Burst, Short, and Sustained Vitamin D3 Applications Differentially Affect Osteogenic Differentiation of Human Adipose Stem Cells

Abstract

Incorporation of 1,25(OH)₂ vitamin D₃ (vitD₃) into tissue-engineered scaffolds could aid the healing of critical-sized bone defects. We hypothesize that shorter applications of vitD₃ lead to more osteogenic differentiation of mesenchymal stem cells (MSCs) than a sustained application. To test this, release from a scaffold was mimicked by exposing MSCs to exactly controlled vitD₃ regimens. Human adipose stem cells (hASCs) were seeded onto calcium phosphate particles, cultured for 20 days, and treated with 124 ng vitD₃, either provided during 30 min before seeding ([200 nM]), during the first two days ([100 nM]), or during 20 days ([10 nM]). Alternatively, hASCs were treated for two days with 6.2 ng vitD₃ ([10 nM]). hASCs attached to the calcium phosphate particles and were viable (~75%). Cell number was not affected by the various vitD₃ applications. VitD₃ (124 ng) applied over 20 days increased cellular alkaline phosphatase activity at Days 7 and 20, reduced expression of the early osteogenic marker RUNX2 at Day 20, and strongly upregulated expression of the vitD₃ inactivating enzyme CYP24. VitD₃ (124 ng) also reduced RUNX2 and increased CYP24 applied at [100 nM] for two days, but not at [200 nM] for 30 min. These results show that 20-day application of vitD₃ has more effect on hASCs than the same total amount applied in a shorter time span.

Keywords: bioactive components; bone; burst release; calcitriol; short stimulation; sustained release; tissue engineering.

Figure 1

Schematic representation of the different vitamin D₃ applications. A total of 124 ng of vitD₃ was added for either 30 min before seeding at a concentration of 200 nM (Short), for two days at a concentration of 100 nM (Burst high) to mimic a burst release, or for 20 days with a concentration of 10 nM (Sustained) to mimic a sustained release. Alternatively, cells were exposed to 10 nM vitD₃ for two days (Burst low).

Figure 2

Effect of vitamin D₃ on the viability of hASCs on BCP particles. Fluorescence microscopy of live/dead staining shows the attachment and viability of hASCs on Day 2 after seeding. Living cells are stained green, while the red-stained cells are dead/dying. (A,B) Sample without vitD₃ treatment, where (B) is a higher magnification of the same sample as (A). (C,D) A representative sample with vitD₃ treatment (highest concentration, i.e., [200 nM]) is depicted. Both (C,D) were made in the same sample. (E) Gene expression of pro-apoptotic marker BAX measured at Days 7 and 20. Gene expression in controls (left) and vitD₃-treated cultures (right) of the same donor are connected by a line. (F) Relative expression of BAX at Days 7 and 20. Data represent mean + SD of four or five independent donors. (G) Gene expression of anti-apoptotic marker BCL-2 measured at Days 7 and 20. Gene expression in controls (left) and vitD₃-treated cultures (right) of the same donor are connected by a line. * Significant effect (p < 0.05) of vitD₃ compared to the control at one time point. ** p < 0.01. All gene expression levels were normalized to HPRT.

Figure 3

Effect of vitamin D₃ on proliferation and protein content of hASCs. hASCs were subjected to the various vitD₃ treatment modalities and Alamar blue was measured at Days 2, 7, and 20; the gene expression of Ki67 was measured at Days 7 and 20; and total protein content was measured at Days 7 and 20. (A) VitD₃ treatment did not affect Alamar blue measurement between any of the groups, at any time point measured. (B) Gene expression of proliferation marker Ki67 did not significantly differ between vitD₃-treated and control groups. Gene expression in controls (left) and vitD₃-treated cultures (right) of the same donor are connected by a line. (C) Total protein content was similar between all vitD₃-treated groups at Day 7, as well as on Day 20. Data represent four or five independent donors (mean + SD). Gene expression data was normalized to HPRT.

Figure 4

Effect of vitamin D₃ on ALP activity and RUNX2 expression. (A) Cellular ALP, as a measure for early osteogenic differentiation, was measured on Days 7 and 20. Sustained-release significantly increased ALP activity compared to all other groups at both time points. (B) RUNX2 expression in general seemed to be higher at Day 20 compared to Day 7, although no statistical comparison was made. VitD₃ seemed to reduce RUNX2 expression in general. This effect of vitD₃ was significant for burst high at Days 7 and 2, and for sustained application at Day 20. Gene expression in controls (left) and vitD₃-treated cultures (right) of the same donor are connected by a line. (C) Average relative expression of RUNX2 at Days 7 and 20. Data represent four or five independent donors (mean + SD). All gene expression levels were normalized to HPRT. * Significant difference (p < 0.05) between control and vitD₃ treatment at the same time point ** p < 0.01.

Figure 5

Gene expression of VDR and CYP24a1. (A) VDR expression. Data of controls (left) and vitD₃ treated cells (right) from the same donor are connected with a line. (B) Average relative expression of VDR at Days 7 and 20. Data represent four or five independent donors (mean + SD). (C) Burst high, Sustained, and Burst low application of vitD₃ significantly increased CYP24a1 (note the 10-fold differences in axis scale). Data of controls (left) and vitD₃ treated cells (right) from the same donor are connected with a line. (D) Average relative expression of CYP24a1 at Days 7 and 20. Data represent four or five independent donors (mean + SD). All gene expression data were normalized to HPRT. (A,C) * Significant difference (p < 0.05) between control and vitD₃ treatment at the same time point; *** p < 0.001. (D) * Significant difference (p < 0.001) between Sustained vitD₃ treatment and all other groups.