3.1 Chemical characterization of PGS/PLLA scaffold
FTIR spectroscopy was performed to determine the presence of PGS, PLLA polymers and two substances, heparin and PRP. Figure (1-a) shows the FTIR spectrum of PGS pre-polymer. The peaks at 2917 cm-1 and 2844 cm-1 related to alkane and methyl groups, the peak at 1181 cm-1 related to bond C-O and 1743 cm-1 related to bonds (C=O) confirm the successful synthesis of PGS (23,24). Figure (1-b) shows the FTIR spectrum of PGS/PLLA scaffold, where all the characteristic peaks of PGS polymer were observed in this spectrum. The peak at 1088 cm-1, which is related to the stretch bond (C-O) and also the peaks at 1453 cm-1 and 1381 cm-1 are related to the bond (C-H) and (C-C), prove the presence of PLLA polymer. Also, the peak at 1740 cm-1 is related to the bond (C=O), which due to overlap with the peak of PGS, an increase in the intensity of the peak compared to the FTIR spectrum of the PGS pre-polymer was observed (25,26). Figure (1-c) shows the FTIR spectrum of PGS/PLLA scaffold containing heparin. The peaks at 1264 cm -1 , 1417 cm-1 and 1043 cm-1 correspond to (C-O), (C-H) and (C-N) bonds in heparin, respectively (27,28). Figure (1-d) shows the FTIR spectrum of PGS/PLLA scaffold containing PRP. The characteristic peak at 3316 cm-1 is related to the OH bond in PRP. Also, this peak is related to amide A. The peak at 1416 cm-1 shows the amide III group. Stretch (C-O-C) for pure PRP is in the range of 2800 cm-1 to 2950 cm -1, which peak of 2846 cm-1, which overlaps with PGS, confirms the presence of PRP. Also, the sharp peak at 1729 cm-1 indicates the bond (C=O) in PRP (29,30).
3.2 Scaffolds morphology study
Figure 2 shows the electron microscopic images of fibers related to three layers containing PGS/PLLA compound with different ratios of this compound in two magnifications of 1000 and 5000. As can be seen in the figures, the scaffolds do not have willows and the fibers are uniform.
Adding heparin and platelet-rich plasma, we see fibers in Figure 3. Various parameters in the electrospinning process such as polymer concentration, solution viscosity, voltage level, polymer solution amount and solution conductivity are effective on the fiber diameter. Ionic salts that have an electrical charge affect the solution. The available electrical charges increase the charge density of the solution and as a result the fiber becomes more elongated and ultimately reduces the diameter. Therefore, by adding heparin, which is a charged salt, as shown in Table 2, the diameter of the fibers has decreased (31). But the introduction of PRP into the scaffold has increased the diameter of the scaffold. By adding PRP to the scaffold, the diameter of the nanofibers expands. So by adding PRP, which is a solution itself, the total concentration of the solution increases and the diameter of the fibers increases. The electrospinning method provides us with high porosity in fibers. According to the studies of Yi Tang, Hui Zhang, et al., it was found that adding PRP to chitosan-collagen-hydroxyapatite scaffolds increased the diameter of nanofibers (32). As we can see in Table 1, the percentage of porosity for all samples is a good amount (20).
Table 2 Average fiber diameter and porosity percentage for electrospun scaffolds
Porosity (%)
|
(µm) Average fiber diameter
|
samples
|
54/90
|
.1.2± 0.4
|
PGS/PLLA (1:1)
|
5/90
|
1.2± 0.3
|
PGS/PLLA (2:1)
|
95/89
|
1.5± 0.1
|
PGS/PLLA (3:1)
|
56/89
|
0.03±0.38
|
PGS/PLLA +heparin
|
95/90
|
0.9±1.9
|
PGS/PLLA+PRP
|
3.3. Contact angle of electrospun samples
To check the hydrophilicity of electrospun PGS/PLLA scaffolds, PGS/PLLA containing heparin and PGS/PLLA containing PRP, the water droplet contact angle measurement test was used. As can be seen in Figure 4, the PGS/PLLA sample with a contact angle of 50.72± 5.09 is somewhat hydrophilic. In another group, by adding heparin to PGS/PLLA with a ratio of 2:1, the amount of hydrophilicity increased significantly and the contact angle decreased to 38.06±10.28. The reason for this change is the presence of hydrophilic drugs such as heparin in the structure which, due to having functional groups with negative charge COO- or SO3-, not only inhibit non-specific protein absorption, but can mediate adhesion and absorption of cells. On the other hand, increasing the amount of PGS in the scaffold has also contributed to this issue (33,34). In the last layer, the contact angle decreased to 28±1.66 degrees and the hydrophilicity increased again. The reason for the increase in hydrophilicity is the increase in the amount of PGS compared to the other two layers and the addition of PRP, which increases the hemocompatibility and cell compatibility due to the increase in hydrophilicity and make it suitable for cardiovascular applications. Hydrophilicity of is an important factor in electrospun scaffolds for cell growth and cell adhesion followed by cell proliferation and differentiation that is one of the basic steps after scaffold implantation in in vitro environment (13, 31,35)
3.4 Degradability of PGS/PLLA scaffold
Figure 4 shows the degradation diagram for samples. As we can see in the graph, the single-layer sample has a uniform degradation rate during 60 days. While in the second and third samples, by adding medicine to the scaffold and also increasing the share of PGS, we saw a significant weight loss in the first 3-5 days and the slope of the weight loss curve was higher, which is related to the release of drugs. Also, within 5 to 30 days, when the drug is removed and water molecules penetrate into the polymer scaffold, the polymer chains are broken, and with the slow release of these chains and the replacement of water molecules, the degradation of the layers takes place slowly. Finally, after 30 days, as the chains become smaller and accelerate their exit, the speed of degradation has also increased. The degradation rate of PGS/PLLA and PGS/PLLA samples containing heparin on the 60th day was 54.96±8.6 and 78.95±4.7%, respectively. Also, the degradation rate in the third sample is 80.9±2.1 and has a significant difference especially in the first 40 days with the first and second samples. The degradation results showed that single-layer PGS/PLLA samples, two-layer PGS/PLLA samples containing heparin, and three-layer PGS/PLLA samples containing heparin and platelet-rich plasma were significantly different from each other (p>0.05). 81% by weight of the final electrospun sample is destroyed within 60 days, which is a suitable degradation rate for vascular tissue engineering applications. According to the studies of Renato S. Navarro et al. on PLLA scaffolds alone and with heparin, they found that PLLA has a very slow degradation rate and in laboratory conditions it has a degradation rate of 6- 12 months and it may not correspond favorably with the speed of regeneration of blood vessels. By including heparin on scaffolds containing PLLA, they found that the scaffold started to disintegrate in the first 35 days, but in the sample of PLLA scaffold without heparin, only a small amount of the scaffold was lost after 35 days (36). Also, according to the study of Soodabeh Gorgani and Anousheh Zargar Kharazi, they found that by combining PLLA polymer with PGS, a suitable degradation rate can be achieved for vascular applications. Also, they observed that PLLA polymer can slow down the access of functional groups of PGS to PBS solution, and as a result reduce the amount of degradation (10) .
3.5 In vitro heparin release rate from electrospun PGS/PLLA scaffold
Figure 6 shows the percentage release of heparin from the three-layer PGS/PLLA scaffold containing heparin. The amount of drug in the final structure was evaluated, which is equal to 3.32 micrograms/ml. In the first 3 hours, 21% of heparin is released at a high and uniform rate, and after that, heparin is released at a slower rate. Heparin is a water soluble polysaccharide. As can be seen, in the first 12 hours 28±2.64% of heparin is released in PBS solution and then the release continues with almost the same slope. So that in 24, 48 and 72 hours, 46±3.65, 71±2.44 and 87±2.41% heparin is released, respectively. According to the studies of Kharaziha et al., the rapid release of heparin immediately prevents the formation of clots at the injury site, and in general, the gradual release of heparin in the early days of the injury increases hemocompatibility (37). According to Su et al.'s studies on heparin-releasing scaffolds, the controlled release of heparin in the graft prevents the occurrence of intimal hyperplasia (38).
3.6 Platelet-rich plasma release from PGS/PLLA scaffold
As shown in Figure 7, the release process of PRP is such that this substance is released at a high speed in the first moments after implantation and in the first two hours. Then, its release rate decreases for the first 5 hours of release, and after that, it continues with a slow slope until the complete release of this substance. The effect of PRP in vascular tissue engineering scaffolds can lead to increased cell growth and proliferation. On the other hand, due to the fact that PRP is a hydrophilic substance, it has an effect on increasing the degradation process of scaffolds, which then leads to the release of this agent from electrospinning scaffolds (39). The rapid release of this factor in vascular tissue engineering can also lead to the improvement of the growth process and cell proliferation in the very early moments. PRP consists of a set of growth factors such as vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF) and transforming growth factor Beta-stimulator (TGF-β). By releasing them, they promote the migration and proliferation of cells, and finally, by inducing angiogenesis, they lead to the regeneration of the target tissue (40). Controlled release of growth factors is essential in many tissue engineering applications, as it leads to appropriate cell responses such as proliferation, differentiation, and angiogenesis to regenerate functional tissues (41). As Gomes et al. showed, the presence of PRP and its release in the in vitro environment exponentially improves the process of cell growth and proliferation, and on the other hand, it leads to an increase in angiogenesis and the expression of appropriate growth factors for the repair of damaged tissues and also increases the healing process (13). The studies of Radyum Ikono et al showed that the total release rate of PRP protein encapsulated in chitosan in PBS was explosive in the first 7 13 hours. Then a constant release was performed, then about 60% of it was released up to 96 hours (13).
3.7 Mechanical properties of electrospun scaffold
Figure 8 and 9 show the effect of polymer composition and drug loaded in electrospun layers on young modulus and ultimate tensile strength(UTS) of the scaffold. As can be seen by adding PGD ratio and drugs, UTS and elastic modulus decreased while elongation slightly increased. In the outer layer, the UTS is 1.17±0.06 MPa and the elastic modulus is 24.4±2.5 MPa. These parameters at the middle layer show a decrease and are o.595±0.07MPa for UTS and 9.8±1.2 MPa for the elastic modulus. This reduction in mechanical properties is due to the increase of PGS ratio in the composition of the layer (PGS:PCL 2:1) andadding PRP into the fibers, which leads to change in the diameter of the fibers according figure2. In the inner layer, the UTS and elastic modulus were 0.335±0.04 MPa and 5.2±0.8 MPa respectively. At the inner layer, PGS:PLA ratio was 3:1 and heparin was added to the composition that affected fiber diameters and fiber hetrogenisity(figure 2).Luong-Van E et al has been reported that by adding hydrophilic drugs (such as heparin) to electrospun fibers, the mechanical properties greatly reduced because of the change in fiber diameter. Moreover disruption in polymer chains continuity due to the addition of other factors (hence Heparin and PRP) could be another reason for the decrease in mechanical properties (17,42) Result of tensile test on the tri-layer scaffold containing drugs revealed that its UTS and elastic modulus were 0.798±0.103 Mpa and 13.8±1.11 MPa respectively. According to previous researches, these values are within the optimal range for vascular tissue engineering applications in such a way that Young's modulus in the range of 4-18 MPa is reported to be favorable for the construction of artificial vessels (43,44). The suitable mechanical properties of the tri-layer scaffold can be related to the proper adhesion between the layers of the scaffold. This hypothesis could also be approved by the figure 8 that showed a gradual change in stress-strain curve. Moreover, during degradation test, the separation between the layers did not occur until complete degradation that indicates the proper interconnectedness of the layers.
Mechanical and biochemical gradients are found throughout the body and provide structural integrity and expected functions. Natural vessels are consisted of three layers and the mechanical properties increase from the inner layer to the outer layer(45). A biomimetic scaffold, requires complex materials to provide suitable replacement. Functionally graded materials are designed to mimic naturally occurring structure in the body. In this research, gradually changes in the composition of the vascular scaffold could modeled tri-layer of the native vessels. Previous research show that PGS:PLA elecectospan membrane in the ratio of 3:1 show mechanical properties like intima layer that is suitable for housing and proliferation of endothelial cells(10). Reducing the ratio of PGS in the composition of the middle and outer layers of the scaffold leads to a gradual increase in the strength and stiffness of these layers and provides a protective role for the inner layer. In addition, the stiffness amount remains within the appropriate range of the vessels. This slight and gradual change prevents from layers separation during loading.
3.8 Blood compatibility assessment
3.8.1 Platelet adhesion ratio
The amount of absorbed platelets on different samples was investigated by lactate dehydrogenase (LDH) method. According to the results presented in Figure 10, there is a significant difference between the positive control group (glass) and the drug-free sample, as well as polyglycerol subcategory and poly-L-lactic acid scaffolds along with heparin compounds and platelet-rich plasma. (P0.05).The results of platelet adhesion based on lactate dehydrogenase enzyme activity show that platelet adhesion in the group without heparin and PRP is 12. 9±0.56%. Meanwhile, the amount of platelet adhesion in negative control and positive control samples is 8.55±0.5 and 178±9.89%, respectively. In this first part, it can be concluded that the desired electrospun scaffold has an ideal compatibility compared to blood glass and Teflon, and on the other hand, the amount of platelet adhesion in the target sample is close to Teflon as a blood substance. After examining the sample without any drug or biological agent, the platelet adhesion process in the samples containing heparin and the sample containing PRP is 18.51±0.69 and 24.4±0.85%, respectively. The results show that with the addition of heparin to the structure, there is no significant difference between the drug-free and heparin-containing samples. On the other hand, with the addition of a biological agent such as PRP to the electrospun structure, the amount of platelet adhesion has not changed much. Considering that there is a significant difference between the samples and the positive control sample, it can be concluded that the studied samples are a good option for vascular tissue engineering applications. Dunn and his colleagues showed in their study that poly (vinylidene fluoridetrifluoroethylene)/oxyhydrogen nanocomposite scaffold has a lower platelet adhesion rate compared to the positive control sample. As a result, it can be a good option for tissue engineering applications. In this study, the percentage of platelet adhesion was reported between 20 and 30% (46). In another study, Liu et al evaluated the platelet adhesion of electrospun polylactic acid/chitosan scaffolds in order to fabricate scaffolds for cardiovascular applications. In this study, it was shown that samples with a platelet adhesion rate of less than 30% are good options for preventing the activation of coagulation cascades and preventing platelet adhesion (47).
3.8.2 The hemolysis ratio
According to Figure 11, the electrospun scaffold of polyglycerol sebacate and poly-L-lactic acid without drugs has a hemolysis percentage of 1.67±0.16%. Meanwhile, the scaffold containing heparin has a hemolysis rate of 22.2±0.17% and the sample containing platelet-rich plasma has a hemolysis rate of 2.17±0.13%. All these results show that the sample without and containing drugs and platelet-rich plasma has a suitable percentage of hemolysis and compared to the positive control sample with 6% hemolysis, they can have a good interaction with blood 15 cells for vascular applications and do not lead to their damage. One of the factors influencing the interaction of scaffolds with blood cells is their morphological structure. As the results of the present study showed, the presence of uniform fibers with suitable diameter and porosity leads to the absence of lysis of blood cells and blood compatibility of the scaffold. In this context, Wang and his colleagues, by examining the blood compatibility of electrospun polyethylene glycol/polyurethane hybrid scaffold, proved that hemolysis rate less than 3% indicates no damage of the biomaterial sample to red blood cells and can be a suitable option for vascular tissue engineering applications (48). The degree of hemolysis of red blood cells is an important parameter to evaluate the destructive potential of foreign substances to red blood cells based on the concentration of hemoglobin released from ruptured red blood cells. Blood compatibility standards show that biomaterials are classified into three general categories based on the percentage of hemolysis. The first category is the non-hemolytic group (0-2% hemolysis), the second category is slightly hemolytic (2-5% hemolysis) and the third category is hemolytic blood, which shows more than 5% hemolysis. In general, materials with a hemolysis percentage of less than 4% of blood are compatible and are suitable options for vascular tissue engineering applications (49).
3.8.3 Blood clotting ratio
Figure 12 shows the blood coagulation time in electrospun polyglycerol sebacate and poly-L-lactic acid scaffolds. The results indicate that compared to the positive control group (glass), blood coagulation time in samples without drugs, scaffold containing heparin and samples containing PRP show a much longer coagulation time. On the other hand, the results show that the samples containing heparin and PRP have better conditions compared to the negative control and it takes more time for the coagulation process to occur in this group. For example, in the first 5 minutes, the absorption rate for three scaffolds without drugs, containing heparin, and samples containing PRP, the hemoglobin absorption rate is 1.72, 2.18, and 2.7, respectively which compared to glass as a positive control with hemoglobin absorption of 0.7, it shows a high absorption which causes no clot formation. The main reason for this high capability of electrospun scaffolds is the presence of hydrophilic PGS, the relative release of heparin and their ideal surface structure. In the study of Yi Ping et al., they showed that in the electrospun polycaprolactone/gelatin scaffold along with silk protein, the trend of anti-coagulant properties of the sample is such that after 30 minutes, the complete sample has a gentler slope compared to other samples and the slower the reduction of hemoglobin absorption, the better the anticoagulation effect. On the other hand, the results of their investigations showed that if a sample shows hemoglobin absorption greater than 1 after 30 minutes, in other words, it is an anti-coagulant sample (50). Considering the results of previous studies, it can be said that all three study groups in the present study have ideal properties in the field of blood coagulation and blood compatibility and are ideal options for cardiovascular engineering applications.
In order to investigate the cytotoxicity of PGS/PLLA, PGS/PLLA/Heparin, PGS/PLLA/PRP, PGS/PLLA/Heparin/PRP scaffold groups, MTT test was performed in 24 hours, 48 hours and 72 hours. And the results were presented according to Figure 13. According to the results, it can be understood that the cell proliferation in the electrospun fibers has increased in all 4 groups of samples, similar to the control sample, and the said scaffolds act as a suitable substrate for the cultivation of HUVEC cells. Also, the results of the MTT test showed that the PGS/PLLA samples containing heparin and PRP not only did not cause any cytotoxicity, but the cell metabolism and cell viability on days 1, 3, and 5 went through their normal course. In addition, between days 1 and 3 and day 5, no significant difference was observed between pure PGS/PLLA, PGS/PLLA containing heparin, PGS/PLLA containing PRP, and PGS/PLLA containing heparin and PRP (P<0.05).
A suitable scaffold for tissue engineering should be able to provide a suitable substrate for the cells forming the target tissue. In such a way that the cells can adhere to the scaffold, grow and multiply in line with the formation of functional ECM. For this purpose and access to a biomimetic approach in blood vessels, a layer of endothelial cells (EC) and smooth muscle cells that are resistant to thrombosis can be used. Thrombotic obstruction and neo-intimal hyperplasia occur as a result of the lack of endothelial layer coverage (51). Also, the studies of Haizhu Kuang and colleagues on two-layer PC/MSN-PEG composite vascular graft scaffolds with heparin showed that heparin-containing scaffolds are suitable for promoting cell proliferation and improving blood compatibility, and the growth and proliferation of cells (HUVECs) on the membrane Scaffolding is clearly visible (52). In the studies of Kashef Saberi and colleagues on PES/PVA scaffolds, it was observed that the presence of PRP in the scaffold provided favorable biocompatibility for the scaffolds, and also led to an increase in cell proliferation compared to the control scaffold (53). SEM images related to PGS/PLLA scaffolds in Figure 14 showed that the electrospinning of PGS/PLLA scaffolds without and containing heparin and PRP not only did not cause toxicity and cell death, but also caused cells to stick to the scaffold and cell proliferation on the scaffolds can be seen well after 72 hours of culture. This favorable growth process is obtained due to the hydrophilic substrates of the fibers, because hydrophilicity is an important physicochemical phenomenon in scaffolds that affects protein absorption and cell behavior. Cell adhesion increases with decreasing contact angle and cells can significantly adhere to hydrophilic substrates compared to hydrophobic ones (54). Because as seen, PGS/PLLA scaffolds containing and without heparin and PRP are hydrophilic scaffolds and have a suitable contact angle for compatibility with the cells that are in contact with them. The hydrophobic properties of the surface can cause adhesion platelets become significant, which can lead to thrombosis (55).