Endochondral ossification, essential for fetal long bone development, begins with mesenchymal progenitor aggregation at embryonic day (E) 11.5. By E13.5, chondrocytes form a cartilage template, which matures and leads to the primary ossification center (POC) by E15.5. Increased vascularization at the POC replaces hypertrophic chondrocytes with bone, with remaining chondrocytes localizing to the growth plates.

Mechanical stimulation is known to play a pivotal role in endochondral ossification. Fetuses that lack mechanical stimulation, termed fetal akinesia, have impaired bone growth and skeletal deformities. Postnatal treatments for such conditions are unfavorable due to reduced morphogenic plasticity in the mature skeleton. Even when a musculoskeletal condition relating to reduced movements is identified in utero, there is currently no commonly performed treatment that can reduce its severity. Maternal exercise has emerged as a potential in utero therapeutic strategy to enhance embryonic skeletal development by providing compensatory mechanical stimuli during critical periods of bone formation. Subjecting pregnant mice to maternal wheel running exercise could serve as a novel system for studying the effects of in vivo mechanical loading on skeletal development.

Female C57BL/6J mice acclimated to running wheels (minimum 2 weeks), mated, and were caged without wheels until E13.5. Pregnant mice ran for one hour daily from E13.5 to E16.5 (Exercise), while controls (Sham) had locked wheels. Embryos were harvested at E17.5, and forelimb samples were then collected. Isolated forelimbs were embedded in optimal cutting temperature compound for cryosectioning. Serial cryosections (10 µm thickness) of the humerus were then prepared for histological analysis. Sections were stained with collagen 10, phalloidin, and alkaline phosphatase (ALP). Images were captured using the Zeiss Axioscan digital slide scanner, and quantitative analysis was conducted using ImageJ. The Student’s t-test and Kolmogorov Smirnov test (significance set at p < 0.05) were used to determine significance.

Staining for collagen 10 allowed for the quantification of the distal hypertrophic zone length. A significantly decreased length was seen in the Exercise group, indicating more advanced bone growth. Phalloidin intensity was measured along the humerus to determine the effects of maternal exercise on F-actin distribution within the POC. The Exercise group had a significantly greater intensity throughout much of the POC, indicating a greater level of cytoskeletal tension that is essential for mechanoregulated bone formation. ALP staining for osteoblastic bone formation activity revealed no significant differences in ALP intensity for both the POC and BC regions as a result of maternal exercise, although there seems to be a noticeable increase in the intensity of the BC (which may imply a role in early bone formation). Immunofluorescent staining with endomucin may provide additional insight into the POC, since ALP intensity may be confounded by enhanced blood vessel infiltration, which would indicate POC maturity. These findings imply that the exercise-induced mechanical stimuli during pregnancy can positively influence skeletal development by promoting bone formation processes. Future studies will conduct similar histological analyses on embryos from muscular dysgenesis and Pax3 Spd/Spd mice, which are models of fetal akinesia, to assess the therapeutic capacity of maternal exercise on fetal akinesia.