Osteogenic effect of low-temperature-heated porcine bone particles in a rat calvarial defect model Ara Go,1* Se Eun Kim,1* Kyung Mi Shim,1 Sang-Myeong Lee,2 Seok Hwa Choi,3 Jun Sik Son,4 Seong Soo Kang1 1
College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Republic of Korea Division of Biotechnology, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan 570-752, Republic of Korea 3 College of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, Republic of Korea 4 Korea Textile Development Institute, Daegu 703-712, Republic of Korea 2
Received 23 May 2013; revised 4 October 2013; accepted 30 October 2013 Published online 18 November 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35022 Abstract: The current study was designed to investigate the chemical and physical properties of porcine-derived xenografts of different crystallinity (low and high) and to evaluate their osteogenic potential. Porcine femur bone underwent a heat treatment process at 400 C (P400) and 1200 C (P1200) and was then milled into particles of 1 mm or less. In X-ray diffraction, P400 exhibited a low crystallinity compared with that of P1200, as indicated by the relatively wide diffraction peaks. Brunauer-Emmett-Teller analysis revealed that P400 also had a wider surface area than P1200. In micro-CT scan analysis of specimens in a rat calvarial defect model, bone mineral density of the P400 group was significantly higher than that of the P1200 group (p < 0.01). New bone formation was also remarkably higher at 8 weeks in the P400 group,
which showed more new osteocytes in the lacuna compared with the P1200 group. In this study, low crystalline bone particles were obtained at low processing temperature (at temperature of 400 C) and achieved superior new bone formation compared with the high crystalline bone particles created at a higher process temperature (1200 C). It can be concluded that lower process temperature bone particles might provide a more effective graft material for enhancing C 2013 Wiley Periodicals, Inc. J Biomed Mater Res bone formation. V Part A: 102A: 3609–3617, 2014.
Key Words: bone tissue engineering, xenograft, porcine cancellous bone, low crystalline, calvarial defect
How to cite this article: Go A, Kim SE, Shim KM, Lee S-M, Choi SH, Son JS, Kang SS. 2014. Osteogenic effect of lowtemperature-heated porcine bone particles in a rat calvarial defect model. J Biomed Mater Res Part A 2014:102A:3609–3617.
INTRODUCTION
There are many bone loss cases owing to traumatic injury and disease; these lead to bone defects and ultimately result in defects within the skeletal structure. In many of these cases, a substitutionary material must be used to fill the bone defect, and the clinical and economic impacts of treatments of bone defects are increasing.1–3 Xenografts are one of the most common alternatives to autografts. Bone xenografts, such as those of bovine or porcine bone, which may be freeze-dried, demineralized freeze-dried, pressure and heat sterilized or irradiated, are used to substitute other bone grafting materials. However, bovine-derived grafting material is not free of zoonosis, such as bovine spongiform encephalopathy.4 In this aspect, porcine-derived xenografts, with crystalline and morphological structures resembling those of human cancellous bone, seem to be the most
appropriate grafting material for bone grafting procedures and have a relatively low risk of zoonosis.5 However, there are many problems that must yet be resolved, such as immune response or poor biocompatibility. Recently, much attention has been attracted by low crystallinity materials because of their high surface areas, large pore size, and large pore volumes, which are crucial for a marked affinity for osteoblast-like cells and for appreciable degradation of grafting material and bone regeneration. However, we found no reports specifically related to low crystalline porcine-derived graft materials. Therefore, the study aims to describe the difference in chemical and physical properties of two porcine-derived xenografts with different crystallinity (low and high) using a wide range of analytical methods (BET, EDS, XRD), and an evaluation of their osteogenic potential made using a rat calvarial defect model.6–8
*These authors contributed equally to this work. Correspondence to: S. S. Kang; e-mail: [email protected] Contract grant sponsor: Rural Development Administration, Republic of Korea; contract grant number: Next-Generation BioGreen 21 Program (PJ009617)
C 2013 WILEY PERIODICALS, INC. V
3609
MATERIALS AND METHODS
Manufacturing heat-treated porcine cancellous bone particles To collect suitable stock material of porcine cancellous bone tissue, harvested porcine femur bone was surgically extracted and was removed of all soft tissue and cartilage. The cancellous bone was then separated from femur bone and was cut to pieces of 2 cm3 or less in size. To remove the blood and bleach, the cancellous bone was washed with distilled water. After that, the bone was soaked in distilled water for 24 h and 10% H2O2 for 12 h, and distilled water was replaced every 2 h. The bone pieces were then boiled in purified water for 72 h while replacing purified water at intervals of 12 h, thus removing many fatty substances and proteins from the bone. The bone pieces were then completely dried in an oven at 60 C for 24 h and were subsequently milled into particles of 1 mm or less using a grinder (MM400, Retsch, Germany). The bone particles were eliminated of lipids in chloroform/methanol solution (1:1 mixed solution, dynamic stat at 130 rpm, 24 h) and were deproteinized by sodium hypochlorite (4%, 130 rpm, 72 h). To remove the solvent from the bone particles at each stage, the bone particles were added to purified water and were then shaken at 130 rpm for 24 or 72 h. The purified water was replaced with fresh purified water at 2 h intervals to increase water-washing efficiency. The water-washed bone particles were completely dried in the oven at 60 C. Delipidized and deproteinized cancellous bone particles underwent a heat treatment process at 400 C (5 h) and 1200 C (3 h) in a muffle furnace (MF-21G, JeioTech, Korea). The heated cancellous bone particles were immersed in acetone and were cleaned using an ultrasonic cleaner for 30 min. To remove the moisture completely, bone particles were freeze-dried and were then stored at 270 C. Characteristic analysis of heat-treated porcine cancellous bone particles Analysis of the morphological features of heat-treated porcine cancellous bone particles was performed using a scanning electron microscope (SEM, S-4300, Hitachi, Japan). Before examination, samples were gold sputter-coated to render them electrically conductive. The calcium and phosphorus ratio of bone particles was analyzed with an energy dispersive spectrometer (EDS). EDS profiles were analyzed with a field emission gun scanning electron microscope system (Quanta 200 FEG, FEI company, USA) at a voltage of 15 kV, spot size 3, and random 5 point. The crystallinity of heat-treated porcine cancellous bone particles was analyzed with an X-ray diffractometer (XRD; Rigaku, Max-2500, Japan) at 40 kV, 200 mA, 2 /min (2u, 5 – 60 ), and the crystallite size was semiquantitatively estimated by the Scherrer equation.9 D5
3610
GO ET AL.
0:94 k b1=2 cos u
D is the average domain size, k is incidence of x-ray wavelength, b1/2 is the peak width (as full-width at half maximum) in 2h, and h is the diffraction angle of the corresponding reflex. The specific surface area of the samples was determined by nitrogen adsorption-desorption isotherms at liquid nitrogen temperature using the Micromeritics (ASAP 2420, Norcross, USA) instrument with outgas at 1.333 Pa on 300 C. The specific surface area was calculated by the BrunauerEmmett-Teller (BET) method, whereas pore size distributions (pore diameter and pore volume) were calculated by the Barrett-Joyner-Halenda method.7 Calvarial defect model and bone grafting Thirty healthy 7-week-old male Sprague-Dawley rats (Samtaco, Osan, Korea; weight, 250–280 g) were used in this study. The animals were kept in an air-conditioned room with a 12 h light–dark cycle with controlled temperature (23 6 2 C) and relative humidity (60 6 10%). Tap water and commercial rodent diet (Samyang feed, Korea) were provided ad libitum. The experimental and housing protocols were approved by the Chonnam National University. The study and housing protocols were approved by the committee for Chonnam National University. The animals were randomly assigned to three groups (Control group, only critical defect; P400 group, grafted heat-treated porcine cancellous bone particle at 400 C; P1200 group, grafted heat-treated porcine cancellous bone particle at 1200 C). Surgical procedure Animals were given general anesthesia by intraperitoneal injection of Ketamine (Ketamine50s, Yuhan Co., Korea) 40 mg/kg and xylazine (Rompuns, Bayer Korea, Korea) 10 mg/kg. The skin was disinfected and incised, and the periosteum was elevated. Calvarial defect (diameter, 8 mm) formation was then conducted using a surgical micromotor (NSK, Kanuma, Japan), and 0.03 g of each bone particles was implanted into the defect site for P400 and P1200 groups, where the control group had no material transplanted. After that, the periosteum was sutured using 4-0 absorbable suture (Surgisorbs, Samyang Co., Seoul, Korea). The skin was closed with 3-0 non-absorbable suture material (Black Silk, Ailee Co., Seoul, Korea). Animals were sacrificed at 4 and 8 weeks after operation by CO2 suffocation. R
R
R
Evaluation of osteogenic potential Specimens were harvested using a diamond saw at 4 and 8 weeks, and radiography was performed by X-ray (Elytis, Trophy, France) for 70 kVp, 7 mA, 0.031 s, and 15 cm focal film distance. Specimens were analyzed by micro-computed tomography (micro-CT) with Skyscan 1172 Desktop X-ray microtomograph (Skyscan, Aartselaar, Belgium) for 48 kVp, 201 lA. Reconstructions were performed with NRecon software (Skyscan, Aartselaar, Belgium) and region of interest was generated to include the calvarial defect margin. Bone volume (BV) and bone mineral density (BMD) were analyzed
OSTEOGENIC EFFECT OF LOW-TEMPERATURE-HEATED PORCINE BONE PARTICLES
ORIGINAL ARTICLE
FIGURE 1. SEM images of heat-treated porcine cancellous bone particles. (a1–3) P400, and (b1-3) P1200. (a1 and b1: Original magnification 330, a2 and b2: 32000, a3 and b3: 320000).
using CTAn software (Skyscan, Aartselaar, Belgium) at 4 and 8 weeks. Specimens were fixed with 10% buffered formalin and were decalified with Calci-ClearTM Rapid (National diagnostics, Atlanta). Samples were then dehydrated in an ascending series of alcohol rinses, and were embedded in a paraplast (Sherwood Medical Industries, St. Louis). Embedded specimens were sectioned to a thickness of 5 mm with a microtome (Reichet-Jung 820). Slides were stained with hematoxylin and eosin (H&E), and were observed under a microscope. Statistical analysis The data were reported as the mean 6 SD, and one-way analysis of variance (SPSS version 12.0) was used to evaluate the BV and BMD by Bonferroni assay. In all cases, a p value
College of Veterinary Medicine, Chonnam National University, Gwangju 500-757, Republic of Korea Division of Biotechnology, College of Environmental and Bioresource Sciences, Chonbuk National University, Iksan 570-752, Republic of Korea 3 College of Veterinary Medicine, Chungbuk National University, Cheongju 361-763, Republic of Korea 4 Korea Textile Development Institute, Daegu 703-712, Republic of Korea 2
Received 23 May 2013; revised 4 October 2013; accepted 30 October 2013 Published online 18 November 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.a.35022 Abstract: The current study was designed to investigate the chemical and physical properties of porcine-derived xenografts of different crystallinity (low and high) and to evaluate their osteogenic potential. Porcine femur bone underwent a heat treatment process at 400 C (P400) and 1200 C (P1200) and was then milled into particles of 1 mm or less. In X-ray diffraction, P400 exhibited a low crystallinity compared with that of P1200, as indicated by the relatively wide diffraction peaks. Brunauer-Emmett-Teller analysis revealed that P400 also had a wider surface area than P1200. In micro-CT scan analysis of specimens in a rat calvarial defect model, bone mineral density of the P400 group was significantly higher than that of the P1200 group (p < 0.01). New bone formation was also remarkably higher at 8 weeks in the P400 group,
which showed more new osteocytes in the lacuna compared with the P1200 group. In this study, low crystalline bone particles were obtained at low processing temperature (at temperature of 400 C) and achieved superior new bone formation compared with the high crystalline bone particles created at a higher process temperature (1200 C). It can be concluded that lower process temperature bone particles might provide a more effective graft material for enhancing C 2013 Wiley Periodicals, Inc. J Biomed Mater Res bone formation. V Part A: 102A: 3609–3617, 2014.
Key Words: bone tissue engineering, xenograft, porcine cancellous bone, low crystalline, calvarial defect
How to cite this article: Go A, Kim SE, Shim KM, Lee S-M, Choi SH, Son JS, Kang SS. 2014. Osteogenic effect of lowtemperature-heated porcine bone particles in a rat calvarial defect model. J Biomed Mater Res Part A 2014:102A:3609–3617.
INTRODUCTION
There are many bone loss cases owing to traumatic injury and disease; these lead to bone defects and ultimately result in defects within the skeletal structure. In many of these cases, a substitutionary material must be used to fill the bone defect, and the clinical and economic impacts of treatments of bone defects are increasing.1–3 Xenografts are one of the most common alternatives to autografts. Bone xenografts, such as those of bovine or porcine bone, which may be freeze-dried, demineralized freeze-dried, pressure and heat sterilized or irradiated, are used to substitute other bone grafting materials. However, bovine-derived grafting material is not free of zoonosis, such as bovine spongiform encephalopathy.4 In this aspect, porcine-derived xenografts, with crystalline and morphological structures resembling those of human cancellous bone, seem to be the most
appropriate grafting material for bone grafting procedures and have a relatively low risk of zoonosis.5 However, there are many problems that must yet be resolved, such as immune response or poor biocompatibility. Recently, much attention has been attracted by low crystallinity materials because of their high surface areas, large pore size, and large pore volumes, which are crucial for a marked affinity for osteoblast-like cells and for appreciable degradation of grafting material and bone regeneration. However, we found no reports specifically related to low crystalline porcine-derived graft materials. Therefore, the study aims to describe the difference in chemical and physical properties of two porcine-derived xenografts with different crystallinity (low and high) using a wide range of analytical methods (BET, EDS, XRD), and an evaluation of their osteogenic potential made using a rat calvarial defect model.6–8
*These authors contributed equally to this work. Correspondence to: S. S. Kang; e-mail: [email protected] Contract grant sponsor: Rural Development Administration, Republic of Korea; contract grant number: Next-Generation BioGreen 21 Program (PJ009617)
C 2013 WILEY PERIODICALS, INC. V
3609
MATERIALS AND METHODS
Manufacturing heat-treated porcine cancellous bone particles To collect suitable stock material of porcine cancellous bone tissue, harvested porcine femur bone was surgically extracted and was removed of all soft tissue and cartilage. The cancellous bone was then separated from femur bone and was cut to pieces of 2 cm3 or less in size. To remove the blood and bleach, the cancellous bone was washed with distilled water. After that, the bone was soaked in distilled water for 24 h and 10% H2O2 for 12 h, and distilled water was replaced every 2 h. The bone pieces were then boiled in purified water for 72 h while replacing purified water at intervals of 12 h, thus removing many fatty substances and proteins from the bone. The bone pieces were then completely dried in an oven at 60 C for 24 h and were subsequently milled into particles of 1 mm or less using a grinder (MM400, Retsch, Germany). The bone particles were eliminated of lipids in chloroform/methanol solution (1:1 mixed solution, dynamic stat at 130 rpm, 24 h) and were deproteinized by sodium hypochlorite (4%, 130 rpm, 72 h). To remove the solvent from the bone particles at each stage, the bone particles were added to purified water and were then shaken at 130 rpm for 24 or 72 h. The purified water was replaced with fresh purified water at 2 h intervals to increase water-washing efficiency. The water-washed bone particles were completely dried in the oven at 60 C. Delipidized and deproteinized cancellous bone particles underwent a heat treatment process at 400 C (5 h) and 1200 C (3 h) in a muffle furnace (MF-21G, JeioTech, Korea). The heated cancellous bone particles were immersed in acetone and were cleaned using an ultrasonic cleaner for 30 min. To remove the moisture completely, bone particles were freeze-dried and were then stored at 270 C. Characteristic analysis of heat-treated porcine cancellous bone particles Analysis of the morphological features of heat-treated porcine cancellous bone particles was performed using a scanning electron microscope (SEM, S-4300, Hitachi, Japan). Before examination, samples were gold sputter-coated to render them electrically conductive. The calcium and phosphorus ratio of bone particles was analyzed with an energy dispersive spectrometer (EDS). EDS profiles were analyzed with a field emission gun scanning electron microscope system (Quanta 200 FEG, FEI company, USA) at a voltage of 15 kV, spot size 3, and random 5 point. The crystallinity of heat-treated porcine cancellous bone particles was analyzed with an X-ray diffractometer (XRD; Rigaku, Max-2500, Japan) at 40 kV, 200 mA, 2 /min (2u, 5 – 60 ), and the crystallite size was semiquantitatively estimated by the Scherrer equation.9 D5
3610
GO ET AL.
0:94 k b1=2 cos u
D is the average domain size, k is incidence of x-ray wavelength, b1/2 is the peak width (as full-width at half maximum) in 2h, and h is the diffraction angle of the corresponding reflex. The specific surface area of the samples was determined by nitrogen adsorption-desorption isotherms at liquid nitrogen temperature using the Micromeritics (ASAP 2420, Norcross, USA) instrument with outgas at 1.333 Pa on 300 C. The specific surface area was calculated by the BrunauerEmmett-Teller (BET) method, whereas pore size distributions (pore diameter and pore volume) were calculated by the Barrett-Joyner-Halenda method.7 Calvarial defect model and bone grafting Thirty healthy 7-week-old male Sprague-Dawley rats (Samtaco, Osan, Korea; weight, 250–280 g) were used in this study. The animals were kept in an air-conditioned room with a 12 h light–dark cycle with controlled temperature (23 6 2 C) and relative humidity (60 6 10%). Tap water and commercial rodent diet (Samyang feed, Korea) were provided ad libitum. The experimental and housing protocols were approved by the Chonnam National University. The study and housing protocols were approved by the committee for Chonnam National University. The animals were randomly assigned to three groups (Control group, only critical defect; P400 group, grafted heat-treated porcine cancellous bone particle at 400 C; P1200 group, grafted heat-treated porcine cancellous bone particle at 1200 C). Surgical procedure Animals were given general anesthesia by intraperitoneal injection of Ketamine (Ketamine50s, Yuhan Co., Korea) 40 mg/kg and xylazine (Rompuns, Bayer Korea, Korea) 10 mg/kg. The skin was disinfected and incised, and the periosteum was elevated. Calvarial defect (diameter, 8 mm) formation was then conducted using a surgical micromotor (NSK, Kanuma, Japan), and 0.03 g of each bone particles was implanted into the defect site for P400 and P1200 groups, where the control group had no material transplanted. After that, the periosteum was sutured using 4-0 absorbable suture (Surgisorbs, Samyang Co., Seoul, Korea). The skin was closed with 3-0 non-absorbable suture material (Black Silk, Ailee Co., Seoul, Korea). Animals were sacrificed at 4 and 8 weeks after operation by CO2 suffocation. R
R
R
Evaluation of osteogenic potential Specimens were harvested using a diamond saw at 4 and 8 weeks, and radiography was performed by X-ray (Elytis, Trophy, France) for 70 kVp, 7 mA, 0.031 s, and 15 cm focal film distance. Specimens were analyzed by micro-computed tomography (micro-CT) with Skyscan 1172 Desktop X-ray microtomograph (Skyscan, Aartselaar, Belgium) for 48 kVp, 201 lA. Reconstructions were performed with NRecon software (Skyscan, Aartselaar, Belgium) and region of interest was generated to include the calvarial defect margin. Bone volume (BV) and bone mineral density (BMD) were analyzed
OSTEOGENIC EFFECT OF LOW-TEMPERATURE-HEATED PORCINE BONE PARTICLES
ORIGINAL ARTICLE
FIGURE 1. SEM images of heat-treated porcine cancellous bone particles. (a1–3) P400, and (b1-3) P1200. (a1 and b1: Original magnification 330, a2 and b2: 32000, a3 and b3: 320000).
using CTAn software (Skyscan, Aartselaar, Belgium) at 4 and 8 weeks. Specimens were fixed with 10% buffered formalin and were decalified with Calci-ClearTM Rapid (National diagnostics, Atlanta). Samples were then dehydrated in an ascending series of alcohol rinses, and were embedded in a paraplast (Sherwood Medical Industries, St. Louis). Embedded specimens were sectioned to a thickness of 5 mm with a microtome (Reichet-Jung 820). Slides were stained with hematoxylin and eosin (H&E), and were observed under a microscope. Statistical analysis The data were reported as the mean 6 SD, and one-way analysis of variance (SPSS version 12.0) was used to evaluate the BV and BMD by Bonferroni assay. In all cases, a p value
