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
Research background

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 (BTE) have been developed recently. For successful bone tissue regeneration, osteogenesis and vasculogenesis should be supported by an appropriate combination of cells, growth factors and biomaterials, which represent the biological triad components.

Research motivation

Despite emerging developments of various approaches in the field of BTE, an optimal combination of the biological triad factors is still unknown. Bearing that in mind, our motivation was to combine endothelial differentiated and osteogenic differentiated adipose-derived stem cells (ASCs) in the same construct together with platelet-rich plasma (PRP) as a growth factor reservoir on the bone mineral matrix (BMM) as a carrier. This approach was applied in a mouse subcutaneous implantation model to examine the ectopic osteogenic potential of this type of construct, which enables interplay between endothelial cells (ECs) and osteoblasts (OBs) in induction of ectopic osteogenesis.

Research objectives

The objective of our study was to examine the effects of simultaneously applied endothelial and osteogenic differentiated ASCs combined with PRP and delivered on the BMM on vascularization and osteogenesis in ectopically implanted BTE constructs. To examine the significance of in vitro endothelial and osteogenic induction, BTE constructs containing uninduced ASCs, PRP and BMM were also prepared. BMM-only constructs represented a basic group of constructs that served as a carrier control.

Research methods

ASCs obtained from a stromal vascular fraction of visceral epididymal adipose tissue of BALB/c mice were cultivated up to the third passage (P03). At the P03, three types of cell cultures were established: ASCs induced into ECs, ASCs induced into OBs and ASCs expanded in vitro without induction towards any cell type (uninduced ASCs). Upon cultivation and/or differentiation, three types of ectopic BTE constructs were prepared: (1) BPEO constructs: BTE constructs containing simultaneously applied endothelial and osteogenic differentiated ASCs combined with PRP and seeded onto BMM carrier; (2) BPUI constructs: BTE constructs containing uninduced ASCs, combined with PRP and seeded onto BMM carrier; and (3) BC constructs (control): BTE constructs that contained only BMM carrier. BTE constructs extracted 1, 2, 4 and 8 wk after implantation were analyzed regarding relative endothelial-related and bone-related gene expression. Histochemical, immunohistochemical and histomorphometric analyses were performed on constructs extracted 2 and 8 wk after implantation.

Research results

The percentage of vascularization was significantly higher in BC constructs compared to BPUI and BPEO after 2 and 8 wk. However, BC constructs had the lowest endothelial-related gene (Vwf, Egr1, Flt1 and Vcam1) expression among the examined types of constructs. This was followed by significantly weaker osteocalcin immunoexpression in comparison with BPUI and BPEO implants and mostly downregulated Spp1 gene expression. Endothelial-related gene expression was generally higher in BPUI constructs compared to BC and BPEO constructs, which was mostly significant. However, histomorphometric analysis showed the lowest percentage of vascularization in the BPUI group. BPEO constructs had a higher percentage of vascularization compared to BPUI constructs at 2 wk and 8 wk. Endothelial-related gene expression in BPEO constructs had a later onset, but with the exception of Vwf, these genes were upregulated and well-balanced through the whole in vivo experimental period. In addition, CD31 immunoexpression was significantly higher in BPEO compared to BC and BPUI implants at both observation points. This also led to the late onset of Spp1 gene expression, although osteocalcin immunoexpression was pronounced at both observation points. The IHC OD score showed osteocalcin immunoexpression increased in BPEO implants at 8 wk compared to 2 wk. In BPEO constructs, tissue regression was observed between BMM granules after 8 wk.

Research conclusions

Ectopically implanted BPEO constructs have a favorable impact on vascularization and osteogenesis but observed tissue regression at later time points imposes the need for discovering the optimal ratio of ECs/OBs within the BPEO constructs prior to consideration for clinical applications.

Research perspectives

Obtained results unequivocally indicate the potential of such an approach, which could be used for further preclinical evaluations in the orthotopic model regarding optimization of EC/OB ratio within bone tissue-engineered constructs.