Direct and Indirect Culture Methods for Studying Biodegradable Implant Materials In Vitro

Direct and indirect culture methods for studying biodegradable implant materials in vitro. Over the past several decades, biodegradable materials have been extensively explored for biomedical applications, such as orthopedic, dental, and cranial maxillofacial implants. to screen the biodegradable materials for biomedical applications, it is necessary to evaluate these materials in terms of in vitro cell responses, cytocompatibility, and cytotoxicity.

Standards in the International Organization for Standardization have been widely utilized in the evaluations of biomaterials. However, most ISO standards were originally established to assess the style toxicity of non-degradable materials, thus providing limited value for screening biodegradable materials. In this video, we will introduce and discuss three different culture methods namely, direct culture method, direct exposure culture method, and exposure culture method for evaluating the in vitro cytocompatibility of biodegradable implant materials.

Specifically, the direct culture method evaluates the interactions between newly seeded cells and the implants. Direct exposure culture mimics the interactions between the established cells in the body and the implants. Exposure culture characterizes the interactions between established cells and the implants when they are not in direct contact with each other but in the same environment where the materials degrade.

This video provides examples of using these three culture methods for studying the in vitro cytocompatibility of biodegradable implant materials and their interactions with bone marrow-derived mesenchymal stem cells. The in vitro methods described in this video mimic different scenarios of the in vivo environment, broadening the applicability and relevance of the in vitro cytocompatibility testing of different materials for various biomedical applications. We follow a protocol approved by the Institutional Animal Care and Use Community at the University of California at Riverside for cell and tissue harvesting.

Cell culture preparation. The three culture methods described in this video are generally applicable for different cell types that are adherent. Here, BMSCs harvested from rat weanlings will be introduced as an example for cell culture preparation Harvesting bone marrow-derived mesenchymal stem cells from rat weanlings.

The schematic diagram in this figure shows the steps to harvest BMSCs from rat weanlings. Remove the skin and muscle and connective tissues to dissect the femur out of the euthanized rat. Place the femoral bones in a 15 mL conical tube containing cell culture media.

Place the conical tubes on ice until the time of performing cell extraction. Transfer the bones to a Petri dish in the biological safety cabinet. Cut the ends of the bone using a surgical blade and flush the bone marrow into a 50 mL conical tube by washing the bone marrow cavity with cell culture media using a syringe with the 25 1/2 gauge needle.

Filter the cell suspension using a 70 micrometer filter followed by centrifugation at 126 times gravity for five minutes to get the cell pellets Aspirate out supernatant media and replenish with 10 mL of fresh media. Gently pipette up and down to resuspend the cells using a 10 mL serological pipette. Pipette the suspension directly on the inside bottom of a T-75 flask and add media to bring the volume up to 25 milliliters.

Culture the cells in an incubator in a standard sterile cell culture environment that is 37 degrees Celsius, humidified atmosphere with 5%carbon dioxide and 95%air. After three to seven days, rinse away the non-adherent cells by aspiring the old media and replenishing with fresh media. Continue culturing and feeding the cells with fresh media until they are ready for cell passage, freezing, or use in an experiment.

Sample preparation and sterilization. Sterilize or disinfect all of the samples before the cell culture. Sterilization or disinfection methods for different sample types vary depending on the different properties of the materials.

In general, use ultraviolet radiation to disinfect biodegradable metals for in vitro studies. Cell culture methods. The schematic diagram in this figure shows the steps of the direct culture method.

In this video BMSCs were cultured on a magnesium derived plate placed inside the wells of a 12-well tissue-culture treated plate as an example to illustrate the culture method. Use a 90%confluent flask to determine the cell concentration in the cell suspension using a hemocytometer. Dilute the cell suspension using fresh media to a prescribed cell concentration needed for the cell study in vitro.

Place the samples in the center of the 12-well tissue-culture plates. Rinse the culture plates with 2 mL of PBS and 2 mL of DMEM sequentially to calibrate the osmotic pressure under sterile conditions. Add 3 mL of the diluted cell suspension into each well onto the samples of interest.

Culture the cells in an incubator under standard cell culture conditions for 24 hours. The schematic diagram in disfigure shows the steps of direct exposure culture. Prepare the cell suspension with the required concentrations of cells based on the experimental design for different cell types and intended applications.

Rinse the culture plates with 2 mL of PBS and 2 mL of DMEM sequentially to calibrate the osmotic pressure under sterile conditions. Add 3 mL of the diluted cell suspension into each well. Culture the cells in the humidified incubator under standard cell culture conditions for 24 hours or until the cells reach 50%to 80%confluence.

After 24 hours rinse, the cells in the well plate with PBS using a pipette to remove floating dead cells. Place the disinfected or sterilized samples directly on the adhered cells. Add 3 mL of fresh media into each well.

Culture the cells under standard cell culture conditions for another 24 hours. The schematic diagram in this figure shows the steps of the exposure culture method. Initial steps for cell preparation are the same as direct exposure culture.

Afterwards, place the samples in the well inserts with a membrane pore size of 0.4 micrometers and place the well inserts with the samples into each well with the cells. Culture the cells under standard cell culture conditions for another 24 hours. Postculture characterization of cells.

For direct culture and direct exposure culture, fix, stain, image and analyze the cells adherent on both well plates and samples. For exposure culture, analyze the cells adhered to the well plates. Collect the post culture media from each well into a corresponding 15 mL conical tube for further analysis.

Collect all the samples after culture for further analysis. Rinse the cells adherent on both samples and well plates three times using PBS. Add 1 mL of 4%paraformaldehyde into each well plate.

Put the lid back on the well plate and allow the PFA to react for 20 minutes. After 20 minutes, aspirate the PFA and dispense it into a waste bottle. Rinse the well plate three times using PBS to remove the PFA and transfer the waste to the waste bottle.

Prepare the working stocks of the staining agents Following the manufacturer's instructions. Add 200 to 400 microliters of diluted Alexa Fluor 488 Phalloidin staining agent to each well to cover the cells on the well plate and the sample. Wrap the well plate in aluminum foil to prevent light exposure and allow the Alexa Fluor 488 Phalloidin to react for 20 minutes at room temperature.

Collect the Alexa Fluor 488 Phalloidin staining agent and dispense it into the corresponding waste bottle. Rinse the wall plates three times using PBS to remove the excess Alexa Fluor 488 Phalloidin and dispense the used PBS into the corresponding waste bottle. Add 200 to 400 microliters of diluted DAPI to each well to cover the cells in the well and on the sample.

Wrap the well plate in aluminum foil and allow the DAPI to react for five minutes at room temperature. Rinse the well plate three times using PBS and dispense the used PBS into the corresponding waste bottle. After staining, image the cells using a fluorescence microscope.

Whenever possible, take phase-contrast images of cells in addition to fluorescence images. Image the cells on the biodegradable samples as soon as possible or immediately after staining to avoid or reduce possible changes caused by the continuous degradation of samples. For direct culture and direct exposure culture, image and evaluate two types of cells.

One, the cells on the samples in direct contact with the samples, and two, the cells adhered on the well plate surrounding the samples in direct contact with the samples as shown in the figure. For exposure culture, as shown here, use the image guide when taking the fluorescence images of cells to determine if the cell's response would be different in response to dynamic degradation gradient of the samples. Image and analyzes cells located in the area within the inner ring, 3.5 mm away from the center, and the outer ring, which is 7 mm away from the center separately.

For each sample, and while in the culture plates, randomly take at least five images from each area of interest where the cells are either indirect contact or indirect contact with the samples at a predefined distance. From all the cell images obtained from step 4.3, quantify the cell morphology by measuring the cell spreading area and aspect ratio for image analysis. Count the number of cells in each image area.

Calculate the cell adhesion density under direct and indirect contact conditions as the number of cells per unit area. Postculture analyses of media and samples. Before self fixation, collect the postculture media.

Measure the pH values of the postculture media in each well immediately after collection using a precalibrated pH meter. Following the previous step of pH measurement, collect and dilute the media using a desirable dilution factor for optimal measurements of ion concentrations. Measure the concentrations of the ions of interest in the post culture media using an inductively coupled plasma-optical emission spectrometer abbreviated as ICP-OES.

After in vitro cell study, the biodegradable samples may change in dimension, mass, surface morphology, microstructure, and composition. Postculture analysis of samples helps understand the degradation mechanism of samples. After cell culture, take the photographs of the samples to show possible changes in sample dimension, color, morphology, and other visible characteristics.

Dry or dehydrate the postculture samples and measure the sample mass, dimension, and volume to quantify any changes in mass, dimension, and volume. Use a scanning electron microscope to characterize the microstructure and morphology of the samples. Use energy dispersive x-ray spectroscopy and x-ray defraction to characterize the composition and phase of the degradation products on the samples.

Use FTIR or ATR to detect the chemical bonding on sample surfaces. Representative results. Here, the figure shows the representative fluorescence images of bone marrow derived stem cells under direct and indirect contact conditions using different culture methods.

This figure shows the example data for quantified cell adhesion density. As shown in figure A, in the 24 hour direct exposure culture, BMSCs in direct contact with the ZC21 have significantly greater cell adhesion density than any other group. As shown in figure B, in the indirect contact condition of direct exposure culture, BMSC adhesion density is significantly higher for the ZC21 group than the magnesium group.

However, it shows no significant difference compared with the T64 and cells only control groups. Here, figure A shows the pH value of postculture media after the direct exposure culture and direct culture. For the direct exposure culture, the pH values of media range from 8.3 to 8.4 for all samples.

In the direct culture, the pH values of media range from 7.9 to 8 across the groups. Figure B shows the magnesium ion concentration in the postculture media. In both the direct exposure culture and the direct culture, their magnesium ion concentrations in the ZC21 and magnesium groups are significantly higher than any other control groups.

This figure shows the XRD patterns for ZSr41 and pure magnesium after a three day exposure culture. The crystalline phases of different compositions, such as magnesium, zinc oxide, and hydroxyapatite were observed. Here, figure A shows the overlay of SEM images and EDX maps of surface elemental composition for magnesium oxide-coated magnesium, and the control of magnesium substrates and glass after 24 hours of direct culture with BMSCs.

Figure B shows the quantitative surface elemental composition of the sample surfaces, indicating different depositions formed during the cell culture.Conclusions. In this video, we introduced three different in vitro methods for evaluating the cytocompatibility of biodegradable implant materials based on different experimental designs and intended applications. The interactions between materials and various types of cells could be investigated through the direct culture method, direct exposure culture method, and exposure culture method.

This video has presented key in vitro methods to study the effect of biodegradable materials along with their degradation products on cell behaviors for a variety of medical implant applications.