Evaluation of differentiation of the umbilical cord blood-derived mesenchymal stem cells into male germ-line cells in two-dimensional and three-dimensional scaffolds containing naringin

Objective(s): This study aimed to investigate the differentiation potential of human umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) into male germ-like cells using a three-dimensional poly (L-lactic acid)/gelatin nanofiber (PLLA/GNF) scaffold incorporated with naringin (Nar).Materials and Methods: The composite scaffold was fabricated by freeze-drying a PLLA solution containing ۱% Nar and electrospun GNFs. Scaffold characterization included morphological analysis, porosity assessment, drug release profiling, and biodegradation testing. UCB-MSCs were isolated and characterized for surface markers (CD۴۴⁺/CD۹۰⁺/CD۳۴⁻/CD۴۵⁻) via flow cytometry. Cells were seeded onto scaffolds and differentiated using BMP۴ (۲۵ ng/ml) alone or in combination with retinoic acid (۱۰⁻⁶ M). Evaluations included the MTT assay for viability, immunofluorescence for germ cell markers (PLZF, DAZL, OCT۴), and in vivo biocompatibility assessed by subcutaneous implantation in rat models.Results: The scaffold demonstrated optimal physical properties, with a porosity of ۸۱.۱۳ ± ۲.۲۲% and an average pore size of ۱۰۸.۳۱ ± ۹.۲۹ μm. Nar release profile showed sustained delivery with cumulative release of ۳۸.۵ ± ۵.۷۲% over ۱۴ days. Biodegradation rate reached ۵۱.۰۷ ± ۵.۶۴% after ۱۴ days. MTT assay revealed significantly enhanced cell proliferation in Nar-containing scaffolds at both ۲۴ and ۷۲ hr (P< ۰.۰۵). Immunofluorescence analysis demonstrated markedly higher expression of germ cell markers in ۳D cultures compared to ۲D controls. In vivo evaluation confirmed excellent biocompatibility with minimal inflammatory response.Conclusion: The ۳D PLLA/GNF/Nar scaffold creates a favorable microenvironment that effectively promotes UCB-MSC differentiation into male germ-like cells, representing a promising platform for reproductive tissue engineering applications.

کلیدواژه ها:

Biocompatible materials ، Freeze drying ، Germ cells ، Mesenchymal stem cells ، Nanofibers