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Regulation of Angiogenesis Discriminates Tissue Resident MSCs from Effective and Defective Osteogenic Environments - PubMed

  • ️Wed Jan 01 2020

Regulation of Angiogenesis Discriminates Tissue Resident MSCs from Effective and Defective Osteogenic Environments

R J Cuthbert et al. J Clin Med. 2020.

Abstract

Background: The biological mechanisms that contribute to atrophic long bone non-union are poorly understood. Multipotential mesenchymal stromal cells (MSCs) are key contributors to bone formation and are recognised as important mediators of blood vessel formation. This study examines the role of MSCs in tissue formation at the site of atrophic non-union.

Materials and methods: Tissue and MSCs from non-union sites (n = 20) and induced periosteal (IP) membrane formed following the Masquelet bone reconstruction technique (n = 15) or bone marrow (n = 8) were compared. MSC content, differentiation, and influence on angiogenesis were measured in vitro. Cell content and vasculature measurements were performed by flow cytometry and histology, and gene expression was measured by quantitative polymerase chain reaction (qPCR).

Results: MSCs from non-union sites had comparable differentiation potential to bone marrow MSCs. Compared with induced periosteum, non-union tissue contained similar proportion of colony-forming cells, but a greater proportion of pericytes (p = 0.036), and endothelial cells (p = 0.016) and blood vessels were more numerous (p = 0.001) with smaller luminal diameter (p = 0.046). MSCs showed marked differences in angiogenic transcripts depending on the source, and those from induced periosteum, but not non-union tissue, inhibited early stages of in vitro angiogenesis.

Conclusions: In vitro, non-union site derived MSCs have no impairment of differentiation capacity, but they differ from IP-derived MSCs in mediating angiogenesis. Local MSCs may thus be strongly implicated in the formation of the immature vascular network at the non-union site. Attention should be given to their angiogenic support profile when selecting MSCs for regenerative therapy.

Keywords: MSCs; fracture healing; induced periosteum; non-union; osteogenesis; regenerative medicine.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1

Colony formation (A) and multi-lineage differentiation potential (B–E) of non-union (NU)-derived mesenchymal stromal cells (MSCs). Enzymatically digested non-union tissue readily forms similar-sized colonies in vitro (A, left panel) compared with control MSCs derived from bone marrow (BM) aspirates (A, right panel). Culture expanded adherent cells undergo osteogenic differentiation, as shown by alkaline phosphatase staining (B); chondrogenic differentiation, as shown by toluidine blue staining of chondrogenic pellets (C); and adipogenic differentiation, as shown by oil red staining (D). The bottom panels on B–D show cells stained after culturing in normal growth media. Quantitative measurements of chondrogenesis, measured by glycosaminoglycan (GAG) accumulation (E), and osteogenesis, measured by accumulation of Ca2+ (F), were comparable between BM MSCs (BM) (n = 6) and non-union derived MSCs (NU) (n = 6).

Figure 2
Figure 2

A comparison of cellular composition between NU and induced periosteum (IP) tissue. Comparison of the percentage of colony forming cells assessed by colony forming unit fibroblast (CFU-F) assay following enzymatic digestion and in vitro culture in induced periosteum (IP) (n = 8) and non-union (NU) tissue (n = 6) (A). Flow cytometry analysis of the percentage of cells expressing cell surface markers consistent with pericytes (CD45−CD34−CD146+) (B), endothelial cells (CD45−CD31+) (C), and lymphocytes (CD45+side scatter low) (D), following enzymatic digestion of IP (n = 10) and NU tissue (n = 6). * denotes p < 0.05, ** denotes p < 0.01.

Figure 3
Figure 3

Histological examination of NU tissue. Comparison of blood vessel size highlighted by immunohistochemistry (IHC) staining for CD31 expression in induced periosteum (IP) and non-union (NU) tissue, representative photomicrographs (200× magnification, scale bar 100µm) showing blood vessels (left and middle panels), comparison of mean internal vessel area by manual blind scoring (top right), and comparison of mean number of blood vessels per field (bottom right) (A). Comparison of leukocyte content highlighted by IHC staining for CD45 expression in IP and NU tissue, representative photomicrographs (200× magnification, scale bar 100 µm) showing leukocyte content (left and middle panels), and automated comparison showing the average area of positive staining per field (right) (B). Higher magnification images of CD45+ cells in IP and NU tissue are shown in the inserts. IHC staining highlighting perivascular location of stromal-derived factor-1 (SDF-1), vascular endothelial growth factor (VEGF), and bone morphogenetic protein-2 (BMP-2) in NU tissue (scale bar 30 µm) (C). All data IP (n = 8) and NU tissue (n = 9), arrows show regions of positive staining (brown). * denotes p < 0.05, *** denotes p ≤ 0.001.

Figure 4
Figure 4

Differentially expressed transcripts in MSCs derived from IP and NU tissue. Cluster analysis of log 2 transformed relative expression data: 2−ΔCt normalised to HPRT. IP—induced periosteum (n = 7), NU—non-union (n = 8), BM—bone marrow (n = 8), and Fib—skin fibroblasts negative control (n = 3). Green < HPRT, red > HPRT, black = HPRT, and grey is missing data. Genes were analysed when 80% present, complete linkage clustering was performed and viewed using Java Treeview (A). Genes showing marked differences in relative expression between NU MSCs and IP MSCs; BM MSCs included as controls. Genes involved in regulation of endothelial cells (FLT1, PTN, ANGPL4, and MCAM), Wnt signalling genes (FZD4 and WNT2), and genes involved in MSC differentiation (SOX9 and BMP-2) (B). All data IP (n = 7), NU (n = 8), BM (n = 8). R.E.U.—relative expression unit.

Figure 5
Figure 5

MSC from induced periosteum inhibit early in-vitro angiogenesis. Angiogenesis measured by total tube length (A) and total number of tubes (B) shows a significant inhibitory effect of media conditioned by MSCs originating from induced periosteum (IP) compared with non-union (NU) and positive control human umbilical vein endothelial cell (HUVEC) endothelial cell growth media (C). Values are normalised to unconditioned MSC growth media and the number of MSCs is counted at the end of the conditioning period. Photomicrographs showing representative examples of tube formation in induced periosteum, non-union, HUVEC control, and unconditioned media treated wells (C). All data IP (n = 6), NU (n = 7), C (four technical replicates). *** denotes p ≤ 0.001.

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