Calcination and ion substitution improve physicochemical and biological properties of nanohydroxyapatite for bone tissue engineering applications.

Nanohydroxyapatite (nanoHAP) is widely used in bone regeneration, but there is a need to enhance its properties to provide stimuli for cell commitment and osteoconduction. This study examines the effect of calcination at 1200 °C on the physicochemical and biological properties of nanoHAP doped with magnesium (Mg2+), strontium (Sr2+), and zinc (Zn2+). A synergistic effect of dual modification on nanoHAP biological properties was investigated. The materials were characterized by X-ray diffraction (XRD), scanning electron microscopy (SEM), BET analysis, Fourier-transform spectroscopy, and thermal analysis methods. Furthermore, ion release tests and in vitro biological characterization, including cytocompatibility, reactive oxygen species production, osteoconductive potential and cell proliferation, were performed. The XRD results indicate that the ion substitution of nanoHAP has no effect on the apatite structure, and after calcination, ß-tricalcium phosphate (ß-TCP) is formed as an additional phase. SEM analysis showed that calcination induces the agglomeration of particles and changes in surface morphology. A decrease in the specific surface area and in the ion release rate was observed. Combining calcination and nanoHAP ion modification is beneficial for cell proliferation and osteoblast response and provide additional stimuli for cell commitment in bone regeneration.

Effect of Selected Crosslinking and Stabilization Methods on the Properties of Porous Chitosan Composites Dedicated for Medical Applications.

Chitosan is one of the most commonly employed natural polymers for biomedical applications. However, in order to obtain stable chitosan biomaterials with appropriate strength properties, it is necessary to subject it to crosslinking or stabilization. Composites based on chitosan and bioglass were prepared using the lyophilization method. In the experimental design, six different methods were used to obtain stable, porous chitosan/bioglass biocomposite materials. This study compared the crosslinking/stabilization of chitosan/bioglass composites with ethanol, thermal dehydration, sodium tripolyphosphate, vanillin, genipin, and sodium ß-glycerophosphate. The physicochemical, mechanical, and biological properties of the obtained materials were compared. The results showed that all the selected crosslinking methods allow the production of stable, non-cytotoxic porous composites of chitosan/bioglass. The composite with genipin stood out with the best of the compared properties, taking into account biological and mechanical characteristics. The composite stabilized with ethanol is distinct in terms of its thermal properties and swelling stability, and it also promotes cell proliferation. Regarding the specific surface area, the highest value exposes the composite stabilized by the thermal dehydration method.

Senescent cancer cell vaccines induce cytotoxic T cell responses targeting primary tumors and disseminated tumor cells.

BACKGROUND: Immune tolerance contributes to resistance to conventional cancer therapies such as radiation. Radiotherapy induces immunogenic cell death, releasing a burst of tumor antigens, but this appears insufficient to stimulate an effective antitumor immune response. Radiation also increases infiltration of cytotoxic T lymphocytes (CTLs), but their effector function is short lived. Although CTL exhaustion may be at fault, combining immune checkpoint blockade with radiation is insufficient to restore CTL function in most patients. An alternative model is that antigen presentation is the limiting factor, suggesting a defect in dendritic cell (DC) function. METHODS: Building on our prior work showing that cancer cells treated with radiation in the presence of the poly(ADP-ribose) polymerase-1 inhibitor veliparib undergo immunogenic senescence, we reexamined senescent cells (SnCs) as preventative or therapeutic cancer vaccines. SnCs formed in vitro were cocultured with splenocytes and evaluated by scRNA-seq to examine immunogenicity. Immature bone-marrow-derived DCs cocultured with SnCs were examined for maturation and activation by flow cytometry and T cell proliferation assays. Viable SnCs or SnC-activated DCs were injected subcutaneously, and vaccine effects were evaluated by analysis of immune response, prevention of tumor engraftment, regression of established tumors and/or potentiation of immunotherapy or radiotherapy. RESULTS: Murine CT26 colon carcinoma or 4T1 mammary carcinoma cells treated with radiation and veliparib form SnCs that promote DC maturation and activation in vitro, leading to efficient, STING-dependent CTL priming. Injecting mice with SnCs induces antigen-specific CTLs and confers protection from tumor engraftment. Injecting immunogenic SnCs into tumor-bearing mice increases inflammation with activated CTLs, suppresses tumor growth, potentiates checkpoint blockade, enhances radiotherapy and blocks colonization by disseminated tumor cells. Addressing the concern that reinjecting tumor cells into patients may be impractical, DCs activated with SnCs in vitro were similarly effective to SnCs in suppressing established tumors and blocking metastases. CONCLUSIONS: Therapeutic vaccines based on senescent tumor cells and/or SnC-activated DCs have the potential to improve genotoxic and immune therapies and limit recurrence or metastasis.

Dual Modification of Porous Ca-P/PLA Composites with APTES and Alendronate Improves Their Mechanical Strength and Cytobiocompatibility towards Human Osteoblasts.

Synthetic implants are used to treat large bone defects that are often unable to regenerate, for example those caused by osteoporosis. It is necessary that the materials used to manufacture them are biocompatible and resorbable. Polymer-ceramic composites, such as those based on poly(L-lactide) (PLLA) and calcium phosphate ceramics (Ca-P), are often used for these purposes. In this study, we attempted to investigate an innovative strategy for two-step (dual) modification of composites and their components to improve the compatibility of composite components and the adhesion between PLA and Ca-P whiskers, and to increase the mechanical strength of the composite, as well as improve osteological bioactivity and prevent bone resorption in composites intended for bone regeneration. In the first step, Ca-P whiskers were modified with a saturated fatty acid namely, lauric acid (LA), or a silane coupling agent γ-aminopropyltriethoxysilane (APTES). Then, the composite, characterized by the best mechanical properties, was modified in the second stage of the work with an active chemical compound used in medicine as a first-line drug in osteoporosis-sodium alendronate, belonging to the group of bisphosphonates (BP). As a result of the research covered in this work, the composite modified with APTES and alendronate was found to be a promising candidate for future biomedical engineering applications.

Targeting telomerase reverse transcriptase with the covalent inhibitor NU-1 confers immunogenic radiation sensitization.

Beyond synthesizing telomere repeats, the telomerase reverse transcriptase (TERT) also serves multiple other roles supporting cancer growth. Blocking telomerase to drive telomere erosion appears impractical, but TERT's non-canonical activities have yet to be fully explored as cancer targets. Here, we used an irreversible TERT inhibitor, NU-1, to examine impacts on resistance to conventional cancer therapies. In vitro, inhibiting TERT sensitized cells to chemotherapy and radiation. NU-1 delayed repair of double-strand breaks, resulting in persistent DNA damage signaling and cellular senescence. Although NU-1 alone did not impact growth of syngeneic CT26 tumors in BALB/c mice, it dramatically enhanced the effects of radiation, leading to immune-dependent tumor elimination. Tumors displayed persistent DNA damage, suppressed proliferation, and increased activated immune infiltrate. Our studies confirm TERT's role in limiting genotoxic effects of conventional therapy but also implicate TERT as a determinant of immune evasion and therapy resistance.

Far-red Fluorescent Senescence-associated ß-Galactosidase Probe for Identification and Enrichment of Senescent Tumor Cells by Flow Cytometry.

Cellular senescence is a state of proliferative arrest induced by biological damage that normally accrues over years in aging cells but may also emerge rapidly in tumor cells as a response to damage induced by various cancer treatments. Tumor cell senescence is generally considered undesirable, as senescent cells become resistant to death and block tumor remission while exacerbating tumor malignancy and treatment resistance. Therefore, the identification of senescent tumor cells is of ongoing interest to the cancer research community. Various senescence assays exist, many based on the activity of the well-known senescence marker, senescence-associated beta-galactosidase (SA-ß-Gal). Typically, the SA-ß-Gal assay is performed using a chromogenic substrate (X-Gal) on fixed cells, with the slow and subjective enumeration of "blue" senescent cells by light microscopy. Improved assays using cell-permeant, fluorescent SA-ß-Gal substrates, including C12-FDG (green) and DDAO-Galactoside (DDAOG; far-red), have enabled the analysis of living cells and allowed the use of high-throughput fluorescent analysis platforms, including flow cytometers. C12-FDG is a well-documented probe for SA-ß-Gal, but its green fluorescent emission overlaps with intrinsic cellular autofluorescence (AF) that arises during senescence due to the accumulation of lipofuscin aggregates. By utilizing the far-red SA-ß-Gal probe DDAOG, green cellular autofluorescence can be used as a secondary parameter to confirm senescence, adding reliability to the assay. The remaining fluorescence channels can be used for cell viability staining or optional fluorescent immunolabeling. Using flow cytometry, we demonstrate the use of DDAOG and lipofuscin autofluorescence as a dual-parameter assay for the identification of senescent tumor cells. Quantitation of the percentage of viable senescent cells is performed. If desired, an optional immunolabeling step may be included to evaluate cell surface antigens of interest. Identified senescent cells can also be flow cytometrically sorted and collected for downstream analysis. Collected senescent cells can be immediately lysed (e.g., for immunoassays or 'omics analysis) or further cultured.

Effects of Sterilization and Hydrolytic Degradation on the Structure, Morphology and Compressive Strength of Polylactide-Hydroxyapatite Composites.

Composites based on polylactide (PLA) and hydroxyapatite (HA) were prepared using a thermally induced phase separation method. In the experimental design, the PLA with low weight-average molar mass (Mw) and high Mw were tested with the inclusion of HA synthesized as whiskers or hexagonal rods. In addition, the structure of HA whiskers was doped with Zn, whereas hexagonal rods were mixed with Sr salt. The composites were sterilized and then incubated in phosphate-buffered saline for 12 weeks at 37 °C, followed by characterization of pore size distribution, molecular properties, density and mechanical strength. Results showed a substantial reduction of PLA Mw for both polymers due to the preparation of composites, their sterilization and incubation. The distribution of pore size effectively increased after the degradation process, whereas the sterilization, furthermore, had an impact on pore size distribution depending on HA added. The inclusion of HA reduced to some extent the degradation of PLA quantitatively in the weight loss in vitro compared to the control without HA. All produced materials showed no cytotoxicity when validated against L929 mouse skin fibroblasts and hFOB 1.19 human osteoblasts. The lack of cytotoxicity was accompanied by the immunocompatibility with human monocytic cells that were able to detect pyrogenic contaminants.

Examining the Influence of Re-Used Nanofiller-Pyrolyzed Montmorillonite, on the Thermal Properties of Polypropylene-Based Engineering Nanocomposites.

Pyrolysis of the polypropylene/montmorillonite (PP/OMMT) nanocomposites allows for recovery of the filler that can be then re-used to produce PP/pyrolyzed MMT (PMMT) nanostructured composites. In this work, we discuss the thermal properties of PP/PMMT composites investigated by thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and dynamic mechanical analysis (DMA). It has been found that effect of PMMT (5 wt. % and 10 wt. %) on matrix thermal stability occurs at temperatures above 300 °C. Addition of 5 wt. % and 10 wt. % of PMMT into polypropylene system gave good stabilization effect, as confirmed by the overall stabilization effect (OSE) values, which increased by 4% and 7%, respectively, compared to the control sample (PP). Interestingly, the presence of 1 wt. % and 3 wt. % of pyrolyzed clay stabilizes the system better than the same concentrations of organoclay added into polypropylene melt. DSC data revealed that pyrolyzed clay has still the same tendency as organoclay to enhance formation of the α and ß crystalline PP phases only. The pyrolyzed MMT causes an improvement of the modulus in the glassy as well as rubbery regions, as confirmed by DMA results.

Column chromatography as a useful step in purification of diatom pigments.

Fucoxanthin, diadinoxanthin and diatoxanthin are carotenoids found in brown algae and most other heterokonts. These pigments are involved in photosynthetic and photoprotective reactions, and they have many potential health benefits. They can be extracted from diatom Phaeodactylum tricornutum by sonication, extraction with chloroform : methanol and preparative thin layer chromatography. We assessed the utility of an additional column chromatography step in purification of these pigments. This novel addition to the isolation protocol increased the purity of fucoxanthin and allowed for concentration of diadinoxanthin and diatoxanthin before HPLC separation. The enhanced protocol is useful for obtaining high purity pigments for biochemical studies.