Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The overall goal of this procedure is to dissect and culture metatarsals from embryonic mice to provide an ex vivo model to allow for the study of endochondral bone growth and mineralization. This method can help answer key questions about endochondral ossification, the process by which long bones grow. In particular, it is well suited to investigate the mechanisms of the initiation of skeletal mineralization.

The main advantage of this technique is that it provides, through the maintenance of cell to cell and cell to matrix interactions, a more physiological model of endochondral ossification compared to cells in 2D or 3D culture. To begin, prepare the dissection medium by diluting alpha minimum essential media one to 13 in PBS. Next, re-suspend BSA at two milligrams per milliliter and then filter sterilize it through a syringe filter.

Then, prepare the culture medium and filter sterilize it using the same type of syringe filter used for the BSA. After filtering the culture media, load 300 microliters of the culture medium into each well of a 24 well culture plate. Next, place the cell culture media 24 well plate and the dissection media into an incubator for at least one hour to equilibrate.

Then, immerse autoclave dissection equipment in 70%ethanol. Obtain E15 wild type C57 black 6J embryos as described in the accompanying text protocol and place them in a Petri dish containing dissection medium. Following decapitation, wash the skin with 70%ethanol and use Vannas micro scissors to remove the hind limbs from the embryos by incising around the proximal femur.

This ensures as much of the hind limb as possible is removed. Place the hind limbs into a Petri dish and submerge them in dissection medium prior to metatarsal dissection. Then, place them under a dissecting microscope.

Use a number four tweezers to hold the limb steady while using a number five tweezers to remove the skin surrounding the embryonic foot. This is most effectively performed by grasping the skin at around the level of the tibia and pulling towards the phalanges. Next, use the number four tweezers to carefully hold the tarsals of the embryonic foot while using the number five tweezers to remove and discard the first and fifth metatarsals.

With the foot held in the same position, use the number five tweezers to disrupt the connective tissues between the three remaining phalanges and metatarsals. Then, insert the tip of the number five tweezers at the joint between the phalanges and metatarsals to remove the phalanges from the metatarsals. Finally, use the number five tweezers to disrupt the connective tissues between the tarsals and metatarsals.

Again, place the tip of the number five tweezers in the joint space between the tarsals and metatarsals and gently free the metatarsals from the tarsals. Then, gently pick up the now free metatarsals with the number four tweezers and place them into fresh dissection medium. It is crucial that embryonic metatarsals are not damaged or do not dry out during the dissection and handling steps.

Damage to metatarsals affects their growth and mineralization. Any remaining soft tissues will detach from the metatarsals during culture, so don't be overly concerned with their total removal during dissection. When all required metatarsals have been dissected, carefully place metatarsals individually into the pre-warmed 24 well plate containing 300 microliters of culture media.

Next, make initial longitudinal measurements using a microscope with a digital camera attached and image analysis software. Culture the metatarsals for up to 14 days. However, do not change the media of E15 cultures until at least day five of culture as this affects their mineralization capability.

After a few days in culture, measure the length of the central mineralization zone when it appears. After a few days in culture, mineralization of the matrix occurs in the mid-diaphysis of the bone rudiment and is easily observed by light microscopy. The second, third, and fourth metatarsals are often pooled for embryonic studies.

Through seven days of culture, the second, third, and fourth metatarsals revealed no significant differences in the appearance or extent of mineralization, validating this practice. To examine the effects of various mineralization substrates and agonists on metatarsals mineralization capability, E15 metatarsals were cultured in media containing a range of supplements as shown here. The images and length measurements were recorded daily.

Out of all the substrates tested, ascorbic acid increased the mineral length the most and most comparable to the effects of beta-glycerolphosphate. Lentivirus transfection of GFP plasmid into E15 metatarsals resulted in the expression of GFP throughout the metatarsal. This highlights the use of lentivirus to successfully transfect embryonic metatarsals.

This technique may also be used to knock down or overexpress target genes. While attempting this procedure, it is important to remember that E15 metatarsals are extremely fragile. The integrity of the metatarsal is paramount to the success of the downstream culture.

Following the dissection and culture of E15 metatarsals, many downstream analysis can be carried out. For example, mRNA and protein expression can be assessed by RTQPCR and Western blotting, respectively. In addition, immunohistochemistry can be used to localize proteins within the metatarsal.

After watching this video you should have a good understanding of how to successfully dissect the metatarsals from the hind limbs of embryonic day 15 mice. This method can be adapted to embryonic day 17 or 18 metatarsals, which are more useful for the study of bone growth.