Effect of modification degree of nanohydroxyapatite on biocompatibility and mechanical property of injectable poly(methyl methacrylate)-based bone cement Changyun Quan,1* Yong Tang,2* Zhenzhen Liu,1 Minyu Rao,1 Wei Zhang,3 Peiqing Liang,1 Nan Wu,1 Chao Zhang,1 Huiyong Shen,2 Qing Jiang1 1
School of Engineering, Sun Yat-sen University, Guangzhou 510006, People’s Republic of China Department of Orthopedics, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, People’s Republic of China 3 Department of Outpatient, First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510008, People’s Republic of China 2
Received 15 May 2014; revised 13 October 2014; accepted 30 March 2015 Published online 7 May 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33428 Abstract: The objective of this study is to prepare a biocompatible nanohydroxyapatite/poly(methyl methacrylate) (HA/ PMMA) composite bone cement, which has good mechanical property and can be used for vertebroplasty. Up to 40 wt % of nanohydroxyapatite (nano-HA) in the power, which was surface modified with poly(methylmethacrylate-co-g-methacryloxypropyl timethoxysilane) [P(MMA-co-MPS)] copolymer, was incorporated into the composite bone cement. The content of P(MMA-co-MPS) on the surface of nano-HA (18.7%, 22.8%, and 26%) was determined through thermogravimetric analysis (TGA). The morphology of biomineralized surface of composite bone cement was observed under scanning electron microscope (SEM). The mechanical measurements of the composite
cements implied that the interfacial interaction between the HA and PMMA matrix may be greatly enhanced after surface modification of HA. Biochemical assays indicated that the HA/ PMMA bone cement had no cytotoxicity and induced no hemolysis. The cell adhesion and alkaline phosphatase (ALP) activity assays indicated that the biocompatibility of HA/ PMMA bone cement could be promoted, demonstrating that it can be used as an ideal weight-bearing bone repair materials C 2015 Wiley Periodicals, Inc. J Biomed on clinical application. V Mater Res Part B: Appl Biomater, 104B: 576–584, 2016.
Key Words: poly(methyl methacrylate), nanohydroxyapatite, modification degree, mechanical property, biocompatibility
How to cite this article: Quan C, Tang Y, Liu Z, Rao M, Zhang W, Liang P, Wu N, Zhang C, Shen H, Jiang Q. 2016. Effect of modification degree of nanohydroxyapatite on biocompatibility and mechanical property of injectable poly(methyl methacrylate)-based bone cement. J Biomed Mater Res Part B 2016:104B:576–584.
INTRODUCTION
Ideal materials for bone repair should be similar to human bone tissue, which could not only induce bone cells to infiltrate into the implanted materials, but also have good mechanical properties around host bone tissue.1,2 Due to its self-hardening behavior and excellent mechanical properties, polymethylmethacrylate (PMMA) bone cement has been widely used in clinical practice for decades.3 However, the bioinertness of PMMA cement hinders its efficient integration with the host bone tissue and would lead to inferior interfacial strength between bone cement and host bone tissue as well as the implant;4 orthopedic implants would be loosened and cause failure after a few years.5 In order to overcome these problems, researches have focused on
adapting the biocompatibility of PMMA bone cement by introducing bioactive inorganic nanoparticles such as hydroxyapatite (HA),6 Al2O3,7 and SiO2.8 HA has similar chemical composition and crystal structure to apatite in human bone tissue and can bind to the host bone tissue;9 it has been extensively studied as inorganic filler in PMMA bone cement to promote biocompatibility.10 Efforts have been devoted to HA/PMMA cement to meet the mechanical and biochemical requirements of clinical practice.11,12 For example, 3-trimethoxysilyl propyl methacrylate (g-MPS) has been employed to modify the surface of HA in order to achieve better mechanical property of the HA/PMMA cement.13 The surface modification of HA by g-MPS would not only enhance the dispersion and stability
*Both authors contributed equally to this work. Correspondence to: C. Quan; e-mail: [email protected] and [email protected] Contract grant sponsor: National Natural Science Foundation of China (NSFC); contract grant number: 21104098 Contract grant sponsor: The Ph.D Programs Foundation of Ministry of Education of China; contract grant number: 20110171120008 Contract grant sponsor: Fundamental Research Funds for the Central Universities; contract grant number: 121 gpy17 Contract grant sponsor: Science and Technology Planning Project of Guangdong Province; contract grant number: 2011A060901013 Contract grant sponsor: Guangdong Innovative Research Team Program; contract grant number: 2009010057
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of HA in MMA during the preparation of HA/PMMA bone cement, but also minimize the difference of surface properties between HA nanoparticles and PMMA matrix.14 As a consequence of such improvement, the mechanical property of HA/PMMA bone cement could be enhanced.15 Considering the fact that reaction between MPS and MMA monomer might be hindered by the steric hindrance from free PMMA chains or chains growing from adjacent MPS groups, such kind of modification may not be enough to achieve better compatibility between the organic and inorganic phases. An alternative solution to this problem is to modify the surface of HA using a copolymer of MPS and MMA, which not only maintains efficient reactivity between the siloxane groups of MPS with hydroxyl group of HA but also interacts/entangles with the continuous PMMA phase. Herein, the surface of nano-HA was first modified with poly(methyl methacrylate-co-g-methacryloxypropyl timethoxysilane) [P(MMA-co-MPS)] copolymer, followed by being added to the MMA monomer as a nanofiller to prepare a series of injectable HA/PMMA bone cements. After modification of HA, the content of HA in the bone cement could reach up to 40 wt % and the cement can be implanted by injection.16 Taking advantage of MMA structural unit in chain of P(MMA-co-MPS) copolymer, the dispersion of modified HA in PMMA matrix could be greatly improved to obtain a more homogenous system. The mechanical properties and biocompatibility, such as biomineralization, cell adhesion, and ALP activity of the cement were investigated to illuminate its biocompatibility and its potential application in weight-bearing bone regeneration. MATERIALS AND METHODS
Materials Methyl methacrylate (MMA) and poly(ethylene glycol) 2000 (PEG 2000) were purchased from Damao Chemical Reagent Factory (Tianjin, China). MMA was used after distillation under reduced pressure. Poly(vinyl alcohol) (PVA, Mw 30,000–50,000 g/mol) and benzoyl peroxide (BPO) were purchased from Sinopharm Chemical Reagent Co. Ltd. 3trimethoxysilyl propyl methacrylate (g-MPS) was purchased from Dow Corning (Nanjing, China) and used after distillation under reduced pressure. Dimethyltryptamine (DMT) was purchased from Aladdin Reagent Company (Shanghai, China) and used as received without further purification. All other reagents were purchased from Guangzhou Chemical Reagent Factory and used as received. HA nanoparticles were prepared according to reported procedure with minor modifications.17 Simulated body fluid (SBF) was prepared following reported protocol.18 Human fetal osteoblast (hFOB) was purchased from the Cell Bank of the Chinese Academy of Sciences at Shanghai, China. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Hyclone (USA). Geneticin Selective Antibiotic (G-418 Sulfate) was purchased from Notlas (Beijing, China). N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES) was purchased from Amresco (USA).
Synthesis of PMMA and P(MMA-co-MPS) PMMA and P(MMA-co-MPS) were synthesized according to our previous report.19 In detail, PVA (0.35 g), MMA (52.864 g), and BPO (0.13 g) were dissolved in 360 mL of deionized water to prepare PMMA. P(MMA-co-MPS) was synthesized via freeradical copolymerization using SHCH2CH2OH as a chain transfer agent. Briefly, MMA (25.488 g), MPS (7.1466 g), AIBN (0.1186 g), and SHCH2CH2OH (0.2659 g) were reacted in 60 mL of tetrahydrofuran (THF) for 6 h at 70 C to synthesize the P(MMA-co-MPS) copolymer. Weight-average molar mass of the polymerization products was determined via gel permeation chromatography (GPC) system equipped with Waters 1515 separations module, Waters 2414 refractive index detector. The concentration of each copolymer was 6 mg/mL, and THF (HPLC grade) was used as the eluent at a flow rate of 0.3 mL/min. Surface modification of HA The surface modification of nano-HA was carried out in a mixture of methanol and water (9:1, v/v).20,21 After nanoHA (8.0 g) was dispersed in the ethanol/water mixture at pH 3.5–4.0, then varied amount of P(MMA-co-MPS) (1.0, 2.0, and 3.0 g, respectively) in acetone was added dropwise to the mixture. The MPS units in the copolymer were hydrolyzed in the solution at 50 C; 90 min later, the pH was raised to 10.0 with NaOH (10%) solution to promote the condensation reaction. Products with different degree of modification (defined as x-HA, x 5 2, 3, or 4; HA without modification was defined as 1-HA) were collected by filtration and sonicated in an ultrasonic cleaner bath in THF for 30 min to remove unreacted P(MMA-co-MPS). The final product was dried at 80 C in vacuum for 6 h. Characterization of HA nanoparticle The morphology of HA was characterized by transmission electron microscope (TEM, FEI Tecnai G2 Spirit, the Netherlands). The size distribution of HA particles was measured using a Malvern Zetasizer (nano series Zen 3600, Malvern Instruments). The content of P(MMA-co-MPS) on the surface of HA was qualitatively evaluated by Fourier-transformed infrared spectroscopy (FT-IR, Bruker, Germany) based on reported method.19 P(MMA-co-MPS)-HA (0.002 g) was pressed into KBr (0.15 g) pellets. The modification degree of HA was also quantified on a TGA/DSC1 thermogravimetric analysis (TGA, Meteler). In detail, x-HA (8 mg) was heated from 30 C to 700 C with a heating rate of 10 C/min under nitrogen atmosphere. The weight percentage of P(MMA-co-MPS) on the surface of HA was calculated by the formula as follows: Weight Percentage ðwt %Þ ¼
WPolymer =WHA 100% (1)
where Wpolymer is the weight loss of P(MMA-co-MPS) on the surface of HA and WHA is the mass of modified HA nanoparticles.
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Preparation of the bone cement Bone cements were prepared by mixing powder and liquid components at a powder (P) to liquid (L) ratio of 1:3 (w/ w). The powder component consisted of PMMA (0.56 g), xHA (0.40 g) and BPO (initiator, 0.04 g). The liquid component contained two compounds: MMA (monomer, 3 mL) and DMT (accelerator, 0.14 ll). The cement was designated as x-HA/PMMA (x 5 1, 2, 3, or 4). Morphology of fracture surface of x-HA/PMMA bone cement The composite bone cement samples after flexural experiment were soaked in PBS for 3 days, and then, the morphologies of fracture surface of bone cement samples were observed on a JSM-6300F scanning electron microscope (SEM, Japan). Biomineralization of x-HA/PMMA bone cement Specimens of x-HA/PMMA bone cement (5 3 6 3 1 mm) were soaked in 5 mL of simulated body fluid (SBF) for 0, 3, 7, and 14 days. The specimens were washed with distilled water after soaking for 7 days and then dried at room temperature. x-HA/PMMA bone cements before and after biomineralization were characterized by thermal field emission environmental SEM (Quanta 400F, the Netherlands) and energy dispersive X-ray spectrometry (Inca X-act, OXFORD, Britain) Mechanical properties According to the ISO5833 standard, mechanical property of the composite bone cement samples was tested on a Lloyd universal material testing machine (LLOYD, Britain). The samples for the compressive test were fabricated in a cylindrical mold (length 3 diameter: 12 3 6 mm). The fourpoint flexural bending test was performed using rectangular-shaped samples (length 3 width 3 thickness: 75 3 10 3 3.3 mm). Both of the tests were carried out at a crosshead speed of 5 mm/min, the supporting span was 34 mm in the four-point bending test. Samples were tested after being soaked in PBS for 3 days at room temperature. Five duplicates were measured for each sample, and the results were presented as mean 6 STD. In vitro cytotoxicity 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out to evaluate the cytotoxicity of x-HA/PMMA bone cement extracts toward hFOB. The extracts of x-HA/PMMA bone cements were prepared according to the ISO10993 standard. x-HA/PMMA bone cements (diameter 3 length: 5 3 6 mm) were first pretreated in 75% ethanol and sterile phosphate-buffered saline (PBS) for one day respectively and were subsequently soaked in sterile DMEM (the ratio of cement to medium was 1/5 (w/v)) at 34 C for 24 h. This DMEM was then used as the “extract medium” in the following cell culture without further filtration.hFOB cells were seeded into a 48well plate (5.0 3 104 cells/well) containing 1 mL of growth medium in each well. After incubation for 24 h (34 C, 5%
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CO2), the medium was removed. About 400 mL of extract medium from corresponding x-HA/PMMA bone cement was added to each well. After incubation for 24 h, 60 mL of MTT solution (5 mg/mL) in DMEM was added to each well. After incubation for 4 h, the MTT medium was aspirated, and 200 mL of DMSO was added to each well to dissolve the formazan. The optical density (OD) of the obtained formzen solution in DMSO was measured at 570 nm on a plate reader (BioTek Synergy4). The cell viable rate was calculated through the following Eq. (2): Viable cell % 5 ðODtreated =ODcontrol Þ 3 100
(2)
where ODcontrol was obtained in the absence of extract medium and ODtreated was obtained in the presence of extract medium. Hemolysis assay Hemolysis assay was performed using human whole blood (acid-citrate-dextrose, ACD22). About 0.5 mL of ACD blood was added to the tubes containing 1 mL of bone cement extracts, and the obtained solution was incubated at 37 C for 1 h, and then, the tubes were centrifuged for 5 min at 1000 r/min, 0.2 mL of the supernatant was subjected to measurement of the optical density. 1.0 mL of PBS was used as negative control and distilled water was used as positive control. Optical density (OD) of supernatant, positive control, and negative control was calculated at 545 nm. The percentage of hemolysis was calculated as follows: Hemolysis % OD of the test sample 2 OD of the negative control ¼ OD of the positive sample 2 OD of the negative sample 3100
Cell morphology hFOB cells were seeded on the surface of x-HA/PMMA bone cement. After 7 days’ incubation, the surface of cement was rinsed with PBS to remove nonadherent cells. The cells were fixed by 2.5% glutaraldehyde in cacodylate buffer23 and then washed in cacodylate buffer with sucrose. After dehydration with gradient alcohol and sputter coating with gold, the samples were subjected to SEM (Quanta 400, Philips) observation. Alkaline phosphatase activity The ALP activity of the cells on the surface of x-HA/PMMA bone cement (size: 6 3 3 mm, 6 parallel samples for each incubation period) was measured using the following protocol.24 hFOB cells were seeded on surface of the cement at a density of 2.0 3 105 cells/cm2 and cultured in growth medium for 24 h at 34 C and 5% CO2. Medium was removed, and the cell monolayer was washed twice with PBS, then the cells were treated with cold Triton X-100 (Sigma, USA) for 1 h under agitation to extract the ALP enzyme. After agitation, aliquots of the supernatant were taken for determination of protein concentration. The ALP
EFFECT OF MODIFICATION DEGREE OF NANOHYDROXYAPATITE
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FIGURE 1. (a) TEM and (b) size distribution of HA nanoparticles at 25 C.
assay was performed using Alkaline Phosphatase Assay Kit (Beyotime, Shanghai, China).
RESULTS
Characterizations of nano-HA The morphology of nano-HA was visualized by TEM (Figure 1a). The rod-like HA nanoparticles exhibit average diameter around 30 nm and length of approximately 100 nm. These nanoparticles disperse well, and there is no apparent aggregation in the suspension. The size of HA determined by dynamic light-scattering method, as shown in Figure 1b, displays a narrow size distribution with an average diameter of around 258.9 nm (peak 164 nm, PDI 0.22). FT-IR spectra were used to qualitatively analyze the content of P(MMA-co-MPS) (Mw 5 45,500 g/mol) on the surface of HA. As shown in Figure 2a, the peak at 557 cm21 and 603 cm21 can be assigned to PAO bending of phosphate group. And the peak at 1034 cm21 can be attributed to OAPAO phosphate ions of hydroxyl site. The decreasing bands at 3433 cm21 and 1638 cm21, which were assigned to stretching and bending vibrations of AOH group, and the increasing peak at 1732 cm21 (C@O) indicated that the
modification degree of P(MMA-co-MPS) on the surface of HA increased with the increase in the feed ratio of P(MMAco-MPS) to HA.25 The amount of P(MMA-co-MPS) covalently attached to the surface of HA nanoparticles was also determined by TGA (Figure 2b). Obviously, there were two main weight loss stages (0 ; 200 C, 310 ; 510 C) in their thermograms of all these samples. According to the Eq. (1), the modification degree of 2-HA, 3-HA, and 4-HA was about 18.7%, 22.8%, and 26%, respectively. For the prestine HA (1-HA), there is a slight decrease from 100% to 97% till 600 C.
Mechanical properties The mechanical property of composite bone cement was investigated in terms of compressive strength, flexural strength, and flexural modulus. After incorporation of HA into the PMMA matrix, the mechanical property of the cement was significantly enhanced. When increasing the modification degree of P(MMA-co-MPS) on the surface of HA, the compressive strength of composite bone cement increased from 62.59 to 74.55 MPa (Figure 3); the flexural strength and flexural modulus of composite bone cement
FIGURE 2. Modification degree of nano-HA determined by (a) FTIR spectra and (b) TGA.
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samples. As shown in the magnified images (the inserts in Figure 6. a4–d4), the sizes of the agglomerates on the surface of 4-HA/PMMA bone cement became larger and the mineral layer became thicker in the same period. The Ca/P ratio of the minerals on surface of the cement was also analyzed (Figure 6. a4–d4). After soaking in SBF for 14 days, the Ca/P ratio of the minerals on the surface of the bone cement was found to be in the range of 1.93–2.05.
FIGURE 3. Compressive strength of x-HA/PMMA (x 5 1, 2, 3, or 4) bone cement.
(Figure 4) were also significantly improved and reached 35.39 MPa, and 1744.72 MPa, respectively. Morphology of fracture surface of the cement The morphology of the fracture surfaces was observed on SEM after flexural test to study the cracks between HA nanoparticles and PMMA matrix. As shown in Figure 5, some HA nanoparticles agglomerated in the PMMA matrix; and the cracks between HA and PMMA matrix were also observed after incorporation of HA into PMMA matrix. Furthermore, both agglomeration of nano-HA and crack size at the facture surface decreased with increased modification degree of HA. Biomineralization Figure 6 presented the morphology of composite bone cement before and after mineralization. Obviously, the area of the apatite layer increased when increasing the modification degree of HA. The sheet-shaped smooth surface of bone cement became rougher and small pieces of apatite began to coalesce into large agglomerates. After 14 days’ mineralization, more fuzzy apatite deposited on the surface of 4-HA/PMMA bone cement as comapred with the other
Hemolysis assay Hemolytic potential of composite bone cement is one of the major concerns for its application in bone fixation. As shown in Figure 7a, all the composite bone cements exhibited hemolysis ratio of
School of Engineering, Sun Yat-sen University, Guangzhou 510006, People’s Republic of China Department of Orthopedics, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, People’s Republic of China 3 Department of Outpatient, First Affiliated Hospital, Sun Yat-sen University, Guangzhou 510008, People’s Republic of China 2
Received 15 May 2014; revised 13 October 2014; accepted 30 March 2015 Published online 7 May 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33428 Abstract: The objective of this study is to prepare a biocompatible nanohydroxyapatite/poly(methyl methacrylate) (HA/ PMMA) composite bone cement, which has good mechanical property and can be used for vertebroplasty. Up to 40 wt % of nanohydroxyapatite (nano-HA) in the power, which was surface modified with poly(methylmethacrylate-co-g-methacryloxypropyl timethoxysilane) [P(MMA-co-MPS)] copolymer, was incorporated into the composite bone cement. The content of P(MMA-co-MPS) on the surface of nano-HA (18.7%, 22.8%, and 26%) was determined through thermogravimetric analysis (TGA). The morphology of biomineralized surface of composite bone cement was observed under scanning electron microscope (SEM). The mechanical measurements of the composite
cements implied that the interfacial interaction between the HA and PMMA matrix may be greatly enhanced after surface modification of HA. Biochemical assays indicated that the HA/ PMMA bone cement had no cytotoxicity and induced no hemolysis. The cell adhesion and alkaline phosphatase (ALP) activity assays indicated that the biocompatibility of HA/ PMMA bone cement could be promoted, demonstrating that it can be used as an ideal weight-bearing bone repair materials C 2015 Wiley Periodicals, Inc. J Biomed on clinical application. V Mater Res Part B: Appl Biomater, 104B: 576–584, 2016.
Key Words: poly(methyl methacrylate), nanohydroxyapatite, modification degree, mechanical property, biocompatibility
How to cite this article: Quan C, Tang Y, Liu Z, Rao M, Zhang W, Liang P, Wu N, Zhang C, Shen H, Jiang Q. 2016. Effect of modification degree of nanohydroxyapatite on biocompatibility and mechanical property of injectable poly(methyl methacrylate)-based bone cement. J Biomed Mater Res Part B 2016:104B:576–584.
INTRODUCTION
Ideal materials for bone repair should be similar to human bone tissue, which could not only induce bone cells to infiltrate into the implanted materials, but also have good mechanical properties around host bone tissue.1,2 Due to its self-hardening behavior and excellent mechanical properties, polymethylmethacrylate (PMMA) bone cement has been widely used in clinical practice for decades.3 However, the bioinertness of PMMA cement hinders its efficient integration with the host bone tissue and would lead to inferior interfacial strength between bone cement and host bone tissue as well as the implant;4 orthopedic implants would be loosened and cause failure after a few years.5 In order to overcome these problems, researches have focused on
adapting the biocompatibility of PMMA bone cement by introducing bioactive inorganic nanoparticles such as hydroxyapatite (HA),6 Al2O3,7 and SiO2.8 HA has similar chemical composition and crystal structure to apatite in human bone tissue and can bind to the host bone tissue;9 it has been extensively studied as inorganic filler in PMMA bone cement to promote biocompatibility.10 Efforts have been devoted to HA/PMMA cement to meet the mechanical and biochemical requirements of clinical practice.11,12 For example, 3-trimethoxysilyl propyl methacrylate (g-MPS) has been employed to modify the surface of HA in order to achieve better mechanical property of the HA/PMMA cement.13 The surface modification of HA by g-MPS would not only enhance the dispersion and stability
*Both authors contributed equally to this work. Correspondence to: C. Quan; e-mail: [email protected] and [email protected] Contract grant sponsor: National Natural Science Foundation of China (NSFC); contract grant number: 21104098 Contract grant sponsor: The Ph.D Programs Foundation of Ministry of Education of China; contract grant number: 20110171120008 Contract grant sponsor: Fundamental Research Funds for the Central Universities; contract grant number: 121 gpy17 Contract grant sponsor: Science and Technology Planning Project of Guangdong Province; contract grant number: 2011A060901013 Contract grant sponsor: Guangdong Innovative Research Team Program; contract grant number: 2009010057
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of HA in MMA during the preparation of HA/PMMA bone cement, but also minimize the difference of surface properties between HA nanoparticles and PMMA matrix.14 As a consequence of such improvement, the mechanical property of HA/PMMA bone cement could be enhanced.15 Considering the fact that reaction between MPS and MMA monomer might be hindered by the steric hindrance from free PMMA chains or chains growing from adjacent MPS groups, such kind of modification may not be enough to achieve better compatibility between the organic and inorganic phases. An alternative solution to this problem is to modify the surface of HA using a copolymer of MPS and MMA, which not only maintains efficient reactivity between the siloxane groups of MPS with hydroxyl group of HA but also interacts/entangles with the continuous PMMA phase. Herein, the surface of nano-HA was first modified with poly(methyl methacrylate-co-g-methacryloxypropyl timethoxysilane) [P(MMA-co-MPS)] copolymer, followed by being added to the MMA monomer as a nanofiller to prepare a series of injectable HA/PMMA bone cements. After modification of HA, the content of HA in the bone cement could reach up to 40 wt % and the cement can be implanted by injection.16 Taking advantage of MMA structural unit in chain of P(MMA-co-MPS) copolymer, the dispersion of modified HA in PMMA matrix could be greatly improved to obtain a more homogenous system. The mechanical properties and biocompatibility, such as biomineralization, cell adhesion, and ALP activity of the cement were investigated to illuminate its biocompatibility and its potential application in weight-bearing bone regeneration. MATERIALS AND METHODS
Materials Methyl methacrylate (MMA) and poly(ethylene glycol) 2000 (PEG 2000) were purchased from Damao Chemical Reagent Factory (Tianjin, China). MMA was used after distillation under reduced pressure. Poly(vinyl alcohol) (PVA, Mw 30,000–50,000 g/mol) and benzoyl peroxide (BPO) were purchased from Sinopharm Chemical Reagent Co. Ltd. 3trimethoxysilyl propyl methacrylate (g-MPS) was purchased from Dow Corning (Nanjing, China) and used after distillation under reduced pressure. Dimethyltryptamine (DMT) was purchased from Aladdin Reagent Company (Shanghai, China) and used as received without further purification. All other reagents were purchased from Guangzhou Chemical Reagent Factory and used as received. HA nanoparticles were prepared according to reported procedure with minor modifications.17 Simulated body fluid (SBF) was prepared following reported protocol.18 Human fetal osteoblast (hFOB) was purchased from the Cell Bank of the Chinese Academy of Sciences at Shanghai, China. Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were purchased from Hyclone (USA). Geneticin Selective Antibiotic (G-418 Sulfate) was purchased from Notlas (Beijing, China). N-2-hydroxyethylpiperazine-N-ethanesulfonic acid (HEPES) was purchased from Amresco (USA).
Synthesis of PMMA and P(MMA-co-MPS) PMMA and P(MMA-co-MPS) were synthesized according to our previous report.19 In detail, PVA (0.35 g), MMA (52.864 g), and BPO (0.13 g) were dissolved in 360 mL of deionized water to prepare PMMA. P(MMA-co-MPS) was synthesized via freeradical copolymerization using SHCH2CH2OH as a chain transfer agent. Briefly, MMA (25.488 g), MPS (7.1466 g), AIBN (0.1186 g), and SHCH2CH2OH (0.2659 g) were reacted in 60 mL of tetrahydrofuran (THF) for 6 h at 70 C to synthesize the P(MMA-co-MPS) copolymer. Weight-average molar mass of the polymerization products was determined via gel permeation chromatography (GPC) system equipped with Waters 1515 separations module, Waters 2414 refractive index detector. The concentration of each copolymer was 6 mg/mL, and THF (HPLC grade) was used as the eluent at a flow rate of 0.3 mL/min. Surface modification of HA The surface modification of nano-HA was carried out in a mixture of methanol and water (9:1, v/v).20,21 After nanoHA (8.0 g) was dispersed in the ethanol/water mixture at pH 3.5–4.0, then varied amount of P(MMA-co-MPS) (1.0, 2.0, and 3.0 g, respectively) in acetone was added dropwise to the mixture. The MPS units in the copolymer were hydrolyzed in the solution at 50 C; 90 min later, the pH was raised to 10.0 with NaOH (10%) solution to promote the condensation reaction. Products with different degree of modification (defined as x-HA, x 5 2, 3, or 4; HA without modification was defined as 1-HA) were collected by filtration and sonicated in an ultrasonic cleaner bath in THF for 30 min to remove unreacted P(MMA-co-MPS). The final product was dried at 80 C in vacuum for 6 h. Characterization of HA nanoparticle The morphology of HA was characterized by transmission electron microscope (TEM, FEI Tecnai G2 Spirit, the Netherlands). The size distribution of HA particles was measured using a Malvern Zetasizer (nano series Zen 3600, Malvern Instruments). The content of P(MMA-co-MPS) on the surface of HA was qualitatively evaluated by Fourier-transformed infrared spectroscopy (FT-IR, Bruker, Germany) based on reported method.19 P(MMA-co-MPS)-HA (0.002 g) was pressed into KBr (0.15 g) pellets. The modification degree of HA was also quantified on a TGA/DSC1 thermogravimetric analysis (TGA, Meteler). In detail, x-HA (8 mg) was heated from 30 C to 700 C with a heating rate of 10 C/min under nitrogen atmosphere. The weight percentage of P(MMA-co-MPS) on the surface of HA was calculated by the formula as follows: Weight Percentage ðwt %Þ ¼
WPolymer =WHA 100% (1)
where Wpolymer is the weight loss of P(MMA-co-MPS) on the surface of HA and WHA is the mass of modified HA nanoparticles.
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Preparation of the bone cement Bone cements were prepared by mixing powder and liquid components at a powder (P) to liquid (L) ratio of 1:3 (w/ w). The powder component consisted of PMMA (0.56 g), xHA (0.40 g) and BPO (initiator, 0.04 g). The liquid component contained two compounds: MMA (monomer, 3 mL) and DMT (accelerator, 0.14 ll). The cement was designated as x-HA/PMMA (x 5 1, 2, 3, or 4). Morphology of fracture surface of x-HA/PMMA bone cement The composite bone cement samples after flexural experiment were soaked in PBS for 3 days, and then, the morphologies of fracture surface of bone cement samples were observed on a JSM-6300F scanning electron microscope (SEM, Japan). Biomineralization of x-HA/PMMA bone cement Specimens of x-HA/PMMA bone cement (5 3 6 3 1 mm) were soaked in 5 mL of simulated body fluid (SBF) for 0, 3, 7, and 14 days. The specimens were washed with distilled water after soaking for 7 days and then dried at room temperature. x-HA/PMMA bone cements before and after biomineralization were characterized by thermal field emission environmental SEM (Quanta 400F, the Netherlands) and energy dispersive X-ray spectrometry (Inca X-act, OXFORD, Britain) Mechanical properties According to the ISO5833 standard, mechanical property of the composite bone cement samples was tested on a Lloyd universal material testing machine (LLOYD, Britain). The samples for the compressive test were fabricated in a cylindrical mold (length 3 diameter: 12 3 6 mm). The fourpoint flexural bending test was performed using rectangular-shaped samples (length 3 width 3 thickness: 75 3 10 3 3.3 mm). Both of the tests were carried out at a crosshead speed of 5 mm/min, the supporting span was 34 mm in the four-point bending test. Samples were tested after being soaked in PBS for 3 days at room temperature. Five duplicates were measured for each sample, and the results were presented as mean 6 STD. In vitro cytotoxicity 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was carried out to evaluate the cytotoxicity of x-HA/PMMA bone cement extracts toward hFOB. The extracts of x-HA/PMMA bone cements were prepared according to the ISO10993 standard. x-HA/PMMA bone cements (diameter 3 length: 5 3 6 mm) were first pretreated in 75% ethanol and sterile phosphate-buffered saline (PBS) for one day respectively and were subsequently soaked in sterile DMEM (the ratio of cement to medium was 1/5 (w/v)) at 34 C for 24 h. This DMEM was then used as the “extract medium” in the following cell culture without further filtration.hFOB cells were seeded into a 48well plate (5.0 3 104 cells/well) containing 1 mL of growth medium in each well. After incubation for 24 h (34 C, 5%
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CO2), the medium was removed. About 400 mL of extract medium from corresponding x-HA/PMMA bone cement was added to each well. After incubation for 24 h, 60 mL of MTT solution (5 mg/mL) in DMEM was added to each well. After incubation for 4 h, the MTT medium was aspirated, and 200 mL of DMSO was added to each well to dissolve the formazan. The optical density (OD) of the obtained formzen solution in DMSO was measured at 570 nm on a plate reader (BioTek Synergy4). The cell viable rate was calculated through the following Eq. (2): Viable cell % 5 ðODtreated =ODcontrol Þ 3 100
(2)
where ODcontrol was obtained in the absence of extract medium and ODtreated was obtained in the presence of extract medium. Hemolysis assay Hemolysis assay was performed using human whole blood (acid-citrate-dextrose, ACD22). About 0.5 mL of ACD blood was added to the tubes containing 1 mL of bone cement extracts, and the obtained solution was incubated at 37 C for 1 h, and then, the tubes were centrifuged for 5 min at 1000 r/min, 0.2 mL of the supernatant was subjected to measurement of the optical density. 1.0 mL of PBS was used as negative control and distilled water was used as positive control. Optical density (OD) of supernatant, positive control, and negative control was calculated at 545 nm. The percentage of hemolysis was calculated as follows: Hemolysis % OD of the test sample 2 OD of the negative control ¼ OD of the positive sample 2 OD of the negative sample 3100
Cell morphology hFOB cells were seeded on the surface of x-HA/PMMA bone cement. After 7 days’ incubation, the surface of cement was rinsed with PBS to remove nonadherent cells. The cells were fixed by 2.5% glutaraldehyde in cacodylate buffer23 and then washed in cacodylate buffer with sucrose. After dehydration with gradient alcohol and sputter coating with gold, the samples were subjected to SEM (Quanta 400, Philips) observation. Alkaline phosphatase activity The ALP activity of the cells on the surface of x-HA/PMMA bone cement (size: 6 3 3 mm, 6 parallel samples for each incubation period) was measured using the following protocol.24 hFOB cells were seeded on surface of the cement at a density of 2.0 3 105 cells/cm2 and cultured in growth medium for 24 h at 34 C and 5% CO2. Medium was removed, and the cell monolayer was washed twice with PBS, then the cells were treated with cold Triton X-100 (Sigma, USA) for 1 h under agitation to extract the ALP enzyme. After agitation, aliquots of the supernatant were taken for determination of protein concentration. The ALP
EFFECT OF MODIFICATION DEGREE OF NANOHYDROXYAPATITE
ORIGINAL RESEARCH REPORT
FIGURE 1. (a) TEM and (b) size distribution of HA nanoparticles at 25 C.
assay was performed using Alkaline Phosphatase Assay Kit (Beyotime, Shanghai, China).
RESULTS
Characterizations of nano-HA The morphology of nano-HA was visualized by TEM (Figure 1a). The rod-like HA nanoparticles exhibit average diameter around 30 nm and length of approximately 100 nm. These nanoparticles disperse well, and there is no apparent aggregation in the suspension. The size of HA determined by dynamic light-scattering method, as shown in Figure 1b, displays a narrow size distribution with an average diameter of around 258.9 nm (peak 164 nm, PDI 0.22). FT-IR spectra were used to qualitatively analyze the content of P(MMA-co-MPS) (Mw 5 45,500 g/mol) on the surface of HA. As shown in Figure 2a, the peak at 557 cm21 and 603 cm21 can be assigned to PAO bending of phosphate group. And the peak at 1034 cm21 can be attributed to OAPAO phosphate ions of hydroxyl site. The decreasing bands at 3433 cm21 and 1638 cm21, which were assigned to stretching and bending vibrations of AOH group, and the increasing peak at 1732 cm21 (C@O) indicated that the
modification degree of P(MMA-co-MPS) on the surface of HA increased with the increase in the feed ratio of P(MMAco-MPS) to HA.25 The amount of P(MMA-co-MPS) covalently attached to the surface of HA nanoparticles was also determined by TGA (Figure 2b). Obviously, there were two main weight loss stages (0 ; 200 C, 310 ; 510 C) in their thermograms of all these samples. According to the Eq. (1), the modification degree of 2-HA, 3-HA, and 4-HA was about 18.7%, 22.8%, and 26%, respectively. For the prestine HA (1-HA), there is a slight decrease from 100% to 97% till 600 C.
Mechanical properties The mechanical property of composite bone cement was investigated in terms of compressive strength, flexural strength, and flexural modulus. After incorporation of HA into the PMMA matrix, the mechanical property of the cement was significantly enhanced. When increasing the modification degree of P(MMA-co-MPS) on the surface of HA, the compressive strength of composite bone cement increased from 62.59 to 74.55 MPa (Figure 3); the flexural strength and flexural modulus of composite bone cement
FIGURE 2. Modification degree of nano-HA determined by (a) FTIR spectra and (b) TGA.
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samples. As shown in the magnified images (the inserts in Figure 6. a4–d4), the sizes of the agglomerates on the surface of 4-HA/PMMA bone cement became larger and the mineral layer became thicker in the same period. The Ca/P ratio of the minerals on surface of the cement was also analyzed (Figure 6. a4–d4). After soaking in SBF for 14 days, the Ca/P ratio of the minerals on the surface of the bone cement was found to be in the range of 1.93–2.05.
FIGURE 3. Compressive strength of x-HA/PMMA (x 5 1, 2, 3, or 4) bone cement.
(Figure 4) were also significantly improved and reached 35.39 MPa, and 1744.72 MPa, respectively. Morphology of fracture surface of the cement The morphology of the fracture surfaces was observed on SEM after flexural test to study the cracks between HA nanoparticles and PMMA matrix. As shown in Figure 5, some HA nanoparticles agglomerated in the PMMA matrix; and the cracks between HA and PMMA matrix were also observed after incorporation of HA into PMMA matrix. Furthermore, both agglomeration of nano-HA and crack size at the facture surface decreased with increased modification degree of HA. Biomineralization Figure 6 presented the morphology of composite bone cement before and after mineralization. Obviously, the area of the apatite layer increased when increasing the modification degree of HA. The sheet-shaped smooth surface of bone cement became rougher and small pieces of apatite began to coalesce into large agglomerates. After 14 days’ mineralization, more fuzzy apatite deposited on the surface of 4-HA/PMMA bone cement as comapred with the other
Hemolysis assay Hemolytic potential of composite bone cement is one of the major concerns for its application in bone fixation. As shown in Figure 7a, all the composite bone cements exhibited hemolysis ratio of
