- Email: [email protected]
Mechanical-chemical analyses and sub-chronic systemic toxicity of chemical treated organic bovine bone
Accepted Manuscript Mechanical-chemical analyses and sub-chronic systemic toxicity of chemical treated organic bovine bone Kwang-il Lee, Jung-soo Lee, Keun-soo Lee, Hong-hee Jung, Chan-min Ahn, Youngsik Kim, Young-bock Shim, Ju-woong Jang PII:
S0273-2300(15)30111-2
DOI:
10.1016/j.yrtph.2015.10.027
Reference:
YRTPH 3441
To appear in:
Regulatory Toxicology and Pharmacology
Received Date: 17 September 2015 Revised Date:
26 October 2015
Accepted Date: 27 October 2015
Please cite this article as: Lee, K.-i., Lee, J.-s., Lee, K.-s., Jung, H.-h., Ahn, C.-m., Kim, Y.-s., Shim, Y.-b., Jang, J.-w., Mechanical-chemical analyses and sub-chronic systemic toxicity of chemical treated organic bovine bone, Regulatory Toxicology and Pharmacology (2015), doi: 10.1016/ j.yrtph.2015.10.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Cut into small pieces of bone block
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Cleaning with sterile water
1st. 3% hydroxide treatment
Chloroform and methanol (50v%:50v%) treatment
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1st. 70% ethanol treatment
Characterization of materials Scanning electron microscopy X-ray diffraction pattern Inductively coupled llama-mass spectroscopy Mechanical test Chemical treated bone vs. Heat treated bone
Rinsing with sterile water
Freeze drying
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Hematological test Blood biochemistry test Microscopic observation evaluation Implantation in gluteal muscles of SD rats for 12 weeks (Female: 20, Male: 20) bovine bone graft vs. physiological saline
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4% sodium hydroxide treatment
Better mechanical property Chemical and biologically safe material
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2nd. 70% ethanol treatment
The chemical treated organic bovine bone
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2nd. 3% hydroxide treatment
Gamma irradiation at 25kGy
Chemical treated organic bovine bone
Safety and effectiveness tests
Possibility of clinical use
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Mechanical-chemical analyses and sub-chronic systemic toxicity of chemical treated
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organic bovine bone
3 Kwang-il Lee*, Jung-soo Lee, Keun-soo Lee, Hong-hee Jung, Chan-min Ahn, Young-sik Kim, Young-
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bock Shim, Ju-woong Jang*
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The Institute of Biomaterial and Medical Engineering, Cellumed Co., Ltd., Seoul, Republic of Korea
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*Corresponding authors.
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The Institute of Biomaterial and Medical Engineering, Cellumed Co., Ltd.
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#608 Acetechnotower 9th. Gasan-dong, Geumcheon-gu, Seoul, 153-782, Republic of Korea
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E-mail address: [email protected] (K.I. Lee), [email protected] (J.W. Jang)
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Telephone: +82-2-2104-0475
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Fax: +82-2-2104-0474
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Abstract Sequentially chemical-treated bovine bone was not only evaluated by mechanical and chemical analyses but
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also implanted into the gluteal muscles of rats for 12 weeks to investigate potential local pathological effects
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and systemic toxicities. The test (chemical treated bone) and control (heat treated bone) materials were
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compared using scanning electron microscope (SEM), x-ray diffraction pattern, inductively coupled plasma
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analysis, and bending strength test. In the SEM images, the micro-porous structure of heat-treated bone was
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changed to sintered ceramic-like structure. The structure of bone mineral from test and control materials was
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analyzed as100% hydroxyapatite. The ratio of calcium (Ca) to potassium (P), the main inorganic elements, was
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same even though the Ca and P percentages of the control material was relatively higher than the test material.
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No death or critical symptoms arose from implantation of the test (chemical treated bone) and control
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(physiological saline) materials during 12 weeks. The implanted sites were macroscopically examined, with all
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the groups showing non-irritant results. Our results indicate that chemical processed bovine bone has a better
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mechanical property than the heat treated bone and the implantation of this material does not produce systemic
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or pathological toxicity.
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Keywords: Bone graft, Bovine bone, Bending strength, Sub-chronic, Toxicity, Xenograft
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20 21 Abbreviations
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SD, Sprague-Dawley; HTOs, High tibia osteotomies; NP, Not present; WBC, White blood cell; NE,
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Neutrophils; LY, Lymphocyte; MO, Monocyte; EO, Eosinophil; BA, Basophil; RBC, Red blood cell; Hb,
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Hemoglobin; HCTL, Hematocrit; MCV, Mean corpuscular volume; MCH, Mean corpuscular hemoglobin;
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MCHC, Mean corpuscular hemoglobin concentration; RDW, Red cell distribution width; PLT, Platelet; MPV,
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Mean platelet volume; ALB, Albumin; ALP, Alkaline phosphatase; CA, Calcium; CHO, Total cholesterol;
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CRE, Creatinine; GLU, Glucose; AST, Aspartate aminotrasferase; ALT, Alanine aminotransferase; TP, Total
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protein; BUN, Blood urea nitrogen; T-BIL, Total bilirubin; IP, Inorganic phosphorus; TG, Triglycerides; CPK,
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Creatine phosphokinase; Na, Sodium; K, Potassium; Cl, Chloride
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1. Introduction To overcome the increasing demand for autografts or allografts, bone-grafting substitutes have long been
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examined and commercialized in the field of muscular skeletal surgery (Abbas et al., 2007; Zarate-Kalfoqulos
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and Reyes-Sanchez, 2006; Scarborough, 1992). All grafting materials must be approved by ISO10993
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regulations or be specifically validated for safety and effectiveness before commercialization (Galia et al.,
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2008). Xenografts such as bovine bone grafts have been used in dental augmentation surgery and more recently
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in orthopedic surgery (Li et al., 2013; Athanasiou et al., 2010). The manufacturers of commercial xenografts
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perform validation studies, including viral-inactivation studies, toxicity tests, and animal-implantation studies
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for safety assurance purposes (Laurencin and El-Amin, 2008; Sammarco and Chang, 2002). Most xenografts are
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heat treated to prevent viral transmission and immune responses in recipients (Kim et al., 2013; Testori et al.,
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2012). However, applications with heat-treated xenografts are limited because heating changes their mechanical
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or biological properties, making them unsuitable for use in orthopedic surgery (Campana et al., 2014; Yan et al.,
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2008). Therefore, many orthopedic surgeons are concerned about the safety and efficacy of xenografts for
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clinical use in cases involving large bone defects, long bone fractures, spinal fusions, and high tibia osteotomies
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(HTOs) where xenografts need to provide mechanical support.
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Chemical treatment of xenografts has been employed in attempt to circumvent the problems associated with
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heat treatment (Kemper et al., 2011). However, even with chemical treatments, the safety and efficacy of
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xenografts for clinical use remain controversial because residual chemicals in the xenografts may cause adverse
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effects. Thus, long-term animal studies for clinical pathology, including hematology and clinical chemistry,
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need to be performed. Although the methods involved in sub-chronic systemic toxicity testing are routine, the
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results may vary across device manufacturers, as each company uses different processing techniques. The
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purposes of this study were to compare between chemical treated and heat treated bones using chemical and
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mechanical analysis. And to evaluate the potential sub-chronic systemic toxicity of bovine bone grafts after
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sequential chemical processing and propose the optimal treatment conditions for safety and effectiveness.
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2. Materials and methods
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2.1. Animals used for the implantation study Forty 8-week-old Sprague-Dawley (SD) rats (ORIENT BIO Inc., Seongnam, Korea) were used as implant
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recipients. The animal-treatment protocol conformed to the Guidelines for Care and Use of Laboratory Animals
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and was approved by the Committee of Experimental Animal Science (The College of Medicine, Seoul National
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University, Seoul, Korea). Test and control substances were administered separately to 10 male and 10 female
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rats each. Skin marking was used to distinguish between the test and control animals, with blue marking used
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during the acclimation period and black marking used during the main study. All animals were housed in
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polycarbonate cages (150 × 350 × 180 mm; W: L: H) during the quarantine, acclimation, administration, and
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observation periods.
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Natural bovine bone grafts (male, femoral and tibial bone, again less than 24 months) were processed using
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chemical reagents without heating. The graft material was prepared following the standard operating procedure
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of manufacturer (Cellumed Co., Ltd., Seoul, Korea). Briefly, the bovine bones were cut into proximal, middle,
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and distal parts using a band saw. All separated bone shafts were cleaned ultrasonically using 15 mL/g of 3%
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hydroxide solution and then with the same volume of 70% ethanol, for 1 hour during each cleaning step. The
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cleaned some shafts were cut into smaller blocks (10 × 25 × 13 mm; W: L: H) with a band saw and the other
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blocks were ground using a bone-milling machine to yield bone powder with particle sizes ranging from 200 to
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1000 µm. The test substance was cleaned ultrasonically with a 1:1 chloroform: methanol solution (15 mL/g) for
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1 hour. Next, it was treated with the same volume of 3% hydroxide solution, followed by rinsing with deionized
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water for 30 minutes. It was subsequently treated with 15 ml/g of 70% ethanol for 1 hour at room temperature
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and then with the same volume of 4% NaOH for 30 minutes, agitated for 15 minutes, ultrasonicated for 15
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minutes, and rinsed for 30 minutes with deionized water. Finally, it was freeze-dried for 12 hours at-70 ºC to
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ensure a water residue of ≤6%, placed in glass vials, stored at-70 ºC, and sterilized by gamma (Co60) irradiation
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at a dose of 25 kGy.
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2.3. Preparation of test and negative-control substances
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The bovine cortical bone blocks after chemical processing were used for mechanical test and powder was used
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for chemical analysis and animal implantation as described above. For the comparison of mechanical test, the
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2015; Tadic et al., 2004). And the negative-control substance for the implantation was physiological saline
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(Daihan Pharm Co., Ltd., Seoul, Korea). Polyethylene micro-medical tubing (Scientific Commodities, Inc.,
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Seoul, Korea) was sterilized with ethylene oxide gas and used as the implantation tube following the
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international standard (ISO 10993-6: 2007E). For the implantation study, rats were administered 0.025 ml of the
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test or control product via sterile tubing (length: 10 mm; outside diameter: 2.08 mm; inner diameter: 1.57 mm).
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For implantation, rats were anesthetized with 40–50 mg/kg ketamine and 20–30 mg/kg xylazine. The gluteal
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region, where the implantation procedure was conducted, was shaved and disinfected with 70% ethanol and
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povidone. Approximately 2 cm of the gluteal skin was cut to expose the underlying muscle tissue, after which
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the test or control material was implanted into the left and right gluteal muscles. After implantation, the muscle
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and skin were sutured using a standard surgical method and disinfected with povidone. The implants were
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remained in the rats for 12 weeks following the international standard (ISO 10993-11: 2006E).
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15 2.5. Observation points and test items
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2.5.1.Characterization of materials
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The morphology of the obtained bone grafts was examined by a scanning electron microscopy (SEM, S-4800;
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Hitachi, Tokyo, Japan). SEM revealed that the surface of all samples was covered with 7-nm-thick platinum (Pt)
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coating when the instrument was operated at 15 kV.
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X-ray diffraction pattern (XRD) was used to determine the structure of the individual samples. XRD
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measurements were performed with an X-ray diffractometer (D/Max 2500/PC, RIGAKU, Tokyo, Japan) with
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copper (Cu) radiation in the 2θ range from 10° to 70° (scan speed of 0.02° per second).
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Chemical analysis of materials was carried out by the Inductively coupled plasma-mass spectroscopy (ICP-MS)
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OPTIMA 5300DV (PerkinElmer, Waltham, MA, USA).
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2.5.2. Mechanical test
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All freeze dried test blocks were rehydrated by saline for 30 minutes. A bending test was performed by a
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universal testing machine (DUS-200, Oriental TM, Siheung, Korea) with a loading speed of 1 mm/min
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2.5.3. Clinical signs and mortality
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During the observation period, the animals were observed daily for appearance; food intake; water intake;
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histological reactions at acquisition, grouping, and once per week during the observation periods; and before
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implantation and sacrifice. The total and remaining amounts of feed were measured once per week during the
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study period.
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On the day the animals were sacrificed, blood was drawn from all surviving animals, and hematological tests
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were conducted using an automatic analyzer (ADVIA 2120; SIEMENS, USA). Prior to sacrifice, all animals
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were fasted overnight. During the autopsies, the abdomens were cut open in 5% CO2 environment, and blood
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was drawn from the aorta descendens. Ethylenediaminetetraacetic acid dipotassium salt was used as an
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anticoagulant.
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2.5.5. Blood biochemistry
Blood drawn from the aorta descendens during autopsy was centrifuged at 3,000 rpm for 10 min, and the
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resultant serum was used for biochemical tests conducted using an automatic analyzer (Hitachi 7180; Hitachi,
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Japan).
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2.5.6. Macroscopic examination of implantation sites
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The test- and control-substance implantation sites were observed macroscopically. Any changes identified
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visually around the implantation sites, such as inflammation, encystment, bleeding, necrosis, or discoloration,
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were evaluated and scored as shown in Table 1.
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Insert Table 1 here.
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2.6. Statistical analysis
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All statistical analyses were performed using commercially available software (SPSS v.15.0; IBM Corp.,
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Armonk, NY, USA). The independent t tests were used to analyze differences between groups, with significance
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levels set at * p < 0.05 and ** p < 0.01.
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3.1. Characterization analysis of materials
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In SEM images, after heat treatment, the macro-porous structure (50~100 um) was same as chemical treated
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bone. However, the micro-porous structure of heat treated bone was invisible. The structure of heat treated bone
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was changed to sintered ceramic-like structure forming permanent bonds and a dense materials which affects the
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microstructure and mechanical properties (Figure. 1).
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In the result of XRD, the both of chemical treated and heat treated bones were analyzed as same mineral
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appearance of 100% hydroxyapatite (Ca5P3O13H) in Figure 2.
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The result of ICP-MS showed that the main elements of chemical treated bone was Calcium (Ca, 36.9%),
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Potassium (P, 30.2%), and Carbone (C, 14.37%). The rest elements were remained less than 1%. The heat
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treated bone showed Ca (51.9%) and P (42.67%), however, C (0.68%) was significantly decreased after heat
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treatment. There was no difference of calculated Ca/P ratio between the chemical treated bone and heat treated
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bone (Table. 2).
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The average strength for the three point bending test was shown that the heat treated bone of 915 MPa was
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significantly decreased compared with natural bone of 2165.25 MPa. The change to sintered ceramic-like
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structure after heat treatment affected mechanical property of bone tissue. However, there was no significant
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difference between the chemical treated bone of 1749.75 MPa and natural bone. Moreover, the strength of
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chemical treated bone was significantly higher than heat treated bone (Figure 3).
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3.2. Observation of clinical signs and mortality
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In this study, chemically treated bovine bone powder and physiological saline (control) were implanted into the
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left and right gluteal muscles of SD rats. During a 12-week study period, the mortality, clinical symptoms,
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weight change, and amounts of food and water intake were observed in these animals. Subsequently, the
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animals were sacrificed and examined both visually and histopathologically.
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No mortality or clinical symptoms arising from implanting the test material were observed during the study
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period. In addition, no significant difference was observed in the weight, amount of food and water intake, and
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3.3. Hematology test
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Hematological tests such as white and red blood cell counts, and hemoglobin measurements revealed no
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significant differences between the control and test groups, when studying both female and male rats. No
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abnormal signs related to the test substance were observed for the other clinical symptoms tested (Tables 3 and
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Performing a blood biochemistry test for various factors revealed no significant differences between the control
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and test groups (Table 5).
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At 12 weeks post-implantation, all animals were autopsied, and the implanted sites of animals in the test and
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control groups were compared to investigate local reactions (bleeding, necrosis, discoloration, infection, or
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encystment). However, no significant changes were observed (Table 6).
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ACCEPTED MANUSCRIPT 1 4. Discussion
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Bovine bone grafts have been used in dental bone augmentation for decades. Most commercial bovine bone
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grafts are heat treated to prevent viral transmission and to suppress immune responses in recipients by
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deproteinization. However, these heat-treated grafts may not suitable for some orthopedic surgeries requiring
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mechanical supports such as large bone void filling, long bone fractures, spinal fusions, and HTOs. Many long-
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term follow-up clinical studies have shown that commercialized bovine bone grafts, which were heat-treated
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during manufacturing, were not suitable for treating large bony defects or fracture patients. Therefore, the
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effectiveness of mechanical properties should be important character in orthopedic surgery.
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Recently, chemical processing of animal-derived graft materials has been applied to commercial grafts.
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However, residual organic materials may pose potential health risks. Thus, validation of each chemical process
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followed during manufacture is essential for ensuring the safety of xenografts.
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Most studies on xenografts have investigated animal implantation in terms of efficacy, rather than safety.
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Although grafting materials are in great demand for the treatment of muscular skeletal diseases, concerns
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regarding the suitability of xenografts for clinical implantation exist. Moreover, in orthopedic surgery, grafting
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materials must be able to provide mechanical strength for long bone fractures, spinal fusions, and bone fixation.
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For safety approval, xenografts are heat-treated to remove organic materials or inactivate transmissible viruses.
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However, heat treatment can impair the mechanical properties of bone-graft materials because it may cause
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change of micro-porous structure and degeneration of mineral components and denature collagen, both of which
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are related to bone strength (Shin et al., 2005; Wang et al., 2001; Wang et al., 2000).
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To overcome these disadvantages, we employed sequential chemical treatment during bovine bone processing.
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Previously, we confirmed that viral inactivation occurred in bovine-derived grafting materials using these
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conditions (Lee et al., 2012). Three steps were performed during the entire chemical-treatment procedure,
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including sequential treatment with 70% ethanol, 4% sodium hydroxide, and gamma irradiation, which resulted
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in viral inactivation. The upstream steps involved the removal of blood, lipids, proteins, and other organic
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materials using 3% hydroxide, 70% ethanol, and a mixture of chloroform and methanol.
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5. Conclusions To investigate biological safety and chemical-mechanical effectiveness of the chemical treated bovine bone,
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the difference of processing type did not affect main chemical component such as the Ca/P ratio and
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hydroxyapatite, the inorganic material. However, the micro-porous structure and carbon component were
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changed by heat treatment and the mechanical property was decreased even though the chemical treated bone
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did not show any significant difference compared with natural bone. The sub-chronic toxicity of bovine bone
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processed using the conditions described herein was implanted into the gluteal muscles of SD rats for a 12-week
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duration. Implantation of this material did not produce systemic or local pathological toxicity in SD rats; thus,
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our results indicate that chemically treated bovine bone is safe as xenografts for clinical use. Taken together with our previous study of viral inactivation in chemically processed bovine bone grafts, our
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collective findings suggest that the chemical treated organic bovine bone grafts can be used as biologically and
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chemically safe and mechanically effective grafting materials not only in dental surgery but also in orthopedic
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surgery.
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Conflicts of interest statement
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The authors declared no conflicts of interest.
4 Acknowledgement
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This study was supported by World Premier Materials (10037842) funded by the Ministry of Trade, Industry &
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Energy, Republic of Korea.
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References
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Campana, V., Milano, G., Pagano, E., Barba, M., Cicione, C., Lattanzi, W., Logroscino, G., 2014. Bone
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Kemper, N., Davison, N., Fitzpatrick, D., Marshall, R., Lin, A., Mundy, K., Cobb, R.R., 2011. Characterization
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substitutes: A systematic review. Clin. Implant. Dent. Relat. Res. 15, 645-653.
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Kim, Y., Nowzari, H., Rich, S.K., 2013. Risk of prion disease transmission through bovine-derived bone
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Lee, K.I., Lee, J.S., Jung, H.H., Lee, H.Y., Moon, S.H., Kang, K.T., Shim, Y.B., Jang, J.W., 2012. Inactivation
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of enveloped and non-enveloped viruses in the process of chemical treatment and gamma irradiation of
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Shin, S., Yano, H., Fukunaga, T., Ikebe, S., Shimizu, K., Kaku, N., Nagatomi, H., Masumi, S., 2005.
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Legend of figures
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Figure 1. Scanning electron microscopy. Chemical treated bovine bone, (A) x100, (B) x30,000, (C) x100,000,
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and heat treated bovine bone, (D) x100, (E) x30,000, (F) x100,000 Figure 2. X-ray diffraction pattern. Chemical treated (A) and heat treated (B) bovine bone
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Figure 3. Comparison of the groups of bending strength. Each value is expressed as mean (N=18). Values are
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expressed as MPa. * Significantly different from the control group at p < 0.05, ** p < 0.01.
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ACCEPTED MANUSCRIPT 1 Table 1. Specifications of encapsulation capsules Capsule Width
Score
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0.6–1.0 mm
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Not Present
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M AN U
Exceed 2.0 mm
NP
ACCEPTED MANUSCRIPT 1 2
Table 2. Summary of ICP-MC test results with chemical treated bovine bone and heat treated bovine bone.
3
(N=6, ** Significantly different from the control group at p < 0.01.) Heat Treated Bovine Bone
CaO**
36.90
51.90
P2O5**
30.20
C**
14.37
MgO**
0.84
Na2O**
0.42
S
0.08
SrO
0.05
BaO
0.03
ZnO
0.01
Al2O3
0.01
CuO
0.01
Fe2O3
0.01
K2O
0.01
MnO2
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
31.43
3.58
1.22
1.22
SiO2 Ignition Loss**
4 5 6 7 8 9 10 11 12 13
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Ca/P ratio
42.67 0.68 1.11
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0.56 0.08
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0.07
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ZrO
EP
TiO2
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Chemical Treated Bovine Bone
0.04 0.02 0.01 0.01 0.01 0.01
ACCEPTED MANUSCRIPT 1 2
Table 3. Summary of hematological test results from male and female SD rats with bovine bone implants in the
3
left and right gluteal muscles after 12 weeks. (N=10) Summary of hematological tests WBC
NE
LY
MO
EO
BA
NE
LY
MO
EO
Units
K/µL
K/µL
K/µL
K/µL
K/µL
K/µL
%
%
%
%
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Tests
Control Mean
10.38
1.48
8.46
0.21
S.D
1.432
0.434
1.458
0.065
0.045
0.007
Mean
10.14
1.32
8.39
0.20
0.09
S.D
2.693
0.499
2.486
0.079
0.044
Control 7.33
1.04
S.D
2.839
0.978
Test
14.49
81.21
2.05
1.11
4.410
4.640
0.488
0.375
0.01
13.60
82.22
2.01
0.83
0.005
5.699
5.081
0.561
0.343
M AN U
0.01
SEX: FEMALE
5.91
0.22
0.05
0.00
12.95
82.01
2.73
0.68
1.773
0.192
0.025
0.005
6.278
6.647
0.993
0.346
EP
Mean
0.12
TE D
Test
Mean
4.88
0.61
4.05
0.11
0.04
0.00
13.80
81.52
2.33
0.90
S.D
1.712
0.244
1.727
0.042
0.020
0.004
6.909
7.356
0.596
0.411
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4 5
SC
SEX: MALE
6
Abbreviation used: WBC, White blood cell; NE: Neutrophils; LY: Lymphocyte; MO: Monocyte; EO:
7
Eosinophil; BA: Basophil
8 9 10
ACCEPTED MANUSCRIPT 1 2
Table 4. Summary of hematological test results from male and female SD rats with bovine bone implants in the
3
left and right gluteal muscles after 12 weeks (Continued, N=10) Summary of hematological tests BA
RBC
Hb
HCT
MCV
MCH
MCHC
RDW
PLT
MPV
Units
%
M/µL
g/dL
%
fL
pg
g/dL
%
K/µL
fL
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Tests
Mean
0.06
8.87
17.30
47.85
S.D
0.052
0.382
0.533
1.248
1.029
1.004
Mean
0.07
8.71
16.79
46.89
53.81
S.D
0.048
0.339
1.041
1.779
1.250
Control 0.06
8.03
S.D
0.052
0.389
Test
12.80
1028.30
6.47
1.425
0.843
123.208
0.445
19.27
35.77
13.18
1057.60
7.00
0.787
1.199
1.131
119.049
0.663
SEX: FEMALE
44.88
55.91
19.93
35.66
11.61
1062.80
7.59
0.918
2.101
1.436
0.663
0.759
0.451
114.658
0.739
Mean
0.10
8.22
16.82
46.90
57.08
20.49
35.92
11.17
1004.50
7.88
S.D
0.047
0.428
0.666
2.275
1.692
0.768
0.708
0.211
78.869
1.085
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4 5
19.54
16.01
EP
Mean
53.96
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Test
36.18
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Control
SC
SEX: MALE
6
Abbreviation used: RBC: Red blood cell; Hb: Hemoglobin; HCTL Hematocrit; MCV: Mean corpuscular
7
volume; MCH: Mean corpuscular hemoglobin; MCHC: Mean corpuscular hemoglobin concentration; RDW:
8
Red cell distribution width; PLT: Platelet; MPV: Mean platelet volume
9 10
ACCEPTED MANUSCRIPT 1 2
Table 5. Summary of serum biochemical test results from male and female SD rats with bovine bone implants in
3
the left and right gluteal muscles after 12 weeks (N=10) Summary of biochemical test results ALB
ALP
CA
CHO
CRE
GLU
Units
g/dL
IU/L
mg/dL
mg/dL
mg/dL
mg/dL
AST
ALT
TP
mg/dL
IU/L
g/dL
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Tests
Control 2.3
255.4
9.9
70.3
0.6
S.D
0.25
53.87
0.84
14.70
0.07
N
10
10
10
10
Mean
2.3
278.2
10.6
S.D
0.13
36.06
0.41
N
10
10
Tests
BUN
T-BIL
Units
mg/dL
mg/dL
115.9
31.9
6.0
31.77
32.80
7.32
0.61
10
10
10
10
10
835
0.6
210.3
110.1
33.4
6.1
16.93
0.09
20.22
34.75
6.45
0.30 10
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Test
185.6
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Mean
SC
SEX: MALE
10
10
10
10
10
IP
TG
CPK
Na
K
Cl
mg/dL
mg/dL
U/L
mmol/L
mmol/l
mmol/L
EP
10
AC C
Control
SEX: FEMALE
Mean
16.8
0.1
8.5
51.8
541.4
137.8
4.8
100.9
S.D
1.35
0.01
1.22
24.69
358.61
11.55
0.76
7.88
N
10
10
10
10
10
10
10
10
Mean
32.7
0.3
9.2
41.8
363.2
142.5
9.8
103.5
S.D
47.22
0.05
0.70
21.67
243.51
2.76
12.38
2.32
N
10
10
10
10
10
10
10
10
Test
ACCEPTED MANUSCRIPT 1 2 Abbreviation used: ALB: Albumin; ALP: Alkaline phosphatase; CA: Calcium; CHO: Total cholesterol; CRE:
4
Creatinine; GLU: Glucose; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; TP: Total
5
protein; BUN: Blood urea nitrogen; T-BIL: Total bilirubin; IP: Inorganic phosphorus; TG: Triglycerides; CPK:
6
Creatine phosphokinase; Na: Sodium; K: Potassium; Cl: Chloride
7 8
SC
9 10 11
M AN U
12 13 14 15
20 21 22 23 24 25 26 27 28 29 30
EP
19
AC C
18
TE D
16 17
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Table 6. Macroscopic observations of male and female SD rats with bovine bone implants in the left and right
4
gluteal muscles after 12 weeks
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Individual animals, reported by group SEX: MALE
Animal
Control 1/2
3/4
5/6
Left
0/0
0/0
0/0
Right
0/0
0/0
0/0
Test
7/8
9/10
Clinical
21/22
No. 0/0
Right
0/0
Clinical opinion
5 6 7 8
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
Nothing abnormal detected
SEX: FEMALE
25/26
Test
27/28
29/30
31/32
33/34
35/36
37/38
39/40
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
AC C
Left
23/24
19/20
0/0
Control
EP
Animal
17/18
0/0
TE D
Group
15/16
0/0
Nothing abnormal detected
opinion
13/14
M AN U
No.
11/12
SC
Group
Nothing abnormal detected
Nothing abnormal detected
ACCEPTED MANUSCRIPT 1 2 Figure 1.
(D
(E)
4 5
10 11 12 13 14 15 16 17 18 19 20
EP
9
AC C
8
(F)
TE D
6 7
(C)
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(B)
SC
(A)
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Figure 2.
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EP
TE D
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(A)
4 5 6 7 8
(B)
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Figure 3.
7 8 9
EP
6
AC C
5
TE D
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4
ACCEPTED MANUSCRIPT
Highlights
• The chemical treated bovine bone had significantly higher bending strength than the heat treated bone.
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• The chemical appearance of all the materials was same such as 100% hydroxyapatite and Ca/P ratio.
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• There was no significant sub-chronic toxicity of chemical treated bone in the rat muscle during 12 weeks.
S0273-2300(15)30111-2
DOI:
10.1016/j.yrtph.2015.10.027
Reference:
YRTPH 3441
To appear in:
Regulatory Toxicology and Pharmacology
Received Date: 17 September 2015 Revised Date:
26 October 2015
Accepted Date: 27 October 2015
Please cite this article as: Lee, K.-i., Lee, J.-s., Lee, K.-s., Jung, H.-h., Ahn, C.-m., Kim, Y.-s., Shim, Y.-b., Jang, J.-w., Mechanical-chemical analyses and sub-chronic systemic toxicity of chemical treated organic bovine bone, Regulatory Toxicology and Pharmacology (2015), doi: 10.1016/ j.yrtph.2015.10.027. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Cut into small pieces of bone block
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Cleaning with sterile water
1st. 3% hydroxide treatment
Chloroform and methanol (50v%:50v%) treatment
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1st. 70% ethanol treatment
Characterization of materials Scanning electron microscopy X-ray diffraction pattern Inductively coupled llama-mass spectroscopy Mechanical test Chemical treated bone vs. Heat treated bone
Rinsing with sterile water
Freeze drying
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Hematological test Blood biochemistry test Microscopic observation evaluation Implantation in gluteal muscles of SD rats for 12 weeks (Female: 20, Male: 20) bovine bone graft vs. physiological saline
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4% sodium hydroxide treatment
Better mechanical property Chemical and biologically safe material
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2nd. 70% ethanol treatment
The chemical treated organic bovine bone
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2nd. 3% hydroxide treatment
Gamma irradiation at 25kGy
Chemical treated organic bovine bone
Safety and effectiveness tests
Possibility of clinical use
ACCEPTED MANUSCRIPT 1
Mechanical-chemical analyses and sub-chronic systemic toxicity of chemical treated
2
organic bovine bone
3 Kwang-il Lee*, Jung-soo Lee, Keun-soo Lee, Hong-hee Jung, Chan-min Ahn, Young-sik Kim, Young-
5
bock Shim, Ju-woong Jang*
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The Institute of Biomaterial and Medical Engineering, Cellumed Co., Ltd., Seoul, Republic of Korea
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*Corresponding authors.
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The Institute of Biomaterial and Medical Engineering, Cellumed Co., Ltd.
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#608 Acetechnotower 9th. Gasan-dong, Geumcheon-gu, Seoul, 153-782, Republic of Korea
26
E-mail address: [email protected] (K.I. Lee), [email protected] (J.W. Jang)
27
Telephone: +82-2-2104-0475
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Fax: +82-2-2104-0474
29
ACCEPTED MANUSCRIPT 1
Abstract Sequentially chemical-treated bovine bone was not only evaluated by mechanical and chemical analyses but
3
also implanted into the gluteal muscles of rats for 12 weeks to investigate potential local pathological effects
4
and systemic toxicities. The test (chemical treated bone) and control (heat treated bone) materials were
5
compared using scanning electron microscope (SEM), x-ray diffraction pattern, inductively coupled plasma
6
analysis, and bending strength test. In the SEM images, the micro-porous structure of heat-treated bone was
7
changed to sintered ceramic-like structure. The structure of bone mineral from test and control materials was
8
analyzed as100% hydroxyapatite. The ratio of calcium (Ca) to potassium (P), the main inorganic elements, was
9
same even though the Ca and P percentages of the control material was relatively higher than the test material.
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No death or critical symptoms arose from implantation of the test (chemical treated bone) and control
11
(physiological saline) materials during 12 weeks. The implanted sites were macroscopically examined, with all
12
the groups showing non-irritant results. Our results indicate that chemical processed bovine bone has a better
13
mechanical property than the heat treated bone and the implantation of this material does not produce systemic
14
or pathological toxicity.
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Keywords: Bone graft, Bovine bone, Bending strength, Sub-chronic, Toxicity, Xenograft
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20 21 Abbreviations
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SD, Sprague-Dawley; HTOs, High tibia osteotomies; NP, Not present; WBC, White blood cell; NE,
24
Neutrophils; LY, Lymphocyte; MO, Monocyte; EO, Eosinophil; BA, Basophil; RBC, Red blood cell; Hb,
25
Hemoglobin; HCTL, Hematocrit; MCV, Mean corpuscular volume; MCH, Mean corpuscular hemoglobin;
26
MCHC, Mean corpuscular hemoglobin concentration; RDW, Red cell distribution width; PLT, Platelet; MPV,
27
Mean platelet volume; ALB, Albumin; ALP, Alkaline phosphatase; CA, Calcium; CHO, Total cholesterol;
28
CRE, Creatinine; GLU, Glucose; AST, Aspartate aminotrasferase; ALT, Alanine aminotransferase; TP, Total
29
protein; BUN, Blood urea nitrogen; T-BIL, Total bilirubin; IP, Inorganic phosphorus; TG, Triglycerides; CPK,
30
Creatine phosphokinase; Na, Sodium; K, Potassium; Cl, Chloride
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ACCEPTED MANUSCRIPT 1
1. Introduction To overcome the increasing demand for autografts or allografts, bone-grafting substitutes have long been
3
examined and commercialized in the field of muscular skeletal surgery (Abbas et al., 2007; Zarate-Kalfoqulos
4
and Reyes-Sanchez, 2006; Scarborough, 1992). All grafting materials must be approved by ISO10993
5
regulations or be specifically validated for safety and effectiveness before commercialization (Galia et al.,
6
2008). Xenografts such as bovine bone grafts have been used in dental augmentation surgery and more recently
7
in orthopedic surgery (Li et al., 2013; Athanasiou et al., 2010). The manufacturers of commercial xenografts
8
perform validation studies, including viral-inactivation studies, toxicity tests, and animal-implantation studies
9
for safety assurance purposes (Laurencin and El-Amin, 2008; Sammarco and Chang, 2002). Most xenografts are
10
heat treated to prevent viral transmission and immune responses in recipients (Kim et al., 2013; Testori et al.,
11
2012). However, applications with heat-treated xenografts are limited because heating changes their mechanical
12
or biological properties, making them unsuitable for use in orthopedic surgery (Campana et al., 2014; Yan et al.,
13
2008). Therefore, many orthopedic surgeons are concerned about the safety and efficacy of xenografts for
14
clinical use in cases involving large bone defects, long bone fractures, spinal fusions, and high tibia osteotomies
15
(HTOs) where xenografts need to provide mechanical support.
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2
Chemical treatment of xenografts has been employed in attempt to circumvent the problems associated with
17
heat treatment (Kemper et al., 2011). However, even with chemical treatments, the safety and efficacy of
18
xenografts for clinical use remain controversial because residual chemicals in the xenografts may cause adverse
19
effects. Thus, long-term animal studies for clinical pathology, including hematology and clinical chemistry,
20
need to be performed. Although the methods involved in sub-chronic systemic toxicity testing are routine, the
21
results may vary across device manufacturers, as each company uses different processing techniques. The
22
purposes of this study were to compare between chemical treated and heat treated bones using chemical and
23
mechanical analysis. And to evaluate the potential sub-chronic systemic toxicity of bovine bone grafts after
24
sequential chemical processing and propose the optimal treatment conditions for safety and effectiveness.
26 27 28 29 30
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ACCEPTED MANUSCRIPT 1
2. Materials and methods
2
2.1. Animals used for the implantation study Forty 8-week-old Sprague-Dawley (SD) rats (ORIENT BIO Inc., Seongnam, Korea) were used as implant
4
recipients. The animal-treatment protocol conformed to the Guidelines for Care and Use of Laboratory Animals
5
and was approved by the Committee of Experimental Animal Science (The College of Medicine, Seoul National
6
University, Seoul, Korea). Test and control substances were administered separately to 10 male and 10 female
7
rats each. Skin marking was used to distinguish between the test and control animals, with blue marking used
8
during the acclimation period and black marking used during the main study. All animals were housed in
9
polycarbonate cages (150 × 350 × 180 mm; W: L: H) during the quarantine, acclimation, administration, and
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3
observation periods.
12
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11 2.2. Processing of bovine bone
Natural bovine bone grafts (male, femoral and tibial bone, again less than 24 months) were processed using
14
chemical reagents without heating. The graft material was prepared following the standard operating procedure
15
of manufacturer (Cellumed Co., Ltd., Seoul, Korea). Briefly, the bovine bones were cut into proximal, middle,
16
and distal parts using a band saw. All separated bone shafts were cleaned ultrasonically using 15 mL/g of 3%
17
hydroxide solution and then with the same volume of 70% ethanol, for 1 hour during each cleaning step. The
18
cleaned some shafts were cut into smaller blocks (10 × 25 × 13 mm; W: L: H) with a band saw and the other
19
blocks were ground using a bone-milling machine to yield bone powder with particle sizes ranging from 200 to
20
1000 µm. The test substance was cleaned ultrasonically with a 1:1 chloroform: methanol solution (15 mL/g) for
21
1 hour. Next, it was treated with the same volume of 3% hydroxide solution, followed by rinsing with deionized
22
water for 30 minutes. It was subsequently treated with 15 ml/g of 70% ethanol for 1 hour at room temperature
23
and then with the same volume of 4% NaOH for 30 minutes, agitated for 15 minutes, ultrasonicated for 15
24
minutes, and rinsed for 30 minutes with deionized water. Finally, it was freeze-dried for 12 hours at-70 ºC to
25
ensure a water residue of ≤6%, placed in glass vials, stored at-70 ºC, and sterilized by gamma (Co60) irradiation
26
at a dose of 25 kGy.
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2.3. Preparation of test and negative-control substances
29
The bovine cortical bone blocks after chemical processing were used for mechanical test and powder was used
30
for chemical analysis and animal implantation as described above. For the comparison of mechanical test, the
ACCEPTED MANUSCRIPT heat treated test substance was processed at 300℃ for 20 minutes based on the other theoretical values (Li et al.,
2
2015; Tadic et al., 2004). And the negative-control substance for the implantation was physiological saline
3
(Daihan Pharm Co., Ltd., Seoul, Korea). Polyethylene micro-medical tubing (Scientific Commodities, Inc.,
4
Seoul, Korea) was sterilized with ethylene oxide gas and used as the implantation tube following the
5
international standard (ISO 10993-6: 2007E). For the implantation study, rats were administered 0.025 ml of the
6
test or control product via sterile tubing (length: 10 mm; outside diameter: 2.08 mm; inner diameter: 1.57 mm).
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7 2.4. Implantation procedure
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For implantation, rats were anesthetized with 40–50 mg/kg ketamine and 20–30 mg/kg xylazine. The gluteal
10
region, where the implantation procedure was conducted, was shaved and disinfected with 70% ethanol and
11
povidone. Approximately 2 cm of the gluteal skin was cut to expose the underlying muscle tissue, after which
12
the test or control material was implanted into the left and right gluteal muscles. After implantation, the muscle
13
and skin were sutured using a standard surgical method and disinfected with povidone. The implants were
14
remained in the rats for 12 weeks following the international standard (ISO 10993-11: 2006E).
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15 2.5. Observation points and test items
17
2.5.1.Characterization of materials
18
The morphology of the obtained bone grafts was examined by a scanning electron microscopy (SEM, S-4800;
19
Hitachi, Tokyo, Japan). SEM revealed that the surface of all samples was covered with 7-nm-thick platinum (Pt)
20
coating when the instrument was operated at 15 kV.
21
X-ray diffraction pattern (XRD) was used to determine the structure of the individual samples. XRD
22
measurements were performed with an X-ray diffractometer (D/Max 2500/PC, RIGAKU, Tokyo, Japan) with
23
copper (Cu) radiation in the 2θ range from 10° to 70° (scan speed of 0.02° per second).
24
Chemical analysis of materials was carried out by the Inductively coupled plasma-mass spectroscopy (ICP-MS)
25
OPTIMA 5300DV (PerkinElmer, Waltham, MA, USA).
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2.5.2. Mechanical test
28
All freeze dried test blocks were rehydrated by saline for 30 minutes. A bending test was performed by a
29
universal testing machine (DUS-200, Oriental TM, Siheung, Korea) with a loading speed of 1 mm/min
ACCEPTED MANUSCRIPT according to the standard of American society for testing materials (ASTM F-417, USA).
2
2.5.3. Clinical signs and mortality
3
During the observation period, the animals were observed daily for appearance; food intake; water intake;
4
histological reactions at acquisition, grouping, and once per week during the observation periods; and before
5
implantation and sacrifice. The total and remaining amounts of feed were measured once per week during the
6
study period.
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7 2.5.4. Hematology
9
On the day the animals were sacrificed, blood was drawn from all surviving animals, and hematological tests
10
were conducted using an automatic analyzer (ADVIA 2120; SIEMENS, USA). Prior to sacrifice, all animals
11
were fasted overnight. During the autopsies, the abdomens were cut open in 5% CO2 environment, and blood
12
was drawn from the aorta descendens. Ethylenediaminetetraacetic acid dipotassium salt was used as an
13
anticoagulant.
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2.5.5. Blood biochemistry
Blood drawn from the aorta descendens during autopsy was centrifuged at 3,000 rpm for 10 min, and the
17
resultant serum was used for biochemical tests conducted using an automatic analyzer (Hitachi 7180; Hitachi,
18
Japan).
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2.5.6. Macroscopic examination of implantation sites
21
The test- and control-substance implantation sites were observed macroscopically. Any changes identified
22
visually around the implantation sites, such as inflammation, encystment, bleeding, necrosis, or discoloration,
23
were evaluated and scored as shown in Table 1.
24
Insert Table 1 here.
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2.6. Statistical analysis
27
All statistical analyses were performed using commercially available software (SPSS v.15.0; IBM Corp.,
28
Armonk, NY, USA). The independent t tests were used to analyze differences between groups, with significance
29
levels set at * p < 0.05 and ** p < 0.01.
30
ACCEPTED MANUSCRIPT 1 3. Results
3
3.1. Characterization analysis of materials
4
In SEM images, after heat treatment, the macro-porous structure (50~100 um) was same as chemical treated
5
bone. However, the micro-porous structure of heat treated bone was invisible. The structure of heat treated bone
6
was changed to sintered ceramic-like structure forming permanent bonds and a dense materials which affects the
7
microstructure and mechanical properties (Figure. 1).
8
In the result of XRD, the both of chemical treated and heat treated bones were analyzed as same mineral
9
appearance of 100% hydroxyapatite (Ca5P3O13H) in Figure 2.
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The result of ICP-MS showed that the main elements of chemical treated bone was Calcium (Ca, 36.9%),
11
Potassium (P, 30.2%), and Carbone (C, 14.37%). The rest elements were remained less than 1%. The heat
12
treated bone showed Ca (51.9%) and P (42.67%), however, C (0.68%) was significantly decreased after heat
13
treatment. There was no difference of calculated Ca/P ratio between the chemical treated bone and heat treated
14
bone (Table. 2).
15
Insert Table 2 here.
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18
The average strength for the three point bending test was shown that the heat treated bone of 915 MPa was
19
significantly decreased compared with natural bone of 2165.25 MPa. The change to sintered ceramic-like
20
structure after heat treatment affected mechanical property of bone tissue. However, there was no significant
21
difference between the chemical treated bone of 1749.75 MPa and natural bone. Moreover, the strength of
22
chemical treated bone was significantly higher than heat treated bone (Figure 3).
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3.2. Observation of clinical signs and mortality
25
In this study, chemically treated bovine bone powder and physiological saline (control) were implanted into the
26
left and right gluteal muscles of SD rats. During a 12-week study period, the mortality, clinical symptoms,
27
weight change, and amounts of food and water intake were observed in these animals. Subsequently, the
28
animals were sacrificed and examined both visually and histopathologically.
29
No mortality or clinical symptoms arising from implanting the test material were observed during the study
30
period. In addition, no significant difference was observed in the weight, amount of food and water intake, and
ACCEPTED MANUSCRIPT ocular parameters between the test and control groups.
2
3.3. Hematology test
3
Hematological tests such as white and red blood cell counts, and hemoglobin measurements revealed no
4
significant differences between the control and test groups, when studying both female and male rats. No
5
abnormal signs related to the test substance were observed for the other clinical symptoms tested (Tables 3 and
6
4).
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Insert Table 3 and 4 here.
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Performing a blood biochemistry test for various factors revealed no significant differences between the control
11
and test groups (Table 5).
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Insert Table 5 here.
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13 3.5. Macroscopic examination
15
At 12 weeks post-implantation, all animals were autopsied, and the implanted sites of animals in the test and
16
control groups were compared to investigate local reactions (bleeding, necrosis, discoloration, infection, or
17
encystment). However, no significant changes were observed (Table 6).
18
Insert Table 6 here.
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ACCEPTED MANUSCRIPT 1 4. Discussion
3
Bovine bone grafts have been used in dental bone augmentation for decades. Most commercial bovine bone
4
grafts are heat treated to prevent viral transmission and to suppress immune responses in recipients by
5
deproteinization. However, these heat-treated grafts may not suitable for some orthopedic surgeries requiring
6
mechanical supports such as large bone void filling, long bone fractures, spinal fusions, and HTOs. Many long-
7
term follow-up clinical studies have shown that commercialized bovine bone grafts, which were heat-treated
8
during manufacturing, were not suitable for treating large bony defects or fracture patients. Therefore, the
9
effectiveness of mechanical properties should be important character in orthopedic surgery.
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Recently, chemical processing of animal-derived graft materials has been applied to commercial grafts.
11
However, residual organic materials may pose potential health risks. Thus, validation of each chemical process
12
followed during manufacture is essential for ensuring the safety of xenografts.
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Most studies on xenografts have investigated animal implantation in terms of efficacy, rather than safety.
14
Although grafting materials are in great demand for the treatment of muscular skeletal diseases, concerns
15
regarding the suitability of xenografts for clinical implantation exist. Moreover, in orthopedic surgery, grafting
16
materials must be able to provide mechanical strength for long bone fractures, spinal fusions, and bone fixation.
17
For safety approval, xenografts are heat-treated to remove organic materials or inactivate transmissible viruses.
18
However, heat treatment can impair the mechanical properties of bone-graft materials because it may cause
19
change of micro-porous structure and degeneration of mineral components and denature collagen, both of which
20
are related to bone strength (Shin et al., 2005; Wang et al., 2001; Wang et al., 2000).
21
To overcome these disadvantages, we employed sequential chemical treatment during bovine bone processing.
22
Previously, we confirmed that viral inactivation occurred in bovine-derived grafting materials using these
23
conditions (Lee et al., 2012). Three steps were performed during the entire chemical-treatment procedure,
24
including sequential treatment with 70% ethanol, 4% sodium hydroxide, and gamma irradiation, which resulted
25
in viral inactivation. The upstream steps involved the removal of blood, lipids, proteins, and other organic
26
materials using 3% hydroxide, 70% ethanol, and a mixture of chloroform and methanol.
27 28 29 30
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5. Conclusions To investigate biological safety and chemical-mechanical effectiveness of the chemical treated bovine bone,
4
the difference of processing type did not affect main chemical component such as the Ca/P ratio and
5
hydroxyapatite, the inorganic material. However, the micro-porous structure and carbon component were
6
changed by heat treatment and the mechanical property was decreased even though the chemical treated bone
7
did not show any significant difference compared with natural bone. The sub-chronic toxicity of bovine bone
8
processed using the conditions described herein was implanted into the gluteal muscles of SD rats for a 12-week
9
duration. Implantation of this material did not produce systemic or local pathological toxicity in SD rats; thus,
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our results indicate that chemically treated bovine bone is safe as xenografts for clinical use. Taken together with our previous study of viral inactivation in chemically processed bovine bone grafts, our
12
collective findings suggest that the chemical treated organic bovine bone grafts can be used as biologically and
13
chemically safe and mechanically effective grafting materials not only in dental surgery but also in orthopedic
14
surgery.
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Conflicts of interest statement
3
The authors declared no conflicts of interest.
4 Acknowledgement
6
This study was supported by World Premier Materials (10037842) funded by the Ministry of Trade, Industry &
7
Energy, Republic of Korea.
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References
3
Abbas, G., Bali, S.L., Abbas, N., Dalton, D.J., 2007. Demand and supply of bone allograft and the role of
4
orthopaedic surgeons. Acta. Orthop. Belg. 73, 507-511. Athanasiou, V.T., Papachristou, D.J., Panaqopoulos, A., Saridis, A., Scopa, C.D., Megas, P., 2010. Histological
6
comparison of autograft, allogaft-DBM, xenograft, and synthetic grafts in trabecluar bone defect: an
7
experimental study in rabbits. Med. Sci. Monit. 16, BR24-BR31.
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5
Campana, V., Milano, G., Pagano, E., Barba, M., Cicione, C., Lattanzi, W., Logroscino, G., 2014. Bone
9
substitutes in orthopaedic surgery: from basic science to clinical practice. J Mater Sci Mater Med. 25,
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2445-2461.
Galia, C.R., Macedo, C.A., Rosito, R., Mello, T.M., Camarqo, L.M., Moreira, L.F., 2008. In vitro and in vivo
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8
evaluation of lyophilized bovine bone biocompatibility. Clinics (Sao Paulo) 63, 801-806. ISO 10993-6: 2007(E). Biological evaluation of medical devices - Part 6: Tests for local effects after implantation.
ISO 10993-11: 2006(E). Biological evaluation of medical devices - Part 11: Tests for systemic toxicity.
16
Kemper, N., Davison, N., Fitzpatrick, D., Marshall, R., Lin, A., Mundy, K., Cobb, R.R., 2011. Characterization
17
of the mechanical properties of bovine cortical bone treated with a novel tissue sterilization process. Cell
18
Tissue Bank 12, 273-279.
21 22
substitutes: A systematic review. Clin. Implant. Dent. Relat. Res. 15, 645-653.
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Kim, Y., Nowzari, H., Rich, S.K., 2013. Risk of prion disease transmission through bovine-derived bone
Laurencin, C.T., El-Amin, S.F., 2008. Xenotransplantation in orthopaedic surgery. J. Am. Acad. Orthop. Surg. 16, 4-8.
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Lee, K.I., Lee, J.S., Jung, H.H., Lee, H.Y., Moon, S.H., Kang, K.T., Shim, Y.B., Jang, J.W., 2012. Inactivation
24
of enveloped and non-enveloped viruses in the process of chemical treatment and gamma irradiation of
25 26 27 28 29 30
bovine-derived grafting materials. Xenotransplantation. 40, 365-369.
Li, J., Xuan, F., Choi, B.H., Jeong, S.M., 2013. Minimally invasive ridge augmentation using xenogenous bone blocks in an atrophied posterior mandible: a clinical and histological study. Implant. Dent. 22, 112-116. Sammarco, V.J., Chang, L., 2002. Modern issues in bone graft substitutes and advances in bone tissue technology. Foot Ankle Clin. 7, 19-41. Li, Q., Zhou, G., Yu, X., Wang, T., Xi, Y., Tang, Z., 2015. Porous deproteinized bovine bone scaffold with
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three-dimensional localized drug delivery system using chitosan microspheres. BioMed Eng OnLine. 14,
2
1-14.
3
Scarborough, N.L., 1992. Current procedures for banking allograft human bone. Orthopedics. 15, 1161-1167.
4
Shin, S., Yano, H., Fukunaga, T., Ikebe, S., Shimizu, K., Kaku, N., Nagatomi, H., Masumi, S., 2005.
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Biomechanical properties of heat-treated bone grafts. Arch. Orthop. Trauma Surg. 125, 1-5.
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Tadic, D., Epple, M., 2004. A thorough physicochemical characterisation of 14 calcium phosphate-based bone substitution materials in comparison to natural bone. Biomaterials. 25, 987-994.
Testori, T., Iezzi, G., Manzon, L., Fratto, G., Piattelli, A., Weinstein, R.L., 2012. High temperature-treated
9
bovine porous hydroxyapatite in sinus augmentation procedures: a case report. Int. J. Periodontics
14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
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mechanical properties. J. Orthop. Res. 19, 1021-1026.
Wang, X., Bank, R.A., Tekoppele, J.M., Aqrawal, C.M., 2000. Effect of collagen denaturation on the toughness of bone. Clin. Orthop. Relat. Res. 371, 228-239.
Yan, J., Daga, A., Kumar, R., Mecholsky, J.J., 2008. Fracture toughness and work of fracture of hydrated, dehydrated, and ashed bovine bone. J. Biomech. 41, 1929-1936.
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Wang, X., Bank, R.A., Tekoppele, J.M., Aqrawal, C.M., 2001. The role of collagen in determining bone
Zarate-Kalfoqulos, B., Reyes-Sanchez, A., 2006. Bone grafts in orthopedic surgery. Cir. Cir. 74, 217-222
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Restorative Dent. 32, 295-301.
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Legend of figures
3
Figure 1. Scanning electron microscopy. Chemical treated bovine bone, (A) x100, (B) x30,000, (C) x100,000,
4
and heat treated bovine bone, (D) x100, (E) x30,000, (F) x100,000 Figure 2. X-ray diffraction pattern. Chemical treated (A) and heat treated (B) bovine bone
6
Figure 3. Comparison of the groups of bending strength. Each value is expressed as mean (N=18). Values are
7
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expressed as MPa. * Significantly different from the control group at p < 0.05, ** p < 0.01.
8
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ACCEPTED MANUSCRIPT 1 Table 1. Specifications of encapsulation capsules Capsule Width
Score
None
0
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Less than 0.5 mm
1
0.6–1.0 mm
2 3
SC
1.1–2.0 mm
Not Present
3 4 5 6
10 11 12 13 14 15 16 17 18 19 20 21
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Exceed 2.0 mm
NP
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Table 2. Summary of ICP-MC test results with chemical treated bovine bone and heat treated bovine bone.
3
(N=6, ** Significantly different from the control group at p < 0.01.) Heat Treated Bovine Bone
CaO**
36.90
51.90
P2O5**
30.20
C**
14.37
MgO**
0.84
Na2O**
0.42
S
0.08
SrO
0.05
BaO
0.03
ZnO
0.01
Al2O3
0.01
CuO
0.01
Fe2O3
0.01
K2O
0.01
MnO2
0.01
0.01
0.01
0.01
0.01
0.01
0.01
0.01
31.43
3.58
1.22
1.22
SiO2 Ignition Loss**
4 5 6 7 8 9 10 11 12 13
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Ca/P ratio
42.67 0.68 1.11
SC
0.56 0.08
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0.07
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ZrO
EP
TiO2
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Chemical Treated Bovine Bone
0.04 0.02 0.01 0.01 0.01 0.01
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Table 3. Summary of hematological test results from male and female SD rats with bovine bone implants in the
3
left and right gluteal muscles after 12 weeks. (N=10) Summary of hematological tests WBC
NE
LY
MO
EO
BA
NE
LY
MO
EO
Units
K/µL
K/µL
K/µL
K/µL
K/µL
K/µL
%
%
%
%
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Tests
Control Mean
10.38
1.48
8.46
0.21
S.D
1.432
0.434
1.458
0.065
0.045
0.007
Mean
10.14
1.32
8.39
0.20
0.09
S.D
2.693
0.499
2.486
0.079
0.044
Control 7.33
1.04
S.D
2.839
0.978
Test
14.49
81.21
2.05
1.11
4.410
4.640
0.488
0.375
0.01
13.60
82.22
2.01
0.83
0.005
5.699
5.081
0.561
0.343
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0.01
SEX: FEMALE
5.91
0.22
0.05
0.00
12.95
82.01
2.73
0.68
1.773
0.192
0.025
0.005
6.278
6.647
0.993
0.346
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Mean
0.12
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Test
Mean
4.88
0.61
4.05
0.11
0.04
0.00
13.80
81.52
2.33
0.90
S.D
1.712
0.244
1.727
0.042
0.020
0.004
6.909
7.356
0.596
0.411
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SEX: MALE
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Abbreviation used: WBC, White blood cell; NE: Neutrophils; LY: Lymphocyte; MO: Monocyte; EO:
7
Eosinophil; BA: Basophil
8 9 10
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Table 4. Summary of hematological test results from male and female SD rats with bovine bone implants in the
3
left and right gluteal muscles after 12 weeks (Continued, N=10) Summary of hematological tests BA
RBC
Hb
HCT
MCV
MCH
MCHC
RDW
PLT
MPV
Units
%
M/µL
g/dL
%
fL
pg
g/dL
%
K/µL
fL
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Tests
Mean
0.06
8.87
17.30
47.85
S.D
0.052
0.382
0.533
1.248
1.029
1.004
Mean
0.07
8.71
16.79
46.89
53.81
S.D
0.048
0.339
1.041
1.779
1.250
Control 0.06
8.03
S.D
0.052
0.389
Test
12.80
1028.30
6.47
1.425
0.843
123.208
0.445
19.27
35.77
13.18
1057.60
7.00
0.787
1.199
1.131
119.049
0.663
SEX: FEMALE
44.88
55.91
19.93
35.66
11.61
1062.80
7.59
0.918
2.101
1.436
0.663
0.759
0.451
114.658
0.739
Mean
0.10
8.22
16.82
46.90
57.08
20.49
35.92
11.17
1004.50
7.88
S.D
0.047
0.428
0.666
2.275
1.692
0.768
0.708
0.211
78.869
1.085
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19.54
16.01
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Mean
53.96
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Test
36.18
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Control
SC
SEX: MALE
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Abbreviation used: RBC: Red blood cell; Hb: Hemoglobin; HCTL Hematocrit; MCV: Mean corpuscular
7
volume; MCH: Mean corpuscular hemoglobin; MCHC: Mean corpuscular hemoglobin concentration; RDW:
8
Red cell distribution width; PLT: Platelet; MPV: Mean platelet volume
9 10
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Table 5. Summary of serum biochemical test results from male and female SD rats with bovine bone implants in
3
the left and right gluteal muscles after 12 weeks (N=10) Summary of biochemical test results ALB
ALP
CA
CHO
CRE
GLU
Units
g/dL
IU/L
mg/dL
mg/dL
mg/dL
mg/dL
AST
ALT
TP
mg/dL
IU/L
g/dL
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Tests
Control 2.3
255.4
9.9
70.3
0.6
S.D
0.25
53.87
0.84
14.70
0.07
N
10
10
10
10
Mean
2.3
278.2
10.6
S.D
0.13
36.06
0.41
N
10
10
Tests
BUN
T-BIL
Units
mg/dL
mg/dL
115.9
31.9
6.0
31.77
32.80
7.32
0.61
10
10
10
10
10
835
0.6
210.3
110.1
33.4
6.1
16.93
0.09
20.22
34.75
6.45
0.30 10
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Test
185.6
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Mean
SC
SEX: MALE
10
10
10
10
10
IP
TG
CPK
Na
K
Cl
mg/dL
mg/dL
U/L
mmol/L
mmol/l
mmol/L
EP
10
AC C
Control
SEX: FEMALE
Mean
16.8
0.1
8.5
51.8
541.4
137.8
4.8
100.9
S.D
1.35
0.01
1.22
24.69
358.61
11.55
0.76
7.88
N
10
10
10
10
10
10
10
10
Mean
32.7
0.3
9.2
41.8
363.2
142.5
9.8
103.5
S.D
47.22
0.05
0.70
21.67
243.51
2.76
12.38
2.32
N
10
10
10
10
10
10
10
10
Test
ACCEPTED MANUSCRIPT 1 2 Abbreviation used: ALB: Albumin; ALP: Alkaline phosphatase; CA: Calcium; CHO: Total cholesterol; CRE:
4
Creatinine; GLU: Glucose; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; TP: Total
5
protein; BUN: Blood urea nitrogen; T-BIL: Total bilirubin; IP: Inorganic phosphorus; TG: Triglycerides; CPK:
6
Creatine phosphokinase; Na: Sodium; K: Potassium; Cl: Chloride
7 8
SC
9 10 11
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12 13 14 15
20 21 22 23 24 25 26 27 28 29 30
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19
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18
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Table 6. Macroscopic observations of male and female SD rats with bovine bone implants in the left and right
4
gluteal muscles after 12 weeks
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Individual animals, reported by group SEX: MALE
Animal
Control 1/2
3/4
5/6
Left
0/0
0/0
0/0
Right
0/0
0/0
0/0
Test
7/8
9/10
Clinical
21/22
No. 0/0
Right
0/0
Clinical opinion
5 6 7 8
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
Nothing abnormal detected
SEX: FEMALE
25/26
Test
27/28
29/30
31/32
33/34
35/36
37/38
39/40
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
0/0
AC C
Left
23/24
19/20
0/0
Control
EP
Animal
17/18
0/0
TE D
Group
15/16
0/0
Nothing abnormal detected
opinion
13/14
M AN U
No.
11/12
SC
Group
Nothing abnormal detected
Nothing abnormal detected
ACCEPTED MANUSCRIPT 1 2 Figure 1.
(D
(E)
4 5
10 11 12 13 14 15 16 17 18 19 20
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(F)
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(C)
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(B)
SC
(A)
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Figure 2.
AC C
EP
TE D
M AN U
SC
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(A)
4 5 6 7 8
(B)
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Figure 3.
7 8 9
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6
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Highlights
• The chemical treated bovine bone had significantly higher bending strength than the heat treated bone.
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• The chemical appearance of all the materials was same such as 100% hydroxyapatite and Ca/P ratio.
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• There was no significant sub-chronic toxicity of chemical treated bone in the rat muscle during 12 weeks.