A novel multifunctional chitosan-gelatin/carboxymethyl cellulose-alginate bilayer hydrogel containing human placenta extract for accelerating full-thickness wound healing.

The replication of skin's dermal and epidermal morphology within a full-thickness wound using a bi-layer hydrogel to cater to their distinct needs is a compelling pursuit. Moreover, human placenta extract (HPE), containing a diverse array of bioactive agents, has proven to be effective in promoting the wound healing process and enhancing epidermal keratinocytes. This study presents a multifunctional bi-layer hydrogel incorporating HPE for accelerating full-thickness wound healing through sustained HPE release, inhibition of bacteria invasion, and promotion of cell proliferation. The upper layer of the scaffold, known as the dressing layer, is composed of carboxymethyl cellulose and sodium alginate, serving as a supportive layer for cell proliferation. The under layer, referred to as the regenerative layer, is composed of chitosan and gelatin, providing an extracellular matrix-like, porous, moist, and antibacterial environment for cell growth. The scaffold was optimized to replicate the morphology of the dermal and epidermal layers, with suitable fibroblast infiltration and a pore size of approximately 283μm. Furthermore, the degradation rate of the samples matched the wound healing rate and persisted throughout this period. The sustained HPE release rate, facilitated by the degradation rate, was optimized to reach ~98% after 28 days, covering the entire healing period. The samples demonstrated robust antibacterial capabilities, with bacterial inhibition zone diameters of and 2.63±0.12cm for S. aureus and E. coli, respectively. The biocompatibility of the samples remained at approximately 68.33±4.5% after 21 days of fibroblast cell culture. The in vivo experiment indicated that the HPE@Bilayer hydrogel promotes the formation of new blood vessels and fibroblasts during the early stages of healing, leading to the appropriate formation of granulation tissue and a wound contraction rate of (79.31±3.1)%. Additionally, it resulted in the formation of a thick epidermal layer (keratinization) that effectively covered all the impaired areas, achieving a wound contraction rate of 95.83±6.3% at the late stage of wound healing. Furthermore, immunohistochemistry staining for CD31 and TGF-β revealed that the HPE@Bilayer group had 22 blood vessels/field and 34%-66% immunoactive cells, respectively, after 14 days of healing. However, by day 21, angiogenesis and TGF-β expression had declined, demonstrating that the wounds had been successfully treated with minimal scarring.