Chitosan-collagen-G-90 film enhances wound closure and collagen remodeling: FTIR imaging and PCA-kNN analysis

Chitosan-based films containing different bioactive components were developed to assess their effect on wound healing in a dermal rat skin tissue. Four formulations pure chitosan, chitosan-collagen (ACS), chitosan conjugated with glycolipoprotein extract from earth worm, namely chitosan-G-90, and a mixture of three constituents, chitosan-ACS-G-90 were compared with an untreated control. 14 days post-injury, wound closure was greatest in the chitosan-ACS-G-90 group. Ratio mapping (1325-1351 cm^-1/1190-1357 cm^-1) confirmed that the chitosan-ACS-G-90 film provided the most consistent improvement in collagen organization, highlighting its superior role in matrix maturation. Vector-normalized, second-derivative spectra from the protein (1710-1475 cm^-1) and fingerprint (1800-900 cm^-1) regions revealed treatment-dependent variations in amide I/II profiles, collagen triple-helix markers (1338 cm^-1, 1203 cm^-1), and distinct absorptions in the carbohydrate associated region (∼1150-1000 cm^-1). These bands while often attributed to glycosaminoglycans or other ECM polysaccharides may also include overlapping contributions from collagen side chains and nucleic acid phosphate vibrations (DNA/RNA), reflecting the inherently complex biochemical composition of dermal ECM. Principal Component Analysis (PCA) clearly separated the chitosan-ACS-G-90 group from untreated dermis, with loadings associated with cross-link-related (∼1690 cm^-1) and amide I/II bands (∼1658 cm^-1, 1566-1543 cm^-1). Classification of PCA scores using k-Nearest Neighbors (kNN) achieved high accuracy (AUC = 0.993). These results show that the chitosan-ACS-G-90 accelerates wound closure and enhances collagen remodeling.