New technologies and novel biomaterials can help address the prevalent and costly chronic diabetic wounds, enabling customized wound tissue regeneration approaches that can significantly reduce morbidity and mortality. The individual and combined effects of ESM and Ulva lactuca sterile extracts on wound healing in vivo in diabetic-induced rats were characterized and investigated using the following methodologies.
Visual description
High purity product was obtained after extracting ESM and Ulva lactuca using standard procedures, as displayed in Fig. 1. Figure 1A and B show raw waste ESM both before and after treatment and grinding, revealing a typical white hue and a high degree of purity, respectively. Figure 1C and D show the final purity and characteristic green color of Ulva lactuca.
Visual appearance of the raw waste eggshell membrane (ESM) (A and B) and the green seaweed (Ulva lactuca) (C and D) before and after treatment and grinding
Compressive strength
Compressive strength measurements were conducted on tablets of ESM and ESM + Ulva lactuca mixture since it was impossible to form tablets from Ulva lactuca powder due to its brittleness and stickiness. Table 1; Fig. 2 show the flexibility and elasticity ranges for the ESM and ESM + Ulva lactuca mixture. Figure 2 shows that, like other collagen-based systems, the stress-strain curves for both materials have a toe, a heel, and a linear dependence region, as recently described by Torres et al. [16]. Fiber un-folding and stretching at low stiffness due to entropic elasticity characterizes the toe region of collagen-based systems, while increased stiffness associated with membrane elongation characterizes the heel region, and a proportional elongation response to load is displayed in the linear dependence region [37, 38].
Stress-strain graphical behavior for the eggshell membrane (ESM) powder and the ESM + Ulva lactuca mixture samples showing a higher elastic modulus for the mixture at 11.82 MPa as compared to the ESM at 9.49 MPa
When the load levels are low, both materials exhibit a non-linear region in which the strain and applied stress are not directly related, and a linear region at higher levels in which strain and stress variations are directly related. After applying 1676.85 N, the ESM reached a maximum compressive strength of 9.49 MPa, while the ESM + Ulva lactuca mixture reached a maximum of 11.82 MPa after being subjected to 2087.91 N. Thus, the mixture is a biomaterial that is both more elastic and mechanically biocompatible, as evidenced by the average Young’s modulus values of 44.14 MPa for ESM and 27.17 MPa for the mixture [39]. Recent experimental measurements of hen ESM Young’s modulus have shown a wide range of variation between 4.2 and 38.1 MPa, with an average 19.8 ± 14.3 MPa, depending on the medium and the degree of dehydration of the ESM fibers [38]. It was argued that the glycoproteins-rich layer encapsulating a collagen core forms a basis for CaCO3 crystallization that is responsible for the observed differences in ESM Young’s modulus [40].
Most biopolymer materials, including the ESM and Ulva lactuca, exhibit viscoelastic mechanical characteristics that mimic the flexibility and resilience of their natural polymer and tissue counterparts. Collagen’s triple helix bundles provide animal tissues compressive and tensile strength and anchoring to cell adhesion via surface receptors [40]. Individual fibers’ activities and the type of their interactions, as in skin, cornea, tendon, and blood vessels, affect this behavior [16]. Putative mechano-responders in cells include membrane elements, cytoskeletal components, and the nucleus, and surrounding cell positioning in the tissue is determined by the mechanical microenvironment, which includes the extracellular matrix composition and extrinsic loading [41]. Moreover, the substrate stiffness has a direct impact on cell behavior, which in turn can modify the substrate itself and control the process of tissue regeneration [41, 42]. Thus, the inherent cell ability to detect external mechanical forces and substrate stiffness could benefit from biomaterials, such as ESM and ESM + Ulva lactuca, to direct stem or resident cells towards the process of tissue regeneration or repair [39, 42].
Dielectric properties
A material’s dielectric properties are frequency-dependent, among other factors like temperature and moisture content, since dipolar and ionic conduction mechanisms change as the frequency changes [24]. As shown in Fig. 3, the relative permittivity (έ) decreases from 7.35 to 6.62 for ESM and from 8.69 to 6.95 for ESM + Ulva lactuca mixture samples as the frequency increases from 1 kHz to 1 MHz at the same temperature (25 ± 2°C), due in part to the ESM’s low moisture content and to polarization effects [24, 43]. The overall relative permittivity values for both samples, plateauing at 6.62 and 6.95 at 1 MHz, respectively; are relatively small because of ESM porous nature [43], with a slightly higher value for the mixture due to Ulva lactuca. It’s worth noting that the ESM maintained a nearly constant relative permittivity of 6.62 throughout a wide frequency range (3 kHz – 1 MHz), making it an effective biomaterial for tissue engineering applications [6, 11,12,13,14,15,16,17, 25, 38, 40,41,42,43]. It was shown earlier that the hen ESM relative permittivity retained approximately constant values between 8.5 and 6.5 in the temperature range 25–35 °C, before dramatically reducing to 1.0 at 100 °C, at frequencies from 200 MHz to 20 GHz [25]. The relative permittivity tends to approach the value of a vacuum (i.e., 1.0) at very high frequencies because reorientation of the dipoles becomes impossible [44]. Lin et al., have recently measured a transferred charge density of 0.80 mC/m2, a surface potential of 4.5 kV, and a relative permittivity of 2.81 for hen ESM at 1 MHz, arguing that its rough surface morphology and non-flat 3D topography may decrease power production owing to inadequate draping over the friction layer in comparison to other avian species ESM [43].
Variation of dielectric constant for the eggshell membrane (ESM) powder and the ESM + Ulva lactuca mixture samples in the frequency range from 1 kHz to 1 MHz
SEM analysis
SEM micrographs of Fig. 4A and B show the porous structure of the ESM powder samples, with an amorphous structure of irregular elongated particles (2–10 μm wide and 5–20 μm length), which was reported to be constituted of CaCO3 (94%), MgCO3 (1%), Ca3(PO4)2 (1%), and organic material (4%) [45]. The observed pores and cracks may occur and become more pronounced because of mechanical grinding, which also exposes CaCO3 in the form of crystal calcite [45, 46]. Micronized ESM biomaterials have shown a large surface area of 21.2 m2/g and porosity of 0.183 as compared to only 0.5 m2/g and 0.0009 for the raw eggshell [45], leading to enhanced skin contact, which promotes their efficacy as a wound healing treatment [15, 25, 46]. The ESM + Ulva lactuca mixture samples showed a highly homogenous porous structure of woven interconnected filaments (2–10 μm wide and 30–40 μm length) of Ulva lactuca, with ESM rough deposits in the pores among fibers (Fig. 4C and D). It is noteworthy that the rough texture of the mixture due to ESM particles may facilitate cell adhesion and proliferation [15]. In tissue engineering, adequate porosity in the targeted wound area is necessary for cellular uptake, migration, and survival; vascularization, gas exchange, nutrition delivery, and ingrowth into tissues [15, 47].
Scanning electron microscope (SEM) micrographs for the eggshell membrane (ESM) powder (A, B) and the ESM + Ulva lactuca mixture samples (C, D) at magnifications of 1,000× (A and C) and 2,500× (B and D)
Diabetic wound healing in vivo examination
Table 2; Fig. 5 show the size of skin wound area (mm2) for the diabetic untreated rats (negative controls) as compared to those treated with Dermazin cream (positive controls), ESM powder, Ulva lactuca powder, and ESM + Ulva lactuca mixture, at baseline and after 3, 7, 14, and 21 days of treatment. Wound sizes had significantly decreased in all rat groups that had wounds treated, but in the negative control group, it had grown larger by day 14 and day 21 (Fig. 5A). Figure 5B shows the wound healing stages for the positive control group treated with Dermazin, with a significant decrease in wound sizes from baseline to day 21. Dermazin, a 1% silver sulfadiazine cream, is a traditional antiseptic medication used to promote wound healing and maintain low bacterial colonization in burn wounds. It reduces bioburden, treats local infections, and prevents infection spreading throughout the body [48, 49].
Stages of skin wound healing of diabetic untreated rats (negative controls, A) and treated with Dermazin cream (positive controls, B), eggshell membrane (ESM) powder (C), Ulva lactuca powder (D), and ESM + Ulva lactuca mixture (E), at baseline and after 3, 7, 14, and 21 days of treatment
Wound closure quantitative analysis showed that the ESM group healed at a rate of 99.49%, with diabetic wounds completely closed and hair growing again on day 21, which was significantly (p < 0.001) faster than the other groups (Fig. 5C). The negative control group reached a final healing rate of 36.44%, the positive control group 96.79%, the Ulva lactuca group 87.05%, and the mixture group 90.23% (Table 2; Fig. 5). Thus, the ESM treatment outperformed Dermazin, Ulva lactuca, and the mixture treatments by a significant margin (p < 0.001). The ESM treatment results also outperformed those by Choi et al. [50], who showed that raw ESM marginally enhanced skin wound healing compared to negative control rats and that acid-modified ESM could improve tissue regeneration by promoting a full-thickness epidermal layer for faster wound healing. In an excisional wound splinting hairless mice model, soluble ESM accelerated wound closure at days 3, 7, and 10 through fibroblast-to-myofibroblast differentiation, enhancing fibroblast proliferation and the alpha-smooth muscle actin (α-SMA) contractile protein levels in vitro [25]. Granulation tissue myofibroblasts have been shown to promote wound closure by bringing the wound edges closer together through exerting tension and contracting the extracellular matrix [13].
Histological analysis
Successful skin wound healing within a reasonable time frame entails four consecutive and overlapping phases: hemostasis, inflammation, proliferation, and remodeling. During the proliferative phase, the skin undergoes angiogenesis, collagen deposition, tissue granulation, and re-epithelization formation [9, 10, 14, 15, 25, 35,36,37]. The histological examination at a magnification of 200× and the qualitative histopathological analysis of the skin wound healing of diabetic rats after applying Dermazin cream (positive control), ESM powder, Ulva lactuca powder, and ESM + Ulva lactuca mixture as compared to the untreated negative control rats are shown in Fig. 6; Table 3.
Histological evaluation of skin wound healing of diabetic untreated rats (negative controls, A) and treated with Dermazin cream (positive controls, B), eggshell membrane (ESM) powder (C), Ulva lactuca powder (D), ESM + Ulva lactuca mixture (E), and the epidermal thickness (in µm) of each group is shown as bar chart (*p < 0.001 as compared to negative controls, F). [H&E staining original magnification 200×, Keratinocytes (K), Epidermal layer (E), Dermis layer (D), Blood vessel (Bv), Collagen fibers (C), Hair follicles (Hf), Sebaceous gland (S), Edema (Ed), Infiltrating lymphocytes (IF), and Basement membrane (*)]
The negative control rats showed weak wound healing, with irregular re-epithelialization and disassociated keratinocytes lining the upper surface of the epidermal layer (Fig. 6A). The dermal layer had an abundance of blood capillaries, dilated blood vessels, collagen fibrin, and a sparse distribution of edemas. The wounds also had a broad region of edema and pale dark pigments on the epidermis. The positive control group of rats treated with Dermazin cream had significantly better wound healing, with less epithelial necrotic cell infiltration and better re-epithelialization of the epidermal layer (Fig. 6B). The basement membrane was enfolded, and lymphocytes were minimal, with sebaceous gland and hair follicles migrated to the upper epidermal layer. The dermis layer had irregular perpendicular collagen fibrins, and dilated blood vessels and blood capillaries leading to edema.
Wound healing in rats treated with ESM was significantly better than in control rats. This improvement was accompanied by a well-formed basement membrane, well differentiated epithelial cells, and regular thick keratinocytes lining the surface of the epidermal cells (Fig. 6C). The wounds showed moderately dilated blood vessels and blood capillaries perpendicular to the epidermal layer, which was covered with regular collagen fibrins in different directions, and the sebaceous gland migrated to the upper epidermis. These results are consistent with earlier findings showing that soluble ESM topical treatment could enhance skin wound healing in an excisional wound splinting mouse model, by stimulating the synthesis of collagen type III in the papillary dermis of hairless mice [13, 25, 51]. During the early stages of healing, resident and myeloid cell-converted fibroblasts primarily synthesize collagen type III, creating a flexible matrix that supported cell migration and granulation tissue formation. However, it is eventually replaced by collagen type I in healed skin [52]. Lysyl oxidase enzyme-induced covalent cross-linking during granulation tissue formation could promote remodeling and collagen fibril assembly, restoring tensile strength to over 80% of normal tissue for months following wound closure [25, 37, 52, 53]. The elasticity and reversible deformation characteristics of these collagen fibrils were affected by cross-links mediated by disulfide bonds, reducible and mature cross-links, transglutaminase cross-links, and advanced glycation end products [52]. An in vitro investigation found that human dermal fibroblasts cultured with soluble ESM triggered genes encoding collagen type III, which improved the papillary dermis health, increasing skin elasticity and alleviating the wrinkles in humans [54].
Although, ESM per se regulated inflammation and accelerated cell proliferation, angiogenesis, and wound contraction [16], bioactive glass nano-coatings with copper could modify the ESM’s dielectric and physicochemical properties leading to the formation of a continuous and homogeneous layer of the epidermis in vivo [13]. EMS and silver nanoparticle composites also promoted cell proliferation while suppressing inflammation, leading to improved re-epithelialization, granulation tissue formation, and wound healing in Albino mice [12, 13, 17, 55]. ESM powders have been found to stimulate matrix metalloproteinase (MMP) activity in mice dermal fibroblasts, increase MMP-2 protein levels, and accelerate the α-SMA contractile protein synthesis, thereby promoting wound healing and keratinocyte cell proliferation [13, 25, 51]. MMPs are important regulators for angiogenesis, tissue homeostasis, and immune response by modulating the release or activation of chemokines, cytokines, and growth factors [51, 52]. MMP activity, which regulates cell surface recruitment, substrate availability, and protein interactions, is tightly regulated at multiple levels, including transcriptional control, mRNA stability, pro-peptide activation, and proteolysis inhibition by tissue inhibitors [56]. It has been shown that biofilm-mediated microRNAs upregulate MMP-2, leading to a collagenolytic environment in wounds, resulting in a decrease in collagen I/collagen III ratio, which affects the biomechanical properties of repaired skin [52].
ESM is a naturally occurring biopolymer with 90% structural proteins, 3% lipids, and 2% carbohydrates. Proteome analysis, based on total spectral counts of unique peptides, showed that processed ESM powders are primarily composed of cysteine-rich proteins (∼ 30%) and collagen type X (10%), along with the bioactive enzymes lysyl oxidase-like protein 2 (30%) and lysozyme C (10%), involved in collagen processing [25, 53]. These constituents had favored ESM powders to regulate MMPs on fibroblasts in vitro and on epidermal cells in vivo, promoting cell proliferation and myofibroblast differentiation due to re-epithelialization and fibroblasts activation [34, 51]. ESM promoted fibroblast proliferation after 3 days of incubation without affecting cell viability, suggested biocompatibility, and continued stimulation upon further incubation [51]. Fibroblasts, which are normally dormant, play a vital role in wound healing upon activation by producing matrix components essential for wound contraction and closure. After tissue injury, activated fibroblasts proliferate in the peri-wound stroma and migrate into the wound provisional matrix of fibrin and fibronectin [34]. This proliferation, expansion, migration, and myofibroblast differentiation are essential for granulation tissue formation and healing, requiring membrane-bound molecules like integrin and syndecan-4 [35].
On the other hand, the Ulva lactuca treated rats showed weaker wound healing, with few keratinocytes covering the upper surface of a thin epidermal layer (marked by dark pigments), necrotic epithelial and lymphocytic cells infiltrating the basement membrane (Fig. 6D). Multiple necrotic sebaceous glands encircled the hair follicles in the dermis layer, which also had dilated blood vessels, congested capillaries, and migrated clotting collagen fibrins in different directions. In contrast, rats treated with ESM + Ulva lactuca mixture showed mild healing comparable to ESM and Dermazin treatments (Fig. 6E). Secondary metabolites found in Ulva lactuca include steroids, alkaloids, triterpenoids, saponins, phenolic compounds, and flavonoids, which are anti-inflammatory, antioxidant, anticoagulant, and antimicrobial activities and have proven crucial for wound healing [20,21,22, 57]. However, compared to Dermazin or ESM treatments, the rates of re-epithelialization, collagen formation, tissue construction, and wound healing were much lower when using either Ulva lactuca extracts alone or mixed with EMS powder (Table 3). We hypothesize that this is because Ulva lactuca increases the mixture’s dielectric constant to significantly higher levels (Fig. 3), which in turn alters its net negative charges, conduction ionic minerals, and intrinsic phytochemicals necessary for tissue regeneration. Flavonoids are also known to enhance collagen fibril viability, prevent cell damage, stimulate DNA synthesis, and promote fibroblast cell proliferation and differentiation into specialized myofibroblasts, leading to wound contraction and closure [58]. Triterpenes, which are absent in Ulva lactuca, have been shown to induce cell migration, proliferation, and collagen deposition by regulating reactive oxygen species generation, thus accelerating the wound healing process [59, 60]. Moreover, Ulva lactuca has a higher lipid and a lower protein concentration compared to ESM, which exhibited weak antiproliferative capacities [61]. These reasons may explain why rats treated with Ulva lactuca alone or with ESM + Ulva lactuca mixture had a slower healing rate and wound closure compared to those treated with Dermazin or ESM.
Finally, ESM treatment resulted in a significantly thicker epidermal layer as compared to Dermazin, ESM + Ulva lactuca mixture, and Ulva lactuca treatments and to negative controls (i.e., 8.30 ± 0.70 vs. 6.17 ± 1.44, 6.29 ± 0.68, 4.41 ± 0.77, and 4.17 ± 0.73 μm, p < 0.001, respectively) (Fig. 6F). In a recent study conducted on male Albino rats, the use of ESM powders and dressing significantly improved tissue regeneration after superficial open wounds. Compared to conventional treatment using hydrogen peroxide, which resulted in a length reduction rate of 0.852 mm/day and healing in 16 days, the ESM and dressing treatments resulted in a length reduction rate of 1,009 and 1,020 mm/day, respectively, and shortened the healing time to 12 days [14]. Thus, ESM powder is a powerful biomaterial for tissue engineering and chronic wounds healing, such as diabetic venous, foot, and pressure ulcers [3, 4, 16].
In conclusion, nonhealing diabetic wounds cause global morbidity and poor quality of life. Understanding wound healing mechanisms is crucial for developing effective treatments. ESM, a natural waste product with proteinaceous meshwork, is a potential biomaterial for treating chronic wounds. In the present study, ESM, Ulva lactuca, and their mixture micronized powders were evaluated for compressive strengths, dielectric properties, and SEM surface morphology, which showed stable favorable characteristics that could promote in vivo wound healing in a rat model of induced diabetes. Wound closure was significantly hastened on days 3, 7, and 14 with ESM treatment, and epidermal layer was much thicker with enhanced re-epithelization and collagen deposition on day 21, compared to conventional Dermazin and Ulva lactuca treatments. It is therefore postulated that ESM’s structural extracellular matrix-like components promoted re-epithelialization and neovasculature development, leading to better wound healing. Thus, ESM is a practical biomaterial for the design of innovative wound healing remedies that are both economical and effective.
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