Structure–property–function relationships of supercritical CO2-processed amniotic membrane: An ex vivo ocular proof of concept

• Supercritical carbon dioxide alters amniotic membrane ultrastructure. • Correlations among elasticity, bioactivity, and matrix composition are demonstrated. • The scCO 2 process is a promising strategy for sterile off-the-shelf amniotic membrane. Human amniotic membrane (AM) exhibits a range of biological and structural properties that support its clinical use in regenerative medicine, particularly for ophthalmologic applications. This study aims to develop a readily available, sterile AM using modified (m)-Supercrit® process. Compared with freeze-drying, m-Supercrit® processing produces a transparent, thin, and hydrophilic membrane, key attributes for corneal applications. Mechanistically, m-Supercrit® processing induces marked alterations in collagen and glycosaminoglycan composition, which are associated with a significant increase in the membrane’s linear elastic modulus. Although m-Supercrit®-processed AM shows a reduction in bioactivity and cell migration, including a significant decrease in HGF and b-FGF levels, it retains essential biological functions such as hemocompatibility, cytocompatibility, cell adhesion/proliferation, anti-inflammatory, and anticancer activities. Importantly, the clinical feasibility of handling and suturing m-Supercrit®-processed AM is demonstrated in an ex vivo ocular surface model. Overall, these findings support the m-Supercrit® process as a promising strategy for developing an off-the-shelf, sterile amniotic membrane suitable for ophthalmologic clinical applications.