Studies has shown that the BMSCs derived from cranial facial bone exhibit superior capabilities than those from axial and appendicular bones. Therefore, mandibular BMSCs are considered to be the preferred resource for developing future therapeutics of craniofacial diseases. This protocol combines the advantages of whole bone marrow adherence and fluorescent cell sorting to obtain significant numbers of purified mandibular BMSCs in a short time.
If you are trying this protocol for the first time I think it's very important to understand the anatomic structure and the functional movement of the rat mandible, which can be achieved by watching this video or reading related literature. After euthanizing the rat, place it in a clean fume hood in a supine position. Incise the skin and buccinator muscles from bilateral angulus oris to the posterior region of the mandible.
Open the mouth of the rat by pushing the maxillary and mandibular incisors towards the opposite direction with both thumbs, to expose the mandibular teeth, which are attached to the mandible. Completely disconnect the buccal muscles and the tendons attached to the coracoids and the inferior border of the mandible. Then press the mandibular posterior teeth and rotate them back and downwards until the condyles on both sides are clearly exposed.
Separate the mandible body from the skull, and clean the adherence soft tissue on the bone surface using a wet gauze. Place the bone in a 10 centimeter sterile glass dish filled with precooled minimum essential medium alpha, or PBS on ice, to preserve the viability. Cut off the anterior bone along the mesial edge of the first molar from the mandibular body, then remove the mandibular ramus including coracoid and condyle along the distal edge of the third molar to expose the marrow cavity.
Fill a 10 centimeter sterile glass dish with 10 milliliters of alpha-MEM with 10%FBS. Aspirate the medium with a 10 milliliter syringe, then insert the needle into the bone marrow cavity and repeatedly flush the bone marrow into the dish. Flush bone cavity at least three times from both mesial and distal sides of the bone respectively, until the bone turns white.
Transfer the media containing the flushed cells into a 15 milliliter centrifuge tube, and centrifuge it at 800 RPM and for five minutes. Discard the supernatant, and resuspend the cells with three milliliters of alpha-MEM with 10%FBS. Plate the cells in a new 10 centimeter culture dish and incubate them at 37 degrees Celsius in a 5%carbon dioxide incubator.
Aspirate the culture medium and wash the dish with PBS. Add two milliliters of 0.25%trypsin with 0.02%EDTA in the dish and digest the cells at 37 degrees Celsius for five minutes. Then add four milliliters of alpha-MEM with 10%FBS to stop the reaction.
Transfer the cell suspension into a 15 milliliter centrifuge tube and centrifuge it at 800 RPM for five minutes. Resuspend the cells in 120 microliters of PBS with 10%FBS after centrifugation. Transfer 100 microliters of the cell suspension into a new micro centrifuge tube.
Block the cell suspensions with one microliter of antibody against CD16 through CD32 at four degrees Celsius for 15 minutes, and stain the cells with PE-conjugated antibody against CD45, FITC-conjugated antibody against CD90, and APC-antibody against CD29, at four degrees Celsius for one hour in the dark. Use the other 20 microliters of cell suspension as unstained negative control. Centrifuge the tubes at 800 RPM for five minutes.
Discard the supernatant and resuspend the cells in 0.5 milliliters of PBS with 10%FBS. Add 10 microliters of 0.01 milligrams per milliliter DAPI 10 minutes before analysis. Use 40 nanometer filters placed on centrifuge tubes to filter the cells, then analyze the cells on a fluorescence activated cell sorter.
First remove dead cells from total cell count by gating DAPI negative cells, then gate for CD29 positive, CD90 positive, CD45 negative, in the selected cells as targeted mBMSCs. Collect the sorted mBMSCs into a 15 milliliter centrifuge tube with five milliliters of alpha-MEM with 10%FBS. Centrifuge the tubes at 800 RPM for five minutes and remove the collection buffer.
Add one milliliter of fresh medium to resuspend the cells, then plate them in a six centimeter culture dish. Using this protocol, a large proportion of cells adhered to the plate on the third day after the initial culture. After an additional three to four days of culture, the cell confluence reached 70 to 80%Fluorescent cell sorting was used to purify mBMSCs which accounted for about 81.1%in the P0 cells.
After culturing P2 mBMSCs in a six well plate for a week, a significant amount of colony forming units were observed. To assess the multi-lineage differentiation ability of the mBMSCs they were induced into osteo-chondro-and adipo-lineages. Increased activity of ALP, red calcific nodules under alizarin red staining, and increased expression of osteogenic specific genes Runx2, Alp, Bsp and Ocn, indicated osteogenic induction.
Oil-red-O staining was used to identify adipogenesis. Numerous lipid rich vacuoles were evident after nine days of induction. Furthermore expression of adipogenic specific genes Ppar-gamma-1 and Cebpa was increased.
The samples showed positive staining for Alcian blue during microscopic observation of chondrogenic differentiation. In addition, immunostaining with anti-type II collagen antibody showed enhanced accumulation of cartilage matrix. Ensuring the viability of mBMSCs is very important in this protocol.
This purpose can be achieved by choosing proper experimental animal, operating on ice, and shortening the experimental time. Utilizing this in vitro model, one can obtain a high number of proliferative mandibular BMSCs, which may facilitate the study of the biological characteristics, the subsequent reaction to the microenvironment, and other applications of mandibular BMSCs.
