Dispersion of TiO2 nanoparticles improves burn wound healing and tissue regeneration through specific interaction with blood serum proteins

Abstract

Burn wounds are one of the most important causes of mortality and especially morbidity around the world. Burn wound healing and skin tissue regeneration remain thus one of the most important challenges facing the mankind. In the present study we have addressed this challenge, applying a solution-stabilized dispersion TiO₂ nanoparticles, hypothesizing that their ability to adsorb proteins will render them a strong capacity in inducing body fluid coagulation and create a protective hybrid material coating. The in vitro study of interaction between human blood and titania resulted at enhanced TiO₂ concentrations in formation of rather dense gel composite materials and even at lower content revealed specific adsorption pattern initiating the cascade response, promising to facilitate the regrowth of the skin. The subsequent in vivo study of the healing of burn wounds in rats demonstrated formation of a strongly adherent crust of a nanocomposite, preventing infection and inflammation with quicker reduction of wound area compared to untreated control. The most important result in applying the TiO₂ dispersion was the apparently improved regeneration of damaged tissues with appreciable decrease in scar formation and skin color anomalies.

Figure 1

TEM image of the dried dispersion of the applied TiO₂ nanoparticles produced using modified methodology from ref. (A). Hydrodynamic size of the particles in water (B) and in isotonic salt solution ((C), 0.5 ml of dispersion diluted by 10 ml isotonic NaCl), both by DLS.

Figure 2

Accelerated blood clotting on addition of sol-gel titania, represented by average values of 3 measurements for each of the curves.

Figure 3

SEM images illustrating interaction of nano titania dispersion with human blood and skin samples: Natural blood clot and its EDS analysis (A), enlarged structure of the inner part of the blood clot forming on interaction with the TiO₂ dispersion and its EDS analysis (B), TiO2 crust on the surface of a treated blood clot and its EDS analysis (C). The integrations of EDS spectra are presented in Table S1 (Supplementary). Untreated skin sample (D) coating on the surface of skin sample (E), enlarged view of the titania film on a skin sample (F).

Figure 4

(A) Remaining platelets in whole blood after contact with the titania (TiO₂), polystyrene (PS) and Corline heparin (CHS) surfaces for 60 min at 37 °C. Data represent mean ± SEM (n = 4). (B) Formation of Thrombin-Antithrombin complex in contact with the titania (TiO₂), polystyrene (PS) and Corline heparin (CHS) surfaces. Data represent mean ± SEM (n = 4). Significance was determined by One Way ANOVA analysis using Dunnett’s multiple comparison test. Significant differences are indicated as **p < 0.01; ***p < 0.001; ****p < 0.0001; n.s = non-significant. Results were considered statistically significant for p < 0.05.

Figure 5

(A) Wound surface reduction: Group 1 – healing of untreated 2^nd degree burns, Group 2 – second degree burns treated with nano titania, Group 3 – untreated 4^th degree burns, Group 4–4^th degree burns treated with nano titania. Each value is an average of 3 rats/group (for details, please, see Supplementary Table. S2). All animals survived until the end of the experiment (day 19). (B) General appearance of the wounds through the healing process for one representative animal from each group.

Figure 6

The optical images of healed skin samples, Group 1, second degree burns, non-treated, – (A) and (B) (x40 and × 100 magnification respectively), Group 2, second degree burns, treated with 0.1 mL TiO₂ sol daily – (C) and (D) (x40 and × 100 magnification respectively), Group 3, fourth degree burns, untreated, – (E) and (F) (x40 and × 100 magnification respectively), and Group 4, fourth degree burns, treated with 0.1 mL TiO₂ sol daily, – (G) and (H) (x40 and × 100 magnification respectively). Numbers are tissue structural elements as follows: 1- epidermis; 2 - sweat glands; 3 – scar; 4 – derma; 5 – hypodermis; 6 -hair follicles; 7 - sebaceous glands; 8 – de-epithelialized scar tissue; 9 – scar vessels; 10 - inflammatory infiltration in the scar.

Figure 7

Concentrations of TiO₂ in the liver, spleen, kidney and lung of the rats untreated, control (to the left), and treated (to the right) by nano dispersion in the course of burn wound healing on 1,14 and 28 days respectively.