Differentiation of Mononuclear Cells From Cord Blood in Endothelial Cells Forming Colony Onto Bioactive Poly(ethylene terephthalate) Film for In Situ Endothelialization

Caroline Royer (Département de Génie des Mines, de la Métallurgie et des Matériaux, CERMA, Québec, Canada)
Andrée-Anne Guay Bégin (Laboratoire d’Ingénierie de Surface, Centre de recherche du CHU de Québec, Canada)
Pascale Chevallier (Laboratoire d’Ingénierie de Surface, Centre de recherche du CHU de Québec, Canada)
Laurent Plawinski (CNRS UMR5248 CBMN, Université de Bordeaux, Bordeaux INP, France)
Christel Chanseau (CNRS UMR5248 CBMN, Université de Bordeaux, Bordeaux INP, France)
Marie-Christine Durrieu (CNRS UMR5248 CBMN, Université de Bordeaux, Bordeaux INP, France)
Gaétan Laroche (Département de Génie des Mines, de la Métallurgie et des Matériaux, CERMA, Québec, Canada)

Introduction

Cardiovascular diseases are one of the leading causes of mortality worldwide. When the replacement of diseased arteries is needed, vascular surgeons often have no choice but to use synthetic arterial prostheses made either of poly(ethylene terephthalate) (PET or Dacron®) or expanded tetrafluoroethylene (ePTFE or Teflon®). However, the patency rate of these devices is poor due to the lack of endothelialization. Accordingly, several researches focus on promoting the endothelialization of vascular grafts through the conjugation of bioactive molecules on their surfaces to target mature cells and, more recently, endothelial progenitor cells (EPCs). In this context, the strategy used in this work consisted in the immobilization of innovative bioactive molecules onto biomaterial surface. We opted for four bioactive molecules to be conjugated on PET surfaces to promote and enhance recruitment, adhesion and the differentiation of mononuclear cells isolated from the cord blood into endothelial progenitor cells. The aforementioned biomolecules were selected for their ability to enhance adhesion (GRGDS1 and GHM2) peptides or induce differentiation of endothelial progenitor cells with sitagliptin3 and SFLLRN4 peptide. These latter molecules are specific for endothelial progenitor cells and have never been grafted on surfaces. For this purpose, these four bioactive molecules were homogenously conjugated to mimic the extracellular matrix (ECM) organization with the emphasis put on the assessment of these bioengineered materials on mononuclear and endothelial progenitor cells fate. This work will focus on the biological characterization, the analysis of identity and the state of differentiation of cells emerging from modified surfaces.

Materials and Methods

PET commercial films (thickness=100 mm) were purchased from GoodFellow (France). Sitagliptin was from Biovision, Milpitas, CA. The three peptides were custom-made by Genecust (Luxembourg). PET films were first oxidized, then functionalized according to a protocol previously described by Chollet et al1. All materials were characterized using XPS, AFM, and contact angle measurements. The activity of sitagliptin-grafted surfaces was ascertained by analyzing ftheir capability to inhibit DPP4 with or without cells. Mononuclear cells were isolated from cord blood according to the procedure reported by Smadja4. They were then sorted out by attaching them to anti-CD34+ conjugated microbeads (Miltenyi Biotec, Germany) and characterized by flow cytometry prior seeding them on the abovementioned bioengineered materials. The formation of endothelial cell colonies was observed by optical microscopy. Additional tests were performed in matrigel® in order to ascertain that the isolated cells were functional EPCs. The endothelial progenitor phenotype was confirmed by qRT-PCR, by analysing CD34, CD133, KDR, vWF, eNOS markers, and GAPDH as a positive control.

Results

According to XPS and contact angle, bioactive molecules are covalently grafted on our surface. All surfaces exhibited similar roughness, as measured by AFM (Ra= 11±3nm). DPP4 test enable to evidence that sitagliptin remains active, even after its surface conjugation. After selection, the purity of CD34+ cells obtained is 90%. These stem cells were then seeded on the bioactive materials and endothelial progenitor cells appeared approximately after the 11th day. The first colonies were observed at day 11 on sitagliptin surfaces and at day 13 for all other samples. Three out of 4 surfaces (SFLLRN, GRGDS, sitagliptin) gave rise to confluent colonies after 21 days of culture while none or very small endothelial progenitor colonies were observed on oxidized PET and PET-GHM, respectively (figure 1). EPCs emerging from these surfaces can form tube-like structure in matrigel®(figure 2). Optical microscopy enables to evidence the presence of EPCs colonies while qRT-PCR experiments were performed to confirm cell phenotype. CD34 and CD133 expression was lower on SFLLRN and grafted surfaces as opposed to other conditions. Endothelial markers (vWF and KDR) expression increased at 10 days after seeding and after 14 days in culture, eNOS expression increased in a significant way, leading us to conclude that EPCs are functional.

Discussion and Conclusion

PET materials were homogeneously functionalized with selected adhesion and differentiation molecules. Mononuclear cells were differentiated into endothelial progenitors on some of the bioengineered surfaces. GRGDS and SFLLRN surfaces seem to be the more promising couple for both adhesion and differentiation as compared to GHM and sitagliptin. The cells obtained from the differentiation of CD34+ cells are able to form tube-like structure on matrigel®, therefore attesting of the functionality of EPCs. Fluorescence microscopy and shear stress surfaces will be tested to determine the influence of the flow on the attachment, differentiation, and alignment of endothelial progenitor cells.


Figure 1: Optical microscopy images of EPCs obtained from mononuclear cells on our modified surfaces (magnification x20).

Figure 2: Optical microscopy images of tube-like structure obtained with EPCs on matrigel® (magnification x10).

Acknowledgements

The authors thank the « Région Aquitaine » and the National Science and Engineering Research Council (NSERG, Canada), for financial supports.

References

1. Chollet, C. et al, The Effect of RGD Density on Osteoblast and Endothelial Cell Behavior on RGD-Grafted Polyethylene Terephthalate Surfaces. Biomaterials 2009, 30, 711–720

2. Veleva, A. N. et al, Selection and Initial Characterization of Novel Peptide Ligands That Bind Specifically to Human Blood Outgrowth Endothelial Cells. Biotechnol. Bioeng. 2007, 98, 306–312

3. Brenner, C. et al, Short-Term Inhibition of DPP-4 Enhances Endothelial Regeneration after Acute Arterial Injury via Enhanced Recruitment of Circulating Progenitor Cells. Int. J. Cardiol. 2014, 177, 266–275

4. Smadja, D. PAR-1 Activation on Human Late Endothelial Progenitor Cells Enhances Angiogenesis In Vitro With Upregulation of the SDF-1/CXCR4 System. A. T. V. B. 2005, 25, 2321–2327

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