Titanium Dioxide Nanoparticle/Gelatin: A Potential Burn Wound Healing Biomaterial

Introduction. Over the past few decades, the application of nanotechnology has gained progressive interest for the regeneration of injured and burned tissues. Objective. This study evaluates the effects of titanium dioxide (TiO₂) nanoparticle (NP)/gelatin on burn wound healing in mice. Materials and Methods. Sixty healthy male BALB/c mice with a full-thickness burn wound were randomized into 4 experimental groups of 15 animals each: (1) control group was treated with normal saline; (2) gelatin group was treated with gelatin-based ointment; (3) silver sulfadiazine group was treated with silver sulfadiazine 1% ointment; and (4) TiO₂ NP/gelatin group (TNG) received TiO₂ NP/gelatin. Wound size was measured on postoperative days 2, 6, 10, 14, 18, and 21, and histopathological studies of tissue samples were performed on postop days 7, 14, and 21. Results. The wound area reduction indicated that there was a significant difference between the TNG and other groups (P < .05). Quantitative histological and morphometric studies and the mean rank gained from the qualitative studies demonstrated that there was a significant difference between the TNG and other groups (P < .05). Conclusions. In this study, TNG offered potential advantages in burn wound healing acceleration and improvement through angiogenesis stimulation, fibroblast proliferation, and granulation tissue formation in the early phases of healing. In addition, factors such as accelerated wound repair associated with earlier wound contraction and stability of the damaged area by rearrangement of granulation tissue and collagen fibers were also advantages of TNG.

Severe acute and chronic wounds, such as burns, mechanical trauma, pressure and leg ulcers, congenital skin diseases, and cancer excision, pose a challenge to surgeons. Burns and skin ulcers are especially problematic issues that require more attention.¹ Burn injuries and infections are the most common reason for patient mortality globally; due to this, medications that improve the healing process and possess antimicrobial effects are important.^2,3 Various medications (eg, chemical and herbal remedies) have been utilized with the goal of accelerating burn wound healing.^4,5 

The application of nanotechnology has gained progressive interest for regeneration of injured and burned tissue. Collagen/gold nanocomposites and nanoparticles (NPs) such as zinc, zinc oxide, silver, titanium dioxide (TiO₂), and zeolite can act as efficient drug delivery vehicles for controlled and targeted release or as covering scaffolds in combination with synthetic and natural compounds, including chitin, chitosan, collagen, keratin, polyethylene, and gelatin.^6-8 Gelatin, the natural polymer of bone and skin collagen, is known for its potential advantage as a nanomaterial scaffold due to its bioavailability, nontoxicity, biodegradability, and biocompability.⁹ Moreover, gelatin possesses strong hydrophilic activity and prevents fluid loss in wounded areas that can ultimately result in preserving moisture in the wound and create positive effects on regeneration of burned tissues.¹⁰

Titanium dioxide, a mineral oxide, bears particular and unique characteristics (eg, electrical and photocatalytic effects) and has numerous other applications.¹¹ The most important application areas are purification, disinfection, and destroying tumor cells’ protection against ultraviolet (UV) as well as usages in cosmetics.¹² It also is an ideal photo catalyst due to its UV absorption feature.¹² Considering the UV absorption effect and photocatalytic properties of TiO₂ NP, the nanoscale material creates an antibacterial coat on outer surfaces and prevents the transition of rays.¹³ 

The promising effects of TiO₂ on the burn wound healing process through antimicrobial and cell growth stimulation features have been reported in other studies.^3,9,14 Priorities of burn wound healing include acceleration of the healing process and prevention of infections, both of which are available in TiO₂ NP and optimize its application for a functional recovery.³ 

A review of the exisiting literature revealed a gap in the application of TiO₂ NP for burn wound dressings. Thus, the current study focused on evaluating the effects of TiO₂/gelatin nanocomposites on burn wound healing in a murine model. The assessments were based on planimetric, histologic, and histomorphometic features of the burn wounds.

Study design and animals
Sixty male BALB/c mice aged an average of 8 weeks and weighed a mean of 30 g ± 2 g were used in this study. Two weeks before the study, the animals were housed in individual plastic cages and had chew pellets and fresh water ad libitum. The animals were kept in a temperature of 22°C ± 3°C, 60% ± 5% humidity, and a 12-hour light/dark cycle. The procedures were carried out based on the guidelines of the Ethics Committee of the International Association for the Study of Pain (Washington, DC).¹⁵ All experiments were approved by the Research Ethics Committee of University of Tabriz, Tabriz, Iran.

The animals were randomized into 4 experimental groups of 15 animals each: (1) control group (control) was treated with normal saline, (2) gelatin group (gelatin) was treated with gelatin-based ointment, (3) silver sulfadiazine group (SSD) was treated with silver sulfadiazine 1% ointment, and (4) TiO₂NP/gelatin group (TNG) received TiO₂/gelatin nanocomposite. The animals were treated for 3 weeks and were sampled at the end of days 7, 14, and 21. The animals received treatment once daily until the day of sampling.

All mice were closely observed for any infection and were immediately separated and excluded from the study in case of infection.

Antibacterial testing
The antibacterial activity of TiO₂ NP, TiO₂NP/gelatin, gelatin, and SSD were tested qualitatively. Staphylococcus aureus and Pseudomonas aeroginosa were used for testing the antimicrobial activity of the samples. Samples were cultured on nutrient agar and incubated at 37°C for 48 hours. The antibacterial activity was tested using modified agar diffusion assay (well diffusion method). The plates were examined for possible inhibition zones after 48 hours of incubation. The presence of any clear zone around samples on the plates was recorded as an inhibition against the microbial species. The antibacterial activities of each sample were repeated 3 times.

Synthesis of TiO₂ NP by hydrothermal method
All materials were obtained from Merck (Kenilworth, NJ). For preparing TiO₂ NP by a typical hydrothermal method, 11.3 mL of tetrabutylammonium hydroxide and 10 mL of ethanol were mixed for 1 hour. Then, 10 mL of tetraethyl orthotitanate and 60 mL of deionized water were added to the mixure at 80°C. After aging for 2 hours, the slurry was transferred to a stainless steel autoclave and placed in the oven at 180°C for 48 hours. The autoclave was then cooled to room temperature and annealed at 450°C for 3 hours before further characterization.

Preparation of TiO₂/gelatin 
To prepare the TiO₂/gelatin, 100 mL of aqueous solution of gelatin (20%) was prepared and 0.5% TiO₂ NP was added; the mixture was ultrasounded for 2 hours at 50°C. Then, the homogenous mixture was transferred to the ice bath to form the TiO₂/gelatin gel.

Characterization of TiO₂ NPs
The structural analysis of TiO₂ NPs was carried out using a x-ray diffractometer (XRD) instrument. The diffractogram was recorded in 2-range of 4° to 70°. Figure 1A shows a representative XRD pattern of TiO₂ NP in anatase phase. All observed peaks are in good agreement with the standard spectrum (JCPDS no. 21-1272). The morphology and size distribution of the prepared TiO₂ NP was observed by scanning electron microscope and transmission electron microscopy, which is shown in Figure 1B and 1C. The shape of the particles were seen as a sphere-like morphology 15 nm to 25 nm in size.

MTT assays
L929 murine fibroblast cells (Cell Bank of Pastur Institute, Tehran, Iran) were incubated in 96-well plates (11 x 10³ cells/well) each containing 200 µL of supplemented cell culture media for 24 hours at 37°C and 5% CO₂. The cells were divided into 3 groups: blank, TiO₂ NP, and TiO₂NP/gelatin, with 5 concentrations (0.01, 0.05, 0.1, 0.25, and 0.5 mg/mL) for both the TiO₂ NP and TiO₂NP/gelatin groups. After an incubation period of 24 hours, the spent media were removed and the plate wells were washed with phosphate-buffered solution. Briefly, 50 μL of 2-mg/mL MTT assay and 150 μL of culture media were added to each well. The cells were incubated at 37°C and 5% CO₂ for 4 hours, then the media were discarded and dimethyl sulfoxide and Sorensen’s buffer were added to each well as solubilizer buffers. Finally, absorbance rate was read using an ELISA plate reader (BioTek Instruments, Inc, Winooski, VT) at 570-nm wavelength. Three independent experiments were performed for each study, and all measurements were performed in triplicate. Results were expressed as the percentage of proliferation with respect to vehicle-treated cells. The growth inhibition of at least 50% was considered as cytotoxic.

Procedures for third-degree burn wound creation 
General anesthesia was performed by an intraperitoneal injection of ketamine 10% (50 mg/kg) and xylazine 2% (5 mg/kg). Afterwards, the dorsal surface of the animal was shaved and prepared for burn creation; the burn wounding process was the same for all mice. They were placed on the plate of a standard burn device (Iran Intellectual Property Office registration no. 86664; University of Tabriz) in the dorsal recumbent position, and a full-thickness burn wound was created on the dorsal lumbar surface (measuring 1 cm x 1 cm) in a 10-second period of time at 95°C (Figure 2). The animals were monitored daily for general health, appetite, weight, depression, and inflammation of wound areas.

Planimetric studies
Third-degree wound healing properties were evaluated by the percentage of wound contraction and the time of wound closure. Photographs were taken immediately after wounding (day 0) and postoperative days 2, 6, 10, 14, 18, and 21 with a digital camera (Cyber-shot DSC-W350; Sony Corporation, Tokyo, Japan) with a standard ruler placed near the wounds (Figure 3). The wound areas were analyzed by Adobe Acrobat 9 Pro Extended Measuring Tool (Adobe Systems Inc, San Jose, CA) and wound contraction percentage was calculated using the Formula:

In the Formula, where A₀ is the original wound area and A_t is the wound area at the time of imaging. 

Histological preparation and quantitative morphometric studies
Tissue samples were taken on days 7, 14, and 21 after wound creation from the wound periphery along with normal skin and fixed in 10% buffered formalin, dehydrated and embedded in paraffin wax, sectioned at 5 µm, and stained with hematoxylin and eosin. Photomicrographs were obtained under light microscope to assess the predominant stage of wound healing. Three parallel sections were obtained from each specimen. Cellular infiltration, including the number of polymorphonuclear cells and fibroblastic aggregation, granulation tissue thickness, blood vessel numbers, secondary inflection rate, reepithelialization, and total pathological grade, were quantitatively evaluated.

Statistical analysis
Statistical analysis was completed using GraphPad Prism, Version 5.05 (GraphPad Software, Inc, La Jolla, CA). The analysis of parametric data was performed by 1-way analysis of variance and repeated measure test followed by Tukey post-hoc test (parametric methods). In addition, nonparametric methods included Kruskal-Wallis multivariate analysis followed by Dunn’s post-hoc test. Results were expressed as mean ± standard deviation. In all analyses, P < .05 was considered to be statistically significant.

Antibacterial testing 
Antibacterial testing results in different groups during the course of study is shown in the Table. As presented, TiO₂NP showed bacteriostatic properties and adding gelatin to TiO₂NP lowered the bacteriostatic property (because gelatin does not bear bacteriostatic properties). The inhibition zone in TNG was significantly larger than that of gelatin and of SSD (P < .05). The inhibition zones were 14 mm and 12 mm for TiO₂NP/gelatin against S aureus and P aeruginosa, respectively (Figure 4).

MTT assay findings
The cytotoxicity of the TiO₂NP and TiO₂ NP/gelatin were investigated using the L929 cell line. Figure 5 shows the relative cell viability ([C_r/C₀, where C_r refers to the viable cell numbers treated with the nanomaterials and C₀ refers to the viable cell numbers of the control sample] 100%) versus various concentrations of nanocompounds determined by MTT assay. The error bars are the calculated standard deviations. The relative viabilities (%) of cells treated with 0.01 mg/mL, 0.05 mg/mL, 0.1 mg/mL, 0.25 mg/mL, and 0.5 mg/mL of TiO₂ NP and 0.1 mg/mL, 0.5 mg/mL, 1 mg/mL, 2.5 mg/mL, and 5.0 mg/mL of TiO₂ NP/gelatin are illustrated in Figure 5 after 24 hours of incubation. The results indicated that no significant harmful effect is imposed to the cells up to 0.05 mg/mL and 1 mg/mL of TiO₂ NP and TiO₂NP/gelatin respectively. Thus, it was found that TiO₂ NP/gelatin is more cytocompatible than TiO₂ NP alone.

Reduction in wound area
Figure 6 illustrates the percentage of wound contraction in different groups during days 7, 14, and 21. On day 7, the highest rate of contraction was observed in the SSD group (16% ± 2.28%) and the lowest rate in the control group (10.83% ± 2.56%) (P = .011). On day 14, the highest rate of contraction was observed in the TNG group (68% ± 2.75%) and the lowest rate in the control group (35.67% ± 5.98%) (P = .001). On day 21, the highest rate of contraction was observed in the TNG group (97.17% ± 2.31%) and the lowest rate in the control group (58.33% ± 2.87 %) (P = .0005). The healing rate of wounds in the TNG group was statistically significant compared with the other experimental groups (P < .05).

Histological and morphometric findings
There were significant differences between the TNG and other groups, particularly in terms of cellular infiltration, including the number of polymorphonuclear cells, fibroblastic aggregation, and granulation tissue thickness (P < .05).

On day 7, the highest level of angiogenesis (29 ± 2.34) was observed in the TNG group compared with the other groups (P = .0001). On day 21, the lowest level of angiogenesis (11.2 ± 1.8) belonged to the TNG group in comparison with other groups (P = .0001). In addition, on day 14, the highest number of fibroblasts (74.2 ± 4.97) was observed in the TNG group compared with the others (P = .0001). On day 7, the highest thickness of granulation tissue was observed in the TNG group (1042 ± 85.56 µm) compared with the other groups (P = .0001). Animals in the control group showed the highest granulation tissue thickness at 932 ± 96.8 µm on day 21 compared with the other groups (P = .0001). The least number of cells was observed in the TNG group (1.6 ± 0.54) on day 21 (P = .0001), and the highest percentage of reepithelialization was observed on days 7, 14, and 21 in the TNG group compared with the others (P = .0001). There were significant differences between the TNG and control groups in total pathological grade (P = .0001) (Figures 7, 8, 9). 

The 3 phases of the wound healing process (inflammation, proliferation, and tissue remodeling) try to repair tissue damage to be as close to its original pre-wounded state. The healing process is activated when platelets come into contact with exposed collagen, leading to platelet aggregation and the release of clotting factors, which results in the deposition of a fibrin clot at the site of injury. The fibrin clot serves as a provisional matrix and sets the stage for the subsequent events of healing. Inflammatory cells arrive along with the platelets at the injury site, providing key signals known as growth factors. The fibroblast is the connective tissue cell responsible for collagen deposition required to repair the tissue injury. Collagen is the main constituent of extracellular tissue, which is responsible for support and strength.¹⁶

Nanoparticles have become significant in regenerative medicine over the last 2 decades.¹⁷ Many biological processes occur through mechanisms that fundamentally act at the nanometer scale. Thus, materials such as NPs can be used as unique tools for drug delivery, imaging, sensing, and probing biological processes.¹⁸ In the context of wound healing, the special properties of NPs (eg, electric conductivity, antimicrobial activity, and high surface to volume ratio, swelling, and contraction) make NPs versatile resources.

Deepachitra et al¹⁰ reported that TiO₂NP governs the progress of the wound healing process through enhancement of skin moisture (hydrophilic activity) and antimicrobial properties. In the present study, the healing effects of TiO₂NP/gelatin on burn wounds were studied. The findings showed that in the TNG animals, the wounds were significantly contracted in the second and third weeks of the experiment compared with the other groups.

The highest levels of wound contraction in week 3 were observed in the TNG group (97.17%)  compared with the control group (58.33%), representing a promising effect of TiO₂ NP in wound contraction and closure. These results are in agreement with other studies.^3,14 High and significant contraction levels in the gelatin group in comparison with the control might be associated with the hydrophilic effects of gelatin and subsequent preservation of wound moisture that improved wound healing.¹⁰

Angiogenesis and the expansion of blood vessels is an essential factor in wound healing that can increase blood expansion through biochemical and pharmacological mechanisms, consequently leading to successful accelerated healing.¹⁹ Angiogenesis also provides an outline for connective tissue formation in the early days of the wound healing process. According to the present results, maximum angiogenesis was observed in the TNG group on day 7 with significant difference compared with the control group (P = .0001), showing the stimulating effect of TiO₂ NP on angiogenesis. Tan et al²⁰ suggested that a reduction in immigration and proliferation of endothelial cells and apoptosis of unessential blood vessels can occur by establishing adequate blood circulation in granulation tissue.Acceleration in blood vessel reduction of the TNG group showed the positive effect of TiO₂ NP in wound healing. 

The number of fibroblasts is a well-known index for quality assessment of healing in connective tissues.²¹ The presence and early proliferation of fibroblasts in the TNG group demonstrated an increase in stimulation of fibroblasts by TiO₂ NP. Others^14,22 conducted a survey on the effects of nanocomposite of chitosan-TiO₂ on excisional wounds and suggested an improvement in wound healing by cell growth stimulation from TiO₂ NP; fibroblasts create extracellular matrix and collagen, which play an essential role in the wound healing phases.²³

Granulation tissue formation in the early days of the healing process is considered a momentous factor in accelerating healing.²⁴ The more fibroblasts and blood vessels form, the more granulation tissue develops. Maturation of granulation tissue and a decrease in its thickness for the TNG group initiated on day 7 and was followed by a minimum thickness on day 21. At the end of the study period, the maximum thickness of granulation tissue was observed in the control group, with significant difference with the TNG group (P = .0001). This was in agreement with the results of angiogenesis and fibroblast proliferation that were responsible for the maturation of blood vessels, fibroblasts, and healing enhancement in the TNG group.

The inflammatory phase is the first step of wound repair and is determined by the presence of inflammatory cells. This phase is critical due to the secreted cytokines associated with proliferation, reepithelization, contraction, and wound closure.²⁵ In the present study, the TNG group showed the least number of inflammatory cells on day 7, which could be associated with anti-inflammatory and antibacterial activity by TiO₂ NP. This was done through reducing polymorphonuclear neutrophil (PMN) cells via reduction in secondary infection and acceleration in inflammation initiation and termination.¹⁰ The presence of low PMN cell numbers and high numbers of blood vessels and fibroblasts on day 7 in the TNG group indicated accelerated healing in the TNG animals.

Reepithelization creates a barrier between the wound and environment during healing.²⁶ Newly formed epithelium is characterized by more cells and layers compared with normal epithelium. When the wound surface is coverd by new epidermal cells, differentiation initiates, cell shape changes to normal, and rearrangement and reduction of the cell layer occurs.²⁷ Based on the results of the present study, epithelization in the TNG group was nearly complete (96.6%) on day 14 (1 week earlier than the other groups); on day 21 of the experiment, complete epithelization (100%) was achieved. This outcome was indicative of the epithelization stimulation effect of TiO₂, which is in accordance with findings of a previous study.¹⁴

Despite the sterility of burn wounds upon creation, due to the existence of vascular necrotic tissue and loss of epithelial integrity, the wound is disposed to infection, which can reduce the chance of healing and increase mortality in severe cases. The presence of neutrophils in histopathological sections is an indicator of wound infection²⁸; this was the basis of the grading system of secondary infection, as follows: grade 0, absence of neutrophils in the epidermal surface; grade 1 (mild), presence of slight neutrophils in both the ulcer and epidermal surfaces; grade 2 (moderate), presence of dense neutrophils in both the ulcer and epidermal surfaces; and grade 3 (severe), dense neutrophils associated with neutrophilic debris and secrection in the epidermal and dermal surfaces.

According to the results of the present study, the TNG group showed the least amount of infection on days 7 and 14 on the scab and epidermis, and no infection was observed on day 21. It could be concluded that TiO₂ NP/gelatin had antibacterial properties, which is in agreement with other studies.^3,29 In the present study, antibacterial effects were detected more in the TNG group than the SSD. Early epithelialization in the TNG group prevented the wound from penetration of microorganisms to the healing tissue. Both TiO₂ and gelatin, as the carrier, exhibited hydrophilic features and accelerated the healing process. 

Enhancement of burn wounds is associated with reepithelialization, fibroblast proliferation, and angiogenesis.³⁰ Since the pathological phases of the wound healing process have dynamic features and the results of the inflammatory, proliferative, and remodeling phases are dependent on each other, all healing factors should be considered together for a valid conclusion. Therefore, the histomorphometric results of this study were scored on a grade of 1 to 5, in which higher grades represented an improvement in healing. Consequently, the highest grade indicated the best healing activity by enhancing the inflammatory, proliferative, and remodeling phases. Based on the results of the present study, high percentage of wound contraction and total pathological grade as well as insignificant or absence of infection were observed in TiO₂ NP/gelatin group, which is in agreement with findings in other studies.^14,23

A limitation of the present study is that the authors did not measure the biomechanical properties of the healed skin. 

Based on the results of the present study, it can be summized that TiO₂ NP/gelatin offered potential advantages in accelerating and improving burn wound healing through angiogenesis stimulation, fibroblast proliferation, and granulation tissue formation in the early phases of wound healing. In addition, an acceleration in wound repair, associated with earlier wound contraction and stability of damaged areas by rearrangement of granulation tissue and collagen fibers, was noted with TiO₂NP/gelatin. Likewise, antibacterial activity of TiO₂ NP prevented wound infection. Therefore, TiO₂ NP/gelatin could be considered as a therapeutic option in burn wound treatment. 

Authors: Sara Javanmardi, DVM, DVSc; Amaneh Ghojoghi, DVM; Baharak Divband, PhD; and Javad Ashrafi, PhD

Note: This study was conducted for a doctorate of veterinary medicine thesis at the University of Tabriz, Veterinary Medicine Faculty (Tabriz, Iran). 

Affiliation: University of Tabriz, Tabriz, Iran

Correspondence: Sara Javanmardi, DVM, DVSc, Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Tabriz, 29 Bahman Avenue, Tabriz, Azarbaycane- khavari 5166616471 Iran; sarahjavanmardi@yahoo.com; s.javanmardi@tabrizu.ac.ir

Disclosure: The authors disclose no financial or other conflicts of interest. This research was supported by a grant from the University of Tabriz.