ORIGINAL ARTICLE
Comparison of Silkworm-Cocoon–Derived Silk Membranes of Two Different Thicknesses for Guided Bone Regeneration Hyun Seok, DDS,* Min Keun Kim, DDS,* Seong-Gon Kim, DDS, PhD,* and HaeYong Kweon, PhD† Abstract: The objective of this study was to compare the effectiveness of silk membranes (SMs) of different thicknesses for guided bone regeneration. Two kinds of SMs were prepared (SM1: 0.01 mm thickness, SM2: 0.5 mm thickness). Before use in animal experiments, scanning electron microscope images were taken to examine the gross morphology of each membrane. Ten New Zealand white rabbits were used for this study. Bilateral round-shaped defects were created in the parietal bone (diameter: 8.0 mm) and each defect was covered with SM1 or SM2. Animals were killed at 4 weeks and 8 weeks. Bone regeneration was analyzed in each specimen by micro-computed tomography (μ-CT) and histological analysis. In the μ-CT analysis, the average amount of newly formed bone in the SM2 group was greater than that in the SM1 group. There was a significant difference at 4 weeks after surgery (P = 0.004). In the histological analysis, the amount of formed lamellar bone was much greater in the SM2 group than in the SM1 group at 8 weeks after surgery (P = 0.021). In conclusion, the thick SM was much more effective for bone regeneration of bone defects than the thin SM. Key Words: Guided bone regeneration, silk membrane, rabbit calvarial defect, thickness (J Craniofac Surg 2014;25: 2066–2069)
T
he principle of guided bone regeneration (GBR) has been to promote bone regeneration in osseous defects using a membrane.1 The membrane acts as a barrier to allow the migration of osteoblasts into the osseous defect and to prevent the growth of epithelial cells.2 For successful GBR, the membrane should have the following properties: biocompatibility, biodegradation, space maintenance, ability to stabilize the bone graft material, tissue integration of cells derived from bone, cell occlusion for exclusion of soft tissue cells, and clinical manageability.3 Membranes can be From the *Department of Oral and Maxillofacial Surgery, College of Dentistry, Gangneung-Wonju National University, Gangneung; and †Sericultural and Apicultural Materials Division, National Academy of Agricultural Science, Suwon, Korea. Received March 3, 2014. Accepted for publication June 19, 2014. Address correspondence and reprint requests to Seong-Gon Kim, DDS, PhD, Department of Oral and Maxillofacial Surgery, College of Dentistry, Gangneung-Wonju National University, Gangneung, Gangwondo 210-702, Korea; E-mail: [email protected] This study was supported by a grant from the Next-Generation BioGreen21 Program (No. PJ009013), Rural Development Administration, Republic of Korea. The authors report no conflicts of interest. Copyright © 2014 by Mutaz B. Habal, MD ISSN: 1049-2275 DOI: 10.1097/SCS.0000000000001151
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classified as bioresorbable and non-resorbable. Collagen membranes are mainly derived from porcine collagen and are bioresorbable.4 They have good biocompatibility and biodegradability; however, they have disadvantages such as poor mechanical strength and insufficient space maintenance.5 Compared with bioresorbable membranes, non-resorbable membranes have better mechanical strength. Titanium-reinforced expanded polytetrafluoroethylene has excellent mechanical stability and space maintenance properties to prevent collapse of the osseous defect.6,7 A titanium mesh can be applied to large bone defects that require large bone grafts or vertical bone augmentation.8,9 However, non-resorbable membranes have the risk of membrane exposure during the healing period, which would lead to failure of bone grafting and unsuccessful implantation.10 Silk is a natural macromolecule produced by the silkworm Bombyx mori. It is composed of fibroin and sericin.11 Sericin, a glue-like material, has been reported to trigger inflammatory and immune reactions in the human body.12 However, several studies have shown that sericin has a great advantage in wound healing by enhancing fibroblast growth and collagen production.13 Silk fibroin (SF) has good biocompatibility, oxygen and water permeability, and slow biodegradability.11 Because of these properties, SF has been used in many biomedical materials, for example, as scaffold for bone regeneration, drug delivery material, artificial skin, or vessel.14–16 SF films are used as artificial tympanic membrane.17 Silk is an inexpensive material compared to other sources for the production of GBR membranes. Therefore, efforts have been made to apply SF films as GBR membranes.18 However, SF films are less flexible and they are fragile in the absence of moisture and easily break away.19 A drawback of SF films for application in GBR is their poor mechanical properties. Silk from the cocoons of silkworms has excellent mechanical properties (ie, strength, toughness, and stability).20 Membranes of different thicknesses can be easily obtained from the silk cocoon by peeling.21 The layers that are mechanically separated from the silk cocoon could be considered for GBR membranes. As silk cocoons consist of multiple layers, each layer could be considered as silk membrane (SM). In this study, we prepared SMs of 0.01 mm and 0.5 mm thickness. For the evaluation of the gross morphology of SMs, we analyzed each SM using a scanning electron microscope (SEM). The objective of this study was to compare the effectiveness of SMs of different thicknesses for GBR in a rabbit calvarial defect model. Micro-computed tomography (μ-CT) and histomorphometric analysis were used for the evaluation of new bone formation.
MATERIALS AND METHODS SEM Imaging SEM images of SMs of 0.01 mm and 0.5 mm thickness were taken with an electron microscope (Hitachi, SU-70, Japan).
The Journal of Craniofacial Surgery • Volume 25, Number 6, November 2014
Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
The Journal of Craniofacial Surgery • Volume 25, Number 6, November 2014
SMs for Guided Bone Regeneration
Animals and Surgical Procedure
Histomorphometric Evaluation
Ten 10-week-old New Zealand white rabbits with an average weight of 2.3 kg (range 2.0–2.5 kg) were used in this study. This study was approved by the Institutional Animal Care and Use Committee of the Gangneung-Wonju National University, Gangneung, Korea (IACUC GWNU-2013-14). General anesthesia was administered by intramuscular injection of 0.5 mL of a combination of tiletamine and zolazepam (125 mg/mL; Zoletil; Bayer, Seoul, Korea) and 0.5 mL xylazine hydrochloride (10 mg/kg body weight; Rompun; Bayer). The cranial area was shaved and disinfected with povidone-iodine; then, 2% lidocaine with epinephrine (1:100,000) was applied to the cranial area. A longitudinal incision was made from the nasal bone to the occipital protuberance of the skull. Then, a midline incision was made through the periosteum. Sharp subperiosteal dissection reflected the pericranium from the outer table of the cranial vault, exposing the parietal bones. A trephine bur was used under saline irrigation to create a bilateral calvarial defect in parietal bones. Two 8-mm-diameter defects were created, one on each side of the midline. Then, right parietal bone defects were covered with SMs of 0.01 mm thickness (SM1 group) and left parietal bone defects were covered with SMs of 0.5 mm thickness (SM2 group) (Fig. 1). None of the animals received the same graft for both calvarial defects. After treatment, pericranium and skin were closed in layers with 3-0 black silk (AILEE, Busan, Korea). After surgery, the rabbits received gentamycin at 1 mg/kg (Kookje, Seoul, Korea) and pyrin at 0.5 mL/kg (Green Cross Veterinary Products, Seoul, Korea) intramuscularly 3 times daily for 3 days. Each rabbit was individually caged and received food and water. Five animals were killed at 4 weeks and 8 weeks, respectively. Specimens were separated and fixed in 10% formalin. After μ-CT analysis, histological analysis was performed.
At 4 and 8 weeks after surgery, the calvarial samples were harvested, decalcified in 5% nitric acid for 2 weeks, and dehydrated in ethyl alcohol and xylene. After separation of the parietal bones through the midline sagittal suture, they were embedded in paraffin blocks. The paraffin blocks were sliced into sections that were then stained with Masson trichrome. The section that showed the largest defect area and sections 50 μm proximal and distal to that one were selected. Digital images of the selected sections were taken with a digital camera (DP-73; Olympus, Tokyo, Japan). The images were analyzed by Sigma Scan pro (SPSS, Chicago, IL, USA). The total amount of newly formed bone was calculated as percentage of the total region of the defect.
Micro-CT Analysis
Micro-CT Analysis
The prepared specimens were analyzed by μ-CT using an animal PET/CT/SPECT system (Inveon; Siemens, Erlangen, Germany) at the Ochang Center in Korea Basic Science Institute. The μ-CT scanner was set to 80 kVp voltage for the x-ray tube, 500 μA current for the x-ray source, and 210 ms exposure time. The detector and x-ray source were rotated through 360 degrees in 360 steps. The number of calibration exposures was 30. System magnification was set to produce an axial field of view (FOV) of 30.74 mm and a transaxial FOV of 30.74 mm. The scanned images were reconstructed using the Inveon Research Workplace software (Siemens). Gross profiles of the specimens were obtained from reconstructed 3-dimensional images. Because the initial defect was round in shape with an 8.0-mm diameter, the setting of the region of interest (ROI) was considered to reflect the initial defect size and shape. The ROI of each specimen was analyzed for bone volume (BV).
FIGURE 1. Silk membranes of 0.01 mm thickness were grafted into right parietal bone defects; left parietal bone defects were covered with silk membranes of 0.5 mm thickness.
Statistical Analysis As the same animal received 2 different types of membrane, the paired t test was used for comparison of the groups. The level of statistical significance was set at P less than 0.05.
RESULTS SEM Results SEM images of SMs of 0.01 mm and 0.5 mm thickness are shown in Figure 2. The assembly of silk fibers was similar in both types of SM. That had a cross-linked structure and each silk fiber was attached by a glue-like substance. However, fibers of 0.01-mm-thick SMs were more loosely connected and thinner than those of 0.5-mm-thick SMs (Fig. 2A). Fibers of 0.5-mm-thick SMs had a denser structure and formed a thicker layer than those of 0.01-mm-thick SMs (Fig. 2B).
Results of the μ-CT analysis are presented in Table 1. The BV was 3.24 ± 2.05 mm3 in the SM1 group and 7.08 ± 3.72 mm3 in the SM2 group at 4 weeks after surgery (Figs. 3A, B). There was a significant difference between both groups (P = 0.004). The BVof the SM1 group was 12.53 ± 10.69 mm3 at 8 weeks after surgery, whereas that of the SM2 group was 20.13 ± 7.22 mm3 (Figs. 3C, D). The difference between both groups was not significant (P > 0.05).
FIGURE 2. Scanning microscopic images. A, Silk membrane of 0.01 mm thickness. B, Silk membrane of 0.5 mm thickness.
© 2014 Mutaz B. Habal, MD
Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
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The Journal of Craniofacial Surgery • Volume 25, Number 6, November 2014
Seok et al
TABLE 1. Micro-Computed Tomographic Analysis 4 wk Group Bone volume, mm3
SM1 3.24 ± 2.05
SM2 7.08 ± 3.72
TABLE 2. Histomorphometric Analysis 8 wk
SM1 12.53 ± 10.69
4 wk SM2 20.13 ± 7.22
SM1, silk membrane of 0.01 mm thickness; SM2, silk membrane of 0.5 mm thickness.
Histomorphometric Analysis The histomorphometric results are presented in Table 2. The total amount of newly formed bone was 11.06 ± 12.48% in the SM1 group and 8.83 ± 5.36% in the SM2 group at 4 weeks after surgery (Figs. 4A, B). The difference was not significant (P > 0.05). The total amount of newly formed bone was 18.98 ± 11.70% in the SM1 group and 44.26 ± 15.00% in the SM2 group at 8 weeks after surgery (Figs. 4C, D). The difference between both groups was significant (P = 0.021). Well-organized lamellar bone islands were formed in both groups at 8 weeks after surgery (Figs. 4E, F).
Group Total new bone, %
SM1 11.06 ± 12.48
8 wk SM2 8.83 ± 5.36
SM1 18.98 ± 11.70
SM2 44.26 ± 15.00
SM1, silk membrane of 0.01 mm thickness; SM2, silk membrane of 0.5 mm thickness.
still maintain their own tensile properties.21 In a previous study, an effort was made to use a SF film as GBR membrane.18 However, the SF film was fragile and easily dissolved in aqueous solution. Application and fixation of the SF film to the bone defect was nearly impossible. Compared to the SF film, mechanically separated silk
DISCUSSION The principle of GBR is to promote the migration of desired cells into the bone defect using a barrier membrane.22 An ideal GBR membrane should have the following properties: biocompatibility, biodegradability, and sufficient mechanical stability to maintain the space required for bone regeneration.10 To achieve bone regeneration of large bone defects, the mechanical stability of the membrane is very important.8,23,24 The membrane should maintain the intended form of the alveolar bone; furthermore, it should protect the bone graft material and blood clot, which should not be broken down and washed out.8,25 In this study, greater bone regeneration was achieved with the thicker SM. In the μ-CT analysis, the average amount of newly formed bone in the SM2 group was greater than that in the SM1 group at 4 weeks after surgery (P = 0.004, Figs. 3A, B). In the histological analysis, the amount of formed lamellar bone was much greater in the SM2 group than in the SM1 group at 8 weeks after surgery (P = 0.021, Figs. 4C, D). To the best of our knowledge, this is the first report in which bone regeneration is compared on the basis of the thickness of the SM. Silk cocoons can be easily separated into layers of different thickness by mechanical peeling. The obtained separated silk layers
FIGURE 3. Micro-computed tomography. A, Silk membrane of 0.01 mm thickness at 4 weeks after surgery. B, Silk membrane of 0.5 mm thickness at 4 weeks after surgery. C, Silk membrane of 0.01 mm thickness at 8 weeks after surgery. D, Silk membrane of 0.5 mm thickness at 8 weeks after surgery.
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FIGURE 4. Histological section (Masson trichrome stain). A, Silk membrane of 0.01 mm thickness at 4 weeks after surgery (original magnification 10). B, Silk membrane of 0.5 mm thickness at 4 weeks after surgery (original magnification 10). C, Silk membrane of 0.01 mm thickness at 8 weeks after surgery (original magnification 10). D, Silk membrane of 0.5 mm thickness at 8 weeks after surgery (original magnification 5). E, F, Well-organized lamellar bone in (C, D original magnification 100).
© 2014 Mutaz B. Habal, MD
Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
The Journal of Craniofacial Surgery • Volume 25, Number 6, November 2014
sheets have sufficient strength under both dry and wet conditions.26 Its yield strength under wet conditions is even higher than that of commercially available collagen membranes.26 We used SMs of 2 different thicknesses to compare the effectiveness for bone regeneration. Results of the μ-CT analysis showed that the BV in the SM2 group was greater than that in the SM1 group at 4 weeks after surgery (Figs. 3A, B; P = 0.004). In the histomorphometric analysis, the total amount of newly formed bone in the SM2 group was greater than that in the SM1 group at 8 weeks after surgery (Figs. 4C, D; P = 0.021). In the histological assessment of the SM2 group, the SMs maintained their form, provided support to maintain the space, and prevented infiltration of soft tissue cells (Figs. 4B, D). On the other hand, the SMs in the SM1 group did not maintain their form and collapsed into the bone defects (Figs. 4A, C), thereby decreasing the amount of space available for new bone formation. For use as GBR membrane, the membrane should maintain its original form and be sufficiently stable to prevent collapse into the bone defect.8,25 In this respect, SMs of 0.5 mm thickness functioned properly as GBR membrane because they maintained their form and provided sufficient space for bone growth compared to SMs of 0.01 mm thickness. As seen in the SEM image, SM2 had a denser structure compared to SM1 (Figs. 2A, B), which might provide a more conducive environment for osteoblast differentiation and prevent infiltration of soft tissue cells. SF has osteogenic cell attachment and differentiation properties.27,28 In a previous study, SF was shown to suppress the notch signaling pathway and to induce the differentiation of osteoblasts.29 As shown in the SEM image, SM2 had more silk fibers that were densely arranged (Fig. 2). Each fiber consists of fibroin and sericin.20 Though the total amount of SF in each SM cannot be accurately calculated, the thicker membrane is thought to contain more SF. In conclusion, covering the bone defect with a thick SM resulted in greater bone regeneration than covering it with a thin SM. The mechanical properties and dense fiber structure of SM2 are thought to contribute to greater bone regeneration. However, silk is produced by insects and, thus, a foreign protein. Considering the immune response to foreign proteins, SMs of unlimited thickness might not always be suitable for bone regeneration. The optimal thickness of SMs for clinical application should be determined in future studies.
REFERENCES 1. Hämmerle CH, Lang NP. Single stage surgery combining transmucosal implant placement with guided bone regeneration and bioresorbable materials. Clin Oral Implants Res 2001;12:9–18 2. Hämmerle CH, Jung RE, Feloutzis A. A systematic review of the survival of implants in bone sites augmented with barrier membranes (guided bone regeneration) in partially edentulous patients. J Clin Periodontol 2002;29:226–231 3. McAllister BS, Haghighat K. Bone augmentation techniques. J Periodontol 2007;78:377–396 4. Rothamel D, Schwarz F, Sculean A, et al. Biocompatibility of various collagen membranes in cultures of human PDL fibroblasts and human osteoblast‐like cells. Clin Oral Implants Res 2004;15:443–449 5. Lundgren D, Sennerby L, Falk H, et al. The use of a new bioresorbable barrier for guided bone regeneration in connection with implant installation. Case reports. Clin Oral Implants Res 1994;5:177–184 6. Fontana F, Santoro F, Maiorana C, et al. Clinical and histologic evaluation of allogeneic bone matrix versus autogenous bone chips associated with titanium-reinforced e-PTFE membrane for vertical ridge augmentation: a prospective pilot study. Int J Oral Maxillofac Implants 2007;23:1003–1012 7. Cornelini R, Cangini F, Covani U, et al. Simultaneous implant placement and vertical ridge augmentation with a titanium-reinforced membrane: a case report. Int J Oral Maxillofac Implants 2000;15:883
SMs for Guided Bone Regeneration
8. Roccuzzo M, Ramieri G, Spada MC, et al. Vertical alveolar ridge augmentation by means of a titanium mesh and autogenous bone grafts. Clin Oral Implants Res 2004;15:73–81 9. Louis PJ, Gutta R, Said-Al-Naief N, et al. Reconstruction of the maxilla and mandible with particulate bone graft and titanium mesh for implant placement. J Oral Maxillofac Surg 2008;66:235–245 10. Dimitriou R, Mataliotakis GI, Calori GM, et al. The role of barrier membranes for guided bone regeneration and restoration of large bone defects: current experimental and clinical evidence. BMC Med 2012;10:81 11. Cao Y, Wang B. Biodegradation of silk biomaterials. Int J Mol Sci 2009;10:1514–1524 12. Dewair M, Baur X, Ziegler K. Use of immunoblot technique for detection of human IgE and IgG antibodies to individual silk proteins. J Allergy Clin Immunol 1985;76:537–542 13. Aramwit P, Kanokpanont S, De-Eknamkul W, et al. Monitoring of inflammatory mediators induced by silk sericin. J Biosci Bioeng 2009;107:556–561 14. Altman GH, Diaz F, Jakuba C, et al. Silk-based biomaterials. Biomaterials 2003;24:401–416 15. Sofia S, McCarthy MB, Gronowicz G, et al. Functionalized silk‐based biomaterials for bone formation. J Biomed Mater Res 2001;54:139–148 16. Kweon H, Yeo JH, Lee KG, et al. Semi-interpenetrating polymer networks composed of silk fibroin and poly (ethylene glycol) for wound dressing. Biomed Mater 2008;3:034115 17. Kim J, Kim CH, Park CH, et al. Comparison of methods for the repair of acute tympanic membrane perforations: silk patch vs. paper patch. Wound Rep Reg 2010;18:132–138 18. Song JY, Kim SG, Lee JW, et al. Accelerated healing with the use of a silk fibroin membrane for the guided bone regeneration technique. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2011;112:e26–e33 19. Lee SW, Park YT, Kim SG, et al. The effects of tetracycline-loaded silk fibroin membrane on guided bone regeneration in a rabbit calvarial defect model. J Korean Assoc Maxillofac Plast Reconstr Surg 2012;34:293–298 20. Vepari C, Kaplan DL. Silk as a biomaterial. Prog Polym Sci 2007;32:991–1007 21. Zhao HP, Feng XQ, Yu SW, et al. Mechanical properties of silkworm cocoons. Polymer 2005;46:9192–9201 22. Retzepi M, Donos N. Guided bone regeneration: biological principle and therapeutic applications. Clin Oral Implants Res 2010;21:567–576 23. Maiorana C, Santoro F, Rabagliati M, et al. Evaluation of the use of iliac cancellous bone and anorganic bovine bone in the reconstruction of the atrophic maxilla with titanium mesh: a clinical and histologic investigation. Int J Oral Maxillofac Implants 2000;16:427–432 24. Merli M, Migani M, Esposito M. Vertical ridge augmentation with autogenous bone grafts: resorbable barriers supported by ostheosynthesis plates versus titanium-reinforced barriers. A preliminary report of a blinded, randomized controlled clinical trial. Int J Oral Maxillofac Implants 2007;22:373 25. Kostopoulos L, Karring T. Augmentation of the rat mandible using guided tissue regeneration. Clin Oral Implants Res 1994;5:75–82 26. Lee JM, Kim SW, Joo YY, et al. The physical property and composition analysis of the detached silk cocoon from Bombyx mori. In: Proceedings of the conference of the Korean Society of Sericultural Science. 2014: 45 27. Wongputtaraksa T, Ratanavaraporn J, Pichyangkura R, et al. Surface modification of Thai silk fibroin scaffolds with gelatin and chitooligosaccharide for enhanced osteogenic differentiation of bone marrow‐derived mesenchymal stem cells. J Biomed Mater Res B Appl Biomater 2012;100:2307–2315 28. Tien LW, Gil ES, Park SH, et al. Patterned silk film scaffolds for aligned lamellar bone tissue engineering. Macromol Biosci 2012;12:1671–1679 29. Jung SR, Song NJ, Yang DK, et al. Silk proteins stimulate osteoblast differentiation by suppressing the Notch signaling pathway in mesenchymal stem cells. Nutr Res 2012;33:162–170
© 2014 Mutaz B. Habal, MD
Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
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Comparison of Silkworm-Cocoon–Derived Silk Membranes of Two Different Thicknesses for Guided Bone Regeneration Hyun Seok, DDS,* Min Keun Kim, DDS,* Seong-Gon Kim, DDS, PhD,* and HaeYong Kweon, PhD† Abstract: The objective of this study was to compare the effectiveness of silk membranes (SMs) of different thicknesses for guided bone regeneration. Two kinds of SMs were prepared (SM1: 0.01 mm thickness, SM2: 0.5 mm thickness). Before use in animal experiments, scanning electron microscope images were taken to examine the gross morphology of each membrane. Ten New Zealand white rabbits were used for this study. Bilateral round-shaped defects were created in the parietal bone (diameter: 8.0 mm) and each defect was covered with SM1 or SM2. Animals were killed at 4 weeks and 8 weeks. Bone regeneration was analyzed in each specimen by micro-computed tomography (μ-CT) and histological analysis. In the μ-CT analysis, the average amount of newly formed bone in the SM2 group was greater than that in the SM1 group. There was a significant difference at 4 weeks after surgery (P = 0.004). In the histological analysis, the amount of formed lamellar bone was much greater in the SM2 group than in the SM1 group at 8 weeks after surgery (P = 0.021). In conclusion, the thick SM was much more effective for bone regeneration of bone defects than the thin SM. Key Words: Guided bone regeneration, silk membrane, rabbit calvarial defect, thickness (J Craniofac Surg 2014;25: 2066–2069)
T
he principle of guided bone regeneration (GBR) has been to promote bone regeneration in osseous defects using a membrane.1 The membrane acts as a barrier to allow the migration of osteoblasts into the osseous defect and to prevent the growth of epithelial cells.2 For successful GBR, the membrane should have the following properties: biocompatibility, biodegradation, space maintenance, ability to stabilize the bone graft material, tissue integration of cells derived from bone, cell occlusion for exclusion of soft tissue cells, and clinical manageability.3 Membranes can be From the *Department of Oral and Maxillofacial Surgery, College of Dentistry, Gangneung-Wonju National University, Gangneung; and †Sericultural and Apicultural Materials Division, National Academy of Agricultural Science, Suwon, Korea. Received March 3, 2014. Accepted for publication June 19, 2014. Address correspondence and reprint requests to Seong-Gon Kim, DDS, PhD, Department of Oral and Maxillofacial Surgery, College of Dentistry, Gangneung-Wonju National University, Gangneung, Gangwondo 210-702, Korea; E-mail: [email protected] This study was supported by a grant from the Next-Generation BioGreen21 Program (No. PJ009013), Rural Development Administration, Republic of Korea. The authors report no conflicts of interest. Copyright © 2014 by Mutaz B. Habal, MD ISSN: 1049-2275 DOI: 10.1097/SCS.0000000000001151
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classified as bioresorbable and non-resorbable. Collagen membranes are mainly derived from porcine collagen and are bioresorbable.4 They have good biocompatibility and biodegradability; however, they have disadvantages such as poor mechanical strength and insufficient space maintenance.5 Compared with bioresorbable membranes, non-resorbable membranes have better mechanical strength. Titanium-reinforced expanded polytetrafluoroethylene has excellent mechanical stability and space maintenance properties to prevent collapse of the osseous defect.6,7 A titanium mesh can be applied to large bone defects that require large bone grafts or vertical bone augmentation.8,9 However, non-resorbable membranes have the risk of membrane exposure during the healing period, which would lead to failure of bone grafting and unsuccessful implantation.10 Silk is a natural macromolecule produced by the silkworm Bombyx mori. It is composed of fibroin and sericin.11 Sericin, a glue-like material, has been reported to trigger inflammatory and immune reactions in the human body.12 However, several studies have shown that sericin has a great advantage in wound healing by enhancing fibroblast growth and collagen production.13 Silk fibroin (SF) has good biocompatibility, oxygen and water permeability, and slow biodegradability.11 Because of these properties, SF has been used in many biomedical materials, for example, as scaffold for bone regeneration, drug delivery material, artificial skin, or vessel.14–16 SF films are used as artificial tympanic membrane.17 Silk is an inexpensive material compared to other sources for the production of GBR membranes. Therefore, efforts have been made to apply SF films as GBR membranes.18 However, SF films are less flexible and they are fragile in the absence of moisture and easily break away.19 A drawback of SF films for application in GBR is their poor mechanical properties. Silk from the cocoons of silkworms has excellent mechanical properties (ie, strength, toughness, and stability).20 Membranes of different thicknesses can be easily obtained from the silk cocoon by peeling.21 The layers that are mechanically separated from the silk cocoon could be considered for GBR membranes. As silk cocoons consist of multiple layers, each layer could be considered as silk membrane (SM). In this study, we prepared SMs of 0.01 mm and 0.5 mm thickness. For the evaluation of the gross morphology of SMs, we analyzed each SM using a scanning electron microscope (SEM). The objective of this study was to compare the effectiveness of SMs of different thicknesses for GBR in a rabbit calvarial defect model. Micro-computed tomography (μ-CT) and histomorphometric analysis were used for the evaluation of new bone formation.
MATERIALS AND METHODS SEM Imaging SEM images of SMs of 0.01 mm and 0.5 mm thickness were taken with an electron microscope (Hitachi, SU-70, Japan).
The Journal of Craniofacial Surgery • Volume 25, Number 6, November 2014
Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
The Journal of Craniofacial Surgery • Volume 25, Number 6, November 2014
SMs for Guided Bone Regeneration
Animals and Surgical Procedure
Histomorphometric Evaluation
Ten 10-week-old New Zealand white rabbits with an average weight of 2.3 kg (range 2.0–2.5 kg) were used in this study. This study was approved by the Institutional Animal Care and Use Committee of the Gangneung-Wonju National University, Gangneung, Korea (IACUC GWNU-2013-14). General anesthesia was administered by intramuscular injection of 0.5 mL of a combination of tiletamine and zolazepam (125 mg/mL; Zoletil; Bayer, Seoul, Korea) and 0.5 mL xylazine hydrochloride (10 mg/kg body weight; Rompun; Bayer). The cranial area was shaved and disinfected with povidone-iodine; then, 2% lidocaine with epinephrine (1:100,000) was applied to the cranial area. A longitudinal incision was made from the nasal bone to the occipital protuberance of the skull. Then, a midline incision was made through the periosteum. Sharp subperiosteal dissection reflected the pericranium from the outer table of the cranial vault, exposing the parietal bones. A trephine bur was used under saline irrigation to create a bilateral calvarial defect in parietal bones. Two 8-mm-diameter defects were created, one on each side of the midline. Then, right parietal bone defects were covered with SMs of 0.01 mm thickness (SM1 group) and left parietal bone defects were covered with SMs of 0.5 mm thickness (SM2 group) (Fig. 1). None of the animals received the same graft for both calvarial defects. After treatment, pericranium and skin were closed in layers with 3-0 black silk (AILEE, Busan, Korea). After surgery, the rabbits received gentamycin at 1 mg/kg (Kookje, Seoul, Korea) and pyrin at 0.5 mL/kg (Green Cross Veterinary Products, Seoul, Korea) intramuscularly 3 times daily for 3 days. Each rabbit was individually caged and received food and water. Five animals were killed at 4 weeks and 8 weeks, respectively. Specimens were separated and fixed in 10% formalin. After μ-CT analysis, histological analysis was performed.
At 4 and 8 weeks after surgery, the calvarial samples were harvested, decalcified in 5% nitric acid for 2 weeks, and dehydrated in ethyl alcohol and xylene. After separation of the parietal bones through the midline sagittal suture, they were embedded in paraffin blocks. The paraffin blocks were sliced into sections that were then stained with Masson trichrome. The section that showed the largest defect area and sections 50 μm proximal and distal to that one were selected. Digital images of the selected sections were taken with a digital camera (DP-73; Olympus, Tokyo, Japan). The images were analyzed by Sigma Scan pro (SPSS, Chicago, IL, USA). The total amount of newly formed bone was calculated as percentage of the total region of the defect.
Micro-CT Analysis
Micro-CT Analysis
The prepared specimens were analyzed by μ-CT using an animal PET/CT/SPECT system (Inveon; Siemens, Erlangen, Germany) at the Ochang Center in Korea Basic Science Institute. The μ-CT scanner was set to 80 kVp voltage for the x-ray tube, 500 μA current for the x-ray source, and 210 ms exposure time. The detector and x-ray source were rotated through 360 degrees in 360 steps. The number of calibration exposures was 30. System magnification was set to produce an axial field of view (FOV) of 30.74 mm and a transaxial FOV of 30.74 mm. The scanned images were reconstructed using the Inveon Research Workplace software (Siemens). Gross profiles of the specimens were obtained from reconstructed 3-dimensional images. Because the initial defect was round in shape with an 8.0-mm diameter, the setting of the region of interest (ROI) was considered to reflect the initial defect size and shape. The ROI of each specimen was analyzed for bone volume (BV).
FIGURE 1. Silk membranes of 0.01 mm thickness were grafted into right parietal bone defects; left parietal bone defects were covered with silk membranes of 0.5 mm thickness.
Statistical Analysis As the same animal received 2 different types of membrane, the paired t test was used for comparison of the groups. The level of statistical significance was set at P less than 0.05.
RESULTS SEM Results SEM images of SMs of 0.01 mm and 0.5 mm thickness are shown in Figure 2. The assembly of silk fibers was similar in both types of SM. That had a cross-linked structure and each silk fiber was attached by a glue-like substance. However, fibers of 0.01-mm-thick SMs were more loosely connected and thinner than those of 0.5-mm-thick SMs (Fig. 2A). Fibers of 0.5-mm-thick SMs had a denser structure and formed a thicker layer than those of 0.01-mm-thick SMs (Fig. 2B).
Results of the μ-CT analysis are presented in Table 1. The BV was 3.24 ± 2.05 mm3 in the SM1 group and 7.08 ± 3.72 mm3 in the SM2 group at 4 weeks after surgery (Figs. 3A, B). There was a significant difference between both groups (P = 0.004). The BVof the SM1 group was 12.53 ± 10.69 mm3 at 8 weeks after surgery, whereas that of the SM2 group was 20.13 ± 7.22 mm3 (Figs. 3C, D). The difference between both groups was not significant (P > 0.05).
FIGURE 2. Scanning microscopic images. A, Silk membrane of 0.01 mm thickness. B, Silk membrane of 0.5 mm thickness.
© 2014 Mutaz B. Habal, MD
Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
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The Journal of Craniofacial Surgery • Volume 25, Number 6, November 2014
Seok et al
TABLE 1. Micro-Computed Tomographic Analysis 4 wk Group Bone volume, mm3
SM1 3.24 ± 2.05
SM2 7.08 ± 3.72
TABLE 2. Histomorphometric Analysis 8 wk
SM1 12.53 ± 10.69
4 wk SM2 20.13 ± 7.22
SM1, silk membrane of 0.01 mm thickness; SM2, silk membrane of 0.5 mm thickness.
Histomorphometric Analysis The histomorphometric results are presented in Table 2. The total amount of newly formed bone was 11.06 ± 12.48% in the SM1 group and 8.83 ± 5.36% in the SM2 group at 4 weeks after surgery (Figs. 4A, B). The difference was not significant (P > 0.05). The total amount of newly formed bone was 18.98 ± 11.70% in the SM1 group and 44.26 ± 15.00% in the SM2 group at 8 weeks after surgery (Figs. 4C, D). The difference between both groups was significant (P = 0.021). Well-organized lamellar bone islands were formed in both groups at 8 weeks after surgery (Figs. 4E, F).
Group Total new bone, %
SM1 11.06 ± 12.48
8 wk SM2 8.83 ± 5.36
SM1 18.98 ± 11.70
SM2 44.26 ± 15.00
SM1, silk membrane of 0.01 mm thickness; SM2, silk membrane of 0.5 mm thickness.
still maintain their own tensile properties.21 In a previous study, an effort was made to use a SF film as GBR membrane.18 However, the SF film was fragile and easily dissolved in aqueous solution. Application and fixation of the SF film to the bone defect was nearly impossible. Compared to the SF film, mechanically separated silk
DISCUSSION The principle of GBR is to promote the migration of desired cells into the bone defect using a barrier membrane.22 An ideal GBR membrane should have the following properties: biocompatibility, biodegradability, and sufficient mechanical stability to maintain the space required for bone regeneration.10 To achieve bone regeneration of large bone defects, the mechanical stability of the membrane is very important.8,23,24 The membrane should maintain the intended form of the alveolar bone; furthermore, it should protect the bone graft material and blood clot, which should not be broken down and washed out.8,25 In this study, greater bone regeneration was achieved with the thicker SM. In the μ-CT analysis, the average amount of newly formed bone in the SM2 group was greater than that in the SM1 group at 4 weeks after surgery (P = 0.004, Figs. 3A, B). In the histological analysis, the amount of formed lamellar bone was much greater in the SM2 group than in the SM1 group at 8 weeks after surgery (P = 0.021, Figs. 4C, D). To the best of our knowledge, this is the first report in which bone regeneration is compared on the basis of the thickness of the SM. Silk cocoons can be easily separated into layers of different thickness by mechanical peeling. The obtained separated silk layers
FIGURE 3. Micro-computed tomography. A, Silk membrane of 0.01 mm thickness at 4 weeks after surgery. B, Silk membrane of 0.5 mm thickness at 4 weeks after surgery. C, Silk membrane of 0.01 mm thickness at 8 weeks after surgery. D, Silk membrane of 0.5 mm thickness at 8 weeks after surgery.
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FIGURE 4. Histological section (Masson trichrome stain). A, Silk membrane of 0.01 mm thickness at 4 weeks after surgery (original magnification 10). B, Silk membrane of 0.5 mm thickness at 4 weeks after surgery (original magnification 10). C, Silk membrane of 0.01 mm thickness at 8 weeks after surgery (original magnification 10). D, Silk membrane of 0.5 mm thickness at 8 weeks after surgery (original magnification 5). E, F, Well-organized lamellar bone in (C, D original magnification 100).
© 2014 Mutaz B. Habal, MD
Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
The Journal of Craniofacial Surgery • Volume 25, Number 6, November 2014
sheets have sufficient strength under both dry and wet conditions.26 Its yield strength under wet conditions is even higher than that of commercially available collagen membranes.26 We used SMs of 2 different thicknesses to compare the effectiveness for bone regeneration. Results of the μ-CT analysis showed that the BV in the SM2 group was greater than that in the SM1 group at 4 weeks after surgery (Figs. 3A, B; P = 0.004). In the histomorphometric analysis, the total amount of newly formed bone in the SM2 group was greater than that in the SM1 group at 8 weeks after surgery (Figs. 4C, D; P = 0.021). In the histological assessment of the SM2 group, the SMs maintained their form, provided support to maintain the space, and prevented infiltration of soft tissue cells (Figs. 4B, D). On the other hand, the SMs in the SM1 group did not maintain their form and collapsed into the bone defects (Figs. 4A, C), thereby decreasing the amount of space available for new bone formation. For use as GBR membrane, the membrane should maintain its original form and be sufficiently stable to prevent collapse into the bone defect.8,25 In this respect, SMs of 0.5 mm thickness functioned properly as GBR membrane because they maintained their form and provided sufficient space for bone growth compared to SMs of 0.01 mm thickness. As seen in the SEM image, SM2 had a denser structure compared to SM1 (Figs. 2A, B), which might provide a more conducive environment for osteoblast differentiation and prevent infiltration of soft tissue cells. SF has osteogenic cell attachment and differentiation properties.27,28 In a previous study, SF was shown to suppress the notch signaling pathway and to induce the differentiation of osteoblasts.29 As shown in the SEM image, SM2 had more silk fibers that were densely arranged (Fig. 2). Each fiber consists of fibroin and sericin.20 Though the total amount of SF in each SM cannot be accurately calculated, the thicker membrane is thought to contain more SF. In conclusion, covering the bone defect with a thick SM resulted in greater bone regeneration than covering it with a thin SM. The mechanical properties and dense fiber structure of SM2 are thought to contribute to greater bone regeneration. However, silk is produced by insects and, thus, a foreign protein. Considering the immune response to foreign proteins, SMs of unlimited thickness might not always be suitable for bone regeneration. The optimal thickness of SMs for clinical application should be determined in future studies.
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© 2014 Mutaz B. Habal, MD
Copyright © 2014 Mutaz B. Habal, MD. Unauthorized reproduction of this article is prohibited.
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