Eggshell‐Based Unconventional Biomaterials for Medical Applications

Abstract

Eggshells are one of the most abundant byproducts of food processing waste. Each discarded eggshell represents a missed opportunity to convert a no‐cost waste material into a valuable product. Beyond their economic practicality and widespread availability, eggshells possess unique biological and chemical properties that support cell differentiation. Their composition includes biologically active compounds, essential trace elements, and collagenous and noncollagenous elements, mimicking the components of bones, teeth, and skin. Additionally, eggshells serve as a suitable precursor for synthesizing hydroxyapatite, calcium carbonate (CaCO₃), and β‐tricalcium phosphate. Eggshells can be utilized on their own or as derived materials to produce regenerative biocomposite scaffolds for tissue engineering. These scaffolds often exhibit high porosity, excellent biocompatibility, degradability, and mechanical properties. Eggshells and their derivatives have also been employed as carriers for targeted drug delivery systems and in electrochemical biosensors. Eggshells serve as a versatile biomaterial, adept at not only addressing practical gaps but also bridging the divide between sophistication and ease of production. In this review, the chemical composition of eggshells and their numerous applications in hard and soft tissue regeneration, biomolecule delivery, and biosensor development are discussed highlighting their innovative and unconventional use as a natural biomaterial providing solutions for unmet clinical needs.

Introduction

The widespread health challenge posed by tissue defects, arising from various causes such as trauma, congenital anomalies, tissue diseases, or functional atrophy, is a significant global concern.^[1,2] After blood transfusion, bone grafting is the second most common tissue transplantation procedure worldwide.^[3] With over two million annual surgeries and repairs exceeding half a million patients in the United States and Europe, the associated costs exceed $3 billion.^[3–6] Autograft bone has been the preferred choice for various procedures due to its unique qualities: osteogenesis, which involves the formation of new bone by the cells; osteoinduction, the process of recruiting and stimulating undifferentiated cells into bone-forming cells; and osteoconduction, which allows new bone formation on a surface.^[7–9] However, limitations such as procurement morbidity and limited availability have hindered the widespread use of autografts.^[10] In 1996, autografts made up ≈60% of bone grafts in the United States, with allografts and other materials accounting for 34% and 7%, respectively.^[11] Allografts, the primary alternative to autografts, come with their own challenges, notably the risk of transferring contaminants, toxins, or infections from the donor.^[10] While processing allograft tissue does help mitigate this risk, it unfortunately often leads to significant degradation of the biological and mechanical properties originally present in the donated tissue.^[10] Furthermore, there is no guarantee of an ample supply; processed and banked donor bone may not be readily available at the time of surgery.^[12,13]

Nonhealing wounds are also among the multitude of challenges within the healthcare system, requiring an annual expenditure surpassing $25 billion and fueled by rising medical costs, an aging population, and a notable increase in global rates of diabetes and obesity.^[14] Intensive research is focused on developing effective treatments for both acute and chronic skin wounds. Wounds refer to the breakdown of tissue or cellular structure resulting from mechanical, physical, or metabolic causes, including those primarily linked to diabetes mellitus.^[15,16] Wound healing is a complex biological process that progresses through four interconnected and overlapping phases: hemostasis, inflammation, proliferation, and tissue remodeling.^[17] Wound healing occurs through three distinct mechanisms: primary intention, where epithelialization occurs; secondary intention, involving granulation formation, contraction, and epithelialization; and tertiary intention (delayed primary closure or secondary suture), where wounds are initially left open and later closed, typically within 4 or 5 days, through approximation or with the use of tissue grafts like skin grafts or flaps.^[18,19] In terms of wound coverage using skin grafts, autograft skin transplantation is the primary preference. Acknowledged widely as the gold standard, it provides both split-thickness and full-thickness skin grafts, recognized for their highly effective therapeutic outcomes.^[20] However, autografts do present certain limitations, including less-than-desirable functional and cosmetic outcomes resulting from hypertrophic scarring, decreased skin elasticity, and restrictions on joint mobility due to scar contracture.^[21]

Addressing unmet clinical demands requires the use of innovative biomaterials tailored to repair and regenerate tissues.^[22] Eggshells are rich in CaCO₃ and eggshell membrane in collagen-like proteins, providing promising avenues for regenerating hard and soft tissues. These natural materials could enhance patient outcomes by facilitating tissue repair and healing while potentially offering cost-effective and accessible medical treatments.^[23]

In the past 30 years, there has been a 150% surge in global egg production, leading to a significant increase in the disposal of eggshells.^[17] Asia, in particular, has seen a fourfold increase in egg production, greatly impacting global growth. China stands as the top producer with a staggering 604.7 billion eggs, surpassing the combined production of the other top five egg-producing countries. India, its closest competitor, produces 114.4 billion eggs, which is 5.3 times less than China.^[24] Eggs are utilized both domestically and industrially for food production and processing, serving as a crucial source of nutrition worldwide. The rise in egg production leads to a correspondingly greater generation of eggshell waste, typically thrown away and ending up in landfills that are already at maximum capacity. The abundance of eggshells provides a valuable resource for supplying free eggshells to support research endeavors. Various studies have shown the versatile use of eggshells as effective components for medical applications, due to their abundance, lightweight nature, and suitability for implantable composite applications.^[25] Incorporating chicken eggshell microparticles (ESP) into tissue-engineered scaffolds offers an exciting opportunity to enhance osteogenic differentiation and support bone regeneration, especially in hard tissues.^[22,26,27]

For bone regeneration, conventional scaffolds frequently incorporate a diverse range of bioactive inorganic compounds, such as hydroxyapatite (HA), bioactive glass, calcium silicate (CSi), β-tricalcium phosphate (β-TCP), tetracalcium phosphate, monocalcium phosphate, dicalcium phosphate, and amorphous calcium phosphate.^[28,29] Beyond bioactivity and biocompatibility, these materials must demonstrate robust mechanical strength and a porous architecture to ensure successful integration within the human body.^[30,31] Additionally, achieving optimal osteoconductive, osteoinductive, and osteointegrative properties is crucial for their performance and effectiveness.^[31] Eggshells confer significant advantages over bioactive ceramics across a variety of tissue engineering applications. For example, the process to obtain both synthetic and naturally derived HA is known for its resource-intensive nature, necessitating substantial investments of time and labor.^[32–35] Chemically synthesized HA exhibits reduced biological activity, which may slow the processes of bone regeneration and bone resorption, possibly due to lack of essential ions.^[36–39] Additionally, HA is more expensive when compared to eggshells, which can be sourced for free from households, restaurants, and farms. The distinct chemical profile and biophysical qualities of eggshells establish them as a highly suitable biomaterial for applications in bone tissue engineering. The primary chemical components of eggshells encompass CaCO₃, HA (Ca₁₀(PO₄)₃(OH)), sodium oxide (Na₂O), magnesium oxide (MgO), iron (III) oxide (Fe₂O₃), strontium oxide (SrO), ovocalyxin-32 (OCX-32), and phosphate anions.^[22,40] CaCO₃ facilitates mineralization and bone regeneration as an osteoinductive agent, while its presence in eggshells forms intricate bioceramic structures and their pores enable the selective diffusion of ions and metabolic gases, vital for osteoregeneration and enhancing the skeletal strength.^[22,41] Maximizing the potential of eggshells is essential for successful clinical translation and commercial adoption in regenerative medicine, as it fulfills the criteria for biocompatibility, bioactivity, cost-efficiency, and practicality.^[42–44]

Leveraging on the potential of integrating ESP within tissue-engineered scaffolds, Gezek et al. (2024) investigated the use of ESP as fillers in 3D-printed thermoplastic scaffolds for potential applications in bone tissue engineering.^[45] Using extrusion-based 3D printing, the researchers aimed to maintain the physicochemical properties of the scaffolds while introducing ESP at various concentrations (0%, 5%, 15%, and 50% w/w). The integration of ESP led to higher porosity in the scaffolds and had a direct impact on their mechanical properties, a finding that aligns with Huang et al. (2019), who also demonstrated that the addition of eggshells can affect mechanical performance.^[46] ESP were shown to be evenly distributed within the scaffolds and retained their morphology, suggesting that the pellet preparation and 3D printing processes did not damage the ESP. The addition of ESP enhanced thermal stability and altered the degradation rate of the scaffolds, with higher concentrations of ESP increasing the degradation rate of the composite scaffolds. Additionally, ESP-reinforced scaffolds supported cell adhesion and proliferation, as well as osteoinductive properties, which were observed in early differentiation markers such as RUNX2 and alkaline phosphatase (ALP). RUNX2 is a transcription factor critical for osteoblast differentiation and bone formation, while ALP is an enzyme involved in mineralization, making them key indicators of the scaffolds’ ability to support bone tissue engineering. The study validated the idea that ESP can provide a cost-effective and promising method for reinforcing thermoplastic scaffolds in bone tissue engineering, paving the way for future progress in regenerative medicine.

Eggshell membranes exhibit significant potential in soft tissue engineering due to their abundant composition of collagen, growth factors, and glycosaminoglycans (GAGs), which promote cellular growth and tissue repair, making it ideal for regenerative applications.^[47,48] Zhang et al. (2024) developed eggshell-based copper iron oxide (CuFe₂O₄) nanocomposites, which were incorporated into a multifunctional hydrogel made from a material consisting of oxidized starch hydrogel. This hydrogel demonstrated strong antimicrobial activity and significantly improved diabetic wound healing by enhancing collagen deposition, stimulating angiogenesis, and reducing inflammation.^[49] Similarly, in a different study conducted by Zhang et al. (2020), copper sulfide (CuS) nanoparticles are evenly coated on the surface of a porous eggshell to form CaCO₃/CuS nanocomposites that demonstrated excellent antibacterial properties when activated by near-infrared light.^[50] In combination with other materials, eggshell membranes promote angiogenesis while also exhibiting antimicrobial, anti-inflammatory, biocompatible, and biodegradable properties.^[51] In addition to their regenerative applications, eggshell-based biomaterials have been explored as efficient carriers for delivering therapeutic agents due to their biocompatibility, porous structure, and ability to encapsulate various compounds. For example, Tao et al. (2022) demonstrated the use of chicken eggshell powder as a carrier for the delivery of low-dose silver nanoparticles (AgNPs), significantly enhancing their antimicrobial activity against foodborne pathogens and biofilms.^[52] The versatility of eggshell membranes extends to biosensing and diagnostic tools, as eggshell membranes provide a porous and stable platform that facilitates the immobilization of biomolecules. A study by Kamel et al. (2024) developed a copper-eggshell nanocomposite with chitosan and carbon nanoparticles as electrodes in an electrochemical biosensor for l-tyrosine (l-Tyr) analysis for the detection and monitoring of liver cirrhosis and hepatocellular carcinoma, as elevated levels of l-Tyr amino acid are altered due to the impaired function of the liver. The research team utilized the porous hierarchical structure of eggshell nanoparticles to efficiently entrap metal nanoparticles and prevent their aggregation while providing abundant 3D active sites for high loading capacity. The use of eggshell nanoparticles enhances the electrochemical response due to their porous surface and improves the stability of the synthesized copper nanoparticles, making the biosensor ideal for determining l-Tyr concentrations in the serum of both healthy and diseased individuals.^[53]

Recent advances in eggshell research reveal their remarkable potential, especially in the medical field, where their unique biological and physicochemical properties position them as promising candidates for tissue repair and regeneration.^[54] With their rich composition of calcium carbonate and biologically active compounds, eggshells have demonstrated osteoinductive properties, making them particularly suited for bone regeneration applications. Moreover, eggshell-derived materials offer a sustainable, cost-effective alternative to traditional biomaterials, which are often expensive and resource intensive to produce. This article provides an in-depth review of the latest research on eggshell-based biomaterials, emphasizing their application in tissue regeneration, wound healing, biomolecule delivery, and biosensor systems, as displayed in Figure 1. By evaluating the relevant research on eggshells, this review supports their potential to address critical gaps in current approaches used for clinical applications, such as the risk of immunogenic reaction and bacterial infection with allografts, limited availability of autographs, and the need for more effective and affordable materials for treating nonhealing wounds and bone defects.^[55,56]

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Structure and Function of Eggshells

The primary function of the eggshell is to provide a protective barrier for the embryo against external threats, while also allowing for easy internal breakage to facilitate hatching. Additionally, the structural design of the eggshell allows for the exchange of water and gases between the developing embryo and the environment during development outside the uterus. The eggshell also serves as a crucial reservoir of calcium for the embryo's growth.^[57–59] This is possible due to the eggshell's intricate design as a calcitic bioceramic, which exhibits complexity and precision in its daily reproducible formation within the uterus, specifically the shell gland, of the distal oviduct.^[60,61] This complex structure is rapidly created within the oviduct in less than 24 h, occurring at physiological temperatures ranging between 37 and 38 °C.^[62–65] The outer layer of the eggshell features a strong structure of large crystals, absorbing external impacts with the assistance of intercrystalline organic layers that impede crack propagation.^[66,67] In contrast, the inner region is composed of spherulitic-textured microcrystals of calcite, facilitating crack propagation during pipping, the process through which the embryo breaks out, while also aiding in calcium mobilization for embryo nourishment through the dissolution of reactive microcrystals.^[68]

The avian eggshell represents a composite structure, featuring both an organic matrix and mineral components, primarily the trigonal phase of CaCO₃, commonly known as calcite. The mineralized shell is composed of ≈96% calcite, with the remaining 4% consisting of the organic matrix, magnesium, phosphorus, and various trace elements.^[66,69] The ultrastructure of the calcified eggshell reveals a complex architecture with variations across specific zones, encompassing the mammillary zone, the palisade layer, and the cuticle.^[70–72] These components collectively form the shell membranes, contributing to the intricate structure of the eggshell, as shown in Figure 2.

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The mammillary layer is integral to successful embryogenesis, serving as a significant calcium reservoir.^[73] Comprising an inner region with microcrystals of calcite arranged in a spherulitic texture, it facilitates mineral dissolution and calcium mobilization for embryo nourishment.^[71,74] In eggshell formation, the mammillary knob layer, as the innermost calcified portion, initiates calcite crystal formation through cones or knobs containing cores of concentrated organic material, including “mammillan”, a keratan sulfate proteoglycan.^[68] These structures act as epitactic centers, eventually forming the basal cap portion of the mammillary knob layer.^[71,74] In avian eggshells, the mammillary knob and associated palisade column are fundamental units, emphasizing the organizational role of primary calcified structures in determining shell quality.^[75] These sites serve as the initial points for shell calcification, and their spacing could potentially influence the morphology of shell crystals.^[76] A study provided evidence for this notion through microscopic examinations of the outer membrane layer obtained from both weak and strong eggshells, revealing that weak and thin eggshells exhibited abnormal organic mammillary cores, supporting the idea that the arrangement of these sites plays a role in shell structure.^[77]

Numerous experimental observations provide support for the significance of eggshell matrix proteins in determining the eggshell formation and function, thereby influencing its mechanical properties.^[62] Certain proteins within the eggshell matrix are distinct to the calcification process and are specifically secreted in the relevant regions of the oviduct.^[78] One such example is Ovocleidin-17 (OC-17), the pioneer eggshell protein, exhibiting both glycosylated and nonglycosylated forms.^[62] Ovocleidin-116 (OC-116), the first cloned eggshell matrix protein, serves as the core for the dermatan sulfate proteoglycan ovoglycan.^[79] OCX-36, a 36 kDa protein, is uniquely found in the shell gland during eggshell calcification, with heightened expression during this process.^[80] Notably, OCX-36 predominantly localizes in the inner part of the shell and shares homology with innate immune response proteins.^[80] Lastly, OCX-21 is a recently identified eggshell-specific protein.^[81]

The palisade layer is a fundamental component, crucial for the structural integrity and strength of the eggshell.^[82] Composed primarily of CaCO₃ calcite crystals, which make up two-thirds of the total thickness of the eggshells, this layer forms a substantial part of the calcified shell.^[72,83,84] Its ability to resist external impacts is attributed to a robust outer structure characterized by large crystals, complemented by thin intercrystalline organic layers that impede crack propagation, enhancing the overall durability of the ESP.^[85] Essential components such as proteins (70%) and polysaccharides, including GAGs, contribute to the composition of the palisade layer.^[86] The intricate interdigitation of calcite columnar structures and the stability of the underlying mammillary knob layer play pivotal roles in determining the intrinsic strength of this layer. Within this context, OC-116 assumes a central role as the core of a significant 200 kDa shell dermatan sulfate proteoglycan, named ovoglycan, seamlessly incorporated throughout the palisade region.^[79,87] The ESP cuticle, serving as the outermost layer of the mineralized ESP, directly engages with the surrounding environment.^[88] Comprising HA crystals, polysaccharides, lipids, and glycoproteins, this slender layer displays variable thickness, ranging from 0.5 to 12.8 μm.^[87,89,90] Applied to the eggshell surface, this organic coating plays a pivotal role in regulating water exchanges and establishing a protective barrier against microorganisms by sealing eggshell pores.^[90] Functioning as a safeguard, the eggshell cuticle markedly reinforces the antimicrobial defense, serving as a physical impediment to microbial contamination.^[89] Additionally, OCX-32, a 32 kDa uterine-specific protein, is concentrated in the outer calcified region and cuticle.^[91,92]

Emerging as a notable phosphoprotein within the intricate matrix of eggshells, osteopontin (OPN) holds significance as a prevalent mineralized tissue protein found in bone and teeth.^[93,94] OPN is situated within the core of non-mineralized shell membrane fibers, at the base of mammillae, and in the outermost portion of the palisade structure.^[95] Notably relevant to bone metabolism, OPN assumes a crucial role in the calcification process by enhancing the adhesion of osteoblasts to the matrix and forming bonds with HA.^[96] McKee et al. (2011) showed that OPN plays a pivotal role in the intricate process of bone regeneration subsequent to surgical procedures.^[97] Their research suggests that following macrophage-mediated clearance of bone debris via phagocytosis, OPN augments this process through opsonization. Consequently, a cement line forms at the wound margins, facilitating the seamless integration of newly formed bone with the surrounding damaged bone tissue.

Eggshell-Based Biomaterials for Hard-Tissue Regeneration

In humans, there are four distinct hard tissues: bone, cementum, dentin, and enamel.^[98] The term “hard tissue”, often used interchangeably with bone, describes mineralized structures such as bone or tooth.^[99] Its distinction from soft tissues in mammals lies in its hardness.^[100] Carbonate HA nanocrystals constitute ≈69% of the mineral content in natural bone, while the organic matrix comprises around 22%, predominantly consisting of proteins such as type I collagen (accounting for 90% of the organic matrix), along with non-collagenous proteins like proteoglycans, lipids, and osteogenic factors including bone morphogenetic proteins (BMPs) and vascular endothelial growth factors (VEGFs).^[101–105] The remaining percentage consists of water, which typically accounts for ≈5–10% of the total weight of bone tissue.^[106,107] Table 1 presents the various biomacromolecules found in human bone and their functions, along with a column showing the correlation between these biomolecules and eggshells.

Table 1 The fundamental and intricate functions of organic components in the complex process of bone mineralization.

Biomacromolecule	Functions	Eggshell correlation	References
Type I collagen	Plays a vital role in enhancing toughness, contributing to its capacity to absorb energy, while bone stiffness is predominantly influenced by its mineral content.	Collagen-like proteins, resembling types I and V collagens, have been identified in the thick outer and thin inner layers of hen eggshell membranes.	[207,208]
Bone sialoprotein (BSP)	A cell-binding protein containing arginine–glycine–aspartic acid (Arg–Gly–Asp, RGD), which emerges as a late characteristic in the development of the osteoblast phenotype.	BSP was found in the eggshell matrix.	[209–211]
Osteonectin (ON)	It possesses a robust affinity for both collagen and mineral, leading to the hypothesis that it serves as a bone-specific nucleator of mineralization.	SPARC (secreted protein, acidic, cysteine-rich), known as osteonectin, is a 34 kDa glycoprotein found in moderate abundance in mammillary cones. Initially identified in bone extracellular matrix, osteonectin binds hydroxyapatite calcium crystals and collagen type I in vitro, suggesting its role in initiating mineral formation and promoting crystal attachment to a collagenous matrix. Its presence in eggshell mammillary cones indicates a possible link to mineralization and crystal attachment in the eggshell structure.	[212,213]
Osteopontin (OPN)	Enhances the adhesion of osteoblasts to the matrix and facilitates the formation of bonds with hydroxyapatite.	Osteopontin has been identified in the avian eggshell and oviduct.	[95,96]
Chondroitin sulfate (ChS) and keratan sulfate	Contribute to the attachment and adhesion between osteoblastic cells and the extracellular matrix.	Comparative analyses of mineralization models, such as bones and teeth, highlight the involvement of glycosaminoglycans (GAGs) and proteoglycans (PGs) in the mineralization process. The mammillary layer, with mammillae enriched in mammillan, a highly sulfated keratan sulfate proteoglycan, points to keratan sulfate's crucial role in reinforcing structural integrity and facilitating mineralization within the eggshell matrix.	[214,215]
Thrombospondin and fibronectin	Glycoproteins within the matrix that bind to integrins and various extracellular matrix (ECM) components, including collagen, fibrin, and others.	The eggshell membrane exhibits a structural resemblance to the ECM of bone and is predominantly composed of ≈90% protein and includes complex carbohydrates such as glycosaminoglycans and N-glycans. It comprises cysteine-rich proteins (CREMPs), type X collagen, glycoproteins, and calcium-regulating proteins, along with ADAMs enzymes featuring a thrombospondin type 1 motif.	[47,216]

Amid the advancements in biomedical technology over the past few decades, the emergence of advanced biomaterials has been notable. Specifically, various ceramic materials have been developed for bone repair and reconstruction. These materials, known as “bioceramics,” have garnered considerable attention.^[108] Bioceramics, characterized by their remarkable biological and osteoinductive properties, stand out for their ability to serve as scaffolds conducive to bone tissue regeneration, self-adhesion, and differentiation.^[109] Moreover, their exceptional chemical and mechanical attributes, including superior biocompatibility, wear resistance, and enhanced osteoconductivity, have positioned bioceramics as viable alternatives for bone regeneration.^[110,111] Bioceramics have a wide range of uses, including substituting bone grafts, providing sustained-release drug delivery, and assisting in protein purification.^[33] Table 2 displays widely used bioceramics and their biomedical applications. Eggshells, as renewable bioceramics, serve as a biocompatible grafting material.^[112] They can be utilized alone or in conjunction with other substances listed later to amplify their efficacy.^[113]

Table 2 Commonly utilized bioceramics and their biomedical applications.

Bioceramics	Type	Applications	References
Eggshells	Natural	Bone graft substitutes, dental restorations, wound dressings, drug delivery, and bioactive coatings.	[217]
Zirconia (ZrO2)	Bioinert nonabsorbable	Dental implants, prostheses, orthodontic brackets, endodontic posts, implant abutments, implant-supported overdentures, and potentially in temporomandibular joint (TMJ) implants.	[218]
Alumina (Al2O3)	Bioinert nonabsorbable	Orthopedic implants, dental implants, surgical tools, prosthetic devices, and bioactive coatings.	[219]
Hydroxyapatite (HA) and hydroxycarbonate apatite (HCA)	Bioactive	Bone substitutes, dental implants, coatings for orthopedic implants, drug delivery systems, tissue engineering scaffolds, dental fillers and restorative materials, and biomedical coatings.	[220]
Calcium phosphate	Biodegradable resorbable	Biomedical coatings, bone void fillers, periodontal treatments, guided bone regeneration, spinal fusion, augmentation of alveolar ridge, and repair of craniofacial defects.	[221]
Calcium sulfate	Biodegradable resorbable	Bone grafting procedures, including filling bone defects.	[222]
Calcium silicate	Biodegradable resorbable	Bone tissue engineering, endodontics, drug delivery, and wound healing.	[223]
Bioglass	Scaffolds of biologically active molecules	Bone regeneration, dental implants, tissue engineering scaffolds, controlled drug delivery systems, and wound healing applications.	[224]

The upcoming wave of biomaterial advancements is anticipated to seamlessly facilitate the effective mobilization of mesenchymal stem cells (MSCs).^[114,115] These cells will populate the scaffold, undergoing differentiation into bone tissue that meets the specific criteria of shape, structure, and resilience desired for optimal healing. Bones exhibit four major cell types: osteoblasts, bone lining cells, osteocytes, and osteoclasts. Osteoblasts are derived from MSCs and osteoclasts are derived from hematopoietic stem cells.^[116] MSCs are the cells of choice for in vitro investigation of biomaterial osteoconductivity, osteoinductivity, or osteogenicity, since they are the precursors to the mineral-secreting osteoblasts.^[117]

For scaffolds to be effective, they must satisfy stringent criteria across biological, physical, and mechanical characteristics.^[88] Biologically, this encompasses biocompatibility, sterility, and absence of cytotoxicity, along with biodegradability.^[118,119] This involves supporting cell adhesion, proliferation, osteoinduction, osteoconduction, and osseointegration. Physically, scaffolds should offer optimal microstructure, particularly with respect to porosity. Mechanically, they must provide adequate tensile strength and stiffness to ensure structural integrity.

Eggshells present as a biomaterial with the potential to fulfill the major requirements for bone scaffolds. For instance, eggshells offer excellent biocompatibility with mild inflammatory response.^[120,121] The organic components, especially proteins within the eggshell, play a significant role in osteointegration, cell migration, and cell proliferation, thereby facilitating important steps in bone regeneration.^[96] Furthermore, eggshells are porous and can provide a suitable environment for cell proliferation and nutrient exchange.^[22] Figure 3 illustrates the fabrication of different eggshell-derived scaffolds, including 3D-printed scaffolds and hydrogels, for applications in bone tissue engineering.

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In Vitro Applications of Eggshell-Based Biomaterials

The versatility of eggshells as a biomaterial can be illustrated by various avenues for usage either in their complete form or as a source for deriving valuable compounds such as HA and CaCO₃. A common approach to utilizing whole eggshells is by first sterilizing them, then crushing the ESP into a powder and sieving them.^[122] The pioneering study in size-controlled ESP was conducted by Wu et al. (2019), where the average size ranged from 47 to 53 μm, proven to promote osteoinductivity.^[22] The ESPs are typically integrated into scaffolds, with the main objective being to create an optimal design capable of mimicking the components of the ECM of the bone.^[123] PCL is a common polymer used to fabricate tissue engineering scaffolds. It showcases a gradual degradation profile suitable for bone remodeling applications, alongside exceptional attributes such as biodegradability, biocompatibility, and mechanical properties, despite its intrinsic lack of osteogenic potential.^[124] Gezek et al. (2024) conducted a study to develop ESP-reinforced PCL thermoplastic scaffolds.^[45] An Alamar blue assay was utilized to analyze the metabolic activity of MC3T3-E1 preosteoblast cells. Alamar blue reduction provides a convenient index of cell proliferation and has been used in assays measuring in situ cell response to irradiation. Additionally, the study also utilized a membrane-bound glycoprotein, ALP, well known as an early osteogenic marker of bone formation and bone calcification. ALP activity assays can determine the differentiation of osteoblastic cells.^[125,126] Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was also used to quantify gene expression and require standardization with reference genes. Osteoinductivity can be investigated by performing a RT-qPCR on messenger RNA (mRNA) that was isolated from the 3D encapsulated preosteoblasts after 14 days of culture. The incorporation of ESP into PCL influenced the rate of mass loss overtime, with higher concentrations of ESP correlating to increased degradation ratios. Utilizing MC3T3-E1 preosteoblast cells, the study investigated the cytocompatibility and osteoinductive properties of ESP-reinforced PCL scaffolds. By day 14, the cells exhibited adherence, forming a confluent layer with elongated and spread morphology on the scaffold surface, as shown in Figure 4. These findings show the suitability of ESP-reinforced composite scaffolds for cell adhesion and highlight their potential for supporting preosteoblast cell growth. Furthermore, a significant increase in the expression level of RUNX2 was observed in preosteoblasts seeded on ESP-reinforced composite scaffolds, indicating early osteogenic differentiation. In another study, Biscaia et al. (2015) explored the integration of eggshell-derived materials into composite PCL scaffolds for bone tissue engineering.^[124] Their investigation, utilizing scanning electron microscopy (SEM), demonstrates the successful dispersion of ESP within the scaffold matrix, ensuring uniform distribution and robust interfacial bonding. Moreover, mechanical testing, encompassing tensile strength and compressive modulus assessments, highlights notable improvements in scaffold properties attributed to ESP incorporation, crucial for stability and tissue integration.

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A different study conducted by Huang et al. (2019) provides insightful depth into the development of biomimetic hybrid hydrogels incorporating egg white and eggshell components for bone regeneration. Notably, the integration of eggshells within the hydrogels not only enhanced mechanical properties but also maintained excellent cytocompatibility, suggesting a synergistic effect. Moreover, the biomimetic hybrid hydrogels exhibit a remarkable ability to influence macrophage behavior and regulate osteogenic differentiation in human dental pulp stem cells (hDPSCs), indicating their potential to actively modulate cellular responses crucial for bone regeneration.^[46] In vivo studies have the capacity to substantiate the claims that were reported by literature investigations.^[127,128]

In Vivo Applications of Eggshell-Based Biomaterials

Wu et al. (2021) investigated the use of ESP-reinforced gelatin methacryloyl (GelMA) hydrogel scaffolds for regenerating critical-sized cranial defects.^[41] The scaffolds reinforced with ESP were studied for the extent of cellular proliferation, early differentiation, and mineral deposition behavior in vitro. Moving on to in vivo studies, the ESP-reinforced scaffolds, blank scaffolds, and pristine GelMA scaffolds were evaluated. The findings showed that the ESP-reinforced scaffolds facilitated optimal bone formation and exhibited superior healing of critical-sized cranial defects 12 weeks post implantation. This outcome can be attributed to the mechanical reinforcement of the GelMA matrix through the incorporation of ESPs, as well as the mineral content within the scaffolds, which supports osteogenic differentiation.^[129,130] Additionally, the inclusion of ESP in the scaffolds promoted vascularization and bone remodeling, as evidenced by VEGF and Tartrate-resistant acid phosphatase (TRAP) staining, which are crucial for successful bone healing.

Subcutaneous implantation in rats is a common model for assessing the biocompatibility and biodegradation of materials intended for bone regeneration applications. In a study conducted by Wu et al. (2019), ESP-reinforced hydrogels were developed and comprehensively evaluated for their effectiveness in supporting osteogenic cell cultures and in vivo biocompatibility.^[22] The fabricated hydrogels exhibited highly tunable material properties, demonstrating osteoinductive behavior in vitro with significant improvements in differentiation and mineralization of preosteoblasts. The incorporation of ESPs into GelMA hydrogels resulted in enhanced mechanical properties, as indicated by a positive correlation between increasing ESP concentrations and compressive strength. In addition to favorable in vitro results, in vivo subcutaneous implantation experiments in a rat model revealed the biodegradation potential of ESP-reinforced hydrogels within 14 days, with no significant inflammatory response, as displayed in Figure 5. The implants exhibited excellent acceptance by the host, allowed cellular infiltration in three dimensions, and were highly vascularized.

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Eggshell-Derived Mineral Components

Eggshell-derived CaCO₃ nanoparticles are unique compared to their chemically synthesized counterparts due to their porous nature, which allows for efficient drug loading.^[131] Moreover, their inherent nontoxic and biocompatible characteristics make them well suited for a wide range of biomedical applications. A study by Huang et al. (2020) introduces an innovative approach to bone regeneration by incorporating eggshell-derived CaCO₃/MgO nanocomposites and chitosan (CS) into a biomimetic scaffold.^[132] The authors successfully constructed a porous, bioactive scaffold using natural products such as eggshell powder and carboxymethyl chitosan (CMC) through simple impregnation, calcination, and chemical crosslinking methods. This scaffold, known as CaCO₃/MgO/CMC/BMP2, showed significant improvement in new bone formation and mineralization, both at the cellular level and in animal models. Furthermore, the study revealed the osteogenic mechanism of the scaffold, demonstrating the release of Mg²⁺ and BMP2 played crucial roles in promoting osteogenic differentiation of the seeded cells through the ERK1/2 and Akt pathways. Overall, the newly developed composite scaffold proved to be a promising alternative in bone tissue engineering, providing valuable insights into advanced strategies for enhancing bone regeneration.

HA is frequently obtained from eggshells and in contrast to HA derived from commercially available synthetic reagents, eggshell-derived HA demonstrates superior bioactivity, exceptional osteoconductivity, osteoinductivity, and enhanced cell proliferation.^[133,134] These features are frequently attributed to the inclusion of trace elements (such as Mg, P, Si, Sr, Na) originating from the eggshell source.^[134] The bioactive ions and trace elements present in the eggshell-derived HA promote cell adhesion, proliferation, and differentiation, contributing to the formation of new bone tissue within the defect site.^[135] The incorporation of eggshell-derived nano-HA into polymer-based composite membranes or scaffolds also results in notable enhancements in thermal, mechanical, and biological characteristics when compared to membranes or scaffolds made solely from the polymer.^[136–138] Increased cell viability, proliferation, and osteogenic differentiation of MSCs cultured within an eggshell-derived HA-incorporated composite hydrogel, compared to a pure polymer matrix, demonstrate the scaffold's bioactivity and osteoinductive potential.^[135] Increased cell viability in eggshell-derived bone substitutes is also supported in a different study by Prathap et al. (2024).^[139] In another study conducted by Patel et al. (2020) the biocompatibility of eggshell-derived HA was assessed through the evaluation of cytotoxicity and osteogenic potential using hMSCs.^[140] Results indicated no significant cytotoxicity in the presence of HA, underlining its biocompatibility. Furthermore, the presence of HA powder led to enhanced osteogenesis of hMSCs, affirming its potential in osteogenic treatments.

In a study performed by Acharjee et al. (2023), they investigated the potential of titanium-doped waste eggshell-derived HA (Ti-WESHA) for bone regeneration through both in vitro and in vivo assessments.^[141] The methods involved synthesizing Ti-WESHA and characterizing its physicochemical properties, followed by in vitro evaluation of its biocompatibility and osteogenic potential using cell culture assays. The in vivo assessment utilized an animal model to study bone regeneration upon implantation of Ti-WESHA scaffolds. The results revealed favorable biocompatibility and osteogenic properties of Ti-WESHA, as evidenced by cell proliferation, differentiation, and ECM deposition in vitro. Moreover, the in vivo experiments demonstrated significant bone regeneration and integration with host tissues, highlighting the potential of Ti-WESHA as a promising biomaterial for bone tissue engineering applications. This comprehensive evaluation indicated the suitability of Ti-WESHA as a viable option for promoting bone regeneration and underscores its potential clinical relevance in the field of biomaterials science and engineering. A different study by Apalangya et al. (2019) explored the development of tissue scaffolds using eggshell-based nano-engineered HA (EnHA) and poly(lactic acid) (PLA) electrospun fibers.^[136] The methods involved the successful coelectrospinning of EnHA with PLA to form composite fibers (PLA/EnHA). Characterization of the composite fibers revealed improved thermal and mechanical properties compared to pristine PLA fibers, particularly with lower EnHA loading. Notably, the maximum tensile stress was achieved in composite fibers containing 5 wt% EnHA. In vitro cell culture experiments demonstrated successful adhesion and proliferation of osteoblast cells on the PLA/EnHA fibers. Immuno-histochemistry staining further indicated that cells could grow on the fibers to confluence and secrete components of the extracellular matrix. Overall, the study suggests that PLA/EnHA composite fibers hold promise as potential tissue scaffolds, offering enhanced mechanical properties and biocompatibility for various biomedical applications.

Another commonly derived mineral from eggshells is β-TCP. When employed as a bone graft material, the β-form of TCP, which maintains thermal stability below 1180 °C, is preferred over its α-form (which exhibits thermal stability in the range of ≈1180–1400 °C), given its favorable combination of mechanical strength and appropriate bioresorption rate.^[142] Specifically, β-TCP proves effective as a resorbable osteogenic filler and scaffold material for bone tissue regeneration.^[143] Roopavath et al. (2019) employed a mechanochemical synthesis approach to produce phase-stable and biocompatible β-TCP from avian eggshell.^[144] The results indicated successful transformation of eggshell-derived CaCO₃ into β-TCP, confirmed through X-ray diffraction analysis, which revealed the desired crystalline structure. SEM images demonstrated the formation of porous structures with particle sizes in the range suitable for tissue ingrowth applications. Biocompatibility assessments using osteoblast-like cells revealed favorable cell adhesion, proliferation, and viability on the synthesized β-TCP, indicative of its potential for promoting tissue ingrowth. Moreover, in vivo experiments in animal models showed enhanced bone regeneration and tissue integration at the implant site, further validating the suitability of mechanochemically synthesized β-TCP from avian eggshell for tissue ingrowth systems.

Eggshell-Based Biomaterials for Wound Healing

Soft tissues constitute a significant portion of the body and are vulnerable to a range of injuries, from minor skin scratches to severe burns and trauma.^[145,146] While many tissues possess an inherent ability to self-repair, this process often fails short in severe cases, necessitating the use of natural or synthetic substitutes that are biocompatible, biodegradable, and mechanically robust.^[147] Among these substitutes, eggshell-based biomaterials have emerged as a promising solution, extending beyond hard tissue regeneration for use in soft tissue engineering and biomolecule delivery due to their porous structure and composition. Improving soft tissue repair is a critical research interest, addressing the economic burdens associated with chronic wounds and enhancing recovery outcomes for patients.^[148,149] For instance, recent advancements in bioceramic-based wound dressings have supported the use of naturally derived materials such as eggshells. Eggshell powder and eggshell-derived HA (EHA) are increasingly employed in a range of medical applications, from soft tissue regeneration to targeted drug delivery.^[150–153] Eggshells and their derivatives are effective for soft tissue repair due to their biocompatibility, biodegradability, and tunability.^[154]

Bioceramic-based wound dressings have been investigated for their potential in enhancing tissue healing. Composites consisting of bioceramic particles infused into alginate and CS hydrogels have shown improved wound closure rates and reduced infection risks.^[155–157] Among bioceramics, CaCO₃ has emerged as a notable contributor to wound healing, as it supports the migration and proliferation of keratinocytes—essential for the reepithelialization of wounds—and has shown antimicrobial properties, adhesion to wet tissue, hemostatic efficiency, and ability to serve as a carrier for other therapeutic agents.^[158–161] Wang et al. (2023) used amorphous calcium carbonate (ACC) stabilized with polyphosphate (polyP) to accelerate healing for chronic wounds.^[158] The presence of polyP and serum proteins inhibits the transformation of ACC into its crystalline form, maintaining the solubility that allows calcium ions to participate in cellular functions, including cell migration, angiogenesis, enzyme activation, extracellular matrix remodeling, and signal transduction. In vitro experiments demonstrated that ACC stabilized with polyP significantly stimulated endothelial cell migration and increased granulation tissue formation and maturation. These findings indicate that CaCO₃-based composites can provide a sustained release of bioavailable calcium and support cellular activities in tissue regeneration.

The potential use of eggshells in this domain is promising due to their primary composition of CaCO₃, sustainability, and cost-effectiveness.^[162] CaCO₃ can be derived from eggshells, with ongoing research aimed at enhancing its adsorptive properties by enlarging pores, increasing surface area and volume, and creating new pores.^[163,164] Furthermore, processed and sterilized eggshell powder can be integrated into bioceramic-based dressings to harness the beneficial properties of eggshells, such as the collagen in the membrane, which supports skin tissue structure and strengthens wound healing.^[17] This approach aligns with the objectives of tissue engineering, where scaffolds are designed to mimic the biochemical and mechanical properties of natural tissue.^[165]

Studies that directly use eggshells in wound dressings are limited, but they have shown promising results for wound healing similar to those of CaCO₃. Deilami et al. (2022) created scaffolds made of CS alone and a combination of eggshell with CS (ES/CS) to support cutaneous wound healing.^[166] The ES/CS scaffolds were synthesized by dissolving 1 g of CS in 10 mL of acetic acid (1% v/v) to a final volume of 100 mL, to which 0.1 g of eggshell powder and 12.5 mL of Tween 20 were added. CS scaffolds were synthesized using the same composition without the eggshell powder. The ES/CS scaffolds showed better healing results than the CS scaffolds due to the incorporation of eggshells, which led to higher viability of cultured L929 fibroblasts, enhanced epithelialization, collagen production, and wound healing, as shown in Figure 6 and 7. This study revealed that incorporating eggshells creates an optimal healing environment that speeds up the natural wound healing process.

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Compared to the application of eggshells, pristine HA is more commonly used in developing wound dressings, likely due to the relatively unexplored potential of eggshells. HA's capacity to expedite wound healing is attributed to its biocompatibility and angiogenic potential.^[167] Additionally, the release of Ca²⁺ ions from HA plays a crucial role in maintaining mammalian skin homeostasis and influences the proliferation and differentiation of keratinocytes.^[168] These properties, along with increased mechanical strength, are evident in scaffolds made from composites containing HA for wound healing applications.^[169,170] Notably, similar benefits are observed with EHA, which has been utilized in composite scaffolds for wound healing.^[171]

Although the exploration of eggshells and eggshell-derived materials, particularly EHA, in wound healing is still in its early stages, these materials have demonstrated significant potential as sustainable and effective components of bioceramic-based wound dressings. Studies indicate that EHA is valuable for wound dressing applications due to its ability to support cell adhesion, proliferation, and overall healing processes, while also being eco-friendly and cost-effective. This suggests that eggshells could play a pivotal role in advancing affordable regenerative medicine, particularly in the development of innovative wound healing solutions.

Eggshell-Based Biomaterials for Biomolecule Delivery

Biomaterials synthesized from eggshells have been studied for biomolecule delivery, demonstrating their potential beyond tissue engineering applications.^[172] For instance, EHA exhibits outstanding biocompatibility and bioactivity, positioning it as an ideal candidate for drug delivery systems.^[173] Composite materials made from eggshells can be tailored for targeted drug delivery, leveraging the distinctive porous structure and chemical composition of eggshells to enhance biomolecule encapsulation and release.^[174,175]

Protein Delivery

Protein delivery systems utilizing bioceramics focus on delivering therapeutic proteins essential for tissue engineering, such as cytokines and enzymes, which are vital for tissue regeneration and repair. Cytokines play a crucial role in regulating bone resorption by affecting the formation and resorptive activity of osteoclasts, necessary for maintaining bone homeostasis and remodeling.^[176] Enzymes are important for degrading the organic matrix and facilitating osteoclast resorption, which is key for remodeling and integrating engineered bone tissues with native structures.^[105] The controlled release of these proteins from carriers is critical for their effective therapeutic action. Bovine serum albumin (BSA) is often used as a model protein for evaluating protein delivery due to its well-characterized properties and stability.^[177]

EHA nanoparticles used as carriers can be characterized by their morphology and calcium-to-phosphorus ratio (Ca/P).^[178] The synthesis of EHA nanoparticles requires controlled pH and temperature to achieve the desired morphology. Spherical nanoparticles can be prepared at a pH of 12.25 and a temperature of 298 K, rod-shaped nanoparticles form at a pH of 9.5 and a temperature of 303 K, and fibroid nanoparticles are synthesized at a pH of 5.25 and a temperature of 353 K. These different morphologies exhibit varying surface-area-to-volume ratios, mechanical properties, and porosities, which can be tailored for specific applications. The Ca/P ratio, which typically ranges from 1.5 to 1.67 in HA nanoparticles, can significantly affect the density of positively charged calcium sites relative to negatively charged phosphate groups on the surface. Higher ratios (up to 1.67) increase the density of calcium sites, enhancing calcium-rich phases, while lower ratios (down to 1.5) may increase phosphate dominance. These changes influence the electrostatic properties of materials and interactions with biomolecules, altering protein interactions with the surface. For instance, a higher Ca/P ratio (1.67) results in greater BSA adsorption.

Kumar et al. (2014) synthesized eggshell-derived calcium-deficient HA (ECDHA) nanoparticles to enhance bone tissue engineering by improving BSA delivery.^[150] Their study compared the loading efficiency of ECDHA nanoparticles with synthetic counterparts, focusing on the incorporation and release of BSA. Nanoparticles with various Ca/P molar ratios were synthesized, and ECDHA exhibited higher BSA-loading efficiency and release rates than synthetic calcium-deficient hydroxyapatite (SCDHA), achieving a maximum BSA loading of 57% at a Ca/P ratio of 1.51, compared to 37% for SCDHA, as observed in Figure 8. In a subsequent study, Kumar et al. (2021) further attributed ECDHA's superior BSA binding to the presence of trace elements like magnesium and strontium, which facilitated better protein interaction due to ionic composition and lattice distortion.^[151] These findings indicate the potential advantages of using natural sources for biomedical applications. The investigation of eggshell-derived bioceramics, particularly ECDHA nanoparticles, highlights their benefits compared to synthetic alternatives. Economically sourced eggshell-derived materials possess unique ionic compositions and enhanced interactions with therapeutic proteins, making them widely available, accessible, and cost-effective for EHA synthesis and biomolecular delivery. The use of eggshells as effective carriers opens new avenues for bone tissue engineering and regenerative medicine, offering promising prospects for future research and applications. These findings, combined with the superior BSA binding efficiency of ECDHA due to trace elements like magnesium and strontium, suggest that eggshell-derived materials can significantly enhance the delivery of therapeutic proteins essential for bone repair and regeneration.

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Therapeutic Delivery

Eggshell-based biomaterials are a promising substrate for the fabrication of carriers for therapeutic drug delivery. Numerous studies have explored the potential of using eggshells as a source of CaCO₃ for EHA and novel composites to deliver various therapeutics, ranging from anti-inflammatory drugs to cancer treatments.^[179–181] EHA and its derivatives have consistently demonstrated effectiveness as vehicles for biomolecule delivery, emphasizing their versatility and significant potential impact in the field of medicine.

Anti-Inflammatory Therapeutics

In a study on therapeutic release using EHA, Ibrahim et al. (2015) synthesized EHA as a carrier for drug delivery, using ibuprofen as the model drug.^[152] The EHA was produced through a two-step process, where calcium ions from eggshells combined with phosphate ions to form HA. The resulting EHA exhibited a large pore volume (1.4 cm³ g⁻¹) and a high surface area (284.1 m² g⁻¹), which enhanced its drug-loading capacity and efficiency. The loaded ibuprofen demonstrated improved dissolution and controlled release, marking a significant advancement in utilizing waste materials for high-value applications in medicine.

CaCO₃ in eggshells can act as a precursor for a range of eggshell-derived materials beyond EHA, which can serve as carriers for therapeutics. Priyadarsini et al. (2021) developed a biocomposite called pectin-g-P(AN-co-AM)/chicken eggshell by incorporating nano-calcium oxide (CaO) derived from chicken eggshells as a bio-filler.^[182] This biocomposite exhibited superabsorbent properties similar to a hydrogel and demonstrated swelling and deswelling behaviors in response to pH variations. To assess its drug-loading capacity, the biocomposite was immersed in a solution containing ibuprofen, and the concentration of ibuprofen remaining in the solution after immersion was measured. Additionally, the release of ibuprofen from the biocomposite was studied in vitro at pH 2 and 8 to mimic stomach and intestinal conditions, respectively. The concentration of the^[183] released drug was measured using UV–visible spectrophotometry at 222 nm over different time intervals, and the percentage of drug release was calculated. The biocomposite exhibited a higher drug release rate under alkaline conditions due to increased swelling. The incorporation of chicken-derived nano-CaO enhanced the water and drug retention capabilities of the biocomposite through interactions with other components such as pectin, acrylonitrile, and monomer acrylamide.

Anticancer Therapeutics

The innovative use of eggshells in anticancer therapeutics has gained significant attention in recent years, highlighting their multifaceted roles in drug delivery systems and their potential to enhance the efficacy and specificity of anticancer therapies. Extensive research has been conducted using EHA in combination with other materials to optimize the entrapment and release of cancer treatments. Wang et al. (2018) developed selenium (Se)-doped EHA (Se-EHA) nanorods as a nanocarrier for antitumor drug delivery.^[184] Waste eggshells were converted into calcium oxide through sintering at 900 °C for 2 h. The Se-EHA nanorods were synthesized via a coprecipitation method, where a solution of sodium phosphate and sodium selenite was added to a calcium chloride (CaCl₂) solution derived from the CaO. The pH was adjusted to around 11.0 using sodium hydroxide, causing the precipitation of selenium-doped HA. This mixture was then subjected to hydrothermal treatment at 121 °C under autogenous pressure to form crystalline nanorods. These nanorods had a high curcumin-loading capacity, encapsulating up to 66.96 mg g⁻¹ of curcumin, and demonstrated a slow, stable, controlled release of 1.38% of curcumin in physiological buffers over 159 h. Furthermore, the incorporation of selenium increased the degradation rate by 17.86% over 136 h and enhanced entrapment efficiency. These properties indicate that Se-EHA nanorods hold promise for postoperative bone tumor therapy, providing a platform for localized, controlled drug delivery.

Ganesan et al. (2022) synthesized etoposide-loaded eggshell-derived HA (EHA-ET) using a wet precipitation method.^[153] Their research demonstrated that EHA-ET facilitated sustained etoposide release, with 48% of the drug released within the first 24 h and ≈93% over 168 h. The study confirmed the excellent bioactivity and compatibility of EHA-ET, showing over 90% cell viability in L929 fibroblast cells at concentrations up to 300 μg mL⁻¹. Additionally, EHA-ET exhibited significant cytotoxicity against human osteosarcoma cell line MG-63, reducing cell viability to around 60% even at a concentration of 100 μg mL⁻¹. The intrinsic bioactivity and osteoconductivity of EHA, along with its ability to effectively deliver and release therapeutic agents, underscore its potential as a multifunctional material. This innovative approach to drug delivery, utilizing a chemotherapeutic drug and a biowaste-derived material, holds promise for more effective and targeted treatments of bone-related diseases such as osteosarcoma and osteomyelitis. The development of dual- or multifunctional bone graft materials could significantly enhance bone regeneration while simultaneously reducing the risk of infection and tumor recurrence.

Innovative eggshell-derived materials are also being utilized for targeted delivery to specific cancer-susceptible areas of the body. Render et al. (2016) developed an enteric drug delivery system using CaCO₃ nanoparticles derived from eggshells, demonstrating effective controlled drug release in the intestines when tested in rabbits.^[131] This study aimed to enhance drug delivery to specific gastrointestinal tract areas by engineering nanoparticles that remain stable in the stomach's acidic environment and disintegrate under the intestines’ alkaline conditions. The solubility of CaCO₃ changes with pH, remaining relatively insoluble at a low pH (2.0) and becoming more soluble in the pH range of ≈6–7.4 (duodenum to ileum). CaCO₃ sourced from eggshells and loaded with the model drug 5-fluorouracil (5-FU) formed the basis of five distinct tablet formulations using a blend of drug-loaded CaCO₃ nanoparticles, starch, hydroxypropyl methylcellulose (HPMC), and silicified microcrystalline cellulose (SMCC). When administered to rabbits, the optimal formulation remained intact in the stomach for up to 3 h and then gradually disintegrated, demonstrating effective gastric retention and controlled drug release for enteric delivery. The radiopaque nature of compacted CaCO₃ nanoparticles facilitated gastric transit studies, allowing researchers to track the tablet's progression through the rabbit's digestive system using radiography. CaCO₃ nanoparticles showed excellent drug-loading capacity (1 mg of 5-FU per 100 mg of nanoparticles), and their biocompatible and porous nature contributed to the efficacy of the tablets in drug delivery. While the study itself did not target a specific illness or disease, using 5-FU as a model drug indicates its potential application in treating colon cancer. The exploration of eggshells in anticancer therapies represents a significant step in medical research, particularly in developing sophisticated drug delivery systems. Utilizing the unique characteristics of eggshells, research has led to innovative drug carriers that improve the precision and effectiveness of anticancer treatments.

Growth Factor Delivery

The derivation of materials from eggshells has shown considerable promise in growth factor delivery studies, providing substantial evidence for the efficacy of eggshell-derived substances such as CaCO₃ and nano-EHA (nEHA) as effective carriers. Due to their intrinsic properties of biocompatibility, porosity, and ability to maintain a stable pH, these materials facilitate the encapsulation and controlled release of various growth factors, thereby modulating cell behaviors such as growth and differentiation. CaCO₃ sourced from eggshells has emerged as a medium for growth factor delivery, marking advancements in therapeutic applications. Schliephake et al. (2015) utilized CaCO₃ as a key component in developing composite scaffolds for the controlled release of osteogenic growth factors.^[185] CaCO₃ was integrated into poly(DL-lactic acid) (PDLLA), where DL indicates a racemic mixture of D and L lactic acid enantiomers, to form PDLLA/CaCO₃ composites at a weight ratio of 1:0.25 (PDLLA to CaCO₃), providing a consistent and effective medium for pH stabilization. The addition of CaCO₃ plays a crucial role in neutralizing the acidic degradation products of PDLLA, maintaining a stable physiological pH essential for preventing tissue irritation and inflammation that can occur if the environment becomes too acidic during scaffold degradation.^[186] This study demonstrates that CaCO₃ not only aids in maintaining pH balance but also enhances the biological efficacy of the growth factors released from the scaffolds, making it a valuable component in developing bone regeneration materials.

Building on the innovative use of CaCO₃ for growth factor delivery, EHA has emerged as a promising material. Baskar et al. (2022) developed and assessed a composite scaffold made of nEHA and CMC for dentine regeneration.^[187] These components were mixed in various ratios, ranging from 0:5 to 1:1 w/w%, and formed into scaffolds through magnetic stirring and freeze-drying. Growth factors such as VEGF and dentin sialophosphoprotein (DSPP) were used to evaluate the scaffold's effects on hDPSCs. The cells cultured on the 1:5 nEHA–CMC scaffold exhibited a more than threefold increase in DSPP expression by day 21, and VEGF levels also increased at day 21, indicating a significant enhancement compared to the control. These results highlight the scaffold's capability to support cell viability, proliferation, and differentiation, making it a viable option for dentine pulp engineering. The use of nEHA, which closely resembles the natural HA found in human bone and teeth, provides an promising structural environment for cell attachment and proliferation. The scaffold's ability to support cell growth and differentiation underlines its potential as a promising material for engineering dentin pulp. Advancements in utilizing eggshell-derived materials for growth factor delivery are noteworthy, signifying a burgeoning area of research with the potential for significant contributions to tissue engineering and regenerative medicine, and potentially expanding the understanding and utilization of this abundant and versatile resource.

Antimicrobial Properties of Eggshell-Based Biomaterials

Eggshell-derived materials are increasingly being utilized as carriers for antibiotics, with EHA emerging as a novel carrier for controlled antibacterial drug delivery. Its porous structure and surface chemistry effectively target and treat infections, allowing for a consistent therapeutic level at the infection site over a prolonged period and reducing the risk of developing antibiotic resistance. Mobarak et al. (2023) synthesized nEHA as a delivery system for the antibiotic doxycycline hyclate (DOXh).^[188] The synthesis involved calcination at four different temperatures (100, 300, 600, and 900 °C) to investigate the effects of temperature on the material's physicochemical properties and drug-loading capability. nEHA samples were used to adsorb DOXh from a 50 ppm solution, with 100 mg of nEHA per 50 mL of solution. The study monitored DOXh release in simulated body fluid at 37 °C and pH 7.4, using spectrophotometric analysis at 273 nm to measure antibiotic concentration at predetermined intervals. The release kinetics revealed that nEHA calcined at 900 °C demonstrated the highest drug-loading efficiency with an entrapment efficacy of 38% and the most favorable loading capacity. This high-calcination sample also provided a more sustained release than those calcined at lower temperatures. These findings highlight nEHA's potential to significantly reduce systemic side effects by delivering antibiotics directly to the infection site, thereby enhancing therapeutic efficacy and minimizing the required dosage. In a similar study, Ain et al. (2019) explored the use of nano-eggshell-derived HA (nEHA) loaded with vancomycin, a glycopeptide antibiotic, to enhance antibacterial properties.^[189] The nEHA demonstrated a loading content of 23.9% for vancomycin and an encapsulation efficiency of 95.6%, highlighting its high efficiency as a drug carrier. The study reported that nEHA nanoparticles loaded with vancomycin significantly improved antibacterial activity, with inhibition zones of 11.5 ± 0.5 mm against Escherichia coli and 15 ± 0.4 mm against Staphylococcus aureus, compared to smaller inhibition zones produced by vancomycin alone (10 ± 0.5 mm for E. coli and 13 ± 0.7 mm for S. aureus). The increased efficacy was attributed to the slow and sustained release of vancomycin facilitated by the porous structure of the nanoparticles, allowing for targeted delivery. This method enhances not only the potency of antibiotics but also their retention time at the infection site, which is crucial for treating persistent infections such as osteomyelitis. Additionally, other antibacterial drugs like ciprofloxacin have shown similar targeted drug delivery and sustained release capabilities, acting as a bone filler and aiding in bone regeneration at the site of osteomyelitis.^[173,190]

In addition to serving as carriers for antimicrobial agents, eggshells possess inherent antimicrobial properties derived from their role as a natural barrier. The eggshell cuticle acts as an effective shield against microbial attachment and penetration, primarily due to its physical structure and chemical composition.^[191] Antimicrobial proteins identified within the cuticle can inhibit the growth of bacteria and fungi.^[192] Furthermore, the high calcium content in eggshells can alter the microbial growth environment from acidic to alkaline.^[193] These properties can be harnessed in eggshell-containing composites, enhancing their antimicrobial efficacy.

Several studies have explored the incorporation of eggshells in composites to leverage the antimicrobial properties of their cuticle layers. Nath et al. (2021) derived calcium oxide (CaO) from eggshells to produce antibacterial composites.^[194] While this study primarily focused on the antimicrobial construction of composites, these materials hold potential for biomedical applications, such as antimicrobial coatings for medical implants or components in tissue grafts where both antimicrobial properties and biocompatibility are essential. The antimicrobial properties of eggshells, particularly from their cuticle layer, have emerged as significant in medical science. Eggshell-derived materials, notably EHA, have proven effective carriers of antibiotics due to their targeted and prolonged release capabilities. Ongoing research in this field continues to reveal the potential of natural resources to address critical challenges in regenerative medicine, especially in combating antibiotic resistance.

Eggshells as Biosensor Systems

Biosensors are becoming increasingly important in various sectors, such as healthcare, environmental monitoring, and food safety. This is because they have the ability to convert biological responses into measurable signals.^[195,196] The integration of nanostructured materials has significantly improved the sensitivity and efficiency of biosensors. These advancements are crucial for developing highly responsive biosensors capable of quickly and accurately detecting low analyte concentrations, thereby expanding their applications in disease diagnosis and environmental safety.^[197] Eggshells and their derivatives can be utilized as signal probes in biosensors to enhance electrochemical signals necessary for detecting various biological and chemical species. This is because they can conduct ions, facilitate electron transfer in electrochemical reactions, and are biocompatible.^[198]

In an application utilizing eggshell particles, Tasaltin et al. (2020) developed a novel K-carrageenan/PVA/nano-eggshell biocomposite-based nonenzymatic electrochemical biosensor for detecting low levels of urea in phosphate-buffered solution.^[199] This biosensor offers a cost-effective, simple, and portable method for urea detection. The biocomposite was synthesized using ultrasonic sonochemistry, beginning with multiple washes of the eggshells, followed by microwaving at 720 W for 15 min, as displayed in Figure 9. The treated eggshells were then ground to a particle size of 200 μm and mixed with ethanol and acetic acid solutions. The ground eggshells were combined with 5 g of PVA dissolved in 50 mL of water at 80 °C, sonicated with 0.1 g of eggshell for 1 h at 25 W, and then mixed with 0.1 g of κ carrageenan in 50 ml of water. The resulting mixture was sonicated, filtered, and stored at room temperature before being applied to electrochemical gold transducers for sensor fabrication.

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Similar to their applications in biomolecule delivery, eggshell-derived CaCO₃ and nEHA can be utilized as components within biosensors. Ren et al. (2024) demonstrated that CaCO₃ microparticles prepared from discarded eggshells could detect Listeria monocytogenes, a harmful foodborne pathogen, using an environmentally friendly technique involving a micro-orifice resistance assay (MORA) and a DNA signal amplification strategy.^[200] This method was combined with a ligation-mediated branched hybridization chain reaction (LB-HCR) for signal amplification, enabling the sensitive detection of Listeria monocytogenes DNA. To prepare the CaCO₃ microparticles, eggshells were dissolved in hydrochloric acid, and sodium hydroxide was added to adjust the pH and precipitate magnesium ions. The resultant calcium ions were converted into calcium chloride, which was then mixed in a coprecipitation reaction with 965 μL of 0.33 m sodium carbonate in a 5:1 ethanol solution to form CaCO₃ microparticles. These particles were washed, centrifuged, and subsequently coated with 1 mL streptavidin solution. The CaCO₃ with streptavidin binds to biotinylated DNA probes, which are part of an LB-HCR designed to amplify the DNA signal of Listeria monocytogenes. Aggregation changes the electrical resistance measured by the assay, signaling the presence of the pathogen. The microparticles, combined with LB-HCR, demonstrated enhanced sensitivity in detecting Listeria monocytogenes, with a detection limit of 21.0 CFU mL⁻¹, significantly lower than that of conventional MORA. This enhanced sensitivity, supported by a detailed optimization of reaction conditions such as probe concentrations and reaction times, established that CaCO₃ from eggshells can be used as a signal probe in MORA and combined with LB-HCR as a more effective and environmentally sustainable method for pathogen detection in food safety applications, as presented in Figure 9. The synthesized biosensor exhibited a sensitivity of 0.018 μA μM⁻¹ cm⁻², within a linear urea detection range of 250–1000 μM, and a lower detection limit of 60 μM at room temperature. The sensor preparation allowed for a stable and reproducible detection platform, attributed to the high surface area and effective functional groups of the biocomposite that interact with urea.

Using nEHA, Derkus et al. (2016) developed an improved method for immobilizing aptamers in biosensors, with a focus on detecting thrombin, a key biomarker in neurological diseases.^[201] Through a sonochemical process, nEHA particles were synthesized from eggshells treated with a 1:3 hydrochloric acid solution to release calcium ions. These ions were then mixed with a 0.156 m diammonium hydrogen phosphate solution under ultrasonic conditions, promoting the nucleation of HA into uniform, spherical nanoparticles ≈60–80 nm in diameter. This spherical morphology demonstrated superior adsorption properties compared to traditionally synthesized needle-shaped nEHA nanoparticles, which were 50–60 nm in length and 9–10 nm in width. The aptasensor was constructed using a jellyfish atelo collagen matrix integrated with nEHA at a concentration of 0.42 mg mL⁻¹ in a weight ratio of 1:10 (nEHA to collagen). The functionality of the aptasensor was assessed using electrochemical impedance spectroscopy (EIS), revealing a detection limit of 0.25 nM for thrombin and a responsive calibration range from 0.25 to 1000 nM. This low detection limit is particularly advantageous for clinical applications that require precise measurement of thrombin levels in biological fluids. The effectiveness of the aptasensor was confirmed through clinical trials involving blood and cerebrospinal fluid samples, showing performance comparable to commercial ELISA kits.

The use of eggshells and eggshell-derived bioceramics in biosensors not only demonstrates a sustainable approach to managing biowaste but also opens the door for advanced biomedical applications. The creative use of eggshell-derived CaCO₃ and nEHA in biosensor systems highlights their ability to improve sensitivity and accuracy in detecting a wide range of analytes, from foodborne pathogens to crucial biomarkers in clinical settings. By integrating eco-friendly materials into biosensor design, these studies show the potential for developing cost-effective, highly responsive diagnostic tools.

Conclusions and Future Perspectives

The pervasive health challenges presented by tissue defects, stemming from trauma, congenital anomalies, tissue diseases, degenerative conditions, and the aging population, continue to drive the demand for novel materials in tissue regeneration, biomolecule delivery, and biosensor development. This ongoing need calls for the creation of innovative and unconventional biomaterials, pushing the boundaries of current research and technology. The exploration of eggshell-based biomaterials in medical applications represents a promising frontier in tissue engineering and regenerative medicine. Recent advancements in biomedical technologies have unveiled substantial opportunities for integrating whole eggshells and egg-derived materials into various medical applications, such as bone grafts and wound dressings. Eggshells have the microstructure, biocompatibility, and bone-forming ability to overcome the limitations of autologous bone, while the eggshell membrane promotes wound healing by facilitating the formation of new connective tissue and microscopic blood vessels. Eggshell-derived bioinspired scaffolds are nontoxic, biodegradable, and bioactive, significantly enhancing natural extracellular matrix dynamics to promote skin progenitor cell activity and accelerate differentiation. They exhibit minimal or mild inflammatory or immunological reactions and produce no harmful byproducts at the implantation site. Additionally, eggshells serve as biocompatible grafting materials with bone formation capabilities, possessing physical and mechanical characteristics reminiscent of the bone organic mineralized matrix structure. Their suitable architecture provides stability without loss of bioactivity and maintains porosity, allowing for cell migration, angiogenesis, and the transport of nutrients and waste. Furthermore, the use of eggshells as a source of calcium phosphate, specifically HA and β-TCP, holds promise for bone tissue engineering, addressing the rising demand for bone replacements due to conditions such as bone cancer, trauma, and aging.

Eggshell-derived materials, especially their cuticle layer, have shown antimicrobial properties and can be adapted for use as carriers in drug delivery systems. This allows for targeted and extended release of antibiotics to combat antibiotic resistance, presenting new opportunities to address key challenges in regenerative medicine and drug delivery. Furthermore, eggshell-derived CaCO₃ and nEHA improve the sensitivity and accuracy of biosensors, offering cost-effective and eco-friendly diagnostic tools. The effectiveness of eggshell membranes in biosensor development further highlights the versatility and sustainability of eggshell-based biomaterials in advancing biomedical technologies.

It is anticipated that future research will focus on refining methods for mass production and developing cost-effective, simple fabrication techniques for porous scaffolds with controlled macro- and microstructures. Engineering technologies for screening, separation, washing, sterilization, and processing are crucial steps to generate high-quality eggshells for value-added applications. Standardizing the source and processing conditions of chicken eggs is essential to address the inherent variability in their composition. This could involve using a single species of chicken with a controlled, consistent diet to ensure uniformity in eggshell composition, as the quality of the eggshell can be influenced by various internal and external factors, such as oviposition, age, genotype, housing conditions, and nutrition.^[202] Further developments also need to address the use of eggshells in combination with different elements to enhance its properties and the integration of eggshell- or eggshell-membrane-derived materials with cutting-edge technologies such as 3D bioprinting and nanofabrication, leading to the development of sophisticated tissue-engineered constructs.^[203] These combinations have the potential to revolutionize regenerative medicine and personalized healthcare by producing functional and viable tissues and organs, ultimately improving patient outcomes and expanding the horizons of biomedical research. Additionally, future studies should investigate the similarities between eggs across different species to determine whether they offer the same or improved benefits compared to chicken eggshells, given that the structure, crystallinity, and composition of eggshells are species dependent.^[202] For example, eggshells of laying hens have the following CaCO₃ contents: white eggshells 93.72%, light-brown eggshells 94.44%, and dark-brown 95.48%, while ostrich eggshells have a CaCO₃ content of 98.79%.^[204] Additionally, the uniformity of shell thickness contributes toward an overall increase in shell strength, due to a tight crystallographic control of calcite formation.^[202,204] While the literature on eggshell-based biomaterials is promising, there remains a clear lack of robust clinical trials to draw definitive conclusions for healthcare applications. Future directions in eggshell research must focus on filling this gap to validate their potential in clinical practice.

Acknowledgements

This work was funded by the University of Massachusetts Lowell.

Conflict of Interest

The authors declare no conflict of interest.

Author Contributions

Maria Eduarda Torres Gouveia: Investigation: (lead); Writing—original draft: (lead); Writing—review & editing: (lead). Charles Milhans: Investigation: (supporting); Writing—original draft: (supporting); Writing—review & editing: (supporting). Mert Gezek: Investigation: (supporting); Visualization: (lead); Writing—review & editing: (supporting). Gulden Camci-Unal: Conceptualization: (lead); Investigation: (supporting); Project administration: (lead); Resources: (lead); Supervision: (lead); Visualization: (supporting); Writing—review & editing: (supporting).

References

N. Xue, X. Ding, R. Huang, R. Jiang, H. Huang, X. Pan, W. Min, J. Chen, J. A. Duan, P. Liu, Y. Wang, Pharmaceuticals 2022, 15, 879.

E. Jabbarzadeh, J. Blanchette, T. Shazly, A. Khademhosseini, G. Camci‐Unal, Curr. Angiog. 2012, 1, 180.

V. Campana, G. Milano, E. Pagano, M. Barba, C. Cicione, G. Salonna, W. Lattanzi, G. Logroscino, J. Mater. Sci., Mater. Med. 2014, 25, 2445.

H. J. Haugen, S. P. Lyngstadaas, F. Rossi, G. Perale, J. Clin. Periodontol. 2019, 46, 92.

W. Wang, K. W. K. Yeung, Bioact. Mater. 2017, 2, 224.

M. Rupp, L. Klute, S. Baertl, N. Walter, G. K. Mannala, L. Frank, C. Pfeifer, V. Alt, M. Kerschbaum, J. Biomed. Mater. Res. Part B: Appl. Biomater. 2022, 110, 350.

M. E. Elsalanty, D. G. Genecov, Craniomax. Trauma Reconstr. 2009, 2, 125.

W. Ahmed, M. A. Asim, A. Ehsan, Q. Abbas, J. Coll. Phys. Surg. Pak. 2018, 28, 17.

T. Albrektsson, Johansson C. Osteoinduction, Eur. Spine J. 2001, 10, S96.

R. R. Betz, Orthopedics. 2002, 25, S561.

T. Boyce, J. Edwards, N. Scarborough, Orthop. Clin. 1999, 30, 571.

M. J. Broom, J. V. Banta, T. S. Renshaw, JBJS. 1989, 71, 32.

P. Henman, D. Finlayson, J. Arthroplasty 2000, 15, 368.

C. K. Sen, G. M. Gordillo, S. Roy, R. Kirsner, L. Lambert, T. K. Hunt, F. Gottrup, G. C. Gurtner, M. T. Longaker, Wound Repair Regen. 2009, 17, 763.

H. Bannasch, M. Föhn, T. Unterberg, A. D. Bach, B. Weyand, G. B. Stark, Clin. Plast. Surg. 2003, 30, 573.

A. A. Chaudhari, K. Vig, D. R. Baganizi, R. Sahu, S. Dixit, V. Dennis, S. R. Singh, S. R. Pillai, Int. J. Mol. Sci. 2016, 17, 1974.

H. J. Choi, Y. M. Kim, J.‐Y. Suh, J. Y. Han, Mater. Sci. Eng. C 2021, 128, 112350.

T. A. E. Ahmed, H.‐P. Suso, A. Maqbool, M. T. Hincke, Mater. Sci. Eng. C 2019, 95, 192.

H.‐J. You, S.‐K. Han, J. Korean Med. Sci. 2014, 29, 311.

Comparison of Skin Substitutes for Acute and Chronic Wound Management (Eds: C. Bay, Z. Chizmar, E. M. Reece, J. Z. Yu, J. Winocour, J. Vorstenbosch, S. Winocour), Thieme Medical Publishers, Inc. New York, NY 2021.

J. H. Min, I. S. Yun, D. H. Lew, T. S. Roh, W. J. Lee, Arch. Plast. Surg. 2014, 41, 330.

X. Wu, S. I. Stroll, D. Lantigua, S. Suvarnapathaki, G. Camci‐Unal, Biomater. Sci. 2019, 7, 2675.

Y. Shi, K. Zhou, D. Li, V. Guyonnet, M. T. Hincke, Y. Mine, Foods. 2021, 10, 2178.

M. Sinclair, N. Y. P. Lee, M. J. Hötzel, M. C. T. de Luna, A. Sharma, M. Idris, M. A. Islam, O. S. Iyasere, G. Navarro, A. A. Ahmed, M. Curry, Front. Animal Sci. 2022, 3, 995430.

N. Tangboriboon, R. Kunanuruksapong, A. Sirivat, Mater. Sci. 2012, 30, 313.

B. P. Chan, K. W. Leong, Eur. Spine J. 2008, 17, 467.

M. Hosseinkhani, D. Mehrabani, M. H. Karimfar, S. Bakhtiyari, A. Manafi, R. Shirazi, World J. Plast. Surg. 2014, 3, 3.

K. S. Almulhim, M. R. Syed, N. Alqahtani, M. Alamoudi, M. Khan, S. Z. Ahmed, A. S. Khan, Materials. 2022, 15, 6864.

X. Wu, K. Walsh, S. Suvarnapathaki, D. Lantigua, C. McCarthy, G. Camci‐Unal, Biotechnol. Bioeng. 2021, 118, 1411.

Y.‐T. Huang, M. Imura, Y. Nemoto, C.‐H. Cheng, Y. Yamauchi, Sci. Technol. Adv. Mater. 2011, 12, 045005.

W. Xiao, B. S. Bal, M. N. Rahaman, Mater. Sci. Eng. C. 2016, 60, 324.

N. A. S. M. Pu'ad, J. Alipal, H. Z. Abdullah, M. I. Idris, T. C. Lee, Mater. Today Proc. 2021, 42, 172.

W.‐F. Ho, H.‐C. Hsu, S.‐K. Hsu, C.‐W. Hung, S.‐C. Wu, Ceram. Int. 2013, 39, 6467.

S. Mahdavi, A. Amirsadeghi, A. Jafari, S. V. Niknezhad, S. A. Bencherif, Ind. Eng. Chem. Res. 2021, 60, 17348.

N. I. Agbeboh, I. O. Oladele, O. O. Daramola, A. A. Adediran, O. O. Olasukanmi, M. O. Tanimola, Heliyon 2020, 6, e03765.

P. Arokiasamy, M. M. A. B. Abdullah, S. Z. Abd Rahim, S. Luhar, A. V. Sandu, N. H. Jamil, M. Nabiałek, Ceram. Int. 2022, 48, 14959.

E. A. Ofudje, A. Rajendran, A. I. Adeogun, M. A. Idowu, S. O. Kareem, D. K. Pattanayak, Adv. Powder Technol. 2018, 29, 1.

A. R. Toibah, F. Misran, A. Shaaban, Z. Mustafa, J. Mech. Eng. Sci. 2019, 13, 4958.

E. Barua, A. Das, D. Pamu, A. B. Deoghare, P. Deb, S. D. Lala, S. Chatterjee, Ceram. Int. 2019, 45, 23265.

T.‐S. Kang, S.‐J. Lee, T.‐S. Kang, S.‐J. Lee, J. Korean Ceram. Soc. 2017, 54, 395.

X. Wu, O. Gauntlett, T. Zhang, S. Suvarnapathaki, C. McCarthy, B. Wu, G. Camci‐Unal, ACS Appl. Mater. Interfaces 2021, 13, 60921.

F.‐M. Chen, X. Liu, Prog. Polym. Sci. 2016, 53, 86.

I. Abdulrahman, H. I. Tijani, B. A. Mohammed, H. Saidu, H. Yusuf, M. N. Jibrin, S. Mohammed, J. Mater. 2014, 2014, 802467.

K. Kongpaopong, N. Ratchatawatanapipat, N. Tangboriboon, J. Appl. Polym. Sci. 2021, 138, 50439.

M. Gezek, M. Altunbek, M. E. Torres Gouveia, G. Camci‐Unal, ACS Appl. Mater. Interfaces 2024.

K. Huang, J. Hou, Z. Gu, J. Wu, ACS Biomater. Sci. Eng. 2019, 5, 5384.

S. B. Rønning, R. S. Berg, V. Høst, E. Veiseth‐Kent, C. R. Wilhelmsen, E. Haugen, H. P. Suso, P. Barham, R. Schmidt, M. E. Pedersen, Int. J. Mol. Sci. 2020, 21, 8130.

M. M. Pillai, R. Saha, P. Tayalia, J. Mater. Sci. 2023, 58, 6865.

Y. Zhang, C. Xue, Y. Zhang, Q. Zhang, K. Zhang, Y. Liu, Z. Shan, W. Qiu, G. Chen, N. Li, H. Zhang, Chin. Chem. Lett. 2024, 35, 109196.

X. Zhang, M. Liu, Z. Kang, B. Wang, B. Wang, F. Jiang, X. Wang, D. P. Yang, R. Luque, Chem. Eng. J. 2020, 388, 124304.

H. Elkhenany, N. Eldebany, E. Said, A. Elsayed, M. George, M. Gamal, M. El‐Kammar, R. Abdel‐Wahed, A. Korittum, H. Abu‐Ahmed, S. Nouh, Alex. J. Vet. Sci. 2022, 73.

M. Tao, K. Huang, ACS Appl. Mater. 2022, 5, 4390.

R. M. Kamel, F. A. M. Abdel‐aal, F. A. Mohamed, A. Abdeltawab, M. O. Abdel‐Malek, A. A. Othman, A.‐M. I. Mohamed, Microchem. J. 2024, 201, 110703.

H. Herman, R. Minhajat, M. Muis, B. T. Umar, M. H. Cangara, A. A. Zainuddin, Malays. J. Med. Health Sci. 2023, 19, 278.

M. A. Nguyen, G. Camci‐Unal, Trends Biotechnol. 2020, 38, 178.

J. Dhillon, M. J. Kraeutler, J. W. Belk, E. C. McCarty, P. C. McCulloch, A. J. Scillia, Arthrosc. Sports Med. Rehabil. 2022, 4, e1513.

Y. Nys, M. T. Hincke, J. L. Arias, J. M. Garcia‐Ruiz, S. E. Solomon, Avian Poult. Biol. Rev. 1999, 10, 143.

A. Ar, C. V. Paganelli, R. B. Reeves, D. G. Greene, H. Rahn, Condor 1974, 76, 153.

H. Rahn, A. Ar, C. V. Paganelli, Sci. Am. 1979, 240, 46.

M. T. Hincke, J. Gautron, M. Panheleux, J. Garcia‐Ruiz, M. D. McKee, Y. Nys, Matrix Biol. 2000, 19, 443.

N. Le Roy, L. Stapane, J. Gautron, M. T. Hincke, Front. Genet. 2021, 12, 672433.

J. Gautron, L. Stapane, N. Le Roy, Y. Nys, A. B. Rodriguez‐Navarro, M. T. Hincke, BMC Mol. Cell Biol. 2021, 22, 11.

J. E. Dennis, S. Q. Xiao, M. Agarwal, D. J. Fink, A. H. Heuer, A. I. Caplan, J. Morphol. 1996, 228, 287.

S. Yalcin, S. Özkan, T. Shah, Front. Physiol. 2022, 13.

M. Halgrain, N. Bernardet, M. Crepeau, N. Même, A. Narcy, M. Hincke, S. Réhault‐Godbert, Poult. Sci. 2022, 101, 101622.

Y. Nys, J. Gautron, J. M. Garcia‐Ruiz, M. T. Hincke, C. R. Palevol. 2004, 3, 549.

J. L. Arias, D. J. Fink, S.‐Q. Xiao, A. H. Heuer, A. I. Caplan, Int. Rev. Cytol. 1993, 145, 217.

M. T. Hincke, Y. Nys, J. Gautron, K. Mann, A. B. Rodriguez‐Navarro, M. D. McKee, Front. Biosci. 2012, 17, 1266.

Y. C. Chien, M. T. Hincke, H. Vali, M. D. McKee, J. Struct. Biol. 2008, 163, 84.

Y. Li, Y. Li, S. Liu, Y. Tang, B. Mo, H. Liao, J. Struct. Biol. 2018, 203, 162.

A. H. Parsons, Poult. Sci. 1982, 61, 2013.

J. L. Qiu, Q. Zhou, J. M. Zhu, X. T. Lu, B. Liu, D. Y. Yu, G. Lin, T. Ao, J. M. Xu, Poult. Sci. 2020, 99, 1483.

B. Liao, H. G. Qiao, X. Y. Zhao, M. Bao, L. Liu, C. W. Zheng, C. F. Li, Z. H. Ning, Poult. Sci. 2013, 92, 2236.

Y. C. Chien, M. T. Hincke, M. D. McKee, J. Struct. Biol. 2009, 168, 527.

M. J. Bunk, S. L. Balloun, Poult. Sci. 1978, 57, 639.

R. M. Leach Jr, Poult. Sci. 1982, 61, 2040.

D. S. Robinson, N. R. King, Br. Poult. Sci. 1970, 11, 39.

V. Jonchère, S. Réhault‐Godbert, C. Hennequet‐Antier, C. Cabau, V. Sibut, L. A. Cogburn, Y. Nys, J. Gautron, BMC Genomics 2010, 11, 57.

M. T. Hincke, J. Gautron, C. P. W. Tsang, M. D. McKee, Y. Nys, J. Biol. Chem. 1999, 274, 32915.

J. Gautron, E. Murayama, A. Vignal, M. Morisson, M. D. McKee, S. Ré, M. Belghazi, M. L. Vidal, Y. Nys, M. T. Hincke, J. Biol. Chem. 2007, 282, 5273.

R. Huopalahti, M. Anton, R. López‐Fandiño, R. Schade, in Bioactive Egg Compounds, Springer, New York 2007.

K. M. Carnarius, K. M. Conrad, M. G. Mast, J. H. Macneil, Poult. Sci. 1996, 75, 656.

C. A. Lunam, J. Ruiz, Br. Poult. Sci. 2000, 41, 584.

M. M. Fathi, A. Z. El‐Dein, S. A. El‐Safty, L. M. Radwan, Int. J. Poult. Sci. 2007, 6, 236.

M. T. Hincke, Y. Nys, J. Gautron, J. Poult. Sci. 2010, 47, 208.

Y. Dauphin, G. Luquet, A. Perez‐Huerta, M. Salomé, Connect. Tissue Res. 2018, 59, 67.

M. S. Fernandez, A. Moya, L. Lopez, J. L. Arias, Matrix Biol. 2001, 19, 793.

A. Torres‐Mansilla, M. Hincke, A. Voltes, E. López‐Ruiz, P. A. Baldión, J. A. Marchal, P. Álvarez‐Lloret, J. Gómez‐Morales, Polymers. 2023, 15, 1342.

O. Wellman‐Labadie, J. Picman, M. T. Hincke, Comp. Biochem. Physiol. B Biochem. Mol. Biol. 2008, 149, 640.

I. Mikšík, P. Ergang, J. Pácha, Anal. Bioanal. Chem. 2014, 406, 7633.

J. Gautron, M. T. Hincke, K. Mann, M. Panhéleux, M. Bain, M. D. McKee, S. E. Solomon, Y. Nys, J. Biol. Chem. 2001, 276, 39243.

M. T. Hincke, K. Mann, M. Bain, M. D. McKee, S. E. Solomon, in Ovocalyxin‐32, A Novel Chicken Eggshell Matrix Protein, Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology (ASBMB), Rockville, MD 2001.

M. T. Hincke, Y.‐C. Chien, L. C. Gerstenfeld, M. D. McKee, J. Histochem. Cytochem. 2008, 56, 467.

J. Sodek, B. Ganss, McKee M. D. Osteopontin, Medicine. 2000, 11, 279.

M. S. Fernandez, C. Escobar, I. Lavelin, M. Pines, J. L. Arias, J. Struct. Biol. 2003, 143, 171.

J. Neunzehn, T. Szuwart, H.‐P. Wiesmann, Head Face Med. 2015, 11, 12.

M. D. McKee, C. E. Pedraza, M. T. Kaartinen, Cells Tissues Organs 2011, 194, 313.

D. Arola, D. Bajaj, J. Ivancik, H. Majd, D. Zhang, Int. J. Fatigue 2010, 32, 1400.

J. L. Katz, A. Misra, O. Marangos, Q. S. P. Ye, Biomed. Eng. Handbook 1995, 1, 1.

A. Merolli, P. T. Leali, in Biomimetic, Bioresponsive, and Bioactive Materials: An Introduction to Integrating Materials with Tissues, Wiley, Hoboken, NJ 2012, pp. 81–94.

M. Vallet‐Regi, J. M. González‐Calbet, Prog. Solid State Chem. 2004, 32, 1.

A. J. Salinas, P. Esbrit, M. Vallet‐Regí, Biomater. Sci. 2013, 1, 40.

R. Z. LeGeros, Chem. Rev. 2008, 108, 4742.

G. H. Bourne, in The Biochemistry and Physiology of Bone, Elsevier, Amsterdam 2014.

J. R. Porter, T. T. Ruckh, K. C. Popat, Biotechnol. Prog. 2009, 25, 1539.

X. Feng, Curr. Chem. Biol. 2009, 3, 189.

A. M. Mohamed, Malay. J. Med. Sci. 2008, 15, 4.

S. M. Best, A. E. Porter, E. S. Thian, J. Huang, J. Eur. Ceram. Soc. 2008, 28, 1319.

T. J. Blokhuis, J. J. C. Arts, Injury 2011, 42, S26.

F. Al Bayaty, M. M. J. Al‐Obaidi, A. Lokman, S. Yazid, O. E. Ibrahim, Arab Gulf J. Sci. Res. 2023.

S.‐C. Wu, H.‐C. Hsu, H.‐C. Yu, C.‐E. Shen, W.‐F. Ho, Adv. Powder Technol. 2023, 34, 103909.

D. R. Kadhim, T. I. Hamad, A. A. Fatalla, Int. J. Biomater. 2022, 2022, 8722283.

H. Opris, C. Dinu, M. Baciut, G. Baciut, I. Mitre, B. Crisan, G. Armencea, D. A. Prodan, S. Bran, Biology 2020, 9, 476.

A. Szwed‐Georgiou, P. Płociński, B. Kupikowska‐Stobba, M. M. Urbaniak, P. Rusek‐Wala, K, Szustakiewicz, P. Piszko, A. Krupa, M. Biernat, M. Gazińska, M. Kasprzak, ACS Biomater. Sci. Eng. 2023, 9, 5222.

K. M. Sadek, S. El Moshy, I. A. Radwan, D. Rady, M. M. S. Abbass, A. A. El‐Rashidy, C. E. Dörfer, K. M. Fawzy El‐Sayed, Int. J. Mol. Sci. 2023, 24, 4599.

P. A. Downey, M. I. Siegel, Phys. Ther. 2006, 86, 77.

D. W. Buck, G. A. Dumanian, Plast. Reconstr. Surg. 2012, 129, 1314.

A. Johnston, A. Callanan, Biomimetics 2023, 8, 205.

D. Suhag, S. Kaushik, V. B. Taxak, in Scaffold Design in Tissue Engineering. Handbook of Biomaterials for Medical Applications, Vol. 1, Fundamentals, Springer, New York 2024, pp. 155–184.

L. Dupoirieux, D. Pourquier, F. Souyris, J. Cranio‐Maxillofac. Surg. 1995, 23, 187.

R. Salama, M. Khashaba, D. El Rouby, Saudi Dent. J. 2019, 31, 375.

N. D. Calvert, S. Proulx, A. Rodriguez‐Navarro, T. Ahmed, E. A. Lehoux, M. T. Hincke, I. Catelas, J. Biomed. Mater. Res. Part B: Appl. Biomater. 2024, 112.

E. Yüce‐Erarslan, B. İzbudak, A. A. Kızılkurtlu, M. Topal, A. Akpek, A. Bal‐Öztürk, J. Appl. Polym. Sci. 2023, 140, e54310.

S. I. Biscaia, T. F. Viana, H. A. Almeida, P. J. Bártolo, Mater. Today Proc. 2015, 2, 208.

U. Sharma, D. Pal, R. Prasad, Ind. J. Clin. Biochem. 2014, 29, 269.

S. Anoopkumar‐Dukie, J. B. Carey, T. Conere, E. O’Sullivan, F. N. Van Pelt, A. Allshire, The Br. J. Radiol. 2005, 78, 945.

A. Ünsal, E. Durmuş, İ. Çelik, Iran. Biomed. J. 2023, 27, 366.

D. Fuadiyah, R. Ratnawati, S. Kurniawati, R. Sidharta, V. Sutanti, A. Swastirani, R. G. Pongrekun, I. I. Farahdiba, Y. Pertiwi, J. Christi, L. Christie, Malay. J. Med. Health Sci. 2023, 19.

D. Lantigua, X. Wu, S. Suvarnapathaki, M. A. Nguyen, G. Camci‐Unal, Bioengineering 2021, 8, 169.

S. Suvarnapathaki, M. A. Nguyen, X. Wu, S. P. Nukavarapu, G. Camci‐Unal, RSC Adv. 2019, 9, 13016.

D. Render, T. Samuel, H. King, M. Vig, S. Jeelani, R. J. Babu, V. Rangari, J. Nanomater. 2016, 2016, 3170248.

Y.‐Z. Huang, Y.‐R. Ji, Z.‐W. Kang, F. Li, S.‐F. Ge, D.‐P. Yang, J. Ruan, X. Q. Fan, Chem. Eng. J. 2020, 395, 125098.

D. Muthu, G. S. Kumar, M. Gowri, M. Prasath, V. Viswabaskaran, V. S. Kattimani, E. K. Girija, Ceram. Int. 2022, 48, 1326.

V. H. Ingole, K. Hany Hussein, A. A. Kashale, K. Ghule, T. Vuherer, V. Kokol, J. Y. Chang, Y. C. Ling, A. Vinchurkar, H. N. Dhakal, A. V. Ghule, J. Biomed. Mater. Res. A 2017, 105, 2935.

P. Chuysinuan, P. Nooeaid, T. Thanyacharoen, S. Techasakul, P. Pavasant, K. Kanjanamekanant, Int. J. Biol. Macromol. 2021, 193, 799.

V. A. Apalangya, V. K. Rangari, B. J. Tiimob, S. Jeelani, T. Samuel, Int. J. Biomater. 2019, 2019, 6762575.

S. K. Padmanabhan, L. Salvatore, F. Gervaso, M. Catalano, A. Taurino, A. Sannino, A. Licciulli, J. Nanosci. Nanotechnol. 2015, 15, 504.

Y. E. Arslan, T. S. Arslan, B. Derkus, E. Emregul, K. C. Emregul, Colloids Surf. B: Biointerfaces 2017, 154, 160.

S. Prathap, K. S. Rajesh, N. G. Thomas, J. Venkateshan, M. S. Prathap, S. Surya, J. Pharm. Bioallied Sci. 10, 4103.

D. K. Patel, B. Jin, S. D. Dutta, K. T. Lim, J. Biomed. Mater. Res. Part B: Appl. Biomater. 2020, 108, 1953.

D. Acharjee, S. Mandal, S. K. Samanta, M. Roy, B. Kundu, S. Roy, B. Kundu, S. Roy, P. Basak, S. K. Nandi, ACS Biomater. Sci. Eng. 2023, 9, 4673.

R. Meenambal, R. K. Singh, P. N. Kumar, S. Kannan, Mater. Sci. Eng. C 2014, 43, 598.

S. N. Rath, G. Pryymachuk, O. A. Bleiziffer, C. X. F. Lam, A. Arkudas, S. T. B. Ho, J. P. Beier, R. E. Horch, D. W. Hutmacher, U. Kneser, J. Mater. Sci. Mater. Med. 2011, 22, 1279.

U. K. Roopavath, M. K. Sah, B. B. Panigrahi, S. N. Rath, Ceram. Int. 2019, 45, 12910.

G. A. Holzapfel, in Handbook of Materials Behavior Models, Vol. 3, Elsevier, Amsterdam, Netherlands 2001, p. 1049.

D. S. Masson‐Meyers, L. Tayebi, J. Tissue Eng. Regen. Med. 2021, 15, 747.

S. Kargozar, R. K. Singh, H.‐W. Kim, F. Baino, Acta Biomater. 2020, 115, 1.

K. A. Al‐Gharibi, S. Sharstha, M. A. Al‐Faras, Sultan Qaboos Univ. Med. J. 2018, 18, e433.

J. J. He, C. McCarthy, G. Camci‐Unal, Adv. Nanobiomed. Res. 2021, 1, 2100004.

T. S. Sampath Kumar, K. Madhumathi, B. Rajkamal, S. Zaheatha, A. Rajathi Malar, S. Alamelu Bai, Colloids Surf. B: Biointerfaces 2014, 123, 542.

T. S. S. Kumar, K. Madhumathi, Trends Biomater. Artif. Org. 2021, 35, 30.

A.‐R. Ibrahim, X. Li, Y. Zhou, Y. Huang, W. Chen, H. Wang, J. Li, Int. J. Mol. Sci. 2015, 16, 7960.

V. Ganesan, G. Meiyazhagan, M. Devaraj, S. Kandasamy, P. Manogaran, G. Suresh Kumar, G. Raji, V. S. Kattimani, G. Easwaradas Kreedapathy, ACS Biomater. Sci. Eng. 2022, 8, 682.

K. M. Salleh, N. F. Abd Rashid, in Polymer Composites Derived from Animal Sources, Elsevier, Amsterdam, Netherlands 2024, pp. 103–123.

Y. Chen, A. Mahmood, Y. Xu, S. Wang, Z. Yin, Z. Jin, Ceram. Int. 2023, 49, 33500.

K. Elakkiya, P. Bargavi, S. Balakumar, Chemosphere 2023, 341, 140131.

N. Manjubaashini, P. Bargavi, S. Balakumar, Biomater. Sci. 2023, 11, 3921.

S. Wang, M. Neufurth, H. Schepler, R. Tan, Z. She, B. Al‐Nawas, X. Wang, H. C. Schröder, W. E. G. Müller, Pharmaceutics 2023, 15.

A. S. Kumar, D. Prema, R. G. Rao, J. Prakash, P. Balashanmugam, T. Devasena, G. D. Venkatasubbu, Int. J. Biol. Macromol. 2024, 254, 128060.

W. Yang, X. Kang, X. Gao, Y. Zhuang, C. Fan, H. Shen, Y. Chen, J. Dai, Adv. Funct. Mater. 2023, 33, 2211340.

M. H. Azarian, T. Junyusen, W. Sutapun, ACS Omega 2024, 9, 955.

M. T. Hincke, Front. Biosci. 2012, 17, 1266.

R. A. Ningrum, S. Humaidi, S. Sihotang, D. Bonardo, Estananto, J. Phys. Conf. Ser. 2022, 2193, 012009.

H. B. Sembiring, N. Pasaribu, J. Sitepu, AIP Conf. Proc. 2021, 2342, 070005.

T. Courtney, M. S. Sacks, J. Stankus, J. Guan, W. R. Wagner, Biomaterials 2006, 27, 3631.

A. Deilami, Z. Niknam, A. Golchin, V. Niazi, H. Zali, M. Omidi, Polym. Bull. 2022, 79, 3971.

J. Gao, L.‐S. Hao, B.‐B. Ning, Y.‐K. Zhu, J.‐B. Guan, H.‐W. Ren, H. P. Yu, Y. J. Zhu, J. L. Duan, Coatings 2022, 12.

M. Li, Q.‐F. Ke, S.‐C. Tao, S.‐C. Guo, B.‐Y. Rui, Y.‐P. Guo, J. Mater. Chem. B. 2016, 4, 6830.

N. Ribeiro, A. Sousa, C. Cunha‐Reis, A. L. Oliveira, P. L. Granja, F. J. Monteiro, S. R. Sousa, Nanomed. Nanotechnol. Biol. Med. 2021, 33, 102353.

L. C. Espitia‐Quiroz, A. L. Fernández‐Orjuela, L. M. Anaya‐Sampayo, A. P. Acosta‐Gómez, L. G. Sequeda‐Castañeda, S. J. Gutiérrez‐Prieto, N. S. Roa‐Molina, D. A. García‐Robayo, Dent. J. 2022, 10.

M. Mehedi Hasan, M. Nuruzzaman Khan, P. Haque, M. M. Rahman, Int. J. Biol. Macromol. 2018, 117, 1110.

N. A. S. Mohd Pu'ad, J. Alipal, H. Z. Abdullah, M. I. Idris, T. C. Lee, Mater. Today Proc. 2021, 42, 172.

S. Sultana, M. S. Hossain, M. Mahmud, M. B. Mobarak, M. H. Kabir, N. Sharmin, S. Ahmed, RSC Adv. 2021, 11, 3686.

V. A. Apalangya, V. K. Rangari, B. J. Tiimob, S. Jeelani, T. Samuel, Int. J. Biomater. 2019, 2019, 6762575.

A. Kazemi, M. Abdellahi, A. Khajeh‐Sharafabadi, A. Khandan, N. Ozada, Mater. Sci. Eng. C. 2017, 71, 604.

G. D. Roodman, Calcif. Tissue Int. 1993, 53, S94.

P. Yadav, A. B. Yadav, Futur. J. Pharm. Sci. 2021, 7, 200.

S. K. Swain, D. Sarkar, Appl. Surf. Sci. 2013, 286, 99.

C. Lin, M. Akhtar, Y. Li, M. Ji, R. Huang, Pharmaceutics 2024, 16, 275.

X. Yang, Y. Sun, H. Zhang, F. Liu, Q. Chen, Q. Shen, Z. Kong, Q. Wei, J. Shen, Y. Guo, Nanoscale 2024.

D. B. Trushina, T. N. Borodina, S. Belyakov, M. N. Antipina, Mater. Today Adv. 2022, 14, 100214.

M. Priyadarsini, T. Biswal, S. Dash, Poly. Bull. 2021, 78, 6337.

M. H. Azarian, W. Sutapun, Front. Mater. 2022, 9, 1024977.

Y. Wang, W. He, H. Hao, J. Wu, N. Qin, J. Sol‐Gel Sci. Technol. 2018, 87, 600.

H. Schliephake, M. Vucak, J. Boven, S. Backhaus, T. Annen, M. Epple, Oral Maxillofac. Surg. 2015, 19, 133.

H. M. Torres, K. M. Arnold, M. Oviedo, J. J. Westendorf, S. R. Weaver, Curr. Osteoporos. Rep. 2023, 21, 842.

K. Baskar, B. Saravana Karthikeyan, I. Gurucharan, S. Mahalaxmi, G. Rajkumar, V. Dhivya, A. Kishen, Int. Endodont. J. 2022, 55, 89.

M. Bin Mobarak, M. N. Islam, F. Chowdhury, M. N. Uddin, M. S. Hossain, M. Mahmud, U. S. Akhtar, N. I. Tanvir, A. M. Rahman, S. Ahmed, RSC Adv. 2023, 13, 36209.

Q.‐U. Ain, H. Munir, F. Jelani, F. Anjum, M. Bilal, Mater. Res. Express 2019, 6, 11.

P. Agalya, S. Cholan, K. M. Prabu, G. Suresh Kumar, G. Karunakaran, M. Shkir, E. Kolesnikov, S. Ramalingam, Inorg. Chem. Commun. 2022, 143, 109788.

L. D’Alba, R. Torres, G. I. N. Waterhouse, C. Eliason, M. E. Hauber, M. D. Shawkey, Physiol. Biochem. Zool. 2017, 90, 588.

J. R. Roberts, S. Samiullah, J. World's Poult. Sci. 2014, 70, 693.

P. Shekhawat, G. Sharma, R. M. Singh, Constr. Build. Mater. 2019, 223, 1112.

D. Nath, K. Jangid, A. Susaniya, R. Kumar, R. Vaish, Compos. C: Open Access 2021, 5, 100123.

A. R. Ahmed, O. C. Gauntlett, G. Camci‐Unal, ACS Omega 2020, 6, 46.

X. Wu, S. Suvarnapathaki, K. Walsh, G. Camci‐Unal, MRS Commun. 2018, 8, 1.

V. Naresh, N. Lee, Sensors 2021, 21.

Y. W. Hartati, I. Irkham, S. Zulqaidah, R. S. Syafira, I. Kurnia, A. R. Noviyanti, S. N. Topkaya, Sens. Bio‐Sens. Res. 2022, 38, 100542.

N. Taşaltın, E. Aydın, S. Karakuş, A. Kilislioğlu, Appl. Phys. A 2020, 126, 827.

L. Ren, F. Hong, N. Feng, J. Lin, J. Wang, S. Ruan, Y. Chen, Sens. Actuators B: Chem. 2024, 405, 135312.

B. Derkus, Y. E. Arslan, K. C. Emregul, E. Emregul, Talanta 2016, 158, 100.

Evaluation of Calcium Carbonate Content in Eggshells of Avian, Turtle, Snail, and Ostrich Using Chemical Analysis and Scanning Electron Microscopy (Eds: I. Umbraško, A. Petjukevičs, A. Batjuka, N. Harlamova), Rezekne Academy of Technologies, Latvia 2021.

A. Abaci, G. Camci‐Unal, M. Guvendiren, MRS Bull. 2023, 48, 624.

A. Pérez‐Huerta, J.‐P. Brugal, M. Salomé, C. N. Z. Schmitt, Y. Dauphin, Minerals 2023, 13, 481.

V. Trakoolwannachai, P. Kheolamai, S. Ummartyotin, Compos. B: Eng. 2019, 173, 106974.

A. Deilami, Z. Niknam, A. Golchin, V. Niazi, H. Zali, M. Omidi, Polym. Bull. 2021, 1.

S. Viguet‐Carrin, P. Garnero, P. Delmas, Osteoporosis Int. 2006, 17, 319.

M. Wong, M. J. C. Hendrix, Dev. Biol. 1984, 104, 28.

L. Malaval, N. M. Wade‐Guéye, M. Boudiffa, J. Fei, R. Zirngibl, F. Chen, N. Laroche, J. P. Roux, B. Burt‐Pichat, F. Duboeuf, G. Boivin, J. Exp. Med. 2008, 205, 1145.

M. L. H. Rose, M. T. Hincke, Cell. Mol. Life Sci. 2009, 66, 2707.

M. Horvat‐Gordon, F. Yu, D. Burns, R. M. Leach Jr, Poult. Sci. 2008, 87, 1618.

M. F. Young, Osteoporosis Int. 2003, 14, 35.

M. Rose‐Martel, S. Smiley, M. T. Hincke, J. Proteomics 2015, 116, 81.

H. Nakamura, F. Butz, L. Saruwatari, T. Ogawa, J. Dent. Res. 2007, 86, 147.

Y. W. Ha, M. J. Son, K. S. Yun, Y. S. Kim, Comp. Biochem. Physiol., Part A: Mol. Integr. Physiol. 2007, 147, 1109.

J. Lahav, J. Lawler, M. A. Gimbrone, Eur. J. Biochem. 1984, 145, 151.

T. S. S. Kumar, K. Madhumathi, R. Jayasree, J. Ind. Inst. Sci. 2022, 102, 599.

C. M. Cristache, M. Burlibasa, G. Cristache, S. Drafta, I. A. Popovici, A. A. Iliescu, S. Zisi, L. Burlibasa, Metal. Int. 2011, 16, 18.

M. Mohanty, Trans. Ind. Ceram. Soc. 1995, 54, 200.

E. Fiume, G. Magnaterra, A. Rahdar, E. Verné, F. Baino, Ceramics 2021, 4, 542.

N. Eliaz, N. Metoki, Materials 2017, 10, 334.

H. J. Hsu, R. A. Waris, M. Ruslin, Y. H. Lin, C. S. Chen, K. L. Ou, J. Am. Ceram. Soc. 2018, 101, 419.

P. Zadehnajar, M. H. Mirmusavi, S. Soleymani Eil Bakhtiari, H. R. Bakhsheshi‐Rad, S. Karbasi, Int. J. Appl. Ceram. Technol. 2021, 18, 1901.

W. Vogel, W. Höland, Angew. Chem. Int. Ed. 1987, 26, 527.