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Materials used in the study were Chitosan poly [D-glucosamine] powder form from shrimp shells [medium molecular weight, deacetylated chitin 75-85%, viscosity 200-800 cP], Sigma-Aldrich company [St. Louis, MO, USA]; 448877-50G, Bioglass, laboratory of faculty of chemistry, Rennes university1, France; Acetic acid [C2H4O2] [99.8%], Sds company, France [Pyongtack, Gyeonggi, South Korea]; P0070515 LOT: P0E022250E, Sodium hydroxide [NaOH], Sds company, France [Pyongtack, Gyeonggi, South Korea]; Simulated body fluid [SBF], laboratory of faculty of chemistry, Rennes university1, France; and Potassium bromide [KBr] from Sigma-Aldrich corporation [St. Louis, MO, USA], CASNº: 7758-02-3.
The equipment utilized was Lyophilizer, Bio block Christ, Alpha 1-2 LD plus, Sciquip. Co. UK; incubator, Memmert, Single DISPLAY, Universal oven UN / UF / UNplus / UFplus, German; Panalytical XPERT PRO powder diffractometer, D 8 Advance, Karlsruhe, Germany; Fourier transformed infrared spectroscopy [FTIR], Bruker Equinox 55Corporation, International Equipment Trading [IET], Vernon Hills, Illinois 60061 USA; mercury intrusion pore sizer, Model: 9320 V2.08, Micrometrics Inc., USA; and scanning electron microscope [SEM], Jeol company JSM 6301, Japan.
2.2.2. Chitosan /Bioacive Glass Composite Scaffold PreparationChitosan solution as natural polymeric material was prepared from medium molecular weight chitosan powder that was extracted from shrimp shells [MW= 480.000 and degree of acetylation [DA]= 85%]. The chitosan powder was dissolved on a magnetic stirrer at room temperature in a 1% acetate solution. A produced homogenous chitosan solution was poured into custom made cylindrical Teflon molds [10mm diameter×10mm thickness] for obtaining the chitosan scaffold [C] composition. The same chitosan solution was again elaborated as a polymeric dispersion medium for compositional preparation of different proportions of composite scaffolds, where the bioactive glass was gradually added as a dispersed phase.
A thermally induced phase separation [TIPS], i.e., Freeze-drying technique, was implemented in order to elaborate C as well as biocomposite scaffolds with four different compositional proportions of chitosan [C]/bioactive glass 46S6 [M], which was prepared in the laboratory by melting method. Those fabricated four other compositional scaffold groups were C, 1C:1M, 1C:2M, and 2C:1M by weight. Finally, the scaffolds were dried inside an incubator adjusted at 37°C before proceeding to their physicochemical characterization.
2.3. Physicochemical Characterization of the Fabricated Chitosan-based ScaffoldsA factorial design was performed for the physicochemical characterization tests of the constructed chitosan-based scaffolds, where n=five. The differently prepared biocompo- sites [C, 1C:1M, 1C:2M, and 2C:1M] were investigated with the aid of XRD analysis and Fourier transformed infrared spectroscopy [FTIR] for detection of phases and molecular structures of prepared scaffolds, respectively. In addition, microstructural analyses of those scaffolds were accomplished using a scanning electron microscope [SEM] to study their external and internal micro-morphologies.
2.3.1. X-ray Diffraction Analysis [XRD]XRD patterns of the various fabricated chitosan-based scaffolds [C, 1C:1M, 1C:2M, and 2C:1M] were achieved to identify the existing crystalline phases in the constructed bio compositions and to track the alterations that might develop in the structural characteristics of those biomaterials. As the pure 46S6 bioactive glass, the XRD pattern of pure hydroxyapatite was essentially established. A Panalytical XPERT PRO powder diffractometer was used with wide-angle [WA] XRD patterns for analysis of the different synthesized biocomposite scaffolds. The scaffolds XRD were performed using Cu Kα radiation and operated at an electrical voltage of 40 kV at room temperature. The scaffold XRD patterns were investigated at angle 2 ϴ with a range of 5-60 º, scanned at a speed of 2º/ min., and data of the XRD analysis were computed based on Bragg’s equation [20]:
n λ = 2 d sin ϴ
Where, n = an integral number
λ = wave length
d = interplanar spacing
ϴ = diffraction angle
2.3.2. Fourier Transformed Infrared Spectroscopy [FTIR]FTIR identified functional groups of elaborated different biocomposite scaffold compositions [C, 1C:1M, 1C:2M, and 2C:1M] and the intermolecular interaction between the components in each scaffolding system. For each prepared scaffold composition, two milligrams of powder were mixed with 198 mg KBr [potassium bromide] powder press to give 1% concentration, which was suitable for obtaining proper IR transmission spectral curves. The mixtures were then subjected to 8 tons /cm2 load to get the required discs with a resolution of 2 cm-1. FTIR collected spectra were detected to be ranging between 400 and 4000 cm-1.
2.3.3. Scanning Electron Microscopic Analysis [SEM]All chitosan scaffolds [C, 1C:1M, 1C:2M, and 2C:1M] were coated with a gold-palladium layer for the examination of surface morphology as well as microstructure of the different scaffolds using the scanning electron microscope [SEM].
2.3.4. Porosity MeasurementMercury intrusion pore sizer [MIP] was used as a porosimeter to evaluate the 3D pore structure of the different synthesized [C, 1C:1M, 1C:2M, and 2C:1M] scaffolds. Mercury had a contact angle with the specimen's material being more significant than 90°.
2.3.5. Steps of the Porosity TestingBefore placement in the glass bulb of the penetrometer, the scaffolds were weighed [Ws]. Applying a low-pressure cycle, the capillary stem and the space around the specimen were filled with mercury [Hg]. Mercury was then gradually forced into the specimen's pores during the high-pressure cycle, in which the pressure was raised up to 207 MPa [30.000 psia]. The Hg volume penetrating into the pores was identified by monitoring the level of the receding Hg column in the capillary stem while raising the pressure [21]. Afterward, envelope [Bulk] density, apparent density, and percent porosity of the prepared different scaffold compositions were calculated as follows:
Scaffold’s envelope [Bulk] density [ρse] was determined by dividing the initial scaffold’s weight [Ws] by the scaffold’s envelope volume [Vse], which was the total volume of the specimen, including the volume of its open pores. At the end of the low-pressure cycle, Vse was obtained by subtraction of the volume of Hg present in the penetrometer [VHg] from total penetrometer volume [Vp]. Envelope density for each scaffold composition was calculated from the following equation:
ρse= Ws/ Vse = Ws/ [Vp - VHg][22]
Where, ρse; specimen's envelope density
Ws: specimen’s weight
Vse: specimen’s envelope volume
Vp: total penetrometer volume
VHg: volume of mercury in penetrometer.
Scaffold’s apparent density [ρsa] was identified by dividing the initial scaffold’s weight [Ws] by its apparent volume [Vsa], which was the volume of the scaffold per se after excluding the volume of its open and interconnected pores. Then, the scaffold's apparent volume was identified by subtracting the volume of Hg that filled the scaffold's connected pores [V] from the specimen's envelope volume [Vse]. At the end of the high-pressure cycle, Hg volume penetrating into the open pores [V] corresponded to the volume of Hg that receded from the capillary stem. Thus, the scaffold's apparent density was determined from the following equation:
ρsa = Ws / Vsa = Ws/ [Vse– V] [22]
Where, ρsa: scaffold’s apparent density
Ws: scaffold’s weight
Vsa: scaffold’s apparent volume
Vse: scaffold’s envelope volume
V: volume of mercury into open pores.
Percent porosity of each scaffold was calculated from envelope density [ρse] and apparent density [ρsa] according to the following equation:
Percent porosity = 100 [22]
Where: ρse: scaffold’s envelope density
ρsa: scaffold’s apparent density.
In order to determine the scaffold's pore volume distribution [i.e., incremental intrusion versus pore diameter], the Hg pressure was gradually increased until the high-pressure cycle aimed to force the Hg into the scaffold’s open pores. Washburn equation [23, 24] correlated the entry diameter of the intruded pore [D] with the applied pressure that forced Hg into that pore [P]:
D = - 4 µ cos θ / P
Where, D: entry diameter of scaffold pore
µ: surface tension of Hg [485 dynes/cm] [23]
θ: mercury’s contact angle on scaffold’s surface [130o] [24]
P: applied pressure
Since both surface tension and Hg’s contact angle were constant, each pressure value corresponded to a pore diameter calculated from the Washburn equation. Then, an incremental intrusion versus diameter curve was plotted, describing the distribution of the total pore volume distribution over the pore diameter range and determining at which diameter the pore volume was mostly concentrated [22].
2.3.6. Compressive StrengthAt room temperature, uniaxial compression tests were carried out for all scaffold compositions [C, 1C:1M, 1C:2M, and 2C:1M] [thickness ~3 mm and diameter ~8 mm] by a computer-controlled Universal testing machine at a static load cell of 5 KN and at a crosshead speed of 1mm/min until failure. Obtained data were recorded through computer software. Moreover, the maximum failure load of each scaffold was recorded in N, and the compressive strength value was computed from the peak load record divided by scaffold specimen surface according to the following equation [25]:
Compressive strength [CS] = 4P/πd2
Where, P: load [in N] at the fracture point
d: diameter [in mm] of the cylindrical specimen
Compressive strength values of different scaffolds were collected, computed, and were calculated in MPa, and then statistically analyzed as means ± standard deviation [SD].
2.3.7. Ex- vivo Biodegradation in SBF for the Different Chitosan-based ScaffoldsEx vivo biodegradation for all scaffold biomaterials [C, 1C:1M, 1C:2M, and 2C:1M] was investigated by immersing each scaffold individually in a simulated body fluid [SBF] solution. In the chemical laboratory, freshly prepared SBF solution was elaborated by the homogenous dissolution of NaHCO3, NaCl, K2HPO4.3H2O, KCl, CaCl2, and MgCl2.6H2O in de-ionized water. Afterward, the elaborated SBF solution was buffered using Tris-buffer [CH2OH]3 CNH2 and HCl [6M = 6mol/L] to adjust the pH at 7.4; therefore, the composition of the prepared SBF solution was simulating that of human blood plasma Table 1 [26].
Table 1.Composition of the freshly prepared SBF solution in comparison to that of human blood plasma.
Ionic concentrations [10-3mol.L-1] Na+ K+ Ca+2 Mg+2 Cl- HCO-3 HPO4-2 SBF 142 5 2.5 1.5 148.8 4.2 1 Plasma 142 5 2.5 1.5 103.0 27.0 1 2.3.8. Statistical AnalysisNumerical data of the elaborated study were presented in the form of means and standard deviation [±SD] statistical values. The One-way ANOVA test was implemented for comparison between the different scaffolds [C, 1C:1M, 1C:2M, and 2C:1M]. Repeated measures ANOVA test was applied to investigate the time-dependent changes within each scaffold. Tukey’s post-hoc test was applied for pair-wise comparisons, whenever the ANOVA test was found significant. Kruskal-Wallis test was carried out for comparison between the fabricated scaffolds. Mann-Whitney U test was performed for studying pair-wise scaffold comparisons, when obtained; the results of the Kruskal-Wallis test were found to be significant. Friedman’s test was also used to monitor the changes within each scaffold over time. Moreover, Wilcoxon signed-rank test with Bonferroni’scorrection was implemented for pair-wise scaffold comparisons in case of significant Friedman’s test findings. The significance level was defined at P ≤ 0.05. Statistical analysis was conducted using IBM [IBM Corporation, NY, USA] SPSS[SPSS, Inc., an IBM Company] Statistics Version 20 for Windows.
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