Mesenchymal stem cell function on hybrid organic/inorganic microparticles in vitro

Abstract

The aim of this study was to investigate mesenchymal stem cell (MSC) function on novel type hybrid organic/inorganic microparticles (MPs) for application to bone regeneration. The MPs were based on chitosan (CS) and consisted of inorganic components, such as dibasic calcium phosphate (CaHPO(4)) or calcium carbonate (CaCO(3)). The MPs were crosslinked using tripolyphosphate. Four types of hybrid MPs were fabricated: CS; CS-10% CaHPO(4); CS-20% CaHPO(4); and CS-10% CaCO(3). The MSCs were attached to all the types of MPs at day 1 and started to spread and proliferate further by days 2 and 7, as analysed by fluorescence microcopy. Cell proliferation was measured at days 7, 14, 21 and 28 by counting the cells attached on the MPs. The number of proliferated cells increased significantly for all types of MPs as time increased. MSC differentiation was analysed using osteoblast (OB) phenotype markers, including alkaline phosphatase activity (ALP), collagen I (COLLI) and osteocalcin (OCN) at days 7, 14, 21 and 28, using quantitative real-time PCR. The normalized mRNA expression of ALP for all MPs was observed only at day 7. The normalized mRNA expression of COLLI and OCN was significantly increased for all types of hybrid MPs at each time point compared to the control samples. Collectively, our results proved that hybrid organic/inorganic MPs were non-cytotoxic and supported MSC attachment, spreading, proliferation and differentiation into the OB phenotype. These hybrid MPs have great potential for application as bone-void fillers or bone tissue engineering scaffolds in bone regeneration.

Figure 1.

(A) Digital camera image of the CS MPs. SEM images of surfaces of hybrid MPs: (B) CS MPs; (C) CS–10% CaHPO₄ MPs; (D) CS–20% CaHPO₄ MPs; (E) CS–10% CaCO₃ MPs

Figure 2.

Florescent microcopy images of MSCs attached on controls and four types of MPs when treated with live/dead assay (original magnification, ×200). (A) Control wells; (B) CS-MPs; (C) CS–10% CaHPO4 MPs; (D) CS–20% CaHPO₄ MPs, and (E) CS-CaCO₃ MPs. The subscripts indicate day 1, 2 and 7 time points. Bar = 100 μm

Figure 3.

Normalized number of MSCs proliferated on four different types ofMPs and controls as counted by hemacytometer on days 7, 14, 21 and 28. *Significant difference between the cell number on MPs and control well. **Significant increase in cell number with respect to day 7 cell numbers

Figure 4.

Amount of DNA of MSCs seeded on four different types of MPs and controls as determined by DNA quantitation assay on days 7, 14, 21 and 28. *Significant difference between the cell number on MPs and control well. **Significant increase in cell number with respect to day 7 cell numbers

Figure 5.

Normalized mRNA expression of ALP for MSCs seeded on four different types of MPs and controls on days 7, 14, 21, and 28. The mRNA levels were normalized to housekeep gene GAPDH. *Significant difference in normalized mRNA expression between the MPs and control group

Figure 6.

Normalized mRNA expression of COLLI for MSCs seeded on four different types of MPs and controls on days 7, 14, 21 and 28. The mRNA levels were normalized to housekeep gene GAPDH. *Significant difference in normalized mRNA expression between theMPs and control group. **Significant difference with respect to day 7 expression

Figure 7.

Normalized mRNA expression of OCN for MSCs seeded on four different types of MPs and controls on days 7, 14, 21 and 28. The mRNA levels were normalized to housekeep gene GAPDH. *Significant difference in normalized mRNA expression between theMPs and control group. **Significant difference with respect to day 7 expression