Materials Science and Engineering C 58 (2016) 180–186
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Physicochemical properties and bioactivity of freeze-cast chitosan nanocomposite scaffolds reinforced with bioactive glass Masoud Pourhaghgouy a, Ali Zamanian a,⁎, Mostafa Shahrezaee b, Milad Pourbaghi Masouleh a a b
Department of Nanotechnology & Advanced Materials, Materials & Energy Research Center, Karaj, P.O. Box: 13145-1659, Iran Department of Orthopedic Surgery, AJA University of Medical Sciences, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 11 February 2015 Received in revised form 8 July 2015 Accepted 31 July 2015 Available online 22 August 2015 Keywords: Bioactive glass nanoparticles Chitosan Freeze casting Nanocomposite Scaffold Bone tissue engineering
a b s t r a c t Chitosan based nanocomposite scaffolds were prepared by freeze casting method through blending constant chitosan concentration with different portions of synthesized bioactive glass nanoparticles (BGNPs). Transmission Electron Microscopy (TEM) image showed that the particles size of bioactive glass (64SiO2.28CaO.8P2O5) prepared by sol–gel method was approximately less than 20 nm. Fourier Transform Infrared Spectroscopy (FT-IR) and X-ray Diffraction (XRD) analysis showed proper interfacial bonding between BGNPs and chitosan polymers. Scanning Electron Microscopy (SEM) images depicted a unidirectional structure with homogenous distribution of BGNPs among chitosan matrix associated with the absence of pure chitosan scaffold's wall pores after addition of only 10 wt.% BGNPs. As the BGNP content increased from 0 to 50 wt.%, the compressive strength and compressive module values increased from 0.034 to 0.419 MPa and 0.41 to 10.77 MPa, respectively. Biodegradation study showed that increase in BGNP content leads to growth of weight loss amount. The in vitro biomineralization studies confirmed the bioactive nature of all nanocomposites. Amount of 30 wt.% BGNPs represented the best concentration for absorption capacity and bioactivity behaviors. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Bone tissue engineering is a developing field, whereby cells are allowed to proliferate and organize their extracellular matrix in a three-dimensional lattice to form a functional tissue, exhibiting properties identical to native, healthy tissue [1]. Structural geometry of scaffolds is one of the important characteristics which provides an appropriate environment for cell attachment, growth and finally formation of new bone tissue [2]. Freeze casting or ice templating method is one of the promising ways to produce interconnected porous materials containing unidirectional channels as a result of unidirectional freezing of liquid suspension (aqueous or non-aqueous) which have some special advantages like controlled pore size distribution, environmentally friendliness, slight processing contraction and high mechanical strength [3–5]. However, some scaffold's material properties still need to be studied to fulfill the requirements of bioactivity, degradability and adequate biological responses in order to be applied in hard tissues [6,7]. Based on the fact that bone is a naturally mineralized composite, it is obvious that one biomaterial type does not possess all the mechanical/ chemical properties that are necessary for such applications [8].
⁎ Corresponding author. E-mail addresses: [email protected] (M. Pourhaghgouy), [email protected] (A. Zamanian), [email protected] (M. Shahrezaee), [email protected] (M.P. Masouleh).
http://dx.doi.org/10.1016/j.msec.2015.07.065 0928-4931/© 2015 Elsevier B.V. All rights reserved.
Flexibility of some polymers which facilitate implantation process is an advantage over more rigid materials with inherent brittleness and low toughness features that are unsuitable for load-bearing applications. Hence, biocomposites based on flexible biodegradable polymers and inorganic elements like bioactive glasses or bio ceramics, which exhibit the required mechanical properties and bioactivity, compared with polymeric scaffolds alone, have been developed for applications in bone repair and reconstruction [9]. Chitosan which is produced by deacetylation of chitin is a linear polysaccharide, based on glucosamine units, that can be found in sub-product of shellfish such as crabs and shrimps [10]. It is considered as a suitable functional polymer for biomedical applications due to its good biocompatibility, biodegradability, anti-inflammatory effect, protein adsorption properties and ability of accelerating wound healing. Its degradation products are non-toxic, non-antigenic, non-immunogenic and non-carcinogenic [11–13]. Furthermore, the positive surface charge of this biomaterial and its structural similarities with glycosaminoglycans, a major component of bone and cartilage, enable it to effectively enhance cell adhesion, proliferation, and differentiation [14,15]. However, chitosan by itself is not an ideal material for bone regeneration; its osteoconductivity and bio mineralization capability need to be improved. In general no apatite can precipitate on the surface of chitosan scaffold [16]. The addition of appropriate inorganic components such as bioactive glass to chitosan matrix leads to inducing biomineralization capability to prepare composite. This ability results in formation of apatite layer on bioactive glass which allows creation of safe chemical bonding between
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composite and living bone [17–19]. Since bioactive glasses are of surface reactive biomaterials, physical properties such as particle size, morphology and surface area can facilitate bone healing. Therefore, their addition in nanoscale range is more preferred [20–23]. In this study, bioactive glass nanoparticles (BGNPs) were synthesized through sol–gel method to fabricate chitosan based nanocomposite scaffolds by freeze casting method. Pure chitosan scaffold also was prepared to investigate the effect of BGNP content on morphology, physicochemichal characteristics, absorption behavior and mechanical properties of chitosan scaffold. Furthermore, in vitro biodegradation and biomineralization studies were investigated to evaluate the potential bone integration ability of each porous nanocomposites. 2. Materials and method 2.1. Materials TEOS: C8H20O4Si, nitric acid: HNO3, ethanol: C2H6O, TEP: C6H15O4P, calcium nitrate tetrahydrate: (Ca(NO3)2.4H2O) and ammonia: NH3 for synthesis of BGNPs and also acetic acid were purchased from Merck Company (Darmstadt, Germany). NaCl, KCl, NaHCO3, MgCl2 · 6H2O, KH2PO4, TRIS: C4H11NO3, CaCl2 and HCl for preparation of simulated body fluid (SBF) and also chitosan (MMW: 190–310 kDa, DD: 75– 85%) were purchased from Sigma Aldrich (Darmstadt, Germany). 2.2. Synthesis of BGNPs based on SiO2–CaO–P2O5 BGNPs with formula of SiO2:CaO:P2O5 ≈ 64:28:8 (mol) were synthesized through sol–gel method. Concisely, mixture of TEOS (43.67 g) and distilled water (174.67 ml) was poured into a mixture of nitric acid (5.5 ml, 2 mol/L) and ethanol (87.33 ml) and stirred for 30 min. 8.38 ml of TEP was added to solution and stirred for another 30 min. The solution was stirred for also another 30 min after addition of 20.16 g calcium nitrate tetrahydrate. After that, the solution was aged for 1 h on stirrer. Finally, ammonia solution (2 mol/L) was added drop wisely to the solution under vigorous stirring until conversion of solution to gel was observed. The obtained gel was dried in oven (at 70 °C) to eliminate the residual water and ethanol. Then, dried powders were placed in furnace, heat treated at 700 °C for 2 h with heating rate of 3 °C/min to remove organics in order to form of glass particles (stabilization process). After that the powders were cooled slowly down in the furnace [24]. 2.3. Preparation of scaffolds The pure chitosan and chitosan-BGNPs nanocomposite scaffolds were prepared by freeze casting method. Chitosan (3 g) was dissolved in
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deionized water containing 2% of acetic acid by volume, at 30 °C, while stirring for 8 h to produce a homogenous solution of 3 wt.% chitosan. Synthesized BGNPs with different percentages of 10, 30 and 50 wt.% (toward amount of chitosan) were homogenously suspended in chitosan solution using ultrasonicator and a magnetic stirrer instrument. After freeze casting process, porous chitosan and chitosan-BGNPs nanocomposites were obtained. Freeze casting of prepared solutions was performed by pouring the solutions into a polytetrafluoroethylene (PTFE) mold with an inner diameter of 20 mm located on a copper cold finger. While liquid nitrogen was poured into the container, the cooling rate was controlled by a ring heater and a thermocouple which both were connected to a proportional-integral-derivative (PID) controller. A schematic illustration of freeze casting setup and prepared scaffolds is shown in Fig. 1. The cooling rate applied in this study was 1 °C/min. After a very careful removal of frozen samples from the mold, samples were dried in the freeze dryer (FD-10, Pishtaz Engineering Co. Tehran, Iran) at temperature of −55 °C and the pressure of 2.1 Pa for 48 h, in order to sublime the ice crystals. All samples were neutralized with 0.1 N NaOH for 1 h. Then, the excess base agents was removed by repeated washing with deionized water until the pH returned to the physiological range [25] and then freeze dried again. Prepared samples were maintained in a silica gel desiccator for further characterizations. 2.4. Transmission electron microscopy (TEM) observation The morphology of the BGNPs was observed by TEM instrument (GM200, PEG, Philips), operated at an accelerating voltage of 200 kV. For TEM analysis, the BGNPs were ultrasonically dispersed in ethanol for 15 min and then few drops were placed on the carbon-coated copper grids. 2.5. Porosity of composite The porosity value of the nanocomposites with different BGNP contents was calculated by following formula: Porosity ð%Þ ¼ ð½V t –ðW Chi =ρChi Þ–ðW BG =ρBG Þ=V t Þ 100ð1Þ; where Vt is the total volume of nanocomposite scaffold (cm3), WChi and WBG are the mass of chitosan and BGNPs in the scaffold (g), ρChi and ρBG are the density of chitosan and synthesized BGNPs which were measured by automatic density analyzer (Micrometrics, AccuPyc 1330 Pycnometer, USA) with amount of 1.4499 ± 0.0083 (g/cm3 ) and 1.35 ± 0.0034 (g/cm3), respectively. Triplicate measurements were carried out for each nanocomposites.
Fig. 1. Schematic illustration of freeze casting setup (Left side) and prepared scaffolds (right side). For example Chi-BGNPs10 code is indicative of 3 wt.% chitosan which contains 10 wt.% BGNP and freeze cast with cooling rate of 1 °C/min.
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2.6. Mechanical behavior Mechanical behavior of prepared scaffolds was considered by compression strength test (SANTAM Eng. Design. Co. Tehran, Iran). The cylindrical samples with a diameter and height of ∼ 20 mm were placed between two compression plates and compressed with a 100 kN load cell and cross head speed of 0.5 mm/min. The compressive module was calculated as the slope of the initial linear portion of each stress–strain curves. The maximum height of curves after initial linear portion was considered as compressive strength. Five samples of each type of scaffold were used to obtain reliable data (n = 5).
The absorption capacity of each composition was also measured by removing each nanocomposites after being held in PBS for almost 2 h. Surface of samples was wiped with filter papers and they were weighted afterward (Ww). Absorption capacity for each composition was calculated based on the following formula: Absorption capacityð%Þ ¼ ½ðW w –W i Þ=W i 100ð3Þ;
3. Results and discussions 3.1. Porous Chi-BGNPs nanocomposites
2.7. In vitro bioactivity evaluation In vitro bioactivity test was carried out by immersion of each nanocomposite in 10 ml of prepared SBF solution in cylindrical flasks, and placing the samples in an oven at 37 °C for 3, 7, 14 and 21 days. Every 3 days, samples were taken out, washed with distilled water and soaked again in fresh SBF solution. Samples were freeze dried before any analysis. For preparation of SBF solution, NaCl (8.06 g), KCl (0.224 g), NaHCO3 (0.353 g), MgCl2 · 6H2O (0.301 g) and KH2PO4 (0.174 g) were firstly dissolved into distilled water while stirring. After 10 min, 6.05 g of TRIS (C4H11NO3) and 0.28 g of CaCl2 with an interval of 15 min was added to the solution, respectively. Finally, water based solution of HCl was made and added dropwisely to the solution until pH value reached to 7.4 [21]. 2.8. SEM analyses SEM images were obtained by Stereoscan S 360-Leica microscope (Cambridge, England). A thin layer of gold was coated over the surface of prepared scaffolds to eliminate poor conductivity of sample's current before testing. 2.9. Phase analysis XRD (Unisantis, XMD 300, Germany) patterns were obtained with a Cu Kα radiation (λ = 1.5418 A°) under the operating conditions of 40 kV and 30 mA. Data were collected from 2θ = 10° to 60°. 2.10. Physicochemical characterization FT-IR spectroscopy (Perkin–Elmer, spectrum 400, USA) was performed in the wavenumber range of 400–4000 cm−1 using KBr pellets technique. Each FT-IR spectrum was obtained from 40 scans at a resolution of 2 cm−1.
Fig. 2 shows the TEM image of BGNPs with spherical morphology and the size of approximately under 20 nm, with two conditions of distribution of nanoparticles and agglomerates. Agglomeration phenomenon occurs because of nanoparticles' tendency to formation of larger clusters due to their high specific surface area and surface energy [26]. The SEM images of chitosan scaffold and Chi-BGNPs nanocomposites with different BGNP contents, taken from both perpendicular and parallel directions of ice growth, are shown in Fig. 3. As it can be seen, the representative morphology of scaffolds shows a typical unidirectional microstructure of porous scaffolds prepared by freeze-casting method. The creation of unidirectional structure was completely discussed in previous work [27]. Also, as concentration of BGNP increased, no change was observed in pore channels size of the scaffolds (distance of chitosan walls from each other), while the addition of nanoparticles to ceramic slurry containing micro-particles, makes larger pores in scaffolds structure [5,28]. This phenomenon may be due to the relatively high viscosity of the polymer solution compared to ceramic suspensions. This means that BGNPs which are distributed in the chitosan solution can be moved only with movement of chitosan macromolecules, so that they would lose their mobility and free movement. Accordingly, ice crystals are still faced with the same resistance of viscose chitosan solution. Hence, no change in ice growth limits or size of the ice crystals (size of the pore channels) was observed. Furthermore, according to high-magnification images of Fig. 3 (B' series), the surfaces of the chitosan walls were smooth and included pores contrary to the surfaces of nanocomposites walls which represent roughness (partly exposed BGNPs) and contain no pore. About wall pores, as mentioned in previous work [27], it seems that low surface tension of chitosan walls leads to growth and penetration of some ice crystals in perpendicular direction of preferable ice growth, even in the presence of the thermal gradient, which results in perforation of the thin chitosan walls. But after addition of just 10% of BGNPs, it seems that the surface tension of walls improves and limits growth of
2.11. In vitro biodegradation and absorption evaluations Biodegradability evaluation of samples was studied by soaking three samples of each composition in separate cylindrical flasks, each one containing 10 ml of phosphate-buffered saline (PBS) solution (pH 7.4), and placing them in an thermoshake instrument (Gerhardt) at 37 °C for 21 and 42 days. The PBS solution was replaced every 3 days and each time the samples washed with distilled water in order to eliminate soluble inorganic salt. After each time point, samples were freeze dried and the degradation of scaffolds was calculated using the following formula: Degradationð%Þ ¼
W i –W f =W i 100ð2Þ;
where Wi and Wf were noted as initial weight of the scaffold before soaking in PBS and final weight of freeze dried scaffold after soaking in PBS, respectively.
Fig. 2. TEM image of synthesis BGNPs with two conditions of distribution of a) nanoparticles and b) agglomerates.
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Fig. 3. The SEM images taken from both (A) perpendicular and (B & B') parallel directions to the ice growth during freeze-casting process. The images inserted in the B' series are the highmagnification pictures of B series which illustrate scaffolds' wall surfaces and distribution of BGNPs on them. The subscripts indicate the BGNP contents of each scaffold (0, 10, 30 and 50 wt.%).
ice crystals in perpendicular way which will lead to elimination of voids. Also, as the BGNP content enlarged, more partly exposed BGNPs on the surface of the chitosan walls were seen. Moreover, distribution of BGNPs in nanocomposites was uniform and that leads to similar properties in all body of them. FT-IR and XRD patterns of BGNPs, chitosan polymers and ChiBGNPs30 nanocomposite are presented in Fig. 4. For FT-IR patterns, the spectrum of Chi-BGNPs30 nanocomposite showed the characteristic
peaks of both chitosan polymers and BGNPs. However, several characteristic peaks were shifted, deformed or disappeared. All these changes are evidences to some chemical interactions between the BGNPs and the chitosan polymers [29]. For example, about chitosan characteristic peaks, absorption bonds at 2878 cm−1 (C–H stretch), 1649 cm−1 (C_O stretch Amide I), 1379 cm−1 (C–CH3 deformation), 1260 cm−1 (δs(CH3) in NHCOCH3 group Amide III) and 895 cm−1 (C–O–C stretch in saccharide structure) were deformed and also the characteristic
Fig. 4. The FT-IR (left side) and XRD (right side) patterns of BGNPs, chitosan polymers and Chi-BGNPs30 nanocomposite.
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Fig. 5. Porosity measurements (left side) and mechanical compressive properties (right side) of chitosan and nanocomposites containing 10, 30 and 50 wt.% of BGNP.
bond at 1599 cm−1 (N–H band Amide II) was shifted a little to the short waves. Likewise characteristic bond of BGNPs at 3642 cm−1 was disappeared and two bonds at 804 cm−1 (Si–O bend) and 470 cm−1 (Si–O–Si bend) were observed with slight intensity [29–34]. Also about XRD patterns, synthesized BGNPs did not indicate any proof of crystalline phase which confirms the amorphous characteristic of it and makes it potentially suitable for using in bone tissue engineering. Crystallization of BGNP reduces its bioactivity and even turns it into an inert material [35,36]. Also chitosan polymers displayed one characteristic peak at about 20° which shows type II orthorhombic crystallization of chitosan [37]. Finally, the diffraction diagram of Chi-BGNPs30 indicated only the characteristic peak of chitosan which had reduction in intensity and shifted a little to the left. This shift and decline of the peak emphasize the chemical interactions between BGNPs and chitosan polymers. Based on these evidences obtained by FT-IR and XRD analyses, BGNPs have well combined into the matrix of chitosan polymers. To support tissue regeneration at the site of implantation and sustain sufficient integrity during both in vitro and in vivo cell growth, scaffolds need to have enough mechanical strength for these purposes [27]. From a mechanical standpoint, good interfacial bonding between the nanoparticles and polymer has great importance. Powerful interfacial bonding leads to improve mechanical properties, while weak interactions result to reduction in mechanical properties compared to polymer scaffold alone [38]. Fig. 5 shows porosity measurements and compressive stress–strain curves of chitosan scaffold and nanocomposites containing different amounts of BGNP. As it depicts, after addition of only 10 wt.% of BGNP to chitosan scaffold, significant improvement on both chitosan's compressive strength (about 11 times from 34 to 363 kPa) and compressive modulus (about 24 times from 0.41 to 10.04 MPa) and also significant decline on chitosan porosity value (from 96.60% to 92.22%) can be observed. The main reason of this effective change is due to the absence of chitosan wall pores after addition of only 10 wt.% of BGNP (according to Fig. 3-B'0 and B'10 images). Furthermore, slight improvement of mechanical properties of nanocomposite after addition of
more BGNPs (30 and 50 wt.%) is due to higher solid volume of them (according to porosity measurements) associated with homogeneous distribution of BGNPs on chitosan polymers (according to Fig. 3-B' series images) and also good interfacial bonding between BGNPs and chitosan polymers (according to FT-IR and XRD analyses) which all were mentioned in other literatures [39,40].
3.2. In vitro degradation and absorption capacity studies In order to study the effect of BGNP content on the degradability of the nanocomposite scaffolds and their absorption capacity, measurements of weight loss and absorption characteristics were carried out in PBS medium. These measurements are summarized in Fig. 6. About the absorption capacity values, as the curve illustrates, after adding 10% BGNPs, a sharp drop in PBS absorption was observed due to absence of chitosan pores in their walls (according to Fig. 3-B'0 and B'10 images). The PBS absorption rose after increase in amount of BGNPs from 10% to 30% owing to the strong hydrophilic nature of BGNPs. Finally, addition of 50 wt.% BGNPs into polymeric scaffold led to reduction of absorption capacity due to overcoming the influence of porosity of nanocomposite (Fig. 5-left side) on the hydrophilic nature of BGNPs. Degradation of Chi-BGNPs nanocomposites seems to be the result of three mechanisms. First, degradation of the chitosan; second, degradation of the BGNPs and third; separation of BGNPs from chitosan surfaces due to degradation of interfacial areas [41,42]. Since the chitosan with medium molecular weight and 75–85% degree of deacetylation has very low degradation speed [41], large part of weight loss is result of second and third mechanisms. Accordingly, as it can be seen in Fig. 6right side, passage of time and augmentation in BGNPs concentration led to further weight loss. The degradation rate of scaffolds should be coordinated with the bone tissue regeneration process, which can be adjusted with changes in chitosan deacetylation degree, molecular weight of chitosan and BGNP content [9,41,42].
Fig. 6. The absorption capacity (left side) and weight loss measurements (right side) of the scaffolds after immersion in PBS solution.
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Fig. 7. The SEM images show the evolution of hydroxyapatite appearance on the surface of Chi-BGNPs30 nanocomposite after incubation in SBF for different time periods (A series) and also distribution of 14 days old hydroxyapatites on each nanocomposite respectively Chi-BGNPs30 (A14), Chi-BGNPs50 (B14) and Chi-BGNPs10 (C14). The subscripts indicate the incubation time in SBF (days).
3.3. In vitro bio mineralization studies Hydroxyapatite (Ca10(PO4)6(OH)2) is a major component of bone materials which composed of about 60% of it. Hence, it is vital to ensure the formation of apatite on the surface of the scaffolds prepared with the aim of repairing bone tissue [43]. Mechanism of apatite formation on the surface of biomaterials in SBF was expressed by Clark and Hench [44]. The SEM images in Fig. 7 show the evolution of hydroxyapatite appearance on the surface of Chi-BGNPs30 nanocomposite over immersion time respectively 0, 3, 7 and 14 days in SBF solution and also distribution of 14-day old hydroxyapatite on each nanocomposites. About Fig. 7-A series, as it can be seen, after 3 days of immersion, the surface of the scaffold was completely covered by uniform flakyshaped apatite layer. After 7 days, nucleation of cauliflower-shaped apatite with numerous small apatite crystals was observed. As incubation prolonged, after 14 days, the size of cauliflower-like apatite was increased along the c-axis direction [26]. So, as it can be seen, over immersion time in SBF, the morphology of the apatite which formed onto bioactive surfaces was changed from flaky-liked structure to typical cauliflower-liked structure. SEM images taken from surfaces of each nanocomposites after 14 days of immersion in SBF medium are depicted in Fig. 7 (A14, B14, C14). Images show that the appearance of the 14 days old apatite was the same on each scaffold in the case of shape and size. Therefore, the difference in BGNPs amount did not change the appearance of apatite and so the rate of apatite formation. But the quantity of apatite in scaffold containing 30% BGNPs (A14) is further which could be due to the
more absorption capacity of this scaffold. The rate of apatite formation is considered to be descriptive of a material's bioactive (bone-bonding) potential. It seems that the rate of apatite formation is directly related to the type of bioactive glass or preparation procedure of it [45]. It is also important to note that chitosan is not one of bioactive materials, so the apatite formation on the surfaces of these nanocomposites is only due to presence of BGNPs on them [46]. Furthermore, Fig. 8 depicts the FT-IR and XRD analyses which were performed on Chi-BGNPs30 nanocomposite after immersion in SBF for 0, 3, 7, 14 and 21 days in order to analyze changes in the surface chemical composition related to the mineralization process. As it can be seen, the IR spectrum of Chi-BGNPs30 nanocomposite revealed two well defined phosphate bonds at 550 and 600 cm− 1 3 assigned to P–O bending vibration of PO− group in calcium phos4 phate crystalline phases, after just 3 days of soaking in SBF solution (left side). These bonds are the major evidence for the presence of hydroxyapatite on the surface of the nanocomposite [47]. Furthermore, over soaking time, the intensity of the P–O bonds developed toward the Si–O–Si bond. Also, XRD patterns (right side) clearly showed precipitation of hydroxyapatite on the surface of Chi-BGNPs30 nanocomposite after 3 days of immersion which reflected as a sharp peak at 31.7°, which was matched with standard hydroxyapatite diffraction pattern (ICDD PDF-2, ref. code 01-076-0694). Likewise, with increasing the soaking time, the intensity of HA peaks developed toward the only characteristic peak of nanocomposite. These results are confirmation of more apatite deposition over the surfaces of nanocomposites through increasing
Fig. 8. The FT-IR (left side) and XRD (right side) patterns of Chi-BGNPs30 nanocomposite after soaking in SBF solution for different periods (days).
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immersion time in SBF solution which are in accordance with the SEM images in Fig. 7. 4. Conclusions In this research, different amounts of synthesized BGNPs were blended with constant concentration of chitosan solution to produce porous nanocomposite scaffolds with unidirectional aligned pore channels by freeze casting method. Strong interfacial bonding between chitosan polymers and BGNPs were detected by XRD and FT-IR analysis. BGNPs were homogenously distributed on chitosan matrix. Increase in BGNP content did not change scaffold's pore channels size, but resulted in absence of the pores which were located on chitosan walls and accordingly reduced porosity amount of nanocomposites. The compressive mechanical properties and degradation behavior of scaffolds were also increased by augmentation of BGNP content. The nanocomposite containing 30 wt.% BGNP represented the more acceptable absorption capacity and bioactivity behavior. References [1] D.J. Griffon, M.R. Sedighi, D.V. Schaeffer, J.A. Eurell, A.L. Johnson, Chitosan scaffolds: interconnective pore size and cartilage engineering, Acta Biomater. 2 (2006) 313–320. [2] G. Chen, T. Ushida, T. Tateishi, Scaffold design for tissue engineering, Macromol. Biosci. 2 (2002) 67–77. [3] S. Deville, Freeze-casting of porous biomaterials: structure, properties and opportunities, Materials 3 (2010) 1913–1927. [4] S. Farhangdoust, A. Zamanian, M. Yasaei, M. Khorami, The effect of processing parameters and solid concentration on the mechanical and microstructural properties of freeze-casted macroporous hydroxyapatite scaffolds, Mater. Sci. Eng. C 33 (2013) 453–460. [5] S.M.H. Ghazanfari, A. Zamanian, Phase transformation, microstructural and mechanical properties of hydroxyapatite/alumina nanocomposite scaffolds produced by freeze casting, Ceram. Int. 39 (2013) 9835–9844. [6] S.M.H. Ghazanfari, A. Zamanian, Effect of nanosilica addition on the physicomechanical properties, pore morphology, and phase transformation of freeze cast hydroxyapatite scaffolds, J. Mater. Sci. 49 (2014) 5492–5504. [7] S. Hesaraki, A. Zamanian, F. Moztarzadeh, Effect of adding sodium hexametaphosphate liquefier on basic properties of calcium phosphate cements, J. Biomed. Mater. Res. Part A 88 (2009) 314–321. [8] G.M. Luz, J.F. Mano, Mineralized structures in nature: examples and inspirations for the design of new composite materials and biomaterials, Compos. Sci. Technol. 70 (2010) 1777–1788. [9] S. Roohani-Esfahani, S. Nouri-Khorasani, Z. Lu, R. Appleyard, H. Zreiqat, Effects of bioactive glass nanoparticles on the mechanical and biological behavior of composite coated scaffolds, Acta Biomater. 7 (2011) 1307–1318. [10] H.S. Kas, Chitosan: properties, preparations and application to microparticulate systems, J. Microencapsul. 14 (1997) 689–711. [11] N.M. Alves, J.F. Mano, Chitosan derivatives obtained by chemical modifications for biomedical and environmental applications, Int. J. Biol. Macromol. 43 (2008) 401–414. [12] A. Lahiji, A. Sohrabi, D.S. Hungerford, C.G. Frondoza, Chitosan supports the expression of extracellular matrix proteins in human osteoblasts and chondrocytes, J. Biomed. Mater. Res. 51 (2000) 586–595. [13] H.H. Lu, S.F. El-Amin, K.D. Scott, C.T. Laurencin, Three-dimensional, bioactive, biodegradable, polymer–bioactive glass composite scaffolds with improved mechanical properties support collagen synthesis and mineralization of human osteoblast-like cells in vitro, J. Biomed. Mater. Res. Part A 64A (2003) 465–474. [14] P.R. Klokkevold, L. Vandemark, E.B. Kenney, G.W. Bernard, Osteogenesis enhanced by chitosan (poly-N-acetyl glucosaminoglycan) in vitro, J. Periodontol. 67 (1996) 1170–1175. [15] P. Sudha, “Chitin, chitosan, oligosaccharides and their derivatives”, in: SeKwon Kim (Ed.) CRC Press, 2010. [16] Y. Zhang, M. Zhang, Synthesis and characterization of macroporous chitosan/ calcium phosphate composite scaffolds for tissue engineering, J. Biomed. Mater. Res. 55 (2001) 304–312. [17] L.L. Hench, R.J. Splinter, W. Allen, T. Greenlee, Bonding mechanisms at the interface of ceramic prosthetic materials, J. Biomed. Mater. Res. 5 (1971) 117–141. [18] S. Hesaraki, A. Zamanian, H. Nazarian, Physical and physicochemical evaluation of calcium phosphate cement made using human derived blood plasma, Adv. Appl. Ceram. 108 (2009) 253–260. [19] A. Zamanian, F. Moztarzadeh, S. Kordestani, S. Hesaraki, M. Tahriri, Novel calcium hydroxide/nanohydroxyapatite composites for dental applications: in vitro study, Adv. Appl. Ceram. 109 (2010) 440–444.
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Contents lists available at ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Physicochemical properties and bioactivity of freeze-cast chitosan nanocomposite scaffolds reinforced with bioactive glass Masoud Pourhaghgouy a, Ali Zamanian a,⁎, Mostafa Shahrezaee b, Milad Pourbaghi Masouleh a a b
Department of Nanotechnology & Advanced Materials, Materials & Energy Research Center, Karaj, P.O. Box: 13145-1659, Iran Department of Orthopedic Surgery, AJA University of Medical Sciences, Tehran, Iran
a r t i c l e
i n f o
Article history: Received 11 February 2015 Received in revised form 8 July 2015 Accepted 31 July 2015 Available online 22 August 2015 Keywords: Bioactive glass nanoparticles Chitosan Freeze casting Nanocomposite Scaffold Bone tissue engineering
a b s t r a c t Chitosan based nanocomposite scaffolds were prepared by freeze casting method through blending constant chitosan concentration with different portions of synthesized bioactive glass nanoparticles (BGNPs). Transmission Electron Microscopy (TEM) image showed that the particles size of bioactive glass (64SiO2.28CaO.8P2O5) prepared by sol–gel method was approximately less than 20 nm. Fourier Transform Infrared Spectroscopy (FT-IR) and X-ray Diffraction (XRD) analysis showed proper interfacial bonding between BGNPs and chitosan polymers. Scanning Electron Microscopy (SEM) images depicted a unidirectional structure with homogenous distribution of BGNPs among chitosan matrix associated with the absence of pure chitosan scaffold's wall pores after addition of only 10 wt.% BGNPs. As the BGNP content increased from 0 to 50 wt.%, the compressive strength and compressive module values increased from 0.034 to 0.419 MPa and 0.41 to 10.77 MPa, respectively. Biodegradation study showed that increase in BGNP content leads to growth of weight loss amount. The in vitro biomineralization studies confirmed the bioactive nature of all nanocomposites. Amount of 30 wt.% BGNPs represented the best concentration for absorption capacity and bioactivity behaviors. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Bone tissue engineering is a developing field, whereby cells are allowed to proliferate and organize their extracellular matrix in a three-dimensional lattice to form a functional tissue, exhibiting properties identical to native, healthy tissue [1]. Structural geometry of scaffolds is one of the important characteristics which provides an appropriate environment for cell attachment, growth and finally formation of new bone tissue [2]. Freeze casting or ice templating method is one of the promising ways to produce interconnected porous materials containing unidirectional channels as a result of unidirectional freezing of liquid suspension (aqueous or non-aqueous) which have some special advantages like controlled pore size distribution, environmentally friendliness, slight processing contraction and high mechanical strength [3–5]. However, some scaffold's material properties still need to be studied to fulfill the requirements of bioactivity, degradability and adequate biological responses in order to be applied in hard tissues [6,7]. Based on the fact that bone is a naturally mineralized composite, it is obvious that one biomaterial type does not possess all the mechanical/ chemical properties that are necessary for such applications [8].
⁎ Corresponding author. E-mail addresses: [email protected] (M. Pourhaghgouy), [email protected] (A. Zamanian), [email protected] (M. Shahrezaee), [email protected] (M.P. Masouleh).
http://dx.doi.org/10.1016/j.msec.2015.07.065 0928-4931/© 2015 Elsevier B.V. All rights reserved.
Flexibility of some polymers which facilitate implantation process is an advantage over more rigid materials with inherent brittleness and low toughness features that are unsuitable for load-bearing applications. Hence, biocomposites based on flexible biodegradable polymers and inorganic elements like bioactive glasses or bio ceramics, which exhibit the required mechanical properties and bioactivity, compared with polymeric scaffolds alone, have been developed for applications in bone repair and reconstruction [9]. Chitosan which is produced by deacetylation of chitin is a linear polysaccharide, based on glucosamine units, that can be found in sub-product of shellfish such as crabs and shrimps [10]. It is considered as a suitable functional polymer for biomedical applications due to its good biocompatibility, biodegradability, anti-inflammatory effect, protein adsorption properties and ability of accelerating wound healing. Its degradation products are non-toxic, non-antigenic, non-immunogenic and non-carcinogenic [11–13]. Furthermore, the positive surface charge of this biomaterial and its structural similarities with glycosaminoglycans, a major component of bone and cartilage, enable it to effectively enhance cell adhesion, proliferation, and differentiation [14,15]. However, chitosan by itself is not an ideal material for bone regeneration; its osteoconductivity and bio mineralization capability need to be improved. In general no apatite can precipitate on the surface of chitosan scaffold [16]. The addition of appropriate inorganic components such as bioactive glass to chitosan matrix leads to inducing biomineralization capability to prepare composite. This ability results in formation of apatite layer on bioactive glass which allows creation of safe chemical bonding between
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composite and living bone [17–19]. Since bioactive glasses are of surface reactive biomaterials, physical properties such as particle size, morphology and surface area can facilitate bone healing. Therefore, their addition in nanoscale range is more preferred [20–23]. In this study, bioactive glass nanoparticles (BGNPs) were synthesized through sol–gel method to fabricate chitosan based nanocomposite scaffolds by freeze casting method. Pure chitosan scaffold also was prepared to investigate the effect of BGNP content on morphology, physicochemichal characteristics, absorption behavior and mechanical properties of chitosan scaffold. Furthermore, in vitro biodegradation and biomineralization studies were investigated to evaluate the potential bone integration ability of each porous nanocomposites. 2. Materials and method 2.1. Materials TEOS: C8H20O4Si, nitric acid: HNO3, ethanol: C2H6O, TEP: C6H15O4P, calcium nitrate tetrahydrate: (Ca(NO3)2.4H2O) and ammonia: NH3 for synthesis of BGNPs and also acetic acid were purchased from Merck Company (Darmstadt, Germany). NaCl, KCl, NaHCO3, MgCl2 · 6H2O, KH2PO4, TRIS: C4H11NO3, CaCl2 and HCl for preparation of simulated body fluid (SBF) and also chitosan (MMW: 190–310 kDa, DD: 75– 85%) were purchased from Sigma Aldrich (Darmstadt, Germany). 2.2. Synthesis of BGNPs based on SiO2–CaO–P2O5 BGNPs with formula of SiO2:CaO:P2O5 ≈ 64:28:8 (mol) were synthesized through sol–gel method. Concisely, mixture of TEOS (43.67 g) and distilled water (174.67 ml) was poured into a mixture of nitric acid (5.5 ml, 2 mol/L) and ethanol (87.33 ml) and stirred for 30 min. 8.38 ml of TEP was added to solution and stirred for another 30 min. The solution was stirred for also another 30 min after addition of 20.16 g calcium nitrate tetrahydrate. After that, the solution was aged for 1 h on stirrer. Finally, ammonia solution (2 mol/L) was added drop wisely to the solution under vigorous stirring until conversion of solution to gel was observed. The obtained gel was dried in oven (at 70 °C) to eliminate the residual water and ethanol. Then, dried powders were placed in furnace, heat treated at 700 °C for 2 h with heating rate of 3 °C/min to remove organics in order to form of glass particles (stabilization process). After that the powders were cooled slowly down in the furnace [24]. 2.3. Preparation of scaffolds The pure chitosan and chitosan-BGNPs nanocomposite scaffolds were prepared by freeze casting method. Chitosan (3 g) was dissolved in
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deionized water containing 2% of acetic acid by volume, at 30 °C, while stirring for 8 h to produce a homogenous solution of 3 wt.% chitosan. Synthesized BGNPs with different percentages of 10, 30 and 50 wt.% (toward amount of chitosan) were homogenously suspended in chitosan solution using ultrasonicator and a magnetic stirrer instrument. After freeze casting process, porous chitosan and chitosan-BGNPs nanocomposites were obtained. Freeze casting of prepared solutions was performed by pouring the solutions into a polytetrafluoroethylene (PTFE) mold with an inner diameter of 20 mm located on a copper cold finger. While liquid nitrogen was poured into the container, the cooling rate was controlled by a ring heater and a thermocouple which both were connected to a proportional-integral-derivative (PID) controller. A schematic illustration of freeze casting setup and prepared scaffolds is shown in Fig. 1. The cooling rate applied in this study was 1 °C/min. After a very careful removal of frozen samples from the mold, samples were dried in the freeze dryer (FD-10, Pishtaz Engineering Co. Tehran, Iran) at temperature of −55 °C and the pressure of 2.1 Pa for 48 h, in order to sublime the ice crystals. All samples were neutralized with 0.1 N NaOH for 1 h. Then, the excess base agents was removed by repeated washing with deionized water until the pH returned to the physiological range [25] and then freeze dried again. Prepared samples were maintained in a silica gel desiccator for further characterizations. 2.4. Transmission electron microscopy (TEM) observation The morphology of the BGNPs was observed by TEM instrument (GM200, PEG, Philips), operated at an accelerating voltage of 200 kV. For TEM analysis, the BGNPs were ultrasonically dispersed in ethanol for 15 min and then few drops were placed on the carbon-coated copper grids. 2.5. Porosity of composite The porosity value of the nanocomposites with different BGNP contents was calculated by following formula: Porosity ð%Þ ¼ ð½V t –ðW Chi =ρChi Þ–ðW BG =ρBG Þ=V t Þ 100ð1Þ; where Vt is the total volume of nanocomposite scaffold (cm3), WChi and WBG are the mass of chitosan and BGNPs in the scaffold (g), ρChi and ρBG are the density of chitosan and synthesized BGNPs which were measured by automatic density analyzer (Micrometrics, AccuPyc 1330 Pycnometer, USA) with amount of 1.4499 ± 0.0083 (g/cm3 ) and 1.35 ± 0.0034 (g/cm3), respectively. Triplicate measurements were carried out for each nanocomposites.
Fig. 1. Schematic illustration of freeze casting setup (Left side) and prepared scaffolds (right side). For example Chi-BGNPs10 code is indicative of 3 wt.% chitosan which contains 10 wt.% BGNP and freeze cast with cooling rate of 1 °C/min.
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2.6. Mechanical behavior Mechanical behavior of prepared scaffolds was considered by compression strength test (SANTAM Eng. Design. Co. Tehran, Iran). The cylindrical samples with a diameter and height of ∼ 20 mm were placed between two compression plates and compressed with a 100 kN load cell and cross head speed of 0.5 mm/min. The compressive module was calculated as the slope of the initial linear portion of each stress–strain curves. The maximum height of curves after initial linear portion was considered as compressive strength. Five samples of each type of scaffold were used to obtain reliable data (n = 5).
The absorption capacity of each composition was also measured by removing each nanocomposites after being held in PBS for almost 2 h. Surface of samples was wiped with filter papers and they were weighted afterward (Ww). Absorption capacity for each composition was calculated based on the following formula: Absorption capacityð%Þ ¼ ½ðW w –W i Þ=W i 100ð3Þ;
3. Results and discussions 3.1. Porous Chi-BGNPs nanocomposites
2.7. In vitro bioactivity evaluation In vitro bioactivity test was carried out by immersion of each nanocomposite in 10 ml of prepared SBF solution in cylindrical flasks, and placing the samples in an oven at 37 °C for 3, 7, 14 and 21 days. Every 3 days, samples were taken out, washed with distilled water and soaked again in fresh SBF solution. Samples were freeze dried before any analysis. For preparation of SBF solution, NaCl (8.06 g), KCl (0.224 g), NaHCO3 (0.353 g), MgCl2 · 6H2O (0.301 g) and KH2PO4 (0.174 g) were firstly dissolved into distilled water while stirring. After 10 min, 6.05 g of TRIS (C4H11NO3) and 0.28 g of CaCl2 with an interval of 15 min was added to the solution, respectively. Finally, water based solution of HCl was made and added dropwisely to the solution until pH value reached to 7.4 [21]. 2.8. SEM analyses SEM images were obtained by Stereoscan S 360-Leica microscope (Cambridge, England). A thin layer of gold was coated over the surface of prepared scaffolds to eliminate poor conductivity of sample's current before testing. 2.9. Phase analysis XRD (Unisantis, XMD 300, Germany) patterns were obtained with a Cu Kα radiation (λ = 1.5418 A°) under the operating conditions of 40 kV and 30 mA. Data were collected from 2θ = 10° to 60°. 2.10. Physicochemical characterization FT-IR spectroscopy (Perkin–Elmer, spectrum 400, USA) was performed in the wavenumber range of 400–4000 cm−1 using KBr pellets technique. Each FT-IR spectrum was obtained from 40 scans at a resolution of 2 cm−1.
Fig. 2 shows the TEM image of BGNPs with spherical morphology and the size of approximately under 20 nm, with two conditions of distribution of nanoparticles and agglomerates. Agglomeration phenomenon occurs because of nanoparticles' tendency to formation of larger clusters due to their high specific surface area and surface energy [26]. The SEM images of chitosan scaffold and Chi-BGNPs nanocomposites with different BGNP contents, taken from both perpendicular and parallel directions of ice growth, are shown in Fig. 3. As it can be seen, the representative morphology of scaffolds shows a typical unidirectional microstructure of porous scaffolds prepared by freeze-casting method. The creation of unidirectional structure was completely discussed in previous work [27]. Also, as concentration of BGNP increased, no change was observed in pore channels size of the scaffolds (distance of chitosan walls from each other), while the addition of nanoparticles to ceramic slurry containing micro-particles, makes larger pores in scaffolds structure [5,28]. This phenomenon may be due to the relatively high viscosity of the polymer solution compared to ceramic suspensions. This means that BGNPs which are distributed in the chitosan solution can be moved only with movement of chitosan macromolecules, so that they would lose their mobility and free movement. Accordingly, ice crystals are still faced with the same resistance of viscose chitosan solution. Hence, no change in ice growth limits or size of the ice crystals (size of the pore channels) was observed. Furthermore, according to high-magnification images of Fig. 3 (B' series), the surfaces of the chitosan walls were smooth and included pores contrary to the surfaces of nanocomposites walls which represent roughness (partly exposed BGNPs) and contain no pore. About wall pores, as mentioned in previous work [27], it seems that low surface tension of chitosan walls leads to growth and penetration of some ice crystals in perpendicular direction of preferable ice growth, even in the presence of the thermal gradient, which results in perforation of the thin chitosan walls. But after addition of just 10% of BGNPs, it seems that the surface tension of walls improves and limits growth of
2.11. In vitro biodegradation and absorption evaluations Biodegradability evaluation of samples was studied by soaking three samples of each composition in separate cylindrical flasks, each one containing 10 ml of phosphate-buffered saline (PBS) solution (pH 7.4), and placing them in an thermoshake instrument (Gerhardt) at 37 °C for 21 and 42 days. The PBS solution was replaced every 3 days and each time the samples washed with distilled water in order to eliminate soluble inorganic salt. After each time point, samples were freeze dried and the degradation of scaffolds was calculated using the following formula: Degradationð%Þ ¼
W i –W f =W i 100ð2Þ;
where Wi and Wf were noted as initial weight of the scaffold before soaking in PBS and final weight of freeze dried scaffold after soaking in PBS, respectively.
Fig. 2. TEM image of synthesis BGNPs with two conditions of distribution of a) nanoparticles and b) agglomerates.
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Fig. 3. The SEM images taken from both (A) perpendicular and (B & B') parallel directions to the ice growth during freeze-casting process. The images inserted in the B' series are the highmagnification pictures of B series which illustrate scaffolds' wall surfaces and distribution of BGNPs on them. The subscripts indicate the BGNP contents of each scaffold (0, 10, 30 and 50 wt.%).
ice crystals in perpendicular way which will lead to elimination of voids. Also, as the BGNP content enlarged, more partly exposed BGNPs on the surface of the chitosan walls were seen. Moreover, distribution of BGNPs in nanocomposites was uniform and that leads to similar properties in all body of them. FT-IR and XRD patterns of BGNPs, chitosan polymers and ChiBGNPs30 nanocomposite are presented in Fig. 4. For FT-IR patterns, the spectrum of Chi-BGNPs30 nanocomposite showed the characteristic
peaks of both chitosan polymers and BGNPs. However, several characteristic peaks were shifted, deformed or disappeared. All these changes are evidences to some chemical interactions between the BGNPs and the chitosan polymers [29]. For example, about chitosan characteristic peaks, absorption bonds at 2878 cm−1 (C–H stretch), 1649 cm−1 (C_O stretch Amide I), 1379 cm−1 (C–CH3 deformation), 1260 cm−1 (δs(CH3) in NHCOCH3 group Amide III) and 895 cm−1 (C–O–C stretch in saccharide structure) were deformed and also the characteristic
Fig. 4. The FT-IR (left side) and XRD (right side) patterns of BGNPs, chitosan polymers and Chi-BGNPs30 nanocomposite.
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Fig. 5. Porosity measurements (left side) and mechanical compressive properties (right side) of chitosan and nanocomposites containing 10, 30 and 50 wt.% of BGNP.
bond at 1599 cm−1 (N–H band Amide II) was shifted a little to the short waves. Likewise characteristic bond of BGNPs at 3642 cm−1 was disappeared and two bonds at 804 cm−1 (Si–O bend) and 470 cm−1 (Si–O–Si bend) were observed with slight intensity [29–34]. Also about XRD patterns, synthesized BGNPs did not indicate any proof of crystalline phase which confirms the amorphous characteristic of it and makes it potentially suitable for using in bone tissue engineering. Crystallization of BGNP reduces its bioactivity and even turns it into an inert material [35,36]. Also chitosan polymers displayed one characteristic peak at about 20° which shows type II orthorhombic crystallization of chitosan [37]. Finally, the diffraction diagram of Chi-BGNPs30 indicated only the characteristic peak of chitosan which had reduction in intensity and shifted a little to the left. This shift and decline of the peak emphasize the chemical interactions between BGNPs and chitosan polymers. Based on these evidences obtained by FT-IR and XRD analyses, BGNPs have well combined into the matrix of chitosan polymers. To support tissue regeneration at the site of implantation and sustain sufficient integrity during both in vitro and in vivo cell growth, scaffolds need to have enough mechanical strength for these purposes [27]. From a mechanical standpoint, good interfacial bonding between the nanoparticles and polymer has great importance. Powerful interfacial bonding leads to improve mechanical properties, while weak interactions result to reduction in mechanical properties compared to polymer scaffold alone [38]. Fig. 5 shows porosity measurements and compressive stress–strain curves of chitosan scaffold and nanocomposites containing different amounts of BGNP. As it depicts, after addition of only 10 wt.% of BGNP to chitosan scaffold, significant improvement on both chitosan's compressive strength (about 11 times from 34 to 363 kPa) and compressive modulus (about 24 times from 0.41 to 10.04 MPa) and also significant decline on chitosan porosity value (from 96.60% to 92.22%) can be observed. The main reason of this effective change is due to the absence of chitosan wall pores after addition of only 10 wt.% of BGNP (according to Fig. 3-B'0 and B'10 images). Furthermore, slight improvement of mechanical properties of nanocomposite after addition of
more BGNPs (30 and 50 wt.%) is due to higher solid volume of them (according to porosity measurements) associated with homogeneous distribution of BGNPs on chitosan polymers (according to Fig. 3-B' series images) and also good interfacial bonding between BGNPs and chitosan polymers (according to FT-IR and XRD analyses) which all were mentioned in other literatures [39,40].
3.2. In vitro degradation and absorption capacity studies In order to study the effect of BGNP content on the degradability of the nanocomposite scaffolds and their absorption capacity, measurements of weight loss and absorption characteristics were carried out in PBS medium. These measurements are summarized in Fig. 6. About the absorption capacity values, as the curve illustrates, after adding 10% BGNPs, a sharp drop in PBS absorption was observed due to absence of chitosan pores in their walls (according to Fig. 3-B'0 and B'10 images). The PBS absorption rose after increase in amount of BGNPs from 10% to 30% owing to the strong hydrophilic nature of BGNPs. Finally, addition of 50 wt.% BGNPs into polymeric scaffold led to reduction of absorption capacity due to overcoming the influence of porosity of nanocomposite (Fig. 5-left side) on the hydrophilic nature of BGNPs. Degradation of Chi-BGNPs nanocomposites seems to be the result of three mechanisms. First, degradation of the chitosan; second, degradation of the BGNPs and third; separation of BGNPs from chitosan surfaces due to degradation of interfacial areas [41,42]. Since the chitosan with medium molecular weight and 75–85% degree of deacetylation has very low degradation speed [41], large part of weight loss is result of second and third mechanisms. Accordingly, as it can be seen in Fig. 6right side, passage of time and augmentation in BGNPs concentration led to further weight loss. The degradation rate of scaffolds should be coordinated with the bone tissue regeneration process, which can be adjusted with changes in chitosan deacetylation degree, molecular weight of chitosan and BGNP content [9,41,42].
Fig. 6. The absorption capacity (left side) and weight loss measurements (right side) of the scaffolds after immersion in PBS solution.
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Fig. 7. The SEM images show the evolution of hydroxyapatite appearance on the surface of Chi-BGNPs30 nanocomposite after incubation in SBF for different time periods (A series) and also distribution of 14 days old hydroxyapatites on each nanocomposite respectively Chi-BGNPs30 (A14), Chi-BGNPs50 (B14) and Chi-BGNPs10 (C14). The subscripts indicate the incubation time in SBF (days).
3.3. In vitro bio mineralization studies Hydroxyapatite (Ca10(PO4)6(OH)2) is a major component of bone materials which composed of about 60% of it. Hence, it is vital to ensure the formation of apatite on the surface of the scaffolds prepared with the aim of repairing bone tissue [43]. Mechanism of apatite formation on the surface of biomaterials in SBF was expressed by Clark and Hench [44]. The SEM images in Fig. 7 show the evolution of hydroxyapatite appearance on the surface of Chi-BGNPs30 nanocomposite over immersion time respectively 0, 3, 7 and 14 days in SBF solution and also distribution of 14-day old hydroxyapatite on each nanocomposites. About Fig. 7-A series, as it can be seen, after 3 days of immersion, the surface of the scaffold was completely covered by uniform flakyshaped apatite layer. After 7 days, nucleation of cauliflower-shaped apatite with numerous small apatite crystals was observed. As incubation prolonged, after 14 days, the size of cauliflower-like apatite was increased along the c-axis direction [26]. So, as it can be seen, over immersion time in SBF, the morphology of the apatite which formed onto bioactive surfaces was changed from flaky-liked structure to typical cauliflower-liked structure. SEM images taken from surfaces of each nanocomposites after 14 days of immersion in SBF medium are depicted in Fig. 7 (A14, B14, C14). Images show that the appearance of the 14 days old apatite was the same on each scaffold in the case of shape and size. Therefore, the difference in BGNPs amount did not change the appearance of apatite and so the rate of apatite formation. But the quantity of apatite in scaffold containing 30% BGNPs (A14) is further which could be due to the
more absorption capacity of this scaffold. The rate of apatite formation is considered to be descriptive of a material's bioactive (bone-bonding) potential. It seems that the rate of apatite formation is directly related to the type of bioactive glass or preparation procedure of it [45]. It is also important to note that chitosan is not one of bioactive materials, so the apatite formation on the surfaces of these nanocomposites is only due to presence of BGNPs on them [46]. Furthermore, Fig. 8 depicts the FT-IR and XRD analyses which were performed on Chi-BGNPs30 nanocomposite after immersion in SBF for 0, 3, 7, 14 and 21 days in order to analyze changes in the surface chemical composition related to the mineralization process. As it can be seen, the IR spectrum of Chi-BGNPs30 nanocomposite revealed two well defined phosphate bonds at 550 and 600 cm− 1 3 assigned to P–O bending vibration of PO− group in calcium phos4 phate crystalline phases, after just 3 days of soaking in SBF solution (left side). These bonds are the major evidence for the presence of hydroxyapatite on the surface of the nanocomposite [47]. Furthermore, over soaking time, the intensity of the P–O bonds developed toward the Si–O–Si bond. Also, XRD patterns (right side) clearly showed precipitation of hydroxyapatite on the surface of Chi-BGNPs30 nanocomposite after 3 days of immersion which reflected as a sharp peak at 31.7°, which was matched with standard hydroxyapatite diffraction pattern (ICDD PDF-2, ref. code 01-076-0694). Likewise, with increasing the soaking time, the intensity of HA peaks developed toward the only characteristic peak of nanocomposite. These results are confirmation of more apatite deposition over the surfaces of nanocomposites through increasing
Fig. 8. The FT-IR (left side) and XRD (right side) patterns of Chi-BGNPs30 nanocomposite after soaking in SBF solution for different periods (days).
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