Functionalized hydroxyapatite with anti-oxidant and bone repair properties

摘要

Introduction: Functionalization of hydroxyapatite with bioactive ions, molecules and drugs has proven to be a successful strategy to couple the beneficial effect of the calcium phosphate on promotion of new bone formation with the specific action of the functionalizing agent. Quercetin (Que) is a flavonoid that displays anti-cancer, anti-oxidant, anti-inflammatory and cardiovascular protection properties. Moreover, it is a free radical scavenger able to reduce the production of oxidation-derived free radicals from the bone resorbing osteoclasts, which suggests a potential role of quercetin in the treatment of diseases related to bone loss. However, its low solubility in aqueous media, poor permeability, oral bioavailability and biodegradation can affect its pharmacological applications. In this work we synthesized hydroxyapatite (HA) functionalized with Que in order to develop a strategy for the local administration of the flavonoid and provide composite materials where the peculiar properties of Que add to the excellent biocompatibility and bioactivity of HA. Materials and Methods: The synthesis of HA in the presence of Que was carried out in a solution of water/ethanol following two different procedures: (1) direct synthesis of HA and (2) phase transition from Monetite (DCPA), in presence of different amounts (2.8 mM, 5.6 mM and 8.4 mM) of quercetin dehydrate. The products were characterized by X-ray powder diffraction (XRD), FT-IR and ATR/IR spectroscopy, transmission electron microscopy (TEM), and spectrophotometric techniques. In vitro tests were performed using an innovative triculture model involving osteoblast, osteoclast and endothelial cells. Results and Discussion: Composite crystals obtained through direct synthesis display reduced crystals mean dimensions and length of the coherently scattering domains, as shown by XRD and TEM results (Figure 1, top) and contain up to about 3.1 wt% Que. Que incorporation does not significantly affect HA crystal structure, most likely because of a good structural fit with the molecular structure of the flavonoid. However, during the synthesis Que undergoes a partial oxidation, which provokes a significant reduction of its radical scavenging activity (RSA). At variance, Que-functionalized HA crystals (containing up to about 1.3 wt% Que) obtained through phase transition display higher crystallinity and greater dimensions (figure 1, bottom), as well as relevant anti-oxidant activities, as testified by their high RSA levels. The results of the triculture in vitro tests mimicking bone microenviroment indicate that the presence of quercetin in the composite materials enhances osteoblast proliferation and differentiation, whereas it downregulates osteoclasteogenesis, and supports proliferation and differentiation of endothelial cells. Conclusions: Functionalization of HA with quercetin through phase transition from monetite turned out to be a successful strategy to maintain a good radical scavenging activity. The composite crystals positively influence bone repair microenvironment, stimulating osteoblast proliferation and activity, inhibiting osteoclast proliferation, and supporting micrc-angiogenetic processes necessary for new bone formation.