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Recent evidence suggests that protecting human amnion and chorion matrices (HACM) during processing enhances the performance of HACM for wound repair and tissue regeneration. We utilised a diabetic (db/db) delayed wound healing mouse model. Treatment of db/db full‐thickness excisional wounds with HACM, processed with a polyampholyte preservative accentuated the proliferative phase of wound healing that decreased the time necessary to heal wounds. Polyampholyte protection improved the preservation of growth factors and cytokines during room temperature storage following E‐beam sterilisation and improved its function in wound healing applications. Our findings indicate protected HACM tissue up‐regulated MIP2, NF‐kB, TNF‐α, KI‐67, and Arg1 (0.6‐fold to 1.5‐fold) but those changes were not statistically significant. Immunofluorescent assessment identifying cell activity illustrated an induction of the proliferative phase of wound healing and a switch from an inflammatory macrophage phenotype (M1) to a pro‐regenerative macrophage phenotype (M2a). Genomic profiling of 282 genes was performed using Nanostring from co‐cultures of human macrophages and fibroblasts. The polyampholyte + HACM‐treated group, compared with the HACM or polyampholyte alone groups, had a statistically significant up‐regulation (32–368 fold) of 12 genes primarily involved in macrophage plasticity including CLC7, CD209, CD36, HSD11B1, ICAM1, IL1RN, IL3RA, ITGAX, LSP1, and PLXDC2 (adj. p‐value < 0.05). The polyampholyte alone group demonstrated statistically significant down‐regulation of four genes ADRA2, COL7A1, CSF3, and PTGS2 (adj. p < 0.05). The HACM alone group up‐regulated four genes ATG14, CXCL11, DNMT3A, and THBD, but the results were not statistically significant. Biomechanical measurements indicated that wounds treated with polyampholyte‐protected HACM had more tensile integrity compared with wounds treated with HACM alone. These findings indicate that better protection of HACM during processing stabilises the HACM matrix, which may lead to improved wound healing outcomes. [ABSTRACT FROM AUTHOR]