An Ex Vivo Tissue Culture Model of Cartilage Remodeling in Bovine Knee Explants

Cartilage implants represent a translational easily accessible method for interrogating tissue remodeling in the cartilage matrix. The method may provide early insight into how potential new treatments affect joint structure. The main advantage of this technique is that that cartilage remain in a native 3D environment, which allow profiling of the interaction between cells and the surrounding matrix using biomarkers.

The principle in the method is not unique to cartilage. Explants can be extracted from other tissues such as an ovium, liver, and lung and interrogated using matrix derived biomarkers. Demonstrating the procedure will be Amalie Engstrom, a grad student from our laboratory.

Perform the entire tissue sourcing process outside of a laminar flow hood in an aseptic environment. From a local slaughterhouse, obtain an entire fresh bovine tibial femoral knee joint from calves between 1.5 and two years of age. Then, gentle dissect the calf knee by first removing the excess flesh, uncovering the condyles, meniscus, tendons, and synovial membrane.

Cut the tendons and synovial membrane allowing the joint to dismember. Remove the meniscus to expose the femoral condyles. Use a three millimeter biopsy puncher to isolate explants from the load bearing area of the femoral condyles and release them from auricular surface by cutting with a scalpel parallel to and as close to the subchondral bone as possible.

Immediately store and mix the explants in DMEM F12 glutimax culture medium containing 1%penicillin and streptomycin in a 50 milliliter tube or Petri dish. To begin tissue culturing, transfer the explants to a sterile 96 well plate in a laminar flow hood placing all replicates within each group diagonally to minimize the variation induced by evaporation. Add PBS to the outer wells of the culture plate to further avoid evaporation of the supernatant.

Then, wash the explants three times in either culture medium or PBS. Culture the explants in 200 microliters of culture medium per well until the start of the experiment for up to 10 weeks in an incubator at 37 degrees Celsius with 5%carbon dioxide. If applying any treatments, prepare these to the wanted concentration in the explant wells by dilution in the culture medium prior to changing the medium.

Change the culture medium every two to three days in a laminar flow hood. Gently remove the supernatant from each well and transfer it to a new 96 well plate for storage. Immediately add 200 microliters of fresh culture medium or treatment to each well.

Do not let the explants dry out during the medium change and ensure that all of the explants are completely submerged in the new medium. Seal the new plate with sealing tape and store it at minus 20 degrees Celsius for biomarker analysis of tissue turnover and protein expression. To begin a resazurin test, make a solution of culture medium containing 10%resazurin.

Harvest the supernatant as previously described and immerse the explants in the 10%resazurin solution at 37 degrees Celsius for three hours or until the supernatants turn purple. Then, transfer the conditioned and background control resazurin solution to a black microtiter plate and measure the fluorescence at an excitation of 540 nanometers and an emission of 590 nanometers. After this, submerge the explants in wash medium for five to 10 minutes to allow the resazurin to completely diffuse out and add new culture medium or treatments if used.

Continue measuring the metabolic activity once weekly as an indirect measurement of cell viability. In this study, bovine full depth explants are isolated, cultured, and treated. Explants are divided into four groups for treatment, including those treated with Oncostatin M and tumor necrosis factor alpha, those treated with Oncostatin M and tumor necrosis factor alpha with GM 6001, those treated with insulin growth factor one, and a control without treatment.

The metabolic activity is relatively stable throughout the three weeks for all four groups. There was a tendency for IGF-1 to increase and O plus T groups to decrease compared to control. O plus T is then applied to culture wells three times a week to investigate O plus T mediated cartilage degradation.

O plus T is seen to increase type two collagen degradation from day seven to 21 and aggrecan degradation from days three to 14 compared to the control group. Adding GM 6001 in combination with O plus T blocks the release of the O plus T mediated C2M, but only has limited affect on AGN X1 with levels similar to O plus T group at day 10. Insulin like growth factor one is applied three times weekly to bovine full depth explants to investigate how anabolic stimulation modules the cartilage ECM turnover.

The effect of IGF-1 on the cartilage explants was mainly observed on measurements of type two collagen formation as expected for anabolic stimuli. When treating with IGF-1, the the pro-C2 levels decrease less than those observed in the control group, indicating that IGF-1 stimulates type two collagen formation from day seven. Explants may deteriorate over time and thus careful pipetting is critical in all treatment and washing steps in order to not lose or destroy explants in the different media exchange steps.

Results generated using this model should not stand alone, but be supported by data generated in human tissue as well as cellular and in newer models to further validate findings. To shed light on the underlying mechanism in the tissue and not just the outcomes, it's possible to further analyze both tissue and supernatants using other molecular biology techniques, such as gene and protein expression analysis, or immunohistochemistry, or other permanent molecular biology techniques.