Description:Field of the Invention:
Present invention pertains to a formulation of an oral gel and the process of making thereof to reverse the course of areca nut-induced oral submucous fibrosis. More particularly the formulation is enriched with lyophilised dental pulp-derived mesenchymal stem cells secretome (DPMSCs-S) which helps to increase the mouth opening while improving fibrosis-related characteristics such as epithelial atrophy, tissue degeneration, and collagen buildup.
Background of the Invention:
Oral submucous fibrosis (OSMF) is a precancerous condition of the submucosa that produces inflammation and progressive fibrosis of the submucosal tissues, resulting in severe stiffness and eventually, the inability to open the mouth. OSMF is regarded as a potentially malignant condition with a high chance of progressing to oral cancer. According to reports, the malignant transformation rate is roughly 7.6%. Chewing areca nut is one of the main cause of OSMF. OSMF has been observed to affect people aged 11 to 60 years, with a prevalence of 0.2% to 2.3% in males and 1.2% to 4.6% in females in India, where areca nut chewing is common. Areca nut is classified as a class I carcinogen that causes cytotoxicity and genotoxicity by disrupting cell cycle control, mitochondrial membrane potential, glutathione levels, and H2O2 synthesis. It promotes oral fibrosis by autophagy, EMT transition, and pro-inflammatory mechanisms. Additionally, cellular senescence plays a key role in the pathophysiology of OSMF where prolonged cellular senescence promotes fibrosis and eventually malignant transformation. Early identification and treatment are critical in controlling OSMF and preventing it from progressing to oral cancer.
Although, OSMF is quite common and has major repercussions, the effectiveness of existing treatment approaches is limited. Current pharmacological treatment includes anti-inflammatory medications, antioxidants and vitamins and immunomodulators such as, pentoxifylline improves blood flow to affected tissues and reduce fibrosis. Enzyme therapy of collagenase and chymotrypsin is also a known option, where collagenase helps reduce the density of fibrotic tissue, improving flexibility and mouth opening. Apart from this, regular mouth-opening exercises, as well as low-energy ultrasound therapy, can help to reduce inflammation and fibrosis. Surgical alternatives for advanced cases with severe fibrosis and functional impairment include fibrotomy, coronoidectomy, flap reconstruction, and laser surgery.
In recent years, mesenchymal stem cells (MSCs), particularly those derived from dental pulp (DPMSCs), have emerged as a promising tool in regenerative medicine due to their self-renewal capacity, multi-lineage differentiation potential, antigenicity and immunomodulatory potential. Stem cells secrete a wide range of growth factors, cytokines, and extracellular vesicles, including exosomes, together known as the secretome, which possesses anti-fibrotic, anti-inflammatory, and immunomodulatory effects. MSC’s and secretome have been demonstrated in preclinical and clinical investigations to reverse fibrosis in the lung, liver, kidney, and heart, but there have been no reports of them repairing OSMF in patient-derived oral tissues.
In the current invention, DPMSCs-S fortified gel is developed to reverse OSMF.
Object of the Invention:
The primary object of the invention is to reverse the progression of areca nut-induced oral submucous fibrosis.
The secondary object of the invention is to understand how growth factors and bioactive substances rich secretome gel influences the anti-fibrosis and tissue regeneration pathways.
Further object of the invention is to avoid the likely need of the surgeries at an advanced stage of OSMF.
Summary of the Invention:
The purpose of this innovation is to examine the ability of DPMSCs-S gel to reverse areca nut extract (ANE)-induced OSMF.
According to the present disclosure, the gel formulation comprises reconstituted DPMSCs-S (1-5 µg of proteins/ ml), hydroxypropyl methylcellulose (HPMC) (1-3%), polyethylene glycol 400 (PEG400) (3-5%) and carboxymethyl chitosan (1-1.5 % w/v) (Santa Cruz Biotechnology) subsequently dissolved in 1% acetic acid.
The study was initiated by DPMSCs isolation, characterization and tri- lineage differentiation potential. In the next step, DPMSCs-S is prepared and growth factor analysis is carried out. In DPMSCs-S, there are higher levels of interleukin-6, interleukin-8, angiopoietin-1, angiopoietin-2, fibroblast growth factor basic, platelet endothelial cell adhesion molecule (PECAM-1), placenta growth factor (PIGF), vascular endothelial growth factor (VEGF), and tumor necrosis factor (TNF-α) (Figure 2a). Then, DPMSCs-S is lyophilized (L-DPMSCs-S) and stored at -80oC. Further, angiogenic potential of DPMSC-S and L-DPMSC-S is investigated by In-ovo Yolk Sac Membrane (YSM) assay for the quantitative assessment of the angiogenic potential in terms of total vessels area, total vessels length, vessels thickness and branching point. The proteome study of DPMSCs-S and L-DPMSCs-S demonstrated that lyophilization concentrates bioactive molecules, resulting in higher peak intensities and more distinct chromatographic profiles (figure 2(c) A).
Areca Nut Extract (ANE) is prepared and is examined using liquid chromatography-tandem mass spectrometry (LC-MS/MS). Using Agilant Technologies' mass hunter compound discovery software, five compounds (Arecoline and Retronecine) (D), Homoarecoline (E), Catechin, and Epicatechin (F) related with the progression of OSMF are discovered in areca nut extract (figure 3). Further, cytotoxicity of the ANE is assessed by MTT assay and confirms the presence of Arecoline (ARC), a primary cytotoxic phytochemical. ANE did not exert any cytotoxic effect on fibroblast at 10-1000 µg/ml concentration. On the contrary, arecoline exhibited a significant and dose-dependent decrease in cell viability. The MTT assay shows DPMSCs-S efficiently protect cells from ARC's cytotoxicity at doses ranging from 50 to 1000 µg/ml (figure 4a(b)). Also, it has been observed that the fibroblasts cultured in the presence of ANE and ARC and 50% DPMSCs-S showed the absence of apoptotic features like cell shrinkage and blebbing.
The generation of reactive oxygen species (ROS) is then assessed to determine DPMSCs-S's protective impact against oxidative stress generated by the ANE and ARC. Treatment with ANE and ARC at 10, 50, and 100 µg/ml greatly increased ROS generation, as determined by fluorescence microscopic investigations as shown in (figure 5(a)). Whereas, figure 5 (b) shows that DPMSCs-S treatment significantly reduces ANE and ARC-mediated ROS in fibroblast cells.
The β-galactosidase assay is used to further evaluate cellular senescence. Figure 6 illustrates how 50 µg/ml of ANE and ARC significantly increased cellular senescence in fibroblast cells and how DPMSCs-S therapy successfully shields fibroblasts from ANE and ARC-mediated cellular senescence.
Immunostaining of COL1 and Ki67 in fibroblasts subjected to ANE and ARC enhanced COL1 and Ki67 expression relative to untreated fibroblasts, while DPMSCs-S significantly reduced ANE-induced COL1 and Ki67 expression, as evidenced by confocal microscopy images shown in fig 7a and 7b.
The therapeutic efficacy of L-DPMSCs-S gel in reversing ANE-induced OSMF in Swiss albino mice (in vivo model) is further investigated. Fibrosis-related features, including epithelial atrophy, hyperinflammation, connective and muscle tissue degeneration, and collagen accumulation in ANE-administered Swiss albino mice are confirmed by H & E and Masson’s trichrome staining of buccal tissue (Figure 8(b)). Furthermore, therapy with DPMSCs-S and L-DPMSCs-S gel enhanced mouth opening in OSMF mice while dramatically reducing fibrosis-related histological alterations such as epithelial atrophy, hyper-inflammation, connective and muscle tissue degeneration, and collagen buildup (Figure 9(a)). L-DPMSCs-S gel therapy resulted in a more organized epithelium, a well-structured collagen matrix with low density, and less inflammation (Figure 9(b)).
Oxidative stress plays an important role in the pathogenesis of OSMF, with elevated serum lactate dehydrogenase (LDH) levels found in both OSMF and oral cancer. L-DPMSCs-S gel significantly reduced LDH and MDA levels while enhancing superoxide dismutase (SOD), indicating improved antioxidant activity and effective oxidative stress reduction (Figure 9(c)).
Intervention with L-DPMSCs-S gel in OSMF mice led to considerable downregulation of fibrosis-related genes, such as COLI, COLIII, and α-SMA, demonstrating its anti-fibrotic properties (Figure 9(d)).
Also, it has been observed that the DPMSCs-S treatment decreased the mRNA expression of COL15A1, and COL3 in OSMF patient derived buccal mucosa tissues.
Detailed description of the drawings
The present invention can be understood in detail, particularly the description of the invention, briefly summarized above may be had by reference to embodiments, some of which are illustrated in the appended drawings.
It is to be noted, however, that the appended drawings illustrate only typical embodiments of this invention and are therefore not to be considered limiting of its scope, the invention may admit to other equally effective embodiments. These and other features, benefits and advantages of the present invention will become apparent by reference to the following text figure, with like reference numbers referring to like structures across the views, wherein:
Figure 1a illustrates the explant culture of DPMSCs on day 0(i), day 5(ii) and day 7 (iii), and the day when substantial outgrowth of fibroblast-like cells was seen at the periphery of the explant (iv).
Figure 1b shows characterization of Isolated DPMSCs through flow cytometry analysis of cell surface markers demonstrates positive expression of CD73 (97.94%), CD90 (91.74%), and CD105 (99.43%) and negative expression of CD34 (2.40%), CD45 (0.09%), and HLA-DR (0.20%).
Figure 1c depicts in-vitro differentiation of DPMSCs in adipogenic, osteogenic and chondrogenic lineages stained with oil red o, alizarin red and safranin O respectively. (Images shown are of 10X resolution).
Figure 2a shows the concentration of bioactive factors (Angiopoietin-2 (Ang-2), Angiopoietin-1 (Ang-1), basic Fibroblast Growth Factor (bFBF), Platelet Endothelial Cell Adhesion Molecule (PECAM-1), Vascular Endothelial Growth Factor (VEGF), Epidermal Growth Factor (EGF), Tumor Necrosis Factor (TNF-α), Placenta Growth Factor (PlGF)) found in DPMSCs-S and L-DPMSCs-S. The data is expressed as mean±SD (n=3, *p<0.05, **p<0.01, ***p<0.001)
Figure 2b shows the angiogenic potential of DPMSCs-S and L-DPMSCs-S by YSM assay. A. Original and analysed images of the yolk sac membrane assay analyzed with CAM Assay tool (IKOSA). B. Angiogenic analysis of control, DPMSCs-S and L-DPMSCs-S for quantitative assessment of the angiogenic potential in terms of total vessels area, total vessels length, vessels thickness and branching point. ٭p < 0.05, ٭٭p < 0.01, and ٭٭٭p < 0.001 were considered to be statistically significant (n-3).
Figure 2(c) depicts the proteomic analysis of dental pulp mesenchymal stem cells secretome (DPMSCs-S) and lyophilized secretome (L-DPMSCs-S). (A) Extracted ion chromatogram of DPMSCs-S and L-DPMSCs-S by LC-MS/MS, (B) Total pathway hits, (C) Molecular pathway hits, (D) Biological pathway hits, (E) Protein class hits, (F) Panther pathway hits.
Figure 3 illustrates the extracted ion chromatogram of Areca Nut Extract (ANE) by LC-MS/MS (A-C).
Figure 4a depicts viability of DPMSCs treated with (a) Areca Nut Extract (ANE), and (b) Arecoline (ARC) alone with and without 50% DPMSCS-S by MTT assay. Data represents mean absorbance measured at 570 nm. (n=3, *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001).
Figure 4b shows morphological evaluation of fibroblast cells treated with (c) ANE and (d) ARC, with and without DPMSCS-S. The DPMSCS-S inhibited the production of apoptotic cell characteristics such as cell shrinkage in ANE-treated cells while allowing cell shrinkage and blebbing in ARC-treated cells.
Figure 5 depicts intracellular reactive oxygen species (ROS) monitoring using fluorescence microscopy: The generation of ROS (green fluorescence) was significantly enhanced by the treatment of ANE (a (i)) and ARC (a (ii)) at 10, 50, 100 µg/ml concentration. DPMSCS-S protected the fibroblast from ANE and ARC induced ROS. (b) Quantitative estimation of the fluorescence intensity revealed the similar trend as fluorescence microscopy results. (n=3, *p <0.05, **p <0.01, ***p <0.001, ****p <0.0001).
Figure 6 shows cellular senescence in the ANE, ARC and DPMSCS-S treated fibroblast cells and shows significant increase in the formation of blue hue in the ANE and ARC treated fibroblast at 50µg/ml concentration.
Figure 7 illustrates (a) Immunostaining of the intracellular collagen-1 in fibroblast exposed to ANE for 48h with and without 50% DPMSCS-S. Fixed cells were subjected to immunostaining to detect expression of Collagen-I (labeled with red fluorescence-Alexa fluor 568) and nucleic acid (labeled with blue fluorescence-DAPI), (b) Ki67 in fibroblast cells exposed to ANE for 48h with and without 50% DPMSCS-S. Fixed cells were subjected to immunostaining to detect expression of Ki67 (labeled with green fluorescence-Alexa fluor 488) and nucleic acid (labeled with blue fluorescence-DAPI).
Figure 8 shows the conformation of the ANE induced OSMF in the Swiss albino mice. (A) H & E staining of the buccal mucosa showed well-organized epithelial layer (orange arrow) and normal connective tissue (blue arrow). ANE administered animals showed disorganized epithelium and thickened connective tissue, (B) Masson’s trichome staining revealed thicker epithelial layer and less intense collagen (green arrow) in control animals whereas buccal mucosa of ANE administered animals showed increase in collagen deposition and potentially increase in denser and fibrotic tissue, (C) Mouth opening in ANE administered mice in comparison with the control group. ٭p < 0.05 were considered to be statistically significant (n-7).
Figure 9(a) depicts (A) representative photographic images of the mouth openings of mice. (B) Mouth opening of the experimental animals upon treatment with DPMSCs-S and L-DPMSCs-S gel taken by Vernier calliper. Data shown are mean ±SD. *,#, $p < 0.05, *, #, $p < 0.01, and *, #, $p < 0.001. n=7 animals.
Figure 9 (b) shows (A) H and E, and Masson Trichrome Staining of the buccal mucosa tissues of the DPMSCs-S and L-DPMSCs-S administered OSMF mice. (B) Total fibrosis area quantified from Masson’s Trichrome-stained histological images using ImageJ analysis shows that the Treatment with DPMSCs-S and L-DPSCs-S gel significantly reduced the fibrotic area compared to the disease control group.
Figure 9(c) illustrates the serum levels of (A) Lactate Dehydrogenase (LDH), (B) Malondialdehyde (MDA), (C) Superoxide Dismutase Activity (SOD) in the DPMSCs-S and L-DPMSCs-S administered OSMF mice. Data represents mean ± SEM, n=7, *p<0.05, **p<0.01, ***p<0.001. Figure 9(d) depicts the mRNA expression of collagen type I (Col I), collagen type III (Col III) and α-SMA in buccal tissue. Data shown are mean±SD (n=3, *p<0.05, **p <0.01, ***p <0.001).
Detailed description of the Invention:
The present invention is described hereinafter by various embodiments with reference to the accompanying drawing, wherein reference numerals used in the accompanying drawing correspond to the like elements throughout the description.
While the present invention is described herein by way of example using embodiments and illustrative drawings, those skilled in the art will recognize that the invention is not limited to the embodiments of drawing or drawings described and are not intended to represent the scale of the various components. Further, some components that may form a part of the invention may not be illustrated in certain figures, for ease of illustration, and such omissions do not limit the embodiments outlined in any way. It should be understood that the drawings and detailed description thereto are not intended to limit the invention to the particular form disclosed, but on the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the scope of the present invention as defined by the appended claims. As used throughout this description, the word "may" is used in a permissive sense (i.e., meaning having the potential to), rather than the mandatory sense, (i.e., meaning must). Further, the words "a" or "an" mean "at least one” and the word “plurality” means “one or more” unless otherwise mentioned. Furthermore, the terminology and phraseology used herein is solely used for descriptive purposes and should not be construed as limiting in scope. Language such as "including," "comprising," "having," "containing," or "involving," and variations thereof, is intended to be broad and encompass the subject matter listed thereafter, equivalents, and additional subject matter not recited, and is not intended to exclude other additives, components, integers or steps. Likewise, the term "comprising" is considered synonymous with the terms "including" or "containing" for applicable legal purposes. Any discussion of documents, acts, materials, devices, articles and the like is included in the specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention.
This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiment set forth herein. Rather, the embodiment is provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art. In the following detailed description, numeric values and ranges are provided for various aspects of the implementations described. These values and ranges are to be treated as examples only and are not intended to limit the scope of the claims. In addition, a number of materials are identified as suitable for various facets of the implementations. These materials are to be treated as exemplary and are not intended to limit the scope of the invention.
Arecoline, the principal cytotoxic phytochemical found in ANE, is known to have cytotoxic effects on gingival fibroblasts, cementoblasts, and endothelial cells. ANE can upregulate specific survival signals, increasing the probability of malignant transformation. Furthermore, the cytotoxic effect may result in chromosomal instability, which can lead to carcinogenesis.
An embodiment of the present invention studies the use of an oral chitosan gel formulation of DPMSC-derived secretome to treat areca nut-induced oral submucous fibrosis. The study is focused on the ability of DPMSCs-S for its prospective use as a therapy for OSMF and helps to prevent the DPMSCs cells from the cytotoxic effect of ANE. The oral gel is utilized for topical administration; the composition may display multipurpose therapeutic capabilities, serving as an anti-inflammatory, antibacterial, and impacts the fibrosis and tissue regeneration pathways.
This study employed an established technique from M. Shekatkar et al. 2022 to investigate the therapeutic efficiency of L-DPMSCs-S gel in treating ANE-induced OSMF in Swiss albino mice.
The study began with approval from the Institutional Ethics Committee for Stem Cell Research at Dr. D. Y. Patil Dental College and Hospital (Ref. No. DYPV/EC/618/20) in Pune, India. Primary buccal tissues were obtained from patients undergoing OSMF surgery with previous informed consent. DPMSCs were isolated using the explant culture method, and they were characterized using laboratory-adapted methodology that included cell surface marker analysis and tri-lineage differentiation capacity. The cells started to multiply on days 5–6, as shown in figure 1a (i), (ii), and (iii). After being further sub-cultured, the cells displayed fibroblast-like morphology under a microscope as show in figure 1a (iv). Further, DPMSC’s were put through a cell surface marker examination using a flow cytometer in order to evaluate the purity of the cultivated cells. As illustrated in figure 1(b), DPMSCs showed positive expression of CD73 (97.94%), CD90 (91.74%), and CD105 (99.43%) and negative expression of CD34 (2.40%), CD45 (0.09%), and HLA-DR (0.20%).
Additionally, as shown in figure 1(c) their capacity for tri-lineage differentiation isolated cells were able to develop into osteogenic, adipogenic, and chondrogenic lineages which is stained with oil red o, alizarin red and safranin O respectively.
DPMSC’s were planted at a density of 1x106 cells in a T75 cell culture flask to prepare their secretome (DPMSCs-S). When the cells achieved 90-95% confluence, the culture media was removed and rinsed with DPBS (Gibco). Fresh α-MEM medium with 1% antibiotic-antimycotic solution (without serum) was added and cultured for 48 hours. The material was collected, filtered with 0.22 µm syringe filters, and kept at -800C. Further, the LEGEND plexTM Human Angiogenesis Panel 1 (10-plex, Biolegend, USA) was used to assess growth factor concentration in DPMSCs-S according to the manufacturer's instructions.
MSC’s release growth factors, bioactive proteins, cytokines, and chemokines, which operate as paracrine effectors. Figure 2a demonstrates elevated levels of Interleukin-6, Interleukin-8, Angiopoietin-1, Angiopoietin-2, Fibroblast growth factor basic, Platelet endothelial cell adhesion molecule (PECAM-1), Placenta growth factor (PIGF), Vascular endothelial growth factor (VEGF), and Tumor necrosis factor (TNF-α) in DPMSCs-S. Data shown are Mean±SEM (n=3).
Furthermore, DPMSCs-S (100 ml) were frozen at -80°C overnight. The next day, it was placed in a freeze dryer (iGENE) at -37°C for 24 hours. The resulting lyophilized powder was stored at -80°C for further experiments (Sandonà et al., 2021).
Further, angiogenic potential of DPMSCs-S and L-DPMSCs-s investigated by In-ovo Yolk Sac Membrane (YSM) assay. Fertilized eggs procured from Venkateshwara Hatcheries and incubated for 48 hours. The broad end was cleaned with 70% ethanol, and 4-5 ml of albumin was removed. DPMSCs-S and L-DPMSCs-S (100 µl) were added, and the eggs were incubated at 37°C. After 48 h, images of blood vessel sprouting were captured, using α-MEM medium as a control. Figure 2b shows the angiogenic analysis of control, DPMSCs-S and L-DPMSCs-S for quantitative assessment of the angiogenic potential in terms of total vessels area, total vessels length, vessels thickness and branching point. ٭p < 0.05, ٭٭p < 0.01, and ٭٭٭p < 0.001 were considered to be statistically significant (n-3). IKOSA software with the CAM assay tool is used for the analysis.
In the next step, proteomic analysis of DPMSCs-S and L-DPMSCs-S was performed using LC-MS/MS at CAMS, Pune, India(Jain et al., 2018). Data acquisition and processing were conducted using Mass Hunter Workstation Software on the Agilent 6540 UHD QTOF MS. The resulting mass spectrometry data were analysed using panther analysis software (www.pantherdb.org)(Li et al., 2022) corresponding to the Homo sapiens species with default parameters, enabling the identification and characterization of the proteomic profiles and pathway hits of both secretome. The proteomic analysis of DPMSCs-S and L-DPMSCs-S revealed that lyophilization concentrates bioactive chemicals, leading to increased peak intensities and clearer chromatographic profiles (figure 2(c) A). The PANTHER classification revealed that both secretome contain diverse bioactive compounds affecting various biological pathways, molecular functions, protein classes, and cellular components. Notably, L-DPMSCs-S exhibited superior activity, showing higher counts across pathways (1,698 vs. 1,025), molecular functions (2,470 vs. 1,603), protein classes (2,041 vs. 1,365), and biological processes (4,405 vs. 2,853) compared to DPMSCs-S (figure 2(c) B).
The essential molecular functions associated with fibrosis, as indicated in square brackets, encompass antioxidant activity, structural molecular activity, ATP-dependent activity, binding, and cytoskeletal motor activity, which are illustrated in (figure 2(c)C).
Additionally, biological processes associated with fibrosis—such as biological regulation, cellular processes, developmental processes, growth, homeostasis, immune processes, and metabolism linked to both secretome are shown in (figure 2(c)D).
Proteins associated with calcium binding, cell adhesion, cell junctions, chaperone activity, cytoskeletal functions, immunity, extracellular matrix dynamics, membrane trafficking, structural roles, and transmembrane signalling were also identified (figure 2(c) E). Notably, pathways including TGF-beta signalling, VEGF signalling, oxidative stress response, apoptosis signalling, and angiogenesis exhibited significant associations with both DPMSCs-S and L-DPMSCs-S, as illustrated in (figure 2(c) F). The highlighted pathways (shown in bracket) are those actively engaged in the advancement and alleviation of OSMF, highlighting the therapeutic potential of the secretome formulations.
The subsequent stage involves the preparation of an oral gel produced from DPMSCs-S (w/v). The oral gel is made using reconstituted DPMSCs-S (1-5 µg of proteins/ml), hydroxypropyl methylcellulose (HPMC) (1-3%), polyethylene glycol 400 (PEG400) (3-5%), and carboxymethyl chitosan (1-1.5% w/v) (Santa Cruz Biotechnology), diluted in 1% acetic acid. In this formulation, a stock solution of 4mg/ml carboxymethyl chitosan is produced in phosphate buffered saline. The resulting gel is stored at 4° Celsius.
In the next step, Areca Nut Extract (ANE) is prepared at the GLP facility of Dr. D. Y. Patil College of Ayurved and Research Centre, Pune, India. ANE is examined using liquid chromatography-tandem mass spectrometry (LC-MS/MS) at the Center for Applications of Mass Spectrometry (CAMS), Venture Center in Pune (Jain et al., 2018). Data collection and processing are performed using Mass Hunter Workstation Software. LC-MS/MS analysis of ANE identified a complex mixture of compounds based on mass-to-charge ratios (m/z) and retention times. Peaks from 1 to 30 minutes indicated smaller polar molecules (1-5 minutes) and larger less polar compounds (10-30 minutes), with notable peaks at 1.5, 4.5, and 10.1 minutes. From 753 potential compounds, 684 were identified using the METLIN PCDL database.
Using Mass Hunter Compound Discovery Software (Agilant Technologies), five chemicals (Arecoline and Retronecine) (D), Homoarecoline (E), Catechin, and Epicatechin (F) associated with the advancement of OSMF were found in areca nut extract. The extracted ion chromatogram and compound structures are shown in figure 3 and summarized in Table 01.
The viability of DPMSCs treated with ANE and ARC (Sigma, USA) is assessed using the (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium Bromide (MTT) test. DPMSCs were seeded at a density of 1x104 cells/well in a 96-well cell culture plate and incubated at 37°C in a humidified CO2 incubator for 24 hours. Cells were treated with ANE, ARC, and 50% DPMSCs-S at concentrations of 5, 10, 20, 50, 80, 100, 200, 300, 400, 500, 800, and 1000 µg/ml, respectively. After 48 hours, remove the medium and add 50 µl of MTT solution (5 mg/mL in PBS) (Hi Media). Incubate for 4 hours at 37°C. The absorbance was measured at 570 nm using an ELISA plate reader. Cells that had not been treated with ANE or ARC were used as controls. Interestingly, the cells cultured in presence of ANE and DPMSCs-S showed significantly higher viability as compared to the ANE alone as evident by MTT assay figure 4a(a). Data indicate that ANE has no cytotoxic effect on fibroblasts at doses ranging from 10 to 1000 µg/ml (fig 3 (a)). In contrast, ARC induced a significant and dose-dependent decrease in cell viability, as seen in figure 4a(b). DPMSCs-S efficiently protect cells from ARC's cytotoxicity at doses ranging from 50 to 1000 µg/ml.
Furthermore, the effects of ANE and ARC on DPMSC morphology were investigated. DPMSCs were seeded at 1 x 104 cells/well in a 6-well cell culture plate and incubated for 24 hours in a humidified CO2 incubator. After reaching 80% confluence, cells were treated with 10, 50, and 100 µg/ml of ANE and ARC, with or without 50% DPMSCs-S. After 48 hours, cell morphology was studied under the phase contrast microscope. Normally, fibroblast apoptosis is a critical process in the pathophysiology of tissue fibrosis. During fibrosis, cells exhibit a typical feature of apoptotic cells such as cell shrinkage, blebbing, and vacuolation. Fibroblasts treated with ARC at 10, 50, and 100 µg/ml concentration showed a dose-dependent increase in apoptotic characteristics like cell shrinkage and blebbing. Interestingly, cells grown with ANE and DPMSCs-S had considerably higher vitality than cells cultured with ANE alone, as demonstrated by the MTT experiment figure 4(a). The impact was significant in ARC-treated cells at a dosage of 100 µg/ml. Interestingly, fibroblasts cultivated with ANE or ARC and 50% DPMSCs-S demonstrated a lower incidence of fibroblasts with apoptotic characteristics such as cell shrinkage and blebbing, as seen in figure 4b(c, d).
The experiment indicates significant variations in fibroblast morphology between ANE and ARC with and without DPMSCs-S. ANE treatment at 50 and 100 µg/ml dosage resulted in cell shrinkage, while untreated cells exhibited normal fibroblast shape.
The next phase involves estimating the formation of reactive oxygen species (ROS). Oxidative stress is a major driver of inflammation, myofibroblast differentiation, and fibrogenesis during tissue fibrosis. Excessive ROS generation, combined with an ineffective anti-oxidative defence system, is known to produce excessive fibrosis and scarring of the healing tissue. Thus, the formation of reactive oxygen species (ROS) is measured to investigate DPMSCs-S's protective effect against oxidative stress caused by the ANE and ARC. DPMSCs are sown in a 48-well cell culture plate at 3x103 cells per well. After reaching 80-90% confluence, cells are treated with 10, 50, and 100 µg/ml of ANE or ARC, with and without 50% DPMSCs-S, over 48 hours. ROS levels is assessed using the Enzolife ROS-ID ROS/RNS Detection Kit (Enzo Life Science, NY, USA) in accordance with the manufacturer's instructions. The photos are captured using an Olympus fluorescence microscope with excitation and emission wavelengths of 490/525 nm. The fluorescence intensity is quantitatively estimated using Image J software. Treatment with ANE and ARC at concentrations of 10, 50, and 100 µg/ml significantly increased ROS production, as seen by fluorescence microscopic examinations (figure 5(a)). Despite the general consensus that ANE might have lethal effects on cultured cells, observation suggest that some concentrations of ANE could also upregulate some survival signals, raising the risk of malignant transformation (figure 5a). Figure 5 (b) demonstrates that DPMSCs-S treatment dramatically reduced ANE and ARC-mediated ROS in fibroblast cells. Quantitative estimation of fluorescence intensity demonstrated a similar pattern to the fluorescence microscopy results. (n=3) *p < 0.05, **p <0.01, ***p < 0.001, ****p < 0.0001.
The cellular senescence is further assessed using the β-galactosidase assay. Cellular senescence is an irreversible cell cycle arrest in which cells cease to divide. In-vitro studies have shown that alkaloids in ANE can cause fibroblast senescence and permanent DNA damage. Excessive oxidative stress may induce fibroblast cells to undergo early senescence in clinical circumstances like OSMF, both in vitro and in vivo. To determine replicative cellular senescence, a β-galactosidase staining solution kit (Cell Signaling Technology, USA) is used according to manufacturer recommendations.
DPMSCs (passage 9) were planted in a 48-well cell culture plate and allowed to reach 50-60% confluence. They were administered with 50 µg/ml of ANE or ARC, with or without 50% DPMSCs-S, during 48 hours. Cells that had not been treated with ANE, ARC, or DPMSCs-S were used as controls. After 48 hours, cells were frozen and treated overnight with β-galactosidase solution and 50 µl of 20 mg/ml X-gal solution at 37oC. The blue color development was detected using an olympus microscope. The quantitative estimation of blue colour generation is performed using Image J program. Figure 6 shows that 50 µg/ml of ANE and ARC dramatically promoted cellular senescence in fibroblast cells. The results of DPMSCs-S treatment show that it effectively protects fibroblasts from ANE and ARC-mediated cellular senescence.
The immunostaining of COL1 and Ki67 in fibroblasts exposed to ANE with and without DPMSCS-S is further examined. OSMF is associated with the deposition of collagen Fiber in the connective tissue of the oral cavity, and multiple research studies suggest that densely packed collagen Fiber deposition increases as disease develops from early to advanced stages. In order to study, immunostaining of COL1 and Ki67, DPMSCs are seeded in 8-well micro chamber plate at a density of 3x103 cells/well. Once 70-80 % confluence is achieved, cells are treated with ANE or ARC with or without 50% DPMSCs-S for 48h. Post incubation, cells are fixed with 100% methanol for 5 mins and permealized with 0.1% Triton X-100 for 5 mins. Further, cells are blocked with 1% BSA for 1 h and incubated with 0.4 µg/ml Anti-COL1 (Ab138492) and 1:1000 Recombinant Alexa Fluor 488 Anti-Ki67 (Ab197234) overnight at 4°C. Then, cells are washed 3-4 times with 1%BSA/PBS to remove unbound primary antibody and further incubated with goat anti-rabbit Alexa flour 568 antibody (1:750) for 1 h. Further, cells were washed with PBS, and counter stained with DAPI (0.1mg/ml) visualize nuclear DNA. Images were taken under confocal laser scanning microscopy (Zeiss). The results shows that ANE and ARC therapy increased the expression of COL1 in human fibroblast cells. Interestingly, DPMSCs-S greatly reduced COL1 protein expression, as shown by immunostaining with COL1 antibodies (fig 7(a)). Ki67 is a proliferative marker that indicates malignant transformation and carcinogenesis in oral premalignant lesions. Several human studies have revealed a strong connection between Ki67 expression and disease progression from dysplasia to OSMF to OSCC. The results demonstrated that ANE and ARC treatment of fibroblasts increased Ki67 expression relative to untreated fibroblasts. Interestingly, the treatment of DPMSCs-S dramatically reduced the ANE-induced expression of Ki67, as revealed by confocal microscopy images shown in (fig 7(b)).
Further, the therapeutic efficacy of L-DPMSCs-S gel in reversing ANE-induced OSMF in Swiss albino mice is investigated. An in vivo mice model of OSMF is established and the study is carried out as per M. Shekatkar et. al., 2022 model. Swiss Albino mice (male and female ratio 1:1), 3 months old, 25-35 g, n=40, are housed in polypropylene cages in a controlled environment with access to standard pellet diet and water ad libitum at CCSEA approved animal house facility of Dr. D. Y. Patil Institute of Pharmaceutical Sciences. Mice that displayed symptoms of illness or physical harm were eliminated. Mice were first separated into two groups: Group 1 (n=12) got intraoral PBS (50 µl) twice daily, whereas Group 2 (n=28) received intraoral ANE (50 µl of 50 mg/ml) twice daily for three months. Following this period, 7 mice from each group were sacrificed using CO2 inhalation for OSMF confirmation. Buccal tissues were fixed in 10% paraformaldehyde for histopathological analysis. Fibrosis-related features, including epithelial atrophy, hyperinflammation, connective and muscle tissue degeneration, and collagen accumulation in ANE-administered Swiss albino mice are confirmed by H & E and Masson’s trichrome staining of buccal tissue (Figure 8(b) (M. Shekatkar et al., 2022). H&E staining showed a normal epithelial layer (orange arrow) and connective tissue (blue arrow) in controls, while ANE-treated mice displayed disorganized epithelium and thickened connective tissue. Masson’s trichrome staining revealed increased collagen deposition and fibrotic tissue (green arrow) in ANE-treated mice compared to controls, which showed reduced collagen intensity (green arrow).
After 90 days, the mouth opening was measured with a Vernier calliper. ANE delivery resulted in decreased mouth opening (1.69±0.09 mm vs. 2.00±0.01 mm in controls, p<0.05) due to collagen buildup.
After three months of ANE administration, mice's buccal mucosa developed fibrosis-associated alterations such as disordered epithelium, thicker connective tissue, and increased collagen accumulation, resulting in denser, more fibrotic tissue. Following the conformation of ANE-induced OSMF in mice, they were administered DPMSCs-S and L-DPMSCs-S gel intra orally. The remaining 21 animals in Group 2 were randomly assigned to three treatment groups: (1) Disease Control (n-7) (100 µl PBS), (2) DPMSCs-S (n-7) (100 µl), and (3) L-DPMSCs-S gel. Treatments were administered intraorally twice a week for one month.
To avoid potential confounders, we used randomization for treatment allocation and kept the animals in separate cages to eliminate location bias. The researchers who measured the physical and biochemical markers were unaware that the animals had been randomly assigned. All procedures were completed in compliance with the ARRIVE standards. 2.0
After 30 days of treatment with DPMSCs-S and L-DPMSCs-S gels, mice are euthanized via CO₂ inhalation and cervical dislocation. The right buccal mucosa was excised, extending from the mouth corner to the midline of the eye, with an incision from 2 mm below the eye to the lower jaw for precise tissue removal. Samples were preserved in Trizol (Gibco) at -80°C for RNA isolation, while others were fixed in 10% formalin for histopathology.
Samples were treated for Hematoxylin and Eosin (H&E) and Masson's trichrome staining using the previously reported methodology (M. Shekatkar et al., 2022). The slides were examined under a compound light microscope (Olympus CX21i). Control animals had complete epithelial layers and well-organized connective tissue, whereas disease control had classic fibrosis-related characteristics such as thick collagen bundles and a thinning epithelial layer. DPMSCs-S therapy reduced fibrosis and partially restored the epithelial layer and vascular structures. Interestingly, treatment of these animals with DPMSCs-S and L-DPMSCs-S gel resulted in a significant improvement in fibrosis-associated alterations, including reasonably well-organized epithelium and reduced collagen density, as indicated by H and E and Masson Trichrome staining (figure 9(b) A).
Also, both treatments effectively improved mouth opening (2.14±0.02, 2.26±0.02 versus 1.83±0.01 mm) in OSMF mice, as demonstrated by the photographic images shown in (figure 9(a) A) as well as measurements taken with the Vernier calliper (figure 9(a) B).
Furthermore, Image J program used to make quantitative assessment of the fibrotic area stained in Masson Trichorme. Figure 9(b) B shows that L-DPMSCs-S gel is more effective in reducing fibrotic area (2.14±0.02, 0.82±0.12 vs 10.35±0.16%) and mouth opening as compared to the DPMSCs-S.
LDH, MDA, and SOD levels are known biomarkers of the oxidative stress and are known to be altered in OSMF. In order to understand how DPMSCs-S and L-DPMSCs-S gel ameliorate oxidative stress in ANE induced OSMF mice: Lactate dehydrogenase (LDH) levels in mice serum were measured using the UltiChem LDH-P Reagent kit (modified IFCC method; Yucca Diagnostics, Kolhapur, India) (Pagani, Bonora, & Panteghini, 2003) and Serum malondialdehyde (MDA) levels were quantified by absorbance extrapolation against a standard calibration curve, expressed as µmol/mg protein(Khairnar, Kulkarni, & Singh, 2024). Also, superoxide dismutase (SOD) activity was determined as U/mg protein based on half-maximal inhibition of nitro blue tetrazolium (NBT) reduction(McCord & Fridovich, 1969). Analysis data shows that the DPMSCs-S and DMPMSCs-S gel treatment significantly reduced LDH levels (171.99 ±132.64, 261.61±108.35 vs 843.66 ±105.1 IU/L) in serum of the animals (p < 0.001) compared to the disease control group, suggesting its protective effect against ANE induced cellular damage. MDA levels, a marker of lipid peroxidation was markedly elevated in the disease control group (p < 0.001). Treatment with DPMSCs-S and L-DPMSCs-S Gel significantly reduced MDA levels (5.12±1.3, 5.37±0.70 vs 31.75±3.26 U/mg) (p < 0.001) compared to the disease control group, indicating a reduction in oxidative stress. Also, SOD activity was significantly decreased in the disease control group (p < 0.001), reflecting diminished antioxidant defence. Both DPMSCs-S and L-DPMSCs-S Gel treatment significantly increased SOD activity (1.95± 0.22, 1.16±0.06 Vs 0.14±0.02 U/mg) (p < 0.001), indicating enhanced antioxidant defence mechanisms and effective reduction of oxidative stress. Except for the SOD levels, there was no significant difference between the DPMSCs-S and L-DPMSCs-S treatments, as illustrated in figure 9(c).
After collecting oral mucosal tissues in Trizol, the samples were processed for RNA extraction. Reverse transcription of 2 μg of total RNA was performed using a high-capacity cDNA synthesis kit (Takara, Japan). Quantitative real time PCR reactions were performed in conjunction with a gene-specific primer pair. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as housekeeping gene. The reaction was performed using SYBR Premix Ex Taq (Takara, Japan) following the manufacturer's protocol (Wen et al., 2017). Data was analysed by using delta CT method and expressed as mean ± SD of fold expression. Result shows that the treatment of DPMSCs-S and L-DPMSCs-S gel leads to significant down regulation of COLI (6423.50±94.86, 9594.95±640.23 fold), COLIII (806.88±45.38, 84679.53±16947.91 fold), and α-SMA (183.52±12.63, 35.73±12.63 fold) mRNA expression as compared to the disease control animals (figure 9 (d)). It confirms the anti-fibrotic effects.
In the following stage, primary buccal mucosa tissues (n=3) were acquired from patients clinically diagnosed with OSMF (Clinical grade III) and undergoing surgery at the Department of Oral Surgery, Dr. D. Y. Patil Dental College and Hospital, Pune (India). Tissue from the buccal mucosa was kept in DPBS (Gibco) with 1% antibiotic-antimycotic solution (Gibco). After being sliced into tiny 10 mm pieces, the tissue was cleaned 3-4 times using DPBS. For 48 hours, the tissues were cultivated in a CO2 incubator in α-MEM medium containing DPMSCs-S in a 6-well cell culture plate. Tissues not treated with DPMSCs-S were used as controls.
Further, molecular expression analysis of tissues by RT-PCR is carried out. The trizol technique (Thermo) was used to extract total cellular RNA from cells and tissues following the previously reported experimental protocol. RNA quantity and purity were determined using a Nano Drop spectrophotometer. Further, 1000 ng of RNA was reverse transcribed into cDNA using a high-capacity cDNA reverse transcription kit (TAKARA) according to the manufacturer's instructions. The gene expression of COL15A1, COL3, α-SMA, TGFβ1, and TGFβ2 was analysed using Quant Studio 5 (Applied Biosystems). Primer sequences are provided in Supplementary file 1. The mRNA expression levels were adjusted to those of GAPDH. All experiments were performed in triplicates. The relative expression was estimated using the ΔΔCt technique.
The presented data convincingly demonstrated the cytoprotective and antifibrotic action of DPMSCs-S, as proven by a decrease in blebbing, cell mortality, senescence, collagen expression, and oxidative stress. DPMSCs-S generated oral gel has the potential to be a novel therapeutic for reversing areca nut-induced OSMF, which can be investigated further in preclinical and human clinical studies. , Claims:We claim,
1. A formulation of an oral gel for treating areca nut-induced oral submucous fibrosis comprises of reconstituted DPMSCs-S, hydroxypropyl methylcellulose (HPMC), polyethylene glycol 400 (PEG400), and carboxymethyl chitosan, diluted in 1% acetic acid.
2. The formulation of an oral gel for treating areca nut-induced oral submucous fibrosis as claimed in claim 1, wherein predetermined concentration of DPMSCs-S is in the range of 1-5 µg of proteins/ml.
3. The formulation of an oral gel for treating areca nut-induced oral submucous fibrosis as claimed in claim 1, wherein predetermined
percentage of hydroxypropyl methylcellulose (HPMC) is in the range of 1-3%.
4. The formulation of an oral gel for treating areca nut-induced oral submucous fibrosis as claimed in claim 1, wherein predetermined
percentage of polyethylene glycol 400 (PEG400) is in the range of 3-5%.
5. The formulation of an oral gel for treating areca nut-induced oral submucous fibrosis as claimed in claim 1, wherein predetermined
quantity of carboxymethyl chitosan is in the range of 1-1.5% w/v.
6. The formulation of an oral gel for treating areca nut-induced oral submucous fibrosis as claimed in claim 1, wherein the therapeutic potential of the gel is investigated in vitro, in vivo and ex-vivo in regulating the course of areca nut-induced OSMF in Swiss albino mice and in OSMF patient derived buccal mucosa tissues.
7. The formulation of an oral gel for treating areca nut-induced oral submucous fibrosis as claimed in claim 1 and 7, wherein L-DPMSCs-S gel enhanced mouth opening in OSMF mice while dramatically reducing fibrosis-related histological alterations such as epithelial atrophy, hyper-inflammation, connective and muscle tissue degeneration, and collagen buildup.
8. The formulation of an oral gel for treating areca nut-induced oral submucous fibrosis as claimed in claim 1 and 7, wherein L-DPMSCs-S gel significantly reduced LDH and MDA levels while enhancing superoxide dismutase (SOD), indicating improved antioxidant activity and effective oxidative stress reduction.
9. The formulation of an oral gel for treating areca nut-induced oral submucous fibrosis as claimed in claim 1 and 7, wherein L-DPMSCs-S gel in OSMF mice led to considerable downregulation of fibrosis-related genes, such as COLI, COLIII, and α-SMA, demonstrating its anti-fibrotic properties.
Dated this the 12th day of February 2025.