Bladder tissue engineering is a promising approach to generating new tissues to replace damaged or diseased organs, breaking the limitations of current therapies using reconfigured autogenous bowel segments. Adipose tissue extract (ATE) is a non-cellular three-dimensional macromolecular network that provides an appropriate local microenvironment for cells and affects cellular metabolism, proliferation, differentiation, or organoid formation. This study integrated electrospun nanofibers mats and hyaluronic acid colloidal solution to constitute a multilayered nanofibrous patch, where ATE was added as a biological additive. In vitro study, we observed an increase in the pluripotent marker proteins (Oct-4, Sox-2, and Nanog) of adipose-derived stem cells (ADSCs) and bone mesenchymal stem cells (BMSCs) when cultured with ATE. The CCK-8 test, scratch assay, transwell migration assay, and tube formation assay demonstrated the positive effect of ATE on endothelial cells (ECs) proliferation, migration, and angiogenesis. The feasibility and effectiveness of the patch were investigated in a rat bladder defect model. The primary ideal results revealed the patches' great potential for tissue engineering repair and regenerative medicine. (c) 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).