Orbital shaking conditions augment human nasoseptal cartilage formation in 3D culture.

Introduction: This study aimed to determine whether a dynamic orbital shaking culture system could enhance the cartilage production and viability of bioengineered nasoseptal cartilage. Methods: Human nasal chondrocytes were seeded onto nanocellulose-alginate biomaterials and cultured in static or dynamic conditions for 14 days. Quantitative polymerase chain reaction for chondrogenic gene expression (type 2 collagen, aggrecan and SOX9) was performed, demonstrating a transient rise in SOX9 expression at 1 and 7 days of culture, followed by a rise at 7 and 14 days in Aggrecan (184.5-fold increase, p < 0.0001) and Type 2 Collagen (226.3-fold increase, p = 0.049) expression. Samples were analysed histologically for glycosaminoglycan content using Alcian blue staining and demonstrated increased matrix formation in dynamic culture. Results: Superior cell viability was identified in the dynamic conditions through live-dead and alamarBlue assays. Computational analysis was used to determine the shear stress experienced by cells in the biomaterial in the dynamic conditions and found that the mechanical stimulation exerted was minimal (fluid shear stress <0.02 mPa, fluid pressure <48 Pa). Conclusion: We conclude that the use of an orbital shaking system exerts biologically relevant effects on bioengineered nasoseptal cartilage independently of the expected thresholds of mechanical stimulation, with implications for optimising future cartilage tissue engineering efforts.

The effect of chronic dosing and p53 status on the genotoxicity of pro-oxidant chemicals in vitro.

In this study, we have studied the cytotoxicity and genotoxic potency of 3 pro-oxidants; H2O2, menadione and KBrO3 in different dosing scenarios, namely acute (1-day dosing) and chronic (5-days). For this purpose, relative population doubling (RPD%) and mononucleated micronucleus (MN) test were used. TK6 cells and NH32 were employed in in vitro experiments. In the study, the total acute dose was divided into 5 days for each prooxidant chemicals by dose fractionation (1/5th per day) method. Acute dosing was compared to chronic dosing. The oxidative stress caused by the exposure of cells with pro-oxidant chemicals to the cells was determined by an optimized 2',7'-dichlorofluorescein diacetate (DCFHDA) test method. The antioxidant levels of the cell lines were altered with buthionine sulfoxide (BSO) and N-acetyl cysteine (NAC), and the effect of antioxidant capacity on the MN formation in the cells was observed with this method. In the case of H2O2 and menadione, fractional dosing has been observed to result in lower toxicity and lower genotoxicity. But in the case of KBrO3, unlike the other 2 pro-oxidants, higher MN induction was observed with fractionated doses. DCFHDA test clearly demonstrated ROS induction with H2O2 and menadione but not with KBrO3. Unexpectedly, DCFHDA test demonstrated that KBrO3 did not cause an increase ROS levels in both acute and chronic dosing, suggesting an alternative ROS induction mechanism. It was also observed that, treatment with BSO and NAC, caused increasing and decreasing of MN fold change respectively, allowing further ROS specific mechanisms to be explored. Hence, dose fractionation expectedly caused less MN, cytotoxicity and ROS formation with H2O2 and menadione exposure, but not with KBrO3. This implies a unique mechanism of action for KBrO3 induced genotoxicity. Chronic dosing in vitro may be a valuable approach allowing better understanding of how chemicals damage DNA and pose human hazards.

Chromosome breakage induced by the genotoxic agents mitomycin C and cytosine arabinoside is concentration and p53 dependent.

The p53 tumor suppressor protein plays an essential role in cellular integrity and inactivation of the TP53 gene by mutation is the most frequent alteration in human cancer. As loss of p53 function is associated with increased genetic instability, it is important in genotoxicity testing to explore the role of p53 competency. In vitro model systems for genotoxicity testing are sometimes prone to misleading positive results; some of this loss of predictivity may be caused by p53 inactivation in some cell models. To explore whether impaired p53 function plays a role in mutation sensitivity, TK6 cells (p53 competent) and NH32 cells (p53 deficient) were treated with two known genotoxicants, mitomycin C (MMC) and cytosine arabinoside (araC). Chromosomal damage was assessed in the low dose region by an automated micronucleus system and p53 activity was investigated by gene and protein expression analysis. Cell cycle progression studies were also assessed. Low levels of micronucleus and p53 induction were observed in TK6 cells treated with MMC. On the other hand, higher levels of micronucleus and p53 induction were shown in TK6 cells treated with araC and a G1/S arrest was observed after araC treatment. p53 deficient NH32 cells showed an increased sensitivity of micronucleus (MN) induction after araC treatment compared with TK6 cells and less of an active G1/S phase checkpoint. Thus, impaired p53 function sensitizes cells to genotoxicants and plays a central role in the DNA damage response. This data has clear importance for safety assessment of genotoxicity and shows how crucial p53 competence is.