Nano-hydroxy apatite/chitosan/gelatin scaffolds enriched by a combination of platelet-rich plasma and fibrin glue enhance proliferation and differentiation of seeded human dental pulp stem cells.

The purpose of this study was to investigate the effects of Fibrin Glue (FG) and activated platelet-rich plasma (a-PRP) on the proliferation and osteogenic differentiation of human dental pulp stem cells (h-DPSCs). Therefore, we planned to investigate in vitro behavior of porous composite scaffolds based on chitosan-gelatin/nanohydroxyapatite (CS-G/nHA) treated with FG and a-PRP. The porous structure of CS-G/nHA was prepared using combination of particle leaching and freeze-drying methods. The a-PRP was prepared from the centrifugation of whole blood activated with calcium chloride. Four groups of composite scaffolds were fabricated to seed h-DPSCs: (1) a-PRP-FG/CS-G/nHA; (2) FG/CS-G/nHA; (3) a-PRP/CS-G/nHA; (4) CS-G/nHA. The 14 days SEM image reveled organized fibrin network on scaffolds surface. All groups treated with FG and a-PRP, showed improved adhesion of seeded h-DPSCs compared to CS-G/nHA. Cytotoxicity of the composite scaffolds was assessed by MTT. Alizarin red staining confirmed the formation of bone minerals by h-DPSCs after 21 days of cell seeding. In addition, the a-PRP-FG treated scaffolds exhibited significantly elevated bone gamma-carboxyglutamic acid-containing protein (BGLAP), bone morphogenetic protein 2 (BMP2), and runt-related transcription factor 2 (RUNX2) gene expression. The present result the composite scaffolds treated with FG and a-PRP showed a fibrin network, preferentially on the surface of composite scaffold increasing the mineralization and osteoblastic differentiation of harvested cells. In addition, a-PRP-FG/ CS-G/nHA scaffold increased bone marker gene expressions from day 7 to day 21.

Carbonate Apatite Nanoparticles Act as Potent Vaccine Adjuvant Delivery Vehicles by Enhancing Cytokine Production Induced by Encapsulated Cytosine-Phosphate-Guanine Oligodeoxynucleotides.

Vaccine adjuvants that can induce not only antigen-specific antibody responses but also Th1-type immune responses and CD8+ cytotoxic T lymphocyte responses are needed for the development of vaccines against infectious diseases and cancer. Of many available adjuvants, oligodeoxynucleotides (ODNs) with unmethylated cytosine-phosphate-guanine (CpG) motifs are the most promising for inducing the necessary immune responses, and these adjuvants are currently under clinical trials in humans. However, the development of novel delivery vehicles that enhance the adjuvant effects of CpG ODNs, subsequently increasing the production of cytokines such as type-I interferons (IFNs), is highly desirable. In this study, we demonstrate the potential of pH-responsive biodegradable carbonate apatite (CA) nanoparticles as CpG ODN delivery vehicles that can enhance the production of type-I IFNs (such as IFN-α) relative to that induced by CpG ODNs and can augment the adjuvant effects of CpG ODNs in vivo. In contrast to CpG ODNs, CA nanoparticles containing CpG ODNs (designated CA-CpG) induced significant IFN-α production by mouse dendritic cells and human peripheral blood mononuclear cells in vitro; and production of interleukin-12, and IFN-γ was higher in CA-CpG-treated groups than in CpG ODNs groups. In addition, treatment with CA-CpG resulted in higher cytokine production in draining lymph nodes than did treatment with CpG ODNs in vivo. Furthermore, vaccination with CA-CpG plus an antigen, such as ovalbumin or influenza virus hemagglutinin, resulted in higher antigen-specific antibody responses and CD8+ cytotoxic T lymphocyte responses in vivo, in an interleukin-12- and type-I IFN-dependent manner, than did vaccination with the antigen plus CpG ODNs; in addition, the efficacy of the vaccine against influenza virus was higher with CA-CpG as the adjuvant than with CpG ODNs as the adjuvant. These data show the potential of CA nanoparticles to serve as CpG ODN delivery vehicles that increase the production of cytokines, especially IFN-α, induced by CpG ODNs and thus augment the efficacy of CpG ODNs as adjuvants. We expect that the strategy reported herein will facilitate the design and development of novel adjuvant delivery vehicles for vaccines.

Bioresorbable Microspheres with Surface-Loaded Nanosilver and Apatite as Dual-Functional Injectable Cell Carriers for Bone Regeneration.

Bone defect repair, especially in terms of infection, remains a big challenge in clinical therapy. To meet clinical requirements, the dual-functional strategy including antibacterial activity and strong osteogenesis effect is a promising approach in bone tissue engineering. In the present study, bioresorbable porous-structured microspheres are fabricated from amphiphilic block copolymer composed of poly(l-lactide) and poly(ethyl glycol) blocks. After being surface coated with mussel-inspired polydopamine, the microspheres are loaded with nanosilver via reduction of silver nitrate and apatite via biomineralization in sequence. The resulting composite microspheres are systematically characterized by in vitro coculturing with rat bone mesenchymal stromal cells and Staphylococcus aureus to evaluate cytotoxicity and antibacterial activity. In vivo evaluation is carried out by filling the composite microspheres into infected cranial defects in a rat model. The primary results indicate that the dual-purpose microspheres have the capacity to defeat infection while promoting bone regeneration.

Photodynamic Therapy Using Indocyanine Green Loaded on Super Carbonate Apatite as Minimally Invasive Cancer Treatment.

Minimally invasive treatment is getting more and more important in an aging society. The purpose of this study was to explore the possibility of ICG loaded on super carbonate apatite (sCA) nanoparticles as a novel photodynamic therapy (PDT) against cancers. Using colon cancer cells, ICG uptake and anti-tumor effects were examined between the treatments of ICG and sCA-ICG. Reactive oxygen species (ROS) production and temperature rise were also evaluated to explore the underlying mechanism. Atomic force microscopy revealed that the size of sCA-ICG ranged from 10 to 20 nm. In aqueous solution with 0.5% albumin, the temperature increase after laser irradiation was 27.1°C and 23.1°C in sCA-ICG and ICG, respectively (control DW: 5.7°C). A significant increase in ROS generation was noted in cell cultures treated with sCA-ICG plus irradiation compared with those treated with ICG plus irradiation (P < 0.01). Uptake of ICG in the tumor cells significantly increased in sCA-ICG compared with ICG in vitro and in vivo The fluorescence signals of ICG in the tumor, liver, and kidney faded away in both treatments by 24 hours. Finally, the HT29 tumors treated with sCA-ICG followed by irradiation exhibited drastic tumor growth retardation (P < 0.01), whereas irradiation of tumors after injection of ICG did not inhibit tumor growth. This study shows that sCA is a useful vehicle for ICG-based PDT. Quick withdrawal of ICG from normal organs is unique to sCA-ICG and contrasts with the other nanoparticles remaining in normal organs for a long time. Mol Cancer Ther; 17(7); 1613-22. ©2018 AACR.

Knockdown of ROS1 gene sensitizes breast tumor growth to doxorubicin in a syngeneic mouse model.

Treatment of breast cancer, the second leading cause of female deaths worldwide, with classical drugs is often accompanied by treatment failure and relapse of disease condition. Development of chemoresistance and drug toxicity compels compromising the drug concentration below the threshold level with the consequence of therapeutic inefficacy. Moreover, amplification and over-activation of proto-oncogenes in tumor cells make the treatment more challenging. The oncogene, ROS1 which is highly expressed in diverse types of cancers including breast carcinoma, functions as a survival protein aiding cancer progression. Thus we speculated that selective silencing of ROS1 gene by carrier-mediated delivery of siRNA might sensitize the cancer cells to the classical drugs at a relatively low concentration. In this investigation we showed that intracellular delivery of c-ROS1-targeting siRNA using pH-sensitive inorganic nanoparticles of carbonate apatite sensitizes mouse breast cancer cells (4T1) to doxorubicin, but not to cisplatin or paclitaxel, with the highest enhancement in chemosensitivity obtained at 40 nM of the drug concentration. Although intravenous administrations of ROS1-loaded nanoparticles reduced growth of the tumor, a further substantial effect on growth retardation was noted when the mice were treated with the siRNA- and Dox-bound particles, thus suggesting that silencing of ROS1 gene could sensitize the mouse breast cancer cells both in vitro and in vivo to doxorubicin as a result of synergistic effect of the gene knockdown and the drug action, eventually preventing activation of the survival pathway protein, AKT1. Our findings therefore provide valuable insight into the potential cross-talk between the pathways of ROS1 and doxorubicin for future development of effective therapeutics for breast cancer.

Current Applications of Facial Volumization with Fillers.

LEARNING OBJECTIVES: After reading this article and watching the accompanying videos, the participant should be able to: 1. Assess patients seeking facial volumization and correlate volume deficiencies anatomically. 2. Identify appropriate fillers based on rheologic properties and anatomical needs. 3. Recognize poor candidates for facial volumization. 4. Recognize and treat filler-related side effects and complications. SUMMARY: Facial volumization is widely applied for minimally invasive facial rejuvenation both as a solitary means and in conjunction with surgical correction. Appropriate facial volumization is dependent on patient characteristics, consistent longitudinal anatomical changes, and qualities of fillers available. In this article, anatomical changes seen with aging are illustrated, appropriate techniques for facial volumization are described in the setting of correct filler selection, and potential complications are addressed.

Collagen Stimulators: Poly-L-Lactic Acid and Calcium Hydroxyl Apatite.

Over the last decade, many studies of the structural changes observed in the aging face (in bone, fat pads, facial ligaments, muscle, skin) have increased our understanding that facial rejuvenation is more complex and nuanced than simply filling lines and folds or cutting and lifting soft tissue and skin. This, in addition to the many new products introduced to the marketplace over the same period, has fueled the evolution of panfacial rejuvenation and restoration using fillers. This article discusses current techniques used with calcium hydroxylapatite and poly-l-lactic acid to safely and effectively address changes observed in the aging face.

CPP-ACP and CPP-ACFP versus fluoride varnish in remineralisation of early caries lesions. A prospective study.

AIM: To evaluate the effects of novel casein phosphopeptide (CPP) formulations CPP-amorphous calcium phosphate (CCP- ACP) and CPP-amorphous calcium fluoride phosphate (CPP-ACFP) versus fluoride varnish on the remineralisation of enamel white spot lesions (WSLs) over a 12-week follow-up period. STUDY DESIGN: Double-blind prospective study. Eligibility criteria were patients between 6 and 14 years old and have WSLs on their permanent teeth. We evaluated 786 WSLs. Participants were divided randomly into three groups. Groups A and B were instructed to daily topical application of GC Tooth Mouse (CPP-ACP) or Mi Paste Plus (CPP-ACFP), Group C received a monthly professional application of Duraphat fluoride varnish. WSLs were categorised according to the International Caries Detection and Assessment System (ICDAS II; grades 0-3) and assessed by laser fluorescence (DIAGNOdent) at baseline and at 4, 8 and 12 weeks. Changes in mineralisation before and after treatment were analysed using two-way analysis of variance, with post hoc Bonferroni's non-parametric tests for multiple comparisons. RESULTS: DIAGNOdent values were significantly reduced in Group B at 4 weeks, and in Groups A and C at 8 weeks. Mean values in Group B were lower than in Groups A and C at 4 weeks, and lower than Group C at 8 weeks. CPP-ACFP appeared to have a specific effect on smooth-surface caries, but no significant effect on caries in pits and fissures. CONCLUSION: At 4 weeks, CPP-ACFP is superior to fluoride varnish at remineralising smooth-surface WSLs. CPP-ACP is not superior to fluoride varnish by any of the measures studied.

A glucose carbonate apatite complex exhibits in vitro and in vivo anti-tumour effects.

Tumour targeting nanotechnology has recently made therapeutic progress and several therapeutic nanoparticles have been approved for clinical application. However, an ideal nanotechnology based therapeutic for solid tumours, particularly for systemic administration, still remains a challenge in clinical cancer therapy. We previously reported a pH sensitive in vivo delivery system of doxorubicin, or microRNA, using carbonate apatite (CA) nanoparticles. To further explore utility of CA in cancer therapy, we attempted to transport excess glucose into tumour cells by conjugating glucose (Glc) to the nanoparticle. Despite the non-toxicity of CA and Glc, the complex (CA-[Glc]) exhibited an unexpected anti-cancer effect in vitro and in vivo. CA-[Glc] significantly reduced the growth of colon cancer cell lines. Intravenous injections successfully suppressed solid tumour growth. In mice and monkeys, intravenously injected CA-[Glc] complex resulted in no serious abnormalities in body weight or blood chemistry. Because cancer cells intensively metabolise glucose than normal cells, treatment of cancer using glucose seems paradoxical. However, with the aid of CA, this safe and 'sweet' complex may be a novel anti-cancer reagent.

Effect of a new combined therapy with nano-carbonate apatite and CO2 laser on dentin hypersensitivity in an in situ model.

OBJECTIVE: The aim of this study was to determine the occluding effects of a combination of dentifrice containing nano-carbonate apatite (n-CAP) and CO2 laser on dentinal tubules, and to evaluate the acid resistance of this combination after 4 days of treatment, by using an in situ model. BACKGROUND DATA: The synergic effect of this combination was demonstrated in an in vitro study. METHODS: This was a two period crossover, single-blind, randomized, four-treatment, split-mouth study. Ten healthy participants wore lower intraoral appliances during the treatment period. Specimens were divided into the following four groups: no treatment (control group), tooth-brushing using 20% n-CAP dentifrice (n-CAP group), CO2 laser irradiation (laser group), and laser irradiation after n-CAP application (combined group). Occluding effects were evaluated on 2 days (days 1 and 2), and then acid challenge was performed using grape juice on 2 days (days 3 and 4). All of the specimen surfaces were evaluated by a scanning electron microscope. RESULTS: The combined group showed a better occluding effect than control group compared with other treatment groups, and this effect was 20% higher than that in the n-CAP group. Also, the combined group had the smallest open dentinal tubular area among all of the treatment groups. CONCLUSIONS: The combined therapy is a promising method for ensuring a long-lasting effect of dentin hypersensitivity treatment in clinical practice.

Methotrexate- and cyclophosphamide-embedded pure and strontiumsubstituted carbonate apatite nanoparticles for augmentation of chemotherapeutic activities in breast cancer cells.

Most of the classical drugs used today to destroy cancer cells lead to the development of acquired resistance in those cells by limiting cellular entry of the drugs or exporting them out by efflux pumps. As a result, higher doses of drugs are usually required to kill the cancer cells affecting normal cells and causing numerous side effects. Accumulation of the therapeutic level of drugs inside the cancer cells is thus required for an adequate period of time to get drugs' complete therapeutic efficacy minimizing the side effects on normal cells. In order to improve the efficacy of chemotherapeutic drugs, nanoparticles of carbonate apatite and its strontium (Sr(2+))-substituted derivative were used in this study to make complexes with three classical anticancer drugs, methotrexate, cyclophosphamide and 5-flurouracil. The binding affinities of these drugs to apatite were evaluated by absorbance and HPLC analysis and the therapeutic efficacy of drug-apatite complexes was determined by cell viability assay. Carbonate apatite demonstrated significant binding affinity towards methotrexate and cyclophosphamide leading to more cellular toxicity than free drugs in MCF-7 and 4T1 breast cancer cells. Moreover, Sr(2+) substitution in carbonate apatite with resulting tiny particles less than 100 nm in diameter further promoted binding of methotrexate to the nanocarriers indicating that Sr(2+)-substituted apatite nanoparticles have the high potential for loading substantial amount of anti-cancer drugs with eventual more therapeutic effectiveness.

Controlled release of simvastatin from biomimetic ß-TCP drug delivery system.

Simvastatin have been shown to induce bone formation and there is currently a urgent need to develop an appropriate delivery system to sustain the release of the drug to increase therapeutic efficacy whilst reducing side effects. In this study, a novel drug delivery system for simvastatin by means of hydrothermally converting marine exoskeletons to biocompatible beta-tricalcium phosphate was investigated. Furthermore, the release of simvastatin was controlled by the addition of an outer apatite coating layer. The samples were characterized by x-ray diffraction analysis, fourier transform infrared spectroscopy, scanning electron microscopy and mass spectroscopy confirming the conversion process. The in-vitro dissolution of key chemical compositional elements and the release of simvastatin were measured in simulated body fluid solution showing controlled release with reduction of approximately 25% compared with un-coated samples. This study shows the potential applications of marine structures as a drug delivery system for simvastatin.

A delivery system of linezolid to enhance the MRSA osteomyelitis prognosis: in vivo experimental assessment.

Staphylococcus aureus, a major responsible microorganism of osteomyelitis, represents a challenge to treat because of the poor penetration of antibiotics in bone and increasing minimum inhibitory concentrations (MICs) to glycopeptides. The calcium-deficient apatites (CDA), closer to the biological components found in bone and other calcified tissues, have osteoconductive properties. So, to process severe osseous infections, CDA can be used to deliver in the infectious site antibiotics like linezolid. The acute experimental osteomyelitis due to methicillin-resistant Staphylococcus aureus (MRSA) was induced in rabbit's femurs and surgery mimicking human procedures was performed at day three after inoculation. Animals were randomly assigned to treatment groups: L((IV)) [4-day linezolid IV infusion, human-equivalent dose of 10 mg/kg/12 h], L((CDA50%)) (100 mg CDA with linezolid 500 µg/mg) and L((CDA50%)) + L((IV)). Surviving bacteria were counted in bone marrow (BM) and bone (Bo) at day 3 (before treatment), day 7 (4-day treatment) or day 17 (14-day treatment). L(iv) was effective after a 4-day treatment with a log(10)CFU/g decrease of -2.63 ± 1.92 and -2.17 ± 1.58 in bone marrow and bone, respectively. CDA loaded with linezolid enhance the efficacy of the IV linezolid regimen by more than one log(10)CFU/g.

Injectable collagen/α-tricalcium phosphate cement: collagen-mineral phase interactions and cell response.

A bone inspired material was obtained by incorporating collagen in the liquid phase of an α-tricalcium phosphate cement, either in solubilized or in fibrilized form. This material was able to set in situ, giving rise to a calcium deficient hydroxyapatite (CDHA)/collagen composite. The morphology and distribution of collagen in the composite was shown to be strongly affected by the collagen pre-treatment. The interactions between collagen and the inorganic phase were assessed by FTIR. A red shift of the amide I band was indicative of calcium chelation by the collagen carbonyl groups. The rate of CDHA formation was not affected when diluted collagen solutions (1 mg/ml) were used, whereas injectability improved. The presence of solubilized collagen, even in low amount (1 %), increased cell adhesion and proliferation on the composites. Still in the absence of osteogenic medium, significant ALP activity was detected both in the inorganic and the collagen-containing cements. The maximum ALP activity was advanced in the collagen-containing cement as compared to the inorganic cement.

Dissolution medium responsive simvastatin release from biodegradable apatite cements and the therapeutic effect in osteoporosis rats.

Bio-convertible artificial bone with slow release of anti-osteoporosis drug is useful to treat osteoporosis. Apatite cement containing 6% simvastatin (APD) had lower crystallinity than natural bone. In-vitro drug release tests in simulated body fluid (pH 7.8) and acetate buffer (pH 4.5) were performed at 37.0 C as physical models of osteoblast and osteoclast conditions (SOB and SOC). The device had lower drug release rate under SOB, but significantly higher rates under SOC. The simvastatin release rate changed depending on dissolution media, it repeated twice, and the rate under SOC was 15 times higher than under SOB. The device showed dissolution medium responsive drug release. After implantation of the APD in osteoporosis rats, the bone mineral density was evaluated by the x-ray computed tomography. The result indicated that the bone mineral density of APD implanted rat was significantly higher than that of control diseased. The result indicated that the device was therapeutically useful to bone regeneration.

DNA complex-releasing system by injectable self-setting apatite cement.

BACKGROUND: The therapeutic effect of plasmid DNA complexes is not satisfactory because of the short duration of their gene expression. However, an efficient DNA slow-release system has not been established owing to the low dispersion stability of the DNA/polycation (or cationic lipid) complexes. We have found that hyaluronan (HA) could deposit onto the DNA/polycation complexes, stabilize their dispersion and prepare very small particles. We have also reported that the injectable self-setting apatite cement has high biocompatibility and biodegradability. Thus, durable gene expression systems using injectable apatite cement, including DNA/polycation/HA complexes, were expected. METHODS: Small DNA/polyethyleneimine (PEI)/HA complex particles were prepared by a lyophilizing and rehydration process, and the in vitro release ratio of DNA complex from the apatite cements by MLC-6 cells was examined. Apatite cement slurry with collagen including plasmid DNA (pDNA) complex [encoding granulocyte macrophage-colony stimulating factor (GM-CSF)] was injected close to the tumor subcutaneously inoculated into mice. The therapeutic effect and degradation ratio of the apatite cement were evaluated. RESULTS: Very fine DNA/PEI/HA complex particles kept being dispersed in the apatite cement. The DNA complexes were continuously released from the apatite cement by MLC-6 cells. Single injection of the apatite cement-including pDNA-GM-CSF complex induced complete disappearance of tumor in 60% of mice. Smooth degradation of the apatite cement was observed in the mice in which a high therapeutic effect was seen. CONCLUSIONS: Single injection of the apatite cement-including pDNA-GM-CSF complex showed a high therapeutic effect on solid tumor, and thus appears to be promising as a sustained gene expression device.

Effect of fluoride-substituted apatite on in vivo bone formation.

Biological apatites are characterized by the presence of minor constituents such as magnesium (Mg), chloride (Cl), or fluoride (F) ions. These ions affect cell proliferation and osteoblastic differentiation during bone tissue formation. F-substituted apatites are being explored as potential bonegraft materials. The aim of the present study is to investigate the mechanism of bone formation induced by fluoride-substituted apatite (FAp) by analyzing the effect of FAp on the process of in vivo bone formation. FAps containing different F concentrations (l-FAp: 0.48 wt%, m-FAp: 0.91 wt%, h-FAp: 2.23 wt%) and calcium-deficient apatite (CDA), as positive control, were implanted in rat tibia and bone formation was evaluated by histological examination, immuhistochemistry, in situ hybridization and tartrate-resistant acid phosphatase examinations. The results showed that l-FAp, m-FAp, h-FAp, and CDA biomaterials allowed migration of macrophages, attachment, proliferation, and phenotypic expression of bone cells leading to new bone formation in direct apposition to the particles. However, the l-FAp preparation allowed faster bone conduction compared to the other experimental materials. These results suggest that FAp with low F concentration may be an efficient bonegraft material for dental and medical application.

Mineralization, biodegradation, and drug release behavior of gelatin/apatite composite microspheres for bone regeneration.

Gelatin microspheres are well-known for their capacity to release growth factors in a controlled manner, but gelatin microspheres do not calcify in the absence of so-called bioactive substances that induce deposition of calcium phosphate (CaP) bone mineral. This study has investigated if CaP nanocrystals can be incorporated into gelatin microspheres to render these inert microspheres bioactive without compromising the drug releasing properties of gelatin microspheres. Incorporation of CaP nanocrystals into gelatin microspheres resulted into reduced biodegradation and drug release rates, whereas their calcifying capacity increased strongly compared to inert gelatin microspheres. The reduced drug release rate was correlated to the reduced degradation rate as caused by a physical cross-linking effect of CaP nanocrystals dispersed in the gelatin matrix. Consequently, these composite microspheres combine beneficial drug-releasing properties of organic gelatin with the calcifying capacity of a dispersed CaP phase.

Failure of a glass ionomer to remineralize apatite-depleted dentin.

Remineralization of demineralized dentin lesions adjacent to glass-ionomer cements (GICs) has been reported in the literature. This study tested the hypothesis that a strontium-based GIC can remineralize completely demineralized dentin by nucleation of new apatite crystallites within an apatite-free dentin matrix. Human dentin specimens were acid-etched, bonded with Fuji IX(GP), and immersed in a calcium-and-phosphate-containing 1.5X simulated body fluid (SBF) for 1-4 months. Polyacrylic acid and polyvinylphosphonic acid biomimetic analogs were added to the SBFs to create 2 additional remineralization media. Specimens were processed by transmission electron microscopy (TEM). No apatite deposition could be identified in the completely demineralized dentin in any of the specimens immersed in the 3 remineralization media, despite TEM/EDX evidence of diffusion of ions specific to the strontium-based GIC into the demineralized dentin. The hypothesis was rejected; mineral concentration alone is not a sufficient endpoint for assessing the success of contemporary remineralization strategies.