Calcification of primary human osteoblast cultures under flow conditions using polycaprolactone scaffolds for intravascular applications

Abstract

Total atherosclerotic occlusion is a leading cause of death. Recent animal models of this disease are devoid of cell-mediated calcification and arteries are often not occluded gradually. This study is part of a project with the objective of developing a new model featuring the above two characteristics, using a tissue-engineering scaffold. The amount and distribution of calcium deposits in primary human osteoblast (HOB) cultures on polycaprolactone (PCL) scaffolds under flow conditions were investigated. HOBs were cultured on PCL scaffolds with TGF-β1 loadings of 0 (control), 5 and 50 ng. HOB-PCL constructs were cultured in spinner flasks. Under flow conditions, cell numbers present in HOB cultures on PCL scaffolds increased from day 7 to day 14, and most calcification was induced at day 21. TGF-β1 loadings of 5 and 50 ng did not show a significant difference in ALP activity, cell numbers and amount of calcium deposited in HOB cultures, but calcium staining showed that 50 ng TGF-β1 had higher calcium deposited on both days 21 and 28 under flow conditions compared with 5 ng of loading. Amount of calcium deposited by HOBs on day 28 showed a decrease from their levels on day 21. PCL degradation may be a factor contributing to this loss. The results indicate that cell-induced calcification can be achieved on PCL scaffolds under flow conditions. In conclusion, TGFβ1-HOB loaded PCL can be applied to create a model for total atherosclerotic occlusion with cell-deposited calcium in animal arteries.

Keywords: PCL; calcification; dynamic flow; polycaprolactone; primary human osteoblast; scaffold; tissue engineering; total atherosclerotic occlusion.

Figure 1

Schematic views of spinner flask bioreactors from side and the top

Figure 2

Intracellular ALP activity of HOBs on PCL scaffolds coated with 0 (control), 5 ng, and 50 ng of TGF-β1 under dynamic conditions (*p<0.05, **p<0.01).

Figure 3

Cell number of HOBs on PCL scaffolds with none, 5, and 50 ng loading of TGF-β1 in dynamic conditions (*p<0.05, **p<0.01).

Figure 4

HOBs were cultured on PCL scaffolds with different doses of TGF-β1 coatings at 0 (control), 5 ng, and 50 ng respectively under flow conditions. The amount of calcium deposited in cultures was measured at 7, 14, 21, and 28 days (*p<0.05, **p<0.01).

Figure 5

Calcification staining (green fluorescence) of HOB cultures on PCL scaffolds under dynamic cultures. HOB nuclei were counterstained with DAPI (blue fluorescence). HOBs were cultured on 0 (A, B), 5 (C, D), and 50 ng (E, F) of TGF-β1 loaded PCL scaffolds, and calcification was imaged at day-21. At day-28, calcium was examined in HOB cultures with 0 (G, H), 5 (I, J), and 50 ng (K, L) of TGF-β1 loading on PCL scaffolds. First column indicates calcium staining. Second column shows the nuclei of HOBs on PCL scaffolds from the same location. Scale bar was 250 μm in A, and this applies from A to R. All arrows point to calcium deposits.

Figure 6

Quantification of calcification area on PCL scaffolds under dynamic conditions based on fluorescent calcium images. HOBs were cultured on 0 ng (control), 5 ng and 50 ng of TGF- β1 loaded scaffolds for 21 and 28 days (* p<0.05, ** p<0.01).

Figure 7

Percentage mass remaining of PCL scaffolds over 35 days in PBS immersion under static conditions.

Figure 8

Alizarin Red staining of HOBs cultured on PCL films for 21 days (A), and 28 days (B). Media was composed of 10⁻¹⁰ M dexamethasone and without TGF-β1. Scale bar was 1 mm in A and this applies to B.