3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

The overall goal of this procedure is to perform a 3D biomimetic hydrogel culture to expand articular chondrocytes and generate human articular cartilage. This is accomplished by first dissecting the femoral condyles obtained from total knee arthroplasty, removing cartilage tissue from the subc chondral bone, and isolating chondrocytes from the extracellular matrix by enzymatic dissociation. The second step is to synthesize chondroitin sulfate, methacrylate polymer for the fabrication of a biomimetic hydrogel.

Next, the chondrocytes are encapsulated in the 3D biomimetic hydrogels, and the constructs are cultured for three to six weeks in vitro. Ultimately, results can be obtained that show growth and maturation of articular chondrocytes using both biochemical and mechanical testing. The main advantage of utilizing a 3D biometric hydrogen platform for culturing chondrocytes is that it provides a physiologically relevant microenvironment because it incorporates the native components of cartilage.

This is the advantage that chondrocytes can maintain their physiology and phenotype for a longer period of time. The inclusion of a bioactive polymer, the chondroitin surface into the three dimensional matic hydrogel enables chondrocyte mediated metrics, degradation turnover. In addition, the mechanical and biochemical properties of the hydrogel matrix can be easily modified.

This technique is compatible with conventional assays to evaluate cell phenotype and functional outcome. It can be used to study chondrocyte intrinsic features associated with ancient disease stage and to evaluate cell maturation and regenerative potential in a physiological context. To begin prepared dissociation medium by combining one part collagenase two at 125 units per milliliter with one part collagenase four at 160 units per milliliter in choy growth medium, using fresh cartilage tissue that is typically discarded during a total knee arthroplasty.

Take a sterile scalpel and remove a biopsy from the smooth surface of one of the femoral condiss. Then shave the biopsy into thin slices. Add the cartilage slices to the combined dissociation medium in a one to three ratio by volume, and place the dish at 37 degrees Celsius overnight to release the choys from the extracellular matrix the following day.

Filter the digested tissue containing the dissociated cells through a 70 micron pore size nylon mesh. Then wash the cells twice with 20 milliliters of prewarm D-M-E-M-F 12, medium and pellet the cells at 600 G for five minutes. Next count the isolated chondrocytes on a he cytometer plate.

1 million cells onto a 60 millimeter culture dish and cover with supplemented D-M-E-M-F 12 medium. Maintain the primary choy cultures as a high density monolayer by following standard culture techniques. Synthesize chondroitin sulfate methacrylate by adding 1.952 grams of two morph eth ethane sul acid, and 5.84 grams of sodium chloride in 200 milliliters of deionized water and stir the solution until completely dissolved.

Next, dissolve five grams of chondroitin sulfate sodium salt into the buffered solution. Then add 0.532 grams of N hydroxy CIN amide, and 1.771 grams of one ethyl three three dimethyl amino propyl carbo ide hydrochloride to the solution and stir for five minutes. Once dissolved, add 0.765 grams of two amino ethyl methacrylate to the solution and maintain the reaction at room temperature for 24 hours covered with aluminum foil.

Purify the final mixture by dialysis against deionized water for four days, using 12 to 14 kilodalton molecular weight cutoff dialysis tubing. Then filter the purified solution through a 0.22 micron filter and freeze at minus 20 degrees Celsius. Place the open tubes containing the solution in a desiccate.

Protect them from light by covering with aluminum foil and apply a vacuum overnight. Store the dissolved polymer at minus 20 degrees Celsius in Falcon tubes, wrapped with parfum and foil to protect from light and moisture the day prior to cell encapsulation. Remove the media from cultured cytes and cover the cells with dissociation medium.

Incubate the cells in the collagenase solution overnight at 37 degrees Celsius autoclave PCR film and cylindrical rods, and then sterilize the custom made cylindrical gel mold by submersing it in 0.2 micron filtered 70%ethanol placed within a tissue culture hood under UV light overnight the following day, remove the molds from the ethanol and allow them to dry within the tissue culture hood. Then place the mold into a sterile 150 millimeter plate. Once dry, seal the bottom of the molds using autoclave PCR film.

Avoid any air bubbles or gaps in order to prevent leaks. Next, take a 50 milliliter tube and add 10%of the prepared chondroitin sulfate methacrylate, 10%polyethylene glycol di acrl, and 5%weight per volume. Photo initiator in DPBS to a final concentration of 3%5%and 0.05%respectively.

Protect the tube from light and briefly vortex the mixture. Collect the dissociated cells in a 50 milliliter falcon tube and then count them using a hemo cytometer. Pellet the cells at 460 times G for five minutes and resuspend them in the mixed gel material at a density of 15 million cells per milliliter.

Mix the solution 30 times taking care to avoid bubble formation. Next pipette 72 microliters of the cell hydrogel suspension or hydrogel alone into the custom made cylindrical gel. Mold induce gelian by exposing the gels to 365 nanometer UV light at three milliwatts per square meter for five minutes.

Once jelled, use a scalpel to cut the film and carefully remove it. With the help of the cylindrical rods, push out the gel into a six well plate with five milliliters of sterile DPBS to wash away any remaining un polymerized gel and loose cells. Next, transfer the washed hydrogels into wells of a 24 well plate containing 1.5 milliliters of Chondra site growth media in each.

Well incubate the hydrogels at 37 degrees Celsius and change the media every two days. Assess cell viability by live dead staining 24 hours post encapsulation using standard techniques culture, the cell laden hydrogels, and the empty hydrogels for three to six weeks before harvesting and analysis. After the desired number of days of in vitro culture, remove the samples from the incubator to perform the compression test on the cell hydrogel scaffold and the acellular control hydrogels.

Place the samples one at a time at room temperature onto the PBS bath attached to a mechanical testing system fit with a 10 Newton load cell compress at a rate of 1%strain per second to a maximum strain of 15%To analyze the mechanical tests, create stress versus strain curves for each sample and curve fit. Using a third order polynomial equation, determine the compressive tangent modulus from the curve fit equation at strain values of 15%after three weeks of culture In 3D Biomimetic hydrogels the gene expression analysis of normal chondrocytes, both juvenile and adult, showed an increase in the expression of the chondrocyte genes collagen two A one and collagen six A one. On the contrary, diseased chondrocytes showed a dramatic decrease in collagen two A one while maintaining the expression of collagen six A one showing a loss of congenic phenotype despite being cultured in a favorable biomimetic environment.

Chocy expansion after three weeks of culture can be estimated by quantification of DNA with the pico green dye. Comparative analysis of the three groups of cells show that the cell density of juvenile and adult populations was unchanged while osteoarthritic chondrocytes exhibited a dramatic decrease compared to day one of culture. The secreted chondrocyte matrix was quantified as sulfated gag content after three weeks of culture.

As shown here, the chondrocytes deposited a significant amount of gag during three weeks of culture, which may have contributed to the improved mechanical properties of cell laden hydrogels compared to controls. Besides applying this procedure for individual studies, the 3D biomimetic hydrogel culture model can be easily translated to a full thickness cartilage defect model and to characterize the functional and biochemical outcome in long-term articular cartilage regeneration. After watching this video, you should have a good understanding of how to isolate human chondrocyte from articular knee specimens and produce three dimensional hydrogen scaffold for long-term cony culture and cutlet generation Utilization of the 3D OME hydrogels will allow us and other researchers to compare the congenic potential of different mature chondrocyte populations, as well as novel stem and progenitor cell populations.

In future, these techniques can be extended to incorporate additional mimetic factors in order to formulate an optimal scaffold which can be utilized for cartilage regeneration and repair.