Regenerative repair of bone defects with ... - Springer Link

J Orthop Sci (2012) 17:484–489 DOI 10.1007/s00776-012-0235-7

ORIGINAL ARTICLE

Regenerative repair of bone defects with osteoinductive hydroxyapatite fabricated to match the defect and implanted with combined use of computer-aided design, computer-aided manufacturing, and computer-assisted surgery systems: a feasibility study in a canine model Koichi Yano • Takashi Namikawa • Takuya Uemura Masatoshi Hoshino • Shigeyuki Wakitani • Kunio Takaoka • Hiroaki Nakamura

•

Received: 20 October 2011 / Accepted: 11 April 2012 / Published online: 27 April 2012 Ó The Japanese Orthopaedic Association 2012

Abstract Background Currently, regenerative repair of large bone defects that result from bone tumor resection or severe trauma is a challenging issue because of the limited regenerative potential of bone and treatment modalities. The aim of this study was to achieve repair of large bone defects to the original three-dimensional (3D) anatomical state by combining computer-aided technologies and local delivery of bone morphogenetic protein (BMP) in a canine model. Methods Computed tomography (CT) images of the pelvic bone of each dog were obtained, and an imaginary spherical malignant bone tumor of 15-mm diameter was placed in the left ilium of a canine on the 3D CT image. Resection of the whole tumor with a 10-mm margin of healthy bone was planned preoperatively by using computer-aided design (CAD) software. In addition, an image of the implant to be used to fill the resulting bone defect was constructed on the computer image. A porous hydroxyapatite (HA) implant identical to the imaged bone defect was made by shaving a tetragonal porous apatite block (40 9 20 9 10 mm) with a computer-aided manufacturing system operated by using the CT-image data of the bone defect obtained from the CAD system. To resect the iliac bone as planned preoperatively on the 3D CT image, computer-aided surgery was performed using the CT data. The defect was filled with the HA implant fabricated as described and coated with a putty carrier either K. Yano (&) T. Namikawa T. Uemura M. Hoshino S. Wakitani K. Takaoka H. Nakamura Department of Orthopaedic Surgery, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan e-mail: [email protected]

123

with BMP-2 (BMP group, n = 6) or without BMP-2 (control group, n = 6). Results In the BMP group, new bone formation was noted around each implant on CT images at 3 weeks after surgery and was remodeled to restore the original anatomy of the ilium on serial CT images. At 12 weeks, the implant was enclosed within new bone, and histological analysis revealed bone formation on and within the implant. Little bone formation was noted in the control group. Conclusions This new method may enable efficacious and precise regenerative repair of large bone defects without bone grafting.

Introduction Reconstruction of large skeletal defects to restore the original anatomy and function remains a challenge in orthopedic surgery. Such bone defects are often the result of bone tumor resection, chronic infection, or severe trauma to the bone, and are usually treated with auto- or allotissue grafts, or artificial biomaterials [1]. However, positive outcomes from these methods have been limited because of donor site morbidity, limitation of the graft mass in autogenous bone grafting, poor osteogenic potential, risk of infectious disease transmission in allogeneic bone grafting, and lack of osteoinductive potential and incompatible mechanical properties in artificial materials [2–4]. One of the approaches to overcome the issues associated with large bone defect reconstruction would be the efficacious use of bone morphogenetic proteins (BMPs), which are produced by DNA recombination techniques, in combination with biocompatible materials or cultured mesenchymal stem cells [5, 6].

Reconstruction of large bone defect

485

Materials and methods

with free access to food and water. We collected CT image data (1-mm slice thickness) of the whole pelvis of each dog with a CT scanner (GE Yokogawa, Tokyo, Japan) and recorded them as Digital Imaging and Communication in Medicine (DICOM) data a few days before surgery. These data were then transferred to computer-aided design (CAD) software (Mimics, Magics; Materialise Japan, Yokohama, Japan) to convert them from the DICOM format to the Standard Triangulated Language (STL) format in order to construct 3D pelvis images. Subsequently, an imaginary (i.e., computer-generated) spherical bone tumor (15 mm in diameter) was placed in the left iliac bone, just inferior to the anterior superior iliac spine (Fig. 1, orange area). Then, an assumptive resection surgery was performed along a marginal line 10 mm away from the tumor margin (Fig. 1a, green area, b) in order to generate a bone defect on the 3D CT image. The real size of the bone defect was determined by subtracting the image of the left ilium with the bone defect from the mirror image of the right ilium (Fig. 1c).

Iliac bone tumor resection model in dogs

Fabrication of the implant to fill the defect

Twelve Beagle dogs (male; 10 months old; weight 9–11 kg) were purchased from Shimizu Laboratory Supplies Co., Ltd. (Kyoto, Japan), and housed in separate cages

An interconnected porous calcium hydroxyapatite (IPCHA) ceramic block (tetragonal, 40 9 20 9 10 mm) for each animal was provided by MMT Co., Ltd. (Osaka,

In the case of wide resection of malignant bone tumors, the resection margin should be outside the tumor and planned. Preoperative determination using computed tomography (CT) image data also might be utilized to drive a computer-aided surgery (CAS) system, and to estimate the accurate size and shape of the bone defect to be repaired. If three-dimensional (3D) CT data of the bone defect are obtained, a biomaterial block to be compatible with the bone defect might be fabricated preoperatively with a computer-aided manufacturing (CAM) system. In addition, if the biomaterial block and its delivery system are coated with BMP, the bone defect might be repaired much more efficaciously. In this experimental study, we tested the feasibility of such an advanced bone regeneration system in a canine iliac bone tumor resection model.

Fig. 1 Preoperative planning on computer-aided design (CAD) software. a An imaginary spherical tumor (orange 15 mm in diameter) on the left ilium was placed on the three-dimensional computed tomography (3D CT) image reconstruction of the canine pelvis. The margin of the resection was set in green and 10 mm away from the tumor margin on the 3D CT image. b After resection of bone tumor. c CAD image of defected bone generated by subtracting the left iliac bone from the mirror image of the right iliac bone

123

486

Japan). The physicochemical characteristics of this porous ceramic and its function as a scaffold for bone ingrowth have been reported previously [7]. CT image data of the defected bone were transferred to the CAM system (Modela player, Roland DG Co, Shizuoka, Japan) in order to fabricate IP-CHA blocks of identical size and shape as the iliac bone defect by using a 3D drilling machine (MDX-20, Roland DG Co.). Furthermore, multiple dimples were made in the surface of the fabricated IP-CHA blocks with a high-speed surgical burr to facilitate retention of the BMP-retaining putty described below. Preparation of BMP-retaining biodegradable putty material Recombinant human BMP-2 (rhBMP-2) was kindly provided by Osteopharma Inc. (Osaka, Japan). The rhBMP-2, which retains bone-inducing capacity, was produced by dimerizing Escherichia coli-derived monomer BMP-2 molecules [8]. The bioactive rhBMP-2 was dissolved in 0.01 N HCl at 1 lg/ll. Poly-D,L-lactic acid-polyethylene glycol block copolymer (PLA-PEG) (MW: 9100, PLA/ PEG molar ratio: LA/EO.60/40) was synthesized and provided by Taki Chemical Co., Ltd. (Kakogawa, Japan) [9]. b-Tricalcium phosphate (b-TCP) powder (particle size \100 mm in diameter) was provided by Olympus Biomaterial Corp. (Tokyo, Japan). To prepare a bone-inducing putty material for each dog, 200 mg of b-TCP powder, 200 mg of PLA-PEG, and 100 lg of rhBMP-2 were mixed in a small ware and stirred thoroughly with a metal rod at 50 °C to maintain liquidity of the polymer for several minutes [10]. We have reported the physicochemical characteristics of this composite as a carrier material for BMP [11, 12]. Putty without rhBMP-2 was also made to be used as control material. The putty material samples were stored at -30 °C until use. Surgery to generate a preoperatively designed bone defect and to fill it with implant For guidance by CAS, the CT image data of the pelvis with defect were converted to the DICOM format and transferred to a CT-based computed navigation system (Stealth Station Tria; Medtronic Navigation, Louisville, CO, USA). Each animal was anesthetized by intramuscular injection of ketamine (10 mg/kg) and xylazine (1.2 mg/kg), and maintained under anesthesia by intravenous injection of pentobarbital (25 mg/kg). For surgery, the left ilium was exposed through a lateral approach in the right lateral position by standard sterile techniques. The left iliac bone surface was exposed to the acetabular rim. Two thread pins were set just above the hip joint and posterior to the ilium

123

K. Yano et al.

to obtain a fixed reference frame. Five points, i.e., the anterior superior iliac spine, posterior superior iliac spine, top of the iliac crest, top of the acetabular rim, and posterior rim of the ilium, were registered with the probe. After paired-point matching, surface matching was also performed. After bone resection guided by CAS, the bone defect in six dogs was filled with an implant with BMPretaining putty (BMP group). Putty material was coated all around the implant except the side of the ilium. In contrast, implants coated with putty without BMP-2 were placed in the bone defects of control animals (n = 6). All implants were fixed to the iliac bone with 2 or 3 thin (0.6 mm in diameter) Kirschner wires (Synthes Co., Ltd., Tokyo, Japan). The implants were carefully inserted so that they were in contact with the covering muscles. Prophylactic antibiotics (10 mg/body weight) were administered preand postoperatively. This animal experimental protocol was approved by the Animal Care and Oversight Committee of our institution (date of issue: 1 April 2008, registration no. 08060). Evaluation CT scans were taken immediately after surgery and serially at 3-week intervals to monitor new bone formation around the implant. Time-sequential CT images of the pelvis (1-mm slice thickness) of each dog were reconstructed with reconstruction software (Aze, Tokyo, Japan). Twelve weeks after surgery, all animals were killed by an overdose of anesthetics, and the left pelvises containing the implants were harvested and fixed in 70 % ethanol for histological examinations. The undecalcified histological sections (10 lm thickness) were analyzed using Villanueva bone staining [10]. Midtransverse CT slice images of the implant and two 1-mm additional slices were analyzed. The high density area on CT scans around the implant was considered to be new bone area and measured manually (cm2) using ImageJ software (Wayne Rasband, National Institutes of Health, Bethesda, MD). Mann-Whitney U tests were used to determine significant differences (P \ 0.05).

Results Accuracy of computer-aided surgery to the left iliac bone The imaginary bone tumor resection under navigation guidance was successfully performed in accordance with preoperative planning on CT images in all animals. Furthermore, the IP-CHA implants, which were fabricated with CAD and CAM systems driven by CT image data,


487

Fig. 2 Time sequential axial computed tomography (CT) images of the bone defect with implant. a In the bone morphogenetic protein (BMP) group, new bone encasing the implant was observed at 3 weeks after surgery. At 12 weeks, new remodeled bone connected to the original iliac bone was seen. b No new bone was noted in the control group

fitted well to the bone defects. Over the experimental period of 12 weeks, all animals survived without any fatal complications. Assessment of bone defect repair on reconstructive CT and 3D CT images Time-sequential axial images of a pelvis at the midsection of the bone defect are shown in Fig. 2. Implants in both BMP and control groups of dogs on reconstructed axial CT slices are shown. In the BMP group animals implanted with IP-CHA coated with BMP-2-retaining putty, radiopaque images on the implant surface were noted at 3 weeks. Thereafter, the radiopaque mass on the implant surface decreased with increasing density, until the implant was fully covered with dense bone (at 12 weeks). In contrast, small radiopaque shadows were seen throughout the experimental period in the control group animals. Macroscopic and histological assessment According to macroscopic examination, the whole implants from the BMP group animals were covered adequately with new bone connected to the original iliac bone and showed restoration of normal anatomy (Fig. 3a). In contrast, no bone tissue was noted on the implant surfaces of the control group (Fig. 3b). Representative undecalcified sections of experimental and control samples with Villanueva bone stain are shown in Fig. 4. In the BMP group, the implants were covered with bone connected to the original iliac bone, and new bone had grown into the pores of the implants

Fig. 3 Macroscopic views of implants harvested at 12 weeks after surgery. a The defect implanted with a bone morphogenetic protein (BMP)-2-retaining putty-coated interconnected porous calcium hydroxyapatite (IP-CHA) block was repaired with new bone anatomically, and the implant surface was not seen from outside. b In the control pelvis, no obvious new bone formation was noted, and the implant surface was exposed

(Fig. 4a). However, no putty material was noted on the sections at 12 weeks after surgery because of its degradable nature. Inflammatory and foreign body reactions were not

123

488

K. Yano et al.

Fig. 5 New bone formation areas around the implant at the axial plane. The new bone area of the bone morphogenetic protein (BMP) group was significantly larger than that of the control group in axial planes at all time periods examined (closed columns significant difference, P \ 0.05). Values are mean ± SD

Fig. 4 Histological sections at 12 weeks after surgery [lower (920) and higher magnification (9200)]. a, b Marked area of reconstructed axial computed tomography (CT) image indicates histological section of c and d, respectively. c In the bone morphogenetic protein (BMP) group, abundant bone formation was noted around the implant, at the junction with ilium, and in the interconnection pores of the implant. d In the control group, fibrous tissue was seen at those areas [arrowhead new bone, asterisk interconnected porous calcium hydroxyapatite (IP-CHA) implant, cross ilium, arrow fibrous tissue, circle osteoid, box calcified bone]

seen. In the control group, the implants were covered with fibrous tissue without bone (Fig. 4b). Temporal change of new bone mass In the BMP group, the largest new bone mass was noted around the implant at 3 weeks postoperatively, which reduced with time, eventually taking on the anatomical appearance of the iliac bone (Fig. 5). In contrast, little bone formation was noted throughout the experimental period in the control group.

Discussion BMPs are established potent bone-inducing factors. In particular, rhBMP-2 and BMP-7 have been synthesized by DNA transfection to mammalian cells (Chinese hamster ovarian cells, CHO) and are currently available for spinal fusion, repair of nonunion fractures, and treatment of open

123

tibial fractures with combined use of animal-derived collagen as a carrier material [13–17]. These cytokine therapies have achieved high rates of bone union and minimized the frequency of bone grafting. Recently, some methods have combined BMP-transduced cells and artificial materials, and cell sheet transplantation of cultured mesenchymal stem cells could reconstruct bone defects of rabbit mandibles or achieve union in a rat nonunion model [5, 6]. However, cell culture systems applicable to human clinical settings need special institution with special high-cost facilities for culture systems; meanwhile, BMPs can be used anywhere and easily if the preservation technique is adequate. IP-CHA was used as scaffold material in this study because previous reports indicated that IP-CHA has superior osteoconductive characteristics because of its interconnected porous structure, which allows growth of BMP-2-induced bone into the material [7]. However, IP-CHA remained unresorbed in the regenerated bone, likely because of its nondegradable characteristic. If the IP-CHA material was replaced by a degradable material like b-TCP, the defect might have been completely resorbed by the host, and the defect might ultimately be regenerated by new bone mass alone. However, because of the brittleness of b-TCP, we could not use porous b-TCP as the scaffold material in this study. If a new degradable material with acceptable mechanical strength becomes available, bone defects might be filled with regenerated bone without remaining biomaterials [11, 12]. This study demonstrated the essential role of using a biomaterial as scaffold to control 3D figures of BMP-2-induced regenerated bone. With regard to the change in new bone area, the maximum value was observed at 3 weeks after surgery, and the


area decreased gradually with time. The reduction of the new bone mass would be induced by bone remodeling. One limitation of this study was that BMP-2 was used at a fixed dose (100 lg), and dose-dependent effects of BMP2 were not presented. Furthermore, we have not explored the potential adverse events caused by excessive doses of BMP-2, including potential damage to neurovascular tissue by BMP-2-induced large bone mass. The appropriated ratio and dose of putty carrier material should be examined. In addition, the bone defect was also fixed to the anterior part of the iliac bone; thus, repair of bone defects in regions with more complex anatomies should be examined to elucidate the efficacy of this computer-aided bone repair system. Once these issues have been resolved and the BMP-2 dose has been optimized for humans, this new method might be used as an efficacious tool to repair large and complicated bone defects in clinical practice. Acknowledgments This work was supported in part by a Grant-inAid from the Ministry of Education, Cultures, Sports, Science and Technology of Japan (project grant nos. 18591640 and 21591922 to KT). Conflict of interest The authors declare that they have no conflict of interest. The authors did not receive and will not receive any benefits or funding from any commercial party related directly or indirectly to the subject of this article.

References 1. Buck BE, Malinin TI. Human bone and tissue allografts. Preparation and safety. Clin Orthop Relat Res. 1994;303:8–17. 2. Arrington ED, Smith WJ, Chambers HG, Bucknell AL, Davino NA. Complications of iliac crest bone graft harvesting. Clin Orthop Relat Res. 1996;329:300-309. 3. Butler D. Last chance to stop and think on risks of xenotransplants. Nature. 1998;391:320–4. 4. DeLustro F, Dasch J, Keefe J, Ellingsworth L. Immune responses to allogeneic and xenogeneic implants of collagen and collagen derivatives. Clin Orthop Relat Res. 1990;270:263–279. 5. Li J, Li Y, Ma S, Gao Y, Zuo Y, Hu J. Enhancement of bone formation by BMP-7 transduced MSCs on biomimetic nanohydroxyapatite/polyamide composite scaffolds in repair of mandibular defects. J Biomed Mater Res A. 2010;95:973–81. 6. Nakamura A, Akahane M, Shigematsu H, Tadokoro M, Morita Y, Ohgushi H, Dohi Y, Imamura T, Tanaka Y. Cell sheet transplantation of cultured mesenchymal stem cells enhances bone formation in a rat nonunion model. Bone. 2010;46:418–24. 7. Yoshikawa H, Tamai N, Murase T, Myoui A. Interconnected porous hydroxyapatite ceramics for bone tissue engineering. J R Soc Interface. 2009;6(Suppl 3):S341–8.

489 8. Yano K, Hoshino M, Ohta Y, Manaka T, Naka Y, Imai Y, Sebald W, Takaoka K. Osteoinductive capacity and heat stability of recombinant human bone morphogenetic protein-2 produced by Escherichia coli and dimerized by biochemical processing. J Bone Miner Metab. 2009;27:355–63. 9. Saito N, Okada T, Horiuchi H, Murakami N, Takahashi J, Nawata M, Ota H, Miyamoto S, Nozaki K, Takaoka K. Biodegradable poly-D,L-lactic acid-polyethylene glycol block copolymers as a BMP delivery system for inducing bone. J Bone Joint Surg Am. 2001;83-A(Suppl 1):S92–8. 10. Hoshino M, Namikawa T, Kato M, Terai H, Taguchi S, Takaoka K. Repair of bone defects in revision hip arthroplasty by implantation of a new bone-inducing material comprised of recombinant human BMP-2, Beta-TCP powder, and a biodegradable polymer: an experimental study in dogs. J Orthop Res. 2007;25:1042–51. 11. Kato M, Namikawa T, Terai H, Hoshino M, Miyamoto S, Takaoka K. Ectopic bone formation in mice associated with a lactic acid/dioxanone/ethylene glycol copolymer-tricalcium phosphate composite with added recombinant human bone morphogenetic protein-2. Biomaterials. 2006;27:3927–33. 12. Namikawa T, Terai H, Suzuki E, Hoshino M, Toyoda H, Nakamura H, Miyamoto S, Takahashi N, Ninomiya T, Takaoka K. Experimental spinal fusion with recombinant human bone morphogenetic protein-2 delivered by a synthetic polymer and beta-tricalcium phosphate in a rabbit model. Spine. 2005;30:1717–22. 13. Wozney JM, Rosen V, Celeste AJ, Mitsock LM, Whitters MJ, Kriz RW, Hewick RM, Wang EA. Novel regulators of bone formation: molecular clones and activities. Science. 1988;242:1528–34. 14. Boden SD, Kang J, Sandhu H, Heller JG. Use of recombinant human bone morphogenetic protein-2 to achieve posterolateral lumbar spine fusion in humans: a prospective, randomized clinical pilot trial: 2002 Volvo Award in clinical studies. Spine. 2002;27:2662–73. 15. Burkus JK, Transfeldt EE, Kitchel SH, Watkins RG, Balderston RA. Clinical and radiographic outcomes of anterior lumbar interbody fusion using recombinant human bone morphogenetic protein-2. Spine. 2002;27:2396–408. 16. Friedlaender GE, Perry CR, Cole JD, Cook SD, Cierny G, Muschler GF, Zych GA, Calhoun JH, LaForte AJ, Yin S. Osteogenic protein-1 (bone morphogenetic protein-7) in the treatment of tibial nonunions. J Bone Joint Surg Am. 2001;83-A(Suppl 1):S151–8. 17. Govender S, Csimma C, Genant HK, Valentin-Opran A, Amit Y, Arbel R, Aro H, Atar D, Bishay M, Borner MG, Chiron P, Choong P, Cinats J, Courtenay B, Feibel R, Geulette B, Gravel C, Haas N, Raschke M, Hammacher E, van der Velde D, Hardy P, Holt M, Josten C, Ketterl RL, Lindeque B, Lob G, Mathevon H, McCoy G, Marsh D, Miller R, Munting E, Oevre S, Nordsletten L, Patel A, Pohl A, Rennie W, Reynders P, Rommens PM, Rondia J, Rossouw WC, Daneel PJ, Ruff S, Ruter A, Santavirta S, Schildhauer TA, Gekle C, Schnettler R, Segal D, Seiler H, Snowdowne RB, Stapert J, Taglang G, Verdonk R, Vogels L, Weckbach A, Wentzensen A, Wisniewski T. Recombinant human bone morphogenetic protein-2 for treatment of open tibial fractures: a prospective, controlled, randomized study of four hundred and fifty patients. J Bone Joint Surg Am. 2002;84-A: 2123–34.

123