Bovine Placental Cotyledon-Derived Hydrogel: Methodology to Produce a Substrate for Tissue Engineering

Abstract

Bioengineering aims to develop biomaterials that closely mimic the native extracellular matrix (ECM) to support tissue regeneration. This study presents a detailed protocol for producing hydrogels derived from decellularized bovine placental cotyledons. Bovine placentas at 4-5 months of gestation (<i>n</i> = 10) were subjected to vascular perfusion with increasing concentrations of sodium dodecyl sulfate (0.01-1%) and Triton X-100 (1%), which effectively removed cellular components. Decellularization efficacy was confirmed by histological (hematoxylin and eosin and 4',6-diamidino-2-phenylindole [DAPI] staining), molecular, and structural analyses, including residual genomic DNA quantification averaging 9.1 ng/mg of dry tissue. The ECM scaffolds were enzymatically digested using 0.1% (w/v) pepsin in 0.01 M HCl and reconstituted with sodium alginate at concentrations of 5%, 8%, 10%, and 12% (w/v). Crosslinking was achieved with 1% calcium chloride. Among the tested formulations, hydrogels containing 12% alginate demonstrated greater mechanical stability and preserved three-dimensional architecture, including interconnected porosity, as evidenced by scanning electron microscopy. Cytocompatibility was evaluated by culturing canine adipose-derived mesenchymal stem cells on both decellularized biomaterials and hydrogels. DAPI staining revealed nuclei after 7 and 25 days of culture, indicating cell presence and distribution throughout the constructs. These results indicate that bovine cotyledon-derived ECM hydrogels maintain structural and biochemical features favorable for cell interaction and may serve as adaptable platforms for tissue engineering, dermal repair, and three-dimensional cell culture.