Biomedical Potential of Keratin-Biphalin Wound Dressing in Diabetic Mice: In Vitro and In Vivo Studies

ABSTRACT

Strategies for successful healing of diabetic wounds are lacking. Natural-derived dressings have gained much attention due to their promising properties. We hypothesized that keratin-biphalin fiber-dressing accelerates skin wound healing in a mouse model of diabetes. Keratin fibers were obtained from mouse fur and soaked in a biphalin solution. Studies on NIH/3T3 cells showed that keratin-biphalin fibers increase cell viability. In vivo studies were made on C57BL/6J mice with iatrogenically induced diabetes. Two full-thickness wounds were created on the back of the mice, one untreated and the other treated with an experimental dressing. Western blot analysis, histopathological and immunohistochemical staining were performed. Biphalin was slowly released from keratin fibers. The obtained keratin-biphalin fibers were biocompatible and supported cell growth. Western blot analysis showed that cells treated with our dressing had an increasing expression level of mTOR, and p-AKT 72 h post-treatment compared to 24 h and 48 h. Animal studies showed that the keratin-biphalin dressing accelerated wound healing on days 5 and 15. Treated wounds showed faster reepithelization and developed thicker epidermis. Histopathological and immunohistochemical studies confirmed that keratin-biphalin fibers stimulate macrophage infiltration, which promotes tissue remodeling and regeneration. Exogenous keratin-biphalin fibers p-AKT/mTOR expression protein in vitro and accelerate skin wound healing in diabetic mice.

摘要

缺乏成功治愈糖尿病伤口的策略. 天然敷料因其良好的性能而备受关注. 在糖尿病小鼠模型中，我们假设角蛋白双页蛋白纤维敷料可以加速皮肤伤口愈合. 角蛋白纤维从小鼠毛皮中获得，并浸泡在联苯林溶液中. 对NIH/3T3细胞的研究表明，角蛋白双页蛋白纤维可提高细胞活力. 对患有医源性糖尿病的C57BL/6J小鼠进行了体内研究. 在小鼠背部造成两个全厚伤口，一个未经治疗，另一个用实验敷料治疗. 进行蛋白质印迹分析、组织病理学和免疫组织化学染色. Biphalin从角蛋白纤维中缓慢释放. 所获得的角蛋白双页蛋白纤维具有生物相容性并支持细胞生长. Western印迹分析显示，与24小时和48小时相比，用我们的敷料处理的细胞在处理后72小时具有增加的mTOR和p-AKT表达水平. 动物研究表明，角蛋白联苯胺敷料在第5天和第15天加速了伤口愈合. 治疗后的伤口表现出更快的再上皮化和更厚的表皮. 组织病理学和免疫组织化学研究证实，角蛋白双页蛋白纤维刺激巨噬细胞浸润，促进组织重塑和再生. 外源性角蛋白biphalin纤维p-AKT/mTOR在体外表达蛋白并加速糖尿病小鼠皮肤伤口愈合.

KEYWORDS:

• Keratin fibers
• natural-derived dressing
• wound healing
• biphalin
• diabetic mouse model

关键词:

• 角蛋白纤维
• 天然衍生敷料
• 伤口愈合
• 糖尿病小鼠模型

Introduction

Delayed wound healing is one of the most common medical problems in today’s world. Due to increasing incidence of diabetes mellitus, millions of patients will need treatment of chronic wounds in the nearest future. Chronic wounds in patients with diabetes are mainly caused by ischemia, impaired angiogenesis, and prolonged inflammation (Capó et al. 2023). Thus, therapeutic process is often very expensive and requires a multidisciplinary approach. This is why researchers are looking for wound dressing that will increase the effectiveness of chronic wound therapy. Fibrous dressings are increasingly being used for medical purposes. They can be both modified with active substances that will be secreted during the healing process and affect the hemostasis essential for wound healing (Yang et al. 2022). Various natural-derived dressings possess the ability to accelerate wound healing (Konop, Rybka, and Drapała 2021; Mazurek et al. 2022; Qu et al. 2018). Our team proposes experimental dressing made of keratin fibers. Keratin-based biomaterials derived from wool, chicken feather, hair and bristle have gained much attention lately and represent a novel approach to wound management (Chen et al. 2021; Jull et al. 2020; Konop et al. 2023; Pakdel et al. 2022; Sadeghi et al. 2020; Sanchez Ramirez et al. 2022). Several studies showed that keratin fibers possess a unique set of properties, including tissue biocompatibility, biodegradability and the ability to support cell growth (Gao et al. 2019; Konop et al. 2021). Due to the extensive surface morphology, it is possible to incorporate compounds with anti-inflammatory, antibacterial properties (Bochynska-Czyz et al. 2020; Sanchez Ramirez et al. 2022). Dressings made of keratins are effective in wound healing, both in healthy and diabetic conditions (Chen et al. 2020; Konop et al. 2023; Ye et al. 2022). However, in today’s world it is also necessary to provide a therapy that delivers as little pain as it is possible. It is well known that the analgesic effect of the dressing improves the compliance and life quality of patients (Graziottin et al. 2011). For this reason, both antinociceptive agents and the primary dressings in which they can be incorporated gain much attention in chronic wound therapy. Natural keratin fibers do not possess pain-relief effect at the sufficient level. Fortunately, the keratin scaffold architecture enables researchers to modify it with additional substances and can release them in a predictable manner (Konop et al. 2020; Konop, Rybka, and Drapała 2021; Sadeghi et al. 2020). The gold standard for treating pain are opioids, but the impact of natural or synthetic opioids in wound healing is yet to be evaluated (Berthézène et al. 2021; Price et al. 2008; Rook and McCarson 2007; Shanmugam et al. 2017). Biphalin is a dimeric enkephalin with a potent analgesic effect that was synthesized by Lipkowski in 1982 (Lipkowski, Konecka, and Sroczyńska 1982). It is distinguished by its strong analgesic properties with relatively low dependence potential (Shen and Crain 1995). However, despite the desired analgesic effect, the impact of biphalin on the healing of full-thickness wounds is not fully understood (Muchowska et al. 2020; Yıldız et al. 2018). In this study, we have decided to develop an experimental dressing created based on an insoluble fraction of keratin, enriched with biphalin. The main goal was to assess the effect of keratin-biphalin fibrous scaffolds as a new therapeutic approach in full-thickness skin wounds in a diabetic mice model. In addition, the impact of the experimental dressing on Akt/mTOR signaling pathway was studied, as data suggest that its dysfunction plays a crucial role in impaired wound healing of diabetic wounds (Huang et al. 2015).

Materials and methods

Preparation of experimental wound dressings

The mouse fur keratin-derived powder (FKDP) was prepared as described previously (Konop et al. 2017, 2021). All technical details are described in supplementary materials. FKDP was suspended in 0.1% solution of biphalin (5 g of FKDP/10 ml of 0.1% biphalin) by 24 h continuous blending and then left to create a sediment layer. The final product (FKDP +0.1%Biph) was lyophilized and powdered.

In vitro drug release from the wound scaffold and electrophoretic measurements

The release of biphalin from keratin dressing (FKDP +0.1%Biph) was examined according to the method described previously (Konop et al. 2021). All technical details are described in supplementary materials.

Cell culture

NIH/3T3 fibroblasts (ATTC CRL1658) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Sigma-Aldrich, St. Louis, USA) with 10% (v/v) heat-inactivated, fetal bovine serum (FBS, Sigma-Aldrich, St. Louis, USA), 2 mM L-glutamine (Sigma-Aldrich, St. Louis, USA), 1 mM sodium pyruvate (Sigma-Aldrich, St. Louis, USA) and 1% (v/v) penicillin-streptomycin solution (Sigma-Aldrich, St. Louis, USA). Cell cultures were kept at 37°C and 5% CO2.

Cell proliferation assay

Cells were seeded into 96-well plates at a density of 3 × 10³ ­­ cells/well and incubated for 24 h. Next, different concentrations of tested dressing or opioids were added to the cells and incubated for 24 and 48 h. Cell viability was evaluated with Cell Proliferation Kit I (MTT) (Roche, Basel, Switzerland). All experiments were performed according to the manufacturer’s protocol and described in the supplementary materials.

Hemolysis assay

Compound (biphalin or morphine) or dressing induced (FKDP +0.1%Biph or FKDP + 0.1%Mor) hemolysis was evaluated according to Mazzarino et al. (Mazzarino et al. 2015) with a modification described in supplementary materials.

In vitro wound-healing assay

A “scratch assay” was created in a monolayer of NIH/3T3 cells that were seeded in 24-well plates at a density of 2 × 10­⁴ per well (ATCC CRL1658, MA, USA). Cells were then incubated with experimental keratin dressings for up to 48 h. The rate of migration was calculated at each time point by measuring the distance between the edges of the wound and described in supplementary materials.

Immunoblotting

To determine Akt-1, Akt-1 phospho S473, mTOR and RpS6 protein levels, 10 µg of protein extracts from cell culture was tested by electrophoresis on 10% (Akt-1, Akt-1 phospho, Rps6) and 8% (mTOR) SDS-PAGE gels. Resolved proteins were transferred onto PVDF membranes (Bio-Rad, Hercules, CA, USA), blocked with skim milk and incubated with the primary and secondary antibodies. The primary and secondary antibodies used for immunoblotting experiments are described in supplementary materials (see Supplementary Table 1, Additional File 1). For quantitative analysis of protein content, reactive bands were quantified relatively to actin using a Molecular Imager with Quantity One software (Bio-Rad, Hercules, CA, USA).

Animals

Twenty 12–15-week-old male C57BL6/J mice (23–25 g body weight) were acquired from the animal house. The experimental design of the study was approved by the 4th Local Ethics Committee for Experiments on Animals at the National Medicines Institute, Warsaw, Poland (Certificate of Approval No. 58/2012). Procedures adhered to guidelines published in European Directive 2010/63/EU on the protection of animals used for scientific purposes.

Iatrogenically induced diabetes

Diabetes in mice was induced with intraperitoneal injection of streptozotocin at the dose 80 mg/kg (STZ, Sigma-Aldrich, St. Louis, USA). A mouse was considered diabetic if three consecutive measurements of a blood glucose level taken from the lateral saphenous vein were above 250 mg/dL.

Surgical procedure

Surgical procedure was performed according to the protocol described previously (Konop et al. 2021). Skin biopsies were taken from control and FKDP + 0.1%Biph treated wounds at the relevant time points for histological and immunofluorescence examination by dermatopathologist. Technical details are described in supplementary materials. After the procedure, one wound was covered with the experimental dressing, and the other was left untreated. Once applied, the dressing was not changed until the end of the experiment.

Histopathological and immunofluorescence analysis

The preparation of histological specimens was identical for all examined tissue samples according to the standard procedure at the Histology Laboratory at the Department of Dermatology, as described previously (Konop et al. 2018, 2023). Immunofluorescence staining for macrophages, Nuclear Factor kappa B (NF-κβ; subunits: p65 and p50), Tumor protein P53, VEGF and neutrophils were performed with the appropriate primary and secondary antibodies. Skin biopsies were examined using a light microscope or Optiphot-2 Nikon fluorescent microscope (Nicon Instruments Inc., Toki, Japan) equipped with the appropriate filters and recorded with a Model DS-L1 Nikon camera. All technical details are described in supplementary materials.

Cytokine analysis

On days 5, 8 and 15 post-injury, the blood samples were taken from each mouse. Blood was collected from the right ventricle of the heart for tubes without anticoagulant for serum analysis. To determine the serum concentrations (pg/ml) of interleukin (IL)-1β, IL-6, IL-10, IL-17A, interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α) using Bio-Plex Pro Mouse Cytokine Panel (lot #5030291, Bio-Rad, Poland) according to the manufacturer’s protocol. Magnetic beads were analyzed at a Bio-Plex 200 (Luminex, Bio-Rad, Hercules, CA, USA).

Statistical analysis

All data are presented as means ± standard error of the mean (SEM). Data were analyzed with the use of GraphPad Prism v.5.0 software (GraphPad Software, San Diego, USA). Kolmogorov–Smirnov test was made to confirm the normal distribution before applying all parametric tests. The statistical analyses were performed using two-way ANOVA followed by the Bonferroni post-hoc test. Student’s t-tests for analysis of dependent and independent variables were also used (Konop et al. 2017). In all cases, a p-value of <0.05 was considered significant.

Results

Preparation of experimental dressing and monitoring of the release of biphalin from the examined dressing using capillary electrophoresis

In order to describe the structure of the obtained FKDP, an SEM study was conducted. SEM images show the fiber structure before and after pepsin digestion (See Supplementary Figure S1, Additional File 1). In the electropherogram obtained for FKDP dressing (basic dressing), no signal derived from an opioid was found. The electropherograms obtained for the examined FKDP + 0.1%Biph demonstrated the slow release of biphalin for 5 consecutive days (See Supplementary Figure S2, Supplementary Table 2, Additional File 1).

Cell viability assay

We evaluated the effect of opioids and tested dressings on NIH/3T3 cells (Suplementary Figure S3 a-d, Additional File 1). When treated with biphalin or morphine alone, murine fibroblasts showed no significant change in cell viability when compared to untreated cells (Supplementary Figure S3 a, b, Additional File 1). In contrast to opioid-treated cells, the viability of cells treated with all tested dressings was significantly higher compared to control cells. After 24 h, cells treated with FKDP grew better than cells treated with FKDP + 0.1%Biph. However, after extended incubation, cells treated with FKDP + 0.1%Biph showed higher viability compared to FKDP-treated cells (p < .05). When exposed to FKDP + 0.01%Mor for 48 h significant increase in cell viability was found (p < .05). After 24 h incubation, cells exposed to FKDP + 0.1%Mor showed higher viability (Supplementary Figure S3 c, Additional File 1) compared to FKDP + 0.1%Biph-treated cells. However, after 48 h incubation, higher viability was observed for cells treated with FKDP + 0.1%Biph (Fig S3 d). For all dressings, the highest increase in cell viability was noted at a concentration of 0.01% wound dressing (m/v) after both 24 h and 48 h of treatment.

Haemolysis assay

There was no statistically significant difference in the level of hemolysis obtained for biphalin and morphine (See Supplementary Figure S4a, Additional File 1). In opioid-incrusted dressings, the level of hemolysis did not exceed 2% for all dressings. Among tested dressings, FKDP + 0.1%Biph showed the lowest level of degradation of RBCs, whereas the level of FKDP-induced hemolysis was the highest (See Supplementary Figure S4 b, Additional File 1).

In vitro wound healing

Cells treated with opioid-incrusted dressings (1 mg/ml) and untreated cells migrated at a similar rate. The highest rate of migration was observed for cells treated with FKDP. There was no significant difference in migration rate for cells treated with FKDP + 0.1%Biph and FKDP + 0.1%Mor (See Supplementary Figure S5, 6 a, Additional File 1). The rate of cellular migration for biphalin and morphine was similar. After 24 h, cells treated with 10 µM morphine migrated faster than cells treated with 10 µM biphalin (p < .05) (See Supplementary Figure S5, 6 b, 6 c, Additional File 1). The migration rate was not concentration dependent (See Supplementary Figure S5, Additional File 1).

Immunoblotting

We observed a significantly increased mTOR protein level at 72 h post-treatment compared to 24 h and 48 h in FKDP, FKDP + 0.1%Biph and Biph treated cells. On the other hand, rapamycin as per assumption inhibits the mTOR protein level 24 h post-treatment (Figure 1). Interestingly, the combination of FKDP + 0.1%Biph+Rapamycin 100 µM did not inhibit mTOR protein 24 h post-treatment (p < .05).

Figure 1. The protein expression levels of RPS6, akt, p-akt, and mTOR were examined by Western blot analysis. The protein expression levels are displayed as the mean ± SEM (n = 3). The data were only tested by the t-test for dependent samples (p-value <.05 was considered significant, mean ± SEM).

In cells treated with 0.1% of FKDP or/and FKDP + 0.1%Biph the highest AKT protein level was observed at 48 h (p < .05) and decreased 72 h post-treatment (Figure 1). Decreased AKT protein level was observed at 24 h post-treatment in the biphalin and rapamycin group. However, it significantly increased at 72 h post-treatment (p < .05 24 h vs. 72 h). The combination of FKDP + 0.1%Biph+Rapamycin 100 µM did not induce inhibition of AKT protein level at 24 h post-treatment (p < .05). The influence of the examined substances on the phosphorylation of AKT (Figure 1). FKDP at concentration 0.1% upregulated the protein level of p-AKT after 48 h compared with 24 h and 72 h post-treatment (p < .05). The FKDP + 0.1% biphalin activated phosphorylation of AKT at all time points. Biphalin alone increased p-AKT protein level at 72 h post-treatment. The combination of FKDP + 0.1%Biph+Rapamycin 100 µM did not inhibit p-AKT protein expression at 24 h post-treatment (p < .05). Moreover, the inhibition of phosphorylation slowly decreased. The highest protein level of ribosomal protein S6 (RPS6) was observed in 72 h post-treatment in cells treated with 0.1% of FKDP and 100 µM of biphalin (p < .05) alone. For rapamycin-treated cells, a reduction in RPS6 protein level was observed 72 h after treatment. In cells treated with FKDP + 0.1%Biph, the up regulation (p < .05) of RPS6 protein was observed at 48 h compared with 24 h and 72 h time points respectively. Cells treated with FKDP + 0.1%Biph with the addition of 100 µM of rapamycin showed up-regulation of RPS6 protein level after 24 h. Statistically significant inhibition of RPS6 protein expression was observed at 48 h and 72 h compared to 24 h.

Diabetes study

A dose of STZ 80 mg/kg destroyed pancreatic β-cells, and thus mice developed diabetes (BGL ~520 mg/dL). Control mice receiving citrate buffer had an average BGL of 200 mg/dL. It was observed that after intraperitoneal (i.p.) injection of STZ, mice lost weight despite having unlimited access to food and water (p < .05) (See Supplementary Figure 7a, b, Additional File 1).

Effect of keratin dressing on the skin wound healing in diabetic mice

Wounds treated with FKDP + 0.1%Biph dressing healed significantly faster (p < .05) on days 5 and 15 compared to the control wound (Figure 2, Supplementary Figure 8). Eight-day post-injury wounds treated with keratin dressing still healed faster compared to untreated wounds; however, the difference was not statistically significant. At the end of the experiment (day 15 post-injury), wounds treated with FKDP + 0.1%Biph were healed by 78.66% if compared to the control wounds (62.25%).

Figure 2. The effect of fur keratin-derived powder containing biphalin on skin wound healing. Because of the decreasing number of surviving mice, the data (mean ± SEM) were only tested by the t-test for dependent samples (for each post-wounding day separately), with no prior two-way analysis of variance. * - p < .05, ** p < .01.

Histopathological studies

On the fifth day after surgery, a thin layer of the epidermis was visible on the top of both wounds. The treated wound was characterized by a slightly higher density of cells in the dermis in comparison to the untreated wound. FKDP + 0.1%Biph treated wounds had fewer voids and discontinuities than the control wound. Numerous fields with extravasated erythrocytes were observed in both wounds (Figure 3). On the eighth day, the treated wounds showed a visible thicker dermal layer than the untreated ones. The epidermis was restored in both wounds. Few areas with extravasated erythrocytes were present. The dressed wound showed fewer tissue gaps compared to the untreated wound.

Figure 3. a) microscopic view of control and fur keratin-derived powder containing biphalin (FKDP +0.1%Biph)-treated wounds during the course of healing; hematoxylin-eosin. Original magnification: 100 × .Legend: - epidermis; - hair follicle; - empty zones; - blood extravasation, (color online, black and white in print). b) microscopic view of the control and FKDP + 0.1%Biph-treated wounds during the healing process. H&E. white arrow – neutrophils, black arrow – macrophages, green arrow – multinucleated giant cells, black & white arrow – lymphocytes; magnification 100× (color online, black & white in print). c) type of cells infiltrating FKDP + 0.1%Biph and control wound during tissue recovery visible under the light microscopy (the data were only tested by the t-test for dependent samples, p-value <.05 was considered significant) (color online, black and white in print).

Fifteen days post-injury wounds treated with FKDP + 0.1%Biph had higher cell density in the dermis with the morphology of fibroblasts/fibrocytes compared to the untreated wound. The applied dressing was fully incorporated into the regenerating tissue and surrounded by histiocytes, multinucleated giant cells and fibroblasts without suppuration and granuloma formation (Figure 3). In both wounds, a layer of the reconstituted epidermis was visible. The epidermis was thicker in the dressed wound; the dermis layer showed a normal structure with uniform cell distribution. In untreated wounds, there was bleeding into the wound area and a significant number of “tissue discontinuities” and “loose spaces’’ compared to the dressed wound. On the fifth day after the injury, in the control wound, increased neutrophilic content was observed with accompanying lymphocytes and occasionally visible monocytes (Figure 3). Wounds treated with FKDP + 0.1%Biph showed less exudate. In skin biopsies taken from the dressed wound, predominantly macrophages infiltrated with only a few histiocytes found in the proximity to the keratin/biphalin dressing. Eight days after surgery, the control wound was dominated by neutrophils and occasionally visible macrophages. However, in dressed wounds, multiple macrophages with few histiocytes were observed in treated wounds. Additionally, in FKDP + 0.1%Biph treated wounds some multinucleated giant cells (MGCs) showed engulfment of keratin in their cytoplasm with the characteristic black color corresponding to foreign body granulomas. This foreign body reaction was typically deprived of neutrophils. Two weeks after the surgery, in the control wounds, neutrophilic inflammatory response decreased showing mostly macrophages and histiocytes. In FKDP + 0.1%Biph treated wounds histiocytes, MGCs and lymphocytes were present. An inflammatory response consisting of macrophages and histiocytes at an early stage of healing is more favorable for tissue remodeling and regeneration.

Histological sections of tissue from FKDP + 0.1%Biph treated wounds showed collagen fibers packed more densely with more fibroblasts in comparison to the control group. On the 8th and 15th days post-injury, Masson staining revealed thicker, denser organized and more abundant collagen bundles in the keratin-biphalin-treated wounds. Contrarily, the control wounds presented more irregularly arranged and thinner collagen fibers (See Supplementary Figure 9a, 9b, Additional File 1). Statistical analysis demonstrated a significant effect of FKDP + 0.1%Biph dressing on the reduction of the number of areas with extravasated erythrocytes in a time-dependent manner. An interaction (p = .012) was observed between the applied dressing and the time from the injury to the reduction of the number of areas with extravasated erythrocytes during the healing process (See supplementary Table 3, Additional File 1).

Immunohistochemical studies

In control wounds at day 5 post-injury, neutrophils predominated, and decreased in the further investigated post-injury time points (8, 15 days). On the other hand, in FKDP + 0.1%Biph treated wounds lesser numbers of neutrophils were observed during the whole post-injury period (Figure 4). In the next step, we examined the wound infiltration by lymphocytes (Figure 4). On 5 and 8-days post-injury, stronger labeling for lymphocytes was seen in keratin-biphalin-treated wounds compared with untreated controls. However, 2 weeks after surgery, immunolabeling was similar in both wounds.

Figure 4. a) tissue biopsy taken from FKDP + 0.1%Biph (3 upper panels) treated and control wounds (lower panel) immunolabeled for neutrophils (green), nucleus (blue). b) tissue biopsy taken from FKDP + 0.1%Biph (3 upper panels) treated and control wounds (lower panel) immunolabeled for lymphocytes (green), nucleus (blue). Note the changes in immunoreactive cell numbers during the healing course (color online, black & white in print) (color online, black and white in print).

Macrophage infiltration in control and keratin-biphalin-treated wounds were also examined. Stronger labeling of macrophages was observed in FKDP + 0.1%Biph treated wounds at all-time points, compared with untreated controls. All cells were positive also for p53 (Figure 5).

Figure 5. Tissue biopsy taken from FKDP + 0.1%Biph (3 upper panels) treated and control wounds (lower panel) immunolabeled for macrophages (red), p53 (green). Note the changes in immunoreactive cell numbers during the healing course (color online, black & white in print).

In addition, we examined the presence of NF-κβ and we found that in both FKDP + 0.1%Biph treated wounds and controls the immunolabeling for NF-κβ increased during recovery. However, immunolabeling was weaker in treated wounds than in undressed wounds (See Supplementary Figure 10, Additional File 1). We observed slightly stronger VEGF labeling in keratin-biphalin dressed wounds on days 5, 8 and 15 post-surgery compared with controls (See Supplementary Figure 11, Additional File 1).

Changes in cytokine level during the healing course

A statistically significant increase in the concentration of pro-inflammatory cytokines: IL-1β, IL-6, IL-17A and TNF-α was seen during the experiment (p < .05) (See Supplementary Figure 12, Additional File 1). The concentration of IL-1β slowly decreased compared to day 5 post-injury. The highest concentration of IFN-γ was observed on day 5 compared with day 8 and 15 post-injury (p < .05). At the same time, a slight and non-significant increase in IL-10 was observed.

Discussion

Our study confirmed that keratin fibrous scaffolds containing biphalin are safe and significantly accelerate skin wound healing in diabetic mice. In vitro examination revealed that keratin/biphalin fibrous scaffolds are nontoxic and increase NIH/3T3 viability. In an in vivo model, the dressing was incorporated into regenerated tissues without significant inflammatory reaction. The FKDP + 0.1%Biph dressing induced infiltration of macrophages, monocytes, and histiocytes-promoted tissue remodeling and new collagen synthesis. This study is the first that utilizes capillary electrophoresis (CE) for the monitoring of biphalin release from the wound dressing. Previously, the CE was used by Hettiarachchi et al. (2001), only for the determination of the purity of the peptide after synthesis. Lazarczyk et al. (2010) showed that biphalin used at micromolar quantities stimulated fibroblast proliferation. However, our in vitro examination has shown that FKDP + 0.1%Biph increased proliferation after 48 h incubation but did not increase cell viability when biphalin was tested alone. This phenomenon may be related to the synergistic effect of keratin fibers and biphalin. Similar results were observed with keratin scaffolds or keratin casomorphin scaffolds (Konop et al. 2017, 2018, 2021). We confirmed that FKDP + 0.1%Biph dressing is tissue biocompatible, nontoxic and accelerates wound healing (Bochynska-Czyz et al. 2020; Konop et al. 2017, 2020). Cuvas Apan et al. (2016) showed that topical tramadol application on the cornea did not cause any side effects, and wound healing was not affected. On the other hand, Yıldız et al. (2018) suggested that biphalin at concentration 1 µM had positive effects on epithelial wound healing. Statistically significant differences in the concentration of pro-inflammatory cytokines IL-1β, IL-6, IL-17A, IFN-γ and TNF-α were observed during the whole experiment period. IL-1β may delay wound closure and IL-6 main task is to coordinate the inflammatory process (Johnson et al. 2020; Tan et al. 2021). The reduction of IFN-γ and IL-17A during wound healing may be related to the ongoing tissue remodeling process and decreased inflammatory phase (Hadian et al. 2019). Increasing levels of anti-inflammatory cytokine IL-10 can be associated with the infiltration of macrophages to the wound as confirmed by immunohistochemical staining. Histopathological and immunofluorescence studies of FKDP + 0.1%Biph-treated wounds showed increased infiltration of macrophages and lymphocytes compared with control wounds which were preoccupied with neutrophils. The presence of macrophages in the inflammatory infiltrate contributes to the angiogenic process by producing a broad array of angiogenic growth factors and cytokines promoting remodeling and angiogenesis (Martin et al. 2010). Our study found that from day 5 post-injury in FKDP + 0.1%Biph treated wound a stronger Vascular Endothelial Growth Factor (VEGF) immunolabeling was seen compared to the untreated side (Bao et al. 2009). VEGF is a major proangiogenic mediator during wound healing process. Its expression increases from the initial stages of wound healing and is most likely to be elevated until the process is complete (Johnson and Wilgus 2014). Increased expression of VEGF is a characteristic sign of improved healing in diabetic wounds. The primary antidiabetic drug metformin in its palette of actions can activate the vascular endothelial growth factor pathway. Exosome/metformin-loaded hydrogels were shown to accelerate wound healing in diabetic mice (He et al. 2023; Zhang et al. 2023). We also observed stronger immunolabeling for p53 in biopsies taken from FKDP + 0.1%Biph treated wounds, similar results were observed in our previous studies (Konop et al. 2021). In response to inflammation induced by various factors such as infection, injury, or tissue damage, p53 is activated to suppress inflammation (Uehara and Tanaka 2018). NF-ĸB is associated with cell proliferation, adhesion, inflammation, and elimination of reactive oxygen species (Johnson and Wilgus 2014; Park et al. 2018; Qin et al. 2015). In the proximity to the FKDP + 0.1%Biph dressing, an increased fibroblast-rich cellular infiltrate was seen. Waters et al. (Waters, VandeVord, and Van Dyke 2018) showed that keratin biomaterials made from human hair stimulate anti-inflammatory responses from native macrophages and polarize them toward the M2 phenotype. It is important to highlight that NF-κβ signals play a crucial role in the healing of various wound types including corneal epithelial and cutaneous wounds (Chen et al. 2016; Konop et al. 2021; Poranki et al. 2014; Sando et al. 2010; Wang et al. 2013). Bigliardi et al. (2002) showed that opioid peptides released in wound tissue were crucial for re-epithelialization and tissue regeneration. Poonawala et al. (2005) showed that fentanyl was the most effective in assisting wound closure, with hydromorphone and morphine being weaker, but still having significant positive effects if compared to placebo. Gupta et al. (2015) showed that topically applied fentanyl promotes the closure of ischemic wounds in diabetic rats. Possible adverse effects of opioids on wound healing were also suggested, Martin et al. (2010) showed that the chronic morphine treatment resulted in a marked delay in wound closure, tissue disintegrity, and increased bacterial infections. In our study, increased activation and better organization of collagen fibers were found in treated wounds compared to the controls. This is in accordance with a previous animal-based study that suggested opioids might enhance collagen deposition and thus tensile strength in incisional wounds (Chang et al. 2010; Tang et al. 2012). Our in vitro examination correlated with western blot analysis and confirmed activation of the AKT/mTOR pathway demonstrating that keratin-treated wounds healed faster when compared to the undressed side. Activation of the Akt/mTOR pathway can promote cell proliferation and migration, angiogenesis and collagen synthesis (Bujor et al. 2008; Clark and Pavlis 2009; Goren et al. 2009; Wullschleger, Loewith, and Hall 2006). Huang et al. (Huang et al. 2015) showed that topical application of GM-CSF markedly improved diabetic wound healing through the activation of the Akt/mTOR signaling pathway, which presents a novel clinical intervention strategy to improve diabetic wound healing. Similar results were obtained by Xing et al. (2015) in cutaneous wound healing in mice. In conclusion, our findings indicate that keratin dressing containing biphalin is a safe, non-toxic, tissue-biocompatible scaffold, preventing prolonged inflammatory reactions, promoting tissue remodeling and accelerating full-thickness skin wound healing in diabetic mice. This is one of the first studies demonstrating that exogenous keratin protein-containing synthetic opioid – biphalin up-regulates the expression of AKT/mTOR protein in vitro during wound healing process.

Authors’ contributions

MK – investigation, data curation, formal analysis, funding acquisition, writing – original draft, and writing – review & editing, JC, AK, MS, ŁM, MR, DS, EK, AD – investigation, formal analysis, writing – original draft, ZR – formal analysis, writing – original draft, RAS – supervision, writing – original draft, JC, MK – supervision, formal analysis, writing – original draft, and writing – review & editing.

Highlights

• Keratin fibers have been used to prepare a wound dressing with promising properties.

• Keratin-biphalin dressing is biodegradable, biocompatible and nontoxic.

• Biphalin is slowly relased from scaffolds and support cell growth.

• Increased healing rate was seen in wounds treated with keratin-biphalin dressing.

• p-AKT/mTOR protein was upregulated in cells treated with experimental dressing.

Ethical approval

We confirm that all the research meets ethical guidelines and adheres to the legal requirements of the study country. The experimental design of the study was approved by the 4th Local Ethics Committee for Experiments on Animals at the National Medicines Institute, Warsaw, Poland (Certificate of Approval No. 58/2012). The study was conducted under the approval of the Bioethics Committee – Commission for the Supervision of the Research on People and Animals at CSK MSWiA in Warsaw (no. 67/2017)

Acknowledgments

The authors would like to express their gratitude to the Prof. Lidia Rudnicka, Head of the Department of Dermatology, Medical University of Warsaw, for her valuable tips and Agnieszka Bujanowska of the Department of Dermatology, Medical University of Warsaw, for her assistance in the preparation of the histological slides. We thank dr Anna Leśniak, Department of Pharmacodynamics, Medical University of Warsaw, for her valuable suggestions on working with biphalin.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary data

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15440478.2023.2287647

Additional information

Funding

This research was supported by Warsaw Medical University under Student mini-grants (1S7/3/M/MG/N/22, 1S7/1/M/MG/N/22, 1S7/1/M/MG/N/21, 1S7/2/M/MG/N/21) and the Young Research Grant (1S7/2/M/MB/N/20/20:MAT, WLS14/1/M/MB/N/23).