Characterizing electrospun PLGA/PCL matrices for reconstructive pelvic surgery: A role of fiber diameter in new matrix formation and fibrosis

Abstract

Event Abstract Back to Event Characterizing electrospun PLGA/PCL matrices for reconstructive pelvic surgery: A role of fiber diameter in new matrix formation and fibrosis Mahshid Vashaghian1, 2, Behrouz Zandieh-Doulabi3, Theo Smit2 and Jan-Paul Roovers1 1 Academic Medical Center (AMC), University of Amsterdam, Department of Obstetrics and Gynecology, Netherlands 2 Vrij University medical center Amsterdam, Department of orthopedic surgery, Netherlands 3 ACTA, Department of Oral Cell Biology,, Netherlands Introduction: the use of braided meshes for reconstructive pelvic surgery is sometimes hampered by the post-implantation complications they cause, in which the microstructure of the mesh is suggested to play a role[1]. Nanofibrous electrospun matrices resemble the architecture of natural proteins in extracellular matrix, thus could support cellular interactions. Of course, geometrical topography of the matrices, such as fiber diameter, significantly influences the cellular responses[2]-[4] and should be well-defined for optimal clinical outcomes and prevention of fibrosis. Aim of the Study: we aimed to explore the role of fiber diameter of PLGA/PCL electrospun matrices on i) mechanical properties, ii) cell infiltration, iii) new matrix deposition and integration. Also, we characterized the newly-made matrix on different fiber sizes for micro-stiffness, amount of collagenous proteins, myofibroblastic differentiation and collagen type I. We studied these criteria as indications of potential fibrosis in vivo. Methodology: electrospun PLGA/PCL (75/25 Wt/Wt) matrices with different fiber size (1 and 8 μm) were subjected to uni-axial tensile test or cultured with human vaginal fibroblasts for 28 days. Cellular infiltration was measured by an optical sectioning fluorescent microscope on DAPI-stained cells. Immunohistochemistry was performed using α-smooth muscle actin (α-SMA) and collagenI antibodies. Samples were then imaged with an optical sectioning fluorescent microscope, and the integration depth of collagen I and amount of positive α-SMA were semi-quantified on images using ImageJ software (NIH). Total amount of collagenous proteins deposited on both matrices were measured using picrosirius red/fast green kit. Micro-indentation tests were performed on fresh samples to measure the micro-stiffness of the newly-formed extracellular matrix on both matrices. Results and Discussion: larger pores, in large-fiber samples, enhanced cell penetration significantly (Figure.1) but had limited effect on the penetration of collagenI protein through the thickness of the matrices. Perhaps, all the newly-produced proteins will eventually infiltrate but the new tissue can only mature when new ECM is produced by cells and vascularized in depth of the matrices. Fiber size had no effect on the ultimate tensile strength. Large-fiber samples were almost two times stiffer with significantly less ultimate strain than small-fiber samples (Table1). After 28 days of culture, cells on the small-fiber samples were almost 100% myofibroblastic as identified with positive α-SMA (Figure.2), produced significantly more collagen I, more collagenous proteins (Figure.2) and subsequently a matrix which was 1.5 fold stiffer than that produced on large-fiber matrices (Figure.3). This shows that cells on small-fiber samples are more triggered by a larger contact surface, and as a result their metabolic activity and myofibrobaltic differentiation[5],[6] is higher with indications of fibrosis in vitro which might also appear in vivo. Thus, fiber diameter of an electrospun matrix should be designed in such a way that the cell and tissue integration is optimized while fibrosis is prevented in vivo. Significance: electrospun matrices may be potential alternative to improve clinical results in reconstructive surgery of pelvic disorders, provided that its structural characteristics are properly designed.