Poster AA6 - Citrate, A Signaling Molecule, Facilitates Bone Regeneration via Modulation of Energy Metabolism and Signaling Pathway

Introduction: Bone regeneration is a complex process involving osteogenesis, osteoclastogenesis, angiogenesis, and more, which collectively create a unique microenvironment crucial for successful regeneration. Bone-marrow derived mesenchymal stem cells (BMSCs) are central to this process, differentiating into various lineages to facilitate repair and regeneration. Their osteogenic differentiation is regulated by intricate signaling pathways, with the mTORC1 pathway playing a key role in cell proliferation and differentiation. Additionally, energy metabolism shifts from glycolysis to oxidative phosphorylation (OXPHOS) to meet the high energy demands of this process. AMPK, an energy sensor, is perceived as an antagonist to mTORC1 and is activated under nutrient scarcity, but their interaction during osteogenesis is not fully understood. The roles of metabolic regulators like citrate are also unclear. Citrate, a significant component of bone and cellular energy metabolism, enhances energy levels and protein synthesis, thus promoting osteogenesis. While incorporating citrate into biomaterials has been shown to enhance bone formation in previous studies, its precise mechanism remains elusive.
Here, we identify citrate as a signaling molecule that activates both mTORC1 and AMPK pathways, promoting osteogenic differentiation. Inspired by this, we design a citrate-based orthopedic biomaterial enriched with glutamine and magnesium. Glutamine and magnesium optimize energy metabolism and signaling pathways synergistically with citrate. Additionally, magnesium reinforces mechanical strength of the materials. Utilizing a citrate-based polymer platform, we synthesize a biomaterial releasing bioactive molecules (citrate, glutamine, and magnesium) to support osteogenesis. With these properties, we hypothesize that citrate, glutamine, and magnesium can intensify the osteogenic promotive effect for a better bone regeneration.

Materials and

Methods: Various concentrations of citrate, glutamine, and magnesium were supplemented individually and together during osteogenic differentiation of human BMSCs to access the individual and synergistic effect of these molecules on osteogenesis, respectively. ALP activity, bone-related gene expression and calcium nodule formation were evaluated. Similarly, to assess the effects of citrate, glutamine, and magnesium on energy metabolism, intracellular ATP and oxygen consumption rate were measured after 7 days of incubation with citrate, glutamine, and magnesium supplementation during osteogenesis of hMSCs. Western blot was conducted to study the effect of citrate, glutamine, and magnesium on mTORC1 and AMPK signaling pathway during osteogenesis. BPLP-Gln-Mg were synthesized via a one-pot, catalyst free polycondensation reaction using citric acid, 1,8-octanediol, glutamine, and magnesium. Resulting prepolymers were then purified via precipitation in DI water followed by lyophilization and dissolution in organic solvent. The glutamine and magnesium incorporation were characterized by NMR, FTIR, and HPLC. Films and composites were fabricated via solvent casting and physical mixing with hydroxyapatite ceramic (HA), respectively, followed by thermal crosslinking to obtain the final crosslinked thermoset materials. The effect of glutamine and magnesium on mechanics and degradation rate was thoroughly studied. Biocompatibility and cell proliferation was assessed on films and completely degraded products using L929 cells and hMSCs. Osteogenic potential was assessed via ALP detection, and RT-PCR. Finally, bone regeneration in vivo was assessed in a rat critical size cranial defect model using BPLP-Gln-Mg composite scaffolds compared to empty defect, POC, and PLGA controls, with bone regeneration measured via CT and histological staining.

Results, Conclusions, and Discussions: Our study found that even though citrate supplementation during osteogenesis increased intracellular ATP level, AMPK phosphorylation also increased. In addition, citrate supplementation also enhanced mTORC1 activity during osteogenesis. Here, we identify citrate, acting independently of its role in upregulating energy metabolism, as a signaling molecule that concurrently activates mTORC1 and AMPK pathways to promote osteogenic differentiation. When combined with glutamine and magnesium, they further enhance mTORC1 and AMPK activities and intracellular ATP levels during osteogenesis compared to citrate alone. Importantly, we found that the upregulation of AMPK by citrate, glutamine, and magnesium occurs via calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2), another upstream signaling pathway of AMPK besides energy metabolism. This suggests that the effect of citrate, glutamine, and magnesium on CaMKK2 overrides their effect on energy metabolism in regulating AMPK activity. Consistent with in vitro results, BPLP-Gln-Mg/HA supports osteogenesis. CT image showed more bone growth in BPLP-Gln-Mg/HA group compared to control groups. New bone formed in BPLP-Gln-Mg/HA group also showed significantly higher bone mineral density compared to control groups. The presence of collagen component in newly formed bone tissue was further confirmed with Masson’s Trichrome staining.
Previous studies have shown that citrate-based materials promote bone regeneration in animal models, yet the underlying mechanism remains unclear. In this study, we gained further insights into the role of citrate in osteogenesis, from energy metabolism to signaling pathways. Importantly, we identified citrate, glutamine, and magnesium as multifaceted regulators involved not only in energy production but also in signaling pathways during osteogenic differentiation. Additionally, we obtained a better understanding and new insights into the roles of mTORC1 and AMPK signaling pathways during osteogenesis. These findings may significantly impact the design of next-generation biomimetic orthopedic materials.

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