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Thursday, 23 February 2017
Fabrication of highly porous biodegradable biomimetic nanocomposite as advanced bone tissue scaffold
Published Date
Arabian Journal of Chemistry February 2017, Vol.10(2):240–252, doi:10.1016/j.arabjc.2016.09.021 Open Access, Creative Commons license, Funding information Original article
Author
Abdalla Abdal-hay a,b
Khalil Abdelrazek Khalil c,d,,
Abdel Salam Hamdy e
Fawzi F. Al-Jassirf,g
aDept. of Engineering Materials and Mechanical Design, Faculty of Engineering, South Valley University, Qena, Egypt
bDept. of Bionano System Engineering, College of Engineering, Chonbuk National University, Jeonju 561-756, Republic of Korea
cDepartment of Mechanical Engineering, College of Engineering, University of Sharjah, P.O. Box 27272, Sharjah, United Arab Emirates
dDept. of Mechanical Engineering, Faculty of Energy Engineering, Aswan University, Aswan, Egypt
eDept. of Manufacturing and Industrial Engineering, College of Engineering and Computer Science, University of Texas Rio Grande Valley, 1201 West University Dr., Edinburg, TX 78541-2999, USA
fFRCSC, College of Medicine, King Saud University, Riyadh 11461 Saudi Arabia
gOrthopedic Surgery Research Chair, King Saud University, Riyadh 11461 Saudi Arabia
Received 24 April 2016. Accepted 20 September 2016. Available online 11 October 2016.
Abstract Development of bioinspired or biomimetic materials is currently a challenge in the field of tissue regeneration. In-situ 3D biomimetic microporous nanocomposite scaffold has been developed using a simple lyophilization post hydrothermal reaction for bone healing applications. The fabricated 3D porous scaffold possesses advantages of good bonelike apatite particles distribution, thermal properties and high porous interconnected network structure. High dispersion bonelike apatite nanoparticles (NPs) rapidly nucleated and deposited from surrounding biological minerals within chitosan (CTS) matrices using hydrothermal technique. After that, freeze-drying method was applied on the composite solution to form the desired porous 3D architecture. Interestingly, the porosity and pore size of composite scaffold were not significantly affected by the particles size and particles content within the CTS matrix. Our results demonstrated that the compression modulus of porous composite scaffold is twice higher than that of plain CTS scaffold, indicating a maximization of the chemical interaction between polymer matrix and apatite NPs. Cytocompatibility test for MC3T3-E1 pre-osteoblasts cell line using MTT-indirect assay test showed that the fabricated 3D microporous nanocomposite scaffold possesses higher cell proliferation and growth than that of pure CTS scaffold. Collectively, our results suggest that the newly developed highly porous apatite/CTS nanocomposite scaffold as an alternative of hydroxyapatite/CTS scaffold may serve as an excellent porous 3D platform for bone tissue regeneration. Graphical abstract
Keywords
Tissue engineering
Nanocomposite scaffold
Bioactive materials
Bioinspired materials
1 Introduction
Clinically, the gold standard for bone defect repair is the use of autografts and allografts. Autografts are bone grafts, which are taken from a donor site, such as the iliac crest or fibula, implanted to a recipient site during surgery. However, limited supply and donor site morbidity is a major concern. Other factors, e.g. infection, immune rejection, and pathogen transfer caused by allografts have also been reported (Hutmacher, 2000 and Sánchez-Salcedo et al., 2008). Tissue engineering scaffold, which acts as an extracellular matrix (ECM) to interact with the living human cells, seems to be an alternative platform for bone defect repair and bone regeneration. It has been generally accepted that the chemical composition and physical structural properties of scaffolds are major concerns in fabricating ideal three-dimensional (3D) porous composite scaffold for bone tissue regeneration applications (Wu et al., 2014). In addition, the bone composite scaffold should possess good mechanical properties to overcome the collapse during handling and to well-match those tissues that need to be replaced and increase the cell functionality at the site of implantation (Shimojo et al., 2016). It should also overcome the particles agglomeration within the polymer matrices (Abdal-hay et al., 2014a) and within the pore walls and cavities (Wu et al., 2014 and Das et al., 2016). In fact, NPs agglomeration results in weaken molecular interaction and poor dispersion which can affect negatively the porous structure, NPs loading capacity, and the mechanical properties of the resultant composite scaffold, in addition to unfavorable cellular responsiveness (Zhao et al., 2002, Li et al., 2007 and Abdal-hay et al., 2014a). Incorporation of nano-scaled inorganic bone minerals (such as, hydroxyapatite, HA, Ca10(PO4)6(OH)2) by conventional direct-mixing was found to significantly decrease the pore size and porosity of the scaffolds, particularly, in case of large HA content (Mohammad et al., 2015, Omran et al., 2015 and Esfahani et al., 2016).
Hence, in more specific, bone tissue engineering scaffolds should possess sufficient porosity which plays a critical role in cell seeding, proliferation and new tissue formation in 3D (Halake et al., 2014, Nasiri-Tabrizi et al., 2014 and Wu et al., 2014). 3D composite scaffolds with high porosity (such as ⩾90%) and well-interconnectivity are required to minimize the amount of implanted polymer and to increase the specific surface area for cell attachment and tissue regeneration (Maghdouri-White et al., 2016), facilitating a uniform distribution of cells and adequate transport of nutrients and metabolic waste products. Specific cells require 3D scaffolds with interconnected high porosities for optimal attachment and growth (Ranucci et al., 2000 and Wu et al., 2014). The big challenge of the present approach is to fabricate polymeric matrices containing bonelike NPs as advanced nanocomposite platform with uniform particles size, particles distribution, high porosity and good pore interconnectivity.
Currently, there are two major simple and common methods, which can be used to create porous tissue scaffold including, electrospinning and self-assembly (Ramadass et al., 2014 and Chen et al., 2016). Although numerous attempts have been paid to apply these common methods in clinical settings, these methods are still lacking the specific properties of uniform dispersion of inorganic NPs within the 3D porous substitute fabricated scaffolds as well as low interfacial bonding between the organic molecules and inorganic NPs. For example, electrospinning is limited to create only two-dimensional (2D) porous composite mat in addition to non-uniform distribution of deposited HA NPs within the polymer matrix (Armentano et al., 2010). The most reported self-assembled scaffolds were made from gels, which lack mechanical integrity and are not adequate structural tissue. Compared to aforementioned methods, our study offers the possibility of optimization of the 3D porous architecture of organic-inorganic (bonelike), biocomposite scaffolds in bone tissue regeneration in relation to the specific cells and tissues and the convenient preparation of such 3D porous scaffolds by a simple freeze-drying post hydrothermal nucleation of apatite NPs. In this paper, a new methodology is presented for the preparation of porous 3D polymeric composite with desirable scaffold requirements and a homogeneous distribution of biological apatite NPs as an alternative to HA within the polymeric matrix may offer novel insights in the field of bone implants development. The scaffold has been developed using hydrothermal route (based on convection theory in heat transfer) to rapidly nucleate and homogenously distribute the apatite NPs within polymeric matrix in a short time (3 h). A commonly used method for preparation of 3D porous scaffolds, known as lyophilization (freeze-drying) was exploited. Preliminary results of nucleation and in situ deposition of biological apatite NPs within polymer matrix by controlling hydrothermal processing conditions have recently been reported by our group (Abdal-hay et al., 2014a). The fabricated scaffold possesses the advantages of mineralized NPs homogeneous dispersion, and subsequently high chemical interfacial bonding between the NPs and CTS matrix is achieved. The hydrothermal reaction could rapidly in situ nucleate and uniformly distribute the bonelike-apatite NPs within the CTS matrix as well as maximize the electrostatic attraction forces between the apatite inorganic particles and polymer organic matrices.
The present study systematically examined the effects of concentration of composite suspension (concentration of mineralized bonelike-apatite ions) on phase composition, pore size, pore wall morphology, porosity, water absorbance capacity, thermal and mechanical characteristics of the composite scaffolds and these properties were compared with plain CTS 3D scaffold. The ability of the nanocomposite apatite/CTS scaffold to promote MC3T3-E1 osteoblasts cell growth was also investigated.
2 Experimental procedures
2.1 Porous nanocomposite scaffolds preparation
Chitosan from crab shells with a degree of deacetylation (>85%) and acetic acid (HAc, 99.8%) was supplied by Sigma-Aldrich Co., Ltd. Two different CTS porous scaffolds were prepared by controlling the volume fraction of water in the solution system. 3.0 g from CTS polymer was dissolved in 75 and 150 mL aqueous HAc. Bonelike apatite NPs were in situ nucleated and subsequently precipitated from the surrounding simulated body fluids in the presence of CTS molecules. A comparison between ion concentration in SBF solution used in the present study and the human blood plasma is illustrated in Table 1.
Table 1. Ion concentrations of simulated body fluid (SBF) solutions used in the present study and Human blood plasma (Cüneyt Tas, 2000).
Ion
Human blood plasma (mM)
SBF in present work (mM) (1 × SBF)
SBF in present work (mM) (3 × SBF)
Na+
142.0
142.0
426.0
Cl−
103.0
125.0
375.0
HCO3−
27.0
27.0
81.0
K+
5.0
5.0
15.0
Mg2+
1.5
1.5
4.5
Ca2+
2.5
2.5
7.5
HPO42−
1.0
1.0
3.0
SO42−
0.5
0.5
1.5
The reagent grade chemical composition of 1 × SBF solution used to nucleate biological compounds (bonelike apatite) was dissolved in 1.0 l distilled water in sequentially until complete dissolution of each salt in the following order: NaCl (6.547 g/l), NaHCO3 (2.268 g/l), KCl (0.373 g/l), Na2HPO4 2H2O (0.178 g/l), MgCl2·6H2O (0.305 g/l), CaCl2 2H2O (0.368 g/l), Na2SO4 (0.071 g/l), (CH2OH)3CNH2 (6.057 g/l) (Cüneyt Tas, 2000 and Chen et al., 2006). The pH of SBF solution was adjusted with HCl (25 ml/l) and NH2C(CH2OH)3 to 7.54 at 37.5 °C to mimic physiological conditions before the addition of CaCl2. To prepare composite scaffolds with higher apatite concentrations (with no more than 3 times SBF concentration as recommended by Kokubo which induces a maximum growth rate (Kokubo, 1996)), 3 × SBF salts grade chemicals were dissolved in one litter distilled water. For 3 × SBF solution, the grade chemicals were prepared by sequentially dissolving CaCl2, MgCl2. 6H2O, NaHCO3, and K2HPO4. 3H2O in distilled water. Solution pH was lowered to 6 by adding HCl to increase the solubility of the salts. Na2SO4, KCl and NaCl were added until the previous solution becomes transparent. The pH of the solution was adjusted with 1 M NaOH or 1 M HCl to 7.54 at 37.5 °C.
25 ml from 1 × SBF and 3 × SBF solution was added separately to 25 ml of CTS solution to obtain two 50 ml apatite/CTS composite solutions. After magnetic stirrer mixing of the two solutions for 10 min, the 50 ml of apatite/CTS solution was transferred to a Teflon®-lined autoclave container and heat-treated at 110 °C for 3 h in an oven. The nanocomposites and pure CTS solutions were then used to fabricate a porous (3D) scaffold. After the reaction process, apatite/CTS solution was first cast in a Petri dish polypropylene container. Then, the casting solution which still contains a small quantity of solvent, was lyophilized at −20 °C for 20 h, rinsed with diethyl ether, and then dried under reduced pressure at room temperature for 24 h to completely eliminate the solvent and obtain the apatite/CTS nanocomposite 3D porous scaffolds as illustrated in Scheme S1 of supplementary materials. The apatite/CTS composite scaffolds with the two-apatite grade concentrations, 1 × SBF and 3 × SBF, were naked as 1-apatite/CTS and 3-apatite/CTS composite scaffold, respectively. All the prepared samples were reserved under dry conditions.
2.2 Characterization
XRD patterns of different samples were recorded at ambient temperature on an X’pert Pro® Diffractometer (PW3050/60, Philips, Holland). Samples were irradiated with a monochromatized Cu Kα (1.54056 Å) X-ray source at a scanning rate of 0.02°/min (2θ). Operating voltage and current used were 40 kV and 30 mA, respectively, with a beam size of 20 mm. Average crystal grain sizes (t) were estimated using the Scherrer equation (Eq. (1)) (Choi et al., 2004), assuming spherical and stress free crystals:
equation1
where B is the half width of the characterized peak, θB is the maximum intensity angle, λ is the wavelength (Cu Kα = 1.5059 Ǻ), and t is the crystal size. The coexistence of the inorganic Ca-P phase and organic CTS molecules was also investigated using Fourier transform infrared spectroscopy (FTIR, Nicolet Avatar 380) in transmission mode and the spectra were recorded over the range of 4000–400 cm−1. The Ca/P ratios in the as-prepared composite porous scaffolds were measured using JEOL JSX 3222 elemental analyzer. TGA-DSC, Q-20 Perkin-Elmer Inc., USA instrument system was used to determine the melting temperature (Tm) and the melting enthalpy (ΔHm) of the fabricated samples. For the DSC tests, sample sizes ranged from 5 to 10 mg and the melting curves were recorded from 0 to 250 °C scanned at a heating rate of 10 °C min−1.
The pore size of the scaffolds was measured by fully automated Capillary Flow Porometry (CFP, 1200-AE capillary flow porometer, PMI Inc., Ithaca, NY, USA) as previously described (Ju et al., 2010). Briefly, the scaffolds were cut in circular shape (Diameter 30 mm) to place inside a sample holder. Then, the prepared disk specimens were saturated with wetting liquid of known surface tension. Once the specimen was wetted, the specimen holder was screwed tightly. Next, using instrument software, the starting pressure (lowest pressure) and the total ending pressure were set. The lowest pressure and the highest pressure (the ending pressure) determine the relative pore size of the scaffold. Finally, the pore sizes of the tested fibrous scaffolds were calculated by the software equipped with the CFP machine by applying the following equation (Eq. (2)) Ranucci et al., 2000:
equation2
where D is the pore diameter; is the wetting liquid surface tension; θ is the contact angle of the wetting liquid and P is the differential pressure. The porous morphology of plain and composite scaffolds was examined using a scanning electron microscope (SEM, Hitachi S-7400, Japan) at an accelerating voltage of 15–20 kV. SEM specimens were sputter-coated with platinum to avoid discharge. The surface topography (2D and 3D images) of the cross section of composite scaffolds was observed by atomic force microscopy (AFM, MultiMode/BioScope, CJ10). Topographic and phase images were recorded simultaneously with a standard silicon tip on a cantilever beam.
2.3 Water absorption capacity
The water absorption properties of the 3D fabricated porous scaffolds were tested according to the previous demonstrated method (Ramadass et al., 2014). Briefly, the pure and composite 3D porous scaffolds were dried in oven at 40 °C overnight to get constant weight (dry weight, W0) and subsequently, the dried scaffolds were immersed in Petri dishes containing same amount of distilled water. The 3D scaffolds were withdrawn at different immersion times as follows: 3, 8, 15, 30, 45 and 60 min, and the samples were placed on clean tissue to allow drain off any excess water. After that, the scaffolds were weighted and considered as wet weight (W1). The water absorption percentage (W) of the 3D porous scaffolds was then calculated with the following equations (Eq. (3)) and the average value (W%) was obtained from five parallel specimens.
equation3
2.4 Mechanical properties
The compression properties of 3D microporous plain and apatite/CTS composite scaffolds were measured using an Instron mechanical tester (Lloyd Instruments, LR5Kplus, UK). Specimen thickness was measured using a digital micrometer with a precision of 1.0 μm. Compression properties of pure and composite samples were obtained under the following conditions: sample length between jaws, 15 mm; width, 10 mm; cross head speed, 1 mm min−1; and test temperature, 22 °C. The initial cross section was used to calculate the compression strengths and compression moduli. Four specimens were tested and their values were averaged for each scaffold.
2.5 Cytotoxicity test
The cytotoxicity properties of the fabricated pure and composite scaffolds were evaluated using a method described in reference (Abdal-hay et al., 2013). Briefly, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St Louis, MO, USA) assay was used which was prepared in phosphate buffered saline (PBS) at a final concentration of 5 mg/ml. Extracts of different materials were prepared using Dulbecco’s Modified Eagle’s Medium (DMEM; Invitrogen, Carlsbad, CA, USA) serum-free under condition of 37 °C/120 r/min, according to a standard ratio of 0.2 g/ml of culture medium. Then, the supernatant was withdrawn and centrifuged to prepare the conditioned extracts before the cytotoxicity test. MC3T3-E1 cells, osteoblast-like cell line (American Type Culture Collection (ATCC, Manassas, VA, USA) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA) and 1% penicillin/streptomycin (P/S; Gibco, Grand Island, NY, USA) under a physiological condition (37 °C; 5% CO2; 95% relative humidity). MC3T3-E1 cells were seeded in 24-well plates with a cell density of 10 × 103 cells per ml of the medium and incubated for 1-day in a humidified incubator at 37 °C and 5% CO2 to ensure proper cell adhesion. The medium was subsequently replaced with the extracts as well as with the negative control (cell culture medium alone) and the MC3T3-E1 cell lines were incubated again under physiological conditions for 3, 5 and 7 days. At each cultured time, 50 μl of MTT solution and 450 μl of a-MEM containing FBS and antibiotic were added to each well. After 4 h of incubation, the medium was aspirated, and 350 μl DMSO was added to each well to dissolve the formazan. Then the absorbance was recorded at a test wavelength of 570 nm and a reference wavelength of 630 nm (Benchmark™ microplate reader, BIO-RAD, California, USA), whereas, reference wavelength in colorimetry is a correction wavelength. Then, the optical density (OD) was recorded from the obtained results. The cell number was determined with a standard curve. The relative growth rate of the cells was calculated as a function of negative control according to previous study (Abdal-hay et al., 2013). The cell morphology and extent of cell growth after the stipulated period of cell culture followed by crystal violet staining were assessed using an inverted phase contrast microscope. The data obtained were evaluated for statistical significance using the Student’s t-test. The results are reported as mean ± standard deviation (SD), and the differences observed between composites results were considered significant when p < 0.05.
3 Results and discussion
3.1 Structure and composition of prepared composite scaffolds
Apatite or bonelike has an excellent biocompatibility due to their close chemical and crystal resemblance to bone mineral (Rezwan et al., 2006). A common characteristic of synthesized bioactive apatite particles is a time-dependent complete nucleation and precipitation of its minerals that occurs upon implantation. Several factors, such as temperature and ion concentration, could influence the nucleation and the formation of the apatite crystals. In our previous report (Abdal-hay et al., 2013), we studied the influences of temperature on growth rate of bonelike apatite deposited onto polymer fibers. Herein, our concern is to investigate the variable concentrations of inorganic ion of simulated body fluid (1 and 3 times SBF, Table. 1) on the growth rate and formation of the bonelike minerals within CTS matrices at constant elevated temperature. XRD spectra, FTIR profiles and Elemental analysis were used to investigate and confirm the coexistence, complete nucleation and deposition of apatite inorganic phase within the CTS organic matrix at different apatite contents.
XRD analysis of the prepared apatite/CTS nanocomposite and plain CTS scaffolds is shown in Fig. 1A. For plain CTS scaffold, the natural state of CTS is a semi-crystalline biopolymer, with broad diffraction peaks at 2 − θ = 19.8° (curve 1 of Fig. 1A). The existence of 2θ peaks at approximately 31.7° (the strongest intensity peak), 46.1° and 56.4° corresponds to the diffraction planes (2 1 1), (2 0 3) and (3 2 2) of the apatite structure, respectively, confirming the formation of the bonelike apatite phase structure within the CTS matrix (Ghahremani et al., 2013 and Rusu et al., 2005). After the formation of apatite/CTS composites, the crystalline characteristics of the CTS’s constituents cannot be observed. Absence of respective typical crystalline peaks and formation of the sharp and strong apatite peaks even at low apatite concentration is the clear evidence of formation of apatite/CTS composites (1-apatite/CTS scaffold, curve 2). This can also be documented by the nucleation and growth of the apatite phase structure via CTS matrix. Accordingly, not all diffraction planes were clearly identified due to the initial peak overlap and hydrothermal nucleation of the apatite phase and due to the molecular interactions between apatite compounds and CTS molecules (Ghahremani et al., 2013, Zhang et al., 2008 and Abdal-hay et al., 2014a). It was also noted that peaks in the precipitated 3-apatite/CTS scaffold were sharper and stronger compared to the 1-apatite/CTS scaffold, implying the growth of a Ca-P phase in a higher biological mineral content within the CTS matrix. The XRD peaks, (2 1 1), (2 0 3) and (3 2 2) were shifted to lower 2θ values in case of 3-apatite/CTS, composites compared to 1-apatite/CTS (curves 2 and 3). This could be due to compression from the contracting CTS matrix through interfacial bonding as explained in our previous report (Abdal-hay et al., 2013). The shift and change in crystallinity of each peak of the polymer or apatite in composite scaffolds clearly indicate the presence of bonding between apatite inorganic particles and polymer organic molecules. The peak at (2 1 1) plan of apatite is strengthen at higher concentration, which also indicates the further growth of apatite particles within CTS matrix. High concentrations of Ca-P ions enhance the condition of super-saturation within CTS matrix, thus avail more nuclei of apatite within the polymer matrix.
Figure 1. (A) XRD profiles, (B) FTIR spectra and (C) DSC curves of pure and apatite/CTS composite scaffolds.
However, the macromolecule phase analysis using IR of the prepared composite compact film scaffolds (free porosity) has been previously investigated (Abdal-hay et al., 2014a); meanwhile, the phase composition and interfacial bonding of the as-prepared 3D porous plain and composite scaffolds at different apatite concentrations were examined using IR spectra. From IR results, Fig. 1B, it was found that the characteristic bands at 1645 cm−1 correspond to hydroxyl group, OH, vibration and distinctive bands at about 435 cm−1 and 550–600 cm−1 for phosphate vibrational bands, , were noticed, consistent with the presence of apatite phase within CTS matrix. In addition, the 1645 cm−1 band of pure CTS scaffold was slightly shifted to lower value after incorporation of apatite NPs at higher concentration. This indicates that strong interfacial bonding was created between the organic molecules (CTS) and inorganic NPs (apatite). The results also are in good agreement with previous reports, whereas the symmetrical bending vibration of the carboxylic groups (–COO−) is located in the region of 870 cm−1. This indicates the precipitated apatite phase contained few carbonate ions, which were derived from carbon dioxide in the air. The different concentration of apatite mineral phases deposited within CTS matrix can be distinguished from one another (Fig. 1B) by the relative intensity, position and shapes of the related bands in the Raman spectra. These XRD and FTIR results support the coexistence of both apatite and CTS components in the composite 3D porous scaffolds. From the results of XRD and FTIR, it can be deduced that the induced apatite was a carbonated HA (Kong et al., 2006).
Further, the crystal size of apatite/CTS nanocomposites does not show significant change, indicating the complete nucleation of the apatite phase at lower apatite concentration. This was consistent with the theoretical calculations in which the crystal grain sizes for 1-apatite/CTS and 3-apatite/CTS composite scaffolds, within the nanocomposites, should be 23 nm and 26.2 nm, respectively. These nanocrystal biomimetic apatite particles are closer to that in the natural bone in dimension than the apatite particles formed in the classical process (Wu et al., 2014). XRD patterns of the two composite samples indicate that the method of preparation has a crucial influence on apatite nucleation and precipitation within CTS matrix. Our previous analysis (Abdal-hay et al., 2013 and Abdal-hay et al., 2014a) showed that the main elements of the synthesized composite film were carbon, oxygen, calcium, phosphorus, magnesium, and sodium. Carbon and oxygen could have originated from both CTS and the bonelike apatite, but the other elements could only have formed from the bonelike apatite. These results suggest that the in situbiomineralization approach could completely nucleate and precipitate all elements of biological apatite during hydrothermal reaction in 3 h. Thus, using hydrothermal approach results in a rapid nucleation of the bonelike apatite phase within the CTS matrix in a short time. In addition, on the basis of these data, it is suggested that amorphous Ca/P is present within the CTS treated scaffold.
Elemental analysis was carried out to investigate the effect of Ca and P compounds concentration within the CTS matrix of the two composite scaffolds (1-apatite/CTS and 3-apatite/CTS) and the results are summarized in Table. 2. The results present that the loading amount of Ca and P nucleated and deposited within the CTS matrix is completely proportional to biological apatite concentration in the composite. However, the contribution of Ca-P is increased within the matrix as the concentration of SBF increased; but, Ca/P ratio is not the same of stoichiometric hydroxyapatite (HA) (Tang et al., 2009 and Wu et al., 2014). This difference between Ca/P ratio (Table. 2) obtained from biomineralization in situ rapid synthesis process and stoichiometric HA is because the composition of bone mineral is much more complex and contains additional ions such as, Mg, Zn, K and Co3− (Abdal-hay et al., 2014a and Wu et al., 2014). Thus, mimicking the bone mineral composition is of prime concern in order to elicit specific cellular responses and provide an ideal environment for bone formation. In addition, it was observed that with the increase of SBF minerals concentration, the amount of inorganic particles, which were nucleated and deposited within the CTS matrix increased correspondingly. This preliminary work shows how the ions concentration of SBF significantly influences the rate of apatite formation within CTS matrices in a biomimetic solution. These observations can confirm that the nucleated and deposited compounds within CTS matrices are bonelike apatite. Du et al. (Maghdouri-White et al., 2016) suggested that better osteoconductivity would be accomplished if synthetic bioceramic material could resemble bone minerals more in composition. A possible explanation of the nucleation and formation of biological apatite NPs has already been reported previously (Wu et al., 2014).
Table 2. Results obtained for element composition of the composite samples.
The thermal properties of the as-prepared scaffolds were investigated by the DSC curves. Fig. 1B shows the exothermal temperature of plain CTS scaffold at 103.4 °C, which was shifted toward a higher temperature than that of 1-apatite/CTS (106.3 °C), and a lower temperature than that of 3-apatite/CTS (99.2 °C) composite scaffolds. It was noticed that the peak intensity and melting area were increased in the composite porous scaffold of lower apatite amount than that of higher apatite amount. It is likely that lower apatite content and smaller particle size (higher surface area) can maximize the interaction bonding between apatite NPs and CTS molecules. This favorable bonding between inorganic particles and organic matrix may improve the mechanical characteristics of the composites. In addition, the problem of migration of inorganic NPs can also be readdressed (Rezwan et al., 2006). This observation shows that the crystallinity of apatite/CTS composite scaffold is sufficiently higher compared to plain CTS porous scaffold. The melting enthalpy of the plain scaffold increased from 339.4 J/g to 443.5 J/g when a low apatite amount was applied into the CTS matrix, which indicates that the bonelike apatite NPs and fabrication process induced crystallinity changes in the polymer (Baji et al., 2010). Indeed, it can be concluded that the interaction between the CTS matrix and apatite is sufficiently strong.
3.3 Fabrication of porous polymeric composite scaffold
The porosity of the porous polymeric scaffolds could be well controlled by changing the concentration of the solvent-polymer systems. The water content in the solution system has a remarkable effect on the pore size and pore morphology of plain CTS scaffold (Ramadass et al., 2014 and Esfahani et al., 2016) and the resulting 3D architecture. Based on the present findings, two different proportions of water content in CTS solution were used to obtain plain CTS scaffolds with different pore structures. Fig. 2A and B, shows a remarkable difference in the morphologies of CTS scaffolds fabricated with different contents of water. This confirms that the proportion of water in the CTS scaffold had a remarkable effect on the pore size formation and overall morphology of the CTS scaffold. At a high water content (3.0 g of CTS was dissolved in 150 ml aqueous solution), the average pore size and porosity significantly increased from 33 ± 18 μm and 45% ± 3.48 to about 250 ± 41 μm and 95% ± 1.5 and random pore structure replaced with the leaf-like pore structure. The pore structure morphology of matrices generated from the plain polymeric solution using freeze-drying process often is not uniform throughout the matrix due to degassing of the water content and a subsequent rich phase (pore wall) and poor phase (porous structure) are formed (Hutmacher, 2000). Another remarkable change was the texture of pore wall surface. Rezwan et al. in their review showed that at least 90% porosity and 100 μm pore size are known to be compulsory for cell penetration and a proper vascularization of the ingrown tissues (Rezwan et al., 2006). Taking into account that larger pore size is desired in tissue regeneration, therefore the sample with large water content would be considered for further investigation in the present study in order to incorporate biological apatite NPs at different concentrations within the CTS matrix.
Figure 2. Scanning electron micrographs of; (A) pure CTS at high water content, (B) pure CTS at low water content, (C) 1-apatite/CTS and (D) 3-apatite/CTS composite porous scaffolds.
The method of fabricating porous polymeric composite structures by precipitation of apatite NPs within CTS matrices homogeneously using a hydrothermal reaction and subsequent freeze-drying under control parameters was applied to form 3D porous structures which overcomes the disadvantages of the previous methods (Hutmacher, 2000). A multilevel freeze-drying technique was employed to transform the apatite/CTS nanocomposites into 3D porous scaffold. In the present work, we studied the influence of the precipitated apatite NPs within the CTS matrix on porosity deformation performance. 3D porous composite scaffolds with a larger pore sizes and porosities were successfully prepared. The results obtained from the cross section area of composite scaffolds with two apatite NPs contents (Fig. 3A and B) indicate that nano-sized apatite nanoparticles were distributed homogeneously within the pore walls of the CTS matrix. The diameters of apatite NPs deposited within CTS matrix are about 39.7 ± 11.16 and 260 ± 45.22 nm for 1-apatite/CTS and 1-apatite/CTS composite scaffolds, respectively. In addition, the apatite particle sizes deposited within CTS matrix are uniform suggesting a new approach for preventing the particles agglomeration (Abdal-hay et al., 2014b).
Figure 3. Scanning electron micrographs of fracture surfaces of resultant composites: (A) 1-apatite/CTS and (B) 3-apatite/CT.
The surface topography of composite scaffolds cross section in 2D and 3D topographies (Fig. 4A and B) was investigated. The images display dispersed bonelike apatite nanoparticles in the CTS matrix. At low apatite concentration (1-apatite/CTS), the scaffold has small NPs embedded within polymer matrix, Fig. 4A. With further increase in apatite concentration (3-apatite/CTS), the scaffold possesses coarse NPs with uniform size and good distribution, which contributes the further nucleation (assembled of the NPs) of apatite particles within CTS matrix, Fig. 4B. It is expected that the further nucleation of apatite allows the particles to rearrange uniformly beside each other and deposited within the polymer chain of CTS in a layer-by-layer structure. This is likely due to high specific surface energy of apatite particles during hydrothermal reaction resulting in their arrangement layer by layer within the polymer chain of CTS (Wu et al., 2014). It seems that the deposition of apatite particles layer by layer within the CTS matrix results in inhibition of aggregates and agglomerates within the polymer matrix, which is in agreement with previous study (Abdal-hay et al., 2013). This result suggests that the particle size of biomimetic apatite can be controlled by the biopolymer chain in composite as depicted in the AFM micrograph, (Fig. 4). Based on the present findings, to add various contents of apatite particles within CTS and to make diversity and from these achieved results, it would be understood that the place is where maximum value of apatite particles nucleated and deposited within polymeric chain. This is clear from the growth of particles, whereas the particle sizes are almost larger due to the increasing values of apatite (Kokubo, 1996). In addition to the perfect homogeneity of NPs, they are also shown to be well embedded in the pore wall layer where there are no voids or structure defects observed within the pore wall structure (Fig. 2C and D). The pore walls are composed of homogeneous inorganic NPs and organic matrices, compared with conventional biomedical 3D porous scaffolds prepared from either polymers or inorganic bioceramic NPs, or mechanically mixture of these two phases (Rezwan et al., 2006). Accordingly, our results present a new approach for fabrication of defect-free, 3D porous scaffolds structure with well-interconnected pore using controlled freeze-drying method. Unidirectional freezing and strict degassing of the composite solution system are necessary to prohibit cracking and void formation in the resulting porous structure (Varshosaz et al., 2012). In addition, shrinking velocity during fabrication process of the composite porous scaffold might induce a positive effect and could be controlled with the incorporation of inorganic NPs within polymeric matrix. Higher inorganic particles contents induce lower shrinking velocity and subsequently, cracking could be avoided. Fig. 2C and D shows anisotropic microtubules morphology with an internal ladder like structure. The resultant structures possess pores of irregular shapes varying from 190 to 240 μm in size. The maximum pore size obtained by the Hou et al. (Hou et al., 2003) was about 100 μm. The 1-apatite/CTS and 3-apatite/CTS composite scaffolds have high porosity of about 93 and 91%, respectively, which is considered beneficial for cell ingrowth and survival. The large pore size with well-interconnectivity fabricated in the present study using lyophilization post hydrothermal reaction is desired in bone tissue engineering, as the exchange of cell waste products and nutrients is easily transferred and could enhance cell viability (Gentile et al., 2016). Channels morphology was formed and found to be parallel to the direction of solidification (heat transfer direction). Each channel has repeating partitions with uniform spacing perpendicular to the solidification direction. Incorporation of apatite NPs did not show significant effect on the porosity properties indicating that the apatite NPs is well-dispersed within the CTS pore wall. This demonstrates that nano-sized apatite particles are distributed uniformly within the pore walls of the scaffolds and do not appear to aggregate in pores.
Figure 4. AFM images of (A) 1-apatite/CTS and (B) 3-apatite/CT composite scaffolds.
3.4 Water absorption capacity study of prepared scaffolds
The water uptake capacity of plain CTS and apatite/CTS composite scaffolds is displayed in Fig. 5. As shown from the obtained results, all scaffolds showed fast water absorption within 15 min from the immersion starting time. The water absorption uptake of the scaffolds was ranged from 150% to 720%, which indicated that the pores structure and composition of the 3D scaffold were the mechanism for water uptake capacity (Ramadass et al., 2014). More specifically, composite scaffolds induced higher water uptake in comparison with pure CTS scaffold. This contributes that the as-prepared scaffolds could facilitate the entrapment of water in the scaffold matrix. In addition, the deposition of apatite NPs could contribute to increase water absorption capacity, despite the composite scaffold possesses lesser porosity. It is hypothesized that the swelling behavior of CTS composite scaffold is excellent which may be due to the presence of a large number of narrow pores which entrap and hold water through capillary action (Ramadass et al., 2014). Collectively, the samples could adsorb and desorb same amount of water by immersion or applying force on scaffold’s surface, respectively.
Figure 5. Water absorption capacity of the as-prepared 3D porous scaffolds at different immersion times. Inset shows 2-D optical image of the scaffold during immersion test.
3.5 Effect of the bonelike apatite NPs on the mechanical properties
The nanometer size of the inorganic bonelike apatite in natural bone is considered to be critical to the mechanical properties of the bone (Rho et al., 1998). In the composite scaffolds, the presence of biological mineral compounds within nested CTS interconnected porosity enhance the compressive modulus and compressive strength of the as-obtained scaffold via matrix reinforcement (Pant et al., 2011). Selected compressive stress-strain curves of pure and composite porous scaffolds are shown in Fig. 6 and their average values are listed in the table inset of Fig. 6. While assessing the mechanical properties of plain CTS scaffold, we observed that the addition of apatite significantly increased the compressive strength of composite scaffolds. These results are consistent with our previous results (Abdal-hay et al., 2013), in which the addition of inorganic nanofillers was found to enhance the mechanical properties when compared with pure polymeric material. Herein, we found that the compressive properties of apatite/CTS samples depend on the concentration of apatite NPS within the porous scaffold. Fig. 6 shows the mechanical properties measured for apatite/CTS composites with the two different apatite concentrations, where the strength exhibited decreases with the increasing apatite concentrations (from 3.15 to 2.52 MPa). Despite this effect, apatite/CTS composite porous scaffold lasted longer than that of plain CTS scaffold (1.45 MPa). The increased compressive strength provides evidence that the precipitated apatite NPs interacted well within pore wall of CTS matrix. Furthermore, these results confirm that the excellent dispersion of NPs within the polymer matrix has a remarkable effect on the improvement of the mechanical properties of the porous composite scaffolds. The highest mechanical properties (particularly, the compressive modulus) was obtained from the samples of low apatite content, inset of Fig. 6. However, the scaffold with low apatite NPs loading possesses larger pore size and porosity. Gibson and Ashby (Gibson and Ashby, 1999) illustrated that the compressive properties (compressive modulus) of the polymeric scaffolds decrease with increasing pore size and porosity. However, in the present case, the dependence of the compression modulus on the pore size is negligible. This further verifies the good distribution of NPs and well interconnectivity of the pore network of the composite scaffolds fabricated by this strategy. A close combination of the nanosized apatite NPs and organic matrices can enhance the retention of mechanical properties, and maintain a tissue space of prescribed size and shape than polymer scaffold, which has enough flexibility, but lack of sufficient mechanical resistance. The smaller particle size of 1-apatite/CTS compared to that of 3-apatite/CTS composite porous scaffold (Figure 2 and Figure 4) provides further benefits for the enhancement of mechanical properties. In addition to aforementioned benefits, a greater interface and connection with the matrix play a crucial role on enhancement the mechanical properties. Hence, the composite scaffold 1-apatite/CTS scaffold has good interfacial bonding between apatite nanofillers and CTS matrices and possesses higher porosity, which resulted in noteworthy higher mechanical properties when compared to that of 1-apatite/CTS composite scaffold. Accordingly, our study provides a novel insight toward optimizing the 3D microporous composite scaffold for a desired bone tissue engineering application by controlling the bioactive bioceramic particles distribution, the porosity, and the mechanical properties. Overall, composites of apatite/CTS composite porous scaffolds appear to possess promising bone tissue clinical applications for future biomaterials.
Figure 6. Selected compressive stress-strain curves of the fabricated porous scaffolds. Inset shows the mean values and STD of compressive strength and compressive modulus of as-prepared scaffolds.
3.6 Cytocompatibility of the prepared scaffolds
A crucial factor in the success of a tissue scaffold, especially in bone tissue engineering, is the combination of physiochemical properties and biological activities of the implanted material, all of which play critical roles in cell seeding, proliferation, and new tissue formation. In tissue engineering, a large number of cells are always needed in order to produce enough extracellular matrices for the successful formation of neo-tissue constructs. Rapid screening of cell growth and proliferation using in vitro cytotoxicity studies is a common procedure followed in the evaluation of biomaterials. Fig. 7A shows the proliferation of MC3T3-E1 cells and preosteoblast cells after cell culture using indirect extract of pure CTS, 1-apatite/CTS and 3-apatite/CTS composite porous scaffolds for 3, 5 and 7 days. Our results showed that the cytotoxicity of pure and composite porous scaffolds could be classified non-toxic compounds to MC3T3-E1 cells as per ISO 10993-5 standard. All scaffolds fabricated in this study showed higher growth rate than that of negative control (Fig. 7A). The relatively lower growth rate observed for pure CTS porous scaffold suggests lower cell proliferation when compared to the apatite/CTS composite scaffolds. More specifically, the composite scaffold loaded with higher apatite NPs contents (3 times SBF) showed the highest cell density at longer culture of time, 7-days, compared to scaffold loaded with less apatite NPs. The cell morphology and the extent of cell growth after cell seeding for 7 days using indirect extract are illustrated in Fig. 7B–E. The morphological results are in accordance with MTT cell density. In general, it can be observed from these images that the cell morphologies in different scaffolds were normal and healthy, similar to that of the negative control, evidencing that the CTS based porous scaffolds have free toxicity with cells. It was also evident that the cell growth is relatively much better on apatite/CTS composite scaffolds, particularly at 3 times SBF concentration than the pure scaffold one. Hence, the presence of apatite phase has a significant influence on the cell growth rate in cell culture medium at different concentrations.
Figure 7. (A) Cytotoxicity evaluation as a function in cell density using MTT-assay of MC3T3-E1 cells after cell culture using indirect extracts for 3, 5 and 7 days (p < 0.05) and cell microscopical observation and the extent of cell growth on, (B) control cells, (C) pure CTS, (D) 1-apatite/CTS and (E) 3-apatite/CTS nanocomposite porous scaffolds after cell culture for 7 days indirect cytotoxicity test. Similar cellular morphology was observed in three independent experiments (n = 3). Scale bar = 20 μm.
Simulated body fluid (1 and 3 times SBF) was initially designed to test the bioactivity of artificial bone material in vitro because its composition is very close to human blood plasma. Wu et al. (Yu et al., 2009), reported the influence of biomimetic apatite/CTS composite scaffold on osteoblast viability and proliferation. In this study, the apatite precipitation on the CTS surface caused different cell responses. Hence, it is likely that the ionic dissolution from the inorganic compounds, particularly in concentration (3 × SBF), might play an important role in such cellular processes as bone cell growth and proliferation. This contribution may be related to the free calcium and phosphate ionic concentration levels in the apatite microenvironment, specifically at higher apatite NPs concentration, 3 × SBF (Chou et al., 2005). These relative influences are beyond the scope of the present preliminary study, which only suggests that different apatite loadings can induce different proliferation response under specified conditions. Accordingly, as explained previously, the materials chemistry is responsible for the observed behavior. Rice et al. (2003) investigated human pre-osteoblast behavior on Ti oxide and concluded that materials chemistry was playing an important role in osteoblasts response. Another plausible mechanism may involve the high porosity of the fabricated composite scaffolds which might help fast release of apatite compounds into the physiological medium and subsequently effects on cell responses (Abdal-hay et al., 2012). Experiments are underway to answer the question of whether these porous composite scaffolds-induced variations are significant on osteoblast differentiation in vitro and bone formation in vivo. The main element of apatite composition (Ca2+) at various levels has been reported to control every aspect of cell and tissue physiology (Demaurex and Distelhorst, 2003). This attributes to protein adsorption and subsequent integrin-mediated signaling with cells. The higher cell proliferation on 3-apatite/CTS scaffold may be attributed to the completion of NPs dissolution, or altered local microenvironment, such as the phase transformation of the synthesized apatite NPs to a more compatible apatite. These fluffy variations in the local microenvironment can result in changes in cell behavior. The likely thing exists that the variations may be partially regulated by calcium phosphate levels, in which case engineering of apatite microenvironment should balance osteoblast behavior. It is not known yet whether and how apatite concentration as well as the pore properties associated upregulation of osteoblasts growth and proliferation, and is currently being investigated. Based on the present findings, the results suggest higher cell proliferation to subtle changes in apatite/CTS nanocomposite microenvironment at higher apatite concentration, 3 × SBF.
Biomimetic approach could offer a practical method of controlling surface topography and chemistry within porous tissue engineering scaffolds. Therefore, an important factor for a successful implantation in orthopedic applications is considered a close contiguity between bone and the implanted surface, which is known as osteointegration. This is very important with regard to developing materials for bone repair or bone tissue engineering. Many materials are biocompatible but often require addition of growth factors and other regents to improve the cell response. Based on the results obtained from in vitro testing, it is believed that the incorporation of bonelike apatite NPs into the CTS matrix induced good cytocompatibility and enhances the development of the connections between CTS-based implant materials and natural bone. Although observationally this appears to be correct, further works are needed to confirm this idea.
4 Conclusion
A 3D highly porous and well-interconnected apatite/CTS nanocomposite scaffold was fabricated using an in situ biomineralization and subsequently lyophilization process. In the CTS system, scaffolds with trapped calcium ions enhanced the creation of the nucleation sites that interact with phosphate ions hydrothermally treated in SBF to produce bonelike apatite crystals. The effect of SBF concentration on the crystal growth within CTS matrix was investigated. This new fabrication strategy of 3D porous composite scaffold overcomes an important problem, which is the structural integrity of the structure during the direct-mixing of polymer solution and inorganic NPs. At high particle content and high porosities, the scaffolds are prone to agglomeration, i.e., weakness of the structures at high porosities. We successfully fabricated new defect-free, porous structures with well interconnectivity through careful control of the hydrothermal reaction and freeze-drying parameters of composite scaffolds. Herein, high porosity (above 94%) polymeric composite structures have been prepared, where the porosity and pore size are easily formed and are independently on the bonelike apatite NPs loadings within the polymer matrix. Our results showed that the incorporation of deposited apatite NPs layer by layer as a second phase in the CTS matrix significantly improved the mechanical properties as well as MC3T3-E1 cells growth rate of the CTS scaffold. The new strategy described in this paper might be of great interest in bone tissue engineering for treatment of bone that is defective, lost, or needs fixation. Fabrication of biodegradable and osteoconductive, organic-inorganic, nanocomposite materials combined with the high porosities, dispersion and interaction of inorganic NPs and adequate mechanical strength is a formidable challenge. The developed 3D highly porous apatite/CTS nanocomposite scaffold is strongly suggested as promising next generation biomaterials for bone tissue engineering and regeneration. We expect that the highly porous apatite/CTS composite scaffold may induce good bioactivity, and high cell ingrowth and proliferation. Accordingly, it is our belief that this new class of highly porous 3D composite scaffolds with their unique thermal and mechanical properties would have a great potential for biomedical applications. We suggest that the new 3D microtubular architecture fabricated in this study could serve as superior scaffolds for the bone tissue engineering. A detailed study about the morphology and structure as well as biological functions, such as cell differentiation, attachment and migration, of the fabricated scaffolds will be an emerging topic to be covered soon by our group.
Acknowledgment
The paper was financially supported by King Saud University, Vice Deanship of Research Chairs.
P. Gentile, V.K. Nandagiri, J. Daly, V. Chiono, C. Mattu, C. Tonda-Turo, G. Ciardelli, Z. Ramtoola
Localised controlled release of simvastatin from porous chitosan–gelatin scaffolds engrafted with simvastatin loaded PLGA-microparticles for bone tissue engineering application
J.M. Rice, J.A. Hunt, J.A. Gallagher, P. Hanarp, D.S. Sutherland, J. Gold
Quantitative assessment of the response of primary derived human osteoblasts and macrophages to a range of nanotopography surfaces in a single culture model in vitro
Biomaterials, Volume 24, Issue 26, 2003, pp. 4799–4818
Corresponding author at: Department of Mechanical Engineering, College of Engineering, University of Sharjah, P.O. Box 27272, Sharjah, United Arab Emirates. Tel: +971065052610.
is the wetting liquid surface tension; θ is the contact angle of the wetting liquid and P is the differential pressure. The porous morphology of plain and composite scaffolds was examined using a scanning electron microscope (SEM, Hitachi S-7400, Japan) at an accelerating voltage of 15–20 kV. SEM specimens were sputter-coated with platinum to avoid discharge. The surface topography (2D and 3D images) of the cross section of composite scaffolds was observed by atomic force microscopy (AFM, MultiMode/BioScope, CJ10). Topographic and phase images were recorded simultaneously with a standard silicon tip on a cantilever beam.
, were noticed, consistent with the presence of apatite phase within CTS matrix. In addition, the 1645 cm−1 band of pure CTS scaffold was slightly shifted to lower value after incorporation of apatite NPs at higher concentration. This indicates that strong interfacial bonding was created between the organic molecules (CTS) and inorganic NPs (apatite). The results also are in good agreement with previous reports, whereas the symmetrical bending vibration of the carboxylic groups (–COO−) is located in the region of 870 cm−1. This indicates the precipitated apatite phase contained few carbonate ions, which were derived from carbon dioxide in the air. The different concentration of apatite mineral phases deposited within CTS matrix can be distinguished from one another (Fig. 1B) by the relative intensity, position and shapes of the related bands in the Raman spectra. These XRD and FTIR results support the coexistence of both apatite and CTS components in the composite 3D porous scaffolds. From the results of XRD and FTIR, it can be deduced that the induced apatite was a carbonated HA (Kong et al., 2006).
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