Unleashing the healing potential: Exploring next-generation regenerative protein nanoscaffolds for burn wound recovery

Severe burn injuries pose a significant global health problem, resulting in high morbidity and mortality rates. Approximately 11 million people experience severe burns annually, requiring extensive medical care. Burns are categorized by severity and depth, with third-degree burns representing the most severe, involving complete destruction of all skin layers and posing significant challenges for treatment. The limited availability of autografts necessitates the development of effective wound dressings to facilitate fibroblast cell adhesion, migration, proliferation, and angiogenesis, promoting efficient wound healing. Nanofibrous dressings have emerged as promising candidates for managing severe burn wounds due to their high surface area, nano-porosity, excellent air permeability, and barrier properties against infection and dehydration. Electrospinning is a widely used technique for producing these nanofibrous scaffolds (ENs), offering scalability, simplicity, cost-effectiveness, and versatility. The resulting ENs mimic the extracellular matrix (ECM) structure, providing support for cell adhesion and migration. While synthetic polymers like polycaprolactone (PCL) are often used in EN fabrication due to their spinnability and mechanical properties, their hydrophobic nature limits cell adhesion. To address this, hydrophilic proteins are often incorporated to create composite ENs. Previous research has demonstrated the potential of α-lactalbumin (ALA)-based nanofiber dressings in promoting deep second-degree burn wound healing. However, the underlying mechanisms remain unclear. ALA, a tryptophan-rich whey protein, serves as a serotonin precursor. Serotonin has been shown to enhance skin wound healing by promoting fibroblast proliferation, migration, and angiogenesis. This study aims to compare the burn wound healing activity of ALA with other regenerative proteins like lysozyme (LZM), bovine serum albumin (BSA), and collagen type I (COL), focusing on serotonin production and neovascularization to confirm ALA's superior efficacy.

The literature review section extensively cites previous research on burn wound management, nanofibrous dressings, electrospinning techniques, and the bioactivity of various proteins used in tissue regeneration. It highlights the use of PCL as a biocompatible polymer in wound dressings and the need to improve its hydrophilicity for better cell interaction. The review also discusses the individual properties and roles of lysozyme, bovine serum albumin, and collagen type I in wound healing. Specific studies focusing on the potential of α-lactalbumin in wound healing and the role of serotonin in promoting tissue repair are cited to support the study's rationale and hypotheses. The review establishes the existing knowledge gap that this research addresses: a comprehensive comparison of ALA's wound-healing properties compared to other regenerative proteins when incorporated into nanofibrous scaffolds, focusing on the mechanisms involved.

The study involved the fabrication of electrospun nanofibrous scaffolds (ENs) using various regenerative proteins blended with poly(ε-caprolactone) (PCL). Specifically, four composite ENs were prepared: LZM/PCL, BSA/PCL, COL/PCL, and ALA/PCL, with a protein/PCL weight ratio of 1:3. The electrospinning process parameters were carefully controlled to produce bead-free, homogeneous nanofibers. The morphology and fiber diameter of the ENs were analyzed using field emission scanning electron microscopy (FESEM). Physicochemical characterizations included powder X-ray diffraction (XRD), differential scanning calorimetry (DSC), and Fourier transform infrared spectroscopy (FTIR) to examine crystalline/amorphous states, thermal properties, and intermolecular interactions between proteins and PCL. Circular dichroism (CD) spectroscopy determined the secondary structure changes of the proteins during electrospinning. The wettability of the ENs was assessed using water contact angle (WCA) measurements, and water absorption rate (WAR) and water vapor transmission rate (WVTR) were also determined. Mechanical properties, such as tensile strength, elongation, and Young's modulus, were evaluated using a texture analyzer. In vitro studies evaluated fibroblast proliferation using Cell Counting Kit-8 (CCK-8) assays and fibroblast adhesion using laser scanning confocal microscopy (LSCM) and FESEM. In vivo experiments utilized a third-degree burn wound rat model. Rats were treated with different ENs, and wound healing was assessed by measuring wound area over time. Histological analysis using H&E staining evaluated tissue regeneration. ELISA determined serotonin levels in wound tissues. Masson's trichrome and Sirius red staining assessed collagen deposition and collagen type ratios. Immunofluorescence staining (CD31 and α-SMA) evaluated angiogenesis. Statistical analysis was performed using one-way ANOVA and Bonferroni's multiple comparison tests.

The electrospinning process successfully produced bead-free, homogenous nanofibrous scaffolds with different proteins blended into the PCL matrix. The composite ENs exhibited significantly improved wettability compared to pure PCL ENs. ALA/PCL ENs showed the most significant reduction in water contact angle, indicating enhanced hydrophilicity. The mechanical properties varied among the different composite ENs, but all exhibited suitable properties for wound dressing applications. In vitro cell studies demonstrated that ALA/PCL ENs significantly promoted fibroblast proliferation and adhesion compared to other ENs. The liquid extracts from ALA/PCL ENs and BSA/PCL ENs significantly enhanced fibroblast proliferation after 72 hours of incubation, exceeding the effects observed with other protein extracts. ALA demonstrated the highest fibroblast proliferation potential among all tested proteins. In vivo studies using a third-degree burn wound rat model showed that ALA/PCL ENs significantly accelerated wound closure compared to the control group and other ENs. The ALA/PCL ENs achieved a wound closure rate comparable to the positive control (commercial collagen sponge). Histological analysis revealed that ALA/PCL ENs reduced inflammation and promoted faster epithelialization. The ALA/PCL group showed significantly higher levels of serotonin at the wound site compared to other groups, indicating that ALA promotes serotonin production. Masson's trichrome and Sirius red staining revealed enhanced collagen deposition and improved collagen I/III ratios in the ALA/PCL treated group, indicating better collagen maturation. Immunofluorescence staining indicated that ALA/PCL ENs also enhanced angiogenesis compared to other ENs and the control group. The body weight changes during the study were not significantly different among groups.

The findings demonstrate the superior efficacy of ALA/PCL ENs in promoting burn wound healing compared to other regenerative protein-based ENs and pure PCL ENs. The improved wound healing with ALA/PCL ENs can be attributed to several factors, including enhanced wettability, increased fibroblast proliferation and adhesion, and the promotion of serotonin production at the wound site. Serotonin is known to play a crucial role in wound healing by stimulating cell proliferation, collagen deposition, and angiogenesis. The increased collagen deposition, improved collagen I/III ratio, and enhanced angiogenesis observed in the ALA/PCL group further support this conclusion. The study successfully linked the improved wound healing effects of ALA/PCL ENs to the increased serotonin levels in wound tissues. This mechanism of action distinguishes ALA from other regenerative proteins studied. The results suggest that ALA’s unique properties, potentially related to its tryptophan content and ability to influence serotonin production, are responsible for its superior performance in burn wound healing. These findings have significant implications for the development of novel and effective burn wound dressings.

This study demonstrates that ALA/PCL electrospun nanofibrous scaffolds exhibit superior efficacy in third-degree burn wound healing compared to other regenerative protein-based scaffolds. The enhanced healing is linked to ALA's ability to promote serotonin production, resulting in improved collagen maturation, angiogenesis, and faster wound closure. ALA emerges as a promising candidate for next-generation burn wound treatments. Future research should explore the precise mechanisms of ALA action at the molecular level, investigating whether the active component is intact ALA or its degradation products. Further investigation is also warranted to evaluate the long-term effects and potential for scar reduction.

The study primarily focused on a rat model of third-degree burns, and the results may not be directly translatable to human burn wounds. Further studies are required to assess the efficacy of ALA/PCL ENs in human clinical trials. The study did not investigate potential adverse effects associated with the use of ALA/PCL ENs in the long term. The sample size in the animal study could be considered relatively small for some of the analyses. More comprehensive analyses examining the interactions between ALA and other components of the wound microenvironment could provide additional insights.