Three-dimensionally decellularized human amniotic membrane scaffold: structure, processing, and biological properties.

Tissue engineering (TE) combines cells, biomaterials, and bioactive molecules to create functional tissue constructs aimed at restoring tissue function and improving patient outcomes. The human amniotic membrane (HAM) is a widely studied biological scaffold for various biomedical applications. Decellularization of HAM (dHAM) is necessary to reduce graft rejection but depletes stem cells and growth factors, potentially limiting regenerative potential. This study investigates the recellularization of dHAM with adipose-derived mesenchymal stem cells (AdMSCs) to enhance its bioactivity using a novel 3D seeding technique. Decellularized HAM (dHAM) was recellularized with AdMSCs employing a novel 3D seeding method to achieve uniform cell distribution within the scaffold. The viability, differentiation potential, and morphology of AdMSCs were assessed in both 2D and 3D culture systems. Flow cytometry was used to evaluate the differentiation capacity of AdMSCs into osteogenic, chondrogenic, and adipogenic lineages. Field emission scanning electron microscopy (FESEM) was utilized to analyze cell morphology and penetration depth within the scaffold. AdMSC viability was comparable between 2 and 3D cultures, indicating that dHAM scaffolds effectively support cell survival regardless of the culture technique. The composition and properties of dHAM preserved cell functions in both culture systems. Flow cytometry confirmed the multilineage differentiation potential of AdMSCs. FESEM imaging revealed AdMSCs with extending filopodia on the scaffold surface and cell penetration up to 17.68 µm into the dHAM matrix. The successful 3D recellularization of dHAM with AdMSCs demonstrates its potential as a biological scaffold for stem cell delivery. This approach holds promise for tissue repair and wound healing applications, enhancing the regenerative efficacy of dHAM-based constructs.