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Experimental study on demineralized bone matrix (DBM) and coral as bone graft substitutes in maxillofacial surgery
Int. J. Oral Maxillofac. Surg. 1994; 23. 395 398 Printed in Denmark. All rights reserved
Copyright ©Munksgaard 1994 lntemationa]]ottmd of
Oral8c MaxillofacialSurgery ISSN 0901-5027
Experimental study on demineralized bone matrix (DBM) and coral as bone graft substitutes maxllofacval surgery
L. Dupoirieux 1, V. Costes 2, P. Jammet 1, F. Souyris 1 Departments of 1Oral and Maxillofacial Surgery and 2pathology, H6pital Lapeyronie, Montpellier, France
L. Dupoirieux, V. Costes, P. Jammet, F. Souyris: Experimental study on demineralized bone matrix ( D B M ) and coral as bone graft substitutes in maxillofacial surgery. Int. J. Oral Maxillofac. Surg. 1994; 23:395 398. © Munksgaard, 1994 Abstract. Three different bone substitutes were implanted in a standardized nasal bone defect in 30 Wistar rats. The results were assessed at 2 months by macroscopic examination, contact radiography, and histologic analysis. Demineralized osseous implants sterilized by ethylene oxide induced bone formation in 90% of the the cases, as no heat-treated graft showed any bone formation. Coralline grafts were osteointegrated in 50% of the cases, but osteoconduction was not sufficient to achieve complete bone repair. This study implies that ethylene oxide sterilization does not impair biologic properties of demineralized grafts, but further studies on more evolved animal species are necessary before human implantation.
Bone reconstruction is a daily problem for the maxillofacial surgeon involved in traumatology, tumor, or preprosthetic surgery. Although autogenous bone remains the best material for bone grafting 6, many bone substitutes have been proposed to decrease donor site morbidity and operative time. For wide clinical acceptance, the ideal bone substitute should be biocompatible, have good mechanical properties, and show osteoinductive or at least osteoconductive capacities. Osteoconduction is the most recognized process for bone repair. The implant acts as a scaffold for bone ingrowth and is supposed to be replaced by bone. Osteoinduction has been more recently investigated as a possible mechanism of bone regeneration. Since the first report of URIST24 in 1965, many studies have focused on the
use of demineralized bone as an osteoinductive bone substitute. In 1972, REDDI & HUGGINS 17 described the histologic sequences of bone formation from demineralized bone. Bone morphogenetic protein, which has been extracted from bone, dentin, and osteosarcoma tissue, can induce differentiation of mesenchyma-type cells into cartilage and bone I. Bone differentiation has been reproduced in an in vitro model of rat connective-tissue cell cultures 12. Experimental studies on demineralized bone matrix as a bone substitute are very promising, but methods of storage and sterilization of these grafts are still unclear 1°,14. This study compared two methods of sterilization of demineralized bone with coralline grafts, because this bone substitute has been recognized as a safe and effective
Key words: demineralized bone matrix; coral; osteoinduction; animal experiment.
Accepted for publication 1 January 1994
material for reconstruction of the maxillofacial skeleton ls'2°'21.
Material and methods Material Thirty adult Wistar rats were used in this study. The demineralized osseous implants were obtained from the iliac crest of killed rats. The grafts were prepared according to the method of REDDI c~5 HUGGINS 17. The corticospongiose disks were demineralized for 3 h in O. 5 M HC1, followed by sequential washing in saline water, absolute ethanol, and anhydrous ether. In group I (n = 10), the grafts were sterilized by autoclaving for 1 h at 80°C. In group II (n 10), the grafts were sterilized with ethylene oxide gas. The specimens were aerated for 48 h before implantation. In group III (n = 10), natural coralline grafts with a void volume of 50% and mean pore size of 150-400 /2m were used. The
396
Dupoirieux et al.
"6
HT-DBM EO-DBM COaL Fig. 1. Results on graft mobility.
Fig. 4. Postoperative radiograph of HT-DBM graft (arrowhead) showing no bone mineralization.
Fig. 2. Postoperative view of HT-DBM graft.
"6
E E
HT-DBM
EO-DBM
CORAL
Fig. 3. Results on radiopacity.
grafts were sterilized by the same method as in group I.
Surgical procedure Animals were anesthetized with intramuscular ketamine (150 mg/kg of body weight). Under aseptic conditions, a midline incision was made on the nose, and the periosteum was reflected laterally. A round, 6-mm-wide, full-thickness defect was created on the nasal bone with a dental bur under saline irrigation. A similar defect was made on the frontal bone. The graft was inserted in the nasal defect because the frontal defect was left empty for absolute control. The incision was closed in layers. All animals were killed at 2 months by ether inhalation. Results were analyzed by three methods. At gross examination, graft mobility was tested by gentle pal-
pation. Contact radiographs were obtained by placing the calvarial specimens on a 5×7 cm Kodak Ektaspeed intraoral film. The radiograph apparatus (Siemens Heliodent, Siemens, Germany) was set at 70 kV, with an exposure time of 0.4 s. The film was then treated in an automatic x-ray processor (Dfirr-Periomat, Bietigheim, Germany). The radiopacity was analyzed in a semiquantitative way by comparison with the control site. After radiography, the mandible specimens were fixed in 10% buffered formalin, decalcified in formic acid, and embedded in hard paraffin. Serial 7-#m-thick sections were made in the coronal plane and stained with hematoxylin-eosin. The histologic variables observed were osteoblastic apposition and bone-marrow development. The extent of bone ingrowth was assessed in a qualitative, comparative fashion only and categorized in three classes (no bone formation, marginal bone formation, and complete ossification).
conclusion on bone f o r m a t i o n can be drawn from radiographic results on coralline grafts because this material is naturally dense. In the two coralline specimens showing no radiopacity, macroscopic examination revealed complete resorption of the graft. In the the EOD B M grafts, partial vertical resorption was found in four cases. Histologic results are graphed in Fig. 5. N o bone formation was found in the H T - D B M grafts. Histologic section of the grafts showed the presence of an a m o r p h o u s substance without living cells. Five of the coralline grafts showed bone ingrowth from the margins, but none of them were completely replaced by bone. Complete bone repair was obtained in five EOD B M grafts (Fig. 6), as four other grafts showed only marginal ossification.
Results At gross examination, no graft showed signs of infection or encapsulation. The control defects showed no bone healing, and the cavities were filled with connective tissue. Results on graft mobility are graphed in Fig. 1. All heat-treated demineralized grafts (HT-DBM) were mobile, and the interface between bone and implant was well delineated. At inspection, these grafts had a cartilaginous-like aspect (Fig. 2). Coralline grafts were twice as mobile as ethylene oxide-demineralized grafts (EO-DBM). Results on radiopacity are shown in Fig. 3. N o radiopacity could be detected in H T - D B M grafts (Fig. 4). N o
Discussion Determination of a critical-size defect is necessary for valid results on a new bone substitute ]9. Our model appeared to be satisfactory, as no bone healing
numero, 10 °
[ ] no bone formation
cases
[ ] marginal
4
ossification
[ ] complete ossification
HT-
EO-
DBM
DBM
CORAL
Fig. 5. Results on bone regeneration.
B o n e g r a f t substitutes
397
In conclusion, demineralized bone graft may be favorably compared with coral as a bone substitute in our experimental model. Sterilization by ethylene oxide was found to preserve the osteogenic capacity of the grafts, although complete bone repair was achieved in only 50% of the cases. However, possible clinical applications of demineralized bone should be demonstrated by further studies on higher animal species. References
Fig. 6. Histologic result of EO-DBM graft specimen showing complete bone regeneration
(HE, × 100).
was present at the control site. Macroscopic examination revealed that the best results were obtained with EODBM grafts. These results were consistent with previous studies 14 using a calvarial model, although the size of the defect was too small to draw firm conclusions. Other studies 5'8 have related better results with demineralized bone powder because the surface area of the graft exposed to the recipient bed is increased. The embryogenic origin of the graft does not influence its biologic properties 1°. Contradicting the original study of URIST24, our results indicate that osteogenesis was altered in HTDBM grafts. Although URIST stated that only collagen fibers were denatured by heating, .our radiologic studies have confirmed that HT-DBM grafts were not mineralized. Heat treatment of the graft also impaired revascularization from the recipient bed, as reported for nondemineralized grafts 22. A refrigerating liquid is thus recommended to prevent mechanical trauma during graft preparation s. Our results on coralline grafts as inlay bone grafts were disappointing because graft incorporation was obtained in only 60% of the cases. Histologic results have also confirmed limited bone regeneration. In previous studies9,11,1s, resorbable porite-type coralline grafts appeared to promote initial bone formation, but osteoconduction remains limited even after long-term follow-up. Natural or artificial coralline grafts have also been used as onlay bone
graft in different sites with uncertain resuits24. These studies support the choice of a nonresorbable coralline graft in these clinical situations because osteoconduction cannot produce complete bone regeneration2j. Histologic results have confirmed osteointegration of EODBM grafts, as already reported in other experimental studies I°,14,24. Bone differentiation of mesenchymal cells has been attributed to osteoinductive proteins, but none of these factors have been completely purified7'23. The success of demineralized implants is also explained by acid treatment that decreases bone antigenicity. Acid might be considered to be a sterilizing agent, but penetration of the solution into dense cortical bone is not sufficient to achieve complete decontamination of the graft. This study has demonstrated that oxide ethylene treatment has no adverse effect on demineralized bone matrix. This is a safe method currently used for allograft bone banking16. Alternative methods have been shown to have deleterious effects on biologic characteristics of demineralized grafts. Soaking the grafts in 1% povidone-iodine inhibits the osteogenic capacity of the graft 5. Irradiated DBM grafts have been used for human implantation5, but experimental studies 13 have found a reduced osteogenic capacity with this process. Chemosterilization seems to be an effective method to prevent bacterial contamination, but this procedure is long and expensive25.
1. BESSHOK, TAGAWAT0 MURATAM. Purification of rabbit bone morphogenetic protein derived from bone, dentin, and wound tissue after tooth extraction. J Oral Maxillofac Surg 1990: 48: 162-9. 2. BUTTS TE, PETERSON LJ, ALLEN CM. Early soft tissue ingrowtb into porous block hydroxyapafite. J Oral Maxillofac Surg 1989: 47:475 9. 3. EL DEEB M, ROSZKOWSKI M. Hydroxylapatite granules and blocks as an extracranial augmenting material in rhesus monkeys. J Oral Maxillofac Surg 1988: 46: 33-40. 4. EL DEEBM, HOLMESRE. Zygomatic and mandibular augmentation with proplast and porous hydroxyapatite in rhesus monkeys. J Oral Maxillofac Surg 1989: 47:480 8. 5. GLOWACKI J, KABANLB, MURRAYJE, FOLKMANJ, MULLIKENJB. Application of the biological principle of induced osteogenesis for craniofacial defects. Lancet 1981: i: 959-63. 6. GOLDBERGVM, STEVENSON S. Natural history of autografts and allografts. Clin Orthop 1987: 225: %16. 7. HOLLINGER J, MARK DE, BACH DE, REDOI AH, SBYFERAE. Calvarial bone regeneration using osteogenin. J Oral Maxillofac Surg 1989: 47:1182-6. 8. HOLLINGERJO, MARKDE, GOCOR QuIGLEYN, DESVERREAUXRW,,BACHDE. A comparison of four particulate bone derivatives. Clin Orthop 1991: 267:255 63. 9. HOLMES RE, HAGLERHK. Porous hydroxyapatite as a bone graft substitute in cranial reconstruction: a histometric study. Plast Reconstr Surg 1988: 81:662 71. t0. ISAKSSONS, ALBERIUSP. Comparison of regenerative capacity elicited by demineralized bone matrix of different embryogenic origins. J Craniomaxillofac Surg 1992: 20: 73-80. 11. KLINGE B, ALBERIUS E ISAKSSON S, JONSSON J. Osseous response to implanted natural bone mineral and synthetic hydroxylapatite ceramic in the repair of experimental skull bone defects. J Oral Maxillofac Surg 1992: 50: 241-9. 12. K~BLERN, gRISTM. Allogenicbone and cartilage morphogenesis. Rat BMP in vivo and in vitro. J Craniomaxillofac Surg 1991: 19: 283-8.
398
Dupoirieux et al.
13. LORENTE CA, SOnG BZ, DONOFF RB. Healing of bony defects in the irradiated and unirradiated rat mandible. J Oral Maxillofac Surg 1992: 50: 1305-9. 14. MULLIKEN JB, GLOWACKI J. Induced osteogenesis for repair and construction in the craniofacial region. Plast Reconstr Surg 1980: 65: 553-9. 15. PATAT JL, GUILLEMIN G. Le corail naturel utilis6 comme biomat6riau de substitution g la greffe osseuse. Ann Chir Plast Esthet 1989: 34: 221-5. 16. PROLO DJ, PEDROTTI PW, WHITE DH. Ethylene oxide sterilization of bone, dura mater, and fascia lata for human transplantation. Neurosurgery 1980: 6: 52939. 17. REDDI AH, HUGGINS C. Biochemical sequences in the transformation of normal fibroblasts in adolescent rats. Proc Natl Acad Sci U S A 1972: 69: 1601-5. 18. ROSEN HM, McEAgLAND MM. The bio-
logic behavior of hydroxyapatite implanted into the maxillofacial skeleton. Plast Reconstr Surg 1990: 85: 71853. 19. SCHNITZ JR HOLUNOER JO. The critical size defect as an experimental model for craniomandibulofacial nonunions. Clin Orthop 1986: 205: 29%308. 20. SOUYRISF, CHEVALIERJR PAYROTC, PELLEQUER C, GARY-BOBo A, MERLr~R C. Bilan apr~s quatre ans d'exp6rimentation du corail au titre d'implant osseux. Ann Chir Plast Esthet 1984: 29: 256-60. 21. SouYRIS F, PELLEQUERC, PAYROTC, SERVERA C. Coral, a new biomedical material. Experimental and first clinical investigations on Madreporaria. J Maxillofac Surg 1985: 13: 64-9. 22. SULLIVANWG, SZWmKUNPR. Revascularization of cranial versus iliac crest bone grafts in the rat. Plast Reconstr Surg 1991: 87: 1105-9. 23. TAKAGI K, URIST MR. The reaction of
the dura to bone morphogenetic protein (BMP) in repair of skull defects. Ann Surg 1982: 196: 100-9. 24. URlST MR. Bone formation by autoinduction. Science 1965: 150:893 9. 25. URIST MR. Chemosterilized antigen-extracted-surface demineralized autolysed allogeneic (AAA) bone for arthrodesis. In: FRIEDLANDERGE, ed.: Osteochondral allografts. Biology, banking and clinical applications. Boston: Little, Brown, 1981: 193-201.
Address:
Dr L. Dupoirie;tx Department of Oral and Maxillofacial Surgery H6pital Lapeyronie 34295-Montpellier Cedex 5 France
Copyright ©Munksgaard 1994 lntemationa]]ottmd of
Oral8c MaxillofacialSurgery ISSN 0901-5027
Experimental study on demineralized bone matrix (DBM) and coral as bone graft substitutes maxllofacval surgery
L. Dupoirieux 1, V. Costes 2, P. Jammet 1, F. Souyris 1 Departments of 1Oral and Maxillofacial Surgery and 2pathology, H6pital Lapeyronie, Montpellier, France
L. Dupoirieux, V. Costes, P. Jammet, F. Souyris: Experimental study on demineralized bone matrix ( D B M ) and coral as bone graft substitutes in maxillofacial surgery. Int. J. Oral Maxillofac. Surg. 1994; 23:395 398. © Munksgaard, 1994 Abstract. Three different bone substitutes were implanted in a standardized nasal bone defect in 30 Wistar rats. The results were assessed at 2 months by macroscopic examination, contact radiography, and histologic analysis. Demineralized osseous implants sterilized by ethylene oxide induced bone formation in 90% of the the cases, as no heat-treated graft showed any bone formation. Coralline grafts were osteointegrated in 50% of the cases, but osteoconduction was not sufficient to achieve complete bone repair. This study implies that ethylene oxide sterilization does not impair biologic properties of demineralized grafts, but further studies on more evolved animal species are necessary before human implantation.
Bone reconstruction is a daily problem for the maxillofacial surgeon involved in traumatology, tumor, or preprosthetic surgery. Although autogenous bone remains the best material for bone grafting 6, many bone substitutes have been proposed to decrease donor site morbidity and operative time. For wide clinical acceptance, the ideal bone substitute should be biocompatible, have good mechanical properties, and show osteoinductive or at least osteoconductive capacities. Osteoconduction is the most recognized process for bone repair. The implant acts as a scaffold for bone ingrowth and is supposed to be replaced by bone. Osteoinduction has been more recently investigated as a possible mechanism of bone regeneration. Since the first report of URIST24 in 1965, many studies have focused on the
use of demineralized bone as an osteoinductive bone substitute. In 1972, REDDI & HUGGINS 17 described the histologic sequences of bone formation from demineralized bone. Bone morphogenetic protein, which has been extracted from bone, dentin, and osteosarcoma tissue, can induce differentiation of mesenchyma-type cells into cartilage and bone I. Bone differentiation has been reproduced in an in vitro model of rat connective-tissue cell cultures 12. Experimental studies on demineralized bone matrix as a bone substitute are very promising, but methods of storage and sterilization of these grafts are still unclear 1°,14. This study compared two methods of sterilization of demineralized bone with coralline grafts, because this bone substitute has been recognized as a safe and effective
Key words: demineralized bone matrix; coral; osteoinduction; animal experiment.
Accepted for publication 1 January 1994
material for reconstruction of the maxillofacial skeleton ls'2°'21.
Material and methods Material Thirty adult Wistar rats were used in this study. The demineralized osseous implants were obtained from the iliac crest of killed rats. The grafts were prepared according to the method of REDDI c~5 HUGGINS 17. The corticospongiose disks were demineralized for 3 h in O. 5 M HC1, followed by sequential washing in saline water, absolute ethanol, and anhydrous ether. In group I (n = 10), the grafts were sterilized by autoclaving for 1 h at 80°C. In group II (n 10), the grafts were sterilized with ethylene oxide gas. The specimens were aerated for 48 h before implantation. In group III (n = 10), natural coralline grafts with a void volume of 50% and mean pore size of 150-400 /2m were used. The
396
Dupoirieux et al.
"6
HT-DBM EO-DBM COaL Fig. 1. Results on graft mobility.
Fig. 4. Postoperative radiograph of HT-DBM graft (arrowhead) showing no bone mineralization.
Fig. 2. Postoperative view of HT-DBM graft.
"6
E E
HT-DBM
EO-DBM
CORAL
Fig. 3. Results on radiopacity.
grafts were sterilized by the same method as in group I.
Surgical procedure Animals were anesthetized with intramuscular ketamine (150 mg/kg of body weight). Under aseptic conditions, a midline incision was made on the nose, and the periosteum was reflected laterally. A round, 6-mm-wide, full-thickness defect was created on the nasal bone with a dental bur under saline irrigation. A similar defect was made on the frontal bone. The graft was inserted in the nasal defect because the frontal defect was left empty for absolute control. The incision was closed in layers. All animals were killed at 2 months by ether inhalation. Results were analyzed by three methods. At gross examination, graft mobility was tested by gentle pal-
pation. Contact radiographs were obtained by placing the calvarial specimens on a 5×7 cm Kodak Ektaspeed intraoral film. The radiograph apparatus (Siemens Heliodent, Siemens, Germany) was set at 70 kV, with an exposure time of 0.4 s. The film was then treated in an automatic x-ray processor (Dfirr-Periomat, Bietigheim, Germany). The radiopacity was analyzed in a semiquantitative way by comparison with the control site. After radiography, the mandible specimens were fixed in 10% buffered formalin, decalcified in formic acid, and embedded in hard paraffin. Serial 7-#m-thick sections were made in the coronal plane and stained with hematoxylin-eosin. The histologic variables observed were osteoblastic apposition and bone-marrow development. The extent of bone ingrowth was assessed in a qualitative, comparative fashion only and categorized in three classes (no bone formation, marginal bone formation, and complete ossification).
conclusion on bone f o r m a t i o n can be drawn from radiographic results on coralline grafts because this material is naturally dense. In the two coralline specimens showing no radiopacity, macroscopic examination revealed complete resorption of the graft. In the the EOD B M grafts, partial vertical resorption was found in four cases. Histologic results are graphed in Fig. 5. N o bone formation was found in the H T - D B M grafts. Histologic section of the grafts showed the presence of an a m o r p h o u s substance without living cells. Five of the coralline grafts showed bone ingrowth from the margins, but none of them were completely replaced by bone. Complete bone repair was obtained in five EOD B M grafts (Fig. 6), as four other grafts showed only marginal ossification.
Results At gross examination, no graft showed signs of infection or encapsulation. The control defects showed no bone healing, and the cavities were filled with connective tissue. Results on graft mobility are graphed in Fig. 1. All heat-treated demineralized grafts (HT-DBM) were mobile, and the interface between bone and implant was well delineated. At inspection, these grafts had a cartilaginous-like aspect (Fig. 2). Coralline grafts were twice as mobile as ethylene oxide-demineralized grafts (EO-DBM). Results on radiopacity are shown in Fig. 3. N o radiopacity could be detected in H T - D B M grafts (Fig. 4). N o
Discussion Determination of a critical-size defect is necessary for valid results on a new bone substitute ]9. Our model appeared to be satisfactory, as no bone healing
numero, 10 °
[ ] no bone formation
cases
[ ] marginal
4
ossification
[ ] complete ossification
HT-
EO-
DBM
DBM
CORAL
Fig. 5. Results on bone regeneration.
B o n e g r a f t substitutes
397
In conclusion, demineralized bone graft may be favorably compared with coral as a bone substitute in our experimental model. Sterilization by ethylene oxide was found to preserve the osteogenic capacity of the grafts, although complete bone repair was achieved in only 50% of the cases. However, possible clinical applications of demineralized bone should be demonstrated by further studies on higher animal species. References
Fig. 6. Histologic result of EO-DBM graft specimen showing complete bone regeneration
(HE, × 100).
was present at the control site. Macroscopic examination revealed that the best results were obtained with EODBM grafts. These results were consistent with previous studies 14 using a calvarial model, although the size of the defect was too small to draw firm conclusions. Other studies 5'8 have related better results with demineralized bone powder because the surface area of the graft exposed to the recipient bed is increased. The embryogenic origin of the graft does not influence its biologic properties 1°. Contradicting the original study of URIST24, our results indicate that osteogenesis was altered in HTDBM grafts. Although URIST stated that only collagen fibers were denatured by heating, .our radiologic studies have confirmed that HT-DBM grafts were not mineralized. Heat treatment of the graft also impaired revascularization from the recipient bed, as reported for nondemineralized grafts 22. A refrigerating liquid is thus recommended to prevent mechanical trauma during graft preparation s. Our results on coralline grafts as inlay bone grafts were disappointing because graft incorporation was obtained in only 60% of the cases. Histologic results have also confirmed limited bone regeneration. In previous studies9,11,1s, resorbable porite-type coralline grafts appeared to promote initial bone formation, but osteoconduction remains limited even after long-term follow-up. Natural or artificial coralline grafts have also been used as onlay bone
graft in different sites with uncertain resuits24. These studies support the choice of a nonresorbable coralline graft in these clinical situations because osteoconduction cannot produce complete bone regeneration2j. Histologic results have confirmed osteointegration of EODBM grafts, as already reported in other experimental studies I°,14,24. Bone differentiation of mesenchymal cells has been attributed to osteoinductive proteins, but none of these factors have been completely purified7'23. The success of demineralized implants is also explained by acid treatment that decreases bone antigenicity. Acid might be considered to be a sterilizing agent, but penetration of the solution into dense cortical bone is not sufficient to achieve complete decontamination of the graft. This study has demonstrated that oxide ethylene treatment has no adverse effect on demineralized bone matrix. This is a safe method currently used for allograft bone banking16. Alternative methods have been shown to have deleterious effects on biologic characteristics of demineralized grafts. Soaking the grafts in 1% povidone-iodine inhibits the osteogenic capacity of the graft 5. Irradiated DBM grafts have been used for human implantation5, but experimental studies 13 have found a reduced osteogenic capacity with this process. Chemosterilization seems to be an effective method to prevent bacterial contamination, but this procedure is long and expensive25.
1. BESSHOK, TAGAWAT0 MURATAM. Purification of rabbit bone morphogenetic protein derived from bone, dentin, and wound tissue after tooth extraction. J Oral Maxillofac Surg 1990: 48: 162-9. 2. BUTTS TE, PETERSON LJ, ALLEN CM. Early soft tissue ingrowtb into porous block hydroxyapafite. J Oral Maxillofac Surg 1989: 47:475 9. 3. EL DEEB M, ROSZKOWSKI M. Hydroxylapatite granules and blocks as an extracranial augmenting material in rhesus monkeys. J Oral Maxillofac Surg 1988: 46: 33-40. 4. EL DEEBM, HOLMESRE. Zygomatic and mandibular augmentation with proplast and porous hydroxyapatite in rhesus monkeys. J Oral Maxillofac Surg 1989: 47:480 8. 5. GLOWACKI J, KABANLB, MURRAYJE, FOLKMANJ, MULLIKENJB. Application of the biological principle of induced osteogenesis for craniofacial defects. Lancet 1981: i: 959-63. 6. GOLDBERGVM, STEVENSON S. Natural history of autografts and allografts. Clin Orthop 1987: 225: %16. 7. HOLLINGER J, MARK DE, BACH DE, REDOI AH, SBYFERAE. Calvarial bone regeneration using osteogenin. J Oral Maxillofac Surg 1989: 47:1182-6. 8. HOLLINGERJO, MARKDE, GOCOR QuIGLEYN, DESVERREAUXRW,,BACHDE. A comparison of four particulate bone derivatives. Clin Orthop 1991: 267:255 63. 9. HOLMES RE, HAGLERHK. Porous hydroxyapatite as a bone graft substitute in cranial reconstruction: a histometric study. Plast Reconstr Surg 1988: 81:662 71. t0. ISAKSSONS, ALBERIUSP. Comparison of regenerative capacity elicited by demineralized bone matrix of different embryogenic origins. J Craniomaxillofac Surg 1992: 20: 73-80. 11. KLINGE B, ALBERIUS E ISAKSSON S, JONSSON J. Osseous response to implanted natural bone mineral and synthetic hydroxylapatite ceramic in the repair of experimental skull bone defects. J Oral Maxillofac Surg 1992: 50: 241-9. 12. K~BLERN, gRISTM. Allogenicbone and cartilage morphogenesis. Rat BMP in vivo and in vitro. J Craniomaxillofac Surg 1991: 19: 283-8.
398
Dupoirieux et al.
13. LORENTE CA, SOnG BZ, DONOFF RB. Healing of bony defects in the irradiated and unirradiated rat mandible. J Oral Maxillofac Surg 1992: 50: 1305-9. 14. MULLIKEN JB, GLOWACKI J. Induced osteogenesis for repair and construction in the craniofacial region. Plast Reconstr Surg 1980: 65: 553-9. 15. PATAT JL, GUILLEMIN G. Le corail naturel utilis6 comme biomat6riau de substitution g la greffe osseuse. Ann Chir Plast Esthet 1989: 34: 221-5. 16. PROLO DJ, PEDROTTI PW, WHITE DH. Ethylene oxide sterilization of bone, dura mater, and fascia lata for human transplantation. Neurosurgery 1980: 6: 52939. 17. REDDI AH, HUGGINS C. Biochemical sequences in the transformation of normal fibroblasts in adolescent rats. Proc Natl Acad Sci U S A 1972: 69: 1601-5. 18. ROSEN HM, McEAgLAND MM. The bio-
logic behavior of hydroxyapatite implanted into the maxillofacial skeleton. Plast Reconstr Surg 1990: 85: 71853. 19. SCHNITZ JR HOLUNOER JO. The critical size defect as an experimental model for craniomandibulofacial nonunions. Clin Orthop 1986: 205: 29%308. 20. SOUYRISF, CHEVALIERJR PAYROTC, PELLEQUER C, GARY-BOBo A, MERLr~R C. Bilan apr~s quatre ans d'exp6rimentation du corail au titre d'implant osseux. Ann Chir Plast Esthet 1984: 29: 256-60. 21. SouYRIS F, PELLEQUERC, PAYROTC, SERVERA C. Coral, a new biomedical material. Experimental and first clinical investigations on Madreporaria. J Maxillofac Surg 1985: 13: 64-9. 22. SULLIVANWG, SZWmKUNPR. Revascularization of cranial versus iliac crest bone grafts in the rat. Plast Reconstr Surg 1991: 87: 1105-9. 23. TAKAGI K, URIST MR. The reaction of
the dura to bone morphogenetic protein (BMP) in repair of skull defects. Ann Surg 1982: 196: 100-9. 24. URlST MR. Bone formation by autoinduction. Science 1965: 150:893 9. 25. URIST MR. Chemosterilized antigen-extracted-surface demineralized autolysed allogeneic (AAA) bone for arthrodesis. In: FRIEDLANDERGE, ed.: Osteochondral allografts. Biology, banking and clinical applications. Boston: Little, Brown, 1981: 193-201.
Address:
Dr L. Dupoirie;tx Department of Oral and Maxillofacial Surgery H6pital Lapeyronie 34295-Montpellier Cedex 5 France