J Neurosurg 76:275-279, 1992
Regeneration of calvarial defects by a composite of bioerodible polyorthoester and demineralized bone in rats EmIK SOLHEIM,M.D., ELSE M. PINHOLT, D.D.S., GISLE BANG, PH.D., AND EINAR SUDMANN, PH.D.
Institutefor Surgical Research, Rikshospitalet, University of Oslo, Oslo, Department of Oral Pathology and Forensic Odontology and Hagavik Orthopaedic Hospital, University of Bergen, Bergen, Norway ~" A study was performed to evaluate regeneration of defects in rat calvaria either unfilled or filled with a bioerodible polyorthoester only, demineralized bone only, or a composite of both. At 4 weeks, histological and radiographic studies showed that defects filled with a composite ofbioerodible polyorthoester and demineralized bone or demineralized bone alone were bridged by bone. Unfilled defects or defects filled with polyorthoester only did not heal. The polyorthoester caused slight inflammation that subsided by 3 weeks, and only traces of the filler could be detected at 4 weeks. The polyorthoester provided local hemostasis when used either alone or in composites with demineralized bone. The composite implant was moldable, easily contoured, and technically easier to use than demineralized bone alone. KEY WORDS cranioplasty 9 demineralized bone bioerodible polyorthoester rat '
UTOGENOUS split-skull, 9,~s iliac-crest, 17 and splitrib 2 bone grafts are considered the standard materials for cranioplasty. Autogenous bone may, however, be difficult to obtain in sufficient amount, and the use of it requires that the patient undergo additional surgical procedures. Demineralized bone and dentin produce osseous healing in the craniomaxillofacial region by osteoinduction,37 both experimentally ~3'19,26.33and clinically.7" 14,21.22 When used as chips or powder, demineralized bone is technically difficult to apply for reconstruction and bridging of bone defects since the particles may become displaced intra- and postoperatively; thus, incorporating the demineralized bone in a bioerodible carrier seems warranted. 8 In addition, when osteoinductors that cause bone bridging of cranial defects experimentally~ 1,~6,28,36,38have been further purified, characterized, and become readily available, a delivery system for their sustained release is needed. 28'36Alzamer is a bioerodible polyorthoester (poly(2,2-dioxy-cis,trans- 1,4-cyclohexane dimethylene tetrahydrofuran)) that has been developed for use as the vehicle in a sustained drug-release system. 3 The purpose of the present study was to evaluate regeneration of bone in rat calvarial defects induced by a composite of bioerodible polyorthoester and demineralized lyophilized bone.
J. Neurosurg. / Volume 76/February, 1992
.
bone regeneration
Materials and Methods
Preparation of Implant Material Demineralized bone was prepared by sterile technique from the long bones of male Wistar rats, each weighing 400 to 500 gin. Dissected diaphyses were crushed and the marrow was removed. The cortex was cut into chips, demineralized in 0.2 N HCI for 48 hours at 4~ and flushed with saline, j The demineralized bone was suspended in liquid nitrogen, lyophilized for 22 hours, and pulverized at room temperature to particles 0.1 to 2.0 sq m m in size. The size was assessed by area measurements on photomicrographs of random samples. The demineralized lyophilized bone particles were sterilized in ethylene oxide* for 3 hours, maintained in sterile containers at 4~ and implanted within 48 hours. Alzamer,t the bioerodible polyorthoester (poly(2,2dioxy-cis,trans-l,4-cyclohexane dimethylene tetrahydrofuran)) was used alone and in composites with demineralized bone. The polyorthoester is a result of condensation of 2,2-diethoxytetrahydrofuran and cis, * Ethylene oxide manufactured by Alcon Universal Ltd., Fort Worth, Texas. t Alzamer polyorthoester manufactured by Alza Corporation, Palo Alto, California. 975
E. Solheim, E. M. Pinholt, G. Bang, and E. Sudmann TABLE 1
Osteoinduction and bone bridging of defects in rats* Week2
Week 3
Week4
Total
Experimental Group 0 1 2 0 1 2 0 1 2 0 1 2 A(composite) 0 2 1 B(demineralizedbone) 1 l 1 C(polyorthoester) 3 0 0 D (no implant) 3 0 0
0 0 3 3
0 0 0 0
3 3 0 0
0 0 3 3
0 0 0 0
3 0 2 7~ 3 1 1 7"~ 0 9 0 0 0 9 0 0
* The data indicate the number of rats with a positive treatment response (osteoinductionat Week 2 or 3 or bone bridgingat Week 4) in none (0), one ( 1), or both (2) of the calvarial defects. ~"Significantlydifferent from the groupswith no implant or filling with polyorthoesteronly (p = 0.0001).
trans- 1,4-bis(hydroxymethyl)cyclohexane (CHDM) and can be formulated with different physical properties; in this study, the polyorthoester was soft and moldable. The composites were prepared by manually mixing demineralized bone and polyorthoester at room temperature under sterile conditions immediately before implantation.
Surgical Procedure A total of 36 male Wistar rats, each weighing 231 + 13 gm (mean _+ standard deviation), were randomly assigned to one of four groups of nine rats each. The animals were fed standard rat laboratory food and water ad libitum. Anesthesia was induced by Hypnorm-Dormicum at a dosage of0.15 ml/100 gm given intramuscularly. Under sterile conditions, the pericranium was exposed through a sagittal incision and stripped laterally. Two 4-mm burr holes were made; one in each parietal bone. The burr holes were irrigated with sterile saline during and after drilling to cool the tissue and to wash out the bone particles. Both defects in all Group A to C rats were filled: with a composite of 7 mg polyorthoester and 3 mg demineralized bone in Group A, with 3 mg demineralized bone in Group B, and with 7 mg polyorthoester in Group C. The defects were not filled in the Group D rats.
Evaluation of Specimens At 2, 3, and 4 weeks following the surgical procedure, three randomly selected animals in each group were killed by an overdose of ether inhalation; the dissected skull vaults were fixed in 4% neutral formalin. Radiographs were taken using dental x-ray equipment. The specimens were demineralized in 17% formic acid, dehydrated, and embedded in paraffin. Serial sections were cut at 5 um and stained with Harris' hematoxylin and eosin. Host-tissue responses, osteoinduction, and bone bridging of the defects were evaluated by light microscopy. Regeneration of bone defects by osteoinduction is a multistep process. Thus, a positive treatment response was defined as osteoinduction, indicated by the presence of bone formation within the defect and not in 276
FIG. 1. Photomicrograph showing a calvarial defect at Week 2 after filling with a composite of polyorthoester and demineralized bone. Parietal bone with new bone formation at the edge and cartilage, hypertrophied chondrocytes, calcified cartilage matrix, and incipient bone formation can be seen within the defect. H & E, x 70.
contact with the bone edges, at Weeks 2 to 3 as seen on histological examination and bone bridging of the defects at Week 4 as seen on histological and radiographic examinations. The treatment response of each rat was recorded as 0 (no osteoinduction or bone bridging), l (osteoinduction or bone bridging in one defect), or 2 (osteoinduction or bone bridging in both defects). To permit statistical analysis, the treatment response of all rats in each group studied at Weeks 2, 3, and 4 was compared collectively. The Kruskal-Wallis test was used to determine the null hypothesis (Ho) that there is no significant difference in treatment response by the different implants. Two sample Mann-Whitney tests were used to determine in which group the mean results were significantly different from each other when Ho was rejected. Because comparing multiple groups introduces a greater probability of type I error, the required p value (0.05) was divided by the number of comparisons (Bonferroni's correction); p < 0.008 (0.05/0.06) was regarded as significant. Results
There were no intra- or postoperative deaths. The animals gained weight and showed no signs of unhealthiness. The polyorthoester provided local hemostasis when used either alone or in composites with demineralized bone. The composite implant was moldable and easily contoured and technically easier to use than demineralized bone alone.
New Bone Formation At 2 weeks, cartilage, hypertrophied chondrocytes, calcified cartilage matrix, and incipient bone formation were present in a total of four defects implanted with
J. Neurosurg. / Volume 76 /February, 1992
Regeneration of calvarial defects by polyorthoester composite
FIG. 2. Photomicrographs showing calvarial defects at Week 4 after filling with a composite of polyorthoester and demineralized bone (left) and with demineralized bone only (right). Bridging of the defect by bone with bone marrow can be seen. H & E, x 2l.
the composite in three Group A rats (Fig. 1 and Table 1) and in a total of three defects implanted with demineralized bone alone in two Group B rats (Table 1). In both groups, additional new bone formation and some bone marrow were present within all defects on histological and radiographic study at 3 weeks. At 2 and 3 weeks, new bone had developed within all parts of the defects in Group A and B rats, whereas new bone formation in Group D rats with no defect filling and in Group C rats with polyorthoester filling only was sparse and confined to the bone edges (Table I). At 4 weeks, histological and radiographic study showed that all defects filled with the composite or demineralized bone alone were bridged by bone tissue (Fig. 2). No defects filled with polyorthoester only or left open were found to be healed on histological or radiographic examination during the observation period (Fig. 3). A positive response (osteoinduction at Week 2 or 3 or bone bridging at Week 4) was significantly greater in rats with defects filled with the composite or demineralized bone only (Groups A and B) compared to those with the defects filled with polyorthoester only or left open (Groups C and D) (p = 0.0001), whereas no significant difference was found between the two former groups (p = 0.6) or between the two latter (p = 1.0).
Host- Tissue Response In defects filled with the composite or polyorthoester only, the polyorthoester was present and slight inflammation with monocytes and some giant cells was seen at Week 2. The inflammation subsided by Week 3 and only traces of the polyorthoester could be detected at Week 4. In defects filled with demineralized bone only or left open, the inflammation at Week 2 was milder and no giant cells were detected. Discussion The low potential for regeneration of cranial defects in both animals and humans, 29 especially in adults, has clinical and experimental implications. Clinically, reconstruction of cranial defects resulting from trauma, infection, and craniectomies performed for access to intracranial abnormalities and tumor resection has presented challenging problems requiring the use of cranioplasty materials; bone grafts from various sites and alloplastic materials have been used. 4'5'~2'23 The ideal cranioplasty material should fulfill several criteria; 25,39 it should be: biocompatible, not inducing an inflammatory response; capable of stimulating osteogenesis; capable of being vascularized; radiolucent; moldable and easily contoured; and thermally nonconductive.
FIG. 3. Photomicrographs showing calvarial defects at Week 4 after filling with polyorthoester only (left) and without filling (right). Sparse new bone formation at the edges of the defect and loose connective tissue within the defect can be seen. H & E, x 21.
J. Neurosurg. / Volume 76 /February, 1992
277
E. Solheim, E. M. Pinholt, G. Bang, and E. Sudmann Critical-Size Defect Experimentally, healing of large skull defects in animals has been a commonly used test system for autogenous bone substitutes for more than a century.. 33 A defect that does not heal during the animals' lifetime is termed a "critical-size defect," and it has been proposed that evaluation of craniomaxillofacial bone repair materials should be initiated in critical-size defects in rat calvaria. 29 The critical-size calvarial defect varies according to species and age. It has been shown that 2mm parietal defects in Wistar rats weighing 500 gin, ~ 4-mm parietal defects in 28-day-old CD strain rats, j9 and 8-mm parietal defects in 6-month-old SpragueDawley rats 36 do not heal during 12 weeks, 6 months, and 12 weeks, respectively. We used 4-ram defects in 8-week-old male Wistar rats and healing did not occur during the observation period in the animals with the defects left open, whereas all defects that were filled with the composite or demineralized bone alone were bridged with new bone within 4 weeks.
Osteoinduction In rodents, osteoinduction consists of chemotaxis of mesenchymal ceils, mitosis, differentiation of cartilage, vascular invasion, and bone formation. 27'37In composites of osteoinductor and carrier, the carrier may inhibit the osteoinduction by two mechanisms. In the first possible mechanism, unabsorbed carrier will physically inhibit bone growth and closure of a bone defect. It has been stated that the ideal delivery system would be resorbed and replaced by preosseous tissues within 2 weeks, cartilage within 3 weeks, and bone within 4 to 6 weeks. 3~ Some existing delivery systems are resorbed more slowly than the time required for the demineralized bone-induced regeneration of the defect to take place. In one study of rabbits, ~ residual copolymer was still present 24 weeks after implantation of a composite of biodegradable copolymer of polylactide-polyglycolide and demineralized bone in calvarial defects. In another study, 38 beta tricalcium phosphate was still present 4 months after implantation as a composite with bone morphogenetic protein in calvarial defects in dogs. In the second possible mechanism, osteoinduction may be inhibited by a bioincompatible carrier that interferes physiologically with the multistep cascade of bone induction (for example, by inducing a chronic inflammation 3~ or by immunological mechanisms20).
Delivery System The delivery system should provide sustained, controlled release of the active substance. ~oAlzamer polyorthoester is designed to be used as a local sustained drug-release system? Zero-order drug release from polyorthoesters may be accomplished under certain conditions, 3'~~possibly because the rate of drug release is determined by erosion and not by simple diffusion since the polyorthoester is hydrophobic and may erode 978
heterogeneously from the surface first. ~5 Biodegradation of the polyorthoester takes place by hydrolysis to the ultimate products 4-hydroxybutyrate and CHDM; 4-hydroxybutyrate is further metabolized in the tricarboxylic acid cycle, with CO2 and H20 as the end products, and CHDM is excreted in urine. 32 In the present study, the polyorthoester did not inhibit osteoinduction and caused only a slight inflammation that subsided within 3 weeks. The results are consistent with those of a previous study where a composite of polyorthoester and demineralized bone implanted in the abdominal muscle of 89 Wistar rats induced cartilage and bone at the same rate as demineralized bone alone, as evaluated histologically and by ~SSruptake. 24By Week 4, the polyorthoester was mostly resorbed and all defects filled with a composite of polyorthoester and demineralized bone were bridged by new bone. These results support the contention that the polyorthoester is resorbed and does not physically obstruct bone formation. 3~ Our results indicate that the bridging of defects by the composite or demineralized bone alone was caused by bone induction and not merely osteoconduction; bone formation from the edges of the defect, as the typical pattern of bone induction, could be seen on histological examination (for example, cartilage at Week 2). J9.~6Furthermore, the regeneration progressed at an equal rate within all parts of the implant and not centripetally as with osteoconduction. Alzamer polyorthoester of the present formulation has a plastic consistency and can be molded intraoperatively according to present needs. The polyorthoester adheres to bone surfaces and provides local hemostasis by plugging spongy bone and secondarily promoting concentration of platelets and coagulation factors?4'35 Assorted formulations of the polyorthoester with different physical properties ranging from gel to solid can be manufactured.
Conclusions The composite implant used in this study was moldable, easily contoured, and technically easier to use than demineralized bone alone. The polyorthoester provided local hemostasis when used either alone or in a composite with demineralized bone, induced minor transient reaction, did not inhibit osteoinduction, and was resorbed as new bone formed within 4 weeks. As the polyorthoester may provide controlled, sustained release of the incorporated active substance, it seems promising as a carrier of purified inductors and/or growth factors.
Acknowledgments We thank Katrine Hove, Inger Liv Nordli, and Gunnvor ~3ijordsbakken for technical assistance and the Alza Corporation, Palo Alto, California, for providing the Alzamer polyorthoester. J. Neurosurg. / Volume 76/February, 1992
Regeneration of calvarial defects by polyorthoester composite References 1. Bang G: Induction of heterotopic bone formation by demineralized dentin: an experimental model in guinea pigs. Scand J Dent Surg 81:240-250, 1973 2. Brown RC: The repair of skull defects. Med J Aust 2: 409-411, 1917 3. Capozza R, Sendelbeck L, Balkenhol W: Preparation and evaluation of a bioerodible naltrexone delivery system, in Kostelnick RJ (ed): Polymeric Delivery Systems. Midland Maeromolecular Monographs. New York: Gordon & Breach, 1978, pp 59-73 4. Courtemanche AD, Thompson GB: Silastic cranioplasty following cranio-facial injuries. Hast Reconstr Surg 41: 165-170, 1968 5. Elkins CW, Cameron JE: Cranioplasty with acrylic plates. J Neurosurg 3:199-205, 1946 6. Freeman E, Turnbull RS: The value of osseous coagulum as a graft material. J Periodont Res 8:229-236, 1973 7. Glowacki J, Kaban LB, Murray JE, el at: Application of the biological principle of induced osteogenesis for craniofacial defects. Lancet 1:959-962, 1981 8. Glowacki J, Mulliken JB: Demineralized bone implants. Clin Hast Snrg 12:233-241, 1985 9. Guyuron B, Shafron M, Column B: Management of extensive and difficult cranial defects. J Neurosurg 69: 210-212, 1988 I0. Heller J, Penhale DWH, Helwing RF, etal: Release of norethindrone from poly(ortho esters). Polym Eng Sci 21: 727-731, 1981 11. Hollinger J, Mark DE, Bach DE, etal: Calvarial bone regeneration using osteogenin. J Oral Maxillofac Surg 47:1182-1186, 1989 12. Holmes RE: Bone regeneration within a coraUine hydroxyapatite implant. Hast Reconstr Surg 63:626-633, 1979 13. Kaban LB, Glowacki J: Augmentation of rat mandibular ridge with demineralized bone implants. J Dent Res 63: 998-1002, 1984 14. Ktimmel H: Ueber Knoch enimplantation. Dtsch Med Wochenschr 17:389-392, 1891 15. Langer R: Biopolymers in controlled release systems, in Piskin E, Hoffman AS (eds): Polymeric Biomaterials. Dordrecht: Martinus Nijhoff, 1986, pp 161-169 16. Lindholm TC, Lindholm TS, Alitalo I, et al: Bovine bone morphogenetic protein (bBMP) induced repair of skull trephine defects in sheep. Clln Orthop 227:265-268, 1988 17. Mauclaire P: Autogreffe cr~nienne empruntre ~ la tubrrosit6 iliaque, et homogreffe srreuse intermrningo-encOphalique. Bull Mere Soe Chit Paris 40:113-I 15, 1914 18. Miiller W: Zur Frage der tempor/iren Schfidelresektion an Stelle der Trepanation. Zentralbl Chir 17:65-66, 1890 19. Mulliken JB, Glowacki J: Induced osteogenesis for repair and construction in the craniofacial region. Plast Reconstr Surg 65:553-559, 1980 20. Nathan RM, Benlz H, Armstrong RM, et al: Osteogenesis in rats with an inductive bovine composite. J Orthop Res 6:324-334, 1788 21. Nordenram A, Bang G, Bernhoft CH: A clinical-radiographic study of allogenic demineralized dentin implants in cysticjaw cavities. Int J Oral Maxillofac Surg 4:61-64, 1975 22. Ousterhout DK: Clinical experience in cranial and facial reconstruction with demineralized bone. Ann Plast SuN 15:367-373, 1985
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23. Peyton WT, Hall HB: The repair of a cranial defect with a vitallium olate. Surgery 10:711-715, 1941 24. Pinholt EM, Solheim E, Bang G, et al: Bone induction by composite of bioerodible polyorthoester and demineralized bone matrix in rats. Acta Orthop Seam 62:476-48G, 1991 25. Rawlings CE III, Wilkins RH, Hanker JS, et al: Evaluation in cats of a new material for cranioplasty: a composite of plaster of Paris and hydroxylapatite, J Neurnsurg 69: 269-275, 1988 26. RayRD, Holloway JA: Bone implants. Preliminary report of an experimental study, d Bone Joint Surg (Am) 39: 1119-1128, 1957 27. Reddi AH, Huggins C: Biochemical sequences in the transformation of normal fibroblasts in adolescent rats. Proc Natl Acad Sci USA 69:1601-1605, 1972 28. Sato K, Urist MR: Induced regeneration of calvaria by bone morphogenetic protein (BMP) in dogs. Clin Orthop 197:301-311, 1985 29. Schmitz JP, Hollinger JO: The critical size defect as an experimental model for craniomandibulofacial nonunions. Clin Orthop 205:299-308, 1986 30. Schmitz JP, Hollinger JO: A preliminary study of the osteogenic potential of a biodegradable alloplastic-osteoinductive alloimplant. Clin Orthop 237:245-255, 1988 31. Sela J, Applebaum J, Uretzky G: Osteogenesis induced by bone matrix is inhibited by inflammation. Biomater Med Devices Artif Organs 14:227-237, 1986 32. Sendelbeck SL, Girdis CL: Disposition of a 14C-labeled bioerodible polyorthoester and its hydrolysis products, 4-hydroxybutyrate and cis,trans-l,4-bis(hydroxymethyl) cyclohexane, in rats. Drug Metab Dispos 13:291-295, 1985 33. Senn N: On the healing of aseptic bone cavities by implantation of antiseptic decalcified bone. Am J Meal Sci 98:219-243, 1989 34. Solheim E, Anfinsen OG, Holmsen H, et al: Effect of local hemostatics on platelet aggregation. Eur Surg Res 23:45-50, 1991 35. Sudmann B, Anfinsen OG, Rail M, etal: Use of a new hemostatic, bioerodible polymer versus bone wax made of beeswax - - a clinical and experimental study. Acta Orthop Scand 61 (Suppl 237):63-64, 1990 (Abstract) 36. Takagi K, Urist MR: The reaction of the dura to bone morphogenetie protein (BMP) in repair of skull defects. Ann Surg 196:100-109, 1982 37. Urist MR: Bone: formation by autoinduction. Science 150:893-899, 1965 38. Urist MR, Nilsson O, Rasmussen J, et al: Bone regeneration under the influence of a bone morphogenetic protein (BMP) beta tricalcium phosphate (TCP) composite in skull trephine defects in dogs. Clin Orthop 214: 295-304, 1987 39. Yamashima T: Cranioplasty with hydroxylapatite ceramic plates that can easily be trimmed during surgery. A preliminary report, Acta Neurochir 96:149-153, 1989 Manuscript received January 2, 1991. Accepted in final form June 3, 1991. This study was supported by grants from the J. E. Isberg Foundation. Address reprint requests to: Eirik Solheim, M.D., Hagavik Orthopaedic Hospital, N-5220 Hagavik, Norway.
279
Regeneration of calvarial defects by a composite of bioerodible polyorthoester and demineralized bone in rats EmIK SOLHEIM,M.D., ELSE M. PINHOLT, D.D.S., GISLE BANG, PH.D., AND EINAR SUDMANN, PH.D.
Institutefor Surgical Research, Rikshospitalet, University of Oslo, Oslo, Department of Oral Pathology and Forensic Odontology and Hagavik Orthopaedic Hospital, University of Bergen, Bergen, Norway ~" A study was performed to evaluate regeneration of defects in rat calvaria either unfilled or filled with a bioerodible polyorthoester only, demineralized bone only, or a composite of both. At 4 weeks, histological and radiographic studies showed that defects filled with a composite ofbioerodible polyorthoester and demineralized bone or demineralized bone alone were bridged by bone. Unfilled defects or defects filled with polyorthoester only did not heal. The polyorthoester caused slight inflammation that subsided by 3 weeks, and only traces of the filler could be detected at 4 weeks. The polyorthoester provided local hemostasis when used either alone or in composites with demineralized bone. The composite implant was moldable, easily contoured, and technically easier to use than demineralized bone alone. KEY WORDS cranioplasty 9 demineralized bone bioerodible polyorthoester rat '
UTOGENOUS split-skull, 9,~s iliac-crest, 17 and splitrib 2 bone grafts are considered the standard materials for cranioplasty. Autogenous bone may, however, be difficult to obtain in sufficient amount, and the use of it requires that the patient undergo additional surgical procedures. Demineralized bone and dentin produce osseous healing in the craniomaxillofacial region by osteoinduction,37 both experimentally ~3'19,26.33and clinically.7" 14,21.22 When used as chips or powder, demineralized bone is technically difficult to apply for reconstruction and bridging of bone defects since the particles may become displaced intra- and postoperatively; thus, incorporating the demineralized bone in a bioerodible carrier seems warranted. 8 In addition, when osteoinductors that cause bone bridging of cranial defects experimentally~ 1,~6,28,36,38have been further purified, characterized, and become readily available, a delivery system for their sustained release is needed. 28'36Alzamer is a bioerodible polyorthoester (poly(2,2-dioxy-cis,trans- 1,4-cyclohexane dimethylene tetrahydrofuran)) that has been developed for use as the vehicle in a sustained drug-release system. 3 The purpose of the present study was to evaluate regeneration of bone in rat calvarial defects induced by a composite of bioerodible polyorthoester and demineralized lyophilized bone.
J. Neurosurg. / Volume 76/February, 1992
.
bone regeneration
Materials and Methods
Preparation of Implant Material Demineralized bone was prepared by sterile technique from the long bones of male Wistar rats, each weighing 400 to 500 gin. Dissected diaphyses were crushed and the marrow was removed. The cortex was cut into chips, demineralized in 0.2 N HCI for 48 hours at 4~ and flushed with saline, j The demineralized bone was suspended in liquid nitrogen, lyophilized for 22 hours, and pulverized at room temperature to particles 0.1 to 2.0 sq m m in size. The size was assessed by area measurements on photomicrographs of random samples. The demineralized lyophilized bone particles were sterilized in ethylene oxide* for 3 hours, maintained in sterile containers at 4~ and implanted within 48 hours. Alzamer,t the bioerodible polyorthoester (poly(2,2dioxy-cis,trans-l,4-cyclohexane dimethylene tetrahydrofuran)) was used alone and in composites with demineralized bone. The polyorthoester is a result of condensation of 2,2-diethoxytetrahydrofuran and cis, * Ethylene oxide manufactured by Alcon Universal Ltd., Fort Worth, Texas. t Alzamer polyorthoester manufactured by Alza Corporation, Palo Alto, California. 975
E. Solheim, E. M. Pinholt, G. Bang, and E. Sudmann TABLE 1
Osteoinduction and bone bridging of defects in rats* Week2
Week 3
Week4
Total
Experimental Group 0 1 2 0 1 2 0 1 2 0 1 2 A(composite) 0 2 1 B(demineralizedbone) 1 l 1 C(polyorthoester) 3 0 0 D (no implant) 3 0 0
0 0 3 3
0 0 0 0
3 3 0 0
0 0 3 3
0 0 0 0
3 0 2 7~ 3 1 1 7"~ 0 9 0 0 0 9 0 0
* The data indicate the number of rats with a positive treatment response (osteoinductionat Week 2 or 3 or bone bridgingat Week 4) in none (0), one ( 1), or both (2) of the calvarial defects. ~"Significantlydifferent from the groupswith no implant or filling with polyorthoesteronly (p = 0.0001).
trans- 1,4-bis(hydroxymethyl)cyclohexane (CHDM) and can be formulated with different physical properties; in this study, the polyorthoester was soft and moldable. The composites were prepared by manually mixing demineralized bone and polyorthoester at room temperature under sterile conditions immediately before implantation.
Surgical Procedure A total of 36 male Wistar rats, each weighing 231 + 13 gm (mean _+ standard deviation), were randomly assigned to one of four groups of nine rats each. The animals were fed standard rat laboratory food and water ad libitum. Anesthesia was induced by Hypnorm-Dormicum at a dosage of0.15 ml/100 gm given intramuscularly. Under sterile conditions, the pericranium was exposed through a sagittal incision and stripped laterally. Two 4-mm burr holes were made; one in each parietal bone. The burr holes were irrigated with sterile saline during and after drilling to cool the tissue and to wash out the bone particles. Both defects in all Group A to C rats were filled: with a composite of 7 mg polyorthoester and 3 mg demineralized bone in Group A, with 3 mg demineralized bone in Group B, and with 7 mg polyorthoester in Group C. The defects were not filled in the Group D rats.
Evaluation of Specimens At 2, 3, and 4 weeks following the surgical procedure, three randomly selected animals in each group were killed by an overdose of ether inhalation; the dissected skull vaults were fixed in 4% neutral formalin. Radiographs were taken using dental x-ray equipment. The specimens were demineralized in 17% formic acid, dehydrated, and embedded in paraffin. Serial sections were cut at 5 um and stained with Harris' hematoxylin and eosin. Host-tissue responses, osteoinduction, and bone bridging of the defects were evaluated by light microscopy. Regeneration of bone defects by osteoinduction is a multistep process. Thus, a positive treatment response was defined as osteoinduction, indicated by the presence of bone formation within the defect and not in 276
FIG. 1. Photomicrograph showing a calvarial defect at Week 2 after filling with a composite of polyorthoester and demineralized bone. Parietal bone with new bone formation at the edge and cartilage, hypertrophied chondrocytes, calcified cartilage matrix, and incipient bone formation can be seen within the defect. H & E, x 70.
contact with the bone edges, at Weeks 2 to 3 as seen on histological examination and bone bridging of the defects at Week 4 as seen on histological and radiographic examinations. The treatment response of each rat was recorded as 0 (no osteoinduction or bone bridging), l (osteoinduction or bone bridging in one defect), or 2 (osteoinduction or bone bridging in both defects). To permit statistical analysis, the treatment response of all rats in each group studied at Weeks 2, 3, and 4 was compared collectively. The Kruskal-Wallis test was used to determine the null hypothesis (Ho) that there is no significant difference in treatment response by the different implants. Two sample Mann-Whitney tests were used to determine in which group the mean results were significantly different from each other when Ho was rejected. Because comparing multiple groups introduces a greater probability of type I error, the required p value (0.05) was divided by the number of comparisons (Bonferroni's correction); p < 0.008 (0.05/0.06) was regarded as significant. Results
There were no intra- or postoperative deaths. The animals gained weight and showed no signs of unhealthiness. The polyorthoester provided local hemostasis when used either alone or in composites with demineralized bone. The composite implant was moldable and easily contoured and technically easier to use than demineralized bone alone.
New Bone Formation At 2 weeks, cartilage, hypertrophied chondrocytes, calcified cartilage matrix, and incipient bone formation were present in a total of four defects implanted with
J. Neurosurg. / Volume 76 /February, 1992
Regeneration of calvarial defects by polyorthoester composite
FIG. 2. Photomicrographs showing calvarial defects at Week 4 after filling with a composite of polyorthoester and demineralized bone (left) and with demineralized bone only (right). Bridging of the defect by bone with bone marrow can be seen. H & E, x 2l.
the composite in three Group A rats (Fig. 1 and Table 1) and in a total of three defects implanted with demineralized bone alone in two Group B rats (Table 1). In both groups, additional new bone formation and some bone marrow were present within all defects on histological and radiographic study at 3 weeks. At 2 and 3 weeks, new bone had developed within all parts of the defects in Group A and B rats, whereas new bone formation in Group D rats with no defect filling and in Group C rats with polyorthoester filling only was sparse and confined to the bone edges (Table I). At 4 weeks, histological and radiographic study showed that all defects filled with the composite or demineralized bone alone were bridged by bone tissue (Fig. 2). No defects filled with polyorthoester only or left open were found to be healed on histological or radiographic examination during the observation period (Fig. 3). A positive response (osteoinduction at Week 2 or 3 or bone bridging at Week 4) was significantly greater in rats with defects filled with the composite or demineralized bone only (Groups A and B) compared to those with the defects filled with polyorthoester only or left open (Groups C and D) (p = 0.0001), whereas no significant difference was found between the two former groups (p = 0.6) or between the two latter (p = 1.0).
Host- Tissue Response In defects filled with the composite or polyorthoester only, the polyorthoester was present and slight inflammation with monocytes and some giant cells was seen at Week 2. The inflammation subsided by Week 3 and only traces of the polyorthoester could be detected at Week 4. In defects filled with demineralized bone only or left open, the inflammation at Week 2 was milder and no giant cells were detected. Discussion The low potential for regeneration of cranial defects in both animals and humans, 29 especially in adults, has clinical and experimental implications. Clinically, reconstruction of cranial defects resulting from trauma, infection, and craniectomies performed for access to intracranial abnormalities and tumor resection has presented challenging problems requiring the use of cranioplasty materials; bone grafts from various sites and alloplastic materials have been used. 4'5'~2'23 The ideal cranioplasty material should fulfill several criteria; 25,39 it should be: biocompatible, not inducing an inflammatory response; capable of stimulating osteogenesis; capable of being vascularized; radiolucent; moldable and easily contoured; and thermally nonconductive.
FIG. 3. Photomicrographs showing calvarial defects at Week 4 after filling with polyorthoester only (left) and without filling (right). Sparse new bone formation at the edges of the defect and loose connective tissue within the defect can be seen. H & E, x 21.
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E. Solheim, E. M. Pinholt, G. Bang, and E. Sudmann Critical-Size Defect Experimentally, healing of large skull defects in animals has been a commonly used test system for autogenous bone substitutes for more than a century.. 33 A defect that does not heal during the animals' lifetime is termed a "critical-size defect," and it has been proposed that evaluation of craniomaxillofacial bone repair materials should be initiated in critical-size defects in rat calvaria. 29 The critical-size calvarial defect varies according to species and age. It has been shown that 2mm parietal defects in Wistar rats weighing 500 gin, ~ 4-mm parietal defects in 28-day-old CD strain rats, j9 and 8-mm parietal defects in 6-month-old SpragueDawley rats 36 do not heal during 12 weeks, 6 months, and 12 weeks, respectively. We used 4-ram defects in 8-week-old male Wistar rats and healing did not occur during the observation period in the animals with the defects left open, whereas all defects that were filled with the composite or demineralized bone alone were bridged with new bone within 4 weeks.
Osteoinduction In rodents, osteoinduction consists of chemotaxis of mesenchymal ceils, mitosis, differentiation of cartilage, vascular invasion, and bone formation. 27'37In composites of osteoinductor and carrier, the carrier may inhibit the osteoinduction by two mechanisms. In the first possible mechanism, unabsorbed carrier will physically inhibit bone growth and closure of a bone defect. It has been stated that the ideal delivery system would be resorbed and replaced by preosseous tissues within 2 weeks, cartilage within 3 weeks, and bone within 4 to 6 weeks. 3~ Some existing delivery systems are resorbed more slowly than the time required for the demineralized bone-induced regeneration of the defect to take place. In one study of rabbits, ~ residual copolymer was still present 24 weeks after implantation of a composite of biodegradable copolymer of polylactide-polyglycolide and demineralized bone in calvarial defects. In another study, 38 beta tricalcium phosphate was still present 4 months after implantation as a composite with bone morphogenetic protein in calvarial defects in dogs. In the second possible mechanism, osteoinduction may be inhibited by a bioincompatible carrier that interferes physiologically with the multistep cascade of bone induction (for example, by inducing a chronic inflammation 3~ or by immunological mechanisms20).
Delivery System The delivery system should provide sustained, controlled release of the active substance. ~oAlzamer polyorthoester is designed to be used as a local sustained drug-release system? Zero-order drug release from polyorthoesters may be accomplished under certain conditions, 3'~~possibly because the rate of drug release is determined by erosion and not by simple diffusion since the polyorthoester is hydrophobic and may erode 978
heterogeneously from the surface first. ~5 Biodegradation of the polyorthoester takes place by hydrolysis to the ultimate products 4-hydroxybutyrate and CHDM; 4-hydroxybutyrate is further metabolized in the tricarboxylic acid cycle, with CO2 and H20 as the end products, and CHDM is excreted in urine. 32 In the present study, the polyorthoester did not inhibit osteoinduction and caused only a slight inflammation that subsided within 3 weeks. The results are consistent with those of a previous study where a composite of polyorthoester and demineralized bone implanted in the abdominal muscle of 89 Wistar rats induced cartilage and bone at the same rate as demineralized bone alone, as evaluated histologically and by ~SSruptake. 24By Week 4, the polyorthoester was mostly resorbed and all defects filled with a composite of polyorthoester and demineralized bone were bridged by new bone. These results support the contention that the polyorthoester is resorbed and does not physically obstruct bone formation. 3~ Our results indicate that the bridging of defects by the composite or demineralized bone alone was caused by bone induction and not merely osteoconduction; bone formation from the edges of the defect, as the typical pattern of bone induction, could be seen on histological examination (for example, cartilage at Week 2). J9.~6Furthermore, the regeneration progressed at an equal rate within all parts of the implant and not centripetally as with osteoconduction. Alzamer polyorthoester of the present formulation has a plastic consistency and can be molded intraoperatively according to present needs. The polyorthoester adheres to bone surfaces and provides local hemostasis by plugging spongy bone and secondarily promoting concentration of platelets and coagulation factors?4'35 Assorted formulations of the polyorthoester with different physical properties ranging from gel to solid can be manufactured.
Conclusions The composite implant used in this study was moldable, easily contoured, and technically easier to use than demineralized bone alone. The polyorthoester provided local hemostasis when used either alone or in a composite with demineralized bone, induced minor transient reaction, did not inhibit osteoinduction, and was resorbed as new bone formed within 4 weeks. As the polyorthoester may provide controlled, sustained release of the incorporated active substance, it seems promising as a carrier of purified inductors and/or growth factors.
Acknowledgments We thank Katrine Hove, Inger Liv Nordli, and Gunnvor ~3ijordsbakken for technical assistance and the Alza Corporation, Palo Alto, California, for providing the Alzamer polyorthoester. J. Neurosurg. / Volume 76/February, 1992
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23. Peyton WT, Hall HB: The repair of a cranial defect with a vitallium olate. Surgery 10:711-715, 1941 24. Pinholt EM, Solheim E, Bang G, et al: Bone induction by composite of bioerodible polyorthoester and demineralized bone matrix in rats. Acta Orthop Seam 62:476-48G, 1991 25. Rawlings CE III, Wilkins RH, Hanker JS, et al: Evaluation in cats of a new material for cranioplasty: a composite of plaster of Paris and hydroxylapatite, J Neurnsurg 69: 269-275, 1988 26. RayRD, Holloway JA: Bone implants. Preliminary report of an experimental study, d Bone Joint Surg (Am) 39: 1119-1128, 1957 27. Reddi AH, Huggins C: Biochemical sequences in the transformation of normal fibroblasts in adolescent rats. Proc Natl Acad Sci USA 69:1601-1605, 1972 28. Sato K, Urist MR: Induced regeneration of calvaria by bone morphogenetic protein (BMP) in dogs. Clin Orthop 197:301-311, 1985 29. Schmitz JP, Hollinger JO: The critical size defect as an experimental model for craniomandibulofacial nonunions. Clin Orthop 205:299-308, 1986 30. Schmitz JP, Hollinger JO: A preliminary study of the osteogenic potential of a biodegradable alloplastic-osteoinductive alloimplant. Clin Orthop 237:245-255, 1988 31. Sela J, Applebaum J, Uretzky G: Osteogenesis induced by bone matrix is inhibited by inflammation. Biomater Med Devices Artif Organs 14:227-237, 1986 32. Sendelbeck SL, Girdis CL: Disposition of a 14C-labeled bioerodible polyorthoester and its hydrolysis products, 4-hydroxybutyrate and cis,trans-l,4-bis(hydroxymethyl) cyclohexane, in rats. Drug Metab Dispos 13:291-295, 1985 33. Senn N: On the healing of aseptic bone cavities by implantation of antiseptic decalcified bone. Am J Meal Sci 98:219-243, 1989 34. Solheim E, Anfinsen OG, Holmsen H, et al: Effect of local hemostatics on platelet aggregation. Eur Surg Res 23:45-50, 1991 35. Sudmann B, Anfinsen OG, Rail M, etal: Use of a new hemostatic, bioerodible polymer versus bone wax made of beeswax - - a clinical and experimental study. Acta Orthop Scand 61 (Suppl 237):63-64, 1990 (Abstract) 36. Takagi K, Urist MR: The reaction of the dura to bone morphogenetie protein (BMP) in repair of skull defects. Ann Surg 196:100-109, 1982 37. Urist MR: Bone: formation by autoinduction. Science 150:893-899, 1965 38. Urist MR, Nilsson O, Rasmussen J, et al: Bone regeneration under the influence of a bone morphogenetic protein (BMP) beta tricalcium phosphate (TCP) composite in skull trephine defects in dogs. Clin Orthop 214: 295-304, 1987 39. Yamashima T: Cranioplasty with hydroxylapatite ceramic plates that can easily be trimmed during surgery. A preliminary report, Acta Neurochir 96:149-153, 1989 Manuscript received January 2, 1991. Accepted in final form June 3, 1991. This study was supported by grants from the J. E. Isberg Foundation. Address reprint requests to: Eirik Solheim, M.D., Hagavik Orthopaedic Hospital, N-5220 Hagavik, Norway.
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