Decellularized bone extracellular matrix as a promising natural substrate for bone tissue engineering

Abstract

Aim: The decellularization process aims to remove cellular components from the tissues while preserving the ultrastructural composition of the extracellular matrix (ECM). Decellularization of bone is gaining attention as a biological scaffold due to its unique histoarchitecture, which consists of both organic and inorganic compounds. This study aims to develop a biological bone ECM using a novel decellularization method for bone regeneration. Methods: Rabbit and rat bone tissues were decellularized using a novel process that combines physical, chemical, and enzymatic methods with 0.1% SDS. Bone tissues were evaluated in terms of histology, biochemistry, and biomechanical tests, both before and after decellularization. Additionally, decellularized bone substitutes were recellularized with preosteoblast cells to assess the cytotoxic effect of the decellularization process. Results: Our method effectively removes cellular components while preserving both organic and inorganic compounds. We achieved a 95% in DNA content for rabbit bone and 92% for rat bone. The biochemical and biomechanical properties remained unchanged, and mineralization features were preserved after decellularization. The cell culture results revealed that decellularized bone extracellular matrix (dbECM) is biocompatible, bioactive, and provides a suitable environment for cell growth. Conclusions: This study demonstrates that our novel decellularization method effectively develops biological bone ECM containing both organic and inorganic compounds while utilizing minimal chemical concentration and incubation time. It is foreseen that the resulting decellularized bone could serve as a biological substitute, providing a favorable microenvironment for bone regeneration.

Keywords

decellularization / bone tissue / bone extracellular matrix / bone tissue engineering / mineralization

References

[1] Ansari M. Bone tissue regeneration: biology, strategies and interface studies. Prog Biomater. 2019; 8: 223-37.

[2] Calori GM, Mazza E, Colombo M, Ripamonti C. The use of bone-graft substitutes in large bone defects: any specific needs? Injury. 2011; 42: S56-63.

[3] Oryan A, Kamali A, Moshiri A, Eslaminejad MB. Role of Mesenchymal Stem Cells in Bone Regenerative Medicine: What Is the Evidence? Cells Tissues Organs. 2017; 204: 59-83.

[4] Bracey DN, Seyler TM, Jinnah AH, Lively MO, Willey JS, Smith TL, et al. A Decellularized Porcine Xenograft-Derived Bone Scaffold for Clinical Use as a Bone Graft Substitute: A Critical Evaluation of Processing and Structure. J Funct Biomater. 2018; 9: 45.

[5] Bracey DN, Jinnah AH, Willey JS, Seyler TM, Hutchinson ID, Whitlock PW, et al. Investigating the Osteoinductive Potential of a Decellularized Xenograft Bone Substitute. Cells Tissues Organs. 2019; 207: 97-113.

[6] Rossi F, Santoro M, Perale G. Polymeric scaffolds as stem cell carriers in bone repair. J Tissue Eng Regen Med. 2015; 9: 1093-119.

[7] Qu H, Fu H, Han Z, Sun Y. Biomaterials for bone tissue engineering scaffolds: a review. RSC Adv. 2019; 9: 26252-62.

[8] Rajangam T, An SSA. Fibrinogen and fibrin based micro and nano scaffolds incorporated with drugs, proteins, cells and genes for therapeutic biomedical applications. Int J Nanomedicine. 2013; 8: 3641-62.

[9] Kruger TE, Miller AH, Wang J. Collagen scaffolds in bone sialoprotein-mediated bone regeneration. Sci World J. 2013; 2013: 812718.

[10] Denry I, Kuhn LT. Design and characterization of calcium phosphate ceramic scaffolds for bone tissue engineering. Dent Mater. 2016; 32: 43-53.

[11] Gloria A, Santis RD, Ambrosio L. Polymer-based composite scaffolds for tissue engineering. J Appl Biomater Biomech. 2010; 8: 57-67.

[12] Nikolova MP, Chavali MS. Recent advances in biomaterials for 3D scaffolds: A review. Bioact Mater. 2019; 4: 271-92.

[13] Lin X, Patil S, Gao Y, Qian A. The Bone Extracellular Matrix in Bone Formation and Regeneration. Front Pharmacol. 2020; 11: 757.

[14] Badylak SF, Kropp B, McPherson T, Liang H, Snyder PW. Small intestinal submucosa: a rapidly resorbed bioscaffold for augmentation cystoplasty in a dog model. Tissue Eng. 1998; 4: 379-87.

[15] Badylak SF, Freytes DO, Gilbert TW. Extracellular matrix as a biological scaffold material: Structure and function. Acta Biomater. 2009; 5: 1-13.

[16] Badylak SF, Freytes DO, Gilbert TW. Reprint of: Extracellular matrix as a biological scaffold material: Structure and function. Acta Biomater. 2015; 23: S17-26.

[17] Fernández-Pérez J, Ahearne M. The impact of decellularization methods on extracellular matrix derived hydrogels. Sci Rep. 2019; 9: 14933.

[18] Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole organ decellularization processes. Biomaterials. 2011; 32: 3233-43.

[19] Chen F, Yoo JJ, Atala A. Acellular collagen matrix as a possible “off the shelf” biomaterial for urethral repair. Urology. 1999; 54: 407-10.

[20] Verstegen MMA, Willemse J, van den Hoek S, Kremers G, Luider TM, van Huizen NA, et al. Decellularization of Whole Human Liver Grafts Using Controlled Perfusion for Transplantable Organ Bioscaffolds. Stem Cells Dev. 2017; 26: 1304-15.

[21] Armour AD, Fish JS, Woodhouse KA, Semple JL. A comparison of human and porcine acellularized dermis: interactions with human fibroblasts in vitro. Plast Reconstr Surg. 2006; 117: 845-56.

[22] Luc G, Charles G, Gronnier C, Cabau M, Kalisky C, Meulle M, et al. Decellularized and matured esophageal scaffold for circumferential esophagus replacement: Proof of concept in a pig model. Biomaterials. 2018; 175: 1-18.

[23] Gilbert TW, Sellaro TL, Badylak SF. Decellularization of tissues and organs. Biomaterials. 2006; 27: 3675-83.

[24] Faulk DM, Badylak SF. Natural biomaterials for regenerative medicine applications. In: Orlando G, Lerut J, Soker S, Stratta RJ, editors. Regenerative medicine applications in organ transplantation. Boston: Academic Press; 2014. pp. 101-12.

[25] Chen G, Lv Y. Decellularized Bone Matrix Scaffold for Bone Regeneration. Methods Mol Biol. 2018; 1577: 239-54.

[26] Woods T, Gratzer PF. Effectiveness of three extraction techniques in the development of a decellularized bone-anterior cruciate ligament-bone graft. Biomaterials. 2005; 26: 7339-49.

[27] Cartmell JS, Dunn MG. Effect of chemical treatments on tendon cellularity and mechanical properties. J Biomed Mater Res. 2000; 49: 134-40.

[28] Kim YS, Majid M, Melchiorri AJ, Mikos AG. Applications of decellularized extracellular matrix in bone and cartilage tissue engineering. Bioeng Transl Med. 2018; 4: 83-95.

[29] Cartmell JS, Dunn MG. Development of cell-seeded patellar tendon allografts for anterior cruciate ligament reconstruction. Tissue Eng. 2004; 10: 1065-75.

[30] Kara A. Development of Meniscus Tissue Bio-Compatibility Using Allograft Native Scaffold [dissertation]. Izmir (Turk): Dokuz Eylül University; 2017.

[31] Porzionato A, Stocco E, Barbon S, Grandi F, Macchi V, De Caro R. Tissue-Engineered Grafts from Human Decellularized Extracellular Matrices: A Systematic Review and Future Perspectives. Int J Mol Sci. 2018; 19: 4117.

[32] Sawkins MJ, Bowen W, Dhadda P, Markides H, Sidney LE, Taylor AJ, et al. Hydrogels derived from demineralized and decellularized bone extracellular matrix. Acta Biomater. 2013; 9: 7865-73.

[33] Lee DJ, Diachina S, Lee YT, Zhao L, Zou R, Tang N, et al. Decellularized bone matrix grafts for calvaria regeneration. J Tissue Eng. 2016; 7: 2041731416680306.

[34] Rindone AN, Nyberg E, Grayson WL. 3D-Printing Composite Polycaprolactone-Decellularized Bone Matrix Scaffolds for Bone Tissue Engineering Applications. Methods Mol Biol. 2018; 1577: 209-26.

[35] Paduano F, Marrelli M, Alom N, Amer M, White LJ, Shakesheff KM, et al. Decellularized bone extracellular matrix and human dental pulp stem cells as a construct for bone regeneration. J Biomater Sci Polym Ed. 2017; 28: 730-48.

[36] Abedin E, Lari R, Shahri NM, Fereidoni M. Development of a demineralized and decellularized human epiphyseal bone scaffold for tissue engineering: A histological study. Tissue Cell. 2018; 55: 46-52.

[37] Mattioli-Belmonte M, Montemurro F, Licini C, Iezzi I, Dicarlo M, Cerqueni G, et al. Cell-Free Demineralized Bone Matrix for Mesenchymal Stem Cells Survival and Colonization. Materials (Basel). 2019; 12: 1360.

[38] Fielding G, Bose S. SiO₂ and ZnO dopants in three-dimensionally printed tricalcium phosphate bone tissue engineering scaffolds enhance osteogenesis and angiogenesis in vivo . Acta Biomater. 2013; 9: 9137-48.

[39] Hsu EL, Ghodasra JH, Ashtekar A, Nickoli MS, Lee SS, Stupp SI, et al. A comparative evaluation of factors influencing osteoinductivity among scaffolds designed for bone regeneration. Tissue Eng Part A. 2013; 19: 1764-72.

[40] Kara Özenler A. Development and characterization of novel bioink by using decellularized extracellular matrix for bone tissue engineering applications [dissertation]. Izmir (Turk): Izmir Institute of Technology; 2023.

[41] American Veterinary Medical Association. AVMA Guidelines for the Euthanasia of Animals: 2020 Edition [Internet]. Schaumburg (IL): AVMA; c2020 [cited 2025 Jul 18]. Available from: https://www.avma.org/sites/default/files/2020-02/Guidelines-on-Euthanasia-2020.pdf

[42] Bagi CM, Berryman E, Moalli MR. Comparative bone anatomy of commonly used laboratory animals: implications for drug discovery. Comp Med. 2011; 61: 76-85.

[43] Dong C, Lv Y. Application of Collagen Scaffold in Tissue Engineering: Recent Advances and New Perspectives. Polymers (Basel). 2016; 8: 42.

[44] Gupta SK, Mishra NC, Dhasmana A. Decellularization Methods for Scaffold Fabrication. Methods Mol Biol. 2018; 1577: 1-10.

[45] Zhang W, Zhu Y, Li J, Guo Q, Peng J, Liu S, et al. Cell-Derived Extracellular Matrix: Basic Characteristics and Current Applications in Orthopedic Tissue Engineering. Tissue Eng Part B Rev. 2016; 22: 193-207.

[46] Kuna VK, Xu B, Sumitran-Holgersson S. Decellularization and Recellularization Methodology for Human Saphenous Veins. J Vis Exp. 2018; 137: 57803.

[47] Laker L, Dohmen PM, Smit FE. Synergy in a detergent combination results in superior decellularized bovine pericardial extracellular matrix scaffolds. J Biomed Mater Res B Appl Biomater. 2020; 108: 2571-8.

[48] Xing S, Liu C, Xu B, Chen J, Yin D, Zhang C. Effects of various decellularization methods on histological and biomechanical properties of rabbit tendons. Exp Ther Med. 2014; 8: 628-34.

[49] Lovati AB, Bottagisio M, Moretti M. Decellularized and Engineered Tendons as Biological Substitutes: A Critical Review. Stem Cells Int. 2016; 2016: 7276150.

[50] Hoshiba T, Yamaoka T. Extracellular matrix scaffolds for tissue engineering and biological research. In: Yamaoka T, Hoshiba T, editors. Decellularized extracellular matrix: characterization, fabrication and applications. Cambridge: The Royal Society of Chemistry; 2019. pp. 1-14.

[51] Li M, Fu X, Gao H, Ji Y, Li J, Wang Y. Regulation of an osteon-like concentric microgrooved surface on osteogenesis and osteoclastogenesis. Biomaterials. 2019; 216: 119269.

[52] Yates P, Thomson J, Galea G. Processing of whole femoral head allografts: validation methodology for the reliable removal of nucleated cells, lipid and soluble proteins using a multi-step washing procedure. Cell Tissue Bank. 2005; 6: 277-85.

[53] Ibrahim T, Qureshi A, McQuillan TA, Thomson J, Galea G, Power RA. Intra-operative washing of morcellised bone allograft with pulse lavage: how effective is it in reducing blood and marrow content? Cell Tissue Bank. 2012; 13: 157-65.

[54] McIntosh L, van der Walle C. Control of Mammalian Cell Behaviour Through Mimicry of the Extracellular Matrix Environment. In: On Biomimetics. InTech; 2011.

[55] Rozario T, DeSimone DW. The extracellular matrix in development and morphogenesis: a dynamic view. Dev Biol. 2010; 341: 126-40.

[56] Smith CA, Richardson SM, Eagle MJ, Rooney P, Board T, Hoyland JA. The use of a novel bone allograft wash process to generate a biocompatible, mechanically stable and osteoinductive biological scaffold for use in bone tissue engineering. J Tissue Eng Regen Med. 2015; 9: 595-604.

[57] Safdari M, Bibak B, Soltani H, Hashemi J. Recent advancements in decellularized matrix technology for bone tissue engineering. Differentiation. 2021; 121: 25-34.

[58] Mendoza-Novelo B, Avila EE, Cauich-Rodríguez JV, Jorge-Herrero E, Rojo FJ, Guinea GV, et al. Decellularization of pericardial tissue and its impact on tensile viscoelasticity and glycosaminoglycan content. Acta Biomater. 2011; 7: 1241-8.

[59] Petersen TH, Calle EA, Colehour MB, Niklason LE. Matrix composition and mechanics of decellularized lung scaffolds. Cells Tissues Organs. 2012; 195: 222-31.

[60] Xu H, Xu B, Yang Q, Li X, Ma X, Xia Q, et al. Comparison of decellularization protocols for preparing a decellularized porcine annulus fibrosus scaffold. PLoS One. 2014; 9: e86723.

[61] Albrektsson T, Johansson C. Osteoinduction, osteoconduction and osseointegration. Eur Spine J. 2001; 10: S96-101.

[62] Ibsirlioglu T, Elçin AE, Elçin YM. Decellularized biological scaffold and stem cells from autologous human adipose tissue for cartilage tissue engineering. Methods. 2020; 171: 97-107.

[63] Wolf MT, Daly KA, Brennan-Pierce EP, Johnson SA, Carruthers CA, D’Amore A, et al. A hydrogel derived from decellularized dermal extracellular matrix. Biomaterials. 2012; 33: 7028-38.

[64] Bordbar S, Lotfi Bakhshaiesh N, Khanmohammadi M, Sayahpour FA, Alini M, Baghaban Eslaminejad M. Production and evaluation of decellularized extracellular matrix hydrogel for cartilage regeneration derived from knee cartilage. J Biomed Mater Res A. 2020; 108: 938-46.

[65] Kara A, Koçtürk S, Bilici G, Havitcioglu H. Development of biological meniscus scaffold: Decellularization method and recellularization with meniscal cell population derived from mesenchymal stem cells. J Biomater Appl. 2021; 35: 1192-207.

[66] Seo Y, Jung Y, Kim SH. Decellularized heart ECM hydrogel using supercritical carbon dioxide for improved angiogenesis. Acta Biomater. 2018; 67: 270-81.

[67] Sackett SD, Tremmel DM, Ma F, Feeney AK, Maguire RM, Brown ME, et al. Extracellular matrix scaffold and hydrogel derived from decellularized and delipidized human pancreas. Sci Rep. 2018; 8: 10452.

[68] Blair HC, Larrouture QC, Li Y, Lin H, Beer-Stoltz D, Liu L, et al. Osteoblast Differentiation and Bone Matrix Formation In Vivo and In Vitro. Tissue Eng Part B Rev. 2017; 23: 268-80.

[69] Murray E, Provvedini D, Curran D, Catherwood B, Sussman H, Manolagas S. Characterization of a human osteoblastic osteosarcoma cell line (SAOS-2) with high bone alkaline phosphatase activity. J Bone Miner Res. 1987; 2: 231-8.

[70] Fu C, Yang X, Tan S, Song L. Enhancing Cell Proliferation and Osteogenic Differentiation of MC3T3-E1 Pre-osteoblasts by BMP-2 Delivery in Graphene Oxide-Incorporated PLGA/HA Biodegradable Microcarriers. Sci Rep. 2017; 7: 12549.

[71] Nakamura A, Dohi Y, Akahane M, Ohgushi H, Nakajima H, Funaoka H, et al. Osteocalcin secretion as an early marker of in vitro osteogenic differentiation of rat mesenchymal stem cells. Tissue Eng Part C Methods. 2009; 15: 169-80.

[72] Saldaña L, Bensiamar F, Boré A, Vilaboa N. In search of representative models of human bone-forming cells for cytocompatibility studies. Acta Biomater. 2011; 7: 4210-21.

[73] Prewitz MC, Seib FP, von Bonin M, Friedrichs J, Stißel A, Niehage C, et al. Tightly anchored tissue-mimetic matrices as instructive stem cell microenvironments. Nat Methods. 2013; 10: 788-94.

[74] Golebiowska AA, Intravaia JT, Sathe VM, Kumbar SG, Nukavarapu SP. Decellularized extracellular matrix biomaterials for regenerative therapies: Advances, challenges and clinical prospects. Bioact Mater. 2023; 32: 98-123.

[75] Leferink AM, Santos D, Karperien M, Truckenmüller RK, van Blitterswijk CA, Moroni L. Differentiation capacity and maintenance of differentiated phenotypes of human mesenchymal stromal cells cultured on two distinct types of 3D polymeric scaffolds. Integr Biol (Camb). 2015; 7: 1574-86.

[76] Kara Özenler A, Distler T, Tihminlioglu F, Boccaccini AR. Fish scale containing alginate dialdehyde-gelatin bioink for bone tissue engineering. Biofabrication. 2023; 15: 025012.

[77] Yamada S, Yassin MA, Weigel T, Schmitz T, Hansmann J, Mustafa K. Surface activation with oxygen plasma promotes osteogenesis with enhanced extracellular matrix formation in three-dimensional microporous scaffolds. J Biomed Mater Res A. 2021; 109: 1560-74.