Analysis of Macromolecular Structures
Accurate Identification of synthesized primarily polymers is one of the most challenging and sensitive steps to start work. Figure 2 shows the results of FTIR, 1H-NMR, and GPC of r-PGIt and PCL-diol. According to FTIR analysis of r-PGIt (Fig. 2A), ester bonds at 1600 Cm− 1 and 1100 Cm− 1 have appeared. That indicated that the interaction between glycerol and itaconic acid had occurred. To create a better document in confirming the synthesis of r-PGIt, 1H-NMR was reported and presented in Fig. 2B. Identification of chemical shifts of this sample is explained below with high accuracy:
1H-NMR (400 MHz, DMSO) δ 6.70 (dd, J = 27.0, 8.6 Hz, 13H), 6.19 (q, J = 18.9 Hz, 57H), 5.85 (s, 22H), 4.80 (dt, J = 116.2, 87.4 Hz, 140H), 5.83–4.04 (m, 406H), 4.60 (t, J = 57.0 Hz, 47H), 4.60 (t, J = 58.1 Hz, 47H), 5.20–4.04 (m, 322H), 4.17 (ddd, J = 45.3, 32.4, 30.3 Hz, 182H), 4.04–3.60 (m, 292H), 3.51–2.96 (m, 560H), 3.01 (s, 9H), 3.11–2.60 (m, 21H), 2.55 (s, 6H), 2.51 (s, 58H), 2.45–2.18 (m, 243H), 2.16 (s, 22H), 1.39 (d, J = 88.3 Hz, 261H), 1.24 (s, 491H), 0.84 (d, J = 8.8 Hz, 16H).
PCL-diol macromolecules were synthesized based on ROP polymerization. FTIR and 1H NMR techniques have been used to check the correctness in the synthesis of PCL-diol, and its results are shown in Fig. 2C and Fig. 2D, respectively. Investigations revealed that this material was well synthesized, and its analysis of 1H-NMR is given below:
1H-NMR (400 MHz, DMSO) δ 3.96 (s, 3H), 3.35 (s, 4H), 2.50 (s, 1H), 2.25 (s, 3H), 1.79–1.27 (m, 8H), 0.89 (d, J = 28.1 Hz, 1H).
The numerical molecular weight (Wn) of r-PGIt and PCL-diol were measured by GPC analysis, and their results are presented in Fig. 2E. Their results indicated that the molecular weight of r-PGIt and PCL-diol are around 300 and 20000 gmol− 1 respectively. Interactions between blended samples are shown in Fig. 2F. Tiny changes can be detected in the location of essential peaks in all samples. These results depicted that there is a weak interaction between these two materials. The presence of nanoparticles in those samples and their role in the interactions between the two polymers is shown in Fig. 2G. Examining the results showed slight changes in the amount of peaks seen in all of the samples.
Microstructures analysis
The microstructure evaluation of all of the prepared samples and distribution of the nanoparticles into their nanocomposites have been presented in Fig. 3. From cross-section SEM images of the samples can be seen that the morphology of the PGIt100 is totally smooth and very uniform (Fig. 3A). At the same time, presence PCL-diol with different weight fractions have been caused to formation coarse surfaces in PGIt70PCLdiol30 and PGIt50PCLdiol50 samples (Fig. 3B and Fig. 3C). By summarizing the results for the samples, it can be concluded that the two substances are incompatible. On the other hand, the presence of nanoparticles in the above samples can be helped to form good compatibility and reduction of surface energy between two polymers (Fig. 3D, Fig. 3E, and Fig. 3F).
Mapping and EDX analysis of nanocomposite samples have been shown in a part of Fig. 3. From those results, the distribution of the nanoparticles is proper within the polymer textures. It can be concluded that the two polymers have formed good chemical and physical bonds between functional groups on the clay surface.
Analysis of Physical and Mechanical Properties
The XRD results at low and normal angles for studied samples have been shown in Fig. 4A and Fig. 4B. XRD analysis at low angles for characterization distance between clay layers is shown in Fig. 4A Bragg’s equation was used to quantify this parameter[26, 27]. The distance between layers in pure Clay-Na+ is 1.77nm, while this parameter in the PGIt70PCLdiol30Clay5 and PGIt50PCLdiol50Clay5 nanocomposite samples are around 2.21 and 2.07nm, respectively. These results confirmed that almost an intercalated structure of clay layers could be assumed in these two samples[26]. On the other hand, it is possible that the presence of polymer chains PCL-diol, due to the high incompatibility between PGIt, has provided a barrier against penetration between clay layers. It can also be seen that without the presence of PCL-diol pair, an exfoliated structure has been obtained in sample PGIt100Clay5.
The crystal structures of the samples are evaluated with XRD analysis, and their results are presented in Fig. 4B. According to the observation results, polymer PGIt100 has a transparent crystal plane (001) that shows a sharp peak at the 2ɵ=20o [19, 26, 28]. The crystal structure of sample PGIt70PCLdiol30 is almost decreased with 30 wt.% of PCL-diol and 50 wt.% of PCL-diol, causing an increase in the crystal plans. This observation is probably due to the formation of separate phases of two polymers and their non-interference in the degree of crystallinity of each [15, 18]. The changes in the crystal plans of clay nanoparticles have also been investigated to examine the degree of crystallinity in the nanocomposite samples. In nanocomposite samples, the crystal structures of nanoparticles are clearly defined among the crystal planes of polymers due to the presence of nanoparticles. The better dispersion of clay nanoparticles in the samples does not show the samples' peak crystal structures in the XRD spectrum. Therefore, it seems that the clay crystal plates in nanocomposite samples were not completely removed, which was also proved in Fig. 4A.
The TGA analysis of all samples in a neutral atmosphere is given in Fig. 4C. Based on this analysis, three different degradation steps can be found, which is probably related to using other materials to prepare primary polymers. Investigation about of initial degradation temperature for all of the samples is obtained from Fig. 4D. The changes in this temperature for samples PGIt100 and PGIt100Clay5 have a steep downward slope compared to the other samples. This observation may be due to the presence of weak ester bonds, the creation of chains with shorter lengths, and the high absorption of water molecules in the nanocomposite structure. Besides this behavior, the presence of PCL-diol has relatively improved the thermal stability of the other samples. The presence of clay has also shown a strange behavior in nanocomposites because their initial degradation temperature has concentrated the thermal stability.
The DTG analysis with high clarity is depicted in Fig. 4E. As seen in the results, a very chaotic degradation behavior for all samples in the temperature range of 100 and 200oC was visible. This observation can be related to humidity absorption, the current low molecular weight of polymer chains, and some unreacted monomers. The first stage of degradation at the temperature of 300oC is related to the chain scission of ester bonds in the PGIt matrix. In the second stage of degradation at 450oC, the ester bonds on the PCL-diol phase. The third stage of degradation happened at 550oC; in this stage, clay nanoparticles are also located beside the polymer chains and obstruct their degradation process. At a glance, the changes in the amount of remaining char at 700oC alongside the first maximum degradation temperature for all of the samples are shown in Fig. 4F. The highest amount of char was observed in samples PGIt100 and PGIt70PCLdiol30Clay5. This result may be due to the interaction between PGIt and PCL-diol chains and the presence of clay nanoparticles beside these polymer materials. On the other hand, the maximum degradation temperature in the samples with the presence of PCL-diol and clay nanoparticles has been improved.
The stress-strain curves for all of the samples are presented in Fig. 4G. According to the presented results, PGIt100 has a high percentage of elongation (around 160%) and low tensile strength (around 7 MPa), which is related to the soft structure of this material. The observed behavior has already been mentioned in previous research [19, 26, 29, 30]. It can be seen that the presence of 5 wt.% of nanoparticles has had a significant effect on the mechanical properties of PGIt. The mechanical properties of blended samples with 30 and 50 wt.% of PCL-diol have been changed. Also, the presence of clay nanoparticles has significantly altered the mechanical properties of nanocomposite samples. A comparison between the changes in Young's modulus and elongation at break for all of the samples is shown in Fig. 4H. Young's modulus has increased with the increasing amount of PCL-diol compared to the pure sample, and these changes have grown in their nanocomposite samples. Contrary to the behavior of Young's modulus, changes in the amount of elongation at break in the samples show a reverse trend. Variations against Young's modulus and maximum stress in all samples are shown in Fig. 4I. Young's modulus and maximum stress in nanocomposite samples are higher than those without nanophase due to the reinforcing effect of nanoparticles in the polymer substrate.
Mechanical behavior of samples at a different range of temperatures and frequency modes were analyzed by DMTA analysis, and all of the results have been presented in Fig. 5. Trend changes in the storage module E’ and loss module E’’ as a function of temperature are shown in Fig. 5A and Fig. 5B respectively. In the glassy region, the storage modulus of PGIt100 is higher than in other samples. This observation indicates that PCL-diol inside PGIt has influenced the movement of polymer chains at low temperatures. The presence of PCL-diol as a long-chain crosslinker agent can be effective in forming long network structures, and the presence of these materials can create a much more flexible structure than PGIt with a uniform network structure in a linear viscoelastic section. It is expected that the presence of nanoparticles has increased the stability of the structure and strengthened the viscoelastic behavior of nanocomposites. The behavior of samples in the rubbery region is entirely different. In this area, the PCL-diol phase controls the viscous behavior of the samples, and PGIt70PCLdiol30 and PGIt50PCLdiol50 have significant modulus. Figure 5B shows the behavior of the loss modulus of prepared samples at a selected range of temperatures. As can be observed from the comparison behavior of samples, it is clear that PGIt100 has the highest loss modulus and viscous behavior among all of the samples. This manner may be due to the presence of free volumes between the polymer chains. 30 and 50 wt.% of PCL-diol blending with PGIt has significantly reduced the samples' viscous behavior. In this situation, due to the addition of hydroxyl groups, it is possible to create more crosslinking, and on the other hand, chains of PCL-diols can limit the free volumes for the movement of PGIt’s chains. The presence of nanoparticles as a challenging phase has a significant effect on the shake of polymer chains and has caused a remarkable decrease in the loss modulus of nanocomposites. Examining the amount of changes in the Tg of all samples is presented in Fig. 5C. With a glance; it is clear that the Tg of PGIt100 is lower than that of other samples. Also, the presence of nanoparticles inside the polymer matrix has increased the Tg due to the decrease in the movement of the polymer chains.
In Fig. 5D, a comparison between Tg and storage modulus at 37oC has been presented. The comparison results of Tg showed that this parameter is higher for PGIt50PCLdiol50 than other samples, and the presence of nanoparticles has caused the increase of this parameter for additional samples. For a better understanding of the amount of changes made in the samples, a mechanism is proposed in Fig. 5E, which describes the amount of interaction of the polymer chains with each other in the samples.
The frequency behaviors of the samples have been evaluated in Fig. 5F, Fig. 5G, Fig. 5H, and Fig. 5I. As can be seen, the behavior of the samples against the selected frequencies was appropriate. At temperatures ranging between 0 and 10oC, the behavior of the storage modulus for all samples has almost the same value (Fig. 5F). In nanocomposite samples (Fig. 5G), this range has disappeared, and the modulus reduction slope has decreased. In these samples, the presence of clay between the polymer chains has caused more development of the rubbery area.
Figure 6 shows the results related to frequency's effect on the samples' viscoelastic behavior. In Fig. 6A and 6B, changes in the glass transition temperature against frequencies and changes in storage modulus at the glass transition temperatures of the samples have been evaluated. These results show that with the increase in frequency, slight changes have occurred in increasing the glass transition temperature for all samples. On the other hand, at the glass transition temperature, the storage module has shown different behavior against the frequency. The presence of clay nanoparticles inside the samples has reduced the modulus reduction process against the frequency. Shift factor values for all samples were obtained based on the WLF equation (valid within a specific range (Tg, Tg+100oC), and the results are shown in Fig. 6C. In this figure, the reference temperature line is also shown. Based on this, PGIt100 is placed in the reference temperature range of 37oC. To obtain the master curve of the tan(delta) for sample PGIt100, in Fig. 6D, the changes of this parameter at the selected temperatures are plotted against the specified frequencies. Figure 6E shows the master curve at a temperature of 37oC.
For the calculation of shift factor parameters C1 and C2 should be calculated. These two parameters change based on the selected reference temperature. The C1 and C2 were calculated according to the following relations used[31]:
$${C}_{1}^{o}=\frac{{C}_{1}^{g}{C}_{2}^{g}}{{C}_{2}^{g}+\left({T}_{r}-{T}_{g}\right)} \left(1\right)$$
$${C}_{2}^{o}={C}_{2}^{g}+\left({T}_{r}-{T}_{g}\right) \left(2\right)$$
Moreover, obtained results are reported in Table 2; the values of these coefficients are reported for each sample.
Table 2
Calculation of C1 and C2 for all of samples
Samples | Tg (oC) | Tr (oC) | C1 | C2 (K) |
---|
PGIt100 | 29.68 | 37 | 15.23 | 58.92 |
PGIt70PCLdiol30 | 40.34 | 47 | 15.41 | 58.26 |
PGIt50PCLdiol50 | 63.26 | 67 | 16.22 | 55.34 |
PGIt100Clay5 | 30.54 | 37 | 15.46 | 58.06 |
PGIt70PCLdiol30Clay5 | 63.66 | 67 | 16.34 | 54.94 |
PGIt50PCLdiol50Clay5 | 89.41 | 97 | 15.17 | 59.19 |
Biological Studies and In-Vitro Analysis
The interaction of the surface of samples with cells is one of the most critical biomaterials used in tissue engineering. The biological degradation of representatives at the body temperature and during 60 days was performed as a preliminary test, and their results are shown in Fig. 7A. The results show that PGIt100 has good degradability behavior and that more than 60% of its neat weight has been degraded within 60 days. On the other hand, the presence of PCL-diol within the PGIt matrix has reduced the rate of degradability. Because PCL-diol has a weak interaction with water molecules, it is a barrier against hydrolyzed ester bonds. The presence of nanoparticles due to their mineral nature has increased the amount of water exchange with the surface of the samples, and hence the rate of their degradation increased.
The contact angle analysis of all samples is shown in Fig. 7B. Generally, the contact angle values of all samples are in a suitable range. The addition of PCL-diol within the PGIt has caused slight changes in the contact angle. On the other hand, the presence of nanoparticles has a positive effect on reducing the contact angle. PGIt50PCLdiol50Clay5 has higher contact angle values among all the contact angle behaviors. This result may be related to clay layers and enhanced hydrophilicity.
The results of MTT analysis for selected samples PGIt100, PGIt70PCLdiol30, and PGIt70PCLdiol30Clay5 on days 1, 3, and 5 are presented in Fig. 7C. Examining the effects showed that all the samples have low toxicity, and it seems that the surface of these samples is suitable for cell adhesion and growth. Therefore, cell adhesion analysis on the surface of the samples on the 5th day is given in Fig. 7D, Fig. 7E, and Fig. 7F. In these figures; it can be seen that the cells are well spread on the surface of the scaffolds, which shows the excellent interaction of the cells with these polymer materials. Also, in the nanocomposite sample PGIt70PCLdiol30Clay5, cells on the surface are more suitable for morphology.
The Dapi and Alizarin red results on selected samples are examined and shown in Fig. 8. A general look at the Dapi images shows that almost all the samples behave well against the distribution of cells on the surfaces.
These observations emphasized that prepared samples can be used for various tissue engineering applications. Moreover, the presence of clay nanoparticles can also increase the ability of this sample (PGIt70PCLdiol30Clay5) to interact with the desired cells because it seems that clay can provide the necessary additive for increasing hydrophilicity and cell attachment.
Calcium deposition on the surface of selected samples was stained by using Alizarin Red S staining at 3, 8, and 11 days and their results, as shown in Fig. 8. At a glance on day 3, can be found that increasing clay nanoparticles showed the excellent effect on calcium formation. On the other hand, on the 11th day, sample PGIt70PCLdiol30Clay5 showed better behavior than different samples.