Page 103 - Physico-Chemical Niche Conditions for Bone Cells

Chapter 4
(Fig. 4G). One h PFF increased Dmp1 (Fig. 4H) and Col1⍺1 mRNA levels after 1 h post- incubation (Fig. 4I). Two-way ANOVA revealed significant interaction effects between without and with PFF treatment (Runx2, Ocn, Ki67, Fgf2, and Dmp1), and post-incubation time (0, 1, 3, and 6 h; Fgf2 and Dmp1; Table 1).
Long-term down-stream impact of PFF (days)
ALP protein (long-term)
ALP protein was quantified in control and 1 h PFF-treated MC3T3-E1 pre-osteoblast cultures after 21 days (Fig. 5A, B). PFF did not affect ALP protein in cells that were post-incubated without medium refreshment for 1, 3, or 6 h compared to static controls and similar post- incubation conditions (Fig. 5A, B).
Collagen secretion
Collagen secretion by MC3T3-E1 pre-osteoblasts treated without or with PFF was visualized by picrosirius red staining using light microscopy, and quantified at day 21 (Fig. 5C, D). The images showed abundant collagen secretion by cells that were post-incubated without medium refreshment for 0, 1, 3, and 6 h, after 21 days of culture as shown by the intense red color. PFF did not stimulate collagen secretion compared to static control (Fig. 5C, D).
Matrix mineralization
Images of the alizarin red-stained cultures showed cellular calcium deposition resulting in ECM mineralization (Fig. 5E). Static control cultures showed slightly more red-stained mineralization dots (no significance) than PFF treated cultures at 0 h post-incubation. Interestingly, PFF increased matrix mineralization in cells that were post-incubated for 3 h, as shown by the presence of abundant mineralization nodules compared to static control cultures (Fig. 5E). Quantification of the ECM mineralization levels confirmed this observation, i.e. PFF significantly increased mineral deposition in cells post-incubated for 3 h (Fig. 5F).
101
4