As alternatives to autografts, allografts, and xenografts, calcium phosphate (CaP)-based bone-defect-filling materials (e.g., deproteinized bovine bone (DBB, Bio-Oss^®)) are widely used to repair large-volume bone defects (LVBDs) in clinic. However, most of these materials show a very low degradability due to a sintering process in their production. In this study, we synthesized a novel type of granules—biomimetically precipitated nanocrystalline calcium phosphate (BpNcCaP) by developing our previous biomimetic protocol. We evaluated the cytotoxicity of BpNcCaP by assessing the viability of L929 mouse fibroblasts using a 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay. To characterize the physicochemical properties of the novel BpNcCaP granules, we first compared the morphology and composition of BpNcCaP with those of Bio-Oss^® using scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and energy dispersive X-ray spectroscopy (EDS). We further compared the surface area, pore size distribution, hydrophilicity behavior, and hardness of BpNcCaP with those of Bio-Oss^® granules using specific surface area, contact angle, and Vickers hardness as parameters, respectively. BpNcCaP showed no obvious cytotoxicity. In-vitro characterization data showed that BpNcCaP and Bio-Oss^® granules were both comprised of nanocrystalline hydroxyapatite (HAp). The Ca/P ratios of BpNcCaP and Bio-Oss^® calculated from the EDS results were 1.34 and 1.66, respectively. Hence, BpNcCaP and Bio-Oss^® were Ca-deficient HAp. Compared with Bio-Oss^®, synthetic BpNcCaP had better hydrophilicity, higher specific surface area, lower crystallinity, and hardness. These data suggested a good performance of BpNcCaP granules in clinical applications.

Nanocrystalline hydroxyapatite has been extensively used for biomedical field, such as drug carrier, antitumor agent, surface coating, and bone-regenerating material. The wet chemical method is one of the most commonly used methods in the synthesis of nanosized hydroxyapatite due to its relatively low costs and more customizable control of the final product. Herein, we updated the most recent advances in artificial nano-hydroxyapatite prepared from biomimetic precipitation throughout context. Furthermore, micron-sized particles and macro-sized scaffolds made of nano-hydroxyapatite deposition are introduced. Their outstanding physicochemical properties and potential clinical application are highlighted. This article reviews the latest progress on preparing biomimetic nano-hydroxyapatite and provides inspiration to promote new breakthrough in regenerative therapy and clinical translation.

Human salivary histatin 1 (Hst1) exhibits a series of cell-activating properties, such as promoting cell spreading, migration, and metabolic activity. We recently have shown that fluorescently labeled Hst1 (F-Hst1) targets and activates mitochondria, presenting an important molecular mechanism. However, its regulating signaling pathways remain to be elucidated. We investigated the influence of specific inhibitors of G protein-coupled receptors (GPCR), endocytosis pathways, extracellular signal-regulated kinases 1/2 (ERK1/2) signaling, p38 signaling, mitochondrial respiration and Na+/K+-ATPase activity on the uptake, mitochondria-targeting and -activating properties of F-Hst1. We performed a siRNA knockdown (KD) to assess the effect of Sigma-2 receptor (S2R) /Transmembrane Protein 97 (TMEM97)—a recently identified target protein of Hst1. We also adopted live cell imaging to monitor the whole intracellular trafficking process of F-Hst1. Our results showed that the inhibition of cellular respiration hindered the internalization of F-Hst1. The inhibitors of GPCR, ERK1/2, phagocytosis, and clathrin-mediated endocytosis (CME) as well as siRNA KD of S2R/TMEM97 significantly reduced the uptake, which was accompanied by the nullification of the promoting effect of F-Hst1 on cell metabolic activity. Only the inhibitor of CME and KD of S2R/TMEM97 significantly compromised the mitochondria-targeting of Hst1. We further showed the intracellular trafficking and targeting process of F-Hst1, in which early endosome plays an important role. Overall, phagocytosis, CME, GPCR, ERK signaling, and S2R/TMEM97 are involved in the internalization of Hst1, while only CME and S2R/TMEM97 are critical for its subcellular targeting. The inhibition of either internalization or mitochondria-targeting of Hst1 could significantly compromise its mitochondria-activating property.