Calcium Phosphate Coating Prepared by Microarc Oxidation Affects hTERT Expression, Molecular Presentation, and Cytokine Secretion in Tumor-Derived Jurkat T Cells

Abstract

Calcium phosphate (CaP) materials are among the best bone graft substitutes, but their use in the repair of damaged bone in tumor patients is still unclear. The human Jurkat T lymphoblast leukemia-derived cell line (Jurkat T cells) was exposed in vitro to a titanium (Ti) substrate (10 × 10 × 1 mm3) with a bilateral rough (average roughness index (Ra) = 2–5 μm) CaP coating applied via the microarc oxidation (MAO) technique, and the morphofunctional response of the cells was studied. Scanning electron microscopy (SEM), X-ray diffraction (XRD), and energy dispersive X-ray spectroscope (EDX) analyses showed voltage-dependent (150–300 V) growth of structural (Ra index, mass, and thickness) and morphological surface and volume elements, a low Ca/PaT ratio (0.3–0.6), and the appearance of crystalline phases of CaHPO4 (monetite) and β-Ca2P2O7 (calcium pyrophosphate). Cell and molecular reactions in 2-day and 14-day cultures differed strongly and correlated with the Ra values. There was significant upregulation of hTERT expression (1.7-fold), IL-17 secretion, the presentation of the activation antigens CD25 (by 2.7%) and CD95 (by 5.15%) on CD4+ cells, and 1.5–2-fold increased cell apoptosis and necrosis after two days of culture. Hyperactivation-dependent death of CD4+ cells triggered by the surface roughness of the CaP coating was proposed. Conversely, a 3.2-fold downregulation in hTERT expression increased the percentages of CD4+ cells and their CD95+ subset (by 15.5% and 22.9%, respectively) and inhibited the secretion of 17 of 27 test cytokines/chemokines without a reduction in Jurkat T cell survival after 14 days of coculture. Thereafter, cell hypoergy and the selection of an hTERT-independent viable CD4+ subset of tumor cells were proposed. The possible role of negative zeta potentials and Ca2+ as effectors of CaP roughness was discussed. The continuous (2–14 days) 1.5–6-fold reductions in the secretion of vascular endothelial growth factor (VEGF) by tumor cells correlated with the Ra values of microarc CaP-coated Ti substrates seems to limit surgical stress-induced metastasis of lymphoid malignancies.

Amorphous–Crystalline Calcium Phosphate Coating Promotes In Vitro Growth of Tumor-Derived Jurkat T Cells Activated by Anti-CD2/CD3/CD28 Antibodies

“…An acidic suspension electrolyte (pH = 1-2) incorporating an undissolved nano-dispersed HA was obtained. The MAO process was carried out in an anodic potentiostatic regime with following parameters: pulse frequency of 50 Hz; pulse duration of 100 µs; time of 10 min; and varied applied voltage of 200, 250, and 300 V [39,43,44].…”

“…After culturing, cells were harvested to measure membrane antigen presentation, apoptosis, necrosis, and hTERT gene expression. Spontaneous secretion induced by CaP coating was measured in cell culture supernatants as described earlier [39].…”

“…MAb was labeled with violet blue (VioBlue), fluorescein (FITC), phycoerythrin (PE), allophycocyanin (APC), or phycoerythrin cyanine 7 (PE-Cy7), as described elsewhere [39]. Before staining, Jurkat T cells were washed with phosphate-buffered saline (pH = 7.2).…”

“…For this, an automated processing system (Bio-Plex Protein Assay System, Bio-Rad, Hercules, CA, USA) and a cytokine analysis system (Bio-Plex Pro Human Cytokine 27-Plex Panel, Bio-Rad, Hercules, CA, USA) were used. This test system measures the concentration (pg/mL) of human cytokines and chemokines such as tumor necrosis factor alpha (TNFα); basic fibroblast growth factor (FGFb); vascular endothelial growth factor (VEGF); platelet growth factor (PDGF-BB); granulocytemacrophage colony-stimulating factor (GM-CSF); granulocyte colony-stimulating factor (G-CSF); interleukins: (IL)-1β, IL-1Ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17; interferon gamma (IFN-γ); gamma-induced interferon protein 10 (IP-10; chemokine 10 with motif CXC (CXCL10)); chemoattractant protein-1 of monocytes (MCP-1; chemokine (motif CC) ligand 2 (CCL2)); inflammatory macrophage protein 1 alpha (MIP-1α; CCL3); MIP-1β (CCL4); regulated upon activation, and normal T cell expressed and secreted (RANTES; CCL5); and eotaxin [39]. All work was carried out following the manufacturer's instructions.…”

“…Little is known about the interactions of CaP materials with leukemic immune cells. We have recently demonstrated in vitro downregulation of morphofunctional reaction of human Jurkat T lymphoblast-like leukemia-derived cell line (Jurkat T cells) and the possible role of negative surface charge of micro-arc CaP coating on titanium (Ti) implants for the development of tumor cell hypoergy [39]. Jurkat T cells change their electrostatic charge when the cells are activated with microbeads coated with anti-CD3/CD28 antibodies [40].…”