Introduction: Calcium phosphates, such as hydroxyapatite (Ca10(PO4)6(OH)2; HAp) are one of the important key materials in the field of biomaterials. Recently, immunotherapy without side effects has been expected as a novel medical treatment for a cancer. Thus, we focused on an inositol phosphate (IP6) that has both anti-cancer activity and immunopotentiative action[1],[2]. In the present work, in order to create the novel cell-culture substrates for enhancing the activity of immune cells, we fabricated the HAp ceramics surface-modified with IP6 (hereafter, IP6-HAp ceramics), and examined the cell population of immunocytes (helper T, killer T, B cells) derived from mouse spleen to IP6-HAp ceramics. In addition, in order to clarify the reason why the population of immunocytes (ex, T cells) changed, we observed the morphology of cells attached on the IP6-HAp ceramics.
Materials and Methods: As previously reported[3], the HAp ceramics were fabricated by firing the compacts at 1200 °C for 5 h at the heating rate of 10 °C･min-1. The IP6 solution for surface modification was prepared at the concentration of 0, 1000, 2000, 3000, and 5000 ppm and adjusted to pH 7.3. The IP6-HAp ceramics were obtained by soaking the HAp ceramics into IP6 solution for 24 h. Splenocytes (1 x 106 cells·cm-3) were seeded on three kinds of the specimens: polystyrene plate (control), HAp ceramics or IP6-HAp ceramics. Splenocytes taken out from the mouse (C57BL/6N) were collected and seeded on the specimens. After 1 day of seeding, we analyzed the activation of the splenocytes by a flow cytometry, together with the observation of the morphology of ones by scanning electron microscopy (SEM) and by immunostaining.
Results and Discussion: The responses of splenocytes to the IP6-HAp ceramics with various IP6 concentrations were examined, together with control of polystyrene. The antibodies used in flow cytometry analysis were CD3 and CD4 for helper T cells, CD3 and CD8 for killer T cells, and CD19 for B cells. These antibodies were selected to clarify the presence of helper T cells, killer T cells and B cells, respectively. We focused the population of cells expressed both CD3 and CD4 for helper T cells, both CD3 and CD8 for killer T cells, and CD19 for B cells. Especially, the percentage (%) of T cells on the IP6-HAp(3000) and IP6-HAp(5000) significantly increased compared to the control. These results indicate that high-dosage IP6-HAp ceramics seem to enhance the activity of immunocytes. Figure 1(a) shows the SEM image of the splenocytes cultured on the IP6-HAp(3000) ceramics. Several kinds of cells adhered on the ceramics. In order to examine the expressions of CD3 (identification of T cells) and vinculin (adaptor protein), we observed the cells attached on the ceramics by a confocal laser microscopy. The cells immunostained by anti-vinculin antibody (ab) (red colour) and anti-CD3 ab (green one) are shown in Fig. 1(b). CD3-positive cells were attached on the surface of the ceramics. This indicates that immune cells were stimulated by the interaction between cells and IP6 immobilized-HAp ceramics.
Conclusion: When the splenocytes derived from mouse spleen were co-cultured with the IP6-HAp ceramics, the IP6-HAp ceramics increased population of helper and killer T cells, compared with pure HAp ceramics without IP6 and control (polystyrene). Therefore, the IP6-HAp ceramics may be expected as a useful biomaterial for cancer immunity medical treatment.
References:
[1] Y. Okazaki, T. Katayama, J. Jpn. Soc. Nutr. Food Sci., 58, 151-56 (2005).
[2] M. Verghese, D.R. Rao, C.B. Chawan, L.T. Walker, L. Shackelford, LWT, 39, 1093-98 (2006).
[3] K. Yamada, M. Nakamura, S. Nagai, M. Honda, M. Aizawa, Bioceramics 26, Barcelona, Spain, 6th-8th, November 2014.