Comparison of physical, chemical and cellular responses to nano- and micro-sized calcium silicate/poly(epsilon-caprolactone) bioactive composites

Abstract

In this study, we fabricated nano-sized calcium silicate/poly(epsilon-caprolactone) composite (n-CPC) and micro-sized calcium silicate/poly(epsilon-caprolactone) composite (m-CPC). The composition, mechanical properties, hydrophilicity and degradability of both n-CPC and m-CPC were determined, and in vitro bioactivity was evaluated by investigating apatite forming on their surfaces in simulated body fluid (SBF). In addition, cell responses to the two kinds of composites were comparably investigated. The results indicated that n-CPC has superior hydrophilicity, compressive strength and elastic modulus properties compared with m-CPC. Both n-CPC and m-CPC exhibited good in vitro bioactivity, with different morphologies of apatite formation on their surfaces. The apatite layer on n-CPC was more homogeneous and compact than on m-CPC, due to the elevated levels of calcium and silicon concentrations in SBF from n-CPC throughout the 14-day soaking period. Significantly higher levels of attachment and proliferation of MG63 cells were observed on n-CPC than on m-CPC, and significantly higher levels of alkaline phosphatase activity were observed in human mesenchymal stem cells (hMSCs) on n-CPC than on m-CPC after 7 days. Scanning electron microscopy observations revealed that hMSCs were in intimate contact with both n-CPC and m-CPC surfaces, and significantly cell adhesion, spread and growth were observed on n-CPC and m-CPC. These results indicated that both n-CPC and m-CPC have the ability to support cell attachment, growth, proliferation and differentiation, and also yield good bioactivity and biocompatibility.

Figure 1

Scanning electron micrographs of (a,b) nano-calcium silicate and (c,d) micro-calcium silicate with different magnifications.

Figure 2

X-ray diffraction patterns of (a) n-CS and (b) m-CS.

Figure 3

Change of weight loss of (a) n-CPC and (b) m-CPC with 40 wt% CS content in PBS at different incubated times and (c) pure PCL as a control. Data represent the mean±s.d., n=3.

Figure 4

Scanning electron micrographs of the surface morphology of (a) n-CPC and (b) m-CPC before immersion in SBF.

Figure 5

n-CPC specimens immersed in SBF for 14 days (a–d) at different magnifications.

Figure 6

m-CPC specimens immersed in SBF for 14 days (a–d) at different magnifications.

Figure 7

EDS spectra of surfaces of (a) n-CPC and (b) m-CPC samples in SBF for 14 days.

Figure 8

Concentration change of Ca, Si and P after immersion of (a) n-CPC and (b) m-CPC samples in SBF at different times (filled diamonds, Ca; filled squares, P; open triangles, Si).

Figure 9

The early attachments of MG63 on n-CPC and m-CPC. In particular, the n-CPC showed a significantly higher value than the other group (n=5, *p<0.05).

Figure 10

Proliferation of MG63 cells cultivated on m-CPC (light grey) and n-CPC (dark grey) for 1, 4 and 7 days. Data were represented as mean±s.d., n=6. Asterisks indicate that the proliferation rate of cells seeded on n-CPC was significantly higher than that of m-CPC at 4 and 7 days (p<0.05).

Figure 11

Phase contrast microscopy photographs of MG63 cells cultured on (a–c) n-CPC and (d–f) m-CPC at (a,b) 1 day, (c,d) 4 days and (e,f) 7 days.

Figure 12

Alkaline phosphatase activity of hMSCs cultured on the n-CPC (light grey) and m-CPC (dark grey) samples for 1, 4 and 7 days. Alkaline phosphatase activity on n-CPC was significantly higher than that on m-CPC at 7 days (n=5, *p<0.05).

Figure 13

SEM micrographs of hMSCs seeded on the surfaces of the (a,b) n-CPC and (c,d) m-CPC at (a,c) 4 days and (b,d) 7 days.