Published online Jan 26, 2021. doi: 10.4252/wjsc.v13.i1.91
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 (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.
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.
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.
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.
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.
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.
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.