4-1- Bio-composites of step 1
FE-SEM
FE-SEM micrographs of bio-composites prepared in step 1 are illustrated in Fig. 1. The larger FE-SEM images are shown in 10µm magnification and the images in circles are the 2µm magnification. As it is clearly depicted in the FE-SEM images, in low BG/C ratios fibril like structures related to bioactive glass were not clearly observed (Fig. 1a). The morphology of this sample is mostly governed by chitosan particles left after solvent evaporation. This topographic view is similar to other reported morphologies assigned to chitosan particles resulted after solvent evaporation methods [25]. By increasing BG/C ratio fibril like structures gradually began to appear (Fig. 1b and c) in an unordered pattern. In higher BG/C ratios (compounds 8 and 9 with 60 and 65 wt.% BG) the condensed fibril like structures demonstrate a compact ordered structure. Chitosan morphology is completely dependent to its preparation method [21,22]. Since the morphology of the compound 2 is mostly governed chitosan, the observed morphology is similar to the previous reports in articles.
Zeolite component does not play an important role in the morphology of the surface images due to its low percentage among other composite components. FE-SEM images of bio-composites containing higher amount of chitosan indicated rough surface changes.
EDS diagrams (Fig. 1) of the bio-composites confirmed the elemental existence of Ca, P, Al, Si, C in the surface composition of all samples, exhibiting the presence of BG, C and Z in the sample contents [26].
Figure 1- FE-SEM images and EDS diagrams of compounds a)2, b)3, c)5, d)8, and e)9
The particle size distribution from the surface of the bio-composites was investigated by the histogram obtained from FE-SEM images. According to the histogram (Fig. 2) the narrowest and the broadest particle size distribution belongs to compounds 3 and 9, respectively.
Figure 2- Histogram of compounds 2, 3, 5, 8, and 9
FTIR
FTIR spectra of nanocomposites prepared with changing BG/C ratio are illustrated in Fig. 3. The vibration wavenumber value of the bio-composites and components are listed in Table 3. Vibration bands at 478 and 1063 cm− 1 are related to the symmetric and asymmetric vibration bands of SiO4 tetrahedra [26,27]. As it is depicted in Fig. 3, by increasing the amount of BG in the bio-composite content, the intensity of these vibration band is increased. The observed shift in the symmetric vibration of Si-O-Si is attributed to the presence of Ca2+ and Na+ ions in the bioactive glass [28]. The Si-O-Si symmetric vibration band of pure silica appear commonly at 450 cm− 1 [29]. The vibration bond appeared at 478 cm− 1 is attributed to Si-O-Si vibration bending of silicate anion (SiO44−) indicating the presence of silicate with amorphous crystalline structure [30–32] Increasing the BG content in the bio-composite is presented by gradual decrease in the transmittance intensity of this band samples [30,33].
Chitosan and crosslinked chitosan have several main differences in FT-IR spectra that proved chitosan crosslinking. The vibration band at 1559 cm− 1 is assigned to N-H bending were observed in FT-IR of crosslinked chitosan but not in the FT-IR of chitosan [34]. The band at 1150 cm− 1 corresponds to C-O-C which presented in FT-IR spectra of chitosan while not found in FT-IR of crosslinked chitosan [34].
A broad band in 3500 − 3000 cm− 1 consisting of 3 main bands at ~ 3441, 3280 and 3180 cm− 1 are attributed to the –NH2 and –OH groups stretching vibrations in chitosan and natural zeolite [35]. This pattern in the FTIR spectra of samples matched with the presence of chitosan particles routed from the preparation procedure of chitosan in the composite [36]. The vibration bands at 1559 and 1420 cm− 1 are assigned to the N-H and C-N stretching of chitosan functional groups [34,35]. The stretching vibration of C = O appeared at 1645 cm− 1 and the band at 2910 cm− 1 is ascribed to C-H bond asymmetric and symmetric stretching [31,33,36]. By increasing the amount of BG, the vibration intensity of these bands decreases and the vibration intensity of Si-O-Si stretching at ~ 1063 increases [37–39]. The vibration band at 796 cm− 1 and 495 cm− 1 are attributed to Si-O/Al-O and P-O bonds, respectively [40–42]
Figure 3- FTIR spectra of a) components and b) bio-composites containing compounds 2, 3, 4, 5, 7, 8 and 9
Comparing the FT-IR spectra of the component (Fig. 3-a) and FT-IR spectra of the fabricated bio-nanocomposites (Fig. 3-b) confirmed the existence of bioactive glass, chitosan and zeolite in the manufactured bio-composites. FT-IR vibration bands of the components and the manufactured bio-composites indicated in the Table 3.
Table 3- FT-IR vibration bands of the components and the bio-composites
XRD
XRD patterns of initial components (C, BG and Z) are shown in Fig. 4. As it is illustrated in the XRD pattern of Chitosan, a broad peak at 20˚ exhibits a semi-crystalline structure [43]. Bioactive glass also possessed a broad peak characteristic of amorphous materials [42]. The XRD pattern of natural zeolite displays some characteristic peaks between 2θ of 10–40ᵒ [13]. The diffraction peaks of the zeolite could not be detected in XRD pattern of bio-composites because of the low quota of zeolite.
Figure 4- XRD pattern of pure BG, C and Z, the components of bio-composite
Figure 5- XRD patterns of prepared samples with different BG/C ratios
XRD patterns of the bio-composites prepared in step 1 are depicted in Fig. 5. As can be detected the broad peak related to chitosan appeared at 2θ ~ 20˚ gradually disappeared by decreasing chitosan amount in the bio-composite content. Reflections attributed to bioactive glass increased by rising BG/C ratio.
Mechanical Tests
Calculated amount of compressive strength, fractional strength and young modulus of prepared samples in the step 1 are listed in Table 4.
Table 4
Mechanical properties of the bio-composites prepared by different BG/C percentages.
Compound
|
BG/C percentage
(Constant 5wt% Z)
|
Young Modulus (MPa)
|
Fractional Strength (MPa)
|
Compressive strength (MPa)
|
2
|
30/65
|
78.83
|
18.62
|
28.37
|
3
|
35/60
|
102.39
|
102.39
|
23.8
|
4
|
40/55
|
82.06
|
82.06
|
19.92
|
5
|
45/50
|
83.64
|
83.64
|
30.11
|
6
|
50/45
|
622.0
|
22.18
|
56.35
|
7
|
55/40
|
153.56
|
20.60
|
47.91
|
8
|
60/35
|
245.65
|
39.01
|
56.5
|
9
|
65/30
|
151.51
|
37.32
|
64.8
|
Based on the mechanical properties listed in Table 3, bio-composite composed of 65wt% BG and 30wt% C (compound 9) has the highest compressive strength. Therefore, BG/C percentage ratio of 2.16 was chosen as the ratio with best mechanical strength. Due to the low strength of chitosan, bioactive glasses (BGs) have been widely added to pure chitosan to improve the mechanical properties. According to the literature [26,27,29,44] increasing of bioactive glass, increased mechanical properties. Pure chitosan scaffold showed 0.11 MPa compressive strength, while enhancing bioactive glass to pure chitosan increased compressive strength (2–12 MPa) [7]. Also, based on research of D. Sierra et al. chitosan/bioactive glass composite exhibited 56.1 MPa compressive strength [45]. H. Faqhiri et al. reported that mechanical properties of chitosan/bioactive glass composite (with 5 wt%, 15 wt% and 30 wt% of bioactive glass content) increased with increasing bioactive glass content to 15 wt% [46].
4-2- Bio-composites Of Step 2
FE-SEM images
FE-SEM micrographs and EDS spectra for samples prepared in step 2, with constant BG/C ratio of 2.16 and varying zeolite percentage are depicted in Fig. 6. Fibril like structures indicated the presence of high amount of BG in the composites. As it is illustrated in the FE-SEM images, by increasing the zeolite content to 20wt%, BG structure disappear and plate-like morphologies related to the presence of Z began to appear.
Figure 6- FE-SEM images and EDS of samples prepared with 10, 15 and 20 wt.% Zeolite
As it is depicted in EDS diagrams, the existence of Na, P, Al, Ca, Si, C, and O are confirmed in the bio-composites. Therefore, all the three components, bioactive glass, chitosan and zeolite, are present in the composites structure. The high intensity of peaks attributed to Ca and Si and P confirmed the high amounts of BG on the bio-composite surface.
FTIR
FTIR analysis confirmed that changing zeolite weight percentage did not change the nature of chemical bonds existing in the groups, but increasing zeolite content raises the intensity of vibration bands corresponding to Si-O and Al-O existing in zeolite structure [41,42]. The illustration of this investigation is depicted in Fig. 7.
Figure 7- FTIR spectra for bio-composites prepared with 2.16 BG/C ratio and different zeolite weight percentages
Mechanical properties
Mechanical properties of the step 2 bio-nanocomposites in terms of young modulus, fractural strength, and compressive strength, in comparison to other structurally similar ones, have been given in Table 5. Composite number 12 with 15 wt.% zeolite (entry 3) has the highest mechanical properties among the other prepared bio-composites. Comparing entries 1–4 shows that the compressive strength has increased with increasing the percentage of the zeolite from 5 wt.% to 15 wt.%. Increasing the percentage of the zeolite to more than 15 wt.% causes a decrease in mechanical properties [47]. This means that the optimal amount of zeolite in this composite structure is 15% and our optimal composite with the best mechanical properties is composite number 12 with the formula 58 wt.% BG/ 27 wt.% C/15 wt.% Z. This composite not only exhibits the highest compressive strength, but also has the highest fractional strength, least deformation, and highest rigidity.
Table 5
Comparing the mechanical properties of the bio-composites step 2 with structurally similar published ones.
Entry
|
Composition of compounds
|
Young modulus (MPa)
|
Fractural strength (MPa)
|
Compressive Strength (MPa)
|
Reference
|
1
|
65 wt.% BG/ 30 wt.% C/5 wt.% Z (Composition number 10)
|
151.51
|
37.32
|
64.8
|
Present work
|
2
|
61.5 wt.% BG/ 28.5 wt.% C/10 wt.% Z (Composition number 11)
|
239.09
|
47.8
|
69.3
|
Present work
|
3
|
58 wt.% BG/ 27 wt.% C/15 wt.% Z (Composition number 12)
|
597.48
|
59.62
|
75.2
|
Present work
|
4
|
55 wt.% BG/ 25 wt.% C/20 wt.% Z (Composition number 13)
|
634.2
|
38.44
|
61.5
|
Present work
|
5
|
50wt.%BG/45wt.%C/5 wt.% Z
|
704.4
|
22.18
|
56
|
[23]
|
6
|
25wt.%BG/70wt.%C/5 wt.% Z
|
264.8
|
19.79
|
32
|
[23]
|
7
|
Zeolite/chitosan (20–55 wt.% of zeolite)
|
-
|
-
|
1.51–3.2
|
[14]
|
8
|
PLGA/45S5 Bioactive glass scaffold (10 wt.% BG)
|
-
|
-
|
0.42
|
[48]
|
9
|
Chitosan/bioactive glass (20 wt.% BG)
|
460
|
-
|
7.68
|
[50]
|
10
|
Chi-BG hybrid scaffolds (0–30 wt.% of BG)
|
-
|
-
|
2–12
|
[7]
|
11
|
60 wt.% Chitosan/40 wt.% BG
|
-
|
-
|
54
|
[45]
|
12
|
70 wt.% Collagen/30 wt.% BG
|
|
|
5.80 ± 1.60
|
|
[49]
|
The composites of entries 5 and 6 are the bio-composites manufactured in our previous article that was prepared by the same method used in this work (liquid phase) [23]. A comparison of the composite entry 3 with composites entries 5 and 6 shows that by optimizing the BG/C ratio and then increasing the amount of zeolite from 5 wt.% to 15 wt.%, the compressive strength increases. Increasing compressive strength in this study is due to the optimization of the percentage of bio-composite components (determining the ratio of BG/C and then determining the optimal percentage of zeolite). Also, a comparison of entries 7–12 with entry 3 shows that the compressive strength of composite entry 3 is much higher than similar samples. This difference in compressive strength could be due to the differences in fabrication method and the percentage and the type of consistent materials of composites. Entry 7 is a composite made of chitosan/zeolite, which has a lower compressive strength compared to entry 3. This difference could be the effect of using bioactive glass together with chitosan and zoelite [14]. It seems that the reason for the lower compressive strength of the composites of entry 8 [48] and entry 12 [49] compared to entry 3 is the difference in the component used next to the bioactive glass. Chitosan/bioactive glass scaffold (entry 9) has a lower compressive strength than composite entry 3 that could be due to the using small amounts of bioactive glass and the absence of zeolite [50]. According to the literature, bioactive glass has high compressive strength, and zeolite as a silica-based material could increase the mechanical properties [7,14,16,50]. The composites of entries 10 [7] and 11 [45] are prepared in a different method and with different percentages of bioactive glass and chitosan components, so their strength is lower compared to entry 3. Considering that the preparation method, type, and percentage of components affect the mechanical properties of the composite, it seems that by optimizing the manufacturing method (in our previous work, [23]) and optimizing the percentage of components in the present work, we have been able to achieve an optimal formulation with favorable mechanical properties.
Evaluation Of Ha Layer On The Surface
To evaluate the growth of the hydroxyapatite layer on the surface of compound 12 (with 15wt% Z), a combination of FE-SEM, EDS, and XRD characterization techniques was applied. Figure 8 illustrates the SEM images of samples prepared in step 2.
FE-SEM observations exhibit that after the first day of exposure (Fig. 8,b), the initial tiny strip like hydroxyapatite particles is formed on the surface of the bio-composite. Forming of lamellar structures after 3 days of exposure to SBF solution (Fig. 8,c), confirmed the formation hydroxyapatite on the surface of the sample. Based on literature, through the study of the correlation of in vitro bioactivity of materials with their in vivo behavior, we can conclude that the manufactured bio-composites behave like biomaterials [26,42]
Figure 8- SEM images of bio-composite with 2.16 BG/C ratio and 15 wt.% zeolites, before (a) and after 1st day (b) and 3rd day (c) of exposure
Xrd
In order to find out hydroxyapatite growth to confirm biocompatibility, the sample with the best physical properties (compound 12) was selected. XRD patterns of compound 12 after 1, 3 and 7 days of exposure to SBF solution were investigated. The characteristic peaks of hydroxyapatite appeared at 2θ = 30–33 (ICDD 9-432) [51]. Comparison of the bio-composites XRD patterns before and after immersing in the SBF solution (Fig. 9), illustrated the formation of hydroxyapatite in the composite after exposure to SBF. Results show that upon 1 day immersing in SBF, hydroxyapatite layer formed in the sample surface. The intensity of the peaks in the XRD pattern indicated the amount of the hydroxyapatite growth on the surface of the sample [29,36]. Comparison of the peaks relating to hydroxyapatite confirmed that the more SBF exposure resulted in more hydroxyapatite formation.
Figure 9- XRD patterns of compound 12 before and after immersing in SBF for 1, 3 and 7 days