Designing Multifonctionnal Nanofiber Scaffold for Endothelial Cells Adhesion and Proliferation on Vascular Substitutes

Sabbatier Gad (Université Laval/ Centre de recherche du CHU de Québec)
Larrañaga Aitor (Department of Mining-Metallurgy Engineering and Materials Science, University of the Basque Country)
Ko Na Re (Department of Chemistry and Biochemistry, Concordia University)
Cunningham Alexander (Department of Chemistry and Biochemistry, Concordia University)
Guay-Bégin Andrée-Anne (Centre de recherche du CHU de Québec)
Fernandez Jorge (Department of Mining-Metallurgy Engineering and Materials Science, University of the Basque Country)
Oh Jung Kwon (Department of Chemistry and Biochemistry, Concordia University)
Sarasua Jose-Ramon (Department of Mining-Metallurgy Engineering and Materials Science, University of the Basque Country)
Laroche Gaétan (Université Laval/ Centre de recherche du CHU de Québec)

Introduction

The absence of neo-endothelium on the intimal surface of vascular artificial substitutes is known to be one cause of failure upon implantation of these prostheses in humans. As coating with proteins (collagen, albumin, etc) does not improve the endothelialization capability of textile prostheses, it was sought to replace these traditional proteins with a poly(lactide) (PLA) nanofiber scaffold obtained by an innovative air spinning system. The ultimate goal of this fine-tuned interface between blood and textile threads is to provide an adequate scaffold for endothelial cells to proliferate as monolayer [1]. On one hand, degradation features of PLA-based materials enable the progressive replacement of the scaffold by the extracellular matrix of endothelial cells. On another hand, the grafting of hydrophilic molecules on hydrophobic PLA nanofiber surface would improve prostheses integration in vivo. In this context, the objective of this work is to understand mechanisms of degradation of PLA-based nanofiber, characterize their cytotoxicity as a function of degradation time, and functionalize the surface of these nanofibers to promote cell adhesion.

Materials and Methods

Poly(L-lactide) (PLLA) (Mw = 100kDa) and poly(lactide-co-ε-caprolactone) (PLCL) (Mw = 100kDa) were dissolved in chloroform and air-spun as described in a previous study [2]. Control polymer film samples were also made through hot pressing. Films and nanofiber meshes were immersed into phosphate buffer solution (PBS) and degraded during 70 days at 37°C. PLLA samples were also hydrolysed at 50°C to accelerate degradation process. Degradation was followed using gel permeation chromatography (GPC) and differential scanning calorimetry (DSC) every 7 days. Human saphenous vein endothelial cells adhesion (1 hour) and proliferation (5 days) assays were achieved after 0, 35 and, 70 days of degradation. These cell culture experiments as a function of polymer degradation time was performed in contact with fibrous and film surfaces as well as degradation mediums. In parallel, PLA labeled with disulfide-bromine linkages (PLA-SS-Br) were synthesized by ring opening polymerization and air-spun. Then, bromine end-bounds enabled grafting hydrophilic pendant oligo(ethylene oxide)-containing polymethacrylate (POEOMA) by atom transfer radical polymerization (ATRP). ATRP has been performed in an aqueous medium that preserve nanofiber structure. Finally, these new PLA-SS-POEOMA nanofibers were surrounded in reductive media to cleave disulfide linkages and release the POEOMA in media, then forming PLA-SH fibers. Syntheses and releases steps were followed by proton nuclear magnetic resonance spectroscopy (1H-NMR), GPC, scanning electron microscopy (SEM) and dynamic water contact angle.

Results

Figure 1 shows the evolution of molecular weights of the various polymers measured by GPC during degradation process for both films and fibers. These data reveal that the film degradation rate is higher than nanofibers for both temperature conditions investigated. Generally, bulk degradation mechanism of polyesters leads to the formation of acidic by-products entrapped in the polymer structure which, in turn, autocatalyze chain scissions. Consequently, it is likely that the porosity inherent to the structure of nanofiber scaffolds has enabled the diffusion of degradation products outside the polymer assembly. Moreover, DSC thermograms of fibers (not shown) highlighted strong relaxation and crystallization peaks as compared with films, which is characteristic of a low free-volume and a more packed distribution with a higher content of amorphous structure. This particular organization leads to decreasing water absorption and accordingly, to a slower degradation. Figure 2 shows that air spun PLA-SS-Br polymer fibers can be grafted with a hydrophilic polymer through atom transfer radical polymerization. This was also confirmed by GPC and SEM (not shown), which evidenced that nanofibers made of PLA-SS-POEOMA exhibit a higher molecular weight and a larger fiber diameter as compared to PLA nanofibers [3]. Finally, dynamic contact angle of PLA-SS-Br, PLA-SS-POEOMA and PLA-SH confirmed that POEOMA have been released in reductive conditions (Figure 3). Furthermore, PLA-SH highlighted a decreased of contact angle as a function of time that provides a higher hydrophilic surface than PLA-SS-Br nanofibers following the POEOMA cleavage [3]. Surface features (roughness, hydrophilicity, microstructure, etc.) are directly related to immune response and coagulation activation. Nanofiber surface modification with a hydrophilic polymer would decrease these non-specific interactions between blood and biomaterials. Moreover, grafting and release of POEOMA was used as model toward grafting other biomolecules via ATRP for biomedical applications.

Discussion and Conclusion

On one hand, these studies shed more light on PLLA and PLCL nanofiber scaffolds degradation mechanisms for biomedical applications. This model will therefore constitute a step toward to design PLCL nanofibrous scaffold with tailored degradation behavior for specific biomaterial applications. On the other hand, air-spun PLA nanofiber with releasable POEOMA functionalities has been synthetized for enhancing PLA hydrophilicity. SI-ATRP method is a promising tool for grafting and releasing bioactive molecules such as peptides, growth factors, or drugs to improve PLA biocompatibility.

Figure 1: Evolution of molecular weight in function of time for PLCL samples. The table summarizes GPC results for PLCL and PLLA fibers and films, where k is the kinetic constant and t1/2 half-life time.

Figure 2: 1H-NMR spectra of PLA fibers (A), PLA-SS-POEOMA (B), and PLA-SS-POEOMA fibers (C) in CDCl3 (adapted from [3] with permission).

Figure 3: Evolution of contact angle on PLA-SS-Br nanofibers, PLA-SS-POEOMA nanofibers and PLA-SH nanofibers (from [3] with permission).

Acknowledgements

The authors acknowledge the Centre Québécois sur les Matériaux Fonctionnels (CQMF) for its financial support.

References

1. FranÁ§ois S.; Chakfé N.; Durand B.; Laroche G. A poly(l-lactic acid) nanofibre mesh scaffold for endothelial cells on vascular prostheses. Acta Biomater. 2009, 5, 2418–2428. 2. Sabbatier G.; Abadie P.; Dieval F.; Durand B.; Laroche G. Evaluation of an air spinning process to produce tailored biosynthetic nanofibre scaffolds. Mater. Sci. Eng. C 2014, 35, 347–353. 3. Ko NR.; Sabbatier G.; Cunningham A.; Laroche G.; Oh JK.; Air-Spun PLA Nanofibers Modified with Reductively Sheddable Hydrophilic Surfaces for Vascular Tissue Engineering: Synthesis and Surface Modification. Macromol. Rapid Commun. 2014, 35, 447–453.

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