Alveolar ridge augmentation by osteoinductive materials in goats
Else Marie Pinholt'•^ Hans Reidar Haanaes^ Magnus Roervik^, Karl Donath", Gisle Bang^ Institute for Surgical Research, Rikshospitalet. Llniversity of Osio', Department of Oral Surgery and Oral Medicine, Dental Faculty. University of Oslo^, Tfie Norwegian College of Veterinary Medicine, Oslo^ Department of Oral Pathology, Universify of Hamburg, Federal Republic of Germany*, and Department of Oral Pathology and Forensic Dentistry, University of Bergen^, Norway
PitthoU EM, Haanacs HR, Rocrvik M, Donath K, Bang G: Alveolar ridge augmentation by osteoinduetive materials in goats, Scand J Dettt Res 1992: 100: 361-5.
•
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/-
•-•'^
•
,,.
-
-.-.;•-:-
•::.-:.
The purpose of the present study was to determine whether alveolar ridge augmentation could be induced in goats. In 12 male goats allogenic, demineralized, and lyophilized dentin or bone was implanted subperiosteally on the buccal sides of the natural edentulous regions of the alveolar process of the mandible. Light microscopic evaluation revealed fibrous encapsulation, a few multinuclcar giant cells, little inflammatory reaction, and no osteoinduction. It was concluded that no osteoinduction took place in goats.
Demineralized bone and dentin induce osteogenesis (1, 2) in certain mammal species, Osteoinduction consists of chemotaxis, mitosis, and differentiation of mesenchymal cells into chondroblasts and osteoblasts (3, 4). The local stimulating factor initiating the process is bone morphogenetic protein (BMP), a noncollagenous protein (1, 5). Osteoconduction, on the other hand, is a mode of healing consisting of creeping substitution. In this process the implanted material acts as a scaffold which gradually is resorbed while preosteoblasts and osteoblasts migrate from the adjacent vital bone (6, 7). Osteoinduction has mainly been studied in rodents. They appear to be a suitable animals for this purpose (1, 2, 8). Alveolar ridge augmentation by osteoinduction has been studied in rats by PINHOLT et al. (9), and new bone formation was observed in all animals. The purpose of this study was to evaluate alveolar ridge augmentation by osteoinduction, using allogenic, demineralized, and lyophilized dentin or bone, in goats. This animal ranks relatively high on the phylogenetic ladder, and its physiology resembles the human more than does the rodent. Material and methods Castrated Norwegian male goats weighing 35-50 kg (2.5 yr of age) were used for the two experimental groups (I = six animals; 11 = six animals), the
Key words: alveolar ridge augmentation; jaws, therapy: osteoinduction E. M. Pinholt, Institute for Surgical Research, University of Oslo, Rikshospitalet, 0027 Oslo 1, Norway Accepted for publication 31 December 1991
control group (111 = four animals), and the donor group (three animals). They were fed a standard diet (standard diet for goats, Norwegian College of Veterinary Medicine, Oslo, Norway) and given water ad libitum. Incisors and long bones from allogenic goats were harvested, cleaned, demineralized in 0,2 mol HCl for 48 h at 4 X , lyophilized, and sterilized in ethylene oxide gas for 3 h, as previously described (2, 9). In Groups I and II, implants were placed in the mandible and in one anterior limb. The surgery was done under sedation and local anesthesia by Rompun vet. (xylazin, 20 mg/ml) 0.17 mg/kg body weight intramuscular and 1% Lidocain with adrenaline 1/200000 submucosally. Prophylactic antibiotic treatment was initiated 1 h preoperatively with Proca-Mycin vet. 5 ml (benzylpenicillinprocaine 200 mg/ml+ dehydrostreptoiTiycin 250 mg/ml) intramuscular, and repeated daily for 3 days postoperatively. A fluorochrome (oxytetracycline, 20 mg/ kg body weight) was injected intravenously 4 days prior to the experiment for bone marking (10). A vertical mandibular incision close to the midline was made in the inferior buccal sulcus. Bilaterally subperiosteal dissections were carried out on the buccal sides at the level of the base of the mandible. Ridge augmentation was performed bilaterally in these tunnels at the buccal aspect of the edentulous regions of the mandible between the incisors and the first molar. In Group I, 0.5 ml (150 mg) granules of allogenic, demineralized, and
362
Pinholt et al.
lyophilized bone was implanted bilaterally with a custom-made syringe. The surgical site was closed with Prolene monofil suture. The same procedure was performed in Group II where 0.5 cm^ (100 mg) granules of allogenic, demineralized, and lyophilized dentin was deposited. In both Group I and Group II, a skin incision was made down to the muscle sheet of a randomized anterior limb. Blunt dissection was performed into the muscle, and 0.5 ml allogenic, demineralized, and lyophilized granules of bone was implanted in the muscles in the animals of Group I, and 0.5 ml of allogenic, demineralized, and lyophilized granules of dentin was deposited in Group II. The incision was closed in layers with Prolene. In Group III the same procedures were carried out as sham operations without implantation. To observe wound healing, we performed clinical evaluation daily the first 3 postoperative days, and thereafter weekly. After 16 wk the animals were killed. The mandibles and the muscle from the anterior limb were immediately dissected from the animal. The specimens were fixed in 4% neutral formalin and radiographically examined to detect possible hard tissues. They were then divided into halves, one half being demineralized in 17% formic acid, dehydrated, and, embedded in paraffin wax. Serial sections were cut at 5 \\.m and stained with Harris hematoxylin-eosin. The other half was dehydrated and embedded in metamethacrylate. Sections were cut at 10-20 \im and stained with toluidine blue (11). ;;/;..;?v :,--:.; / . ; : : . .•'.C'i':-"
Results
All animals gained weight during the 16 wk of observation. No signs of infection were observed, but local dehiscence of some incisions was detected at the time of suture removal, 2 wk postoperatively. No communication with implanted areas dorsal to incisions were seen. In Groups I and II, all implants in the alveolar areas were found, but none of the implants in the muscles of the anterior limbs were detected. Eibrous connective tissue with a few multinuclear giant cells and a minimum of infiammatory reaction encapsulated almost all implants of Group I and Group II (Eigs. 1 and 2), and only a few of the implants were in direct contact with the recipient bone. A sparse ingrowth of vascular connective tissue in the implants was observed, and it was noted that mononuclear phagocytes had dissolved the structure of some of the demineralized dentin or bone (Eig. 3). However, a large part of the implants showed lack of reaction, and no osteoinduction was seen in any of the animals (Eig. 2). Remineralization of the implanted material was only seen when the demineralized dentin or bone was in contact with the recipient bone (Eigs. 1, 4, 5). In these locations new bone developed by osteoconduction (Eigs. 5 and 6). The recipient bone was resorbed at the surface (Eigs. 5 and 6), especially when no thick capsule of fibrous connective tissue surrounded the implant. Reversal lines of new bone were observed next to the surface resorption of the operated sites of Groups I, II, and III
DD
DD
Fig. 1. Goat alveolar bone (AB) augmentation by allogenic, demineralized, and lyophilized dentin (DD). Encapsulation by fibrous connective tissue (CT). Remineralization of implant remote from. «. and when in contact, A, with alveolar bone. See Fie. 5 for details. H&E, x 2.5. Fig. 2. Allogenic, demineralized, and lyophilized dentin (DD) enveloped in fibrous connective tissue (CT) with few giant cells (GC) and absence of infiammatory reaction. No osteoinduction is observed. H&E, x 25.
Augmentation by osteoinductive materials
363
V ^
AB
Fig. 3. AUogenic, demineralized, and lyophilized bone (DB) separated from goat alveolar bone (AB) by fibrous connective tissue (CT). Structure of demineralized bone is dissolved, but no new bone formation is generated. H&E, x 10. Fig. 4. Matrix around dentin canals, | , of implanted allogenic, demineralized, and lyophilized dentin (DD) is remineralized (RD). H&E, x 10.
and where the implant material was remineralized. This was confirmed in all three groups by uptake of tetracycline. The sham operations on the anterior limb revealed no osteoinduction. Discussion The present study reveals that alveolar ridge augmentation is not obtained by osteoinduction when implanting allogenic, demineralized, and lyophilized dentin or bone in goats. Therefore quantification of the results was not performed. Instead a fibrous encapsulation and minimal resorption of the implant material was seen. This contrasts with our findings in rodents where massive new bone was formed. The allogenic dentin and bone of the rodents and of the goats was demineralized and lyophilized in exactly the same way. In the present study the often seen dense fibrous connective tissue capsule seerns to create a barrier to the implant, and, although there was minimal lymphocytic infiltration, the tissue reaction resembles a rejection process. This is in accordance with the findings of DoNATH in a study on different implant materials (12). He concluded that the body's response to an implant material, whether it is allogenic, xenogenic, or alloplastic, is always an immunologic rejection (12). It has previously been reported (13) that demineralization and freeze-drying lower the antigenicity of allogenic tissue, which otherwise gives rise to a cell-mediated immunologic response upon implantation (14). In our study the bone and dentin
was harvested from goats that were known to be genetically nonidentical with the recipients. It is also known that histocompatibility matching improves acceptance of frozen bone allografts in dogs (15, 16). Since tissue response in our study seemed to be weak, incornpatibility must have been moderate. BANG (17) showed in rodents that the secondary immunologic response was minimal after implantation of allogenic dentin treated in the way the allogenic implants were handled in this study. It is, therefore, not expected that the allogenic dentin or bone implanted into the muscles of the extremities caused initiation of any serious immune reaction to the implantations on the jaws. Furthermore, the implants were placed on the mandibles and in the muscles at the same titne, and this does not allow time for a delayed hypersensitivity reaction to take place. Animals ranking high on the phylogenetic ladder have a low metabolic activity index (18, 19). They are also reported to react less to osteoinductive stimuli and to have lower content of BMP in bone and dentin (20, 21). Furthermore, osteoinduction is dose related (1, 3, 22) and is dependent on solubility of the implant (5). In the present investigation, only minimal resorption of the implanted material was observed. Therefore only traces of BMP and growth factors were available in the tissue for contact with the precocious cells for a sufficient length of time. This may refiect the lack of new bone formation in the goat. This could have been confirmed by isolatitig BMP from goat dentin atid
364
Pinholt et al.
Fig. 5. Enlargement of Fig. I. Goat alveolar bone (AB) augmented with allogenic, demineralized, and lyophilized dentin which is remineralized (RD). New bone formation (NB) by osteoconduction is observed on implant and on surface of resorbed recipient bone, |. H&E, x25. Fig. 6. Resorption, j , of goat alveolar bone (AB) augmented with allogenic, demineralized. and lyophilized bone (DB) which, in contact with recipient, is remineralized (RB) and acts as a scaffold for new bone formation by osteocoinduction (NB). No reactions are noted around remnants of demineralized bone (DB) remote from alveolar bone. H&E, x 10.
bone for examination, as was done by SAMPATH & REDDI (23) with BMP from other species. However, this is considered a separate project. Castration, the age of the goats, insufficient subperiosteal blood supply, lack of function of implant, and minor movements of the implant material because of constant chewing may also have resulted in less new bone formation. It is not likely that new bone development was impaired because of possible low-grade infection, since no connection between the oral cavity and the implant was recognized. This is in accordance with other studies (12, 24). Locally, in the muscle of the anterior limb, the blood supply probably was sufficient. If new bone had been formed, it might have been resorbed because of lack of function, and the observation time might therefore have been too long. However, lack of extraskeletal osteoinduction was also observed after implantation of BMP in sheep and dogs by LiNDHOLM (25). In order to approximate experimental osteoinduction to expected results in human beings, researchers have performed investigations in monkeys. Implanting allogenic, demineralized bone in primates has yielded conflicting reports on new bone formation (26, 27). In human beings, reports on allogenic, demineralized, and lyophilized bone utilized within orthopedic and cranio-maxillofacial surgery are diverting (28-31), and the investigations have mainly been clinical and radiologic, with only a few histologic sections performed. Osteo-
conduction and recalcification might be part of the healing process, leading to satisfactory findings clinically and radiologically. Therefore the investigators might have reported positive findings which were due only to osteoinduction. Conclusion - allogenic, demineralized, and lyophilized bone and dentin were tested for alveolar ridge augmentation purposes in goats. Minimal osteoconduction was noted and no osteoinduction was observed. This may be due to lack of resorption of the implant material, together with subsequent low amounts of available BMP, the initiating factor of osteoinduction. It may also be due to possible low osteoinductive ability in the goat, an animal relatively high on the phylogenetic ladder. References 1. URIST MR. Bone. Formation by autoinduction. Science 1965; 150: 893-9. 2. BANG G , URIST MR. Bone induction in excavation chambers in matrix of decalcified dentin. Arcli Sur^ 1967; 94: 781-9. 3. URIST MR,
SILVERMAN BF, BURING K , ROSENHERG J M .
The bone induction principle. Clin Orttiop 1967; 53: 243-83. 4. REDDI AH,
WIHNTROUB S, MUTHUKUMARAN N . Biologic
principles of bone induction. Orttiop Ctin North Atn 1987; 18: 207-12. 6. URIST MR, NILSSON OS, HUDAK R, RASMUSSEN J, HIROTA
W, LiETZE A Immunologic evidence of a bone morphogenetic protein in the niitieu interieur Ann Biot Ctiti 1985; 43: 755-66. 6. AxHAUSEN G. Uber histologischen Vorgang bei der Transplantation von Gelenkenden. Arch Ktin Ctiir 1912; 99: 1-50.
Augmentation by osteoinductive materials
365
7. PHEMISTER DB. The fate of transplanted bone and regenerative power of its various constituents. Surg Gynecol Obstet 1914; 19: 303-33. 8. KABAN LB, GLOWACKI J. Induced osteogenesis in the repair of experimental mandibular defects in rats. J Dent Res 1981; 60; 1356-64. 9. PiNHOt.T EM, BANG G , HAANAES HR. Alveolar ridge augmentation by osteoinduction in rats. Scand J Dent Res 1990; 98; 434-41. 10. FROST H M . Tetracycline labelling of bone and the zone of demarcation of osteoid seams. Can J Biochem PItys 1962; 40; 485-9. 11. DONATH K. Die Trenn-Dunnschliff-Technik zur Herstellung histologischer Praeparate von nicht schneidbaren Geweben und Materialien. Der Praeparator 1988; 34; 197-206. 12. DoNATH K. Histopathologische Befunde von Hydroxylapatit-Keraniiken im Kieferbereich. In; WATZEK G VON, MATEJKA M , eds. Der zalmlose Unterkiefer Seine chirurgisch-prothetisehe Rehabilitation. Vienna; Springer 1988, 177-85. 1 3. BuRCHARDT H. The biology of bone grafi repair. Clin Orthop 1983; 174; 28-42.
Academy of Science; National Research Council. Philadelphia; W. B. Saunders, 1956; 258. 20. SATO K , URIST MR. Induced regeneration of caivaria by bone morphogenetic protein (BMP) in does. Clin Orthop 1985; 197; 301-11.
14. BuRWELL RG, FRIEDLANDER GE, MANKIN HJ. Current
eralized dentin implants in jaw defects in Java monkeys. Int J Oral Surg 1972; 1; 126-36. 27. HosNY M, SHARAWY M . Osteoinduction in rhesus monkeys using demineralized bone powder allografts. J Oral Maxillofae Surg 1985; 43; 837-44.
perspectives and future directions; The 1983 invitational conference on osteochondral allografts. Clin Orthop 1985197; 141-57. 1 5. Bos GD, GOLDBERG V M , POWELL A E , HEIPLE KG, ZIKA
JM. The effect of histocompatibility matching on canine frozen bone allografts. / Bone Jt Surg 1983; 65A; 89-96. 16. GOLDBERG VM, POWELL A, SHAEFER JW, ZIKA J, Bos GD.
HEIPLE KG. Bone grafting; role of histoeompatibility in transplantation. J Orthop Res 1985; 3; 389--404. 17. BANG G. Induction of heterotopic bone formation by demineralized dentin in guinea pigs; antigenicity of the dentin matrix. J Oral Pathol 1972; 1; 172-85. 18. CouLSON RA. Relationship between fiuid fiow and O, demand in tissues in vivo and in vitro. Penspect Biol Med 1983; 27; 121-6. 19. SPECTOR W, Ed. Handbook of biological data. National
: v^
-^i',
21. URIST MR, MIKULSKI A, BOYD SD. A chemosterilized anti-
gen extracted autodigested alloimplant for bone banks. Arch Surg 1975; 110; 416-28. 22. ASPENBERG P, LOHMANDER LS, THORNGREN KG. Failure of bone induction by bone matrix in adult monkeys. J Bone Jt Surg 1988; 70B; 625-7. 23. SAMPATH T K , REDDI AH. Homology of bone-inductive
proteins from human, monkey, bovine, and rat extracellular matrix. Proc Natl Acad Sci USA 1983; 80; 6591-5. 24. PiNiiOLT EM, KwoN PHJ. Alveolar ridge augmentation in dogs, using hydroxylapatite, and the effect of therapeutic radiation. J Oral Maxillofae Surg 1992; 50; 250-4. 25. LiNDHOLM TC, LiNDHOLM TS, ALITALO I, URIST MR. Bo-
vine bone morphogenetic protein (bBMP) induced repair of skull trephine defects in sheep. Clin Orthop 1988; 227; 265-8. 26. BANG G , NORDENRAM A, ANNEROTH G . Allogenic demin-
28. KNUDSEN GE, BANG G , KRISTOFFERSEN T. Implantitig of
allogenic demineralized dentin in human gingival tissue. J Clin Periodontol 1974; 1; 153-9. 29. NORDENRAM A, BANG G . Reconstruction of the alveolar
process by implantation of allogenic, demineralized dentin. Oral Surg Oral Med Oral Pathol 1975; 40; 48-50. 30. KABAN LB. MULLIKEN JB, GLOWACKI J. Treatment of jaw
defects with demineralized bone implants. J Oral Maxillofae Surg 1982; 40; 623-6. 31. OusTERHOUT DK. Clinical experience in cranial and facial reconstruction with demineralized bone. Plast Reeonstr Surg 1985; 15; 367-73.
Else Marie Pinholt'•^ Hans Reidar Haanaes^ Magnus Roervik^, Karl Donath", Gisle Bang^ Institute for Surgical Research, Rikshospitalet. Llniversity of Osio', Department of Oral Surgery and Oral Medicine, Dental Faculty. University of Oslo^, Tfie Norwegian College of Veterinary Medicine, Oslo^ Department of Oral Pathology, Universify of Hamburg, Federal Republic of Germany*, and Department of Oral Pathology and Forensic Dentistry, University of Bergen^, Norway
PitthoU EM, Haanacs HR, Rocrvik M, Donath K, Bang G: Alveolar ridge augmentation by osteoinduetive materials in goats, Scand J Dettt Res 1992: 100: 361-5.
•
^i-
/-
•-•'^
•
,,.
-
-.-.;•-:-
•::.-:.
The purpose of the present study was to determine whether alveolar ridge augmentation could be induced in goats. In 12 male goats allogenic, demineralized, and lyophilized dentin or bone was implanted subperiosteally on the buccal sides of the natural edentulous regions of the alveolar process of the mandible. Light microscopic evaluation revealed fibrous encapsulation, a few multinuclcar giant cells, little inflammatory reaction, and no osteoinduction. It was concluded that no osteoinduction took place in goats.
Demineralized bone and dentin induce osteogenesis (1, 2) in certain mammal species, Osteoinduction consists of chemotaxis, mitosis, and differentiation of mesenchymal cells into chondroblasts and osteoblasts (3, 4). The local stimulating factor initiating the process is bone morphogenetic protein (BMP), a noncollagenous protein (1, 5). Osteoconduction, on the other hand, is a mode of healing consisting of creeping substitution. In this process the implanted material acts as a scaffold which gradually is resorbed while preosteoblasts and osteoblasts migrate from the adjacent vital bone (6, 7). Osteoinduction has mainly been studied in rodents. They appear to be a suitable animals for this purpose (1, 2, 8). Alveolar ridge augmentation by osteoinduction has been studied in rats by PINHOLT et al. (9), and new bone formation was observed in all animals. The purpose of this study was to evaluate alveolar ridge augmentation by osteoinduction, using allogenic, demineralized, and lyophilized dentin or bone, in goats. This animal ranks relatively high on the phylogenetic ladder, and its physiology resembles the human more than does the rodent. Material and methods Castrated Norwegian male goats weighing 35-50 kg (2.5 yr of age) were used for the two experimental groups (I = six animals; 11 = six animals), the
Key words: alveolar ridge augmentation; jaws, therapy: osteoinduction E. M. Pinholt, Institute for Surgical Research, University of Oslo, Rikshospitalet, 0027 Oslo 1, Norway Accepted for publication 31 December 1991
control group (111 = four animals), and the donor group (three animals). They were fed a standard diet (standard diet for goats, Norwegian College of Veterinary Medicine, Oslo, Norway) and given water ad libitum. Incisors and long bones from allogenic goats were harvested, cleaned, demineralized in 0,2 mol HCl for 48 h at 4 X , lyophilized, and sterilized in ethylene oxide gas for 3 h, as previously described (2, 9). In Groups I and II, implants were placed in the mandible and in one anterior limb. The surgery was done under sedation and local anesthesia by Rompun vet. (xylazin, 20 mg/ml) 0.17 mg/kg body weight intramuscular and 1% Lidocain with adrenaline 1/200000 submucosally. Prophylactic antibiotic treatment was initiated 1 h preoperatively with Proca-Mycin vet. 5 ml (benzylpenicillinprocaine 200 mg/ml+ dehydrostreptoiTiycin 250 mg/ml) intramuscular, and repeated daily for 3 days postoperatively. A fluorochrome (oxytetracycline, 20 mg/ kg body weight) was injected intravenously 4 days prior to the experiment for bone marking (10). A vertical mandibular incision close to the midline was made in the inferior buccal sulcus. Bilaterally subperiosteal dissections were carried out on the buccal sides at the level of the base of the mandible. Ridge augmentation was performed bilaterally in these tunnels at the buccal aspect of the edentulous regions of the mandible between the incisors and the first molar. In Group I, 0.5 ml (150 mg) granules of allogenic, demineralized, and
362
Pinholt et al.
lyophilized bone was implanted bilaterally with a custom-made syringe. The surgical site was closed with Prolene monofil suture. The same procedure was performed in Group II where 0.5 cm^ (100 mg) granules of allogenic, demineralized, and lyophilized dentin was deposited. In both Group I and Group II, a skin incision was made down to the muscle sheet of a randomized anterior limb. Blunt dissection was performed into the muscle, and 0.5 ml allogenic, demineralized, and lyophilized granules of bone was implanted in the muscles in the animals of Group I, and 0.5 ml of allogenic, demineralized, and lyophilized granules of dentin was deposited in Group II. The incision was closed in layers with Prolene. In Group III the same procedures were carried out as sham operations without implantation. To observe wound healing, we performed clinical evaluation daily the first 3 postoperative days, and thereafter weekly. After 16 wk the animals were killed. The mandibles and the muscle from the anterior limb were immediately dissected from the animal. The specimens were fixed in 4% neutral formalin and radiographically examined to detect possible hard tissues. They were then divided into halves, one half being demineralized in 17% formic acid, dehydrated, and, embedded in paraffin wax. Serial sections were cut at 5 \\.m and stained with Harris hematoxylin-eosin. The other half was dehydrated and embedded in metamethacrylate. Sections were cut at 10-20 \im and stained with toluidine blue (11). ;;/;..;?v :,--:.; / . ; : : . .•'.C'i':-"
Results
All animals gained weight during the 16 wk of observation. No signs of infection were observed, but local dehiscence of some incisions was detected at the time of suture removal, 2 wk postoperatively. No communication with implanted areas dorsal to incisions were seen. In Groups I and II, all implants in the alveolar areas were found, but none of the implants in the muscles of the anterior limbs were detected. Eibrous connective tissue with a few multinuclear giant cells and a minimum of infiammatory reaction encapsulated almost all implants of Group I and Group II (Eigs. 1 and 2), and only a few of the implants were in direct contact with the recipient bone. A sparse ingrowth of vascular connective tissue in the implants was observed, and it was noted that mononuclear phagocytes had dissolved the structure of some of the demineralized dentin or bone (Eig. 3). However, a large part of the implants showed lack of reaction, and no osteoinduction was seen in any of the animals (Eig. 2). Remineralization of the implanted material was only seen when the demineralized dentin or bone was in contact with the recipient bone (Eigs. 1, 4, 5). In these locations new bone developed by osteoconduction (Eigs. 5 and 6). The recipient bone was resorbed at the surface (Eigs. 5 and 6), especially when no thick capsule of fibrous connective tissue surrounded the implant. Reversal lines of new bone were observed next to the surface resorption of the operated sites of Groups I, II, and III
DD
DD
Fig. 1. Goat alveolar bone (AB) augmentation by allogenic, demineralized, and lyophilized dentin (DD). Encapsulation by fibrous connective tissue (CT). Remineralization of implant remote from. «. and when in contact, A, with alveolar bone. See Fie. 5 for details. H&E, x 2.5. Fig. 2. Allogenic, demineralized, and lyophilized dentin (DD) enveloped in fibrous connective tissue (CT) with few giant cells (GC) and absence of infiammatory reaction. No osteoinduction is observed. H&E, x 25.
Augmentation by osteoinductive materials
363
V ^
AB
Fig. 3. AUogenic, demineralized, and lyophilized bone (DB) separated from goat alveolar bone (AB) by fibrous connective tissue (CT). Structure of demineralized bone is dissolved, but no new bone formation is generated. H&E, x 10. Fig. 4. Matrix around dentin canals, | , of implanted allogenic, demineralized, and lyophilized dentin (DD) is remineralized (RD). H&E, x 10.
and where the implant material was remineralized. This was confirmed in all three groups by uptake of tetracycline. The sham operations on the anterior limb revealed no osteoinduction. Discussion The present study reveals that alveolar ridge augmentation is not obtained by osteoinduction when implanting allogenic, demineralized, and lyophilized dentin or bone in goats. Therefore quantification of the results was not performed. Instead a fibrous encapsulation and minimal resorption of the implant material was seen. This contrasts with our findings in rodents where massive new bone was formed. The allogenic dentin and bone of the rodents and of the goats was demineralized and lyophilized in exactly the same way. In the present study the often seen dense fibrous connective tissue capsule seerns to create a barrier to the implant, and, although there was minimal lymphocytic infiltration, the tissue reaction resembles a rejection process. This is in accordance with the findings of DoNATH in a study on different implant materials (12). He concluded that the body's response to an implant material, whether it is allogenic, xenogenic, or alloplastic, is always an immunologic rejection (12). It has previously been reported (13) that demineralization and freeze-drying lower the antigenicity of allogenic tissue, which otherwise gives rise to a cell-mediated immunologic response upon implantation (14). In our study the bone and dentin
was harvested from goats that were known to be genetically nonidentical with the recipients. It is also known that histocompatibility matching improves acceptance of frozen bone allografts in dogs (15, 16). Since tissue response in our study seemed to be weak, incornpatibility must have been moderate. BANG (17) showed in rodents that the secondary immunologic response was minimal after implantation of allogenic dentin treated in the way the allogenic implants were handled in this study. It is, therefore, not expected that the allogenic dentin or bone implanted into the muscles of the extremities caused initiation of any serious immune reaction to the implantations on the jaws. Furthermore, the implants were placed on the mandibles and in the muscles at the same titne, and this does not allow time for a delayed hypersensitivity reaction to take place. Animals ranking high on the phylogenetic ladder have a low metabolic activity index (18, 19). They are also reported to react less to osteoinductive stimuli and to have lower content of BMP in bone and dentin (20, 21). Furthermore, osteoinduction is dose related (1, 3, 22) and is dependent on solubility of the implant (5). In the present investigation, only minimal resorption of the implanted material was observed. Therefore only traces of BMP and growth factors were available in the tissue for contact with the precocious cells for a sufficient length of time. This may refiect the lack of new bone formation in the goat. This could have been confirmed by isolatitig BMP from goat dentin atid
364
Pinholt et al.
Fig. 5. Enlargement of Fig. I. Goat alveolar bone (AB) augmented with allogenic, demineralized, and lyophilized dentin which is remineralized (RD). New bone formation (NB) by osteoconduction is observed on implant and on surface of resorbed recipient bone, |. H&E, x25. Fig. 6. Resorption, j , of goat alveolar bone (AB) augmented with allogenic, demineralized. and lyophilized bone (DB) which, in contact with recipient, is remineralized (RB) and acts as a scaffold for new bone formation by osteocoinduction (NB). No reactions are noted around remnants of demineralized bone (DB) remote from alveolar bone. H&E, x 10.
bone for examination, as was done by SAMPATH & REDDI (23) with BMP from other species. However, this is considered a separate project. Castration, the age of the goats, insufficient subperiosteal blood supply, lack of function of implant, and minor movements of the implant material because of constant chewing may also have resulted in less new bone formation. It is not likely that new bone development was impaired because of possible low-grade infection, since no connection between the oral cavity and the implant was recognized. This is in accordance with other studies (12, 24). Locally, in the muscle of the anterior limb, the blood supply probably was sufficient. If new bone had been formed, it might have been resorbed because of lack of function, and the observation time might therefore have been too long. However, lack of extraskeletal osteoinduction was also observed after implantation of BMP in sheep and dogs by LiNDHOLM (25). In order to approximate experimental osteoinduction to expected results in human beings, researchers have performed investigations in monkeys. Implanting allogenic, demineralized bone in primates has yielded conflicting reports on new bone formation (26, 27). In human beings, reports on allogenic, demineralized, and lyophilized bone utilized within orthopedic and cranio-maxillofacial surgery are diverting (28-31), and the investigations have mainly been clinical and radiologic, with only a few histologic sections performed. Osteo-
conduction and recalcification might be part of the healing process, leading to satisfactory findings clinically and radiologically. Therefore the investigators might have reported positive findings which were due only to osteoinduction. Conclusion - allogenic, demineralized, and lyophilized bone and dentin were tested for alveolar ridge augmentation purposes in goats. Minimal osteoconduction was noted and no osteoinduction was observed. This may be due to lack of resorption of the implant material, together with subsequent low amounts of available BMP, the initiating factor of osteoinduction. It may also be due to possible low osteoinductive ability in the goat, an animal relatively high on the phylogenetic ladder. References 1. URIST MR. Bone. Formation by autoinduction. Science 1965; 150: 893-9. 2. BANG G , URIST MR. Bone induction in excavation chambers in matrix of decalcified dentin. Arcli Sur^ 1967; 94: 781-9. 3. URIST MR,
SILVERMAN BF, BURING K , ROSENHERG J M .
The bone induction principle. Clin Orttiop 1967; 53: 243-83. 4. REDDI AH,
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