Fabrication of Biomimetic Bone Tissue Using Mesenchymal Stem Cell-Derived Three-Dimensional Constructs Incorporating Endothelial Cells

<div><p>The development of technologies to promote vascularization of engineered tissue would drive major developments in tissue engineering and regenerative medicine. Recently, we succeeded in fabricating three-dimensional (3D) cell constructs composed of mesenchymal stem cells (MSCs). However, the majority of cells within the constructs underwent necrosis due to a lack of nutrients and oxygen. We hypothesized that incorporation of vascular endothelial cells would improve the cell survival rate and aid in the fabrication of biomimetic bone tissues <i>in vitro</i>. The purpose of this study was to assess the impact of endothelial cells combined with the MSC constructs (MSC/HUVEC constructs) during short- and long-term culture. When human umbilical vein endothelial cells (HUVECs) were incorporated into the cell constructs, cell viability and growth factor production were increased after 7 days. Furthermore, HUVECs were observed to proliferate and self-organize into reticulate porous structures by interacting with the MSCs. After long-term culture, MSC/HUVEC constructs formed abundant mineralized matrices compared with those composed of MSCs alone. Transmission electron microscopy and qualitative analysis revealed that the mineralized matrices comprised porous cancellous bone-like tissues. These results demonstrate that highly biomimetic bone tissue can be fabricated <i>in vitro</i> by 3D MSC constructs incorporated with HUVECs.</p></div

<i>In silico</i> analyses of the reticulate process of HUVECs.

<p>The HUVEC model (red polygons) was set in those of the hMSCs models (blue polygons) at 0 MCS. In absence of VEGF, HUVECs proliferated and colonized near their initial position (A). By contrast, in the presence of VEGF, HUVECs responded and spread across a wide area with the reticulate structure (B).</p

Mineral deposition in the hMSC/HUVEC 3D constructs.

<p>(A) Mineralized matrices within the cell constructs were detected by von Kossa staining as black aggregates, and (B) semi-quantitative assessment was performed (n = 4).</p

SEM and TEM images of 3D cell constructs.

<p>Cavities (arrows) were formed within the 95:5 cell constructs (B) but not in the 100:0 constructs (A). TEM images revealed that tight structures formed in the 100:0 cell constructs (C). By contrast, void spaces (arrowhead) between the cells were observed in 95:5 cell constructs (D).</p

Immunofluorescence stained images of hMSC/HUVEC 3D constructs for angiogenic factors.

<p>(A–D) VEGF and (E–H) HGF were diffused within each cell construct. White dotted lines indicate outline of cell construct.</p

Self-organization of mineralized matrices in the 3D cell constructs after 50 days of culture.

<p>(A) Mineral deposition (M) was generated in the cytoplasm in the 100:0 cell constructs. (B) Mineral deposition resulted in a cancellous bone-like porous structure in the 95:5 cell constructs.</p

Fabrication of hMSC/HUVEC 3D cell constructs.

<p>Cell constructs were formed using a thermo-responsive poly-NIPAAm hydrogel (A) and were obtained as uniform sizes (B, C).</p

CD31 immunofluorescence stained images of hMSC/HUVEC 3D constructs.

<p>(A) Stained area of HUVECs (CD31-positive cells; green), which were more diffuse with the increasing ratio of HUVECs. The hMSCs only stained positive for Hoechst33342 (nucleus; red) (B) Representative magnified image of a hMSC/HUVEC (99:1) construct showing that HUVECs formed a reticulated structure within the 3D cell construct. White dotted lines indicate outline of cell construct.</p

Characterization of mineralized matrices in hMSC/HUVEC 3D constructs.

<p>Mineralized matrices were evaluated by calcium content (A) and Vickers hardness (B). Both values increased following incorporation of HUVECs into the hMSC constructs.</p

ALP activity of hMSC/HUVEC 3D constructs.

<p>HUVEC incorporated hMSC constructs (99:1–95:5) showed higher ALP activity compared with the hMSC only construct (100:0) at 7 and 14 days of culture (* <i>p</i> < 0.05; ** <i>p</i> < 0.01).</p