International Journal of Biological Macromolecules 74 (2015) 155–161
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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Carboxymethyl cellulose/silica hybrids as templates for calcium phosphate biomimetic mineralization Ahmed Salama ∗ , Ragab E. Abou-Zeid, Mohamed El-Sakhawy, Ahmed El-Gendy Cellulose and Paper Department, National Research Center, El-Tahrir Street, Dokki, Cairo, Egypt
a r t i c l e
i n f o
Article history: Received 21 October 2014 Received in revised form 20 November 2014 Accepted 22 November 2014 Available online 16 December 2014 Keywords: Hybrid Sol–gel Bioactivity Carboxymethyl cellulose Calcium phosphate
a b s t r a c t Multiphase hybrid materials were synthesized using carboxymethyl cellulose (CMC) as bioactive polymer, silica gel as matrix assisted networks and calcium phosphate as inorganic mineral phase. These hybrids were investigated with infrared spectroscopy, X-ray diffraction, scanning electron microscopy, energy dispersive X-ray spectroscopy and transmission electron microscopy. Biomimetic crystal growth nucleated from the CMC/silica hybrids was suggested as amorphous calcium phosphate with an evidence that hydroxyapatite, the mineralized component of bone, may be formed at high CMC content. This study provides an efficient approach toward bone-like hybrids with potential bone healing applications. © 2014 Elsevier B.V. All rights reserved.
1. Introduction More than 2.2 million bone grafting process is carried out every year for regenerative bone surgery [1]. However, due to the limitations associated with these bone surgery, searching for new alternative biocompatible regenerative scaffolds for bone defects becomes a clinical challenge. Bone tissue engineering is emerging as a novel solution that can be used to promote nearnatural materials for using in implantology and traumatology. Last decade, significant efforts have been devoted to develop bone graft substitutes from biomaterials that can augment or regenerate injured bone [2–6]. The ideal biomaterials are required to be bioactive, biodegradable, have sufficient mechanical integrity for implantation in load bearing defects, and have a highly porous and interconnected architecture for bone and vascular ingrowth. Low-bioactive materials need firstly surface modifications by incorporation of functional groups to be mineralized by the biomimetic method. Some reports revealed that the introduction of compounds have bioactive functional groups such as proteins [7,8], charged polysaccharides [9,10] and amino acids [11] as initiations for calcium ions nucleation can promote hydroxyapatite formation when immersed in a biological fluid containing ion concentrations nearly equal to those of human blood plasma [12]. A
∗ Corresponding author. Tel.: +20 1008842629. E-mail address: ahmed
[email protected] (A. Salama). http://dx.doi.org/10.1016/j.ijbiomac.2014.11.041 0141-8130/© 2014 Elsevier B.V. All rights reserved.
recent study investigated the effects of anionic polysaccharides like alginate and phosphorylated alginate on the rate of hydroxyapatite growth and mineralization. The results of the study showed that alginate had no large effect on development of calcium phosphate crystals comparing to phosphorylated alginate which exhibited strong nonspecific binding to the crystals [3]. Carboxymethyl cellulose (CMC) has gained increasing interest in the recent years as a candidate material for bone tissue engineering due to its biocompatibility, biodegradability and anionic properties [13–17]. In most cases, CMC was mechanically mixed with pre-prepared hydroxyapatite for hybrid material formation. The additions of nanohydroxyapatite to carboxymethyl cellulose/chitosan film had improved the mechanical properties, swelling behavior and bioactivity of CMC/chitosan films [14]. However, few attempts have been made for studying bioactivity and biomematic mineralization of ionic polysaccharides like chitosan [18], and carboxymethyl cellulose [5] in simulated body fluids. Silica has numerous favorable properties such as biocompatibility, adjustable surface area, and easy modification with a large number of functional molecules. In addition, the effect of silicon on bone formation has been studied early and the results revealed that silicon deficiency in animal trials results in abnormal bone formation with reduced precipitation of bone apatite [19]. Also, there is evidence that the presence of polysilanol groups of the amorphous silica may promote the role of collagen as a template for intrafibrillar apatite precipitation. The fragility and difficult processing in the form of 3D scaffold architecture of sol–gel derived bioactive
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Table 1 Sample codes for the prepared CMC/silica hybrids. Samples
CMC% *
Pure silica CMC/silicaA CMC/silicaB CMC/silicaC CMC/silicaD *
Table 2 Composition of double concentration simulated body fluid.
0 5 10 15 20
Prepared with the same condition at 8 ml TEOS.
Tris Na2 SO4 ·10H2 O NaHCO3 K2 HPO4 NaCl KCl MgCl2 ·6H2 O CaCl2 ·2H2 O *
glasses restricted their application for bone regeneration. Several attempts have been carried out to combine organic polymers like polysaccharides with silica [20–26] to improve the mechanical properties [22,24], washing durability [27] and thermal stability [28] of the produced hybrid. Porous bioactive glass microspheres contained chitosan as biomolecular template initiate the precipitation of apatite crystals on their surface when immersed in SBF for 24 h at 37 ◦ C [22]. The hydrogen bonding between the polysaccharides and the inorganic mineral has a positive effect in avoiding phase separation and producing transparent free standing hybrids [29]. The threshold task of this article was to study the development of a novel CMC-silica-calcium phosphate multiphase biocomposite. CMC could be used as assistant reagent for silica gel formation at low silica precursor concentration, and as a material for improving mechanical properties and a template for biomimatic mineralization of bone like inorganic. Moreover, silica gel was used as a network matrix for preparing of bioactive water insoluble CMC/silica hybrids. Additionally, the study aims to understand the growth of bioactive calcium phosphate crystals as a function of CMC concentration.
2. Materials and methods 2.1. Materials Carboxymethyl cellulose sodium salt (>99.5%) with high viscosity was purchased from Fluka Biochemika. The viscosity of 4% CMC in water at 25 ◦ C is 1000–1500 mPa s. Tetraethyl orthosilicate (TEOS), (99.9%) was purchased from Sigma Aldrich. The other chemicals, such as calcium chloride dihydrate, dibasic potassium phosphate, Magnesium chloride hexahydrate and other simulated body fluid chemicals were analytical grade and used as received without further purification.
Phosphate part* [gm]
Calcium part [gm]
1.2110 0.0644 0.1411 0.0696 – – – –
1.2110 – – – 3.1980 0.0880 0.1220 0.1470
Every part dissolves in 100 ml double distilled water.
2.3. Biomimetic mineralization of calcium phosphate on the hybrids All solutions were prepared just before use. The in vitro bioactivity of the CMC/silica hybrid disk samples (10 mm × 10 mm) was carried out by immersing in double concentration simulated body fluid (2 × SBF). This solution was prepared in two parts as shown in Table 2, to accelerate the hydroxyapatite formation [30]. CMC/silica hybrids in swellable form were incubated in 50 ml 2 × SBF (25 ml from each part) in Falcon tubes for 5 days. The solution was renewed after centrifugation every 24 h and the pH was checked on samples regularly. pH values were maintained at constant physiological pH (7.4) over the entire course of the mineralization to minimize problems associated with SBF preparation and stabilization [31]. Finally, the samples were washed with double distilled water and dried at room temperature for further analysis. 2.4. Cyclic water absorbance of CMC/silica hybrid Water absorbance for the hybrid samples was done with double distilled water. Discs of the hybrid materials with a defined weight were immersed in water. The weights of the hydrated samples were recorded over various time intervals after removal of the surface liquid using Whitman filter paper until reach equilibrium. The percent of water uptake was calculated by the following equation: Water absorption% =
W − W t 0 W0
× 100
where W0 is the initial weight and Wt the final weight of the hybrid samples at time t. The study was continued for 4 times to observe the stability of the hybrids toward absorption and drying cycles. 2.5. Characterization methodology
2.2. CMC/silica hybrids preparation Organic-inorganic hybrids with different weight ratios of CMC:TEOS (w:w) were synthesized via a sol–gel process. As shown in Table 1, a given amount of CMC was dissolved in double distilled water at room temperature under continuous stirring for 24 h. Then, 4 ml TEOS and 4 ml acetic acid solution (acetic acid:water 2:1 v/v) were added to form a mixture of pH 4 with a total volume 32 ml. The former was used as a silica precursor and the latter as a catalyst. Subsequently, the mixture was stirred at 60 ◦ C for 2 h to ensure a complete hydrolysis of TEOS molecules. The resulting homogenous solution was cast in 15 ml Falcon tube in a water bath at 40 ◦ C for 48 h to form a homogenous gel. The unreacted residue was extracted from the hybrid materials via washing for three days with distilled water and subsequently dried at 40 ◦ C for 24 h in a vacuum oven.
2.5.1. ATR–FTIR Attenuated total reflection–Fourier transform infrared spectroscopy (ATR–FTIR) was done on a Thermo Nicolet FT-IR Nexus 470 with a diamond crystal. Spectra were recorded from 500 to 4000 cm−1 with a resolution of 2 cm−1 . 2.5.2. XRD X-ray diffraction (XRD) patterns were recorded with an Empyrean Powder Diffractometer (Cu K␣ , 0.154 nm) between 3 and 70◦ 2 with a step size of 0.01◦ s−1 . Samples were mounted on a silicon support. 2.5.3. Compressive strength test Hybrids (diameter: 20 mm; height/diameter ratio: 4/1) were tested using a LLOYD universal testing machine with a 500 N load cell by simultaneously determining force and
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corresponding length variation. The rate of strain was set by adjusting the cross-head speed to 2 mm/min until 50% compression ultimately. Stress–strain relationships were determined in dry state for at least three samples per composition and condition. Apparent density of the scaffolds was calculated as the quotient of the weight and the cylindrical volume. Compressive modulus was calculated from the slope of a linear fit to the elastic range of the stress–strain curve.
2.5.4. SEM and EDX Scanning electron microscopy was done on a JEOL JXA-840A Electron probe microanalyzer with tungsten filament (30 kV). For EDX experiments an Oxford INCAx-sight SN detector with a resolution of 128 eV at 5.9 keV was used.
2.5.5. TEM Transmission electron microscope (TEM) images were taken with a JEOL JEM-2100 electron microscopy at 100k× magnification, with an acceleration voltage of 120 kV.
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3. Results and discussion 3.1. ATR–FTIR In the present study, CMC was used with low concentrations to enforce the silica gel network matrix and also to functionalize the produced hybrid materials by providing carboxylic and hydroxyl active sites to create biocompatible, biodegradable and bioactive hybrid material. Attenuated total reflection Fourier transform infrared spectroscopy of pure silica gel and CMC/silica hybrids are shown in Fig. 1A. The pure silica sample exhibits major bands at 1078 and 950 cm−1 from Si O Si symmetric stretching and Si O stretching vibrations of the silanol groups. Moreover, the characteristic bands at 1637, 2908, and 3435 cm−1 are related to carboxyl group, asymmetric C H stretching and O H stretching vibration in CMC, respectively. The intensity of these bands increases gradually with increasing CMC concentration from 5 to 20%. In addition, the FTIR peak in the range of 3000–3700 cm−1 and centered at 3400 cm−1 assigned to the hydroxyl stretching vibrations of the self-associated silanol groups. The width of this peak reflects the wide frequency distribution of the hydrogen bonded OH groups. The increase in CMC concentration exhibits broadening for the OH peak which reflects the formation of hydrogen bond interactions between silanol hydroxyls (Si OH) from incomplete polycondensation of the TEOS [32] and hydroxyl groups of CMC. Furthermore, the carboxylic groups of CMC are capable of forming intermolecular hydrogen-bonding with the silanol groups (Si OH) on the surface of the inorganic network produced in the sol–gel process [33]. The CMC/silica hybrid materials were mineralized through immersion in simulated body fluids with ion concentrations higher than those present in human blood serum for 72 h. As seen in Fig. 1B, new characteristic peaks for phosphate group appeared. The characteristic bands at 1075 and 1153 cm−1 (P O 3 mode), 559 cm−1 (P O 4 mode), and 947 cm−1 (P O 1 mode) are assigned to different vibrations modes of PO4 3− group. In accordance with some data, these modes could be related to the presence of crystalline calcium phosphates on the soaked hybrid surface. 3.2. Mechanical properties The choice of CMC in this work was to enhance biocompatibility and to optimize the mechanical properties of the hybrid systems.
Fig. 1. FTIR spectra of CMC/silica hybrid materials at different CMC concentrations (panel A) and mineralized hybrids (panel B).
Fig. 2. Representative stress–strain curves for mineralized CMC/silica hybrid samples.
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Fig. 3. Water uptake/drying cycles of CMC/silica hybrids.
The compression test results of the CMC/silica hybrids are illustrated in Fig. 2. The mean values of strain at failure, compressive modulus and load at rupture of the CMC/silica hybrids obtained from the current study are summarized in Table 3. Up to 15% CMC concentration, the results showed that the incorporation of CMC within the inorganic silica network significantly increased the compression modulus from 769 for CMC/silicaA to 1432 for CMC/silicaC . In addition, the strain at failure for the hybrid samples was increased from 1.01 for CMC/silicaA to 3.64 for CMC/silicaC . In contrast, increasing CMC up to 20% showed to decrease the compressive modulus and strain at failure for the CMC/silicaD . Load at rupture show the same trend as compression modulus. These results clearly indicate that CMC may enforce the silica network matrix at concentrations up to 15% but at high concentrations it may disrupt the network of the silica matrix.
3.3. Cyclic water absorption Water absorbency and structure stability of the hybrids are critical factor for their practical use in medical applications. Although pure silica gel is brittle with no affinity for water absorbance, most of hybrids with natural polymers, including CMC, swell readily in biological fluids with saving molded form. CMC/silica hybrids are allowed to absorb distilled water until equilibrium and re-dried for several times. The water absorption cycles of these hybrids are summarized in Fig. 3. The results of the absorbability in the first cycle reach 24, 33, 48 and 68% for the hybrid materials CMC/silicaA , CMC/silicaB , CMC/silicaC , CMC/silicaD , respectively. These increase accompanied by increasing CMC weigh ratio and consequently increasing the carboxylic group content which is assigned to the degree of the hydrophilicity. Moreover, the hybrids have the ability to dry and re-water absorption several times without any weight loss.
Fig. 4. XRD patterns of neat CMC and mineralized CMC/silica hybrids at different concentrations of CMC.
3.4. XRD The XRD patterns of CMC and the mineralized CMC/silica hybrids at different CMC concentrations are displayed in Fig. 4. Pattern of neat carboxymethyl cellulose shows reflections at 9.5 and 20.2◦ 2; these can be attributed to the crystalline structure of the carboxymethyl cellulose. XRD patterns of the mineralized hybrid samples show that the order of the carboxymethyl cellulose decreases after construction the hybrid network because the carboxymethyl cellulose reflections are significantly broader after hybrid formation which may be assigned to the amorphous glassy structure of the hybrid materials. Moreover, a peak at 44.7 which refers to (0 2 2) index of silicon oxide tetragonal could be distinguished for the CMC/silica hybrid patterns. In addition, the broad scattering peak located between 20 and 25 in all mineralized CMC/silica hybrids may revealed to the presence of amorphous calcium phosphate within 2 × SBF. However, the hybrids with high CMC concentration (CMC/silicaD ) illustrate a development of apatite crystallites. The characteristic peaks of this apatite form located at 22.9, 31.85, 39.2, 42.1 and 46.81 which can be indexed as the (1 1 1), (1 2 1), (1 2 2), (3 1 1) and (2 2 2) planes of hydroxyapatite, respectively. In most cases of calcium-phosphate formation, amorphous calcium phosphate was firstly formed as non-crystalline form, which have strong tendency to transform into thermodynamically stable crystalline apatite by dissolution and recrystalization [34,35]. 3.5. SEM and EDX The SEM micrographs for CMC/silica hybrid and the mineralized CMC/silica hybrids at different CMC concentrations after mineralization in 2 × SBF solution are shown in Fig. 5. Before mineralization, the CMC/silicaB sample shows homogenous rough surface with smoothness degree, which is an indication for a homogenous
Table 3 Summary of the mechanical properties of the CMC/silica hybrids as obtained from the compressive testing. Sample codes
Strain at failure [%]
CMC/silicaA CMC/silicaB CMC/silicaC CMC/silicaD
1.01 1.24 1.26 1.32
± ± ± ±
0.09 0.14 0.11 0.22
Compressive modulus [MPa] 769 1340.0 1432.5 622.3
± ± ± ±
54.34 134.50 145.61 80.93
Load at rupture [N] 97 117.20 597.04 244.89
± ± ± ±
9.91 16.69 80.04 30.60
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Fig. 5. SEM micrographs and corresponding representative EDX patterns (below) for CMC/silicaB hybrid (A), and mineralized hybrids at CMC concentration 5% (B), 10% (C), 15% (D) and 20% (E). (Scale bar = 10 m.).
distributions of CMC fibers in silica-gel network. After immersing CMC/silica hybrid materials in 2 × SBF for 72 h, the samples of lower CMC concentration (CMC/silicaA and CMC/silicaB ) revealed formation of calcium phosphate layer comprised of spherical globules ranging in diameter from few nanometers to 2 m as seen in Fig. 5. These types of spherical globules, which exhibit no distinct surface features, are normal for amorphous calcium phosphate [34] which can be converted, under hydrolysis-conversion process, to other crystalline phases [34,35]. On the other hand, the hybrids contain higher CMC concentrations (CMC/silicaC and CMC/silicaD ) form calcium phosphate layer with distinct surface features consisting of micrometer range surface spherical calcium phosphate. These spherical particles connect together to form a uniform cluster from calcium phosphate layers. The above SEM results indicate that increasing CMC concentration in the hybrids may significantly accelerate the formation rate of
calcium phosphate and reduce the mineralization time. Moreover, the SEM results clearly indicate that the size and shape of the calcium phosphate particles are CMC concentration-dependent. These results agree with the recent results which state that the polymer concentration strongly influences the rate of calcium phosphate aggregation [36]. Before mineralization, the EDX spectrum of CMC/silicaB (Fig. 5) showed that the surface of the sample had a large amount of silicon (48 at%). However, after mineralization in 2X SBF, the EDX analysis detected the presence of Ca and P elements as the major constituents with Ca/P ratio 1.1, 1.22, 1.3 and 1.35 for CMC/silicaA , CMC/silicaB , CMC/silicaC and CMC/silicaD , respectively. Moreover, EDX shows sharp decrease of Si content on the surface of the hybrids, likely due to the consequence of the thickness of the newly formed calcium phosphate layer and the limitation on the X-ray beam penetration depth. In addition, the Ca/P atomic ratios of
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Fig. 6. Representative TEM images of the mineralized CMC/silicaC hybrid.
the mineralized surfaces which range from 1.1 to 1.35 are below the stoichiometric value of 1.5 and 1.67 for amorphous calcium phosphate and hydroxyapatite, respectively. Deviation from the stoichiometic values are known in the literature and may be due to cationic substitutions at the Ca2+ sites by Mg2+ or Na+ or anionic substitution at PO4 3− sites by CO3 2− or HPO4 2− or a combination of these substitutions [34]. In conclusion, increasing the CMC concentration in the hybrid materials had a significant effect on the Ca/P ratio of the calcium phosphate mineralized layer. The Ca/P ratio was increased gradually with increasing CMC concentration which
in so far exhibits the role of anionic carboxylate group in CMC to adsorb Ca2+ and hence initiate the nucleation process. The results from SEM and EDX studies clearly confirmed that the hybrid surfaces have new surface enriched by Ca and P, which are the main constituents of hydroxyapatite. 3.6. TEM To provide better qualitative understanding of the internal structure and give more detailed information about biomimetic
Fig. 7. Illustration of the CMC/silica hybrids and biomimetic mineralization processes using 2 × SBF.
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growth of calcium phosphate onto CMC/silica hybrid materials, TEM observation for the mineralized CMC/silicaC was recorded. As shown in the TEM images in Fig. 6, calcium phosphate nano and microparticles are tightly closed to CMC fibers which confirm the previous suggestion that CMC may act as mineralization sites for calcium phosphate rather than silica.
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Acknowledgement This work was supported by the National Research Center in Cairo, Egypt. Project of cellulose and paper department (No. 10130101). References
3.7. Growth mechanism of calcium phosphate on CMC/silica hybrid The effect of polymer concentration, surfaces and molecular weight on the calcium phosphate crystal phases, particle sizes and crystal shapes were investigated [37]. This study showed that CMC can be used as an effective template for biomimetic mineralization to synthesize micro and nano-spherical calcium phosphate with rough surface. The construction of CMC/silica hybrids and the subsequent biomimetic mineralization mechanism are illustrated in Fig. 7. Calcium phosphate layer coating formation from 2 × SBF onto the surface of the biocompatible CMC/silica hybrids was promoted by negatively charged functional carboxylate groups [34]. These negatively charged functional groups act as nucleation sites which attract Ca2+ ions on the hybrid surface. After that, calcium ions adsorb PO4 3− ions from simulated body fluid solution which in turn starts to form a nucleus of calcium phosphate. These nuclei are formed on the hybrid surface and grow into a dense calcium phosphate layer by additional precipitation of calcium and phosphate ions from the SBF. Since the main factor for calcium phosphate precipitation is the density of carboxylate ion groups, increasing the CMC concentration was accompanied by increasing the bioactivity of the hybrids and also by increasing the Ca/P ratio. The XRD, FTIR, and EDX data establish that various shapes and composition of calcium phosphate layers are mineralized on CMC/silica hybrid after immersing in 2 × SBF. 4. Conclusion In vitro biomimetic calcium phosphate formation ability on CMC/silica hybrids was investigated. It was shown that the bioactivity of sol–gel derived hybrids is mainly governed by the CMC content. After immersing in 2 × SBF, the hybrids with lower CMC content (5 and 10%) showed amorphous calcium phosphate formation. In contrast, the hybrids with higher CMC content (15 and 20%) showed amorphous calcium phosphate and/or hydroxyapatite growing. The compressive modulus and strain at failure increased with an increase in CMC up to 15% content then decreased at 20% concentration. These results suggest that the formed Ca/P ratio and the morphology of the formed calcium phosphate layers are governed by the active carboxylate sites which are represented by CMC concentration. CMC can be incorporated directly with the biomimetic mineralization process of calcium phosphate, which is useful to prepare new categories of hybrid materials.
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