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Bioactive hydrogel-nanosilica hybrid materials: a potential injectable scaffold for bone tissue engineering
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 Biomed. Mater. 10 015020 (http://iopscience.iop.org/1748-605X/10/1/015020) View the table of contents for this issue, or go to the journal homepage for more Download details: IP Address: 132.239.1.231 This content was downloaded on 28/04/2017 at 17:43 Please note that terms and conditions apply.
You may also be interested in: Development of novel silk fibroin/polyvinyl alcohol/sol-gel bioactive glass composite matrix by modified layer by layer electrospinning method for bone tissue construct generation B N Singh and K Pramanik Biopolymer-based hydrogels as injectable materials for tissue repair scaffolds Sylwia Fiejdasz, Krzysztof Szczubiaka, Joanna Lewandowska-acucka et al. Synthesis, characterization and cytocompatibility of spherical bioactive glass nanoparticles for potential hard tissue engineering applications Agda Aline Rocha de Oliveira, Dickson Alves de Souza, Luisa Lima Silveira Dias et al. Biological and mechanical evaluation of poly(lactic-co-glycolic acid)-based composites reinforced with 1D, 2D and 3D carbon biomaterials for bone tissue regeneration Tejinder Kaur, Senthilguru Kulanthaivel, Arunachalam Thirugnanam et al. Chemical characteristics and hemostatic performances of ordered m-SXC Xiaohui Wu, Jie Wei, Xun Lu et al. Calcium phosphate-based ceramic and composite materials for medicine Sergei M Barinov Hierarchically biomimetic scaffold of a collagen–mesoporous bioactive glass nanofiber composite for bone tissue engineering Fu-Yin Hsu, Meng-Ru Lu, Ru-Chun Weng et al. Preparation and characterization of bioactive glass nanoparticles prepared by sol–gel forbiomedical applications Gisela M Luz and João F Mano
Biomed. Mater. 10 (2015) 015020
doi:10.1088/1748-6041/10/1/015020
Paper
received
20 August 2014
Bioactive hydrogel-nanosilica hybrid materials: a potential injectable scaffold for bone tissue engineering
re vised
19 December 2014 accep ted for publication
Joanna Lewandowska-Łańcucka, Sylwia Fiejdasz, Łucja Rodzik, Marcin Kozieł and Maria Nowakowska
5 January 2015
Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Kraków, Poland
published
E-mail: [email protected]
10 February 2015
Keywords: nanosilica particles, hydrogels, hybrid materials, bioactivity, simulated body fluid, bone tissue engineering
Abstract Novel bioactive organic–inorganic hybrid materials that can serve as injectable hydrogel systems for bone tissue regeneration were obtained. The silica nanoparticles (SiNP) prepared in situ by the Stöber method were dispersed in collagen, collagen-chitosan or chitosan sols, which were then subsequently crosslinked. Laser scanning confocal microscopy studies, in which fluorescent SiNP were applied, and SEM images indicated that the nanosilica particles were distributed in the whole volume of the hydrogel matrix. In vitro studies on fibroblast cell viability indicated that the hybrid materials are biocompatible. The silica nanoparticles dispersed in the biopolymer matrix had a positive effect on cell viability. Studies on the mineralization process under simulated body fluid (SBF) conditions confirmed the bioactivity of prepared materials. SEM images revealed mineral phase formation in the majority of the hybrid materials developed. EDS analysis indicated that these mineral phases are mainly composed of calcium and phosphorus. The XRD studies confirmed that mineral phases formed during SBF incubation of hybrid materials based on collagen are bone-like apatite minerals. The silica nanoparticles added to the hydrogel at the stage of synthesis induced the occurrence of mineralization. This process occurs not only at the surface of the material but in its entire volume, which is important for the preparation of scaffolds for bone tissue engineering. The ability of these materials to undergo in situ gelation under physiological temperature and their bioactivity as well as biocompatibility make them interesting candidates for bioactive injectable systems.
1. Introduction Bone tissue engineering is a newly emerging biomedical technology with clinical applications in bone replacement on orthopedic defects, pseudoarthrosis treatment, bone neoplasia and tumors [1, 2]. Providing novel solutions for developing new bone tissue may reduce the risk and expenses related to procedures based on using allografts, autografts and metals [3, 4]. Biomaterials play an important role in bone tissue engineering, since they mimic the structure and composition of human tissue and provide mechanical stability [5–7]. The biodegradable biopolymers often serve as the scaffolds in the development of hybrid materials for bone replacement and regeneration [8, 9]. Chitosan and collagen are the most widely used for that purpose. Chitosan is a linear, semi-crystalline polysaccharide composed of (1 → 4)-2-acetamido-2-deoxy-β-D-glucan (N-acetyl D-glucosamine) and (1 → 4)-2-amino-2deoxy-β-D-glucan (D-glucosamine) units [10]. Chitosan has many unique advantages such as biodegradability, © 2015 IOP Publishing Ltd
mucoadhesivity and permeation enhancing properties [11, 12]. Collagen is the protein polymer [13]. The most abundant type I collagen constitutes the major component of bony tissue [14]. Collagen is an important biomaterial for medical applications due to its high biocompatibility, biodegradability, low toxicity and immunogenicity compared to other natural polymers. Its structure and interactivity with tissue make collagen an excellent matrix to use as a scaffold in bone tissue engineering [15]. Bioactivity is a desired property in the case of scaffolds designed for bone tissue regeneration. Hydroxyapatite (HAp) [Ca10(PO4)6(OH)2] is one of the major inorganic components of bones and teeth [16]. It has been used clinically due to its good biocompatibility and efficacy in promoting biointegration for implants—mostly in hard but also in the soft tissues [17]. To evaluate the in vitro progress of HAp mineralization in scaffold materials under physiological conditions, Kokubo et al developed a simulated body fluid (SBF) with similar inorganic ion concentrations as
Biomed. Mater. 10 (2015) 015020
J Lewandowska-Łańcucka et al
those in human plasma [18]. The results of the in vitro experiments carried out using SBF can be used to predict the in vivo bioactivity of the scaffold materials [19]. Silica is a well-known component of bioactive glasses, found to be active in the stimulation of apatite formation in SBF [20]. Silica is very attractive as a biomaterial as it is non-toxic, easy to fabricate as well as stable in most chemical and biological environments [21–23]. Silica particles have a negative surface charge under physiological pH, imitating most biological species. Additionally, the silanol groups make the surfaces of particles lyophilic, thus enhancing the stability of their suspensions in aqueous media [24] but also inducing apatite formation in hybrid systems. The mechanism of silica to induce apatite formation was under investigation. It was suggested that the silica can chelate and provide sufficient atomic distance required by the crystal structure of bone apatite. It was proposed that hydrated silica formed at the surface of glass ceramics provides sites for favorable apatite nucleation [25]. In terms of the chemical bonding between Si– OH and apatite, it was speculated that under the SBF condition the Si–OH group can be negatively charged (Si–O−) and interact via electrostatic forces with positively charged Ca2+ ions to form a Carich positive thin layer, which can then combine with negatively charged PO43− ions to create amorphous calcium phosphate, that eventually transforms into the apatite structures [26]. It was reported that addition of silica can improve the bioactivity and biocompatibility of the polymeric materials. The incorporation of silica into the chitin scaffold resulted in obtainment of a bioactive scaffold, which can be useful for bone tissue engineering applications [8]. It was also shown that silanol (Si–OH) groups present on the surface of polymeric materials can induce apatite formation in SBF. Lee et al [27] examined the hybrid membrane of chitosansilica xerogel which was applied to guided bone regeneration (GBR). They found that such a chitosan-silica xerogel membrane, when compared with a chitosan membrane exhibited high bioactivity in vitro. By incorporation of the Si–OH groups onto chitosan microparticles Leonor et al [28] obtained the bioactive materials used for designing an injectable bone substitute system. The presence of Si–OH groups on the surface of chitosan microparticles gave them bone-bonding ability and accelerated tissue integration. Kawai et al has shown that the modification of an organic polymer with silanol groups in combination with calcium salts enhances hydroxyapatite formation after exposure to 1.5 SBF with ion concentrations 1.5 times higher than SBF prepared according to Kokubo’s recipe. In this study aromatic polyamide-containing carboxyl (–COOH) groups were modified with different amounts of –SiOH groups in biomimetic solutions. It was reported that the rate of apatite formation increased with increasing silanol groups on the polymer surface [29]. 2
A the nanoscale, bone is a natural composite. The organic matrix is composed of collagen fibers that are mineralized with apatite nanocrystals [30]. In the current study we have focused our attention on the preparation and study of the bioactivity of novel hybrid organic–inorganic materials based on collagen, collagen-chitosan and chitosan hydrogels containing silica nanoparticles. These materials were prepared as potentially useful injectable scaffolds for bone tissue regeneration. The effect of the type of hydrogel matrix used, the presence of SiNP in the hybrid material, and the size of the added NPs on the promotion of the formation of apatite-like mineral structures under physiological conditions (incubation in a simulated body fluid (SBF)) was evaluated and discussed. The structures of the resulting minerals were examined using scanning electron microscopy (SEM), energy dispersive x-ray spectroscopy (EDS), and x-ray diffraction (XRD) techniques.
2. Materials and methods 2.1. Materials Collagen type I rat tail (3.9 mg ml −1 solution, BD Biosciences), genipin (Challenge Bioproducts Co., 98%), chitosan (low molecular weight, Aldrich), ß-Glycerophosphate disodium salt hydrate (≥99%, Sigma), tetraethoxysilane (TEOS, ≥98%, Fluka), ethanol (99,8%, spectroscopic grade), ammonium hydroxide (25%, pure p.a., Chempur) and tris(1,10phenanthroline) ruthenium(II) chloride, ([Ru(phen)3] Cl 2, Sigma Aldrich, 98%), sodium chloride, NaCl (POCh, p.a.), sodium hydrogen carbonate, NaHCO3 (POCh, p.a.), potassium chloride, KCl (Chempur, p.a.), di-potassium hydrogen phosphate trihydrate, K2HPO4 3H2O (Sigma Aldrich, 99%), magnesium chloride hexahydrate, MgCl2 6H 2O (POCh, p.a.), calcium chloride, CaCl2 (Sigma Aldrich, 93%), sodium sulfate, Na 2SO 4 (POCh, p.a.), tris-hydroxymethyl aminomethane, ((HOCH 2) 3CNH 2) (Tris) (Sigma Aldrich, 99,8%), hydrochloric acid 1 M, HCl (POCh). All reagents were used as received without further purification. Millipore-quality water was used during the experiments. Primary Dermal Fibroblasts; Normal, Human, Adult, Organism: Homo sapiens, human / Tissue: skin (ATCC® PCS-201-012 ™), Minimum Essential Medium Eagle With Earle’s salts, L-glutamine and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture (Sigma), Sodium pyruvate solution 100 mM, sterile-filtered, BioReagent, suitable for cell culture (Sigma), MEM Non-essential Amino Acid Solution (100×) without L-glutamine, liquid, sterilefiltered, BioReagent, suitable for cell culture (Sigma), Research Grade FETAL BOVINE SERUM, SouthAmerican origin, Triple 0.1 μm Sterile Filtered, HyClone (Thermo Scientific), Penicilin–Streptomycin Solution 10.00 units ml−1, Penicilin/10.000 µg ml−1, Streptomycin 0.1 µm Sterile Filtered, In Vitro Toxicology Assay Kit, XTT based (Sigma).
Biomed. Mater. 10 (2015) 015020
J Lewandowska-Łańcucka et al
2.2. Apparatus 2.2.1. Dynamic light scattering (DLS) and zeta potential measurements A Malvern Nano ZS light-scattering apparatus (Malvern Instrument Ltd., Worcestershire, UK) was used for dynamic light scattering (DLS) and zeta potential measurements. The time-dependent autocorrelation function of the photocurrent was acquired every 10 s, with 15 acquisitions for each run. The sample of solutions was illuminated by a 633 nm laser, and the intensity of light scattered at an angle of 173° was measured by an avalanche photodiode. The z-averaged hydrodynamic mean diameters (dz), polydispersity (PDI) and distribution profiles of the samples were calculated using the software provided by Malvern. The zeta potential was measured using the technique of laser Doppler velocimetry (LDV). 2.2.2. Laser scanning confocal microscopy (LSCM) The samples in the gel state were put on the glass plate and were directly imaged under a Nikon A1 confocal laser scanning microscope attached to an inverted microscope Nikon Ti (Japan). A 100 × objective lens (Nikon Plan Apo VC /1.40 oil) was used. Samples were excited with a diode laser (405 or 488 nm). Fluorescence spectra were collected using a 32-channel spectral detector. 2.2.3. Scanning electron microscopy (SEM) SEM observations were carried out using a cold field emission scanning electron microscope (FESEM) HITACHI S-4700 equipped with a NORAN Vantage energy dispersion spectrometer. Materials after the mineralization experiments were dried at 37 °C and, in the case of the model hydrogel samples, were frozen in liquid nitrogen, lyophilized and then the solid materials obtained were stuck to the carbon tape on the silicone plate. Finally, the thin film of carbon was deposited on the sample by sputtering. 2.2.4. X-ray diffraction (XRD) X’Pert PRO MPD diffractometer by PANalytical with a Bragg–Brentano geometry was used for all XRD measurements. A copper x-ray sealed tube was used as the radiation source. A graphite monochromator was applied to select only Cu Kα (1.540598 Å—Kα1 and Kα2-1.544426 Å) radiation. The results were crossreferenced with material identification database PDF4+ [31] using X’Pert HighScore commercial software. The samples for XRD experiments (after mineralization experiment) were soaked in water several times, left in 37 °C to dry and next disintegrated in the mortar. Thus the resulting XRD diffraction patterns were taken as the average from the minerals created both inside and on the surface. 2.2.5. Optical microscopy (OM) The cultures were analyzed under phase-contrast light microscope Nikon Eclipse TS 100, 10x. 3
2.3. Preparation of the silica particles, the hydrogels and the hybrid materials 2.3.1. Silica particles Silica particles with the diameter of about 210 nm (SiO2-S1) and particles with the diameter being about 438 nm (SiO 2-S2) were obtained using the same synthetic procedure differing only in the amounts of the reagents used. The procedure was as follows: 1.0 mL/0.65 mL of TEOS was dissolved in ethanol (1.5 mL/1.85 mL) and then a mixture consisting of ethanol (11.5 mL/7.5 mL), water (0.25 mL/1.5 mL) and ammonium hydroxide (30%, 0.75 mL/3.5 mL) was added. The solution was stirred for one hour at room temperature. The formation of the milky dispersion was observed. After that the solution was sonicated for 10 min. The resulting particles were centrifuged and washed with ethanol and this procedure was repeated four times. The obtained materials were dried in a vacuum chamber at 60 °C. 2.3.2. Fluorescent silica particles To the 0.65 mL of TEOS dissolved in ethanol (1.85 mL) the mixture of 7.5 mL ethanol, 3.5 mL ammonium hydroxide and 1.5 mL of aqueous solution of [Ru(phen)3]Cl2 (0.5 mg mL−1) was added. The solution was stirred for one hour at room temperature. After that the solution was sonicated for 10 min. The resulting particles were centrifuged and washed with ethanol and this procedure was repeated four times. The obtained materials were dried in a vacuum chamber at 60 °C. 2.3.3. Hydrogels The hydrogels were obtained using the procedure developed by us and described earlier [32]. Collagen hydrogels (Col) were obtained by adding 200 μl of genipin solution in PBS buffer (pH = 7.4) to 800 μl of the stock collagen solution and then incubated at 37 °C till a hydrogel was formed. Collagen hydrogels named Col1 and Col2 were obtained using 2 or 10 mM genipin solution, respectively. Collagen/chitosan hydrogel (ColCh) was prepared by mixing 690 μl of the stock collagen solution and 138 μl of 2 wt% chitosan solution in 1% acetic acid, 172 μl of 20 mM genipin solution. The formulation was incubated at 37 °C until gel formation was achieved. The collagen/chitosan weight ratio in the hydrogels prepared was 50:50. Chitosan hydrogel (Ch) synthesis was carried out as follows: 800 μl of 2 wt% chitosan solution in 1% acetic acid was mixed with 200 μl of 20 mM genipin solution and the mixtures were vortexed and incubated at 37 °C until gel formed. 2.3.4. Hybrid materials The hydrogels were prepared as previously described but before incubation the water dispersion of silica particles (0.3 mL, 16.6 mg mL−1) was added to the sol (1 ml). The mixture was then vigorously vortexed and incubated at 37 °C until gel formed. In our previous work [32] we investigated the gelation time of hydrogels based on collagen, collagen-chitosan and chitosan at
Biomed. Mater. 10 (2015) 015020
J Lewandowska-Łańcucka et al
physiological temperature 37 °C using fluorescence anisotropy measurements. The resulted gelation time was in the range of 2–4 min depending on the type of hydrogel. We observed that the addition of nanosilica did not effect gelation time significantly. Using these hydrogels and introducing silica particles of different sizes (S1-210 nm and S2-440 nm) the following hybrid materials were prepared: collagen with a lower degree of crosslinking—silica— named Col1S1, Col1S2, collagen with higher degree of crosslinking—silica named Col2S1, Col2S2, collagen-chitosan-silica named ColChS1, ColChS2,and chitosan-silica named ChS1, ChS2. 2.3.5. Model hybrid materials: fluorescent silica particles in chitosan hydrogels crosslinked with βglycerophosphate disodium salt hydrate 800 μl of 2 wt% chitosan solution in 1% acetic acid was mixed with 200 μl of β-glycerophosphate disodium salt solution (1 g L−1) and then 0.3 mL of aqueous dispersion of the fluorescent silica particles (16.6 mg mL−1) was added. In the next step the mixtures were vortexed and incubated at 37 °C until gel formed. The gel was cut into small sections for microscope observations. 2.4. Mineralization in simulated body fluid (SBF) 2.4.1. SBF preparation Simulated body fluid was prepared according to Kokubo’s method [18]. Briefly, reagents: NaCl (8.035 g), NaHCO3 (0.355 g), KCl (0.225 g), K2HPO4 ·3H2O (0.231 g), MgCl2 ·6H2O (0.311 g), 1.0 M HCl (39 ml), CaCl2 (0.292 g) and Na2SO4 (0.072 g) were added to 1 L of distilled water in the order listed above. The pH of the solution was adjusted to 7.4 by the addition of Tris/HCl. The temperature was controlled during the preparation process and kept at around 37 °C. SBF was prepared and stored in a plastic container to avoid apatite nucleation at the surface. 2.4.2. Incubation of hybrid materials in SBF Prepared hybrid materials were transferred into sixwell plates and 8 ml of freshly prepared SBF was added to each well. The materials in the well plates were then placed in a shaker table and incubated in 37 °C for 21 d. After that SBF was removed and the materials were rinsed with deionised water. When water was removed the materials were left in the incubator for drying. 2.4.3. Evolution of the mineralization process The incubation of hybrid materials in SBF—shortterm experiments. The procedure was analogous to that described in section 2.4.2 but the time at which the materials were immersed in SBF was as follows: 6 h or 1, 3 and 7 d. 2.5. Cytotoxicity testing 2.5.1. Preparation of the cell culture scaffolds First, 24- and 96-well plates containing the studied materials were prepared. Briefly, selected samples (Col1, Col1S1, Col1S2, Col2, Col2S1, Col2S2, ColCh, 4
ColChS1, ColChS2, Ch, ChS1, ChS2) in the sol state (n = 3) were transferred into the well plate and kept at 37 °C until the gelling process occurred. After that the plates were sterilized with UV. The prepared wells were next filled with the cell culture medium without serum and left for about two hours in the incubator (37 °C, 5% CO2). The medium was removed directly before cell culture. 2.5.2. Cell isolation and culture on hydrogel scaffolds Primary dermal fibroblasts (normal, human, adult) in the cell culture medium containing 90 vol% of MEM (500 ml of MEM + 5 ml of MEM non-essential amino acid solution (100×) + 5 ml of sodium pyruvate solution + 5 ml of antibiotics: Penicilin –Streptomycin solution) and 10 vol% of serum were seeded in the plate at a density of 6.9 × 104 cells per cm2 (in the case of the 96well plate) and 3.6 × 104 cells per cm2 (in the case of the 24-well plate) on the investigated materials prepared in the wells. The plates were placed in the incubator which maintained optimal cell culture conditions (37 °C, 5% CO2). The experiment lasted 3 d. The culture medium was replaced directly before the XTT test. 2.5.3. Cell viability assay (XTT test) On day 3 of the cell culture the cell viability was studied using the XTT test. The medium from the 96-well plate was removed and a fresh one was added (100 μl per well), next into each well 20 μl (20% of the culture medium volume) of XTT stock solution (1 mg ml−1 in PBS) was added. The cells were then incubated for three hours in the incubator (37 °C, 5% CO2) and then the media from each well were transferred separately to 96-well plates, and their absorbance was measured at 450 nm. As a control, the cells seeded on the tissue culture polystyrene (TCPS) were used. 2.5.4. Statistical analysis The experiments were repeated three times and results expressed as a mean ± standard deviation. The statistical significance was calculated using the analysis of variance (ANOVA). A comparison between two means was analyzed using Tukey’s test with statistical significance level set at p
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Bioactive hydrogel-nanosilica hybrid materials: a potential injectable scaffold for bone tissue engineering
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 Biomed. Mater. 10 015020 (http://iopscience.iop.org/1748-605X/10/1/015020) View the table of contents for this issue, or go to the journal homepage for more Download details: IP Address: 132.239.1.231 This content was downloaded on 28/04/2017 at 17:43 Please note that terms and conditions apply.
You may also be interested in: Development of novel silk fibroin/polyvinyl alcohol/sol-gel bioactive glass composite matrix by modified layer by layer electrospinning method for bone tissue construct generation B N Singh and K Pramanik Biopolymer-based hydrogels as injectable materials for tissue repair scaffolds Sylwia Fiejdasz, Krzysztof Szczubiaka, Joanna Lewandowska-acucka et al. Synthesis, characterization and cytocompatibility of spherical bioactive glass nanoparticles for potential hard tissue engineering applications Agda Aline Rocha de Oliveira, Dickson Alves de Souza, Luisa Lima Silveira Dias et al. Biological and mechanical evaluation of poly(lactic-co-glycolic acid)-based composites reinforced with 1D, 2D and 3D carbon biomaterials for bone tissue regeneration Tejinder Kaur, Senthilguru Kulanthaivel, Arunachalam Thirugnanam et al. Chemical characteristics and hemostatic performances of ordered m-SXC Xiaohui Wu, Jie Wei, Xun Lu et al. Calcium phosphate-based ceramic and composite materials for medicine Sergei M Barinov Hierarchically biomimetic scaffold of a collagen–mesoporous bioactive glass nanofiber composite for bone tissue engineering Fu-Yin Hsu, Meng-Ru Lu, Ru-Chun Weng et al. Preparation and characterization of bioactive glass nanoparticles prepared by sol–gel forbiomedical applications Gisela M Luz and João F Mano
Biomed. Mater. 10 (2015) 015020
doi:10.1088/1748-6041/10/1/015020
Paper
received
20 August 2014
Bioactive hydrogel-nanosilica hybrid materials: a potential injectable scaffold for bone tissue engineering
re vised
19 December 2014 accep ted for publication
Joanna Lewandowska-Łańcucka, Sylwia Fiejdasz, Łucja Rodzik, Marcin Kozieł and Maria Nowakowska
5 January 2015
Faculty of Chemistry, Jagiellonian University, Ingardena 3, 30-060 Kraków, Poland
published
E-mail: [email protected]
10 February 2015
Keywords: nanosilica particles, hydrogels, hybrid materials, bioactivity, simulated body fluid, bone tissue engineering
Abstract Novel bioactive organic–inorganic hybrid materials that can serve as injectable hydrogel systems for bone tissue regeneration were obtained. The silica nanoparticles (SiNP) prepared in situ by the Stöber method were dispersed in collagen, collagen-chitosan or chitosan sols, which were then subsequently crosslinked. Laser scanning confocal microscopy studies, in which fluorescent SiNP were applied, and SEM images indicated that the nanosilica particles were distributed in the whole volume of the hydrogel matrix. In vitro studies on fibroblast cell viability indicated that the hybrid materials are biocompatible. The silica nanoparticles dispersed in the biopolymer matrix had a positive effect on cell viability. Studies on the mineralization process under simulated body fluid (SBF) conditions confirmed the bioactivity of prepared materials. SEM images revealed mineral phase formation in the majority of the hybrid materials developed. EDS analysis indicated that these mineral phases are mainly composed of calcium and phosphorus. The XRD studies confirmed that mineral phases formed during SBF incubation of hybrid materials based on collagen are bone-like apatite minerals. The silica nanoparticles added to the hydrogel at the stage of synthesis induced the occurrence of mineralization. This process occurs not only at the surface of the material but in its entire volume, which is important for the preparation of scaffolds for bone tissue engineering. The ability of these materials to undergo in situ gelation under physiological temperature and their bioactivity as well as biocompatibility make them interesting candidates for bioactive injectable systems.
1. Introduction Bone tissue engineering is a newly emerging biomedical technology with clinical applications in bone replacement on orthopedic defects, pseudoarthrosis treatment, bone neoplasia and tumors [1, 2]. Providing novel solutions for developing new bone tissue may reduce the risk and expenses related to procedures based on using allografts, autografts and metals [3, 4]. Biomaterials play an important role in bone tissue engineering, since they mimic the structure and composition of human tissue and provide mechanical stability [5–7]. The biodegradable biopolymers often serve as the scaffolds in the development of hybrid materials for bone replacement and regeneration [8, 9]. Chitosan and collagen are the most widely used for that purpose. Chitosan is a linear, semi-crystalline polysaccharide composed of (1 → 4)-2-acetamido-2-deoxy-β-D-glucan (N-acetyl D-glucosamine) and (1 → 4)-2-amino-2deoxy-β-D-glucan (D-glucosamine) units [10]. Chitosan has many unique advantages such as biodegradability, © 2015 IOP Publishing Ltd
mucoadhesivity and permeation enhancing properties [11, 12]. Collagen is the protein polymer [13]. The most abundant type I collagen constitutes the major component of bony tissue [14]. Collagen is an important biomaterial for medical applications due to its high biocompatibility, biodegradability, low toxicity and immunogenicity compared to other natural polymers. Its structure and interactivity with tissue make collagen an excellent matrix to use as a scaffold in bone tissue engineering [15]. Bioactivity is a desired property in the case of scaffolds designed for bone tissue regeneration. Hydroxyapatite (HAp) [Ca10(PO4)6(OH)2] is one of the major inorganic components of bones and teeth [16]. It has been used clinically due to its good biocompatibility and efficacy in promoting biointegration for implants—mostly in hard but also in the soft tissues [17]. To evaluate the in vitro progress of HAp mineralization in scaffold materials under physiological conditions, Kokubo et al developed a simulated body fluid (SBF) with similar inorganic ion concentrations as
Biomed. Mater. 10 (2015) 015020
J Lewandowska-Łańcucka et al
those in human plasma [18]. The results of the in vitro experiments carried out using SBF can be used to predict the in vivo bioactivity of the scaffold materials [19]. Silica is a well-known component of bioactive glasses, found to be active in the stimulation of apatite formation in SBF [20]. Silica is very attractive as a biomaterial as it is non-toxic, easy to fabricate as well as stable in most chemical and biological environments [21–23]. Silica particles have a negative surface charge under physiological pH, imitating most biological species. Additionally, the silanol groups make the surfaces of particles lyophilic, thus enhancing the stability of their suspensions in aqueous media [24] but also inducing apatite formation in hybrid systems. The mechanism of silica to induce apatite formation was under investigation. It was suggested that the silica can chelate and provide sufficient atomic distance required by the crystal structure of bone apatite. It was proposed that hydrated silica formed at the surface of glass ceramics provides sites for favorable apatite nucleation [25]. In terms of the chemical bonding between Si– OH and apatite, it was speculated that under the SBF condition the Si–OH group can be negatively charged (Si–O−) and interact via electrostatic forces with positively charged Ca2+ ions to form a Carich positive thin layer, which can then combine with negatively charged PO43− ions to create amorphous calcium phosphate, that eventually transforms into the apatite structures [26]. It was reported that addition of silica can improve the bioactivity and biocompatibility of the polymeric materials. The incorporation of silica into the chitin scaffold resulted in obtainment of a bioactive scaffold, which can be useful for bone tissue engineering applications [8]. It was also shown that silanol (Si–OH) groups present on the surface of polymeric materials can induce apatite formation in SBF. Lee et al [27] examined the hybrid membrane of chitosansilica xerogel which was applied to guided bone regeneration (GBR). They found that such a chitosan-silica xerogel membrane, when compared with a chitosan membrane exhibited high bioactivity in vitro. By incorporation of the Si–OH groups onto chitosan microparticles Leonor et al [28] obtained the bioactive materials used for designing an injectable bone substitute system. The presence of Si–OH groups on the surface of chitosan microparticles gave them bone-bonding ability and accelerated tissue integration. Kawai et al has shown that the modification of an organic polymer with silanol groups in combination with calcium salts enhances hydroxyapatite formation after exposure to 1.5 SBF with ion concentrations 1.5 times higher than SBF prepared according to Kokubo’s recipe. In this study aromatic polyamide-containing carboxyl (–COOH) groups were modified with different amounts of –SiOH groups in biomimetic solutions. It was reported that the rate of apatite formation increased with increasing silanol groups on the polymer surface [29]. 2
A the nanoscale, bone is a natural composite. The organic matrix is composed of collagen fibers that are mineralized with apatite nanocrystals [30]. In the current study we have focused our attention on the preparation and study of the bioactivity of novel hybrid organic–inorganic materials based on collagen, collagen-chitosan and chitosan hydrogels containing silica nanoparticles. These materials were prepared as potentially useful injectable scaffolds for bone tissue regeneration. The effect of the type of hydrogel matrix used, the presence of SiNP in the hybrid material, and the size of the added NPs on the promotion of the formation of apatite-like mineral structures under physiological conditions (incubation in a simulated body fluid (SBF)) was evaluated and discussed. The structures of the resulting minerals were examined using scanning electron microscopy (SEM), energy dispersive x-ray spectroscopy (EDS), and x-ray diffraction (XRD) techniques.
2. Materials and methods 2.1. Materials Collagen type I rat tail (3.9 mg ml −1 solution, BD Biosciences), genipin (Challenge Bioproducts Co., 98%), chitosan (low molecular weight, Aldrich), ß-Glycerophosphate disodium salt hydrate (≥99%, Sigma), tetraethoxysilane (TEOS, ≥98%, Fluka), ethanol (99,8%, spectroscopic grade), ammonium hydroxide (25%, pure p.a., Chempur) and tris(1,10phenanthroline) ruthenium(II) chloride, ([Ru(phen)3] Cl 2, Sigma Aldrich, 98%), sodium chloride, NaCl (POCh, p.a.), sodium hydrogen carbonate, NaHCO3 (POCh, p.a.), potassium chloride, KCl (Chempur, p.a.), di-potassium hydrogen phosphate trihydrate, K2HPO4 3H2O (Sigma Aldrich, 99%), magnesium chloride hexahydrate, MgCl2 6H 2O (POCh, p.a.), calcium chloride, CaCl2 (Sigma Aldrich, 93%), sodium sulfate, Na 2SO 4 (POCh, p.a.), tris-hydroxymethyl aminomethane, ((HOCH 2) 3CNH 2) (Tris) (Sigma Aldrich, 99,8%), hydrochloric acid 1 M, HCl (POCh). All reagents were used as received without further purification. Millipore-quality water was used during the experiments. Primary Dermal Fibroblasts; Normal, Human, Adult, Organism: Homo sapiens, human / Tissue: skin (ATCC® PCS-201-012 ™), Minimum Essential Medium Eagle With Earle’s salts, L-glutamine and sodium bicarbonate, liquid, sterile-filtered, suitable for cell culture (Sigma), Sodium pyruvate solution 100 mM, sterile-filtered, BioReagent, suitable for cell culture (Sigma), MEM Non-essential Amino Acid Solution (100×) without L-glutamine, liquid, sterilefiltered, BioReagent, suitable for cell culture (Sigma), Research Grade FETAL BOVINE SERUM, SouthAmerican origin, Triple 0.1 μm Sterile Filtered, HyClone (Thermo Scientific), Penicilin–Streptomycin Solution 10.00 units ml−1, Penicilin/10.000 µg ml−1, Streptomycin 0.1 µm Sterile Filtered, In Vitro Toxicology Assay Kit, XTT based (Sigma).
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2.2. Apparatus 2.2.1. Dynamic light scattering (DLS) and zeta potential measurements A Malvern Nano ZS light-scattering apparatus (Malvern Instrument Ltd., Worcestershire, UK) was used for dynamic light scattering (DLS) and zeta potential measurements. The time-dependent autocorrelation function of the photocurrent was acquired every 10 s, with 15 acquisitions for each run. The sample of solutions was illuminated by a 633 nm laser, and the intensity of light scattered at an angle of 173° was measured by an avalanche photodiode. The z-averaged hydrodynamic mean diameters (dz), polydispersity (PDI) and distribution profiles of the samples were calculated using the software provided by Malvern. The zeta potential was measured using the technique of laser Doppler velocimetry (LDV). 2.2.2. Laser scanning confocal microscopy (LSCM) The samples in the gel state were put on the glass plate and were directly imaged under a Nikon A1 confocal laser scanning microscope attached to an inverted microscope Nikon Ti (Japan). A 100 × objective lens (Nikon Plan Apo VC /1.40 oil) was used. Samples were excited with a diode laser (405 or 488 nm). Fluorescence spectra were collected using a 32-channel spectral detector. 2.2.3. Scanning electron microscopy (SEM) SEM observations were carried out using a cold field emission scanning electron microscope (FESEM) HITACHI S-4700 equipped with a NORAN Vantage energy dispersion spectrometer. Materials after the mineralization experiments were dried at 37 °C and, in the case of the model hydrogel samples, were frozen in liquid nitrogen, lyophilized and then the solid materials obtained were stuck to the carbon tape on the silicone plate. Finally, the thin film of carbon was deposited on the sample by sputtering. 2.2.4. X-ray diffraction (XRD) X’Pert PRO MPD diffractometer by PANalytical with a Bragg–Brentano geometry was used for all XRD measurements. A copper x-ray sealed tube was used as the radiation source. A graphite monochromator was applied to select only Cu Kα (1.540598 Å—Kα1 and Kα2-1.544426 Å) radiation. The results were crossreferenced with material identification database PDF4+ [31] using X’Pert HighScore commercial software. The samples for XRD experiments (after mineralization experiment) were soaked in water several times, left in 37 °C to dry and next disintegrated in the mortar. Thus the resulting XRD diffraction patterns were taken as the average from the minerals created both inside and on the surface. 2.2.5. Optical microscopy (OM) The cultures were analyzed under phase-contrast light microscope Nikon Eclipse TS 100, 10x. 3
2.3. Preparation of the silica particles, the hydrogels and the hybrid materials 2.3.1. Silica particles Silica particles with the diameter of about 210 nm (SiO2-S1) and particles with the diameter being about 438 nm (SiO 2-S2) were obtained using the same synthetic procedure differing only in the amounts of the reagents used. The procedure was as follows: 1.0 mL/0.65 mL of TEOS was dissolved in ethanol (1.5 mL/1.85 mL) and then a mixture consisting of ethanol (11.5 mL/7.5 mL), water (0.25 mL/1.5 mL) and ammonium hydroxide (30%, 0.75 mL/3.5 mL) was added. The solution was stirred for one hour at room temperature. The formation of the milky dispersion was observed. After that the solution was sonicated for 10 min. The resulting particles were centrifuged and washed with ethanol and this procedure was repeated four times. The obtained materials were dried in a vacuum chamber at 60 °C. 2.3.2. Fluorescent silica particles To the 0.65 mL of TEOS dissolved in ethanol (1.85 mL) the mixture of 7.5 mL ethanol, 3.5 mL ammonium hydroxide and 1.5 mL of aqueous solution of [Ru(phen)3]Cl2 (0.5 mg mL−1) was added. The solution was stirred for one hour at room temperature. After that the solution was sonicated for 10 min. The resulting particles were centrifuged and washed with ethanol and this procedure was repeated four times. The obtained materials were dried in a vacuum chamber at 60 °C. 2.3.3. Hydrogels The hydrogels were obtained using the procedure developed by us and described earlier [32]. Collagen hydrogels (Col) were obtained by adding 200 μl of genipin solution in PBS buffer (pH = 7.4) to 800 μl of the stock collagen solution and then incubated at 37 °C till a hydrogel was formed. Collagen hydrogels named Col1 and Col2 were obtained using 2 or 10 mM genipin solution, respectively. Collagen/chitosan hydrogel (ColCh) was prepared by mixing 690 μl of the stock collagen solution and 138 μl of 2 wt% chitosan solution in 1% acetic acid, 172 μl of 20 mM genipin solution. The formulation was incubated at 37 °C until gel formation was achieved. The collagen/chitosan weight ratio in the hydrogels prepared was 50:50. Chitosan hydrogel (Ch) synthesis was carried out as follows: 800 μl of 2 wt% chitosan solution in 1% acetic acid was mixed with 200 μl of 20 mM genipin solution and the mixtures were vortexed and incubated at 37 °C until gel formed. 2.3.4. Hybrid materials The hydrogels were prepared as previously described but before incubation the water dispersion of silica particles (0.3 mL, 16.6 mg mL−1) was added to the sol (1 ml). The mixture was then vigorously vortexed and incubated at 37 °C until gel formed. In our previous work [32] we investigated the gelation time of hydrogels based on collagen, collagen-chitosan and chitosan at
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physiological temperature 37 °C using fluorescence anisotropy measurements. The resulted gelation time was in the range of 2–4 min depending on the type of hydrogel. We observed that the addition of nanosilica did not effect gelation time significantly. Using these hydrogels and introducing silica particles of different sizes (S1-210 nm and S2-440 nm) the following hybrid materials were prepared: collagen with a lower degree of crosslinking—silica— named Col1S1, Col1S2, collagen with higher degree of crosslinking—silica named Col2S1, Col2S2, collagen-chitosan-silica named ColChS1, ColChS2,and chitosan-silica named ChS1, ChS2. 2.3.5. Model hybrid materials: fluorescent silica particles in chitosan hydrogels crosslinked with βglycerophosphate disodium salt hydrate 800 μl of 2 wt% chitosan solution in 1% acetic acid was mixed with 200 μl of β-glycerophosphate disodium salt solution (1 g L−1) and then 0.3 mL of aqueous dispersion of the fluorescent silica particles (16.6 mg mL−1) was added. In the next step the mixtures were vortexed and incubated at 37 °C until gel formed. The gel was cut into small sections for microscope observations. 2.4. Mineralization in simulated body fluid (SBF) 2.4.1. SBF preparation Simulated body fluid was prepared according to Kokubo’s method [18]. Briefly, reagents: NaCl (8.035 g), NaHCO3 (0.355 g), KCl (0.225 g), K2HPO4 ·3H2O (0.231 g), MgCl2 ·6H2O (0.311 g), 1.0 M HCl (39 ml), CaCl2 (0.292 g) and Na2SO4 (0.072 g) were added to 1 L of distilled water in the order listed above. The pH of the solution was adjusted to 7.4 by the addition of Tris/HCl. The temperature was controlled during the preparation process and kept at around 37 °C. SBF was prepared and stored in a plastic container to avoid apatite nucleation at the surface. 2.4.2. Incubation of hybrid materials in SBF Prepared hybrid materials were transferred into sixwell plates and 8 ml of freshly prepared SBF was added to each well. The materials in the well plates were then placed in a shaker table and incubated in 37 °C for 21 d. After that SBF was removed and the materials were rinsed with deionised water. When water was removed the materials were left in the incubator for drying. 2.4.3. Evolution of the mineralization process The incubation of hybrid materials in SBF—shortterm experiments. The procedure was analogous to that described in section 2.4.2 but the time at which the materials were immersed in SBF was as follows: 6 h or 1, 3 and 7 d. 2.5. Cytotoxicity testing 2.5.1. Preparation of the cell culture scaffolds First, 24- and 96-well plates containing the studied materials were prepared. Briefly, selected samples (Col1, Col1S1, Col1S2, Col2, Col2S1, Col2S2, ColCh, 4
ColChS1, ColChS2, Ch, ChS1, ChS2) in the sol state (n = 3) were transferred into the well plate and kept at 37 °C until the gelling process occurred. After that the plates were sterilized with UV. The prepared wells were next filled with the cell culture medium without serum and left for about two hours in the incubator (37 °C, 5% CO2). The medium was removed directly before cell culture. 2.5.2. Cell isolation and culture on hydrogel scaffolds Primary dermal fibroblasts (normal, human, adult) in the cell culture medium containing 90 vol% of MEM (500 ml of MEM + 5 ml of MEM non-essential amino acid solution (100×) + 5 ml of sodium pyruvate solution + 5 ml of antibiotics: Penicilin –Streptomycin solution) and 10 vol% of serum were seeded in the plate at a density of 6.9 × 104 cells per cm2 (in the case of the 96well plate) and 3.6 × 104 cells per cm2 (in the case of the 24-well plate) on the investigated materials prepared in the wells. The plates were placed in the incubator which maintained optimal cell culture conditions (37 °C, 5% CO2). The experiment lasted 3 d. The culture medium was replaced directly before the XTT test. 2.5.3. Cell viability assay (XTT test) On day 3 of the cell culture the cell viability was studied using the XTT test. The medium from the 96-well plate was removed and a fresh one was added (100 μl per well), next into each well 20 μl (20% of the culture medium volume) of XTT stock solution (1 mg ml−1 in PBS) was added. The cells were then incubated for three hours in the incubator (37 °C, 5% CO2) and then the media from each well were transferred separately to 96-well plates, and their absorbance was measured at 450 nm. As a control, the cells seeded on the tissue culture polystyrene (TCPS) were used. 2.5.4. Statistical analysis The experiments were repeated three times and results expressed as a mean ± standard deviation. The statistical significance was calculated using the analysis of variance (ANOVA). A comparison between two means was analyzed using Tukey’s test with statistical significance level set at p
