This method adapts the metatarsal culture protocol to bigger bones, allowing direct bone manipulation to be combined with complex genetic models to address processes related to bone development. This technique allows manipulation and analysis of bone growth that are not possible in vivo, such as live imaging or direct physical and pharmacological manipulation. This ex vivo culture method allows the specific study of the local effects of the genetic inset in the bone separating it from the systemic effect the treatment has on the organism.
Begin by sterilizing the abdominal region of a pregnant gestational day 14.5 to 18.5 mouse with 80%ethanol and using small scissors to open the skin and abdominal muscles to access the uterine horns. To extract the uterus from the abdominal cavity, remove the mesometrium and cut the base of the horns. Then, transfer the uterus to a 60 millimeter petri dish containing ice-cold dissection medium on ice.
In a biosafety cabinet cut between the sacks with scissors to separate the individual fetuses, and transfer an individual sack into a new 60 millimeter dish with fresh dissection medium under a dissection stereo microscope. Use tweezers to separate the fetuses from the placentas and to clean the fetuses from the membranes. Use a trimmed plastic pipette to transfer the body into a new 60 millimeter dish, and decapitate the embryo before further dissection.
Then use tweezers to remove the skin, starting from the back and peeling out to the toes. Use the tweezers to cut close to the spine and separate the hind limbs from the body. Transfer the hind limbs to a clean dish containing ice-cold dissection medium, and insert the tweezers between the surface cartilage of the distal femur and the proximal tibia to separate the tibia from the femur.
Remove the hip bones from the proximal femur and the calcaneus bone and the fibula from the tibia. Carefully nip and pull the soft tissues from the femurs and tibias and use a sterile one-milliliter pipette to transfer all four leg bones into the first well of a 24-well plate containing fresh dissection medium. It is important to show that the soft tissue that connects the pulse of the bone is removed.
Otherwise, the bone will bend and not achieve proper growth. When the bones have been dissected from the appropriate experimental number of fetuses, image the bones in each well at times zero under a light microscope with a good contrast and replace the dissection medium in each well with one milliliter of culture medium per well taking extra care not to aspirate the bones. To observe the effect of growth inhibition in the culture conditions, treat the left tibias with 500 nanomolar retinoic acid and incubate the right tibias with an equivalent volume of vehicle as a control.
Then, place the plate in a cell culture incubator under standard cell culture conditions. After two days, treat the bones with an appropriate thymidine analog for one to two hours to assess bone cell proliferation before transferring the bones to individual two-milliliter tubes of 4%paraformaldehyde for 10 minutes. Then, transfer the bones to PBS to image the bones at the final time point before returning the samples to the paraformaldehyde for overnight fixation at four degrees Celsius.
To measure the length and mineralized region of the bones, open an appropriate imaging software program, and taking into account the scale of the image, measure both the total length of the bone, and the mineralized region. Starting the measurements from the first dark cells at one end until the last dark cells at the other end. To calculate the growth rate which is defined as the average increase in length per day, divide the difference between the final length of the bone and the initial length by the number of days in culture.
Here, a comparison between cultured tibia and freshly-extracted tibia at equivalent stages is shown. After up to two days of culture, the size achieved is comparable to the in vivo bone growth for both the cartilage and mineralized bone. Longer culture periods lead to bigger differences between the cultured bones and freshly extracted bones.
Note, the tibia grown with an incomplete removal of the soft tissue can result in bending of the cultured tibia. Treatment with retinoic acid has a robust effect on the tibial growth as early as after two days of treatment. Although there is a consistent increase in the total length of the tibia after two days in culture, this growth corresponds to an approximate increase of only nine to 29%from initial length.
Less than the bone growth increase that would be observed in vivo. Indeed, thymidine analog labeling shows fewer positive cells in this region after culture compared to freshly extracted bones of an equivalent stage. Further, in in vitro cultured tibia, the total length of the skeletal element increases substantially, while almost no increase in the mineralized region is observed.
In addition to histological and molecular analysis, clearing and 3D imaging can be performed after the culture to characterize cellular effect of the treatments applied to the cultured bones. The technique allows us to address questions related to interorganal communication. For example, by co-culturing bones from the different genetic models in some sort of pain-free parabiosis experiment.
