Assessment of Melatonin-Cultured Collagen/Chitosan Scaffolds Cross-Linked by a Glyoxal Solution as Biomaterials for Wound Healing

Abstract

Chitosan (CTS) and collagen (Coll) are natural biomaterials that have been extensively used in tissue engineering or wound healing applications, either separately or as composite materials. Most methods to fabricate CTS/Coll matrices employ chemical crosslinking to obtain solid and stable scaffolds with the necessary porosity and mechanical properties to facilitate regeneration. In this study, we comparatively assessed the physicochemical properties of 3D scaffolds loaded with a cross-linker, glyoxal. Using a scanning electron microscope, we evaluated the microstructure of resultant matrices and their mechanistic testing by the determination of the compressive modulus (E_mod), the maximum force (F_max), thermogravimetric analysis (TG), Fourier Transform Infrared Spectroscopy-Attenuated Total Reflectance (FTIR-ATR), and proliferation rate in vitro using human epidermal keratinocytes and dermal fibroblasts cultured in presence of melatonin solution (10^-5 M). We observed that enhanced content of collagen (50CTS/50Coll or 20CTS/80Coll compared to 80CTS/20Coll) significantly elevated the physicochemical capacities of resultant materials. Besides, presence of 5% glyoxal increased porosity, E_mod and F_max, compared to scaffolds without glyoxal. Finally, keratinocytes and dermal fibroblasts cultured on subjected matrices in presence of melatonin revealed a prominently enhanced growth rate. This indicates that the combination of glyoxal and melatonin make it imperative to consider these materials as a promising approach for targeting skin tissue engineering or regenerative dermatology.

Keywords: biopolymers; chitosan; cutaneous cells; glyoxal; melatonin; scaffolds; wound healing.

Figure 1

SEM images of 80CTS/20Coll (A), 80CTS/20Coll+5%G (B), 50CTS/50Coll (C), 50CTS/50Coll+5%G (D), 20CTS/80Coll (E), and 20CTS/80Coll+5%G (F) (magnification: 150×).

Figure 2

The compressive modulus (E_mod) (A) and maximum force (F_max) (B) obtained for the scaffold during the compression; * p < 0.001.

Figure 3

FTIR-ATR spectra obtained for all tested samples where the characteristic peaks are visible upon incorporation of 5% glyoxal (arrows).

Figure 4

Human epidermal keratinocytes (NHEK) and human dermal fibroblasts (NHDF) were seeded on CTS/Coll scaffolds alone or containing 5% glyoxal in comparison to the culture medium including 10⁻⁵ M melatonin, cultured for 96 h, and viability was assessed using the MTT assay as described in Materials and Methods. Data are presented as mean + S.D. (n = 6), expressed as a percentage of the control sample (cells cultured on scaffolds without glyoxal and without melatonin). Statistically significant differences were indicated as follows; ^§ p < 0.01: vs. 50CTS/50Coll sample, # p < 0.01: comparison of the corresponding samples containing 5% glyoxal and scaffolds alone, and corresponding statistical comparison as * p < 0.05, *** p < 0.001, n.s.-not significant.