Regenerative Capacity of Eggshell Membrane in Management of Critical-Sized Bone Defect: An Experimental Study

Regenerative Capacity of Eggshell Membrane in Management of Critical-Sized Bone Defect: An Experimental Study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Regenerative Capacity of Eggshell Membrane in Management of Critical-Sized Bone Defect: An Experimental Study Hossam Ibrahem Abdelrasoul, Enas Alaa Eldin AbdElaziz, Eman Alaa Eldin AbdElaziz, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7122120/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objectives: This study aimed to evaluate the regenerative potential of eggshell membrane (ESM) in critical-sized bone defects (CSBD) and its synergistic effect with xenograft bone through histological and histomorphometric analysis. Materials and Methods: Thirty-two male Wistar rats were used, each receiving bilateral femoral CSBDs (2 mm diameter/depth). Defects were divided into: Group I (Control): Filled with xenograft alone (n=32 defects). Group II (Test): Filled with ESM-xenograft composite (n=32 defects). Rats were sacrificed at 4- and 8-weeks post-operation. Bone regeneration was assessed via H&E and Masson’s trichrome staining, with histomorphometric analysis using ImageJ software. Results: The Test group showed significantly higher bone regeneration (27.7 ± 1.57% at 4 weeks; 86.53 ± 3.81% at 8 weeks) compared to the Control group (15.44 ± 2.12% and 55.98 ± 2.53%, respectively; p<0.001). Collagen deposition was also greater in the Test group (54.79 ± 1.52% vs. 24.48 ± 1.73% at 4 weeks). Histologically, the Test group exhibited mature lamellar bone by 8 weeks, while the Control group showed slower maturation. Conclusions: ESM enhances bone regeneration synergistically with xenograft, offering a promising, cost-effective biomaterial for CSBD management. Unstructured Abstract: This experimental study assessed eggshell membrane (ESM) combined with xenograft for critical-sized bone defect (CSBD) repair in 32 rats. CSBDs were created bilaterally in femurs, treated with xenograft alone (Control) or ESM-xenograft (Test), and evaluated histologically at 4/8 weeks. Results demonstrated superior bone regeneration and collagen synthesis in the Test group (p<0.001), with 86.53% new bone formation by 8 weeks versus 55.98% in Controls. ESM’s osteoconductive properties suggest clinical potential for bone defect therapies. Biological sciences/Biotechnology Physical sciences/Materials science Health sciences/Medical research Biological sciences/Stem cells Eggshell membrane (ESM) Xenograft Critical-sized bone defect (CSBD) Bone regeneration Histomorphometry Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Bone abnormalities resulting from trauma, bone tumours, or infection are prevalent and significant clinical manifestations. Bone tissue has significant challenges in self-repair and regeneration without external intervention. In recent decades, bone transplantation has consistently been regarded as the "gold standard" for addressing bone abnormalities (1). A critical bone defect is characterized as one that will not mend without intervention, with segmental abnormalities above 60mm often considered significant defects. Treatment success was characterized by bone union (2). Numerous operations necessitate the utilization of bone grafts to restore or augment bone volume that has been diminished owing to systemic diseases, periodontal abnormalities, tooth loss, or other disorders. Progress in contemporary medicine has resulted in a greater availability of novel biomaterials that can augment bone volume restoration. These biomaterials can be sourced from the patient's own body, other persons, animals, or generated synthetically. Notwithstanding these advancements, minimal study has been undertaken about patients' perspectives on various bone transplants (3). Xenograft materials are extensively utilized and sourced from several animal species, predominantly from bovine origins. Anorganic bovine bone is one of the most often utilized xenografts due to its similarity to human bone. It demonstrates favourable biocompatibility, osteoconductivity, and volumetric stability (4). Bovine bone grafts are extensively utilized in dentistry for directed tissue regeneration and facilitate new bone development in direct contact with the graft. ESM is a plentiful industrial and home waste that is ecologically benign, non-toxic, composed of various natural proteins, and amenable to modification by carbonization and dissolution. It has been employed in many technical applications, including as capacitors, batteries, solar cells, catalysis, biosensors, cell culture, wound healing, and bone replacements (6). ESM functions as a biopolymer network in shell mineralization during oviposition, facilitating mineral precipitation within 24 hours. The extracellular matrix fibres consist of collagens (I, V, and X), comprising approximately 10% of the total protein composition, glycosaminoglycans, egg white proteins, and eggshell matrix proteins, with collagens constituting the core of the fibres. Natural Eggshell Membrane (NEMÒ) is an innovative dietary component that comprises naturally occurring glycosaminoglycans and proteins vital for the preservation of healthy joints and connective tissues. NEMÒ underwent safety assessment through in vitro and in vivo toxicology investigations. This encompassed assessments for cytotoxicity, genotoxicity, and acute oral toxicity (8). The objective of this study was to assess the possible impact of eggshell membrane on bone regeneration in critical-sized bone lesions by histological and histomorphometric analysis. It also examines the efficacy of ESM in conjunction with a xenograft to improve bone repair in the identical animal. Materials and Methods Ethical Regulation All experiments were conducted at the animal house of the Faculty of Pharmacy, Minia University, Egypt, following approval from the Ethics Committee of the Faculty of Dentistry, Minia University (774/2023). Sample Size Calculation and Grouping A sample size of 32 rats was calculated using G*Power software (version 3.1.9.7) to detect an effect size of 0.30 with 90% power at p ff 0.05. Thirty-two male Wistar albino rats (150–350 g) were housed under controlled conditions (21–24°C, 40–60% humidity, 12-hour light–dark cycle) with access to standard food and water. Each rat received bilateral femoral critical-sized defects (2 mm depth, 2 mm width), divided into two groups: Group I (control, n=32) filled with xenograft alone (left femur), and Group II (test, n=32) filled with ESM combined with xenograft (right femur). Sixteen rats were sacrificed at 4 weeks and 16 at 8 weeks post-operation. Material Preparation Chitosan-H (Lot 337) and eggshell (Lot 65H0427) (Sigma Chem. Co., St. Louis, MO) were used to prepare ESM gel. Chitosan-H (1%) was dissolved in 1% acetic acid, mixed with eggshell (500 mg) fig (1). at 400 rpm, and cross-linked with 0.1% tripolyphosphate pentasodium salt (TPP). Xenograft was prepared with saline for the control group and mixed with ESM gel for the test group. Anesthetic Protocol Rats fasted for 12 hours before surgery. Anesthesia was induced with intramuscular ketamine (50 mg, Troikaa Pharmaceuticals Ltd) and xylazine (20 mg, ADWIA, Egypt), supplemented with local 2% lidocaine (Alexandria Co. for Pharmaceuticals, Egypt). Surgical Procedure The femur was exposed via a 7-cm incision, and defects (2 mm depth, 2 mm width) Fig (2) were created using a contra-angle low-speed handpiece with a round bur under saline irrigation. Control defects received xenograft alone Fig (3,A), while test defects received ESM-xenograft mix Fig (3,B). Flaps were sutured with 3/0 black silk, and the area was disinfected with iodine and 70% ethanol. Post-Surgical Care Post-operatively, rats received intramuscular oxytetracycline (1.0 cm³/kg, Pfizer, Egypt) for 3 days and an analgesic (0.05 mg/kg). Full weight-bearing was permitted immediately.in addition to the administration of analgesics, animal discomfort and distress were monitored through a comprehensive set of behavioral and physiological indicators. Trained personnel performed daily observations (or more frequently as needed) to assess clinical signs including, but not limited to: reduced grooming, altered posture or locomotion, changes in appetite or water intake, vocalization, social withdrawal, and abnormal respiratory patterns. Body weight was measured regularly as a quantitative measure of well-being, with a loss of more than 15–20% from baseline triggering intervention or consideration for humane euthanasia. If any animal exhibited persistent or severe signs of distress unrelieved by analgesics, humane endpoints were applied in accordance with IACUC/ethical guidelines, prioritizing animal welfare. Assessment Femurs were harvested after sacrifice (Nembutal overdose) at 4 and 8 weeks. Samples were fixed in 4% paraformaldehyde, decalcified in 10% EDTA for 5 weeks, and embedded in paraffin. Sections (5 mm) were stained with hematoxylin and eosin (H&E) and Masson trichrome (MT). Histomorphometric analysis was performed using ImageJ software to measure the area percentage of new bone and collagen at 10X magnification. Statistical Analysis Data were analyzed using one-way ANOVA followed by post hoc tests or equivalent nonparametric tests. Paired and independent T-tests assessed intra- and inter-group differences (p 0.05). Pearson correlation tested the relationship between bone regeneration and collagen percentage. Results Histological Examination No inflammation was observed in either group. At 4 weeks, control defects showed minimal bone regeneration with dense fibrovascular tissue, while test defects exhibited more woven bone and fibrous tissue. At 8 weeks, control defects displayed increased trabecular bone, while test defects showed abundant mature lamellar bone Fig. (4). Masson trichrome staining revealed higher collagen deposition in the test group at 4 weeks, with more mature bone (red staining) at 8 weeks in both groups, though immature bone persisted Fig. (5) . Histomorphometric Analysis New bone formation increased significantly from 4 to 8 weeks in both groups. The control group showed 15.44 ± 2.12% at 4 weeks and 55.98 ± 2.53% at 8 weeks, while the test group achieved 27.7 ± 1.57% at 4 weeks and 86.53 ± 3.81% at 8 weeks (p < 0.001). Collagen percentage was higher in the test group at 4 weeks (54.79 ± 1.52% vs. 24.48 ± 1.73%) and 8 weeks (23.01 ± 1.69% vs. 11.43 ± 1.61%) (p < 0.001). A strong negative correlation between bone regeneration and collagen percentage was observed (r = -0.989 for control, r = -0.954 for test, p < 0.001). Table (1 ): Mean ± SD and Percentage of change of regeneration % for the two groups at the two-time intervales. 4 Weeks 8 Weeks Percentage of Change P-value* Control 15.44 ± 2.12 55.98 ± 2.53 -270.1 ± 60.8 < 0.001 HS Test 27.7 ± 1.57 86.53 ± 3.81 -213.1 ± 19.7 < 0.001 HS P-value** < 0.001 HS < 0.001 HS < 0.001 HS -* P-value for Intra-group comparison between the two-time intervals (Paired T-test). -** Overall P-value for Inter-group comparison between the two groups (ANOVA Test). - S = Statistically significant at P ≤ 0.05 - NS = Non-significant P < 0.05. - HS = Highly significant at P ≤ 0.001 . II) Assessment of Collagen fibers percentage (Collagen %) Both the Control and Test groups demonstrated a significant decrease in bone collagen over time, with the Test group consistently outperforming the Control group at both 4 and 8 weeks. The differences between the groups were statistically highly significant at all measured intervals, as confirmed by the Paired T-test and Independent T-test as showen in Table 2) Table (2): Mean ± SD and Percentage of change of collagen % for the two groups at the two-time intervales. 4 Weeks 8 Weeks Percentage of Change P-value* Control 24.48 ± 1.73 11.43 ± 1.61 53.2 ± 6.5 < 0.001 HS Test 54.79 ± 1.52 23.01 ± 1.69 58.0 ± 2.4 < 0.001 HS P-value** < 0.001 HS < 0.001 HS 0.432 NS III) Correlation between Regeneration % and Collagen % Results The result of the Pearson correlation test for the change from 4 to 8 weeks between the Regeneration % and Collagen % are tabulated in Table 3.; from the results we can conclude the following: There was a strong negative correlation between Regeneration % and Collagen % in the two groups and r values were − 0.989 and − 0.954 for Control and Test groups respectively, and this correlation was statistically highly significant (P < 0.001) as shown in Table (3 ) Table (3 ): Correlation between Regeneration % and Collagen % from 4 to 8 weeks for the two studied groups. r** P-value Correlation type Control − 0.989 < 0.001 HS Strong negative Test − 0.954 < 0.001 HS Strong negative - ** Pearson Correlation value - HS Highly significant (Correlation is significant at the 0.01 level). Discussion Critical-size bone loss can be caused by trauma, pathologies, tumor resections, or tooth loss affecting patients' esthetic and function. Thus, another point of debate raised is what is the best biomaterial to treat critical-size defects. Several biomaterials are available and have different characteristics, divided into autogenous/autograft, allogenous/allograft, xenograft (mainly bovine), and alloplastic/synthetic ( 9 ) . Synthetic and bovine bones have been options as substitute biomaterials in trying to overcome possible adversities of the autograft (donor site, increased morbidity, limited bone volume, and postoperative pain). They are considered to have only osteoconduction capacity ( 9 ) . Eggshell waste is a readily available biological waste product that can be repurposed for medical applications. Utilizing ESM for bone regeneration aligns with sustainable development by reducing waste and offering a low-cost alternative to synthetic biomaterials ( 10 ) . ESM, when combined with other regenerative strategies (e.g., growth factors, stem cells, or 3D scaffolds), can significantly enhance osteogenesis and angiogenesis in critical bone defects. This suggests its potential in clinical applications for orthopedic and maxillofacial reconstructions ( 11 ) . We use experimental rats in my research is due to their genetic, biological, and behavioural similarities to humans. Scientists use rats in experiments to study diseases, test drugs, and explore genetic functions. Here are some key reasons why rats are widely used in scientific research ( 12 ) . Our results show an increase in bone regeneration in both groups over time because Eggshell membrane is a natural biomaterial rich in collagen, glycosaminoglycans, and bioactive proteins, which are essential for bone healing. Studies suggest that ESM has osteoconductive properties, making it a promising material for bone regeneration ( 13 ) . Our result is matching with study by Kavarthapu and Malaiappan(14) who assessed demineralized bone matrix with a collagen membrane against eggshell powder and its membrane in a rat model. Histological evaluations revealed significant new bone formation in both groups, with no notable differences between them. This suggests that eggshell powder, combined with its membrane, could serve as a potential graft material ( 14 ) . Wu et al. ( 15 ) reveal that effects of microparticles of whole eggshells, eggshells without a membrane, and a pristine eggshell membrane on osteogenic differentiation in protein-derived hydrogels. The in vitro studies showed that gels reinforced with eggshells with and without a membrane demonstrated comparable cellular proliferation, osteogenic gene expression, and osteogenic differentiation. Subsequently, in vivo studies were performed to implant eggshell microparticle-reinforced composite hydrogel scaffolds into critical-sized cranial defects in Sprague Dawley (SD) rats for up to 12 weeks to study bone regeneration ( 15 ) . Rachna et al. ( 16 ) reveals that eggshell membrane has good regenerative properties and excellent osteogenic capacity when used to assess the effect of the eggshell membrane on alveolar bone regeneration after tooth extraction ( 16 ) . In contrast the our study, Arias et al. ( 17 ) demonstrate the first time that eggshell membranes as interpositional material in rabbit osteotomized ulnar experiments acted as an active barrier against bone bridging ( 17 ) . The research by Dupoirieux et al ( 18 ) investigated the use of eggshell powder in rat calvarial defects. The study found that while the eggshell powder was biocompatible, it did not enhance bone regeneration compared to controls ( 18 ) . Conclusion Our study demonstrated that eggshell membrane (ESM) significantly enhances bone regeneration when combined with xenograft in critical-sized bone defects. The histological and histomorphometric analyses revealed that the ESM-xenograft group exhibited faster and more extensive bone formation compared to xenograft alone, with 86.53% new bone formation at 8 weeks versus 55.98% in the control group. The collagen-rich composition of ESM, along with its osteoconductive properties, contributed to improved bone healing, making it a promising, cost-effective, and sustainable biomaterial for bone defect repair. Declarations Ethical Approval: All experimental procedures were approved by the Ethics Committee of the Faculty of Dentistry, Minia University (Approval No. 774/2023) and conducted in accordance with international guidelines for the care and use of laboratory animals.This study investigating the regenerative potential of eggshell membrane in the treatment of critical-sized bone defects in [animal model, e.g., rats] was conducted and reported in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. All experimental procedures were ethically approved by [name of institutional ethics committee], and efforts were made to minimize animal suffering. The manuscript includes detailed information on the study design, sample size calculation, randomization, blinding, outcome measures, statistical methods, and ethical considerations to ensure transparency and reproducibility. Data availability Data availability statement: Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the corresponding author ( [email protected] ) upon reasonable request. Funds: No fund References Shi W, Jiang Y, Wu T, Zhang Y, Li T. Advancements in drug-loaded hydrogel systems for bone defect repair. Regen Ther. 2024;25:174-185. Tsang STJ, van Rensburg AJ, van Heerden J, et al. The management of critical bone defects. Eur J Orthop Surg Tra Fernández RF, Bucchi C, Navarro P, Beltrán V, Borie E. Bone grafts utilized in dentistry: an analysis of patients' preferences. BMC Med Ethics. 2015;16(1):1-6. Bakry S. Evaluation of bone quality and quantity by using bovine xenograft versus β-TCP for maxillary sinus augmentation. Egypt Dent J. 2019;65(4):3329-3338. Soares MQS, Van Dessel J, Jacobs R, et al. Morphometric evaluation of bone regeneration in segmental mandibular bone defects. Int J Implant Dent. 2019;5:1-9. Opris H, Baciut M, Moldovan M, et al. Comparison of the eggshell and the porcine pericardium membranes for guided tissue regeneration. Biomedicines. 2023;11(9):2529. Torres-Mansilla A, Hincke M, Voltes A, et al. Eggshell membrane as a biomaterial for bone regeneration. Polymers. 2023;15(6):1342. Ruff KJ, Endres JR, Clewell AE, Szabo JR, Schauss AG. Safety evaluation of a natural eggshell membrane-derived product. Food Chem Toxicol. 2012;50(3-4):604-611. Fernandes GVO, Castro F, Pereira RM, et al. Critical-size defects reconstruction with four different bone grafts associated with e-PTFE membrane. Clin Oral Implants Res. 2024;35(2):167-178. Torres-Mansilla A, Hincke M, Voltes A, et al. Eggshell membrane as a biomaterial for bone regeneration. Polymers. 2023;15(6):1342. Polo-Corrales L, Latorre-Esteves M, Ramirez-Vick JE. Scaffold design for bone regeneration. J Nanosci Nanotechnol. 2014;14(1):15-56. Sengupta P. The laboratory rat: relating its age with human's. Int J Prev Med. 2013;4(6):624. Sah MK, Rath SN. Eggshell membrane: A natural scaffold for bone tissue engineering. J Biomater Sci Polym Ed. 2016;27(7):629-648. Kavarthapu A, Malaiappan S. Comparative evaluation of demineralized bone matrix and type II collagen membrane versus eggshell powder. Indian J Dent Res. 2019;30(6):877-880. Wu X, Gauntlett O, Zhang T, et al. Eggshell microparticle reinforced scaffolds for regeneration of critical sized cranial defects. ACS Appl Mater Interfaces. 2021;13(51):60921-60932. Rachna M, Nandita S, Rashmi KS, et al. Eggshell membrane as a regenerative material in alveolar bone grafting. Clin Ter. 2024;175(4):219-225. Arias JL, Gonzalez A, Fernandez MS, Gonzalez C, Saez D, Arias JI. Eggshell membrane as a biodegradable bone regeneration inhibitor. J Tissue Eng Regen Med. 2008;2(4):228-235. Dupoirieux L, Pourquier D, Picot MC, Neves M. Comparative study of three different membranes for guided bone regeneration of rat cranial defects. Int J Oral Maxillofac Surg. 2001;30(1):58-62. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7122120","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":489851602,"identity":"941e7949-4dda-43b7-a0b6-0bd8c07cd6ce","order_by":0,"name":"Hossam Ibrahem Abdelrasoul","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/0lEQVRIiWNgGAWjYBACAwglkcDAwAOkKyAiEiRoOUO8FgaIFsY2IrSYs7c//HSDwSLPvP3swUc3591J3M7AfPA2D4ONXQMOLZY9Z4ylcxgkimXO5CUb5257lrizgS3ZmochLRmXFoMbOQwgLYkzGHLMpHO3HU7ccIDHTJqH4XAyTr/cSH/8G6yF/43579w5IC3834Ba/uPRkmAGsUUix4w5twFsCxtQywE7nFrOnDGzzjGQKJaQeAP01LHDxjub2Ywt5xgkJ+DUcrz98e2ciro8Cf4cw885NYdlt7M3P7zxpsLOHpcWqEZkDjNEJLEBvx4sgIAto2AUjIJRMIIAAFK+VE41E2j7AAAAAElFTkSuQmCC","orcid":"","institution":"Nahda University","correspondingAuthor":true,"prefix":"","firstName":"Hossam","middleName":"Ibrahem","lastName":"Abdelrasoul","suffix":""},{"id":489851603,"identity":"02d59096-2ef4-42b7-98b1-75a73a49789f","order_by":1,"name":"Enas Alaa Eldin AbdElaziz","email":"","orcid":"","institution":"Minia University","correspondingAuthor":false,"prefix":"","firstName":"Enas","middleName":"Alaa Eldin","lastName":"AbdElaziz","suffix":""},{"id":489851605,"identity":"53ae1263-011e-4d27-bb7e-75f985453f5e","order_by":2,"name":"Eman Alaa Eldin AbdElaziz","email":"","orcid":"","institution":"Minia University","correspondingAuthor":false,"prefix":"","firstName":"Eman","middleName":"Alaa Eldin","lastName":"AbdElaziz","suffix":""},{"id":489851607,"identity":"c8ed9778-d53a-4598-b0ae-8d497b0ca2ef","order_by":3,"name":"Aya Allah kamal Abd Elal","email":"","orcid":"","institution":"Minia University","correspondingAuthor":false,"prefix":"","firstName":"Aya","middleName":"Allah kamal Abd","lastName":"Elal","suffix":""},{"id":489851608,"identity":"2e30fdcb-aa72-4544-b6cb-ecadd69db010","order_by":4,"name":"Ahmed Abd Allah Khalil","email":"","orcid":"","institution":"Minia University","correspondingAuthor":false,"prefix":"","firstName":"Ahmed","middleName":"Abd Allah","lastName":"Khalil","suffix":""}],"badges":[],"createdAt":"2025-07-14 14:23:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7122120/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7122120/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":87574371,"identity":"a5102cc7-a33c-4537-adf7-fd15e1b6d61e","added_by":"auto","created_at":"2025-07-25 11:25:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":86852,"visible":true,"origin":"","legend":"\u003cp\u003eNEM Natural Eggshell Membrane 500 mg\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7122120/v1/c6aaa67a2beecbcdcb6e9ec8.png"},{"id":87574373,"identity":"fbc3c6b9-4cc2-48f7-b86f-4942fda618c8","added_by":"auto","created_at":"2025-07-25 11:25:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":334182,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7122120/v1/8237ca2f49ebd6a99b32a8b7.png"},{"id":87574374,"identity":"ea1ff566-6c18-4bda-a0d0-a15f22276c88","added_by":"auto","created_at":"2025-07-25 11:25:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":312950,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7122120/v1/b9408df7183fb53a1f326f59.png"},{"id":87574372,"identity":"91ce8a64-ffed-4c88-b363-38526947b86f","added_by":"auto","created_at":"2025-07-25 11:25:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":552081,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7122120/v1/b6cdca44b35136623e078fc4.png"},{"id":87574381,"identity":"ba70602f-4366-4cc6-93d7-386e9a971ffa","added_by":"auto","created_at":"2025-07-25 11:25:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":674476,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-7122120/v1/b710044544ac104bde87c33b.png"},{"id":90153680,"identity":"52c6363f-8062-4f74-8d4a-a7492adb9b6c","added_by":"auto","created_at":"2025-08-29 07:39:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2669728,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7122120/v1/56ec76ef-a520-4d02-9630-2f8a8b1c89c9.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Regenerative Capacity of Eggshell Membrane in Management of Critical-Sized Bone Defect: An Experimental Study","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBone abnormalities resulting from trauma, bone tumours, or infection are prevalent and significant clinical manifestations. Bone tissue has significant challenges in self-repair and regeneration without external intervention. In recent decades, bone transplantation has consistently been regarded as the \"gold standard\" for addressing bone abnormalities (1).\u003c/p\u003e\u003cp\u003eA critical bone defect is characterized as one that will not mend without intervention, with segmental abnormalities above 60mm often considered significant defects. Treatment success was characterized by bone union (2).\u003c/p\u003e\u003cp\u003eNumerous operations necessitate the utilization of bone grafts to restore or augment bone volume that has been diminished owing to systemic diseases, periodontal abnormalities, tooth loss, or other disorders. Progress in contemporary medicine has resulted in a greater availability of novel biomaterials that can augment bone volume restoration. These biomaterials can be sourced from the patient's own body, other persons, animals, or generated synthetically. Notwithstanding these advancements, minimal study has been undertaken about patients' perspectives on various bone transplants (3).\u003c/p\u003e\u003cp\u003eXenograft materials are extensively utilized and sourced from several animal species, predominantly from bovine origins. Anorganic bovine bone is one of the most often utilized xenografts due to its similarity to human bone. It demonstrates favourable biocompatibility, osteoconductivity, and volumetric stability (4). Bovine bone grafts are extensively utilized in dentistry for directed tissue regeneration and facilitate new bone development in direct contact with the graft.\u003c/p\u003e\u003cp\u003eESM is a plentiful industrial and home waste that is ecologically benign, non-toxic, composed of various natural proteins, and amenable to modification by carbonization and dissolution. It has been employed in many technical applications, including as capacitors, batteries, solar cells, catalysis, biosensors, cell culture, wound healing, and bone replacements (6). ESM functions as a biopolymer network in shell mineralization during oviposition, facilitating mineral precipitation within 24 hours. The extracellular matrix fibres consist of collagens (I, V, and X), comprising approximately 10% of the total protein composition, glycosaminoglycans, egg white proteins, and eggshell matrix proteins, with collagens constituting the core of the fibres.\u003c/p\u003e\u003cp\u003eNatural Eggshell Membrane (NEM\u0026Ograve;) is an innovative dietary component that comprises naturally occurring glycosaminoglycans and proteins vital for the preservation of healthy joints and connective tissues. NEM\u0026Ograve; underwent safety assessment through in vitro and in vivo toxicology investigations. This encompassed assessments for cytotoxicity, genotoxicity, and acute oral toxicity (8).\u003c/p\u003e\u003cp\u003eThe objective of this study was to assess the possible impact of eggshell membrane on bone regeneration in critical-sized bone lesions by histological and histomorphometric analysis. It also examines the efficacy of ESM in conjunction with a xenograft to improve bone repair in the identical animal.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cem\u003e\u003cu\u003e\u0026nbsp;Ethical Regulation\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;All experiments were conducted at the animal house of the Faculty of Pharmacy, Minia University, Egypt, following approval from the Ethics Committee of the Faculty of Dentistry, Minia University (774/2023).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eSample Size Calculation and Grouping\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;A sample size of 32 rats was calculated using G*Power software (version 3.1.9.7) to detect an effect size of 0.30 with 90% power at p ff 0.05. Thirty-two male Wistar albino rats (150\u0026ndash;350 g) were housed under controlled conditions (21\u0026ndash;24\u0026deg;C, 40\u0026ndash;60% humidity, 12-hour light\u0026ndash;dark cycle) with access to standard food and water. Each rat received bilateral femoral critical-sized defects (2 mm depth, 2 mm width), divided into two groups: Group I (control, n=32) filled with xenograft alone (left femur), and Group II (test, n=32) filled with ESM combined with xenograft (right femur). Sixteen rats were sacrificed at 4 weeks and 16 at 8 weeks post-operation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eMaterial Preparation\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Chitosan-H (Lot 337) and eggshell (Lot 65H0427) (Sigma Chem. Co., St. Louis, MO) were used to prepare ESM gel. Chitosan-H (1%) was dissolved in 1% acetic acid, mixed with eggshell (500 mg) fig (1). at 400 rpm, and cross-linked with 0.1% tripolyphosphate pentasodium salt (TPP). Xenograft was prepared with saline for the control group and mixed with ESM gel for the test group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eAnesthetic Protocol\u0026nbsp;\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eRats fasted for 12 hours before surgery. Anesthesia was induced with intramuscular ketamine (50 mg, Troikaa Pharmaceuticals Ltd) and xylazine (20 mg, ADWIA, Egypt), supplemented with local 2% lidocaine (Alexandria Co. for Pharmaceuticals, Egypt).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eSurgical Procedure\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The femur was exposed via a 7-cm incision, and defects (2 mm depth, 2 mm width)\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eFig (2)\u0026nbsp;were created using a contra-angle low-speed handpiece with a round bur under saline irrigation. Control defects received xenograft alone\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eFig (3,A), while test defects received ESM-xenograft mix\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eFig (3,B). Flaps were sutured with 3/0 black silk, and the area was disinfected with iodine and 70% ethanol.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003ePost-Surgical Care\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Post-operatively, rats received intramuscular oxytetracycline (1.0 cm\u0026sup3;/kg, Pfizer, Egypt) for 3 days and an analgesic (0.05 mg/kg). Full weight-bearing was permitted immediately.in addition to the administration of analgesics, animal discomfort and distress were monitored through a comprehensive set of behavioral and physiological indicators. Trained personnel performed daily observations (or more frequently as needed) to assess clinical signs including, but not limited to: reduced grooming, altered posture or locomotion, changes in appetite or water intake, vocalization, social withdrawal, and abnormal respiratory patterns. Body weight was measured regularly as a quantitative measure of well-being, with a loss of more than 15\u0026ndash;20% from baseline triggering intervention or consideration for humane euthanasia. If any animal exhibited persistent or severe signs of distress unrelieved by analgesics, humane endpoints were applied in accordance with IACUC/ethical guidelines, prioritizing animal welfare.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eAssessment\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Femurs were harvested after sacrifice (Nembutal overdose) at 4 and 8 weeks. Samples were fixed in 4% paraformaldehyde, decalcified in 10% EDTA for 5 weeks, and embedded in paraffin. Sections (5 mm) were stained with hematoxylin and eosin (H\u0026amp;E) and Masson trichrome (MT). Histomorphometric analysis was performed using ImageJ software to measure the area percentage of new bone and collagen at 10X magnification.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cu\u003eStatistical Analysis\u003c/u\u003e\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Data were analyzed using one-way ANOVA followed by post hoc tests or equivalent nonparametric tests. Paired and independent T-tests assessed intra- and inter-group differences (p 0.05). Pearson correlation tested the relationship between bone regeneration and collagen percentage.\u0026nbsp;\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eHistological Examination\u003c/span\u003e\u003c/p\u003e\u003cp\u003eNo inflammation was observed in either group. At 4 weeks, control defects showed minimal bone regeneration with dense fibrovascular tissue, while test defects exhibited more woven bone and fibrous tissue. At 8 weeks, control defects displayed increased trabecular bone, while test defects showed abundant mature lamellar bone Fig.\u0026nbsp;(4). Masson trichrome staining revealed higher collagen deposition in the test group at 4 weeks, with more mature bone (red staining) at 8 weeks in both groups, though immature bone persisted Fig.\u0026nbsp;(5) .\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eHistomorphometric Analysis\u003c/span\u003e\u003c/p\u003e\u003cp\u003eNew bone formation increased significantly from 4 to 8 weeks in both groups. The control group showed 15.44\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12% at 4 weeks and 55.98\u0026thinsp;\u0026plusmn;\u0026thinsp;2.53% at 8 weeks, while the test group achieved 27.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.57% at 4 weeks and 86.53\u0026thinsp;\u0026plusmn;\u0026thinsp;3.81% at 8 weeks (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). Collagen percentage was higher in the test group at 4 weeks (54.79\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52% vs. 24.48\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73%) and 8 weeks (23.01\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69% vs. 11.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.61%) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). A strong negative correlation between bone regeneration and collagen percentage was observed (r = -0.989 for control, r = -0.954 for test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001).\u003c/p\u003e\u003cp\u003e\u003cb\u003eTable\u0026nbsp;(1\u003c/b\u003e): Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and Percentage of change of regeneration % for the two groups at the two-time intervales.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4 Weeks\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8 Weeks\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePercentage of Change\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eP-value*\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e15.44\u0026thinsp;\u0026plusmn;\u0026thinsp;2.12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e55.98\u0026thinsp;\u0026plusmn;\u0026thinsp;2.53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-270.1\u0026thinsp;\u0026plusmn;\u0026thinsp;60.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003csup\u003eHS\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTest\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e27.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1.57\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e86.53\u0026thinsp;\u0026plusmn;\u0026thinsp;3.81\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e-213.1\u0026thinsp;\u0026plusmn;\u0026thinsp;19.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003csup\u003eHS\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP-value**\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003csup\u003eHS\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003csup\u003eHS\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003csup\u003eHS\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003e-* P-value for Intra-group comparison between the two-time intervals (Paired T-test).\u003c/em\u003e\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003e-** Overall P-value for Inter-group comparison between the two groups (ANOVA Test).\u003c/em\u003e\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003e- S\u0026thinsp;=\u0026thinsp;Statistically significant at P\u0026thinsp;\u0026le;\u0026thinsp;0.05 - NS\u0026thinsp;=\u0026thinsp;Non-significant P\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/em\u003e\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003e\u003cem\u003e- HS\u0026thinsp;=\u0026thinsp;Highly significant at P\u0026thinsp;\u0026le;\u0026thinsp;0.001\u003c/em\u003e\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003e.\u003c/p\u003e\u003cp\u003eII) Assessment of Collagen fibers percentage (Collagen %)\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eBoth the Control and Test groups demonstrated a significant decrease in bone collagen over time, with the Test group consistently outperforming the Control group at both 4 and 8 weeks.\u003c/p\u003e\u003c/li\u003e\u003cli\u003e\u003cp\u003eThe differences between the groups were statistically highly significant at all measured intervals, as confirmed by the Paired T-test and Independent T-test as showen in Table\u0026nbsp;2)\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eTable\u0026nbsp;(2): Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD and Percentage of change of collagen % for the two groups at the two-time intervales.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4 Weeks\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8 Weeks\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePercentage of Change\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eP-value*\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e24.48\u0026thinsp;\u0026plusmn;\u0026thinsp;1.73\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e11.43\u0026thinsp;\u0026plusmn;\u0026thinsp;1.61\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e53.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003csup\u003eHS\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTest\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e54.79\u0026thinsp;\u0026plusmn;\u0026thinsp;1.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e23.01\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e58.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003csup\u003eHS\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP-value**\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003csup\u003eHS\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003csup\u003eHS\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.432\u003csup\u003eNS\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eIII) Correlation between Regeneration % and Collagen % Results\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eThe result of the Pearson correlation test for the change from 4 to 8 weeks between the Regeneration % and Collagen % are tabulated in Table\u0026nbsp;3.; from the results we can conclude the following: There was a strong negative correlation between Regeneration % and Collagen % in the two groups and r values were \u0026minus;\u0026thinsp;0.989 and \u0026minus;\u0026thinsp;0.954 for Control and Test groups respectively, and this correlation was statistically highly significant (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) as shown in \u003cb\u003eTable\u0026nbsp;(3\u003c/b\u003e)\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTable\u0026nbsp;(3\u003c/b\u003e): Correlation between Regeneration % and Collagen % from 4 to 8 weeks for the two studied groups.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabc\" border=\"1\"\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003er**\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eCorrelation type\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eControl\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026minus;\u0026thinsp;0.989\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003csup\u003eHS\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eStrong negative\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTest\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e\u0026minus;\u0026thinsp;0.954\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u0026lt;\u0026thinsp;0.001\u003csup\u003eHS\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eStrong negative\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e- ** Pearson Correlation value -\u003csup\u003eHS\u003c/sup\u003e Highly significant (Correlation is significant at the 0.01 level).\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eCritical-size bone loss can be caused by trauma, pathologies, tumor resections, or tooth loss affecting patients' esthetic and function. Thus, another point of debate raised is what is the best biomaterial to treat critical-size defects. Several biomaterials are available and have different characteristics, divided into autogenous/autograft, allogenous/allograft, xenograft (mainly bovine), and alloplastic/synthetic \u003csup\u003e(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eSynthetic and bovine bones have been options as substitute biomaterials in trying to overcome possible adversities of the autograft (donor site, increased morbidity, limited bone volume, and postoperative pain). They are considered to have only osteoconduction capacity \u003csup\u003e(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eEggshell waste is a readily available biological waste product that can be repurposed for medical applications. Utilizing ESM for bone regeneration aligns with sustainable development by reducing waste and offering a low-cost alternative to synthetic biomaterials \u003csup\u003e(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eESM, when combined with other regenerative strategies (e.g., growth factors, stem cells, or 3D scaffolds), can significantly enhance osteogenesis and angiogenesis in critical bone defects. This suggests its potential in clinical applications for orthopedic and maxillofacial reconstructions \u003csup\u003e(\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWe use experimental rats in my research is due to their genetic, biological, and behavioural similarities to humans. Scientists use rats in experiments to study diseases, test drugs, and explore genetic functions. Here are some key reasons why rats are widely used in scientific research \u003csup\u003e(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOur results show an increase in bone regeneration in both groups over time because Eggshell membrane is a natural biomaterial rich in collagen, glycosaminoglycans, and bioactive proteins, which are essential for bone healing. Studies suggest that ESM has osteoconductive properties, making it a promising material for bone regeneration \u003csup\u003e(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eOur result is matching with study by Kavarthapu and Malaiappan(14) who assessed demineralized bone matrix with a collagen membrane against eggshell powder and its membrane in a rat model. Histological evaluations revealed significant new bone formation in both groups, with no notable differences between them. This suggests that eggshell powder, combined with its membrane, could serve as a potential graft material \u003csup\u003e(\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWu et al. \u003csup\u003e(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e)\u003c/sup\u003e reveal that effects of microparticles of whole eggshells, eggshells without a membrane, and a pristine eggshell membrane on osteogenic differentiation in protein-derived hydrogels. The in vitro studies showed that gels reinforced with eggshells with and without a membrane demonstrated comparable cellular proliferation, osteogenic gene expression, and osteogenic differentiation. Subsequently, in vivo studies were performed to implant eggshell microparticle-reinforced composite hydrogel scaffolds into critical-sized cranial defects in Sprague Dawley (SD) rats for up to 12 weeks to study bone regeneration \u003csup\u003e(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eRachna et al. \u003csup\u003e(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e)\u003c/sup\u003e reveals that eggshell membrane has good regenerative properties and excellent osteogenic capacity when used to assess the effect of the eggshell membrane on alveolar bone regeneration after tooth extraction \u003csup\u003e(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn contrast the our study, Arias et al. \u003csup\u003e(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e)\u003c/sup\u003e demonstrate the first time that eggshell membranes as interpositional material in rabbit osteotomized ulnar experiments acted as an active barrier against bone bridging \u003csup\u003e(\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe research by Dupoirieux et al \u003csup\u003e(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e)\u003c/sup\u003e investigated the use of eggshell powder in rat calvarial defects. The study found that while the eggshell powder was biocompatible, it did not enhance bone regeneration compared to controls \u003csup\u003e(\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e)\u003c/sup\u003e.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study demonstrated that eggshell membrane (ESM) significantly enhances bone regeneration when combined with xenograft in critical-sized bone defects. The histological and histomorphometric analyses revealed that the ESM-xenograft group exhibited faster and more extensive bone formation compared to xenograft alone, with 86.53% new bone formation at 8 weeks versus 55.98% in the control group. The collagen-rich composition of ESM, along with its osteoconductive properties, contributed to improved bone healing, making it a promising, cost-effective, and sustainable biomaterial for bone defect repair.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental procedures were approved by the Ethics Committee of the Faculty of Dentistry, Minia University (Approval No. 774/2023) and conducted in accordance with international guidelines for the care and use of laboratory animals.This study investigating the regenerative potential of eggshell membrane in the treatment of critical-sized bone defects in [animal model, e.g., rats] was conducted and reported in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. All experimental procedures were ethically approved by [name of institutional ethics committee], and efforts were made to minimize animal suffering. The manuscript includes detailed information on the study design, sample size calculation, randomization, blinding, outcome measures, statistical methods, and ethical considerations to ensure transparency and reproducibility.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData availability statement: Data underlying the results presented in this paper are not publicly available at this time but may be obtained from the corresponding author ([email protected]) upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunds:\u0026nbsp;\u003c/strong\u003eNo fund\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eShi W, Jiang Y, Wu T, Zhang Y, Li T. Advancements in drug-loaded hydrogel systems for bone defect repair. Regen Ther. 2024;25:174-185.\u003c/li\u003e\n \u003cli\u003eTsang STJ, van Rensburg AJ, van Heerden J, et al. The management of critical bone defects. Eur J Orthop Surg Tra\u003c/li\u003e\n \u003cli\u003eFern\u0026aacute;ndez RF, Bucchi C, Navarro P, Beltr\u0026aacute;n V, Borie E. Bone grafts utilized in dentistry: an analysis of patients\u0026apos; preferences. BMC Med Ethics. 2015;16(1):1-6.\u003c/li\u003e\n \u003cli\u003eBakry S. Evaluation of bone quality and quantity by using bovine xenograft versus \u0026beta;-TCP for maxillary sinus augmentation. Egypt Dent J. 2019;65(4):3329-3338.\u003c/li\u003e\n \u003cli\u003eSoares MQS, Van Dessel J, Jacobs R, et al. Morphometric evaluation of bone regeneration in segmental mandibular bone defects. Int J Implant Dent. 2019;5:1-9.\u003c/li\u003e\n \u003cli\u003eOpris H, Baciut M, Moldovan M, et al. Comparison of the eggshell and the porcine pericardium membranes for guided tissue regeneration. Biomedicines. 2023;11(9):2529.\u003c/li\u003e\n \u003cli\u003eTorres-Mansilla A, Hincke M, Voltes A, et al. Eggshell membrane as a biomaterial for bone regeneration. Polymers. 2023;15(6):1342.\u003c/li\u003e\n \u003cli\u003eRuff KJ, Endres JR, Clewell AE, Szabo JR, Schauss AG. Safety evaluation of a natural eggshell membrane-derived product. Food Chem Toxicol. 2012;50(3-4):604-611.\u003c/li\u003e\n \u003cli\u003eFernandes GVO, Castro F, Pereira RM, et al. Critical-size defects reconstruction with four different bone grafts associated with e-PTFE membrane. Clin Oral Implants Res. 2024;35(2):167-178.\u003c/li\u003e\n \u003cli\u003eTorres-Mansilla A, Hincke M, Voltes A, et al. Eggshell membrane as a biomaterial for bone regeneration. Polymers. 2023;15(6):1342.\u003c/li\u003e\n \u003cli\u003ePolo-Corrales L, Latorre-Esteves M, Ramirez-Vick JE. Scaffold design for bone regeneration. J Nanosci Nanotechnol. 2014;14(1):15-56.\u003c/li\u003e\n \u003cli\u003eSengupta P. The laboratory rat: relating its age with human\u0026apos;s. Int J Prev Med. 2013;4(6):624.\u003c/li\u003e\n \u003cli\u003eSah MK, Rath SN. Eggshell membrane: A natural scaffold for bone tissue engineering. J Biomater Sci Polym Ed. 2016;27(7):629-648.\u003c/li\u003e\n \u003cli\u003eKavarthapu A, Malaiappan S. Comparative evaluation of demineralized bone matrix and type II collagen membrane versus eggshell powder. Indian J Dent Res. 2019;30(6):877-880.\u003c/li\u003e\n \u003cli\u003eWu X, Gauntlett O, Zhang T, et al. Eggshell microparticle reinforced scaffolds for regeneration of critical sized cranial defects. ACS Appl Mater Interfaces. 2021;13(51):60921-60932.\u003c/li\u003e\n \u003cli\u003eRachna M, Nandita S, Rashmi KS, et al. Eggshell membrane as a regenerative material in alveolar bone grafting. Clin Ter. 2024;175(4):219-225.\u003c/li\u003e\n \u003cli\u003eArias JL, Gonzalez A, Fernandez MS, Gonzalez C, Saez D, Arias JI. Eggshell membrane as a biodegradable bone regeneration inhibitor. J Tissue Eng Regen Med. 2008;2(4):228-235.\u003c/li\u003e\n \u003cli\u003eDupoirieux L, Pourquier D, Picot MC, Neves M. Comparative study of three different membranes for guided bone regeneration of rat cranial defects. Int J Oral Maxillofac Surg. 2001;30(1):58-62.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Eggshell membrane (ESM), Xenograft, Critical-sized bone defect (CSBD), Bone regeneration, Histomorphometry","lastPublishedDoi":"10.21203/rs.3.rs-7122120/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7122120/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjectives:\u003c/strong\u003e This study aimed to evaluate the regenerative potential of eggshell membrane (ESM) in critical-sized bone defects (CSBD) and its synergistic effect with xenograft bone through histological and histomorphometric analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials and Methods:\u003c/strong\u003e Thirty-two male Wistar rats were used, each receiving bilateral femoral CSBDs (2 mm diameter/depth). Defects were divided into:\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cstrong\u003eGroup I (Control):\u003c/strong\u003e Filled with xenograft alone (n=32 defects).\u003c/li\u003e\n \u003cli\u003e\u003cstrong\u003eGroup II (Test):\u003c/strong\u003e Filled with ESM-xenograft composite (n=32 defects).\u003cbr\u003e\n\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp;Rats were sacrificed at 4- and 8-weeks post-operation. Bone regeneration was assessed via H\u0026amp;E and Masson’s trichrome staining, with \u0026nbsp;histomorphometric analysis using ImageJ software.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003eResults:\u003c/strong\u003e The Test group showed significantly higher bone regeneration (27.7 ± 1.57% at 4 weeks; 86.53 ± 3.81% at 8 weeks) compared to the Control group (15.44 ± 2.12% and 55.98 ± 2.53%, respectively; p\u0026lt;0.001). Collagen deposition was also greater in the Test group (54.79 ± 1.52% vs. 24.48 ± 1.73% at 4 weeks). Histologically, the Test group exhibited mature lamellar bone by 8 weeks, while the Control group showed slower maturation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions:\u003c/strong\u003e ESM enhances bone regeneration synergistically with xenograft, offering a promising, cost-effective biomaterial for CSBD management.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUnstructured Abstract:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis experimental study assessed eggshell membrane (ESM) combined with xenograft for critical-sized bone defect (CSBD) repair in 32 rats. CSBDs were created bilaterally in femurs, treated with xenograft alone (Control) or ESM-xenograft (Test), and evaluated histologically at 4/8 weeks. Results demonstrated superior bone regeneration and collagen synthesis in the Test group (p\u0026lt;0.001), with 86.53% new bone formation by 8 weeks versus 55.98% in Controls. ESM’s osteoconductive properties suggest clinical potential for bone defect therapies.\u003c/p\u003e","manuscriptTitle":"Regenerative Capacity of Eggshell Membrane in Management of Critical-Sized Bone Defect: An Experimental Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-25 11:25:52","doi":"10.21203/rs.3.rs-7122120/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a5a973dd-3768-414b-a929-9cca786a5404","owner":[],"postedDate":"July 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":52007625,"name":"Biological sciences/Biotechnology"},{"id":52007626,"name":"Physical sciences/Materials science"},{"id":52007627,"name":"Health sciences/Medical research"},{"id":52007628,"name":"Biological sciences/Stem cells"}],"tags":[],"updatedAt":"2025-08-29T07:38:50+00:00","versionOfRecord":[],"versionCreatedAt":"2025-07-25 11:25:52","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7122120","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7122120","identity":"rs-7122120","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}