Scand J Plast Reconstr Hand Surg 24: 13-19. 1990
HEALING OF MAXILLARY AND MANDIBULAR BONE DEFECTS USING A MEMBRANE TECHNIQUE Scand J Plast Surg Recontr Surg Hand Surg Downloaded from informahealthcare.com by Washington University Library on 01/21/15 For personal use only.
An Expcv-imental Stitdy in Monkeys
Christer Dahlin,' Jan Gottlow,' Anders Linde' and Sture Nyman' From ihe 'Deparirnenf of Oral Biorhernisfry rind the 'Deparfmenf of Periodonfology. Fncrtlfv of Odonfology. Gofhenhrrrg Uniuersity. Gofhenhrrrg. Sweden (Submitted for publication May 17, 1989)
A h s f r a c f . Cyst-like cavities in the jaw bone often heal incompletely owing to ingrowth of connective tissue, thus preventing osteogenesis from occurring. In the present study. a new membrane technique has been utilized in an attempt to improve bone healing. By means of an inert, porous membrane. placed in close contact with the bone surface, a secluded space is created which can only be repopulated by cells from the adjacent bone. Thus, osteogenesis is able to occur without interference from other tissue types. Through-and-through bone defects were produced bilaterally ( I ) in edentulous areas of monkey ( n = 5 ) mandibles, and (2) in conjunction with apicectomy of the lateral maxillary incisors, also in monkeys (n=7) . On one side, the defects were covered buccally as well as lingually/palatally with expanded PTFE membranes, whereas the defects on the other side served as controls (no membrane). In the mandible, complete bone healing was seen at all test sites after a healing period of 3 months. On the control side. 3 experimental sites showed bone discontinuity with a transosseous core of connective tissue. whereas some bone healing had occurred lingually at 2 sites. but with massive soft tissue ingrowth from the buccal side. In the maxillary periapical defects. all the membrane-covered defects had healed with bone closure after 3 months but with a minute portion of connective tissue, probably derived from the periodontal ligament. around the tooth apices. None of the control defects (no membrane) healed spontaneously. but all were filled with connective tissue to varying degrees. Seen in the context of previous results from our laboratory, this study gives further evidence for a clinical applicability of the membrane technique for bone healing.
Key words: bone healing, cystic defects, expanded PTFE membranes, monkeys.
Incomplete bone healing is a major problem following reconstructive surgery of large jaw bone defects caused by cysts or tumours (10). One of the main reasons is that connective tissue from surrounding soft tissues rapidly grows into the defect, thereby preventing ingrowth of bone-forming cells and os-
teogenesis (9). In order t o overcome such problems, bone grafting is often used. However, harvesting of autogenous bone grafts is traumatic, and resorption of the bone grafts is often noted (1, 11). Bone allografts, commercially available, act mainly as a passive scaffold for bone formation, and incorporation of the specimen with the host bone is uncertain and unpredictable ( 2 ) . Incomplete bone healing is also observed following endodontic treatment of periapical lesions which have penetrated both the buccal and lingual/ palatal cortical bone plates, i.e. have created a through-and-through defect in the alveolar bone (3, 12). Despite the fact that the pulpal bacterial infection is eliminated as a result of the treatment, bone healing does not always occur, owing to ingrowth of connctive tissue into the defect. In addition, radiographic follow-ups may not give a definitive answer as to whether or not the pathological process has ceased. This often leads to a need for a surgical re-entry, both in the case of periapical lesions and when treating other cyst-like cavities in the jaws. In a recent study, we have described a new membrane technique for improving bone regeneration (5). This technique is based on the hypothesis that different cellular components in the tissue have varying rates of migration into a wound area during healing. By placing an inert membrane barrier over the defect, in close contact with the bone tissue, a secluded space is created between the bone and the membrane. Since the membrane acts as a mechanical barrier, preventing the ingrowth of connective tissue into the defect, the only cells which are allowed to repopulate the wound area are cells originating from the surrounding bone. Thus, osteo.Srnnd J Plost R e < o n r f r Hund Sitry 24
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genesis is able t o occur without interference from other tissue types. In the studv by Dahlin e t al. (51, through-andthrough rat mandibular defects were found t o heal within a few weeks' time with complete regeneration of bone using the membrane technique. This was in contrast t o sites where n o membranes had been placed. T h e methodology has also been used in connection with dental implants in a study in rabbbits (6). B y placing porous membranes around titanium implants ad modurn B r i n e m a r k with t o o little bone coverage, it was possible t o create new bone which completely covered the exposed parts of the implants. The present investigation was designed t o evaluate whether improved bone regeneration can be achieved, using the membrane technique, following treatment of ( a ) wide through-and-through bone defects in edentulous areas of the monkey mandible, and ( b )through-and-through maxillary defects produced in conjunction with apicectomy in monkeys. These situations cause significant clinical problems in dentistry. Bone regeneration in the defects may not occur. Instead, the defects are filled mainly with connective tissue. Since our earlier results were obtained in relatively low-order phylogenetic species with characteristically high potential for osteogenesis (41, another aim of the present study was to evaluate the efficacy of the membrane technique in an animal more close t o the human. MATERIAL A N D METHODS Animals and anaesthesia. Seven adult monkeys (Macaca cynomolgus) were used in the study. In 2 monkeys no defects were made in the lower jaw, giving a total of 10 defects ( 5 test and 5 controls) in the mandible. In the maxilla, a total of 14 defects (7 test and 7 controls) were created. During surgical procedures, the animals were sedated with intramuscularly injected Ketalap (10 mg/kg body weight; Parke Davis, USA). Local anaesthesia at the sites of surgery was given using 1 ml 2% lidocain/adrenaline solution (Astra, Sodertalje, Sweden). Pre-experimental treatment. In the mandible, all the premolars and the first molars were extracted bilaterally. The sites were then allowed to heal for 3 months in order to re-establish normal continuity of the bone. During this period, the maxillary lateral incisors were treated endodontically and the root canals sealed with guttapercha using a conventional endodontic technique. Experimental procedure-mandible. After 3 months of healing, mucoperiosteal flaps were raised bilaterallv in the premolar regions of the mandible, both buccally and lingually. Transosseous defects with a vertical diameter of 8 Sc.ond J Plasr Reconsrr Hand Siirp 24
Fig. 1 . Transosseous defect in the mandible on the test side. The lingually placed membrane can be seen through the defect.
mm (k 1 mm) and a horizontal diameter of 12 mm ( 2 1 mm) were created on both sides of the jaw using a round bur and under extensive cooling with saline. Care was taken not to interfere with the neuro-vascular bundle emerging from the mental foramen. On the right side (test site). expanded PTFE membranes (Gore-TexTM, W. L. Gore & Assoc., Flagstaff, AZ, USA), with a pore size of 0.45 pm, were placed over the entrances of the defect in direct contact with the bone surface, both buccally and lingually, and extending 2-3 mm over the margins of the defect (Fig. 1). The flaps were thereafter re-sutured (C-4 silk sutures; Nobel Medica, Sundbyberg, Sweden) on the outer side of the membranes, aiming at full coverage. The left side (control site) served as a control. as no membranes were placed there. Experimental procedure-maxilla. In the maxilla, mucoperiosteal flaps were raised bilaterally in the region of the lateral incisors. Transosseous defects were made on both sides, using a 10 mm trephine bur under vigorous cooling with saline. Apicectomy was performed on both teeth. On the right side (rest site), expanded PTFE membranes were placed both buccally and palatally, totally covering the entrances to the defect (Fig. 21. The membranes were adjusted to extend about 3 mm over the margins of the bone defect. The flaps were re-sutured in order to obtain total coverage of the wound area. The contralateral side (left) served as a control, as no membranes were placed there. Post-surgical procedures. The sutures were removed after 14 d. Post-operative care included polishing and scaling of the teeth as well as cleaning of the wound area with 0.2 % chlorhexidine digluconate solution once every 14 d during a healing period of 3 months, after which the animals were sacrificed with an overdose of Pentotalsodium". The jaws were removed, and specimens containing the regions of the test and control defects were dissected free and placed in 10% buffered formalin. The specimens were decalcified in EDTA. dehydrated and embedded in paraffin. Serial sectioning in a bucco-lingual direction in the mandible and a bucco-palatal direction in the maxilla was performed. The sections (8 pm) were stained with Hema-
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Bone heuling with a membrane technique
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Fig. 2. Buccal aspect of a maxillary transosseous defect in the region of the lateral incisor. On the animal’s right side ( a ) . the defect was covered. both buccally and palatally with an expanded PTFE membrane in direct contact with the bone before repositioning of the muco-periosteal flap. On the contralateral side ( b ) . no membrane was placed.
toxylin-Eosin or van Gieson’s stain. In order to reveal any cartilage formation, specific staining was performed with Alcian Blue. From each specimen, five sections, representing the mid-portion of the defect, were used for light microscopic analysis.
RESULTS Owing to technical failures in the histological preparation procedures, one test and one control defect in the maxillary jaw had to be excluded from analysis. The material available for histological examinations thus comprised 6+6 (test and control) defects in the maxilla and 5 + 5 (test and control) defects in the mandible. At one mandibular site a perforation through the mucosa was seen buccally and a patch of the membrane was exposed. Minor inflammatory signs were seen clinically in this area. Except for this site, healing had occurred uneventfully, and the membranes were found to be in proper position under the soft tissue at all test sites. No clinical signs of inflammatory reactions were seen around these membranes. Owing to pronounced remodelling of the bone in the wound areas, the exact margins of the original defects were sometimes difficult to define. Mandibular defects
In the mandible, complete closure of the defects with new bone was seen at all test sites ( n = 5 ) (Fig.
3). In 4 cases a substantial apposition of new bone was also found on the surface of the intact parts of the mandibular bone within the boundaries of the membrane. Generally, the membranes were found histologically to be tightly attached to the bone surface. At 3 sites a slight apposition of bone was even found on the outer surface of the membrane. The membranes were well accepted by the host tissue, and no inflammatory cells or signs of foreign-body reactions were seen. Also at that particular test site where a patch of the buccal membrane had penetrated the oral mucosa. complete osseous healing of the defect was seen histologically. A layer of connective tissue had developed, however, between the new I y-formed bone and the inner surface of the membrane. At the contralateral control sites, 3 defects showed a non-union situation with no continuity of the bone. A transosseous core of soft connective tissue was seen at these sites (Fig. 4). Collagen fibers were seen throughout the defect a t right angles to the surface of the mandible. At 2 sites, the lingual cortical bone had achieved continuity, whereas a massive ingrowth of connective tissue into the defect had occurred from the buccal aspect. A marked buccal depression was seen and only small amounts of new bone were discovered in these areas. Scrind
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Fig. 4. Control side (no membrane) in the mandible after a
Fig. 3, Photomicrograph showing complete bone healing on the test (membrane) side in the mandible after a healing period of months. The original margins of the defect are indicated by arrOw heads, Note also the apposition of new bone on the outside of the mandible (left) beneath the membrane. H & E stain (x25).
No cartilage formation was seen in any of the test
or control specimens. Maxillary defects The test sites ( n = 6 ) in the maxilla presented a similar picture as the mandibular test sites. At all sites. the membranes were found to be in proper position in the tissue, and the defects had healed with almost complete closure by newly formed bone (Fig. 5 ) . However, around the apices of the lateral incisors, a small portion of connective tissue could be seen to dip into the defects. Virtually no inflammatory cells were present. A new cementum-like tissue. with inserting collagen fibers. had formed covering the cut root surfaces (Fig. 6). In 5 out of the 6 test sites, apposition of new bone had also occurred on the outer side of the membranes. At these sites.
3-month healing period. Arrow heads indicate the original margins of the defect. Marked depressions in the compact bone were seen both buccally and lingually. A transosseous core of fibrous connective tissue can be seen. with collagen fibers running from the buccal to the lingual mucosa, stain (x30),
collagen fibers could be seen inserting from the mucosa into the membrane surface. At the control sites available for histological analysis (n=6), none of the defects had healed with bone closure. The defects were, to varying extents. filled with a fibrous connective tissue core. The fibrous tissue was characterized by collagenous fibers running in parallel to the root surface and only few cellular elements could be seen (Fig. 7). The bone compacta had neither regained its continuity buccally. nor palatally, except at one site where the palatal cortical bone plate had healed to some extent. In some control defects, the trabecular bone had remodelled and created a bony wall around the through-and-through connective tissue core. N o newly-formed cementum layer was present on the cut root surfaces of the teeth at the control sites.
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Fig. 5 . Histological section of a through-and-through osseous defect (test site) in the upper jaw. Arrow heads indicate the original, buccal entrance to the defect. Complete bone healing is evident, except for a small soft tissue portion periapically (arrow). H & E stain (x80).
Fig. 6. Apical region of a lateral incisor at a test (membrane) site in the maxilla. Note
the newly formed cementum-like substance with inserting collagen fibers on the cut root surface. Minor infiltration of inflammatory cells can be seen. H & E stain ( X 135).
No root resorption was found at the lateral incisors at the test or control sites. N o signs of cartilage formation was seen in any of the specimens, test or contro I. 2-908221
DISCUSSION The present study demonstrates in monkey jaws that it is possible to achieve virtually complete regeneration of bone in a bone defect by preventing Scnnd J Pln.rt Reumstr Hand Sirrg 24
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Fig. 7. Control site (no membrane) in the maxilla. Arrow heads indicate the buccal entrance to the defect. After a 3-
month healing period, the defect was filled with fibrous connective tissue, indicating a stabilization of the healing in the region. Note the depressions in the compact bone both buccally and palatally. H & E stain (X70).
other types of cells than those derived from the surrounding bone from repopulating the wound area. Using a membrane technique, in which a biologically inert expanded PTFE membrane with a pore size that does not allow for the penetration of cells is placed around the defect, a secluded environment is created in which osteogenesis may take place relatively unimpeded. As shown by the control sites with no membrane, the defects would not have healed otherwise. It should be noted that the periosteum is sutured on the outer surface of the membrane. The results are in agreement with our previous findings in rats and rabbits ( 5 , 6 ) and give additional evidence for the potential clinical value of this osteo-permissive membrane technique. The mandibular bone defects demonstrated complete healing after 3 months with the use of the Scund J Plusr Reconsrr Hond
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membrane technique, whereas none of the control defects showed complete regeneration of bone. The 3 sites, where a core of fibrous tissue was found extending from the buccal to the lingual mucosa, probably represented a steady-state situation, since the direction of the collagen fibers was at right angles to the surface of the bone, i.e. the defect will probably not heal spontaneously with bone during the lifetime of the animal. It is not possible to compare our results in this respect with those of other investigations, since monkey mandibles seem to have been little used, if at all, to study experimental bone defects (13). The bone defects, created in the maxilla, had slightly different conditions for healing, since the through-and-through defects involved the apices of the lateral incisors, thereby providing a larger variation of cell types in the wound area. All the test sites showed complete healing with newly formed bone except for a small soft tissue portion, which had formed at the apices of the roots. Collagen fibers of this tissue were inserted into a newly formed cementum-like substance, which seemed to be attached to and totally covered the cut root surface. This fibrous tissue and the newly formed root cementum were probably produced by cells migrating in from the adjacent periodontal ligament (7). The maxillary control sites demonstrated incomplete healing with a transosseous core of fibrous tissue. This is in good agreement with the clinical appearance after endodontic therapy in humans, where severe defects, especially in the maxillary lateral incisor region, are frequently found to be filled with fibrous scar tissue rather than with bone (12). Attempts have been made to replace bone, lost in cystic jaw lesion, using various kinds of bone grafts (8). However, only limited success has been reported. The present results suggest that a pronounced improvement in bone regeneration is possible to achieve predictably, both in the maxilla and the mandible, using the membrane technique. The number of surgical procedures can be reduced, and the potential risk for wound infections, donor site morbidity, as well as foreign body reactions can be diminished. ACKNOWLEDGEMENTS This study was supported by the Swedish Medical Research Council and the Swedish National Board for Technical Development.
Bone heciling with
REFERENCES 1 . Adell R, Lekholm U , Grondahl K. Brinemark P-I.
Scand J Plast Surg Recontr Surg Hand Surg Downloaded from informahealthcare.com by Washington University Library on 01/21/15 For personal use only.
2.
3. 4.
5.
6.
Lindstrom J , Jakobsson M . Fresh autogenous bone grafts combined with fixtures in the treatment of severely resorbed edentulous upper jaws. In press (1989). Amler MH. Osteogenic potential of nonvital tissues and synthetic implant materials. J Periodontol 1987; 58: 758-763. Andreasen JO. Rud J. Modes of healing histologically after endodontic surgery in 70 cases. Int J Oral Surg 1972; I: 148-160. Bays RA. In: WB Irby, DW Shelton, eds. Current advances in oral and maxillofacial surgery: Current concepts in bone grafting. St. Louis: C . V . Mosby. 1983. Vol. 4, p. 109. Dahlin C, Linde A,, Gottlow J , Nyman S . Healing of bone defects by guided tissue regeneration. Plast Reconstr Surg 1988: 81: 672-676. Dahlin C. Sennerby L , Lekholm U. Linde A. Nyman S. Generation of new bone around titanium implants using a membrane technique. An experimental study
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memhrune techniqrie
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in rabbits. J Oral Maxillofac Implants 1989; 4: 19-25. 7. Gottlow J , Nyman S , Karring T. Lindhe J. New attachment formation as a result of controlled tissue regeneration. J Clin Periodontol 1984; 1 1 : 494-503. 8. Hjorting-Hansen E. Studies on implantation of anorganic bone in cystic jaw lesions. Thesis. Copenhagen: Munksgaard, 1970. 9. Hjorting-Hansen E. Andreasen JO. Incomplete bone healing of experimental cavities in dog mandibles. Br J Oral Surg 1971: 9: 33-40. 10. Kent JN, Zide MF. Wound healing: Bone and biomaterials. Otolaryngol Clin North Am 1984; 17: 273-319. I I. Manchester WM. Some technical improvements in the reconstruction of the mandible and temporomandibular joint. Plast Reconstr Surg 1972; 50: 249-256. 12. Rud J . Andreasen JO, Moller-Jensen JE. A multivariate analysis of the influence of various factors upon healing after endodontic surgery. Int J Oral Surg 1972: I : 258-27 I . 13. Schmitz JP, Hollinger JO. The critical size defect as an experimental model for craniomandibulofacial nonunions. Clin Orthop 1986: 205: 299-309.
HEALING OF MAXILLARY AND MANDIBULAR BONE DEFECTS USING A MEMBRANE TECHNIQUE Scand J Plast Surg Recontr Surg Hand Surg Downloaded from informahealthcare.com by Washington University Library on 01/21/15 For personal use only.
An Expcv-imental Stitdy in Monkeys
Christer Dahlin,' Jan Gottlow,' Anders Linde' and Sture Nyman' From ihe 'Deparirnenf of Oral Biorhernisfry rind the 'Deparfmenf of Periodonfology. Fncrtlfv of Odonfology. Gofhenhrrrg Uniuersity. Gofhenhrrrg. Sweden (Submitted for publication May 17, 1989)
A h s f r a c f . Cyst-like cavities in the jaw bone often heal incompletely owing to ingrowth of connective tissue, thus preventing osteogenesis from occurring. In the present study. a new membrane technique has been utilized in an attempt to improve bone healing. By means of an inert, porous membrane. placed in close contact with the bone surface, a secluded space is created which can only be repopulated by cells from the adjacent bone. Thus, osteogenesis is able to occur without interference from other tissue types. Through-and-through bone defects were produced bilaterally ( I ) in edentulous areas of monkey ( n = 5 ) mandibles, and (2) in conjunction with apicectomy of the lateral maxillary incisors, also in monkeys (n=7) . On one side, the defects were covered buccally as well as lingually/palatally with expanded PTFE membranes, whereas the defects on the other side served as controls (no membrane). In the mandible, complete bone healing was seen at all test sites after a healing period of 3 months. On the control side. 3 experimental sites showed bone discontinuity with a transosseous core of connective tissue. whereas some bone healing had occurred lingually at 2 sites. but with massive soft tissue ingrowth from the buccal side. In the maxillary periapical defects. all the membrane-covered defects had healed with bone closure after 3 months but with a minute portion of connective tissue, probably derived from the periodontal ligament. around the tooth apices. None of the control defects (no membrane) healed spontaneously. but all were filled with connective tissue to varying degrees. Seen in the context of previous results from our laboratory, this study gives further evidence for a clinical applicability of the membrane technique for bone healing.
Key words: bone healing, cystic defects, expanded PTFE membranes, monkeys.
Incomplete bone healing is a major problem following reconstructive surgery of large jaw bone defects caused by cysts or tumours (10). One of the main reasons is that connective tissue from surrounding soft tissues rapidly grows into the defect, thereby preventing ingrowth of bone-forming cells and os-
teogenesis (9). In order t o overcome such problems, bone grafting is often used. However, harvesting of autogenous bone grafts is traumatic, and resorption of the bone grafts is often noted (1, 11). Bone allografts, commercially available, act mainly as a passive scaffold for bone formation, and incorporation of the specimen with the host bone is uncertain and unpredictable ( 2 ) . Incomplete bone healing is also observed following endodontic treatment of periapical lesions which have penetrated both the buccal and lingual/ palatal cortical bone plates, i.e. have created a through-and-through defect in the alveolar bone (3, 12). Despite the fact that the pulpal bacterial infection is eliminated as a result of the treatment, bone healing does not always occur, owing to ingrowth of connctive tissue into the defect. In addition, radiographic follow-ups may not give a definitive answer as to whether or not the pathological process has ceased. This often leads to a need for a surgical re-entry, both in the case of periapical lesions and when treating other cyst-like cavities in the jaws. In a recent study, we have described a new membrane technique for improving bone regeneration (5). This technique is based on the hypothesis that different cellular components in the tissue have varying rates of migration into a wound area during healing. By placing an inert membrane barrier over the defect, in close contact with the bone tissue, a secluded space is created between the bone and the membrane. Since the membrane acts as a mechanical barrier, preventing the ingrowth of connective tissue into the defect, the only cells which are allowed to repopulate the wound area are cells originating from the surrounding bone. Thus, osteo.Srnnd J Plost R e < o n r f r Hund Sitry 24
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genesis is able t o occur without interference from other tissue types. In the studv by Dahlin e t al. (51, through-andthrough rat mandibular defects were found t o heal within a few weeks' time with complete regeneration of bone using the membrane technique. This was in contrast t o sites where n o membranes had been placed. T h e methodology has also been used in connection with dental implants in a study in rabbbits (6). B y placing porous membranes around titanium implants ad modurn B r i n e m a r k with t o o little bone coverage, it was possible t o create new bone which completely covered the exposed parts of the implants. The present investigation was designed t o evaluate whether improved bone regeneration can be achieved, using the membrane technique, following treatment of ( a ) wide through-and-through bone defects in edentulous areas of the monkey mandible, and ( b )through-and-through maxillary defects produced in conjunction with apicectomy in monkeys. These situations cause significant clinical problems in dentistry. Bone regeneration in the defects may not occur. Instead, the defects are filled mainly with connective tissue. Since our earlier results were obtained in relatively low-order phylogenetic species with characteristically high potential for osteogenesis (41, another aim of the present study was to evaluate the efficacy of the membrane technique in an animal more close t o the human. MATERIAL A N D METHODS Animals and anaesthesia. Seven adult monkeys (Macaca cynomolgus) were used in the study. In 2 monkeys no defects were made in the lower jaw, giving a total of 10 defects ( 5 test and 5 controls) in the mandible. In the maxilla, a total of 14 defects (7 test and 7 controls) were created. During surgical procedures, the animals were sedated with intramuscularly injected Ketalap (10 mg/kg body weight; Parke Davis, USA). Local anaesthesia at the sites of surgery was given using 1 ml 2% lidocain/adrenaline solution (Astra, Sodertalje, Sweden). Pre-experimental treatment. In the mandible, all the premolars and the first molars were extracted bilaterally. The sites were then allowed to heal for 3 months in order to re-establish normal continuity of the bone. During this period, the maxillary lateral incisors were treated endodontically and the root canals sealed with guttapercha using a conventional endodontic technique. Experimental procedure-mandible. After 3 months of healing, mucoperiosteal flaps were raised bilaterallv in the premolar regions of the mandible, both buccally and lingually. Transosseous defects with a vertical diameter of 8 Sc.ond J Plasr Reconsrr Hand Siirp 24
Fig. 1 . Transosseous defect in the mandible on the test side. The lingually placed membrane can be seen through the defect.
mm (k 1 mm) and a horizontal diameter of 12 mm ( 2 1 mm) were created on both sides of the jaw using a round bur and under extensive cooling with saline. Care was taken not to interfere with the neuro-vascular bundle emerging from the mental foramen. On the right side (test site). expanded PTFE membranes (Gore-TexTM, W. L. Gore & Assoc., Flagstaff, AZ, USA), with a pore size of 0.45 pm, were placed over the entrances of the defect in direct contact with the bone surface, both buccally and lingually, and extending 2-3 mm over the margins of the defect (Fig. 1). The flaps were thereafter re-sutured (C-4 silk sutures; Nobel Medica, Sundbyberg, Sweden) on the outer side of the membranes, aiming at full coverage. The left side (control site) served as a control. as no membranes were placed there. Experimental procedure-maxilla. In the maxilla, mucoperiosteal flaps were raised bilaterally in the region of the lateral incisors. Transosseous defects were made on both sides, using a 10 mm trephine bur under vigorous cooling with saline. Apicectomy was performed on both teeth. On the right side (rest site), expanded PTFE membranes were placed both buccally and palatally, totally covering the entrances to the defect (Fig. 21. The membranes were adjusted to extend about 3 mm over the margins of the bone defect. The flaps were re-sutured in order to obtain total coverage of the wound area. The contralateral side (left) served as a control, as no membranes were placed there. Post-surgical procedures. The sutures were removed after 14 d. Post-operative care included polishing and scaling of the teeth as well as cleaning of the wound area with 0.2 % chlorhexidine digluconate solution once every 14 d during a healing period of 3 months, after which the animals were sacrificed with an overdose of Pentotalsodium". The jaws were removed, and specimens containing the regions of the test and control defects were dissected free and placed in 10% buffered formalin. The specimens were decalcified in EDTA. dehydrated and embedded in paraffin. Serial sectioning in a bucco-lingual direction in the mandible and a bucco-palatal direction in the maxilla was performed. The sections (8 pm) were stained with Hema-
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Fig. 2. Buccal aspect of a maxillary transosseous defect in the region of the lateral incisor. On the animal’s right side ( a ) . the defect was covered. both buccally and palatally with an expanded PTFE membrane in direct contact with the bone before repositioning of the muco-periosteal flap. On the contralateral side ( b ) . no membrane was placed.
toxylin-Eosin or van Gieson’s stain. In order to reveal any cartilage formation, specific staining was performed with Alcian Blue. From each specimen, five sections, representing the mid-portion of the defect, were used for light microscopic analysis.
RESULTS Owing to technical failures in the histological preparation procedures, one test and one control defect in the maxillary jaw had to be excluded from analysis. The material available for histological examinations thus comprised 6+6 (test and control) defects in the maxilla and 5 + 5 (test and control) defects in the mandible. At one mandibular site a perforation through the mucosa was seen buccally and a patch of the membrane was exposed. Minor inflammatory signs were seen clinically in this area. Except for this site, healing had occurred uneventfully, and the membranes were found to be in proper position under the soft tissue at all test sites. No clinical signs of inflammatory reactions were seen around these membranes. Owing to pronounced remodelling of the bone in the wound areas, the exact margins of the original defects were sometimes difficult to define. Mandibular defects
In the mandible, complete closure of the defects with new bone was seen at all test sites ( n = 5 ) (Fig.
3). In 4 cases a substantial apposition of new bone was also found on the surface of the intact parts of the mandibular bone within the boundaries of the membrane. Generally, the membranes were found histologically to be tightly attached to the bone surface. At 3 sites a slight apposition of bone was even found on the outer surface of the membrane. The membranes were well accepted by the host tissue, and no inflammatory cells or signs of foreign-body reactions were seen. Also at that particular test site where a patch of the buccal membrane had penetrated the oral mucosa. complete osseous healing of the defect was seen histologically. A layer of connective tissue had developed, however, between the new I y-formed bone and the inner surface of the membrane. At the contralateral control sites, 3 defects showed a non-union situation with no continuity of the bone. A transosseous core of soft connective tissue was seen at these sites (Fig. 4). Collagen fibers were seen throughout the defect a t right angles to the surface of the mandible. At 2 sites, the lingual cortical bone had achieved continuity, whereas a massive ingrowth of connective tissue into the defect had occurred from the buccal aspect. A marked buccal depression was seen and only small amounts of new bone were discovered in these areas. Scrind
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Fig. 4. Control side (no membrane) in the mandible after a
Fig. 3, Photomicrograph showing complete bone healing on the test (membrane) side in the mandible after a healing period of months. The original margins of the defect are indicated by arrOw heads, Note also the apposition of new bone on the outside of the mandible (left) beneath the membrane. H & E stain (x25).
No cartilage formation was seen in any of the test
or control specimens. Maxillary defects The test sites ( n = 6 ) in the maxilla presented a similar picture as the mandibular test sites. At all sites. the membranes were found to be in proper position in the tissue, and the defects had healed with almost complete closure by newly formed bone (Fig. 5 ) . However, around the apices of the lateral incisors, a small portion of connective tissue could be seen to dip into the defects. Virtually no inflammatory cells were present. A new cementum-like tissue. with inserting collagen fibers. had formed covering the cut root surfaces (Fig. 6). In 5 out of the 6 test sites, apposition of new bone had also occurred on the outer side of the membranes. At these sites.
3-month healing period. Arrow heads indicate the original margins of the defect. Marked depressions in the compact bone were seen both buccally and lingually. A transosseous core of fibrous connective tissue can be seen. with collagen fibers running from the buccal to the lingual mucosa, stain (x30),
collagen fibers could be seen inserting from the mucosa into the membrane surface. At the control sites available for histological analysis (n=6), none of the defects had healed with bone closure. The defects were, to varying extents. filled with a fibrous connective tissue core. The fibrous tissue was characterized by collagenous fibers running in parallel to the root surface and only few cellular elements could be seen (Fig. 7). The bone compacta had neither regained its continuity buccally. nor palatally, except at one site where the palatal cortical bone plate had healed to some extent. In some control defects, the trabecular bone had remodelled and created a bony wall around the through-and-through connective tissue core. N o newly-formed cementum layer was present on the cut root surfaces of the teeth at the control sites.
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Bone healing with a membrane technique
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Fig. 5 . Histological section of a through-and-through osseous defect (test site) in the upper jaw. Arrow heads indicate the original, buccal entrance to the defect. Complete bone healing is evident, except for a small soft tissue portion periapically (arrow). H & E stain (x80).
Fig. 6. Apical region of a lateral incisor at a test (membrane) site in the maxilla. Note
the newly formed cementum-like substance with inserting collagen fibers on the cut root surface. Minor infiltration of inflammatory cells can be seen. H & E stain ( X 135).
No root resorption was found at the lateral incisors at the test or control sites. N o signs of cartilage formation was seen in any of the specimens, test or contro I. 2-908221
DISCUSSION The present study demonstrates in monkey jaws that it is possible to achieve virtually complete regeneration of bone in a bone defect by preventing Scnnd J Pln.rt Reumstr Hand Sirrg 24
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C. Dahlin et al.
Fig. 7. Control site (no membrane) in the maxilla. Arrow heads indicate the buccal entrance to the defect. After a 3-
month healing period, the defect was filled with fibrous connective tissue, indicating a stabilization of the healing in the region. Note the depressions in the compact bone both buccally and palatally. H & E stain (X70).
other types of cells than those derived from the surrounding bone from repopulating the wound area. Using a membrane technique, in which a biologically inert expanded PTFE membrane with a pore size that does not allow for the penetration of cells is placed around the defect, a secluded environment is created in which osteogenesis may take place relatively unimpeded. As shown by the control sites with no membrane, the defects would not have healed otherwise. It should be noted that the periosteum is sutured on the outer surface of the membrane. The results are in agreement with our previous findings in rats and rabbits ( 5 , 6 ) and give additional evidence for the potential clinical value of this osteo-permissive membrane technique. The mandibular bone defects demonstrated complete healing after 3 months with the use of the Scund J Plusr Reconsrr Hond
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membrane technique, whereas none of the control defects showed complete regeneration of bone. The 3 sites, where a core of fibrous tissue was found extending from the buccal to the lingual mucosa, probably represented a steady-state situation, since the direction of the collagen fibers was at right angles to the surface of the bone, i.e. the defect will probably not heal spontaneously with bone during the lifetime of the animal. It is not possible to compare our results in this respect with those of other investigations, since monkey mandibles seem to have been little used, if at all, to study experimental bone defects (13). The bone defects, created in the maxilla, had slightly different conditions for healing, since the through-and-through defects involved the apices of the lateral incisors, thereby providing a larger variation of cell types in the wound area. All the test sites showed complete healing with newly formed bone except for a small soft tissue portion, which had formed at the apices of the roots. Collagen fibers of this tissue were inserted into a newly formed cementum-like substance, which seemed to be attached to and totally covered the cut root surface. This fibrous tissue and the newly formed root cementum were probably produced by cells migrating in from the adjacent periodontal ligament (7). The maxillary control sites demonstrated incomplete healing with a transosseous core of fibrous tissue. This is in good agreement with the clinical appearance after endodontic therapy in humans, where severe defects, especially in the maxillary lateral incisor region, are frequently found to be filled with fibrous scar tissue rather than with bone (12). Attempts have been made to replace bone, lost in cystic jaw lesion, using various kinds of bone grafts (8). However, only limited success has been reported. The present results suggest that a pronounced improvement in bone regeneration is possible to achieve predictably, both in the maxilla and the mandible, using the membrane technique. The number of surgical procedures can be reduced, and the potential risk for wound infections, donor site morbidity, as well as foreign body reactions can be diminished. ACKNOWLEDGEMENTS This study was supported by the Swedish Medical Research Council and the Swedish National Board for Technical Development.
Bone heciling with
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Lindstrom J , Jakobsson M . Fresh autogenous bone grafts combined with fixtures in the treatment of severely resorbed edentulous upper jaws. In press (1989). Amler MH. Osteogenic potential of nonvital tissues and synthetic implant materials. J Periodontol 1987; 58: 758-763. Andreasen JO. Rud J. Modes of healing histologically after endodontic surgery in 70 cases. Int J Oral Surg 1972; I: 148-160. Bays RA. In: WB Irby, DW Shelton, eds. Current advances in oral and maxillofacial surgery: Current concepts in bone grafting. St. Louis: C . V . Mosby. 1983. Vol. 4, p. 109. Dahlin C, Linde A,, Gottlow J , Nyman S . Healing of bone defects by guided tissue regeneration. Plast Reconstr Surg 1988: 81: 672-676. Dahlin C. Sennerby L , Lekholm U. Linde A. Nyman S. Generation of new bone around titanium implants using a membrane technique. An experimental study
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in rabbits. J Oral Maxillofac Implants 1989; 4: 19-25. 7. Gottlow J , Nyman S , Karring T. Lindhe J. New attachment formation as a result of controlled tissue regeneration. J Clin Periodontol 1984; 1 1 : 494-503. 8. Hjorting-Hansen E. Studies on implantation of anorganic bone in cystic jaw lesions. Thesis. Copenhagen: Munksgaard, 1970. 9. Hjorting-Hansen E. Andreasen JO. Incomplete bone healing of experimental cavities in dog mandibles. Br J Oral Surg 1971: 9: 33-40. 10. Kent JN, Zide MF. Wound healing: Bone and biomaterials. Otolaryngol Clin North Am 1984; 17: 273-319. I I. Manchester WM. Some technical improvements in the reconstruction of the mandible and temporomandibular joint. Plast Reconstr Surg 1972; 50: 249-256. 12. Rud J . Andreasen JO, Moller-Jensen JE. A multivariate analysis of the influence of various factors upon healing after endodontic surgery. Int J Oral Surg 1972: I : 258-27 I . 13. Schmitz JP, Hollinger JO. The critical size defect as an experimental model for craniomandibulofacial nonunions. Clin Orthop 1986: 205: 299-309.
