADIAS,A. X. and TRUETA, J. 1965. An experimental study of the vascular contribution to the callus of fracture. Surg. Gyxec. Obst. 120, 731-747. DEYSSON, G. 1968. Antimitotic substances. In: International Review of Cytology. Vol. 24. (Eds. G. H. BOIJRNEand J. F. DANIELLI) pp. 99-148. Academic Press, New York. FULLMER,H. M. 1967. Connective tissue components of the periodontium. In: Structural and Chemical Organization of Teeth (edited by MILES, A. E. W.) Vol 11, pp. 349-414. Academic Press, New York. GIRGIS, F. G. and PRITCHARD,J. J. 1958. Experimental production of cartilage during the repair of fractures of the skull vault in rats. J. Bone Jt Surg. 4OB, 274-281. HAM, A. W. 1930. An histological study of the early phases of bone repair. J. Bone Jt Surg. 12, 827-844. HAMMER,H. 1955. Replantation and implantation of teeth. Znt. dent. J. 5,439-457. HUEBSCH,R., COLEMAN,R. D., FRANDSEN,A. M. and BECKS,H. 1952. The healing process following molar extraction. 1. Normal male rats (Long-Evans strain) Oral Surg. $864-876. -rovA, M. and MATENA,V. 1962. Blood vessels of the rat molar. J. dent. Res. 41,650-660. KNESE, K. H. 1964. A histochemical study of the polysaccharides in osteogenic areas. In: Bone and Tooth (edited by BLACKWOOD,H.) pp. 283-287. Pergamon Press, Oxford. LINGHORNE,W. J. and O’C~NNEL, D. C. 1950. Studies in the regeneration and reattachment of supporting structures of the teeth. J. dent. Res. 29,419-428. LYE. H. and WAERHAUG.J. 1961. Exoerimental renlantation of teeth in dogs and monkeys. Archs oral Bio[. 3, 176-184. MACAPANPAN,L. C., WEINMANN,J. P. and BRODIE, A. G. 1954. Early tissue changes following tooth movement in rats. Angle Orthodont. 24,79-95. MCLEAN, F. C. and URIST, M. R. 1968. Bone, 3rd edn, p. 223. University of Chicago Press, London.

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MELCHER.A. H. 1969. Role of the periosteum in repair of wounds of the parietal bone of the rat. Archs oral Biol. 14,1101-l 109. MELCHER,A. H. and IRVING, J. T. 1962. The healing mechanism in artificially created circumscribed defects in the femora of albino rats. J. Bone Jt Surg. 44B, 928-936. MORRIS, M. L. and THOMPSON.R. H. 1963. Healing of human periodontal tissues following surgical detachment: factors related to the deposition of new cementum on dentine. Periodontics 1, 189-195. PFEIFFER,J. S. 1963. The growth of gingival tissue over denuded bone. J. Periodont. 34, 10-16. PRITCHARD,J. J. 1964. Histology of fracture repair. In: Modern Trends in Orthopaedics. Vol. 4, Science of Fractures (edited by CLARKE,J. M. P.) pp. 69-90. Butterworths. London. PRITCHARD,J. J. and RUZICKA, A. J. 1950. Comparison of fracture repair in frog, lizard and rat. .I. Anat., Lond. 84,236261. RADDEN,H. G. 1959. Local factors in healing of the alveolar tissues. Ann. Roy. CON. Surg. Eng. 24, 366-386. RAMFJORD,S. P., ENGLER,W. 0. and HINIKER,J. J. 1966. A radioautographic study of healing following simple gingivectomy. II. The connective tissue. J. Periodont. 37, 179-189. RETIEF, D. H. and DREYER,C. J. 1967. Effects of neural damage on the repair of bony defects in the rat. Archs oral Biol. 12,1035-1039. RUEDI, T. P. and BASSETT,C. A. L. 1967. Repair and remodelling in Millipore-isolated defects in cortical bone. Acta Amt. 68,509-531. SANDSTEDT,C. 1904. Einige Beitrage zur Theorie der Zahn-Regulierung. Nordisk. Tandl. Tidsk. No. 4 (Quoted by MACAPANPANet aI., 1954). SHERMAN,P. JR. 1968. Intentional replantation of teeth in dogs and monkeys. J. dent, Res. 47. 1066-1071. SLEDGE, C. B. 1968. Biochemical events in the epiphyseal plate and their physiological control, Clin. Orthopaed. 61,37-47. STALLARD,R. E. and I-IIA~~, W. H. 1968. The induction of new bone and cementum formation. 1. Retention of mineralized fragments within the flap. J. Periodont. 39, 273-277. WEISS,R., STAHL.S. S. and TONNA,E. A. 1968. Functional demands on the cell proliferative activity of the rat periodontium studied autoradiographically. J. dent, Res. 47, 1153-l 157. WILDERMAN,M. N. 1963. Repair after a periosteal retention procedure. J. Periodont. 34,487-503.

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FIG. 2. Wounded area 4 days after operation illustrating the junction (arrowed) between the half of the wound from which the periodontal ligament had been extirpated (E) and the half of the wound in which it was exposed but left intact (P). Ccmentocytes are present in the cementum underlying the part of the wound where periodontal ligament is present, but the lacunae in the cellular cementum underlying the other half of the wound are mostly empty. Haematoxylin and eosin. x 220 FIG. 3. A wound 4 days after operation. C-cementum; B-alveolar bone; W-wound; S-space from which periodontal ligament had been extirpated; E-exposed periodontal ligament; D-bone debris. The wound is filled with fibrin and polymorphonuclear leukocytes. Note the difference between the number of connective tissue cells in the exposed periodontal ligament and the adjacent undisturbed ligament (P), and in the ligament before operation illustrated in Fig. 4. Haematoxylin and eosin. x 55 FIG. 4. The appearance of a part of the operative site before operation. C-cementum; P-periodontal ligament; B-alveolar bone. Haematoxylin and eosin. x 220

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PLATE2 FIG. 5. Periodontal ligament exposed by wounding 4 days after operation. There are a number of cells in the field that have been arrested in metaphase (arrowed). Haematoxylin and eosin. x 530 FIG. 6. Undisturbed periodontal ligament adjacent to the wound 4 days after operation, Two of the cells in the field which had been arrested in metaphase are arrowed. Ccementum. This photomicrograph illustrates a part of the wound similar to that labelled ‘P’ in Fig. 3. Haematoxylin and eosin. x 530 FIG. I. A wound 7 days after operation. W-wound; D-denture; kenturn; S-space from which the periodontal ligament has been extirpated; E-exposed periodontal ligament; B-alveolar bone. The wound is being organized by vascular, young connective tissue. Haematoxylin and eosin. x 50

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PLATE 3 FIG. 8. Vascular, young connective tissue organizing the wound in the overlying soft tissues 1 week after operation. A number of cells have been arrested in metaphase, two of which are arrowed. Haematoxylin and eosin. x 530

Pro. 9. Periodontal ligament 1 week after operation. E-periodontal ligament exposed in the wound; P-cellular undisturbed periodontal ligament adjacent to the wound, from which cells appear to be migrating into the exposed periodontal ligament; U-undisturbed periodontal ligament remote from the wound; C-cementum; B-alveolar bone; A-artefact. Haematoxylin and eosin. x 75

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PLAlX 4 FIG. 10. Ankylosis (arrowed) between bony callus (BC) laid down in the repair process and the cementum (C) of the tooth 2 weeks after operation. The callus has proliferated into the periodontal space from which the periodontal ligament had been extirpated. Note that the lacunae in the cementum are empty. P-periodontal ligament; Arelatively acellular periodontal ligament; B-alveolar bone constituting the wall of the wound; D-inclusions of bone debris. Haematoxylin and eosin. x 220 FIG. 11. Ankylosis (arrowed) between bony callus (BC) and cementum (C) 2 weeks after operation. In this wound there is evidence that multinucleated giant cells (G) are engaged in resorption of cementurn. D-dentine. Haematoxyhn and eosin. x220

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PLATES FIG. 12. Ankylosis (arrowed) between endosteal callus (E) and cementum (C) in the apical aspect of the wound 2 weeks after operation. Note the relatively acellular (A) and more cellular periodontal ligament (P). B-alveolar bone. The periosteal bony callus (S) contains a nodule of cartilage (CA). The junction between endosteal callus and overlying soft connective tissue is clearly discernible. Haematoxylin and eosin. x75 FIG.13. High power view of Fig. 12 illustrating the hypertrophic cartilage cells (c) in the periosteal callus (p>. Haematoxylin and eosin. x 530 FIG. 14. Alveolar bone (B) forming the coronal wall of a wound 2 weeks after operation. Note the nodule of cartilage (CA) exhibiting hypertrophic chondrocytes in the periosteal callus (S). The bony callus has proliferated coronal to the crest of the alveolar bone. Haematoxylin and eosin. x 90

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FIG. 1. A wound 2 weeks after operation. Endosteal callus (E), whose junction with adjacent soft connective tissue (S) is not clearly discernible. Some of the osteoblasts (0) associated with the callus are spindle-shaped or epithelioid. Haematoxylin and eosin. x220 FIG. 16. A wound 3 weeks after operation. C-cementum; D-dentine. The bony callus (B) has proliferated external to the periodontal space but has not occluded the wound entirely. The periodontal ligament that was exposed at operation (p) has regained much of its cellularity, and is of ordered appearance. The connective tissue constituting the periodontal ligament that has replaced ligament extirpated at operation (E) is not as well ordered. The junction of the two halves of the wound (arrowed) is indicated by the presence or absence of cementocytes in the cementum. (see also Figs. 17 and 18). Haematoxylin and eosin. x 65 FrG. 17. Higher magnification of the periodontal ligament exposed at operation which is illustrated in Fig. 16. The junction between periodontal ligament and alveolar bone is clearly delineated. Haematoxylin and eosin. x 180

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FrG. 18. Higher magnification than in Fig. 16 of new connective tissue occupying the periodontal space from which periodontal ligament was extirpated at operation. This connective tissue is not as well ordered as that in the other half of the wound, and there are localized areas (at-rowed) where it is not easy to discern the periodontal liits of the new alveolar bone. Haematoxylin and eosin. x 180 FIG. 19. A remnant of periodontal ligament (A) exposed at operation that has not regained its cellularity 3 weeks after operation. Haematoxylin and eosin. x 130 FIG. 20. A periodontal space from which periodontal ligament has been extirpated, 4 weeks after operation. Endosteal callus (E) has proliferated into the space, which is now of irregular width, and in one situation (arrowed) it is separated from the *mentum of the tooth by only a thin sliver of connective tissue. A study of serial sections revealed that ankylosis had not occurred in this wound. Note that almost all the lacunae in the cementum are empty. Haematoxylin and eosin. x 220

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