Potential of Biofermentative Unsulfated Chondroitin and Hyaluronic Acid in Dermal Repair

Abstract

Chondroitin obtained through biotechnological processes (BC) shares similarities with both chondroitin sulfate (CS), due to the dimeric repetitive unit, and hyaluronic acid (HA), as it is unsulfated. In the framework of this experimental research, formulations containing BC with an average molecular size of about 35 KDa and high molecular weight HA (HHA) were characterized with respect to their rheological behavior, stability to enzymatic hydrolysis and they were evaluated in different skin damage models. The rheological characterization of the HHA/BC formulation revealed a G' of 92 ± 3 Pa and a G″ of 116 ± 5 Pa and supported an easy injectability even at a concentration of 40 mg/mL. HA/BC preserved the HHA fraction better than HHA alone. BTH was active on BC alone only at high concentration. Assays on scratched keratinocytes (HaCaT) monolayers showed that all the glycosaminoglycan formulations accelerated cell migration, with HA/BC fastening healing 2-fold compared to the control. In addition, in 2D HaCaT cultures, as well as in a 3D skin tissue model HHA/BC efficiently modulated mRNA and protein levels of different types of collagens and elastin remarking a functional tissue physiology. Finally, immortalized human fibroblasts were challenged with TNF-α to obtain an in vitro model of inflammation. Upon HHA/BC addition, secreted IL-6 level was lower and efficient ECM biosynthesis was re-established. Finally, co-cultures of HaCaT and melanocytes were established, showing the ability of HHA/BC to modulate melanin release, suggesting a possible effect of this specific formulation on the reduction of stretch marks. Overall, besides demonstrating the safety of BC, the present study highlights the potential beneficial effect of HHA/BC formulation in different damage dermal models.

Keywords: ECM biomarkers; chondroitin; hyaluronan gels; skin model; striae distensae; wound healing.

Figure 1

Mechanical spectrum (a) and complex viscosity as a function of the oscillation frequency (b), recorded at 37 °C, 1% strain.

Figure 2

Sensitivity of HHA and HHA/BC preparations to enzymatic hydrolysis. Residual (%) sample fraction with M_w higher than 1 MDa after 2 h and 4 h of incubation with BTH (1 U/mL) (§ p < 0.05 vs. HHA).

Figure 3

Wound healing experiments/assays. (a) Representative fields of view of scratched HaCaT over time in the presence of the different product formulations. CTR, control; BC, biotechnological chondroitin; HHA, high molecular weight hyaluronic acid. Scale bar, 100 μm. (b) Quantitative analysis of wound closure [(A₀ − A/A₀) × 100] vs. the time. (c) Average time (±SD) to achieve a specific percentage of wound closure, namely 40 and 80% for untreated samples (CTR) and in presence of the products.

Figure 3

Wound healing experiments/assays. (a) Representative fields of view of scratched HaCaT over time in the presence of the different product formulations. CTR, control; BC, biotechnological chondroitin; HHA, high molecular weight hyaluronic acid. Scale bar, 100 μm. (b) Quantitative analysis of wound closure [(A₀ − A/A₀) × 100] vs. the time. (c) Average time (±SD) to achieve a specific percentage of wound closure, namely 40 and 80% for untreated samples (CTR) and in presence of the products.

Figure 4

RNA was extracted from HaCat cells. and qRT-PCR were performed to determine the gene expression of COLI, COL III, COLIV, COL VII and ELS at 4 h (a) and (b) 24 h. * p < 0.01; § p < 0.05 vs. CTR. Data are presented as mean ± SD.

Figure 5

Cell viability of HDF-hTERT exposed to desiccation under no protective conditions (CTR−) and after the potentially protective pre-treatment with HHA/BC, BC and HHA. Cell viability was reported as % in respect to unstressed cells (CTR+). * p < 0.01; § p < 0.05 vs. CTR.

Figure 6

Effect of HHA, BC and HHA/BC on 3D FT-Skin. Data are presented as mean ± SD. At 24 h and 7 days, total RNA was extracted, and qRT-PCR was performed to determine the expression levels of the genes coding for COLI, COL III, COLIV, COL VII and ELS at 24 h (a,b) 7 days. * p < 0.01; § p < 0.05 vs. CTR.

Figure 7

Immunofluorescence elastin staining 7 days after the treatments on 3D skin. Higher elastin expression in the presence of HHA/BC treatments, mainly localized at dermal layer respect to the CTR and HHA and BC alone. Scale bar 100 µm.

Figure 8

Histological cross section of the full thickness human skin model. Black arrow indicates the injected materials. Mag 20×; scale bar 100 µm.

Figure 9

Effect of HHA, BC and HHA/BC on HDF insulted in vitro with TNFα. Data are presented as mean ± SD. qRT-PCR was performed to determine the gene expression of (a) Col I, (b) Col III, (c) ELS and (d) FIB-1 after 4 h and 24 h of incubation. * p < 0.01; § p < 0.05 vs. CTR.

Figure 10

(a) Fibrillin, type I collagen and elastin expression determined by western blotting analyses in HDF treated with TNF-α and HHA, BC and HHA/BC samples. (b) The expression of each protein was normalized in respect to actin used as housekeeping internal control. Data are reported as average ± SD (n = 2).

Figure 11

HFD cells treated with TNFα and then treated with HHA, BC and HA/BC samples. Protein expression of cytokines IL-6 by ELISA assay. Samples are in triplicates. * p < 0.05 vs. TNFα.

Figure 12

Melanin content in lysed HaCat/HEMa-LP co-colture after 24 and 48 h of HHA, BC and HHA/BC treatments. § p < 0.05 vs. CTR.

Figure 13

Schematic diagram that shows the experimental set up.