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collagen and bioactive glass fibers for bone tissue engineering
Materials Science and Engineering C 59 (2016) 533–541
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Preparation of a biomimetic composite scaffold from gelatin/collagen and bioactive glass fibers for bone tissue engineering Esmaeel Sharifi a, Mahmoud Azami a, Abdol-Mohammad Kajbafzadeh a,b, Fatollah Moztarzadeh c, Reza Faridi-Majidi d, Atefeh Shamousi a, Roya Karimi a, Jafar Ai a,e,⁎ a
Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran Pediatric Urology Research Center, Section of Tissue Engineering and Stem Cells Therapy, Department of Pediatric Urology, Children's Hospital Medical Center, Tehran, Iran (IRI) Department of Biomedical Engineering, Amirkabir University of Technology (Tehran Polytechnic), Tehran, Iran d Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran e Brain and Spinal Injury Research Center (BASIR), Imam Khomeini Hospital, Tehran University of Medical Sciences, Tehran, Iran b c
a r t i c l e
i n f o
Article history: Received 23 May 2015 Received in revised form 30 August 2015 Accepted 7 September 2015 Available online 11 September 2015 Keywords: Bone tissue engineering Bioglass fibers Composite Copper Biomimetic
a b s t r a c t Bone tissue is a composite material made of organic and inorganic components. Bone tissue engineering requires scaffolds that mimic bone nature in chemical and mechanical properties. This study proposes a novel method for preparing composite scaffolds that uses sub-micron bioglass fibers as the organic phase and gelatin/collagen as the inorganic phase. The scaffolds were constructed by using freeze drying and electro spinning methods and their mechanical properties were enhanced by using genipin crosslinking agent. Electron microscopy micrographs showed that the structure of composite scaffolds were porous with pore diameters of approximately 70–200 μm, this was again confirmed by mercury porosimetery. These pores are suitable for osteoblast growth. The diameters of the fibers were approximately 150–450 nm. Structural analysis confirmed the formation of desirable phases of sub-micron bioglass fibers. Cellular biocompatibility tests illustrated that scaffolds containing copper ion in the bioglass structure had more cell growth and osteoblast attachment in comparison to copper-free scaffolds. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Tissue engineering is an emerging field which aims to fabricate biological substitutes to restore and improve deteriorated tissues or organ's functionality [1,2]. An interesting branch of tissue engineering is biomimetic tissue engineering. Biomimetic tissue engineering utilizes bioartificial scaffolds which mimic natural tissue structure and biophysical and mechanical properties [3]. Highly porous scaffolds can be used as a temporary three dimensional substitute for cell adhesion, cell proliferation, cell migration, vascularization and formation of new tissues [4,5]. Important criteria in porous scaffolds are pore size and interconnectivity of pore network to allow cell migration and transportation of nutrient and metabolites through the scaffold [4]. In addition a suitable scaffold for bone tissue engineering should have porosity more than 80% and pore size between 100 and 500 μm depending on the application of the scaffold [6–9]. Porous scaffolds such as composite hydrogel containing bioactive glass (BG) and glass-ceramics (GC) fabricated by freeze drying method are scaffolds with excellent biocompatibility and adequate biomechanical properties. BG and GC can enhance bone ⁎ Corresponding author. E-mail address: [email protected] (J. Ai).
http://dx.doi.org/10.1016/j.msec.2015.09.037 0928-4931/© 2015 Elsevier B.V. All rights reserved.
formation and bonding to the surrounding bone tissue in vivo [10], In composite scaffolds BG and GC act as mineralization agents, provide support for osteoblast cells and possess osteoconductive properties. 45S5 BGs (in wt%: 45% SiO2, 24.5% Na2O, 24.4% CaO and 6% P2O5) exposes critical concentrations of Ca, Si, Na and P ions, which have been shown to activate genes in osteoblast cells, stimulating new bone formation in vivo [11–13]. In addition some essential ions, such as copper (Cu), have stimulating effects on osteogenesis, angiogenesis, wound healing and also possess anti-inflammatory, anti-infectious and antibacterial potentials [14–16]. It was shown that, during in vitro culture, Cu ions stimulated endothelial cell proliferation in a dose dependent manner, and promoted wound healing in rats by up regulation of the VEGF expressed by stimulated cells. Thus, Cu has been combined with biomaterials in order to improve their biological effects. Previous studies suggest that direct mixing of Cu ions with bioactive materials is a feasible way to improve angiogenesis [11,17] Therefore, in this study we aimed to prepare Cu-containing composite scaffolds and investigate their effects on properties of scaffolds. It is expected that incorporation of Cu into scaffolds will improve their angiogenesis potential and will also offer composite scaffolds additional antibacterial property [17]. Copper depletion leads to reduction of bone mineral density (BMD). However, Osteoblast activity is inhibited at a concentration of
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1 mM Cu, so the concentration of the Cu in scaffold should be optimized [15]. In order to enhance the biological performance of BGs and GCs, Cu was incorporated in the glass matrix. Cu-containing mesoporous BG scaffolds can stimulate angiogenesis and osteogenesis [15]. Different methods for cross-linking gelatin (or collagen gel) have been described. Due to limited usage in cellular tissues, physical treatments such as ultraviolet (UV), dehydrothermal and γ-irradiation treatments are not practical. Chemical treatments using aldehydes are often used to maintain and tighten tissues but these treatments are highly toxic. Genipin has been proved to increase mechanical properties of collagen and gelatin. Li et al. used genipin to crosslink a gelatin coated 45S5 bioglass scaffolds with high porosity (N 90%). They showed
26-fold higher compressive strength for the coated scaffold compare with un-coated scaffolds [18]. Genipin, extracted from Gardenia Jasminoides fruit, crosslinks polypeptide structure through nucleophilic attack of amine I located on lysine and arginine residues to the C3 atom of genipin, causing gel strength comparable to glutaraldehyde, while genipin is 10,000- fold less cytotoxic. After crosslinking, the color of gelatin changes to blue, and when exited at 590 nm, emits fluorescence at 630 nm [19]. There are three main methods for making fibrous scaffolds, electrospinning (ES), phase separation and self-assembly [20,21] ES is an affordable method to make fibrous structures of nano and microscale diameters, with very high specific surface areas. Specifically, the
Fig. 1. DSC-TGA of a. 45S5 sub-micron BG fibers and b. copper containing 45S5 sub-micron BG fibers.
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diameter of the fiber produced by ES is usually more than a few hundred nanometers [20,22]; however, these fibers are often called “nanofibers”. Nanofibers have different applications in tissue engineering and these purposes are achieved by controlling the three-dimensional structure and chemical/physical properties of the fibers. Most organs and tissues such as bone, skin, tendons and cartilage formations are highly organized fibrous structures [20,21,23]. Traditional methods for constructing porous bio-polymer scaffolds include gas foaming, freeze drying, solvent casting, two-phase and particulate leaching. Through these methods, a wide range of architecture and geometry settings with interconnected pores are created. Compared to others, freeze-drying is the simplest method for making a porous scaffold [22]. The diameter of the pores in the scaffold may be uniformed by freeze-drying method. Molecular weight of components and materials can severely affect the rate of biodegradation of these scaffolds [24]. A number of studies have been conducted to produce fibrillar BG mimicking the ECM, in which sol–gel derived BG nanofibers were produced by ES method [25,26]. and nanocomposite scaffolds composed of nanofibrillar BG and reconstituted collagen were developed for bone regeneration matrix [27]. In this study, we prepared a novel composite scaffold by combining BG fibers (with and without Cu) within gelatin-collagen hydrogel. We designed a composite including sub-micron fibers inside a polymeric matrix of gelatin and collagen which mimics natural bone structure. Although, the selected composition for the prepared scaffold is not exactly the same as bone, but it is not so far from natural bone composition, as the organic phase, gelatin is known as the denatured structure of collagen; and as the mineral part, BG fibers will turn to apatite fibers or at least fibers with apatite coating after being exposed to body environment. In this study by using Human osteoblast-like cell line SaOS-2, cell growth and viability were investigated and compared in three scaffolds; gelatin-collagen hydrogel scaffold, composite scaffolds containing 45S5 BG fibers and composite scaffolds containing Cu-doped 45S5 BG fibers. 2. Materials and methods
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the precursor through stirring. The following reagents were added separately after 45 min, during stirring, in the following sequence: 2.9 ml triethyl phosphate, 20.13 g calcium nitrate tetrahydrate and 13.52 g sodium nitrate [10,28], in the case of Cu-45S5 19.84 g calcium nitrate tetrahydrate and 0.61 g Cu(II) nitrate trihydrate. The sol mixtures were stirred for 36 h and aged without stirring at 25 °C for 24 h, followed by a further 24 h at 40 °C. Prior to ES the 12% wt. solution of PVA was prepared, and mixed with sol-solution in ratio of 1:1.5. 5 ml of this solution was loaded in syringe and under controlled condition (voltage: 20 kV, distance: 10 cm, and injection rate: 1 mL/h) ES process was done. The sub-micron fibers were subsequently heat-treated at 700 °C for 1 h in air with a heating rate of 4 °C/min and cooling rate of 5 °C/min. The heat-treatment temperature was determined to be high enough to eliminate organic sources and nitrates completely [10,28]. 2.1.2. Fabrication of composite hydrogel Porous GEL/COL composites and porous GEL/COL/BG fibers (45S5 and Cu containing 45S5) composites were fabricated as follows: At first, collagen/gelatin solution with the ratio of 1:9 (Merck, microbiology grade, catalog number 104,070) was prepared, for this purpose, collagen was added to deionized water containing 50 mM acetic acid, and gelatin was added to deionized water in a stirrer associated with heating. After 1 h, collagen and gelatin solutions were mixed to make a 10% (w/v) solution. In the case of GEL/COL/sub-micron BG fibers (45S5 and Cu containing 45S5) composites the ratio of GEL/COL/BG fibers was 70:30% [29]. In the next step, the fiber was mixed with hydrogel through gentle vortexing. Then the prepared hydrogel and the composite hydrogel were poured into a cylindrical mold and kept at 4 °C for 2 h until physical gelation occurred. Afterward, the gel was extracted and kept at − 20 °C for about 24 h. The resultant composite was extracted and freeze dried to create a porous structure. Scaffolds were incubated in a 0.5% genipin solution for 16 h. Samples were washed with ethanol, PBS and deionized water to remove remnants of the genipin. The provided scaffolds were kept at a dry place.
2.1. Fabrication and characterization of composite scaffold
2.2. Scaffold characterization
2.1.1. Fabrication of sub-micron BG fibers The sub-micron 45S5 BG fibers and Cu containing sub-micron 45S5 BG fibers are composed of: (45 wt% SiO2, 6 wt% P2O5, 24.5 wt% CaO, 24.5 wt% Na2O) and (45 wt% SiO2, 6 wt% P2O5, 23.5 wt% CaO, 1 wt% CuO, 24.5 wt% Na2O) (TEOS, Si(OC4H9)4, 99.99%, Sigma Aldrich), triethylphosphite (P(OEt)3, P(C2H5O)3, 99.5%, Sigma Aldrich), calcium nitrate tetrahydrate (Ca(NO3)2?4H2O, 99.60%, Sigma Aldrich) and sodium nitrate (NaNO3, 100.40% Sigma Aldrich). Gel-derived 45S5 and Cu containing 45S5 were prepared as follows: initially 33.5 ml tetraethyl orthosilicate (TEOS) was added to 1 M nitric acid, with H2O: TEOS molar ratio equal to 18; to prepare the required amount of 1 M nitric acid solution, 3.26 mL of 69% nitric acid was mixed with 47.6 mL distilled water. The solution was allowed to react for 60 min for hydrolysis of
2.2.1. Differential scanning calorimetry and thermogravimetric analysis (DSC-TGA) DSC-TGA analysis was used to determine the transition temperature and mass loss of the samples. It was applied on the sol-derived 45S5 and Cu containing 45S5 sub-micron fibers before stabilization. This test was accomplished to assess the transformations and their temperatures including weight loss from ambient temperature up to 1000 °C with the heating rate of 5 °C/min. 2.2.2. Mercury porosimetery To measure the distribution of prepared scaffold pores, porosity percentage and mean value of pore diameter, mercury porosimetery was used.
Fig. 2. Scanning electron microscopy (SEM) micrograph of the fibers before heating. a. 45S5 sub-micron BG fibers, b. 45S5 sub-micron BG fibers containing Cu.
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Fig. 3. Scanning electron microscopy (SEM) micrograph of the fibers after heating at 700 °C. a. 45S5 sub-micron BG fibers, b. 45S5 sub-micron BG fibers containing Cu.
In this method mercury penetrates through pores with various pressures in an autoclave instrument, the smaller the pores are, the higher the pressure will be. With mercury porosimetry not only the total porosity can be measured, but also the distribution of the pores can be measured. Mercury porosimetry method is able to distinguish a wide range of pore sizes depending on the Hg pressure applied on the sample, the lower pressure senses bigger pores. In this case, we applied pressure between 0.1–400 kPa which can show pore size ranging from 3 nm to 200 μm. In this study Finnigan mercury intrusion porosimeter PASCAL140 (Germany) was used. In this method the sample was dried initially and then exposed with mercury inside high vacuum chamber. Mercury pressure was changed incrementally from 0.1 to 336 KPa. Pore diameter in which mercury infiltrates are determined by Washburn equation [L = γDt/4η]. Where, L: infiltrated mercury volume, γ: surface tension, D: diameter of pores, T: Time of mercury infiltration through pores, μ: mercury viscosity. 2.2.3. Scanning electron microscopy (SEM) The morphology of sub-micron fibers and composite hydrogel scaffolds was assessed by scanning electron microscopy (SEM; Philips XL30 microscope). The images were obtained using an accelerating
voltage of 15 kV. All samples were coated with a gold sputtering device, before being investigated under the SEM. The obtained images were used to evaluate the sub-micron BG fibers morphology and pore size of the composites. 2.3. Structural analysis 2.3.1. Fourier transform infrared spectroscopy (FTIR) The functional groups of composite samples and the sub-micron BG fibers were examined by FTIR with Bomem MB 100 spectrometer. For IR analysis, at first 1 mg of the powder samples were carefully mixed with 300 mg of KBr (infrared grade) and palletized under vacuum. Then the pellets were analyzed in the range of 400–4000 cm−1 at a scan speed of 23 scan/min with 4 cm−1 resolution. 2.3.2. X-ray diffraction (XRD) Crystallographic properties of synthesized BG fibers were assessed using X-ray diffraction method. For this aim, X-ray diffraction patterns of fibers were recorded on a Philips X-ray diffractometer with Co-Kα radiation (λ = 1.78901 Å). The scans of the selected diffraction peaks were carried out in step mode (step size 0.02°, measurement time 1 s, measurement temperature 25 °C, and standard: Si powder).
Fig. 4. Scanning electron microscopy (SEM) micrograph of the composite hydrogel, a. gelatin/collagen scaffold, b. gelatin/collagen/45S5 fibrillar BG, c, d gelatin/collagen/45S5 fibrillar BG containing Cu.
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Fig. 5. Porosimetry of the composite scaffolds, a. gelatin/collagen scaffold, b. gelatin/collagen/45S5 fibrillar BG, c. gelatin/collagen/45S5 fibrillar BG containing Cu.
Crystallographic identification of the phases of synthesized fibers was accomplished by comparing experimental XRD patterns to standards compiled by the International Center for Diffraction Data (ICDD).
2.4. Cells attachment Human osteoblast-like cell line SaOS-2 was used to evaluate in vitro cytocompatibility of the composite scaffolds. These cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and streptomycin/penicillin 100 U/ml (1%). For seeding, the cells were trypsinized (0.05% trypsin/0.53 mM EDTA in 0.1 M PBS without calcium or magnesium), centrifuged and resuspended in complete culture medium. Medium containing 3 × 105 cells were then seeded on top of each scaffold sample before they were immersed in the medium. Cells/scaffold constructs were incubated for 4 days at 37 °C in a humidified atmosphere containing 5% CO2. Then, the samples were washed twice with PBS before fixation. For fixation, samples were soaked in 2.5% glutaraldehyde for 1 h. Post-fixation was performed in 1% osmium tetroxide, followed by dehydration in a series of graded acetone solutions. The samples were freeze-dried and
kept dry with silica gel. Finally, they were kept in a hood for air drying and then used for SEM observation [30]. 2.5. MTT assay Cell viability was analyzed using 3- [4, 5-dimethylthiazol-2-YL] -2, 5 diphenyl tetrazolium bromide (MTT) assay on days 1, 2 and 4. For this test 5 mg/ml MTT solution was prepared by dissolving the MTT powder (Sigma) in warm PBS (37 °C) [31]. SaOS-2 cell line (1 × 104 cells/well) were cultured in 96 well (NUNC, Denmark), then incubated for 24 h. 20 μ L of MTT solution was added to each well for 4 h. Then Dimethylsulfoxide- 99.5% (DMSO, Sigma) was added to each well and the plate was shaken for 5 min with stirring. The absorbance at 570 nm was measured by Elisa Reader. 3. Results and discussion 3.1. Differential scanning calorimetry and thermogravimetric analysis (DSC-TGA) DSC and TGA analysis of sub-micron fibers were performed to obtain the right heat treatment temperature. Fig. 2 shows the DSC-
Fig. 6. XRD patterns of sol-derived 45S5 sub-micron BG fibers and copper containing 45S5 sub-micron BG fibers after heating at 700 °C and composite scaffolds containing fibrillar BG.
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Fig. 7. FTIR spectrum of composite scaffolds before and after crosslinking with genipin.
TGA curves obtained from the sol-derived 45S5 sub-micron fibers between ambient temperatures to 1000 °C. DSC curve includes both endothermic and exothermic peaks. The first endothermic peak, which initiated at 75 °C, corresponds to the release of physically adsorbed water, which was not removed during drying. TGA trace shows that all water and products from the polycondensation reaction were removed between 75 and 160 °C (12% weight loss). The other two endothermic peaks, starting at 350 °C (41% weight loss), are related to pyrolysis reaction of free organic species and/or release of water from the further condensation of Silanol and P-OH group and PVA burning out. All nitrates were removed at 560 °C (a further 22% of the total weight loss), so the total weight loss was 61%. In addition, two peaks at 350 °C and 582 °C may correspond to the decomposition of nitrate and hydrocarbon molecules respectively. Above 700 °C no significant weight loss was observed (Fig. 1a), this curve confirmed that the residuals can be removed before 700 °C, thus at this temperature the structure will be completely stabilized. Cu was incorporated as Cu2+ in the 45S5 fibrous BG network. Samples containing Cu have thermal patterns similar to fibers containing 45S5 BG, although a sharper peak is observed in 350 °C in Cu containing BG, Further, Cu decreases the glass transition and lowers the melting onset temperature (Fig. 1b). Endothermic peaks correspond to the removal of sodium nitrite and other nitrogen compounds. On the other hand, the exothermic peak indicates the formation of a crystalline phase and phase transformation. The best stabilization temperature, with high bioactivity and removal of all the nitrogen contents is about 700 °C according to a previous study [28].
3.2. Morphological characterization by SEM In this study, morphology of the sub-micron fibers and composite scaffolds were observed using SEM. Low and high magnification SEM micrographs taken from the surface of the composites are shown in Figs. 2–4. Fig. 2 shows photographs taken from the sub-micron fibers before heating at 700 °C, with SEM, as shown in this figure, the diameter of the fibers was between 150 and 450 nm. Fig. 3 shows the sub-micron BG fibers after heating, the approximate diameter of these fibers are 150–450 nm. Obtained glass sub-micron fibers represented coarse unsmooth morphology on the surface and showed fusion to each other after thermal treatment. The surface morphology of the porous composite scaffolds is shown in Fig. 4, as shown in this figure, a network of interconnected pores is shown. This scaffold has a pore diameter in the range of 70–250 μm, which is optimal for bone cell growth [29,30]. The incorporation of submicron BG fibers in composite hydrogel structure can be seen in the micrograph shown in Fig. 4b and d.
3.3. Mercury porosimetery Total porosity and pore distribution were investigated with mercury porosimetery method. The results showed that the porosity percentage of the three prepared scaffolds fell in range of 70–80% and size distribution of the pores was in the range of 10–200 μm for three scaffolds (Fig. 5a-c). The average pore size for the GEL/COL, GEL/COL/BG fibers and GEL/COL/Cu-BG fibers were 72 ± 20, 129 ± 24 and 82 ± 22, respectively. Considering the length of the osteoblasts and the length of mesenchymal stem cells (MSCs), it seems that the achieved distribution of porosity in the samples were enough for MSCs and osteoblasts penetration and immigration into the structures of these composite scaffolds [32–34]. 3.4. XRD analysis crystallographic properties of sub-micron BG fibers Sub-micron BG fibers were analyzed using XRD. Fig. 6 shows diffractograms obtained from XRD for the glass fibers samples. The diffractogram has weak peaks representing a type of amorphous or semi-crystalline nature. These peaks can be ascribed to combeite (marked in this figure), according to the ICCD database. BG in the forms of scaffold and powder were also characterized by X-ray diffraction, These BGs are composed of two sodium calcium silicate phases including combeite (Na2Ca2Si3O9) and silicorhenanite (Na2Ca4(PO4)2SiO4). Chen QZ et al. [35] reported that formation of Na2Ca2Si3O9 phase is favorable because it confers the scaffolds mechanical strength and furthermore. They also showed that this strong crystalline phase transforms into biodegradable and bioactive amorphous calcium phosphate after being implanted in the body [35]. Fig. 6 shows the XRD patterns obtained from different types of BGs. Addition of Cu into the BG structure did not affect the formation of
Table 1 Correlation of peaks detected by Fourier transform infrared spectroscopy (FTIR) for prepared scaffold [10,28,36,39]. Wavenumber (cm−1) ~ 463 ~ 939 ~ 1088 ~ 1663 ~ 1546 ~ 1240 ~ 3300
Assignment
Material
Si-O-Si bending Si-O stretching Si-O-Si stretching (asymmetric) amide I (C = O stretch) amide II peak (N-H bend and C-H stretch) amide III (C-N stretch plus N-H in phase bending) (N-H stretching vibration)
characteristic of silicate network in BG characteristic of gelatin and collagen in scaffolds
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Fig. 8. MTT assay analysis for cell culture on scaffold after 24, 48 and 96 h.
sodium calcium silicate phases, which has been reported previously in the literature [15]. Also, silicorhenanite phase that has been detected in this analysis was isostructural to apatite.
3.5. FTIR analysis Fig. 7 shows the FTIR spectra, in the 500–4000 cm−1 spectral range, for plain gelatin/collagen, cross-linked gelatin/collagen, composite containing heat-treated 45S5 BG fibers and composite scaffold containing Cu ion before and after crosslinking with genipin. FTIR spectra of plain gelatin/collagen exhibited a number of characteristic spectral bands such as: C = O stretch at 1662.1 cm−1 for amide I, N-H bend and C-H stretch at 1546 cm− 1 for amide II, C-N stretch plus N-H in phase bending at 1244 cm−1 for amide III, N-H stretching vibration at 3298 cm− 1 for the amide, which are the distinguishing features of
gelatin and collagen. In cross linked hydrogel we see similar peaks with a little shift as previously reported [36]. All these characteristic bands are also present in the spectrum obtained from composite scaffolds. In addition, composite samples containing 45S5 fibrillar BG and Cu ion containing BG exhibit three infrared bands located at: 464, 940 and 1084 cm−1, which are related to the silicate network and attributed to the Si-O-Si bending vibration, Si-O stretching vibration and asymmetric stretching vibration of Si-O-Si, respectively [28]. Correlation of the peaks, achieved by FTIR, is presented in Table 1. It should be noted that FTIR analysis shows a peak related to chemical bonds formed by the combination of fibrillar BG with hydrogel and subsequent crosslinking with Genipin. The peak is located at about 1334 cm−1 and indicates formation of the chemical bond between carboxyl groups of hydrogel and Ca2+ ions of fibrillar BG, same result has also been mentioned in former studies for Gelatin and hydroxyapatite [37,38].
Fig. 9. Scanning electron microscopy (SEM) micrograph of the cultured cells on composite scaffolds.
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3.6. MTT assay As shown by MTT assay (Fig. 8), scaffolds did not have any negative effects on the proliferation rate of osteoblasts compared with plastic cell culture surfaces. MTT assay was carried out at days 1, 2, and 4 on gelatin-collagen hydrogel and composite scaffolds including fibrillar BG 45S5 and Cu containing fibrillar BG 45S5. No significant differences were observed between composite scaffolds containing Cu-doped fibrillar 45S5 BG and control group in all the mentioned times, which indicates that the scaffolds had no toxicity. In the first 24 h gelatin-collagen hydrogel and composite containing fibrillar 45S5 BG had no toxicity, but after 48 h a significant reduction was observed in the viability of the cells seeded on the hydrogel scaffold, and the scaffold had inhibited the cell growth, but again after 96 h an increase in the cell growth was noticed. In the case of composite containing fibrillar BG 45S5, the results were the same after 48 and 96 h. (p b 0.05). For the scaffold containing fibrillar Cu-doped BG no toxic effects were observed. 3.7. Cell adhesion and proliferation on the scaffold SEM pictures of osteoblast cells were obtained after culturing the cells on the prepared scaffolds. As shown in Fig. 9, osteoblast cells have penetrated into scaffold pores and have completely widespread on the surface of the pores, the filopodia of the cells are also well observed in the images. In the gelatin-collagen hydrogel micrographs cell adhesions are observable. In the composite scaffolds the cells are attached to the scaffold and the cells are expanded, indicating that prepared biomimetic scaffolds could provide suitable environment similar to ECM for the osteoblast cells growth. Review on the research done in the field of fabrication of hydrogel based composite scaffolds reinforced by a ceramic compound shows that use of ceramic fibers as reinforcing phase are one of the novel techniques which has been less studied till now [27]. In addition, previously used calcium phosphate phase like Hydroxyapatite [3,9] has been replaced by bioactive glasses and other silicate based ceramics [35]. The possibility of different elements addition into the structure of bioactive glasses provides the chance of study on the potential effects of these elements in biological environments [11]. According to the above explanations and the fact that the effects of copper have been less investigated in tissue engineering applications, in this study we aimed to use cu-containing bioactive glass fibers to reinforce a hydrogel based scaffold. The results showed that cu-containing bioactive glass fibers can be obtained through described method and together with hydrogel basis can be processed toward making porous bone tissue engineering scaffold with a suitable structure. 4. Conclusion In this study three types of scaffolds were fabricated including, gelatin-collagen hydrogel, gelatin-collagen composite containing submicron 45S5 BG fibers, and Cu-doped composites. Sub-micron BG fibers were fabricated by combination of sol–gel and electrospinning processes, the fibers were then mixed with hydrogel matrix containing gelatin and collagen, freeze dried, followed by genipin crosslinking to fabricate final composite scaffolds. Growth and viability of human osteoblast-like cell line SaOS-2 were investigated on these biomimetic scaffolds. Cellular biocompatibility assays illustrated that scaffolds containing Cu ion in the BG structure had more viability and osteoblast growth in comparison with other scaffolds, the scaffolds containing Cu ion in the 45S5 BG network showed better biocompatibility compared to hydrogel and composite scaffold containing the 45S5 BG. Cu ion increased growth of the osteoblasts on composite scaffold containing sub-micron fibers BG compared to hydrogel without sub-micron fibers BG. Obtained results showed that the prepared scaffolds, especially Cu-doped BG fibers containing scaffold is
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Preparation of a biomimetic composite scaffold from gelatin/collagen and bioactive glass fibers for bone tissue engineering Esmaeel Sharifi a, Mahmoud Azami a, Abdol-Mohammad Kajbafzadeh a,b, Fatollah Moztarzadeh c, Reza Faridi-Majidi d, Atefeh Shamousi a, Roya Karimi a, Jafar Ai a,e,⁎ a
Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran Pediatric Urology Research Center, Section of Tissue Engineering and Stem Cells Therapy, Department of Pediatric Urology, Children's Hospital Medical Center, Tehran, Iran (IRI) Department of Biomedical Engineering, Amirkabir University of Technology (Tehran Polytechnic), Tehran, Iran d Department of Medical Nanotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran e Brain and Spinal Injury Research Center (BASIR), Imam Khomeini Hospital, Tehran University of Medical Sciences, Tehran, Iran b c
a r t i c l e
i n f o
Article history: Received 23 May 2015 Received in revised form 30 August 2015 Accepted 7 September 2015 Available online 11 September 2015 Keywords: Bone tissue engineering Bioglass fibers Composite Copper Biomimetic
a b s t r a c t Bone tissue is a composite material made of organic and inorganic components. Bone tissue engineering requires scaffolds that mimic bone nature in chemical and mechanical properties. This study proposes a novel method for preparing composite scaffolds that uses sub-micron bioglass fibers as the organic phase and gelatin/collagen as the inorganic phase. The scaffolds were constructed by using freeze drying and electro spinning methods and their mechanical properties were enhanced by using genipin crosslinking agent. Electron microscopy micrographs showed that the structure of composite scaffolds were porous with pore diameters of approximately 70–200 μm, this was again confirmed by mercury porosimetery. These pores are suitable for osteoblast growth. The diameters of the fibers were approximately 150–450 nm. Structural analysis confirmed the formation of desirable phases of sub-micron bioglass fibers. Cellular biocompatibility tests illustrated that scaffolds containing copper ion in the bioglass structure had more cell growth and osteoblast attachment in comparison to copper-free scaffolds. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Tissue engineering is an emerging field which aims to fabricate biological substitutes to restore and improve deteriorated tissues or organ's functionality [1,2]. An interesting branch of tissue engineering is biomimetic tissue engineering. Biomimetic tissue engineering utilizes bioartificial scaffolds which mimic natural tissue structure and biophysical and mechanical properties [3]. Highly porous scaffolds can be used as a temporary three dimensional substitute for cell adhesion, cell proliferation, cell migration, vascularization and formation of new tissues [4,5]. Important criteria in porous scaffolds are pore size and interconnectivity of pore network to allow cell migration and transportation of nutrient and metabolites through the scaffold [4]. In addition a suitable scaffold for bone tissue engineering should have porosity more than 80% and pore size between 100 and 500 μm depending on the application of the scaffold [6–9]. Porous scaffolds such as composite hydrogel containing bioactive glass (BG) and glass-ceramics (GC) fabricated by freeze drying method are scaffolds with excellent biocompatibility and adequate biomechanical properties. BG and GC can enhance bone ⁎ Corresponding author. E-mail address: [email protected] (J. Ai).
http://dx.doi.org/10.1016/j.msec.2015.09.037 0928-4931/© 2015 Elsevier B.V. All rights reserved.
formation and bonding to the surrounding bone tissue in vivo [10], In composite scaffolds BG and GC act as mineralization agents, provide support for osteoblast cells and possess osteoconductive properties. 45S5 BGs (in wt%: 45% SiO2, 24.5% Na2O, 24.4% CaO and 6% P2O5) exposes critical concentrations of Ca, Si, Na and P ions, which have been shown to activate genes in osteoblast cells, stimulating new bone formation in vivo [11–13]. In addition some essential ions, such as copper (Cu), have stimulating effects on osteogenesis, angiogenesis, wound healing and also possess anti-inflammatory, anti-infectious and antibacterial potentials [14–16]. It was shown that, during in vitro culture, Cu ions stimulated endothelial cell proliferation in a dose dependent manner, and promoted wound healing in rats by up regulation of the VEGF expressed by stimulated cells. Thus, Cu has been combined with biomaterials in order to improve their biological effects. Previous studies suggest that direct mixing of Cu ions with bioactive materials is a feasible way to improve angiogenesis [11,17] Therefore, in this study we aimed to prepare Cu-containing composite scaffolds and investigate their effects on properties of scaffolds. It is expected that incorporation of Cu into scaffolds will improve their angiogenesis potential and will also offer composite scaffolds additional antibacterial property [17]. Copper depletion leads to reduction of bone mineral density (BMD). However, Osteoblast activity is inhibited at a concentration of
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1 mM Cu, so the concentration of the Cu in scaffold should be optimized [15]. In order to enhance the biological performance of BGs and GCs, Cu was incorporated in the glass matrix. Cu-containing mesoporous BG scaffolds can stimulate angiogenesis and osteogenesis [15]. Different methods for cross-linking gelatin (or collagen gel) have been described. Due to limited usage in cellular tissues, physical treatments such as ultraviolet (UV), dehydrothermal and γ-irradiation treatments are not practical. Chemical treatments using aldehydes are often used to maintain and tighten tissues but these treatments are highly toxic. Genipin has been proved to increase mechanical properties of collagen and gelatin. Li et al. used genipin to crosslink a gelatin coated 45S5 bioglass scaffolds with high porosity (N 90%). They showed
26-fold higher compressive strength for the coated scaffold compare with un-coated scaffolds [18]. Genipin, extracted from Gardenia Jasminoides fruit, crosslinks polypeptide structure through nucleophilic attack of amine I located on lysine and arginine residues to the C3 atom of genipin, causing gel strength comparable to glutaraldehyde, while genipin is 10,000- fold less cytotoxic. After crosslinking, the color of gelatin changes to blue, and when exited at 590 nm, emits fluorescence at 630 nm [19]. There are three main methods for making fibrous scaffolds, electrospinning (ES), phase separation and self-assembly [20,21] ES is an affordable method to make fibrous structures of nano and microscale diameters, with very high specific surface areas. Specifically, the
Fig. 1. DSC-TGA of a. 45S5 sub-micron BG fibers and b. copper containing 45S5 sub-micron BG fibers.
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diameter of the fiber produced by ES is usually more than a few hundred nanometers [20,22]; however, these fibers are often called “nanofibers”. Nanofibers have different applications in tissue engineering and these purposes are achieved by controlling the three-dimensional structure and chemical/physical properties of the fibers. Most organs and tissues such as bone, skin, tendons and cartilage formations are highly organized fibrous structures [20,21,23]. Traditional methods for constructing porous bio-polymer scaffolds include gas foaming, freeze drying, solvent casting, two-phase and particulate leaching. Through these methods, a wide range of architecture and geometry settings with interconnected pores are created. Compared to others, freeze-drying is the simplest method for making a porous scaffold [22]. The diameter of the pores in the scaffold may be uniformed by freeze-drying method. Molecular weight of components and materials can severely affect the rate of biodegradation of these scaffolds [24]. A number of studies have been conducted to produce fibrillar BG mimicking the ECM, in which sol–gel derived BG nanofibers were produced by ES method [25,26]. and nanocomposite scaffolds composed of nanofibrillar BG and reconstituted collagen were developed for bone regeneration matrix [27]. In this study, we prepared a novel composite scaffold by combining BG fibers (with and without Cu) within gelatin-collagen hydrogel. We designed a composite including sub-micron fibers inside a polymeric matrix of gelatin and collagen which mimics natural bone structure. Although, the selected composition for the prepared scaffold is not exactly the same as bone, but it is not so far from natural bone composition, as the organic phase, gelatin is known as the denatured structure of collagen; and as the mineral part, BG fibers will turn to apatite fibers or at least fibers with apatite coating after being exposed to body environment. In this study by using Human osteoblast-like cell line SaOS-2, cell growth and viability were investigated and compared in three scaffolds; gelatin-collagen hydrogel scaffold, composite scaffolds containing 45S5 BG fibers and composite scaffolds containing Cu-doped 45S5 BG fibers. 2. Materials and methods
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the precursor through stirring. The following reagents were added separately after 45 min, during stirring, in the following sequence: 2.9 ml triethyl phosphate, 20.13 g calcium nitrate tetrahydrate and 13.52 g sodium nitrate [10,28], in the case of Cu-45S5 19.84 g calcium nitrate tetrahydrate and 0.61 g Cu(II) nitrate trihydrate. The sol mixtures were stirred for 36 h and aged without stirring at 25 °C for 24 h, followed by a further 24 h at 40 °C. Prior to ES the 12% wt. solution of PVA was prepared, and mixed with sol-solution in ratio of 1:1.5. 5 ml of this solution was loaded in syringe and under controlled condition (voltage: 20 kV, distance: 10 cm, and injection rate: 1 mL/h) ES process was done. The sub-micron fibers were subsequently heat-treated at 700 °C for 1 h in air with a heating rate of 4 °C/min and cooling rate of 5 °C/min. The heat-treatment temperature was determined to be high enough to eliminate organic sources and nitrates completely [10,28]. 2.1.2. Fabrication of composite hydrogel Porous GEL/COL composites and porous GEL/COL/BG fibers (45S5 and Cu containing 45S5) composites were fabricated as follows: At first, collagen/gelatin solution with the ratio of 1:9 (Merck, microbiology grade, catalog number 104,070) was prepared, for this purpose, collagen was added to deionized water containing 50 mM acetic acid, and gelatin was added to deionized water in a stirrer associated with heating. After 1 h, collagen and gelatin solutions were mixed to make a 10% (w/v) solution. In the case of GEL/COL/sub-micron BG fibers (45S5 and Cu containing 45S5) composites the ratio of GEL/COL/BG fibers was 70:30% [29]. In the next step, the fiber was mixed with hydrogel through gentle vortexing. Then the prepared hydrogel and the composite hydrogel were poured into a cylindrical mold and kept at 4 °C for 2 h until physical gelation occurred. Afterward, the gel was extracted and kept at − 20 °C for about 24 h. The resultant composite was extracted and freeze dried to create a porous structure. Scaffolds were incubated in a 0.5% genipin solution for 16 h. Samples were washed with ethanol, PBS and deionized water to remove remnants of the genipin. The provided scaffolds were kept at a dry place.
2.1. Fabrication and characterization of composite scaffold
2.2. Scaffold characterization
2.1.1. Fabrication of sub-micron BG fibers The sub-micron 45S5 BG fibers and Cu containing sub-micron 45S5 BG fibers are composed of: (45 wt% SiO2, 6 wt% P2O5, 24.5 wt% CaO, 24.5 wt% Na2O) and (45 wt% SiO2, 6 wt% P2O5, 23.5 wt% CaO, 1 wt% CuO, 24.5 wt% Na2O) (TEOS, Si(OC4H9)4, 99.99%, Sigma Aldrich), triethylphosphite (P(OEt)3, P(C2H5O)3, 99.5%, Sigma Aldrich), calcium nitrate tetrahydrate (Ca(NO3)2?4H2O, 99.60%, Sigma Aldrich) and sodium nitrate (NaNO3, 100.40% Sigma Aldrich). Gel-derived 45S5 and Cu containing 45S5 were prepared as follows: initially 33.5 ml tetraethyl orthosilicate (TEOS) was added to 1 M nitric acid, with H2O: TEOS molar ratio equal to 18; to prepare the required amount of 1 M nitric acid solution, 3.26 mL of 69% nitric acid was mixed with 47.6 mL distilled water. The solution was allowed to react for 60 min for hydrolysis of
2.2.1. Differential scanning calorimetry and thermogravimetric analysis (DSC-TGA) DSC-TGA analysis was used to determine the transition temperature and mass loss of the samples. It was applied on the sol-derived 45S5 and Cu containing 45S5 sub-micron fibers before stabilization. This test was accomplished to assess the transformations and their temperatures including weight loss from ambient temperature up to 1000 °C with the heating rate of 5 °C/min. 2.2.2. Mercury porosimetery To measure the distribution of prepared scaffold pores, porosity percentage and mean value of pore diameter, mercury porosimetery was used.
Fig. 2. Scanning electron microscopy (SEM) micrograph of the fibers before heating. a. 45S5 sub-micron BG fibers, b. 45S5 sub-micron BG fibers containing Cu.
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Fig. 3. Scanning electron microscopy (SEM) micrograph of the fibers after heating at 700 °C. a. 45S5 sub-micron BG fibers, b. 45S5 sub-micron BG fibers containing Cu.
In this method mercury penetrates through pores with various pressures in an autoclave instrument, the smaller the pores are, the higher the pressure will be. With mercury porosimetry not only the total porosity can be measured, but also the distribution of the pores can be measured. Mercury porosimetry method is able to distinguish a wide range of pore sizes depending on the Hg pressure applied on the sample, the lower pressure senses bigger pores. In this case, we applied pressure between 0.1–400 kPa which can show pore size ranging from 3 nm to 200 μm. In this study Finnigan mercury intrusion porosimeter PASCAL140 (Germany) was used. In this method the sample was dried initially and then exposed with mercury inside high vacuum chamber. Mercury pressure was changed incrementally from 0.1 to 336 KPa. Pore diameter in which mercury infiltrates are determined by Washburn equation [L = γDt/4η]. Where, L: infiltrated mercury volume, γ: surface tension, D: diameter of pores, T: Time of mercury infiltration through pores, μ: mercury viscosity. 2.2.3. Scanning electron microscopy (SEM) The morphology of sub-micron fibers and composite hydrogel scaffolds was assessed by scanning electron microscopy (SEM; Philips XL30 microscope). The images were obtained using an accelerating
voltage of 15 kV. All samples were coated with a gold sputtering device, before being investigated under the SEM. The obtained images were used to evaluate the sub-micron BG fibers morphology and pore size of the composites. 2.3. Structural analysis 2.3.1. Fourier transform infrared spectroscopy (FTIR) The functional groups of composite samples and the sub-micron BG fibers were examined by FTIR with Bomem MB 100 spectrometer. For IR analysis, at first 1 mg of the powder samples were carefully mixed with 300 mg of KBr (infrared grade) and palletized under vacuum. Then the pellets were analyzed in the range of 400–4000 cm−1 at a scan speed of 23 scan/min with 4 cm−1 resolution. 2.3.2. X-ray diffraction (XRD) Crystallographic properties of synthesized BG fibers were assessed using X-ray diffraction method. For this aim, X-ray diffraction patterns of fibers were recorded on a Philips X-ray diffractometer with Co-Kα radiation (λ = 1.78901 Å). The scans of the selected diffraction peaks were carried out in step mode (step size 0.02°, measurement time 1 s, measurement temperature 25 °C, and standard: Si powder).
Fig. 4. Scanning electron microscopy (SEM) micrograph of the composite hydrogel, a. gelatin/collagen scaffold, b. gelatin/collagen/45S5 fibrillar BG, c, d gelatin/collagen/45S5 fibrillar BG containing Cu.
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Fig. 5. Porosimetry of the composite scaffolds, a. gelatin/collagen scaffold, b. gelatin/collagen/45S5 fibrillar BG, c. gelatin/collagen/45S5 fibrillar BG containing Cu.
Crystallographic identification of the phases of synthesized fibers was accomplished by comparing experimental XRD patterns to standards compiled by the International Center for Diffraction Data (ICDD).
2.4. Cells attachment Human osteoblast-like cell line SaOS-2 was used to evaluate in vitro cytocompatibility of the composite scaffolds. These cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and streptomycin/penicillin 100 U/ml (1%). For seeding, the cells were trypsinized (0.05% trypsin/0.53 mM EDTA in 0.1 M PBS without calcium or magnesium), centrifuged and resuspended in complete culture medium. Medium containing 3 × 105 cells were then seeded on top of each scaffold sample before they were immersed in the medium. Cells/scaffold constructs were incubated for 4 days at 37 °C in a humidified atmosphere containing 5% CO2. Then, the samples were washed twice with PBS before fixation. For fixation, samples were soaked in 2.5% glutaraldehyde for 1 h. Post-fixation was performed in 1% osmium tetroxide, followed by dehydration in a series of graded acetone solutions. The samples were freeze-dried and
kept dry with silica gel. Finally, they were kept in a hood for air drying and then used for SEM observation [30]. 2.5. MTT assay Cell viability was analyzed using 3- [4, 5-dimethylthiazol-2-YL] -2, 5 diphenyl tetrazolium bromide (MTT) assay on days 1, 2 and 4. For this test 5 mg/ml MTT solution was prepared by dissolving the MTT powder (Sigma) in warm PBS (37 °C) [31]. SaOS-2 cell line (1 × 104 cells/well) were cultured in 96 well (NUNC, Denmark), then incubated for 24 h. 20 μ L of MTT solution was added to each well for 4 h. Then Dimethylsulfoxide- 99.5% (DMSO, Sigma) was added to each well and the plate was shaken for 5 min with stirring. The absorbance at 570 nm was measured by Elisa Reader. 3. Results and discussion 3.1. Differential scanning calorimetry and thermogravimetric analysis (DSC-TGA) DSC and TGA analysis of sub-micron fibers were performed to obtain the right heat treatment temperature. Fig. 2 shows the DSC-
Fig. 6. XRD patterns of sol-derived 45S5 sub-micron BG fibers and copper containing 45S5 sub-micron BG fibers after heating at 700 °C and composite scaffolds containing fibrillar BG.
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Fig. 7. FTIR spectrum of composite scaffolds before and after crosslinking with genipin.
TGA curves obtained from the sol-derived 45S5 sub-micron fibers between ambient temperatures to 1000 °C. DSC curve includes both endothermic and exothermic peaks. The first endothermic peak, which initiated at 75 °C, corresponds to the release of physically adsorbed water, which was not removed during drying. TGA trace shows that all water and products from the polycondensation reaction were removed between 75 and 160 °C (12% weight loss). The other two endothermic peaks, starting at 350 °C (41% weight loss), are related to pyrolysis reaction of free organic species and/or release of water from the further condensation of Silanol and P-OH group and PVA burning out. All nitrates were removed at 560 °C (a further 22% of the total weight loss), so the total weight loss was 61%. In addition, two peaks at 350 °C and 582 °C may correspond to the decomposition of nitrate and hydrocarbon molecules respectively. Above 700 °C no significant weight loss was observed (Fig. 1a), this curve confirmed that the residuals can be removed before 700 °C, thus at this temperature the structure will be completely stabilized. Cu was incorporated as Cu2+ in the 45S5 fibrous BG network. Samples containing Cu have thermal patterns similar to fibers containing 45S5 BG, although a sharper peak is observed in 350 °C in Cu containing BG, Further, Cu decreases the glass transition and lowers the melting onset temperature (Fig. 1b). Endothermic peaks correspond to the removal of sodium nitrite and other nitrogen compounds. On the other hand, the exothermic peak indicates the formation of a crystalline phase and phase transformation. The best stabilization temperature, with high bioactivity and removal of all the nitrogen contents is about 700 °C according to a previous study [28].
3.2. Morphological characterization by SEM In this study, morphology of the sub-micron fibers and composite scaffolds were observed using SEM. Low and high magnification SEM micrographs taken from the surface of the composites are shown in Figs. 2–4. Fig. 2 shows photographs taken from the sub-micron fibers before heating at 700 °C, with SEM, as shown in this figure, the diameter of the fibers was between 150 and 450 nm. Fig. 3 shows the sub-micron BG fibers after heating, the approximate diameter of these fibers are 150–450 nm. Obtained glass sub-micron fibers represented coarse unsmooth morphology on the surface and showed fusion to each other after thermal treatment. The surface morphology of the porous composite scaffolds is shown in Fig. 4, as shown in this figure, a network of interconnected pores is shown. This scaffold has a pore diameter in the range of 70–250 μm, which is optimal for bone cell growth [29,30]. The incorporation of submicron BG fibers in composite hydrogel structure can be seen in the micrograph shown in Fig. 4b and d.
3.3. Mercury porosimetery Total porosity and pore distribution were investigated with mercury porosimetery method. The results showed that the porosity percentage of the three prepared scaffolds fell in range of 70–80% and size distribution of the pores was in the range of 10–200 μm for three scaffolds (Fig. 5a-c). The average pore size for the GEL/COL, GEL/COL/BG fibers and GEL/COL/Cu-BG fibers were 72 ± 20, 129 ± 24 and 82 ± 22, respectively. Considering the length of the osteoblasts and the length of mesenchymal stem cells (MSCs), it seems that the achieved distribution of porosity in the samples were enough for MSCs and osteoblasts penetration and immigration into the structures of these composite scaffolds [32–34]. 3.4. XRD analysis crystallographic properties of sub-micron BG fibers Sub-micron BG fibers were analyzed using XRD. Fig. 6 shows diffractograms obtained from XRD for the glass fibers samples. The diffractogram has weak peaks representing a type of amorphous or semi-crystalline nature. These peaks can be ascribed to combeite (marked in this figure), according to the ICCD database. BG in the forms of scaffold and powder were also characterized by X-ray diffraction, These BGs are composed of two sodium calcium silicate phases including combeite (Na2Ca2Si3O9) and silicorhenanite (Na2Ca4(PO4)2SiO4). Chen QZ et al. [35] reported that formation of Na2Ca2Si3O9 phase is favorable because it confers the scaffolds mechanical strength and furthermore. They also showed that this strong crystalline phase transforms into biodegradable and bioactive amorphous calcium phosphate after being implanted in the body [35]. Fig. 6 shows the XRD patterns obtained from different types of BGs. Addition of Cu into the BG structure did not affect the formation of
Table 1 Correlation of peaks detected by Fourier transform infrared spectroscopy (FTIR) for prepared scaffold [10,28,36,39]. Wavenumber (cm−1) ~ 463 ~ 939 ~ 1088 ~ 1663 ~ 1546 ~ 1240 ~ 3300
Assignment
Material
Si-O-Si bending Si-O stretching Si-O-Si stretching (asymmetric) amide I (C = O stretch) amide II peak (N-H bend and C-H stretch) amide III (C-N stretch plus N-H in phase bending) (N-H stretching vibration)
characteristic of silicate network in BG characteristic of gelatin and collagen in scaffolds
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Fig. 8. MTT assay analysis for cell culture on scaffold after 24, 48 and 96 h.
sodium calcium silicate phases, which has been reported previously in the literature [15]. Also, silicorhenanite phase that has been detected in this analysis was isostructural to apatite.
3.5. FTIR analysis Fig. 7 shows the FTIR spectra, in the 500–4000 cm−1 spectral range, for plain gelatin/collagen, cross-linked gelatin/collagen, composite containing heat-treated 45S5 BG fibers and composite scaffold containing Cu ion before and after crosslinking with genipin. FTIR spectra of plain gelatin/collagen exhibited a number of characteristic spectral bands such as: C = O stretch at 1662.1 cm−1 for amide I, N-H bend and C-H stretch at 1546 cm− 1 for amide II, C-N stretch plus N-H in phase bending at 1244 cm−1 for amide III, N-H stretching vibration at 3298 cm− 1 for the amide, which are the distinguishing features of
gelatin and collagen. In cross linked hydrogel we see similar peaks with a little shift as previously reported [36]. All these characteristic bands are also present in the spectrum obtained from composite scaffolds. In addition, composite samples containing 45S5 fibrillar BG and Cu ion containing BG exhibit three infrared bands located at: 464, 940 and 1084 cm−1, which are related to the silicate network and attributed to the Si-O-Si bending vibration, Si-O stretching vibration and asymmetric stretching vibration of Si-O-Si, respectively [28]. Correlation of the peaks, achieved by FTIR, is presented in Table 1. It should be noted that FTIR analysis shows a peak related to chemical bonds formed by the combination of fibrillar BG with hydrogel and subsequent crosslinking with Genipin. The peak is located at about 1334 cm−1 and indicates formation of the chemical bond between carboxyl groups of hydrogel and Ca2+ ions of fibrillar BG, same result has also been mentioned in former studies for Gelatin and hydroxyapatite [37,38].
Fig. 9. Scanning electron microscopy (SEM) micrograph of the cultured cells on composite scaffolds.
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3.6. MTT assay As shown by MTT assay (Fig. 8), scaffolds did not have any negative effects on the proliferation rate of osteoblasts compared with plastic cell culture surfaces. MTT assay was carried out at days 1, 2, and 4 on gelatin-collagen hydrogel and composite scaffolds including fibrillar BG 45S5 and Cu containing fibrillar BG 45S5. No significant differences were observed between composite scaffolds containing Cu-doped fibrillar 45S5 BG and control group in all the mentioned times, which indicates that the scaffolds had no toxicity. In the first 24 h gelatin-collagen hydrogel and composite containing fibrillar 45S5 BG had no toxicity, but after 48 h a significant reduction was observed in the viability of the cells seeded on the hydrogel scaffold, and the scaffold had inhibited the cell growth, but again after 96 h an increase in the cell growth was noticed. In the case of composite containing fibrillar BG 45S5, the results were the same after 48 and 96 h. (p b 0.05). For the scaffold containing fibrillar Cu-doped BG no toxic effects were observed. 3.7. Cell adhesion and proliferation on the scaffold SEM pictures of osteoblast cells were obtained after culturing the cells on the prepared scaffolds. As shown in Fig. 9, osteoblast cells have penetrated into scaffold pores and have completely widespread on the surface of the pores, the filopodia of the cells are also well observed in the images. In the gelatin-collagen hydrogel micrographs cell adhesions are observable. In the composite scaffolds the cells are attached to the scaffold and the cells are expanded, indicating that prepared biomimetic scaffolds could provide suitable environment similar to ECM for the osteoblast cells growth. Review on the research done in the field of fabrication of hydrogel based composite scaffolds reinforced by a ceramic compound shows that use of ceramic fibers as reinforcing phase are one of the novel techniques which has been less studied till now [27]. In addition, previously used calcium phosphate phase like Hydroxyapatite [3,9] has been replaced by bioactive glasses and other silicate based ceramics [35]. The possibility of different elements addition into the structure of bioactive glasses provides the chance of study on the potential effects of these elements in biological environments [11]. According to the above explanations and the fact that the effects of copper have been less investigated in tissue engineering applications, in this study we aimed to use cu-containing bioactive glass fibers to reinforce a hydrogel based scaffold. The results showed that cu-containing bioactive glass fibers can be obtained through described method and together with hydrogel basis can be processed toward making porous bone tissue engineering scaffold with a suitable structure. 4. Conclusion In this study three types of scaffolds were fabricated including, gelatin-collagen hydrogel, gelatin-collagen composite containing submicron 45S5 BG fibers, and Cu-doped composites. Sub-micron BG fibers were fabricated by combination of sol–gel and electrospinning processes, the fibers were then mixed with hydrogel matrix containing gelatin and collagen, freeze dried, followed by genipin crosslinking to fabricate final composite scaffolds. Growth and viability of human osteoblast-like cell line SaOS-2 were investigated on these biomimetic scaffolds. Cellular biocompatibility assays illustrated that scaffolds containing Cu ion in the BG structure had more viability and osteoblast growth in comparison with other scaffolds, the scaffolds containing Cu ion in the 45S5 BG network showed better biocompatibility compared to hydrogel and composite scaffold containing the 45S5 BG. Cu ion increased growth of the osteoblasts on composite scaffold containing sub-micron fibers BG compared to hydrogel without sub-micron fibers BG. Obtained results showed that the prepared scaffolds, especially Cu-doped BG fibers containing scaffold is
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