Background: Carbon nanotubes (CNTs) have been considered highly successful and proficient
in terms of their mechanical, thermal and electrical functionalization and biocompatibility. In regards
to their significant extent in bone regeneration, it has been determined that CNTs hold the capability
to endure clinical applications through bone tissue engineering and orthopedic procedures. In the
present study, we report on a composite preparation, involving the use of CNT-chitosan as scaffold for
bone repair and regeneration. Through the use of water-soluble tetrazolium salt (WST-1) and double
staining methods, the cytotoxic, necrotic, and apoptotic effects of chitosan-multiwalled carbon nanotube
nanocomposites on the chondrocyte ATTC cell line have been exhibited.
Methods: The chitosan-multiwalled carbon nanotube scaffolds were prepared. Chondrocytes differentiation
tool (ATCC) cell line was prepared. WST-1 assay for cytotoxicity studies were performed by
using chondrocytes cells in 12.5-200 μL concentration range. The samples of membranes (chitosan–
multiwalled carbon nanotube scaffold) were measured at 2 mg/mL and further prepared amongst chitosan–
multiwalled carbon nanotube scaffold’s which were placed into separate wells. While in the process
of incubation, in the four-hour time range, the plates were immediately read in an Elisa microplate
Reader. To predict the number of apoptotic and necrotic cells in culture, the technique of double staining
with Hoechst dye was performed with PI on the basis of scoring cell nuclei. The mechanical properties
such as tensile strength and elongation at break values of the chitosan only and chitosan/CNT
scaffolds were evaluated on Texture Analyzer.
Results: Based on the results of the WST-1 assay procedure, the amount of cell viability was not significantly
affected by nanocomposite concentrations and the lowest mortality rate of cells was obtained
at a concentration of 12.5 μg/mL, whereas the highest mortality rate was obtained at a rate of 200 μg/mL.
In addition, the effects of chitosan-CNT nanocomposites were not found to cytotoxic on chondrocyte
cells. The double staining method has been able to determine the apoptotic and necrotic effects of chitosan
MWCNT nanocomposites. The apoptotic and necrotic effects of the combined compounds had
varied within the concentrations. In a similar manner to the outcome of the control groups, apoptosis
was obtained at a percentage of 2.67%. Under a fluorescent inverted microscope, the apoptotic cell nuclei
were stained with a stronger blue fluorescence in comparison to non-apoptotic cells, which may
have had an effect. We also compared the strain-stress curve measurements results. The results indicated
that the mechanical properties of scaffold were not different. Elongation at break values increased
by addition of CNT.
Conclusion: CNTs as a biomaterial hold the potential to be used for applications in future regenerative
medicine. By using the components of chondrocytes (ATTC) cell lines, the cytotoxicity evaluations
were made for the chitosan-multiwalled carbon nanotube scaffold. The chitosan-MWCNT nanocomposites
do not seem to induce drastic cytotoxicity to the chondrocyte cells.