With the explosion of new materials for medical applications, the initial screening should be made systematically, so this protocol can be a fundamental tool for researchers aiming to develop and characterize novel biomaterials. The methodology allows the quantitative and qualitative evaluation of cytotoxicity and bioactivity of biomaterials and pharmaceutical formulations, both solid and liquid. Following cell exposure, several complementary assays can be performed.
The establishment of biomaterial cytotoxicity can have relevant implications for clinical practice. For instance, this knowledge may empower the clinician to choose the most appropriate biomaterial for a specific patient or condition. This protocol allows the characterization of novel biomaterials in different areas such as dentistry, orthopedics, surgery, ophthalmology, cardiology, among others.
The evaluation performance should allow concluding on the biomaterial bioactivity in living tissues besides cytotoxicity. As close as possible to the beginning of the experiment, use a spatula to load the freshly prepared biomaterial into the PVC molds, and let the material set for the appropriate amount of time at room temperature. When the material has solidified, remove the pellets from the molds, and place them in an appropriately sized container.
Then sterilize the pellets under an ultraviolet light lamp for 20 minutes per side. To extract the biomaterials from the pellets, place the sterilized pellets in a 50 milliliter tube, and add the appropriate cell culture medium to the pellets. Place the tubes at 37 degrees Celsius for 24 hours.
The next day, use fresh medium to dilute the extract to the appropriate volumes of interest. To evaluate the cell morphology in response to biomaterial exposure, use sterile tweezers to place an appropriately sized sterile glass coverslip into each well of a multi-well plate. Add cells to each coverslip at an adequate concentration, and place in the incubator overnight.
Add the appropriate volume of the extract dilution of interest to each well, and return the cells to the cell culture incubator for the appropriate incubation period. At the end of the incubation, aspirate the supernatant from each well to allow the coverslips to dry at room temperature. When the samples have dried, cover each coverslip with a sufficient volume of May-Grunwald solution for a three minute incubation at room temperature.
After removing the dye, wash the coverslips with an appropriate volume of distilled water for one minute, followed by a 15 minute incubation in a sufficient volume of Giemsa solution. At the end of the incubation, wash the coverslips in running water, and image the cells by light microscopy. To assess the cell function by protein expression evaluation after exposure to the biomaterial of interest as demonstrated, wash each well of cells with PBS before fixing the cells with 3.7%paraformaldehyde for 30 minutes at room temperature.
At the end of the incubation, wash the cells two times with PBS, and permeabilize the cells with 0.5%Triton in PBS for 15 minutes. At the end of the permeation, block the peroxidase with 0.3%hydrogen peroxide in PBS for five minutes, followed by two washes with PBS. After the second wash, wash the cells two times with 0.5%BSA before blocking the non-specific binding with 2%BSA for 45 minutes.
At the end of the incubation, wash the cells one time with 0.5%BSA before incubating the cells with a primary antibody against the protein of interest for 60 minutes at room temperature, followed by five washes with fresh 0.5%BSA. After the last wash, incubate the cells with the appropriate secondary antibody of interest for 90 minutes at room temperature, followed by five one minute washes in 0.5%BSA. After the last wash, incubate the cells with an appropriate substrate and chromogen mixture at 20 microliters of chromogen per milliliter of substrate concentrations for 25 minutes.
At the end of the incubation, wash the cultures two times with 0.5%BSA in PBS, and counterstain the cells with hematoxylin for 15 minutes. Wash the counterstained cells for five minutes with 0.037 moles per liter of ammonia. Then wash for five minutes with distilled water to remove any excess dye, and use glycerol to mount the coverslips onto slides.
To assess the formation of mineralized deposits within the extract treated cell cultures, wash each well three times with PBS before fixing the cells with 4%paraformaldehyde for 15 minutes at room temperature. After the last wash, stain the cells with an appropriate volume of alizarin red staining solution for 20 minutes at 37 degrees Celsius in the dark. At the end of the incubation, wash the wells with PBS until the excess dye has been completely removed, and image the cells by light microscopy.
Then add extraction solution to each well for a 40 minute incubation with stirring at room temperature, and measure the absorbance in each well on a spectrophotometer at a 490 nanometer wavelength. An MTT assay can be used to obtain an overview of the cytotoxicity of the materials in a quick and straightforward manner as measured by its effects on metabolic activity. Cell viability analyses also allow evaluation of the cytotoxicity of a material of interest, as materials with higher cytotoxicity induce a higher percentage of cell death at the same concentration.
Reductions of more than 30%are considered to be critical, and define materials as at risk of low biocompatibility. In addition, more cytotoxic materials are characterized by an accentuated decrease in cell viability and a late apoptosis and necrosis cell death profile, while less cytotoxic materials induce less cell death and a more apoptotic and late apoptotic profile. Morphologic evaluation compliments the cell viability evaluation, as changes in the cell morphology can indicate an apoptotic or necrotic profile, as well as reveal the presence of material particles.
In addition, the effects of materials of interest on protein expression can be observed independently of effects on viability. For example, in this analysis, one material induced an increase in protein expression, while the other promoted a significant decrease. In both cases, the concentration of the extracts directly influenced the protein expression.
In the extracts preparation, appropriate biomaterial surface to medium volume ratios is critical. Additionally, the samples must be sterilized using methods that do not alter their properties. Another detail to remember is extracts pH, that should be registered without adjustment.
If needed, perform additional controls. The biomaterial extracts can be used for chemistry and biochemistry studies, as well as for several other molecular biology techniques. Obtained results provides the basis for future in vivo biocompatibility testing.
Cytotoxicity evaluation is crucial for the development of any material intended for medical use. Therefore, this protocol provides a systematic and comprehensive approach for evaluating new biomaterials or new applications of others already existing into different medical areas.
