The Fixation Effect of a Silk Fibroin–Bacterial Cellulose Composite Plate in Segmental Defects of the Zygomatic Arch: An Experimental Study

Importance Bioresorbable fixation systems have been popular for the treatment of facial fractures. However, their mechanical properties are uncertain and complications have been reported. To overcome these problems, we developed a bioresorbable fixation plate using a composite of silk fibroin and bacterial cellulose (SF-BC) with biodegradability and increased biocompatibility.

Objective To investigate the regenerative effect of the bioresorbable SF-BC fixation plate on zygomatic arch defects in rats.

Design In vivo animal study. The SF-BC composite plate had a tensile strength similar to that of a polylactic acid plate and a tight, pore-free microstructure. Bilateral segmental bone defects (2 mm in length) were created in the zygomatic arches of adult rats. One side was fixed with the SF-BC composite plate, and the other side was left without fixation.

Setting Academic research laboratory.

Participants Fifteen adult Sprague-Dawley rats.

Interventions Fixation of the zygomatic arch defect with the SF-BC composite plate.

Main Outcomes and Measures Micro–computed tomography and histological evaluation of bone samples.

Results Gross inspection revealed no specific complication. At 1, 2, 4, and 8 postoperative weeks, the zygomatic arches were explored by micro–computed tomography and histological examination. Control sides did not heal completely and showed bony degeneration and necrosis during the 8-week follow-up. However, we observed new bone formation in sides treated with the SF-BC composite plate, and bony defects were completely healed within 8 weeks.

Conclusions and Relevance The SF-BC composite plate is a potential candidate for a new bioresorbable fixation system. Our composite material could considerably shorten bone regeneration time. Additional study of the control of biodegradability and mechanical properties of SF-BC composite plates and a comparative study with the resorbable plates currently in use should be undertaken.

Facial fractures with bone displacement cause functional and cosmetic problems. Management includes repositioning and fixation of the fractured bones to restore function and facial appearance of the affected site. Traditionally, metallic plates and screws have been used to fix the fractured segments. However, these metallic fixation systems have disadvantages. First, their extreme stiffness may cause stress shielding of the underlying bone; second, the plates may be removed after fracture healing is complete.^1,2 To avoid these drawbacks of metal plate systems, bioresorbable fixation systems have been developed during the past 20 years.² Polylactic acid was one of the first bioresorbable materials studied in this regard.^3,4 Polyglycolic acid, poly-L-lactic acid, poly-D-lactic acid, and poly(lactic-co-glycolic acid) are other materials used in resorbable systems. However, the currently available resorbable fixation systems can develop an inflammatory foreign body reaction and postoperative infection.^5,6

Silk fibroin (SF) is a potential candidate material for biomedical applications because it has several attractive properties, including good biocompatibility, good oxygen and water vapor permeability, and biodegradability.⁷ The SF matrix is known to support the attachment, growth, and differentiation of adult human, progenitor bone marrow stromal cells and to enhance bone regeneration.^7,8 In addition, because SF has excellent strength and durability, it may provide sufficient stability during bone ingrowth. However, SF has some disadvantages, such as brittleness, easy fragmentation, and difficulty in creating a uniform thickness.

Bacterial cellulose (BC) is an organic compound with the formula (C₆H₁₀O₅)n that is synthesized by the genera Acetobacter, Rhizobium, Agrobacterium, and Sarcina.⁹ However, only the Acetobacter species produce enough cellulose to justify commercial interest. In particular, cellulose biosynthesis by Acetobacter xylinum has been widely investigated owing to its ability to produce relatively high levels of polymer from a wide range of carbon and nitrogen sources.¹⁰ In general, BC is biocompatible and chemically pure, contains no hemicellulose or lignin, and has high water-binding capacity and hydrophilicity, greater tensile strength resulting from a larger amount of polymerization, and an ultrafine network architecture. Furthermore, BC can be produced in almost any shape because of its high moldability during formation.¹¹ Therefore, BC offers a wide range of applications, especially medical applications, such as wound dressing of second- or third-degree burn ulcers, artificial microvessels, and tissue engineering of cartilage.^12-14 However, BC degrades slowly and can remain in the body. Thus, we hypothesized that SF-BC composite plates could provide a stronger material that would overcome this drawback.

We developed SF-BC composite plates and assessed their mechanical and healing properties by micro–computed tomography (micro-CT) and histological examination in an animal model of segmental defects of the zygomatic arch.

Bombyx mori cocoons were used to obtain SF. Polyethylene oxide with an average molecular weight of 200 000 (Sigma-Aldrich) was used in blending. The cocoons were boiled for 30 minutes in an aqueous solution of 0.02M sodium carbonate and then rinsed thoroughly with water to extract the gluelike sericin proteins. The extracted silk was then dissolved in calcium chloride solution. This solution was then filtered through a miracloth (Calbiochem; EMD Biosciences) and dialyzed for 3 days to remove the salt. The final concentration of aqueous silk solution was 8 weight percentage (wt%), which was determined by weighing the remaining solid after drying. The SF solutions were stored at 4°C before use to avoid premature precipitation.

The BC pellicles were synthesized from A xylinum BRC5 in Hestrin and Schramm medium, which consists of 2% (weight to volume ratio [w/v]) glucose, 0.5% (w/v) yeast extract, 0.5% (w/v) enzymatic digest (Bacto-Peptone; BD Biosciences), 0.27% (w/v) disodium phosphate, and 0.115% (w/v) citric acid. All cells were precultured in a test tube for 3 days to maximize bacterial activity before BC pellicles were prepared. Fifty microliters of active bacteria were then injected into a culture dish with 10 mL of Hestrin and Schramm medium and incubated at 30°C for 10 days. The fabricated BC pellicles were purified by immersing them in 0.25M aqueous sodium hydroxide solution for 48 hours at room temperature. Then, the sodium hydroxide–treated BC was neutralized by repeated washing with deionized water. Cultivated BC was homogenized using a sonicator (Ultra-Turrax T25; IKA-Labortechnik). The homogenized BC was strained through a sieve (pore size, approximately 200 nm) and dried in a vacuum oven at 60°C for 24 hours.

To produce the SF-BC composite plates, 10 mL of aqueous SF solution was added to the BC mats in a petri dish, and they were dried together for 2 days at room temperature, followed by drying in a vacuum oven. The structures of the SF-BC composite plates were sputter coated with gold palladium and characterized by scanning electron microscopy (S3500N; Hitachi).

The width and thickness of the narrow section of each specimen were measured using an electronic digital caliper. Tensile strength was measured at room temperature using a universal test machine according to the standard test method for tensile properties of plastics (test D638; ASTM International). Five samples were tested. The stress-strain curve of the specimen was calculated by the attached computer. Tensile strength, elongation at breaking, and the tensile modulus were calculated from the stress-strain curve.

Fifteen adult Sprague-Dawley rats weighing 250 to 270 g each were housed in plastic cages with free access to laboratory chow and water under suitable environmental conditions, such as room temperature and exposure to 12 hours of daylight. Each animal was anesthetized with a single intraperitoneal injection of xylazine hydrochloride (5-10 mg/kg) and intramuscular ketamine hydrochloride (40-80 mg/kg).

The surgical areas were shaved and disinfected, and a skin incision was made in the zygomatic arch areas. Facial muscles were retracted and bilateral bone defects of 2-mm lengths were formed in the zygomatic arches using a surgical nipper (Figure 1A and B). One side was covered with the SF-BC composite plate and fixed using a 4-0 nylon suture, followed by skin suturing (Figure 1C). The skin of the other side was closed with no additional procedure in the bone defect.

Groups of rats were killed at 1, 2, 4, and 8 weeks with a lethal dose of urethane and thiopental sodium. Complete zygomatic arches were removed and fixed in 4% paraformaldehyde for 3 days. This study was approved by the institutional review board of Hallym University.

Following the period of bone regeneration, grafted regions of specimens were imaged with micro-CT (MicroCAT II; Siemens Medical Solutions). The micro-CT data acquisition scanning protocol consisted of x-ray settings of 50 peak kV and 400 μA, with 400-millisecond exposure. Data were processed using a commercially available software package (Amira 4.1; Mercury Computer Systems/TGS).

The rats were killed and the removed zygomatic bone samples were placed in fixative (Cal-Ex; Fisher Chemical) for 12 hours, decalcified, dehydrated through an ascending graded alcohol series (50%, 70%, 90%, and 100% ethanol), cleared in 2 changes of xylene, and embedded in tissue-embedding medium (57°C melting point [Paraplast; Sigma Aldrich]). Serial sections of 5-μm thickness were stained by Harris hematoxylin-eosin. Histological observation was performed under a light microscope (Eclipse 80i; Nikon).

The SF-BC composite plates had a thickness of 0.3 mm and exhibited flexibility (Figure 2A-C). They had a mean (SD) tensile strength of 68.28 (6.71) MPa, elongation at break of 5.84% (1.65%), and tensile modulus of 722.56 (56.15) MPa. The scanning electron microscopy images showed that the plates had a tight, pore-free structure (Figure 2D and E).

No rat experienced a specific complication after SF-BC plate implantation. No gross evidence of inflammation, abscess formation, seroma accumulation, or surrounding soft-tissue necrosis due to the implanted SF-BC plate was observed. Adhesion of SF-BC composite plates to the surrounding soft tissues increased with time. The SF-BC composite plates were fixed for 8 weeks, but their preimplanted shapes were almost completely preserved (Figure 3).

We examined the healing process in control and SF-BC composite plate–implanted zygomatic arches. Two- and 3-dimensional images were acquired by micro-CT. No healing was observed on 2- or 3-dimensional images of the control side during the 8-week period (Figure 4A and B). In the zygomatic arches fixed with SF-BC composite plates (Figure 4C and D), formation of a hard callus was detected on 2- and 3-dimensional images in the first week and was more evident in the second week. In the fourth week after fracture, calcification of the soft callus was apparent, indicating endochondral ossification. In addition, the new bone bridged the fracture gap. Defects of the zygomatic arch were completely healed with bridging and remodeling of the callus in the eighth week. Continued remodeling of the callus and the original zygomatic arch at the fracture site was noticeable.

On the control side (Figure 5A), no healing was observed during the 8-week follow-up. At the end of the first week, some inflammatory cells and overgrowth of soft tissue were observed at the periphery of the bone defect. Necrotic bony ends with minimal inflammatory reaction and minimal granulation tissue formation were seen after the second week, and some soft tissue appeared as a result of bone degeneration at the end of week 4. Necrosis of the bony ends was more distinct at the end of the eighth week.

In contrast, the SF-BC composite plate side showed complete healing of bony defects by the eighth week (Figure 5B). After the first week, the SF-BC plate served as a shield, and minimal inflammatory reaction was observed. New bone formation was observed at the edges of bony defects, and new bone in the form of bony islands was frequently observed in the central defect areas after the second week. We also noted new bone formation—which indicates osteoblast activity—under the SF-BC composite plate. The fracture ends were connected with new bone after the fourth week; however, hard tissue was mixed with soft tissue. At the end of the eighth week, bony defects were filled with hard tissue and completely healed.

In this study, segmental defects of the rat zygomatic arch fixed with SF-BC composite plates were completely healed with new bone that grew in continuation with both ends of the zygomatic arch within 8 weeks. The ideal fixation materials require sufficient strength to maintain fracture reduction, resist compression forces in the early postoperative course, and enable bone healing without causing a foreign body reaction in the later period. Resorbable plates and screws for rigid internal fixation were naturally developed from biodegradable suture materials already available for many years. The polymers of high-molecular-weight α-hydroxy acids, including polylactic acid and polyglycolic acid, are commonly used to manufacture resorbable plating systems.¹⁵ However, Laine et al¹⁶ reported an 8.6% rate of complications, including foreign body reactions, wound dehiscence, granulation tissue growth, and device exposure, and a postoperative infection rate of 0.6% during 10 years in patients who underwent craniofacial surgery using a self-reinforced polymer consisting of poly-L-lactic acid and poly-D-lactic acid. Our previous study⁶ evaluating the effectiveness of resorbable plate-and-screw systems in the treatment of zygomaticomaxillary fractures found complication rates of 7% with resorbable skeletal fixation systems and 4% with metal plate-and-screw systems. Therefore, the new resorbable fixation system, which is more biocompatible, is required.

Although SF has low tissue reactivity, noninfectivity, biodegradability, and very high strength in combination with excellent elasticity,^7,17 SF plates are inappropriate for resorbable plate-and-screw systems because of their brittleness and uneven thickness. Bacterial cellulose also is biocompatible and has excellent mechanical properties and high moldability. However, BC can be incompletely degraded in the body.¹¹ We thought that SF and BC could complement each other. The new SF-BC composite plate has a tight structure and excellent mechanical properties. The composite is highly flexible, and its tensile strength (68.28 MPa) is similar to those of polylactic acid, poly-L-lactic acid, and polyglycolic acid (60-80 MPa). This result indicates that SF-BC composite plates had appropriate mechanical properties for application as resorbable plates.

We found no gross evidence of inflammation, infection, or necrosis in segmental defects in rat zygomatic arches fixed with the SF-BC plate. Micro-CT and histological evaluation show that these defects fixed with SF-BC plates heal completely within 8 weeks, with no microscopic inflammatory reaction or infection. The new bone began growing in the bony islands in week 2, filled the defect after week 4, and subsequently became scleroid. In contrast, the control side with no plate showed no evidence of new bone formation or healing. For comparison, bone regeneration was observed at 1 year in 10-mm segmental defects in rabbit radii treated with a poly-L-lactic acid–poly-D-lactic acid membrane.¹⁸ Although this animal model and defect site differed from those used in our study, our new SF-BC composite could considerably shorten the bone regeneration time. Similarly, Kim et al⁸ reported that SF nanofiber membranes had good biocompatibility and enhanced bone regeneration in a rabbit calvarial model, suggesting that the SF membrane could be useful in guided bone regeneration. Their findings indicate that cells preserve their phenotypic characteristics for bone formation throughout an entire culture period, as demonstrated by the production of alkaline phosphatase and osteocalcin and by calcification.⁸ Other studies^7,19,20 have demonstrated that SF induces bone tissue growth of osteoblasts or stem cells in vitro.

Previous data estimate the total resorption time of SF plates to be about 2 to 4 years.^21,22 However, an additional study of the control of biodegradability and mechanical properties of SF-BC composite plates should be undertaken. Also, a comparative study should be performed with the resorbable plates currently in use.

In conclusion, the SF-BC composite plate enhances new bone regeneration with excellent biocompatibility in an animal study. These results suggest that SF-BC composite plates are good candidates for new fixation systems.

Correspondence: Chan Hum Park, MD, PhD, Department of Otorhinolaryngology–Head and Neck Surgery, Chuncheon Sacred Heart Hospital, Hallym University College of Medicine, 153, Kyo-Dong, Chuncheon, Ganwon-do 200-704, South Korea (hlpch@paran.com).

Submitted for Publication: October 25, 2012; final revision received February 19, 2013; accepted March 21, 2013.

Author Contributions: Mr Lee and Drs Park and Lee had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: All authors. Acquisition of data: All authors. Analysis and interpretation of data: J. M. Lee, Kim, and Park. Drafting of the manuscript: J. M. Lee. Critical revision of the manuscript for important intellectual content: All authors. Statistical analysis: J. M. Lee and Kim. Obtained funding: Park. Study supervision: Park.

Funding/Support: This study was supported by grants 111100-03-02-SB010 and 112007-05-1-SB010 for Bio-industry Technology Development from the Ministry for Food, Agriculture, Forestry, and Fisheries. Bombyx mori cocoons were supplied by the Rural Development Administration.