Biofunctional Nanofibrous Substrate Comprising Immobilized Antibodies and Selective Binding of Autologous Growth Factors

The immobilization of biomolecules at the surface of different biomedical devices has attracted enormous interest in order to enhance their biological functionality at the cellular level. This work aims to develop a biofunctional polymeric substrate capable of selectively binding growth factors (GFs) of interest from a pool of proteins present in a biological fluid: platelet lysate (PL). To achieve this goal, the surface of electrospun PCL nanofibers needs to be activated and functionalized to be able to insert chemical groups for the immobilization of antibodies. After determining the maximum immobilization capacity of each antibody, TGF-β1 (12 μg mL^–1), bFGF (8 μg mL^–1), and VEGF (4 μg mL^–1), the next step was to confirm their bioavailability using recombinant proteins. The binding efficiency of PL-derived GFs was of 84–87% for TGF-β1, 55–64% for bFGF, and 50–59% for VEGF. Cellular assays confirmed the biological activity of the bound VEGF (both recombinant and PL-derived). Multiple antibodies (i.e., bFGF and VEGF) were also immobilized over the same structure in a mixed or side-by-side fashion. Using both autologous biological fluids and cells, it is possible to use this platform to implement very effective and personalized therapies that can be tailored to specific medical conditions

Flow cytometry analysis of hASCs.

<p>The expression pattern of specific antigens on the surface of the hASCs is depicted with representative histograms and the expression of each marker. The cell population expressed CD29, CD44, CD73 and HLA-ABC, and did not express CD34, CD45 and HLA-DR.</p

Proliferation and viability of hASCs cultured on TCPS and electrospun PHB/PHB-HV fiber mesh.

<p>(A) MTT proliferation assays performed 7, 14 and 21 days after the cell seeding and cultured in two specific medium: the basal medium and the endothelial differentiation medium. The results are expressed as the means ± SD, (*) indicating a significant difference with p<0.05, (**) p<0,01, (***) p<0,001; (B) cell viability after 21 days of cell culture with the basal medium and (C) the endothelial differentiation medium on the electrospun PHB/PHB-HV fiber mesh, as analyzed by Calcein-AM staining.</p

RT-PCR analysis of VEGFR2 mRNA expression during endothelial differentiation on the electrospun PHB/PHB-HV fiber mesh and TCPS.

<p>Total RNA was extracted from cells cultured on basal medium and endothelial differentiation medium for analysis of VEGFR2 mRNA expression. The cells were cultivated up to 21 days.</p

Protein expression of hASCs during endothelial differentiation.

<p>Confocal images of the expression of the VE-Cadherin (A) and the vWF factor (B) after 21 days. A.1 and B.1 - hASCs cultured with the basal medium on the TCPS coverslips, A.2 and B.2 - hASCs cultured with the endothelial differentiation medium on the TCPS coverslips, A.3 and B.3 - hASCs cultured with the basal medium on the electrospun PHB/PHB-HV fiber mesh, A.4 and B.4 - hASCs cultured with the endothelial differentiation medium on the electrospun PHB/PHB-HV fiber mesh. Scale bar 20 µm. The images, A.2, A.4, B.2 and B.4 represent the overlay of bright-field and confocal images for visualization of the fiber mesh. (C) Fluorescence intensity of the expression of VE-Cadherin and the vWF factor in cells differentiated on TCPS coverslip and electrospun PHB/PHB-HV fiber mesh. The results are expressed as the means ± SD, (*) significant difference for p<0,05. (a.u): arbitrary units.</p

Regulation of Human Mesenchymal Stem Cell Osteogenesis by Specific Surface Density of Fibronectin: a Gradient Study

The success of synthetic bone implants requires good interface between the material and the host tissue. To study the biological relevance of fibronectin (FN) density on the osteogenic commitment of human bone marrow mesenchymal stem cells (hBM-MSCs), human FN was adsorbed in a linear density gradient on the surface of PCL. The evolution of the osteogenic markers alkaline phosphatase and collagen 1 alpha 1 was monitored by immunohistochemistry, and the cytoskeletal organization and the cell-derived FN were assessed. The functional analysis of the gradient revealed that the lower FN-density elicited stronger osteogenic expression and higher cytoskeleton spreading, hallmarks of the stem cell commitment to the osteoblastic lineage. The identification of the optimal FN density regime for the osteogenic commitment of hBM-MSCs presents a simple and versatile strategy to significantly enhance the surface properties of polycaprolactone as a paradigm for other synthetic polymers intended for bone-related applications