Evaluation of the neuro-modulatory potential of odontogenic derived mesenchymal stem cells - conditioned medium against cuprizone-induced multiple sclerosis in mice

Introduction

Multiple sclerosis (MS) is a chronic inflammatory autoimmune demyelinating disease of the central nervous system and a common cause of disability in young adults (Carotenuto et al., 2022) [11] . In MS, inflammation and demyelination drive astrocytic proliferation, leading to the formation of glial scars. The increase of the intermediate filament protein glial fibrillary acidic protein (GFAP) is a marker of this reactive process (García-Domínguez, 2025) [20] . In MS, the blood-brain barrier is compromised, peripheral immune infiltrate CNS with lesions in the brain and spinal cord. Pro-inflammatory cytokines disturb the blood brain barrier (BBB). Activated macrophages and microglia worsen tissue damage by releasing oxidative burst mechanisms, reactive oxygen species, and proinflammatory cytokines (Woo et al., 2024). Astrogliosis is a stress-induced reaction in the central nervous system that causes the release of GFAP and its breakdown products into the cerebrospinal fluid and circulation. Accordingly, GFAP levels in CSF and blood (Park et al., 2024) [47] . Interleukin-6 plays a pivotal role in neuroinflammation by activating astrocytes, which attract immune cells to injured sites by secreting pro-inflammatory cytokines and chemokines. IL-6 also induces structural changes in astrocytes, amplifying the inflammatory response (Kerkis et al., 2024) [30] International Journal of Advanced Biochemistry Research 2026; 10 (3): 415-424 and activation of microglial lead to produces inflammatory mediators and reactive oxygen species (Zhang et al., 2024). Several studies have reported a substantial reduction in catalase activity in MS patients and experimental MS models, including cuprizone-induced demyelination (Abo Taleb and Alghamdi, 2020; Ghahremani et al., 2025) [1,22] . This reduction in CAT reflects impaired antioxidant defenses and excessive generation of ROS (Omotoso et al., 2018) [46] which oxidatively damage lipids, proteins, and nucleic acids (Shiri et al., 2021) [58] . Dental pulp stem cells(DPSCs) originate from the ectomesenchyme during embryonic development, making them more potent than other MSC sources such as bone marrow (Grawish et al., 2021) [24] . In addition they can also differentiate into neurogenic cell lineages (Salkin et al., 2022) [54] . Conditioned medium (CM) derived from stem cells may offer superior therapeutic advantages over exosome. It is rich in potent immunomodulatory and anti-inflammatory cytokines (Kim et al., 2025), [31] in addition to a wide variety of bioactive compounds, including hormones, chemokines, cytokines, proteins, nucleic acids, and lipids (Ventura et al., 2025) [64] .

Materials and methods

Materials Chemicals

Bis-cyclohexanone-oxaldihydrazone (Cuprizone): White to off white powder,(purity ≥ 95%; CAS no. 370-81-0, Product number C9012), was purchased from(Sigma-Aldrich), administrated via dietary supplementation, by mixing with standard mice chow at concentration of 0.2%(w/w).

Animals & ethical approval

The present study was carried out on 30 male C57BL/6J mice, weighted 40 to 50 gm, housed in cages and were given free access to water and food. All animal care and experimental methods were carried out in accordance with the guidelines of ethical committee of Medical Research, Faculty of Medicine, Tanta University, Egypt(Approval no 36264MS664/8/24).

Experimental design and animal grouping

After being acclimatized for one week, thirty mice were categorized randomly into three equal groups: Group I(control): Normal mice were fed standard mice chow. Group II (MS-model): mice were fed standard mice chow mixed with 0.2% cuprizone (w/w) for 6 weeks. Group III (Cuprizone treated with Odontogenic derived-mesenchymal Stem cells-Conditioned media): Mice were fed standard mice chow mixed with 0.2% cuprizone (w/w), After confirmation of the MS model development by assessment of behavioral, motor test and histopathological examination of the brain tissue, the animals were injected by 500 µl of P2 odontogenic MSCs-CM (200 μg/ml) intravenously in the tail vein once weekly for 3 successive weeks. After one week of the end of the experiment (one week after the last dose of CM), mice were reassessed by behavioral, motor test and the mice were euthanized by injecting 30 mg/kg of pentobarbital intraperitoneally and blood and brain tissues were collected.

Methods

In vitro work: Isolation, culturing and identification of dental pulp-mesenchymal stem cells (BM-MSCs) in culture (Aziz et al., 2008) [5] a) Isolation and culturing of DP-MSCs (enzymatic digestion): Dental pulp was isolated from disinfected healthy permanent teeth, enzymatically digested with collagenase, and centrifuged to obtain cells. The cells were cultured in DMEM with fetal bovine serum under standard conditions, with regular medium changes. Spindle-shaped cells appeared within 5-10 days, and second-passage cells at 80-85% confluence were used for experiments (Aziz et al., 2008; Gatti et al., 2011) [5,21] . b) Characterization of DP-MSCs: Characterization of cultured MSCs was done using morphology: MSCs exhibited a spindle-shaped, fibroblast-like appearance in culture. Plastic adherence, and Phenotypic Fluorescence-Activated Cell Sorting(FACS): using the criteria of CD105 positive and CD45 negative (Tondreau et al., 2004; Dominici et al., 2006; Mageed et al., 2007; Szatanek et al., 2017) [17,38,62,63] .

c) Preparation of Mesenchymal Stem Cells (MSCs)-CM

At 80-85% confluence, cells were washed, trypsinized, and centrifuged to obtain first-passage cultures, which were then re-cultured under standard conditions. After reaching confluence again, cells were washed and incubated in serum-free, low-glucose, low-oxygen DMEM for 48 hours to produce conditioned medium. The collected medium was centrifuged, filtered, aliquoted, stored at -80 °C (Simundic, 2014) [59] , and its total protein content was measured using the Bradford assay (Bradford, 1976) [8] . All CM preparations were obtained from DP-MSCs at the 2nd passage and under identical culture conditions to minimize batch-to-batch variability (Dominici et al., 2006; Pawitan, 2014) [48] .Cell viability was confirmed at the time of CM collection (Strober, 1997) [61] .

In vivo work: Animal groups were assessed three times for body weight, motor clinical scoring, assessment working memory by Delayed-non-matching to sample(DNMS) modified T-maze test, biochemical and histopathological changes; at the start of experiment, after induction of MS model to confirm the demyelination and after treatment period.

Tissue sampling: After the animals were sacrificed, brain tissues were carefully dissected, weighed, rinsed three times with ice-cold saline, blotted on ash-free filter paper, and cooled on ice. Tissues were fixed in 10% neutral buffered formalin for 72 h, processed through graded ethanol (95%), cleared in xylene, and embedded in Paraplast. Sagittal sections(5 µm) were cut using a rotary microtome and stained with Harris Hematoxylin and Eosin(H&E) for general examination and Luxol Fast Blue(LFB) to quantify myelinated fibers in the corpus callosum, following the protocol of Culling, 2013 [14] . Microscopic analysis: At least six random fields from each corpus callosum sample were analyzed for LFB-positive myelinated fibers. Images were captured using a Leica Full HD microscope and quantified with Alpha-Imager and Odyssey scanners (Mustafa et al., 2024) [41] .

Biochemical assessment

 The GFAP level and IL6 levels in blood were evaluated by enzyme-linked immunosorbent assay. A commercially available GFAP and IL-6 assay kit (Cat. Nos. 201-11-0971 and 201-12-0091) respectively; was obtained from Sun Red Biotechnology Company Ltd., China.  Level of catalase enzyme in plasma was determined through colorimetric method by commercial assay kit supplied by bio-diagnostic company, Egypt.

Statistical analysis:

The data were presented as mean± standard deviation (SD), and p-values were calculated. Differences among groups were analyzed using one-way ANOVA followed by Tukey's post hoc test. A p-value less than 0.05 was considered significant. These deficits are commonly assessed using motor clinical scoring, with a widely used scale ranges from 0 to 5 described by Aharoni et al. (2019) [3] . Notably, these neurological deficits were significantly ameliorated in group III after treatment with odontogenic-derived mesenchymal stem cell conditioned medium injection compared to group II (P<0.001) (Fig Statistical analysis is carried out using one-way ANOVA followed by Tukey's test. Statistical analysis is carried out using one-way ANOVA followed by Tukey's test. H&E results (figure 9): Control group demonstrated normal histological architecture of corpus callosum. It was formed of densely backed nerve fibers as well as normally organized oligodendrocytes with small dense nuclei arranged in short chains. There were number of glial cells with deeply stained nuclei (A&B). Cuprizone treated group showed disrupted histological architecture of corpus callosum with loosely arranged degenerated nerve fibers. There was decrease in the number of oligodendrocytes with scattered small dense pyknotic nuclei. In addition, the number of glial cells increased together with interstitial hemorrhage(C&D). Odontogenic derived mesenchymal stem cell -CM treated group showed a significant improvement in the histological architecture of the corpus callosum with densely packed nerve fibers except for localized vacuolation of a few axons. There was increased number of oligodendrocytes with small dense nuclei arranged in short chains together with glial cells with deeply stained nuclei (E&F).

Results

Luxol fast blue results (figure 10)

Control group demonstrated strong positive reaction in closely packed myelinated nerve fibers (A&B). Cuprizone treated group showed weak positive reaction in lightly myelinated nerve fibers(C&D). Odontogenic derived mesenchymal stem cell -CM treated group demonstrated strong positive reaction in closely packed myelinated nerve fibers more or less similar to control group (E&F). (Luxol Fast Blue, scale bar 500 and 50µm) Figure (11) shows statistical analysis of the mean area% of myelinated nerve fibers showed that Cuprizone treated group revealed statistically highly significant decrease in the mean area percentage of myelinated nerve fibers compared with control and CM treated groups CM treated group showed non-significant change in the mean area percentage of myelinated nerve fibers when compared with control group. There was a highly significant increase in the mean area percentage of myelinated nerve fibers in control and odontogenic mesenchymal stem cell -CM treated groups compared to the Cuprizone treated group. [39,40,44,45] .In this study, the neuro-modulatory and immunoregulatory potential of odontogenic mesenchymal stem cell-conditioned medium was investigated using CPZinduced mouse model of MS. Their effects are mediated by secretome which includes cytokines, growth factors, enzymes, lipids and extracellular vesicles such as exosomes (Hade et al., 2021) [28] .Even though exosome-based methods have been extensively researched, CM provides better therapeutic benefits because of its composition of bioactive ingredients that can target several disease pathways(Das and Sloan, 2023; Ghasemi et al., 2023) [16,23] .

Discussion

In the present study, cuprizone administration successfully induced an MS-like demyelinating phenotype characterized by body weight loss, increased motor clinical scores, and neurological impairments, consistent with previous reports (Li et al., 2019; Aharoni et al., 2019) [3,36] . These effects are mainly attributed to cuprizone's copper-chelating action, which disrupts oligodendrocyte function, increases oxidative stress, and induces demyelination (Zirngibl et al., 2022; Yong and Yong, 2022). Neurobehavioral assessment using the modified T-maze showed reduced correct responses and prolonged latency in cuprizone-treated mice, indicating cognitive and motor impairment related to cortico-hippocampal damage and neuroinflammation (Navarro et al., 2023; Omotoso et al., 2018) [43,46] . Treatment with odontogenic mesenchymal stem cell-conditioned medium significantly improved body weight and neurobehavioral performance, consistent with previous studies demonstrating functional recovery following MSC-derived secretome therapy(Zhang et al., 2022; Brown et al., 2021; Farinazzo et al., 2018) [10,19] . On a histopathological level, brain tissue examination of MS mice(group II) using hematoxylin and eosin(H&E) and Luxol Fast Blue(LFB) revealed disorganized morphology in the midbody zones of the corpus callosum, with demyelination of nerve fibers and significant oligodendrocyte loss. These findings are consistent with previous studies (Pyka-Fosciak et al., 2023; Hassani, 2021) [29, 50] , which reported axonal damage, myelin loss, and inflammatory lesions in MS models. The observed demyelination can be explained by the recruitment of activated CD4 T cells and monocyte-derived macrophages, which phagocytize myelin and promote inflammatory lesions (Hassani, 2021) [29] . OMSC-CM-treated mice (group III) showed improved myelin integrity, higher nerve fiber density, and partial restoration of normal corpus callosum architecture, with abundant oligodendrocytes and minimal focal infiltrates. These results align with earlier findings of (Kråkenes et al., 2023; Gugliandolo et al., 2021) [26,34] and are likely due to MSCs' ability to migrate to affected brain regions, reduce inflammation, and enhance myelin content. Additionally, MSC-derived extracellular vesicles secrete neurotropic factors and exert paracrine effects on subventricular zone oligodendrocytes, promoting oligodendrogenesis, remyelination, and oligodendrocyte progenitor differentiation(Cruz-Martinez et al., 2016) [13] . Secretome components also likely downregulate microglial inflammatory activity, contributing to structural recovery in the corpus callosum (Kråkenes et al., 2023) [34] . GFAP is a key marker of astrocyte activation and reactive astrogliosis, central to MS pathology. In CPZ-treated mice(group II), GFAP levels were significantly elevated compared to controls, consistent with previous studies (Azzolini et al., 2022; Saraste et al., 2021; Zahoor et al., 2025) [6,55] . This increase likely results from astrocyte structural and functional changes, BBB disruption, and glymphatic transport of GFAP into circulation (Dalenogare et al., 2023) [15] . OMSC-CM group show significantly reduction in GFAP, indicating decreased astrocytic reactivity and restored homeostasis. These findings align with Allegretta et al., 2022 [4] and Laso-García et al., 2018 [35 ] who reported that MSC-derived extracellular vesicles (EVs) reduce GFAP levels, reduce astrocyte hypertrophy, modulate inflammatory responses and promote a neuroprotective astrocytic phenotype.CM may provide additional benefits over isolated EVs due to soluble factors that exert anti-inflammatory, neurotrophic, and angiogenic effects(Chen et al., 2020) [12] . IL-6, a pleiotropic cytokine, promotes Th17 differentiation, inhibits regulatory T cells, activates astrocytes and microglia, and disrupts BBB integrity (Grebenciucova and VanHaerents, 2023) [25] . In group II, IL-6 was significantly elevated, reflecting a pro-inflammatory state, which is consistent with the findings of (Brown et al., 2021; Haase and Linker, 2021) [10,27] . This elevation contributes to glial scar formation and M1 microglial polarization, enhancing demyelination (Brambilla, 2019) [9] . [33,42,49,56] . CPZ-induced oxidative stress impairs antioxidant defenses by generating ROS that exceed the capacity of SOD and catalase(CAT), damaging myelin, mitochondria, and cytoskeletal proteins (Omotoso et al., 2018; Shiri et al., 2021) [46,58] . In this study, catalase activity was significantly reduced in MS-model mice, consistent with previous reports (Ghahremani et [1,18,22,32,46] . Astrocytes and other CNS and immune cells further contribute to ROS/RNS production, aggravating oxidative damage in demyelinating lesions (Brambilla, 2019) [9] . OMSC-CM treatment may help restore antioxidant balance and protect against ROS-mediated CNS damage. In contrary, research by Shen et al., 2024 [57] revealed that active MS lesions have been shown to destroy the oxidative system, as seen by the increase levels of glutathione, catalase, superoxide dismutase. The observed increase in catalase levels in multiple sclerosis may represent a compensatory antioxidant response to ongoing neuroinflammation and oxidative stress. (Baud et al., 2004; Qi et al., 2007) [7,51] . According to the current study, catalase activity increased after OMSC-CM treatment, indicating that endogenous antioxidant capacity had been restored. MSCs have been proposed to reduce oxidative injury via scavenging free radicals, enhancing host antioxidant defenses and modulating the inflammatory response (Stavely and Nurgali, 2020) [60] . The antioxidative effects of MSCs often occur in a paracrine manner. These findings support the potential of MSC-CM to modulate neuroinflammation effectively. The therapeutic benefits of MSCs in neurological disorders, including multiple sclerosis, are largely mediated through paracrine mechanisms. Under conditions of excessive oxidative stress, as occurs in the cuprizone-induced MS model, hydrogen peroxide H₂O₂ is markedly increased leading to allosteric and oxidative modifications of the catalase enzyme that impair enzyme kinetics and reduce catalytic efficiency, even when total enzyme protein levels are preserved or upregulated as a compensatory response. Moreover, inflammatory mediators such as TNF-α, IL-1β, and nitric oxide derivatives can further compromise catalase function through post-translational modifications causing disruption of enzyme conformation, substrate binding, and turnover rate, resulting in decreased enzymatic activity independent of protein abundance. Therefore, the reduced catalase activity observed in our MS model does not contradict earlier findings but rather complements them by highlighting that oxidative stress in MS impairs antioxidant defense at the functional level, even when antioxidant enzymes are upregulated. This functional insufficiency contributes to the persistence of oxidative damage and disease progression. Restoration of catalase activity following therapeutic intervention would thus indicate not only enhanced antioxidant expression, but also recovery of enzyme functionality, representing a more meaningful indicator of redox homeostasis. This study is among the first to demonstrate that mesenchymal stem cell-conditioned medium derived from teeth can serve as a cell-free therapy in a cuprizone-induced demyelination model. Unlike prior studies relying on direct stem cell transplantation or EAE models, this work combines a secretome-based approach with a non-immune demyelination paradigm. By simultaneously assessing oxidative stress (catalase), neuroinflammation(IL-6), astrocytic activation(GFAP), and histology, the study shows that dental stem cell-derived CM can modulate multiple MS-related pathological pathways, highlighting its translational therapeutic potential

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