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Restoration of mandibular nonunion bone defects
Copyright © Munksgaard 1994
Int. J. Oral MaxilloJac. Surg. 1994; 23:237-242 Printed in Denmark. All rights reserved
[ntemationalJoumal of
Oral& MaxillofacialSurgery ISSN 0901-5027
Restoration of mandibular nonunion bone defects
C. Dahlin 1, E. Sandberg 1, P. Alberius 2, A. Linde 1 1Department of Oral Biochemistry, G~teborg University; 2Department of Plastic Surgery, Maim6 General Hospital, Sweden
An experimental study in rats using an osteopromotive membrane method C. Dahlin, E. Sandberg, P. Alberius, A. Linde: Restoration of mandibular nonunion bone defects. An experimental study in rats using an osteopromotive membrane method. Int. J. Oral Maxillofac. Surg. 1994; 23." 237-242. © Munksgaard, 1994 Abstract. Standardized through-and-through critical size defects were created in rat mandibles. After 12 weeks, the sites revealed a massive ingrowth of soft connective tissue, forming a transosseous core filling the defects. Upon reentry, the soft tissue inside the remaining bone defects was removed. On one side of the jaw, the defect was covered both buccally and lingually with an expanded polytetrafluoroethylene (e-PTFE) membrane, but on the other side no membrane was placed. Histologic analysis after 6 weeks revealed an essentially complete healing with bone of the membrane-covered defects. No cartilage was present in any of the specimens. At the control sites (no membrane), the amount of newly produced bone showed variations, most through defects revealing the presence of a remaining central portion of connective tissue. This investigation thus showed that predictable and successful bone regeneration can be achieved by the osteopromotive membrane method in treatment of nonunion defects filled with mature connective tissue.
Jawbone defects often occur as the result of cysts, infection, or tumors. Bone regeneration to bridge such defects is a complex process influenced by age, bone structure, vascularization, and soft-tissue environment, as well as by the size of the defect ~8. Although bone defects normally heal by bony union, incomplete healing often ensues clinically. This is largely due to rapid ingrowth into the wound area of connective tissue from the surroundings. Histologically, nonunions are characterized by fibrous connective tissue ~9, and in long-bone fractures hyaline cartilage may develop between the segments 2. Such tissue reactions delay or prevent the formation of new bone, causing anatomic aberrations and functional disturbances. The formation of scar tissue is thus a major problem after bone
surgery ~3, frequently necessitating reoperation. In a series of experimental investigations, we have described an osteopromotive membrane method which has consistently improved bone regeneration (for review, see Reference 14). The principle of accomplishing unimpeded osteogenesis is mechanically to prevent soft-tissue ingrowth into the bone defect. We have utilized an inert, porous, expanded polytetrafluoroethylene (ePTFE) membrane, which is placed as a barrier over the defect, thus preventing the rapidly migrating connective-tissue cells from interfering with the osteogenic process. The studies have clearly shown that this membrane method is a valuable tool in clinical practice 6,8. However, it has not yet been established whether the osteopromotive
Key words: bone regeneration; osteogenesis; maxillofacial surgery; reconstructive Surgery; polytetrafluoroethylene (PTFE); artificial membrane. Accepted for publication 10 March 1994
membrane method after second stage reentry, contributes to successful treatment of nonunion bone defects filled with fibrous connective tissue. The purpose of the present study was, firstly, to develop an experimental model exhibiting reproducible nonunion osseous defects filled with mature fibrous connective tissue, and, secondly, to study the potential of the osteopromotive membrane method to restore such nonunion bone defects.
Material and methods Animals and anesthesia
Thirteen male albino Sprague-Dawley rats (body Weigtit450-550 g) were used. The animals were kept in plastic cages in a room with 12-h light-dark cycles, and an ambient temperature of 21 °C. They were allowed free
238
Dahlm et al. raised bilaterally at both the buccal and the lingual aspects of the mandibular angles in 13 animals. A standardized through-andthrough osseous defect (5 m m in diameter) was created bilaterally by a trephine burr 9 (Fig. 1). The flaps were repositioned carefully and sutured to obtain full coverage of the surgical site. All animals seemed to have tolerated the surgical procedure well, and they all gained weight postoperatively. They were allowed to heal for a period of 12 weeks, after which a reentry operation was performed.
Fig. 1. Preparation of 5-mm transosseous defect, through mandibular angle of rat, at surgery I.
access to drinking water and standard laboratory food pellets. Before surgery, the animals were anesthetized with an intraperitoneal injection (3.3 ml/kg body weight) consisting of 25% Hypnorm Vet.~ (Janssen Pharmaceuticals, Beerse, Belgium), 25% Dormicum ~ (Hoffman-LaRoche, Basel, Switzerland), and 50% sterile water. The skin overlying the mandibular angles was shaved and cleaned with 70% ethanol. During surgery, supplemental seda-
tion was administered as needed. Local anesthetic, 1 ml of 2% lidocaine/adrenalin solution (Astra, S6dert~lje, Sweden), was given at the site of surgery. Postoperatively, the animals were given daily doses of Temgesic ® (Reckitt & Colman, Hull, UK) for 1 week for pain relief and fed crushed standard pellets dispersed in water. The experimental outline was approved by the Animal Research Ethics Committee at GSteborg University, Sweden.
Surgical procedures Surgery I (12 weeks before surgery II). Musculoperiosteal flaps, based superiorly, were
Surgery H (12 weeks after surgery I). The anesthesia was done in the same manner as described above. Again, buccal and lingual musculoperiosteal flaps were raised bilaterally over the angles of the rat mandible. The regenerative result after the previous surgery was evaluated macroscopically after the bone had been dissected free from soft tissues, and, subsequently, photographs were taken (Fig. 2). The animals were divided into three groups to receive different surgical procedures. Procedure A. In this group (n = 4), the soft tissue inside the remaining bone defect was gently removed bilaterally so as not to cause damage or interfere with the established regenerated bone layer. On the right side, the remaining defect was covered both buccally and lingually by an e-PTFE membrane (Gore-Tex®; W. L. Gore & Associates, Flagstaff, AZ, USA), as previously described 9. The membrane was extended 2 3 mm beyond the margins of the defect to adhere tightly to the denuded bone surface (Fig. 3). Fixation was obtained by means of a transosseous ePTFE suture (Gore-Tex®). The buccal and lingual flaps, including the periosteum, were carefully repositioned superfically on the
Table 1. Results of bone healing 12 weeks after surgery I and 6 weeks after surgery II, respectively, in different groups Surgery I: macroscopic evaluation after 12 weeks of healing No. of sites
Complete healing
Persistent defect (3-5 mm)
Persistent defect (5 mm)
25
1
18
6
Surgery II: membrane-treated defects Surgical procedure II A II B II C
Complete healing
Remaining defect (< 1 mm)
4
4
0
0
4
3
1
0
5
5
0
0
Surgery II: control defects Surgical procedure
Fig. 2. Photograph showing reentry at surgery II after 12 weeks of initial healing. Note transosseous connective tissue core through created defect. Only minute apposition of new bone had occurred.
II A II B II C
results after 6 weeks of healing
No. of sites
Remaining defect (3-5 mm)
results after 6 weeks of healing
No. of sites
Complete healing
Remaining defect ( < 1 ram)
Remaining defect (3-5 mm)
4 4 4*
2 0 0
2 1 4
0 3 0
* One site was excluded from this group because of infection.
Osteopromotion in bone defects
239
identical, except that no membrane was applied. The postoperative treatment was identical to that used after surgery I. All animals were allowed to heal for 6 weeks. All animals gained weight during this period. Tissue processing
Fig. 3. e-PTFE membrane placed buccally and lingually over bone defect. Membrane is tightly adapted to denuded bone surface and fixed by transosseous suture.
Six weeks after the second surgical procedure, the animals were killed by an overdose of CO2, and block biopsies, comprising the bone defects with surrounding soft tissues, were prepared. Fixation by immersion in 4% buffered formalin was followed by demineralization in EDTA, dehydration, and embedding in bard paraffin. Serial horizontal sections (4 /m 0 were taken tbrougbout the defects. After staining with hematoxylin and eosin, the specimens were examined microscopically. The three most central sections of each experimental site were histologically evaluated by light microscopy. Results
Fig. 4. Histologic section showing complete healing of membrane (M)-treated nonunion defect 6 weeks after surgery II. Newly formed bone (NB) comprises mixture of woven and sparse lamellar bone. Arrowheads indicate original margins of nonunion defect (HE, × 40).
outer side of the membrane to achieve complete coverage of the membrane. The periosteum was thus located on the superficial surface of the membrane without direct contact with the bone defect. Treatment on the contratateral side was done as described above except that no membrane was placed over the defect.
Procedure B. In this group (n = 4), the bilaterally remaining bone defects were cleansed of the ingrowing connective tissue by curettage, corresponding to the more vigorous manner required in most clinical surgical situations. Through the defect, a new penetration,
baving the size of the original defect (5 mm in diameter), was re-created witb a trepbine burr (Fig. 1). On the right mandibular side, an e-PTFE membrane was placed over the defect and fixed as described above, while no membrane was used on the left side.
Procedure C. In the third group (n = 5), the soft tissue was removed from the persisting bilateral jaw defects in the same manner as in group A. On the right side, an e-PTFE membrane was applied over the cavity, but extra care was taken to ensure that the bone defect was completely filled with a blood clot. On the left side, the surgical procedure was
T h e results o f b o n e healing 12 weeks after surgery I a n d 6 weeks after surgery II, respectively, are s u m m a r i z e d in Table 1. The results after surgery I were evaluated macroscopically and photographically. T h e 25 sites revealed m a ture connective tissue filling the defects. N i n e t e e n sites showed varying degrees o f b o n e f o r m a t i o n along the m a r g i n s o f the defect, but, still, only one a n i m a l s h o w e d a r e m a i n i n g defect smaller t h a n 3 m m in diameter. In this defect, a continuous, very thin, bone-like tissue with a h e m a t o p o i e t i c c h a r a c t e r was found. N o ossification h a d occurred in the rem a i n i n g six animals, a n d the defects retained virtually the original d i a m e t e r o f 5 m m . Macroscopically, a massive ing r o w t h of soft connective tissue was generally found, f o r m i n g a transosseous core (Fig. 2). W h e n the rats were killed after the second healing period, local w o u n d complications were seen at two sites. O n e (in g r o u p C) was excluded f r o m f u r t h e r analysis, while the o t h e r showed a m o d e s t i n f l a m m a t o r y reaction a r o u n d a suture r e m n a n t , which was a s s u m e d to be o f late date. Generally, the m e m b r a n e s were f o u n d to be retained in their p r o p e r position b e n e a t h the soft tissue a n d tightly a d h e r i n g to the surface o f the b o n e formed. In general, the e - P F T E m e m b r a n e s were tolerated well. Foreignb o d y or i n f l a m m a t o r y reactions adj a c e n t to the m e m b r a n e s were virtually absent.
240
Dahlin et al.
Fig. 5. Persisting bone defect after removal of transosseous connective tissue core (Surgery
II, procedure C).
produced bone showed great variation (Fig. 5). In the sites operated according to procedure B, in which the original dimension of the defects (5 mm) was reestablished, three of four sites showed no or minimal new bone formation (Fig. 6). Conversely, in groups A and C, in which the new bone margins formed during the initial 12 weeks of healing after surgery I were preserved, a different picture was seen. Two of eight sites had healed completely with new bone (Fig. 7), whereas in six sites a substantial regeneration of new bone with a central portion of connective tissue measuring less than 1 mm was observed (Fig. 8). The soft-tissue core basically comprised a mixture of dense connective tissue with collagen fibers running parallel to the bone surface. Muscular tissue and adipose tissue were found to a limited extent. Generally, the newly formed bone in control defects was thinner than the surrounding bone (Figs. 7 and 8). Discussion
Fig. 6. Control defect, 6 weeks after currettage and reestablishment of original defect size (5 mm) (procedure II B). Only minor apposition of new bone is seen at margins of defect (arrows). Major part of defect is filled with dense connective tissue (CT) (HE, x 40).
The membrane-treated sites showed complete healing of the defects in 12 of 13 sites (Fig. 4), i.e., regardless of whether the sites were treated according to procedures A, B, or C at surgery II. One site showed a local infection external to the membrane. However, complete bone healing was seen within the margin of this particular defect. At another site, a central portion of connective tissue, with a width of less than 1 mm, was observed, containing islands of bone with active osteoblasts cen-
trally. In general, when compared with the original mandibular bone, the regenerated bone was found to exhibit increased buccolingual dimensions. The newly formed bone comprised a mixture of woven and sparse lamellar bone. Marrow spaces were generally found to a greater extent in the newly formed bone than the adjacent original bone. No cartilage was found at any membrane-treated site. At the control sites, where no membrane was applied, the amount of newly
Reentry procedures, to deal with incompletely healed bone defects, are common in oral and maxillofacial surgery as well as orthopedic surgery ~,3,r7. One example of such defects, which are filled with fibrous scar tissue rather than regenerated bone, is the defect observed in the maxillary lateral incisor region after endodontic therapy. Treatment, however, is often unsuccessful. This investigation, performed on standardized critical size defects in the rat mandible, has demonstrated that predictable and successful bone regeneration can be achieved by the osteopromotive membrane method in treatment of nonunion defects filled with mature connective tissue. Similar successful results in bone regeneration have ea,rlier been achieved by the membrane method in other experimental animal models TM. The transosseous defect in the rat mandible has earlier been used as an experimental model in several investigations9 11. The membrane method has also been found to give complete healing of transosseous mandibular defects in monkeys7 and of cranial defects in adult rats 5. All defects studied were "critical size defects ''18, a term implying that the size of the defect precludes spontaneous healing during the lifetime of the animal. The osteopromotion membrane method has also been successfully applied clinically in
Osteopromotion in bone dejects
Fig. 7. Histologic section, showing control defect from procedure A group. In this group, bone newly formed during initial 12-week healing period was preserved at reentry operation (arrows). Complete bone union (NB) can be seen after 6 additional weeks of healing after reentry (surgery II) (HE, x 40).
Fig. 8. Representative control defect from procedure C group. Six of eight sites in groups A and C showed substantial regeneration of new bone (NB) (as shown in Fig. 7) but with remaining central defect, less than 1 mm, filled with connective tissue (CT) (HE, x 40). placement of endosseous implants4'6'~'16. The efficacy of the method to accomplish complete bone healing at sites containing scar tissue has hitherto not been investigated. In most studies on the regeneration of bone defects, such defects have been created and immediately treated by membrane placement or application of some other osteogenesis-stimulating treatment. However, it is debatable how far conclusions from such experiments
are justified. It is generally believed that one way to initiate bone formation from bone wound margins is by the liberation of specific molecules with a capacity to cause multipotent, mesenchymal-type cells to differentiate into osteoblasts for production of new bone2a. Presumably, a more ideal situation for release of such growth factors is achieved in experimental models with freshly created bone defect margins than in quiescent, longstanding defects filled with scar tissue
241
and, thus, having inactive bone sur 2 faces H. Presumably, the rate of bone healing is determined by the degree of bone damage relative to the amount of soft tissue present. If the volume of soft tissue predominates, mesenchymal cells may acquire a fibroblastic phenotype instead of developing into cells with an osteogenic capacity 2'11. In the present study, all sites except one were filled with fibrous scar tissue 12 weeks after the initial surgical procedure. The direction of the collagen fibers was at right angles to the bone surface, and the margins of the defects showed rounded bone edges. These findings clearly imply that a steady-state situation had been reached; that is, the defect would not, by itself, heal any further by ossification. This corroborates previous studies using the same experimental model 9 II, The results of the present study showed that virtually complete osseous healing had taken place at all sites where occlusive membranes were placed. Thus, a mechanical hindrance of connective-tissue proliferation into the persistent bone defects is of profound importance for unimpeded bone healing. Conversely, in the control groups receiving no membranes, varying healing patterns were found. In groups A and C, where the bone margins were carefully preserved during scar tissue removal, two sites healed completely and the remaining six sites showed almost complete bone healing except for a sparse connective-tissue core in the center of the defects. This represents a higher frequency of spontaneous healing than found in earlier studies9 ~. However, this could be explained by the fact that these control defects had so diminished in diameter during the initial healing phase that the defects did not fulfill the criteria for a critical size defect ~8 at the second procedure. Nevertheless, the findings clearly showed that repeated removal of tissues interfering with normal bone regeneration and the reproduction of optimal healing environments may substantially contribute to a recovered regenerative capacity. In control group B, where the original 5-mm diameter of the defect was re-created by drilling, still another response was found; three of four sites showed little or no new bone formation in the nonunion defect. This seems to be logical, since the defects again fulfilled the requirements for a critical size defect.
242
Dahlin et aL
Establishing a b l o o d clot in j a w b o n e defects has been considered to be crucial to optimize initial b o n e healing 4'~6, alt h o u g h experimental l o n g - b o n e fracture healing seems n o t to be i m p a i r e d after the early clot has b e e n r e m o v e d b y asp i r a t i o n 2°. In g r o u p C, extra care was t a k e n to secure such a b l o o d clot within the b o n e defect. Presumably, a similar a m o u n t of bleeding occurred in the other groups also, because n o difference could be f o u n d f r o m g r o u p A. In conclusion, this study shows t h a t the o s t e o p r o m o t i o n m e m b r a n e m e t h o d m a y be beneficial to the t r e a t m e n t o f n o n u n i o n b o n e defects where a less osteogenic e n v i r o n m e n t is present t h a n in fresh defects. This finding m a y p r o v e to be essential in various clinical situations, such as cystic b o n e defects, periapical lesions, a n d possibly pseudoarthrosis as a result o f n o n h e a l i n g o f b o n e fractures.
Acknowledgments. The authors gratefully acknowledge the technical assistance given by Ms Kerstin Bjurstam and Ms Yvonne Sundin. This study was supported by the Swedish Medical Research Council (grant 2789) and the Faculty of Odontology, G6teborg University, Sweden.
References
I. ANDREASENJO, RUD J. Modes of healing histologically after endodontic surgery in 70 cases. Int J Oral Surg 1972: 1:148 60. 2. ASHHORSTDE. The influence of mechanical conditions on the healing of experimental fractures in the rabbit: a micro-
scopic study. Philos Trans R Soc Lond [Biol] 1986: 313: 271-302. 3. AUSTXNRT. Fractures of the tibial shaft: is medical audit possible? Injury 1977: 9: 93 101. 4. BUSER D, BR,~GGERU, LANG N, NYMAN S. Regeneration and enlargement of jawbone using guided tissue regeneration. Clin Oral Implant Res 1990: 1:22 32. 5. DAHLIN C, ALBERIUSP, LINDE A. Osteopromotion for cranioplasty. An experimental study in rats using a membrane technique. J Neurosurg 1991: 74: 48791. 6. DAHLIN C, ANDERSSONL, L1NDEA. Bone augmentation at fenestrated implants by an osteopromotive membrane technique. A controlled clinical study. Clin Oral Implant Res 1991: 2: 159-65. 7. DAHLIN C, GOTTLOWJ, LINDEA, NYMAN S. Healing of maxillary and mandibular bone defects by a membrane technique: an experimental study in monkeys. Scand J Plast Reconstr Surg 1990: 24:13 19. 8. DAHLIN C, LEKHOLM U, LINDE A. Induced bone augmentation at titanium implants using a membrane technique: a report on l0 fixtures followed 1 to 3 years after loading. J Periodont Rest Dent 1991: 11: 273-81. 9. DAHL1N C, LINDE A, GOTTLOWJ, NYMAN S. Healing of bone defects by guided tissue regeneration. Plast Reconstr Surg 1988: 81: 672-6. 10. GLOWACKI J, ALTOBELLI D, MULLIKEN JB. Fate of mineralized and demineralized osseous implants in cranial defects. Calcif Tissue Int 1981: 33:71-6. 11. HULTH A. Fracture healing: a concept of competing healing factors. Acta Orthop Scand 1980: 51: 5-8. 12. KABAN LB, GLOWACKIJ. Induced osteogenesis in the repair of experimental mandibular defects in rats. J Dent Res 1981: 60: 1356-64.
13. KENTJN, ZIDE M E Wound healing: bone and biomaterials. Otolaryngol Clin North Am 1984: 17: 273-319. 14. LINDE A, ALBERIUSP, DAHLIN C, BJURSTAM K, SUNDIN Y. Osteopromotion. A soft-tissue exclusion principle using a membrane for bone healing and bone neogenesis. J Periodontol 1993: 64: 1116-28. 15. MULLER WE. Bases exp6rimentales et principes de l'ost6osynth&es par compression. Int Orthop 1978: 2:115 25. 16. NYMAN S, LANG N, BUSER D, BR,~GGER U. Bone regeneration adjacent to titanium dental implants using guided tissue regeneration: a report of two cases. Int J Oral Maxillofac Implant 1990: 5: 9 14. 17. RUb J, ANDREASEN JO, M6LLER-JENSEN JE. A multivariate analysis of the influence of various factors upon healing after endodontic surgery. Int J Oral Surg 1972: 1: 258-71. 18. SCHMITZ JP, HOLLINGER JO. The critical size defect as an experimental model for craniomandibulofacial nonunions. Clin Orthop 1979: 205: 299-308. 19. SCHMITZ JP, SCHWARTZ Z, HOLLINGER JO, BOYAN BD. Characterization of rat calvarial nonunion defects. Acta Anat (Basel) 1990: 138: 185-92. 20. SIMMONS DJ. Fracture healing perspectives. Clin Orthop 1985: 200:100 13. 21. URIST MR, McLEAN FC. Recent advances in the physiology of bone. I. J. Bone Joint Surg [Am] 1964: 45:1305 13.
Address: Dr Anders Linde Department of Oral Biochemistry Medicinaregatan 7B S-413 90 Gothenburg Sweden
Int. J. Oral MaxilloJac. Surg. 1994; 23:237-242 Printed in Denmark. All rights reserved
[ntemationalJoumal of
Oral& MaxillofacialSurgery ISSN 0901-5027
Restoration of mandibular nonunion bone defects
C. Dahlin 1, E. Sandberg 1, P. Alberius 2, A. Linde 1 1Department of Oral Biochemistry, G~teborg University; 2Department of Plastic Surgery, Maim6 General Hospital, Sweden
An experimental study in rats using an osteopromotive membrane method C. Dahlin, E. Sandberg, P. Alberius, A. Linde: Restoration of mandibular nonunion bone defects. An experimental study in rats using an osteopromotive membrane method. Int. J. Oral Maxillofac. Surg. 1994; 23." 237-242. © Munksgaard, 1994 Abstract. Standardized through-and-through critical size defects were created in rat mandibles. After 12 weeks, the sites revealed a massive ingrowth of soft connective tissue, forming a transosseous core filling the defects. Upon reentry, the soft tissue inside the remaining bone defects was removed. On one side of the jaw, the defect was covered both buccally and lingually with an expanded polytetrafluoroethylene (e-PTFE) membrane, but on the other side no membrane was placed. Histologic analysis after 6 weeks revealed an essentially complete healing with bone of the membrane-covered defects. No cartilage was present in any of the specimens. At the control sites (no membrane), the amount of newly produced bone showed variations, most through defects revealing the presence of a remaining central portion of connective tissue. This investigation thus showed that predictable and successful bone regeneration can be achieved by the osteopromotive membrane method in treatment of nonunion defects filled with mature connective tissue.
Jawbone defects often occur as the result of cysts, infection, or tumors. Bone regeneration to bridge such defects is a complex process influenced by age, bone structure, vascularization, and soft-tissue environment, as well as by the size of the defect ~8. Although bone defects normally heal by bony union, incomplete healing often ensues clinically. This is largely due to rapid ingrowth into the wound area of connective tissue from the surroundings. Histologically, nonunions are characterized by fibrous connective tissue ~9, and in long-bone fractures hyaline cartilage may develop between the segments 2. Such tissue reactions delay or prevent the formation of new bone, causing anatomic aberrations and functional disturbances. The formation of scar tissue is thus a major problem after bone
surgery ~3, frequently necessitating reoperation. In a series of experimental investigations, we have described an osteopromotive membrane method which has consistently improved bone regeneration (for review, see Reference 14). The principle of accomplishing unimpeded osteogenesis is mechanically to prevent soft-tissue ingrowth into the bone defect. We have utilized an inert, porous, expanded polytetrafluoroethylene (ePTFE) membrane, which is placed as a barrier over the defect, thus preventing the rapidly migrating connective-tissue cells from interfering with the osteogenic process. The studies have clearly shown that this membrane method is a valuable tool in clinical practice 6,8. However, it has not yet been established whether the osteopromotive
Key words: bone regeneration; osteogenesis; maxillofacial surgery; reconstructive Surgery; polytetrafluoroethylene (PTFE); artificial membrane. Accepted for publication 10 March 1994
membrane method after second stage reentry, contributes to successful treatment of nonunion bone defects filled with fibrous connective tissue. The purpose of the present study was, firstly, to develop an experimental model exhibiting reproducible nonunion osseous defects filled with mature fibrous connective tissue, and, secondly, to study the potential of the osteopromotive membrane method to restore such nonunion bone defects.
Material and methods Animals and anesthesia
Thirteen male albino Sprague-Dawley rats (body Weigtit450-550 g) were used. The animals were kept in plastic cages in a room with 12-h light-dark cycles, and an ambient temperature of 21 °C. They were allowed free
238
Dahlm et al. raised bilaterally at both the buccal and the lingual aspects of the mandibular angles in 13 animals. A standardized through-andthrough osseous defect (5 m m in diameter) was created bilaterally by a trephine burr 9 (Fig. 1). The flaps were repositioned carefully and sutured to obtain full coverage of the surgical site. All animals seemed to have tolerated the surgical procedure well, and they all gained weight postoperatively. They were allowed to heal for a period of 12 weeks, after which a reentry operation was performed.
Fig. 1. Preparation of 5-mm transosseous defect, through mandibular angle of rat, at surgery I.
access to drinking water and standard laboratory food pellets. Before surgery, the animals were anesthetized with an intraperitoneal injection (3.3 ml/kg body weight) consisting of 25% Hypnorm Vet.~ (Janssen Pharmaceuticals, Beerse, Belgium), 25% Dormicum ~ (Hoffman-LaRoche, Basel, Switzerland), and 50% sterile water. The skin overlying the mandibular angles was shaved and cleaned with 70% ethanol. During surgery, supplemental seda-
tion was administered as needed. Local anesthetic, 1 ml of 2% lidocaine/adrenalin solution (Astra, S6dert~lje, Sweden), was given at the site of surgery. Postoperatively, the animals were given daily doses of Temgesic ® (Reckitt & Colman, Hull, UK) for 1 week for pain relief and fed crushed standard pellets dispersed in water. The experimental outline was approved by the Animal Research Ethics Committee at GSteborg University, Sweden.
Surgical procedures Surgery I (12 weeks before surgery II). Musculoperiosteal flaps, based superiorly, were
Surgery H (12 weeks after surgery I). The anesthesia was done in the same manner as described above. Again, buccal and lingual musculoperiosteal flaps were raised bilaterally over the angles of the rat mandible. The regenerative result after the previous surgery was evaluated macroscopically after the bone had been dissected free from soft tissues, and, subsequently, photographs were taken (Fig. 2). The animals were divided into three groups to receive different surgical procedures. Procedure A. In this group (n = 4), the soft tissue inside the remaining bone defect was gently removed bilaterally so as not to cause damage or interfere with the established regenerated bone layer. On the right side, the remaining defect was covered both buccally and lingually by an e-PTFE membrane (Gore-Tex®; W. L. Gore & Associates, Flagstaff, AZ, USA), as previously described 9. The membrane was extended 2 3 mm beyond the margins of the defect to adhere tightly to the denuded bone surface (Fig. 3). Fixation was obtained by means of a transosseous ePTFE suture (Gore-Tex®). The buccal and lingual flaps, including the periosteum, were carefully repositioned superfically on the
Table 1. Results of bone healing 12 weeks after surgery I and 6 weeks after surgery II, respectively, in different groups Surgery I: macroscopic evaluation after 12 weeks of healing No. of sites
Complete healing
Persistent defect (3-5 mm)
Persistent defect (5 mm)
25
1
18
6
Surgery II: membrane-treated defects Surgical procedure II A II B II C
Complete healing
Remaining defect (< 1 mm)
4
4
0
0
4
3
1
0
5
5
0
0
Surgery II: control defects Surgical procedure
Fig. 2. Photograph showing reentry at surgery II after 12 weeks of initial healing. Note transosseous connective tissue core through created defect. Only minute apposition of new bone had occurred.
II A II B II C
results after 6 weeks of healing
No. of sites
Remaining defect (3-5 mm)
results after 6 weeks of healing
No. of sites
Complete healing
Remaining defect ( < 1 ram)
Remaining defect (3-5 mm)
4 4 4*
2 0 0
2 1 4
0 3 0
* One site was excluded from this group because of infection.
Osteopromotion in bone defects
239
identical, except that no membrane was applied. The postoperative treatment was identical to that used after surgery I. All animals were allowed to heal for 6 weeks. All animals gained weight during this period. Tissue processing
Fig. 3. e-PTFE membrane placed buccally and lingually over bone defect. Membrane is tightly adapted to denuded bone surface and fixed by transosseous suture.
Six weeks after the second surgical procedure, the animals were killed by an overdose of CO2, and block biopsies, comprising the bone defects with surrounding soft tissues, were prepared. Fixation by immersion in 4% buffered formalin was followed by demineralization in EDTA, dehydration, and embedding in bard paraffin. Serial horizontal sections (4 /m 0 were taken tbrougbout the defects. After staining with hematoxylin and eosin, the specimens were examined microscopically. The three most central sections of each experimental site were histologically evaluated by light microscopy. Results
Fig. 4. Histologic section showing complete healing of membrane (M)-treated nonunion defect 6 weeks after surgery II. Newly formed bone (NB) comprises mixture of woven and sparse lamellar bone. Arrowheads indicate original margins of nonunion defect (HE, × 40).
outer side of the membrane to achieve complete coverage of the membrane. The periosteum was thus located on the superficial surface of the membrane without direct contact with the bone defect. Treatment on the contratateral side was done as described above except that no membrane was placed over the defect.
Procedure B. In this group (n = 4), the bilaterally remaining bone defects were cleansed of the ingrowing connective tissue by curettage, corresponding to the more vigorous manner required in most clinical surgical situations. Through the defect, a new penetration,
baving the size of the original defect (5 mm in diameter), was re-created witb a trepbine burr (Fig. 1). On the right mandibular side, an e-PTFE membrane was placed over the defect and fixed as described above, while no membrane was used on the left side.
Procedure C. In the third group (n = 5), the soft tissue was removed from the persisting bilateral jaw defects in the same manner as in group A. On the right side, an e-PTFE membrane was applied over the cavity, but extra care was taken to ensure that the bone defect was completely filled with a blood clot. On the left side, the surgical procedure was
T h e results o f b o n e healing 12 weeks after surgery I a n d 6 weeks after surgery II, respectively, are s u m m a r i z e d in Table 1. The results after surgery I were evaluated macroscopically and photographically. T h e 25 sites revealed m a ture connective tissue filling the defects. N i n e t e e n sites showed varying degrees o f b o n e f o r m a t i o n along the m a r g i n s o f the defect, but, still, only one a n i m a l s h o w e d a r e m a i n i n g defect smaller t h a n 3 m m in diameter. In this defect, a continuous, very thin, bone-like tissue with a h e m a t o p o i e t i c c h a r a c t e r was found. N o ossification h a d occurred in the rem a i n i n g six animals, a n d the defects retained virtually the original d i a m e t e r o f 5 m m . Macroscopically, a massive ing r o w t h of soft connective tissue was generally found, f o r m i n g a transosseous core (Fig. 2). W h e n the rats were killed after the second healing period, local w o u n d complications were seen at two sites. O n e (in g r o u p C) was excluded f r o m f u r t h e r analysis, while the o t h e r showed a m o d e s t i n f l a m m a t o r y reaction a r o u n d a suture r e m n a n t , which was a s s u m e d to be o f late date. Generally, the m e m b r a n e s were f o u n d to be retained in their p r o p e r position b e n e a t h the soft tissue a n d tightly a d h e r i n g to the surface o f the b o n e formed. In general, the e - P F T E m e m b r a n e s were tolerated well. Foreignb o d y or i n f l a m m a t o r y reactions adj a c e n t to the m e m b r a n e s were virtually absent.
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Fig. 5. Persisting bone defect after removal of transosseous connective tissue core (Surgery
II, procedure C).
produced bone showed great variation (Fig. 5). In the sites operated according to procedure B, in which the original dimension of the defects (5 mm) was reestablished, three of four sites showed no or minimal new bone formation (Fig. 6). Conversely, in groups A and C, in which the new bone margins formed during the initial 12 weeks of healing after surgery I were preserved, a different picture was seen. Two of eight sites had healed completely with new bone (Fig. 7), whereas in six sites a substantial regeneration of new bone with a central portion of connective tissue measuring less than 1 mm was observed (Fig. 8). The soft-tissue core basically comprised a mixture of dense connective tissue with collagen fibers running parallel to the bone surface. Muscular tissue and adipose tissue were found to a limited extent. Generally, the newly formed bone in control defects was thinner than the surrounding bone (Figs. 7 and 8). Discussion
Fig. 6. Control defect, 6 weeks after currettage and reestablishment of original defect size (5 mm) (procedure II B). Only minor apposition of new bone is seen at margins of defect (arrows). Major part of defect is filled with dense connective tissue (CT) (HE, x 40).
The membrane-treated sites showed complete healing of the defects in 12 of 13 sites (Fig. 4), i.e., regardless of whether the sites were treated according to procedures A, B, or C at surgery II. One site showed a local infection external to the membrane. However, complete bone healing was seen within the margin of this particular defect. At another site, a central portion of connective tissue, with a width of less than 1 mm, was observed, containing islands of bone with active osteoblasts cen-
trally. In general, when compared with the original mandibular bone, the regenerated bone was found to exhibit increased buccolingual dimensions. The newly formed bone comprised a mixture of woven and sparse lamellar bone. Marrow spaces were generally found to a greater extent in the newly formed bone than the adjacent original bone. No cartilage was found at any membrane-treated site. At the control sites, where no membrane was applied, the amount of newly
Reentry procedures, to deal with incompletely healed bone defects, are common in oral and maxillofacial surgery as well as orthopedic surgery ~,3,r7. One example of such defects, which are filled with fibrous scar tissue rather than regenerated bone, is the defect observed in the maxillary lateral incisor region after endodontic therapy. Treatment, however, is often unsuccessful. This investigation, performed on standardized critical size defects in the rat mandible, has demonstrated that predictable and successful bone regeneration can be achieved by the osteopromotive membrane method in treatment of nonunion defects filled with mature connective tissue. Similar successful results in bone regeneration have ea,rlier been achieved by the membrane method in other experimental animal models TM. The transosseous defect in the rat mandible has earlier been used as an experimental model in several investigations9 11. The membrane method has also been found to give complete healing of transosseous mandibular defects in monkeys7 and of cranial defects in adult rats 5. All defects studied were "critical size defects ''18, a term implying that the size of the defect precludes spontaneous healing during the lifetime of the animal. The osteopromotion membrane method has also been successfully applied clinically in
Osteopromotion in bone dejects
Fig. 7. Histologic section, showing control defect from procedure A group. In this group, bone newly formed during initial 12-week healing period was preserved at reentry operation (arrows). Complete bone union (NB) can be seen after 6 additional weeks of healing after reentry (surgery II) (HE, x 40).
Fig. 8. Representative control defect from procedure C group. Six of eight sites in groups A and C showed substantial regeneration of new bone (NB) (as shown in Fig. 7) but with remaining central defect, less than 1 mm, filled with connective tissue (CT) (HE, x 40). placement of endosseous implants4'6'~'16. The efficacy of the method to accomplish complete bone healing at sites containing scar tissue has hitherto not been investigated. In most studies on the regeneration of bone defects, such defects have been created and immediately treated by membrane placement or application of some other osteogenesis-stimulating treatment. However, it is debatable how far conclusions from such experiments
are justified. It is generally believed that one way to initiate bone formation from bone wound margins is by the liberation of specific molecules with a capacity to cause multipotent, mesenchymal-type cells to differentiate into osteoblasts for production of new bone2a. Presumably, a more ideal situation for release of such growth factors is achieved in experimental models with freshly created bone defect margins than in quiescent, longstanding defects filled with scar tissue
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and, thus, having inactive bone sur 2 faces H. Presumably, the rate of bone healing is determined by the degree of bone damage relative to the amount of soft tissue present. If the volume of soft tissue predominates, mesenchymal cells may acquire a fibroblastic phenotype instead of developing into cells with an osteogenic capacity 2'11. In the present study, all sites except one were filled with fibrous scar tissue 12 weeks after the initial surgical procedure. The direction of the collagen fibers was at right angles to the bone surface, and the margins of the defects showed rounded bone edges. These findings clearly imply that a steady-state situation had been reached; that is, the defect would not, by itself, heal any further by ossification. This corroborates previous studies using the same experimental model 9 II, The results of the present study showed that virtually complete osseous healing had taken place at all sites where occlusive membranes were placed. Thus, a mechanical hindrance of connective-tissue proliferation into the persistent bone defects is of profound importance for unimpeded bone healing. Conversely, in the control groups receiving no membranes, varying healing patterns were found. In groups A and C, where the bone margins were carefully preserved during scar tissue removal, two sites healed completely and the remaining six sites showed almost complete bone healing except for a sparse connective-tissue core in the center of the defects. This represents a higher frequency of spontaneous healing than found in earlier studies9 ~. However, this could be explained by the fact that these control defects had so diminished in diameter during the initial healing phase that the defects did not fulfill the criteria for a critical size defect ~8 at the second procedure. Nevertheless, the findings clearly showed that repeated removal of tissues interfering with normal bone regeneration and the reproduction of optimal healing environments may substantially contribute to a recovered regenerative capacity. In control group B, where the original 5-mm diameter of the defect was re-created by drilling, still another response was found; three of four sites showed little or no new bone formation in the nonunion defect. This seems to be logical, since the defects again fulfilled the requirements for a critical size defect.
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Establishing a b l o o d clot in j a w b o n e defects has been considered to be crucial to optimize initial b o n e healing 4'~6, alt h o u g h experimental l o n g - b o n e fracture healing seems n o t to be i m p a i r e d after the early clot has b e e n r e m o v e d b y asp i r a t i o n 2°. In g r o u p C, extra care was t a k e n to secure such a b l o o d clot within the b o n e defect. Presumably, a similar a m o u n t of bleeding occurred in the other groups also, because n o difference could be f o u n d f r o m g r o u p A. In conclusion, this study shows t h a t the o s t e o p r o m o t i o n m e m b r a n e m e t h o d m a y be beneficial to the t r e a t m e n t o f n o n u n i o n b o n e defects where a less osteogenic e n v i r o n m e n t is present t h a n in fresh defects. This finding m a y p r o v e to be essential in various clinical situations, such as cystic b o n e defects, periapical lesions, a n d possibly pseudoarthrosis as a result o f n o n h e a l i n g o f b o n e fractures.
Acknowledgments. The authors gratefully acknowledge the technical assistance given by Ms Kerstin Bjurstam and Ms Yvonne Sundin. This study was supported by the Swedish Medical Research Council (grant 2789) and the Faculty of Odontology, G6teborg University, Sweden.
References
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Address: Dr Anders Linde Department of Oral Biochemistry Medicinaregatan 7B S-413 90 Gothenburg Sweden