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Effect of gelatin sponge with colloid silver on bone healing in infected cranial defects
Materials Science and Engineering C 70 (2017) 371–377
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Effect of gelatin sponge with colloid silver on bone healing in infected cranial defects Yuliang Dong a, Weiqing Liu a, Yiling Lei b, Tingxi Wu c, Shiwen Zhang a, Yuchen Guo a, Yuan Liu a, Demeng Chen c, Quan Yuan a,b, Yongyue Wang a,b,⁎ a b c
State Key Laboratory of Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu, China Dental Implant Center, West China Hospital of Stomatology, Sichuan University, Chengdu, China Division of Oral Biology and Medicine, School of Dentistry, University of California Los Angeles (UCLA), Los Angeles, California, USA
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Article history: Received 12 May 2016 Received in revised form 17 August 2016 Accepted 6 September 2016 Available online 7 September 2016 Keywords: Infected bone defect Bone healing Gelatin sponge with colloid silver Methicillin-resistant Staphylococcus aureus (MRSA)
a b s t r a c t Oral infectious diseases may lead to bone loss, which makes it difficult to achieve satisfactory restoration. The rise of multidrug resistant bacteria has put forward severe challenges to the use of antibiotics. Silver (Ag) has long been known as a strong antibacterial agent. In clinic, gelatin sponge with colloid silver is used to reduce tooth extraction complication. To investigate how this material affect infected bone defects, methicillin-resistant Staphylococcus aureus (MRSA) infected 3-mm-diameter cranial defects were created in adult female Sprague-Dawley rats. One week after infection, the defects were debrided of all nonviable tissue and then implanted with gelatin sponge with colloid silver (gelatin/Ag group) or gelatin alone (gelatin group). At 2 and 3 days after debridement, significantly lower mRNA expression levels of IL-6 and TNF-α and lower plate colony count value were detected in gelatin/Ag group than control. Micro-CT analysis showed a significant increase of newly formed bone volume fraction (BV/TV) in gelatin/Ag treated defects. The HE stained cranium sections also showed a faster rate of defect closure in gelatin/Ag group than control. These findings demonstrated that gelatin sponge with colloid silver can effectively reduce the infection caused by MRSA in cranial defects and accelerate bone healing process. © 2016 Published by Elsevier B.V.
1. Introduction Oral infectious diseases, such as periodontitis, peri-implantitis, dry socket and osteomyelitis of the jaws, often manifest as pain, bleeding and pus, and subsequently lead to bone loss. Local treatment of infection and promoting bone healing are of great significance for further oral restoration. Management of these infections often requires multiple staged surgeries and the use of antibiotics as a supportive therapy for eradication [1,2]. However, in recent years, the rise of multidrug resistant bacteria has put forward severe challenges to the use of antibiotics [3,4]. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common pathogens of oral infection. Facing the urgent need for the development of novel therapeutic agents, silver (Ag), which has long been known as a strong antibacterial agent of a wide range of microbes [5–8], has attracted much attention [9–11]. Many medical instruments and products, such as venal catheters [12,13], wound and burn bandages are silver-coated [14–18]. Both in vitro [19] and in vivo [20] experiments confirmed its good antibacterial activity. And its ⁎ Corresponding author at: Dental Implant Center, West China Hospital of Stomatology, Sichuan University, 14 Third Section, Renmin Nan Road, Chengdu 610041, China. E-mail address: [email protected] (Y. Wang).
http://dx.doi.org/10.1016/j.msec.2016.09.015 0928-4931/© 2016 Published by Elsevier B.V.
antibacterial effect was further demonstrated as causing irreversible damage on bacterial cells [21–23]. In recent years, gelatin is widely used for tissue engineering. It is obtained by a controlled hydrolysis of collagen, which is a major component of skin, bones and connective tissue. It exhibits excellent qualities such as biocompatibility, biodegradability [24], low antigenicity [25– 29], and is more economical than collagen. Gelatin's easily modified ability also makes it a good material for drug delivery [30,31]. A wide variety of gelatin-based composites have been developed for tissue engineering, such as gelatin-siloxane hybrids [32], β-TCP/chitosan/gelatin [33], chitosan-gelatin-alginate-hydroxyapatite [34], gelatin-chitosannanobioglass 3D porous scaffold [35] and hybrid macroporous gelatin/ bioactive-glass/nanosilver scaffolds [36]. In oral treatment, gelatin sponge with colloidal silver has been used in clinic to prevent the complication of tooth extraction [37]. Its porous structure can promote blood clotting and stabilize blood coagulation. Along its absorption, it continuously releases silver ions against almost all microbes found in the oral environment even antibiotic resistance bacteria. Existing researches worked mostly on the antibacterial effect of Ag, but few of these studies focused on how it affect infected wound healing. The purpose of this study was to investigate the effect of gelatin sponge with colloid silver on bone healing in infected cranial defects.
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2. Materials and methods
2.6. Plate colony count
2.1. Characterization of gelatin sponge
2 and 3 days after material implantation, rats were sacrificed. The granulation tissue in the defect was obtained using a curette. The surface moisture was wiped off using filter paper and the tissue was weighted. Then the tissue was homogenized and diluted by 20 mL saline. 100 μL of each sample was separately inoculated on sterile plates (35 mm in diameter) containing appropriate amount of plate count agar. The samples were stored at 37 °C in incubator for 24 h. Then the colony-forming units grown were counted.
The characterization of functional groups on the gelatin sponge (with or without Ag) were performed by Fourier-transform infrared spectroscopy (FTIR) on a Varian 680-IR spectrometer (Agilent Tech, USA). The FTIR spectra were recorded using 16 scans/min, with a resolution of 1 cm−1 in the 400–4000 cm−1 wave number region. X-ray photoelectron spectroscopy (XPS) measurement was done using DAR400-XM 1000 (OMICRON Nanotechnologies, Germany) equipped with dual Al/Mg anodes as the X-ray source. The Al anode was used to attain the survey and elemental spectra. All spectra were calibrated using C1 s peak at 284.5 eV to exclude the charging effect on the sample. 2.2. Animals 10-week-old female Sprague-Dawley rats weighing 250–300 g were obtained from Experimental Animal Center of Sichuan University. All the experiments performed were approved by the Subcommittee on Research and Animal Care (SRAC) of Sichuan University. The experimental procedures were in accordance with the Care and Use of Laboratory Animals published in the National Institute of Health Guide (1996). All efforts were made to minimize animal suffering. These animals were kept in standard conditions (22 ± 2 °C; 50–70% relative humidity) and a 12 h light–dark cycle (lights on at 08:30 a.m.). 2.3. Bacteria preparation MRSA (ATCC 43300) was purchased from the American Type Culture Collection (ATCC) (Manassas, VA). MRSA was grown overnight in brain heart infusion broth at 37 °C with shaking at 200 rpm. The numbers of colony-forming units (CFUs) of the inoculum were determined using turbidimetry, then the MRSA solution was diluted with sterile saline to 1 × 107 CFUs. 2.4. Creation of cranial defects and bacteria inoculation The infected cranial defects were created according to our previous work [38]. Rats were anesthetized by an intraperitoneal injection of ketamine (75.0 mg/kg) (Ketaset; Aveco, Fort Dodge, IA, USA)/ dexmedetomidine (0.25 mg/kg). After shaving and disinfection, the scalp was incised along the midline. The skin and periosteum was reflected laterally to the muscle attachment. Full-thickness cranial defects were created bilaterally between the coronal suture and lambdoid suture using a trephine (3 mm in diameter). At the same time, normal saline irrigation was used to prevent heat damage. The defect edge was examined to confirm no residual bone left. Then gelatin sponges (without Ag) were wetted by 0.1 mL MRSA solution or saline (used as control) and inserted into the defects. The periosteum was sutured with 5–0 polyglactin-910 suture to fix the sponges. The skin was approximated along the midline and closed with 3–0 polyglactin-910 suture.
2.7. Quantitative real-time PCR Total RNA from the granulation tissue was extracted using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. For qRT-PCR, cDNA was prepared using QuantiTec reverse transcription kit (Qiagen, Valencia, CA) and analyzed with SYBR GreenMaster Mix (SABiosciences, Valencia, CA) in the iCycler (BioRad, Hercules, CA) using specific primers designed for each targeted gene (Table 1) [39]. Relative expression was calculated using the 2–ΔΔCt method by normalizing with β-actin gene expression, and presented as fold increase relative to control. F-forward; R-reverse.
2.8. Micro-CT analysis The craniums were harvested 2 and 4 weeks after debridement, then immediately fixed in 10% buffered formalin overnight and stored in 70% ethanol at 4 °C. Micro-computed tomography (Micro-CT) analysis with three-dimensional reconstructions were performed as previous described [40,41] using Skyscan 1176μCT imaging system (Bruker, Kontich, Belgium) at a spatial resolution of 18 μm (1 mm aluminum filter, 100 kV, 100 μA). The data were analyzed with NRecon 1.6 and CTAn 1.8 to determine the degree of ectopic bone formation in healed area. Three-dimensional (3D) images of the samples were also reconstructed using CTvox (SkyScan).
2.9. Histology The specimens were then decalcified with 17% EDTA for 8 weeks and processed for paraffin embedding. The specimens were sectioned in the coronal plane to a thickness of 5 μm and stained with HE staining. The sections were observed by Nikon Eclipse 80i microscope (Nikon, Yokohama, Japan) and digital images were generated using NIS-Elements software.
2.10. Statistical analysis All values were expressed as mean ± SD. Statistically significant differences were evaluated by Student's t-test. A p value of less than 0.05 was considered to be statistically significant.
2.5. Debridement and material implantation One week after MRSA inoculation, the cranium were re-exposed through the same midline scalp incision. The wound was treated with debridement and thorough irrigation with saline. The defects were filled with gelatin sponge with colloid silver (Gelatamp, Roeko, Coltene whaledent, Langenau, Germany) (gelatin/Ag group). The control defects were implanted with gelatin sponge (Gelfoam, Pfizer, New York, USA) (Gelatin group). The skin was approximated along the midline and closed with 3–0 polyglactin-910 suture.
Table 1 Primers designed for each target gene. Target gene
Direction
Primer sequence (5′-3′)
TNF-α
F R F R F R
5′-GGCAATGGCATGGATCTCA-3′ 5′-ATGGCAAATCGGCTGACGG-3′ 5’ACTTCCAGCCAGTGCCTTCT-3′ 5′-GGTCTGTTGTGGGGTGTATCCT-3′ 5′-ACGGTCAGGTCATCACTATCG-3′ 5′-GGCATAGAGGTCTTTACGGATG-3′
IL-6 β-actin
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3. Results
3.4. Gelatin/Ag improves bone healing
3.1. Characterization of gelatin sponge
As shown in Fig. 4A, moths-eaten bone destruction was observed at the margin of the infected bone defects from the 3D reconstruction images. Bone formation was observed solely along the margins of the defect, exhibiting absence of central bone formation. 2 weeks after debridement, the gelatin group showed negligible amount of new bone formation in the defect area, while the defects of gelatin/Ag group had larger area occupied by bone tissue.4 weeks after debridement, the defects of gelatin group remained almost unfilled. While new bone tissue had almost closed the defect of gelatin/ Ag group. The results were consistent with the BV/TV value of 34.10 ± 8.50% in gelatin/Ag group compared with 10.60 ± 6.22% in gelatin group at 2 weeks after debridement (p b 0.05), and 42.16 ± 9.27% in gelatin/Ag group compared with 21.44 ± 7.52% in gelatin group at 4 weeks after debridement (p b 0.05) (Fig. 4B). Histological sections were also performed to evaluate the quality of bone tissue that filled the defect sites. As shown in Fig. 4C, cross sections of decalcified cranium showed that the connective tissue (stained blue) occupied most defect sites of control, while the gelatin/Ag group showed greater proportion of bone tissue (stained red). Distinct differences in the organization of the mineralized tissue can be observed between original and new bone. No noticeable material was found suggesting that the material had been fully degraded. The appearance of newly formed bone in rats treated with Ag was similar to that of control, suggesting that the use of colloid silver did not have deleterious effect on tissue formation. 2 weeks after debridement, new bone in the gelatin group was thin with irregular surfaces. In gelatin/Ag group, new bone formed from the edge to the center of the original defects and on the outer and inner surfaces of the original lamellar bone, occupied 1/3 to 1/2 thickness of the tissue. The difference was more obvious at 4 weeks after debridement. The newly formed bone in gelatin group was still thinner than that in gelatin/Ag group. In contrast, a bony bridge formed in the defect area in gelatin/Ag group, with new bone occupying 2/3 to 4/5 of the defect. Several small blood vessels distributed in new bone, with abundant plump cells arranged along the vascular-bone interface, which were assumed to be osteoblasts. New bone had irregular structure, different from the original lamellar bone. External and internal surfaces of the original bone were covered by remodeling bone and became smooth, which was consistent with the Micro-CT results.
The FTIR spectra of gelatin sponge with and without Ag are basically consistent (Fig. 1A). The absorption peak at 3600–2300 cm−1, 1656– 1644 cm−1, 1560–1335 cm− 1, 1240–670 cm−1 and 1239 cm− 1 revealed the presence of Amide A, Amide I, Amide II, Amide III and Amide V respectively which are characteristic peaks of gelatin. That means the two materials used in this study mainly consist of gelatin with similar constituent. As shown in Fig. 1B, the survey spectrum shows the presence of carbon, nitrogen, oxygen and silver. The absorbance band at about 370 eV revealed the presence of Ag which can be found in gelatin sponge with colloid silver (b) but not in the control (a). 3.2. Creation of infected cranial defects The workflow of the study was shown in Fig. 2A. All rats survived the experimental period. One week after MRSA implantation, the defect exhibited severe inflammatory hyperplasia, characterized as local tissue edema, adhesion with surrounding tissue and greater brittleness (Fig. 2B). In contrast, the defect margin of saline treated ones was neat and no significant secretion was found. Images of 3D reconstruction showed that the margin of the defect could not be clearly identified with decreased radio opacity of the surrounding bone, indicating the ‘moths-eaten’ destruction by the infection (Fig. 2C right). On contrary, the margin of saline treated bone defects (negative control) was regular and the remaining bone surface was smooth and complete (Fig. 2C left). 3.3. The antibacterial effect of gelatin/Ag To test the antibacterial effect, we collected the granulation tissue in the center of defects 2 and 3 days after sponge implantation, and cultured tissue dilutions on agar plates for 24 h. As shown in Fig. 3A and Fig. 3B, significant lower number of bacteria colonies was observed in the gelatin/Ag group (7.35 ± 1.39 CFUs/cm2) as compared to that of control (10.36 ± 4.91 CFUs/cm2) on day 2. Ag treated defects also exhibited significantly lower plate colony count value (0.22 ± 0.11 CFUs/ cm2) than control (21.71 ± 9.29 CFUs/cm2) at 3 days after debridement. To further confirm the decreased inflammation in the gelatin/Ag group, we also isolated the RNA from the granulation tissue and investigated the mRNA expression of IL-6 and TNF-α, which are common inflammatory factors. As shown in Fig. 3C, the level of IL-6 in gelatin/Ag is significantly lower than control on day 2, and same with the level of TNF-α on day 2&3. In addition, the defects treated with gelatin sponge exhibited tissue congestion and a large amount of inflammatory cell infiltration (Fig. 3D). Abscess formations with central necrosis were also observed. However, the defects received gelatin/Ag treatment were mostly filled with connective tissue, with much less neutrophils infiltration.
4. Discussion The treatment of infected bone defects remains a challenge in clinic. Thus, biomaterial with both bone regeneration and infection control capabilities is urgently needed. Gelatin sponge with colloid silver has been widely used in clinic to prevent the complication of tooth extraction, but how this material affects infected bone defect is few studied. Therefore, we created infected cranial defects and treated them with gelatin/Ag or gelatin alone to investigate its antibacterial and bone healing effect. Significantly lower mRNA expression levels of IL-6 and TNF-α were observed in gelatin/Ag group at 3 days after debridement, and lower
Fig. 1. FTIR (A) and XPS (B) spectra of gelatin sponge (a) and gelatin sponge with colloid silver (b).
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Fig. 2. Creation of infected cranium defects. (A) Illustration of the workflow. “n” represents the number of animals for each group/analysis; (B) Macroscopic appearance of the defects at main time points; (C) Representative images of micro-CT reconstruction after one week of MRSA inoculation. Red circle indicates the defect margin.
plate colony count value than control both 2 and 3 days after debridement. Micro-CT analysis showed a significant increase of BV/TV value in gelatin/Ag treated defects. The HE stained cranium sections showed similar healing trend with micro-CT. These findings demonstrated that gelatin sponge with colloid silver can effectively reduce the infection caused by MRSA in cranial defects while accelerate bone healing process. To the best of our knowledge, animal models are powerful tools for experimental research. Segmental femur defect model is often used to investigate how antimicrobial agents act in an infected wound [42]. As we all know, cranium and maxillofacial bones are homologous, cranium bones would be a better choice while studying maxillofacial diseases. Gary E et al. reported a novel infected cranial defect model in rabbit [43]. They created a 15 × 15 mm, square, cranium defect, and inoculated the bone flap with S. aureus, then replaced it into the cranium defect. The model establishment was successful, however, rabbits as experimental animals are difficult to reach a large sample size. On this point, rats had natural advantages in case of experiments in large quantities. Moreover, regarding the defect size, this model is under pathological state, the infection has adverse effects on osteogenesis. Based on the expectation of full closure of the defects treated with Ag within a certain period of time, we used subcritical defects (3 mm in diameter) [44] for this novel rat model. Based on the infected bone defect model, we investigated how gelatin sponge with colloid silver affected the wound and found it not only control the infection but also promote bone healing. MRSA infection caused local inflammatory reaction, we could find soft tissue swelling, adhesion and abscess formation around the defect sites. With Ag treatment, fewer colony-forming units and lower expression of IL-6 and TNFα were observed in gelatin/Ag group than control, indicating lighter local inflammation. Studies have demonstrated that silver ions can electrostatically bind with negative charged cell wall structure, form pits on it, which increases membrane permeability, interferes membrane transport and leads to cell rupture [23,45–47]. It is also reported that silver ions can bind with disulphide of membrane proteins, make it easy to penetrate the membrane [48,49]. The osmotic pressure changes lead to membrane deformation, decreased viability and cell death. Silver ions inside cell react with sulphydryl groups and cysteine, affect enzyme activity and result in metabolism disorder [50,51]. Moreover, Wysor et al.
reported that silver ions could stop DNA replication by attaching to guanine [52]. Gelatamp contains 5% (in weight) finely dispersed colloid silver. It releases silver ions in moist conditions, which continuously acted against the infection at the defect area, helping reduce bone resorption. Its long-lasting antibacterial effect may due to the “zombies” effect [53]. Bacteria cells rupture after death and release silver ions to “infect” other surviving bacteria. This kind of chain reaction contributes to its longlasting antibacterial effect. With regard to the promoted bone healing process, some possible reasons for this effect might be: Firstly, the control of infection reduced bone destruction. During inflammation, macrophages and osteoclasts were activated to participate in the bone resorption, resulting in the moths-eaten destruction. Besides, the coagulase produced by MRSA could gather bacteria, as the blue crowd stained by HE we found in the nutrient vessel distributed between the cortical bones, leading to small artery embolism, which aggravated bone resorption. In gelatin/Ag group, Ag continuously acted against the infection at the defect area, helping reduced bone resorption. Additionally, Gelatamp's porous structure allowed it to hold blood as much as 55–75 times than its weight while keeping its shape. It provides a stable scaffold for clot formation, prevents tissue collapse and maintains the space for bone healing. Recently, Sun.'s study showed that a collagen scaffold encapsulated silver nanoparticles and bone morphogenetic protein 2 possessed strong antibacterial properties and enhanced the differentiation of BMSCs toward osteoblasts [54]. Yazdimamaghani et al. found that the addition of silver nanoparticles into a gelatin/bioactive-glass scaffolds could enhance the antibacterial activity against both gram-negative Escherichia coli and gram-positive Staphylococcus aureus [55]. However, both their studies are in vitro, an in vivo test should be performed to further investigate the antibacterial activity and biocompatibility of the silver nanoparticles. In 2010, Zheng et al. [56] fabricated a BMP-2 coupledNanosilver-PLGA composite graft and demonstrated that nanosilver particles had strong antibacterial properties and did not affect the osteoinductivity of BMP-2 in an infected critical femoral segmental defects model. However, their model is quite complicated. In addition, this model is under pathological state, which the infection has adverse effects on osteogenesis, a subcritical defect size would be better. In our study, we first generated a subcritical infected defect model in rat cranium with 3 mm in diameter. Based on the infected bone defect model,
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Fig. 3. Antibacterial effect of gelatin/Ag on 2nd and 3rd day after debridement. (A) Bacteria culture of each group; (B) Plate colony count of each group; (C) Levels of mRNA expression of IL6 and TNF-α gene in the granulation tissue quantified by qRT-PCR analysis; (D) Representative images of HE staining sections at 2 days after debridement. *: p b 0.05. The dashed line showed the estimated borders of the defect. “★” represented region of connective tissue. “☆” represented region of neutrophils infiltration. White arrow represented the center of abscess. Bar = 500 μm left, 100 μm right.
we found that gelatin sponge with colloid silver exhibited good antibacterial and bone healing effect without adding extra factor for promoting osteogenesis. In clinic, recent studies suggest the application feasibility of Gelatamp used in early implant at anterior area [57]. They implanted Gelatamp in the extraction sockets of anterior teeth and performed early-implanting operations after 4 weeks. They described its advantages as easier wound closure, hemostasis effect, preventing infection and less soft tissue depression. Our findings indicated that Gelatamp may also promote bone
healing in these cases. In the future, the application of gelatin sponge with colloid silver may help reduce the risk of restoration of infected bone loss area, shorten the treatment and healing time of wounds and improve the restoration stability and aesthetic effect. 5. Conclusions In the study, we had created infected cranial defects in rats, and treated them with gelatin sponge with colloid silver or gelatin alone
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Fig. 4. Bone healing assay at 2 and 4 weeks. (A) Three-dimensional reconstructions of cranium; Red circle indicates the original defect margin. (B) Bone volume fraction (BV/TV) at each time point; (C) Representative images of HE stained sections of defects. Right panel shows the high magnification of the defect margin. Dash lines indicate the defect margin. *: p b 0.05. nb: new bone. ob: original bone. Bar = 500 μm left, 50 μm right.
to investigate how Ag influenced infected bone healing process. Our work has demonstrated that gelatin/Ag treatment could effectively reduce the infection caused by MRSA and accelerate infected bone healing process. This material may help in the treatment of infected bone defects. Acknowledgments The authors would like to acknowledge, Xiaobo Duan, Yuan Wang and Liyan Zhou for technique support. This study was supported by the National Natural Science Foundation of China (NSFC 81571001).
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Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Effect of gelatin sponge with colloid silver on bone healing in infected cranial defects Yuliang Dong a, Weiqing Liu a, Yiling Lei b, Tingxi Wu c, Shiwen Zhang a, Yuchen Guo a, Yuan Liu a, Demeng Chen c, Quan Yuan a,b, Yongyue Wang a,b,⁎ a b c
State Key Laboratory of Oral Diseases, West China School of Stomatology, Sichuan University, Chengdu, China Dental Implant Center, West China Hospital of Stomatology, Sichuan University, Chengdu, China Division of Oral Biology and Medicine, School of Dentistry, University of California Los Angeles (UCLA), Los Angeles, California, USA
a r t i c l e
i n f o
Article history: Received 12 May 2016 Received in revised form 17 August 2016 Accepted 6 September 2016 Available online 7 September 2016 Keywords: Infected bone defect Bone healing Gelatin sponge with colloid silver Methicillin-resistant Staphylococcus aureus (MRSA)
a b s t r a c t Oral infectious diseases may lead to bone loss, which makes it difficult to achieve satisfactory restoration. The rise of multidrug resistant bacteria has put forward severe challenges to the use of antibiotics. Silver (Ag) has long been known as a strong antibacterial agent. In clinic, gelatin sponge with colloid silver is used to reduce tooth extraction complication. To investigate how this material affect infected bone defects, methicillin-resistant Staphylococcus aureus (MRSA) infected 3-mm-diameter cranial defects were created in adult female Sprague-Dawley rats. One week after infection, the defects were debrided of all nonviable tissue and then implanted with gelatin sponge with colloid silver (gelatin/Ag group) or gelatin alone (gelatin group). At 2 and 3 days after debridement, significantly lower mRNA expression levels of IL-6 and TNF-α and lower plate colony count value were detected in gelatin/Ag group than control. Micro-CT analysis showed a significant increase of newly formed bone volume fraction (BV/TV) in gelatin/Ag treated defects. The HE stained cranium sections also showed a faster rate of defect closure in gelatin/Ag group than control. These findings demonstrated that gelatin sponge with colloid silver can effectively reduce the infection caused by MRSA in cranial defects and accelerate bone healing process. © 2016 Published by Elsevier B.V.
1. Introduction Oral infectious diseases, such as periodontitis, peri-implantitis, dry socket and osteomyelitis of the jaws, often manifest as pain, bleeding and pus, and subsequently lead to bone loss. Local treatment of infection and promoting bone healing are of great significance for further oral restoration. Management of these infections often requires multiple staged surgeries and the use of antibiotics as a supportive therapy for eradication [1,2]. However, in recent years, the rise of multidrug resistant bacteria has put forward severe challenges to the use of antibiotics [3,4]. Methicillin-resistant Staphylococcus aureus (MRSA) is one of the most common pathogens of oral infection. Facing the urgent need for the development of novel therapeutic agents, silver (Ag), which has long been known as a strong antibacterial agent of a wide range of microbes [5–8], has attracted much attention [9–11]. Many medical instruments and products, such as venal catheters [12,13], wound and burn bandages are silver-coated [14–18]. Both in vitro [19] and in vivo [20] experiments confirmed its good antibacterial activity. And its ⁎ Corresponding author at: Dental Implant Center, West China Hospital of Stomatology, Sichuan University, 14 Third Section, Renmin Nan Road, Chengdu 610041, China. E-mail address: [email protected] (Y. Wang).
http://dx.doi.org/10.1016/j.msec.2016.09.015 0928-4931/© 2016 Published by Elsevier B.V.
antibacterial effect was further demonstrated as causing irreversible damage on bacterial cells [21–23]. In recent years, gelatin is widely used for tissue engineering. It is obtained by a controlled hydrolysis of collagen, which is a major component of skin, bones and connective tissue. It exhibits excellent qualities such as biocompatibility, biodegradability [24], low antigenicity [25– 29], and is more economical than collagen. Gelatin's easily modified ability also makes it a good material for drug delivery [30,31]. A wide variety of gelatin-based composites have been developed for tissue engineering, such as gelatin-siloxane hybrids [32], β-TCP/chitosan/gelatin [33], chitosan-gelatin-alginate-hydroxyapatite [34], gelatin-chitosannanobioglass 3D porous scaffold [35] and hybrid macroporous gelatin/ bioactive-glass/nanosilver scaffolds [36]. In oral treatment, gelatin sponge with colloidal silver has been used in clinic to prevent the complication of tooth extraction [37]. Its porous structure can promote blood clotting and stabilize blood coagulation. Along its absorption, it continuously releases silver ions against almost all microbes found in the oral environment even antibiotic resistance bacteria. Existing researches worked mostly on the antibacterial effect of Ag, but few of these studies focused on how it affect infected wound healing. The purpose of this study was to investigate the effect of gelatin sponge with colloid silver on bone healing in infected cranial defects.
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2. Materials and methods
2.6. Plate colony count
2.1. Characterization of gelatin sponge
2 and 3 days after material implantation, rats were sacrificed. The granulation tissue in the defect was obtained using a curette. The surface moisture was wiped off using filter paper and the tissue was weighted. Then the tissue was homogenized and diluted by 20 mL saline. 100 μL of each sample was separately inoculated on sterile plates (35 mm in diameter) containing appropriate amount of plate count agar. The samples were stored at 37 °C in incubator for 24 h. Then the colony-forming units grown were counted.
The characterization of functional groups on the gelatin sponge (with or without Ag) were performed by Fourier-transform infrared spectroscopy (FTIR) on a Varian 680-IR spectrometer (Agilent Tech, USA). The FTIR spectra were recorded using 16 scans/min, with a resolution of 1 cm−1 in the 400–4000 cm−1 wave number region. X-ray photoelectron spectroscopy (XPS) measurement was done using DAR400-XM 1000 (OMICRON Nanotechnologies, Germany) equipped with dual Al/Mg anodes as the X-ray source. The Al anode was used to attain the survey and elemental spectra. All spectra were calibrated using C1 s peak at 284.5 eV to exclude the charging effect on the sample. 2.2. Animals 10-week-old female Sprague-Dawley rats weighing 250–300 g were obtained from Experimental Animal Center of Sichuan University. All the experiments performed were approved by the Subcommittee on Research and Animal Care (SRAC) of Sichuan University. The experimental procedures were in accordance with the Care and Use of Laboratory Animals published in the National Institute of Health Guide (1996). All efforts were made to minimize animal suffering. These animals were kept in standard conditions (22 ± 2 °C; 50–70% relative humidity) and a 12 h light–dark cycle (lights on at 08:30 a.m.). 2.3. Bacteria preparation MRSA (ATCC 43300) was purchased from the American Type Culture Collection (ATCC) (Manassas, VA). MRSA was grown overnight in brain heart infusion broth at 37 °C with shaking at 200 rpm. The numbers of colony-forming units (CFUs) of the inoculum were determined using turbidimetry, then the MRSA solution was diluted with sterile saline to 1 × 107 CFUs. 2.4. Creation of cranial defects and bacteria inoculation The infected cranial defects were created according to our previous work [38]. Rats were anesthetized by an intraperitoneal injection of ketamine (75.0 mg/kg) (Ketaset; Aveco, Fort Dodge, IA, USA)/ dexmedetomidine (0.25 mg/kg). After shaving and disinfection, the scalp was incised along the midline. The skin and periosteum was reflected laterally to the muscle attachment. Full-thickness cranial defects were created bilaterally between the coronal suture and lambdoid suture using a trephine (3 mm in diameter). At the same time, normal saline irrigation was used to prevent heat damage. The defect edge was examined to confirm no residual bone left. Then gelatin sponges (without Ag) were wetted by 0.1 mL MRSA solution or saline (used as control) and inserted into the defects. The periosteum was sutured with 5–0 polyglactin-910 suture to fix the sponges. The skin was approximated along the midline and closed with 3–0 polyglactin-910 suture.
2.7. Quantitative real-time PCR Total RNA from the granulation tissue was extracted using Trizol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. For qRT-PCR, cDNA was prepared using QuantiTec reverse transcription kit (Qiagen, Valencia, CA) and analyzed with SYBR GreenMaster Mix (SABiosciences, Valencia, CA) in the iCycler (BioRad, Hercules, CA) using specific primers designed for each targeted gene (Table 1) [39]. Relative expression was calculated using the 2–ΔΔCt method by normalizing with β-actin gene expression, and presented as fold increase relative to control. F-forward; R-reverse.
2.8. Micro-CT analysis The craniums were harvested 2 and 4 weeks after debridement, then immediately fixed in 10% buffered formalin overnight and stored in 70% ethanol at 4 °C. Micro-computed tomography (Micro-CT) analysis with three-dimensional reconstructions were performed as previous described [40,41] using Skyscan 1176μCT imaging system (Bruker, Kontich, Belgium) at a spatial resolution of 18 μm (1 mm aluminum filter, 100 kV, 100 μA). The data were analyzed with NRecon 1.6 and CTAn 1.8 to determine the degree of ectopic bone formation in healed area. Three-dimensional (3D) images of the samples were also reconstructed using CTvox (SkyScan).
2.9. Histology The specimens were then decalcified with 17% EDTA for 8 weeks and processed for paraffin embedding. The specimens were sectioned in the coronal plane to a thickness of 5 μm and stained with HE staining. The sections were observed by Nikon Eclipse 80i microscope (Nikon, Yokohama, Japan) and digital images were generated using NIS-Elements software.
2.10. Statistical analysis All values were expressed as mean ± SD. Statistically significant differences were evaluated by Student's t-test. A p value of less than 0.05 was considered to be statistically significant.
2.5. Debridement and material implantation One week after MRSA inoculation, the cranium were re-exposed through the same midline scalp incision. The wound was treated with debridement and thorough irrigation with saline. The defects were filled with gelatin sponge with colloid silver (Gelatamp, Roeko, Coltene whaledent, Langenau, Germany) (gelatin/Ag group). The control defects were implanted with gelatin sponge (Gelfoam, Pfizer, New York, USA) (Gelatin group). The skin was approximated along the midline and closed with 3–0 polyglactin-910 suture.
Table 1 Primers designed for each target gene. Target gene
Direction
Primer sequence (5′-3′)
TNF-α
F R F R F R
5′-GGCAATGGCATGGATCTCA-3′ 5′-ATGGCAAATCGGCTGACGG-3′ 5’ACTTCCAGCCAGTGCCTTCT-3′ 5′-GGTCTGTTGTGGGGTGTATCCT-3′ 5′-ACGGTCAGGTCATCACTATCG-3′ 5′-GGCATAGAGGTCTTTACGGATG-3′
IL-6 β-actin
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3. Results
3.4. Gelatin/Ag improves bone healing
3.1. Characterization of gelatin sponge
As shown in Fig. 4A, moths-eaten bone destruction was observed at the margin of the infected bone defects from the 3D reconstruction images. Bone formation was observed solely along the margins of the defect, exhibiting absence of central bone formation. 2 weeks after debridement, the gelatin group showed negligible amount of new bone formation in the defect area, while the defects of gelatin/Ag group had larger area occupied by bone tissue.4 weeks after debridement, the defects of gelatin group remained almost unfilled. While new bone tissue had almost closed the defect of gelatin/ Ag group. The results were consistent with the BV/TV value of 34.10 ± 8.50% in gelatin/Ag group compared with 10.60 ± 6.22% in gelatin group at 2 weeks after debridement (p b 0.05), and 42.16 ± 9.27% in gelatin/Ag group compared with 21.44 ± 7.52% in gelatin group at 4 weeks after debridement (p b 0.05) (Fig. 4B). Histological sections were also performed to evaluate the quality of bone tissue that filled the defect sites. As shown in Fig. 4C, cross sections of decalcified cranium showed that the connective tissue (stained blue) occupied most defect sites of control, while the gelatin/Ag group showed greater proportion of bone tissue (stained red). Distinct differences in the organization of the mineralized tissue can be observed between original and new bone. No noticeable material was found suggesting that the material had been fully degraded. The appearance of newly formed bone in rats treated with Ag was similar to that of control, suggesting that the use of colloid silver did not have deleterious effect on tissue formation. 2 weeks after debridement, new bone in the gelatin group was thin with irregular surfaces. In gelatin/Ag group, new bone formed from the edge to the center of the original defects and on the outer and inner surfaces of the original lamellar bone, occupied 1/3 to 1/2 thickness of the tissue. The difference was more obvious at 4 weeks after debridement. The newly formed bone in gelatin group was still thinner than that in gelatin/Ag group. In contrast, a bony bridge formed in the defect area in gelatin/Ag group, with new bone occupying 2/3 to 4/5 of the defect. Several small blood vessels distributed in new bone, with abundant plump cells arranged along the vascular-bone interface, which were assumed to be osteoblasts. New bone had irregular structure, different from the original lamellar bone. External and internal surfaces of the original bone were covered by remodeling bone and became smooth, which was consistent with the Micro-CT results.
The FTIR spectra of gelatin sponge with and without Ag are basically consistent (Fig. 1A). The absorption peak at 3600–2300 cm−1, 1656– 1644 cm−1, 1560–1335 cm− 1, 1240–670 cm−1 and 1239 cm− 1 revealed the presence of Amide A, Amide I, Amide II, Amide III and Amide V respectively which are characteristic peaks of gelatin. That means the two materials used in this study mainly consist of gelatin with similar constituent. As shown in Fig. 1B, the survey spectrum shows the presence of carbon, nitrogen, oxygen and silver. The absorbance band at about 370 eV revealed the presence of Ag which can be found in gelatin sponge with colloid silver (b) but not in the control (a). 3.2. Creation of infected cranial defects The workflow of the study was shown in Fig. 2A. All rats survived the experimental period. One week after MRSA implantation, the defect exhibited severe inflammatory hyperplasia, characterized as local tissue edema, adhesion with surrounding tissue and greater brittleness (Fig. 2B). In contrast, the defect margin of saline treated ones was neat and no significant secretion was found. Images of 3D reconstruction showed that the margin of the defect could not be clearly identified with decreased radio opacity of the surrounding bone, indicating the ‘moths-eaten’ destruction by the infection (Fig. 2C right). On contrary, the margin of saline treated bone defects (negative control) was regular and the remaining bone surface was smooth and complete (Fig. 2C left). 3.3. The antibacterial effect of gelatin/Ag To test the antibacterial effect, we collected the granulation tissue in the center of defects 2 and 3 days after sponge implantation, and cultured tissue dilutions on agar plates for 24 h. As shown in Fig. 3A and Fig. 3B, significant lower number of bacteria colonies was observed in the gelatin/Ag group (7.35 ± 1.39 CFUs/cm2) as compared to that of control (10.36 ± 4.91 CFUs/cm2) on day 2. Ag treated defects also exhibited significantly lower plate colony count value (0.22 ± 0.11 CFUs/ cm2) than control (21.71 ± 9.29 CFUs/cm2) at 3 days after debridement. To further confirm the decreased inflammation in the gelatin/Ag group, we also isolated the RNA from the granulation tissue and investigated the mRNA expression of IL-6 and TNF-α, which are common inflammatory factors. As shown in Fig. 3C, the level of IL-6 in gelatin/Ag is significantly lower than control on day 2, and same with the level of TNF-α on day 2&3. In addition, the defects treated with gelatin sponge exhibited tissue congestion and a large amount of inflammatory cell infiltration (Fig. 3D). Abscess formations with central necrosis were also observed. However, the defects received gelatin/Ag treatment were mostly filled with connective tissue, with much less neutrophils infiltration.
4. Discussion The treatment of infected bone defects remains a challenge in clinic. Thus, biomaterial with both bone regeneration and infection control capabilities is urgently needed. Gelatin sponge with colloid silver has been widely used in clinic to prevent the complication of tooth extraction, but how this material affects infected bone defect is few studied. Therefore, we created infected cranial defects and treated them with gelatin/Ag or gelatin alone to investigate its antibacterial and bone healing effect. Significantly lower mRNA expression levels of IL-6 and TNF-α were observed in gelatin/Ag group at 3 days after debridement, and lower
Fig. 1. FTIR (A) and XPS (B) spectra of gelatin sponge (a) and gelatin sponge with colloid silver (b).
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Fig. 2. Creation of infected cranium defects. (A) Illustration of the workflow. “n” represents the number of animals for each group/analysis; (B) Macroscopic appearance of the defects at main time points; (C) Representative images of micro-CT reconstruction after one week of MRSA inoculation. Red circle indicates the defect margin.
plate colony count value than control both 2 and 3 days after debridement. Micro-CT analysis showed a significant increase of BV/TV value in gelatin/Ag treated defects. The HE stained cranium sections showed similar healing trend with micro-CT. These findings demonstrated that gelatin sponge with colloid silver can effectively reduce the infection caused by MRSA in cranial defects while accelerate bone healing process. To the best of our knowledge, animal models are powerful tools for experimental research. Segmental femur defect model is often used to investigate how antimicrobial agents act in an infected wound [42]. As we all know, cranium and maxillofacial bones are homologous, cranium bones would be a better choice while studying maxillofacial diseases. Gary E et al. reported a novel infected cranial defect model in rabbit [43]. They created a 15 × 15 mm, square, cranium defect, and inoculated the bone flap with S. aureus, then replaced it into the cranium defect. The model establishment was successful, however, rabbits as experimental animals are difficult to reach a large sample size. On this point, rats had natural advantages in case of experiments in large quantities. Moreover, regarding the defect size, this model is under pathological state, the infection has adverse effects on osteogenesis. Based on the expectation of full closure of the defects treated with Ag within a certain period of time, we used subcritical defects (3 mm in diameter) [44] for this novel rat model. Based on the infected bone defect model, we investigated how gelatin sponge with colloid silver affected the wound and found it not only control the infection but also promote bone healing. MRSA infection caused local inflammatory reaction, we could find soft tissue swelling, adhesion and abscess formation around the defect sites. With Ag treatment, fewer colony-forming units and lower expression of IL-6 and TNFα were observed in gelatin/Ag group than control, indicating lighter local inflammation. Studies have demonstrated that silver ions can electrostatically bind with negative charged cell wall structure, form pits on it, which increases membrane permeability, interferes membrane transport and leads to cell rupture [23,45–47]. It is also reported that silver ions can bind with disulphide of membrane proteins, make it easy to penetrate the membrane [48,49]. The osmotic pressure changes lead to membrane deformation, decreased viability and cell death. Silver ions inside cell react with sulphydryl groups and cysteine, affect enzyme activity and result in metabolism disorder [50,51]. Moreover, Wysor et al.
reported that silver ions could stop DNA replication by attaching to guanine [52]. Gelatamp contains 5% (in weight) finely dispersed colloid silver. It releases silver ions in moist conditions, which continuously acted against the infection at the defect area, helping reduce bone resorption. Its long-lasting antibacterial effect may due to the “zombies” effect [53]. Bacteria cells rupture after death and release silver ions to “infect” other surviving bacteria. This kind of chain reaction contributes to its longlasting antibacterial effect. With regard to the promoted bone healing process, some possible reasons for this effect might be: Firstly, the control of infection reduced bone destruction. During inflammation, macrophages and osteoclasts were activated to participate in the bone resorption, resulting in the moths-eaten destruction. Besides, the coagulase produced by MRSA could gather bacteria, as the blue crowd stained by HE we found in the nutrient vessel distributed between the cortical bones, leading to small artery embolism, which aggravated bone resorption. In gelatin/Ag group, Ag continuously acted against the infection at the defect area, helping reduced bone resorption. Additionally, Gelatamp's porous structure allowed it to hold blood as much as 55–75 times than its weight while keeping its shape. It provides a stable scaffold for clot formation, prevents tissue collapse and maintains the space for bone healing. Recently, Sun.'s study showed that a collagen scaffold encapsulated silver nanoparticles and bone morphogenetic protein 2 possessed strong antibacterial properties and enhanced the differentiation of BMSCs toward osteoblasts [54]. Yazdimamaghani et al. found that the addition of silver nanoparticles into a gelatin/bioactive-glass scaffolds could enhance the antibacterial activity against both gram-negative Escherichia coli and gram-positive Staphylococcus aureus [55]. However, both their studies are in vitro, an in vivo test should be performed to further investigate the antibacterial activity and biocompatibility of the silver nanoparticles. In 2010, Zheng et al. [56] fabricated a BMP-2 coupledNanosilver-PLGA composite graft and demonstrated that nanosilver particles had strong antibacterial properties and did not affect the osteoinductivity of BMP-2 in an infected critical femoral segmental defects model. However, their model is quite complicated. In addition, this model is under pathological state, which the infection has adverse effects on osteogenesis, a subcritical defect size would be better. In our study, we first generated a subcritical infected defect model in rat cranium with 3 mm in diameter. Based on the infected bone defect model,
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Fig. 3. Antibacterial effect of gelatin/Ag on 2nd and 3rd day after debridement. (A) Bacteria culture of each group; (B) Plate colony count of each group; (C) Levels of mRNA expression of IL6 and TNF-α gene in the granulation tissue quantified by qRT-PCR analysis; (D) Representative images of HE staining sections at 2 days after debridement. *: p b 0.05. The dashed line showed the estimated borders of the defect. “★” represented region of connective tissue. “☆” represented region of neutrophils infiltration. White arrow represented the center of abscess. Bar = 500 μm left, 100 μm right.
we found that gelatin sponge with colloid silver exhibited good antibacterial and bone healing effect without adding extra factor for promoting osteogenesis. In clinic, recent studies suggest the application feasibility of Gelatamp used in early implant at anterior area [57]. They implanted Gelatamp in the extraction sockets of anterior teeth and performed early-implanting operations after 4 weeks. They described its advantages as easier wound closure, hemostasis effect, preventing infection and less soft tissue depression. Our findings indicated that Gelatamp may also promote bone
healing in these cases. In the future, the application of gelatin sponge with colloid silver may help reduce the risk of restoration of infected bone loss area, shorten the treatment and healing time of wounds and improve the restoration stability and aesthetic effect. 5. Conclusions In the study, we had created infected cranial defects in rats, and treated them with gelatin sponge with colloid silver or gelatin alone
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Fig. 4. Bone healing assay at 2 and 4 weeks. (A) Three-dimensional reconstructions of cranium; Red circle indicates the original defect margin. (B) Bone volume fraction (BV/TV) at each time point; (C) Representative images of HE stained sections of defects. Right panel shows the high magnification of the defect margin. Dash lines indicate the defect margin. *: p b 0.05. nb: new bone. ob: original bone. Bar = 500 μm left, 50 μm right.
to investigate how Ag influenced infected bone healing process. Our work has demonstrated that gelatin/Ag treatment could effectively reduce the infection caused by MRSA and accelerate infected bone healing process. This material may help in the treatment of infected bone defects. Acknowledgments The authors would like to acknowledge, Xiaobo Duan, Yuan Wang and Liyan Zhou for technique support. This study was supported by the National Natural Science Foundation of China (NSFC 81571001).
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