A comparison of several separation processes for eggshell membrane powder as a natural biomaterial for skin regeneration

Abstract Background Numerous studies have focused on skin damage, the most prevalent physical injury, aiming to improve wound healing. The exploration of biomaterials, specifically eggshell membranes (ESMs), is undertaken to accelerate the recovery of skin injuries. The membrane must be separated from the shell to make this biomaterial usable. Hence, this investigation aimed to identify more about the methods for membrane isolation and determine the most efficient one for usage as a biomaterial. Methods and materials For this purpose, ESM was removed from eggs using different protocols (with sodium carbonate, acetic acid, HCl, calcium carbonate, and using forceps for separation). Consequently, we have examined the membranes' mechanical and morphological qualities. Results According to the analysis of microscopic surface morphology, the membranes have appropriate porosity. MTT assay also revealed that the membranes have no cytotoxic effect on 3T3 cells. The results indicated that the ESM had acquired acceptable coagulation and was compatible with blood. Based on the obtained results, Provacol 4 (0.5‐mol HCl and neutralized with 0.1‐mol NaOH) was better than other methods of extraction and eggshell separation because it was more cell‐compatible and more compatible with blood. Conclusion This study demonstrates that ESMs can be used as a suitable biomaterial in medical applications.

involving several different cell types and cytokine mediators being activated at once. 2 Furthermore, chronic wounds may occur due to diseases like peripheral vascular disease or diabetes mellitus, raising the risk of infection and other problems. 3During abnormal tissue regeneration, impaired angiogenesis and nutrition delivery lead to chronic conditions and the ongoing stimulation of inflammatory cells around the wound area.Hence, wounds are a crucial financial healthcare system burden and a physical, mental, and social problem for the person. 4st superficial wounds heal on their own; on the other hand, fullthickness wounds do not heal on their own and take a very long time to recover.It requires some specific biomaterials to fill the severely damaged area, which helps speed up the healing of the wound. 5The biomaterials for wound healing must be rich in biopolymers/ biomolecules, which can increase the rate of wound healing. 6Today, biomaterials play numerous roles in regenerative medicine, tissue engineering, biosensors, biotemplates, and more 7 ; recent advances in biomaterials have provided opportunities for clinical use.However, the principal challenge of using appropriate biomaterials for biomedical applications is the lack of biocompatibility, often related to allergies or inflammation reactions, blood homeostasis, tumorigenesis, altered viscoelasticity of extracellular matrix (ECM), biodegradability, and multiple drug resistance. 7Eggshell membrane (ESM) has been utilized as a material to accelerate wound healing. 8ESM is a biopolymeric membrane that is one of the most abundant industrial bio-waste materials; this high-value waste can benefit medical applications. 9ESM is the bilayer, a noncalcify membrane between the shell and albumen.The ESM has a fibrous meshwork of two thin submembranes: an outer membrane that is thicker and linked to the shell and an inner membrane that is thinner and attached to the egg white. 10The ESM comprises bioactive compounds, including hyaluronic acids, chondroitin sulfate, dermatan sulfate, and glycoproteins.This structure is also mainly formed by proteins (∼90%), and proteomic examination of it has demonstrated about 500 proteins, containing glycoproteins, elastin, sialoprotein, osteopontin, collagens (I, V, and X), as well as functional proteins like lysozyme and ovotransferrin. 11,12In addition, the ESM has properties essential for wound healing, such as appropriate moisture retention, air permeability, and the ability to adhere to textured surfaces due to its network structure. 13,14Also, ESM is similar to the natural ECM to some degree.Because of this, it is a unique candidate as a biomaterial source for the effective regeneration of tissue and joint pain and for preventing skin aging. 15,169 Glycosaminoglycans (GAGs) in the ESM are intriguing candidates for wound healing mediators because of their anti-inflammatory activities. 20Also, Some investigations have found that solubilized and hydrolyzed ESM has anti-inflammatory effects. 17 and corneal mesenchymal stromal cells (C-MSC). 21In another study, Tram T. Vuong, by examining the function of eggshell in cell activity and the expression of MMP 2,9 that play an important role in the initial phase of wound healing, stated that eggshell powder increases the expression of MMPs, and the constructive effect and positive effects on cell function can play an important role in wound healing. 22cording to another research, ESM exhibits good biocompatibility.
Hence, with the increasing use of ESM, it is essential to develop a separation technology to be used on a large industrial scale.Since using acid solutions to separate minerals from eggshells is common, it is crucial to learn more about these acids' impact on the membrane's chemical composition and biological properties.As a result, to benefit from this biopolymer, the entire membrane must be separated from the mineral shell.While many studies are employing various methods to isolate ESM, more information must be provided to compare the efficacy of different ESM extraction techniques.This paper aims to determine which protocol is better for extracting this valuable biomaterial.To do this, we used six extraction methods to compare the biological characteristics of the natural ESM.Finally, the structural and morphological characteristics of ESM and its compatibility with blood and fibroblast cells were investigated in order to obtain a deeper insight into the optimal extraction method of ESM and to evaluate its performance when used for wound healing in laboratory conditions.

Materials
Chicken eggs were bought from a nearby store.All reagents and

Natural eggshell membrane powder preparation
First, the fresh eggs were washed with deionized (DI) water.Afterwards, a small hole was punctured at the tip of the eggs, and the contents were removed.Subsequently, the inside of the eggs was cleaned three times with DI water.Then the ESM was separated from the shell using the following protocols at room temperature.
Protocol 1 (P1): A 0.05-M solution containing sodium carbonate was used to wash the eggshells.Following that, membranes were given a wash in a solution of distilled water.And the ESMs were separated from the eggshells. 23otocol 2 (P2): After the eggs had been emptied, 5% (v/v) acetic acid was added to them, and they were left to remain for 45 min at 37 • C. Next, the membranes were removed from the calcified portion of the eggshell with great care, and the following three times washed with acetone, and, in the end, the membrane was cleaned with distilled water. 24otocol 3 (P3): When the eggs had been depleted, 24-h immersion in a solution contains 1% acetic acid to remove any eggshell fragments that may still exist in the membranes.Afterwards, membranes were rinsed in DI water. 25otocol 4 (P4): After the albumin had been extracted from the eggs, the emptied eggs were immersed in diluted 0.5-mol HCl for 24 h to get rid of their ES.The membrane of the eggshell was extracted, and the ESM was neutralized with 0.1-mol NaOH for 1 h.Next DI water was used to wash the membranes. 26otocol 5 (P5): After removing the albumin from the eggs, the emptied eggs were soaked in 0.5-M acetic acid for a total of 44 h; once the calcium carbonate shell had been dissolved completely, the extracted membranes were gathered and washed with DI water. 21otocol 6 (P6): As a control group, after albumin had been extracted from eggs, the membranes of the eggshell were separated using forceps from the calcified eggshell and cleaned with DI water.
All collected ESM samples were freeze-dried for 2 days and were subsequently pulverized using liquid nitrogen to a powder for a short time.The preparation powder was stored at 4 • C.

2.3
Characterization of the ESM

Analysis of microscopic surface morphology
The morphology of the surface of the ESM was observed under scanning electron microscopy (SEM; DSM 960A, Zeiss, Germany).Each sample was operated in different sizes after being sputter-coated in gold.Imaging was done at a 15-kV accelerating voltage, and software for image analysis was employed to find the average size of the pores. 27

Functional group identification
Functional groups of ESM were identified using a NICOLET-6700 FTIR spectrophotometer (Thermo Scientific).The study was conducted in 400-4000 cm −1 to find the highest point of O, C, and N. 28

Cytotoxicity assay
The ISO 10993-5 standard was used to conduct the cytotoxicity test.
The MTT analysis was conducted to assess the viability and cytotoxicity of the manufactured ESM.Before cell seeding, ESM was placed for sterility under UV radiation for 20 min.The 3T3-murine fibroblast cell line used in this study and about 1 ×10 4 cells were seeded into ESM in a 96-well plate, which involved Dulbecco's modified Eagle's medium (DMEM) with 10% FBS, and 1% (v/v) pen-strep solution.The incubator's atmosphere was 37 • C and 5% carbon dioxide, and the culture plates were put inside.At 24 and 72 h after cell seeding, 100-µL MTT reagent was applied for every well at a final concentration of 0.5 mg mL −1 .Afterwards, the plates were incubated for another 4 h at 37 • C in an incubator to determine cell viability.Following the removal of the medium, 100 µL of dimethyl sulfoxide (DMSO) solution was put in every well.The optical density of each well at 570 nm was measured with a 96-well microplate reader.The wells in a tissue culture plate with no cells were called "blank," while the wells with cells and without ESM were called "control."Using the following formula, the proportion of viable cells in every sample was calculated. 29llviability% = Sample absorbance value Control absorbance × 100

Wound scratch assay
A wound-healing assay was carried out by scratching the confluent culture to assess the migration of 3T3 cells.At a density of 5 ×10 3 3T3, cells on a six-well plate were seeded.The medium was changed every 2 days, and culture was kept up until about 80% confluence.The medium was removed, and a scratch was formed with a micropipette tip.After that, the cultures were fed with the recommended doses of ESM.As a control, cells with no supplement were employed.An inverted microscope was used to take pictures of the wound site on day 0 (the day after the scratch) and day 2. The migration process was captured in digital images using a computer-connected microscope and Image J software (National Institutes of Health, Bethesda, USA). 30

ESM measurements of moisture content and drying
Determining the amount of moisture in a product requires weighing it, drying it thoroughly, and reweighing it.So for this study, ESM was extracted from eggs, and then the ESM was placed on a plate and weighed.After drying with a freezer dryer for 48 h, the dried ESM were placed on a preweighed plate and reweighed. 31The moisture content of ESM was estimated utilizing the following formula: where W d = weight of the ESM after drying and W w = wet weight of the ESM.

In vitro hemocompatibility assays
In this experiment, 2.5 mL of normal saline was used to dilute 2 mL of fresh anticoagulated human blood.After that, a blood-dilution solution (0.2 mL) was added to the ESM vials.The samples were maintained at a temperature of 37

Blood coagulation index (BCI)
Samples (the produced ESMs powders) weighing 10 mg were put into a beaker in a thermostatic water bath, and100-µL anticoagulated human blood was dumped on them.Next, all specimens were placed at 37

In vitro degradation study
The dried ESMs were placed in a test tube with 5 mL of PBS and separately in trypsin-EDTA liquid for in vitro degradation research.The tubes were maintained for 30 days while kept sterile and at 37 • C.After that, samples were taken, cleaned with purified water, and then dried.
In addition, every sample was also weighed, and the degradation rate was determined using the equation below. 34gradation where W 0 is the primal sample weight and W t represents the weight of the deteriorated samples at various times.

Microenvironmental pH
Fifty milligrams of ESMs was cut and then submerged in 2 mL of 0.9% normal saline solution for 48 h at room temperature.The pH of the immersion solution was analyzed to recognize the microenvironment's pH of the ESM. 35

Statistical analysis
The information was analyzed with the GraphPad Prism application.Each experiment was repeated three times, and the results are shown as the mean SD.To compare the groups, one-factor analysis of variance (ANOVA) with the Tukey's post-hoc test.Data were compared using the Kruskal-Wallis H nonparametric ANOVA test and then the Mann-Whitney U test when significant.The p-value was taken into account and statistical significance was assigned to the value p < 0.05.

Morphological analysis
The morphological properties of ESM were recognized by SEM micrographs (Figure 1).Images demonstrated that the morphology of the fibers was randomly aligned.SEM analysis shows most of the ESM as dense clusters of strong thread-like fibrils due to its intrinsic similarity to the ECM.The spherical structures show the cracks caused by the extraction process in the structure.In addition, the obtained images are similar to other studies. 36Using ImageJ software (NIH, Bethesda, USA), all groups' average fibril diameter was statistically estimated.The membrane's pore morphology, size, interconnectivity of pores, and porosity are crucial characteristics that directly impact cell proliferation.The examination of the pictures revealed that the average size of pores in the ESM was approximately 416.5 nm to 6.952 µm.
The resulting pore size is appropriate for cell invasion and migration.
These data demonstrated that the produced ESM is suitable for tissue regeneration applications, which demand a porous structure with interconnected pores and enough pore size.

Functional groups identification
FTIR-KBr analyses demonstrated the change of the related absorption peaks in the ESMs' organic structure, as demonstrated in Figure 2.
ESMs obtained through different methods of separation yielded FTIR-KBr spectra that varied in the distinctive absorption peaks of the organic contents and also in absorption bands associated with the ESM.
The protein/amide signature bands showed three extremely strong bands.A wide band in the 3400-3500 cm −1 frequency range (amide A: stretching vibration of the NH group) as well as more defined bands in 1600-1670 cm −1 (amide I: stretching vibration of the C = O group) peak at 1530-1540 cm −1 (amide II: NH in-plane bending and CN stretching). 15,37Also, 1234 cm −1 (NH in-plane bending and CN stretching).Amide vibrations of the fiber glycoprotein can be attributed to these regions. 38The spectra of ESMs that were manually separated and those that were separated using an acid dissolution of the membranes exhibited variances in the existence of absorption bands and differences in the shape and intensity of those bands.The membrane

Findings on cell viability
The cytocompatibility of the ESM was tested using an MTT experiment, and the findings were displayed in (Figure 3).After 24 and 72 h, P4 had higher cell viability values than the other groups.Moreover, the rate of cell proliferation was significantly greater than that of the control group.However, the viability of cells in P3 was considerably reduced 72 h after cell seeding.The results demonstrated that the manufactured ESM was cytocompatible and promoted the growth of 3T3 cells.

Wound healing assay
The scratch wound assay was applied to evaluate migration of 3T3 cells in vitro in confluent culture.The rate of cell migration was assessed 2 days after wound creation in 3T3 cell cultures supplemented with ESMs and a control group.The sample images of the migration of 3T3 cells on days 0 and 2 following wounding are shown in Figure 4 in control cultures and cultures supplemented with ESM.The culture supplemented with ESM P4 3T3 cells migrated more quickly, and the wound area recovered substantially faster than in the other groups within 2 days.Compared to control cultures, the migration rate of 3T3 cells in the culture treated with ESM was considerably higher (Figure 5).However, in P2, the rate of migration of 3T3 cells decreased significantly and was the same as in the control culture.These findings show that ESM considerably affects the migration rate of 3T3 cells in vitro, which will benefit wound healing re-epithelization.

Blood compatibility
Blood compatibility of biomaterials is directly related to hemolysis, which reveals the release of hemoglobin into plasma as a result of erythrocyte destruction.One of the significant aspects in the successful treatment of wounds is the produced wound dresser's compatibility with cellular components of the blood, particularly erythrocytes. 39e contact between the wound dresser and erythrocytes in the first phase after implantation ultimately decides the secondary bodily reactions, like inflammation.As in Figure 7, overall, hemolysis rates on all ESMs were lower than the positive control, and the difference was statistically significant.This showed that the membranes were very compatible with blood, which means they can be used in regenerative medicine.

Blood coagulation index (BCI)
The whole blood coagulation index (BCI) primarily represents the effect of dressings on coagulation.With a lower BCI, biomaterials prevent blood clotting.The best protocol was P4, whose BCI value was 43.27 ± 5% (n = 3).However no significant difference between the other groups (Figure 8).The findings showed that the ESM attained a satisfactory level of coagulation.In the presence of trypsin, however, the degradation rate was accelerated.Moreover, the percentages of degradation in all groups were very close together.So the ESM is a degradable material.interior environment of the injury by stimulating fibroblast proliferation. 40As a result, a suitable material should have a pH between 4.0 and 6.8, comparable to that of natural, healthy skin.

ESM's microenvironmental pH
Table 1 demonstrated that all the ESMs had a somewhat acidic pH (between 5.08 and 5.37), which is consistent with pH levels reported by previous researchers for commercial wound care products and there is no significant change in results over time.

DISCUSSION
Tissue engineering disciplines recognize collagen-based biomaterials as a crucial component due to their ability to provide an ECM basis for tissue regeneration.The presence of collagen (type I, V, and X) in chicken ESM makes it a perfect substitute for ECM in the regeneration of tissue. 41cording to this finding, we presented the identification and preparation of the neutral ESM as a biomaterial for skincare products.ESM is a popular unique biomaterial made up of polysaccharides, antimicrobial proteins, glycoproteins, and collagen. 42These components are present in significant concentrations in ESMs compared to other com-mercial sources and may require less processing to extract them. 43The organic material ESM has been proven to boost the activity of cells and collagen formation.Moreover, ESM inhibits skin aging as well as lowers UV light and inflammation-related damage. 43ESM has a rich history of use as a wound-healing product because it increases collagen deposition, which results in thicker granulation tissue and accelerates wound healing. 44Moisturizers, wound healing, skin expansion, and antiwrinkle agents are only some of the applications proposed for ESM. 45so, many studies have examined the ESM as a different biomaterial.Additionally, in vitro and in vivo research ESM has been utilized.
Clinical studies confirmed its efficacy in alleviating pain, reducing joint stiffness, and resolving connective tissue problems.Nevertheless, we anticipate that there will be more ESM commercial products soon due to their great potential.The best fabrication technique is required since even little changes to individual components might result in outputs with wildly varied chemical and mechanical properties. 26nding a suitable extraction technique that maintains the ESM composition and can be applied on a large commercial scale is necessary, given the expanding use of ESM biomaterial.So in this study, a comparison was made between acid solutions often used to separate ESMs, revealing that the dissolution with HCl was the only approach that had blood compatibility, biodegradation, and cell viability.
The FTIR demonstrated that dissolution with HCl, compared to the other methods of ESM separation, could also lead to a shift in the chemical structure, which could affect its qualities as a biomaterial.Acid disintegration of the mineral in eggshells is a standard method utilized by numerous researchers that employ the ESM as a biocompatible material. 38e porous structure in materials is a suitable environment for cellular processes, including adhesion and proliferation. 46It is recognized that the morphology of materials influences cell adhesion.Although the meshwork shape of the surface of ESM could provide a physical framework for cell support, it is also conducive to cell adhesion and growth. 47The SEM pictures were utilized to analyze the porosity structure of the created ESM.It was somewhere between 416.5 nm to 6.952 µm, which is compatible with cellular processes.
At the initial stage of determining a material's biocompatibility, cytotoxicity experiments using cell culture-based techniques are carried out. 7The present investigation demonstrates that the ESMs are bio- scarring. 48The wound healing assay can measure fundamental cell migration factors like speed, persistence, and polarity. 49The results of the wound healing tests revealed that it significantly impacts the in vitro migration rate of 3T3 cells; it will be beneficial for reepithelization during wound healing.Furthermore, our investigation confirms that P4 had a higher migration rate than other protocols and significantly improved wound area in vitro, which can be due to its greater cell compatibility and reproductive potential.
The ASTM F756 hemolytic index says that a material is hemolytic when the percentage of hemolysis is more than 5%, slightly hemolytic when the rate of hemolysis is between 2 and 5%, and not hemolytic when the ratio of hemolysis is <2%. 50As a result, all protocols are hemolytic, suggesting that they are to some extent compatible with blood.However, because the ESM is not directly exposed to blood, The blood clotting index (BCI) calculates the absorption spectrum of hemoglobin freed from nonattached RBCs to assess the formation of stable clots. 51The results indicated that the ESM had acquired acceptable coagulation.Based on the BCI results, P4 is better than other protocols, which can be due to its final washing with NaOH, because based on past studies, sodium hydroxide can lead to the formation of clots and increase the coagulation speed. 52e process of biodegradation of biological materials is a vital feature in wound healing because it leads to the release of effective factors in the structure of the scaffold and biomaterial to the repair site, and on the other hand, the speed of biodegradation must be consistent and proportional with tissue regeneration. 53As time progressed, the percentage of destruction increased in all groups.Therefore, our research confirms that the ESM is a degradable material and according to the timing of the stages of wound healing, especially the homeostasis phase, it can be more effective due to blood compatibility.
Furthermore, microenvironmental pH in all groups revealed that the ESM created in this work could function as an acidic condition to inhibit bacterial invasion and indirectly promote fibroblast proliferation throughout the wound-healing process. 54e of the most important limitations of this study is the lack of examination of the wound healing rate in the animal model, which pro-

CONCLUSIONS
This paper described six valuable methods for extracting the entire membrane from the eggshell.Thus, P4 is superior to other protocols separating the ESM for skin regeneration beacuase more hemo and cell-compatible.The ESM's defined qualities were determined using biocompatibility, biodegradability, and physicomechanical characterization.To evaluate the ESM's efficacy in an animal model, additional in vivo testing will be required.On the whole, the report shows that the ESMs can be used in tissue regeneration to speed up healing after injuries or skin trauma.Currently, there are no commercial products based on ESM designed for specific applications, though ESM supplements are available for purchase online.However, since P4 has been verified via physical, chemical, and biological analysis and is suitable for use on a large industrial scale, we anticipate that ESM commercial skincare products will increase soon.
Also, in the initial stages of wound recovery, Guarderas et al. and Jun et al. demonstrated that ESM enhances wound closure during regeneration.

F I G U R E 1
Morphology of the eggshell membrane.(A) P1, (B) P2, (C) P3, (D) P4, (E) P5, and (F) P6.Magnification: 5.00 K. spectra acquired with HCl treatment showed the organic components' typical absorption bands of the ESM identical to the FTIR spectra produced manually by ESM separation (Figure 2c,e).

Figure 6
Figure 6 displays the ESMs' moisture content.The moisture content of the ESM was identified by drying samples in a freezer dryer until they attained a consistent weight.The maximum moisture content was reported for P1, whereas the lowest was for P2.However, all of the samples had a similar level of moisture content.
Biodegradation of ESM was performed in PBS and trypsin at standard settings (pH 7.4; 37 • C) in the laboratory.The retained percentage weight is displayed in Figure 9. ESM degradation in P4 was faster in PBS than in the other protocols.The degradation speed of the material should correspond with the regeneration rate of the tissue.The slow rate of material degradation hurts the replacement of the growing tissue.

F I G U R E 5
compatible and have no harmful effects on 3T3 cells.The MTT test results indicated that ESMs could boost cell viability.Besides, P4 had the highest cell viability.The utilization of biomimetic scaffolds such as ESM can enhance wound healing, prevent pathogen-mediated infection, and decrease F I G U R E 4 Representative pictures of 3T3 cells grown in control and test conditions healing wounds in vitro (supplemented with eggshell membrane conditions).The lines represent the scratch width.(A) P1, (B) P2, (C) P3, (D) P4, (E) P5, and (F) P6.Migration rate of 3T3 cells cultured supplemented with the eggshell membrane in test conditions and a control group.Image J software calculated the migration rate by measuring the distance traveled by cells.Values represent the mean ± SD, **p < .01 and ***p < 0.001.ns., not significant; SD, standard deviation.

F I G U R E 7
The hemocompatibility assay of eggshell membrane.Values represent the mean ± SD, n = 3. Significance levels are expressed as ****p < .0001,***p < .001,**p < .01,and *p < .05. blood problems will not occur after ESM transplantation on the wound area.

F I G U R E 9
Biodegradation of eggshell membrane.(A) Trypsin and (B) PBS in different time points.Values represent the mean ± SD, n = 3. vides a better and deeper research ability by examining the histology of ESM function.It is suggested that in future studies, the obtained protocol should be used for further investigations in all types of burn, full-thickness, and diabetic wounds.
32croplate Reader.The average of the three readings was determined.Negative controls included 10 mL of normal saline and 0.2 mL of dilute blood, whereas positive controls included 0.2 mL of diluted blood in 10 mL of DI water.The degree of hemolysis was computed as follows.32 • C for 1 h, before being centrifuged at a rate of 1500 rpm for 10 min.The supernatants that were made were put in a 96-well plate, and the absorbance at 545 nm was measured with a