@article {19243, title = {Development of a gene-activated scaffold for long-term stability of capillary-like structures}, journal = {Gene2Skin Winter School}, year = {2017}, month = {2017-11-13 00:00:00}, address = {Porto, Portugal}, abstract = {

Tissue-engineered skin equivalents mimic key aspects of the human skin, and can be employed as wound coverage for large skin defects or as in vitro test systems for alternative to animal models. However, current skin models lack a functional vasculature limiting clinical and research applications. To trigger the formation of capillary-like structures in a 3D environment is necessary the addition of growth factors which are associated with high cost, bolus release and short half-life. Gene-activated scaffolds were shown to induce in vitro and in vivo controlled release of transgenes, while offering structural support for matrix deposition. This study is focused on the development of a combinatorial dual gene-activated scaffold functionalized for delivery of vascular endothelial growth factor (pVEGF) and fibroblast growth factor-2 (pFGF-2) using chitosan (Ch) and polyethyleneimine (PEI) nanoparticles, assessing its efficacy in endothelial cells{\textquoteright} behavior.

Ch-pDNA and PEI-pDNA nanoparticles were formulated for delivery of pVEGF, pFGF-2 and the combination of pVEGF+pFGF-2 to human dermal fibroblasts (hDFbs). In order to establish the optimal vector for each gene individual delivery, cell proliferation and metabolic activity were studied. VEGF and FGF-2 production was quantified using ELISA while functionality was determined using Matrigel{\texttrademark} assay. The optimal nanoparticle formulations were loaded into collagen-glycosaminoglycan scaffolds that were seeded with hDFbs and human dermal microvascular endothelial cells (hDMECs) to assess new vessel formation using immunocytochemistry.

Transfection efficiencies showed that PEI yielded higher levels of transfection than Ch after 3 days. Transfection of hDFbs with PEI and Ch encoding pVEGF or pFGF-2 demonstrated that PEI transfected cells showed higher proliferation. However, Ch transfected cells showed higher metabolic activity over 14 days. In the case of released VEGF concentration, both vectors showed an increased in a range of 5-7 days. On the other hand, FGF-2 released using Ch peaked at day 5 and day 10, while PEI at day 3. hDMECs seeded in Matrigel{\texttrademark} with conditioned media showed microtubule formation in all systems. From these results, three systems were chosen to be incorporated in the scaffolds: PEI-pVEGF, Ch-pFGF-2 and the dual combination. Ongoing studies are focused on the functionality of these systems to induce new vessel formation.

This study has shown that the gene-activated scaffold technology can be used as a therapeutic approach in skin repair and wound healing. Furthermore, it opens the possibility to develop 3D in vitro vascularized skin models to test new compounds.

}, keywords = {Gene delivery, Gene-delivery matrices, skin model, vascularization}, author = {Moreira, H. and Raftery, R. M. and Reis, R. L. and Marques, A. P. and O\&$\#$039;Brien, F. J.} }