Reconstruction of a segmental bone defect using vascularized tissue-engineered bone substitutes: An animal study

RESEARCH ARTICLE Reconstruction of a segmental bone defect using vascularized tissue-engineered bone substitutes: An animal study Yulei Wang1☯, Qian Lv2☯, Xu Zhang3, Jinlong Liang2, Fanzhe Feng2, Jingyuan Li4, Yi Cui 2* 1 Pain Department, Second People’s Hospital of Qujing City, Qujing, China, 2 Department of Orthopedics, 920th Hospital of Joint Logistics Support Force, People’s Liberation Army, Kunming, China, 3 Department of Hand Surgery, Third Hospital of Hebei Medical University, Shijiazhuang, Hebei, China, 4 Department of Traumatology and Orthopedics, The First Affiliated Hospital of Xinjiang Medical University, Urumqi, Xinjiang, China ☯ These authors contributed equally to this work. * Abstract Background Citation: Wang Y, Lv Q, Zhang X, Liang J, Feng F, Li J, et al. (2026) Reconstruction of a segmental bone defect using vascularized tissue-engineered bone substitutes: An animal study. PLoS One 21(6): e0344505. https://doi. org/10.1371/journal.pone.0344505 Repairing large segmental bone defects remains a major challenge in orthopedics, with conventional autografting and allografting limited by donor shortages and high complication rates. Tissue-engineered bone substitutes have emerged as a potential solution. This study aimed to assess the vascular regeneration of dual-vascularized tissue-engineered bone substitutes in the reconstruction of large segmental bone defects in rabbits. Editor: Masahito Yamamoto, Tokai University, School of Medicine, JAPAN Method OPEN ACCESS Received: October 31, 2025 Accepted: February 20, 2026 Published: June 4, 2026 Copyright: © 2026 Wang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data availability statement: All experimental data are available via the following link: https:// data.mendeley.com/datasets/3zf2zhr2z6/1. Funding: This work was supported by the Research on the Remote Microscopic Surgery Assistance System Integrated with MicrometerLevel Robotic Arms (2024YFC2418103, Demineralized bone matrix (DBM) was used as a scaffold, with endothelial progenitor cells (EPCs) seeded onto it. A vascular channel was created within the DBMEPCs composite scaffold, and the radial artery was implanted into this channel. New Zealand white rabbits were used to create a 15-mm critical-sized rabbit radial bone defect model, with animals divided into four groups: DBM, DBM+EPCs, DBM+Vascular Bundle,and DBM+EPCs+Vascular Bundle (n = 9 per group).X-ray examinations,gross morphological observations,and CD31 immunofluorescence staining were conducted at 4, 8,and 12 weeks post-surgery. Micro-CT was used to reconstruct the three-dimensional structures of the defects after 12 weeks. Results The DBM+EPCs+Vascular Bundle group demonstrated the most significant bone regeneration and vascularization across all time points.X-ray,gross morphology, Micro-CT analysis,HE staining,and CD31 immunofluorescence staining all revealed PLOS One | https://doi.org/10.1371/journal.pone.0344505 June 4, 2026 1 / 15 to Y.Cui); Yunnan Provincial Orthopedics and Sports Rehabilitation Clinical Medicine Research Center (202102AA310068, no relevant author); Yunnan Provincial Clinical Orthopedic Trauma Medical Center (no relevant author); the Yunnan Provincial Young and Middle-Aged Academic and Technical Leaders Backup Talent Program (202405AC350079 to Y.Cui); the Science and Technology Program of Yunnan Provincial Department of Science and Technology (202101AY070001-295 to Y.Cui); and the 920 Hospital Science and Technology Program (2019YGB06 to Y.Cui). All funders had no role in the study design, data collection and analysis, interpretation of results, manuscript drafting, or decision to publish this article. All research funds were solely used for experimental implementation, data collection, purchase of experimental reagents and consumables, and manuscript writing related to this study, with no embezzlement or irregular use of funds. Competing interests: The authors declare that they have no competing interests. Abbreviations: DBM, demineralized bone matrix; EPCs, endothelial progenitor cells; BV/ TV, bone volume fraction (bone volume/total volume); Tb.Th, trabecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation; Ct.Th, cortical thickness; VEGF, vascular endothelial growth factor; IHC, immunohistochemistry; IF, immunofluorescence. superior bone regeneration and vascular density in this group compared to the others. Conclusions In conclusion, the dual vascularization strategy significantly enhanced bone regeneration and angiogenesis in the reconstruction of large bone defects. This approach has potential clinical applications for repairing critical-sized bone defects, particularly in anatomical regions with multiple arterial supplies such as the upper limbs and lower legs. Introduction Millions of people worldwide suffer from bone defects due to trauma, tumor, bone diseases, infection, congenital defects, and aging [1]. Because segmental bone defects are difficult to heal spontaneously, Reconstruction of such defects remains challenging due to deranged mechanics and biology [2,3]. Many treatment strategies have been developed. Bone lengthening can be used to treat bone defects based on distraction osteogenesis. However, pain, discomfort, and muscle stiffness may result from the procedure and may be reversible or lifelong [4]. Reconstruction using autografts are the common technique for minor bone loss but is often difficult for large defects due to limited quantities available and donor-site morbidity [5]. Allografting avoids those drawbacks but the downsides are variable osteoinductive properties and possibly transmit diseases [6]. Recently, tissue-engineered bone technology has emerged as a promising solution due to integrating seed cells, scaffold materials, and growth factors [7]. It not only promotes osteogenesis but also supports vascularization and enhances bone regeneration. The only drawback is slow vascular ingrowth from the host into the center of the scaffold, resulting in inadequate internal vascularization and restricting further bone integration [8]. Therefore, improving vascularization in tissue-engineered bone remains a crucial challenge [9,10]. Currently, there are in vivo and in vitro prevascularization methods. In vivo method (establishing flap coverage, arteriovenous loops, arteriovenous bundles, and vascular channels within the scaffold) can effectively promote vascularization in tissueengineered implants, but the downsides include complex procedure, time-consuming vascularization, and postoperative thrombosis and infection [11]. In contrast, in vitro method (employing co-culture techniques, growth factor supplementation, or 3-dimensional printing to create vascularized scaffolds outside the body) offers a simpler approach an (...truncated)