The chicken eggshell membrane: a versatile, sustainable, biological material for translational biomedical applications

Naturally derived materials are often preferred over synthetic materials for biomedical applications due to their innate biological characteristics, relative availability, sustainability, and agreement with conscientious end-users. The chicken eggshell membrane (ESM) is an abundant resource with a defined structural profile, chemical composition, and validated morphological and mechanical characteristics. These unique properties have not only allowed the ESM to be exploited within the food industry but has also led to it be considered for other novel translational applications such as tissue regeneration and replacement, wound healing and drug delivery. However, challenges still exist in order to enhance the native ESM (nESM): the need to improve its mechanical properties, the ability to combine/join fragments of ESM together, and the addition or incorporation of drugs/growth factors to advance its therapeutic capacity. This review article provides a succinct background to the nESM, its extraction, isolation, and consequent physical, mechanical and biological characterisation including possible approaches to enhancement. Moreover, it also highlights current applications of the ESM in regenerative medicine and hints at future novel applications in which this novel biomaterial could be exploited to beneficial use.


Introduction
The chicken eggshell membrane (ESM) is a natural biomaterial that has gained increasing attention in the biomedical field due to its unique properties, versatility, and sustainability (figure 1). It is a thin filmlike structure that lines the interior surface of the eggshell and separates the albumen from the shell (Torres et al 2010, Chai et al 2013, Park et al 2016, Baláž 2014, Chen, Kang and Sukigara 2014, Mensah et al 2021, Shi et al 2021, Zurita-Méndez et al 2022, Torres-Mansilla et al 2023. The ESM is composed of a complex matrix of proteins, glycosaminoglycans, and minerals that confer it with remarkable biodegradability and biocompatibility (Park et al 2016, Ahmed et al 2019a, Torres-Mansilla et al 2023. The ESM has been used for various applications, such as food supplements, nutraceuticals, and cosmetics. However, recent studies have identified its potential for translational biomedical applications, such as wound healing, tissue engineering, drug delivery, and regenerative medicine (Scatena et al 2007, Mensah et al 2021, Mohammadzadeh et al 2021, Mendoza, Chavez and Araya 2022, Webb, Rafferty and Vreugdenhil 2022. The ESM can be easily isolated from waste eggshells generated by the poultry industry, making it a cost-effective and sustainable source of biomaterials (Morooka et al 2009, Vuong et al 2018, Cree and Pliya 2019, Ahmed et al 2019b, Saha et al 2021. The aim of this review is to provide an overview of the chicken ESM as a versatile, sustainable, and biological material for translational biomedical applications. The composition, physical and biological properties, extraction methods and various applications, recent advances and limitations of ESM in the biomedical field are discuss.

Physical properties and components
The ESM lines the inner aspect of the eggshell and has a unique structure and biochemical composition consisting of a porous and fibrous mesh-like membrane composed of three layers: the outer membrane in contact with the eggshell; the inner membrane in contact with the albumen (egg white) and . The ESM is important in the eggshell mineralisation process by preventing the mineralisation of the albumin while inducing the mineralisation of the eggshell (Rose and Hincke 2009, Park et al 2016, Han et al 2023. Likewise, the ability of the ESM to prevent the internalisation of bacteria and thus its antibacterial properties are widely accepted (Ahlborn and Sheldon 2006). The presence of desmosine and isodesmosine crosslinks allows the ESM to be insoluble, allowing it to support the embryo during development (Torres-Mansilla et al 2023).
The inner and outer layers of the ESM differ in their structural morphology and chemical composition. The outer membrane is mainly composed of type I collagen, meanwhile the inner membrane is predominantly composed of type V collagen in addition to type I collagen (Torres et al 2010, Mensah et al 2021, Zurita-Méndez et al 2022. Type X collagen is found in both the inner and outer membrane (Zurita-Méndez et al 2022). In addition to the differences in the type of collagen present, the structural morphology also differs between the layers. The collagen fibres of the outer membrane closest to the eggshell is 1-7 µm in diameter. Meanwhile, the collagen fibres of the inner membrane are smaller with a diameter between 0.1 and 3 µm. Likewise, the general thickness of the three layers vary with the outer layer being the thickest (50-70 µm) followed by the inner membrane with a thickness of 15-30 µm and finally the limiting membrane, the thinnest and is a non-fibrous layer interlaced within the inner membrane (Mensah et al 2021, Shi et al 2021. The collagen fibres of the inner membrane are more densely packed in comparison to the fibres of the outer membrane, which infiltrate the inner surface of the ESM (Chai et al 2013, Shi et al 2021.

Biological properties
It has been widely observed that the ESM is a proteinrich structure containing over 500 different proteins and acts as a natural source of collagen, fibronectin, proteoglycans, glycoproteins, hyaluronic acid and many amino acids such as arginine, glutamic acid, histidine, cystine, and proline which are found in high concentrations (Chi and Zhao 2009, Guru and Dash 2014, Ahmed et al 2019a, Mensah et al 2021. In addition to the proteins and amino acids, CaCO 3 is also present in the ESM, this is due to the presence of a level of mineralisation in the outer membrane (Arias et al 1993, Shi et al 2021, Torres-Mansilla et al 2023. The close resemblance in structure between the ESM and extracellular matrix (ECM) and vast biological constituents allows the ESM to have tremendous usefulness in many applications in material science and tissue engineering. As mentioned previously, collagen is a key constituent of the ESM: however, collagen only makes up 10% of the 80%-85% of the organic matrix the ESM contains (Nakano et al 2003, Kaweewong et al 2013, Shi et al 2021, Han et al 2023. Nonetheless, the collagen fibrils are a major morphological component of the ESM. It provides the ESM with the necessary structural support while also acting as a scaffold for biomineralisation. The presence of collagen is exploited in tissue engineering and biomaterial formation. The ESM provides a natural source of substances vital for tissue engineering and wound healing. For example, the ESM acts as a natural source of collagen but also glucosamine and hyaluronic acid which have important implications in biomaterial development and success. For instance, the collagen fibrils act as a 3D scaffold and can incorporate to form a new matrix which provides anchorage for new cells through cell surface adhesion meanwhile being biocompatible and biodegradable. Additionally, the type V collagen provides great tensile strength and structural support for the new matrix. The ESM also contains fibronectin which in addition to the arginine-glycine aspartic acid tripeptide glycoprotein motif facilitates cell adhesion. Meanwhile the fibronectin also facilitates cell growth, migration and repair which play a key role in wound healing and cell incorporation to ensure the success of the scaffold (Scatena et al 2007, Mensah et al 2021, Mendoza et al 2022. Furthermore, the presence of osteopontin in the ESM facilitates tissue repair and remodelling while also regulating cytokine release and macrophage recruitment (Scatena et al 2007, Han et al 2023).

Historical outlook
Eggs and their products have been historically used for wound healing and beauty (Ohto-Fujita et al 2019). For example, the ESM has been historically used to cover wounds; meanwhile, the egg white has been used as an astringent to help wound closure (Forrest 1982). More recently, eggshell powders are used as dietary calcium supplements (Bartter et al 2018). Eggshells and ESMs can be exploited for use rather than left to be destroyed, reigniting their historical applications in beauty and medicine (Yoo et al 2015, Ohto-Fujita et al 2019. Likewise, in traditional Chinese medicine the ESM was historically used for healing burns, ulcers and tympanic perforations (Jia et al 2011). Meanwhile in Japanese cultures it continues to be used by sumo wrestlers for wound healing (Sah and Pramanik 2014). In the early 2000s, researchers began to explore the potential of ESM in tissue engineering, where it could be used as a scaffold material to support the growth of new tissue (Mohammadzadeh et al 2021). One study published in 2023 demonstrated the ability of ESM to support the growth of bone cells in vitro, leading to the suggestion that it could be used as a bone graft material (Torres-Mansilla et al 2023). Other studies have shown the potential of ESM for cartilage regeneration, nerve regeneration, and other tissue engineering applications (Ninov et al 2015, Kim 2020. More recently, researchers have explored the potential of ESM in drug delivery and as a platform for regenerative medicine (Chen et al 2022b). Studies have shown that ESM can be loaded with drugs and growth factors and used to deliver these agents to targeted tissue (Liu et al 2019). In addition, ESM has been used as a platform for cell transplantation, where cells are seeded onto the membrane and transplanted into the body to regenerate damaged tissue (Vuong et al 2018, Yan et al 2020).

Extraction and isolation
The ESM extraction methods (table 1) can be divided into two major categories: (i) mechanical, and (ii) chemical. The mechanical approach requires manual removal of the eggshell by carefully peeling it from ESM with forceps (Liu et al 2019, Li et al 2019a, Wan et al 2022. However, the procedure is timeconsuming and there is a risk of damage to the ESM or unintentional separation of its layers as the outer side is strongly embedded in the eggshell. To overcome this challenge, an alternative strategy has been proposed which is based on the dissolution of the CaCO 3 -containing shell by placing the egg into highly acidic solution. The most common are acetic acid, hydrochloric acid (HA) and ethylenediaminetetraacetic acid (EDTA) (Farjah et al 2013, Mensah et al 2021, Sheish et al 2021. Mensah et al (2021) showed that the acid treatment resulted in thicker and more porous membranes compared to the manual stripping. Moreover, the membranes exhibited various wettability and swelling profiles depending on the separation method. This could be attributed to the effective preservation of the intact outer layer under the acidic treatment. Additionally, the treatment with acetic acid has been shown to improve biocompatibility of the membranes thanks to the introduction of carboxylic functional groups into the scaffold (Choi et al 2021). However, the efficiency of the chemical approach is strongly affected by the length of incubation, temperature, and the acid concentration (Santana et al 2016). In some studies, the two methods were combined, where the egg is first soaked in weakly concentrated acid solution to weaken the bonds between the ESM and the eggshell, which is then manually stripped (Sun et al 2022).
Nevertheless, the usability of natural ESM is limited by its poor solubility in aqueous conditions, which stems from the strong interactions between cystine, hydroxylysinonorleicine and desmosines present in the ESM fibres (Baker andBalch 1962, Crombie et al 1981). Due to high concentration of disulphide bonds crosslinking the fibres, the manipulation of the shape and size of the membrane becomes a challenge. Therefore, a number of studies attempted to solubilise ESM and produce soluble eggshell membrane protein (SEP) in order to expand the potential of the material. Takahashi et al (1996) successfully obtained SEP by subjecting it to the performic acid oxidation and pepsin digestion, however, the yield was only 16%-39%. Yi et al (2003) proposed a new method for SEP separation, which was based on incubation of ESM in 3-mercaptopropionic acid in the presence of 10% acetic acid. This strategy greatly improved the efficacy of isolation, increasing the yield up to 62%. However, it requires temperatures of at least 80 • C which could lead to protein denaturation.
Nevertheless, it has remained as a prevailing method of SEP extraction (Yang et al 2015, Amirsadeghi et al 2021.

Application dermatology
Millions are estimated to suffer from acute and chronic skin wounds yearly and these various wounds invariably bring aside the obvious health issues, potential emotional and financial implications to patients (Langemo andBrown 2006, Shankaran et al 2013). The centre of interest in intensive research on acute and chronic wounds is to find an effective treatment. The many proteins and peptides found in ESM make it an ideal candidate for wound healing. The application of chicken ESM for skin wound healing was first attempted by Maeda and Sasaki (1982). The initial study was conducted using rabbits. The results revealed that ESM was a suitable material for wound healing. Consequently, the ESM was applied as a skin graft in a patient and after seven days, the wound was well epithelialised. ESMs were further used in two cases, a three-year-old female child with a severe burn on their foot and a three-year-old female child with a scald burn on the elbow joint. In both cases, satisfactory epithelialisation was observed.
In wound management, the dressing must prevent bacterial infection, and stimulate angiogenesis and re-epithelialisation (Raja et al 2007, Kim 2018. In a study conducted by Li et al (2019a), natural ESM was found to exhibit intrinsic antibacterial activity against both Escherichia coli (gram-negative) and Staphylococcus aureus (gram-positive). In order to enhance the bactericidal properties, the membranes were then immersed in a solution of silver nanoparticles, which were adsorbed onto their surface. The composites not only resulted in vastly superior antibacterial properties, but also demonstrated a sustained silver release over four days, which is important for a long-lasting protection. A similar profile was noticed with Briggs et al who further adapted the ESM by modifying it with the thermoresponsive polymer, PNIPAAm (Briggs et al 2022).
In another study, a membrane consisting of polydopamine-modified ESM (mESM) nano/microfibres with KR-12 antimicrobial peptide and HA was generated by Liu et al (2019). Accordingly, the in vitro biological results showed that the membrane had remarkable antibacterial activity and stopped the formation of methicillin-resistant S. aureus biofilm on the membrane surface. In addition, the membrane increased the proliferation of keratinocytes and human umbilical vein endothelial cells and enhanced the secretion of vascular endothelial growth factor (VEGF). The in vivo animal model study revealed that the membrane is a suitable material for wound dressings.
In a quest to generate a cost-effective wound healing product with anti-inflammatory properties, processed ESM powder (PEP) has been explored in  (2018) studied the effect of PEP on matrix metalloproteinase (MMP) activities in vitro dermal fibroblast cell culture and in vivo mouse skin wound healing models. The PEP treatments in both models increased the activity of MMP and the regulation of early cellular functions during wound healing. Ahmed et al (2019b) conducted a study to evaluate PEP for advancement of skin wound healing. A mouse wound model was implemented to assess the impact of the PEP on wound healing. The histopathological assessment of the wound at days 3, 7 and 10 showed that the PEP significantly enhanced the wound closure. Additionally, the histological studies revealed that the granulation tissue in the PEP treated wounds, a bilayered skin substitute was constructed based on PEP-crosslinked gelatine-chitosan cryogel (Saha et al 2021). The dressing exhibited high swelling capacity and porosity as well as enhanced flexibility and biodegradability compared to cryogels traditionally crosslinked by toxic glutaraldehyde. Additionally, the in vitro studies revealed that PEP creates a better microenvironment for fibroblast attachment and proliferation, whereas the in vivo testing showed accelerated wound healing comparable to the commercially available dressing.
Beside wound healing, ESM has been found to possess anti-aging properties. Ohto-Fujita et al (2019) demonstrated that the application of solubilised ESM to the mice skin resulted in increased expression of genes encoding for type III collagen, decorin and MMP2, which resembles the microenvironment of young papillary dermal skin. Moreover, the level of type III collagen was elevated, resulting in higher skin elasticity. Therefore, the ESM might be useful in preventing skin aging and maintaining its healthy state.

Nerve, bone, and cartilage regeneration
Nerve damage represents a major challenge in healthcare due to its devastating impact on the quality of life and the lack of effective treatments. Therefore, in recent years a huge interest has been generated in neural tissue repair and the development of novel regenerative strategies (Schmidt and Leach 2003, Ninov et al 2015, Kim 2020. Due to its biocompatibility and high content of bioactive components, ESM constitutes a promising substrate for nerve regeneration. Farjah et al (2013) developed a conduit made out of an ESM tube that would connect severed nerves and guide their regeneration. The construct was placed between proximal and distal ends of sciatic nerves in rats. The in vivo study revealed that ESM supported the regeneration of peripheral nerves. Moreover, further study revealed that ESM is capable of not only boosting nerve repair, but also encouraging the operational improvement in an injured sciatic nerve of a rat (Farjah et al 2016). It was noticed that on the 90th day post-operation the ESM group exhibited a greater number of regenerated myelinated axons compared to the autograft group. The regenerative capacity of native ESM (nESM) can be enhanced by combining the therapy with lycopene or ibuprofen, which have been shown to further accelerate the functional recovery of sciatic nerves (Raisi andMohammadi 2019, Farjah et al 2020).
In recent years, bone tissue engineering has generated a substantial curiosity among the research community and strong effort has been devoted to the development of a cost-effective bioactive organic/inorganic hybrid materials capable of regulation of bone formation (Yoshikawa et al 2002, Tohma et al 2012. Arias et al (2008) proved the effectiveness of ESM as a biodegradable regulator of bone regeneration, where X-type collagen has been implicated as the main contributor. In this study, dried ESM was interposed into the osteotomy site in the rabbit ulna. The histological and radiographic examination of the ulna after four weeks revealed an intact ESM and lack of bridging of the osteotomised bone ends. After 16 weeks the bone was only partially bridged compared to the complete loss of the fracture line in the control group. This research demonstrated a great potential of biodegradable ESM in preventing the premature closures of bone, which could replace such conventional procedure as the interposition of an autologous fat grafts that require second incisions.
In recent years, several studies attempted to improve the potential of ESM for bone tissue engineering by introducing changes to its nanotopography or by incorporating bioactive agents (Pillai et al 2015, Wan et al 2022. In the study by Park et al, an ESMbased nanopatterned scaffold for bone regeneration was developed . In this study, the disulphide bonds between ESM fibres were broken down by double dissolution and the obtained ESM solution was subjected to nanoimprint lithography to mimic the naturally occurring ECM surrounding osteoblasts. The in vitro studies revealed that the nanopatterned ESM resulted in high attachment and complete alignment of osteoblasts. Moreover, the scaffold promoted growth factor secretion such as VEGF, which is crucial for vascularisation. Further in vivo studies showed that the nanopatterned ESM is capable of accelerating bone regeneration in 3 mm-diameter cranial bone defects in mice . In another study, ESM was used as a base for periosteum-mimicking biomaterial (Wan et al 2022). Cerium (III, IV) oxide-mineralised ESM was fabricated based on the biomimetic mineralisation principle. The cerium (III, IV) oxide provided ESM with enhanced immunomodulatory and neurovascularisation capabilities. Moreover, the construct successfully prevented the infiltration of soft tissue cells and enhanced osteogenesis in vivo.
Chen et al (2019) proved the use of a versatile biomimetic mineralisation procedure to generate ESM/hydroxyapatite composite with the ESM as the model. The findings showed that both sides of ESM proved exceptional biomimetic mineralisation ability, with the hydrophilicity and thermal stability of ESM being efficiently better by the insertion of HA. Furthermore, in vitro experiments on MC3T3-E1 cells showed that the inmost side of the ESM benefited cell proliferation and adhesion more than the outer side. Incredibly, the processes of proliferation, adhesion and multiplying, along with the alkaline phosphatase (ALP) activity and demonstration of bonerelated genes and proteins (runt-related transcription factor 2, ALP, collagen type I, and osteocalcin) on both sides of the ESM composites showed a suggestively advanced as compared to those of the original ESM. These results indicated that ESM-HA composites attained employing biomimetic mineralisation potentially could be new materials for future bone tissue repair.
Likewise, ESM could be combined with chitosan and silk fibroin into a functional hydrogel that could act as an articular cartilage replacement (Adali et al 2019). The hydrogel proved to be suitable to support attachment and promote proliferation of chondrocytes. They were also capable of strong antibacterial response towards gram-positive bacteria. Alternatively, SEP can be incorporated into agarose gel to facilitate cartilage regeneration as proposed by (Been et al 2021). Agarose in itself does not promote cell adhesion, therefore, the addition of SEP resulted in drastically higher numbers of attached and proliferating chondrocytes. Additionally, the presence of ESM resulted in the downregulation of immune response towards the scaffold.
Furthermore, in oral and maxillofacial surgery, periodontitis is a primary cause of tooth loss in adults, and it affects 5%-15% of people worldwide (Petersen 2003). Guided tissue regeneration (GTR) is a technique employed in the regeneration of damaged periodontal tissues (Gentile et al 2011). This technique involves the use of a barrier membrane to eliminate epithelial cells from the damaged surface and repopulate with the periodontal ligament cells (Jia et al 2012, Salonen and Persson 1990, Dupoirieux et al 2001. Synthetic GTR membranes have been shown to have poor biocompatibility and inflammatory effect due to the acidic degradation products (AlGhamdi and Ciancio 2009). In another study by Kalluri and Duan (2022), ESM was electrospun and blended with poly(ε-caprolactone) and bioceramic nano-hydroxyapatite to create a novel GTR membrane. The study was focused on optimisation of parameters that influence the mechanical properties using Taguchi orthogonal arrays. No biological examination was performed of the obtained composite.

Ophthalmology
In ophthalmology, ESM was first utilised by Coover in 1899 for four different eye injuries namely symblepharon, burns on eyeball, cornea ulcer and iritis (Coover 1899). Before then, the ESM was not used due to fear of infection. In that study, raw ESM obtained by manually peeling from the shell was applied in each case study. In the case of symblepharon, after ten days, the eyeballs and lids of the patients were smooth with no adhesions. Similar results were observed in patients with burns on eyeballs. The use of ESM in patients with corneal ulcers experienced no pain or irritation during the treatment. The ulcers were suitably healed after two weeks. Finally, ESM was employed in iridectomy for recurrent iritis and resulted in an effective wound healing with no infection. Mensah et al (2021) further explored nESM as a potential material for corneal wound healing. The study demonstrated that the raw ESM is capable of successfully supporting the attachment and proliferation of immortalised corneal epithelial cells and corneal mesenchymal stromal cells. Additionally, Choi et al (2020) proposed the use of ESM for retinal pigment epithelium (RPE) regeneration. In their study, ESM was incorporated into gellan gum hydrogel, which resulted in improved biocompatibility and biodegradability. Moreover, ESM acted as an antiswelling agent which allowed the implant to retain its shape. The in vitro study with RPE cells extracted from coloured rabbits revealed that ESM enhanced cell proliferation and caused no adverse effect on cell viability. No further studies have reported on the use of ESM in ophthalmic surgery or other eye applications.

Neurosurgery
In neurosurgical operations, it is important to protect the brain tissue from the hazardous effect of the metallic microsurgical instruments (Cokluk and Aydin 2007, Spetzger et al 2011). The experimental study of Gokyar et al (2017) evaluated the use of raw ESM as a therapeutic intervention for the protection of naked brain tissue. In their study, 13.3% of the uncovered fresh cadaveric cow brains operated with ESM were minimally damaged as compared to 60% of the brains without it. According to the findings, ESM has some promising effects as a material for brain tissue protection and essential in neurosurgery.

Otolaryngology
ESM has been shown as an effective patch for the treatment of moderate to large traumatic tympanic membrane perforation (TMP) in human (Jung et al 2017). TMP, a hole in ear drum is a condition that can be caused by infection or trauma (Afolabi et al 2009). In clinical practice, most TMPs have tendencies to heal on their own. Nonetheless, in large perforation, the spontaneous healing fails (Lou et al 2011). Jung et al (2017) evaluated the effects of ESM patches on the healing time for TMP. Sterilised round disc ESM patches moisturised with saline were placed on the surface of perforation in patients. After three months, the healing time for patients with the ESM patches were significantly improved as compared to patients that received perforation edge approximation.

Cardiology
Cardiovascular diseases (CVDs) have become the leading cause of death worldwide, resulting in more than 19 million death per year (Health Intelligence Team 2022). Conventionally, the replacement options for malfunctioning blood vessels are either allografts or autografts, however, they are associated with drawbacks such us availability or high donor morbidity (Fazal et al 2021). Therefore, there is high urgency for the development of new artificial vascular grafts. The intrinsic properties of ESM such as high gas permeability and antibacterial activity make it an attractive biomaterial for investigation in the CVD context. In one study by Yan et al (2020) ESM was used as a material mimicking the vascular intima surface in order to encourage endothelial cell growth. The membrane was incorporated into thermoplastic polyurethane, which provided mechanical support. The constructed vascular graft successfully promoted endothelial cell growth and rapid endothelialisation. Moreover, the grafts that contained heparin also resulted in antithrombotic activity. Further in vivo. study revealed that heparin-conjugated ESM can be successfully used as an arterial patch in a rat aortic angioplasty model (Sun et al 2022).

Bacterial contamination
Widely consumed across the world, eggs are one of the leading causes of food poisoning in the UK (Adak et al 2005), as the warm (42 • C), moist, and nutrient-rich environment of the egg is particularly favourable to rapid bacterial growth. Bacterial contamination of the egg, particularly of Salmonella, is a serious concern within the food industry for its food safety implications. It is therefore well documented how contamination may occur and which pathogens are commonly the causative agents. Trans-shell contamination in the first 30-60 s of laying, whereby eggshells with a wet surface can be penetrated by bacteria, has been heavily researched and confirmed to be the most likely route of infection (Berrang et al 1999). There is currently no literature found, however, which measures bacterial penetration of membranes alone, as for food purposes, the shell and membranes are usually considered together. Although antibiotics are routinely used in egg production and chickens vaccinated against Salmonella, this is only partially protective, and infection of the egg still commonly occurs.
The risk of cross-contamination in vitro or more worryingly in vivo from using the ESM is evident, as it may cause cell death or sepsis, respectively. However, Guarderas et al (2016) reported that their protocol included placing the ESM in solutions of antibiotics eliminates this risk. This seemingly easy solution poses its own risks, primarily, it may contribute to growing antimicrobial resistance which decreases the ability to treat infections. An alternative would be to screen all eggs before the ESM is used for biomedical purposes, but of course, this confers an extra processing step and cost. It should be noted that bacterial contamination is possible during storage of the ESM. Figure 3 shows the bacterial colony found on the inner ESM stored in phosphate buffered saline (PBS) at ambient temperature. This clearly indicates that contamination remains a concern even if the egg is screened and shows no presence of bacteria, these can later be introduced if the ESM is handled in a nonsterile way and stored incorrectly with consequences later during its use.
In summary, ESM has been found to possess antibacterial properties, particularly against Gram-negative bacteria such as E. coli and Pseudomonas aeruginosa (Yoo et al 2015). The antimicrobial activity of ESM is attributed to the presence of lysozyme, a naturally occurring enzyme that breaks down bacterial cell walls by hydrolysing the β-1,4-glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine. It is important to note that the antibacterial properties of ESMs do not provide complete protection against all types of bacteria and should not be relied upon as the sole means of preventing bacterial contamination. Proper handling and storage protocols, such as washing the eggshell before use and storing the ESM under sterile conditions, are necessary to minimise the risk of bacterial contamination. Additionally, using antibiotics to eliminate bacterial contamination in ESMs may contribute to the growing problem of antimicrobial resistance, and should only be used when absolutely necessary.

Variation
Despite the use of the ESM for decades and in a wide variety of applications described herein, there remains a scarcity of biomechanical characterisation in the literature, and any mention is almost always solely of the chicken ESM. This is further compounded by the high degree of heterogeneity within the ESM (Torres et al 2010), as with many other naturally derived materials, which limits the reproducibility of results and the ability to draw significant conclusions. Torres et al (2013) demonstrated that this inhomogeneity resulted in a wide variability of results and made it difficult to accurately define its mechanical and biological properties or conduct further studies. For example, they found it challenging to precisely estimate the cross-sectional area of the ESM, which could explain the variability of ultimate tensile strength and pore volume. The heterogeneous nature of the ESM may also limit its applications as it may not behave in a consistent way each time it is used or depending on which part of the membrane is used. Without a standardised material, application in vivo and in vitro will remain limited to existing uses. However, crosslinking, or other tissue modifications, discussed within this review, are able to overcome this limitation to enhance the native properties of the ESM and generate uniformity in its properties.

Mechanical property
The ESM is fragile and mechanically weak, much like the amniotic membrane and other natural materials used for biomedical applications (Strnková et al 2016, Sari et al 2020. This is an issue for wound healing purposes where a sufficiently strong material that can protect the underlying surface by maintaining a barrier between the wound and the outside environment is needed. Interestingly, Torres et al (2010) reported the ESM to have a higher tensile strength when it is dry than when it is immersed in albumen or water. However, they also found that when dehydrated the structure of the fibrous network is lost so cannot be visualised making it challenging to ascribe the strength of the ESM to a particular structural. They showed that water acted as a plasticiser, interacting with the long-chain polymer molecules of the ESM and reducing the number of hydrogen bonds formed between them. This again is problematic if used as a wound dressing, any exudate that is produced will weaken the structural integrity of the ESM and leave it vulnerable to tearing and allowing infectious agents to access the healing wound, introducing the possibility for infection or development of a chronic wound (Mogoşanu and Grumezescu 2014). Crosslinking of the ESM is a particularly useful avenue that should be further studied to identify the most efficient crosslinker and address the mechanical weakness to produce a more robust material.
The pore properties of the ESM are rarely investigated in literature despite the understanding that pore size is an important parameter in cellular migration, proliferation and nutrient diffusion on growth platforms (Han et al 2021). Added to this, the little that is available, describes porosity in varying ways including as a percentage/volume of the total material ( (2013) reported pore sizes of 3-10 µm. As different cell types have different preferences of pore size, fibroblasts for example prefer 5-10 µm sizes, whilst osteoid and skin regeneration have optimal pores sizes at 20-125 µm (Yang et al 2001), depending on the application of the ESM, the native porosity can be an advantage or disadvantage for its function (Han et al 2021). Fortunately, Hsieh et al (2013) demonstrated that hydrogen peroxide is a useful tool in controlling pore size and was experimentally shown to reduce pore size to 1-5 µm after treatment for 24 h. Where necessary this could be used to achieve the desired porosity in the ESM.

Modification of ESM
Crosslinking has been established as a method to modify tissues, which can improve their mechanical and thermal stability and reduce degradation (Tolinski 2009). In the case of collagen or collagenrich materials, various techniques are used, such as ultraviolet, physical treatment, or chemical processes using 1-ethyl-3-carbodiimide hydrochloride. Caliari and Harley (2011) and Wang et al (2015) suggest that crosslinking can reduce immunogenicity by masking antigenic markers. However, some literature suggests that crosslinking can impede intrinsic crosslinking and inhibit the breakdown of materials (Chapman 2007), and some crosslinking agents are cytotoxic or damage ECM components, such as glycosaminoglycans, which can affect the biocompatibility of treated materials (Hussein et al 2017).
To assess whether the properties and applications of the ESM can be enhanced through crosslinking, analysis of the mechanical properties of the nESM and mESM is required. The tensile strength or toughness of an ECM or growth medium is a factor in cell adhesion, differentiation, and proliferation (Engler et al 2006, Anderson et al 2014. The ability to control the mechanical strength of the ESM through crosslinking could provide influence over cell fate and proliferation rate and could be used in biomedical and clinical applications where specific cell niches are targeted or studied. A mESM could also be used as a platform for drug testing, reducing the dependence on animal models (Grela et al 2020).
Dynamic scanning calorimetry (DSC) is a useful technique to determine the thermal stability of the different crosslinked membranes at differential temperatures (Fessel et al 2014). DSC can show the ESM's behaviour at body temperature for biomedical applications such as wound dressing, as well as the membrane's ability to maintain integrity during storage at freezing (0 • C), refrigerated (2 • C-4 • C), or ambient (23 • C-25 • C) temperatures (FAO and WHO 2009). Water contact angle is another experimental technique that can be used to assess the wettability or hydrophilicity of a material. Hydrophilic membranes are better suited to promoting cell growth and proliferation, but hydrophobicity can be useful for cell detachment and fabric durability, particularly in cancer studies (Ferrari et al 2019). Any tissue modifications to the ESM must consider the impact on hydrophilicity and therefore on protein adsorption.
Nevertheless, there is currently not enough literature or evidence exploring the interplay between the properties discussed herein. There needs to be a greater understanding of how other factors such as surface roughness directly impact the hydrophilicity or toughness both before and following tissue modification. This knowledge will allow for fine tuning of the membrane's properties to suit a wide range of applications and produce the desired outcome. Furthermore, modification will address some of the weaknesses that will be discussed in this review and will increase the efficacy of the ESM for some of the applications described below.
Preliminary studies were conducted to evaluate the effects of physical crosslinking by mechanical, and chemical methods using biologically derived and synthetic agents; genipin and glutaraldehyde respectively. Figure 5 shows the appearance of the membranes following modification.  Each side of the modified membranes were mechanically tested for their tensile strength under strain, changes in physical properties at differential temperatures and their level of hydrophilicity. Once these physical characteristics had been measured, biological characterisation could be done. Gingival cells were seeded on the mESMs and nESM and incubated for one, three and six days under normal physiological conditions.
Initial results suggested that the use of chemical crosslinking agents, namely glutaraldehyde and genipin does indeed enhance the mechanical strength of the ESM. Genipin specifically also modifies the ESM to enhance cell viability and reduce cytotoxicity, making it a suitable construct for supporting cellular adhesion and proliferation. These results conformed with previous evidence reported in literature by Hussein et al (2017).
Further to this, it should be explored if the same crosslinking agent would be equally suited to combining several ESM membranes to form a large matrix. This would require dynamic mechanical analysis (DMA), differencial scanning calorimetry (DSC) experiments and biological assays, particularly looking at the joining sites to determine if these have adequate properties relative to the rest of the membranes. Such a material would be most applicable to the translational purposes of wound dressing and 3D skin modelling for in vitro testing as it would standardise the material and allow custom sizes to be obtainable from one continuous sheet of mESM.

Conclusion
Historically, egg-derived components such as the egg white, eggshell and ESM have been commonly associated with wound healing and beautification strategies in Asian cultures, and suggest a pre-disposed acceptance of the scientific and cultural validation of the material. In addition, the current drive and promotion towards sustainability, ethical resourcing, and anti-animal testing movements have further raised awareness and popularity of alternatives to the current norm in the field of biomedical, clinical therapeutic and drug development pathways. An additional advantage of using this material stems from its encouragement of 'green technology'-the conversion of a low-cost waste material to a product of significantly higher value. The ESM has shown to possess unique characteristics such as high biocompatibility, antimicrobial activity, appropriate mechanical and physical properties as well as additional parameters such as transparency, hydrophilicity/hydrophobicity, and porosity. In addition, the innate structure and composition of the ESM lends itself to additional enhancement which a number of examples have been described (e.g. crosslinking) and could result in a significantly more use in a variety of applications. To this end, the ESM has demonstrated a 'pedigree' of usefulness-even in its native form-and shows promising characteristics which may be further exploited for not only biomedical applications but other areas of interest such as sustainable packaging, filtration systems, and horticultural platforms.

Data availability statement
All data that support the findings of this study are included within the article (and any supplementary files). Chen Y, Feng Y, Deveaux J, Masoud M, Chandra F, Chen H, Zhang D and Feng L 2019b