Functionally multifaceted scaffolds delivering bioactive compounds for treatment of infectious chronic and ischemic wounds

Chronic wounds arise due to delayed healing, disease conditions, bacterial manifestation etc. that are associated with several challenging hallmarks, mainly involving persistent inflammation, oxidative stress, infections, hypoxic conditions occurring at the wound site. Hence, for targeting such critical and multifactorial complication, an advanced combinatorial approach is prerequisite for efficient and timely healing of chronic wounds. In the present study, a versatile bilayer-type cryogel made up of chitosan–gelatin as base layer for housing the bioactive factors (nitric oxide nanoparticles as signaling molecule, cerium oxide microparticles as antioxidant, calcium peroxide microparticles as oxygen releasing moiety) has been fabricated using the cryogelation method along with a top iodine embedded antibacterial layer. The fabricated scaffold and incorporated bioactive molecules are physiochemically characterized for distinctive parameters and validated for the treatment of ischemic and infected chronic wounds model in vivo. The developed scaffolds display a high-water uptake capacity (∼93%-98%), swelling ratio (∼18.3–22.5) and more than 90% of degradation within a period of 15 days. The release kinetics reveals the release of ∼80% (within 3 days), ∼78% (within 10 days) and ∼94% (within 10 days) iodine, oxygen and nitric acid (NO), respectively. The in vitro study shows excellent biocompatibility and proliferative capacity of the scaffolds besides displaying strong antioxidant and cell migration potential in the in vitro models. The scaffold also elucidates efficacious antibacterial action against Escherichia coli and Staphylococcus aureus. Moreover, the in vivo studies demonstrate bilayer scaffold significantly expedites wound closure within a period of 21 days (∼99.6%) and regeneration in comparison to control and other groups. The histological study shows well-organized skin ultrastructure with granulation tissue, blood vessels and hair follicles in the healed wound of bilayer group. Immuno fluorescence staining results showed presence of angiogenesis marker, CD31, in the treatment groups. The results of scaffold treated ischemic wounds signify prevention of necrosis apart from maintaining native skin architecture in the scaffolds with NO and oxygen releasing properties. Thus, the combinatorial approach for chronic wounds proves effective and paves new ways in the advanced treatment strategies for complex wound conditions.