The Spine Journal 14 (2014) 408–415
Basic Science
Enhanced spinal fusion using a biodegradable porous mesh container in a rat posterolateral spinal fusion model Dong-Ah Shin, MD, PhDa, Bo Mi Yang, BSb, Giyoong Tae, PhDb, Young Ha Kim, PhDc, Hyung-Seok Kim, MD, PhDd, Hyoung-Ihl Kim, MD, PhDe,f,* a
Department of Neurosurgery, Yonsei University College of Medicine, 50 Yonsei-Ro, Seodaemun-Gu, Seoul, 120-752, Republic of Korea School of Materials Science and Engineering, Gwangju Institute of Science and Technology, 123 Cheomdan-gwagiro, Buk-gu, Gwangju, 500-712, Republic of Korea c Department of Chemistry, Chung-Ang University, 84 Heukseok-Ro, Dongjak-Gu, Seoul, Republic of Korea d Department of Forensic Medicine, Chonnam National University Medical School, 42 Jebong-Ro, Dong-Gu, Gwangju, 501-757, Republic of Korea e Department of Medical System Engineering, Gwangju Institute of Science and Technology, 123 Cheomdan-gwagiro, Buk-gu, Gwangju, 500-712, Republic of Korea f Department of Neurosurgery, Presbyterian Medical Center, 1-300 Junghwasan-dong, Wansangu, Jeonju, Jeonbuk, Republic of Korea b
Received 12 December 2012; revised 5 July 2013; accepted 23 August 2013
Abstract
BACKGROUND CONTEXT: Posterolateral fusion (PLF) with an autogenous iliac bone graft is the most common procedure for treating various lumbar spinal diseases. However, the limited success and associated morbidity from an iliac crest graft demands new biologically competent graft enhancers or substitutes. PURPOSE: To investigate the feasibility of tubular mesh container made of bioabsorbable sutures (poly-1,4-dioxane-2-one, PDO) for spinal fusion. STUDY DESIGN: Experimental animal study. METHODS: A biodegradable PDO tubular mesh container was used to contain small pieces of bone grafts. Twenty Sprague-Dawley male rats underwent PLF between L4 and L5 transverse processes with bilateral iliac grafts. Experimental animals were assigned into two different groups: autograft-only group (N510) that underwent PLF with autograft-only or mesh container group (N510) that underwent PLF with tubular mesh container filled with autogenous bone grafts. The rats were sacrificed at 8 weeks postoperatively, and the lumbar spines were removed. Spinal fusion was evaluated by manual palpation, microcomputed tomography, three-point bending test, and histological examination. RESULTS: Solid fusion was achieved in all cases of the mesh container group, whereas the autograft-only group showed 60% of solid fusion. New bone mass was higher and more solidly fused in the mesh container group than the autograft-only group (p!.01). Volume of fusion mass and density of bone were significantly higher in the mesh container group (p!.05). In all cases, inflammatory response was minimal. CONCLUSIONS: This study demonstrated that a tubular mesh container made of bioabsorbable suture is useful to hold small pieces of bone grafts and to enhance spinal fusion. Ó 2014 Elsevier Inc. All rights reserved.
Keywords:
Spinal fusion; Bone graft; Biodegration; Polydioxanone; Animal model; Pseudoathrosis
FDA device/drug status: Investigational (Tubular mesh container made of bioabsorbable sutures (poly-1,4-dioxane-2-one)). Author disclosures: D-AS: Grants: The National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2013027782) (E). BMY: Nothing to disclose. GT: Nothing to disclose. YHK: Nothing to disclose. H-SK: Nothing to disclose. H-IK: Grant: National Research Foundation (NRF) funded by Ministry of Education (2011-0010067), Korea, and a grant from the Institute of Medical System 1529-9430/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.spinee.2013.08.038
Engineering (iMSE), GIST, Korea (E, Paid directly to institution). The disclosure key can be found on the Table of Contents and at www. TheSpineJournalOnline.com. * Corresponding author. Department of Medical System Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, Republic of Korea. Tel.: (82) 62-715-3234; fax: (82) 62-715-3244. E-mail address: [email protected] (H.-I. Kim)
D.-A. Shin et al. / The Spine Journal 14 (2014) 408–415
Introduction Spinal fusion surgery is the second most common spinal surgery, and approximately 200,000 spinal fusion procedures are performed every year in the United States [1–3]. The aim of spinal fusion surgery is to eliminate the pain source arising from the instability of a motion segment or to prevent the progression of deformity in patients with instability, deformity, or a degenerative disease of the lumbar spine. Among various spinal fusion techniques, posterolateral fusion (PLF) between transverse processes with autologous bone graft is the most common and standard treatment method [4,5]. A conventional lumbar PLF consists of transpedicular screw fixation, dorsal decortication of transverse processes, and transplantation of bone graft onto the intertransverse bed. The essential parts for the attainment of solid fusion in the PLF include meticulous decortications of the transverse process and placement of an adequate amount of high-quality bone graft. In addition, rigid instrumentation is commonly added to keep the operated-on vertebral segments fixed during the critical period for fusion mass formation [6–8] Although a lumbar PLF can provide more predictive and reproducible results in the treatment of pathological conditions in the lumbar spine, a lumbar PLF has drawbacks inherent to its procedure from the clinical point of view. One of them is nonunion, which can be as high as 35% to 40% in the patients who underwent spinal fusion surgeries [9–11]. Many factors contribute to the increase in the rate of nonunion including biomechanical factors, prior surgery, multilevel arthrodesis, and poor nutrition. Besides the cellular mechanism of osteogenesis, maintenance of a good healing environment for the maturation of bone fusion is important to decrease the rate of nonunion. The prevention of soft-tissue interposition, the easy migration of osteogenic cells, vascularization to the core of fusion mass, and mechanical stress are important physiological factors that facilitate bone fusion [12,13]. Previously, Poyton et al. reported that use of a protective graft container made of a polylactide sheath enhanced the fusion mass volume in a rabbit spinal fusion model [14]. However, because of the undulating space between transverse processes, a more flexible container is preferred to fix it closely to the transverse processes in PLF. In this study, we fabricated a flexible bone container made of biodegradable tubular mesh that met the above mentioned physiological factors for better bone fusion while minimizing the complications of an iliac donor site. The purpose of this study is to evaluate the effect of a flexible, porous tubular mesh container on bone fusion in a rat PLF model. We also investigated the biocompatibility of a tubular mesh container during the process of osteogenesis.
Material and methods Fabrication of porous tubular mesh The tubular mesh container was made of biodegradable poly-1,4-dioxane-2-one (PDO) suture (Samyang Co., Seoul,
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Korea). The cylindrical tubular mesh containers with a diameter of 6 mm and a mesh size of 2 mm were fabricated by using a home-made knitting equipment (S & G Biotech, Seoul, Korea) using PDO monofilament fibers with a diameter of 250 mm (Fig. 1). The mesh container had a large mesh size to allow the free passage of cells and the growth of blood vessels into it while it prevented the interposition of soft tissues between bone pieces. One end of the container was knotted; the other was also knotted after filling the bone grafts (Fig. 1). Although the flexible container could be placed anywhere in the spinal structures, it was firmly attached to the underlying bones by securing it and molded to an undulating, decorticated transverse process.
Animal model and surgical procedure Experiments were conducted following the guidelines of the Animal Care and Use Committee of Gwangju Institute of Science and Technology. A total of 30 male SpragueDawley rats weighing 420615 g (mean6standard deviation) were used for this study. The procedure of PLF in a rat model was described previously [7]. Briefly, the rats were anesthetized and placed prone on the thermocouple pad. After preparing the lumbar region aseptically, two separate 3-mm fascial incisions were mad lateral and parallel to the midline from the spinous process of L3 to the sacral bone. The L4 and L5 transverse processes were exposed by splitting the muscles. A low-speed burr was used to decorticate the dorsal surface of the transverse processes bilaterally. To harvest the graft bones, a lateral oblique incision was made along the iliac crest in both sides. Then, all available corticocancellous bones were harvested and morselized. The volume of graft generated from each iliac crest was 300 mg following morselization. Based on the group assignment, graft materials were implanted between the transverse processes bilaterally in the paraspinal muscle bed and tubular mesh containers were secured to underlying decorticated transverse processes with PDO monofilaments. The wound was closed after irrigation and monitored daily. The rats were sacrificed at 8 weeks postoperatively, and the lumbar spines were harvested immediately from the first lumbar to the first sacral vertebrae with surrounding musculatures intact. The experimental design is outlined in Fig. 1.
Experimental groups The rats were randomly assigned into three different groups: (1) the sham-operated group (N510) that underwent the same operative procedures on the spine without any bone graft; (2) the autograft-only group (N510) that underwent PLF with autograft only; and (3) the mesh container group (N510) that underwent PLF with a container filled with autologous bone grafts. In the bone graft groups,
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Fig. 1. Schematic illustration of rat spinal fusion surgery. Morselized corticocancellous bones were implanted in the space between the L4 and L5 transverse process directly or using mesh container.
300 mg of morselized corticocancellous bone pieces were placed on both sides of the spine. Manual assessment of fusion The explanted spines were immediately palpated to have continuity of fusion mass between the transverse processes. Then they were manually assessed by flexion and extension at the fusion level for intersegmental motion by two different observers and compared with adjacent segments of the vertebral column. The test was conducted with care not to cause any mechanical damage in the fusion mass. The absence of intersegmental motion was assessed as ‘‘solid’’ and if any motion was detected at either side, the spine was assessed as ‘‘not solid.’’ Biomechanical testing All the harvested PLF masses were evaluated by a threepoint bending test using a Universal Tensile Machine (Instron 5567, Instron, MA, USA). Both ends of vertebral bodies were placed with their dorsal sides down on two fulcra. The upper anvil (10.0-mm diameter) was placed in a position to apply a load on the ventral surface of the intervertebral disc perpendicular to the longitudinal spine. Three-point bending tests were performed with a 10.0-mm intersupport distance and a 50-mm/min crosshead speed until the upper anvil was advanced to 15 mm (Fig. 4, Left). The loaddisplacement curves were obtained for the autograft-only group and the mesh container group (N58), and the results were statistically compared.
Microcomputed tomography All the harvested spines were scanned with a highresolution microcomputed tomography (micro-CT; Inveon multimodal CT/positron emission tomography, Simens, Knoxville, TN, USA) to measure the calcified fusion mass at the region of PLF. The scan was performed in the long axis of the spine with an energy of 50 kVp and a current of 100 mA, and a 200-ms exposure time producing a resolution of 100-mm3 voxel size. The images were reconstructed using a Feldkamp cone-beam reconstruction algorithm [15]. The size of the reconstructed image was 1,0241,024 pixels and 700 slices were acquired. The final reconstructed data were converted into the Digital Imaging and Communication in Medicine format. Scout, sagittal, and cross-sectional views were examined using a Digital Imaging and Communication in Medicine viewer to investigate the integrity of bone union. We created three-dimesional image reconstructions to measure the total volume of fusion mass in both sides for each specimen. The quality of fusion and the degree of incorporation to transverse processes were evaluated by measuring the density of fusion mass (% of volume of bone mass/total volume of fusion mass). Integrity of fusion mass was classified into four grades using the method of a modified Lenke’s classification based on the micro-CT findings: grade A: definitely solid fusion with bilateral stout fusion masses present; grade B: possibly solid with asymmetric fusion masses; grade C: probably not solid with a small fusion mass bilaterally; and grade D:
D.-A. Shin et al. / The Spine Journal 14 (2014) 408–415
definitely not solid with bone graft resorption or obvious bilateral pseudarthrosis [16]. Histological examination The decalcified specimens not used for biomechanical test were used for histological examinations. The specimens were fixed in 10% formalin in a neutral buffer solution. The specimens were decalcified by immersion in a citric-buffered formic acid solution and dehydrated in a graded ethanol series. The specimens were then embedded in paraffin and 4-mm-thick sections were stained with Masson’s trichrome and hematoxylin and eosin. The former stain produces high-contrast images with red bone, blue collagen, pink cell cytoplasm, and black cell nuclei. Tiled images were captured with a digital camera system (ProRes C5, Jenoptik, Jena, Germany) mounted on a Leica microscope (Leica DM 2500, Wetzlar, Germany). Combined images were then reconstructed for each slide with an image analyzer (IMT i-Solution Inc., Burnaby, BC, Canada). Statistical analysis The results were analyzed statistically using statistical software (SPSS 13, SPSS, Chicago, IL, USA). Fusion rates as determined by manual palpation were compared using a chi-squared test. A Student t test was performed to analyze the volume of newly formed bone and the results of biomechanical testing. Mann-Whitney U testing was used to evaluate the radiographic fusion status. A value of p!.05 was set to be statistically significant.
Results Postoperative recovery All rats tolerated the surgical procedure well and postoperative recovery was uneventful with no perioperative mortality. They were ambulatory during the entire postoperative
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days. Weight increased after the first postoperative week. Although two rats of the autograft-only group had superficial wound infections, their spines were also assessed because they were fused by manual palpation and study of micro-CT. There was no case of wound infection in the mesh container group. No single case of container-related complication was observed. Gross inspection and manual assessment of fusion At 8 weeks, manual palpation revealed that overall 80% (16/20) of the explanted spines had solid fusion. Four rats of ‘‘not-solid’’ fusion, all of which belonged to the autograft-only group, showed less volume of bony mass on gross inspection and intersegmental motion on manual palpation. In all rats with solid fusion, a continuous bony mass was palpated and no intersegmental motion was noted between the intertransverse processes. The autograft-only group had a solid fusion rate of 60% (6/10), whereas the mesh container group showed significantly higher, complete fusion for all cases (100%, 10/10, p!.01). There was no extended bony growth to invade paravertebral muscle dorsally and psoas muscle ventrally. There was no interobserver difference on the results of gross inspection and manual palpation. Micro-CT findings The mesh container group showed more extensive new bone formation in the fusion mass (Fig. 2). The fusion mass of the mesh container group was fused firmly with adjacent transverse processes, and no crack was observed inside the fusion mass. On the other hand, even though the autograftonly group also showed new bone formation, clefts between the L4 and L5 transverse processes were observed in the subjects showing ‘‘not solid’’ fusion. The fusion mass from the autograft-only group consistently showed a heterogonous fusion mass with not yet remodeled pieces of bone chips. Although unremodeled bone chips were also noted
Fig. 2. Microcomputed tomographic images after 8 weeks of posterolateral fusion. The three-dimensional microcomputed tomography images revealed that the volume of fusion mass was larger in the mesh bone container group (Right) than in the autograft-only group (Left).
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in the mesh container group, cortical rims were more welldefined. The analysis of the micro-CT images revealed that the volume of fusion mass was significantly larger in the mesh container group than in the autograft-only group (137.1626.7 mL vs. 87.0625.6 mL, p!.05, Fig. 3, Left). The bone density of the mesh container group was also higher than that of the autograft-only group (10.662.5 vs. 6.762.3, p!.05, Fig. 3, Right). The difference of posterolateral fusion success according to Lenke’s classification is summarized in the Table. All rats in the mesh container group achieved grade A, definitely solid fusion. In comparison, the autograft-only group showed grade A, solid fusion in four rats (40%); grade B, possibly solid fusion in three (30%); and grade C, not solid fusion in three (30%) (Table). There was a significant statistical difference in fusion success between the two groups (p!.05). Biomechanical testing Figure 4, Right shows the load-displacement curves of the vertebral bodies for the measurement of biomechanical properties. There were no differences in the initial slopes between the tubular mesh container group and the autograft-only group. However, when the displacement was increased to more than 10 mm, the two groups showed statistically significant differences (p!.05); the autograftonly group started to show a nonlinear, yielding behavior whereas the mesh container group continued showing a linear increase in the load with increasing displacement. And, the differences became larger with further increasing the displacement. Because of the geometries of the sample (round) and the crosshead (inverted triangle), the full contact between the sample and the moving crosshead was achieved only after 10 mm of displacement (Fig. 4, Left). So, until the displacement reaches 10 mm, the contact area kept increasing, and the increase in the load seemed to be dominated by the increase in the contact area, which probably resulted in the
initially similar responses between the two groups and the statistically significant different response with the displacement larger than 10 mm. After full contact, the autograftonly group showed a nonlinear increment pattern in the load, possibly because of the fracture of the vertebral bodies, whereas the mesh container group showed a linear increase with increasing displacement. Therefore, the use of a tubular mesh container clearly enhanced the mechanical properties of the vertebral bodies (Fig. 4, Right). The geometry of the experimental setup also limited the maximum displacement up to 15 mm; otherwise, the crosshead would collide with the support. The ultimate strengths of the vertebral bodies therefore could not be obtained because of this limitation in the displacement. Histological examination Histological findings of the fusion mass were consistent with the micro-CT images (Fig. 5). In the autograft-only group, areas of unremodeled cortical autograft bone were widely observed and were encased in cartilage and osteoid. Although a bony bridge was formed, the cortical rim was thin. The defect area was replaced with small amounts of fibrous connective tissues. In the fusion mass, bone growth was more prominent over the transverse processes than was seen centrally in the intertransverse bed. The center of the fusion mass had more cartilage present than did the areas over the transverse processes, where more advanced remodeling with bone marrow was observed. In the autograft-only group, fibrous and chondrified bones were observed in the graft bones and the fusion mass was put in contact with the small surface of the transverse process because of the interruption of soft tissues. In the mesh container group, mature and circuitous bone trabecula structures and a clear medullary cavity were observed. The defected area mostly disappeared and was replaced with fibrous and Masson-Trichrome–positive collagen tissue. The fusion mass was put in contact with the transverse process in a wider area in
Fig. 3. Microcomputed tomography analysis on the volume (Left) and the density (Right) of newly formed bone after 8 weeks of posterolateral fusion. The significantly larger volume and the higher density of fusion mass were observed in the mesh bone container group than the autograft-only group.
D.-A. Shin et al. / The Spine Journal 14 (2014) 408–415 Table Radiographic fusion status assessed by Lenke classification of posterolateral fusion success Grade
Description
Bone pocket group
Autograft-only group
A B C D
Definitely solid Possibly solid Probably not solid Definitely not solid
10 0 0 0
4 3 3 0
the mesh container group than the autograft-only group. The deposition of collagen and the formation of new bone were markedly increased in the bone. Results of a histological analysis of the fusion masses of the mesh container group demonstrated abundant osteoid deposition throughout the fusion mass as well as bone marrow formation. These fusion masses consistently appeared larger than those of the autograft-only group. The mesh container group showed a thicker cortical rim, less cartilage, and more marrow formation than the autograft-only group. PDO suture fibers were half-absorbed, leaving the amorphous vacuoles along the contour of the tubular mesh containers for 8 weeks. There was no acute inflammatory response around the PDO filaments.
Discussion The rat model of posterolateral spinal fusion has been reported to show about 40% fusion rate with an autogenous bone or demineralized bone matrix graft after 6 postoperative weeks [17–19]. Recently, tissue-engineered alternatives to autogenous bone grafts that contain osteoinductive and/ or osteoconductive material were proven to be more effective in enhancing spinal fusion [1,19–23]. In particular, the use of bone morphogenic proteins provided the positive prospect of a higher fusion rate than the conventional method and the possibility to eliminate the complications resulting from iliac crest grafting. However, in spite of their
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remarkable successes, adoption of new techniques is tempered by the high cost of tissue engineering and restriction of accessibility. Further, reported complications warn against the wide use of these alternative graft substitutes [24–26]. Therefore, the device that can increase the successful union rate of autogenous bone graft, using either the usual amount of graft or a smaller amount of bone graft, is still the preferred option for clinical application to eliminate of concerns of graft-induced complications [1]. Therefore, a biodegradable mesh container applied in this study as a ‘‘bone graft enhancer’’ can be a promising tool for promoting spinal fusion. In this study, we achieved a significantly enhanced bone fusion by using a biodegradable tubular mesh container to contain bone graft instead of the direct use of autologous bone graft. Although new bone formation was observed in both groups, significantly larger volume and higher density of new bone were found in the mesh container group compared with the autograft-only group. Furthermore, solid fusion was achieved in all cases of the mesh container group, whereas the autograft-only group showed 60% of solid fusion. Therefore, the spinal fusion was further strengthened and the quality of osteogenesis was improved by the use of a mesh container. These results are quite encouraging because the use of mesh container might reduce the chance of pseudoarthrosis or nonunion, which is reported to occur in 5% to 45% of conventional spinal fusion procedures [27,28]. The success of posterolateral spinal fusion depends on many systemic and local factors, and especially vascularity is one of the critical factors. It is known that the primary blood supply to the fusion mass comes from the decorticated transverse processes [29,30]. The failure to achieve spinal fusion in the absence of extensive decortications also emphasizes the importance of close contacts between the graft bones and transverse processes. In addition, close contact between graft bones is also mandatory to favor chondrocyte formation, which is critical for ensuing
Fig. 4. Biomechanical assessment of fusion mass at 8 weeks of posterolateral fusion (PLF) using a three-point bending test. (Left) Experimental setup of three-point bending measurement. Arrow indicates the direction of force against the fusion mass. (Right) The mesh bone container group showed the higher stiffness than the control group, especially at displacement larger than 10 mm when the full contact between the sample and the crosshead occurred.
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Fig. 5. Histological staining of fusion mass after 8 weeks of posterolateral fusion. (A) Hematoxylin and eosin (H&E) staining showing the mesh bone container containing small pieces of iliac bones (arrowheads) (50). (B) H&E staining showing no acute inflammatory reaction around the PDO fibers (100). (C) Masson-Trichrome staining showing the fusion mass in the autograft-only group. Fibrous and chondrified bones are observed, and fibrous connective tissues fill the space between bones (50). (D) Masson-Trichrome staining showing a mature and circuitous bone trabecula structure and a clear medullary cavity in mesh bone container group. The space between bones is replaced with fibrous and Masson-Trichrome–positive collagen tissue. The collagen tissue extends across the mesh container (arrows) (50). IVD, intervertebral disc; SC, spinal canal; TP, transverse process.
enchondral bone formation [31] in general. The larger the contact area exposed, the greater the availability of potential osteogenic cells and bony bridges to facilitate bone fusion. However, the conventional method of PLF is to place the grafts in the intertransverse space piece by piece; consequently, it does not guarantee the close and wide contacts between the transverse process and graft pieces. In this experiment, histological examination demonstrated that the fusion mass was put in contact with the transverse process in a wider area in the mesh container group because of easy adaptation to the irregular surfaces of the recipient bones and the strong tensile strength of the tubular mesh container to hold the bone pieces together. However, a mesh container is not likely to either provide a stout fixation force or limit the spinal motions as with the screw fixation system. Nonetheless, manual and mechanical testing revealed that the use of the mesh container could enhance bone fusion more strongly than the direct use of autograft bone only. Fabrication of the tubular mesh container was very simple compared wiht other similar containers that usually cost and demand high technology. We used a biodegradable polymer (PDO) suture, approved by the US Food and Drug Administration. Bioabsorbable PDO has been used in repairing the organs, joints, and tissues throughout the body with low immunological response [32–35]. This suture is known to be degraded over 4 to 6 months
with a strength retention up to 6 weeks. Even though this period of strength retention is enough to stabilize the fusion in rat model, it may not be adequate to apply PDO for human spinal fusion because the fusion takes longer than 6 months in humans. For application in humans, materials such as polylactide, which have the strength retention time of longer than 6 months, may be considered alternatively. No inflammatory infiltrations were observed in histological examination around the PDO fibers after 8 weeks of fusion surgery in this study. Previously, it was postulated that a rigid container has advantages for bone fusion because it can prevent muscle interposition and counteract muscle compression [14]. In our experiment, interposition of soft tissue was not observed inside the tubular mesh container. A flexible mesh container seems to be strong enough to prevent soft-tissue interposition and to counteract the compressive force of surrounding muscles in supplying the blood flow to the core of the fusion mass. Conclusion The results of this study support the benefit of a tubular mesh container in spinal fusion. Bone fusion using a biodegradable mesh container was qualitatively and quantitatively significantly better than the autograft-only group. The bone container made of a biodegradable
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PDO-facilitated bone fusion without specific morbidity in a rat model of posterolateral spinal fusion.
References [1] Boden SD. Overview of the biology of lumbar spine fusion and principles for selecting a bone graft substitute. Spine 2002;27:S26–31. [2] Sandhu HS. Anterior lumbar interbody fusion with osteoinductive growth factors. Clin Orthop Relat Res 2000;371:56–60. [3] Andersson GB. Epidemiology of low back pain. Acta Orthop Scand Suppl 1998;281:28–31. [4] Axelsson P, Johnsson R, Stromqvist B, et al. Posterolateral lumbar fusion. Outcome of 71 consecutive operations after 4 (2-7) years. Acta Orthop Scand 1994;65:309–14. [5] Lee CS, Hwang CJ, Lee DH, et al. Fusion rates of instrumented lumbar spinal arthrodesis according to surgical approach: a systematic review of randomized trials. Clin Orthop Surg 2011;3:39–47. [6] McAfee PC, Farey ID, Sutterlin CE, et al. The effect of spinal implant rigidity on vertebral bone density. A canine model. Spine 1991;16:S190–7. [7] Nagel DA, Kramers PC, Rahn BA, et al. A paradigm of delayed union and nonunion in the lumbosacral joint. A study of motion and bone grafting of the lumbosacral spine in sheep. Spine 1991;16:553–9. [8] Wang JC, Mummaneni PV, Haid RW. Current treatment strategies for the painful lumbar motion segment: posterolateral fusion versus interbody fusion. Spine 2005;30:S33–43. [9] Ahlmann E, Patzakis M, Roidis N, et al. Comparison of anterior and posterior iliac crest bone grafts in terms of harvest-site morbidity and functional outcomes. J Bone Joint Surg Am 2002;84-A:716–20. [10] Banwart JC, Asher MA, Hassanein RS. Iliac crest bone graft harvest donor site morbidity. A statistical evaluation. Spine 1995;20: 1055–60. [11] Turner JA, Ersek M, Herron L, et al. Patient outcomes after lumbar spinal fusions. JAMA 1992;268:907–11. [12] Boden SD. The biology of posterolateral lumbar spinal fusion. Orthop Clin North Am 1998;29:603–19. [13] Boden SD. Biology of lumbar spine fusion and use of bone graft substitutes: present, future, and next generation. Tissue Eng 2000;6: 383–99. [14] Poynton AR, Zheng F, Tomin E, et al. Resorbable posterolateral graft containment in a rabbit spinal fusion model. J Neurosurg 2002;97:460–3. [15] Feldkamp L, Davis L, Kress J. Practical cone-beam algorithm. J Opt Soc Am A 1984;1:612–9. [16] Lenke LG, Bridwell KH, Bullis D, et al. Results of in situ fusion for isthmic spondylolisthesis. J Spinal Disord 1992;5:433–42. [17] Grauer JN, Bomback DA, Lugo R, et al. Posterolateral lumbar fusions in athymic rats: characterization of a model. Spine J 2004;4:281–6. [18] Bomback DA, Grauer JN, Lugo R, et al. Comparison of posterolateral lumbar fusion rates of Grafton putty and OP-1 putty in an athymic rat model. Spine 2004;29:1612–7.
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[19] Wang JC, Kanim LE, Yoo S, et al. Effect of regional gene therapy with bone morphogenetic protein-2-producing bone marrow cells on spinal fusion in rats. J Bone Joint Surg Am 2003;85-A:905–11. [20] Morisue H, Matsumoto M, Chiba K, et al. A novel hydroxyapatite fiber mesh as a carrier for recombinant human bone morphogenetic protein-2 enhances bone union in rat posterolateral fusion model. Spine 2006;31:1194–200. [21] Salamon ML, Althausen PL, Gupta MC, Laubach J. The effects of BMP-7 in a rat posterolateral intertransverse process fusion model. J Spinal Disord Tech 2003;16:90–5. [22] Biswas DM, Bible JEM, Whang PHM, et al. Augmented demineralized bone matrix: a potential alternative for posterolateral lumbar spinal fusion. Am J Orthop (Belle Mead NJ) 2010;39:531–8. [23] Tanaka K, Takemoto M, Fujibayashi S, et al. A bioactive and bioresorbable porous cubic composite scaffold loaded with bone marrow aspirate: a potential alternative to autogenous bone grafting. Spine 2011;36:441–8. [24] Shimer AL, Oner FC, Vaccaro AR. Spinal reconstruction and bone morphogenetic proteins: open questions. Injury 2009;40(3 Suppl): S32–8. [25] Benglis D, Wang MY, Levi AD. A comprehensive review of the safety profile of bone morphogenetic protein in spine surgery. Neurosurgery 2008;62:ONS423–31; discussion ONS431. [26] Deutsch H. High-dose bone morphogenetic protein-induced ectopic abdomen bone growth. Spine J 2010;10:e1–4. [27] Steinmann JC, Herkowitz HN. Pseudarthrosis of the spine. Clin Orthop Relat Res 1992;284:80–90. [28] Zdeblick TA. A prospective, randomized study of lumbar fusion. Preliminary results. Spine 1993;18:983–91. [29] Toribatake Y, Hutton WC, Tomita K, Boden SD. Vascularization of the fusion mass in a posterolateral intertransverse process fusion. Spine 1998;23:1149–54. [30] Boden SD, Schimandle JH, Hutton WC. An experimental lumbar intertransverse process spinal fusion model. Radiographic, histologic, and biomechanical healing characteristics. Spine 1995;20: 412–20. [31] Carter DR, Beaupre GS, Giori NJ, Helms JA. Mechanobiology of skeletal regeneration. Clin Orthop Relat Res 1998;355(Suppl): S41–55. [32] Ishaug-Riley SL, Okun LE, Prado G, et al. Human articular chondrocyte adhesion and proliferation on synthetic biodegradable polymer films. Biomaterials 1999;20:2245–56. [33] Sell SA, McClure MJ, Barnes CP, et al. Electrospun polydioxanoneelastin blends: potential for bioresorbable vascular grafts. Biomed Mater 2006;1:72–80. [34] Greiner S, Braunsdorf J, Perka C, et al. Mid to long-term results of open acromioclavicular-joint reconstruction using polydioxansulfate cerclage augmentation. Arch Orthop Trauma Surg 2009;129: 735–40. [35] Zeplin PH, Zahn RK, Meffert RH, Schmidt K. Biomechanical evaluation of flexor tendon repair using barbed suture material: a comparative ex vivo study. J Hand Surg Am 2011;36:446–9.
Basic Science
Enhanced spinal fusion using a biodegradable porous mesh container in a rat posterolateral spinal fusion model Dong-Ah Shin, MD, PhDa, Bo Mi Yang, BSb, Giyoong Tae, PhDb, Young Ha Kim, PhDc, Hyung-Seok Kim, MD, PhDd, Hyoung-Ihl Kim, MD, PhDe,f,* a
Department of Neurosurgery, Yonsei University College of Medicine, 50 Yonsei-Ro, Seodaemun-Gu, Seoul, 120-752, Republic of Korea School of Materials Science and Engineering, Gwangju Institute of Science and Technology, 123 Cheomdan-gwagiro, Buk-gu, Gwangju, 500-712, Republic of Korea c Department of Chemistry, Chung-Ang University, 84 Heukseok-Ro, Dongjak-Gu, Seoul, Republic of Korea d Department of Forensic Medicine, Chonnam National University Medical School, 42 Jebong-Ro, Dong-Gu, Gwangju, 501-757, Republic of Korea e Department of Medical System Engineering, Gwangju Institute of Science and Technology, 123 Cheomdan-gwagiro, Buk-gu, Gwangju, 500-712, Republic of Korea f Department of Neurosurgery, Presbyterian Medical Center, 1-300 Junghwasan-dong, Wansangu, Jeonju, Jeonbuk, Republic of Korea b
Received 12 December 2012; revised 5 July 2013; accepted 23 August 2013
Abstract
BACKGROUND CONTEXT: Posterolateral fusion (PLF) with an autogenous iliac bone graft is the most common procedure for treating various lumbar spinal diseases. However, the limited success and associated morbidity from an iliac crest graft demands new biologically competent graft enhancers or substitutes. PURPOSE: To investigate the feasibility of tubular mesh container made of bioabsorbable sutures (poly-1,4-dioxane-2-one, PDO) for spinal fusion. STUDY DESIGN: Experimental animal study. METHODS: A biodegradable PDO tubular mesh container was used to contain small pieces of bone grafts. Twenty Sprague-Dawley male rats underwent PLF between L4 and L5 transverse processes with bilateral iliac grafts. Experimental animals were assigned into two different groups: autograft-only group (N510) that underwent PLF with autograft-only or mesh container group (N510) that underwent PLF with tubular mesh container filled with autogenous bone grafts. The rats were sacrificed at 8 weeks postoperatively, and the lumbar spines were removed. Spinal fusion was evaluated by manual palpation, microcomputed tomography, three-point bending test, and histological examination. RESULTS: Solid fusion was achieved in all cases of the mesh container group, whereas the autograft-only group showed 60% of solid fusion. New bone mass was higher and more solidly fused in the mesh container group than the autograft-only group (p!.01). Volume of fusion mass and density of bone were significantly higher in the mesh container group (p!.05). In all cases, inflammatory response was minimal. CONCLUSIONS: This study demonstrated that a tubular mesh container made of bioabsorbable suture is useful to hold small pieces of bone grafts and to enhance spinal fusion. Ó 2014 Elsevier Inc. All rights reserved.
Keywords:
Spinal fusion; Bone graft; Biodegration; Polydioxanone; Animal model; Pseudoathrosis
FDA device/drug status: Investigational (Tubular mesh container made of bioabsorbable sutures (poly-1,4-dioxane-2-one)). Author disclosures: D-AS: Grants: The National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2013027782) (E). BMY: Nothing to disclose. GT: Nothing to disclose. YHK: Nothing to disclose. H-SK: Nothing to disclose. H-IK: Grant: National Research Foundation (NRF) funded by Ministry of Education (2011-0010067), Korea, and a grant from the Institute of Medical System 1529-9430/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.spinee.2013.08.038
Engineering (iMSE), GIST, Korea (E, Paid directly to institution). The disclosure key can be found on the Table of Contents and at www. TheSpineJournalOnline.com. * Corresponding author. Department of Medical System Engineering, Gwangju Institute of Science and Technology (GIST), Gwangju, Republic of Korea. Tel.: (82) 62-715-3234; fax: (82) 62-715-3244. E-mail address: [email protected] (H.-I. Kim)
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Introduction Spinal fusion surgery is the second most common spinal surgery, and approximately 200,000 spinal fusion procedures are performed every year in the United States [1–3]. The aim of spinal fusion surgery is to eliminate the pain source arising from the instability of a motion segment or to prevent the progression of deformity in patients with instability, deformity, or a degenerative disease of the lumbar spine. Among various spinal fusion techniques, posterolateral fusion (PLF) between transverse processes with autologous bone graft is the most common and standard treatment method [4,5]. A conventional lumbar PLF consists of transpedicular screw fixation, dorsal decortication of transverse processes, and transplantation of bone graft onto the intertransverse bed. The essential parts for the attainment of solid fusion in the PLF include meticulous decortications of the transverse process and placement of an adequate amount of high-quality bone graft. In addition, rigid instrumentation is commonly added to keep the operated-on vertebral segments fixed during the critical period for fusion mass formation [6–8] Although a lumbar PLF can provide more predictive and reproducible results in the treatment of pathological conditions in the lumbar spine, a lumbar PLF has drawbacks inherent to its procedure from the clinical point of view. One of them is nonunion, which can be as high as 35% to 40% in the patients who underwent spinal fusion surgeries [9–11]. Many factors contribute to the increase in the rate of nonunion including biomechanical factors, prior surgery, multilevel arthrodesis, and poor nutrition. Besides the cellular mechanism of osteogenesis, maintenance of a good healing environment for the maturation of bone fusion is important to decrease the rate of nonunion. The prevention of soft-tissue interposition, the easy migration of osteogenic cells, vascularization to the core of fusion mass, and mechanical stress are important physiological factors that facilitate bone fusion [12,13]. Previously, Poyton et al. reported that use of a protective graft container made of a polylactide sheath enhanced the fusion mass volume in a rabbit spinal fusion model [14]. However, because of the undulating space between transverse processes, a more flexible container is preferred to fix it closely to the transverse processes in PLF. In this study, we fabricated a flexible bone container made of biodegradable tubular mesh that met the above mentioned physiological factors for better bone fusion while minimizing the complications of an iliac donor site. The purpose of this study is to evaluate the effect of a flexible, porous tubular mesh container on bone fusion in a rat PLF model. We also investigated the biocompatibility of a tubular mesh container during the process of osteogenesis.
Material and methods Fabrication of porous tubular mesh The tubular mesh container was made of biodegradable poly-1,4-dioxane-2-one (PDO) suture (Samyang Co., Seoul,
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Korea). The cylindrical tubular mesh containers with a diameter of 6 mm and a mesh size of 2 mm were fabricated by using a home-made knitting equipment (S & G Biotech, Seoul, Korea) using PDO monofilament fibers with a diameter of 250 mm (Fig. 1). The mesh container had a large mesh size to allow the free passage of cells and the growth of blood vessels into it while it prevented the interposition of soft tissues between bone pieces. One end of the container was knotted; the other was also knotted after filling the bone grafts (Fig. 1). Although the flexible container could be placed anywhere in the spinal structures, it was firmly attached to the underlying bones by securing it and molded to an undulating, decorticated transverse process.
Animal model and surgical procedure Experiments were conducted following the guidelines of the Animal Care and Use Committee of Gwangju Institute of Science and Technology. A total of 30 male SpragueDawley rats weighing 420615 g (mean6standard deviation) were used for this study. The procedure of PLF in a rat model was described previously [7]. Briefly, the rats were anesthetized and placed prone on the thermocouple pad. After preparing the lumbar region aseptically, two separate 3-mm fascial incisions were mad lateral and parallel to the midline from the spinous process of L3 to the sacral bone. The L4 and L5 transverse processes were exposed by splitting the muscles. A low-speed burr was used to decorticate the dorsal surface of the transverse processes bilaterally. To harvest the graft bones, a lateral oblique incision was made along the iliac crest in both sides. Then, all available corticocancellous bones were harvested and morselized. The volume of graft generated from each iliac crest was 300 mg following morselization. Based on the group assignment, graft materials were implanted between the transverse processes bilaterally in the paraspinal muscle bed and tubular mesh containers were secured to underlying decorticated transverse processes with PDO monofilaments. The wound was closed after irrigation and monitored daily. The rats were sacrificed at 8 weeks postoperatively, and the lumbar spines were harvested immediately from the first lumbar to the first sacral vertebrae with surrounding musculatures intact. The experimental design is outlined in Fig. 1.
Experimental groups The rats were randomly assigned into three different groups: (1) the sham-operated group (N510) that underwent the same operative procedures on the spine without any bone graft; (2) the autograft-only group (N510) that underwent PLF with autograft only; and (3) the mesh container group (N510) that underwent PLF with a container filled with autologous bone grafts. In the bone graft groups,
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Fig. 1. Schematic illustration of rat spinal fusion surgery. Morselized corticocancellous bones were implanted in the space between the L4 and L5 transverse process directly or using mesh container.
300 mg of morselized corticocancellous bone pieces were placed on both sides of the spine. Manual assessment of fusion The explanted spines were immediately palpated to have continuity of fusion mass between the transverse processes. Then they were manually assessed by flexion and extension at the fusion level for intersegmental motion by two different observers and compared with adjacent segments of the vertebral column. The test was conducted with care not to cause any mechanical damage in the fusion mass. The absence of intersegmental motion was assessed as ‘‘solid’’ and if any motion was detected at either side, the spine was assessed as ‘‘not solid.’’ Biomechanical testing All the harvested PLF masses were evaluated by a threepoint bending test using a Universal Tensile Machine (Instron 5567, Instron, MA, USA). Both ends of vertebral bodies were placed with their dorsal sides down on two fulcra. The upper anvil (10.0-mm diameter) was placed in a position to apply a load on the ventral surface of the intervertebral disc perpendicular to the longitudinal spine. Three-point bending tests were performed with a 10.0-mm intersupport distance and a 50-mm/min crosshead speed until the upper anvil was advanced to 15 mm (Fig. 4, Left). The loaddisplacement curves were obtained for the autograft-only group and the mesh container group (N58), and the results were statistically compared.
Microcomputed tomography All the harvested spines were scanned with a highresolution microcomputed tomography (micro-CT; Inveon multimodal CT/positron emission tomography, Simens, Knoxville, TN, USA) to measure the calcified fusion mass at the region of PLF. The scan was performed in the long axis of the spine with an energy of 50 kVp and a current of 100 mA, and a 200-ms exposure time producing a resolution of 100-mm3 voxel size. The images were reconstructed using a Feldkamp cone-beam reconstruction algorithm [15]. The size of the reconstructed image was 1,0241,024 pixels and 700 slices were acquired. The final reconstructed data were converted into the Digital Imaging and Communication in Medicine format. Scout, sagittal, and cross-sectional views were examined using a Digital Imaging and Communication in Medicine viewer to investigate the integrity of bone union. We created three-dimesional image reconstructions to measure the total volume of fusion mass in both sides for each specimen. The quality of fusion and the degree of incorporation to transverse processes were evaluated by measuring the density of fusion mass (% of volume of bone mass/total volume of fusion mass). Integrity of fusion mass was classified into four grades using the method of a modified Lenke’s classification based on the micro-CT findings: grade A: definitely solid fusion with bilateral stout fusion masses present; grade B: possibly solid with asymmetric fusion masses; grade C: probably not solid with a small fusion mass bilaterally; and grade D:
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definitely not solid with bone graft resorption or obvious bilateral pseudarthrosis [16]. Histological examination The decalcified specimens not used for biomechanical test were used for histological examinations. The specimens were fixed in 10% formalin in a neutral buffer solution. The specimens were decalcified by immersion in a citric-buffered formic acid solution and dehydrated in a graded ethanol series. The specimens were then embedded in paraffin and 4-mm-thick sections were stained with Masson’s trichrome and hematoxylin and eosin. The former stain produces high-contrast images with red bone, blue collagen, pink cell cytoplasm, and black cell nuclei. Tiled images were captured with a digital camera system (ProRes C5, Jenoptik, Jena, Germany) mounted on a Leica microscope (Leica DM 2500, Wetzlar, Germany). Combined images were then reconstructed for each slide with an image analyzer (IMT i-Solution Inc., Burnaby, BC, Canada). Statistical analysis The results were analyzed statistically using statistical software (SPSS 13, SPSS, Chicago, IL, USA). Fusion rates as determined by manual palpation were compared using a chi-squared test. A Student t test was performed to analyze the volume of newly formed bone and the results of biomechanical testing. Mann-Whitney U testing was used to evaluate the radiographic fusion status. A value of p!.05 was set to be statistically significant.
Results Postoperative recovery All rats tolerated the surgical procedure well and postoperative recovery was uneventful with no perioperative mortality. They were ambulatory during the entire postoperative
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days. Weight increased after the first postoperative week. Although two rats of the autograft-only group had superficial wound infections, their spines were also assessed because they were fused by manual palpation and study of micro-CT. There was no case of wound infection in the mesh container group. No single case of container-related complication was observed. Gross inspection and manual assessment of fusion At 8 weeks, manual palpation revealed that overall 80% (16/20) of the explanted spines had solid fusion. Four rats of ‘‘not-solid’’ fusion, all of which belonged to the autograft-only group, showed less volume of bony mass on gross inspection and intersegmental motion on manual palpation. In all rats with solid fusion, a continuous bony mass was palpated and no intersegmental motion was noted between the intertransverse processes. The autograft-only group had a solid fusion rate of 60% (6/10), whereas the mesh container group showed significantly higher, complete fusion for all cases (100%, 10/10, p!.01). There was no extended bony growth to invade paravertebral muscle dorsally and psoas muscle ventrally. There was no interobserver difference on the results of gross inspection and manual palpation. Micro-CT findings The mesh container group showed more extensive new bone formation in the fusion mass (Fig. 2). The fusion mass of the mesh container group was fused firmly with adjacent transverse processes, and no crack was observed inside the fusion mass. On the other hand, even though the autograftonly group also showed new bone formation, clefts between the L4 and L5 transverse processes were observed in the subjects showing ‘‘not solid’’ fusion. The fusion mass from the autograft-only group consistently showed a heterogonous fusion mass with not yet remodeled pieces of bone chips. Although unremodeled bone chips were also noted
Fig. 2. Microcomputed tomographic images after 8 weeks of posterolateral fusion. The three-dimensional microcomputed tomography images revealed that the volume of fusion mass was larger in the mesh bone container group (Right) than in the autograft-only group (Left).
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in the mesh container group, cortical rims were more welldefined. The analysis of the micro-CT images revealed that the volume of fusion mass was significantly larger in the mesh container group than in the autograft-only group (137.1626.7 mL vs. 87.0625.6 mL, p!.05, Fig. 3, Left). The bone density of the mesh container group was also higher than that of the autograft-only group (10.662.5 vs. 6.762.3, p!.05, Fig. 3, Right). The difference of posterolateral fusion success according to Lenke’s classification is summarized in the Table. All rats in the mesh container group achieved grade A, definitely solid fusion. In comparison, the autograft-only group showed grade A, solid fusion in four rats (40%); grade B, possibly solid fusion in three (30%); and grade C, not solid fusion in three (30%) (Table). There was a significant statistical difference in fusion success between the two groups (p!.05). Biomechanical testing Figure 4, Right shows the load-displacement curves of the vertebral bodies for the measurement of biomechanical properties. There were no differences in the initial slopes between the tubular mesh container group and the autograft-only group. However, when the displacement was increased to more than 10 mm, the two groups showed statistically significant differences (p!.05); the autograftonly group started to show a nonlinear, yielding behavior whereas the mesh container group continued showing a linear increase in the load with increasing displacement. And, the differences became larger with further increasing the displacement. Because of the geometries of the sample (round) and the crosshead (inverted triangle), the full contact between the sample and the moving crosshead was achieved only after 10 mm of displacement (Fig. 4, Left). So, until the displacement reaches 10 mm, the contact area kept increasing, and the increase in the load seemed to be dominated by the increase in the contact area, which probably resulted in the
initially similar responses between the two groups and the statistically significant different response with the displacement larger than 10 mm. After full contact, the autograftonly group showed a nonlinear increment pattern in the load, possibly because of the fracture of the vertebral bodies, whereas the mesh container group showed a linear increase with increasing displacement. Therefore, the use of a tubular mesh container clearly enhanced the mechanical properties of the vertebral bodies (Fig. 4, Right). The geometry of the experimental setup also limited the maximum displacement up to 15 mm; otherwise, the crosshead would collide with the support. The ultimate strengths of the vertebral bodies therefore could not be obtained because of this limitation in the displacement. Histological examination Histological findings of the fusion mass were consistent with the micro-CT images (Fig. 5). In the autograft-only group, areas of unremodeled cortical autograft bone were widely observed and were encased in cartilage and osteoid. Although a bony bridge was formed, the cortical rim was thin. The defect area was replaced with small amounts of fibrous connective tissues. In the fusion mass, bone growth was more prominent over the transverse processes than was seen centrally in the intertransverse bed. The center of the fusion mass had more cartilage present than did the areas over the transverse processes, where more advanced remodeling with bone marrow was observed. In the autograft-only group, fibrous and chondrified bones were observed in the graft bones and the fusion mass was put in contact with the small surface of the transverse process because of the interruption of soft tissues. In the mesh container group, mature and circuitous bone trabecula structures and a clear medullary cavity were observed. The defected area mostly disappeared and was replaced with fibrous and Masson-Trichrome–positive collagen tissue. The fusion mass was put in contact with the transverse process in a wider area in
Fig. 3. Microcomputed tomography analysis on the volume (Left) and the density (Right) of newly formed bone after 8 weeks of posterolateral fusion. The significantly larger volume and the higher density of fusion mass were observed in the mesh bone container group than the autograft-only group.
D.-A. Shin et al. / The Spine Journal 14 (2014) 408–415 Table Radiographic fusion status assessed by Lenke classification of posterolateral fusion success Grade
Description
Bone pocket group
Autograft-only group
A B C D
Definitely solid Possibly solid Probably not solid Definitely not solid
10 0 0 0
4 3 3 0
the mesh container group than the autograft-only group. The deposition of collagen and the formation of new bone were markedly increased in the bone. Results of a histological analysis of the fusion masses of the mesh container group demonstrated abundant osteoid deposition throughout the fusion mass as well as bone marrow formation. These fusion masses consistently appeared larger than those of the autograft-only group. The mesh container group showed a thicker cortical rim, less cartilage, and more marrow formation than the autograft-only group. PDO suture fibers were half-absorbed, leaving the amorphous vacuoles along the contour of the tubular mesh containers for 8 weeks. There was no acute inflammatory response around the PDO filaments.
Discussion The rat model of posterolateral spinal fusion has been reported to show about 40% fusion rate with an autogenous bone or demineralized bone matrix graft after 6 postoperative weeks [17–19]. Recently, tissue-engineered alternatives to autogenous bone grafts that contain osteoinductive and/ or osteoconductive material were proven to be more effective in enhancing spinal fusion [1,19–23]. In particular, the use of bone morphogenic proteins provided the positive prospect of a higher fusion rate than the conventional method and the possibility to eliminate the complications resulting from iliac crest grafting. However, in spite of their
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remarkable successes, adoption of new techniques is tempered by the high cost of tissue engineering and restriction of accessibility. Further, reported complications warn against the wide use of these alternative graft substitutes [24–26]. Therefore, the device that can increase the successful union rate of autogenous bone graft, using either the usual amount of graft or a smaller amount of bone graft, is still the preferred option for clinical application to eliminate of concerns of graft-induced complications [1]. Therefore, a biodegradable mesh container applied in this study as a ‘‘bone graft enhancer’’ can be a promising tool for promoting spinal fusion. In this study, we achieved a significantly enhanced bone fusion by using a biodegradable tubular mesh container to contain bone graft instead of the direct use of autologous bone graft. Although new bone formation was observed in both groups, significantly larger volume and higher density of new bone were found in the mesh container group compared with the autograft-only group. Furthermore, solid fusion was achieved in all cases of the mesh container group, whereas the autograft-only group showed 60% of solid fusion. Therefore, the spinal fusion was further strengthened and the quality of osteogenesis was improved by the use of a mesh container. These results are quite encouraging because the use of mesh container might reduce the chance of pseudoarthrosis or nonunion, which is reported to occur in 5% to 45% of conventional spinal fusion procedures [27,28]. The success of posterolateral spinal fusion depends on many systemic and local factors, and especially vascularity is one of the critical factors. It is known that the primary blood supply to the fusion mass comes from the decorticated transverse processes [29,30]. The failure to achieve spinal fusion in the absence of extensive decortications also emphasizes the importance of close contacts between the graft bones and transverse processes. In addition, close contact between graft bones is also mandatory to favor chondrocyte formation, which is critical for ensuing
Fig. 4. Biomechanical assessment of fusion mass at 8 weeks of posterolateral fusion (PLF) using a three-point bending test. (Left) Experimental setup of three-point bending measurement. Arrow indicates the direction of force against the fusion mass. (Right) The mesh bone container group showed the higher stiffness than the control group, especially at displacement larger than 10 mm when the full contact between the sample and the crosshead occurred.
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Fig. 5. Histological staining of fusion mass after 8 weeks of posterolateral fusion. (A) Hematoxylin and eosin (H&E) staining showing the mesh bone container containing small pieces of iliac bones (arrowheads) (50). (B) H&E staining showing no acute inflammatory reaction around the PDO fibers (100). (C) Masson-Trichrome staining showing the fusion mass in the autograft-only group. Fibrous and chondrified bones are observed, and fibrous connective tissues fill the space between bones (50). (D) Masson-Trichrome staining showing a mature and circuitous bone trabecula structure and a clear medullary cavity in mesh bone container group. The space between bones is replaced with fibrous and Masson-Trichrome–positive collagen tissue. The collagen tissue extends across the mesh container (arrows) (50). IVD, intervertebral disc; SC, spinal canal; TP, transverse process.
enchondral bone formation [31] in general. The larger the contact area exposed, the greater the availability of potential osteogenic cells and bony bridges to facilitate bone fusion. However, the conventional method of PLF is to place the grafts in the intertransverse space piece by piece; consequently, it does not guarantee the close and wide contacts between the transverse process and graft pieces. In this experiment, histological examination demonstrated that the fusion mass was put in contact with the transverse process in a wider area in the mesh container group because of easy adaptation to the irregular surfaces of the recipient bones and the strong tensile strength of the tubular mesh container to hold the bone pieces together. However, a mesh container is not likely to either provide a stout fixation force or limit the spinal motions as with the screw fixation system. Nonetheless, manual and mechanical testing revealed that the use of the mesh container could enhance bone fusion more strongly than the direct use of autograft bone only. Fabrication of the tubular mesh container was very simple compared wiht other similar containers that usually cost and demand high technology. We used a biodegradable polymer (PDO) suture, approved by the US Food and Drug Administration. Bioabsorbable PDO has been used in repairing the organs, joints, and tissues throughout the body with low immunological response [32–35]. This suture is known to be degraded over 4 to 6 months
with a strength retention up to 6 weeks. Even though this period of strength retention is enough to stabilize the fusion in rat model, it may not be adequate to apply PDO for human spinal fusion because the fusion takes longer than 6 months in humans. For application in humans, materials such as polylactide, which have the strength retention time of longer than 6 months, may be considered alternatively. No inflammatory infiltrations were observed in histological examination around the PDO fibers after 8 weeks of fusion surgery in this study. Previously, it was postulated that a rigid container has advantages for bone fusion because it can prevent muscle interposition and counteract muscle compression [14]. In our experiment, interposition of soft tissue was not observed inside the tubular mesh container. A flexible mesh container seems to be strong enough to prevent soft-tissue interposition and to counteract the compressive force of surrounding muscles in supplying the blood flow to the core of the fusion mass. Conclusion The results of this study support the benefit of a tubular mesh container in spinal fusion. Bone fusion using a biodegradable mesh container was qualitatively and quantitatively significantly better than the autograft-only group. The bone container made of a biodegradable
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PDO-facilitated bone fusion without specific morbidity in a rat model of posterolateral spinal fusion.
References [1] Boden SD. Overview of the biology of lumbar spine fusion and principles for selecting a bone graft substitute. Spine 2002;27:S26–31. [2] Sandhu HS. Anterior lumbar interbody fusion with osteoinductive growth factors. Clin Orthop Relat Res 2000;371:56–60. [3] Andersson GB. Epidemiology of low back pain. Acta Orthop Scand Suppl 1998;281:28–31. [4] Axelsson P, Johnsson R, Stromqvist B, et al. Posterolateral lumbar fusion. Outcome of 71 consecutive operations after 4 (2-7) years. Acta Orthop Scand 1994;65:309–14. [5] Lee CS, Hwang CJ, Lee DH, et al. Fusion rates of instrumented lumbar spinal arthrodesis according to surgical approach: a systematic review of randomized trials. Clin Orthop Surg 2011;3:39–47. [6] McAfee PC, Farey ID, Sutterlin CE, et al. The effect of spinal implant rigidity on vertebral bone density. A canine model. Spine 1991;16:S190–7. [7] Nagel DA, Kramers PC, Rahn BA, et al. A paradigm of delayed union and nonunion in the lumbosacral joint. A study of motion and bone grafting of the lumbosacral spine in sheep. Spine 1991;16:553–9. [8] Wang JC, Mummaneni PV, Haid RW. Current treatment strategies for the painful lumbar motion segment: posterolateral fusion versus interbody fusion. Spine 2005;30:S33–43. [9] Ahlmann E, Patzakis M, Roidis N, et al. Comparison of anterior and posterior iliac crest bone grafts in terms of harvest-site morbidity and functional outcomes. J Bone Joint Surg Am 2002;84-A:716–20. [10] Banwart JC, Asher MA, Hassanein RS. Iliac crest bone graft harvest donor site morbidity. A statistical evaluation. Spine 1995;20: 1055–60. [11] Turner JA, Ersek M, Herron L, et al. Patient outcomes after lumbar spinal fusions. JAMA 1992;268:907–11. [12] Boden SD. The biology of posterolateral lumbar spinal fusion. Orthop Clin North Am 1998;29:603–19. [13] Boden SD. Biology of lumbar spine fusion and use of bone graft substitutes: present, future, and next generation. Tissue Eng 2000;6: 383–99. [14] Poynton AR, Zheng F, Tomin E, et al. Resorbable posterolateral graft containment in a rabbit spinal fusion model. J Neurosurg 2002;97:460–3. [15] Feldkamp L, Davis L, Kress J. Practical cone-beam algorithm. J Opt Soc Am A 1984;1:612–9. [16] Lenke LG, Bridwell KH, Bullis D, et al. Results of in situ fusion for isthmic spondylolisthesis. J Spinal Disord 1992;5:433–42. [17] Grauer JN, Bomback DA, Lugo R, et al. Posterolateral lumbar fusions in athymic rats: characterization of a model. Spine J 2004;4:281–6. [18] Bomback DA, Grauer JN, Lugo R, et al. Comparison of posterolateral lumbar fusion rates of Grafton putty and OP-1 putty in an athymic rat model. Spine 2004;29:1612–7.
415
[19] Wang JC, Kanim LE, Yoo S, et al. Effect of regional gene therapy with bone morphogenetic protein-2-producing bone marrow cells on spinal fusion in rats. J Bone Joint Surg Am 2003;85-A:905–11. [20] Morisue H, Matsumoto M, Chiba K, et al. A novel hydroxyapatite fiber mesh as a carrier for recombinant human bone morphogenetic protein-2 enhances bone union in rat posterolateral fusion model. Spine 2006;31:1194–200. [21] Salamon ML, Althausen PL, Gupta MC, Laubach J. The effects of BMP-7 in a rat posterolateral intertransverse process fusion model. J Spinal Disord Tech 2003;16:90–5. [22] Biswas DM, Bible JEM, Whang PHM, et al. Augmented demineralized bone matrix: a potential alternative for posterolateral lumbar spinal fusion. Am J Orthop (Belle Mead NJ) 2010;39:531–8. [23] Tanaka K, Takemoto M, Fujibayashi S, et al. A bioactive and bioresorbable porous cubic composite scaffold loaded with bone marrow aspirate: a potential alternative to autogenous bone grafting. Spine 2011;36:441–8. [24] Shimer AL, Oner FC, Vaccaro AR. Spinal reconstruction and bone morphogenetic proteins: open questions. Injury 2009;40(3 Suppl): S32–8. [25] Benglis D, Wang MY, Levi AD. A comprehensive review of the safety profile of bone morphogenetic protein in spine surgery. Neurosurgery 2008;62:ONS423–31; discussion ONS431. [26] Deutsch H. High-dose bone morphogenetic protein-induced ectopic abdomen bone growth. Spine J 2010;10:e1–4. [27] Steinmann JC, Herkowitz HN. Pseudarthrosis of the spine. Clin Orthop Relat Res 1992;284:80–90. [28] Zdeblick TA. A prospective, randomized study of lumbar fusion. Preliminary results. Spine 1993;18:983–91. [29] Toribatake Y, Hutton WC, Tomita K, Boden SD. Vascularization of the fusion mass in a posterolateral intertransverse process fusion. Spine 1998;23:1149–54. [30] Boden SD, Schimandle JH, Hutton WC. An experimental lumbar intertransverse process spinal fusion model. Radiographic, histologic, and biomechanical healing characteristics. Spine 1995;20: 412–20. [31] Carter DR, Beaupre GS, Giori NJ, Helms JA. Mechanobiology of skeletal regeneration. Clin Orthop Relat Res 1998;355(Suppl): S41–55. [32] Ishaug-Riley SL, Okun LE, Prado G, et al. Human articular chondrocyte adhesion and proliferation on synthetic biodegradable polymer films. Biomaterials 1999;20:2245–56. [33] Sell SA, McClure MJ, Barnes CP, et al. Electrospun polydioxanoneelastin blends: potential for bioresorbable vascular grafts. Biomed Mater 2006;1:72–80. [34] Greiner S, Braunsdorf J, Perka C, et al. Mid to long-term results of open acromioclavicular-joint reconstruction using polydioxansulfate cerclage augmentation. Arch Orthop Trauma Surg 2009;129: 735–40. [35] Zeplin PH, Zahn RK, Meffert RH, Schmidt K. Biomechanical evaluation of flexor tendon repair using barbed suture material: a comparative ex vivo study. J Hand Surg Am 2011;36:446–9.
