Avian Egg: A Multifaceted Biomaterial for Tissue Engineering

Abstract

Most components in avian eggs, offering a natural and environmentally friendly source of raw materials, hold great potential in tissue engineering. An avian egg consists of several beneficial elements: the protective eggshell, the eggshell membrane, the egg white (albumen), and the egg yolk (vitellus). The eggshell is mostly composed of calcium carbonate and has intrinsic biological properties that stimulate bone repair. It is a suitable precursor for the synthesis of hydroxyapatite and calcium phosphate, which are particularly relevant for bone tissue engineering. The eggshell membrane is a thin protein-based layer with a fibrous structure and is constituted of several valuable biopolymers, such as collagen and hyaluronic acid, that are also found in the human extracellular matrix. As a result, the eggshell membrane has found several applications in skin tissue repair and regeneration. The egg white is a protein-rich material that is under investigation for the design of functional protein-based hydrogel scaffolds. The egg yolk, mostly composed of lipids but also diverse essential nutrients (e.g., proteins, minerals, vitamins), has potential applications in wound healing and bone tissue engineering. This review summarizes the advantages and status of each egg component in tissue engineering and regenerative medicine, but also covers their current limitations and future perspectives.

1. Introduction

Tissue loss and tissue defects result in approximately eight million surgical interventions each year and represent nearly half of the medical costs in the United States.^{1, 2} Traditionally, tissue failures and defects are mainly treated with prostheses, transplantation of donor organs, or the implantation of mechanical devices. However, the lack of adequate and proper donor organs, and the inability of prostheses and mechanical devices to restore the patient’s normal life highlight the need for alternatives.^3-6 Tissue engineering (TE) has emerged as an attractive alternative to treat tissue malfunctions.^7-9 TE is at the interface of materials science, chemistry, bioengineering, and cell biology. Using principles from these fields, TE aims to develop biological substitutes to maintain, restore, or improve tissue function by combining a scaffold, cells, and biomolecules.^{1, 10, 11} Biomaterials play a vital role in TE because they can provide cells with a microenvironment that mimics their native physiological niche and enable tissue regeneration.^12-14 Biomaterials can be created from synthetic or naturally occurring sources, such as seashells or eggs. Among various sources of materials, those that are naturally derived can recapitulate extracellular matrix (ECM) elements such as cell binding sites, which can ultimately influence cellular behaviors (e.g., spreading, migration, differentiation) needed for tissue growth both in vitro and in vivo.^15-17

Among various biomaterials used for scaffold design, those with high bioactivity, sustainability, easy handling/processability, and low production costs are preferred.^18-20 The multiple components found in avian eggs check all these boxes, prompting interest by many researchers to use them as a source of raw materials.^21-24 Egg constituents are beneficial in the management of various tissue injuries.^25-27 In fact, their use for wound healing and treating burns can be found in manuscripts from ancient Persian, Chinese, Egyptian, and Roman civilizations.^28-30 In addition to their advantageous intrinsic bioactive features, sustainability, ease of handling, and affordability, avian egg elements are naturally degradable.^28-30

The avian egg is composed of four main components: (i) a protective eggshell (ES), which is mainly composed of calcium and phosphate (CP), (ii) a thin ES membrane (ESM), which has a fibrous structure and mainly consists of proteins similar to those found in human ECM, (iii) an egg white (EW), which is a major source of proteins and bioactive peptides, and (iv) an egg yolk (EY), which is mostly composed of lipids, but also contains essential elements such as proteins, polysaccharides, minerals, and vitamins (Figure 1).^31-33 In this review, we describe the utility and advantages of each egg components in TE and regenerative medicine, their current limitations, and future directions for clinical translation.

Figure 1. Major components of a chicken egg include a calcified EG, a thin EGM, a viscous protein-rich EW, and a lipid-rich EY bulge.

The calcified ES is a rigid and semipermeable membrane that provides protection to the egg contents and embryo against physical damage and contamination by microorganisms. The EGM is a thin layer attached to the inner side of shell with a fibrous porous structure that is permeable to oxygen and gases. EW, representing about 60% of the total egg weight, is a viscous fluid covering the EY and is mainly composed of proteins, such as ovalbumin, conalbumin, ovomucoid, and lysozyme. EY, representing about 30% of the total egg weight, is the innermost component and a lipid-rich substance that also contains other vital nutrients (protein, vitamins, and minerals) that play a critical role in embryo development.

2. Eggshell

Properties of ES and its similarities with human bone

Chicken egg farmers, chick hatcheries, egg-breaking plants, food-processing industries, and household consumption produce millions of tons of ES every year.^34-36 Disposal of ES waste not only is an economic burden but it also poses some environmental concerns, including the risk of spreading viral diseases and pathogens.⁸ Recycling and using this naturally derived biomaterial in other industries could reduce this global concern.^{37, 38} Applications of avian ES waste include removal of heavy metals from industrial wastewater,^{39, 40} fabrication of biocatalysts,^{41, 42} production of Li-ion batteries,⁴³ fertilizer in agriculture,⁴⁴ fabrication of ceramics,^{45, 46} production of thermal insulators,⁴⁷ production of composites as a filler,⁴⁸ and biomedical applications such as TE⁴⁹ The ES is a natural composite material that represents about 10% of the total egg weight.⁵⁰ The ES is composed of 94% calcium carbonate (CaCO₃), 4% of organic matter, 1% calcium phosphate [Ca₃(PO₄)₂], 1% magnesium carbonate (MgCO₃), and trace amounts of sodium (Na, 1512 ppm), magnesium (Mg, 3472–4500 ppm), strontium (Sr, 320–411 ppm), potassium (K, 524 ppm), and sulfur (S, 589 ppm).^51-54

Natural human bone consists of two types of calcium phosphate: calcium-deficient hydroxyapatite (HAp) and calcium-deficient β-tricalcium phosphate (βTCP);^55-59 The chemical structure of pure HAp is Ca₅(PO₄)₃(OH) with a calcium to phosphate ratio of 1.67 and that of pure βTCP is β-Ca₃(PO₄)₂ with a calcium to phosphate ratio of 1.5. In bone, these molecules contain trace elements, including Na, Mg, Sr, K, and Si (silica).^{45, 60} Thus, compared to HAp or βTCP synthesized from pure reagents, these compounds isolated from natural sources, like animal bone, coral, seashell, snail shell, pearl, and ES, are non-stoichiometric and calcium-deficient due to the presence of the trace elements. This non-stoichiometry is actually an advantage for bone tissue scaffolds.^{61, 62} The trace elements found in the ES are also present in bone tissues and are known to promote angiogenesis and osteogenesis. ^{60, 63} As a result, ES and ES-derived calcium phosphate, HAp, or βTCP have been used to design bone TE scaffolds.^64-67

ES as a source of calcium phosphate

Dadhich et al. developed three-dimensional (3D)-printed scaffolds using the naturally occurring polymer chitosan and calcium phosphate as the printing material.⁶⁸ One source of calcium phosphate that they investigated was derived from the ES. They compared the characteristics of the fabricated scaffolds with ES-derived calcium phosphate (ES-CaP) or a chemically synthesized calcium phosphate (CS-CaP). To evaluate their biocompatibility, these scaffolds were seeded with primary human mesenchymal stem cells (hMSCs). Compared to CS-CaP scaffolds, cellularized ES-CaP scaffolds exhibited enhanced proliferation, filopodial growth, metabolic activity, and differentiation toward an osteoblast lineage. One explanation for the difference seen in cell seeding and proliferation is the difference in the negative charge between the two scaffolds. Scaffolds with CS-CaP have a highly negative charge and nonspecific adsorption of serum proteins from the cell culture media. Protein coating on the surface of the scaffolds could impair attachment of hMSCs. In contrast, scaffolds with ES-CaP had less of a negative charge, likely due to the presence of trace elements from the ES. Thus, the surface of ES-CaP scaffolds had less nonspecific adsorption of serum proteins and thus provided a favorable environment for cell attachment. Based on the promising in vitro data with the cellularized ES-CaP scaffolds, the authors subcutaneously implanted these biomaterials into rabbits. The ES-CaP scaffolds were biocompatible and provided a suitable environment for tissue growth and vascularization.

In a second study, Huang et al. supplemented an ES powder with magnesium oxide (MgO).⁶³ MgO was added to provide Mg²⁺, an important ion that controls a myriad of cellular processes, including the functional properties of integrins which play a major role in anchoring cells to the ECM. Specifically, Mg²⁺ promotes osteoblast proliferation, matrix mineralization, and vascularization within the scaffolds.^{69, 70} To fabricate a bone TE scaffold, ES powder was combined with MgO to form a nanocomposite material (ES/MgO), which was subsequently mixed with carboxymethyl chitosan (CMC) with or without bone morphogenetic protein 2 (BMP2). BMP2 is a natural signal that promotes osteogenesis and ultimately bone formation.^{71, 72} To obtain a stable scaffold, the composite (ES/MgO/CMC +/− BMP2) was crosslinked through carbodiimide chemistry (Figure 2a). The addition of ES/MgO to CMC significantly improved the young modulus and compressive strength of the resulting scaffold (Figure 2b). Next, the scaffolds were seeded with human adipose-derived mesenchymal stem cells (hADSCs). When implanted in mice, these cellularized scaffolds released BMP2 and Mg²⁺ and over 2 and 4 weeks, respectively, stimulating various signaling pathways that are associated with osteogenesis. Notably, both ES-derived CaO3/MgO/CMC and ES-derived CaO₃/MgO/CMC/BMP2 scaffolds displayed improved bone regeneration when compared to CMC scaffolds (Figure 2c).

Figure 2. The intrinsic similarity of ES to the inorganic component of bones makes it a good candidate material for bone TE.

a) Schematic depicting the preparation and characterization of ES-derived CaO₃/MgO/CMC/BMP2 (CaCO₃/MgO/CMC/BMP2) scaffolds. b) Comparison of the mechanical strength of ES-derived CaO₃/MgO/CMC (CaCO₃/MgO/CMC) and CMC scaffolds. c) High-resolution Micro-CT imaging of scaffold-mediated bone repair in critical-sized rat calvarial defects 8 weeks after implantation: NC (negative control), CMC, CaCO₃/MgO/CMC, and CaCO₃/MgO/CMC/BMP2. (a—c) Reproduced with permission.⁶³ d) Scanning electron microscopy images of (I) pure GelMA hydrogel and (II) GelMA hydrogels reinforced with ES particles (GelMA-ESP). e) Comparison of the compressive modulus of GelMA and GelMA-ESP hydrogels at different ESP concentrations. Reproduced with permission.⁶⁵

In a separate study, Wu et al. combined ES with gelatin methacrylate (GelMA) to form GelMA-reinforced ES particle (GelMA-ESP) hydrogels as potential bone TE scaffolds (Figure 2d).⁶⁵ ES was added to the hydrogels to reinforce the mechanical stiffness of the scaffolds, which would ultimately enhance the adhesion, proliferation, growth, and osteogenic differentiation of pre-osteogenic cells. The compressive moduli of the resulting hydrogels increased proportionally to the amount of incorporated ES (Figure 2e). Prior to photocrosslinking, MC3T3-E1 pre-osteoblast cells were incorporated into the pre-gel solutions and the resulting cellularized hydrogels tested in vitro. Their studies showed that, compared to GelMA hydrogels, GelMA-ESP scaffolds promoted osteogenic differentiation but also enhanced cell proliferation and mineralization. In vivo, the subcutaneous implantations of cell-free hydrogels for 14 days showed that unlike GelMA hydrogels, GelMA-ESP hydrogels had improved biocompatibility and increased biodegradation.

ES as a source of HAp

HAp is a suitable material for bone TE since it can reinforce the mechanical strength of scaffolds and exhibits excellent osteoconductivity (i.e., promotes bone growth) and osteoinductivity (i.e., induces osteogenic differentiation). Not only HAp is bioactive, but it is also biodegradable, biocompatible, and non-immunogenic.^{66, 73, 74} It can be synthesized from synthetic or natural materials. The use of synthetic materials for HAp synthesis is expensive and results in stoichiometric HAp.^{75, 76} Such stoichiometric HAp differs from the calcium-deficient HAp found in human bone.⁷⁷ Thus, natural, readily available sources for the synthesis of calcium-deficient HAp, such as the ES, are desirable. ^{78, 79}

HAp can be produced from the ES by various methods, including but not limited to the sol-gel process, solid-state reaction, aqueous precipitation, hydrothermal technique, and microwave irradiation. To prove that HAp could be sustainability synthesized from biowastes (ES and urine), Ronnan et al. used an electrochemical technique to produce HAp from waste ES and synthetic urine.⁸⁰ The synthesis method substantially influences the morphological properties— shape, size, and crystallinity— of the resulting HAp.^81-83 Hydrothermal synthesis is a direct and widely used method for the synthesis of HAp from the ES. However, it is a time-consuming and arduous technique and does not provide good control over HAp morphology.⁸⁴ Microwave irradiation is an effective method to produce HAp from the ES. Compared with hydrothermal technique, microwave irradiation has several advantages such as scalability, exquisite control over HAp morphology, and faster production turnaround.^{76, 79} HAp particle size is an important feature and several approaches have been developed to control their diameters when synthesized from the ES.^{79, 85} Unlike microsized HAp, nanosized HAp exhibits higher protein adsorption and enhanced cellular responses such as osteoblast adhesion and proliferation.^{82, 84} The biological properties of nanosized HAp may be due to their resemblance to the natural HAp that is typically deposited during bone mineralization.³⁸

Although HAp offers several advantageous features for bone TE, it has several limitations such as slow degradation, low fracture toughness, and high brittleness. These properties have restricted the use of solid HAp in scaffold design, especially for load-bearing bone defects.^{78, 86} To overcome these limitations, HAp ceramic-based and HAp polymer-based composite scaffolds have been investigated.^87-89 Carbon nanotubes and silica have been used to produce HAp ceramic composite scaffolds.^90-92 Natural polymers, such as gelatin, starch, collagen, bacterial cellulose, alginate, and chitosan, as well as synthetic polymers, such as polycaprolactone (PCL), polylactic acid (PLA), and polyvinyl alcohol (PVA) have been used to fabricate HAp polymer composite scaffolds.^{88, 93, 94}

Studies by Trakoolwannachai et al. describe the synthesis and bioactivity of an ES-derived HAp-containing scaffold that is fabricated with a synthetic polymer (PCL)⁹⁵ and chitosan, a naturally derived polymer.⁹⁶ For both applications, ES-derived HAp was synthesized by the wet chemical precipitation method and its biocompatibility was tested with Saos-2 cells, a human osteoblast-like cell line. PCL is an intrinsically hydrophobic polymer that has a low capacity to absorb water and swell and is degraded slowly in vivo (up to 3 years).^{97, 98} The addition of HAp into the PCL matrix slightly increased the swelling and degradation properties of the resulting scaffolds by allowing water to permeate the interface between HAp and PCL. Seeding the HAp-PCL composite scaffolds with Saos-2 cells showed that cells attached to the constructs successfully and remained viable. Unlike PCL, chitosan is a hydrophilic, naturally derived polymer.⁹⁶ The addition of HAp into chitosan scaffolds decreased the swelling ratio and increased the surface roughness. Compared with tissue culture plastic, chitosan and HAp-containing chitosan scaffolds led to a decreased cell-matrix interactions, although the Saos-2 cells proliferated on both scaffold types. For a potential clinical translation, the performance of these ES-derived HAp scaffolds in comparison to pure PCL- or chitosan-based scaffolds needs to be investigated in relevant preclinical models.

ES as a source of TCP

βTCP is another calcium phosphate suitable for TE because of its advantageous mechanical and biodegradable properties. ^{57, 99, 99, 100} Unlike HAp, βTCP has a faster biodegradation rate and, if released into tissue fluids, is resorbed by cells. These properties make βTCP a great candidate in bone TE because it could be replaced by the newly formed bone tissue.^101-103 Similarly to HAp, βTCP is osteoconductive and osteoinductive,¹⁰⁴ and various methods are available for βTCP synthesis from the ES, such as wet precipitation, solid-state synthesis, and thermal conversion.^{99, 100, 102}

Composite scaffolds made with ES-derived βTCP and synthetic polymers such as PCL and PLA have been investigated.¹⁰⁵ For instance, Shafiei et al. incorporated carbon dots, ES-derived βTCP, or both, into nanocomposite scaffolds of PCL and PLA. For the scaffold preparation, they electrospun a PCL/PLA solution containing carbon dots and βTCP with or without carbon dots. In a cell-free degradation assay, the addition of βTCP slightly increased the degradation of the scaffolds. Because βTCP can also be degraded by cells, comparing the degradation in the presence of cells or after implantation could have revealed a greater effect of the inclusion of βTCP on the scaffold stability. In vitro investigations with human buccal fat pad-derived stem cells showed that the carbon dot-containing ES-derived βTCP scaffold led to the highest cell proliferation rate and osteogenic activity.

3. Eggshell membrane

Extraction and Properties of ESM

The ESM is a thin protein-based layer between the ES and the EW. Similar to the human amniotic membrane, this layer protects the growing embryo, has antibacterial properties, and provides a structural foundation for the calcification of the ES during the different stages of embryonic development.^{29, 106} Like the ES, the ESM is considered an industrial waste product that is available in large quantities and at low cost.^{107, 108} The inner part of the ESM is readily separated from the rest of the egg, but the outer portion is adherent to the ES and thus more difficult to separate. In an industrial scale, the efficiency of this process is important and various strategies such as a mechanical separation combined with mild acid treatment can effectively peel the ESM from the ES.^108-110

The ESM is mostly composed of collagenized fibrous proteins (Figure 3). This unique porous interwoven fibrous protein network is biocompatible with high water permeability.^111-113 For centuries the ESM has been used for treating chronic ulcers and bone fractures, and was formally named as “phoenix cloth” in Chinese traditional medicine.^{28, 109, 114, 115}

Figure 3. Microstructure of ESM.

Scanning electron micrograph of an ESM showing (a) side view and (b, c) top view. Reproduced with permission.¹¹⁶

The ESM is mostly constituted of proteins (~90%), including collagen type I, collagen type V, collagen type X, glucosamine, desmosine, and keratin. Lipids comprise ~3% and carbohydrates ~2% with the most abundant the glycosaminoglycan hyaluronic acid.^{110, 117-121} The similarity of the ESM to human ECM, as well as the presence of various functional groups like amines, amides, and carboxylic acids, makes it a promising material for TE, biosensors, and drug delivery.^121-125 Other applications of the ESM have been reported, including removal of heavy metals, fuel cell membranes, dietary supplement, and enzyme immobilization.^126-128

Applications of ESM in TE

The ESM has been used to create scaffolds with potential application in nerve regeneration, skin TE, vascular TE, and bone TE. In each case, the ESM combined with other molecules or polymers was superior to ESM alone. To generate a scaffold with properties compatible with peripheral nerve repair, Golafshan et al. developed a double network composite using polycaprolactone fumarate (PCLF) and manually extracted the ESM.¹²⁹ They used a vacuum infiltration approach to create double network scaffolds by soaking ESM films in PCLF solutions containing either dichloromethane or acetic acid. Acetic acid achieved a greater incorporation of PCLF into ESM films (Figure 4a) and was associated with the lowest swelling rate and highest tensile strength. To evaluate their biocompatibility, pheochromocytoma 12 (PC12) cells, a classical neuronal cell model, were seeded on ESM-PCLF scaffolds. While ESM-PCLF scaffolds prepared in acetic acid exhibited improved cell attachment, cells proliferated similarly on both ESM-PCLF and ESM scaffolds. Furthermore, the PC12 cells grown on ESM-PCLF scaffolds spread rapidly and formed intracellular connections, demonstrating their potential for nerve regeneration.

Figure 4. Leveraging ESM for TE scaffolds.

(a) SEM images showing the microstructure of (I) ESM and PCLF-coated ESM prepared with PCLF dissolved in (II) dichloromethane or (III) acetic acid. Reproduced with permission.¹²⁹ (b) Fluorescent imaging of human dermal fibroblasts showing their proliferation over 1 week incubation on ESM and a nanofiber-coated ESM scaffolds. Reproduced with permission.¹³⁰ (c) Photograph showing the cross section of a bilayered ESM/TPU vascular graft. Reproduced with permission.¹³¹ (d) Schematic depicting in situ mineralization of HAp on the ESM. Reproduced with permission.¹³²

For skin tissue regeneration, smooth bilayered scaffolds constructed on the ESM were superior to plain ESM.¹³⁰ Using direct electrospinning of a PCL and chitosan solution onto the surface an ESM that was extracted with dilute acetic acid, Ray et al. coated the ESM with nanofibers to create smooth bilayered scaffolds.¹³⁰ Unlike non-coated ESM, the nanofiber-coated ESM showed reduced surface roughness, exhibited slower enzyme-mediated degradation, displayed more hydrophobicity, and were associated with increased tensile strength. Water retention and water vapor transmission capacity of the nanofiber-coated ESM were consistent with the profile needed to maintain wound hydration while retaining a stable shape. In vitro investigations with human dermal fibroblasts revealed that the nanofiber coating enhanced cell attachment, spreading, and proliferation at early time points and accelerated cell infiltration into the scaffold at later times (Figure 4b). When compared to non-coated ESM, the nanofiber-coated ESM also demonstrated improved antibacterial activity. Next, non-coated and nanofiber-coated ESMs were tested in full thickness skin wounds in rats. Consistent with their biophysical properties, the wounds treated with nanofiber-coated ESM remained moist. Furthermore, with this biomaterial, wound healing was faster with efficient re-epithelization and hair re-growth.

In another study, nanofiber-coated ESM has been developed for vascular TE. Rather than a sheet-like structure that was used for skin grafts, vascular grafts should be designed with a tubular morphology. Yan et al. designed small-diameter artificial vascular grafts using thermoplastic polyurethane (TPU) nanofibers that were electrospun onto ESM tubes and subsequently coated with polydopamine and heparin via layer-by-layer deposition. In this approach, they combined the bioactivity of the ESM as the internal membrane and the tunable mechanical properties of TPU as the external layer. Furthermore, this strategy led to the formation of wavy small-diameter ESM/TPU tubular-shaped scaffolds with adhesive and anticoagulation properties (Figure 4c).¹³¹ Notably, the wavy structure of these ESM/TPU double-layered vascular grafts reproduced the mechanical behavior of natural blood vessel, an important feature to bear blood pressure when introduced into the body. When seeded with human umbilical vein endothelial cells (HUVECs), the internal membrane supported cell attachment and proliferation, and importantly, the use of heparin reduced platelet attachment, suggesting antithrombogenic properties. Although promising, these biomaterials still need to be tested in relevant animal models to assess their safety, performance, and ultimately clinical potential.

For bone TE, Chen et al. used ESM as a promising template for inducing biomimetic mineralization. HAp was successfully generated when the ESM was incubated with a simulated body fluid containing several ions, including Ca²⁺, Cl⁻, Na⁺, HPO₄²⁻, K⁺, Mg²⁺, SO₄²⁻, and HCO3⁻ (Figure 4d).¹³² Next, they characterized the properties of their HAp-modified and unmodified ESM and compared both the inner (EW side) and outer (ES side) for supporting MC3T3-E1 cell responses.¹³² Unlike unmodified ESM, mineralization occurred on both sides (inner and outer) of the HAp-modified ESM and this platform supported greater cell adhesion and proliferation, but also promoted osteogenic capacity of MC3T3-E1 cells as well as the expression of bone-related genes and proteins. These findings suggest that ESM-based materials have great potential for bone tissue repair.

Applications of soluble ESM-derived proteins in TE

The ESM contains crosslinked disulfide bonds, limiting its solubility in conventional and nontoxic solvents.^{133, 134} Although, the combination of ESM with various polymers has helped, its insolubility makes its processability quite challenging. Even if successful, it is still difficult to control the morphological and mechanical properties of ESM-based scaffolds, impeding their potential use in TE.^135-137 To tackle this problem, cleavage of the disulfide bonds has been used to obtain soluble ESM, mostly composed of soluble ESM proteins (SEP). There are two main methods for ESM solubilization: (i) chemical treatments in strong acidic or basic conditions at high temperatures and (ii) an enzymatic modification, which requires milder conditions.^{109, 138} Depending on the solubilization process, water-soluble or water-insoluble SEP are obtained. Water-insoluble SEP are soluble in organic solvents, such as acetic acid and hexafluoro-2-propanol.^{109, 138} For instance, Yi et al. developed a method to produce water-insoluble SEP.¹⁰⁶ This process used 3-mercaptopropionic acid as a reducing agent in dilute acetic acid, exposure at high temperatures, and neutralization with sodium hydroxide.¹⁰⁶ To improve their mechanical properties, both forms of SEP (water-soluble and water-insoluble) have been blended with synthetic polymers, such as PLA, PVA, PCL, poly (lactic-co-glycolic acid), and poly (ethylene oxide) (PEO) or with naturally derived materials, such as agarose, ES powder, and chitosan.^139-142

A couple of studies using 3-mercaptopropionic acid-extraction method to develop SEP-containing scaffolds for cartilage or skin TE have been reported. ^{143, 142} Been et al. combined SEP with agarose to fabricate a scaffold for cartilage repair and regeneration.¹⁴³ Compared to pure agarose scaffolds, agarose-SEP scaffolds had faster degradation. In vitro studies with rabbit-derived chondrocytes showed that the incorporation of SEP into the scaffold increased cell proliferation and survival, resulting in higher chondrogenic gene expression and increased synthesis and deposition of ECM proteins. Amirsadeghi at al. fabricated a multilayer electrospun scaffold containing SEP mixed with PVA/chitosan in the outer layers and zinc oxide nanoparticles in the inner nanofiber layer. Next, they tested these 3-layered scaffolds with human dermal fibroblasts for skin TE.¹⁴² In vitro studies showed that these 3D scaffolds promoted cell proliferation which was greater than cells cultured in 2D on a tissue culture plastic. This set of data suggests that SEP have great potential for the design of TE scaffolds.

4. Egg white

Properties of EW and EW-derived proteins

The EW, also known as albumen, has multiple applications. It represents approximately 56% of the protein in the egg.^{144, 145} In the food industry, EW is used as a raw material for the preparation of other products and is particularly useful as stabilizer. In medicine, historically, the antibacterial, anti-inflammatory, and healing properties of the EW make it useful for wound healing.^{146, 147} In addition, EW is cheap, abundant, and easily processed.¹⁴⁸ Moreover, it is nontoxic, biodegradable, and bioactive. All of these properties suggest that the EW is a biomaterial of choice for TE and regenerative medicine.^{149, 150} EW or EW-derived proteins are particularly attractive in the design of protein-based hydrogels, as EW-derived proteins are inherently bioactive and possess similar features of the human ECM, such as good porosity and high water uptake.^{151, 152}

The EW is a mixture of proteins, carbohydrates, vitamins, and minerals.³⁰ Among the more than 150 proteins in the EW, the most abundant include ovalbumin (44.5 kDa, ~54%), conalbumin (also known as ovotransferrin, 77.7 kDa, ~12%), and ovomucoid (28 kDa, ~11%), globulins (~8%), ovomucin (~3.5%), lysozyme (~3.4%), and flavoprotein (~0.8%) (Figure 5a).^{148, 153-155} Ovalbumin, conalbumin, and ovomucoid contribute to the EW viscosity, and ovalbumin and conalbumin undergo thermally-induced gelation.^{30, 153}. Furthermore, ovalbumin leads to the foaming properties of EW.^156-158 Conalbumin has antibacterial properties.¹⁵⁹ Ovomucoid has allergenic properties and is extremely resistant to heat, enzymatic denaturation, and degradation.^{160, 161,162, 163} Like conalbumin, lysozyme displays antibacterial properties,^164-166 and contrary to conalbumin, it is thermally stable.^{167, 168} EW-derived components individually or in combination hold great promise in the design of functional biomaterials for TE.

Figure 5. Application of EW in TE.

(a) Structures of EW-derived proteins. Reproduced with permission.³⁰ (b) Infiltration of bone marrow stromal cells into MeGC and lysozyme (MeLyz1)-containing MeGC hydrogels over 14 days. Reproduced with permission.¹⁷⁴ (c) Fluorescent images of HUVECs cultured on different films showing cell spreading on the surface at various time points. Reproduced with permission.¹⁴⁹ (d) SEM images depicting human dental pulp stem cells cultured on EW and ES-loaded EW hydrogels. Reproduced with permission.¹⁷⁵

Applications of EW and EW-derived proteins in TE

Compared with other albumins that are widely used in the biomedical field, including bovine serum albumin (BSA), ovalbumin is more affordable. Comparably to other albumins, ovalbumin binds to various proteins, including growth factors, which is advantageous for TE applications.^{147, 153, 158} Farrar et al. used partially pure, commercially available chicken ovalbumin to develop porous hydrogels for bone TE and showed that, using in vitro studies with MC3T3-E1 cells, the scaffolds supported cell proliferation and promoted their osteogenic differentiation.¹⁶⁹ Other approaches involve combining ovalbumin with synthetic polymers and naturally derived polysaccharides, such as PVA, polyethylene glycol (PEG), cellulose, and starch.^{170, 171}

As a stable enzyme with antimicrobial activity, lysozyme has been incorporated into polymer formulations for skin regeneration or into coatings for prosthetic implants.^{172, 173} Lysozyme has also been used to form hydrogels for bone TE. Kim et al. chemically modified glycol chitosan and lysozyme to form methacrylated glycol chitosan (MeGC) and methacrylated lysozyme (MeLyz), respectively. Next, these biomolecules were crosslinked to fabricate hydrogels with tunable degradation rates.¹⁷⁴ In contrast to pure MeGC hydrogels, the addition of lysozyme allowed MeGC hydrogels to degrade much faster. In vitro studies with bone marrow stromal cells showed that the lysosome-containing hydrogels supported cell proliferation, but also enhanced osteogenic differentiation and infiltration of cells into the constructs (Figure 5b). When tested in vivo for cranial bone repair, the lysozyme-containing hydrogels promoted greater bone regeneration than pure MeGC hydrogels, as indicated by larger bone growth area, the formation of osteoid development at the wound boundary, and by the greater wound closure.¹⁷⁴

The EW also has a potential application in the development of TE scaffolds. EW is incorporated with other naturally derived biomaterials, such as silk fibroin, bone-derived HAp, and ES. You et al. examined the properties of EW-containing films developed with different ratios of silk fibroin and EW.¹⁴⁹ Higher silk content increased their mechanical strength, whereas a higher EW content enhanced elasticity. In vitro studies with HUVECs showed that the presence of EW supported cell adhesion and proliferation on the films (Figure 5c). Owuor et al. created a solution composed of bone-derived HAp, EW, and diethylenetriamine.¹⁷⁶ With this solution, they formed composite scaffolds with layered structures similar to those found in mother-of-pearl. Within these layered structures, the crosslinked EW acted as a glue that provided elastic recovery and low deformability to HAp. The EW/HAp composite scaffolds were exceptionally strong (modulus up to 180 GPa) and exhibited a high tensile strength and load-carrying capacity. As a proof-of-concept, using 3D-printed bone and ear molds, they fabricated tissue-like structures with mechanical properties similar to the native biological tissues.

The ES and EW resemble the inorganic and organic phase of bone, respectively, sparking interest in combining these components together for bone TE. Huang et al. developed hydrogels using photopolymerizable GelMA in combination with EW or ES/EW.¹⁷⁵ Although EW and EW/ES hydrogels had similar mechanical properties, EW hydrogels exhibited a higher welling ratio. Both hydrogels supported attachment and proliferation of human dental pulp stem cells (hDPSCs) (Figure 5d), but unlike EW hydrogels, ES-loaded EW hydrogels promoted osteogenic differentiation to a greater extent. Interestingly, both hydrogels stimulated macrophages, suggesting they could induce an inflammatory response in vivo. Also, they had a fast degradation rate, up to 75% resorption within 10 days. Whether a potential inflammatory response in combination with rapid degradation may promote or impair tissue repair in vivo remain to be tested.

5. Egg yolk

Properties of EY

The EY consists of water (~50%), lipids (~30%), proteins (~16%), carbohydrates (~4%), and traces of important vitamins (A, B, D, E, K, etc.) and essential minerals (calcium, iron, zinc, magnesium, potassium, sodium, etc.).^177-179 EY could be separated into an aqueous phase (plasma) and an insoluble phase (granules). The plasma phase contains ~85% low-density lipoproteins (LDLs) and ~15% livetin, and the granules contain ~70% high-density lipoproteins (HDLs), ~ 16% phosvitin, and ~ 12% LDLs.^{33, 180-182} The combination of these substances, which originally serves as nutrition source for the embryo, makes it a great candidate for TE, regenerative medicine, and drug delivery.^183-185 Phosvitin, known to play a role in osteoblast differentiation and mineralization, is particularly relevant for bone TE.

Applications of EY-derived phosvitin in TE

Phosvitin is a highly phosphorylated protein, resulting in a strong negative charge that enables the protein to bind to metals and minerals.^187-189 These properties also enable phosvitin to function as a mineral crystal nucleation agent.¹⁹⁰ For instance, phosvitin is known to facilitate the transformation of calcium phosphate into HAp, potentially promoting mineralization of pre-cellularized scaffolds for bone tissue repair. Liang et al. fabricated nanofibrous cellulose mats which were subsequently coated layer-by-layer with EY-derived phosvitin and chitosan to create alternating bilayers. Next, these chitosan/phosvitin-coated bilayered mats were tested for their mineralization potential.¹⁸⁶ Incubation in a synthetic body fluid resulted in the complete coverage of HAp on the surface of the nanofibrous mats, especially for those with the highest number of layers (Figure 6a and b). The nanofibrous mats supported the attachment and proliferation of MC3T3-E1 cells, an osteoblast precursor cell line, suggesting their suitability as scaffolds for bone TE. These chitosan/phosvitin-coated nanofibrous mats along with alternative mats coated with tannic acid and phosvitin exhibited antimicrobial properties, demonstrating that these constructs are versatile and could be useful for other biomedical application such as for skin tissue regeneration.^{187, 191}

Figure 6. Exploiting EY for TE.

Transmission electron microscopy images of a) cross section and (b) longitudinal section of a chitosan/phosvitin-coated nanofiber. Reproduced with Permission.¹⁸⁶

6. Conclusions and future perspectives

The avian egg, an affordable food product of high nutritional value for humans, is a remarkable source for biomaterial extraction. Each part of the egg contains bioactive elements with great potential for TE and regenerative medicine (Table 1). Several challenges regarding the use of avian egg components, particularly those from chickens, as a source of biomaterials remain. Similarly to other naturally derived biomaterials, batch-to-batch variations exist and arise through multiple causes such as the type of food eaten by the hens. Consequently, it is necessary to establish protocols and minimize variables that lead to such disparities, ultimately ensuring reproducibility and reliability. Additionally, specific limitations subsist for the current egg-derived scaffolds. ES-based scaffolds lack the mechanical properties required for loadbearing bone defects. Also, the insolubility of ESM restricts its widespread application. Although various methods are available to produce soluble ESM, a mild reaction procedure without the use of toxic chemicals and/or high temperatures are yet to be discovered. With respect to the EY, more work is needed to fully investigate its potential application in TE and beyond. Importantly, many scaffolds containing egg components have yet to be tested in relevant preclinical animal models, which is a critical step for translational research and moving on to the first stage of clinical testing in humans.

Table 1.

Key strategies leveraging egg components in TE. ND: not determined.

Egg Component	Form of Usage	Target Organ	Preparation Methods	Other Materials	Cells	Animal Models	Observations	Ref.
ES	CaP	Bone	3D printing	Chitosan	MSCs	Rabbit	ES-derived CaP showed higher biological activities compared to the synthetic CaP. The 3D printed scaffolds showed Promising biocompatibility in vivo	68
ES	HAp	Bone	Sonochemical isolation for HAp synthesis; freeze drying for scaffold preparation	Keratin and collagen	hADSCs	ND	hADSCs were successfully kept alive on the scaffolds in vitro and subsequently, self-differentiated into osteogenic lineage without any induction agents was seen	66
ES	HAp	Bone	Wet chemical precipitation method for HAp synthesis; blending and film forming for HAp/PCL composite scaffold preparation	PCL	Human osteosarco ma cells	ND	ES-derived HAp improved PCL scaffold swelling and degradation ratio	95
ES	HAp	Bone	Wet chemical precipitation method for HAp synthesis; blending and film forming for chitosan/HAp composite scaffolds	Chitosan	Human osteosarco ma cell line	ND	HAp improved chitosan films roughness and cell viability	96
ES	ES microparticles	Bone	Blending of materials	GelMA	MC3T3-E1 pre-osteoblast cells	Wistar rat	ES particles significantly improved the mechanical properties of GelMA hydrogels and the differentiation and mineralization of pre-osteoblasts. In vivo subcutaneous implantation of ESP hydrogels exhibited suitable biocompatibility	65
ES	HAp	Bone	Sonochemical method for HAp synthesis; blending for HAp/nano-cellulose composite scaffolds	Cellulose nanocrystals; semicrystal line cellulose nanofibrils	Human osteoblast cells	ND	HAp improved the mechanical properties of composite scaffolds and promoted osteoblast cell viability	86
ES	βTCP	Bone	Ball milling process followed by a wet chemical precipitation method for βTCP synthesis; sponge replication method for scaffold preparation	PVA and PU	hADSCs	ND	Scaffolds showed good biocompatibility towards hADSCs	100
ES	HAp	Bone	Wet chemical precipitation method for HAp synthesis; 3D printing for scaffold preparation	PCL	Human osteoblast cells	ND	HAp improved cell adherence and proliferation	8
ES	βTCP	Bone	Ceramic scaffolds using sintering method	HAp	ND	ND	Increasing HAp content increased ceramic hardness	102
ES	βTCP	Bone	Composite nanofibrous scaffolds using electrospinning technique	Carbon dots, PCL, and PVA	Human buccal fat pad-derived stem cells	ND	βTCP significantly improved cell proliferation and osteogenic differentiation	105
ESM	ESM	Skin	Pulsed laser deposition technique over ESM films for scaffold preparation	Copper, bioactive glasses	HUVECs	Mouse	Scaffolds enhanced angiogenesis and wound healing	192
ESM	ESM	Skin	Sulfitolysis method for producing SEP; blending and electron beam irradiation for hydrogel production	PVA	ND	ND	The applied strategy displayed a suitable method for hydrogel production. The scaffolds exhibited excellent absorption capacity	193
ESM	ESM	Nerve	Vacuum infiltration technique to create a double network fibrous scaffold	PCLF	PC12	ND	The ESM/PCLF double network scaffolds showed significant improvement in cell proliferation	129
ESM	ESM	Skin	Acid and alkali treatments for ESM preparation; nanocomposite film for scaffold fabrication	Silver nanoparticl es (Ag-NPs)	Fibroblasts	Mouse	The fabricated scaffolds exhibited good biocompatibility, antibacterial activity, anti-inflammatory properties, and improved wound healing	128
ESM	ESM	Skin	ESM/chitosan blend film	Chitosan	ND	ND	The incorporation of ESM within the scaffold improved wettability, fluid uptake, and antibacterial activity	114
ESM	Processe d ESM Powder (PEP)	Skin	Washed, milled, sieved, and γ sterilized to produce PEP	ND	MMP-2 MMP-9	Mouse	PEP significantly improved wound healing	126
ESM	SEP	Cartilage	Acid treatment for SEP preparation; blending and hydrogel preparation using sodium triphosphate pentabasic	Chitosan, silk fibroin	Normal human articular chondrocyt e cells	ND	These hydrogels showed proper mechanical properties and biocompatibility	124
ESM	ESM	Skin	Acid treatment for SEP extraction Electrospinning for scaffold fabrication	Chitosan and PCL	Human dermal fibroblast cells	Mouse	The scaffolds enhanced cell adhesion and wound healing efficacy	130
ESM	SEP	Skin	Nano fibrous scaffolds using electrospinning technique	PCL, silk fibroin, and aloe vera	Human basal cells	ND	The addition of SEP improved the scaffolds’ wettability and cell viability	136
ESM	ESM	Skin	Dopamine functionalized ESM was coated by hyaluronic acid and KR-12 peptide	Dopamine, hyaluronic acid, and KR-12	HUVECs and human keratinocyt e cells	Rat	The modified ESM enhanced the proliferation of cells in vitro and accelerated re-epithelialization, granulation tissue formation, and wound healing in vivo	194
ESM	SEP	ND	Enzymochemical method for SEP preparation; layer-by-layer film forming for scaffold production	Fe(III)-tannic acid	HeLa cells and primary hippocampal neuron cells	ND	Scaffolds showed great cytocompatibility in vitro	138
ESM	ESM	Bone	HAp was coated on ESM by a biomineralization method	HAp	MC3T3-E1 cells	ND	The prepared scaffolds were suitable for bone TE	132
ESM	PEP	ND	Blending and freeze-drying to fabricate sponge scaffolds	Collagen	Human dermal fibroblasts, myofibroblast, and bovine muscle cells	ND	PEP improved the scaffolds’ mechanical properties and cell adhesion	125
ESM	ESM	Vessel	ESM was coated by poly dopamine and heparin and then PU was electrospunned on its surface	PU, dopamine, heparin	HUVECs	ND	Scaffolds containing ESM exhibited higher biocompatibility	131
ESM	ESM	Vessel	Blending	Expanded polytetraflu oroethylene (ePTFE), heparin, and ethanol	HUVECs	ND	The addition of ESM enhanced the biocompatibility of ePTFE	122
ESM	SEP	Cartilage	Blending	Agarose	Rabbit Cartilage Cells	ND	SEP improved cell proliferation and increased chondrogenic gene expression	143
EW	EW	Soft tissues	Sponge-like scaffolds were prepared via carbodiimide-mediated-mediated crosslinking	1-ethyl-3-(3-dimethylam inopropyl) carbodiimi de (EDC) and N-hydroxysuc cinimide (NHS)	Human dermal fibroblasts	Nude mouse	EW improved angiogenesis and tissue regeneration in vivo	147
EW	Lysozyme	Bone	Coating	PEG and PCL	human osteoblast cells and MSCs	ND	The coating significantly enhanced the composite surface antibacterial activity	173
EW	EW	ND	Blending and film forming	Silk fibroin	HUVECs	ND	The addition of EW improved cell viability	149
EW	EW	Skin	EW adhesive glue was prepared and coated on the PCL nanofibers	PCL	ND	Rat	PCL-coated EW glue showed promising wound healing in vivo	150
EW	Lysozy me	Bone	Lysozyme and Glycol chitosan were methacrylate and crosslinked using riboflavin and visible light	Glycol chitosan	MSCs	Rat	The presence of lysozyme enhanced the scaffold performance in vitro and in vivo	174
EW	Ovotran sferrin	Bone	ND	ND	The murine osteoblastic cell line MC3T3-E1	ND	Ovotransferrin improved cell proliferation, differentiation, and mineralization	195
EW	Albumen	ND	Nanofibrous mat fabricated by electrospinning	PEO	ND	ND	Egg albumen has been successfully formulated with PEO to fabricate fine nanofibers	146
EW	Ovalbu min	Bone	EW ovalbumin was polymerized using diethylenetriamine and injected into a printed negative mold to fabricate 3D scaffolds	HAp	ND	ND	3D EW ovalbumin/HAp composite scaffolds were successfully produced and exhibited promising mechanical properties	176
EW/ES	EW and ES	Bone	GelMA, EW, and ES powder were blended and photocrosslinked using Irgacure 2959 and UV light	GelMA	hDPSCs	ND	The incorporation of EW and ES into GMA hydrogels improved their physical and biological properties	175
EW	EW	ND	EW was blended with gellan gum and then thermally crosslinked to form hydrogels	Gellan gum	ND	ND	The addition of gellan gum reduced the degradation of EW gels	152
EY	Phosvitin	ND	Phosvitin and chitosan were sequentially coated on cellulose acetate nanofibrous mat	Chitosan and cellulose acetate	ND	ND	The fabricated nanocomposite scaffolds possess suitable antibacterial activity	191
EY	EY oil	Skin	Heat EY to 200 °C to gain EY oil and then added the EY oil to chitosan	Chitosan	ND	Rat	The scaffolds showed proper wound healing properties	197
EY	Phosvitin	Skin	Phosvitin and tanic acid were sequentially coated on cellulose acetate nanofibrous mat	Tannic acid and cellulose acetate	ND	ND	Tannic acid/phosvitin-coated cellulose acetate coated nanofibrous mats showed good antioxidant activity	187
EY	EY, plasma and granules	Skin	Protein gels obtained via transglutaminase crosslinking	Transgluta minase	Murine fibroblast	ND	The hydrogels showed good biocompatibility	185
EY	Phosvitin	Bone	Phosvitin and chitosan were sequentially coated on cellulose acetate nanofibrous mat	Chitosan	mouse MC3T3-E1 pre-osteoblast cell line	ND	Phosvitin/chitosan-coated scaffolds displayed enhanced biocompatibility, cell proliferation, adhesion, and spreading as well as improved mineralization	186

Although egg-derived biomaterials are still under-exploited, the latest advancements in the field of TE and other biomedical fiels within the past few years have opened new opportunities for their widespread use. Particularly, bioinspired egg-derived scaffolds may have great applications in the development of organ-on-a-chip systems to further integrate 3D cell culture, microfluidic technology, and TE for disease modelling, drug discovery, and personalized medicine. Lastly, combining these naturally derived biomaterials with state-of-the-art technologies such as 3D bioprinting could lead to the fabrication of sophisticated tissue-engineered constructs, ultimately leading to more functional and viable tissues and organs.