Integrated Bone Formation Through In Vivo Endochondral Ossification Using Mesenchymal Stem Cells

Our research intends to develop a method that improves the lack of blood flow and can be applied to large bone defects. The protocol presented here is one with the approaches we have taken to this end. The approach to bone formation using artificial cartridge, the structure that mimics the natural process of scattered develop during the embryogenesis.

This approach is relatively new in bone regeneration research. It have the advantage of inducing iron neogenesis, which is not existing method currently used in critical practice except for vascularized bone graft. Traditionally, bone regeneration research has used the techniques that combine strength cells, biomaterials, and growth factors alone or in combination.

The method of bone formation by implanting artifical cartilage uses a technique that makes artifical cartilage by mixing stem cells with biomaterials that serve the scaffold material and inducing cartiliage differentiation. Healing a patient's bone defect clinically requires a large quantity of high-quality artificial cartilage. Our protocol addresses the challenge of producing high-quality artificial cartilage and how to make multiple cartilages fuse with each other to form a unified bone.

Our protocol helps generate larger, higher-quality cartilage with fewer cells than protocols that use or do not use other scaffold materials to create artificial cartilage. Furthermore, the protocol can generate one integrated piece of bone, even with multiple pieces implanted in vivo. This is important because preparing clinical cartilage of a shape and size that fits the bone defect is impractical.

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 220 G 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 0.5 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-seeded hydrogels. After three weeks, switch to the hypertrophic differentiation medium, adding 0.5 milliliters per well. Chondrogenic differentiation resulted in sulfated glycosaminogens and type two 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 of 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 in HA constructs due to their larger size.