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Evaluation of a new bioresorbable barrier to facilitate guided bone regeneration around exposed implant threads
Copyright O Munksgaard 1998
Int. J. Oral Maxillofac. Sure. 1998; 27:315-320 Printed in Denmark, All rights reserved
In~t~ttonal]o='nd of
Oral& MaxillafacialSargety ISSN 0901-5027
Evaluation of a new bioresorbable barrier to facilitate guided bone regeneration around exposed implant threads
Marku= B. HOrzeler %=, Raft J. Kohal ~=, Jaffar Naghshbandl =, Lul= F. Mote =, Joohen Conradt 1, D. Hutmacher =, Raul G. Csffesse = 1Department of Prosthodontlcs, AlbertLudwlge-Untverelty, Frelburg, Germany; =Department of Stomatology, Division of Pedodontlcs, Dental Branch, University of Texas-Houston Health Science Center, Houston, TX, USA; SDepartmentof Blornechanlcs, Polytecnlr Offenburg, Offenburg, Germany
An experimentalstudy in the monkey M. R Harzeler, R. J. Kohal, J. Naghshbandi, L. F. Mota, J. Conradt, D. Hutmacher, R. G. Caffesse. Evaluation of a new bioresorbable barrier to facilitate guided bone regeneration around exposed implant threads. An experimental study in the monkey. Int. J, Oral Maxillofac. Surg. 1998; 27: 315320. 9 Munksgaard, 1998 Abstract. The aim of this study was to evaluate the effectiveness of a new bioresorbable barrier alone or in combination with BioOss| for guided bone regeneration around dental implants with exposed implant threads. Five adult Macaca fascicularis monkeys were used in this investigation. After extraction of all premolars and first molars, two endosteal oral implants were installed in each quadrant and the bony defects were randomly treated with either: 1) placement of the new bioresorbable device alone (group 1); 2) placement of the new bioresorbable barrier in combination with BioOss@ (group 2); 3) placement of an ePTFE barrier in combination with BioOss | (group 3); or (4) control (group 4). After a period of six months the animals were killed and the histological processing was performed. There was a significant difference in the amount of new bone regeneration around the implants between the four groups (i.e. groups 1, 2, 3 and 4) (P=0.0122). There was no difference, however, between group 2 and group 3, It can be concluded that the new bioresorbable barrier in combination with BioOss | appears to obtain the same results in this type of bony defects as the grafting material in combination with an ePTFE barrier.
One of the restrictions in using endosteal oral implants has been an insufficient amount of bone in height and width. Reduction of bone volume is the result of ridge atrophy following tooth loss. Several methods have been suggested to improve bone contour including bone grafts~, sinus floor augmentation in the maxilla5,29, and guided bone regeneration1,7.
In the past eight years, the use of guided bone regeneration (GBR) has significantly increased. Many investigators have successfully applied GBR using barrier membranes during implant installation to regenerate bone around exposed implant surfaces resulting from inadequate bone volRme 1'2'7'19 or insufficient bone-to-implant contact, i.e. immediate placement of implants
Key words: bloresorbable membrane; collagen; bovine grafting material; guided bone regeneration; endosteal oral Implants.
Accepted for publication 14 January 1998
into extraction sockets 2'11. Treatment of insufficient bone volume around implants with barrier membranes and various grafting materials has also been used by several authors. DAHLINet al. 9 and JOVANOWC & BUS~2~ supported the expanded polytetrafluoroethylene membranes with autogenous bone, harvested from intraorat sites. K_)aox et al. 22 and S)~m)utr & NYMAN2s used lay-
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droxyapatite in c o m b i n a t i o n with exp a n d e d polytetrafluoroethylene m e m branes. Freezed-dried b o n e a n d m e m b r a n e s for guided b o n e regeneration were evaluated by MELLONIG & TRIPLETT25. All these studies presented good results c o n c e r n i n g the gain o f new bone. However, the m a t e r i a l the b a r r i e r m e m b r a n e s were m a d e of, was always n o n r e s o r b a b l e e x p a n d e d polytetrafluorethylene ( e P T F E ) (Gore-Tex | a u g m e n t a t i o n material, W. L. G o r e & Associates, Flagstaff, A Z , U S A ) . I n the field o f periodontology, studies have already presented successful regeneration o f lost p e r i o d o n t a l tissues by using bioresorbable b a r r i e r m e m b r a n e s m16. This k i n d o f m a t e r i a l does n o t require secondstage surgery for the removal o f the barrier m e m b r a n e . T h e b i o r e s o r b a b l e barrier m e m b r a n e s i n t r o d u c e d for guided b o n e regeneration, however, failed to predictably r e c o n s t r u c t new b o n e form a t i o n a r o u n d dental i m p l a n t s 12,17,24 . Therefore, the p u r p o s e o f this investigation was ~ evaluate the effectiveness o f a new bioresorbable b a r r i e r (BioGide| Biomaterial Geistlich, Wolhusen, Switzerland) a l o n e or in c o m b i n a t i o n with BioOss | (Biomaterial Geistlich) for G B R a r o u n d i m p l a n t s with exposed i m p l a n t threads.
Material and methods A total of five adult male cynomolgus monkeys (Macaca fascicularis), six to eight years of age, were used in this investigation. Housing and feeding of the animals were performed according to standard animal care protocol. The study was approved by the Animal Welfare Committe of the University of Texas-Houston Health Science Center and performed according to the National Institutes of Health Guide for Care and Use of Laboratory Animals. For all surgical procedures, the animals were first sedated with an intramuscular injection of ketamine hydrochloride, 100 mg/ml, 10 mg/kg body weight. General anesthesia was obtained by isoflurane gas intubation, and was supplemented with local administration of 2% xylocaine containing epinephrine (1:50000) in order to reduce hemorrhage in the surgical area. The animals were fed a soft diet for the entire study period.
Surgical procedure After creating full thickness flaps on the buccal and lingual side in all four quadrants, half of the bone over the buccal surfaces of the roots was removed using a carbide bur and saline irrigation. Then, all mandibular and maxillary premolars and first molars
were extracted and the interradicular bone and the bony septa between the premolars and the first molars were removed. The lingual alveolar crest was also reduced to the height of the buccal alveolar crest. This produced a buccolingual ridge defect with an average length of 20 mm mesio-distally, 4-5 mm coronally-apically and 4 mm bucco-lingnally. Further, in each of the four quadrants two endosteal oral implants 8.5 man in length and 3.25 mm in diameter (3i Implant Innovations, West Palm Beach, FL, USA) were installed. The four bony defects were then randomly treated with either: 1) placement of the new bioresorbable BioGide | barrier alone (group 1); 2) placement of the new bioresorbable BioGide | barrier in combination with BioOss | (group 2); 3) placement of a GoreTex| augmentation material barrier in combination with BioOss | (group 3); and 4) control - no placement of BioOss | granulate or barrier (group 4). BioGide | consists of porcine collagen types I and III and is composed of pure collagen fibres without any organic residues or additional chemicals. The membrane is designed to guarantee barrier function for four to five months. The compact layer - which has a smooth surface - is cell-occlusive to prevent connective tissue ingrowth. This side must be turned towards the soft tissue. The other layer of the device is made of collagen fibres in a loose, porous arrangement to enable cell invasion. This side is turned toward the bony defect in order to encourage the integration of bone-forming cells and to stabilize the blood coagulum. The flaps were repositioned after periosteal releasing incisions and sutured. For 7-10 days the wound areas were carefully swabbed with 2% chlorhexidme solution in order to reduce plaque accumulation. After ten days the sutures were removed. During the following six months the soft tissues were monitored. The animals were then killed by exsanguination, under general gas anesthesia.
Histologic processing of the specimens The heads of the animals were fixed by vascular perfusion with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer following a carotid artery cut-down procedure. Following this initial fixation, the segments containing the implants were immersed in halfstrength Karnovsky's fixative21, buffered to a pH of 7.4 with 0.02 M sodium cacodylate for 48 h at 4~ After completion of fixation, the specimens were washed in 0.185 M sodium cacodylate buffer. The blocks were embedded in light-cured composite material (Technovit 7200 VLC, Kulzer, Friedrichsdorf, Germany) and the undemineralized sections were cut in a mesio-distal direction and ground to a thickness of 30 microns according to previously described methods 1~ Subsequently, the sections were stained with toluidine blue and the three most central sections were evaluated histo-
logically, histometrically and histomorphometrically.
Methods of analysis The quantitative evaluation (i.e. histometric and histomorphometric) was performed with the aid of a light microscope (Zeiss| IM, Oberkochen, Germany) equipped with a videotv camera (Sony type MC-3210/PM, Cologne, Germany) coupled to an IBM computer. The transilluminated image from the light microscope was transferred in true colour and real time over the video-tv camera to a frame grabber board where it was digitally converted with graphic resolution of 800x600 pixels, and in 65 000 colours. The use of a semi-automatic computer program (AnalySiS 2.1, Soft-Imaging Software GmbH, MOnster, Germany) allowed direct work on the digital coloured signal on the screen with the mouse and performance of the analyses. The histometric analysis consisted of determining the distance in mm from the most coronal level of old mineralized bone to the most coronal level of new mineralized bone in contact with the implant surface on the mesial and distal aspect of both implants at a magnification of x280 on the screen. The histomorphometric analysis was performed in the newly regenerated bone with BioOss | (i.e. groups 2 and 3) and in the old bone by placing in both areas a template of 2• mm (widthXheight). In the newly regenerated bone area the following tissues were analysed within the template: newly mineralized bone, bone graft particles and fatty bone marrow. In the area of the old bone, the percentages of mineralized bone and bone marrow were determined. Overview pictures were taken with a magnification of • The computer program (AnalySiS 2.1) then converted these pictures automatically in grey scale values between 0 and 255. Each tissue was semi-antomatically assigned to a grey value interval. The percentage of the different tissues within the template was then automatieally calculated by the computer. Descriptive statistics were used to evaluate the results. The Friedman test was selected to test significant differences between the four treatment groups. The sign-test was then used to evaluate the palrwise intra-individual differences between the four treatment groups. The variable analysed was the difference between mean measurements for each treatment.
Results Cllnlcal observations N o n e o f the endosteal oral i m p l a n t s were lost. Soft tissue dehiscences over the b a r r i e r m e m b r a n e were registered in three sites where the b i o r e s o r b a b l e barrier alone was used, whereas in all o t h e r sites n o dehiscence occurred a n d the postoperative healing was uneventful.
New bioresorbable barrier for guided bone regeneration
Histologlc observations
Hlstometrlc analysis
All forty implants placed into the bony defects exhibited hist~ogic evidence o f direct mineralized bone-to-implant contact throughout their surface in the old bone. In the newly regenerated bone, the amount of new mineralized boneto-implant contact was different between the four treatment options. In the control specimens, negligible amounts of new direct bone-to-implant contact had occurred (Fig. 1). In treatment groups 2 (Fig. 2) and 3 (Fig. 3), more new mineralized bone-to-implant contact was observed in comparison to the other two treatments. The use of the xenogenous bone grafting material for the reconstruction of the peri-implant defects demonstrated an intimate contact of new mineralized bone with the BioOss | particles in all sections (Fig. 4). In the specimens of group 3, new lameUar bone was observed up to the nonresorbabte Gore-Tex| barrier (Fig. 4) whereas in group 2 the new bone formation did not occur up to the remants of the collagen barrier (Fig. 5). In these areas, ongoing resorption of the grafting material was detected (Fig. 6). Osteoclastic activity was less observed around the BioOss | in the newly formed bone and the new bone exhibited signs of normal bone remodelling.
Table 1 exhibits the amount of new mineralized bone-to-implant contact in mm for the four treatments. There was a significant difference between the four groups (P=0.0122). In the BioOss| GoreTex | group, a gain of new mineralized bone-to-implant contact of 3.90 mm occurred. The gain of mineralized bone-to-implant contact was less for the BioOss| | group (i.e. 3.26 ram). The difference between these two groups was not significant (P= 0.1875). The other two groups (1 and 4) demonstrated sit,nificantly less new mineralized bone-to-implant contact (i.e. 2.21 nun for the BioGide | alone and 1.36 mm for the control). There was no significant difference between these two groups (P=0.5000). The BioOss| | group and the BioOss| | group yielded a significantly greater amount of new mineralized bone-to-implant contact than the control group (i.e. P=0.0312 for both groups).
Hlstomorphometrlc analysis Table 2 illustrates the percentage of mineralized bone, bone marrow and bone-substituting material in the newly formed bone around the implants and the percentage o f mineralized bone and
Tab& 1. Newdffectmineralizedbone-to-implantcontactforfourtreatmen~inmm(n=5)
group 1 group 2 group 3 group 4
Mean values
SD
Min
Max
2.21B'c 3.26A,c 3.90A 1.36B
0.90 1.87 1.42 0.70
0.71 1.34 2.20 0.79
3.14 6.30 6.00 2.55
Friedman test P=0.0122; within the column, means with same superscript letter are not statistically significant (/'<0.05).
Table 2. Percentage of mineralized bone, bone marrokv and BioOss| particle in newly regenerated bone, and mineralized bone and bone marrow in remaining bone (n=5)
Mean values
SD
Min
Max
Remaining bone bone marrow mineralized bone
69.61 30.39
1.90 1.90
66.75 28.00
72.00 33.25
BioOss|
28.62
3.26
24.00
32.33
Regenerated bone bone marrow mineralized bone
28.62 42.80
4.71 4.86
23.19 36.33
34.50 49.71
Sign-test for parameter "mineralized bone" and "bone marrow". P=0.0312.
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bone marrow in the remaining bone. The amount of mineralized bone was significantly greater in the area of the regenerated bone in comparison to the remaining bone (P=0,0312). Significantly more bone marrow was found in the remaining bone than in the regenerated bone area (/'=0.0312).
Discussion Nonresorbable expanded polytetrafluorethylene membranes have been successfully used clinically and experimentally to regenerate bone around exposed implant threads 1.,19 and after placing iniplants into extraction sockets ~1,3~ The e-PTFE membranes, however, have to be removed after a healing period o f six to eight months and this requires a second surgical pro, cedure. Since the use of two-staged end' osteal oral implants requires surgery for the abutment connection, there will always be a discussion about the need for a bioresorbahle device for guided bone regeneration. Several arguments support the effort for developing bioresorbable or biodegradable devices for this indication: 1) The removal of a nonresorbable barrier causes denudation of immature bone. It is well known that the elevation of a mucoperiosteal flap per se can cause resorption of the underlying bone 27, therefore, the denudation of immature bone underneath the barrier membrane during the second-stage surgery should be prevented. 2) To remove the barrier the surgeon needs to raise a full-thickness flap to expose the barrier membrane, which restricts the possibilities to manage the peri-implant soft tissues during the second-stage surgery 13. 3) The postoperative healing with nonresorbable barrier membranes seems to be unpredictablel9; clinical studies using bioresorbable barriers for guided periodontal regeneration, however, demonstrated less problems postoperatively than the nonresorbable barrier memhranes 4. HI)RZELER & WENG14 clinically evaluated the BioGide | barrier for treatment of dehisced implant sites. They placed a total of 17 bioresorbable collagen barriers to treat peri-implant bony defects. Only in one case did they report exposure of the barrier during the healing period of six months. The results of the present experiment showed that the BioGide | and G T P M | membranes in combination with BioOss | did not become exposed,
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Fig. 1. Control implant demonstrates negli-
gible amount of new mineralized direct boneto-implant contact in defect area (toluidine blue XS).
i
Fig. 2. Implant treated with BioOss| and BioGide| exhibits new mineralized bone-toimplant contact up to implant shoulder (toluidine blue xS).
whereas three out of five sites where the new bioresorbable barrier was used alone, became exposed during the healing period. The poor mechanical prop-
Fig. 3. Implant covered with BioOss| and ePTFE barrier shows new mineralized boneto-implant contact up to cover screw (toluidine blue xS).
Fig. 5. Higher magnification of implant and newly formed bone underneath barrier from Fig. 2, does not demonstrate new mineralized bone up to remants of collagen barrier (toluidine blue x20).
vestigations were performed to evaluate bioresorbable and biodegradable membranes for GBR. L U N D G ~ et al.24 used a double-layered polylactic acid membrane originally designed for periodontal defects. They covered four periimplant defects with the support of autogenous bone chips and two without. Complete bone filling was found in four and partial filling in two of the treated defects. G O D S O N et al.t2 evaluated the use of a polyhydroxybutyrate(PHB)-co-hydroxyvalerate(HV) foil-like device reinforced with polyglactin (P6) fibres over implants placed into fresh extraction sockets in an animal model. After 12 weeks, inflammatory cell infiltrates were seen adjacent to all PHB-HV/PG foils, and frequently the device material was surrounded by a fibrous tissue capsule. An increased inflammatory reaction and significantly Fig. 4. Higher magnification of implant and less marginal bone healing was regisaugmented area showing grafting particles in tered at the membrane sites. They conclose contact with newly formed bone. Newly cluded that the new device used around mineralized bone can be seen up to nonre- immediately placed implants failed to sorbable barrier membrane (toluidine blue generate new bone formation. KOSTOx20). POULUS • KARRING23 explored the possibility of increasing the height of the rat mandible at its inferior border. erties of this new biodegradable barrier They used an occlusive membrane appeared to influence the postoperative made of PHB. The histological data healing events. demonstrated sufficient bone regeneraSeveral experimental and clinical in- tion into the space created by the biore-
New bioresorbable barrier for guided bone regeneration
319
new mineralized bone was found in the regenerated bone area than in the remaining bone. In conclusion, the experimental data indicate that the new bioresorbable barrier cannot be used alone to treat exposed implant threads. The application of the new bioresorbable device in combination with BioOss | appears to enhance new bone formation around exposed implant threads. Further studies, evaluating the clinical long-term stability of the newly regenerated bone, are needed. Such studies are underway.
Fig. 6. Resorption is observed around grafting particles lying directly underneath remnants of bioresorbable barrier (toluidine blue•
sorbable membranes. N o signs of degradation of the brittle PHB membranes were observed after six months of implantation. However, tissue perforation by the membranes as well as poor adaptation to the underlying bone occurred, so the authors concluded that the physical properties of the membrane should be modified before clinical use in maxillofacial surgery could be recommended. HORZELER et al.17 investigated a poly-(D,L-lactid-co-trymethylencarbonate) foil in rhesus monkeys as compared to the ePTFE membrane. Soft tissue dehiscences were only seen on the control side (i.e. ePTFE membrane sites), whereas on the experimental side no dehiscences could be detected. Four months after the augmentation procedures, the histological sections revealed a significant bone gain at the control sites as compared to the test sites. The authors stated that the new bioresorbable device could not be recommended for the purpose of guided bone regeneration. They speculated that the bioresorption process was too fast and that difficulties with the clearance of the bioresorption products could have occurred, resulting in a low pH-value. The low pH-value then interfered with the bone regeneration process. U p to now, the bioresorbable devices have failed to be successful in predicting new bone regeneration underneath the membranes. Collagen is another bioresorbable material. Since collagen is resorbed by an enzymatic process, the problem of the low pH-value can be ignored. Collagen barriers have already been used successfully to treat periodontal defects applying the principle of guided tissue regeneration s', and for regenera-
tion and thickening of the lateral maxillary sinus wall 3. In our experiment, the collagen membrane in combination with a grafting material exhibited the same results as the nonresorbable barrier (ePTFE membrane) in combination with the same grafting material. The new bioresorbable collagen barrier alone revealed significantly less bone formation than both the treatments combining a barrier and a grafting material. Because of the poor mechanical properties of collagen, this material needs to be used in combination with a bone substituting material. Even the combination of the new bioresorbable device with the bone grafting material resulted in no new mineralized bone within the first 1 ram directly underneath the barrier, as demonstrated in the present experiment. The applied bone grafting particles are o f bovine origin. The material is commercially available in two particle sizes (i.e. 250-1000 microns, and 10002000 microns) as well as in two different bone types (i.e. cortical and cancerous bone). It is osteoconductive since all proteins are removed during processing. The surface area of each graft particle is considerably greater than that of porous bioceramics and the modulus of elasticity is similar to natural bone 26. JENS'~ et a l ) s evaluated qualitatively and quantitatively the tissue reaction around two bovine and two coral-derived bone substitutes when implanted into the rabbit tibia. They concluded that BioOss | was osseointegrated to a higher degree than the other biomaterials. The positive osteoconductive features of this grafting material were also demonstrated in our study More
Acknowledgments. The authors wish to thank Maria Bachle and Heike Kr/imer for their technical assistance and valuable laboratory work. The study was supported in part by Biomaterials Geistlich, Wolhusen, Switzerland, and 3i Implant Innovations, West Palm Beach, FL, USA. References 1. BECKERW, BECKERHE, HAND~LSMANM, et al. Bone formation at dehisced dental implant sites treated with implant augmentation material: a pilot study in dogs. Int J Periodont Rest Dent 1990: 10: 92101. 2. BECKERW, B E C ~ BE. Guided tissue regeneration for implants placed into extraction sockets and for implant dehiseenses: surgical techniques and case reports. Surgical and restorative advan9tages. Int J Periodont Rest Dent 1990: 10: 376-91. 3. BECKEgJ, N ~ FW, S c m . m p ~ H. Restoration of the lateral sinus wail using a collagen type I membrane for guided tissue regeneration. Int J Oral Maxillofac Surg 1992: 21: 243-6. 4. B L ~ , NM. A clinical comparison of collagen membranes with ePTFE membranes in the treatment of human mandibular buceal class II furcation defects. J Periodontol 1993: 64: 925-33. 5. BoYl,m PP, JAMESRA. Grafting of the maxillary sinus floor with autogenous marrow and bone. J Oral Surg 1980: 38: 61~6. 6. Bmm~ U, B ~ K P-J. Reconstruction of alveolar jaw bone. An experimental and clinical study of immediate and preformed autologous bone grafts in combination with osseointegrated implants. Scand J Plast Reconstr Surg 1980: 14: 23-48. 7. BUSERD, BP.XGGERU, LANG~ NYMAN S. Regeneration and enlargement of jaw bone using guided tissue regeneration. Clin Oral Impl Res 1990: 1: 22-32. 8. CHUNO. KM, SALKIN LM, FREEDMAN 9 AL. Clinical evaluation of a biodegradable collagen membrane in guided tissue regeneration. J Periodontol 1990: 61: 732-6.
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9. DArIL~ C, ANDERSSONL, Lit,roE A. Bone augmentation at fenestrated implants by an osteopromotive membrane technique. A controlled clinical study. Clin Oral Impl Res 1991: 2: 159--65. 10. DONATH K, BREUNER G. A method for the study of undecalcitied bones and teeth with attached soft tissues. J Oral Pathol 1982: 11: 318-26. 11. GELB DA. Immediate implant surgery: three-year retrospective evaluation of 50 consecutive cases. Int J Oral Maxillofac Impl 1993: 8: 388-99. 12. GOTFREDSEN K, NIMB L, HJORTINGHANSEN E. Immediate implant placement using a biodegradable barrier, polyhydroxybutyrate-hydroxyvalerate reinforeed with Polyglactin 910. Clin Oral Implants Res 1994: 5: 83-91. 13. HORZELER MB, WENG D. A new technique to combine barrier removal at dehisced implant sites with a plastic periodontal procedure. Int J Periodont Rest Dent 1996: 16: 149-63. 14. HORZELER MB, VYINGD. Knochenregeneration um Implantate - eine klinische Studie mit einer neuen resorbierbaren Membran.,Dtsch Zahn/irztl Z 1996: 51: 298-303. 15. HURZELER MB, QU~ONES CR, SCHUPBACH P, CAFFESSE RG. Guided periodontal tissue regeneration in class II furcation defects following treatment with a synthetic bioabsorbable barrier. J Periodontol 1997: 68: 498-505. 16. HORZELER MB, Qul~o~ms CR, SCnt3"pBACH P, CAFFESSE RG. Guided periodontal tissue regeneration in interproximal intrabony defects following treatment with a synthetic bioabsorbable barrier. J Periodontol 1997: 68: 489-97.
17. HtYRZELER MB, QUI~ONEE CR, SCI-I~BACH, P. Guided bone regeneration around dental implants using a bioresorbable membrane in the atrophic alveolar ridge. An experimental study in the monkey. Clin Oral Impl Res 1997: 8: 323--31. 18. JENSL~N SS, AABOE M, PINHOLT EM, HJORTINC,-HANs~ E, Mm,sl~ F, RUYTER E. Tissue reaction and material characteristics of four bone substitutes. Int J Oral Maxillofac Impl 1996: 11: 55-66. 19. JOVANOWCSA, SPmmmMANN H, RIChTER EJ. Bone regeneration around titanium dental implants in dehisced defect sites: a clinical study. Int J Oral Maxillofac Impl 1992: 7: 233-45. 20. JOVAN0VICSA, BUSERD. Guided bone regeneration in dehiscence defects and delayed extraction sites. In: BtlSER D, DAHLIN C, SCHENK RK, eds.: Guided bone regeneration in implant dentistry. Chicago: Quintessence, 1994: 156-88. 21. KARNOVSKYMJ. A formaldehyde-glutaraldehyde fixative of high osmolarity for use in electron microscopy. J Cell Biol 1965: 32: 137A-8A. 22. KNOX R, LEE K, MEFrERT R. Placement of hydroxyapatite-coated endosseous implants in fresh extraction sites: a case report. Int J Periodont Rest Dent 1993: 13: 245-53. 23. KOSTOPOULUSL, KARRING T. Augmentation of rat mandible using guided tissue regeneration. Clin Oral Impl Res 1994: 5: 75-82. 24. LUNDGREND, SENNERBYL, FALK H, FRIBERG B, NYMAN S. The use of a new bioresorbable barrier for guided bone regeneration in connection with implant installation. Clin Oral Implants Res 1994: 3: 177-84.
25. MELLOmG JT, TRIPLETT RG. Guided tissue regeneration and endosseous dental implants. Int J Periodont Rest Dent 1993: 13: 109-19. 26. PAUL C, SCI~XCKEWEI W, KUNER EH, SCI-EN'K RK. Apatit - Wertigkeit beim Knochenersatz. In: PESCH, H J, STOSS H, KUMMER B: Osteologie aktuell. Berlin: Springer, 1993: Bovines S. 288-91. 27. PFmFER J. The reaction of alveolar bone to flap procedures in man. Periodontics 1965: 3: 135---40. 28. SEIBERTJ, NYMANS. Localized ridge augmentation in dogs: a pilot study using membranes and hydroxyapatite. J Periodontol~; 1990: 61: 157~i5. 29. TIDWELU JK, BLUDORP PA, STOELINGA PJW, BROUNS JB, HINDERKS E Composite grafting of the maxillary sinus for placemept of endosteal implants. A preliminary report of 48 patients. Int J Oral Maxillofac Surg 1992: 21: 204-9. 30. WARRER K, GOTFREDSEN K, I-IJoRTrNGHANSEN E, KARRrNG T. Guided tissue regeneration ensures osseointegration of dental implants placed into extraction sockets: an experimental study in monkeys. Clin Oral Impl Res 1991: 2: 166-71.
Address: Markus B. Hfirzeler, DMD, PhD Private Clinic for Periodontology and Implantology Rosenkavalierplatz 18/IV D-81925 Munich Germany Tel: +89 928784-0 Fax: +89 93931847
Int. J. Oral Maxillofac. Sure. 1998; 27:315-320 Printed in Denmark, All rights reserved
In~t~ttonal]o='nd of
Oral& MaxillafacialSargety ISSN 0901-5027
Evaluation of a new bioresorbable barrier to facilitate guided bone regeneration around exposed implant threads
Marku= B. HOrzeler %=, Raft J. Kohal ~=, Jaffar Naghshbandl =, Lul= F. Mote =, Joohen Conradt 1, D. Hutmacher =, Raul G. Csffesse = 1Department of Prosthodontlcs, AlbertLudwlge-Untverelty, Frelburg, Germany; =Department of Stomatology, Division of Pedodontlcs, Dental Branch, University of Texas-Houston Health Science Center, Houston, TX, USA; SDepartmentof Blornechanlcs, Polytecnlr Offenburg, Offenburg, Germany
An experimentalstudy in the monkey M. R Harzeler, R. J. Kohal, J. Naghshbandi, L. F. Mota, J. Conradt, D. Hutmacher, R. G. Caffesse. Evaluation of a new bioresorbable barrier to facilitate guided bone regeneration around exposed implant threads. An experimental study in the monkey. Int. J, Oral Maxillofac. Surg. 1998; 27: 315320. 9 Munksgaard, 1998 Abstract. The aim of this study was to evaluate the effectiveness of a new bioresorbable barrier alone or in combination with BioOss| for guided bone regeneration around dental implants with exposed implant threads. Five adult Macaca fascicularis monkeys were used in this investigation. After extraction of all premolars and first molars, two endosteal oral implants were installed in each quadrant and the bony defects were randomly treated with either: 1) placement of the new bioresorbable device alone (group 1); 2) placement of the new bioresorbable barrier in combination with BioOss@ (group 2); 3) placement of an ePTFE barrier in combination with BioOss | (group 3); or (4) control (group 4). After a period of six months the animals were killed and the histological processing was performed. There was a significant difference in the amount of new bone regeneration around the implants between the four groups (i.e. groups 1, 2, 3 and 4) (P=0.0122). There was no difference, however, between group 2 and group 3, It can be concluded that the new bioresorbable barrier in combination with BioOss | appears to obtain the same results in this type of bony defects as the grafting material in combination with an ePTFE barrier.
One of the restrictions in using endosteal oral implants has been an insufficient amount of bone in height and width. Reduction of bone volume is the result of ridge atrophy following tooth loss. Several methods have been suggested to improve bone contour including bone grafts~, sinus floor augmentation in the maxilla5,29, and guided bone regeneration1,7.
In the past eight years, the use of guided bone regeneration (GBR) has significantly increased. Many investigators have successfully applied GBR using barrier membranes during implant installation to regenerate bone around exposed implant surfaces resulting from inadequate bone volRme 1'2'7'19 or insufficient bone-to-implant contact, i.e. immediate placement of implants
Key words: bloresorbable membrane; collagen; bovine grafting material; guided bone regeneration; endosteal oral Implants.
Accepted for publication 14 January 1998
into extraction sockets 2'11. Treatment of insufficient bone volume around implants with barrier membranes and various grafting materials has also been used by several authors. DAHLINet al. 9 and JOVANOWC & BUS~2~ supported the expanded polytetrafluoroethylene membranes with autogenous bone, harvested from intraorat sites. K_)aox et al. 22 and S)~m)utr & NYMAN2s used lay-
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droxyapatite in c o m b i n a t i o n with exp a n d e d polytetrafluoroethylene m e m branes. Freezed-dried b o n e a n d m e m b r a n e s for guided b o n e regeneration were evaluated by MELLONIG & TRIPLETT25. All these studies presented good results c o n c e r n i n g the gain o f new bone. However, the m a t e r i a l the b a r r i e r m e m b r a n e s were m a d e of, was always n o n r e s o r b a b l e e x p a n d e d polytetrafluorethylene ( e P T F E ) (Gore-Tex | a u g m e n t a t i o n material, W. L. G o r e & Associates, Flagstaff, A Z , U S A ) . I n the field o f periodontology, studies have already presented successful regeneration o f lost p e r i o d o n t a l tissues by using bioresorbable b a r r i e r m e m b r a n e s m16. This k i n d o f m a t e r i a l does n o t require secondstage surgery for the removal o f the barrier m e m b r a n e . T h e b i o r e s o r b a b l e barrier m e m b r a n e s i n t r o d u c e d for guided b o n e regeneration, however, failed to predictably r e c o n s t r u c t new b o n e form a t i o n a r o u n d dental i m p l a n t s 12,17,24 . Therefore, the p u r p o s e o f this investigation was ~ evaluate the effectiveness o f a new bioresorbable b a r r i e r (BioGide| Biomaterial Geistlich, Wolhusen, Switzerland) a l o n e or in c o m b i n a t i o n with BioOss | (Biomaterial Geistlich) for G B R a r o u n d i m p l a n t s with exposed i m p l a n t threads.
Material and methods A total of five adult male cynomolgus monkeys (Macaca fascicularis), six to eight years of age, were used in this investigation. Housing and feeding of the animals were performed according to standard animal care protocol. The study was approved by the Animal Welfare Committe of the University of Texas-Houston Health Science Center and performed according to the National Institutes of Health Guide for Care and Use of Laboratory Animals. For all surgical procedures, the animals were first sedated with an intramuscular injection of ketamine hydrochloride, 100 mg/ml, 10 mg/kg body weight. General anesthesia was obtained by isoflurane gas intubation, and was supplemented with local administration of 2% xylocaine containing epinephrine (1:50000) in order to reduce hemorrhage in the surgical area. The animals were fed a soft diet for the entire study period.
Surgical procedure After creating full thickness flaps on the buccal and lingual side in all four quadrants, half of the bone over the buccal surfaces of the roots was removed using a carbide bur and saline irrigation. Then, all mandibular and maxillary premolars and first molars
were extracted and the interradicular bone and the bony septa between the premolars and the first molars were removed. The lingual alveolar crest was also reduced to the height of the buccal alveolar crest. This produced a buccolingual ridge defect with an average length of 20 mm mesio-distally, 4-5 mm coronally-apically and 4 mm bucco-lingnally. Further, in each of the four quadrants two endosteal oral implants 8.5 man in length and 3.25 mm in diameter (3i Implant Innovations, West Palm Beach, FL, USA) were installed. The four bony defects were then randomly treated with either: 1) placement of the new bioresorbable BioGide | barrier alone (group 1); 2) placement of the new bioresorbable BioGide | barrier in combination with BioOss | (group 2); 3) placement of a GoreTex| augmentation material barrier in combination with BioOss | (group 3); and 4) control - no placement of BioOss | granulate or barrier (group 4). BioGide | consists of porcine collagen types I and III and is composed of pure collagen fibres without any organic residues or additional chemicals. The membrane is designed to guarantee barrier function for four to five months. The compact layer - which has a smooth surface - is cell-occlusive to prevent connective tissue ingrowth. This side must be turned towards the soft tissue. The other layer of the device is made of collagen fibres in a loose, porous arrangement to enable cell invasion. This side is turned toward the bony defect in order to encourage the integration of bone-forming cells and to stabilize the blood coagulum. The flaps were repositioned after periosteal releasing incisions and sutured. For 7-10 days the wound areas were carefully swabbed with 2% chlorhexidme solution in order to reduce plaque accumulation. After ten days the sutures were removed. During the following six months the soft tissues were monitored. The animals were then killed by exsanguination, under general gas anesthesia.
Histologic processing of the specimens The heads of the animals were fixed by vascular perfusion with 2% glutaraldehyde in 0.1 M sodium cacodylate buffer following a carotid artery cut-down procedure. Following this initial fixation, the segments containing the implants were immersed in halfstrength Karnovsky's fixative21, buffered to a pH of 7.4 with 0.02 M sodium cacodylate for 48 h at 4~ After completion of fixation, the specimens were washed in 0.185 M sodium cacodylate buffer. The blocks were embedded in light-cured composite material (Technovit 7200 VLC, Kulzer, Friedrichsdorf, Germany) and the undemineralized sections were cut in a mesio-distal direction and ground to a thickness of 30 microns according to previously described methods 1~ Subsequently, the sections were stained with toluidine blue and the three most central sections were evaluated histo-
logically, histometrically and histomorphometrically.
Methods of analysis The quantitative evaluation (i.e. histometric and histomorphometric) was performed with the aid of a light microscope (Zeiss| IM, Oberkochen, Germany) equipped with a videotv camera (Sony type MC-3210/PM, Cologne, Germany) coupled to an IBM computer. The transilluminated image from the light microscope was transferred in true colour and real time over the video-tv camera to a frame grabber board where it was digitally converted with graphic resolution of 800x600 pixels, and in 65 000 colours. The use of a semi-automatic computer program (AnalySiS 2.1, Soft-Imaging Software GmbH, MOnster, Germany) allowed direct work on the digital coloured signal on the screen with the mouse and performance of the analyses. The histometric analysis consisted of determining the distance in mm from the most coronal level of old mineralized bone to the most coronal level of new mineralized bone in contact with the implant surface on the mesial and distal aspect of both implants at a magnification of x280 on the screen. The histomorphometric analysis was performed in the newly regenerated bone with BioOss | (i.e. groups 2 and 3) and in the old bone by placing in both areas a template of 2• mm (widthXheight). In the newly regenerated bone area the following tissues were analysed within the template: newly mineralized bone, bone graft particles and fatty bone marrow. In the area of the old bone, the percentages of mineralized bone and bone marrow were determined. Overview pictures were taken with a magnification of • The computer program (AnalySiS 2.1) then converted these pictures automatically in grey scale values between 0 and 255. Each tissue was semi-antomatically assigned to a grey value interval. The percentage of the different tissues within the template was then automatieally calculated by the computer. Descriptive statistics were used to evaluate the results. The Friedman test was selected to test significant differences between the four treatment groups. The sign-test was then used to evaluate the palrwise intra-individual differences between the four treatment groups. The variable analysed was the difference between mean measurements for each treatment.
Results Cllnlcal observations N o n e o f the endosteal oral i m p l a n t s were lost. Soft tissue dehiscences over the b a r r i e r m e m b r a n e were registered in three sites where the b i o r e s o r b a b l e barrier alone was used, whereas in all o t h e r sites n o dehiscence occurred a n d the postoperative healing was uneventful.
New bioresorbable barrier for guided bone regeneration
Histologlc observations
Hlstometrlc analysis
All forty implants placed into the bony defects exhibited hist~ogic evidence o f direct mineralized bone-to-implant contact throughout their surface in the old bone. In the newly regenerated bone, the amount of new mineralized boneto-implant contact was different between the four treatment options. In the control specimens, negligible amounts of new direct bone-to-implant contact had occurred (Fig. 1). In treatment groups 2 (Fig. 2) and 3 (Fig. 3), more new mineralized bone-to-implant contact was observed in comparison to the other two treatments. The use of the xenogenous bone grafting material for the reconstruction of the peri-implant defects demonstrated an intimate contact of new mineralized bone with the BioOss | particles in all sections (Fig. 4). In the specimens of group 3, new lameUar bone was observed up to the nonresorbabte Gore-Tex| barrier (Fig. 4) whereas in group 2 the new bone formation did not occur up to the remants of the collagen barrier (Fig. 5). In these areas, ongoing resorption of the grafting material was detected (Fig. 6). Osteoclastic activity was less observed around the BioOss | in the newly formed bone and the new bone exhibited signs of normal bone remodelling.
Table 1 exhibits the amount of new mineralized bone-to-implant contact in mm for the four treatments. There was a significant difference between the four groups (P=0.0122). In the BioOss| GoreTex | group, a gain of new mineralized bone-to-implant contact of 3.90 mm occurred. The gain of mineralized bone-to-implant contact was less for the BioOss| | group (i.e. 3.26 ram). The difference between these two groups was not significant (P= 0.1875). The other two groups (1 and 4) demonstrated sit,nificantly less new mineralized bone-to-implant contact (i.e. 2.21 nun for the BioGide | alone and 1.36 mm for the control). There was no significant difference between these two groups (P=0.5000). The BioOss| | group and the BioOss| | group yielded a significantly greater amount of new mineralized bone-to-implant contact than the control group (i.e. P=0.0312 for both groups).
Hlstomorphometrlc analysis Table 2 illustrates the percentage of mineralized bone, bone marrow and bone-substituting material in the newly formed bone around the implants and the percentage o f mineralized bone and
Tab& 1. Newdffectmineralizedbone-to-implantcontactforfourtreatmen~inmm(n=5)
group 1 group 2 group 3 group 4
Mean values
SD
Min
Max
2.21B'c 3.26A,c 3.90A 1.36B
0.90 1.87 1.42 0.70
0.71 1.34 2.20 0.79
3.14 6.30 6.00 2.55
Friedman test P=0.0122; within the column, means with same superscript letter are not statistically significant (/'<0.05).
Table 2. Percentage of mineralized bone, bone marrokv and BioOss| particle in newly regenerated bone, and mineralized bone and bone marrow in remaining bone (n=5)
Mean values
SD
Min
Max
Remaining bone bone marrow mineralized bone
69.61 30.39
1.90 1.90
66.75 28.00
72.00 33.25
BioOss|
28.62
3.26
24.00
32.33
Regenerated bone bone marrow mineralized bone
28.62 42.80
4.71 4.86
23.19 36.33
34.50 49.71
Sign-test for parameter "mineralized bone" and "bone marrow". P=0.0312.
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bone marrow in the remaining bone. The amount of mineralized bone was significantly greater in the area of the regenerated bone in comparison to the remaining bone (P=0,0312). Significantly more bone marrow was found in the remaining bone than in the regenerated bone area (/'=0.0312).
Discussion Nonresorbable expanded polytetrafluorethylene membranes have been successfully used clinically and experimentally to regenerate bone around exposed implant threads 1.,19 and after placing iniplants into extraction sockets ~1,3~ The e-PTFE membranes, however, have to be removed after a healing period o f six to eight months and this requires a second surgical pro, cedure. Since the use of two-staged end' osteal oral implants requires surgery for the abutment connection, there will always be a discussion about the need for a bioresorbahle device for guided bone regeneration. Several arguments support the effort for developing bioresorbable or biodegradable devices for this indication: 1) The removal of a nonresorbable barrier causes denudation of immature bone. It is well known that the elevation of a mucoperiosteal flap per se can cause resorption of the underlying bone 27, therefore, the denudation of immature bone underneath the barrier membrane during the second-stage surgery should be prevented. 2) To remove the barrier the surgeon needs to raise a full-thickness flap to expose the barrier membrane, which restricts the possibilities to manage the peri-implant soft tissues during the second-stage surgery 13. 3) The postoperative healing with nonresorbable barrier membranes seems to be unpredictablel9; clinical studies using bioresorbable barriers for guided periodontal regeneration, however, demonstrated less problems postoperatively than the nonresorbable barrier memhranes 4. HI)RZELER & WENG14 clinically evaluated the BioGide | barrier for treatment of dehisced implant sites. They placed a total of 17 bioresorbable collagen barriers to treat peri-implant bony defects. Only in one case did they report exposure of the barrier during the healing period of six months. The results of the present experiment showed that the BioGide | and G T P M | membranes in combination with BioOss | did not become exposed,
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Fig. 1. Control implant demonstrates negli-
gible amount of new mineralized direct boneto-implant contact in defect area (toluidine blue XS).
i
Fig. 2. Implant treated with BioOss| and BioGide| exhibits new mineralized bone-toimplant contact up to implant shoulder (toluidine blue xS).
whereas three out of five sites where the new bioresorbable barrier was used alone, became exposed during the healing period. The poor mechanical prop-
Fig. 3. Implant covered with BioOss| and ePTFE barrier shows new mineralized boneto-implant contact up to cover screw (toluidine blue xS).
Fig. 5. Higher magnification of implant and newly formed bone underneath barrier from Fig. 2, does not demonstrate new mineralized bone up to remants of collagen barrier (toluidine blue x20).
vestigations were performed to evaluate bioresorbable and biodegradable membranes for GBR. L U N D G ~ et al.24 used a double-layered polylactic acid membrane originally designed for periodontal defects. They covered four periimplant defects with the support of autogenous bone chips and two without. Complete bone filling was found in four and partial filling in two of the treated defects. G O D S O N et al.t2 evaluated the use of a polyhydroxybutyrate(PHB)-co-hydroxyvalerate(HV) foil-like device reinforced with polyglactin (P6) fibres over implants placed into fresh extraction sockets in an animal model. After 12 weeks, inflammatory cell infiltrates were seen adjacent to all PHB-HV/PG foils, and frequently the device material was surrounded by a fibrous tissue capsule. An increased inflammatory reaction and significantly Fig. 4. Higher magnification of implant and less marginal bone healing was regisaugmented area showing grafting particles in tered at the membrane sites. They conclose contact with newly formed bone. Newly cluded that the new device used around mineralized bone can be seen up to nonre- immediately placed implants failed to sorbable barrier membrane (toluidine blue generate new bone formation. KOSTOx20). POULUS • KARRING23 explored the possibility of increasing the height of the rat mandible at its inferior border. erties of this new biodegradable barrier They used an occlusive membrane appeared to influence the postoperative made of PHB. The histological data healing events. demonstrated sufficient bone regeneraSeveral experimental and clinical in- tion into the space created by the biore-
New bioresorbable barrier for guided bone regeneration
319
new mineralized bone was found in the regenerated bone area than in the remaining bone. In conclusion, the experimental data indicate that the new bioresorbable barrier cannot be used alone to treat exposed implant threads. The application of the new bioresorbable device in combination with BioOss | appears to enhance new bone formation around exposed implant threads. Further studies, evaluating the clinical long-term stability of the newly regenerated bone, are needed. Such studies are underway.
Fig. 6. Resorption is observed around grafting particles lying directly underneath remnants of bioresorbable barrier (toluidine blue•
sorbable membranes. N o signs of degradation of the brittle PHB membranes were observed after six months of implantation. However, tissue perforation by the membranes as well as poor adaptation to the underlying bone occurred, so the authors concluded that the physical properties of the membrane should be modified before clinical use in maxillofacial surgery could be recommended. HORZELER et al.17 investigated a poly-(D,L-lactid-co-trymethylencarbonate) foil in rhesus monkeys as compared to the ePTFE membrane. Soft tissue dehiscences were only seen on the control side (i.e. ePTFE membrane sites), whereas on the experimental side no dehiscences could be detected. Four months after the augmentation procedures, the histological sections revealed a significant bone gain at the control sites as compared to the test sites. The authors stated that the new bioresorbable device could not be recommended for the purpose of guided bone regeneration. They speculated that the bioresorption process was too fast and that difficulties with the clearance of the bioresorption products could have occurred, resulting in a low pH-value. The low pH-value then interfered with the bone regeneration process. U p to now, the bioresorbable devices have failed to be successful in predicting new bone regeneration underneath the membranes. Collagen is another bioresorbable material. Since collagen is resorbed by an enzymatic process, the problem of the low pH-value can be ignored. Collagen barriers have already been used successfully to treat periodontal defects applying the principle of guided tissue regeneration s', and for regenera-
tion and thickening of the lateral maxillary sinus wall 3. In our experiment, the collagen membrane in combination with a grafting material exhibited the same results as the nonresorbable barrier (ePTFE membrane) in combination with the same grafting material. The new bioresorbable collagen barrier alone revealed significantly less bone formation than both the treatments combining a barrier and a grafting material. Because of the poor mechanical properties of collagen, this material needs to be used in combination with a bone substituting material. Even the combination of the new bioresorbable device with the bone grafting material resulted in no new mineralized bone within the first 1 ram directly underneath the barrier, as demonstrated in the present experiment. The applied bone grafting particles are o f bovine origin. The material is commercially available in two particle sizes (i.e. 250-1000 microns, and 10002000 microns) as well as in two different bone types (i.e. cortical and cancerous bone). It is osteoconductive since all proteins are removed during processing. The surface area of each graft particle is considerably greater than that of porous bioceramics and the modulus of elasticity is similar to natural bone 26. JENS'~ et a l ) s evaluated qualitatively and quantitatively the tissue reaction around two bovine and two coral-derived bone substitutes when implanted into the rabbit tibia. They concluded that BioOss | was osseointegrated to a higher degree than the other biomaterials. The positive osteoconductive features of this grafting material were also demonstrated in our study More
Acknowledgments. The authors wish to thank Maria Bachle and Heike Kr/imer for their technical assistance and valuable laboratory work. The study was supported in part by Biomaterials Geistlich, Wolhusen, Switzerland, and 3i Implant Innovations, West Palm Beach, FL, USA. References 1. BECKERW, BECKERHE, HAND~LSMANM, et al. Bone formation at dehisced dental implant sites treated with implant augmentation material: a pilot study in dogs. Int J Periodont Rest Dent 1990: 10: 92101. 2. BECKERW, B E C ~ BE. Guided tissue regeneration for implants placed into extraction sockets and for implant dehiseenses: surgical techniques and case reports. Surgical and restorative advan9tages. Int J Periodont Rest Dent 1990: 10: 376-91. 3. BECKEgJ, N ~ FW, S c m . m p ~ H. Restoration of the lateral sinus wail using a collagen type I membrane for guided tissue regeneration. Int J Oral Maxillofac Surg 1992: 21: 243-6. 4. B L ~ , NM. A clinical comparison of collagen membranes with ePTFE membranes in the treatment of human mandibular buceal class II furcation defects. J Periodontol 1993: 64: 925-33. 5. BoYl,m PP, JAMESRA. Grafting of the maxillary sinus floor with autogenous marrow and bone. J Oral Surg 1980: 38: 61~6. 6. Bmm~ U, B ~ K P-J. Reconstruction of alveolar jaw bone. An experimental and clinical study of immediate and preformed autologous bone grafts in combination with osseointegrated implants. Scand J Plast Reconstr Surg 1980: 14: 23-48. 7. BUSERD, BP.XGGERU, LANG~ NYMAN S. Regeneration and enlargement of jaw bone using guided tissue regeneration. Clin Oral Impl Res 1990: 1: 22-32. 8. CHUNO. KM, SALKIN LM, FREEDMAN 9 AL. Clinical evaluation of a biodegradable collagen membrane in guided tissue regeneration. J Periodontol 1990: 61: 732-6.
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25. MELLOmG JT, TRIPLETT RG. Guided tissue regeneration and endosseous dental implants. Int J Periodont Rest Dent 1993: 13: 109-19. 26. PAUL C, SCI~XCKEWEI W, KUNER EH, SCI-EN'K RK. Apatit - Wertigkeit beim Knochenersatz. In: PESCH, H J, STOSS H, KUMMER B: Osteologie aktuell. Berlin: Springer, 1993: Bovines S. 288-91. 27. PFmFER J. The reaction of alveolar bone to flap procedures in man. Periodontics 1965: 3: 135---40. 28. SEIBERTJ, NYMANS. Localized ridge augmentation in dogs: a pilot study using membranes and hydroxyapatite. J Periodontol~; 1990: 61: 157~i5. 29. TIDWELU JK, BLUDORP PA, STOELINGA PJW, BROUNS JB, HINDERKS E Composite grafting of the maxillary sinus for placemept of endosteal implants. A preliminary report of 48 patients. Int J Oral Maxillofac Surg 1992: 21: 204-9. 30. WARRER K, GOTFREDSEN K, I-IJoRTrNGHANSEN E, KARRrNG T. Guided tissue regeneration ensures osseointegration of dental implants placed into extraction sockets: an experimental study in monkeys. Clin Oral Impl Res 1991: 2: 166-71.
Address: Markus B. Hfirzeler, DMD, PhD Private Clinic for Periodontology and Implantology Rosenkavalierplatz 18/IV D-81925 Munich Germany Tel: +89 928784-0 Fax: +89 93931847