Zn participates in various biochemical processes involved in hard tissue formation in the human body and is also known to exhibit antibacterial activity. The incorporation of Zn into hydroxyapatite (HAP), the main mineral component of teeth, has therefore attracted considerable interest for dental and biomedical applications. In this study, Zn-incorporated fluoridated HAP (F-HAP) was synthesized on a human dentin substrate using a laser-assisted biomimetic process, and the influence of the Zn-addition route on the resulting surface characteristics was investigated. When Zn was added to the supersaturated calcium phosphate solution (reaction medium), F-HAP nanoparticles with a higher Zn content were formed. Contrary, submicrometer-long Zn-containing F-HAP rods were formed on the substrate when Zn was mixed with the light absorber rather than adding to the reaction medium. In mixing with the light absorber, Zn was also readily incorporated into the dentin substrate that was not observed in the addition of Zn to the reaction medium. Both Zn-incorporated dentin surfaces exhibited antibacterial activity against Streptococcus mutans. These results demonstrate that the Zn-addition route significantly influences the morphology and Zn distribution of the formed F-HAP layer and can therefore serve as an important parameter in the design of antibacterial dental coatings.

OBJECTIVE:Regenerative therapy using stem cells to treat cerebral infarction is currently in the research phase. However, this method is costly. It also faces other significant challenges, including optimization of timing, delivery methods, and dosage. Therefore, more practical and effective therapies are required. Bioabsorbable artificial dura mater made from nonwoven Polyglycolic Acid (PGA) fabric is used clinically to treat cerebral infarction. Basic Fibroblast Growth Factor (bFGF) has attracted considerable attention as a potential therapeutic candidate for the treatment of cerebral infarctions. In this study, we aimed to prepare a bFGF-releasing PGA dura mater and investigate its therapeutic efficacy for the recovery of neurological function in a mouse model of focal cerebral infarction. METHODS:An artificial dura mater (Durawave) made from nonwoven PGA fabric was subjected to oxygen plasma treatment, followed by bFGF adsorption. The release of bFGF from the resulting PGA dura mater was evaluated in vitro using enzyme-linked immunosorbent assays. bFGF-releasing PGA dura mater was placed at the site of induced cerebral infarctions in mice. Neurological function was assessed 14 days after insertion, followed by a histological assessment. RESULTS:The prepared PGA dura mater released bFGF in a dose-dependent manner. Neurological function in the bFGF-treated groups was significantly better than that in the control group. bFGFreleasing PGA dura mater also significantly increased the number of neural progenitor cells in the peri-infarct cortex and striatum and showed a trend toward promoting angiogenesis. CONCLUSION:bFGF-releasing PGA dura mater improved neurological function in a mouse model of focal cerebral infarction.

Porous collagen sponges coated with low-crystalline apatite are promising bone tissue regeneration scaffolds owing to their chemical similarity to natural bones. We previously fabricated an apatite-coated collagen sponge loaded with an osteogenic agent L-ascorbic acid 2-phosphate (AS), using a biomimetic coating process, demonstrating its superior functionality. In the present study, this sponge was further functionalized with another osteogenic agent, semaphorin 3A (S3). The collagen sponge was first coated with AS-immobilized apatite by a biomimetic coating process using a supersaturated calcium phosphate solution, and then impregnated with S3 via drop-casting. The resulting sponge showed improved bone regeneration in a rat calvarial defect model via enhanced cell infiltration, angiogenesis, osteogenesis, and bone remodeling, while being resorbed by macrophage-mediated processes, highlighting its potential as a bone tissue regeneration scaffold.

BACKGROUND:Traumatic brain injury harms health, causes disability, and burdens health care systems and economies. Although new treatments for brain injury have been developed, their therapeutic efficacy remains insufficient. Herein, we demonstrate the therapeutic efficacy of artificial dura mater with varying basic fibroblast growth factor (bFGF)-releasing capabilities using a brain injury model. METHODS:Artificial dura mater of lower (FGF-L) and higher (FGF-H) bFGF-releasing capabilities was prepared via oxygen plasma treatment for polyglycolic acid nonwoven fabric followed by bFGF adsorption. Mice received either bFGF-releasing dura mater (FGF-L, FGF-H) or bFGF-free dura mater (FGF-C) at the site of the induced brain injury. RESULTS:Neurological functions significantly improved in the FGF-L and FGF-H groups compared with those in the FGF-C group on Day 14. No significant difference was observed in the brain injury area between the FGF-C group and either the FGF-L or FGF-H group. The number of SRY-box transcription factor 2-positive cells in the cortex was significantly larger in the FGF-L and FGF-H groups than in the FGF-C group on Day 7. The terminal transferase dUTP nick-end labeling-positive cell ratio was significantly lower in the FGF-H group than in the FGF-C group on Day 14. The occludin-positive and ZO-1-positive cell ratios were significantly greater in the FGF-H group than in the FGF-C group on Day 14, suggesting improved blood-brain barrier integrity. CONCLUSION:The bFGF-releasing dura mater enhanced neural progenitor cell proliferation, inhibited apoptosis and blood-brain barrier breakdown, and contributed to neurological function recovery in brain-injured mice.

Lactoferrin is a highly safe antibacterial protein found in the human body and in foods. Calcium phosphate (CaP) nanoparticles with immobilized lactoferrin could therefore be useful as intraoral disinfectants for the prevention and treatment of dental infections because CaP is a mineral component of human teeth. In this study, we fabricated CaP nanoparticles with co-immobilized lactoferrin and heparin using a simple one-step coprecipitation process. Heparin, a negatively charged polysaccharide, was used as both an immobilizing agent for lactoferrin and a particle-dispersing agent. The immobilization efficiency for lactoferrin in the CaP nanoparticles depended on the concentrations of both the lactoferrin and heparin in the reaction solution and was over 90% under optimal conditions. The nanoparticles had a hydrodynamic diameter of about 150–200 nm and could be well dispersed in water, owing to their relatively large negative zeta potential derived from heparin. They were found to exhibit antibacterial activity against Actinomyces naeslundii, which is involved in the initial formation of dental plaque that consequently leads to dental caries and periodontal disease. These results indicate the potential of the proposed nanoparticles as intraoral disinfectants.

A thin-layer of fluoride-incorporated hydroxyapatite (FAp) is useful as an acid-resistant antibacterial mineralized coating to protect a tooth surface. Recently, we achieved rapid and area-specific FAp coating on the surfaces of human tooth substrates via precoating with a light-absorbing agent, indocyanine green, followed by laser irradiation in a fluoride-containing supersaturated calcium phosphate solution. In the present study, this FAp coating technique was applied to a substrate of resin-based composite (RBC), an artificial material with no biomineral component. Similar to the case of tooth substrates, a mineralized layer consisting of needle-like FAp crystals was formed on the RBC surface after laser irradiation for 3 min. The needle-like crystals increased in size with increasing laser power. In the absence of fluoride ions in the supersaturated solution, a layer consisting of platelike octacalcium phosphate crystals was formed on the RBC surface. The present FAp coating technique was finally applied to an RBC-restored tooth substrate. Cross-sectional analyses revealed that the boundary region between the tooth and RBC was coated and sealed with a mineralized layer. The present FAp coating technique is potentially a new tool to seal the tooth-RBC boundary and reduce the risk of secondary caries associated with RBC restorations.

A chemical reaction of disulfiram, an oral anti-alcoholism drug, with copper ions produces bis (diethyldithiocarbamate)-copper complexes (CuETs) having strong anti-cancer efficacy. Water-dispersible calcium phosphate (CaP) nanoparticles with immobilized Cu nanoclusters (Cu-CaP nanoparticles) have the potential for use as a cancer-targeted delivery carrier of Cu for in situ synthesis of CuETs through the reaction with orally administered disulfiram. Herein, we aimed to fabricate such nanoparticles via coprecipitation in a highly supersaturated CaP solution containing Cu nanoclusters and heparin (particle-dispersant) and to demonstrate their cytotoxic effects on cancer cells when used with disulfiram. By tuning Cu concentration of the solution, we successfully fabricated water-dispersible Cu-CaP nanoparticles, which were nearly spherical in shape, were similar to 100 nm in hydrodynamic diameter, and immobilized a large number of Cu nanoclusters (diameter of 1-2 nm) over the matrix of amorphous CaP. The Cu-CaP nanoparticles were non-cytotoxic against murine breast cancer cells (4T1 cells) at a concentration range under 0.1 mM (as Cu). In combination with non-cytotoxic concentration levels of disulfiram (20 ng/mL or more), the Cu-CaP nanoparticles (Cu: 0.05 mM) showed significant cytotoxic efficacy against 4T1 cells most likely due to the production of CuETs. The Cu-CaP nanoparticles would be useful as a Cu-carrier to achieve in situ synthesis of CuETs at the cancer sites.

The formation of hydroxyapatite (Ca-10(PO4)(6)(OH)(2); HAP)-based multilayers containing antibacterial agents at different concentrations on teeth can help control infections against various bacteria in periodontal tissue. However, from a clinical perspective, rapid (within minutes) and simple construction of such multilayers on teeth has not yet been achieved. If realized, such a technique could contribute to oral health by promoting infection control. In this study, crystalline bilayers containing antibacterial agents (Ag and F) at different concentrations were rapidly constructed on dentin. This was achieved by subjecting the dentin substrates precoated with Ag(NH3)(2)F-containing light-absorbing paste to 30-s laser irradiation in a pseudo-physiological calcium phosphate solution. Transmission electron microscopy and energy-dispersive X-ray spectroscopy analyses revealed that the upper layer on the irradiated substrate was composed of F-substituted HAP (F-HAP) rods elongated to the c-axis. The content of Ag (<50 nm in size) in the rods layer was 0.14 +/- 0.03 at.%. The lower layer was composed of particulate F-HAP and CaF2, with the Ag (<50 nm) content of 0.49 +/- 0.17 at.%. X-ray photoelectron spectroscopy measurements indicated two chemical states of Ag on the irradiated substrate surface: Ag-0, corresponding to Ag particles, and Ag delta+, which substituted Ca2+ ions in the F-HAP crystals. The bilayers exhibited antibacterial activity against Streptococcus mutans, highlighting their potential for application in antibacterial dental treatment.

Bioresorbable porous scaffolds capable of promoting osteoregeneration while preventing bacterial infection are needed for regenerative periodontal therapy. Previously, a porous collagen sponge coated with low-crystalline apatite has been shown to possess superior bioresorption and osteogenic properties compared to the uncoated sponge. In this study, we integrated osteogenic and antibacterial dual drugs into the sponge utilizing two types of apatite matrices to achieve further functionalization. First, the collagen sponge was coated with apatite loaded with an osteogenic drug, l-ascorbic acid 2-phosphate (AS), using a metastable supersaturated calcium phosphate (CaP) solution supplemented with AS. Second, the coated sponge was impregnated with apatite particles loaded with an antibacterial drug, ciprofloxacin (CF), which were fabricated using a labile supersaturated CaP solution supplemented with CF. The resulting dual drug-immobilized sponge demonstrated biological activities arising from both AS and CF; it enhanced proliferation of osteoblastic MC3T3-E1 cells and exhibited antibacterial activity against the oral bacterium Actinomyces naeslundii. The proposed technique to fabricate multifunctional scaffolds would offer a solution to provide more effective, patient-tailored regenerative periodontal therapy.

Tamibarotene (Am80) is a promising anti-tumor drug that induces the expression of Meflin (a glycosylphosphatidyl inositol-anchored protein) in cancer-associated fibroblasts, thereby improving the tumor microenvironment. However, Am80, which is approved only for oral administration owing to its poor water solubility, has the challenge of poor tumor penetration. In this study, we developed poly(lactic-co-glycolic acid) nanoparticles loaded with Am80 (Am80–PLGA nanoparticles) as a potential intravenous drug for targeted Am80 delivery to the tumor site. The Am80–PLGA nanoparticles were fabricated using the single-emulsion method in the presence of cationic polyethyleneimine (PEI). The loading efficiency of Am80 in the nanoparticles was controlled by tuning the PEI concentration in the preparation mixture. Nanoparticles with the highest Am80-loading efficiency were dispersible and showed a hydrodynamic diameter of approximately 190 nm in phosphate-buffered saline for up to 2 weeks. The Am80 release from the nanoparticles started in a day and lasted for weeks. The nanoparticles upregulated Meflin expression in human fibroblasts (fHDF/TERT166 cells). These results suggest the potential of Am80–PLGA nanoparticles as a new intravenous anti-tumor drug that can improve the tumor microenvironment, thereby enhancing the efficacy of chemotherapy and immunotherapy.

Functionalization of teeth surfaces has attracted much attention from the perspective of preventive dentistry. Laser irradiation (30 s) of the viscous light-absorber paste coated on the tooth substrate enabled ultrafast coating of orientation-controlled fluoride-substituted hydroxyapatite (HAP) rod layers, 150-500 nm thick, in a calcium phosphate solution. On enamel, the layers perfectly inherited orientations and structures of the crystals in the substrate, exhibiting two types of hierarchies at different scales. One was a submicron-scale hierarchy, in which an approximately 60-nm-wide rod comprised multiple thinner rods, approximately 20-nm wide, oriented along the c-axis. The 20-nm-wide rods exhibited another type of nanoscale hierarchy comprising incompletely assembled nanofibers < 5-nm wide. In dentin and cementum, c-axis-elongated HAP rods grown on the substrates displayed less arrangement in their elongation direction than those grown on enamel. Most rods on dentin were single crystals, unlike those grown on enamel. On the cementum, each polycrystalline rod (100-150 nm wide) comprised multiple single-crystal rods approximately 60-nm wide, forming a sub-micron hierarchy similar to that observed in enamel. The developed method may be useful in dental practice for protecting teeth surfaces and repairing microdefects in enamel, dentin, and cementum.

Calcium phosphate (CaP) coating of zirconia and zirconia-based implants is challenging, due to their chemical instability and susceptibility to thermal and mechanical impacts. A 3 mol% yttrium-stabilized tetragonal zirconia polycrystal was subjected to femtosecond laser (FsL) irradiation to form micro- and submicron surface architectures, prior to CaP coating using pulsed laser deposition (PLD) and low-temperature solution processing. Untreated zirconia, CaP-coated zirconia, and FsL-irradiated and CaP-coated zirconia were implanted in proximal tibial metaphyses of male Japanese white rabbits for four weeks. Radiographical analysis, push-out test, alizarin red staining, and histomorphometric analysis demonstrated a much improved bone-bonding ability of FsL-irradiated and CaP-coated zirconia over CaP-coated zirconia without FsL irradiation and untreated zirconia. The failure strength of the FsL-irradiated and CaP-coated zirconia in the push−out test was 6.2–13.1-times higher than that of the CaP-coated zirconia without FsL irradiation and untreated zirconia. Moreover, the adhesion strength between the bone and FsL-irradiated and CaP-coated zirconia was as high as that inducing host bone fracture in the push-out tests. The increased bone-bonding ability was attributed to the micro-/submicron surface architectures that enhanced osteoblastic differentiation and mechanical interlocking, leading to improved osteointegration. FsL irradiation followed by CaP coating could be useful for improving the osteointegration of cement-less zirconia-based joints and zirconia dental implants.

Bone is a highly dynamic connective tissue that provides structural support, locomotion and acts as a shield for many vital organs from damage. Bone inherits the ability to heal after non-severe injury. In case of severe bone abnormalities due to trauma, infections, genetic disorders and tumors, there is a demand for a scaffold that can enhance bone formation and regenerate the lost bone tissue. In this study, a 3D collagen scaffold (CS) was functionalized and assessed under in vitro and in vivo conditions. For this, a collagen scaffold coated with hydroxyapatite (Ap-CS) was developed and loaded with a peptide LL-37. The physico-chemical characterisation confirmed the hydroxyapatite coating on the outer and inner surfaces of Ap-CS. In vitro studies confirmed that LL37 loaded Ap-CS promotes osteogenic differentiation of human osteosarcoma cells without showing significant cytotoxicity. The efficacy of the LL-37 loaded Ap-CS for bone regeneration was evaluated at 4 and 12 weeks postimplantation by histopathological and micro-CT analysis in rabbit femur defect model. The implanted LL-37 loaded Ap-CS facilitated the new bone formation at 4 weeks compared with Ap-CS without LL-37. The LL-37 loaded Ap-CS incorporating apatite and peptide LL-37 would be useful as a multifunctional scaffold for bone tissue engineering.

Composite nanoparticles of mannose-modified gold nanoclusters (AuMs) and calcium phosphate (CaP), AuM-CaP nanoparticles, have the potential for use as diagnostic and therapeutic agents for tumors. Herein, gold nanoclusters (AuNCs) were surface-modified to various degrees with mannose, a targeting ligand for tumor cells, to prepare AuMs with different amounts of mannose. The prepared AuMs were used to fabricate AuM-CaP nanoparticles via coprecipitation in labile supersaturated CaP solutions containing AuMs. The resulting nano- particles were nearly spherical in shape and a few hundred nanometers in size (hydrodynamic diameter), and showed good dispersibility in water due to the relatively large negative zeta potentials (-16 to-14 mV). The degree of mannose modification in AuMs was a controlling factor affecting the structure and composition of the AuM-CaP nanoparticles; the size and Au content (Au/Ca elemental ratio) of the nanoparticles varied depending on the degree of mannose modification. With a suitable degree of mannose modification in AuMs, the AuM-CaP nanoparticles possessed a size (similar to 100 nm) effective for tumor accumulation, and immobilized a high content of AuMs dispersed densely over the matrix of CaP. Further research is needed to verify the potential of AuM-CaP nanoparticles in tumor theragnostic.

Rapid biomimetic replication of human hard tissues at the nanoscale is an important goal in the field of regenerative medicine. To this end, the repair of human tooth enamel using various techniques has been extensively investigated. In this study, enamel-like hydroxyapatite (HAP) layers were successfully grown on human enamel substrates within 3 min by laser-assisted biomimetic crystallization using indocyanine green (ICG) as a light absorbent. Laser irradiation of ICG-coated substrates in a pseudo-physiological solution enabled enamel regeneration by crystallizing micrometer-thick fluoride-substituted HAP rod layers. The HAP rods formed on the substrates possessed a c-axis-oriented morphology similar to that of natural enamel HAP. The results were reproducible across two substrates derived from two patients; however, the crystallized layers differed in their mesoscale structures. One layer exhibited a mesoscale hierarchical structure, in which approximately 20-nmwide elemental HAP nanorods self-assembled into approximately 230-nm-wide polycrystalline rods with a slight shift in the c-axis direction of each nanorod. The polycrystalline rods were further assembled to form HAP rod groups, generally aligned along the c axis. This layer integrated into the enamel through layers of lownumber-density HAP rods and non-oriented HAP nanoparticles. The layer grown on the other substrate lacked this characteristic mesoscale hierarchy, and a 150-200-nm-wide rod of the HAP single crystals grew without the low-number-density HAP layer. These differences can be attributed to whether or not geometrical selection has occurred at the interface between the enamel substrate and the crystallized layer. The proposed method has the potential to replace conventional resin-based treatments for the repair of early caries lesions.

Coating layers consisting of a crystalline apatite matrix with immobilized basic fibroblast growth factor (bFGF) can release bFGF, thereby enhancing bone regeneration depending on their bFGF content. We hypothesized that the incorporation of fluoride ions into apatite crystals would enable the tailored release of bFGF from the coating layer depending on the layer’s fluoride content. In the present study, coating layers consisting of fluoride-incorporated apatite (FAp) crystals with immobilized bFGF were coated on a porous collagen sponge by a precursor-assisted biomimetic process using supersaturated calcium phosphate solutions with various fluoride concentrations. The fluoride content in the coating layer increased with the increasing fluoride concentration of the supersaturated solution. The increased fluoride content in the coating layer reduced its solubility and suppressed the burst release of bFGF from the coated sponge into a physiological salt solution. The bFGF release was caused by the partial dissolution of the coating layer and, thus, accompanied by the fluoride release. The concentrations of released bFGF and fluoride were controlled within the estimated effective ranges in enhancing bone regeneration. These findings provide useful design guidelines for the construction of a mineralized, bFGF-releasing collagen scaffold that would be beneficial for bone tissue engineering, although further in vitro and in vivo studies are warranted.

Calcium phosphate (CaP) particles immobilizing antibacterial agents have the potential to be used as dental disinfectants. In this study, we fabricated CaP particles with immobilized ciprofloxacin (CF), a commonly prescribed antibacterial agent, via a coprecipitation process using a supersaturated CaP solution. As the aging time in the coprecipitation process increased from 2 to 24 h, the CaP phase in the resulting particles transformed from amorphous to low-crystalline hydroxyapatite, and their Ca/P elemental ratio, yield, and CF content increased. Despite the higher CF content, the particles aged for 24 h displayed a slower release of CF in a physiological salt solution, most likely owing to their crystallized matrix (less soluble hydroxyapatite), than those aged for 2 h, whose matrix was amorphous CaP. Both particles exhibited antibacterial and antibiofilm activities along with an acid-neutralizing effect against the major oral bacteria, Streptococcus mutans, Porphyromonas gingivalis, and Actinomyces naeslundii, in a dose-dependent manner, although their dose–response relationship was slightly different. The aging time in the coprecipitation process was identified as a governing factor affecting the physicochemical properties of the resulting CF-immobilized CaP particles and their functionality as a dental disinfectant.

Current chemotherapy still suffers from unsatisfactory therapeutic efficacy, multi-drug resistance, and severe adverse effects, thus necessitating the development of techniques to confine chemotherapy drugs in the tumor microenvironment. Herein, we fabricated nanospheres of mesoporous silica (MS) doped with Cu (MS-Cu) and polyethylene glycol (PEG)-coated MS-Cu (PEG-MS-Cu) as exogenous copper supply systems to tumors. The synthesized MS-Cu nanospheres showed diameters of 30–150 nm with Cu/Si molar ratios of 0.041–0.069. Only disulfiram (DSF) and only MS-Cu nanospheres showed little cytotoxicity in vitro, whereas the combination of DSF and MS-Cu nanospheres showed significant cytotoxicity against MOC1 and MOC2 cells at concentrations of 0.2–1 μg/mL. Oral DSF administration in combination with MS-Cu nanospheres intratumoral or PEG-MS-Cu nanospheres intravenous administration showed significant antitumor efficacy against MOC2 cells in vivo. In contrast to traditional drug delivery systems, we herein propose a system for the in situ synthesis of chemotherapy drugs by converting nontoxic substances into antitumor chemotherapy drugs in a specific tumor microenvironment.