PTEN Inhibition Suppresses Differentiation in Periodontal Ligament Stem Cells

Abstract

Phosphatase and Tensin Homolog (PTEN) functions in numerous biological processes, encompassing cell proliferation, growth, self-renewal, and differentiation. This study examined the modulatory function of the PTEN inhibitor in periodontal ligament stem cells (PDLSCs). PDLSCs were treated with VO-OHpic at a concentration range from 0.625 to 5 μM. MTT assay and Coomassie Blue staining were conducted to determine cell viability and colony-forming unit ability, respectively. The scratch assay was employed to examine cell migration. Mineral deposition and intracellular lipid accumulation were assessed. The qRT-PCR and immunofluorescence were used to evaluate mRNA and protein expression, respectively. RNA sequencing was employed for transcriptomic analysis. VO-OHpic exposure showed no cytotoxic effects in PDLSCs; however, at 5 μM, it markedly decreased colony-forming efficiency and impaired cell migration. Under osteogenic induction conditions, 5 μM VO-OHpic markedly attenuated mineralisation and downregulated the osteogenic marker gene expression partly through ERK signalling. Indeed, VO-OHpic impaired intracellular lipid accumulation during adipogenic differentiation, as evidenced by reduced expression of adipogenic marker genes. RNA sequencing analysis revealed that VO-OHpic treatment upregulated genes in the TGF-β and calcium signalling pathways, suggesting a regulatory role in PDLSC differentiation. In conclusion, PTEN regulates PDLSC colony formation, migration, and differentiation, suggesting a pivotal role for PTEN in maintaining periodontal tissue homeostasis.

1. Introduction

The periodontal ligament (PDL) is a specialised oral connective tissue that serves to secure the cementum to the alveolar bone in both the maxilla and mandible [1]. It serves as a mechanosensory structure that contributes to tooth support by detecting and transducing mechanical stimuli within its microenvironment, including those arising from physiological processes such as mastication and orthodontic tooth movement. This mechanosensory response is regulated through PDL-mediated signalling, which is integral to the preservation of periodontal tissues’ homeostasis [2,3]. In addition to mechanoreceptive function, PDL comprises diverse cellular subpopulations that play a critical role in preserving the structural and functional integrity of the periodontium.

Periodontal ligament stem cells (PDLSCs) represent a mesenchymal stem cell (MSC) population that possesses the differentiation ability into various cell lineages, encompassing osteoblasts, chondrocytes, cementoblasts, adipocytes, and neuronal cells [4,5,6]. PDLSCs exhibit immunomodulatory properties, which hold promise for the development of new allogeneic stem cell-based therapies. Indeed, PDLSCs modulate immune responses by secreting various biological molecules influencing immune cell functions [7,8].

Stem cell regulatory pathways play a pivotal role in controlling cellular behaviour and responses during physiological and pathological conditions. Phosphatase and tensin homolog (PTEN) exhibits crucial roles in numerous biological processes [9,10]. It functions as a homeostatic negative regulator of major canonical signalling pathways, particularly the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (Akt) pathway, which is involved in cell growth, proliferation, survival signalling, and angiogenesis [11,12]. Importantly, PTEN is expressed in the dental papilla, enamel organ, and cervical loops throughout the phases of cell proliferation and differentiation during tooth development [13], highlighting its significant role in crown and root formation.

PTEN also regulates stem cell function across various cell types, for example, embryonic stem cells, induced pluripotent stem cells, neural stem cells, hematopoietic stem cells (HSCs), retinal progenitor cells, and MSCs [14]. Notably, PTEN expression differs between bone marrow-derived stem cells (BMSCs) and dental pulp stem cells (DPSCs), with significantly greater PTEN pathway activation in DPSCs. This increased PTEN expression in DPSCs has been linked to their multi-lineage differentiation potential, particularly in enhancing osteogenic and adipogenic differentiation, as well as reprogramming their native tissue-specific differentiation potential [15]. In addition, the PTEN pathway regulates MSC differentiation potential, particularly during adipogenesis and osteogenesis [16].

PTEN plays an essential role in maintaining PDL homeostasis, as loss of PTEN function has been shown to accelerate bone turnover in the periodontal tissues. This disruption results in the predominant deposition of cementum and a marked increase in bone volume surrounding the molars, as observed in vivo [17,18]. These findings emphasise the involvement of PTEN in bone remodelling and the maintenance of structural integrity. However, the specific role of PTEN in PDLSCs remains unclear. Thus, this study investigated the effects of PTEN on the differentiation potential of PDLSCs in vitro, using the PTEN inhibitor VO-OHpic (hydroxyl(oxo)vanadium 3-hydroxypyridine-2-carboxylic acid). Furthermore, RNA sequencing was performed to elucidate the regulatory mechanisms underlying PTEN inhibition.

2. Results

2.1. Characterisation of PDLSCs

The PDLSCs were characterised as MSCs by assessing the expression of MSC surface marker proteins and their capacity to differentiate toward osteogenic and adipogenic lineages. PDLSCs demonstrated a marked positive expression of MSC markers (CD44, CD73, CD90, and CD105), whereas they exhibited a negative expression of the haematopoietic surface marker (CD45) (Figure 1A–E). Their differentiation potential was demonstrated by significant calcium nodule formation in osteogenic induction medium (OM) (Figure 1F,G), and clear accumulation of lipid droplets when cultured in adipogenic induction medium (ADM) (Figure 1H,I). These findings confirm that the isolated PDLSCs represent a population of MSCs.

2.2. Effects of VO-OHpic on Cell Viability, Proliferation, and Migration of PDLSCs

The cells were treated with various concentrations of VO-OHpic (0.625, 1.25, 2.5, and 5 μM), as described in a previous study [19]. Cell viability and proliferation were assessed using an MTT assay, cell cycle analysis, and apoptosis analysis. The MTT assay showed no significant differences in the proliferation of PDLSCs in response to VO-OHpic in a dose-dependent manner over days 1, 3, and 7 (Figure 2A). The percentage of apoptotic cells was evaluated via flow cytometry, revealing no significant differences in early or late apoptotic populations following VO-OHpic treatment compared to the control group (Supplementary Data S2). Similarly, cell cycle analysis conducted at 72 h showed no discernible changes in any phase of the cell cycle among PDLSCs treated with varying concentrations of VO-OHpic (Supplementary Data S3). However, the colony-forming ability of PDLSCs decreased in a dose-dependent manner, with 5 μM VO-OHpic treatment resulting in a significant reduction in colony numbers compared to the control group (Figure 2B,C). The quantitative analysis also confirmed that the eluted dye from the 5 μM VO-OHpic treatment group was significantly lower than that of the control (Figure 2D).

Further, PDLSCs treated with 5 μM VO-OHpic demonstrated a marked decrease in wound closure percentage at both 24 and 48 h (Figure 3A,B). These findings suggest that, while VO-OHpic does not exert cytotoxic effects on PDLSCs, it significantly suppresses their colony-forming capacity and migration potential.

2.3. VO-OHpic Suppresses Osteogenic Differentiation Potential of PDLSCs

To investigate the effects of VO-OHpic on osteogenic differentiation, PDLSCs were cultured in OM with various concentrations of VO-OHpic for 14 days. All tested concentrations effectively reduced mineral deposition by PDLSCs, as evidenced by ARS staining (Figure 4A). Quantitative analysis revealed that 1.25 and 5 μM VO-OHpic significantly reduced mineral deposition (Figure 4B). These findings suggest that higher concentrations of VO-OHpic tend to inhibit the osteogenic differentiation capacity of PDLSCs; 5 μM VO-OHpic was therefore selected for subsequent experiments.

The expression of osteogenic marker genes was assessed on days 3 and 7. On day 3, treatment with 5 μM VO-OHpic significantly downregulated the mRNA expression of osterix (OSX), osteocalcin (OCN), bone morphogenetic protein 2 (BMP2), and osteopontin (OPN) (Figure 4C). By day 7, a significant reduction was also observed in the mRNA expression of type I collagen (COL1A1), alkaline phosphatase (ALP), OSX, OCN, and OPN (Figure 4D). Notably, the protein expression of type I collagen in PDLSCs was markedly decreased. In contrast, the expression of OPN showed only minimal changes after 7 days of culture with 5 μM VO-OHpic as determined by immunofluorescence staining (Figure 4E–H).

2.4. VO-OHpic Regulates Osteogenic Differentiation Through the ERK Signalling

To investigate the regulatory signalling pathways underlying the suppressive effect of VO-OHpic on osteogenic differentiation in PDLSCs, specific pathway inhibitors were employed. These included inhibitors targeting the extracellular signal-regulated kinase (ERK), transforming growth factor-beta/phosphoinositide 3-kinase (TGF-β/PI3K), p38 mitogen-activated protein kinase (p38 MAPK), mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (JNK) signalling pathways.

The results indicated that specific signalling pathway inhibitors—including the TGF-β/PI3K inhibitor (SB431542), p38 MAPK inhibitor (SB203580), MEK1/2 inhibitor (U0126), and JNK inhibitor (SP600125)—were unable to reverse the VO-OHpic-suppressed mineralisation in PDLSCs after 14 days under OM conditions (Figure 5A–D). In contrast, inhibition of the ERK signalling using an ERK inhibitor (ERKi, CAS 1049738-54-6) led to an evident enhancement of mineral deposit under VO-OHpic-treated conditions (Figure 5E).

To further investigate the role of ERK signalling as a potential regulatory mechanism, PDLSCs were co-treated with VO-OHpic and ERKi in OM conditions for 7 days. The mRNA expression levels of several osteogenic differentiation markers, including BMP2, COL1A1, DMP1, OCN, OPN, runt-related transcription factor 2 (RUNX2), and OSX, showed an increasing trend compared to VO-OHpic treatment alone (Figure 5F–N). Collectively, these findings suggest that VO-OHpic regulates osteogenic differentiation in PDLSCs, at least in part, through modulation of the ERK signalling pathway.

2.5. VO-OHpic Suppresses Adipogenic Differentiation in PDLSCs

PDLSCs were cultured in ADM supplemented with VO-OHpic at concentrations of 0.625, 1.25, 2.5, and 5 μM for 16 days to investigate its impact on adipogenic differentiation. A marked reduction in intracellular lipid droplets was observed in the 5 μM VO-OHpic treatment group (Figure 6A). The mRNA expression of key adipogenic differentiation genes—peroxisome proliferator-activated receptor gamma (PPARγ), lipoprotein lipase (LPL), and CCAAT enhancer-binding protein alpha (C/EBP-α)—was also analysed. On day 8, treatment with 5 μM VO-OHpic significantly downregulated the mRNA expression of all three adipogenic markers (Figure 6B–D). By day 16, both PPARγ and LPL were significantly downregulated in the 1.25 and 5 μM VO-OHpic groups compared to the control (Figure 6E–G). These results indicate that VO-OHpic modulates the adipogenic differentiation potential of PDLSCs, with the 5 μM concentration markedly attenuating their capacity for adipogenic commitment.

The regulatory signalling pathways involved in adipogenic differentiation were also examined to assess the potential role of VO-OHpic in modulating adipogenic differentiation in PDLSCs. PDLSCs were cultured in ADM with VO-OHpic and each pathway-specific inhibitor for 16 days. However, analysis of lipid droplet accumulation revealed no significant differences among the treatment groups (Figure 7A–E). To confirm these findings, the expression profile of adipogenic markers was further evaluated. The results revealed that inhibition of the ERK pathway did not reverse the effect of VO-OHpic on the expression of PPARγ, LPL, and C/EBP-α (Figure 7F–H). The data suggest that VO-OHpic does not modulate adipogenic differentiation in PDLSCs through these signalling pathways.

2.6. PTEN Inhibition Modulates Osteogenic and Adipogenic Signalling Pathways in PDLSCs

Transcriptomic profiling was conducted on PDLSCs treated with 5 μM VO-OHpic for 24 h, together with untreated controls. Differentially expressed genes were identified based on statistical significance. The top 50 differentially expressed genes were illustrated (Figure 8). Further, the top 20 upregulated genes, including AVIL and NEUROD2, and the top 20 downregulated genes, such as HAS3 and RAMP1, were listed in Supplementary Data S4.

Enrichment pathway analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database revealed that genes were dramatically upregulated in the TGFβ and calcium signalling pathways (Figure 9A). In contrast, significantly downregulated genes were associated with the peroxisome proliferator-activated receptor (PPAR) and tumour necrosis factor (TNF) signalling pathways, both of which are related to lipid metabolism (Figure 9B). A comprehensive list of gene expression data is provided in Supplementary Data S5.

3. Discussion

PTEN is a vital regulator of several fundamental biological functions, including self-renewal, cell growth, proliferation, differentiation, survival, and metabolism [20]. Functionally, it acts as a dual-specificity phosphatase, capable of dephosphorylating both phospholipids and proteins [11]. In the context of MSC regulation, PTEN expression exhibits variability among MSC subpopulations. Notably, DPSCs display significantly elevated PTEN expression and pathway activation compared to BMSCs. This heightened PTEN activity in DPSCs has been implicated in promoting multi-lineage commitment, enhancing both adipogenic and osteogenic differentiation capacities, and reprogramming their intrinsic tissue-specific differentiation potential [15]. However, the specific functional role of PTEN in PDLSCs remains to be fully elucidated.

The present study employed VO-OHpic to elucidate the role of PTEN in PDLSCs. VO-OHpic is a water-soluble, vanadium-based compound that functions as a reversible and competitive inhibitor of PTEN. Notably, it suppresses the enzymatic activity of recombinant cysteine-based phosphatases (CBPs), a group of enzymes that includes PTEN. Previous studies demonstrated that VO-OHpic possesses a strong inhibitory potential against PTEN activity. In vitro analyses have further revealed that VO-OHpic exhibits greater efficacy in inhibiting PTEN compared to other inhibitors [21,22]. Among the various vanadium compounds evaluated, VO-OHpic demonstrated particularly high specificity and selectivity for PTEN inhibition in vitro [23,24]. Our unpublished data suggest altered expression of genes linked to PTEN inhibition. Treatment with VO-OHpic resulted in downregulation of p53 and upregulation of NANOG. Because p53 is a recognised downstream target of PTEN [25] and NANOG has been reported to be upregulated under PTEN-deficient conditions [26], these observations support effective PTEN inhibition by VO-OHpic. Nonetheless, potential off-target effects may confound the interpretation of the present findings. VO-OHpic can inhibit additional phosphatases and, although it appears more specific for PTEN than other inhibitors such as bpVpic [27], off-target activity cannot be fully excluded; therefore, the results should be interpreted with appropriate caution.

The present study demonstrated that VO-OHpic did not affect cell viability, proliferation, or apoptosis in PDLSCs. However, cell migration and colony formation were markedly attenuated. The MTT assay is a method used to determine cell proliferation. The MTT assay reflects mitochondrial activity, which primarily determines cell viability. However, it can be directly employed to determine cell number by assuming that the increase in cell number is proportional to the increase in overall metabolic activity, which converts tetrazolium to formazan. However, it should be noted that the increase in metabolic activity may be caused by other biological events other than cell proliferation. In the present study, we also employed propidium iodide staining and flow cytometry analysis to investigate cell cycle progression. Cell cycle analysis confirmed that VO-OHpic treatment did not alter cell proliferation. Hence, the effects of VO-OHpic on cell migration and colony formation were not directly from the influence on cell viability, proliferation, and apoptosis.

Overexpression of PTEN has been observed in cartilage endplate degeneration and calcification, conditions associated with intervertebral disc degeneration. PTEN is also implicated in the imbalance of oxidative stress-induced chondrocyte apoptosis. Findings indicate that VO-OHpic can attenuate redox imbalance and mitochondrial dysfunction, thereby enhancing cell survival via the Nrf2/HO-1 pathway [28]. Furthermore, DPSCs from patients with Cowden syndrome, a hereditary cancer predisposition disorder strongly associated with germline mutations in the PTEN (phosphatase and tensin homolog) gene, exhibited increased osteogenic differentiation [29]. In contrast, PTEN inhibition has been shown to reduce cell proliferation as well as both osteogenic and adipogenic differentiation in DPSCs. VO-OHpic treatment reduces the mRNA levels of RUNX2, ALP, COL1A1, and OCN, as well as mineral deposition. Additionally, VO-OHpic downregulates the expression of adipogenic marker genes such as LPL and PPARγ, along with the formation of lipid droplets in DPSCs [19]. Elucidating the role of PTEN in these processes may offer valuable insights for regenerative dentistry and could have implications for the management of patients with Cowden syndrome.

In this study, we demonstrated that VO-OHpic treatment reduced mineral deposition in PDLSCs and downregulated the expression of osteogenic differentiation markers. Specifically, on day 3, the expression of OSX, OCN, BMP2, and OPN was decreased, while on day 7, COL1A1, ALP, OSX, OCN, and OPN were significantly downregulated. Additionally, VO-OHpic suppressed the expression of adipogenic markers, including LPL, PPARγ, and C/EBP-α. These findings emphasise the critical role of PTEN in regulating the multipotent differentiation potential of PDLSCs. The regulatory signalling pathway through which VO-OHpic affects osteogenic differentiation in PDLSCs is the ERK signalling pathway. The ERK-specific inhibitor was able to reverse the VO-OHpic-induced suppression of osteogenic gene expression, including BMP2, COL1A1, DMP1, OCN, OPN, RUNX2, and OSX. In contrast, the potential involvement of ERK signalling in PTEN-mediated adipogenic differentiation of PDLSCs remains unclear, and further studies are required to identify the relevant signalling cascades.

Extracellular signal-regulated kinase (ERK) signalling plays a central role in regulating numerous cellular functions [30]. PTEN modulates ERK activity by dephosphorylating and attenuating PIP3-mediated activation of protein kinase C (PKC) and AKT [31]. A previous study indicates that ERK signalling contributes to osteogenic differentiation through modulating the expression of RUNX2, COL1A1, ALP, and OCN, in human adipose-derived stem cells (hASCs) under in vitro conditions [32]. Activation of the ERK pathway has similarly been observed during osteogenic differentiation in BMSCs and preadipocyte cell lines [33,34]. Conversely, ERK signalling has been shown to suppress adipogenic differentiation through the downregulation of PPARγ expression [35]. These findings highlight the critical role of ERK signalling in maintaining the dynamic balance between osteogenesis and adipogenesis in hASCs. Thus, ERK activation is particularly important for enhancing osteogenic differentiation, especially when employing –NH₂-modified substrate materials to facilitate BMSC differentiation for bone tissue engineering [34]. Nevertheless, the downstream molecular targets of ERK in this regulatory context remain to be fully elucidated.

The present study employed RNA sequencing to investigate the differentiation mechanisms of PDLSCs following VO-OHpic treatment. The analysis revealed upregulation of the TGF-β and calcium signalling pathways, both of which are critically involved in regulating odontogenic and osteogenic differentiation in MSCs [36]. Notably, the role of the TGF-β signalling pathway in osteogenesis remains controversial, as its effects appear to be context-dependent, with evidence supporting both promotive and inhibitory roles [37]. PTEN has been identified as a key modulator of the TGF-β/Smad2 signalling axis, which governs the osteogenic differentiation of BMSCs. Under mechanical tension, PTEN downregulation results in the upregulation of TGF-β and Smad2 protein expression, thereby facilitating osteogenic differentiation in BMSCs. Collectively, previous studies and recent findings indicate that modulation of PTEN signalling represents a promising therapeutic strategy for promoting bone regeneration [38]. Additionally, the expression of PPAR and TNF signalling pathways was downregulated following VO-OHpic treatment. The PPAR signalling pathway plays an important role in modulating mesenchymal lineage commitment and has been shown to enhance the de novo differentiation of preadipocytes [39,40]. PTEN is a key regulator of multilineage differentiation potential, including the maintenance of adipose homeostasis [41]. Previous studies have demonstrated that PTEN knockout reduces both the protein and gene expression of RUNX2 and PPARγ in BMSCs, suggesting that the loss of PTEN affects both osteogenic and adipogenic differentiation [42]. These results highlight the pivotal role of PTEN in regulating lineage commitment and underscore its potential as a therapeutic target for promoting bone regeneration in periodontitis and implant dentistry.

A limitation of the present study is that RNA sequencing was performed at only a single time point (24 h) for VO-OHpic treatment, which may restrict comprehensive mechanistic interpretation. The 24 h time point was intentionally selected to capture early molecular responses to acute small-molecule PTEN inhibition, prior to the onset of long-term differentiation or secondary transcriptional effects [43]. The underlying PTEN-mediated regulation in PDLSCs provides mechanistic insights beyond established PI3K/Akt and MAPK signalling pathways. Importantly, PTEN inhibition at 24 h is unlikely to induce a stable quiescent state. Instead, short-term inhibition primarily alters the phosphorylation status of downstream signalling molecules, such as AKT and mTOR, which regulate cell survival, proliferation, and metabolic activity, while suppressing quiescence-associated regulators, such as FOXO transcription factors, and regulating differentiation, such as Glycogen synthase kinase-3 (GSK3) [44,45,46]. Therefore, the observed transcriptional changes likely reflect early priming or modulation of stemness-related pathways, rather than a shift toward cellular quiescence. In contrast, differentiation outcomes were assessed at later time points to evaluate functional consequences of these early molecular [47]. Thus, the temporal separation between RNA sequencing and differentiation assays reflects a progression from early signalling responses to downstream phenotypic effects.

The present study demonstrated that pharmacologic inhibition of PTEN using VO-OHpic suppresses both osteogenic and adipogenic differentiation in PDLSCs, suggesting that targeting of this molecule is unlikely to be a practical strategy for promoting periodontal tissue regeneration. Notably, RNA-sequencing revealed that downregulated transcripts were significantly enriched in inflammation-related pathways, including TNF and NF-κB signalling. These findings raise the possibility that transient PTEN inhibition could be leveraged to modulate periodontal inflammation and infection. Accordingly, the controlled, phase-specific delivery of VO-OHpic warrants investigation to promote local drug accumulation during the inflammatory phase, followed by clearance during subsequent proliferation and differentiation phases, thereby avoiding impairment of regenerative processes and supporting periodontal healing. This proposed therapeutic paradigm remains hypothetical and requires rigorous experimental validation.

4. Materials and Methods

4.1. Isolation and Culture of Periodontal Ligament Stem Cells (PDLSCs)

Human periodontal ligament tissues were harvested from the third molars of healthy individuals scheduled for surgical extraction as part of their treatment plans at the Oral and Maxillofacial Surgery Clinic, Faculty of Dentistry, Chulalongkorn University. Written informed consent was obtained. All human experimental protocols in this study were approved by the Human Research Ethics Committee, Faculty of Dentistry, Chulalongkorn University (approval No. HREC-DCU 2025-006). Cell isolation was conducted according to a previously published protocol [48]. In brief, the periodontal ligaments were scraped from the middle 1/3 of the root surfaces and minced into small pieces. Human periodontal ligament stem cells (PDLSCs) were cultured in complete growth medium containing Dulbecco’s Modified Eagle’s Medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% foetal bovine serum (FBS; Gibco, Grand Island, NY, USA), 2 mM L-glutamine (Gibco, Grand Island, NY, USA), 100 units/mL penicillin, 100 µg/mL streptomycin, and 250 ng/mL amphotericin B (Gibco, Grand Island, NY, USA). PDLSCs were incubated in a humidified atmosphere containing 5% CO₂ at 37 °C, and the culture medium was changed every 2–3 days. Cells were sub-cultured and evaluated for specific MSC markers and their capacity for multi-lineage differentiation before use. All experiments utilised PDLSCs in passages 3–5. For PTEN inhibition, PDLSCs were treated with hydroxyl(oxo)vanadium 3-hydroxypyridine-2-carboxylic acid (Sigma-Aldrich, St. Louis, MO, USA).

4.2. Flow Cytometry

MSC surface marker expression was investigated by flow cytometry [48]. Fluoresceine-conjugated primary antibodies, including FITC-conjugated anti-CD44 (BD Biosciences Pharmingen, San Diego, CA, USA), FITC-conjugated anti-CD73 (BD Biosciences Pharmingen, San Diego, CA, USA), PE-conjugated anti-CD105 (BD Biosciences Pharmingen, San Diego, CA, USA), and APC-conjugated anti-CD90 (ImmunoTools, Friesoythe, Germany) antibodies, were used to label the single cells. An HSC’s surface marker, PerCP-conjugated anti-CD45 antibody (ImmunoTools, Friesoythe, Germany), was also included. All antibodies were diluted 1:25. In brief, PDLSCs cultures were trypsinised (0.25% trypsin/EDTA; Gibco, Grand Island, NY, USA), rinsed with 1X phosphate-buffered saline (PBS), and centrifuged at 2000 rpm at 4 °C. The cell pellet was incubated with the primary antibody for 15 min at room temperature in the dark. Mouse Ig isotype was used as the control (1:25 dilution). The stained cells were subsequently subjected to analysis utilising a FACSCalibur Flow Cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

4.3. Differentiation Assays

The protocol for cell differentiation was conducted according to previously published work [48]. For osteogenic differentiation, PDLSCs (5 × 10⁴ cells/well) were seeded in 24-well plates (Corning, Corning, NY, USA) and cultured in osteogenic induction medium (OM) which consisted of complete growth medium supplemented with 50 µg/mL L-ascorbic acid ((Sigma-Aldrich, St. Louis, MO, USA), 5 mM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO, USA), and 10 µM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA) for 14 days. For adipogenic differentiation, PDLSCs were seeded at density of 2.5 × 10⁴ cells/well in 24-well plates and cultured in an adipogenic induction medium (ADM) which consisted of complete growth medium supplemented with 0.1 mg/mL insulin ((Sigma-Aldrich, St. Louis, MO, USA), 1 µM dexamethasone (Sigma-Aldrich, St. Louis, MO, USA), 1 mM IBMX (Thermo Fisher Scientific, Waltham, MA, USA), and 0.2 mM indomethacin (Sigma-Aldrich, St. Louis, MO, USA). The cultures were maintained in a humidified atmosphere with 5% CO₂ incubator for 16 days. For PTEN inhibition, VO-OHpic at final concentration 0.625, 1.25, 2.5, and 5 μM were added to OM or ADM. PDLSCs cultures were maintained for 7 and 14 days for osteogenic differentiation or 8 and 16 days for adipogenic differentiation conditions. To investigate the involvement of specific regulatory signalling pathways, the following inhibitors were added together with VO-OHpic, including 4 μM SB431542 (TGF-β type I receptor inhibitor; Sigma-Aldrich, St. Louis, MO, USA), 5 μM U0126 (mitogen-activated protein kinase (MAPK) inhibitor; Tocris Bioscience, Minneapolis, MN, USA), 1.5 nM (ERK inhibitor; CAS 1049738-54-6; Sigma-Aldrich, St. Louis, MO, USA), 10 μM SB203580 (p38 inhibitor; Tocris Bioscience, Minneapolis, MN, USA), and 40 nM SP600125 (JNK inhibitor; Sigma-Aldrich, St. Louis, MO, USA).

4.4. Cell Proliferation Assay

To investigate the proliferation of PDLSCs, the cells were seeded at 5 × 10⁵ cells/well in 24-well plates and cultured in complete growth medium overnight. Next, PDLSCs cultures were treated with growth medium overnight containing various concentrations (0.625, 1.25, 2.5, and 5 μM) of VO-OHpic solution. PDLSC cultures were collected at days 1, 3, and 7 post-treatment for evaluation using the MTT assay. Cells were incubated with 0.5 mg/mL of MTT in a serum-free medium for 15 min at 37 °C. The formazan crystals were dissolved using an eluting buffer composed of dimethyl sulfoxide (DMSO) and glycine buffer. The experiments were measured using a microplate reader (Molecular Devices, San Jose, CA, USA) at a wavelength of 540 nm.

4.5. Colony-Forming Unit (CFU) Assay

PDLSCs were seeded in 6-well plates (Corning, Corning, NY, USA) at a density of 500 cells/well and cultured with VO-OHpic solution at concentrations of 0.625, 1.25, 2.5, and 5 μM for 14 days. The culture medium was renewed every 2 days. The samples were then subjected to fixation with 4% formaldehyde (Sigma-Aldrich, St. Louis, MO, USA) for 5 min, washed with deionised (DI) water, and stained with Coomassie Blue (Sigma-Aldrich, St. Louis, MO, USA) for 30 min with gentle shaking, and rinsed with DI water to remove unbound stain and debris. The stained CFUs were visualised by an inverted microscope (Olympus, Center Valley, PA, USA). The eluted dye from the colony formation experiment was measured at an absorbance of 570 nm.

4.6. Migration Assay

Cell migration was evaluated using a scratch wound assay at 24 and 48 h post-treatment. PDLSCs were seeded into 6-well plates at a density of 3 × 10⁵ cells/well and incubated at 37 °C in a humidified atmosphere with 5% CO₂ culture until reaching 100% confluency. The vertical scratch wound was made by using a 200 μL micropipette tip (Corning, Corning, NY, USA), attached cells and cell debris were washed with PBS, and cultured in serum-free culture medium containing 0.625, 1.25, 2.5, and 5 μM VO-OHpic. The wound area was examined and captured using an inverted microscope (Olympus, Center Valley, PA, USA) at 0, 24, and 48 h. The remaining wound area was measured and analysed using ImageJ software (version 1.53d, National Institutes of Health, Bethesda, MD, USA) [49]. The percentage of wound closure was calculated using this formula.

The percentage of wound closure = (T₀ − T_h)/T₀ × 100

T₀ = Initial wound area (0 h)

T_h = Remaining wound area at 24 or 48 h

4.7. Alizarin Red S Staining

The cells were subjected to fixation utilising 4% formaldehyde in PBS for a duration of 10 min, followed by washing with DI water twice. Subsequently, the cells were incubated with Alizarin Red S (ARS) solution (pH 4.1) (Sigma-Aldrich, St. Louis, MO, USA) for 5 min at room temperature. The samples were washed with DI water to remove the excess dyes. Stained mineral nodules were visualised and examined using an inverted microscope (Olympus, Center Valley, PA, USA). For quantitative analysis, the mineral deposits were solubilised using 10% cetylpyridinium chloride monohydrate (Sigma-Aldrich, St. Louis, MO, USA) in a 10 mM sodium phosphate solution for 30 min. The eluted solution from each well was measured using an ELx800 microplate reader (BioTek Instruments, Winooski, VT, USA) at 570 nm.

4.8. Oil Red O Staining

The cells were subjected to fixation utilising 4% formaldehyde in PBS for 15 min, rinsed with DI water, and stained with 0.2% Oil Red O solution Sigma-Aldrich, St. Louis, MO, USA) for 15 min at room temperature. Intracellular lipid droplet accumulation was observed using an inverted microscope (Olympus, Center Valley, PA, USA).

4.9. Immunofluorescence

PDLSCs were seeded at a density of 5 × 10⁴ cells/well in 24-well plates. After reaching 90% confluency, the cells were maintained in OM containing 5 μM VO-OHpic for 7 days. To evaluate the expression of the osteogenic markers, osteopontin (OPN) and type-I collagen, the cells were fixed in 4% formaldehyde in PBS and permeabilised with 0.1% TritonX-100. Non-specific blocking was performed by incubating the cells with 10% foetal bovine serum at 4 °C for 30 min. The cells were then stained with anti-OPN (dilution 1:100, Santa Cruz Biotechnology, Dallas, TX, USA) and anti-type I collagen (dilution 1:100, Abcam, Cambridge, UK)antibodies for 24 h at 4 °C. Secondary antibody, Alexa Flour 488 goat anti-rabbit IgG antibody (1:1000 dilution, Invitrogen, Carlsbad, CA, USA) or Strep-Rhodamine anti-rabbit antibody (1:500 dilution, Invitrogen, Carlsbad, CA, USA) was applied and incubated for 2 h at room temperature. Nuclear counterstaining was performed using DAPI at a dilution of 1:1000 (Invitrogen, Carlsbad, CA, USA) for 20 min at room temperature. Protein expression was visualised and examined using the fluorescence microscope with an ApoTome system (Carl Zeiss, Oberkochen, Germany).

4.10. Real-Time Polymerase Chain Reaction (qRT-PCR)

TRIzol reagent (RiboEx™ solution, GeneAll Biotechnology, Seoul, Republic of Korea). was employed for total RNA extraction procedures according to the manufacturer’s protocol [48,50]. Total RNA (1 µg) was converted to complementary DNA (cDNA) using the ImProm-II Reverse Transcription system (Promega, Madison, WI, USA). A real-time reverse-transcription polymerase chain reaction (qRT-PCR) was performed using a FastStart SYBR Green Master Essential DNA (Roche Diagnostics, Indianapolis, IN, USA) with CFX connect Real-Time PCR (Bio-Rad, Singapore). Melt curve analysis was performed to confirm reaction specificity. The mRNA expression levels of the target genes were normalised to the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) reference gene, and the relative gene expression was quantified by the comparative Ct method (2^−ΔΔCT method). The primer oligonucleotide sequences of the interesting genes are shown in Supplementary Data S1.

4.11. RNA Sequencing Analysis

Cells were treated with 5 μM VO-OHpic in complete growth medium conditions for 24 h. VO-OHpic-untreated cells were used as the control group. Total RNA was extracted using the RNeasy Plus Mini Kit with DNase I treatment (QIAGEN, Germantown, MD, USA). Nanodrop analysis exhibited RNA quality with an OD260/280 ratio of 2.0–2.2 and an RNA concentration exceeding 50 ng/μL. RNA sequencing was conducted by Vishuo Biomedical Pte Ltd., Science Park II, Singapore, using the NextSeq 500 platform (Illumina, San Diego, CA, USA). Data quality was assessed and filtered with FastQC (v0.10.1). Pathway of differentially expressed genes was conducted by WebGestalt (WEB-based Gene Set Analysis Toolkit) using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, used to investigate a pathway of the molecular interaction, reaction, and relation networks [51]. A heatmap of the top 50 genes was performed using the Heatmapper software (version 1.4, University of Alberta, Edmonton, AB, Canada) [52]. The raw sequencing data are available at the NCBI gene expression repository Gene Expression Omnibus GEO295018.

4.12. Cell Cycle Assay

PDLSCs were seeded at 3 × 10⁵ cells/well in 6-well plates for 24 h. The cells were incubated with 0.625, 1.25, 2.5, and 5 μM VO-OHpic for 24 and 48 h. The cells were trypsinised using 0.25% trypsin/EDTA and fixed with cold ethanol for 30 min. RNase A (1 mg/mL) was added to eliminate RNA content, and the mixture was incubated for 30 min with 40 μg/mL propidium iodide (PI) solution (Sigma-Aldrich, St. Louis, MO, USA). The stained samples were assessed using a FACS^Calibur flow cytometer (BD Biosciences Pharmingen, San Diego, CA, USA).

4.13. Apoptosis Assay

PDLSCs were seeded at 3 × 10⁵ cells/well in 6-well plates and cultured overnight. The next day, cultures were treated with 0.625, 1.25, 2.5, and 5 μM VO-OHpic for 24 and 48 h. The cells were harvested by trypsinisation and collected by centrifugation at 2500 rpm for 5 min, and washed with PBS. The pellets were incubated with Annexin V-FITC (BioLegend, San Diego, CA, USA) and PI for 15 min in the dark, then resuspended in Annexin-binding buffer following the manufacturer’s instructions. The stained cells were observed using a FACS^Calibur flow cytometer (BD Biosciences Pharmingen, San Diego, CA, USA).

4.14. Statistical Analysis

All experiments were conducted using primary cells from four different donors. All data are reported as the average and standard deviation. Statistical evaluation was conducted utilising GraphPad Prism version 9.0 (GraphPad Software, San Diego, CA, USA). Kruskal–Wallis tests, followed by Dunn’s post hoc test, were used for multiple comparisons. p-values of less than 0.05 were considered statistically significant.

5. Conclusions

VO-OHpic inhibits both the migration and differentiation of PDLSCs towards osteogenic and adipogenic lineages. VO-OHpic treatment markedly suppressed osteogenic and adipogenic marker gene expression, with a correspondingly pronounced reduction in quantified in vitro mineral deposition and a clear, measurable decrease in intracellular lipid accumulation relative to the untreated control. RNA sequencing analysis revealed upregulation of the TGFβ and calcium signalling pathways, alongside downregulation of the PPAR and TNF signalling pathways. Taken together, the evidence suggests that PTEN regulates PDLSC colony formation, migration, and differentiation toward osteogenic and adipogenic lineages. Clinically, these results support PTEN-directed strategies as a rational approach to maintain periodontal tissue homeostasis and reduce alveolar bone loss in periodontal disease.