Decellularized amniotic membrane hydrogel promotes mesenchymal stem cell differentiation into smooth muscle cells

Previous studies showed that the bladder extracellular matrix (B-ECM) could increase the differentiation efficiency of mesenchymal cells into smooth muscle cells (SMC). This study investigates the potential of human amniotic membrane-derived hydrogel (HAM-hydrogel) as an alternative to xenogeneic B-ECM for the myogenic differentiation of the rabbit adipose tissue-derived MSC (AD-MSC). Decellularized human amniotic membrane (HAM) and sheep urinary bladder (SUB) were utilized to create pre-gel solutions for hydrogel formation. Rabbit AD-MSCs were cultured on SUB-hydrogel or HAM-hydrogel-coated plates supplemented with differentiation media containing myogenic growth factors (PDGF-BB and TGF-β1). An uncoated plate served as the control. After 2 weeks, real-time qPCR, immunocytochemistry, flow cytometry, and western blot were employed to assess the expression of SMC-specific markers (MHC and α-SMA) at both protein and mRNA levels. Our decellularization protocol efficiently removed cell nuclei from the bladder and amniotic tissues, preserving key ECM components (collagen, mucopolysaccharides, and elastin) within the hydrogels. Compared to the control, the hydrogel-coated groups exhibited significantly upregulated expression of SMC markers (p ≤ .05). These findings suggest HAM-hydrogel as a promising xenogeneic-free alternative for bladder tissue engineering, potentially overcoming limitations associated with ethical concerns and contamination risks of xenogeneic materials.