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Open AccessArticle
Oxidative Stress in Long-Term Exposure to Multi-Walled Carbon Nanotubes in Male Rats
by Ewa Florek
1,* [ORCID] , Marta Witkowska
2,3, Marta Szukalska
1 [ORCID] , Magdalena Richter
4 [ORCID] , Tomasz Trzeciak
4, Izabela Miechowicz
5 [ORCID] , Andrzej Marszałek
6 [ORCID] , Wojciech Piekoszewski
7 [ORCID] , Zuzanna Wyrwa
1 and Michael Giersig
3,8
1
Laboratory of Environmental Research, Department of Toxicology, Poznan University of Medical Sciences, 60-631 Poznan, Poland
2
Faculty of Chemistry, Adam Mickiewicz University, 61-614 Poznan, Poland
3
Centre for Advanced Technologies, Adam Mickiewicz University, 61-614 Poznan, Poland
4
Department of Orthopedics and Traumatology, Poznan University of Medical Sciences, 61-545 Poznan, Poland
5
Department of Computer Science and Statistics, Poznan University of Medical Sciences, 60-806 Poznan, Poland
6
Oncologic Pathology and Prophylaxis, Greater Poland Cancer Centre, Poznan University of Medical Sciences, 61-866 Poznan, Poland
7
Department of Analytical Chemistry, Faculty of Chemistry, Jagiellonian University, 30-387 Krakow, Poland
8
Department of Theory of Continuous Media and Nanostructures, Institute of Fundamental Technological Research, Polish Academy of Sciences, 02-106 Warsaw, Poland
*
Author to whom correspondence should be addressed.
Antioxidants 2023, 12(2), 464; https://doi.org/10.3390/antiox12020464
Received: 13 December 2022 / Revised: 7 February 2023 / Accepted: 10 February 2023 / Published: 12 February 2023
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Abstract
Multi-walled carbon nanotubes (MWCNTs) serve as nanoparticles due to their size, and for that reason, when in contact with the biological system, they can have toxic effects. One of the main mechanisms responsible for nanotoxicity is oxidative stress resulting from the production of intracellular reactive oxygen species (ROS). Therefore, oxidative stress biomarkers are important tools for assessing MWCNTs toxicity. The aim of this study was to evaluate the oxidative stress of multi-walled carbon nanotubes in male rats. Our animal model studies of MWCNTs (diameter ~15–30 nm, length ~15–20 μm) include measurement of oxidative stress parameters in the body fluid and tissues of animals after long-term exposure. Rattus Norvegicus/Wistar male rats were administrated a single injection to the knee joint at three concentrations: 0.03 mg/mL, 0.25 mg/mL, and 0.5 mg/mL. The rats were euthanized 12 and 18 months post-exposure by drawing blood from the heart, and their liver and kidney tissues were removed. To evaluate toxicity, the enzymatic activity of total protein (TP), reduced glutathione (GSH), glutathione S–transferase (GST), thiobarbituric acid reactive substances (TBARS), Trolox equivalent antioxidant capacity (TEAC), nitric oxide (NO), and catalase (CAT) was measured and histopathological examination was conducted. Results in rat livers showed that TEAC level was decreased in rats receiving nanotubes at higher concentrations. Results in kidneys report that the level of NO showed higher concentration after long exposure, and results in animal serums showed lower levels of GSH in rats exposed to nanotubes at higher concentrations. The 18-month exposure also resulted in a statistically significant increase in GST activity in the group of rats exposed to nanotubes at higher concentrations compared to animals receiving MWCNTs at lower concentrations and compared to the control group. Therefore, an analysis of oxidative stress parameters can be a key indicator of the toxic potential of multi-walled carbon nanotubes.

Keywords:
multi-walled carbon nanotubes,oxidative stress parameters,rats,long-term toxicity

Abstract:
BACKGROUND: Electrospun nanofibers have widespread putative applications in the field of regenerative medicine and tissue engineering. When compared to naturally occurring collagen matrices, electrospun nanofiber scaffolds have two distinct advantages: they do not induce a foreign body reaction and they are not at risk for biological contamination. However, the exact substrate, structure, and production methods have yet to be defined. MATERIAL AND METHODS: In the current study, tubular-shaped poly(L-lactide-co-caprolactone) (PLCL) constructs produced using electrospinning technology were evaluated for their potential application in the field of tissue regeneration in two separate anatomic locations: the skin and the abdomen. The constructs were designed to have an internal diameter of 3 mm and thickness of 200 μm. Using a rodent model, 20 PLCL tubular constructs were surgically implanted in the abdominal cavity and subcutaneously. The constructs were then evaluated histologically using electron microscopy at 6 weeks post-implantation. RESULTS: Histological evaluation and analysis using scanning electron microscopy showed that pure scaffolds by themselves were able to induce angiogenesis after implantation in the rat model. Vascularization was observed in both tested groups; however, better results were obtained after intraperitoneal implantation. Formation of more and larger vessels that migrated inside the scaffold was observed after implantation into the peritoneum. In this group no evidence of inflammation and better integration of scaffold with host tissue were noticed. Subcutaneous implantation resulted in more fibrotic reaction, and differences in cell morphology were also observed between the two tested groups. CONCLUSIONS: This study provides a standardized evaluation of a PLCL conduit structure in two different anatomic locations, demonstrating the excellent ability of the structure to achieve vascularization. Functional, histological, and mechanical data clearly indicate prospective clinical utilization of PLCL in critical size defect regeneration.

Keywords:
Polymers, Regenerative medicine, Tissue Engineering, Tissue Scaffolds, Urinary Diversion

Abstract:
Objective
Due to the capacity of the amniotic membrane (Am) to support re-epithelisation and inhibit scar formation, Am has a potential to become a considerable asset for reconstructive urology i.e., reconstruction of ureters and urethrae. The application of Am in reconstructive urology is limited due to a poor mechanical characteristic. Am reinforcement with electrospun nanofibers offers a new strategy to improve Am mechanical resistance, without affecting its unique bioactivity profile. This study evaluated biocomposite material composed of Am and nanofibers as a graft for urinary bladder augmentation in a rat model.

Material and Methods
Sandwich-structured biocomposite material was constructed from frozen Am and covered on both sides with two-layered membranes prepared from electrospun poly-(L-lactide-co-E-caprolactone) (PLCL). Wistar rats underwent hemicystectomy and bladder augmentation with the biocomposite material.

Results
Immunohistohemical analysis (hematoxylin and eosin [H&E], anti-smoothelin and Masson’s trichrome staining [TRI]) revealed effective regeneration of the urothelial and smooth muscle layers. Anti-smoothelin staining confirmed the presence of contractile smooth muscle within a new bladder wall. Sandwich-structured biocomposite graft material was designed to regenerate the urinary bladder wall, fulfilling the requirements for normal bladder tension, contraction, elasticity and compliance. Mechanical evaluation of regenerated bladder wall conducted based on Young’s elastic modulus reflected changes in the histological remodeling of the augmented part of the bladder. The structure of the biocomposite material made it possible to deliver an intact Am to the area for regeneration. An unmodified Am surface supported regeneration of the urinary bladder wall and the PLCL membranes did not disturb the regeneration process.

Conclusions
Am reinforcement with electrospun nanofibers offers a new strategy to improve Am mechanical resistance without affecting its unique bioactivity profile.

Keywords:
Bladder, Smooth muscles, Muscle regeneration, Bionanotechnology, Renal system, Urothelium, Urology, Nanomaterials

Abstract:
The aim of this study was to compare two different acellular scaffolds: natural and synthetic, for urinary conduit construction and ureter segment reconstruction. Acellular aortic arch (AAM) and poly(L-lactide-co-caprolactone) (PLCL) were used in 24 rats for ureter reconstruction in both tested groups. Follow-up period was 4 weeks. Intravenous pyelography, histological and immunohistochemical analysis were performed. All animals survived surgical procedures. Patent uretero-conduit junction was observed only in one case using PLCL. In case of ureter segment reconstruction ureters were patent in one case using AAM and in four cases using PLCL scaffolds. Regeneration of urothelium layer and focal regeneration of smooth muscle layer was observed on both tested scaffolds. Obtained results indicates that synthetic acellular PLCL scaffolds showed better properties for ureter reconstruction than naturally derived acellular aortic arch.

Keywords:
Ureter, Muscle regeneration, Kidneys, Collagens, Urine, Surgical and invasive medical procedures, Smooth muscles, Inflammation

Abstract:
The purpose of this study was to compare: a new five-layered poly (L–lactide–co–caprolactone) (PLC) membrane and small intestinal submucosa (SIS) as a control in rat urinary bladder wall regeneration. The five-layered poly (L–lactide–co–caprolactone) membrane was prepared by an electrospinning process. Adipose tissue was harvested from five 8-week old male Wistar rats. Adipose derived stem cells (ADSCs) were seeded in a density of 3×106 cells/cm2 onto PLC membrane and SIS scaffolds, and cultured for 5-7 days in the stem cell culture medium. Twenty male Wistar rats were randomly divided into five equal groups. Augmentation cystoplasty was performed in a previously created dome defect. Groups: (I) PLC+ 3×106ADSCs; (II) SIS+ 3×106ADSCs; (III) PLC; (IV) SIS; (V) control. Cystography was performed after three months. The reconstructed urinary bladders were evaluated in H&E and Masson's trichrome staining. Regeneration of all components of the normal urinary bladder wall was observed in bladders augmented with cell-seeded SIS matrices. The urinary bladders augmented with SIS matrices without cells showed fibrosis and graft contraction. Bladder augmentation with the PLC membrane led to numerous undesirable events including: bladder wall perforation, fistula or diverticula formation, and incorporation of the reconstructed wall into the bladder lumen. The new five-layered poly (L–lactide–co–caprolactone) membrane possesses poorer potential for regenerating the urinary bladder wall compared with SIS scaffold.

Keywords:
urinary bladder regeneration, electrospinning