Our ability to accurately predict how humans respond to drugs during clinical trials is limited due to the lack of physiologically relevant preclinical models. This microphysiological system can address that disparity. This human cell derived system accurately mimics the whole joint disease nature of osteoarthritis in the human knee, which allows for the testing of disease modifying osteoarthritis drugs prior to clinical trials, potentially saving millions of dollars.
Begin preparing methacrylated gelatin, or GelMA, by adding 17 grams of gelatin type B to 500 milliliters of distilled water. Mix it for 30 minutes on a shaker at 37 degrees Celsius and add 13 milliliters of methacrylic anhydride before overnight mixing on a shaker. On the next day, prepare around 60 milliliters of aliquots of the GelMA into dialysis bags.
Place all the dialysis bags in distilled water with a stir bar for seven days. Change the water multiple times per day and leave the bags at four degrees Celsius overnight. On day seven, pour the GelMA in a dish and freeze it at minus 80 degrees Celsius before the lyophilization.
Then, lyophilize the GelMA by placing it in the vacuum chamber of a lyophilizer. Ensure complete drying prior to removal from the lyophilizer. Next, dissolve the lyophilized GelMA in Hanks'balanced salt solution at 15%concentration and adjust the pH to 7.4 using a small amount of sodium hydroxide.
Based on the volume acquired, supplement the solution with antibiotic antimycotic and 0.15%lithium phenyl phosphinate, or LAP. Protect GelMA solution from light and store it at minus 20 degrees Celsius until further usage. Autoclave 3D printed dual flow bioreactor chambers, lids, and inserts in autoclave bags at 121 degrees Celsius for 20 minutes with steam.
And then, for 20 minutes with dry heat. After sterilization, soak the bioreactor chambers, lids, and inserts in 15 milliliters of sterile PBS overnight inside the biosafety cabinet, followed by complete drying. Using a 1000 microliter pipette, resuspend 20 times 10 to the sixth human bone marrow derived mesenchymal stem cells, or MSCs, per milliliter cells in a 15%GelMA solution.
Then, press a sterile, dry silicone mold against a Petri dish, using sterile gloves. Put one insert into each hole of the silicone mold with forceps with the hole side of the insert facing down. Once done, add around 50 microliters of the cell suspension per insert using a 200 microliter pipette.
Cross-link the gel inside the insert by exposing the top to a 395 nanometer wavelength UV flashlight for 1.5 minutes, then illuminating the other side for 30 seconds. With sterile forceps, immediately transfer the inserts into eight milliliters of growth medium in a non-tissue culture six well plate and allow the cells to recover overnight. To initiate the differentiation of adipose tissue, transfer the inserts to eight milliliters of adipogenic medium and culture them for 28 days with daily medium change.
To engineer the osteochondral units, place the inserts into dual flow bioreactor chambers, cap the wells, and infuse the two streams separately at a flow rate of five microliters per minute with 35 milliliters of osteogenic medium and 35 milliliters of chondrogenic medium. Maintain cells for differentiation for 28 days by performing biweekly syringe changes with the respective medium types. To derive the fibroblasts, differentiate the MSCs in 2D culture in a T 150 square centimeter tissue cell culture flask containing 20 milliliters of fibrogenic medium for 21 days.
Maintain the weekly change of the medium. After 21 days, use four milliliters of trypsin to detach the cells. Encapsulate the 3D gels within the inserts.
To obtain synovial-like fibrous tissue, or SFT, follow a similar protocol. Connect the silicone tubing, having a 0.062 inch inner diameter and a 0.125 inch outer diameter, to the minijoint bioreactor bar at one end and F 1/16th lure lock connectors at the other end. Then, autoclave the 3D minijoint bioreactors along with the silicone tubing and lids.
Prepare and load 35 milliliters of medium to be used for the minijoint culture into the medium reservoirs. Use straight forceps to transfer the osteochondral units from the dual flow bioreactor to the right well of the minijoint bioreactor. Transfer the adipose tissue insert and fibrous tissue insert into the left and middle wells, respectively, before covering all the wells with sterilized lids.
Connect the minijoint clip inlets to the medium reservoirs and the outlets to syringes. Then, mount the syringes onto a syringe pump. Transfer the pump and chips to an incubator.
Place the medium reservoirs on ice outside the incubator. Operate the pump in the withdrawal mode, drawing the medium from the medium reservoir into the minijoint bioreactor chamber. Continue this minijoint culture process for 28 days.
To model the joint inflammation and cartilage degeneration, add interleukin one beta to the fibrogenic medium strain at 10 nanograms per milliliter for seven days. Make sure to replace the syringe on the third day before providing fresh interleukin one beta to the fibrogenic medium. For the drug testing, after three days of interleukin one beta treatment, administer the drug either in the shared medium, simulating an intraarticular administration, or in all the medium types, simulating a systemic administration.
After seven days of interleukin one beta treatment, collect individual tissues for the analysis. After removing the inserts using sterile forceps, push a biopsy punch through the center of the insert to remove the gel and place the gel in PBS. Next, cut the osteochondral gels in half while assessing the gene expression.
Collect and centrifuge around 1.5 milliliters of media from each medium source at 14, 000 G for 10 minutes. After discarding the sediment, flash freeze the conditioned media in liquid nitrogen. For histological staining and immuno staining, first, fix the osteochondral and synovial-like fibrous tissue, or SFT samples, in 10%buffered formalin and dehydrate them in ethanol of ascending concentrations.
Clear the sample in xylene, embed them in paraffin, and finally, section the samples into six micrometer thickness. In the case of adipose tissue, stain the 10%buffered formal and fixed samples directly, with oil red O solution or BODIPY. The tissue specific phenotypes were well maintained for the individual micro tissues.
All the tissues of the minijoint were collected to analyze their phenotypes following 28 days of culture. The osseous component of the osteochondral units'micro tissues expressed high levels of osteocalcin. In the cartilage portion of the osteochondral, tissues expressed significantly high levels of collagen type two and aggrecan.
The expression levels of representative adipogenic genes, adiponectin, and leptin were higher in the adipose tissue. The deposition of calcium minerals, alkaline phosphatase, and osteocalcin protein was primarily seen in the bone size of osteochondral micro tissues. Cartilage side of osteochondral tissues showed well retained glycosaminoglycan and collagen type two.
The expression of chondrocyte hypertrophy associated markers, collagen type 10 and Indian hedgehog, was significantly down-regulated after 28 days of culture. Abundant lipid droplet deposition was found in the adipose tissue. The immunofluorescent staining showed robust lubricin and cadherin 11 expression by the SFT after four weeks of culture.
In a disease model, IL one beta treatment resulted in cell apoptosis and elevated MMP-13 levels in the SFT. The cartilage degradation was treated by IL one beta, suggesting the occurrence of crosstalk between the osteochondral tissues and SFT. The therapeutic efficacy of naproxen through systemic administration showed reduced cartilage degradation in IL one beta minijoint.
The realtime PCR indicated that the four potential disease modifying osteoarthritis drugs partially reversed the cartilage loss. The most important steps of this procedure include cross linking of the gels in the inserts and the insert placement into the bioreactor. The chip allows investigation of the contribution of other risk factors offered in osteoarthritis, such as obesity and mechanical injury.
It can also be used to study other joint diseases.
