Tissue Engineering and Regenerative Medicine, Vol. 11, No. 5, pp 341-349 (2014) DOI 10.1007/s13770-014-0016-9

｜Original Article｜

The Effect of Gamma Radiation Sterilization on Dental Biomaterials N.Selcan Türker1, A.Yekta Özer2*, Burak Kutlu3, Rahime Nohutcu3, Arzu Sungur4, Hasan Bilgili5, Melike Ekizoglu6, and Meral Özalp6 1

Hacettepe University, Fac Pharmacy, Dep Radiopharmacy. 1MGH-Harvard Medical School, Dep Radiology 2 Hacettepe University, Fac Pharmacy, Dep Radiopharmacy, 06100, Sihhiye, Ankara 3 Hacettepe University, Fac Dentistry, Dep Periodontology, 06100, Sihhiye, Ankara 4 Hacettepe University, Fac Medicine, Department of Pathology, 06100, Sihhiye, Ankara 5 Ankara University, Faculty of Veterinary Medicine, Department of Orthopaedics and Traumatology, 06110 Diskapi, Ankara 6 Hacettepe University, Fac Pharmacy, Department of Microbiology, 06100 Sihhiye, Ankara (Received: March 17th, 2014; Revision: May 10th, 2014; Accepted: May 29th, 2014)

Abstract : Biomaterials are used in the field of bone and tissue engineering, orthopaedics and dentistry. Dental biomaterials including commercially available biodegradable materials act as physical barriers to help quicker healing while stimulating the regeneration of periodontal tissues, which is defined as Guided Tissue Regeneration (GTR). Amongst natural and synthetic biomaterials, collagen and aliphatic polyesters, such as polylactic acid (PLA) and poly (lactic-co-glycolic) acid (PLGA) are the most frequently used biomaterials for regenerative therapies due to their excellent biocompatibility and biodegradability. Due to their resorption in the body and interaction with biological systems, the GTR membranes must be sterile and pyrogen free. The sterility and apyrogenicity of the GTR membranes before human use is a regulatory requirement, however the sterilization of biomaterials is challenging due to the physicochemical changes and toxic residues with the commonly used sterilization techniques. The purpose of the present study was to evaluate the effect of gamma radiation and ethylene oxide sterilization on dental biomaterials with analytical, microbiological and histological examinations. PLGA-based GTR dental biomaterial is selected as the most gamma stable membrane according to the FTIR, DSC, TGA, and SEM results. This dental membrane was sterilized with ethylene oxide (EtO) and the effect of sterilization method on PLGA-based membrane was also investigated. Animal experiments were carried out to evaluate the regenerative properties and inflammatory responses of gamma and EtO sterilized PLGA-based GTR membrane after implantation. Histological examinations showed that resorption and bone formation of gamma sterilized PLGA-based GTR membrane was completed in 12 weeks without any inflammatory response; while only 60.095 ± 2.019% of new bone formation was observed with EtO sterilized one. Gamma sterilized PLGA membrane had significantly faster (P < 0.05) resorption and bone formation in comparison with EtO sterilization. In conclusion, the PLGA-based biomaterials could be sterilized safely and time- and cost-effectively with validated radiation doses for the tissue engineering applications. Key words: Gamma radiation sterilization, Ethylene oxide sterilization, Guided tissue regeneration, Guided Bone Regeneration, Dental biomaterials

approaches, periodontal tissue regeneration has considerable success in the treatment of periodontal disease and trauma following the use of grafting biomaterials with improved guided tissue regeneration (GTR).3 The clinical applications of GTR in periodontics involve the placement of a physical barrier membrane to enable the bone and tissue to re-grow and to arrest and control periodontal infection and ultimately to regenerate lost periodontal structures.1,4,5 GTR membranes are generally resorbable, which means that they absorbed by the body with no need for a second surgery and allow healing over time.1,6 A wide variety of synthetic and natural

1. Introduction Biomaterials are being used to repair, restore, or replace the functions of living tissues of the human body.1 During the last decade, various regenerative biomaterials have been examined for the treatment of teeth defects, which presents a significant clinical problem in periodontology.2 Among the therapy *Corresponding author Tel: 0090 3123052196; Fax: 0090 3123114777 e-mail: [email protected] (A.Yekta Özer)

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biomaterials are used in dental applications because of their excellent mechanical properties.2,7-9 Poly (lactic acid) (PLA), poly (lactic-co-glycolic acid) (PLGA) and collagenous materials are the most promising bioresorbable GTR membranes and have been widely used during the past several decades.10,11 An ideal GTR membrane should be compatible, safe, nonallergic, non-toxic and have no risk of disease transmission.3,12 GTR membranes are continuously in contact with body fluids and tissues that it is necessary to sterilize the whole product before implantation in patients.13,14 The mechanical and physicochemical properties of the GTR biomaterials, must be stable after sterilization.15 Therefore, it is critical and necessary to analyse the compatibility of the selected sterilization method on GTR biomaterials.16 Among the most widely used sterilization techniques, exposure to the gamma rays is the most commonly used technique to sterilize pharmaceuticals and medical devices because of its high penetrating ability, uniform and time dependent delivery of the required doses without any toxic residue.17 Furthermore, the dosimeters enable parametric release, which provides cost and sterility assurance advantages of gamma radiation sterilization. Depending on the Pharmacopoeias, a minimum dose of 25 kGy was routinely applied for many medical devices, but now, as recommended by the International Organization for Standardization (ISO), the sterilization dose must be set for each type of product depending on its bioburden.18 The purpose of the present study was: a) to investigate the physicochemical and microbiological effects of gamma radiation sterilization on GTR biomaterials and to select the most gamma stable biomaterial, b) to compare the physicochemical, regenerative, microbiological properties of gamma and EtO sterilized GTR biomaterials.

Figure 1. A schematic representation of the experimental design.

Figure 1. The non-sterile biodegradable GTR membranes were sterilized with gamma radiation with different radiation doses. According to the physicochemical test results, the most gamma stable GTR membrane was selected (coded with M). To compare the effect of the sterilization methods, ‘M’ coded non-sterile GTR membrane was sterilized with EtO (METO). The same analytical tests were done on the selected ‘M’ after EtO sterilization. Animal experiments were carried out with gamma sterilized and EtO sterilized membranes (M and MEtO) and histopathological analyses were done to compare the regenerative and inflammatory effect of the sterilization methods.

2. Materials and Methods 2.3 Sterilization of GTR Membranes GTR membranes were sterilized with gamma radiation at four different dose levels (5, 10, 25, 50 kGy) under normal conditions (25oC, 60% relative humidity) in dark. 60Co gamma cell (4523 Ci, Hungary) was used to supply an ionizing radiation source with a dose rate of 1.28 kGy.hr-1 at the Sarayköy Gamma Radiation Facility of Turkish Atomic Energy Agency in Ankara. Unirradiated GTR membranes were used as controls to detect physicochemical and antimicrobial activity changes resulting from the action of ionizing radiation. EtO sterilization (3M Sterivak, USA) was performed at Hacettepe University according to (ISO)10993-7 (Biological Evaluation of MDs-Ethylene Oxide Sterilization Residuals). The gas sterilization process consists of three steps, which the first step was humidity preconditioning at 55oC for 24 h. The

2.1 Materials The biodegradable GTR biomaterials are listed in Table 1. All the GTR membranes were non-sterile and all the chemicals used in experiments were of analytical grade. 2.2 Methods Schematic diagram of the experimental protocol is shown in Table 1. List of biodegradable GTR membranes used in the study. CODE COMPOSITION M1

COMPANY

PLGA

BioMesh® (Sarkcare, India)

M2

PLA

Epi-Guide® (Curusan, USA)

M3

Collagen

BioMend (Zimmer Dental, USA)

M4

Collagen

Collagen AT (Sistema, Italy).

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forming units (cfu) in 1 mL were calculated. The Sterility assurance level (SAL) dose calculations were calculated on the samples, which were infected with Bacillus pumilus spore suspension (6 × 106 cfu.mL-1). SAL 10-6 dose was calculated from the logarithmic microorganism death graphics. The Limulus amebocyte lysate (LAL) test was used to detect and quantify endotoxins, which is based on gel-clot formation to confirm the sterilized membranes are bacterial endotoxin free.

second step was EtO gas cycling in an autoclave with a concentration of 600 mg.L-1 at low pressure and temperature (40-55oC) for 4 h. The last step was degassing period for 72 h at 40oC. EtO sterilization was performed only for the most gamma stable GTR membrane that was selected after the physicochemical analysis. The amount of EtO and ethylene glycol content on sterilized GTR membranes was determined with extraction method based on extraction and colorimetric determination of EtO. The absorbance was read with a spectrophotometer (Shimadzu, Japan).

2.9 Animal Experiments Animal experiments were performed after the approval of the Hacettepe University Ethics Committee (2007/25-8). Skeletally mature male New Zealand rabbits weighing 2,5-3 kg were used for the in vivo experiments including implantation studies and histopathological examinations. Implantation of the dental biomaterials in rabbit cranial defects was performed as described previously.19 Briefly, the rabbits were anesthetized with intramuscular Xylasin® and Ketamin HCl® (1:1, v/v) and skull bone incisions (approximately 4-5 mm) were created by removing cortical bone with an ultrasonic device in the middle of the skull. During the surgical procedure was performed with the caution for the underlying dura mater and sagittal sinus. The chosen diameter corresponds to the critical size defect in the experimental model and has been reported to prevent spontaneous healing during the lifetime of the animal.17,19 The placement of dental membranes was shown in Figure 7. Animals were sacrificed by the injection of 2 mL Lysthenon® Forte (succinyl-bis-cholin 100 mg. 5 mL-1) intracardiacly on day 3, 1st, 2nd, 3rd, 6th and 12th week. The skull specimens were harvested and immersed in a 10% formalin fixative for histological preparation. After staining by haematoxylin and eosin, the sections were examined and graded on a scale.

2.4 FTIR Analysis FTIR spectrum measurements were recorded by Spectrum One FTIR spectrophotometer (Perkin Elmer, England). The membrane samples were directly measured. The main parameters of IR setting were the resolution for 4 cm-1. 2.5 DSC analysis The thermal properties of GTR membranes were analysed with DSC-60 (Shimadzu, Japan). 5 and 10 mg of samples were heated from 40oC to 220oC and cooled down to 40oC and finally reheated back to 220oC with a temperature rate of 10oC.min-1. The information from the DSC curves was used to determine the onset of melting point (Tm). 2.6 TGA Analysis Thermal gravity (TG) analyses were performed with DTG60H (Shimadzu, Japan) under nitrogen atmosphere and a temperature rate of 5oC.min-1. 2.7 SEM Analysis Membranes assigned for scanning electron microscopic (SEM) examinations by using Zeiss EVO and JEOL (SEM 8100, Germany). The samples were coated with gold palladium and studied at high magnification.

2.10 Statistics Statistical analysis was performed using Prism software (Graphpad, San Diego, USA). Differences in resorption times were tested for significance using the Student’s t-test and Pvalues of less than 0.05 were considered significant. Values for the standard error were calculated and are presented.

2.8 Sterility and Apyrogenicity For the sterility test, Fluid Thioglycolate Medium (FTM) and Tryptic Soy Broth (TSB) media was used. Raw samples were shaken in sterile distilled water and 100 µl of water was inoculated to FTM and TSB. They were incubated 14 days, at 35oC for FTM and 25oC for TSB. After 14 days, the cloudy tubes were considered as non-sterile and the clear tubes were considered as sterile. The dental biomaterials were irradiated with various radiation dose levels (1, 5, 10, 25, 50 kGy) and incubated in TSB plates at 35-37oC. Bacillus pumilus colonies were enumerated and colony

3. Results and Discussion An effective sterilization method must guarantee the required sterility assurance level with a minimum deleterious effect on the chemical, physical and biological properties of the biomaterials.20,21 In standard practice gamma radiation and EtO sterilization is the most common sterilization methods used to sterilize the polymeric biomaterials, because dry heat and steam

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addition, the peak intensity around 3440-3650 cm -1 was increased which might be due to the formation of alcohol groups or water molecules depending on oxidation.28,29 The bands at 1239 cm-1 are assigned to amide III, while bands at 1550 cm-1 are assigned to amide II that arises from N-H bending vibrations coupled to C-N stretching vibrations for collagen-based membranes. There are also bands present at 1645 cm-1 in the spectra originates from C=O stretching vibrations coupled to NH bending vibration that assigned to amide I. The process of gamma radiation sterilization has shown change on FTIR spectra of M3 and M4 where the peaks at 1250 cm -1 and 1255 cm-1 were shifted to 1290 cm-1 and 1270 cm-1 respectively. The experimental results of FTIR spectrum indicated that the presence of chemical bonding changes in collagenous intermolecules after gamma radiation. In addition sterilization of collagen with gamma radiation leads to the loss of nitrogen and amino acids and chemical transformation of amino acids, breakdown of peptide bonds, hydrogen bridges along with cross-linking of polypeptide chains of collagen.30 Denaturation of collagen has been found to lead to reduction in the intensity of amide peaks, narrowing of amide I band and cause change between 1243-1269 cm-1. Jiang and co workers10 showed that absorptive peaks of stretching vibration of amide group have changed in IR spectrum of collagen membrane after radiation. They also concluded that the observed changes between 12431269 cm-1 might be due to the denaturation of the collagen. DSC curves of GTR membranes after gamma radiation sterilization are given in Figure 3. In previous studies with synthetic biomaterials, it was observed that gamma radiation causes some changes in DSC curves, especially after 25 kGy. It might be related with changes in polymer matrix and radiolytic degradations such chain scission that leads decrease in molecular weight.22,31 It has been also reported in previous

sterilization can cause rigorous degradation on heat and moisture sensitive polymers.3,22 EtO may retain on the sterilized materials, and EtO and its degradation products have been shown to cause chronic toxicity. Moreover, EtO has mutagenic and carcinogenic effects, due to its interaction ability with DNA.23 The main advantages of gamma radiation sterilization over EtO sterilization are, no toxic residue formed and no areration is needed after gamma radiation sterilization.20,24,25 This current study sought to determine the effect of gamma radiation sterilization on the physicochemical, microbiological and regenerative properties of dental biomaterials and to compare the physicochemical and in vivo results with EtO sterilization. Biodegradable polymers such as PLA, PLGA and collagen are used for a variety of biomedical applications in the pharmaceutical and medical industry during the past decades.26 PLA, PLGA and collagen based dental biomaterials were sterilized with gamma radiation with different radiation doses and the effects of gamma radiation sterilizations were evaluated with physicochemical and microbiological assays. FTIR spectrums of GTR biomaterials after gamma radiation sterilization are shown in Figure 2. The spectrums of M1 were slightly different from the unirradiated one, however these changes were very small. After gamma radiation sterilization, new peak formation at 3320 cm-1 and increase in peak intensity at 3505 cm-1 was discovered. In the 1400-1800 cm-1 spectral region, the dominant bands in the FTIR spectra at 1643 cm-1 and 1540 cm-1 are assigned to the amide I and amide II vibrations respectively. Important modifications appear during the gamma sterilization in the range of 1736-1710 cm-1 corresponding to stretching vibrations of the carbonyl groups.27 New absorption band, which is related with formation of CO2 was determined at 2340 cm-1, after the gamma radiation sterilization of M2. In

Figure 2. The FTIR spectrum of gamma sterilized dental biomaterials with different radiation doses.

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Figure 3. The DSC curves of gamma sterilized dental biomaterials with different radiation doses.

studies that gamma radiation induces free radicals in polymer matrix by chain scission and the extent of molecular weight reduction was radiation dose dependent which causes polymer degradation.22,28 The change in DSC thermographs was more significant on collagen-based GTR biomaterials in comparison with PLGA and PLA, which is assigned to the scission of the collagen polypeptide chain. The peak located around 100oC is a result of the water bonded to molecules and unfolding of triple helical structure. The changes below 200oC are due to the melting of crosslinked part of collagen.10 Previous studies indicated that collagen keeps its physical properties only up to 10 kGy radiation dose.2 Effects of radiation on the thermal stability of GTR biomaterials were determined with TGA analysis as shown in Figure 4. From the TGA curves, it can be clearly concluded that the thermal stability of M4 decreased after gamma radiation sterilization

and three stages of decomposition was observed at 50 kGy radiation dose. The first stage may correspond to the loss of adsorbed and bound water. The second stage of weight loss starts at 230oC and continues up to 330oC during which there was 60% weight loss due to the degradation of collagen. The temperature that causes to 50% total weight loss were compared after sterilization with different radiation doses in Table 2. The gamma radiation sterilization decreased the thermal stability of collagen-based GTR biomaterials due to the 50% weight loss temperatures was shifted to lower temperatures with radiation dose. In previous studies it was shown that gamma radiation leads to significant collagen degradation and to a decrease in denaturation temperature and tensile strength.32 SEM analyzes were performed in order to examine the influence of gamma radiation on the surface and pore structures of the GTR biomaterials as shown in Figure 5. The SEM analysis clearly showed that gamma radiation did not change the pore size and surface characteristics of M1. The pore size of M2 decreased due to the radiation sterilization and the membrane surfaces became rougher probably due to significant Table 2. 50 % weight loss temperatures after gamma radiation sterilization 50 % weight loss temperatures (oC) Radiation dose (kGy)

Code

Figure 4. The TGA curves of gamma sterilized dental biomaterials with different radiation doses.

345

0

5

10

25

50

M1

342

339

340

340

338

M2

348

342

342

338

334

M3

330

311

311

311

310

M4

379

343

337

343

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Figure 6. The physicochemical investigation of PLGA-based dental membrane after sterilization with gamma and EtO sterilization; (A) The FTIR spectrum of gamma and EtO sterilized PLGA-based dental membrane; (B) The DSC curves of gamma and EtO sterilized PLGA-based dental membrane; (C) The TGA curves of gamma and EtO sterilized PLGA-based dental membrane; (D) SEM analysis of EtO sterilized PLGA-based dental membrane.

Figure 5. The SEM analysis of gamma sterilized dental biomaterials before radiation sterilization and with 10 and 25 kGy radiation doses (2500 × magnification). Scale bar represents 20 µm.

scission of polymeric chains. In the literature it was observed that PLA mainly undergoes chain-scissions at doses below 250 kGy while at higher doses of radiation, crosslinking reactions increase as a function of the increasing radiation dose.33 In previous studies, it was found that microstructural changes were significant on PLA and the pore size was dramatically changed after 50 kGy radiation dose.29 The change in surface characteristic and pore structure was the most significant on M3 and M4 collagen-based membranes. The pore size of M3 was increased and the surface became rougher after gamma radiation sterilization. The pore size of M4 was between 510 µm before radiation, and it was increased 5-10 fold after radiation sterilization with even 10 kGy. Additionally, the porotic structure of M4 became much denser due to gamma radiation exposure. It was shown previously that, collagen fibril diameter increased even after low radiation doses.34 Wiegand and co-workers showed that, gamma radiation of collagen results in a loss of bril stability. They also concluded that radiation doses higher than 20 kGy lead to a severe decrease of mechanical strength of the collagen that might be due to the chain scissions via radical formation.35 GTR biomaterials are in direct contact with biological systems and the sterility and apyrogenicity is a regulatory requirement for the GTR biomaterials before human use.16,36,37 According to the sterility test results no microbial growth was observed even after 10 kGy radiation dose. Apyrogenicity was examined with the LAL test, which is an in vitro assay for the detection and quantitation of bacterial endotoxins, which are the most common pyrogens. No clothing was observed, indicating the pyrogen free status of GTR membranes after radiation sterilization. Sterility Assurance Levels (SAL) of M1, M2, M3 and M4 were 14.8, 12.8, 11.5 and 13.3 kGy, respectively. 25 kGy of radiation dose was the recommended sterilization dose by ISO 11137 for

health care products and it was mentioned that the sterilization dose must comply for each kind of the medical product on its bioburden.38 To ensure the sterility with lower radiation doses will help to reach the requirements of sterilization that needs to be safe, time- and cost-saving and not to cause any physicochemical or mechanical damage on materials.14,37,39 GTR membranes were analyzed with different analytical and microbiological examinations and all the results indicated that PLGA-based, M1 was the most gamma stable GTR biomaterial. M1 was also sterilized with EtO to compare the effect of two sterilization methods on physicochemical and histopathological properties of dental membrane. The comparison of two sterilization methods with FTIR, DSC, TGA and SEM on M1 was shown in Figure 6. No change was observed with FTIR, DSC and TGA analysis after EtO sterilization. However, 3-fold increase in pore size was observed after EtO sterilization of M1 and the fibrous structure of the biomaterial became more dense and closer as shown in Figure 6. In a previous report it was mentioned that EtO sterilization resulted deformation of biodegradable polymers and reduced the physical properties of them including the yield stress and break stress.40 Toxic EtO residual was determined colorimetrically and the amount of EtO residual was calculated. The amount of EtO and ethylene glycol were calculated and found less than 10 ppm (mg.l-1), which remained in the official range.23,41 The main problem associated with the EtO sterilization is the toxic residue, which lead potential undesirable effect on patient health and the environment. The series of standards governing on the biologic testing of medical devices include the International Organization

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Figure 7. In vivo experiments on PLGA-based dental biomaterials after gamma and EtO sterilization. (A) The cranial implantation of gamma and EtO sterilized PLGA-based dental biomaterials. 1. Anaesthesia of the rabbits before surgical procedure; 2. Main incision and exposure of the cranial vault; 3. Bone removal and clearance of the surgical site before implantation; 4&5. Implantation of the gamma and EtO sterilized PLGA-based dental biomaterial. (B) SEM picture of the implanted PLGA-based membrane (magnification × 100). Scale bar represents 100 µm. (C) Schematic representation of the implantation in rabbit cranial defects.

Figure 8. The histopathological examination of biodegradable GTR membranes implanted to skull. Bone formation of control (A, D, G), gamma sterilized PLGA-based dental biomaterial (B, E, H) and EtO sterilized M1 (C, F, I) after 2nd, 6th and 12th week of implantation. The arrows in the first raw represent granulation tissue after surgical procedure and the arrows in the second and last raw represent new bone formation.

for Standardization (ISO) 10993-7 (Biological Evaluation of MDs-Ethylene Oxide Sterilization Residuals), which specifies the toxicological risk of the residue to the patient according to the length of the time the patient is likely to be exposed to the device.23,42 The surgical procedure for the implantation of sterilized M1 was shown in Figure 7A. There were four implantation sites one for gamma sterilized M1, one for EtO sterilized M1 and two for defects as control (Fig 7B). From the first week of implantation, no signs of inflammation or discharge in the operation sites were observed and M1 did not migrate from the sites it has been implanted to. According to the histopathological examinations as shown in Figure 8, the implants were covered with normal soft tissue and no implant remnants or foreign body reactions were found. On the first week of the implantation, bleeding and granulation tissue was observed. In addition, very slight histological response was noticed after implantation of the membranes. Bone formation was started at the 2nd week of operation, which was much more faster with gamma sterilized membrane in comparison with control and EtO sterilized one. It was also found that 60.167 ± 3.058% of new bone formation was completed at the end of 6th week with gamma sterilized M1 and it was totally fragmented after 6 weeks and could not be detected at the end of 12 weeks. At the end of 12 weeks, the bone formation was completed with gamma sterilized PLGAbased membrane, however the new bone formation was only 60.095 ± 2.019% for EtO sterilized one and 20.093 ± 1.245% for the defect. Moreover gamma radiation sterilization did not

change the resorption and regenerative properties of M1. The bone formation process was significantly faster (P < 0.05) with gamma sterilized M1, in comparison with EtO sterilized one. The slower bone formation of EtO sterilized M1 might be due to the changes in the dimensions and volume of the dental biomaterial as reported previously.43 Gamma radiation and EtO sterilization on the dimensions, morphology, molecular weight and degradation on PLGA scaffolds were studied and it was shown that scaffolds shrank to 60% of their initial volume and the dimensions were affected dramatically after EtO sterilization.43 In a different study it was indicated that gamma irradiation has no significant effect on the osteoinductivity of demineralised bone matrix. In addition histological evaluation demonstrated that gamma radiation had no negative effects on the new bone formation and did not cause an increase in inflammation.44 The effect of sterilization methods on bone morphogenetic proteins was compared with gamma radiation and EtO sterilization. It was found that the new bone areas were 45% higher in the gamma sterilized groups than EtO sterilized ones, because the bone morphogenetic proteins are more sensitive to EtO sterilization than gamma radiation.45 Our results show that gamma radiation sterilization is the most suitable method for sterilizing PLGA-based devices for tissue engineering applications because it does not alter the properties of the dental biomaterial. The material used as a GTR membrane must be biocompatible, nontoxic, nonantigenic, and easy to be sterilized and use. The barrier function must be established and maintained long enough for bone cells to

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proliferate into the defect. Our results fall within the boundaries of published other GTR membranes. Nieminen and co-workers showed that polymeric GTR membranes degrades completely in vivo with only mild histological responses, being in accordance with the results of the other membranes.17,46,47

membrane: a comparative study in dogs, Clin Oral Implants Res, 22, 802 (2011). 9. JS Son, SG Kim, SC Jin, et al., Development and structure of a novel barrier membrane composed of drug-loaded poly(lactic-coglycolic acid) particles for guided bone regeneration, Biotechnol Lett, 34, 779 (2012). 10. B Jiang, Z Wu, H Zhao, et al., Electron beam irradiation modification of collagen membrane, Biomaterials, 27, 15 (2006). 11. J Li, J Yang, X Zhong, F He, X Wu, G Shen, Demineralized dentin matrix composite collagen material for bone tissue regeneration, J Biomater Sci Poly ed., 24, 1519 (2013). 12. S Kim, KC Oh, DH Han, SJ Heo, IC Ryu, JH Kwon, CH Han, Influence of transmucosal designs of dental implant on tissue regeneration in beagle dogs, Int J Oral Maxillofac Implants, 25, 309 (2010). 13. KG Cornwell, A Landsman, KS James, Extracellular matrix biomaterials for soft tissue repair, Clin Podiatr Med Surg, , 26, 507 (2009). 14. NS Turker, AY Ozer, B Kutlu, R Nohutcu, H Bilgili, D Oztürk, M Ozalp, A Sungur, Gamma irradiation studies I. Dental Grafts, J Med Devices. 5, 031011, (2011). 15. IH Mohamed, Current perspectives of nanoparticles in medical and dental biomaterials, J Biomed Res, 26, 143 (2012). 16. E Rohm-Rodowald, B Jakimiak, Assessment of the sterilization of medical devices--an important challenge to health care in Poland, Przegl Epidemiol, 58, 501 (2004). 17. T Nieminen, I Kallela, J Keranen, et al., In vivo and in vitro degradation of a novel bioactive guided tissue regeneration membrane, Int J Oral Maxillofac Surg, 35, 727 (2006). 18. SJ Khurshid, Bioburden and theoretical sterility dose calculation for radiation sterilization of surgical cotton and bandages, J Pak Med Assoc, 43, 8 (1993). 19. W Ji, F Yang, J Ma, et al., Incorporation of stromal cell-derived factor-1alpha in PCL/gelatin electrospun membranes for guided bone regeneration, Biomaterials, 34, 735 (2013). 20. BP Fairand, N Fidopiastis, Radiation sterilization of aseptically manufactured products, PDA J Pharm Sci Technol, 64, 299 (2010). 21. JA Bushell, M Claybourn, HE Williams, DM Murphy, An EPR and ENDOR study of gamma- and beta-radiation sterilization in poly (lactide-co-glycolide) polymers and microspheres, Journal of controlled release, 110, 49 (2005). 22. CC Chen, JY Chueh, H Tseng, HM Huang, SY Lee, Preparation and characterization of biodegradable PLA polymeric blends, Biomaterials, 24, 1167 (2003). 23. GC Mendes, TR Brandao, CL Silva, Ethylene oxide sterilization of medical devices: a review, American journal of infection control, 35, 547 (2007). 24. RM Brinston, BK Wilson, Converting to gamma-radiation sterilization: an overview for medical device manufacturers, Medical device technology, 4, 18 (1993). 25. M Cignitti, Sterilization by gamma radiation of materials for pharmaceutical use: the problem of its effect on chemical structure, Boll Chim Farm, 131, 71 (1992). 26. N Bitterman, Design of medical devices--a home perspective. Eur J Intern Med, 22, 39 (2011). 27. M Guo, WT Rong, J Hou, et al., Mechanisms of chitosan-coated poly(lactic-co-glycolic acid) nanoparticles for improving oral

4. Conclusions Gamma radiation sterilization is a time and cost-effective sterilization method, which doesn’t have any toxic residue for human health, and environment unlike EtO sterilization. In conclusion, the gamma radiation sterilization could be used to sterilize dental and medical biomaterials for tissue engineering applications after validation of the gamma sterilization dose. Acknowledgement: The authors wish to thank H.U. Research Foundation (Project No: 07.01.301.008) for the valuable financial support. Conflict of Interest: N.Selcan Turker, A.Yekta Ozer, Burak Kutlu, Rahime Nohutcu, Arzu Sungur, Hasan Bilgili, Melike Ekizoglu, and Meral Ozalp declare that they have no conflict of interest.

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