Bifunctional Composite Biomaterials Integrating Bacteriophages and Phytoextracts for Chronic Wound Healing

Chronic wounds are complex and dynamic environments that pose challenges due to impaired healing and bacterial infections, which are increasingly difficult to address because of antimicrobial resistance. Therefore, the infected and damaged tissue at the wound site requires advanced measures to ensure efficient regeneration, highlighting the need for a combinatorial approach. In this direction, this study aims to develop a composite biomaterial using 2-hydroxyethylmethacrylate (HEMA)-chitosan (HC) cryogels as a biomaterial matrix. Further, these HC cryogels will be incorporated with the bacteriophages (phages) against Klebsiella pneumoniae, along with phytoextracts of Berberis aristata (Darvi) in powder (P) formulation, and Trichosanthes dioica (Patola), Azadirachta indica (Neem), and Pongamia pinnata (Karanja) together in oil (O) formulation. The physicochemical properties of the developed composite cryogels were evaluated through swelling studies, mechanical testing, and degradation analysis. The antimicrobial properties against K. pneumoniae were observed in phage-containing cryogels through an agar diffusion assay, colony forming unit assay, and phage release was also determined over a 24-hour period. Further, biological studies showed negligible cytotoxicity, as confirmed by a direct contact test with L929 fibroblast cells. The MTT assay and live-dead staining on days 1, 3, and 7 showed significantly enhanced cell proliferation in the HC-O and HC-PO groups in comparison to the HC and HC-P groups. The wound-healing potential of the composite cryogels, assessed using a scratch assay, further demonstrated the highest healing in the HC-PO group. Therefore, this study provides evidence for the wound-healing potential of the as-developed composite cryogels due to their bifunctional role in phage-mediated antimicrobial properties and enhanced fibroblast migration as well as proliferation under the influence of phytoextracts.