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Orthopedic and Rehabilitation Engineering

Orthopedic and Rehabilitation Engineering Lightning Talks

Effects of Maternal Exercise on Embryonic Bone Formation

Saturday, October 26, 2024
8:30 AM – 8:37 AM EST
Location: Room 339

Introduction:  Endochondral ossification is a critical process for fetal long bone development beginning with mesenchymal progenitor aggregation in the mouse long bones at embryonic day (E) 11.5. This is followed by differentiation into chondrocytes and cartilage template formation by E13.5. Chondrocytes undergo maturation and hypertrophy, leading to the formation of the primary ossification center (POC) by E15.5. At E15.5, the POC experiences increased vascularization, resulting in the replacement of hypertrophic chondrocytes with endochondral bone. As cartilage longitudinally transitions to bone stemming from the POC, hypertrophic chondrocytes become localized to the growth plate regions.

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. Consequently, prenatal interventions are of significant interest. 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. By understanding the impact of maternal exercise on endochondral ossification, this research ultimately seeks to establish a novel prenatal therapeutic approach to address developmental bone defects.

Materials and

Methods:  To establish a foundational understanding of how maternal exercise affects bone development, C57BL/6J mice were initially used in this study. This strain provides a baseline for comparison due to its well-characterized genetic background and absence of genetic mutations. For a minimum duration of 2 weeks, female C57Bl/6 mice were housed separately and given the opportunity to acclimate to a home cage running wheel. After being mated with male C57Bl/6J mice, all female mice were caged without wheels until E13.5, the initiation of cartilage template formation. Pregnant mice received in utero mechanical stimulation through encouraged wheel running exercise for one hour daily from E13.5 to E16.5 (Exercise). Control mice (Sham) were subjected to a locked wheel for the same time period. After embryos were harvested at E17.5, forelimb samples were collected from the offspring and fixed in 4% paraformaldehyde. 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.

Several staining protocols were employed to assess cellular and molecular changes within the humerus. Phalloidin staining was used to visualize actin filament organization. Alkaline phosphatase (ALP) staining was used to assess osteoblast bone formation activity. Images were captured using the Zeiss Axioscan digital slide scanner. Quantitative analysis was conducted using ImageJ software with parameters like fluorescent intensity and relative lengths/areas being used to compare between the Sham and Exercise groups. The Student’s t-test (significance set at p < 0.05) was used to determine significance.

Results, Conclusions, and Discussions: Phalloidin intensity was measured along the humerus length to determine the effects of maternal exercise on F-actin distribution within the POC. While the maximum peaks of phalloidin intensity were similar for Sham and Exercise embryos, the Exercise group had a noticeably greater intensity throughout much of the POC, indicating enhanced bone formation (Fig 1A). We employed ALP staining to further assess bone formation in the POC and bone collar (BC) regions. Effects on the BC give insight into the role of maternal exercise on early bone development, while effects on the POC would reveal the role of maternal exercise on endochondral ossification. There were noticeable increases in ALP intensity in the POC and BC regions as a result of maternal exercise (Fig 1B). These measurements have high variability and likely require larger sample sizes to detect differences (Collins et al., Dev Cell 2023). Immunofluorescent staining with endomucin may provide additional insight into the POC, since alkaline phosphatase intensity may be confounded by enhanced blood vessel infiltration, which would indicate POC maturity. The findings suggest that the mechanical loading provided by maternal exercise, even at supraphysiological levels as seen with C57BL/6J mice, leads to enhanced fetal bone formation. This implies 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. These mouse models, characterized by reduced or absent fetal movements (fetal akinesia), will help assess whether maternal exercise can mitigate skeletal development impairments associated with fetal akinesia, thereby establishing a potential prenatal therapeutic approach for such conditions.

Acknowledgements (Optional):