PSPC1 Inhibition Synergizes with Poly(ADP-ribose) Polymerase Inhibitors in a Preclinical Model of BRCA-Mutated Breast/Ovarian Cancer.

Poly (ADP-ribose) polymerase (PARP) inhibitors are effective against BRCA1/2-mutated cancers through synthetic lethality. Unfortunately, most cases ultimately develop acquired resistance. Therefore, enhancing PARP inhibitor sensitivity and preventing resistance in those cells are an unmet clinical need. Here, we investigated the ability of paraspeckle component 1 (PSPC1), as an additional synthetic lethal partner with BRCA1/2, to enhance olaparib sensitivity in preclinical models of BRCA1/2-mutated breast and ovarian cancers. In vitro, the combined olaparib and PSPC1 small interfering RNA (siRNA) exhibited synergistic anti-proliferative activity in BRCA1/2-mutated breast and ovarian cancer cells. The combination therapy also demonstrated synergistic tumor inhibition in a xenograft mouse model. Mechanistically, olaparib monotherapy increased the expressions of p-ATM and DNA-PKcs, suggesting the activation of a DNA repair pathway, whereas combining PSPC1 siRNA with olaparib decreased the expressions of p-ATM and DNA-PKcs again. As such, the combination increased the formation of γH2AX foci, indicating stronger DNA double-strand breaks. Subsequently, these DNA-damaged cells escaped G2/M checkpoint activation, as indicated by the suppression of p-cdc25C (Ser216) and p-cdc2 (Tyr15) after combination treatment. Finally, these cells entered mitosis, which induced increased apoptosis. Thus, this proves that PSPC1 inhibition enhances olaparib sensitivity by targeting DNA damage response in our preclinical model. The combination of olaparib and PSPC1 inhibition merits further clinical investigation to enhance PARP inhibitor efficacy.

Targeting PEG10 as a novel therapeutic approach to overcome CDK4/6 inhibitor resistance in breast cancer.

BACKGROUND: Breast cancer is the global leading cancer burden in women and the hormone receptor-positive (HR+) subtype is a major part of breast cancer. Though cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitors are highly effective therapy for HR+ subtype, acquired resistance is inevitable in most cases. Herein, we investigated the paternally expressed gene 10 (PEG10)-associated mechanism of acquired resistance to CDK4/6 inhibitors. METHODS: Palbociclib-resistant cells were generated by exposing human HR+ breast cancer cell lines to palbociclib for 7-9 months. In vitro mechanistic study and in vivo xenograft assay were performed. For clinical relevance, public mRNA microarray data sets of early breast cancer were analyzed and PEG10 immunohistochemical staining was performed using pre-CDK4/6 inhibitor tumor samples. RESULTS: We observed that PEG10 was significantly upregulated in palbociclib-resistant cells. Ectopic overexpression of PEG10 in parental cells caused CDK4/6 inhibitor resistance and enhanced epithelial-mesenchymal transition (EMT). On the contrary, PEG10-targeting siRNA or antisense oligonucleotides (ASOs) combined with palbociclib synergistically inhibited proliferation of palbociclib-resistant cells and growth of palbociclib-resistant xenograft in mice and suppressed EMT as well. The mechanistic study confirmed that high PEG10 expression suppressed p21, a natural CDK inhibitor, and SIAH1, a post-translational degrader of ZEB1, augmenting CDK4/6 inhibitor resistance. Then PEG10 siRNA combined with palbociclib suppressed cell cycle progression and EMT via activating p21 and SIAH1, respectively. Consequently, combined PEG10 inhibition and palbociclib overcame CDK4/6 inhibitor resistance. Furthermore, high PEG10 expression was significantly associated with a shorter recurrence-free survival (RFS) based on public mRNA expression data. In pre-CDK4/6 inhibitor treatment tissues, PEG10 positivity by IHC also showed a trend toward a shorter progression-free survival (PFS) with CDK4/6 inhibitor. These results support clinical relevance of PEG10 as a therapeutic target. CONCLUSIONS: We demonstrated a novel PEG10-associated mechanism of CDK4/6 inhibitor resistance. We propose PEG10 as a promising therapeutic target for overcoming PEG10-associated resistance to CDK4/6 inhibitors.

Combined PI3K Inhibitor and Eribulin Enhances Anti-Tumor Activity in Preclinical Models of Paclitaxel-Resistant, PIK3CA-Mutated Endometrial Cancer.

Endometrial cancer stands as the predominant gynecological malignancy in developed nations. For advanced or recurrent disease, paclitaxel-based chemotherapy is the standard front-line therapy. However, paclitaxel resistance eternally develops. Based on the high prevalence of phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) mutation, reaching 50%, in endometrial cancer, we preclinically investigated the effectiveness of a combination of a phosphatidylinositol 3-kinase (PI3K) inhibitor with eribulin, a post-paclitaxel therapy for breast cancer, in treating paclitaxel-resistant, PIK3CA-mutated endometrial cancer. We generated paclitaxel-resistant cell lines from PIK3CA-mutated endometrial cancer cell lines by gradually increasing the concentration of paclitaxel in cell cultures. We observed that the PI3K/AKT and epithelial-mesenchymal transition (EMT) pathways in paclitaxel-resistant cells were significantly upregulated compared with those in parental cells. Then, we demonstrated that the combination of alpelisib (a PI3K inhibitor) and eribulin more effectively suppressed the cellular growth of paclitaxel-resistant cells in in vitro and in vivo xenograft models. Mechanistically, we demonstrated that the effect of the combination could be enhanced by inhibiting both the PI3K/AKT and EMT pathways. Therefore, we suggest that paclitaxel resistance is associated with the activation of the PIK3/AKT pathway in PIK3CA-mutated endometrial cancer, and the combination of a PI3K inhibitor and eribulin merits further clinical investigation.

Preclinical Platform Using a Triple-negative Breast Cancer Syngeneic Murine Model to Evaluate Immune Checkpoint Inhibitors.

BACKGROUND/AIM: To evaluate the feasibility of syngeneic mouse models of breast cancer by analyzing the efficacy of immune checkpoint inhibitors (ICIs) and potential predictive biomarkers. MATERIALS AND METHODS: To establish the murine triple-negative breast cancer (TNBC) models, JC, 4T1, EMT6, and E0771 cells were subcutaneously implanted into female syngeneic mice. When the tumor reached 50-100 mm3, each mouse model was divided into a treatment (using a murine PD-1 antibody) and a no-treatment control group. The treatment group was further divided into the responder and non-responder groups. Potential predictive biomarkers were evaluated by analyzing serum cytokines, peripheral blood T cells and tumor infiltrating immune cells. RESULTS: The EMT6 model showed the highest tumor response rate (54%, 6/11) of the syngeneic models: 4T1 (45%, 5/11), JC (40%, 4/10), or E0771 (23%, 3/13). Early changes in tumor size at 7 days post-PD-1 inhibitor treatment predicted the final efficacy of the PD-1 inhibitor. Peripheral blood CD8+ and CD4+ T cells with or without Ki67 expression at 7 days post-PD-1 inhibitor treatment were higher in the finally designated responder group than in the non-responder group. At the time of sacrifice, analyses of tumor infiltrating lymphocytes consistently supported these results. We also demonstrated that retro-orbital blood sampling procedures (baseline, 7 days post-treatment, time of sacrifice) were safe for serum cytokine analyses, suggesting that our preclinical platform may be used for biomarker research using serum cytokines. CONCLUSION: Our syngeneic mouse model of TNBC is a feasible preclinical platform to evaluate ICI efficacy combined with other drugs and predictive biomarkers in the screening process of immune-oncology drug development.

Dual actions of osteoclastic-inhibition and osteogenic-stimulation through strontium-releasing bioactive nanoscale cement imply biomaterial-enabled osteoporosis therapy.

Repair of defective hard-tissues in osteoporotic patients faces significantly challenges with limited therapeutic options. Although biomedical cements are considered promising materials for healthy bone repair, their uses for healing osteoporotic fracture are clinically limited. Herein, strontium-releasing-nanoscale cement was introduced to provide dual therapeutic-actions (pro-osteogenesis and anti-osteoclastogenesis), eventually for the regeneration of osteoporotic bone defect. The Sr-nanocement hardened from the Sr-doped nanoscale-glass particles was shown to release multiple ions including silicate, calcium and strontium at doses therapeutically relevant over time. When the Sr-nanocement was treated to pre-osteoblastic cells, the osteogenic mRNA level (Runx2, Opn, Bsp, Ocn), alkaline phosphatase activity, calcium deposition, and target luciferase reporter were stimulated with respect to the case with Sr-free-nanocement. When treated to pre-osteoclastic cells, the Sr-nanocement substantially reduced the osteoclastogenesis, such as osteoclastic mRNA level (Casr, Nfatc1, c-fos, Acp, Ctsk, Mmp-9), tartrate-resistant acid trap activity, and bone resorption capacity. In particular, the osteoclastic inhibition resulted in part from the interactive effect of osteoblasts which were activated by the Sr-nanocement, i.e., blockage of RANKL (receptor activator of nuclear factor-κB ligand) binding by enhanced osteoprotegerin and the deactivated Nfatc1. The Sr-nanocement, administered to an ovariectomized tibia defect (osteoporotic model) in rats, exhibited profound bone regenerative potential in cortical and surrounding trabecular area, including increased bone volume and density, enhanced production of osteopromotive proteins, and more populated osteoblasts, together with reduced signs of osteoclastic bone resorption. These results demonstrate that Sr-nanocement, with its dual effects of osteoclastic inhibition and osteogenic-stimulation, can be considered an effective nanotherapeutic implantable biomaterial platform for the treatment of osteoporotic bone defects.

Antibacterial, proangiogenic, and osteopromotive nanoglass paste coordinates regenerative process following bacterial infection in hard tissue.

Bacterial infection raises serious concerns in tissue repair settings involved with implantable biomaterials, devastating the regenerative process and even life-threatening. When hard tissues are infected with bacteria (called 'osteomyelitis'), often the cases in open fracture or chronic inflammation, a complete restoration of regenerative capacity is significantly challenging even with highly-dosed antibiotics or surgical intervention. The implantable biomaterials are thus needed to be armored to fight bacteria then to relay regenerative events. To this end, here we propose a nanoglass paste made of ~200-nm-sized silicate-glass (with Ca, Cu) particles that are hardened in contact with aqueous medium and multiple-therapeutic, i.e., anti-bacterial, pro-angiogenic and osteopromotive. The nanoglass paste self-hardened via networks of precipitated nano-islands from leached ions to exhibit ultrahigh surface area (~300 m2/g), amenable to fill tunable defects with active biomolecular interactions. Also, the nanoglass paste could release multiple ions (silicate, calcium, and copper) at therapeutically relevant doses and sustainably (for days to weeks), implying possible roles in surrounding cells/tissues as a therapeutic-ions reservoir. The osteopromotive effects of nanoglass paste were evidenced by the stimulated osteogenic differentiation of MSCs. Also, the nanoglass paste promoted angiogenesis of endothelial cells in vitro and vasculature formation in vivo. Furthermore, the significant bactericidal effect of nanoglass paste, as assessed with E. coli and S. aureus, highlighted the role of copper played in elevating ROS level and destroying homeostasis, which salvaged tissue cells from co-cultivated bacteria contamination. When administered topically to rat tibia osteomyelitis defects, the nanoglass paste enhanced in vivo bone healing and fracture resistance. The developed nanoglass paste, given its self-setting property and the coordinated therapeutic actions, is considered to be a promising drug-free inorganic biomaterial platform for the regenerative therapy of bacteria-infected hard tissues.

Correction: Dynamic mechanical and nanofibrous topological combinatory cues designed for periodontal ligament engineering.

[This corrects the article DOI: 10.1371/journal.pone.0149967.].

Anti-inflammatory actions of folate-functionalized bioactive ion-releasing nanoparticles imply drug-free nanotherapy of inflamed tissues.

Inflammation prevailing conditions delay healing processes of damaged tissues, leading to a functional impairment. Although anti-inflammatory drugs are clinically available, they often cause unwanted side effects thus being considered suboptimal. Here we report drug-free synthetic nanoparticles that target and internalize pro-inflammatory cells and release ions, ultimately demonstrating profound anti-inflammatory functions. We introduce folate-functionalized bioactive glass nanoparticle BGN(F) that can bind to pro-inflammatory cells to endocytose and release ions. The folate-conjugation significantly enhanced the nanoparticle internalization to LPS-induced pro-inflammatory cells. The direct treatment of BGN(F) at proper doses (80-160 µg/mL) substantially down-regulated pro-inflammatory molecules, including TNF-α, IL-6, iNOS and COX-2, at both gene and protein levels. The phosphorylation of intracellular signaling molecules involved in the inflammatory events, such as p38 MAPK, ERK (1/2), SAPK/JANK, IκBα, and NF-κB, were significantly suppressed by the BGN(F) treatment. Furthermore, BGN(F) was potential to switch the macrophage polarization from M1 to M2. The released ions, not the physical interactions, of nanoparticles were observed to contribute in major part to the anti-inflammatory actions of BGN(F). The BGN(F), when locally administered to a Notexin-induced myoinjury tissue in mice, significantly down-regulated IL-6 and TNF-α, switched the macrophage phenotype from M1 to M2, and accelerated tissue healing. The current findings that demonstrate profound anti-inflammatory actions of BGN(F) in vitro and in vivo support their uses as novel drug-free nanotherapeutic platform for the treatment of inflamed tissues.

Nanocements produced from mesoporous bioactive glass nanoparticles.

Biomedical cements are considered promising injectable materials for bone repair and regeneration. Calcium phosphate composition sized with tens of micrometers is currently one of the major powder forms. Here we report a unique cement form made from mesoporous bioactive glass nanoparticles (BGn). The nanopowder could harden in reaction with aqueous solution at powder-to-liquid ratios as low as 0.4-0.5 (vs. 2.0-3.0 for conventional calcium phosphate cement CPC). The cementation mechanism investigated from TEM, XRD, FT-IR, XPS, and NMR analyses was demonstrated to be the ionic (Si and Ca) dissolution and then reprecipitation to form Si-Ca-(P) based amorphous nano-islands that could network the particles. The nanopowder-derived nanocement exhibited high surface area (78.7 m2/g); approximately 9 times higher than conventional CPC. The immersion of nanocement in simulated body fluid produced apatite nanocrystallites with ultrafine size of 10 nm (vs. 55 nm in CPC). The ultrafine nanocement adsorbed protein molecules (particularly positive charged proteins) at substantial levels; approximately 160 times higher than CPC. The nanocement released Si and Ca ions continuously over the test period of 2 weeks; the Si release was unique in nanocement whereas the Ca release was in a similar range to that observed in CPC. The release of ions significantly stimulated the responses of cells studied (rMSCs and HUVECs). The viability and osteogenesis of rMSCs were significantly enhanced by the nanocement ionic extracts. Furthermore, the in vitro tubular networking of HUVECs was improved by the nanocement ionic extracts. The in vivo neo-blood vessel formation in CAM model was significantly higher by the nanocement implant when compared with the CPC counterpart, implying the Si ion release might play a significant role in pro-angiogenesis. Furthermore, the early bone forming response of the nanocement, based on the implantation in a rat calvarial bone defect, demonstrated a sign of osteoinductivity along with excellent osteocondution and bone matrix formation. Although more studies remain to confirm the potential of nanocement, some of the intriguing physico-chemical properties and the biological responses reported herein support the promise of the new 'nanopowder-based nanocement' for hard tissue repair and regeneration.

Optical imaging and anticancer chemotherapy through carbon dot created hollow mesoporous silica nanoparticles.

Multifunctional nanocarrier-based theranostics is currently considered to solve some key unmet challenges in cancer treatment. Here we report a nanocarrier platform, named carbon dot (CD) created mesoporous hollow organosilica (C-hMOS) nanoparticles, to deliver anticancer drug and to enable optical imaging. The hollow structure was formed by the removal of a nanorod core template, and at the same time, the fluorescent signal was endowed from the heat-treated organosilica network. Thanks to the hollow and mesoporous structure, the C-hMOS effectively loaded doxorubicin (DOX) for cancer chemotherapy. The DOX was released from C-hMOS highly sustainably (over 12days) and pH-dependently (pH 5.0 >pH 7.4). The DOX-loading C-hMOS internalized cancer cells efficiently (>90%), and induced cellular apoptosis including the expression of caspase-3. The treatment of C-hMOS to cancer cells enabled multi-color visualization in vitro, suggesting the possibility of cell tracing. Moreover, when injected intratumorally in mice, the C-hMOS exhibited strong optical signals in vivo along with a high optical stability (over a week). The injected C-hMOS were distributed only a fraction in liver but not in heart, lung, spleen or kidney and displayed good biocompatibility. The DOX-delivering C-hMOS significantly suppressed the in vivo tumor growth associated with apoptotic functions. Taken together, the developed C-hMOS nanoparticles can be a promising nanoplatform for drug delivery and in vivo imaging in cancer treatment. STATEMENT OF SIGNIFICANCE: Multifunctional nanoparticles that combine chemotherapeutic ability with imaging modality comprise promising platform for cancer theranostics. Here we developed a novel theranostic nanoparticle, i.e., carbon-dot created mesoporous hollow silica nanoparticle, to offer unique merit for this purpose. The in vitro and in vivo findings to support this include: i) carbon dots with 1-2nm size in situ generated discretely and uniformly within silica network, ii) hollow and mesoporous structure effective for loading of DOX at high content, iii) release behavior of DOX in a sustainable and pH-dependent manner, iv) chemotherapeutic efficacy in killing cancer cells and suppressing tumor growth through DOX delivery, and v) carbon dot induced multi-color fluorescence imaging within cells and tumor tissues. These collective multifaceted properties may facilitate the novel carbon dot nanocarriers to be a potential candidate for delivering anticancer drug and non-invasive imaging in cancer treatment.

Angiogenic Effects of Collagen/Mesoporous Nanoparticle Composite Scaffold Delivering VEGF165.

Vascularization is a key issue for the success of tissue engineering to repair damaged tissue. In this study, we report a composite scaffold delivering angiogenic factor for this purpose. Vascular endothelial growth factor (VEGF) was loaded on mesoporous silica nanoparticle (MSN), which was then incorporated within a type I collagen sponge, to produce collagen/MSN/VEGF (CMV) scaffold. The CMV composite scaffold could release VEGF sustainably over the test period of 28 days. The release of VEGF improved the cell proliferation. Moreover, the in vivo angiogenesis of the scaffold, as studied by the chick chorioallantoic membrane (CAM) model, showed that the VEGF-releasing scaffold induced significantly increased number of blood vessel complexes when compared with VEGF-free scaffold. The composite scaffold showed good biocompatibility, as examined in rat subcutaneous tissue. These results demonstrate that the CMV scaffold with VEGF-releasing capacity can be potentially used to stimulate angiogenesis and tissue repair.

C-Dot Generated Bioactive Organosilica Nanospheres in Theranostics: Multicolor Luminescent and Photothermal Properties Combined with Drug Delivery Capacity.

Biocompatible nanomaterials that allow for labeling cells and tissues with the capacity to load and deliver drug molecules hold great promise for the therapeutic-diagnostic purposes in tissue repair and disease cure. Here a novel nanoplatform, called C-dot bioactive organosilica nanosphere (C-BON), is introduced to have excellent theranostic potential, such as controlled drug delivery, visible-light imaging, and NIR photothermal activity. C-dots with a few nanometers were in situ generated in the Ca-containing organosilica mesoporous nanospheres through the sol-gel and thermal-treatment processes. The C-BON exhibited multicolor luminescence over a wide visible-light range with strong emissions and high photostability over time and against acidity and the possible in vivo optical imaging capacity when injected in rat subcutaneous tissues. Moreover, the C-BON showed a photothermal heating effect upon the irradiation of near-infrared. The C-BON, thanks to the high mesoporosity and existence of Ca(2+) ions, demonstrated excellent loading capacity of anticancer drug doxorubicin (as high as 90% of carrier weight) and long-term (over a couple of weeks) and pH/NIR-dependent release ability. The C-BON preserved the compositional merit of Ca-Si glass, having excellent bioactivity and cell compatibility in vitro. Taken all, the multifunctional properties of C-BON-multicolor luminescence, photothermal activity, and high drug loading and controlled release-together with its excellent bioactivity and cell compatibility potentiate the future applications in theranostics (chemotherapy and photothermal therapy with optical imaging).

Gene delivery nanocarriers of bioactive glass with unique potential to load BMP2 plasmid DNA and to internalize into mesenchymal stem cells for osteogenesis and bone regeneration.

The recent development of bioactive glasses with nanoscale morphologies has spurred their specific applications in bone regeneration, for example as drug and gene delivery carriers. Bone engineering with stem cells genetically modified with this unique class of nanocarriers thus holds great promise in this avenue. Here we report the potential of the bioactive glass nanoparticle (BGN) system for the gene delivery of mesenchymal stem cells (MSCs) targeting bone. The composition of 15% Ca-added silica, proven to be bone-bioactive, was formulated into surface aminated mesoporous nanospheres with enlarged pore sizes, to effectively load and deliver bone morphogenetic protein-2 (BMP2) plasmid DNA. The enlarged mesopores were highly effective in loading BMP2-pDNA with an efficiency as high as 3.5 wt% (pDNA w.r.t. BGN), a level more than twice than for small-sized mesopores. The BGN nanocarriers released the genetic molecules in a highly sustained manner (for as long as 2 weeks). The BMP2-pDNA/BGN complexes were effectively internalized to rat MSCs with a cell uptake level of ∼73%, and the majority of cells were transfected to express the BMP2 protein. Subsequent osteogenesis of the transfected MSCs was demonstrated by the expression of bone-related genes, including bone sialoprotein, osteopontin, and osteocalcin. The MSCs transfected with BMP2-pDNA/BGN were locally delivered inside a collagen gel to the target calvarium defects. The results showed significantly improved bone regeneration, as evidenced by the micro-computed tomographic, histomorphometric and immunohistochemical analyses. This study supports the excellent capacity of the BGN system as a pDNA-delivery nanocarrier in MSCs, and the engineered system, BMP2-pDNA/BGN with MSCs, may be considered a new promising candidate to advance the therapeutic potential of stem cells through genetic modification, targeting bone defects and diseases.

Effect of Aminated Mesoporous Bioactive Glass Nanoparticles on the Differentiation of Dental Pulp Stem Cells.

Mesoporous bioactive nanoparticles (MBNs) have been developed as promising additives to various types of bone or dentin regenerative material. However, biofunctionality of MBNs as dentin regenerative additive to dental materials have rarely been studied. We investigated the uptake efficiency of MBNs-NH2 with their endocytosis pathway and the role of MBNs-NH2 in odontogenic differentiation to clarify inherent biofunctionality. MBNs were fabricated by sol-gel synthesis, and 3% APTES was used to aminate these nanoparticles (MBNs-NH2) to reverse their charge from negative to positive. To characterize the MBNs-NH2, TEM, XRD, FTIR, zeta(ξ)-potential measurements, and Brunauer-Emmett-Teller analysis were performed. After primary cultured rat dental pulp stem cells (rDPSCs) were incubated with various concentrations of MBNs-NH2, stem cell viability (24 hours) with or without differentiated media, internalization of MBNs-NH2 in rDPSCs (~4 hours) via specific endocytosis pathway, intra or extracellular ion concentration and odontoblastic differentiation (~28 days) were investigated. Incubation with up to 50 µg/mL of MBNs-NH2 had no effect on rDPSCs viability with differentiated media (p>0.05). The internalization of MBNs-NH2 in rDPSCs was determined about 92% after 4 hours of incubation. Uptake was significantly decreased with ATP depletion and after 1 hour of pre-treatment with the inhibitor of macropinocytosis (p<0.05). There was significant increase of intracellular Ca and Si ion concentration in MBNs-NH2 treated cells compared to no-treated counterpart (p<0.05). The expression of odontogenic-related genes (BSP, COL1A, DMP-1, DSPP, and OCN) and the capacity for biomineralization (based on alkaline phosphatase activity and alizarin red staining) were significantly upregulated with MBNs-NH2. These results indicate that MBNs-NH2 induce odontogenic differentiation of rDPSCs and may serve as a potential dentin regenerative additive to dental material for promoting odontoblast differentiation.

Dynamic Mechanical and Nanofibrous Topological Combinatory Cues Designed for Periodontal Ligament Engineering.

Complete reconstruction of damaged periodontal pockets, particularly regeneration of periodontal ligament (PDL) has been a significant challenge in dentistry. Tissue engineering approach utilizing PDL stem cells and scaffolding matrices offers great opportunity to this, and applying physical and mechanical cues mimicking native tissue conditions are of special importance. Here we approach to regenerate periodontal tissues by engineering PDL cells supported on a nanofibrous scaffold under a mechanical-stressed condition. PDL stem cells isolated from rats were seeded on an electrospun polycaprolactone/gelatin directionally-oriented nanofiber membrane and dynamic mechanical stress was applied to the cell/nanofiber construct, providing nanotopological and mechanical combined cues. Cells recognized the nanofiber orientation, aligning in parallel, and the mechanical stress increased the cell alignment. Importantly, the cells cultured on the oriented nanofiber combined with the mechanical stress produced significantly stimulated PDL specific markers, including periostin and tenascin with simultaneous down-regulation of osteogenesis, demonstrating the roles of topological and mechanical cues in altering phenotypic change in PDL cells. Tissue compatibility of the tissue-engineered constructs was confirmed in rat subcutaneous sites. Furthermore, in vivo regeneration of PDL and alveolar bone tissues was examined under the rat premaxillary periodontal defect models. The cell/nanofiber constructs engineered under mechanical stress showed sound integration into tissue defects and the regenerated bone volume and area were significantly improved. This study provides an effective tissue engineering approach for periodontal regeneration-culturing PDL stem cells with combinatory cues of oriented nanotopology and dynamic mechanical stretch.

Synthesis, characterization, and biocompatible properties of alanine-grafted chitosan copolymers.

In order to overcome major problems regarding the lack of affinity to solvents and limited reactivity of the free amines of chitosan, introduction of appropriate spacer arms having terminal amine function is considered of interest. L-Alanine-N-carboxyanhydride was grafted onto chitosan via anionic ring-opening polymerization. The chemical and structural characterizations of L-alanine-grafted chitosan (Ala-g-Cts) were confirmed through Fourier transform infrared spectroscopy and proton nuclear magnetic resonance spectroscopy ((1)H NMR). In addition, the viscoelastic properties of Ala-g-Cts were examined by means of a rotational viscometer, and thermal analysis was carried out with a thermogravimetric analyzer and differential scanning calorimetry. Morphological changes in the chitosan L-alanine moiety were determined by x-ray diffraction. To determine the feasibility of using these films as biomedical materials, we investigated the effects of their L-alanine content on physical and mechanical properties. The biodegradation results of crosslinked Ala-g-Cts films were evaluated in phosphate-buffered solution containing lysozyme at 37℃. Proliferation of MC3T3-E1 cells on crosslinked Ala-g-Cts films was also investigated with use of the CCK-8 assay.

Inhibition of osteoclastogenesis through siRNA delivery with tunable mesoporous bioactive nanocarriers.

Gene silencing through siRNA delivery has shown great promise for treating diseases and repairing damaged tissues, including bone. This report is the first to develop siRNA delivery system in the inhibition of osteoclastic functions which in turn can help turn-over bone mass increase in the diseases like osteoporosis. For this reason, biocompatible and degradable nanocarriers that can effectively load and deliver genetic molecules to target cells and tissues are being actively sought by researchers. In this study, mesoporous bioactive glass nanospheres (MBG), a novel unique biocompatible degradable inorganic nanocarrier, is introduced. Furthermore, siRNA was designed to function by inhibiting the expression of the receptor activator of nuclear factor kappa B (RANK) in order to suppress osteoclastogenesis. Amine-functionalized MBG were synthesized with tunable mesoporosities, showing a strong complexation with siRNA. An in vitro release profile indicated that the siRNA from the MBG was able to achieve a highly sustainable liberation for up to 4 days, confirming a temporary delivery system can be designed to function for that period of time. The intracellular uptake capacity of the complex siRNA(RANK)-MBG was recorded to be around 70%. Furthermore, the RANK-expressing cell population declined down to 29% due to the delivery of siRNA(RANK)-MBG (vs. 86% in control). The expression of osteoclastogenesis-related genes, including c-fos, cathepsin-K, tartrate-resistant acid phosphatase (TRAP), and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1), was substantially down-regulated by the siRNA delivery system. This study reports for the first time on the use of a novel MBG delivery system for siRNA that aims to suppress osteoclastic actions. MBGs may be a potential gene delivery platform for hard tissue repair and disease treatment due to the collective results which indicate a high loading capacity, temporary release kinetics, high intracellular uptake rate, and sufficient gene silencing effects, together with the intrinsic beneficial properties like bone-bioactivity and degradability. STATEMENT OF SIGNIFICANCE: This report is the first to develop siRNA delivery system of biocompatible and degradable nanocarriers made from a unique composition, i.e., mesoporous bioactive glass that can effectively load and deliver genetic molecules to osteoclastic cells. We proved through a series of studies that the biocompatible nanocarriers are effective for the delivery of siRNA in the inhibition of osteoclastic functions which thus might be considered as a nanocarrier platform to help turn-over bone mass increase in the diseases like osteoporosis.

Synthesis of bio-based thermoplastic polyurethane elastomers containing isosorbide and polycarbonate diol and their biocompatible properties.

A new family of highly elastic polyurethanes (PUs) partially based on renewable isosorbide were prepared by reacting hexamethylene diisocyanate with a various ratios of isosorbide and polycarbonate diol 2000 (PCD) via a one-step bulk condensation polymerization without catalyst. The influence of the isorsorbide/PCD ratio on the properties of the PU was evaluated. The successful synthesis of the PUs was confirmed by Fourier transform-infrared spectroscopy and (1)H nuclear magnetic resonance. The resulting PUs showed high number-average molecular weights ranging from 56,320 to 126,000 g mol(-1) and tunable Tg values from -34 to -38℃. The thermal properties were determined by differential scanning calorimetry and thermogravimetric analysis. The PU films were flexible with breaking strains from 955% to 1795% at from 13.5 to 54.2 MPa tensile stress. All the PUs had 0.9-2.8% weight lost over 4 weeks and continual slow weight loss of 1.1-3.6% was observed within 8 weeks. Although the cells showed a slight lower rate of proliferation than that of the tissue culture polystyrene as a control, the PU films were considered to be cytocompatible and nontoxic. These thermoplastic PUs were soft, flexible and biocompatible polymers, which open up a range of opportunities for soft tissue augmentation and regeneration.

Therapeutic-designed electrospun bone scaffolds: mesoporous bioactive nanocarriers in hollow fiber composites to sequentially deliver dual growth factors.

A novel therapeutic design of nanofibrous scaffolds, holding a capacity to load and deliver dual growth factors, that targets bone regeneration is proposed. Mesoporous bioactive glass nanospheres (MBNs) were used as bioactive nanocarriers for long-term delivery of the osteogenic enhancer fibroblast growth factor 18 (FGF18). Furthermore, a core-shell structure of a biopolymer fiber made of polyethylene oxide/polycaprolactone was introduced to load FGF2, another type of cell proliferative and angiogenic growth factor, safely within the core while releasing it more rapidly than FGF18. The prepared MBNs showed enlarged mesopores of about 7 nm, with a large surface area and pore volume. The protein-loading capacity of MBNs was as high as 13% when tested using cytochrome C, a model protein. The protein-loaded MBNs were smoothly incorporated within the core of the fiber by electrospinning, while preserving a fibrous morphology. The incorporation of MBNs significantly increased the apatite-forming ability and mechanical properties of the core-shell fibers. The possibility of sequential delivery of two experimental growth factors, FGF2 and FGF18, incorporated either within the core-shell fiber (FGF2) or within MBNs (FGF18), was demonstrated by the use of cytochrome C. In vitro studies using rat mesenchymal stem cells demonstrated the effects of the FGF2-FGF18 loadings: significant stimulation of cell proliferation as well as the induction of alkaline phosphate activity and cellular mineralization. An in vivo study performed on rat calvarium defects for 6 weeks demonstrated that FGF2-FGF18-loaded fiber scaffolds had significantly higher bone-forming ability, in terms of bone volume and density. The current design utilizing novel MBN nanocarriers with a core-shell structure aims to release two types of growth factors, FGF2 and FGF18, in a sequential manner, and is considered to provide a promising therapeutic scaffold platform that is effective for bone regeneration.

Synthesis of highly elastic biocompatible polyurethanes based on bio-based isosorbide and poly(tetramethylene glycol) and their properties.

Bio-based high elastic polyurethanes were prepared from hexamethylene diisocyanate and various ratios of isosorbide to poly(tetramethylene glycol) as a diol by a simple one-shot bulk polymerization without a catalyst. Successful synthesis of the polyurethanes was confirmed by Fourier transform-infrared spectroscopy and (1)H nuclear magnetic resonance. Thermal properties were determined by differential scanning calorimetry and thermogravimetric analysis. The glass transition temperature was -47.8℃. The test results showed that the poly(tetramethylene glycol)/isosorbide-based elastomer exhibited not only excellent stress-strain properties but also superior resilience to the existing polyether-based polyurethane elastomers. The static and dynamic properties of the polyether/isosorbide-based thermoplastic elastomer were more suitable for dynamic applications. Moreover, such rigid diols impart biocompatible and bioactive properties to thermoplastic polyurethane elastomers. Degradation tests performed at 37℃ in phosphate buffer solution showed a mass loss of 4-9% after 8 weeks, except for the polyurethane with the lowest isosorbide content, which showed an initial rapid weight loss. These polyurethanes offer significant promise due to soft, flexible and biocompatible properties for soft tissue augmentation and regeneration.