The Effect Of Biomimetic Aligned Fiber Stiffness On Macrophage Polarity And Blood Compatibility

In the field of vascular tissue engineering(VTE),small-caliber tissue engineered vascular grafts(TEVGs)with a diameter of less than 6 mm still have the problem of low patency caused by poor biocompatibility of the TEVGs after long-term implantation.This is mainly due to the tissue and blood compatibility issues due to the inadequate biomimicry of the TEVGs to the extracellular matrix of vascular tissues.In view of the anisotropic structural characteristics of natural blood vessels,electrospun biomimetic aligned fibers hold great potential in the construction of TEVGs.Immune cells such as macrophages play an important role in the biocompatibility related response after TEVGs implantation,and their phenotype evolution directly affects the outcome of tissue regeneration.In addition,VTE scaffolds should also have good blood compatibility.However,although current studies have explored the effects of fiber diameter,orientation and pore size of electrospun scaffolds on the macrophage polarization and hemocompatibility,little attention has been paid to the effect of fiber stiffness(especially aligned fibers with structural anisotropy)on the macrophage polarization and blood compatibility.In order to explore the effect of biomimetic aligned fiber stiffness on the polarization of macrophages,a stable jet coaxial electrospinning method(SJCES)was employed to produce four groups of highly aligned fibrous substrates(AFSs,designated as S1,S2,S3 and S4)with adjustable stiffness in the range of 18.05 ± 1.52 ~ 951.92 ± 41.83 MPa,by varying the feeding rate of the rigid polylactic acid(PLLA)core layer and the elastic poly(L-lactide-co-caprolactone)(PLCL)shell layer during the spinning process.Then,RAW264.7 macrophages were seeded on AFSs and incubated for a certain period of time to examine the effect of AFSs stiffness on cell apparent behavior(e.g.,adhesion,morphology,proliferation,and migration)and macrophage polarity.The obtained results are shown as follows: 1)The increase in the AFS stiffness promoted the adhesion and spreading of macrophages with the cell morphology going through a transition from spindle-like shape to extending its two ends along the fiber orientation direction.2)The increase of AFS stiffness enhanced the proliferation capacity of the RAW264.7 cells,showing a positive correlation to the fiber stiffness.3)The results after 24 hours of cell migration showed that increasing the AFS stiffness gave rise to enhanced cell migration ability along the fiber direction;whereas there was no noticeable influence in the direction perpendicular to the fiber alignment.4)After 1 day and 3 days of cell culture,both the genetic and protein level results showed that the effect of fiber stiffness on most gene expression was very limited on day 1,indicating its temporal-dependence of the fiber stiffness effects.On day 3 it was identified that soft substrates were more prone to induce the macrophages differentiate into anti-inflammatory phenotypes compared to the stiffer AFSs.5)By having the macrophages treated with the Rho/ROCK signal pathway inhibitor Y-27632,it was found that ROCK signal pathway partially mediated the stiffness-induced polarization of macrophages.To assess the effect of AFS stiffness on blood compatibility,representative items including platelet adhesion,hemolysis rate,plasma recalcification and whole blood coagulation were evaluated.The results are enumerated as follows: 1)The AFS stiffness affected the number and morphology of the adherent platelets;a smaller number of platelets with a lower degree of activation were observed on soft substrates.2)With a hemolysis rate < 5%,the four groups of AFSs could be considered as non-hemolytic materials.There was no significant difference in the hemolysis rate of these AFSs with varying stiffness.3)The results from plasma recalcification and whole blood coagulation testing revealed that the AFS stiffness had no effect on blood coagulation.In summary,our results showed that the macrophage polarity and platelet adhesion were highly dependent on the AFSs stiffness.The stiffness of AFSs had neglectable effect on hemolysis and coagulation.These results could offer a meaningful guidance for the future design of viable fiberbased TEVGs.