(674c) Heparin-Collagen Layer-By-Layer Coatings Enhance Adhesion and Proliferation of Schwann Cells for Nerve Tissue Regeneration

The use of nerve guide conduits (NGC) is an excellent option to replace the use of autografts in peripheral nerve repair. Autographs are undesirable due to the need for multiple surgeries. However, NGCs fail to repair gaps greater than 1 cm. One approach to promote the repair of large gaps if by including growth factors (GFs)¹. Our work focuses on using the layer-by-layer (LBL) technique to generate polymeric coatings for growth factor presentation, that could be applied to NGCs. This versatile method allows the use of one or more polymers and even the addition of other molecules such as drugs or proteins². In this work, a multilayer combination of heparin and collagen (HEP/COL) was used and its in vitro bioactivity was evaluated by measuring Schwann cells (SC) real-time behavior (using an impedance-based technique), cell morphology (via fluorescence microscopy), viability (PrestoBlue assay), and proliferation (EdU assay) in the presence or absence of the nerve growth factor (NGF) incorporated in the multilayers or in suspension in the culture medium. IR Variable Angle Spectroscopic Ellipsometer (IR-VASE) was used to confirm multilayer construction and the integration of the growth factor to the multilayers. By real-time monitoring of cell behavior for 120 hours, three main regions were observed, an initial attachment region, a cell proliferation region, and a cell confluency stage. The presence of the LbL films enhanced initial cell attachment and proliferation and increase the confluence in the third stage, while the presence of NGF significantly improved cell attachment to HEP/COL films. PrestoBlue Cell viability test showed that coatings of HEP/COL had higher viability as compared to the tissue culture plastic control. Interestingly, the EdU assay showed that the cells had similar proliferation levels on all tested conditions. By fluorescence microscopy, it was observed that the morphology of Schwann cells is not altered and shows a normal behavior (elongated and narrow) and that after 6 days of culture, cells with better morphology were observed in HEP/COL films with NGF in suspension. IR-VASE spectra revealed that the structure of collagen and heparin continue to be present in multilayers created on flat silicone plates and that by observing the spectra of the samples that possess NGF absorbed in the multilayers, some small differences can be found attributed to the contributions of the amide groups I and II of the protein. Overall, our results show that HEP/COL films are a favorable substrate for Schwann cell culture. They can enhance cell attachment which in turns enhances cell proliferation. They can also serve as reservoirs for NGF presentation. These films are promising as coatings for NGCs to enhance peripheral nerve repair.