Fabrication of PES scaffold coated by leech saliva for vascular tissue engineering
Fabrication of PES scaffold coated by leech saliva for vascular tissue engineering
Sarvenaz Torabi,1Babak Akbari,2,*Hossein Noruzy moghadam,3
1. MA student at the University of Tehran 2. Assistant Professor at the University of Tehran 3. Instructor at Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO)
Introduction: Leech saliva has lots of effective components reported previously. Some of the important proteins in leech saliva are Hirudin, Destabilase, Factor Xa inhibitor, Antistasin, HyaIuronidase, and Collagenase. It has anti-inflammatory and analgesic properties, inhibits C1 complement, permits diffusive distribution, improves blood flow, and inhibits platelet adherence. Cardiovascular diseases can be treated with it as a drug according to studies. An artificial blood vessel must contain both the structure and mechanical properties of a real blood vessel. Accordingly, polyether sulfone electrospun fibers were used in this project. Polyether sulfone is a biomaterial extensively utilized in haemodialysis membrane production, owing to its remarkable chemical and mechanical properties.
Methods: The scaffold concentration was 30%, Followed by an electrospinning feed rate of 0.5mL/h, voltage 20kV, and collection distance of 25 cm. To prepare leech saliva, a nauseating solution was prepared by combining sodium chloride and arginine. After feeding the leeches with solution, the leeches were placed in ice and they vomited. Prepared saliva was centrifuged. Different concentrations of 2.5%, 5%, and 7% of protein solution were prepared. Oxygen plasma was used to activate the surface of the scaffolds for coating. Then, the scaffolds were placed in an EDC/NHS solution. Afterward, they were placed in protein solutions.
Results: The concentration of protein solution and constituent proteins were determined using the Bradford and SDS-PAGE method. The concentration of protein was 1.9665±0.053mg⁄ml. The morphologies of the materials were observed via SEM. The obtained fibers were uniform. The average fiber diameter was 0.507±0.136μm and the percentage of porosity calculated was 49.041%. The chemical structure was characterized using ATRI spectroscopy. To verify the developed coating, SEM imaging and ATRI assessments were conducted, and the resulting peaks validated the surface alteration and establishment of a protein coating. Surface wettability was tested with a water contact angle instrument. It was clear that the contact angle of the surface with water decreased with the increase of concentration in the coated protein, which indicated the increase in the hydrophilicity of the fibers. A tensile test was used to check the mechanical properties of the scaffold. Its Young's modulus was 1.40MPa, which is similar to normal coronary arteries, ultimate strength was 3.44MPa, and ultimate strain was 4.39. Anticoagulants in leeches destroy coagulation factors 2 and 10. Considering that coagulation factor 2 is in both the intrinsic and extrinsic pathways, it was expected that a change would be observed in both APTT and PT times. PT and APTT times increased with increasing protein concentration and increased blood compatibility and anticoagulation of the scaffolds were confirmed. To check the antihaemolytic of the scaffolds, Na and K ions, and LDH enzyme were checked. The obtained data didn’t show any hemolysis. MTT test was performed on HUASMC on day 3 and HUVEC on days 1, 3, and 7 and DAPI staining was performed on HUVEC on day 3 to check biocompatibility. Regarding the use of scaffolds for vascular, it is expected to increase the growth and proliferation of the HUVEC and decrease the growth and proliferation of HUASMC with an increase in the concentration of the scaffold coating. An increase in cell growth and viability of HUVEC was observed with increasing coating concentration. Notice that, by increasing the concentration from 5% to 7%, the rate of cell growth and proliferation decreased. This is due to the presence of the hirudin protein, which prevents any sticking to the surface from a certain concentration, which is the property of all anticoagulants that occur from a certain concentration. This shows that the optimal concentration is 5%. As expected, cell growth and viability of HUASMC decreased with increasing concentration. On the third day, DAPI staining was conducted on HUVEC, confirming the findings of the MTT test. According to staining, the number of cells increased from 2.5% to 5% and decreased from 5% to 7%.
Conclusion: In this study, we developed vascular tissue engineering scaffolds via electrospinning PES coated by leech saliva. These scaffolds promoted HUVEC growth and suppressed HUASMC proliferation. Furthermore, these scaffolds displayed excellent blood compatibility via a prolonged blood clotting time. In summary, PES scaffolds coated with leech saliva hold potential applications in vascular tissue engineering due to efficient endothelialization and decreased SMC proliferation.