A polycaprolactone/cuttlefish bone-derived hydroxyapatite composite porous scaffold for bone tissue engineering Beom-Su Kim,1,2 Sun-Sik Yang,1 Jun Lee1 1 2
Wonkwang Bone Regeneration Research Institute, Wonkwang University, Iksan 570-749, Republic of Korea Bonecell Biotech Inc., Dunsan-dong, Seo-gu, Daejeon 302-830, Republic of Korea
Received 8 August 2013; revised 14 October 2013; accepted 29 October 2013 Published online 21 November 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33075 Abstract: Cuttlefish bone (CB) is an attractive natural biomaterial source to obtain hydroxyapatite (HAp). In this study, a porous polycaprolactone (PCL) scaffold incorporating CBderived HAp (CB-HAp) powder was fabricated using the solvent casting and particulate leaching method. The presence of CB-HAp in PCL/CB-HAp scaffold was confirmed by X-ray diffraction (XRD). Scanning electron microscopy (SEM) and porosity analysis showed that the average pore dimension of the fabricated scaffold was approximately 200–300 lm, with 85% porosity, and that the compressive modulus increased after addition of CB-HAp powders. In vitro tests such as cell proliferation assay, cytotoxicity analysis, cell attachment observations, and alkaline phosphatase activity assays showed that the PCL/CB-HAp scaffold could improve
the proliferation, viability, adherence, and osteoblast differentiation rate of MG-63 cells. When surgically implanted into rabbit calvarial bone defects, consistent with the in vitro results, PCL/CB-HAp scaffold implantation resulted in significantly higher new bone formation than did implantation of PCL alone. These findings suggest that addition of CB-HAp powder to the PCL scaffold can improve cellular response and that the PCL/CB-HAp composite scaffold has great C 2013 Wiley potential for use in bone tissue engineering. V Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 102B: 943–951, 2014.
Key Words: cuttlefish bone, hydroxyapatite, polycaprolactone, porous scaffold, bone tissue engineering
How to cite this article: Kim B-S, Yang S-S, Lee J. 2014. A polycaprolactone/cuttlefish bone-derived hydroxyapatite composite porous scaffold for bone tissue engineering. J Biomed Mater Res Part B 2014:102B:943–951.
INTRODUCTION
In bone tissue engineering, the scaffold, along with the cells and stimulation signals, is an important component. The scaffold provides the environment for cell adhesion, proliferation, and differentiation to stimulate bone regeneration.1 During the last decade, calcium-phosphate–based materials have increasingly attracted considerable attention as biomaterials. In particular, hydroxyapatite [HAp, Ca10(PO4)6(OH)2] has been extensively used for bone repair because it is one of the major constituents of natural hard tissues such as bone and teeth.2–4 Moreover, HAp has several beneficial properties such as non-toxicity, osteoconduction, and biocompatibility.5,6 Several sources of analytical grade calcium and phosphate have been used as starting materials to produce HAp. In addition, several groups have reported the synthesis of natural product derived-HAp, which can be produced from natural biomaterials such as eggshell,7 coral,8 and cuttlefish bone (CB).9 CB is a hard organ in cuttlefish and acts as a floating tank. It is mostly composed of calcium carbonate (CaCO3, aragonite), and calcium carbonate materials have been converted into HAp.10,11 Furthermore, CB possesses a unique
interconnected chamber structure that is highly porous.12 For these reasons, several groups have focused on CB use as a scaffold template for bone regeneration and tissue engineering. Through a hydrothermal reaction, Ivankovic et al.9 successfully converted CB into HAp while retaining the porous structure and skeleton architecture. Although, this finding suggested that the CB-derived HAp (CB-HAp) is potentially useful, its brittleness and low strength restricted its application range and the requirement for mechanical enhancement before use as a scaffold in bone tissue engineering remained an obstacle to be overcome. HAp powder has been synthesized from ball-milled CB powder13 and suggested a potential for use as a biomaterial powder in the preparation of the scaffold. To fabricate the highly porous scaffold using a bioceramic powder such as HAp, electron spinning,14 gas forming,15 solvent casting and particulate leaching,16 and the three-dimensional (3D) printing technique17 have been used widely. Among these, the solvent casting and particulate leaching method has been used extensively to produce porous scaffolds. Furthermore, this is one of the most convenient and straightforward strategies for fabricating
Correspondence to: J. Lee (e-mail: [email protected]) Contract grant sponsor: Fishery Commercialization Technology Development Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea; contract grant number: 112092-03-1-CG000
C 2013 WILEY PERIODICALS, INC. V
943
scaffolds using HAp/polymer composites.18,19 In bone tissue engineering, HAp and polymer composites have gained much attention because the mechanical and biological properties of the composites are better than those of their individual components.16 Numerous biopolymers such as poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), and polycaprolactone (PCL) have been investigated for use in tissue engineering.20–22 Among these, PCL is one of the most promising polymers owing to its biocompatibility, nontoxicity, and slow-biodegradable properties.23 In this study, we investigated whether CB-HAp powder could be used in bone tissue engineering. For this, we obtained HAp powder from raw CB through a hydrothermal reaction. Then, a highly porous scaffold was fabricated from a composite of CB-HAp powder and PCL by using the solvent casting and particulate leaching method. Then, the morphological and mechanical properties of the fabricated scaffolds were analyzed and biocompatibility was evaluated using human osteoblast-like cell line (MG-63) in vitro. In addition, the tissue response and bone formation capability were evaluated using the rabbit calvarias defect model in vivo. MATERIALS AND METHODS
Preparation of HAp powder from cuttlefish bone CB was extracted from cuttlefish (Sepia esculenta from Korea western Sea), washed with water, and dried. Then, CB lamella was cut into small pieces by using a lancet. To convert CB into HAp, the hydrothermal reaction method described previously24 was used. Briefly, a small piece of the CB was input in 0.6M (NH4)H2PO4 aqueous solution. The mixture was allowed to settle and was then sealed in a Teflon-lined stainless-steel pressure vessel and heated at 200 C for 24 h. The resultant HAp was washed with distilled water and dried at 100 C. To obtain the powder, the CB-HAp pieces were milled with a zirconia (Y0TZP) ball, and the powder was used for further experiments. Characterization of CB-HAp To characterize the composition of the CB-HAp, X-ray diffraction analysis (XRD; D8, Bruker AXS, Karlsruhe, Germany) was performed with Cu-Ka radiation delivered at 50 kV, at a 0.02 /min scanning rate (2h), and in a 30 mÅ range of 10 to 80 . The microstructure and surface morphology of the CB-HAp granules were examined by scanning electron microscopy (SEM; EM-30, COXEM, Dae-Jeon, Korea). Preparation of PCL and PCL/CB-HAp scaffolds Porous scaffolds were fabricated by the combined solvent casting and particulate leaching method.25 Briefly, PCL (MW 10,000; Sigma-Aldrich, St. Louis, MO was dissolved in chloroform (Sigma-Aldrich), and HAp powder [10% (w/w) of PCL] was added. Then, salt particles 200 to 300 lm in size were added to the PCL/CB-HAp mixture solution to act as porogens. The ratio of PCL to salt was 1:10 (w/w). The composite solution was stirred until most of the organic solvent evaporated. The viscous composite solution was then cast into Teflon molds (6 mm in diameter and 2 mm in
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height) and dried for 24 h. To remove the porogen, the scaffold was immersed in distilled water for 4 days and dried for 48 h before further use. Characterization of the PCL and PCL/CB-HAp scaffolds To evaluate the composition of the PCL and PCL/CB-HAp scaffolds, XRD analysis was performed using Cu-Ka radiation delivered at 50 kV, at a 0.02 /min scanning rate (2h), and in a 30-mÅ range of 20 to 50 . In addition, the microstructure and surface morphology of each scaffold was observed by SEM. The porosity was measured using a mercury porosimetry system (Micromeritics Autopore 9500, Nocross, GA).26 The compressive properties of the scaffolds were measured using a universal testing machine (UTM: 4467; Instron Co., MA). A cylindrical porous scaffold (10 mm in diameter and 5 mm in height) was used as the testing specimen at a cross-head speed of 0.5 mm/min at room temperature. The porosity and compressive modulus of four samples were assessed. Cell culture Human MG-63 pre-osteoblast-like cell line was obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea). Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM; GibcoBRL, Gaithersburg, MD) containing 10% fetal bovine serum (FBS; Gibco-BRL) and 1% antibiotics at 37 C, 5% CO2, and 95% humidity. To induce osteoblast differentiation, cells were cultured in an osteogenic medium supplemented with 1 mM ascorbic acid, 10 mM b-glycerophosphate, and 100 nM dexamethasone (Sigma-Aldrich). The growth medium and osteogenic medium were replaced every 2 days. For three-dimensional culture on the PCL or PCL/CBHAp scaffold, the scaffolds were put onto 48-well plates, sterilized with 70% alcohol for 30 min, and rinsed three times with PBS buffer. Then, the cells were seeded onto the scaffolds in small volumes (20 mL) of maintenance medium at a density of 5 3 104 cells per scaffold. At various time intervals, the cell constructs were assayed to assess cell proliferation, DNA content, viability, cell adherence, and osteoblast differentiation. Cell proliferation Mitochondrial activity of the seeded MG-63 cells was R Aqueous One solution Kit assayed using the CellTiter96V (Invitrogen, Carlsbad, CA). The MG-63 cells were seeded and cultured on the PCL and PCL/CB-HAp scaffolds. At specific time points, 100 mL of [3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) reagent was mixed with 500 mL of growth medium and added to each well. Following 4 h of incubation, the optical density (OD) of the supernatants was measured at 490 nm using a microplate reader (SpectraMAX M3; Molecular Devices, Sunnyvale, CA). Furthermore, to confirm cell proliferation, the DNA content was measured. Briefly, at each time point, the scaffolds containing cultured cells were chopped and sonicated in 1 mL of distilled water for 30 s. Then, the solution was centrifuged for 5 min at 500g to remove the scaffold debris.
PCL/CB-HAP COMPOSITE SCAFFOLD AND BONE TISSUE ENGINEERING
ORIGINAL RESEARCH REPORT
The supernatant (850 mL) was heated with 95 mL of 103 TE buffer containing 1.5M NaCl and 5% sodium dodecyl sulfate. RNA and proteins were removed using RNase and proteinase K, respectively. The total DNA concentration was determined using a ND-1000 spectrophotometer (Nanodrop, Wilmington, DE).
untreated. The subcutaneous tissues and skin were then sutured, the animals were euthanized at 2 or 8 weeks after implantation, and the defect sites along with the surrounding bone areas were dissected from the host bone. Specimens were placed in a 4% paraformaldehyde phosphate buffer solution (pH 7.2) at 4 C for fixation (2 weeks).
Cytotoxicity and viability staining To measure cell viability and cytotoxicity, cells cultured on R Viability/ each scaffold were stained using a Live/DeadV Cytotoxicity kit (Invitrogen). Briefly, cells were seeded and cultured for 3 days. Then, the kit solution was added, and cells were incubated for 40 min in a CO2 incubator. After incubation, the samples were observed using an inverted fluorescence microscope (DM IL LED Fluo; Leica Microsystems, Wetzlar, Germany).
Micro-computed tomography (CT) analysis The 3D images were collected and analyzed using a microfocus CT system (Sky-Scan 1172TM; Skyscan, Kontich, Belgium). The bone specimens were scanned using an X-ray tube potential of 60 kV and 167 lA. After scanning, the images were reconstructed using CT-analyzer (Skyscan). Based on the micro-CT data sets, the newly formed bone volume was calculated by dividing the newly formed bone with the total volume within the region of interest. The value was expressed as a percentage of the bone volume (% BV).28
Cell adherence SEM was used to observe cell adherence and morphology. Cells were seeded onto each scaffold and cultured in the growth media. After 6 days of cultivation, the scaffolds were washed with PBS buffer and fixed in 2.5% glutaraldehyde solution at 4 C for 2 h. Then, the fixative solution was aspirated and the samples were post-fixed with 0.1% osmium tetroxide solution. The scaffolds were then dehydrated using a graded series of ethanol. The dehydrated samples were sputter-coated with gold and observed by SEM. Alkaline phosphatase assay Osteoblast differentiation was evaluated by determining alkaline phosphatase (ALP) activity using p-nitrophenylphosphate as a substrate. Briefly, cells were seeded and cultured on each scaffold for 5 days using the growth medium or the osteogenic medium. The cultured scaffolds were rinsed with PBS and chopped into small pieces. To dissolve the adherent cells and to obtain the proteins from the scaffold, the pieces were sonicated in 1% Triton X-100/PBS for 10 min on ice. The samples were then centrifuged at 12,000 rpm to remove the scaffold debris. Then, the supernatant was tested by the ALP activity assay, as described previously27 and the activity was normalized to the total protein level. Animal experiments All animal experiments were performed according to the guidelines of the Wonkwang University Institutional Animal Care and Use Committee. Six adult male New Zealand 3- to 4-month-old white rabbits were anesthetized with a combination of ketamine (35 mg/kg; Yuhan, Seoul, Korea) and xylazine (5 mg/kg; Bayer Korea, Seoul, Korea) administered through the intramuscular route. Local anesthesia was provided at the surgical site using a 2% lidocaine solution. The calvaria were exposed through a sagittal incision approximately 5 cm long. Four calvarial defects were created by irrigation with 0.9% physiologic saline using an 8-mm outer diameter trephine on a slow-speed electric handpiece. This was followed by scaffold (a random selection of PCL alone or PCL/CB-HAp) implantation. Only one defect was
Histological examination The rabbit skulls were decalcified by immersion in acidic solution (8% formic acid/8% HCl). After dehydration, the tissues were embedded in paraffin. Serial sections (5 lm) were cut longitudinal to the long axis of the calvarial defect with a microtome (HM 325; Microm, Walldorf, Germany). The sections were stained with hematoxylin and eosin, Goldner’s masson trichrome, and immunohistochemical dyes and were evaluated by light microscopy for new bone formation and tissue responses. Statistical analysis Statistical analyses were performed using Origin 8.0 (Originlab, Northampton, MA). Continuous data were compared before and after implantation using paired Student’s t-test. The values were expressed as the mean 6 standard deviation (SD). A p value
Wonkwang Bone Regeneration Research Institute, Wonkwang University, Iksan 570-749, Republic of Korea Bonecell Biotech Inc., Dunsan-dong, Seo-gu, Daejeon 302-830, Republic of Korea
Received 8 August 2013; revised 14 October 2013; accepted 29 October 2013 Published online 21 November 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33075 Abstract: Cuttlefish bone (CB) is an attractive natural biomaterial source to obtain hydroxyapatite (HAp). In this study, a porous polycaprolactone (PCL) scaffold incorporating CBderived HAp (CB-HAp) powder was fabricated using the solvent casting and particulate leaching method. The presence of CB-HAp in PCL/CB-HAp scaffold was confirmed by X-ray diffraction (XRD). Scanning electron microscopy (SEM) and porosity analysis showed that the average pore dimension of the fabricated scaffold was approximately 200–300 lm, with 85% porosity, and that the compressive modulus increased after addition of CB-HAp powders. In vitro tests such as cell proliferation assay, cytotoxicity analysis, cell attachment observations, and alkaline phosphatase activity assays showed that the PCL/CB-HAp scaffold could improve
the proliferation, viability, adherence, and osteoblast differentiation rate of MG-63 cells. When surgically implanted into rabbit calvarial bone defects, consistent with the in vitro results, PCL/CB-HAp scaffold implantation resulted in significantly higher new bone formation than did implantation of PCL alone. These findings suggest that addition of CB-HAp powder to the PCL scaffold can improve cellular response and that the PCL/CB-HAp composite scaffold has great C 2013 Wiley potential for use in bone tissue engineering. V Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 102B: 943–951, 2014.
Key Words: cuttlefish bone, hydroxyapatite, polycaprolactone, porous scaffold, bone tissue engineering
How to cite this article: Kim B-S, Yang S-S, Lee J. 2014. A polycaprolactone/cuttlefish bone-derived hydroxyapatite composite porous scaffold for bone tissue engineering. J Biomed Mater Res Part B 2014:102B:943–951.
INTRODUCTION
In bone tissue engineering, the scaffold, along with the cells and stimulation signals, is an important component. The scaffold provides the environment for cell adhesion, proliferation, and differentiation to stimulate bone regeneration.1 During the last decade, calcium-phosphate–based materials have increasingly attracted considerable attention as biomaterials. In particular, hydroxyapatite [HAp, Ca10(PO4)6(OH)2] has been extensively used for bone repair because it is one of the major constituents of natural hard tissues such as bone and teeth.2–4 Moreover, HAp has several beneficial properties such as non-toxicity, osteoconduction, and biocompatibility.5,6 Several sources of analytical grade calcium and phosphate have been used as starting materials to produce HAp. In addition, several groups have reported the synthesis of natural product derived-HAp, which can be produced from natural biomaterials such as eggshell,7 coral,8 and cuttlefish bone (CB).9 CB is a hard organ in cuttlefish and acts as a floating tank. It is mostly composed of calcium carbonate (CaCO3, aragonite), and calcium carbonate materials have been converted into HAp.10,11 Furthermore, CB possesses a unique
interconnected chamber structure that is highly porous.12 For these reasons, several groups have focused on CB use as a scaffold template for bone regeneration and tissue engineering. Through a hydrothermal reaction, Ivankovic et al.9 successfully converted CB into HAp while retaining the porous structure and skeleton architecture. Although, this finding suggested that the CB-derived HAp (CB-HAp) is potentially useful, its brittleness and low strength restricted its application range and the requirement for mechanical enhancement before use as a scaffold in bone tissue engineering remained an obstacle to be overcome. HAp powder has been synthesized from ball-milled CB powder13 and suggested a potential for use as a biomaterial powder in the preparation of the scaffold. To fabricate the highly porous scaffold using a bioceramic powder such as HAp, electron spinning,14 gas forming,15 solvent casting and particulate leaching,16 and the three-dimensional (3D) printing technique17 have been used widely. Among these, the solvent casting and particulate leaching method has been used extensively to produce porous scaffolds. Furthermore, this is one of the most convenient and straightforward strategies for fabricating
Correspondence to: J. Lee (e-mail: [email protected]) Contract grant sponsor: Fishery Commercialization Technology Development Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea; contract grant number: 112092-03-1-CG000
C 2013 WILEY PERIODICALS, INC. V
943
scaffolds using HAp/polymer composites.18,19 In bone tissue engineering, HAp and polymer composites have gained much attention because the mechanical and biological properties of the composites are better than those of their individual components.16 Numerous biopolymers such as poly(lactic acid) (PLA), poly(lactic-co-glycolic acid) (PLGA), and polycaprolactone (PCL) have been investigated for use in tissue engineering.20–22 Among these, PCL is one of the most promising polymers owing to its biocompatibility, nontoxicity, and slow-biodegradable properties.23 In this study, we investigated whether CB-HAp powder could be used in bone tissue engineering. For this, we obtained HAp powder from raw CB through a hydrothermal reaction. Then, a highly porous scaffold was fabricated from a composite of CB-HAp powder and PCL by using the solvent casting and particulate leaching method. Then, the morphological and mechanical properties of the fabricated scaffolds were analyzed and biocompatibility was evaluated using human osteoblast-like cell line (MG-63) in vitro. In addition, the tissue response and bone formation capability were evaluated using the rabbit calvarias defect model in vivo. MATERIALS AND METHODS
Preparation of HAp powder from cuttlefish bone CB was extracted from cuttlefish (Sepia esculenta from Korea western Sea), washed with water, and dried. Then, CB lamella was cut into small pieces by using a lancet. To convert CB into HAp, the hydrothermal reaction method described previously24 was used. Briefly, a small piece of the CB was input in 0.6M (NH4)H2PO4 aqueous solution. The mixture was allowed to settle and was then sealed in a Teflon-lined stainless-steel pressure vessel and heated at 200 C for 24 h. The resultant HAp was washed with distilled water and dried at 100 C. To obtain the powder, the CB-HAp pieces were milled with a zirconia (Y0TZP) ball, and the powder was used for further experiments. Characterization of CB-HAp To characterize the composition of the CB-HAp, X-ray diffraction analysis (XRD; D8, Bruker AXS, Karlsruhe, Germany) was performed with Cu-Ka radiation delivered at 50 kV, at a 0.02 /min scanning rate (2h), and in a 30 mÅ range of 10 to 80 . The microstructure and surface morphology of the CB-HAp granules were examined by scanning electron microscopy (SEM; EM-30, COXEM, Dae-Jeon, Korea). Preparation of PCL and PCL/CB-HAp scaffolds Porous scaffolds were fabricated by the combined solvent casting and particulate leaching method.25 Briefly, PCL (MW 10,000; Sigma-Aldrich, St. Louis, MO was dissolved in chloroform (Sigma-Aldrich), and HAp powder [10% (w/w) of PCL] was added. Then, salt particles 200 to 300 lm in size were added to the PCL/CB-HAp mixture solution to act as porogens. The ratio of PCL to salt was 1:10 (w/w). The composite solution was stirred until most of the organic solvent evaporated. The viscous composite solution was then cast into Teflon molds (6 mm in diameter and 2 mm in
944
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height) and dried for 24 h. To remove the porogen, the scaffold was immersed in distilled water for 4 days and dried for 48 h before further use. Characterization of the PCL and PCL/CB-HAp scaffolds To evaluate the composition of the PCL and PCL/CB-HAp scaffolds, XRD analysis was performed using Cu-Ka radiation delivered at 50 kV, at a 0.02 /min scanning rate (2h), and in a 30-mÅ range of 20 to 50 . In addition, the microstructure and surface morphology of each scaffold was observed by SEM. The porosity was measured using a mercury porosimetry system (Micromeritics Autopore 9500, Nocross, GA).26 The compressive properties of the scaffolds were measured using a universal testing machine (UTM: 4467; Instron Co., MA). A cylindrical porous scaffold (10 mm in diameter and 5 mm in height) was used as the testing specimen at a cross-head speed of 0.5 mm/min at room temperature. The porosity and compressive modulus of four samples were assessed. Cell culture Human MG-63 pre-osteoblast-like cell line was obtained from the Korean Cell Line Bank (KCLB, Seoul, Korea). Cells were grown in Dulbecco’s Modified Eagle Medium (DMEM; GibcoBRL, Gaithersburg, MD) containing 10% fetal bovine serum (FBS; Gibco-BRL) and 1% antibiotics at 37 C, 5% CO2, and 95% humidity. To induce osteoblast differentiation, cells were cultured in an osteogenic medium supplemented with 1 mM ascorbic acid, 10 mM b-glycerophosphate, and 100 nM dexamethasone (Sigma-Aldrich). The growth medium and osteogenic medium were replaced every 2 days. For three-dimensional culture on the PCL or PCL/CBHAp scaffold, the scaffolds were put onto 48-well plates, sterilized with 70% alcohol for 30 min, and rinsed three times with PBS buffer. Then, the cells were seeded onto the scaffolds in small volumes (20 mL) of maintenance medium at a density of 5 3 104 cells per scaffold. At various time intervals, the cell constructs were assayed to assess cell proliferation, DNA content, viability, cell adherence, and osteoblast differentiation. Cell proliferation Mitochondrial activity of the seeded MG-63 cells was R Aqueous One solution Kit assayed using the CellTiter96V (Invitrogen, Carlsbad, CA). The MG-63 cells were seeded and cultured on the PCL and PCL/CB-HAp scaffolds. At specific time points, 100 mL of [3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) reagent was mixed with 500 mL of growth medium and added to each well. Following 4 h of incubation, the optical density (OD) of the supernatants was measured at 490 nm using a microplate reader (SpectraMAX M3; Molecular Devices, Sunnyvale, CA). Furthermore, to confirm cell proliferation, the DNA content was measured. Briefly, at each time point, the scaffolds containing cultured cells were chopped and sonicated in 1 mL of distilled water for 30 s. Then, the solution was centrifuged for 5 min at 500g to remove the scaffold debris.
PCL/CB-HAP COMPOSITE SCAFFOLD AND BONE TISSUE ENGINEERING
ORIGINAL RESEARCH REPORT
The supernatant (850 mL) was heated with 95 mL of 103 TE buffer containing 1.5M NaCl and 5% sodium dodecyl sulfate. RNA and proteins were removed using RNase and proteinase K, respectively. The total DNA concentration was determined using a ND-1000 spectrophotometer (Nanodrop, Wilmington, DE).
untreated. The subcutaneous tissues and skin were then sutured, the animals were euthanized at 2 or 8 weeks after implantation, and the defect sites along with the surrounding bone areas were dissected from the host bone. Specimens were placed in a 4% paraformaldehyde phosphate buffer solution (pH 7.2) at 4 C for fixation (2 weeks).
Cytotoxicity and viability staining To measure cell viability and cytotoxicity, cells cultured on R Viability/ each scaffold were stained using a Live/DeadV Cytotoxicity kit (Invitrogen). Briefly, cells were seeded and cultured for 3 days. Then, the kit solution was added, and cells were incubated for 40 min in a CO2 incubator. After incubation, the samples were observed using an inverted fluorescence microscope (DM IL LED Fluo; Leica Microsystems, Wetzlar, Germany).
Micro-computed tomography (CT) analysis The 3D images were collected and analyzed using a microfocus CT system (Sky-Scan 1172TM; Skyscan, Kontich, Belgium). The bone specimens were scanned using an X-ray tube potential of 60 kV and 167 lA. After scanning, the images were reconstructed using CT-analyzer (Skyscan). Based on the micro-CT data sets, the newly formed bone volume was calculated by dividing the newly formed bone with the total volume within the region of interest. The value was expressed as a percentage of the bone volume (% BV).28
Cell adherence SEM was used to observe cell adherence and morphology. Cells were seeded onto each scaffold and cultured in the growth media. After 6 days of cultivation, the scaffolds were washed with PBS buffer and fixed in 2.5% glutaraldehyde solution at 4 C for 2 h. Then, the fixative solution was aspirated and the samples were post-fixed with 0.1% osmium tetroxide solution. The scaffolds were then dehydrated using a graded series of ethanol. The dehydrated samples were sputter-coated with gold and observed by SEM. Alkaline phosphatase assay Osteoblast differentiation was evaluated by determining alkaline phosphatase (ALP) activity using p-nitrophenylphosphate as a substrate. Briefly, cells were seeded and cultured on each scaffold for 5 days using the growth medium or the osteogenic medium. The cultured scaffolds were rinsed with PBS and chopped into small pieces. To dissolve the adherent cells and to obtain the proteins from the scaffold, the pieces were sonicated in 1% Triton X-100/PBS for 10 min on ice. The samples were then centrifuged at 12,000 rpm to remove the scaffold debris. Then, the supernatant was tested by the ALP activity assay, as described previously27 and the activity was normalized to the total protein level. Animal experiments All animal experiments were performed according to the guidelines of the Wonkwang University Institutional Animal Care and Use Committee. Six adult male New Zealand 3- to 4-month-old white rabbits were anesthetized with a combination of ketamine (35 mg/kg; Yuhan, Seoul, Korea) and xylazine (5 mg/kg; Bayer Korea, Seoul, Korea) administered through the intramuscular route. Local anesthesia was provided at the surgical site using a 2% lidocaine solution. The calvaria were exposed through a sagittal incision approximately 5 cm long. Four calvarial defects were created by irrigation with 0.9% physiologic saline using an 8-mm outer diameter trephine on a slow-speed electric handpiece. This was followed by scaffold (a random selection of PCL alone or PCL/CB-HAp) implantation. Only one defect was
Histological examination The rabbit skulls were decalcified by immersion in acidic solution (8% formic acid/8% HCl). After dehydration, the tissues were embedded in paraffin. Serial sections (5 lm) were cut longitudinal to the long axis of the calvarial defect with a microtome (HM 325; Microm, Walldorf, Germany). The sections were stained with hematoxylin and eosin, Goldner’s masson trichrome, and immunohistochemical dyes and were evaluated by light microscopy for new bone formation and tissue responses. Statistical analysis Statistical analyses were performed using Origin 8.0 (Originlab, Northampton, MA). Continuous data were compared before and after implantation using paired Student’s t-test. The values were expressed as the mean 6 standard deviation (SD). A p value
