Basic Study
Copyright ©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Stem Cells. Jan 26, 2021; 13(1): 91-114
Published online Jan 26, 2021. doi: 10.4252/wjsc.v13.i1.91
Vascularization and osteogenesis in ectopically implanted bone tissue-engineered constructs with endothelial and osteogenic differentiated adipose-derived stem cells
Jelena G Najdanović, Vladimir J Cvetković, Sanja T Stojanović, Marija Đ Vukelić-Nikolić, Jelena M Živković, Stevo J Najman
Jelena G Najdanović, Sanja T Stojanović, Stevo J Najman, Department of Biology and Human Genetics; Department for Cell and Tissue Engineering, Faculty of Medicine, University of Niš, Niš 18108, Serbia
Vladimir J Cvetković, Department of Biology and Ecology, Faculty of Sciences and Mathematics, University of Niš, Niš 18106, Serbia
Marija Đ Vukelić-Nikolić, Jelena M Živković, Department of Biology and Human Genetics; Scientific Research Center for Biomedicine; Faculty of Medicine, University of Niš, Niš 18108, Serbia
Author contributions: Najman SJ conceptualized the original idea and designed and coordinated the research study; Najdanović JG and Cvetković VJ performed the research and acquired the data; Najdanović JG, Cvetković VJ, Stojanović ST and Najman SJ analyzed and interpreted the data; Stojanović ST and Vukelić-Nikolić MD contributed new analytic tools; Najdanović JG and Cvetković VJ drafted and wrote the manuscript; Najdanović JG, Cvetković VJ, Stojanović ST, Vukelić-Nikolić MD, Živković JM and Najman SJ made critical revisions to the manuscript; All authors have read and approved the final manuscript.
Supported by Ministry of Education, Science and Technological Development of the Republic of Serbia, No. III 41017.
Institutional review board statement: This study was approved by Faculty of Medicine, University of Niš, Laboratory for Cell Culture of Institute for Biomedical Research, Head of the Laboratory for Cell Culture: Prof. Dr. Stevo Najman, approval No. 01/5150 (16.09.08).
Institutional animal care and use committee statement: All animal procedures in this experiment were performed in accordance with the Animal Welfare Act (Republic of Serbia), which is in compliance with European Union guidelines for experimental animals. The animal procedures were approved by the Institutional Ethics Committee of the Faculty of Medicine, University of Niš, Serbia, approval No. 01-2857-8.
Conflict-of-interest statement: The authors have no potential conflicts of interest to declare.
Data sharing statement: Technical appendix, statistical code, and dataset available from the corresponding author at the email address
ARRIVE guidelines statement: The authors have read the ARRIVE Guidelines, and the manuscript was prepared and revised according to the ARRIVE Guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See:
Corresponding author: Stevo J Najman, PhD, Full Professor, Department of Biology and Human Genetics; Department for Cell and Tissue Engineering, Faculty of Medicine, University of Niš, Blvd. Dr Zorana Djindjica 81, Niš 18108, Serbia.
Received: July 12, 2020
Peer-review started: July 12, 2020
First decision: September 24, 2020
Revised: November 1, 2020
Accepted: November 17, 2020
Article in press: November 17, 2020
Published online: January 26, 2021

A major problem in the healing of bone defects is insufficient or absent blood supply within the defect. To overcome this challenging problem, a plethora of approaches within bone tissue engineering have been developed recently. Bearing in mind that the interplay of various diffusible factors released by endothelial cells (ECs) and osteoblasts (OBs) have a pivotal role in bone growth and regeneration and that adjacent ECs and OBs also communicate directly through gap junctions, we set the focus on the simultaneous application of these cell types together with platelet-rich plasma (PRP) as a growth factor reservoir within ectopic bone tissue engineering constructs.


To vascularize and examine osteogenesis in bone tissue engineering constructs enriched with PRP and adipose-derived stem cells (ASCs) induced into ECs and OBs.


ASCs isolated from adipose tissue, induced in vitro into ECs, OBs or just expanded were used for implant construction as followed: BPEO, endothelial and osteogenic differentiated ASCs with PRP and bone mineral matrix; BPUI, uninduced ASCs with PRP and bone mineral matrix; BC (control), only bone mineral matrix. At 1, 2, 4 and 8 wk after subcutaneous implantation in mice, implants were extracted and endothelial-related and bone-related gene expression were analyzed, while histological analyses were performed after 2 and 8 wk.


The percentage of vascularization was significantly higher in BC compared to BPUI and BPEO constructs 2 and 8 wk after implantation. BC had the lowest endothelial-related gene expression, weaker osteocalcin immunoexpression and Spp1 expression compared to BPUI and BPEO. Endothelial-related gene expression and osteocalcin immunoexpression were higher in BPUI compared to BC and BPEO. BPEO had a higher percentage of vascularization compared to BPUI and the highest CD31 immunoexpression among examined constructs. Except Vwf, endothelial-related gene expression in BPEO had a later onset and was upregulated and well-balanced during in vivo incubation that induced late onset of Spp1 expression and pronounced osteocalcin immunoexpression at 2 and 8 wk. Tissue regression was noticed in BPEO constructs after 8 wk.


Ectopically implanted BPEO constructs had a favorable impact on vascularization and osteogenesis, but tissue regression imposed the need for discovering a more optimal EC/OB ratio prior to considerations for clinical applications.

Keywords: Adipose-derived stem cells, Endothelial-related genes, Bone-related genes, Ectopic osteogenesis, Vascularization, Platelet-rich plasma

Core Tip: For successful bone regeneration, osteogenesis and vasculogenesis should be supported by the appropriate combination of cells, growth factors and biomaterials (biological triad). Because the optimal triad composition is unknown, our aim was to combine endothelial and osteogenic differentiated adipose-derived stem cells in the same construct with platelet-rich plasma on bone mineral matrix as a carrier. This construct was examined in a mouse subcutaneous implantation model that enabled interplay between endothelial cells and osteoblasts in induction of ectopic osteogenesis. The results indicated the potential of this approach, but further preclinical evaluations in orthotopic model regarding optimization of ECs/OBs ratio are necessary.