Periodontitis is a chronic inflammatory condition that leads to the destruction of the supporting structures of the teeth, including the periodontal ligament and alveolar bone. A key feature of periodontitis is bone resorption, where the inflammatory process triggers the breakdown of bone tissue, resulting in tooth instability and potential loss. To counteract this destructive process, bone regeneration is essential, and one promising approach is through regenerative therapy. This involves the use of various techniques and materials to promote the formation of new bone. Central to bone regeneration is differentiation of MSCs into osteoblasts, the cells responsible for new bone formation. By enhancing osteoblastogenesis, regenerative therapies aim to restore the lost bone and improve periodontal health, offering a potential solution for patients suffering from the detrimental effects of periodontitis.
Recently, researches about AOX1’s function in MSCs came out, suggesting its function in the regulation and induction of MSCs towards osteogenesis and adipogenesis. Firstly, there was bioinformatic analysis suggested its potential in osteogenic differentiation in hMSCs, identified AOX1 as a potential regulator of osteogenic differentiation in bone marrow-derived mesenchymal stem cells (BMSCs) 14. Furthermore, research in hMSCs revealed that AOX1 silencing induced by miR-513a-5p enhances osteogenic proliferation and differentiation15.
Our experimental findings elucidate the involvement of AOX1 in the osteogenic differentiation of hPDLMSCs through the retinoic acid-retinoid X receptor (RA-RXR) pathway. Initially, AOX1 catalyzes the oxidation of retinaldehyde into retinoic acid, a pivotal product with significant implications in MSCs researches. However, the functional role of retinoic acid in MSCs remains a subject of considerable debate, characterized by divergent findings across different studies, particularly concerning its effects on osteogenesis and adipogenesis22,23.
According to our observations, retinoic acid demonstrates a negative impact on osteogenic differentiation in hPDLMSCs. Subsequent experiments aimed at elucidating the specific receptor-mediated signaling pathways involved revealed that, between the two receptors—RAR and RXR—RXR receptor is predominantly associated with the inhibitory effects exerted by retinoic acid. Suggesting a plausible pathway wherein retinoic acid, oxidized by AOX1, binds to RXR receptors to suppress osteogenesis in hPDLMSCs (Fig. 4D). This novel insight underscores the complexity of the molecular mechanisms governing bone formation processes and highlights the need for further elucidation of the regulatory interactions between AOX1 and RXR-mediated pathways. Such endeavors hold promise for advancing our understanding of periodontal tissue regeneration and may pave the way for the development of targeted therapeutic strategies aimed at enhancing bone formation and regeneration in periodontal disease contexts.
In some studies, RXR activation has been shown to direct mesenchymal stem cells toward an adipogenic lineage18,24. However, our experiments yielded different results. The administration of LG100268 did induce some lipid accumulation in hPDLMSCs, but the overall magnitude of this effect was modest. Additionally, the weak staining intensity observed in the control group suggests that the current adipogenic induction medium may not be fully optimized. Further investigation is warranted to elucidate the specific mechanisms underlying lipid metabolism in hPDLMSCs and to understand the potential implications of RXR pathway modulation on this process.
In this experimental setup, we explored the osteogenic outcomes of two osteoinduction media formulations, each designed to figure out the specific components contributing to the observed effects. Alongside the standard formulation comprising ascorbic acid, βGP and dexamethasone25, a simplified formula containing solely ascorbic acid was employed. Initially, this comparative approach aimed to elucidate the individual contributions of each component to the upregulation of AOX1. However, subsequent PCR analysis revealed no significant differences in AOX1 expression levels between the osteo-inductive groups, prompting the continuation of experiments with both standard and simplified formulations.
The rationale behind this decision was to mitigate any potential confounding effects arising from βGP and dexamethasone, particularly given the multifaceted roles of dexamethasone in cellular pathways. As a synthetic glucocorticoid, dexamethasone is known to modulate pathways associated with metabolism and immune function, thereby influencing crucial cellular processes such as proliferation, apoptosis, and tissue remodeling26,27. Hence, the exclusion of its effects from our experimental analyses was deemed imperative.
Encouragingly, our subsequent experiments using both osteoinduction formulas yielded consistent results, as evidenced by the congruent patterns observed in the data (Supplementary Fig. S1 and S2). This internal consistency provided robust justification for excluding the potential influence of βGP and dexamethasone on our experimental outcomes, thus suggesting specific effects of ascorbic acid on AOX1 expression and osteogenic differentiation in our model system.
Ascorbic acid, commonly known as vitamin C, plays a crucial role in osteogenesis25. This water-soluble vitamin is essential for the synthesis of collagen, a primary structural protein found in bones, cartilage, and connective tissues. It acts as a cofactor for the enzyme prolyl hydroxylase, which catalyzes the hydroxylation of proline residues in procollagen, a precursor to collagen28. This hydroxylation reaction is necessary for the proper folding and stabilization of procollagen molecules, allowing them to form triple helical structures characteristic of collagen fibers.
Moreover, ascorbic acid plays a pivotal role in the regulation of osteoblast differentiation and function. Research has demonstrated that ascorbic acid augments the expression of genes critical for osteogenesis, such as alkaline phosphatase and osteocalcin, thereby providing additional support for bone formation processes. Recent investigations have further elucidated its involvement in epigenetically orchestrating osteogenic differentiation and function29. Specifically, ascorbic acid modulates chromatin accessibility and primes transcriptional activity by regulating histone demethylation, notably targeting H3K9me3 and H3K27me3. Additionally, it promotes TET-mediated 5hmC DNA hydroxymethylation at promoters, enhancers, and super-enhancers proximal to bone-specific genes.
Ascorbic acid-induced AOX1 upregulation may also be linked to these multifaceted functions of ascorbic acid in osteogenesis, particularly its involvement in epigenetic regulation of osteogenic genes by modulating chromatin structure and transcriptional activity. This suggests a potential mechanism through which AOX1 upregulation in hPDLMSCs may be linked to the epigenetic control of osteogenic gene expression by ascorbic acid.
Further investigations into these aspects could yield valuable insights into the regulatory mechanisms and functional significance of AOX1 in hPDLMSCs. By elucidating the interplay between AOX1, ascorbic acid-mediated epigenetic regulation, and osteogenic gene expression, we may uncover novel targets for bone tissue regeneration and repair. Ultimately, such insights could inform the development of targeted therapeutic strategies aimed at enhancing bone regeneration and accelerating the healing process in various clinical contexts.