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Improved mechanical properties of hydroxyapatite whisker-reinforced poly(l -lactic acid) scaffold by surface modification of hydroxyapatite
Materials Science and Engineering C 35 (2014) 190–194
Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Improved mechanical properties of hydroxyapatite whisker-reinforced poly(L-lactic acid) scaffold by surface modification of hydroxyapatite Zhou Fang a, Qingling Feng b,⁎ a b
State Key Laboratory of New Ceramics and Fine Processing, School of Materials Science and Engineering, Tsinghua University, Beijing 100084, China Key Laboratory of Advanced Materials (MOE), School of Materials Science and Engineering, Tsinghua University, Beijing 100084, China
a r t i c l e
i n f o
Article history: Received 19 August 2013 Received in revised form 11 October 2013 Accepted 2 November 2013 Available online 14 November 2013 Keywords: Hydroxyapatite whisker Poly(L-lactide) (PLLA) Surface modification Scaffold Mechanical property
a b s t r a c t To improve the mechanical properties of porous hydroxyapatite/poly(L-lactic acid) (HA/PLLA) composites, HA whiskers with high crystallinity and high aspect ratio were synthesized. HA whiskers were modified with γaminopropyltriethoxysilane (APTES) to improve the interface between HA whiskers and PLLA. The composite scaffold consists of a porous PLLA matrix with HA whiskers distributed homogeneously. The morphology and the distributions of pore sizes of PLLA scaffold was not influenced by introducing HA whiskers, while the mechanical properties were improved. Both the compressive strength and compressive modulus were increased with the weight ratio of APTES-modified HA whiskers up to 30 wt.%, but only up to 15 wt.% for non-modified HA whiskers. With more than 15 wt.% HA whiskers, the mechanical properties of HA/PLLA scaffold were better improved with APTES-modified HA whiskers than non-modified. The HA whisker/PLLA scaffold with high porosity and improved mechanical properties is attractive in the application of tissue engineering. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Biodegradable materials have received wide attention in bone tissue engineering. This kind of material does not need to be removed with additional surgery after bone repair. The most widely used degradable materials are poly(a-hydroxyl acids) such as poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), and poly(D,L-lactic acid-co-glycolic acid) (PLGA) [1]. These polymers were usually fabricated into porous scaffolds, which play an important role in bone repair by supplying space for bone cell growth and differentiation both in vitro and in vivo [2]. But the weak mechanical properties of porous polymer scaffold and the degraded acidic monomers which may cause inflammatory and allergic reactions [3] limit the applications. For example, PLLA has good biocompatibility and degradability with well-controlled molecular weight, but its weak mechanical properties limit its applications in bone tissue engineering. To improve the mechanical properties for bone repair and provide a better environment for cell attachment and proliferation, calcium orthophosphate-based biomaterials and bioceramics are usually considered as fillers and coatings. Furthermore, composite scaffold usually has better osteoconductivity than singleingredient scaffold [4,5]. Hydroxyapatite (Ca10(PO4)6(OH)2) (HA) is the major inorganic component of the bone and teeth. HA has good wear behavior, good bonding ability to the bone directly and most importantly, it has no toxicity but a high biocompatibility [6]. So HA is often considered as medical implants or reinforced phase with different morphologies. Furthermore, the ⁎ Corresponding author. Tel.: +86 10 62782770; fax: +86 10 62771160. E-mail address: [email protected] (Q. Feng). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.11.008
improvement of mechanical properties when introducing different morphologies of HA into the polyanhydride matrix is maximal for whisker, next for sphere, rod and flake [7]. In this research, poly(L-lactic acid) (PLLA) and HA whiskers are mixed to compose a porous scaffold. HA whisker, as a reinforced phase, can improve the mechanical properties and osteoconduction of composite, and the acidity catabolite of PLLA can be neutralized by alkalinity catabolite of HA [8]. The HA whiskers were well-crystallized with controlled morphology. The HA whiskers were modified with silane coupling agent to improve the surface chemical compatibility between HA whiskers and the PLLA matrix. Both the compressive strength and compressive modulus were increased with the weight ratio of HA whiskers. 2. Experimental 2.1. Materials Poly(L-lactide) (PLLA) (i.v. 4.0 dL/g) was purchased from the Institute of Medical Devices of Shandong Province. HA whiskers were synthesized based on a hydrothermal homogeneous precipitation method. γ-aminopropyltriethoxysilane (APTES) and 1,4-dioxane were purchased from Sinopharm Chemical Reagent Co., Ltd. All the reagents were at analytical grade. 2.2. Synthesis of HA whiskers HA whiskers were synthesized by hydrothermal homogeneous precipitation method reported. Briefly, specific concentration of aqueous
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solutions containing calcium and phosphate was prepared by dissolving Ca(NO3)2·4H2O and (NH4)2HPO4 in 0.05 mol/L HNO3, with the Ca/P ratio fixed at 1.67 throughout. Then certain amount of acetamide was added. The initial pH was adjusted to 2.9 with 1:1 ammonium hydroxide. The hydrothermal processing was achieved by loading the prepared solution in autoclave, kept at 180 °C for 10 h in a furnace, and then cooled to room temperature. The mixture was then centrifugally separated and cleaned with deionized water for 3 times. The precipitate was dried at 60 °C to obtain HA whiskers.
samples were cut into cylindrical specimens in 1.8 cm length and 0.8 cm diameter. The compression tests were conducted on more than 3 specimens of each series at a cross-head speed of 1 mm/min on the WDW 3020 electronic universal experiment machine. The initial linear part of stress–strain curve was used to fit compressive modulus and the stresses at 10% compression strain were considered as compressive strength. The porosity data of scaffolds were given by mercury injection apparatus (Autopore IV 9510).
2.3. Silanization of HA whiskers
3. Results and discussion
HA whiskers were modified based on the literature [9] with a more efficient method. First, HA whiskers were dried at 60 °C for 24 h to move the water on the surface. Then HA whiskers were mixed with APTES anhydrous toluene solution (37 mmol/L), after 5 min ultrasonically dispersing, the mixture was kept in a shaker (THZ-C, Taicang City Experimental Equipment Factory) for 48 h at 60 R and 37 °C. Finally, the whiskers were centrifugally separated and cleaned with toluene solution to remove the physically absorbed APTES. The modified HA whiskers were then dried at 60 °C to obtain APTES-modified HA whiskers.
3.1. Preparation of APTES-modified HA whiskers
2.4. Preparation of HA/PLLA composite scaffolds PLLA was dissolved in 1,4-dioxane under stirring until the 5% w/v solution was clarified. Predetermined amounts of non-modified and APTESmodified HA whiskers (marked as HA and a-HA) were suspended in the solution with 5 min ultrasonically dispersing and stirring until homogeneous. The solution was transferred into a freezer at −20 °C in freezing tubes to solidify, and it caused solid–liquid phase separation. Then the composites were transferred into a freeze-dryer under −60 °C and 60 bar for 48 h to obtain the final HA/PLLA scaffold. 2.5. Characterization The crystal phase of HA and a-HA whiskers was characterized by Xray diffraction (D/max 2500 V Rigaku, CuKα, 5°/min, 0.02 per step). After elimination of the background, the crystallinity of HA phase was determined approximately by comparing the sum total intensities of crystalline peaks to the sum total intensities of both crystalline and amorphous peaks using the software Jade 5.0 (Materials Data, Inc.). A X-ray Photoelectron Spectrometer (XPS, ESCALAB 250Xi, 1 eV per step and 0.05 eV per step for narrow scan, C1s peak at 284.6 eV was used for calibration) was used to determine the surface compositions of aHA whiskers. The morphologies of HA and a-HA whiskers were observed by SEM (JSM-2001F). The length and diameter of HA whiskers were measured on SEM image and the data were gained based on more than 80 intact whiskers. The morphology of scaffolds, including PLLA and HA/PLLA composite, was also observed by SEM. All scaffold
Fig. 1 shows the SEM micrographs of HA and a-HA whiskers, respectively. Both HA and a-HA whiskers were uniform and well dispersive. The morphologies of HA whiskers were 60–116 μm in length with the aspect ratio of 68–103. No changes in morphology were observed after the silanization. The powder XRD patterns of both HA and a-HA whiskers are shown in Fig. 2. Despite some variation in peak intensity, all the peaks are in a good agreement with standard hydroxyapatite (Ca10(PO4)6(OH)2, JCPDS No. 54-0022), indicating that both samples are hydroxyapatite phase. The strongest peak is for the (300) rather than the usual (211), which is confirmed by previous report [10]. Furthermore, the spiculate peaks indicate the high HA crystallinity, which is calculated as 97.75% for HA whiskers and 97.33% for a-HA whiskers. Table 1 shows the calculated data based on XPS detail scan results of the surface chemical compositions of a-HA whiskers. Despite the instrument resolution, the data indicate that the surface of HA whiskers are modified with APTES successfully. More detail information is provided by XPS narrow spectra of P2p in Fig. 3. The binding energy of P2p in HA is 133.8 eV [11] as shown in Fig. 3(a), which is mainly related to the group of PO3− 4 . After silanization, the P2p peak of a-HA splits into 2 peaks and a new lower peak is observed. The lower binding energy is contributed by the second-nearest neighbor group, which shows a lower electronegativity than the H in \P\O\H. This illustrates that some of the P\O\H on the surface of HA is replaced by the Si\O\H in APTES and resulting in a covalent bonding of P\O\Si [12] after the hydrolysis and silanization. Considering the lower electronegativity of Si, the XPS narrow spectra of P2p in HA indicates that HA whiskers have been modified with APTES successfully in covalent bonding. This will improve the surface chemical compatibility between HA and PLLA matrix. Briefly, after the silanization of HA whiskers, no differences were observed in the crystalline phase and the crystallinity of HA whiskers. Both HA whiskers and a-HA whiskers were 60–116 μm in length with the aspect ratio of 68–103. The surface of HA whiskers was modified with APTES successfully to improve the surface chemical compatibility between HA and PLLA matrix.
Fig. 1. SEM micrographs of (a) HA whiskers and (b) a-HA whiskers.
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with a mercury injection apparatus (Autopore IV 9510) indicates that the porosities of the scaffolds change a little after mixing with HA whiskers, as shown in Table 2. All the porosities are higher than 89%, which is beneficial for cell growth. The distributions of pore sizes in all samples are mainly around 80 μm and 35 μm. No significant differences in pore size distribution have been observed, indicating that all the scaffolds have the similar porous structure, which does not make contributions to the improvement in the mechanical properties of composite scaffolds. Despite 10 wt.% a-HA series, the porosities of the others are all higher than the pure PLLA scaffold. The increase in the porosity is mainly based on the higher density of HA than PLLA. With a constant total quality of HA/ PLLA matrix, the volume will reduce due to the increase in density with more HA whiskers introduced. So the 1,4-dioxane and HA/PLLA solution contains a larger volume of 1,4-dioxane, which produces more pores after the solid–liquid phase separation.
Fig. 2. XRD patterns of HA whiskers and a-HA whiskers.
3.3. Mechanical properties of HA/PLLA scaffold
Table 1 The surface compositions of a-HA whiskers. Element Atom%
Ca 7.27
P 5.46
O 32.38
Si 8.87
N 6.94
C 39.08
3.2. HA/PLLA composite scaffold Different weight ratios (0, 10 wt.%, 15 wt.%, 20 wt.% and 30 wt.%) of HA whiskers were mixed with PLLA to obtain the HA/PLLA composite scaffolds. The composite scaffolds were cut in the middle and observed the incision by SEM, as shown in Fig. 4. No difference in the morphology is observed between PLLA scaffold and HA (or a-HA)/PLLA composite scaffold. The holes are uniform with about 80 μm and 35 μm in diameter, which is suitable for cell growth. All the holes are interconnected and distributed homogenously. The induced phase separation process in the mixture of HA/PLLA and 1,4-dioxane led to the formation of holes during the freeze drying directly. HA whiskers were dispersed well in the PLLA matrix and no significant fracture of whisker was observed. More HA whiskers were observed with the increasing weight ratio of HA whiskers in the composite. All the HA whiskers were embedded in PLLA matrix, except for the 30 wt.% HA/PLLA scaffold. This rough texture will promote human bone cell attachment [13]. Furthermore, previous research shows that the “point exposure” of HA appeared to provide anchoring sites for cell processes and provide a promising approach for controlling cell density on implant surfaces [14]. The porosity of scaffold plays an important role both in cell growth and the mechanical properties. The porosity data calculated automatically
Compressive properties of the composite scaffolds are shown in Fig. 5. The initial linear part of stress–strain data was used to fit the compressive modulus and the stress at the 10% compression was considered as the compressive strength. The compressive strength of HA/PLLA scaffold increased from 0.18 MPa to 0.28 MPa, while the compressive modulus increased from 3.24 MPa to 5.77 MPa with HA content from 0 to 30%. With regard to a-HA/PLLA scaffold, the compressive strength increased from 0.18 MPa to 0.42 MPa while the compressive modulus increased from 3.24 MPa to 8.03 MPa with HA content from 0 to 30%. The maximum compressive strength of a-HA/PLLA scaffold at 30 wt.% was 47.2% higher than the corresponding HA/PLLA scaffold and the compressive modulus was 39.2% higher than the corresponding HA/PLLA scaffold. According to the mechanical data, the compressive strength of a-HA/PLLA scaffold increased with the increasing weight ratio of aHA whiskers up to 30 wt.%, but the compressive strength of HA/PLLA increased until 15 wt.% HA whiskers and kept around 0.28 MPa until up to 30 wt.% HA whiskers. The ever-increasing improvement in compressive strength can be attributed to the improvement in the surface chemical compatibility between HA whiskers and the PLLA matrix after silanization. With high HA whisker content, HA whiskers were easy to expose and hanged in the holes, while a-HA whiskers were all embedded in the PLLA matrix, as shown in Fig. 6. Fig. 6(a) shows that the PLLA matrix cannot envelop all the HA whiskers when mixed with more than 15 wt.% HA whiskers, so some HA whiskers were extended from the PLLA matrix and hanged in the holes. So the compressive strengths of HA/PLLA scaffolds remained around 0.28 MPa with more than 15 wt.% HA whisker content. After
Fig. 3. XPS narrow spectra of P2p in (a) HA whiskers and (b) a-HA whiskers.
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Fig. 4. SEM micrographs of scaffolds containing (a) 0, (b) 15 wt.% HA whiskers, (c) 15 wt.% a-HA whiskers, (d) 30 wt.% HA whiskers, and (e) 30 wt.% a-HA whiskers (arrows: HA (or a-HA) whiskers).
silanization, the improvement in the surface chemical compatibility resulted in an ever-increasing improvement in the compressive strength of a-HA/PLLA scaffold. More a-HA whiskers were still enveloped in the PLLA matrix, as shown in Fig. 6(b). The matrix could be enhanced with more a-HA whiskers, so the compressive strengths of a-HA/PLLA scaffolds were still increasing with more than 15 wt.% a-HA whiskers. Plenty researches were conducted to develop the ideal scaffold with improvement in mechanical properties. To improve the mechanical properties of polymer scaffolds, most of which are hydrophobic, the hydrophilic HA needs to be modified. These researches on surface modification were mostly focused on the tensile property improvement of the Table 2 The porosity of HA or a-HA whisker scaffolds. Series
HA or a-HA content/wt.%
Porosity/%
HA whisker
0 10 15 10 15 20 30
89.10 94.89 94.17 87.38 89.66 98.12 95.32
a-HA whisker
composites with particles. Wang et al. firstly studied the effects of surface modification on the compressive property of scaffolds [15]. They modified HA nanoparticles with poly(ε-caprolactone) and the mechanical properties were improved due to the enhancement of the interaction between filler and matrix, and the better dispersion of fillers in the matrix. Yang et al. prepared silane-Octadecyltrichlorosilane (OTS)modified HA particle/PLLA porous scaffold with the porosity about 92% [16]. In their work, the highest compressive strength of the scaffold was 0.949 MPa at 50% compression strain with 30 wt.% OTS-modified HA/PLLA scaffold, 70.68% higher than the pure PLLA scaffold. In this work, the compressive strength of the scaffold was 0.42 MPa at 10% compression strain with 30 wt.% APTES-modified HA/PLLA scaffold, 130% higher than the pure PLLA scaffold. Greater improvement in the compressive strength of silane-modified HA/PLLA scaffold is obtained by using HA whiskers rather than HA particles. But compared with HA nanoparticles, the HA whiskers in this research can be exposed from the matrix, resulting in an invalid composite. After the silanization, a-HA whiskers were all enveloped in the PLLA matrix and no exposed whiskers were observed. This is due to the improved surface chemical compatibility between a-HA whiskers and the PLLA matrix and results in an ever-increasing improvement in compressive properties of a-HA/PLLA scaffold.
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Fig. 5. (a) The compressive moduli and (b) compressive strengths of scaffolds.
Fig. 6. (a) 30 wt.% HA whiskers and (b) 30 wt.% a-HA whiskers in HA/PLLA scaffold.
4. Conclusions
References [1] [2] [3] [4]
A new kind of HA/PLLA composite scaffold was prepared. HA whiskers with high crystallinity and high aspect ratio were modified with γaminopropyltriethoxysilane (APTES) to improve its performance as fillers in PLLA matrix. HA whiskers were easy to expose and hanged in the holes with more than 15 wt.% HA whisker content, while a-HA whiskers were all embedded in the PLLA matrix. The morphology of HA/PLLA scaffold was not influenced with different HA whisker contents, while both the compressive strength and compressive modulus were increased with the weight ratio of HA up to 30 wt.% a-HA whiskers, but only up to 15 wt.% HA whiskers. The maximum compressive strength of a-HA/ PLLA scaffold at 30 wt.% was 0.42 MPa, 47.2% higher than the corresponding HA/PLLA scaffold, and 130% higher than the pure PLLA scaffold.
[10] [11] [12]
Acknowledgments
[13] [14]
The authors are grateful for the financial support from the National Natural Science Foundation of China (51072090, 51061130554).
[5] [6] [7] [8] [9]
[15] [16]
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Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Improved mechanical properties of hydroxyapatite whisker-reinforced poly(L-lactic acid) scaffold by surface modification of hydroxyapatite Zhou Fang a, Qingling Feng b,⁎ a b
State Key Laboratory of New Ceramics and Fine Processing, School of Materials Science and Engineering, Tsinghua University, Beijing 100084, China Key Laboratory of Advanced Materials (MOE), School of Materials Science and Engineering, Tsinghua University, Beijing 100084, China
a r t i c l e
i n f o
Article history: Received 19 August 2013 Received in revised form 11 October 2013 Accepted 2 November 2013 Available online 14 November 2013 Keywords: Hydroxyapatite whisker Poly(L-lactide) (PLLA) Surface modification Scaffold Mechanical property
a b s t r a c t To improve the mechanical properties of porous hydroxyapatite/poly(L-lactic acid) (HA/PLLA) composites, HA whiskers with high crystallinity and high aspect ratio were synthesized. HA whiskers were modified with γaminopropyltriethoxysilane (APTES) to improve the interface between HA whiskers and PLLA. The composite scaffold consists of a porous PLLA matrix with HA whiskers distributed homogeneously. The morphology and the distributions of pore sizes of PLLA scaffold was not influenced by introducing HA whiskers, while the mechanical properties were improved. Both the compressive strength and compressive modulus were increased with the weight ratio of APTES-modified HA whiskers up to 30 wt.%, but only up to 15 wt.% for non-modified HA whiskers. With more than 15 wt.% HA whiskers, the mechanical properties of HA/PLLA scaffold were better improved with APTES-modified HA whiskers than non-modified. The HA whisker/PLLA scaffold with high porosity and improved mechanical properties is attractive in the application of tissue engineering. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Biodegradable materials have received wide attention in bone tissue engineering. This kind of material does not need to be removed with additional surgery after bone repair. The most widely used degradable materials are poly(a-hydroxyl acids) such as poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), and poly(D,L-lactic acid-co-glycolic acid) (PLGA) [1]. These polymers were usually fabricated into porous scaffolds, which play an important role in bone repair by supplying space for bone cell growth and differentiation both in vitro and in vivo [2]. But the weak mechanical properties of porous polymer scaffold and the degraded acidic monomers which may cause inflammatory and allergic reactions [3] limit the applications. For example, PLLA has good biocompatibility and degradability with well-controlled molecular weight, but its weak mechanical properties limit its applications in bone tissue engineering. To improve the mechanical properties for bone repair and provide a better environment for cell attachment and proliferation, calcium orthophosphate-based biomaterials and bioceramics are usually considered as fillers and coatings. Furthermore, composite scaffold usually has better osteoconductivity than singleingredient scaffold [4,5]. Hydroxyapatite (Ca10(PO4)6(OH)2) (HA) is the major inorganic component of the bone and teeth. HA has good wear behavior, good bonding ability to the bone directly and most importantly, it has no toxicity but a high biocompatibility [6]. So HA is often considered as medical implants or reinforced phase with different morphologies. Furthermore, the ⁎ Corresponding author. Tel.: +86 10 62782770; fax: +86 10 62771160. E-mail address: [email protected] (Q. Feng). 0928-4931/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.msec.2013.11.008
improvement of mechanical properties when introducing different morphologies of HA into the polyanhydride matrix is maximal for whisker, next for sphere, rod and flake [7]. In this research, poly(L-lactic acid) (PLLA) and HA whiskers are mixed to compose a porous scaffold. HA whisker, as a reinforced phase, can improve the mechanical properties and osteoconduction of composite, and the acidity catabolite of PLLA can be neutralized by alkalinity catabolite of HA [8]. The HA whiskers were well-crystallized with controlled morphology. The HA whiskers were modified with silane coupling agent to improve the surface chemical compatibility between HA whiskers and the PLLA matrix. Both the compressive strength and compressive modulus were increased with the weight ratio of HA whiskers. 2. Experimental 2.1. Materials Poly(L-lactide) (PLLA) (i.v. 4.0 dL/g) was purchased from the Institute of Medical Devices of Shandong Province. HA whiskers were synthesized based on a hydrothermal homogeneous precipitation method. γ-aminopropyltriethoxysilane (APTES) and 1,4-dioxane were purchased from Sinopharm Chemical Reagent Co., Ltd. All the reagents were at analytical grade. 2.2. Synthesis of HA whiskers HA whiskers were synthesized by hydrothermal homogeneous precipitation method reported. Briefly, specific concentration of aqueous
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solutions containing calcium and phosphate was prepared by dissolving Ca(NO3)2·4H2O and (NH4)2HPO4 in 0.05 mol/L HNO3, with the Ca/P ratio fixed at 1.67 throughout. Then certain amount of acetamide was added. The initial pH was adjusted to 2.9 with 1:1 ammonium hydroxide. The hydrothermal processing was achieved by loading the prepared solution in autoclave, kept at 180 °C for 10 h in a furnace, and then cooled to room temperature. The mixture was then centrifugally separated and cleaned with deionized water for 3 times. The precipitate was dried at 60 °C to obtain HA whiskers.
samples were cut into cylindrical specimens in 1.8 cm length and 0.8 cm diameter. The compression tests were conducted on more than 3 specimens of each series at a cross-head speed of 1 mm/min on the WDW 3020 electronic universal experiment machine. The initial linear part of stress–strain curve was used to fit compressive modulus and the stresses at 10% compression strain were considered as compressive strength. The porosity data of scaffolds were given by mercury injection apparatus (Autopore IV 9510).
2.3. Silanization of HA whiskers
3. Results and discussion
HA whiskers were modified based on the literature [9] with a more efficient method. First, HA whiskers were dried at 60 °C for 24 h to move the water on the surface. Then HA whiskers were mixed with APTES anhydrous toluene solution (37 mmol/L), after 5 min ultrasonically dispersing, the mixture was kept in a shaker (THZ-C, Taicang City Experimental Equipment Factory) for 48 h at 60 R and 37 °C. Finally, the whiskers were centrifugally separated and cleaned with toluene solution to remove the physically absorbed APTES. The modified HA whiskers were then dried at 60 °C to obtain APTES-modified HA whiskers.
3.1. Preparation of APTES-modified HA whiskers
2.4. Preparation of HA/PLLA composite scaffolds PLLA was dissolved in 1,4-dioxane under stirring until the 5% w/v solution was clarified. Predetermined amounts of non-modified and APTESmodified HA whiskers (marked as HA and a-HA) were suspended in the solution with 5 min ultrasonically dispersing and stirring until homogeneous. The solution was transferred into a freezer at −20 °C in freezing tubes to solidify, and it caused solid–liquid phase separation. Then the composites were transferred into a freeze-dryer under −60 °C and 60 bar for 48 h to obtain the final HA/PLLA scaffold. 2.5. Characterization The crystal phase of HA and a-HA whiskers was characterized by Xray diffraction (D/max 2500 V Rigaku, CuKα, 5°/min, 0.02 per step). After elimination of the background, the crystallinity of HA phase was determined approximately by comparing the sum total intensities of crystalline peaks to the sum total intensities of both crystalline and amorphous peaks using the software Jade 5.0 (Materials Data, Inc.). A X-ray Photoelectron Spectrometer (XPS, ESCALAB 250Xi, 1 eV per step and 0.05 eV per step for narrow scan, C1s peak at 284.6 eV was used for calibration) was used to determine the surface compositions of aHA whiskers. The morphologies of HA and a-HA whiskers were observed by SEM (JSM-2001F). The length and diameter of HA whiskers were measured on SEM image and the data were gained based on more than 80 intact whiskers. The morphology of scaffolds, including PLLA and HA/PLLA composite, was also observed by SEM. All scaffold
Fig. 1 shows the SEM micrographs of HA and a-HA whiskers, respectively. Both HA and a-HA whiskers were uniform and well dispersive. The morphologies of HA whiskers were 60–116 μm in length with the aspect ratio of 68–103. No changes in morphology were observed after the silanization. The powder XRD patterns of both HA and a-HA whiskers are shown in Fig. 2. Despite some variation in peak intensity, all the peaks are in a good agreement with standard hydroxyapatite (Ca10(PO4)6(OH)2, JCPDS No. 54-0022), indicating that both samples are hydroxyapatite phase. The strongest peak is for the (300) rather than the usual (211), which is confirmed by previous report [10]. Furthermore, the spiculate peaks indicate the high HA crystallinity, which is calculated as 97.75% for HA whiskers and 97.33% for a-HA whiskers. Table 1 shows the calculated data based on XPS detail scan results of the surface chemical compositions of a-HA whiskers. Despite the instrument resolution, the data indicate that the surface of HA whiskers are modified with APTES successfully. More detail information is provided by XPS narrow spectra of P2p in Fig. 3. The binding energy of P2p in HA is 133.8 eV [11] as shown in Fig. 3(a), which is mainly related to the group of PO3− 4 . After silanization, the P2p peak of a-HA splits into 2 peaks and a new lower peak is observed. The lower binding energy is contributed by the second-nearest neighbor group, which shows a lower electronegativity than the H in \P\O\H. This illustrates that some of the P\O\H on the surface of HA is replaced by the Si\O\H in APTES and resulting in a covalent bonding of P\O\Si [12] after the hydrolysis and silanization. Considering the lower electronegativity of Si, the XPS narrow spectra of P2p in HA indicates that HA whiskers have been modified with APTES successfully in covalent bonding. This will improve the surface chemical compatibility between HA and PLLA matrix. Briefly, after the silanization of HA whiskers, no differences were observed in the crystalline phase and the crystallinity of HA whiskers. Both HA whiskers and a-HA whiskers were 60–116 μm in length with the aspect ratio of 68–103. The surface of HA whiskers was modified with APTES successfully to improve the surface chemical compatibility between HA and PLLA matrix.
Fig. 1. SEM micrographs of (a) HA whiskers and (b) a-HA whiskers.
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with a mercury injection apparatus (Autopore IV 9510) indicates that the porosities of the scaffolds change a little after mixing with HA whiskers, as shown in Table 2. All the porosities are higher than 89%, which is beneficial for cell growth. The distributions of pore sizes in all samples are mainly around 80 μm and 35 μm. No significant differences in pore size distribution have been observed, indicating that all the scaffolds have the similar porous structure, which does not make contributions to the improvement in the mechanical properties of composite scaffolds. Despite 10 wt.% a-HA series, the porosities of the others are all higher than the pure PLLA scaffold. The increase in the porosity is mainly based on the higher density of HA than PLLA. With a constant total quality of HA/ PLLA matrix, the volume will reduce due to the increase in density with more HA whiskers introduced. So the 1,4-dioxane and HA/PLLA solution contains a larger volume of 1,4-dioxane, which produces more pores after the solid–liquid phase separation.
Fig. 2. XRD patterns of HA whiskers and a-HA whiskers.
3.3. Mechanical properties of HA/PLLA scaffold
Table 1 The surface compositions of a-HA whiskers. Element Atom%
Ca 7.27
P 5.46
O 32.38
Si 8.87
N 6.94
C 39.08
3.2. HA/PLLA composite scaffold Different weight ratios (0, 10 wt.%, 15 wt.%, 20 wt.% and 30 wt.%) of HA whiskers were mixed with PLLA to obtain the HA/PLLA composite scaffolds. The composite scaffolds were cut in the middle and observed the incision by SEM, as shown in Fig. 4. No difference in the morphology is observed between PLLA scaffold and HA (or a-HA)/PLLA composite scaffold. The holes are uniform with about 80 μm and 35 μm in diameter, which is suitable for cell growth. All the holes are interconnected and distributed homogenously. The induced phase separation process in the mixture of HA/PLLA and 1,4-dioxane led to the formation of holes during the freeze drying directly. HA whiskers were dispersed well in the PLLA matrix and no significant fracture of whisker was observed. More HA whiskers were observed with the increasing weight ratio of HA whiskers in the composite. All the HA whiskers were embedded in PLLA matrix, except for the 30 wt.% HA/PLLA scaffold. This rough texture will promote human bone cell attachment [13]. Furthermore, previous research shows that the “point exposure” of HA appeared to provide anchoring sites for cell processes and provide a promising approach for controlling cell density on implant surfaces [14]. The porosity of scaffold plays an important role both in cell growth and the mechanical properties. The porosity data calculated automatically
Compressive properties of the composite scaffolds are shown in Fig. 5. The initial linear part of stress–strain data was used to fit the compressive modulus and the stress at the 10% compression was considered as the compressive strength. The compressive strength of HA/PLLA scaffold increased from 0.18 MPa to 0.28 MPa, while the compressive modulus increased from 3.24 MPa to 5.77 MPa with HA content from 0 to 30%. With regard to a-HA/PLLA scaffold, the compressive strength increased from 0.18 MPa to 0.42 MPa while the compressive modulus increased from 3.24 MPa to 8.03 MPa with HA content from 0 to 30%. The maximum compressive strength of a-HA/PLLA scaffold at 30 wt.% was 47.2% higher than the corresponding HA/PLLA scaffold and the compressive modulus was 39.2% higher than the corresponding HA/PLLA scaffold. According to the mechanical data, the compressive strength of a-HA/PLLA scaffold increased with the increasing weight ratio of aHA whiskers up to 30 wt.%, but the compressive strength of HA/PLLA increased until 15 wt.% HA whiskers and kept around 0.28 MPa until up to 30 wt.% HA whiskers. The ever-increasing improvement in compressive strength can be attributed to the improvement in the surface chemical compatibility between HA whiskers and the PLLA matrix after silanization. With high HA whisker content, HA whiskers were easy to expose and hanged in the holes, while a-HA whiskers were all embedded in the PLLA matrix, as shown in Fig. 6. Fig. 6(a) shows that the PLLA matrix cannot envelop all the HA whiskers when mixed with more than 15 wt.% HA whiskers, so some HA whiskers were extended from the PLLA matrix and hanged in the holes. So the compressive strengths of HA/PLLA scaffolds remained around 0.28 MPa with more than 15 wt.% HA whisker content. After
Fig. 3. XPS narrow spectra of P2p in (a) HA whiskers and (b) a-HA whiskers.
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Fig. 4. SEM micrographs of scaffolds containing (a) 0, (b) 15 wt.% HA whiskers, (c) 15 wt.% a-HA whiskers, (d) 30 wt.% HA whiskers, and (e) 30 wt.% a-HA whiskers (arrows: HA (or a-HA) whiskers).
silanization, the improvement in the surface chemical compatibility resulted in an ever-increasing improvement in the compressive strength of a-HA/PLLA scaffold. More a-HA whiskers were still enveloped in the PLLA matrix, as shown in Fig. 6(b). The matrix could be enhanced with more a-HA whiskers, so the compressive strengths of a-HA/PLLA scaffolds were still increasing with more than 15 wt.% a-HA whiskers. Plenty researches were conducted to develop the ideal scaffold with improvement in mechanical properties. To improve the mechanical properties of polymer scaffolds, most of which are hydrophobic, the hydrophilic HA needs to be modified. These researches on surface modification were mostly focused on the tensile property improvement of the Table 2 The porosity of HA or a-HA whisker scaffolds. Series
HA or a-HA content/wt.%
Porosity/%
HA whisker
0 10 15 10 15 20 30
89.10 94.89 94.17 87.38 89.66 98.12 95.32
a-HA whisker
composites with particles. Wang et al. firstly studied the effects of surface modification on the compressive property of scaffolds [15]. They modified HA nanoparticles with poly(ε-caprolactone) and the mechanical properties were improved due to the enhancement of the interaction between filler and matrix, and the better dispersion of fillers in the matrix. Yang et al. prepared silane-Octadecyltrichlorosilane (OTS)modified HA particle/PLLA porous scaffold with the porosity about 92% [16]. In their work, the highest compressive strength of the scaffold was 0.949 MPa at 50% compression strain with 30 wt.% OTS-modified HA/PLLA scaffold, 70.68% higher than the pure PLLA scaffold. In this work, the compressive strength of the scaffold was 0.42 MPa at 10% compression strain with 30 wt.% APTES-modified HA/PLLA scaffold, 130% higher than the pure PLLA scaffold. Greater improvement in the compressive strength of silane-modified HA/PLLA scaffold is obtained by using HA whiskers rather than HA particles. But compared with HA nanoparticles, the HA whiskers in this research can be exposed from the matrix, resulting in an invalid composite. After the silanization, a-HA whiskers were all enveloped in the PLLA matrix and no exposed whiskers were observed. This is due to the improved surface chemical compatibility between a-HA whiskers and the PLLA matrix and results in an ever-increasing improvement in compressive properties of a-HA/PLLA scaffold.
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Fig. 5. (a) The compressive moduli and (b) compressive strengths of scaffolds.
Fig. 6. (a) 30 wt.% HA whiskers and (b) 30 wt.% a-HA whiskers in HA/PLLA scaffold.
4. Conclusions
References [1] [2] [3] [4]
A new kind of HA/PLLA composite scaffold was prepared. HA whiskers with high crystallinity and high aspect ratio were modified with γaminopropyltriethoxysilane (APTES) to improve its performance as fillers in PLLA matrix. HA whiskers were easy to expose and hanged in the holes with more than 15 wt.% HA whisker content, while a-HA whiskers were all embedded in the PLLA matrix. The morphology of HA/PLLA scaffold was not influenced with different HA whisker contents, while both the compressive strength and compressive modulus were increased with the weight ratio of HA up to 30 wt.% a-HA whiskers, but only up to 15 wt.% HA whiskers. The maximum compressive strength of a-HA/ PLLA scaffold at 30 wt.% was 0.42 MPa, 47.2% higher than the corresponding HA/PLLA scaffold, and 130% higher than the pure PLLA scaffold.
[10] [11] [12]
Acknowledgments
[13] [14]
The authors are grateful for the financial support from the National Natural Science Foundation of China (51072090, 51061130554).
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