Begin by trypsinizing the human mesenchymal stem cells to create a cell suspension. Next, aliquot the cell suspension into a 1.5-milliliter tube to create 10 constructs. After centrifuging the suspension at 220g for three minutes, use a vacuum pump to remove the supernatant.
Now, place the cell pellet on ice. Next, bring the thiol-modified hyaluronic acid, thiol-reactive crosslinker, polyethylene glycol diacrylate, and degassed water bottles to room temperature. Under sterile conditions, use a needle equipped syringe to add one milliliter of degassed water to the bottle containing hyaluronic acid.
Vortex the solution, occasionally heating it to 37 degrees Celsius until it becomes clear. Then, place the solution on ice. Under sterile conditions, add 5 milliliters of degassed water to the crosslinker bottle.
Dissolve by inverting repeatedly before placing it on ice. Now, add 120 microliters of the dissolved hyaluronic acid solution to the aliquoted cell pellet and resuspend the cell pellet by pipetting the solution back and forth. Add 30 microliters of the dissolved crosslinker solution into the tube with the resuspended cell pellet.
Ensure proper mixing by tapping on the tube. After mixing, briefly spin down the solution. Drop 15 microliters of the combined solution onto a paraffin-coated 24-well plate.
Allow it to solidify at 37 degrees Celsius for 30 minutes. For the in vitro differentiation, add 0.5 milliliters of chondrogenic differentiation medium to the cell seated hydrogels. After three weeks, switch to the hypertrophic differentiation medium, adding 0.5 milliliters per well.
Chondrogenic differentiation resulted in sulfated glycosaminoglycans in type 2 collagen positive extracellular matrix in both the hyaluronic acid or, HA, and collagen constructs. However, the distribution was more homogenous in the hyaluronic acid constructs. The collagen constructs demonstrated peripheral cells with heterogeneous morphology.
The average roundness of cells in the HA constructs was higher than in the collagen constructs. Calcium deposition was detected at the outer edges of both constructs at five weeks. In the in vivo HA constructs, all implants were attached to each other in subcutaneous pockets and formed integrated bone tissue.
40%of the in vivo collagen constructs were independent. Osteoid tissue with lamellar morphology was formed in the outer regions of both in vivo constructs at eight weeks post-implantation. Loss of cartilage was also observed in the HA construct.
Multiple in vivo HA constructs appeared to form integrated bone tissue as indicated by the connection to the bone tissues and the presence of joint fibrous tissue. While 40%of the collagen constructs were united, the remaining constructs existed apart or were only attached without bony tissue connections. Both constructs showed positively labeled vessels in the bone marrow, and the inner cartilage was surrounded by multinucleated osteoclast cells.
The mineral was deposited in the outer osteoid region of both constructs with higher mineral volume and HA constructs due to their larger size.
