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Repair of segmental long bone defect in rabbit femur using bioactive titanium cylindrical mesh cage
ARTICLE IN PRESS
Biomaterials 24 (2003) 3445–3451
Repair of segmental long bone defect in rabbit femur using bioactive titanium cylindrical mesh cage Shunsuke Fujibayashia,*, Hyun-Min Kimb, Masashi Neoa, Masaki Uchidab, Tadashi Kokubob, Takashi Nakamuraa a b
Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Kawahara-Cho 54 Sakyo-Ku, Kyoto 6068507, Japan Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Kawahara-Cho 54 Sakyo-Ku, Kyoto 6068507, Japan Received 7 January 2003; accepted 24 March 2003
Abstract A segmental rabbit femur defect was repaired using an empty bioactive titanium (BAT) mesh cage. A 10 mm long titanium mesh cage was positioned in the bony defect and reinforced by intramedullary fixation. The BAT surface was prepared by chemical and thermal treatment. Pure titanium cages were used as a control. Torsional stiffness of the BAT group at 4 weeks was approximately equal to, and at 8 weeks twice, that of the intact femur. Differences between the torsional stiffness of the control and BAT groups were significant at both time intervals. Histological examinations showed that woven bone appeared around the cage by 4 weeks and transformed to lamella bone by 8 weeks. New bone bonded to the BAT surface without an intervening layer. The BAT cage enhanced the bone repairing process and achieved faster repair of long bone segmental defects. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Titanium; Metal surface treatment; Bone repair; Osseointegration; Animal model
1. Introduction Repair of long bone segmental defects is one of the challenging problems in orthopaedic surgery. Current treatment options include bone grafting (auto- or allogenic), vascularized bone graft and distraction osteogenesis. However, these techniques often involve multistage surgical procedures, inhibit early limb function, and require several revision procedures to maintain acceptable alignment and achieve osseous healing. Disadvantages to bone repair by autologous bone grafts include a significant risk of postoperative complications due to the harvesting procedure, the frequent need for a second operation, and sometimes an inadequate volume of transplant material. Allogenic bone grafts are associated with problems of significant failure rates, poor mechanical stability, and immunological rejection. Vascularized bone grafting can significantly shorten the time for bone healing; however, this
*Corresponding author. Tel.: +81-75-751-3365; fax: +81-75-7518409. E-mail address: [email protected] (S. Fujibayashi).
complex procedure can be technically demanding. Distraction osteogenesis requires considerable surgical skill and experience, as well as exceptional patient compliance. An alternative approach involves the design of biomaterials with specific osteoconductive properties, a geometry, and an internal structure conducive to vascular invasion and homing of autochthonous bone cells, which in principle represent the prerequisites for ideal graft integration and subsequent optimal mechanical performance. Synthetic porous bioceramics represent primary candidate bone substitutes. They display excellent osteoconductive properties, are available in unlimited supply, can have custom-designed shape and size, and are free from risks of rejection or infection. Recently, porous bioactive ceramics were used to repair long bone segmental defects in animal models, and several good results were reported [1–5]. However, the application of porous ceramics for loadbearing conditions is difficult because of their poor mechanical strength. Another approach is the application of osteoinductive agents, including fibroblast growth factor, transforming growth factor, insulin-like growth factors, and bone morphogenetic proteins.
0142-9612/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0142-9612(03)00221-7
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However, the efficacy and adequate doses of these osteoinductive agents are still controversial [6,7]. Titanium and titanium alloys are widely used in the orthopaedic field and can be used in load-bearing conditions because of their good biocompatibilities and superior mechanical properties. Titanium and titanium alloys become bioactive materials by surface chemical and thermal treatment [8]. Surface chemical and thermal treatment forms three-dimensional bioactive sodium titanate on the surface, and bone-like apatite is deposited during soaking in simulated body fluid (SBF) (Fig. 1). From the results of our in vitro and in vivo experiments, bioactive titanium (BAT) is expected to be an ideal biomaterial in high-load conditions [9]. Titanium mesh cage implants were approved by the FDA in 1990 for general marketing for reinforcement of deficient bone and for cement restriction in selected skeletal surgical procedures. Titanium mesh cages filled with bone graft have become quite popular in spine surgery to replace or fuse resected vertebral bodies. Preliminary clinical experience has shown the cage to be safe, mechanically stable and effective in the spine. The efficacy of a titanium cage loaded with osteoinductive agents has been reported in several spinal fusion models [10,11]. In this study, a critical segmental defect of rabbit femur was repaired using an empty BAT cage, and the effects were examined biomechanically and histologically.
Fig. 1. SEM photograph of the BAT surface after soaking in SBF for 3 days, showing apatite depositions on the 3D porous structured surface. (Bar=3 mm).
2. Methods 2.1. Implant The custom-made titanium mesh cage implants were oval shaped cylinders 6.5 8 mm2 in diameter and 10 mm long, with a wall thickness of 1 mm. The BAT surface was prepared by alkali treatment with 5 m NaOH aqueous solution at 60 C for 24 h, subsequent hot water treatment of distilled water at 40 C for 48 h, and then heat treatment of 600 C for 1 h. Pure titanium cages were used as a control (Ti group). Before implantation, the bioactive ability of the treated cage was confirmed by apatite formation after soaking in SBF for 5 days. 2.2. Surgical technique The implants were conventionally sterilized with ethylene oxide gas. Twenty-two adult male Japanese white rabbits weighing 2.8–3.5 kg were used. The rabbits were anaesthetized with an intravenous injection of pentobarbital sodium (0.5 ml/kg), an intramuscular injection of ketamine hydrochloride (10 mg/kg), and local administration of a solution of 1% lidocaine. Under sterile conditions, surgery was performed in the left lateral decubitus position (always on the right leg). The femur was exposed through a lateral approach between the flexor and extensor muscles. A unilateral 10 mm long segmental defect was made at the mid-third of the femoral diaphysis of the rabbit, using a diamond disc with saline irrigation to minimize thermal damage. The length of the segmental bone defect was chosen as 10 mm from the results of Ref. [7], this being the critical defect of femoral diaphyses of mature Japanese white rabbits at 6 weeks. Thin periostium was preserved as much as possible. The implant was positioned in the created bone defect. The reconstructed femur with a titanium cage was reinforced by intramedullary fixation using three titanium Kirshner wires (1.8 mm 2, 2.0 mm 1) (Synthesis, USA) inserted from the distal femoral condyle after realignment of the fragments. The initial stability of the construct was sufficient when three Kirshner wires passed through the cortex of the proximal femur. The distal ends of the Kirshner wires were cut and placed beneath the articular surface. The wound was irrigated, and periosteum and muscles were carefully apposed and the fascia and skin were closed in a routine fashion. The animals were housed individually in standard rabbit cages and fed standard rabbit food and water ad libitum. The Kyoto University guidelines for animal experiments were observed in this study. Animals were killed with an overdose of intravenous pentobarbital sodium at 4 and 8 weeks after implantation. After removal of the internal fixation, the femur was examined by soft X-ray (SEFRON, SRO-M50,
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Soken Co. Ltd., Tokyo, Japan) and the mechanical strength of the repaired constructs was examined by manual palpation and a torsional test. Undecalcified sagittal sections were examined histologically using a light microscope and contact microradiograph (CMR), and bone implant interfaces were examined by scanning electron microscopy (SEM). 2.3. Mechanical test 2.3.1. Manual palpation After removal of the internal fixation, specimens were examined by manual palpation. Constructs that showed abnormal motion were classified as non-union, whereas solid constructs with no motion were classified as union. The specimens were then wrapped and frozen at 20 C for future mechanical testing. 2.3.2. Torsional test Before testing, the specimens were thawed at room temperature for 24 h. Care was taken to keep the segment moist with wet towels throughout the mounting and testing. Each end of the specimen was potted in dental resin and attached to fixation jigs. The torsional test was performed on a material testing device (Maruto MZ500S, Tokyo, Japan). The axis of rotation was through the geometric centre of the femur. A torque rate of 0.3 /s was used. The most linear portion of the torque–angular displacement curve was used to determine the torsional stiffness. The segment was tested to failure in torsion. The failure load was recorded as the point of maximum torque, after which further increase in angular displacement was not accompanied by further increase in torque. Data were recorded as mean7standard deviation (SD) and assessed using a one-way factorial ANOVA and Fisher’s PLSD method as a post hoc test. Differences at po0:05 were considered statistically significant. 2.3.3. Preparation of undecalcified specimen After the torsional test, destructed specimens were fixed in 10% phosphate-buffered formalin at pH 7.4 for 7 days, then dehydrated in serial dilutions of ethanol, and embedded in polyester resin. Sections 500 mm thick were cut with a band saw (BS-3000CP, EXACT cutting system, Norderstedt, Germany) parallel to the axis of the femur, and ground for CMR and Toluidin blue staining to a thickness of 80–120 mm using a grinding– sliding machine (Microgrinding MG-4000, EXACT). Several 500 mm sections were polished with diamond paper and coated with a thin layer of gold for observation by SEM (S-4700, Hitachi Ltd., Tokyo, Japan) attached to an energy-dispersive X-ray microanalyser. CMR were taken using an X-ray generator (SEFRON, SRO-M50) and Kodak high-resolution film (SO-343, Kodak, USA).
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2.3.4. Histomorphometric analysis The length of bridging newly formed bone was calculated from the CMR images. The percentage length of the bridging bone was defined according to a scoring system: grade 0, no new bone was detected in the area of the bone defect; grade 1, o50% of the gap was occupied by newly formed bone; grade 2, X50% of the gap was occupied by newly formed bone but radiolucent discontinuity was present; grade 3, the gap was completely bridged by new bone. The bone formation scores of both the anterior aspect and the posterior aspect of the cage were calculated separately and added to each other. The sum defined the bone formation score (highest score: 6). The total scores were compared between each group using an unpaired t-test.
3. Results 3.1. Mechanical test 3.1.1. Manual palpation Union rates in manual palpation of the Ti group and the BAT group were 20% (1/5) and 60% (3/5) at 4 weeks, 66.7% (4/6) and 100% (6/6) at 8 weeks, respectively. 3.1.2. Torsional test Torsional stiffness of the Ti group and the BAT group was 16.2711.4 and 185.7786.9 N mm/deg at 4 weeks, 142.47141.9 and 329.8771.4 N mm/deg at 8 weeks, respectively. The values for the BAT group at 4 weeks were approximately equal to those of the intact femur (159.6729.4 N mm/deg). Differences between the Ti group and the BAT group were significant at both time intervals (4 weeks: po0:005; 8 weeks: po0:05) (Fig. 2a). The maximum failure loads of the Ti group and the BAT group were 11.878.7 and 12.374.1 N mm at 4 weeks, 23.074.8 and 30.079.8 N mm at 8 weeks, respectively. The value of the BAT group at 8 weeks was close to that of the intact femur (39.8715.8 N mm). The differences between the Ti group and the BAT group were not statistically significant (Fig. 2b). 3.2. Histological examination 3.2.1. Gross inspection Prominent formation of new bone onto the outer surface of the BAT implant was obvious on gross inspection. At 4 weeks, continuous soft callus formation was observed around the BAT cage (Fig. 3a). At 8 weeks, complete integration of the BAT cage and bone was observed for all specimens (Fig. 3b). On the other hand, for the Ti group, no continuous soft callus formation was observed around the cage at 4 weeks, and
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3.2.3. Contact microradiography Formation of new woven bone was readily apparent at subperiosteal sites and within superficial portions of the BAT cage at 4 weeks (Fig. 5a). Areas of primary woven bone were readily recognized in microradiographs, based on greater osteocyte lacuna numbers and higher levels of mineralization. Haversian remodelling was obvious throughout the new cortical bone, which had formed outside the cylinder and across its wall at 8 weeks (Fig. 5b). On the other hand, for the Ti group, although woven bone appeared around the cage, the new bone was not continuous and an apparent defect between the new bone and implant is observable (Figs. 5c and d). 3.2.4. Toluidin blue Almost the same findings as for the CMR study were obtained. In the Ti group, a well-stained fibrous tissue layer existed at the interface between the bone and the cage. 3.2.5. Scanning electron microscopy SEM examination of the interface between new bone and titanium showed direct bone bonding without an intervening layer in the BAT group at both time intervals (Figs. 6a and b). In contrast, a thin intervening layer always existed between the bone and metal surface in the Ti group throughout the experimental period (Fig. 6c).
Fig. 2. Results of mechanical test. (a) The torsional stiffness of the control group and the BAT group is 16.2711.4 and 185.77 86.9 N mm/deg at 4 weeks, 142.47141.9 and 329.8771.4 N mm/deg at 8 weeks, respectively. The value of the BAT group at 4 weeks is approximately equal to that of the intact femur (159.6729.4 N mm/ deg). Differences between the Ti group and the BAT group were significant at both time intervals (4 weeks: po0:005; 8 weeks: po0:05). (b) The maximum failure loads of the Ti group and the BAT group were 11.878.7 and 12.374.1 N mm at 4 weeks, 23.074.8 and 30.079.8 N mm at 8 weeks, respectively. The value of the BAT group at 8 weeks was close to that of the intact femur (39.8715.8 N mm). The differences between the Ti group and the BAT group were not statistically significant.
some specimens showed malalignment with over-callus formation at 8 weeks. 3.2.2. Soft X-ray Alignment of the femur was well maintained and the cage position was stable throughout the experimental period in the BAT group (Figs. 4a and b). Massive new bone appeared around the BAT cage at 4 weeks and well-remodelled thin cortical lamella bone covered the cage and bridged the defect at 8 weeks. On the other hand, some specimens in the Ti group showed malalignment and dislodgment of the cage.
3.2.6. Histomorphometric analysis Bone formation scores for the Ti group and the BAT group were 3.0071.58 and 4.1570.98 at 4 weeks, 5.0071.00 and 5.6070.55 at 8 weeks, respectively. Although a statistically significant difference was not obtained between the bone formation score of the BAT group and that of the Ti group, a strong tendency existed between each group at 4 weeks (p ¼ 0:167).
4. Discussion In the current study, a segmental rabbit femur defect could be effectively repaired using empty BAT cages. BAT enhanced bone-repairing processes and achieved faster repair of the long bone defects. An essential requirement for an artificial material to bond to living bone is the formation of a biologically active bone-like apatite layer on its surface in a body environment [12]. Titanium metal and titanium alloys that have been subjected to NaOH and subsequent heat treatment form a bone-like apatite layer on their surfaces in SBF with ion concentrations nearly equal to those found in human blood plasma. After chemical and thermal treatment, an amorphous sodium titanate hydrogel layer forms on the substrate, and it is believed
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Fig. 3. Gross inspections; (a) BAT at 4 weeks: continuous soft callus formation is observed around the cage; and (b) BAT at 8 weeks: complete integration of BAT cage and bone is observed.
Fig. 4. Soft X-ray photographs: (a) BAT at 4 weeks: massive new bone appears around the cage; and (b) BAT at 8 weeks: well-remodelled thin cortical lamella bone covers the cage and bridges the defect. Alignment of femur is well maintained and the cage position is stable throughout the experimental period.
that a large number of surface Ti–OH groups are essential for apatite nucleation. In our earlier in vitro study, titania gel showed the highest apatiteforming ability when it assumed the anatase structure related to its epitaxy of the apatite crystal, irrespective of the composition [13,14]. The sodium titanate hydrogel changed to a pure titania gel if the sodium ion in the sodium titanate gel layer was released, and such pure titania gel was expected to transform into anatase on being subjected to an appropriate heat
treatment. Our in vivo study actually showed an improvement in the bone-bonding strength of BAT at an early implantation period when its surface crystal structure converted into the anatase structure [15]. In the current study, a BAT cage that has a surface anatase crystal structure also showed superior osseoconductive ability. The torsional stiffness of the BAT group was higher than that of the Ti group; it was equal to that of the intact femur at 4 weeks and twice as high as that of the
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Fig. 5. Contact microradiogram: (a) BAT at 4 weeks: formation of new woven bone is readily apparent at subperiosteal sites and within superficial portions of the cage; (b) BAT at 8 weeks: Haversian remodelling is obvious throughout the new cortical bone, which had formed outside the cylinder and across its wall; (c) control at 4 weeks: although woven bone appears around the cage, new bone is not continuous and an apparent defect between new bone and implant is observed; and (d) control at 8 weeks.
intact femur at 8 weeks. Earlier transfer to lamella bone of the BAT group related to the higher mechanical strength relative to that of the Ti group. For the BAT group, direct bone to implant bonding was revealed from SEM examinations. Subperiosteal woven bone appeared at 4 weeks and bonded directly to the BAT surface. Presumably, femur reconstructed using the BAT cage became stiffer than intact femur by this direct bonding and perfect bone integration between bone and metal implant, because the elastic modulus of titanium metal is much higher than that of cortical bone. Early stabilization between new bone and cage was obtained by this newly formed bone and their direct bonding and integration. A comparison of tissue morphology at early and late time points indicates that subperiosteal formation of woven bone represents the earliest event, detected as early as 4 weeks after surgery. Later events include the completion of primary osteons and the occurrence of Haversian remodelling in the new cortex. This leads to the replacement of woven bone with more mechanically sound lamella bone and to the assembly of a structurally adequate new cortex. On the other hand, stability between the bone and cage was poor in the Ti group because of the existing gap between new bone and the titanium surface, which related to the lower stiffness and higher non-union rate of the construct. Micromovement between bone and implant would hamper the transformation of woven bone to lamella bone. Although the maximum failure load in the BAT group was higher than that of the Ti group, the difference was not significant. This result was also related to the result of histomorphometric analysis.
Because titanium is bioinert material, massive new bone appeared around not only BAT cage but also Ti cage. Moreover, the newly formed bone around the cage penetrated the mesh structure of the cage and achieved mechanical interlocking between cage and bone in both Ti and BAT groups. One of the advantages of this surface bioactive treatment is the capability for application to several meticulous shaped implants. It is difficult to prepare a bioactive surface by the conventional hydroxyapatite plasma spray technique for the inner surface of an implant such as cage or meticulous structure. In contrast, because this treatment is conducted by soaking in aqueous solution and subsequent heat treatment, the inside of the implant or meticulous structure can be treated uniformly and easily [16]. Furthermore, because the thickness of the bioactive treated layer was approximately 1 mm, much thinner than that of conventional hydroxyapatite coatings, this surface treatment does not change the surface morphology of the implant.
5. Conclusion Segmental rabbit femur defect was successfully repaired using an empty BAT mesh cage. Torsional stiffness of the repaired femur of the BAT group at 4 weeks was approximately equal to, and at 8 weeks was twice that of the intact femur. Histological examinations showed that subperiosteal woven bone appeared around the cage by 4 weeks and transformed to lamella bone by
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the clinical area, this surface chemical and thermal treatment will be successfully applied to several orthopaedic implants.
Acknowledgements The authors thank Liang Bojian for skillful support with the animal experiments and also thank Kobe Steel Co. Ltd. for manufacturing the implants.
References
Fig. 6. SEM photographs of the interface between bone and titanium: (a) BAT at 4 weeks ( 250); (b) BAT at 4 weeks ( 1000): direct bone bonding without an intervening layer is observed in the BAT group at both time intervals; and (c) control at 8 weeks ( 250): a thin intervening layer existed between the bone and metal surface in the Ti group throughout the experimental period.
8 weeks, and that new bone directly bonded to the BAT surface. The BAT cage enhanced the bone repairing process and achieved faster repair of long bone segmental defects. From the results of this study, in
[1] Marcacci M, et al. Reconstruction of extensive long-bone defects in sheep using porous hydroxyapatite sponges. Calcif Tissue Int 1999;64:83–90. [2] Johnson KD, et al. Porous ceramics as bone graft substitutes in long bone defects: a biomechanical, histological, and radiographic analysis. J Orthop Res 1996;14:351–69. [3] Wolff D, Goldberg VM, Stevenson S. Histomorphometric analysis of the repair of segmental diaphyseal defect with ceramic and titanium fibermetal implants: effects of bone marrow. J Orthop Res 1994;12:439–46. [4] Hadjipavlou AG, et al. Plaster of Paris as an osteoconductive material for interbody vertebral fusion in mature sheep. Spine 2000;25:10–6. [5] Holmes RE, Bucholz RW, Mooney V. Porous hydroxyapatite as a bone graft substitute in diaphyseal defects: a histometric study. J Orthop Res 1987;5:114–21. [6] Yasko AW, et al. The healing of segmental bone defects, induced by recombinant human BMP-2. J. Bone Jt Surg 1992;74-A: 649–70. [7] Inui K, et al. Local application of basic fibroblast growth factor minipellet induces the healing of segmental bony defects in rabbits. Calcif Tissue Int 1998;63:490–5. [8] Kokubo T, Miyaji F, Kim HM, Nakamura T. Spontaneous apatite formation on chemically surface treated Ti. J Am Ceram Soc 1996;79:1127–9. [9] Nishiguchi S, et al. The effect of heat treatment on bone-bonding ability of alkali-treated titanium. Biomaterials 1999;20:491–500. [10] Boden SD, Martin Jr GJ, Horton WC, Truss TL, Sandhu HS. Laparoscopic anterior spinal arthrodesis with rhBMP-2 in a titanium interbody threaded cage. J Spinal Disorder 1998;11: 95–101. [11] Zdeblick TA. Cervical interbody fusion cages. An animal model with and without bone morphogenetic protein. Spine 1998;23: 758–65. [12] Kokubo T, Kushitani H, Sakka S. Solutions able to reproduce in vivo surface-structure changes in bioactive glass-ceramic A-W. J Biomed Mater Res 1990;24:721–34. [13] Uchida M, Kim HM, Kokubo T, Fujibayashi S, Nakamura T. Structural dependence of apatite formation on titania gel in a simulated body fluid. J Biomed Mater Res 2003;64:161–70. [14] Uchida M, Kim HM, Kokubo T, Fujibayashi S, Nakamura T. Effect of water treatment on the apatite-forming ability of NaOHtreated titanium metal. J Biomed Mater Res 2002;63:522–30. [15] Fujibayashi S, et al. Bioactive titanium: effect of sodium removal on the bone-bonding ability of bioactive titanium prepared by alkali and heat treatment. J Biomed Mater Res 2001; 56:562–70. [16] Kim HM, Kokubo T, Fujibayashi S, Nishiguchi S, Nakamura T. Bioactive macroporous titanium surface layer on titanium substrate. J Biomed Mater Res 2000;52:553–7.
Biomaterials 24 (2003) 3445–3451
Repair of segmental long bone defect in rabbit femur using bioactive titanium cylindrical mesh cage Shunsuke Fujibayashia,*, Hyun-Min Kimb, Masashi Neoa, Masaki Uchidab, Tadashi Kokubob, Takashi Nakamuraa a b
Department of Orthopaedic Surgery, Graduate School of Medicine, Kyoto University, Kawahara-Cho 54 Sakyo-Ku, Kyoto 6068507, Japan Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Kawahara-Cho 54 Sakyo-Ku, Kyoto 6068507, Japan Received 7 January 2003; accepted 24 March 2003
Abstract A segmental rabbit femur defect was repaired using an empty bioactive titanium (BAT) mesh cage. A 10 mm long titanium mesh cage was positioned in the bony defect and reinforced by intramedullary fixation. The BAT surface was prepared by chemical and thermal treatment. Pure titanium cages were used as a control. Torsional stiffness of the BAT group at 4 weeks was approximately equal to, and at 8 weeks twice, that of the intact femur. Differences between the torsional stiffness of the control and BAT groups were significant at both time intervals. Histological examinations showed that woven bone appeared around the cage by 4 weeks and transformed to lamella bone by 8 weeks. New bone bonded to the BAT surface without an intervening layer. The BAT cage enhanced the bone repairing process and achieved faster repair of long bone segmental defects. r 2003 Elsevier Science Ltd. All rights reserved. Keywords: Titanium; Metal surface treatment; Bone repair; Osseointegration; Animal model
1. Introduction Repair of long bone segmental defects is one of the challenging problems in orthopaedic surgery. Current treatment options include bone grafting (auto- or allogenic), vascularized bone graft and distraction osteogenesis. However, these techniques often involve multistage surgical procedures, inhibit early limb function, and require several revision procedures to maintain acceptable alignment and achieve osseous healing. Disadvantages to bone repair by autologous bone grafts include a significant risk of postoperative complications due to the harvesting procedure, the frequent need for a second operation, and sometimes an inadequate volume of transplant material. Allogenic bone grafts are associated with problems of significant failure rates, poor mechanical stability, and immunological rejection. Vascularized bone grafting can significantly shorten the time for bone healing; however, this
*Corresponding author. Tel.: +81-75-751-3365; fax: +81-75-7518409. E-mail address: [email protected] (S. Fujibayashi).
complex procedure can be technically demanding. Distraction osteogenesis requires considerable surgical skill and experience, as well as exceptional patient compliance. An alternative approach involves the design of biomaterials with specific osteoconductive properties, a geometry, and an internal structure conducive to vascular invasion and homing of autochthonous bone cells, which in principle represent the prerequisites for ideal graft integration and subsequent optimal mechanical performance. Synthetic porous bioceramics represent primary candidate bone substitutes. They display excellent osteoconductive properties, are available in unlimited supply, can have custom-designed shape and size, and are free from risks of rejection or infection. Recently, porous bioactive ceramics were used to repair long bone segmental defects in animal models, and several good results were reported [1–5]. However, the application of porous ceramics for loadbearing conditions is difficult because of their poor mechanical strength. Another approach is the application of osteoinductive agents, including fibroblast growth factor, transforming growth factor, insulin-like growth factors, and bone morphogenetic proteins.
0142-9612/03/$ - see front matter r 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0142-9612(03)00221-7
ARTICLE IN PRESS 3446
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However, the efficacy and adequate doses of these osteoinductive agents are still controversial [6,7]. Titanium and titanium alloys are widely used in the orthopaedic field and can be used in load-bearing conditions because of their good biocompatibilities and superior mechanical properties. Titanium and titanium alloys become bioactive materials by surface chemical and thermal treatment [8]. Surface chemical and thermal treatment forms three-dimensional bioactive sodium titanate on the surface, and bone-like apatite is deposited during soaking in simulated body fluid (SBF) (Fig. 1). From the results of our in vitro and in vivo experiments, bioactive titanium (BAT) is expected to be an ideal biomaterial in high-load conditions [9]. Titanium mesh cage implants were approved by the FDA in 1990 for general marketing for reinforcement of deficient bone and for cement restriction in selected skeletal surgical procedures. Titanium mesh cages filled with bone graft have become quite popular in spine surgery to replace or fuse resected vertebral bodies. Preliminary clinical experience has shown the cage to be safe, mechanically stable and effective in the spine. The efficacy of a titanium cage loaded with osteoinductive agents has been reported in several spinal fusion models [10,11]. In this study, a critical segmental defect of rabbit femur was repaired using an empty BAT cage, and the effects were examined biomechanically and histologically.
Fig. 1. SEM photograph of the BAT surface after soaking in SBF for 3 days, showing apatite depositions on the 3D porous structured surface. (Bar=3 mm).
2. Methods 2.1. Implant The custom-made titanium mesh cage implants were oval shaped cylinders 6.5 8 mm2 in diameter and 10 mm long, with a wall thickness of 1 mm. The BAT surface was prepared by alkali treatment with 5 m NaOH aqueous solution at 60 C for 24 h, subsequent hot water treatment of distilled water at 40 C for 48 h, and then heat treatment of 600 C for 1 h. Pure titanium cages were used as a control (Ti group). Before implantation, the bioactive ability of the treated cage was confirmed by apatite formation after soaking in SBF for 5 days. 2.2. Surgical technique The implants were conventionally sterilized with ethylene oxide gas. Twenty-two adult male Japanese white rabbits weighing 2.8–3.5 kg were used. The rabbits were anaesthetized with an intravenous injection of pentobarbital sodium (0.5 ml/kg), an intramuscular injection of ketamine hydrochloride (10 mg/kg), and local administration of a solution of 1% lidocaine. Under sterile conditions, surgery was performed in the left lateral decubitus position (always on the right leg). The femur was exposed through a lateral approach between the flexor and extensor muscles. A unilateral 10 mm long segmental defect was made at the mid-third of the femoral diaphysis of the rabbit, using a diamond disc with saline irrigation to minimize thermal damage. The length of the segmental bone defect was chosen as 10 mm from the results of Ref. [7], this being the critical defect of femoral diaphyses of mature Japanese white rabbits at 6 weeks. Thin periostium was preserved as much as possible. The implant was positioned in the created bone defect. The reconstructed femur with a titanium cage was reinforced by intramedullary fixation using three titanium Kirshner wires (1.8 mm 2, 2.0 mm 1) (Synthesis, USA) inserted from the distal femoral condyle after realignment of the fragments. The initial stability of the construct was sufficient when three Kirshner wires passed through the cortex of the proximal femur. The distal ends of the Kirshner wires were cut and placed beneath the articular surface. The wound was irrigated, and periosteum and muscles were carefully apposed and the fascia and skin were closed in a routine fashion. The animals were housed individually in standard rabbit cages and fed standard rabbit food and water ad libitum. The Kyoto University guidelines for animal experiments were observed in this study. Animals were killed with an overdose of intravenous pentobarbital sodium at 4 and 8 weeks after implantation. After removal of the internal fixation, the femur was examined by soft X-ray (SEFRON, SRO-M50,
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Soken Co. Ltd., Tokyo, Japan) and the mechanical strength of the repaired constructs was examined by manual palpation and a torsional test. Undecalcified sagittal sections were examined histologically using a light microscope and contact microradiograph (CMR), and bone implant interfaces were examined by scanning electron microscopy (SEM). 2.3. Mechanical test 2.3.1. Manual palpation After removal of the internal fixation, specimens were examined by manual palpation. Constructs that showed abnormal motion were classified as non-union, whereas solid constructs with no motion were classified as union. The specimens were then wrapped and frozen at 20 C for future mechanical testing. 2.3.2. Torsional test Before testing, the specimens were thawed at room temperature for 24 h. Care was taken to keep the segment moist with wet towels throughout the mounting and testing. Each end of the specimen was potted in dental resin and attached to fixation jigs. The torsional test was performed on a material testing device (Maruto MZ500S, Tokyo, Japan). The axis of rotation was through the geometric centre of the femur. A torque rate of 0.3 /s was used. The most linear portion of the torque–angular displacement curve was used to determine the torsional stiffness. The segment was tested to failure in torsion. The failure load was recorded as the point of maximum torque, after which further increase in angular displacement was not accompanied by further increase in torque. Data were recorded as mean7standard deviation (SD) and assessed using a one-way factorial ANOVA and Fisher’s PLSD method as a post hoc test. Differences at po0:05 were considered statistically significant. 2.3.3. Preparation of undecalcified specimen After the torsional test, destructed specimens were fixed in 10% phosphate-buffered formalin at pH 7.4 for 7 days, then dehydrated in serial dilutions of ethanol, and embedded in polyester resin. Sections 500 mm thick were cut with a band saw (BS-3000CP, EXACT cutting system, Norderstedt, Germany) parallel to the axis of the femur, and ground for CMR and Toluidin blue staining to a thickness of 80–120 mm using a grinding– sliding machine (Microgrinding MG-4000, EXACT). Several 500 mm sections were polished with diamond paper and coated with a thin layer of gold for observation by SEM (S-4700, Hitachi Ltd., Tokyo, Japan) attached to an energy-dispersive X-ray microanalyser. CMR were taken using an X-ray generator (SEFRON, SRO-M50) and Kodak high-resolution film (SO-343, Kodak, USA).
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2.3.4. Histomorphometric analysis The length of bridging newly formed bone was calculated from the CMR images. The percentage length of the bridging bone was defined according to a scoring system: grade 0, no new bone was detected in the area of the bone defect; grade 1, o50% of the gap was occupied by newly formed bone; grade 2, X50% of the gap was occupied by newly formed bone but radiolucent discontinuity was present; grade 3, the gap was completely bridged by new bone. The bone formation scores of both the anterior aspect and the posterior aspect of the cage were calculated separately and added to each other. The sum defined the bone formation score (highest score: 6). The total scores were compared between each group using an unpaired t-test.
3. Results 3.1. Mechanical test 3.1.1. Manual palpation Union rates in manual palpation of the Ti group and the BAT group were 20% (1/5) and 60% (3/5) at 4 weeks, 66.7% (4/6) and 100% (6/6) at 8 weeks, respectively. 3.1.2. Torsional test Torsional stiffness of the Ti group and the BAT group was 16.2711.4 and 185.7786.9 N mm/deg at 4 weeks, 142.47141.9 and 329.8771.4 N mm/deg at 8 weeks, respectively. The values for the BAT group at 4 weeks were approximately equal to those of the intact femur (159.6729.4 N mm/deg). Differences between the Ti group and the BAT group were significant at both time intervals (4 weeks: po0:005; 8 weeks: po0:05) (Fig. 2a). The maximum failure loads of the Ti group and the BAT group were 11.878.7 and 12.374.1 N mm at 4 weeks, 23.074.8 and 30.079.8 N mm at 8 weeks, respectively. The value of the BAT group at 8 weeks was close to that of the intact femur (39.8715.8 N mm). The differences between the Ti group and the BAT group were not statistically significant (Fig. 2b). 3.2. Histological examination 3.2.1. Gross inspection Prominent formation of new bone onto the outer surface of the BAT implant was obvious on gross inspection. At 4 weeks, continuous soft callus formation was observed around the BAT cage (Fig. 3a). At 8 weeks, complete integration of the BAT cage and bone was observed for all specimens (Fig. 3b). On the other hand, for the Ti group, no continuous soft callus formation was observed around the cage at 4 weeks, and
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3.2.3. Contact microradiography Formation of new woven bone was readily apparent at subperiosteal sites and within superficial portions of the BAT cage at 4 weeks (Fig. 5a). Areas of primary woven bone were readily recognized in microradiographs, based on greater osteocyte lacuna numbers and higher levels of mineralization. Haversian remodelling was obvious throughout the new cortical bone, which had formed outside the cylinder and across its wall at 8 weeks (Fig. 5b). On the other hand, for the Ti group, although woven bone appeared around the cage, the new bone was not continuous and an apparent defect between the new bone and implant is observable (Figs. 5c and d). 3.2.4. Toluidin blue Almost the same findings as for the CMR study were obtained. In the Ti group, a well-stained fibrous tissue layer existed at the interface between the bone and the cage. 3.2.5. Scanning electron microscopy SEM examination of the interface between new bone and titanium showed direct bone bonding without an intervening layer in the BAT group at both time intervals (Figs. 6a and b). In contrast, a thin intervening layer always existed between the bone and metal surface in the Ti group throughout the experimental period (Fig. 6c).
Fig. 2. Results of mechanical test. (a) The torsional stiffness of the control group and the BAT group is 16.2711.4 and 185.77 86.9 N mm/deg at 4 weeks, 142.47141.9 and 329.8771.4 N mm/deg at 8 weeks, respectively. The value of the BAT group at 4 weeks is approximately equal to that of the intact femur (159.6729.4 N mm/ deg). Differences between the Ti group and the BAT group were significant at both time intervals (4 weeks: po0:005; 8 weeks: po0:05). (b) The maximum failure loads of the Ti group and the BAT group were 11.878.7 and 12.374.1 N mm at 4 weeks, 23.074.8 and 30.079.8 N mm at 8 weeks, respectively. The value of the BAT group at 8 weeks was close to that of the intact femur (39.8715.8 N mm). The differences between the Ti group and the BAT group were not statistically significant.
some specimens showed malalignment with over-callus formation at 8 weeks. 3.2.2. Soft X-ray Alignment of the femur was well maintained and the cage position was stable throughout the experimental period in the BAT group (Figs. 4a and b). Massive new bone appeared around the BAT cage at 4 weeks and well-remodelled thin cortical lamella bone covered the cage and bridged the defect at 8 weeks. On the other hand, some specimens in the Ti group showed malalignment and dislodgment of the cage.
3.2.6. Histomorphometric analysis Bone formation scores for the Ti group and the BAT group were 3.0071.58 and 4.1570.98 at 4 weeks, 5.0071.00 and 5.6070.55 at 8 weeks, respectively. Although a statistically significant difference was not obtained between the bone formation score of the BAT group and that of the Ti group, a strong tendency existed between each group at 4 weeks (p ¼ 0:167).
4. Discussion In the current study, a segmental rabbit femur defect could be effectively repaired using empty BAT cages. BAT enhanced bone-repairing processes and achieved faster repair of the long bone defects. An essential requirement for an artificial material to bond to living bone is the formation of a biologically active bone-like apatite layer on its surface in a body environment [12]. Titanium metal and titanium alloys that have been subjected to NaOH and subsequent heat treatment form a bone-like apatite layer on their surfaces in SBF with ion concentrations nearly equal to those found in human blood plasma. After chemical and thermal treatment, an amorphous sodium titanate hydrogel layer forms on the substrate, and it is believed
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Fig. 3. Gross inspections; (a) BAT at 4 weeks: continuous soft callus formation is observed around the cage; and (b) BAT at 8 weeks: complete integration of BAT cage and bone is observed.
Fig. 4. Soft X-ray photographs: (a) BAT at 4 weeks: massive new bone appears around the cage; and (b) BAT at 8 weeks: well-remodelled thin cortical lamella bone covers the cage and bridges the defect. Alignment of femur is well maintained and the cage position is stable throughout the experimental period.
that a large number of surface Ti–OH groups are essential for apatite nucleation. In our earlier in vitro study, titania gel showed the highest apatiteforming ability when it assumed the anatase structure related to its epitaxy of the apatite crystal, irrespective of the composition [13,14]. The sodium titanate hydrogel changed to a pure titania gel if the sodium ion in the sodium titanate gel layer was released, and such pure titania gel was expected to transform into anatase on being subjected to an appropriate heat
treatment. Our in vivo study actually showed an improvement in the bone-bonding strength of BAT at an early implantation period when its surface crystal structure converted into the anatase structure [15]. In the current study, a BAT cage that has a surface anatase crystal structure also showed superior osseoconductive ability. The torsional stiffness of the BAT group was higher than that of the Ti group; it was equal to that of the intact femur at 4 weeks and twice as high as that of the
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Fig. 5. Contact microradiogram: (a) BAT at 4 weeks: formation of new woven bone is readily apparent at subperiosteal sites and within superficial portions of the cage; (b) BAT at 8 weeks: Haversian remodelling is obvious throughout the new cortical bone, which had formed outside the cylinder and across its wall; (c) control at 4 weeks: although woven bone appears around the cage, new bone is not continuous and an apparent defect between new bone and implant is observed; and (d) control at 8 weeks.
intact femur at 8 weeks. Earlier transfer to lamella bone of the BAT group related to the higher mechanical strength relative to that of the Ti group. For the BAT group, direct bone to implant bonding was revealed from SEM examinations. Subperiosteal woven bone appeared at 4 weeks and bonded directly to the BAT surface. Presumably, femur reconstructed using the BAT cage became stiffer than intact femur by this direct bonding and perfect bone integration between bone and metal implant, because the elastic modulus of titanium metal is much higher than that of cortical bone. Early stabilization between new bone and cage was obtained by this newly formed bone and their direct bonding and integration. A comparison of tissue morphology at early and late time points indicates that subperiosteal formation of woven bone represents the earliest event, detected as early as 4 weeks after surgery. Later events include the completion of primary osteons and the occurrence of Haversian remodelling in the new cortex. This leads to the replacement of woven bone with more mechanically sound lamella bone and to the assembly of a structurally adequate new cortex. On the other hand, stability between the bone and cage was poor in the Ti group because of the existing gap between new bone and the titanium surface, which related to the lower stiffness and higher non-union rate of the construct. Micromovement between bone and implant would hamper the transformation of woven bone to lamella bone. Although the maximum failure load in the BAT group was higher than that of the Ti group, the difference was not significant. This result was also related to the result of histomorphometric analysis.
Because titanium is bioinert material, massive new bone appeared around not only BAT cage but also Ti cage. Moreover, the newly formed bone around the cage penetrated the mesh structure of the cage and achieved mechanical interlocking between cage and bone in both Ti and BAT groups. One of the advantages of this surface bioactive treatment is the capability for application to several meticulous shaped implants. It is difficult to prepare a bioactive surface by the conventional hydroxyapatite plasma spray technique for the inner surface of an implant such as cage or meticulous structure. In contrast, because this treatment is conducted by soaking in aqueous solution and subsequent heat treatment, the inside of the implant or meticulous structure can be treated uniformly and easily [16]. Furthermore, because the thickness of the bioactive treated layer was approximately 1 mm, much thinner than that of conventional hydroxyapatite coatings, this surface treatment does not change the surface morphology of the implant.
5. Conclusion Segmental rabbit femur defect was successfully repaired using an empty BAT mesh cage. Torsional stiffness of the repaired femur of the BAT group at 4 weeks was approximately equal to, and at 8 weeks was twice that of the intact femur. Histological examinations showed that subperiosteal woven bone appeared around the cage by 4 weeks and transformed to lamella bone by
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the clinical area, this surface chemical and thermal treatment will be successfully applied to several orthopaedic implants.
Acknowledgements The authors thank Liang Bojian for skillful support with the animal experiments and also thank Kobe Steel Co. Ltd. for manufacturing the implants.
References
Fig. 6. SEM photographs of the interface between bone and titanium: (a) BAT at 4 weeks ( 250); (b) BAT at 4 weeks ( 1000): direct bone bonding without an intervening layer is observed in the BAT group at both time intervals; and (c) control at 8 weeks ( 250): a thin intervening layer existed between the bone and metal surface in the Ti group throughout the experimental period.
8 weeks, and that new bone directly bonded to the BAT surface. The BAT cage enhanced the bone repairing process and achieved faster repair of long bone segmental defects. From the results of this study, in
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