
==== Front
Regen Ther
Regen Ther
Regenerative Therapy
2352-3204
Japanese Society for Regenerative Medicine

S2352-3204(24)00138-X
10.1016/j.reth.2024.08.002
Original Article
Evaluation of decellularized caprine small intestine submucosa encapsulated with nano-formulations of cerium oxide and curcumin in the management of burn wound
Li Juan
Li Hao
Wang Kaikai
Chou Haiyan chy13803863969@outlook.com
⁎
Department of Plastic Surgery, Henan Provincial People's Hospital, Zhengzhou University People's Hospital, Zhengzhou, Henan, 450003, China
⁎ Corresponding author. chy13803863969@outlook.com
14 8 2024
6 2024
14 8 2024
26 578589
20 6 2024
18 7 2024
2 8 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The management of burn injuries presents a significant challenge in clinical settings, yet an optimal solution remains elusive. Therefore, this study aimed to develop a topical therapeutic formulation to address the complex issues hindering burn wound healing. Emphasizing the sustained presence of bioactive principles, we synthesized a bioactive gel derived from decellularized caprine small intestine submucosa (D-CIS) and encapsulated it with nano-formulations of cerium oxide and curcumin to create a burn wound dressing material with enhanced properties. The choice of encapsulated components was guided by their antimicrobial, antioxidant, and immune-modulating characteristics, along with their inherent ability to gradually release bioactive substances. The encapsulated (cerium oxide and curcumin) D-CIS bioactive gel demonstrated a range of properties, including antimicrobial, antioxidant, and anti-inflammatory effects, along with sustained release kinetics of bioactive molecules. These combined effects facilitated accelerated burn wound healing by mitigating oxidative stress, reducing inflammation, and promoting cell recruitment for epithelial and vascular regeneration. This study contributes to the development of a novel bioactive gel incorporating cerium oxide and curcumin, offering a promising approach to enhance burn wound healing.

Keywords

Decellularized caprine small intestine submucosa (D-CIS)
Nano-formulations
Cerium oxide
Curcumin
Burn wound reepithelization
==== Body
pmc1 Introduction

Skin is an Epithelial tissue that performs essential functions which include thermoregulation, and sensory function, and serves as the first line of defense against infection [1]. Once this cutaneous barrier gets injured it results in diverse intricacies ranging from microbial infestation/infection to plasma loss [2]. Beneath it, cutaneous tissue harbors an interconnected blood-vascular network. The majority of blood vascular architecture is established during the prenatal stage of development, but adult tissues can trigger angiogenesis in response to injury, a process regulated by angiogenic factors [3]. Among different types of wounds The healing of burn injuries is a dynamic physiological progression and involves four interrelated phases which include hemostasis, inflammatory reaction, cellular multiplication, and epithelialization [4]. Thermal burns are considered life-threatening burn lesions that can cause the death of the burn victim if not treated properly. As per the latest survey, almost 1.8 × 105 deaths/year are attributed to thermal injuries [[5], [6], [7]]. Diverse interventions which include liniments, auto-grafts, allotransplants, and artificial prosthetics for burn wound management, have significantly improved therapeutic outcomes in patients with burn injuries [8,9]. However, healthcare costs associated with the treatment of burn wounds put an economic burden on patients [10,11]. Researchers have identified decreased vascularization, impaired keratinogenesis, diminished fibroblast migration, and enhanced levels of TNF-α responsible for impaired burn wound healing [12], but still pathogenesis of delayed wound healing remains yet to be understood. Recently some studies have found abnormal macrophage polarization and dysregulation of immune cum inflammatory response which causes delayed wound healing in burn wounds [13,14]. In the preceding decades, researchers have used a wide spectrum of nano-formulations and other traditional medicinal preparations for their therapeutic utility use in the healing of diverse categories of injuries [15,16]. A Plethora's of research findings have reported encouraging results of wound healing, amidst these preparations myriad cerium oxide and curcumin formulations have been reported to have biological effects favorable for the promotion of cellular regeneration in thermal injury microenvironment which includes anti-oxidant potential and amelioration of inflammatory pathways [17]. Furthermore, these studies have reported that these preparations have the potential to promote wound healing by causing enhanced expression of genes associated with the process of wound healing [18]. Results from a plethora of studies have indicated that in delayed burn wound healing, the involvement of oxidative stress and the presence of pro-inflammatory/inflammatory mediators play a crucial role [19]. Taking into account the increasing incidence of burn wounds, exploring novel medications with minimum side effects is needed for an hour. Considering the clue of wound healing potential of cerium oxide and curcumin-based formulation [8,16], the current study was formulated to evaluate regenerative capacity for tissue repair in burn wounds by using cerium oxide and curcumin loaded hydrogel in rabbit model and to explore and understand mechanistic pathways promoted by cerium oxide and curcumin loaded hydrogel in burn wound healing. These findings will further help in fabricating and designing effective topical preparations for various types of chronic non-healing wounds.

2 Materials and methods

2.1 Characterization

2.1.1 Decellularization procedure

Caprine duodenum was collected from the locoregional abattoir and the decellularization procedure was typically performed by involving a series of steps including perfusion, chemical treatment, and physical agitation to remove the cellular components while preserving the ECM. The resulting scaffold was used as a template of biomaterial for drug delivery. Briefly after washing caprine duodenum layers (mucosal layer, serosal layer, and outer muscular layer) were mechanically separated and only the duodenum submucosa layer was retained which was further processed as standard procedure. The Decellularization procedure was performed as per the method [12] and the decellularized duodenum sub mucosa was sterilized in ethanol/H2O2 (70/0.1% (v/v)) solution for 2 h followed by thorough washing to remove the sterilization solution. The decellularized matrix was lyophilized followed by ultrasonic cavitation as per the method of Badylak et al. [13] (Fig. 1, Fig. 2).Fig. 1 (1a) Fabrication of D-CIS and its encapsulation with Cerium Oxide and Curcumin by ultrasonic cavitation and temperature gelation (1b). Storage Modulus (1c). Complex Viscosity (1d). Loss Modulus of different fabricated biomaterials which reveals gradual non-significant decline these parameters from D-CIS to D-CIS/Ceo/Cur.

Fig. 1

Fig. 2 Characterization and physiochemical properties of Fabricated D-CIS encapsulations and SEM characteristics of Nano formulations of Cur and Ceo (2a). Structural characteristics Ceo Nano-particles (2b) structural characteristics of Nano-formulation Curcumin (2c-f) SEM of DG-SIS, DG–SIS–Ceo DG–SIS–Curcumin and DG–SIS–Curcumin/Ceo (2g) Mean Porosity of fabricated biomaterials (2h) Mean Swelling of fabricated biomaterials (2i) Enzymatic Degradation (2j) Non-Enzymatic Degradation (∗p˂0.05).

Fig. 2

2.1.2 Fabrication of bioactive gel with cerium oxide and curcumin nanoparticles

Temperatures depend gelation process was used for the fabrication of bioactive gels enriched with cerium oxide and curcumin nanoparticles (Fig. 1a). These preparations enriched with cerium oxide, curcumin nanoparticles, and a combination of cerium oxide and curcumin nanoparticles were evaluated for storage modulus (Fig. 1b), complex viscosity (Fig. 1c), and loss modulus (Fig. 1d).

2.1.3 Physicochemical characterization of zinc oxide nanoparticles and bioactive hydrogels

The morphology of D-CIS/CeO, D-CIS/Cur, and D-CIS/CeO/Cur bioactive hydrogels was determined using Field Emission Scanning Electron Microscope (FESEM) (TESCAN Mira 3 SEM, USA). The particle sizes of the D-CIS/CeO, D-CIS/Cur, and D-CIS/CeO/Cur and the pore size of the gels were measured by processing FESEM images in the Image J software. FESEM-equipped EDS (energy-dispersive spectroscopy, EDAX, AMTEK, USA) was used to evaluate the elemental composition of synthesized D-CIS/CeO, D-CIS/Cur and D-CIS/CeO/Cur. The FTIR (Fourier transforms infrared) spectrum of D-CIS/CeO, D-CIS/Cur, and D-CIS/CeO/Cur was obtained by PerkinElmer infrared spectrophotometer in potassium bromide (KBr) mode to get the chemical properties. The porosity (P) of the fabricated gels was determined by the liquid displacement method, as explained by Singh et al. [14]. Further, the swelling behavior of the gels was measured by the previously described protocol Furthermore, the in-vitro weight loss rate (under enzymatical and non-enzymatical settings) of the gels was assessed using the ASTM method (F-1635-95), which involved monitoring gradual reduction in mass within simulated physiological conditions at different time intervals.

2.2 In-vitro study

2.2.1 In-vitro anti-oxidant assay

Fabricated bioactive hydrogel was subjected to DPPH (2,2-Diphenyl-1-picrylhydrazyl) assay and H2O2 assay as per the method of [15]. Briefly fabricated bioactive hydrogel was incubated in the presence of DPPH (50 μM) and hydrogen peroxide (50 μM) for 1.5 h under dark laboratory conditions. Next, absorbance was measured at 517 nm and antioxidant activity was measured asAntioxidantActivity(%=Absorbanceofcontrol−AbsorbanceofsampleAbsorbanceofcontrol×100

2.2.2 In vitro anti-inflammatory activity

Protein denaturation assay was used to evaluate in-vitro anti-inflammatory activity [16]. Briefly, 5 mg of albumin was mixed with 5 mg of fabricated biomaterial, 0.98% Nacl, and deionized water were used as negative control and positive control respectively. These mixtures were incubated at room temperature for 240 min and centrifugation supernatants collected were evaluated for absorbance at 545 nm.Hemolysis(%)=OD(biomaterial)−OD(NegativeControl)OD(PositiveControl)−OD(NegativeControl)×100

2.2.3 Anti-microbial assay

For the antimicrobial assay, the disc diffusion method was employed. In brief, a loopful of test microorganisms which includes gram-positive (Staphylococcus aureus) and gram-negative (Escherichia coli) bacteria were inoculated onto nutrient agar petri dishes and incubated until visible colonies formed. Subsequently, 2 mg of test substances (DG-CIS/CeO/Cur, DG-CIS/CeO, DG-CIS/Cur, and DG-CIS) along with positive and negative controls, were placed on the nutrient agar plates and incubated for 24 h. After this period, the diameter of the clear zone (zone of inhibition) around the test substances was measured according to standard procedures.

2.2.4 Cytotoxicity assay

Nano-encapsulated formulations of D-CIS were assessed for cytotoxicity testing utilizing L929 cells. Bioactive hydrogels were positioned within tissue culture plates, which were later inoculated with L929 cells @ density of 5000 cells per square centimeter. The entire process was carried out in a sterile environment. Subsequently, the plates, whether containing a fabricated bioactive hydrogel or not, were placed in Dulbecco's Modified Eagle Medium (DMEM) and incubated at 37 °C with 5% CO2 saturation for 24 h. After the second, fifth, and eighth hours of incubation, cells were trypsinized, and the cell count was determined using a hemocytometer. Moreover, a cell proliferation assessment was performed on the 1st, 3rd, and 5th days following incubation through MTT assay. For the MTT assay, at specified intervals, the media were aspirated, and the cells were rinsed with PBS. Subsequently, 50 μl of MTT dye at a concentration of 5 mg/ml in PBS was applied to each tube, followed by a 4-h incubation period. After introducing the solubilization buffer (DMSO), the absorbance at 540 nm was subsequently monitored. To evaluate cell viability, L929 cells were subjected to staining using fluorescein diacetate/ethylene dibromide for 5 min at 37 °C. Subsequently, the cells were examined under a fluorescent microscope to determine the percentage of live and dead cells [13].

2.2.5 Dermal allergic response

Dermal allergic response was evaluated as per method of [2]. In summary, six rabbits were randomly chosen to assess allergic skin reactions. The dorsal areas of these rabbits were shaved, and the animals were then randomly assigned to two groups (control group and bioactive hydrogel group) of three rabbits in each group. Animals in control group and bioactive hydrogel group were applied with 2 ml topically of distilled water and fabricated bioactive hydrogel respectively. The location where bioactive hydrogel and distilled water were topically applied was monitored at regular intervals of 24, 72, and 94 h. In both sets of animals, there were no indications of irritation or adverse reactions. Throughout the 92-h observation period, all animals exhibited normal behavior, and there were no instances of mortality detected.

2.3 In vivo burn wound healing study

Rabbits obtained from authorized dealers were used as an experimental burn wound model as per method of [7]. The age of rabbits was around 6–8 months, with a weight of about 1.84–2.60 kg. Until the completion of the trial, laboratory animals were housed in separate cages and were offered standard chow feed under standard environmental conditions (25 °C and 55% humidity). All methods used in burn wound healing study strictly followed the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

2.3.1 Analysis of anti-oxidative stress markers and pro-inflammatory cytokines in regenerated granulation tissue

The assessment of anti-oxidant stress parameters was conducted in wet regenerated granulation tissue developed on rabbit wounds following the methodology outlined in Ref. [17]. Briefly, granulation tissue was homogenized and the homogenate was subjected to ultracentrifugation @1500 g, and the supernatant was collected and filtered. The filtrate acquired was employed to assess markers related to antioxidant stress [Nitric oxide (NO), Glutathione Peroxidase (GPx), Malondialdehyde (MDA), Superoxide Dismutase (SOD), Catalase (CAT), Reduced Glutathione Peroxidase (GSH), and Vitamin C] and markers associated with inflammation [IL-1, IL-2, IL-6, and TNF-α] using the methodologies outlined in Refs. [18,19] respectively.

2.3.2 Analysis of connective tissue markers

Regenerated granulation tissue harvested at different time intervals was processed and analyzed for connective markers [Hydroxyproline (HPR), Hexosamine (HXA), and Hexuronic acid (HUA)] as per [14].

2.3.3 Growth markers

The overall DNA amount and the gene activity of growth factors in regenerated granulation samples were examined following the procedures outlined in Refs. [20,21], respectively. In the current experimental framework, the messenger RNA (mRNA) levels of the Epidermal Growth Factor (EGF), Transforming Growth Factor 1-beta (TGF-1β), and Vascular Endothelial Growth Factor (VEGF) were assessed. The expression of these indicative genes was standardized to the reference gene GAPDH using the 2- ΔΔCt method.

2.3.4 Histopathology

Dermal punch biopsy sampling was harvested at specified time intervals, including the 0th, 3rd, and 7th days following the initiation of the full skin burn wound. The dermal specimens were preserved in 10% formalin solution for 3 weeks and stained using hematoxylin-and-eosin (H&E) as per the prescribed method [22,23]. The histopathological specimens from each treatment group were compared by the pathologist (who was blinded to experimental design) and based on re-epithelization and quantum of granulation tissue formed histopathological scoring.

2.4 Statistical analysis

The objective of this study was to assess the effectiveness of a newly developed bioactive hydrogel for the treatment of burn wounds. The data was collected in an excel sheet as per the standard procedure. The main objective of the present study was to assess the decrease in wound area through the application of a mixed-effects model. The analysis of qualitative variables involved the application of the chi-square test, and the results were expressed as odds ratios with a 90% confidence interval (CI). The Shapiro–Wilk test was employed to assess the normal distribution of quantitative variables in the current study. The Kruskal-Wallis test was employed to assess variations among distinct treatment groups. In the statistical tests mentioned above, a significance threshold of P < 0.05 was established. The statistical analysis was conducted following the specified method [24].

3 Results

3.1 Characterization

3.1.1 Physicochemical properties of cerium oxide and curcumin nanoparticles

The cerium oxide and curcumin nanoparticles were spherical and the particle size was approximately 98.32 nm (Fig. 2a) and 97.09 nm (Fig. 2b) respectively. SEM images revealed the presence of cavitated laminar structures in DG-SIS and DG–SIS–Ceo (Fig. 2c Fig. 2d). While as SEM images of DG–SIS–Curcumin (Fig. 2e) and DG–SIS–Curcumin/Ceo (Fig. 2f) revealed presence of laminar cavities with rough peripheries. Elemental profiling of cerium oxide and curcumin nanoparticles revealed the presence of cerium, carbon, oxygen, and hydrogen these elements encompass various aspects of the biological activities associated with wound healing processes, highlighting the multifaceted nature of substances that contribute to antimicrobial, anti-inflammatory, and anti-oxidant activities in the context of tissue repair. Based on the presence of elements having beneficial effects on wound healing it can hypothesized that elements present in cerium oxide and curcumin nanoparticles have a significant role in the regeneration of dermal tissue architecture.

3.1.2 Physicochemical properties of gels

SEM images of D-CIS/CeO, D-CIS/Cur, and D-CIS/CeO/Cur when investigated using Image J analysis, revealed porosity ranged from D-CIS (85%), D-CIS/CeO (89.09%), D-CIS/Cur (84.98%) to D-CIS/CeO/Cur (92.87%) (Fig. 2g). There was a significantly higher degree of porosity in D-CIS/CeO/Cur compared to the other three formulations. In the current study on observation of swelling behavior, we observed that all four gel formulations hydrated within 120 min, and the swelling percentages observed were 2157%, 1733%, 2192% and 2145% for from D-CIS, D-CIS/CeO, D-CIS/Cur and D-CIS/CeO/Cur respectively with no significant difference observed across them (Fig. 2h). Furthermore, the viscosity of the bioactive was found to be decreased with the incorporation of Nano formulations of CeO and Cur in a dose-dependent manner and the lowest degree of viscosity was observed in D-CIS/CeO/Cur compared to the other two fabricated formulations. Degradation of fabricated gels was observed in the presence and absence of the enzymatic preparation and was designated as enzymatic and non-enzymatic degradation. Enzymatic degradation observed was highest for D-CIS which degraded within 180 min and D-CIS, D-CIS/CeO, D-CIS/Cur, and D-CIS/CeO/Cur showed enzymatic degradation within 72 h, 92 h, and 120 h respectively (Fig. 2i). Similarly, non-enzymatic degradation observed within 28 days for D-CIS, D-CIS/CeO, D-CIS/Cur, and D-CIS/CeO/Cur was 86%, 87%, 92% and 84.19% respectively (Fig. 2j).

3.1.3 CeO/Cur release behavior

In the present study from Fig. 3a releasing behavior of bioactive ingredients (CeO/Cur) from D-CIS gel after 50 h of encapsulation was observed and from this it can be observed that from D-CIS/CeO/Cur bioactive nanoformulation of CeO and Cur were released from D-CIS/CeO/Cur bioactive gel were released in sustained fashion compared to other two formulations. These results indicate that co-encapsulation of CeO and Cur in D-CIS results in sustained release of bioactive Nano formulation compared to mono encapsulation of either CeO or Cur. The amount of CeO released within 50 h from D-CIS/CeO, and D-CIS/CeO/Cur was 56% and 32% respectively. Similarly, the quantum of Cur released within 50 h from D-CIS/Cur and D-CIS/CeO/Cur were 49% and 39% respectively.Fig. 3 In-Vitro Evaluation of formulated biomaterials (3a) Cumulative Cur releasing behavior (3b). Cytotoxicity Assay (Hemolysis %) (3c). Apoptosis Assay (Live% and Dead %) (3d). Cell line proliferation Assay (3e) anti-oxidant assay (DHPP assay) (3f). Free radical scavenging assay. (3g) Anti-Inflammatory assay.

Fig. 3

3.2 In-vitro study

3.2.1 In vitro cytotoxicity assay

Fig. 3b and c shows hemocompatibility and apoptosis assay of fabricated bioactive gels. In the current experimental design, we found D-CIS, D-CIS/CeO, D-CIS/Cur and D-CIS/CeO/Cur exhibited erythrocyte-compatibility. Contrarily, D-CIS/CeO induced notable hemolytic effects in contrast to alternative engineered gels. Moreover, L929 fibroblasts exhibited heightened proliferation in the presence of engineered bioactive gels, as evidenced by elevated optical density (OD) values on the 1st, 3rd, and 5th day, as illustrated in Fig. 3d. Comparably, the optical density (OD) values exhibited a noteworthy elevation on the 3rd and 5th days for their corresponding concentrations, demonstrating a significant increase as opposed to the OD values observed on the 1st day. On the third day, examination of cellular viability at various time points revealed a markedly elevated proportion of viable cells (92.57%) within the context of D-CIS/CeO/Cur, in stark contrast to the live cell count observed in D-CIS, D-CIS/CeO, and D-CIS/Cur formulations. Likewise, on the fifth day, viable cells were observed to exhibit a higher proliferation rate when juxtaposed with non-viable cells in all synthesized bioactive hydrogels. (Fig. 3d).

3.2.2 In-vitro antioxidant properties

The in-vitro antioxidant activity of synthesized bioactive gels revealed a decrease in free radical concentrations, with notable reductions observed in D-CIS/CeO/Cur, D-CIS/CeO, and D-CIS/Cur in comparison to D-CIS. This suggests a substantial antioxidant effect in these formulations. All the fabricated bioactive gels showed a significant reduction in free radical levels in juxtaposition to the negative control condition. Likewise, In the H2O2 assay, we noted that the antioxidant efficacy of the engineered bioactive gels closely resembled the results obtained from the 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay (Fig. 3e and f).

3.2.3 In-vitro anti-inflammatory activity

All the fabricated bioactive gels were evaluated for in-vitro suppression of inflammatory responses by evaluating Protein denaturation assay (Fig. 3g). The bioactive gel D-CIS/CeO/Cur and D-CIS/CeO exhibited significant protection against Protein denaturation. These results indicate that the incorporation of Nano formulation in bioactive gels results in the stabilization of the proteins, possibly by stabilizing the lysosome membrane, henceforth reducing the release of lysosome enzymes from lysosome compartments which are known to initiate the inflammatory cascade. An indirect inference can be drawn from this assay that encapsulation of Nano formulations in bioactive causes suppression of inflammatory response in wound healing and promotes the early onset of the proliferative stage, subsequently aiding in the timely healing of wounds.

3.2.4 Antibacterial assay

All the fabricated gels Evidenced significant anti-bacterial potency against reference gram-positive (S. aureus) and gram-negative (E. coli) bacteria. D-CIS/CeO/Cur, D-CIS/CeO, and D-CIS/Cur demonstrated markedly superior antibacterial efficacy in comparison to D-CIS. Moreover, D-CIS/CeO/Cur presented the most powerful and statistically significant antibacterial efficacy in comparison to D-CIS/CeO and D-CIS/Cur (Fig. 4a and b).Fig. 4 (4a) In-vitro anti-microbial assay of various biomaterial formulations (where “s”, “3” “1” “2” “4” and “5” indicates standard anti-microbial, DG-SIS, DG-SIS/Curcumin, DG-SIS/Ceo and DG-SIS/Curcumin/Ceo respectively) (4b) zone of inhibition of different biomaterial formulations and comparison with standard anti-microbial (Enrofloxacin) Dermal allergic response assay (4c) application of DG-SIS and negative control (Distilled Water) (4d) Observation of dermal response after 72 h of topical application (4e). Observation of dermal response after 94 h of topical application.

Fig. 4

3.3 In –vivo study

3.3.1 Gross findings

The site over which fabricated hydrogel was applied was observed periodically on 1st, 3rd and 5th day after application. In both sets of animals, there were no indications of irritation or adverse reactions observed with no allergic response (Fig. 4c–e). Furthermore, site on which fabricated bioactive hydrogel was applied there was no appreciable hyperemic reaction indication that fabricated bioactive hydrogel was immune tolerant on dermal tissue. The physical appearance of dermal tissue was almost similar to that of dermal tissue over which distilled water was applied.

In the current investigation of full-thickness skin wounds, the extent of wound contraction in D-CIS and D-CIS/CeO were 58 % and 62% respectively on the 14th day, and full wound closure (epithelization) was noted on the 27th day and 25th day respectively (Fig. 5). In D-CIS/Cur, a substantial 65.23% of wound closure was evident by the 14th day, with full wound healing observed by the 24th day. In the D-CIS/CeO/Cur group, 79% epithelialization was noted on the 14th day, and full wound closure was observed on the 20th day. Moreover, the formation of granulation tissue was apparent through a substantial rise in the moisture content of granulation tissue per 100 g of body weight, as markedly elevated levels (P < 0.01) of granulation tissue (mg)/100 gms body weight were observed in group D-CIS/Cur (97.56 ± 15.90) and group D-CIS/CeO/Cur (152.89.45 ± 31.12) in comparison to group D-CIS/CeO (61.6 ± 12.34) and group D-CIS (54.67 ± 23.34). It can be seen that encapsulation of Nano-formulation in gels modulated inflammatory response effectively and wound edges in D-CIS/CeO/Cur and D-CIS/Cur groups were found to be well-defined and clear which supports their role in the healing process of the burn wound. On the third day, we extracted granulation tissue for biochemical examination. The isolated granulation tissue underwent assessment for pro-inflammatory cytokine response, connective tissue indicators, anti-oxidative markers, and growth factor markers, as delineated subsequently.Fig. 5 In-Vivo evaluation of biomaterials in full thickness burn wound rabbit model (5a) observation of dermal allergic response for 4 days of study (5b). Induction of burn wound bilaterally on either side of the spine (5c) Topical application of biomaterials on different wounds (5d). Observation of wounds after 7th day of topical application of biomaterials (5e). Observation of wounds after 14th day of topical application of biomaterials.

Fig. 5

3.3.2 Antioxidant status metrics

Numerous investigations have indicated that the existence of free radicals at the wound site leads to a protracted healing process, and treatment approaches that alleviate oxidative stress may prove advantageous for expediting wound recovery. Hence evaluation of oxidative stress mediators at the wound site can provide an essential clue about the progression of wound healing and henceforth fabrication of effective wound healing preparations against burn wounds. In the present study, we found that D-CIS/CeO/Cur, D-CIS/CeO, and D-CIS/Cur exhibited significant anti-oxidant potential compared to D-CIS, furthermore, D-CIS/CeO/Cur showed significant amelioration of anti-oxidant potential compared to D-CIS/CeO and D-CIS/Cur (Fig. 6). In the current investigation, glutathione (GSH) concentrations were found to exhibit a noteworthy (P < 0.0001) elevation in D-CIS/CeO/Cur and D-CIS/Cur in comparison to D-CIS/CeO and D-CIS. SOD levels were observed to have elevated in Nano-formulation encapsulation bioactive gel groups (D-CIS/CeO/Cur 25.67 ± 5.98 U/mg; D-CIS/Cur 15.98 ± 4.67 U/mg) relative to the SOD concentrations in alternative groups (D-CIS/CeO 12.68 ± 3.67 U/mg; D-CIS 10.67 ± 2.81 U/mg), furthermore, the quantum of SOD observed in D-CIS/CeO/Cur was almost twice in other groups considered in the present study. Similarly, when levels of catalase were compared, levels of catalase were found to be threefold increased in Nano-formulation encapsulation bioactive gels compared to the D-CIS group. In addition to these Nano-formulation encapsulation bioactive gels (45.67 ± 12.34% in D-CIS/CeO/Cur and 56.78 ± 15.87% in D-CIS/Cur) demonstrated noteworthy improvement in nitric oxide (NO) suppression in contrast to D-CIS group. In contrast to these observations, levels of markers indicative of cellular damage mediated by free radicals, such as lipid peroxidation (LPO), exhibited a significant reduction in the given context of Nano-formulation encapsulation bioactive gels treated groups compared to the D-CIS group. Results of oxidative stress amelioration potential of Nano-formulation encapsulation bioactive gels indicate that these formulations dampen inflammatory response and hence expedite the resolution of the inflammatory stage in the wound healing process, leading to accelerated healing.Fig. 6 Anti-oxidant assay of granulation tissue harvested from wounded areas, which were applied with different fabricated biomaterials. Parameters estimated included mean peroxidases, Mean LPO, Mean NO inhibition, Mean SOD, Mean CAT and Mean GSH.

Fig. 6

3.3.3 Pro-inflammatory cytokines

In the present study, we compared ILs and TNF-α response on 5th day post application of different bioactive gels with 2nd day of application of various therapeutic bioactive gels. IL-4 was found to vary non-significantly across different days of treatment in all the four treatment groups considered in the present study. IL-6 levels were found to vary significantly in D-CIS/CeO/Cur, D-CIS/Cur and D-CIS/CeO groups, which was found to decrease on 5th day in D-CIS/CeO/Cur (p ≤ 0.001), D-CIS/Cur (p ≤ 0.01) and D-CIS/CeO (p ≤ 0.05) groups compared to values observed on 2nd day, contrarily to this no statistically significant variance was noted in IL-6 levels in D-CIS group (Fig. 7). On similar lines, we observed that IL-1α was significantly reduced on 5th day in all three encapsulation groups (D-CIS/CeO/Cur, D-CIS/Cur, and D-CIS/CeO) compared to values observed on 2nd day, while as the non-significant reduction in values of IL-1α were observed in D-CIS group. Furthermore, on 5th day we observed a significant elevation in IL-1 levels in D-CIS/CeO/Cur (p ≤ 0.001) D-CIS/Cur (p ≤ 0.01) and D-CIS/CeO (p ≤ 0.05) groups compared to values observed in 2nd day while as statistically insignificant rise in IL-1 levels was noted in the D-CIS. Likewise, within the realm of Pro-inflammatory cytokines on the 5th day, comparable outcomes were observed. TNF-α levels were found to be reduced in D-CIS/CeO/Cur, D-CIS/Cur, and D-CIS/CeO groups compared to values observed on 2nd day with the highest degree of reduction observed in D-CIS/CeO/Cur group followed by D-CIS/Cur and D-CIS/CeO groups. Contrary to this TNF-α levels were non-significantly reduced in the D-CIS group on 5th day compared to values on 2nd day of observation.Fig. 7 Pro-inflammatory cytokine response evaluated in full skin burn rabbit model on 2nd and 5th day of application of different biomaterials. The parameters evaluated included IL-4, IL-6, IL-1α, TNF-α, IL-1 and IL-2.

Fig. 7

3.3.4 Connective tissue markers

In the current investigation, we assessed connective tissue indicators within distinct therapeutic interventions on the third day of the experiment. The present study revealed that connective tissue parameters in D-CIS/CeO/Cur (p ≤ 0.001), D-CIS/Cur (p ≤ 0.01), and D-CIS/CeO (p ≤ 0.05) groups were significantly elevated compared to D-CIS. Briefly, significantly higher levels of hydroxyproline were found in D-CIS/CeO/Cur (p ≤ 0.001), D-CIS/Cur (p ≤ 0.01), and D-CIS/CeO (p ≤ 0.05) compared to D-CIS. Furthermore, D-CIS/CeO/Cur was established to manifest considerable disparities compared to values discerned in D-CIS/Cur and D-CIS/CeO groups which indicates significantly higher connective tissue proliferative potential of D-CIS/CeO/Cur compared to other groups (Fig. 8). A comparable pattern was noted in the concentrations of hexosamine and hexuronic acid among various treatment cohorts in the current investigation. We observed almost four-fold increases in Hexosamine and Hexuronic acid levels in D-CIS/CeO/Cur compared to the D-CIS group.Fig. 8 Connective tissue parameters (Hexosamine, Hydroxyproline and Hexuronic acid) and Relative m-RNA expression (VEGF, EGF and TGF-1β) evaluated in granulation tissue isolated from different experimental rabbit wound model.

Fig. 8

3.3.5 Gene expression studies

In the present study, we compared expression levels of growth promoter genes TGF-β1, IGF-I, and EGF across different treatment groups of wounds. The expression studies revealed that only D-CIS/CeO/Cur and D-CIS/Cur caused a significant elevation in gene expression of these growth promoters considered in the present study compared to expression levels in the D-CIS group. In the current investigation, a parallel examination revealed no discernible variance in the genetic transcription profiles of Transforming Growth Factor-β1 (TGF-β1), Insulin-like Growth Factor I (IGF-I), and Epidermal Growth Factor (EGF) between the D-CIS/CeO group and the D-CIS group. Among the parameters of gene expression, EGF showed a more pronounced change while gradual changes were observed in expression of TGF-β1. Results of these studies indicate that fabricated/encapsulation of nanoformulations in D-CIS causes activation of genomic components involved in the expression of growth promoter genes (Fig. 8).

3.3.6 Histopathology

Histopathological evaluation of the tissue samples collected from various groups on 5th day and 14th day of treatment was conducted to get an insight of cellular and subcellular changes of tissue regeneration (Fig. 9). In the present study, we observed that the D-CIS group was seeded with widespread infiltration of pro-inflammatory cells continuation of the inflammatory phase in the healing response in the D-CIS group. Similarly in D-CIS/CeO and D-CIS/Cur we observed a moderate degree of lymphocyte and fibroblast infiltration which indicates concurrent occurrence of the inflammatory phase and proliferative phase of wound healing in these groups. Contrarily to these findings in the D-CIS/CeO/Cur group, we observed that there was prominence of fibroblasts and receding of pro-inflammatory cells, and epitheliogeneis and angiogenesis were observed in this group which indicates progression of the proliferative phase of healing in this group. Histopathological scoring of specimens indicates that D-CIS/CeO/Cur (p ≤ 0.01) and D-CIS/CeO (p ≤ 0.05) treatment caused significant re-epithelization compared to D-CIS and D-CIS/Cur treatment on 5th and 14th-day post application of various topical preparations with no significant difference observed between D-CIS and D-CIS/Cur. Furthermore, significantly higher re-epithelization was observed in D-CIS/CeO/Cur (p ≤ 0.05) group compared to the D-CIS/Cur group on 5th and 14th day.Fig. 9 Histopathological evaluation of tissue specimens harvested from experimental rabbit model full thickness burn wound to evaluate cellular and sub-cellular response of after application of various fabricated biomaterials (LI: indicates Lymphocytic infiltration; G: Sebaceous Glands; NV: Neo-vascularization; NE: Neo-Epithelization; E: Epithelization; BK: Basophilic Keratinocytes).

Fig. 9

4 Discussion

Among various types of wounds burn wounds are considered a challenging task to heal as these wound cause impairments in quality of life, loss of plasma contents, are susceptible to infection, generation of free radicals, and are associated with extreme pain [24]. Owing to these difficulties associated with burn wounds scientists and medical researchers worldwide are grappling with the challenge of formulating a proficient dressing material for effective and precise wound healing of burn wound [25]. Although in the proceeding decades, the focus has been shifted to developing dressing material with the aid of nanotechnology from organic sources but still an effective dressing material is still lacking in clinical settings [26]. Researchers working in this direction have postulated some of the critical characteristics dressing material ought to exhibit requisite scientific and medical attributes to initiate early resolution of burn wound healing and these include biocompatibility, anti-microbial activity, anti-inflammatory activity, and the presence of bioactive agents to reduce duration of inflammatory phase with concurrent early onset of the proliferative phase [27]. Among various dressing materials in consideration, hydrogel-based dressing materials have been found to possess some of these ideal characteristics [5]. Henceforth to incorporate other associated properties in these types of dressing materials, researchers are evaluating various encapsulations in this type of dressing materials to fabricate an effective and potential dressing material for burn wound healing. Some researchers have proposed that ECM-based dressing materials mimic the extracellular conditions and can provide a scaffold for the promotion of early burn wound healing [28]. However, these ECM-based dressing materials lack anti-microbial, anti-oxidant, and anti-inflammatory properties henceforth these ECM-based dressing materials demand fortification with some extrinsic materials to incorporate these properties in this type of dressing materials and enhance their clinical utility. Therefore, relying on the discoveries of these investigators, the current investigation was undertaken to assess the efficacy of Nano formulations containing curcumin and cerium oxide encapsulated in caprine D-CIS for the healing of burn wounds. The study was based on the hypothesis that curcumin and cerium oxide will provide anti-inflammatory, anti-oxidant, and anti-microbial micro-environment which will provide rapid and effective burn wound healing. Although when used as monotheraputic topical preparations both of them have been found to possess properties that help in the promotion of burn wounds, synergistic properties of these components are yet to be established when encapsulated in caprine D-CIS.

In the current study, decellularization was found to be effective and DNA content in decellularized graft was found to be below the threshold levels which are in concurrence with [11]. Preliminary research indicates that DNA levels surpassing the threshold provoke an immunogenic and inflammatory reaction, leading to graft rejection [29]. In addition to this Glycosaminoglycan (GAG) levels in the fabricated graft of the present study were found to be above the threshold level, GAG provides an essential micro-environmental condition conducive for cell-to-cell contact, migration of cells, and transformation of cells which helps in providing micro-environmental conditions that mimic actual subcellular conditions [[12], [13], [14]]. Similarly, another important characteristic of the fabricated bioactive material is porosity, and in the present study, we found that the porosity of bioactive gels was in the nanoscale which helps to serve as cargo for Nano-encapsulation of various types of materials like that of cerium oxide [30]. Furthermore, desirable porosity helps in the sustained release of nano-encapsulation and provides a sustained presence of therapeutically active ingredients at the wound site which helps in early wound healing [16]. In addition to these characteristics, the fabricated bioactive gel needs to be evaluated for swelling properties and enzymatic/non-enzymatic degradation characteristics. An ideal dressing material should possess stable enzymatic/non-enzymatic degradation characteristics and needs to have enhanced swelling characteristics to provide a sustained supply of bioactive ingredients and nutrients at the wound site [[17], [18], [19], [20], [21]]. In the present study, we found that fabricated bioactive gel possesses desirable swelling and stable degradation characteristics which further support their utilization for effective wound healing dressing material.

The sustained release of therapeutically active molecules is supposed to play a pivotal role in wound healing as the sustained presence of therapeutically active molecules at the wound site promotes early and effective wound healing [31]. The property of sustained release depends on the physiochemical properties of the cargo and dispersion medium [32]. In the current study, we observed that CeO and curcumin exhibited sustained and prolonged release from fabricated gels and these properties are of therapeutic interest as earlier studies have proposed that early clearance of therapeutically active molecules from wound sites is responsible for the ineffectiveness of various therapeutic preparations. The sustained release of drug molecules causes effective penetration of drug molecules and ensures effective hydration which subsequently helps in the penetration of drug molecules across dermal barriers [[33], [34]]. In addition to the sustained release of CeO and curcumin from fabricated gels, these molecules possess therapeutically intrinsic properties which include small molecular size, and zwitter ion structure of Ceo which helps in effective penetration through dermal barriers and hence their precise activity [[35], [36]]. Recent studies have attributed the sustained release behavior of CeO and curcumin from biomaterial scaffolds to cause alterations in the kinetics of molecules incorporated into the formulation; previous research has documented the utilization of these molecular entities like CeO and curcumin follows a first-order kinetic model wherein the quantity of drug released exhibits a direct correlation with the concentration of the drug persisting in the depot. Consequently, the therapeutic significance diminishes over time [37]. The incorporation or sequestration of cerium oxide (CeO) and curcumin within a bioactive matrix induces a shift in kinetics from a first-order to zero-order mechanism. In this context, the liberation of the pharmaceutical agent or molecule becomes autonomous of the reservoir's remaining levels, resulting in a sustained and constant quantum of drug release. Such formulations are well-suited for topical administration in biomedical applications [38]. Henceforth it can be postulated physiochemical properties of cargo molecules (CeO and curcumin) and structural characteristics of the scaffold ensure sustained and prolonged availability of therapeutically active molecules at the wound site to promote structural and functional modification of cellular and sub-cellular pathways to induce cellular modeling and henceforth rapid wound healing.

Laboratory animal studies on full-thickness burn wounds revealed that D-CIS/CeO/Cur possesses significant burn wound healing potential as per the molecular and genomic determinants analyzed in the current experimental design. Upon macroscopic inspection, we noted prompt and proficient wound recovery in the D-CIS/CeO/Cur cohort relative to the other therapeutic cohorts. These observations could be ascribed to stimuli provided by bioactive components to genomic determinants and micro-environmental modification at wound sites released from bioactive gels. Furthermore, earlier studies have attributed delayed burn wound healing to the generation of reactive oxygen species (ROS) at the wound site which causes structural and functional alteration at the wound site. Generation of ROS results in the initiation of inflammatory response and hence release of inflammatory mediators which cause the arresting of the wound healing cycle in the inflammatory phase this leads to a postponed process of wound healing. In the present study, we observed that D-CIS/CeO/Cur resulted in modulation of inflammatory response and amelioration of oxidative stress which dampens inflammatory response at the wound site and causes a reduction in the duration of the inflammatory phase of wound healing [39]. Dampening of inflammation results in a shift from the inflammatory stage to the proliferative stage in a biological context of wound healing and henceforth rapid wound healing. Furthermore, curcumin and other anti-oxidants have been reported to cause apoptosis of poly morpho-nuclear lymphocytes henceforth dampening/modulation in the inflammatory pathway, during the proliferation phase, it induces the restructuring of collagen and its appropriate deposition within the wound site [[31], [32], [33], [34], [35], [36]].

In the present study, we found that the application of encapsulated bioactive gel results in accelerated deposition of connective tissue markers like hydroxyproline (HPR), hexosamine (HXA), and hexuronic acid (HUA). These observations align with outcomes from prior investigations that documented the efficacy of anti-oxidative agents such as curcumin and Ceo cause reorganization of collagen and its appropriate placement within the wound site [31]. Moreover, preclinical investigations have indicated that curcumin induces the spatiotemporal manifestation of genes associated with the synthesis of collagen and the deposition of mediators involved in connective tissue formation [40]. Furthermore, recently some studies have found that curcumin and Ceo exhibit potent antioxidant effects in intracellular and extracellular environments which concurrently cause the synthesis of collagen type-I and after prolonged exposure to curcumin fibroblast synthesize collagen type-II, III, and IV. Our results indicate that the inclusion of curcumin and Ceo causes overexpression of growth promoter genes TGF-β1, IGF-I, and EGF. These results further support the inclusion of curcumin and Ceo in bioactive dressing materials which imparts anti-oxidant activity, anti-inflammatory activity, and spatial and selective expression of genomic determinants that promote wound healing [41]. Moreover, earlier research has theorized that immune and inflammatory cellular components actively participate in the healing process, and TGF β1 has been identified as playing a crucial role in the preferential mobilization of these cellular elements [21]. Preclinical investigations have revealed that TGF β1 induces an expedited mobilization of fibroblasts, subsequently leading to a phenotypic transition of fibroblasts into myofibroblasts. Additionally, it diminishes the infiltration of inflammatory cells into the wound region [25]. Based on the findings of the current investigation, it can be hypothesized that the encapsulation of bioactive gels containing curcumin and Ceo creates optimal circumstances for the regeneration of burn wounds, suggesting a prospective topical therapeutic formulation for promoting burn wound healing.

5 Conclusion

The findings of the current investigation suggest that encapsulation of bioactive gels with curcumin and Ceo promotes burn wound healing by providing sustained release of bioactive principles and another pharmacologically active molecule at the wound site. The combinational effect of bioactive gel, curcumin and Ceo causes scavenging of free radicals at the wound site which subsequently causes dampening of the inflammatory pathway by reducing the generation of pro-inflammatory cytokines and increasing the generation of anti-inflammatory cytokines. These changes result in the selective expression of growth-promoting genes temporally and spatially which causes an increase in extracellular matrix at wound site. Collectively these findings indicate that encapsulated cargo of bioactive gel with curcumin and Ceo can serve as an effective therapeutic preparation for burn wound healing. Although the current study is in the preliminary phase, it demands further studies to establish and identify the therapeutic hotspots targeted by these bioactive principles and their potential fabrication in commercial preparation.

Author disclosure and ghostwriting statement

The authors of this article attest that they have directly contributed to the conception, design, and execution of the research work presented herein. All authors have reviewed and provided substantial input into the content of the manuscript. We take full responsibility for the content and originality of this manuscript. All sources of data and materials used in the research have been appropriately cited.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Abbreviations

D-CIS Decellularized caprine small duodenum submucosa

Ceo Cerium Oxide

TNF-α Tumor Necrosis Factor-alpha

ECM Extracellular Matrix

FESEM Field Emission Scanning Electron Microscope

EDAX Energy-dispersive spectroscopy

FTIR Fourier Transform Infrared Spectroscopy

PBS Phosphate-Buffered Saline

DMEM Dulbecco's Modified Eagle Medium

MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromide

HPR Hydroxyproline

HXA Hexosamine

HUA Hexuronic Acid

GSH Glutathione Peroxidase

GPx Glutathione Peroxidase

NO Nitric Oxide

IL-1 IL-2, IL-6, Interleukin-1, Interleukin-2, Interleukin-6

EGF Epidermal Growth Factor

TGF-1β Transforming Growth Factor-1 Beta

VEGF Vascular Endothelial Growth Factor

GAPDH Glyceraldehyde 3-Phosphate Dehydrogenase

c-DNA Complementary Deoxyribonucleic Acid

H&E Hematoxylin and Eosin

CI Confidence Interval.

Peer review under responsibility of the Japanese Society for Regenerative Medicine.
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