Custom-Made Zirconium Dioxide Implants for Craniofacial Bone Reconstruction

Reconstruction of the facial skeleton is challenging for surgeons because of difficulties in proper shape restoration and maintenance of the proper long-term effect. ZrO2 implant application can be a solution with many advantages (e.g., osseointegration, stability, and radio-opaqueness) and lacks the disadvantages of other biomaterials (e.g., metalosis, radiotransparency, and no osseointegration) or autologous bone (e.g., morbidity, resorption, and low accuracy). We aimed to evaluate the possibility of using ZrO2 implants as a new application of this material for craniofacial bone defect reconstruction. First, osteoblast (skeleton-related cell) cytotoxicity and genotoxicity were determined in vitro by comparing ZrO2 implants and alumina particle air-abraded ZrO2 implants to the following: 1. a titanium alloy (standard material); 2. ultrahigh-molecular-weight polyethylene (a modern material used in orbital surgery); 3. a negative control (minimally cytotoxic or genotoxic agent action); 4. a positive control (maximally cytotoxic or genotoxic agent action). Next, 14 custom in vivo clinical ZrO2 implants were manufactured for post-traumatologic periorbital region reconstruction. The soft tissue position improvement in photogrammetry was recorded, and clinical follow-up was conducted at least 6 years postoperatively. All the investigated materials revealed no cytotoxicity. Alumina particle air-abraded ZrO2 implants showed genotoxicity compared to those without subjection to air abrasion ZrO2, which were not genotoxic. The 6-month and 6- to 8-year clinical results were aesthetic and stable. Skeleton reconstructions using osseointegrated, radio-opaque, personalized implants comprising ZrO2 material are the next option for craniofacial surgery.

Imetelstat Sensitizes Hematopoietic Malignancy Cells to Genotoxic Agent Via Suppression of Telomerase Mediated DNA Repair Process

Telomerase is the ribonucleoprotein enzyme that has the main function of extension of telomeric repeat. Telomerase consists of two constructions: telomerase reverse transcriptase protein (hTERT) and RNA template. Inhibition of telomerase activity is considered to be a therapeutic approach for malignant tumors since telomerase is dominantly expressed in germ cells, stem cells, and cancer cells but not in normal cells. We found that (TTAGGG)n repeats could be integrated and repair artificially induced DNA double strand break (DSB) sites (Onozawa M et al. PNAS, 2014). Therefore, we hypothesized that inhibition of telomerase activity may interfere with the DNA repair process and enhance the effects of DNA damaging agents. Imetelstat competitively suppresses telomerase activity. It has a binding site complementary to the RNA template of telomerase and induces specific inhibition. Imetelstat showed clinical benefits in patients with essential thrombocytosis or myelofibrosis used as monotherapy. However, the effectiveness of imetelstat combined with other cytotoxic agent for hematological malignancies is not clear. We therefore evaluated the synergistic anti-tumor effects of imetelstat and cytotoxic agents on hematological malignancies. First, the expression of hTERT mRNA, protein, and activity was confirmed in 8 human cell lines of hematological malignancies (U937, HL60, PALL2, CEM, IM9, RPMI8226, ST1, and SU9T01) but not in peripheral blood mononuclear cells (PBMCs) from a healthy individual. There was a significant correlation between mRNA expression and activity measured by TRAP assay (r=0.839, 95% confidence interval: 0.396-0.965, p=0.00467). The expression of hTERT mRNA in clinical samples was analyzed using primary acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) cells. The expression level of hTERT in ALL cells was significantly higher than that in AML cells (p=0.0093). The telomerase inhibitor, imetelstat had a concentration-dependent effect on telomerase inhibition, while a control oligo did not affect telomerase activity. The expression of hTERT protein was not suppressed by imetelstat treatment. Imetelstat also had a concentration-dependent suppressive effect on cell growth without affecting cell viability. Next, we evaluated the synergistic anti-tumor effects of etoposide and imetelstat at the concentration at which it did not affect cell growth by monotherapy. Cell proliferation at day 4 was inhibited by coadministration of etoposide (1 µM) and imetelstat (1 µM) compared to that with administration of etoposide alone (Figure A). Western blot analysis showed that gamma-H2AX expression level after radiation and imetelstat administration was significantly higher than that of after radiation alone (Figure B). Therefore, imetelstat enhanced DNA-DSB induced by a genotoxic agent. We determined whether the synergistic effect was mediated by shortening of telomere length. Telomere length was analyzed in long-term culture using the flow-FISH method. Imetelstat significantly shortened telomere length compared to a control oligo after day 14, while there was almost no change in telomere length at day 4. We also analyzed the changes of hTERT activity and gamma-H2AX expression after radiation in PBMCs from a healthy individual. Although hTERT activity was not detected in stable state in PBMC, hTERT activity appeared and gradually increased over time and gamma-H2AX expression peaked from 2 to 4 hours after radiation. Images of immunofluorescence staining revealed co-localization of hTERT and gamma-H2AX in the nucleus of PBMCs after radiation (Figure C). Therefore, hTERT induced by DNA damage might be directly involved in the DNA-DSB repair process. We clearly showed synergistic effects of imetelstat and a genotoxic agent in short-term treatment, in which shortening of telomeres was not observed. Potentially, telomerase inhibition could stand as a beneficial addition to other treatment methods when the inhibitor is administered at low non-toxic doses. Our hypothesis needs to be clarified in a clinical trial. Further studies are required to optimize these therapies in a variety of hematopoietic malignancies or other cancers. Figure Disclosures Nakagawa: akeda Pharmaceutical Company Limited: Research Funding. Teshima:Novartis: Honoraria, Research Funding.

Evaluation of genotoxic and cytotoxic effects of hydroalcoholic extract of Euphorbia tirucalli (Euphorbiaceae) in cell cultures of human leukocytes

ABSTRACT Euphorbia tirucalli (L.), commonly known as aveloz, has been indiscriminately used in popular medicine to treat various illnesses. However, some components can have devastating consequences. Injury to a cell's genetic material can cause mutations, cancer, and cell death. Our main goal in this work was to evaluate the genotoxic and cytotoxic effects of E. tirucalli extract on human leukocytes. For this purpose, we performed a phytochemical analysis to evaluate the plant's components. In the second step, we treated cultured human leukocytes with different concentrations of the dry extract of the plant and then evaluated the oxidative and genotoxic profiles of these leukocytes. We found that at 1% and 10% concentrations, the aveloz extract acted as a genotoxic agent that could damage DNA and increase oxidative damage. We conclude that despite its popular use, aveloz can act as a genotoxic agent, especially when it contains phorbol ester. Aveloz's indiscriminate use might actually promote tumors and therefore carry a considerable genetic risk for its users.

Study of chromosomal abnormalities in Channa punctatus exposed to fenvalerate

The chromosomal aberration test in kidney cells of Channa punctatus was conducted to study the genotoxic effect of synthetic pyrethroid fenvalerate, Different chromosomal abnormalities were seen and their frequency was recoded in metaphase spreads obtained from fishes exposed to three sub lethal concentrations (0.0625, 0.0314 and 0.0157 ppm) of this compound. The exposure to fenvalerate caused various structural abnormalities in chromosomes such as chromatid break, fragment, gap, chromatid separation, deletion and ring type chromosomes. Results obtained in present investigations clearly indicate that fenvalerate acts as a genotoxic agent in Channa punctatus.