Anti-Oxidant Multi-Functionalized Materials: Strontium-Substituted Monetite and Brushite as Delivery Systems for Curcumin

Abstract

Curcumin has numerous biological activities and pharmaceutical applications related to its ability to inhibit reactive oxygen species. Herein, strontium-substituted monetite (SrDCPA) and strontium-substituted brushite (SrDCPD) were synthesized and further functionalized with curcumin with the aim to develop materials that combine the anti-oxidant properties of the polyphenol, the beneficial role of strontium toward bone tissue, and the bioactivity of calcium phosphates. Adsorption from hydroalcoholic solution increases with time and curcumin concentration, up to about 5-6 wt%, without affecting the crystal structure, morphology, and mechanical response of the substrates. The multi-functionalized substrates exhibit a relevant radical scavenging activity and a sustained release in phosphate buffer. Cell viability, morphology, and expression of the most representative genes were tested for osteoclast seeded in direct contact with the materials and for osteoblast/osteoclast co-cultures. The materials at relatively low curcumin content (2-3 wt%) maintain inhibitory effects on osteoclasts and support the colonization and viability of osteoblasts. The expressions of Alkaline Phosphatase (ALPL), collagen type I alpha 1 chain (COL1A1), and osteocalcin (BGLAP) suggest that curcumin reduces the osteoblast differentiation state but yields encouraging osteoprotegerin/receptor activator for the NFkB factor ligand (OPG/RANKL) ratio.

Keywords: calcium phosphates; co-cultures; radical scavenging activity.

Figure 1

Photographic images of SrDCPA and SrDCPD crystalline powders before and after immersion in curcumin solutions at 3 mM, 4 mM, and 5 mM for 6 h.

Figure 2

Curcumin content of SrDCPA (a) and SrDCPD samples (b) incubated in hydroalcoholic solutions at different curcumin concentrations and for different time periods.

Figure 3

X-ray diffraction patterns of SrDCPA samples before and after incubation in curcumin solutions with different concentrations for 6 h (a) or 72 h (b), and of SrDCPD samples before and after incubation in curcumin solutions with different concentrations for 6 h (c) or 72 h (d). The peak indicated by (*) is due to the presence of crystalline curcumin.

Figure 4

SEM images of calcium phosphates crystals before (a,b) and after incubation in curcumin solution for 6 h (c,d) and 72 h (e,f).

Figure 5

Curcumin release in Phosphate Buffer Solution up to 14 days.

Figure 6

Antiradical activity, expressed as % RSA, of the different samples and pure curcumin toward DPPH•. Bars represent the mean ± SD of two independent measurements.

Figure 7

AFM images of 4 × 4 µm² calcium phosphates crystals before (a,d) and after incubation in curcumin solution for 6 h (b,e) and 72 h (c,f).

Figure 8

SEM images of the surface of disk-shaped samples obtained after pressing the powders into cylindrical molds: SA0 (a); SA3 (b); SA5 (c); SD0 (d); SD3 (e); SD5 (f).

Figure 9

Cell viability (Alamar Blue Assay) of OCs culture (a), OCs co-cultured with NHOst (b), and NHOst co-cultured with OCs (c) with tested SA and SD disk-shaped samples, expressed as percentage variation from CTR cultures at 7 and 14 days (Mean ± SD, n = 4 duplicates). Dunn’s test (1 symbol, p < 0.05; 2 symbols, p < 0.005; 3 symbols, p < 0.0005): For each experimental time—Disk-shaped SA or SD samples with curcumin versus SA0 or SD0 (*); SA5 or SD5 versus SA3 or SD3, respectively (°); SD versus SA at each curcumin concentration (#); for each disk-shaped SA or SD samples with or without curcumin—14 days versus 7 days (§).

Figure 10

Expression of the ACP5 (a,c), CTSK (b,d) genes in OC (a,b), and OC co-cultured with NHOst (c,d) after 7 and 14 days (Mean ± SD, n = 4 duplicates). Dunn’s test (1 symbol, p < 0.05; 2 symbols, p < 0.005; 3 symbols, p < 0.0005): Each condition versus Control (†); or each experimental time—Disk-shaped SA or SD samples with curcumin versus SA0 or SD0 (*); SA5 or SD5 versus SA3 or SD3, respectively (°); SD versus SA at each curcumin concentration (#); For each disk-shaped SA or SD samples with or without curcumin—14 days versus 7 days (§).

Figure 11

Expression of the ALPL (a), COL1A1 (b), BGLAP (c), and OPG/RANKL (d) genes in NHOst co-cultured with OCs after 7 and 14 days (Mean ± SD, n = 4 duplicates). Dunn’s test (1 symbol, p < 0.05; 2 symbols, p < 0.005; 3 symbols, p < 0.0005): For each experimental time—Disk-shaped SA or SD samples with curcumin versus SA0 or SD0 (*); SD versus SA at each curcumin concentration (#); for each disk-shaped SA or SD samples with or without curcumin—14 days versus 7 days (§).