Evaluation of bone formation guided by DNA/protamine complex with FGF-2 in an adult rat calvarial defect model Yosuke Shinozaki,1 Masako Toda,2 Jun Ohno,3 Minoru Kawaguchi,4 Hirofumi Kido,1 Tadao Fukushima5 1
Department of Oral Rehabilitation, Oral Implantology section, Fukuoka Dental College, Fukuoka, Japan Department of Oral Growth and Development Division of Pediatric Dentistry, Fukuoka Dental College, Fukuoka, Japan 3 Department of Morphological Biology, Pathology section, Fukuoka Dental College, Fukuoka, Japan 4 Department of Dental Engineering, Bioengineering section, Fukuoka Dental College, Fukuoka, Japan 5 Center for Regenerative Medicine, Fukuoka Dental College, Fukuoka, Japan 2
Received 6 November 2013; revised 19 February 2014; accepted 6 March 2014 Published online 24 March 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33143 Abstract: DNA/protamine complex paste (D/P) and D/P complex paste with Fibroblast Growth Factor-2 (FGF-2) (D/P-FGF) were prepared to investigate their new bone formation abilities using an 40-week-old rat calvarial defect model. It was found that D/P could release FGF-2 proportionally in an in vitro experiment with an enzyme-linked immunosorbent assay. It was also found that aging adversely affected selfbone healing of rats by comparison with the results in a previous study using 10-week-old rats. Microcomputed tomography and histopathological examinations showed that new bone formation abilities of D/P and D/P-FGF were superior to that of the control (sham operation). Control, D/P and D/P-FGF showed newly formed bone areas of 6.7, 58.3, and
67.0%, respectively, 3 months after the operation. Moreover, it was found that FGF-2 could support the osteoanagenesis ability of D/P. It was considered that FGF-2 could play an important role in new bone formation at early stages because it induced the genes such as collagen I, CBFA, OSX, and OPN, which are initiated first in the process of osteogenesis. Therefore, D/P-FGF will be a useful injectable biomaterial with biodegradable properties for the repair of bone defects C 2014 Wiley Periodicals, Inc. J Biomed Mater Res in the elderly. V Part B: Appl Biomater, 102B: 1669–1676, 2014.
Key Words: DNA, protamine, fibroblast growth factor-2, bone regeneration, elderly
How to cite this article: Shinozaki Y, Toda M, Ohno J, Kawaguchi M, Kido H, Fukushima T. 2014. Evaluation of bone formation guided by DNA/protamine complex with FGF-2 in an adult rat calvarial defect model. J Biomed Mater Res Part B 2014:102B:1669–1676.
INTRODUCTION
DNA is an interesting candidate for novel bone substitution material because it has the great advantage of having phosphate groups. However, application of DNA alone for a novel bone substitution material is problematic because DNA is water soluble. Water solubility restricts the wide application of biomaterials because of rapid elution of biomaterials from the implantation site in the body.1–3 To improve this disadvantage, Fukushima et al. synthesized DNA/protamine (D/P) complex, which is a water-insoluble white powder, by mixing an aqueous solution of a mean 300 base-pairs of DNA with protamine.4,5 They described that the complex became a paste by kneading with water and that the paste had suitable viscosity for clinical use and showed good cell viability, mild soft tissue responses, and antibacterial effects against Gram-positive bacteria.4–6 It is noted that protamine alone can induce bone sialoprotein transcription.7 Thus, we expected that D/P complex
can induce the formation of new bone in bone defects. In a previous study, we investigated whether this complex could promote new bone formation in a rat cranial defect model experiment with microcomputed tomography (l-CT) and histopathological examination. As expected, the D/P complex induced osteogenesis in the defect.8 The remodeling function of bone generally decreases with aging. We therefore consider that evaluating the potential of new biomaterials as bone regeneration material in aging mouse or rat cranial defect model experiments is necessary. The application of growth factors such as bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2) in tissue regenerative engineering has increased.9,10 It is well known that FGF-2 has shown potential effects on the repair and regeneration of soft tissues.11,12 Recently, it was reported that FGF-2 can promote new bone formation, regardless of age.13 However, FGF-2 is water soluble9; thus, its flow out of the diseased area needs to be controlled by using a container
This article was published online on 24 March 2014. An error was subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected 1 April 2014. Correspondence to: Y. Shinozaki (e-mail: [email protected]) Contract grant sponsor: Grant-in-aid for Scientific Research (B); contract grant number: 23390455
C 2014 WILEY PERIODICALS, INC. V
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FIGURE 1. Schematic illustration of D/P complex (A) and preparation paste of D/P complex (B–D) and preparation disk of D/P complex (E). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
such as gelatin hydrogel. We expect that D/P will be a good container for FGF-2 because the complex paste can be easily mixed with an aqueous solution of FGF-2, in which the desired concentration of FGF-2 is maintained. We also expect that FGF2-containing D/P will promote new bone formation more greatly than D/P alone. It is noted that the complex paste with FGF-2 can be injected directly using a syringe into tissue defects of various shapes. The purpose of this study was to prepare D/P alone and D/P-FGF, and to investigate their effects on new bone formation in Sprague-Dawley aged rat (more than 40 weeks of age) cranial defects by l-CT and histopathological examination.
MATERIALS AND METHODS
Preparation of D/P complex Sterilized salmon testis DNA provided by Maruha-Nichiro (Maruha-Nichiro Holdings, Tokyo, Japan), which was cleaved by nuclease into 300-bp fragments, and 2% sterilized salmon testis protamine sulfate (mol. wt. 5 4500) solution (Maruha-Nichiro Holdings) were used in this study. DNA (500 mg) was dissolved in 100 mL distilled water. The distilled water was added to 2% sterilized salmon testis protamine sulfate solution to prepare 0.5% protamine sulfate solution. DNA in 100 mL distilled water was added to protamine sulfate solution (100 mL) and the mixture was stirred at 20 C for 1 h. The D/P complex was collected by centrifugation at 9000 rpm for 10 min and washed with distilled water. This process was repeated twice. Complexes were cracked with a frozen cell crusher (CRYO-PRESS; Microtec Nition, Funabashi, Japan) after they were frozen in liquid nitrogen and then dried for 24 h in an FD-5N freezedryer (Eyela, Tokyo, Japan). All procedures were carried out under sterile conditions and with sterilized instruments and materials (Figure 1).
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Preparation of D/P complex paste, DNA/complex paste with FGF-2, and paste disks Basic FGF (FGF-2: Lot No.: 545) from Kaken Pharmaceutical (Tokyo, Japan) was used. FGF-2 (1 mg) was dissolved in 300 lL distilled water. Freeze-dried D/P complex powder (35 mg) and 32 lL FGF-2 solution or 32 lL distilled water were kneaded in a mortar with a pestle to prepare D/P containing 0.3% based on the D/P powder of FGF-2 (D/P-FGF paste) and D/P (D/P paste).14 The complex powder/FGF-2 solution ratio or the complex powder/distilled water ratio of each paste were 1.08. For animal experiments and the FGF-2 release test, D/P paste and D/P-FGF paste disks were prepared. D/P paste and D/P-FGF paste were used to fill a syringe with a nozzle of 1mm internal diameter. For disk preparation of D/P paste and D/P-FGF paste, the paste was injected into a silicone mold (8mm internal diameter and 0.8-mm height) on a Teflon plate. The top surface of the complex was covered with a Teflon plate to flatten it and then the covered Teflon plate was removed. Fabricated complex disks (67 mg) were immediately and carefully removed from the Teflon plate and silicone mold (Figure 1).8 All procedures were carried out under sterile conditions and with sterilized instruments and materials. For measurement of the dimensional change of disks, three D/P paste disks and D/P-FGF paste disks were kept at 37 C and 100% humidity for 24 h. Diameter of each disk was measured with a reading microscope (Travelling Microscope Type A; Shimadzu, Kyoto, Japan) before and after 24 h.
FGF-2 release Amounts of FGF-2 released from the D/P-FGF disk were measured by enzyme-linked immunosorbent assay (ELISA; Quantikine ELISA Human FGF basic Immunoassy, R & D Systems, Minneapolis, MN). Five D/P-FGF paste disks were
EVALUATION OF BONE FORMATION GUIDED BY D/P COMPLEX WITH FGF-2
ORIGINAL RESEARCH REPORT
used for the FGF-2 release test. Disks were placed in each well of a 24-well multiplate with 200 mL DMEM (Dulbecco’s Modified Eagle’s Medium; Life Technologies, Carlsbad, CA). After 1-day, 3-day, 5-day, 1-week, 2-week, and 3-week incubation, disk culture supernatants were collected and released FGF-2 was measured by ELISA. Optical density was measured using a plate reader (Multiskan JX; Labsystems Oy, Helsinki, Finland) at an excitation wavelength of 450 nm and an emission reading of 520 nm. New medium was added to each well after each measurement. The amount of FGF-2 released at each time point was the cumulative value. Implantation of paste disk Animal experiments were performed in accordance with the ethics guidelines for animal experiments at Fukuoka Dental College (No. 10017). Forty-week-old male Sprague-Dawley rats were used. Surgery was performed under general anesthesia induced by 2% isoflurane (Abbott Laboratories, Abbott Park, IL) and an air mixture gas flow of 1.0 L/min using an anesthesia gas machine (Anesthesia machine SF-B01; MR Technology, Tsukuba, Ibaraki, Japan). An incision was made in the cranium of the rats, and the exposed periosteum was semicircularly incised and carefully separated from calvarial bone. The 8-mm calvarial defects were created in the central parietal bone with a sinus drill with meticulous care to avoid damaging the underlying dura mater. The defects were treated with an 8-mm diameter paste disk. Defects as sham treatment were used as controls. After the insertion of samples, periosteum and scalp tissues were each closed in separate layers by suturing with intracutaneously resorbable Vicryl 3-0 (Ethicon, Somerville, NJ). The rats (27) were divided into 3 groups (D/P-FGF group, D/P group and control group) for different observation times (1, 2, and 3 months). Three rats were used for each group in each period. It is known from experience that bleeding from the created defect occurs if the dura is damaged during drilling. In the case of bleeding in this study, the created defect in the rat was not used. In this study, three additional rats were used because three rat dura were damaged. l-CT analyses Image analyses of new bone formation were performed using an in vivo l-CT system (Skyscan-1176 microCT; Bruker, Kontich, Belgium) at 50 kVp and 500 lA. The thickness of one l-CT slice was 35 lm. The analyses were performed under general anesthesia induced by 2% isoflurane (Abbott Laboratories) and an air mixture gas flow to 1.0 L/ min using an anesthesia gas machine (Anesthesia machine SF-B01; MR Technology). The percentage of newly bone formed bone in the defect (New-Bone%) was calculate as the area of newly formed bone/area of the defect originally created by trephination. First, the newly formed bone area on l-CT slice images in the horizontal direction was quantified two-dimensionally using WinROOF image analysis software (Mitani, Tokyo, Japan). The analyzed circular area of 8 mm was drawn on each l-CT slice image. Each circular area was closely adjacent to the location where the defect was created while additionally referring to l-CT slice images of coronal and sagittal sections and histological images of a
decalcified sample with hematoxylin and eosin (HE) staining. Ten sequential l-CT slice images showing the highest newly formed bone areas were used for one sample analysis. The percentage of newly bone formed bone in the defect (New-Bone%) was calculated as the total area of newly formed bone per 10 l-CT slice images/total area of the defect per 10 l-CT slice image 3 100. All experiments were performed in quintuplicate. New-Bone% was analyzed statistically using two-way analysis of variance (ANOVA) and Scheffe’s multiple comparison tests to determine statistical differences in cell viability among the samples at the 5% level of significance. Histological evaluation In each period, the animals were sacrificed by injection with an overdose of isoflurane. After sacrificing the animals, cranial tissues containing the implanted samples were immediately excised. The samples were used as decalcified samples stained with HE. For HE staining, samples were fixed in 4% (w/v) paraformaldehyde in phosphate buffer (pH 7.4), decalcified with 0.24M EDTA•4Na•4H2O solution, dehydrated with graded alcohol, cleared in xylene, and embedded in paraffin by routine procedures. The specimens were sectioned at 3 lm. The sections were stained with HE for histological observation using a Nikon Eclipse 55i light microscope (Nikon, Tokyo, Japan). Gene expression Animal experiments were performed using the same methods as for the implantation of the paste disk section. Three rats were used for each group (D/P paste group and D/P-FGF group). The gene expression was observed by real time-PCR using repaired tissue in the defect area where D/P-FGF and D/P were implanted. Two weeks after implantation, tissue was extracted. After the exposed periosteum was carefully separated from cranial bone, the repaired tissue was harvested while avoiding the dura mater. The obtained tissue was washed with PBS buffer solution several times to remove extraneous matter such as blood. After washing, harvested tissue was homogenized (ReadyPrep Mini Grinders Instruction Manual; BIO-RAD, Hercules, CA) and cDNA was synthesized (ReverTra Ace qPCR RT Masyer Mix; Toyobo, Osaka, Japan) after isolation of mRNA (High Pure RNA Tissue Kit; Roche, Tokyo, Japan). Osterix, osteopontin, CBFA, ALP, osteocalcin, collagen I, and sialoprotein gene expression were observed with b-actin as a control. Each target gene expression ratio of D/P-FGF to that of D/P was calculated. RESULTS
Preparation of D/P complex paste, DNA/complex paste with FGF-2, and paste disks, and measurement of dimensional change of disks As shown in Figure 1, D/P and D/P-FGF were prepared from D/P complex powder by kneading with water and FGF-2 solution. Paste disks were prepared by mold fabrication of each paste. The diameter of all disks did not change after storing at 37 C and 100% humidity for 24 h. The range of the diameter change of all disks was within
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FIGURE 2. The cumulative amounts of FGF-2 released from D/P-FGF paste after immersion in DMED (1 D: 1 day, 2D: 2 days, 3D: 3 days, 1 W: 1 week, 2W: 2 weeks, and 3W: 3 weeks).
8.0 6 0.01 mm. Rates of dimensional changes for all disks were less than 1%. FGF-2 release The cumulative amounts of FGF-2 released from the D/PFGF disk are shown in Figure 2. FGF-2 was constantly released from disks and the amounts of released FGF-2 at each time point were 1.15–1.22 ng/100 mL. The cumulative amount of released FGF-2 after 3-week immersion corresponded to 43.0% of the charge quantity. l-CT analyses The l-CT images and new bone formation (New-Bone %) obtained from the l-CT analyses are shown in Figures 3 and 4. For the control, the l-CT images in horizontal and coronal directions showed a little new bone formation in defect areas.
In contrast, for D/P paste and D/P-FGF paste, newly formed bone was observed in defect areas and the areas of newly formed bone increased with the recovery period. Newly formed bone was generated in most of the defect areas 3 months after implantation. Two-way ANOVA, followed by Scheffe’s multiple comparison tests, showed significant differences in the total areas of newly formed bone among D/P, D/P-FGF, and the control (p
Department of Oral Rehabilitation, Oral Implantology section, Fukuoka Dental College, Fukuoka, Japan Department of Oral Growth and Development Division of Pediatric Dentistry, Fukuoka Dental College, Fukuoka, Japan 3 Department of Morphological Biology, Pathology section, Fukuoka Dental College, Fukuoka, Japan 4 Department of Dental Engineering, Bioengineering section, Fukuoka Dental College, Fukuoka, Japan 5 Center for Regenerative Medicine, Fukuoka Dental College, Fukuoka, Japan 2
Received 6 November 2013; revised 19 February 2014; accepted 6 March 2014 Published online 24 March 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33143 Abstract: DNA/protamine complex paste (D/P) and D/P complex paste with Fibroblast Growth Factor-2 (FGF-2) (D/P-FGF) were prepared to investigate their new bone formation abilities using an 40-week-old rat calvarial defect model. It was found that D/P could release FGF-2 proportionally in an in vitro experiment with an enzyme-linked immunosorbent assay. It was also found that aging adversely affected selfbone healing of rats by comparison with the results in a previous study using 10-week-old rats. Microcomputed tomography and histopathological examinations showed that new bone formation abilities of D/P and D/P-FGF were superior to that of the control (sham operation). Control, D/P and D/P-FGF showed newly formed bone areas of 6.7, 58.3, and
67.0%, respectively, 3 months after the operation. Moreover, it was found that FGF-2 could support the osteoanagenesis ability of D/P. It was considered that FGF-2 could play an important role in new bone formation at early stages because it induced the genes such as collagen I, CBFA, OSX, and OPN, which are initiated first in the process of osteogenesis. Therefore, D/P-FGF will be a useful injectable biomaterial with biodegradable properties for the repair of bone defects C 2014 Wiley Periodicals, Inc. J Biomed Mater Res in the elderly. V Part B: Appl Biomater, 102B: 1669–1676, 2014.
Key Words: DNA, protamine, fibroblast growth factor-2, bone regeneration, elderly
How to cite this article: Shinozaki Y, Toda M, Ohno J, Kawaguchi M, Kido H, Fukushima T. 2014. Evaluation of bone formation guided by DNA/protamine complex with FGF-2 in an adult rat calvarial defect model. J Biomed Mater Res Part B 2014:102B:1669–1676.
INTRODUCTION
DNA is an interesting candidate for novel bone substitution material because it has the great advantage of having phosphate groups. However, application of DNA alone for a novel bone substitution material is problematic because DNA is water soluble. Water solubility restricts the wide application of biomaterials because of rapid elution of biomaterials from the implantation site in the body.1–3 To improve this disadvantage, Fukushima et al. synthesized DNA/protamine (D/P) complex, which is a water-insoluble white powder, by mixing an aqueous solution of a mean 300 base-pairs of DNA with protamine.4,5 They described that the complex became a paste by kneading with water and that the paste had suitable viscosity for clinical use and showed good cell viability, mild soft tissue responses, and antibacterial effects against Gram-positive bacteria.4–6 It is noted that protamine alone can induce bone sialoprotein transcription.7 Thus, we expected that D/P complex
can induce the formation of new bone in bone defects. In a previous study, we investigated whether this complex could promote new bone formation in a rat cranial defect model experiment with microcomputed tomography (l-CT) and histopathological examination. As expected, the D/P complex induced osteogenesis in the defect.8 The remodeling function of bone generally decreases with aging. We therefore consider that evaluating the potential of new biomaterials as bone regeneration material in aging mouse or rat cranial defect model experiments is necessary. The application of growth factors such as bone morphogenetic protein-2 (BMP-2) and fibroblast growth factor-2 (FGF-2) in tissue regenerative engineering has increased.9,10 It is well known that FGF-2 has shown potential effects on the repair and regeneration of soft tissues.11,12 Recently, it was reported that FGF-2 can promote new bone formation, regardless of age.13 However, FGF-2 is water soluble9; thus, its flow out of the diseased area needs to be controlled by using a container
This article was published online on 24 March 2014. An error was subsequently identified. This notice is included in the online and print versions to indicate that both have been corrected 1 April 2014. Correspondence to: Y. Shinozaki (e-mail: [email protected]) Contract grant sponsor: Grant-in-aid for Scientific Research (B); contract grant number: 23390455
C 2014 WILEY PERIODICALS, INC. V
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FIGURE 1. Schematic illustration of D/P complex (A) and preparation paste of D/P complex (B–D) and preparation disk of D/P complex (E). [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
such as gelatin hydrogel. We expect that D/P will be a good container for FGF-2 because the complex paste can be easily mixed with an aqueous solution of FGF-2, in which the desired concentration of FGF-2 is maintained. We also expect that FGF2-containing D/P will promote new bone formation more greatly than D/P alone. It is noted that the complex paste with FGF-2 can be injected directly using a syringe into tissue defects of various shapes. The purpose of this study was to prepare D/P alone and D/P-FGF, and to investigate their effects on new bone formation in Sprague-Dawley aged rat (more than 40 weeks of age) cranial defects by l-CT and histopathological examination.
MATERIALS AND METHODS
Preparation of D/P complex Sterilized salmon testis DNA provided by Maruha-Nichiro (Maruha-Nichiro Holdings, Tokyo, Japan), which was cleaved by nuclease into 300-bp fragments, and 2% sterilized salmon testis protamine sulfate (mol. wt. 5 4500) solution (Maruha-Nichiro Holdings) were used in this study. DNA (500 mg) was dissolved in 100 mL distilled water. The distilled water was added to 2% sterilized salmon testis protamine sulfate solution to prepare 0.5% protamine sulfate solution. DNA in 100 mL distilled water was added to protamine sulfate solution (100 mL) and the mixture was stirred at 20 C for 1 h. The D/P complex was collected by centrifugation at 9000 rpm for 10 min and washed with distilled water. This process was repeated twice. Complexes were cracked with a frozen cell crusher (CRYO-PRESS; Microtec Nition, Funabashi, Japan) after they were frozen in liquid nitrogen and then dried for 24 h in an FD-5N freezedryer (Eyela, Tokyo, Japan). All procedures were carried out under sterile conditions and with sterilized instruments and materials (Figure 1).
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Preparation of D/P complex paste, DNA/complex paste with FGF-2, and paste disks Basic FGF (FGF-2: Lot No.: 545) from Kaken Pharmaceutical (Tokyo, Japan) was used. FGF-2 (1 mg) was dissolved in 300 lL distilled water. Freeze-dried D/P complex powder (35 mg) and 32 lL FGF-2 solution or 32 lL distilled water were kneaded in a mortar with a pestle to prepare D/P containing 0.3% based on the D/P powder of FGF-2 (D/P-FGF paste) and D/P (D/P paste).14 The complex powder/FGF-2 solution ratio or the complex powder/distilled water ratio of each paste were 1.08. For animal experiments and the FGF-2 release test, D/P paste and D/P-FGF paste disks were prepared. D/P paste and D/P-FGF paste were used to fill a syringe with a nozzle of 1mm internal diameter. For disk preparation of D/P paste and D/P-FGF paste, the paste was injected into a silicone mold (8mm internal diameter and 0.8-mm height) on a Teflon plate. The top surface of the complex was covered with a Teflon plate to flatten it and then the covered Teflon plate was removed. Fabricated complex disks (67 mg) were immediately and carefully removed from the Teflon plate and silicone mold (Figure 1).8 All procedures were carried out under sterile conditions and with sterilized instruments and materials. For measurement of the dimensional change of disks, three D/P paste disks and D/P-FGF paste disks were kept at 37 C and 100% humidity for 24 h. Diameter of each disk was measured with a reading microscope (Travelling Microscope Type A; Shimadzu, Kyoto, Japan) before and after 24 h.
FGF-2 release Amounts of FGF-2 released from the D/P-FGF disk were measured by enzyme-linked immunosorbent assay (ELISA; Quantikine ELISA Human FGF basic Immunoassy, R & D Systems, Minneapolis, MN). Five D/P-FGF paste disks were
EVALUATION OF BONE FORMATION GUIDED BY D/P COMPLEX WITH FGF-2
ORIGINAL RESEARCH REPORT
used for the FGF-2 release test. Disks were placed in each well of a 24-well multiplate with 200 mL DMEM (Dulbecco’s Modified Eagle’s Medium; Life Technologies, Carlsbad, CA). After 1-day, 3-day, 5-day, 1-week, 2-week, and 3-week incubation, disk culture supernatants were collected and released FGF-2 was measured by ELISA. Optical density was measured using a plate reader (Multiskan JX; Labsystems Oy, Helsinki, Finland) at an excitation wavelength of 450 nm and an emission reading of 520 nm. New medium was added to each well after each measurement. The amount of FGF-2 released at each time point was the cumulative value. Implantation of paste disk Animal experiments were performed in accordance with the ethics guidelines for animal experiments at Fukuoka Dental College (No. 10017). Forty-week-old male Sprague-Dawley rats were used. Surgery was performed under general anesthesia induced by 2% isoflurane (Abbott Laboratories, Abbott Park, IL) and an air mixture gas flow of 1.0 L/min using an anesthesia gas machine (Anesthesia machine SF-B01; MR Technology, Tsukuba, Ibaraki, Japan). An incision was made in the cranium of the rats, and the exposed periosteum was semicircularly incised and carefully separated from calvarial bone. The 8-mm calvarial defects were created in the central parietal bone with a sinus drill with meticulous care to avoid damaging the underlying dura mater. The defects were treated with an 8-mm diameter paste disk. Defects as sham treatment were used as controls. After the insertion of samples, periosteum and scalp tissues were each closed in separate layers by suturing with intracutaneously resorbable Vicryl 3-0 (Ethicon, Somerville, NJ). The rats (27) were divided into 3 groups (D/P-FGF group, D/P group and control group) for different observation times (1, 2, and 3 months). Three rats were used for each group in each period. It is known from experience that bleeding from the created defect occurs if the dura is damaged during drilling. In the case of bleeding in this study, the created defect in the rat was not used. In this study, three additional rats were used because three rat dura were damaged. l-CT analyses Image analyses of new bone formation were performed using an in vivo l-CT system (Skyscan-1176 microCT; Bruker, Kontich, Belgium) at 50 kVp and 500 lA. The thickness of one l-CT slice was 35 lm. The analyses were performed under general anesthesia induced by 2% isoflurane (Abbott Laboratories) and an air mixture gas flow to 1.0 L/ min using an anesthesia gas machine (Anesthesia machine SF-B01; MR Technology). The percentage of newly bone formed bone in the defect (New-Bone%) was calculate as the area of newly formed bone/area of the defect originally created by trephination. First, the newly formed bone area on l-CT slice images in the horizontal direction was quantified two-dimensionally using WinROOF image analysis software (Mitani, Tokyo, Japan). The analyzed circular area of 8 mm was drawn on each l-CT slice image. Each circular area was closely adjacent to the location where the defect was created while additionally referring to l-CT slice images of coronal and sagittal sections and histological images of a
decalcified sample with hematoxylin and eosin (HE) staining. Ten sequential l-CT slice images showing the highest newly formed bone areas were used for one sample analysis. The percentage of newly bone formed bone in the defect (New-Bone%) was calculated as the total area of newly formed bone per 10 l-CT slice images/total area of the defect per 10 l-CT slice image 3 100. All experiments were performed in quintuplicate. New-Bone% was analyzed statistically using two-way analysis of variance (ANOVA) and Scheffe’s multiple comparison tests to determine statistical differences in cell viability among the samples at the 5% level of significance. Histological evaluation In each period, the animals were sacrificed by injection with an overdose of isoflurane. After sacrificing the animals, cranial tissues containing the implanted samples were immediately excised. The samples were used as decalcified samples stained with HE. For HE staining, samples were fixed in 4% (w/v) paraformaldehyde in phosphate buffer (pH 7.4), decalcified with 0.24M EDTA•4Na•4H2O solution, dehydrated with graded alcohol, cleared in xylene, and embedded in paraffin by routine procedures. The specimens were sectioned at 3 lm. The sections were stained with HE for histological observation using a Nikon Eclipse 55i light microscope (Nikon, Tokyo, Japan). Gene expression Animal experiments were performed using the same methods as for the implantation of the paste disk section. Three rats were used for each group (D/P paste group and D/P-FGF group). The gene expression was observed by real time-PCR using repaired tissue in the defect area where D/P-FGF and D/P were implanted. Two weeks after implantation, tissue was extracted. After the exposed periosteum was carefully separated from cranial bone, the repaired tissue was harvested while avoiding the dura mater. The obtained tissue was washed with PBS buffer solution several times to remove extraneous matter such as blood. After washing, harvested tissue was homogenized (ReadyPrep Mini Grinders Instruction Manual; BIO-RAD, Hercules, CA) and cDNA was synthesized (ReverTra Ace qPCR RT Masyer Mix; Toyobo, Osaka, Japan) after isolation of mRNA (High Pure RNA Tissue Kit; Roche, Tokyo, Japan). Osterix, osteopontin, CBFA, ALP, osteocalcin, collagen I, and sialoprotein gene expression were observed with b-actin as a control. Each target gene expression ratio of D/P-FGF to that of D/P was calculated. RESULTS
Preparation of D/P complex paste, DNA/complex paste with FGF-2, and paste disks, and measurement of dimensional change of disks As shown in Figure 1, D/P and D/P-FGF were prepared from D/P complex powder by kneading with water and FGF-2 solution. Paste disks were prepared by mold fabrication of each paste. The diameter of all disks did not change after storing at 37 C and 100% humidity for 24 h. The range of the diameter change of all disks was within
JOURNAL OF BIOMEDICAL MATERIALS RESEARCH B: APPLIED BIOMATERIALS | NOV 2014 VOL 102B, ISSUE 8
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FIGURE 2. The cumulative amounts of FGF-2 released from D/P-FGF paste after immersion in DMED (1 D: 1 day, 2D: 2 days, 3D: 3 days, 1 W: 1 week, 2W: 2 weeks, and 3W: 3 weeks).
8.0 6 0.01 mm. Rates of dimensional changes for all disks were less than 1%. FGF-2 release The cumulative amounts of FGF-2 released from the D/PFGF disk are shown in Figure 2. FGF-2 was constantly released from disks and the amounts of released FGF-2 at each time point were 1.15–1.22 ng/100 mL. The cumulative amount of released FGF-2 after 3-week immersion corresponded to 43.0% of the charge quantity. l-CT analyses The l-CT images and new bone formation (New-Bone %) obtained from the l-CT analyses are shown in Figures 3 and 4. For the control, the l-CT images in horizontal and coronal directions showed a little new bone formation in defect areas.
In contrast, for D/P paste and D/P-FGF paste, newly formed bone was observed in defect areas and the areas of newly formed bone increased with the recovery period. Newly formed bone was generated in most of the defect areas 3 months after implantation. Two-way ANOVA, followed by Scheffe’s multiple comparison tests, showed significant differences in the total areas of newly formed bone among D/P, D/P-FGF, and the control (p
