Acetylsalicylic Acid Promotes Osteogenic Differentiation of Human Dental Pulp Mesenchymal Stem Cells and Regeneration of Alveolar Bone in Experimental Periodontitis Rats

Background . Periodontitis is characterized by bone resorption and periodontal tissue destruction owing to oral microbiota, mechanical stress


Introduction
Periodontitis is a chronic infammatory disease caused by the accumulation of oral microorganisms [1,2].In severe cases, irreversible destruction of the supportive tissues of the gingiva, periodontal ligament, and alveolar bone can occur, leading to tooth loss [3].As one of the most widespread infammatory diseases in humans, periodontitis substantially impacts not only dental care but also systemic disorders such as diabetes [2], cardiovascular diseases [4,5], and dementia [6].Initial nonsurgical periodontal therapy consists of scaling, root planning, and home care review [7,8].Besides, many techniques and procedures have been investigated to restore the lost healthy tissues [9], such as the application of guided tissue regeneration, new biomaterials, and growth factors.However, the clinical applicability of these regenerative therapy approaches is limited.As a consequence, current research trends have been directed towards developing cell-based techniques for periodontal regeneration.
Mesenchymal stem cells have demonstrated a strong ability to diferentiate into osteoblasts, and their paracrine secretion of cytokines and growth factors is also thought to indirectly drive bone formation [10].For instance, Du et al. investigated local injection of allogeneic bone marrow mesenchymal stem cell as a potential noninvasive therapy for clinical periodontitis [11].Another study found that the frst stem cells isolated from the orofacial area were those from the third molar dental pulp [12].Dental pulp mesenchymal stem cells (DPMSCs) have attracted scientifc interest in the feld of tooth tissue engineering due to their noninvasive collection with low morbidity and similarity to target tissue [13].Besides, among bone marrow mesenchymal stem cells (BMMSCs) and periosteal cells, DPSCs had the highest proliferative potential and ability to differentiate into osteoblasts, suggesting an alternative cell source for tissue-engineered bone surrounding dental implants [14][15][16][17].Khorsand et al. [18] also investigated the potential of DPSCs to form hard tissue by evaluating the regeneration of cementum and periodontal ligament (PDL) formed after transplantation of DPSCs combined with Bio-Oss in canine periodontal tissue.
Acetylsalicylic acid (ASA), the most widely used analgesic, antipyretic, and nonsteroidal anti-infammatory drug for decades, has been reported to have a positive impact in regulating mesenchymal stem cells osteogenic diferentiation in several studies [19,20].According to Li et al., ASAtreated human mesenchymal stem cells loaded with a BFP-1 peptide-decorated complex demonstrated increased osteogenic activity [21].
However, to date, no studies have evaluated the impact of ASA on the osteogenic capacity of hDPMSCs on the regeneration of defects in periodontal tissues.Herein, we clarify that ASA-pretreated hDPMSCs is a more efcient method for the treatment of periodontitis and elucidate the involvement of potential pathways.

Animals.
In this study, a total of 35 male Sprague-Dawley (SD) rats (Hunan Slack Jing da Experimental Animal Co., Ltd., Changsha, China) weighing 200-250 g at six weeks were used.Rats were raised and housed under conventional conditions in the Department of Laboratory Animal Science (Central South University, Changsha, Hunan, China).Te experimental protocols were performed in accordance with "Guide for the Care and Use of Laboratory Animals, 8th ed., 2010" (National Institutes of Health, Bethesda, MD) and were approved by the Institutional Animal Care and Use Committee of Central South University (Changsha, China; Permit No. CSU-2022-0001-0047).

Isolation and Cultivation of Human Dental Pulp Mesenchymal Stem Cells.
Human tooth tissues were obtained from impacted/caries-free third molars of patients between 18 and 22 years old, under approved guidelines set by the Changsha Stomatological Hospital, Hunan University of Chinese Medicine.Each patient signed an informed consent document.hDPMSCs were isolated as described previously [12,[22][23][24].
After cleaning the tooth surface with phosphate bufered saline (PBS) (Gibco, USA) containing 5% penicillin/streptomycin (BI, Israel), the pulp was removed and immersed in a digestive solution containing 3 mg/mL of collagenase type I (Sigma-Aldrich, USA) for 1 h at 37 °C.After digestion, singlecell suspensions were collected by passing the cells through a 70 µm strainer (Corning, USA) and cultured in growth media (Dulbecco's modifed Eagle Medium-Ham F-12 (DMEM/F-12; Gibco, USA) supplemented with 10% fetal bovine serum (FBS; BI, Israel), 100 U/mL penicillin, and 0.1 mg/mL streptomycin (BI, Israel)) at 37 °C in a5% CO 2 incubator.Te medium was changed every 2-3 days.When the cells reached 80% confuence, the hDPMSCs were collected and subcultured.Te hDPMSCs from passages 3 to 6 were used for subsequent experiments.

Cell Viability Assay.
Cell Counting Kit-8 (APExBIO, USA) was performed to assess the hDPMSCs viability under the manufacturer's instructions.hDPMSCs (10 4 cells/well) were seeded in a 96-well plate (Corning, USA) and incubated in fresh medium for 24 h.ASA (Sigma-Aldrich, USA) ranging from 0 μg/mL to 200 μg/mL were added to the cell suspension for 24, 48, or 72 h, and the equivalent culture medium was only added for the control.Ten, cells were maintained in 10 μL of CCK-8 solution (APExBIO, USA) at 37 °C for 2 hours [26], and the OD value (450 nm) was detected.

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Alkaline Phosphatase (ALP) Activity and Alizarin Red
Staining (ARS).hDPMSCs were plated at 3 × 10 5 cells/well into six-well plates.When the cell reached 80%-90% confuence, the culture medium was replaced by osteoinductive medium (OriCell, Guangzhou, China) with diferent concentrations of ASA (0, 25, 50, 75, and 100 µg/mL) dissolved.Te cells were cultured for 14 days or 21 days, and the media was changed every 3 days.At day 14, ALP staining was performed using a BCIP/ NBT ALP color development kit (Beyotime, Shanghai, China), following the standard protocol.Te ALP activity of hDPMSCs was determined using an alkaline phosphatase assay kit (Beyotime, Shanghai, China).Total protein concentration was examined by the BCA protein assay kit (Beyotime, Shanghai, China).Te cell lysates (10 µL) were mixed with the ALP assay working solution and assayed following the instructions of the manufacturer for normalization of the results [27].

Animal Model of Periodontitis and Cell Transplantation.
Te rats were given a general anesthesia by ingesting isofurane (R510-22-10, RWD, Shenzhen, China) via an anesthetic machine (R640, RWD, Shenzhen, China).After that, periodontitis was established by placing silk ligature (5-0) bilaterally around the subgingival portion of the second molars of rat maxilla, initiated with the concomitant local injection of 10 µL of PBS dissolution with 2 mg/mL lipopolysaccharide (LPS, L8880, Beijing, Solarbio) into the palatal gingiva, and was repeated every second day [29].After 4 weeks of periodontitis induction, the silks were removed for further experiments [30,31].
ASA-hDPMSCs group was injected with 10 6 hDPMSCs pretreated with 50 µg/mL ASA for 3 days, resuspended in 20 µL PBS at three sites around the second molars, and conducted once per week for two weeks, whereas the hDPMSCs group received the hDPMSCs only during the experimental period.Meanwhile, the ASA group was injected with the same volume of ASA solution, and the control group was injected with the same volume of PBS.Te tip of the needle was stopped at the bottom of the bone defect beneath the periosteum.For systemic injection, the rat received 3 × 10 5 of 1 mL•cell suspension or ASA solution via the tail vein.At 4 weeks after transplantation, all animals were sacrifced.

Efciency Test of Cell Transplantation.
Te in vivo transplantation efciency of the hDPMSCs was evaluated using a lipophilic carbocyanine dye DiR (Mao Kang Bio, Shanghai, China).P5 hDPMSCs were stained with DiR in plastic culture fasks when the cell reaches 80-90%.After 20 min of coculture, the cells were collected for injection in vivo.In the meantime, the control group was injected with PBS.An in vivo imaging system was utilized to assess the biodistribution after transplantation of hDPMSCs for 3 days.

Morphometric Evaluation of Alveolar Bone Loss.
Te right part of the maxilla was processed for alveolar bone morphometry.Maxilla was defeshed and stained with a 1% methylene blue solution (Solarbio, Beijing, China).Te distance between the cementum-enamel junction (CEJ) and the alveolar bone crest (ABC) at six points on the second maxillary region molars was measured by stereomicroscope (Termo Scientifc, USA) and Image J (National Institutes of Health, USA), which was then converted to millimeters as a measure of alveolar bone loss (ABL).
Te calculation formula of vertical bone loss is as follows: ABL � (three buccal + three palatal distances of mesial, central, and distal)/6.All measurements were performed by the same investigator, and all experimental data were sampled three times.

Hematoxylin Eosin Analysis and Immunohistochemical
Staining.Te left axillas were harvested and analyzed for histologic examination posteuthanization.Te maxilla blocks were fxed in 4% paraformaldehyde for 24 hours Journal of Tissue Engineering and Regenerative Medicine before being decalcifed with 26% EDTA (Solarbio, Beijing, China) for 4 weeks and fnally embedded in parafn.Serial parafn tissue sections of 5 µm were stained with hematoxylin and eosin staining (Solarbio, Beijing, China) for observation of tissue morphology.

Enzyme-Linked Immunosorbent (ELISA) Analysis.
ELISA assays were performed as previously described.Te infammatory cytokines levels of tumor necrosis factor-α (TNF-α, CUSABIO, Wuhan, China) and interleukin-1β (IL-1β, Elabscience, Wuhan, China) in gingival tissue samples where the injections were performed were analyzed by the ELISA assay following the manufacturer's instructions.
2.12.Statistical Analysis.All data are shown as the means ± SD from three independent experiments.Statistical analyses were performed using GraphPad Prism7.0 (version 7.0, La Jolla, CA).One-way ANOVA and Tukey's test were used to verify the diferences between groups.Statistical signifcance was defned as P < 0.05.

Isolation and Characterization of hDPMSCs.
Te cells with fbroblast-like morphology were observed in primary culture (Figure 1(a)).Mineralization (Figure 1(b)) and lipid deposits (Figure 1(c)) could be observed under the special induction medium for 21 days in response to osteogenic and adipogenic induction, respectively.Flow cytometry was performed to determine the expression of hDPMSCs surface markers (Figure 1(d)), including CD44, CD90, CD105, CD29, and CD73, while the negative expression of CD34, CD45, CD31, and HLA-DR.Tese data confrmed the stem cell characteristics of these isolated cells.
Terefore, <100 µg/ml ASA was used to treat hDPMSCs in the subsequent experiments.
Next, the hDPMSCs-osteoinductive function of ASA was explored.After 7 days of treatment, ALP activity was higher than in the control group at all assayed doses.When cultured for 14 days, a remarkable elevation of ALP was detected in the 50 µg/mL and 75 µg/mL ASA groups, while other concentrations (25 µg/mL and 100 µg/mL) showed no signifcant efect on ALP levels compared with the 0 µg/mL group (Figures 2(b) and 2(c)).Meanwhile, the staining in the 50 µg/ mL group was the most intense.ARS showed that the staining intensity apparently increased in the 50 µg/mL and 75 µg/mL groups but decreased in the 25 µg/mL group on day 21 (Figures 2(d) and 2(e)).
After subjecting hDPMSCs to osteogenic inductive conditions for 7 days, western blot (WB) analysis was performed to determine the level of bone-related proteins such as OPN and Runx2 (Figure 2(f )).Te results indicated that all ASA doses enhanced OPN and RUNX2 expression, with the 50 µg/mL group achieving the highest levels (Figure 2(g)).
Based on these fndings, ASA at a dose of 50 µg/mL had no signifcant efect on the hDPMSCs viability and showed the greatest efect on the osteogenic diferentiation potential in vitro.Terefore, 50 µg/mL ASA was used in subsequent experiments.

In Vivo Distribution of the hDPMSCs.
Previous studies showed that the local injection of allogeneic bone marrow mesenchymal stem cell produced a certain therapeutic efect on periodontitis.Compared with the control group, the DiRs group showed a high accumulation of the fuorescence signal in the periodontal sites.Results showed that most of the transplanted cells survived in the periodontal tissue (Figure 3).

ASA-Pretreated hDPMSCs Efects Periodontitis in Experimental Rats.
Te distance from the CEJ to the ABC of the maxillary molars was examined 4 weeks after injection in all fve groups to assess bone resorption.Te results showed that the periodontitis group had signifcant root exposure compared with the control, ASA, and hDPMSCs groups.Tere was no signifcant diference between the ASA group and the solo hDPMSCs group; however, the ASA-hDPMSCs treatment showed more bone formation than the other two groups (Figure 4(a)).
Histopathological analyses showed that the untreated periodontitis group had infamed soft tissue, deep periodontal pockets, and signifcant bone resorption.When compared to the ASA and hDPMSC groups, the treatment of ASA-hDPMSCs substantially reduced infammatory cell infltration and promoted periodontal tissue development, including cementum and periodontal ligament (Figure 4(b)).
Te infammation induced both RANKL activation and OPG inhibition in osteoblasts, which has been linked to the balance of alveolar bone formation and resorption [33].IHC showed that treatment with ASA-hDPMSCs or hDPMSCs signifcantly upregulated OPG expression compared to the periodontitis group (Figure 4  diference in expression levels between the control and ASA groups (Figure 4(c)).Relative observations of RANKL expression in the ASA-hDPMSCs group revealed that RANKL was reduced when compared to the ASA or hDPMSCs groups (Figure 4(d)).
Te quantitative analysis revealed that the periodontal ligament OPG/RANKL ratio was increased after ASA-hDPMSCs transplantation, which regulated osteoclast activation and bone resorption in the experimental rats (Figures 4(c  Figure 2: Te efects of ASA on cell proliferation and osteogenic diferentiation in vitro.(a) Proliferation assessment of hDPMSCs after culture with a series of concentrations of ASA (0, 25, 50, 75, 100, 150, and 200 µg/mL) for 24 h, 48 h, and 72 h.48 h: 0 µg/ml vs. 25 µg/ml P � 0.4285; 0 µg/ml vs. 50 µg/ml P � 0.9817; 0 µg/ml vs. 75 µg/ml P � 0.9999; 0 µg/ml vs. 100 µg/ml P � 0.9237; 0 µg/ml vs. 150 µg/ml P � 0.0196 n � 3 for all the groups.(b and c) Staining and quantitative detection of alkaline phosphatase (ALP) activity in hDPMSCs after culture in ASA under osteoinductive medium for 7 and 14 days.n � 3 for all the groups.(d and e) Alizarin red staining of the hDPMSCs under incubation of ASA in osteoinductive medium for 21 days.n � 3 for all the groups.(f ) Western blot analysis for the expression level of RUNX2 and OPN protein after ASA treatment for 7 days.(g) Quantitative analysis of the protein levels of RUNX2 and OPN.n � 3 for all the groups.All values represent the mean ± SD of triplicate experiments.* P < 0.05; * * P < 0.01; * * * P < 0.001.6 Journal of Tissue Engineering and Regenerative Medicine  Journal of Tissue Engineering and Regenerative Medicine

ASA and the Production of Infammation-Associated
Cytokines.TNF-α and IL-1 production are recognized to assist in the stimulation of infammation.Te role of local infammatory mediators in bone resorption has been extensively studied.TNF-α [34] and IL-1 have been shown to signifcantly increase RANKL-mediated osteoclast activity [35][36][37][38].In gingival tissue samples assessed by ELISA, the release of proinfammatory factors was much higher in periodontitis rats than in nondiseased rats.TNF-α and IL-1 levels were signifcantly reduced in the ASA-pretreated hDPMSCs group, but there was no statistically signifcant diference between the ASA and control groups (Figure 5).Furthermore, solo hDPMSC injections had no efcacy on TNF-α levels when compared to the periodontitis group.
We performed WB analysis and found that phospho-JNK and phospho-p38 were upregulated after 10 min (Figures 6(a) and 6(b)).Tat is, ASA treatment increased the ratios of p-JNK/JNK and p-p38/p38 (Figures 6(d) and 6(e)).However, there were no changes identifed in ERK and p-ERK expressions (Figures 6(c) and 6(f )).Tese results indicated that ASA activated the MAPK signaling pathway in hDPMSCs through phosphorylation of p38 MAPK and JNK, but not ERK.

Discussion
According to the Global Burden of Disease Study in 2016, severe periodontal disease was the 11 th most prevalent condition in the world [43], afecting the structure, function, aesthetic, and even the psychological state of the individuals [44].Te high prevalence of periodontal disease increases with age.More research is needed to focus on the burden of this "silent disease" [45].Te ambitious achievement of periodontal therapy is to treat the morphological and functional restoration in the damaged periodontium.
Developmental biology studies state that periodontal tissue is composed of neural crest-derived ectomesenchyme.As the neural crest-derived ecto-mesenchymal stem cell, dental pulp mesenchymal stem cell-based therapeutic strategy is expected to enhance periodontal tissue regeneration.However, after transplantation, the recipient's local infammatory environment limits cell biological behavior such as diferentiation and regulation of related factors.Tus, it is necessary to promote the function of transplanted cells in the tissue.
It is well known that ASA enhances the osteogenic diferentiation of mesenchymal stem cells.In this context, we explored the feasibility of ASA-pretreated hDPMSC injection in a rat periodontitis model.Based on the in vitro fndings, ASA assuredly accelerated osteoblast diferentiation of hDPMSC at a concentration of 50 µg/mL, which was consistent with the expression of osteogenic diferentiationassociated factors (RUNX2 and OPN) detected by WB.
Te destruction of bone induced by an exacerbated immune response is observed in periodontitis, which prevents the acute infammation from being efectively resolved and initiates chronic periodontitis fuence bone-related cells through the secretion of various immune factors [33].Proinfammatory cytokines induce the expression of the receptor activator of nuclear factor-κ B ligand (RANK-L) [46].RANK-L interacts with receptor activator of nuclear factor-κ B (RANK) on osteoclast precursors, resulting in the maturation of osteoclasts and destruction of alveolar bone [7].Te decoy receptor, osteoprotegerin (OPG), which inhibits the entire system by binding RANK-L.Tis is recently referred to as "the convergence hypothesis", whose fnal ratio controls the degree of osteoclast diferentiation,    (a-c) Te protein expression of p-JNK, JNK, p-p38, p38, p-ERK, and ERK was examined by western blotting analysis in 0, 10, 30, and 60 min.(d-f ) Quantifcation of p-JNK/JNK, 0 min vs. 10 min P � 0.0353; 10 min vs. 30 min P � 0.0197; 10 min vs. 60 min P � 0.0051; n � 3 for all the groups.p-p38/p38, 0 min vs. 10 min P � 0.0248; 10 min vs. 30 min P � 0.0029; n � 4 for all the groups.p-ERK/ERK, 0 min vs. 10 min P � 0.9899; 10 min vs. 30 min P � 0.8478; 10 min vs. 60 min P � 0.9998, n � 3 for all the groups.Te results were expressed as the mean ± SD. * P < 0.05; * * P < 0.01.activation, and apoptosis [47].In accordance with our results, a higher OPG/RANKL ratio was observed in the ASA-hDPMSCs administration group, indicating that osteoclast activity was inhibited and tissue repair was promoted.Besides, CEJ-ABC measurements showed very limited bone formation in the hDPMSCs group; however, improved bone tissue regeneration was observed following ASA-hDPMSCs transplantation.Tese results are consistent with previous reports [38,48] suggesting that the recipient's local microenvironment, which includes immune cells and cytokines, may infuence MSC-mediated bone regeneration capacity.Furthermore, the use of solo ASA for treatment reduced bone resorption.We hypothesized that ASA could regulate the infammatory microenvironment, resulting in moderate infammation that promotes tissue regeneration.
TNF-α and IL-1β are potent proinfammatory cytokine secreted by various cell types [49].In diabetic rats with periodontitis, incubation of osteocytes with IL-1β upregulated RANKL and downregulated OPG gene expression in static osteocytes [50].Osteocytic RANKL/sclerostin expression and osteoclast development are adversely afected by TNF-α antagonist, with osteoid formation recovering [51].In accordance with ELISA, HE staining results showed that ASA-hDPMSCs accelerated infammation resolution in rat damaged gingival tissue and redressed the microenvironment imbalance.In this regard, alveolar bone regeneration in periodontitis can thus be directly or indirectly induced by the inhibition of cellular infammatory infltrate.In the future, we will further investigate the impact of ASA on MSCs in terms of immunological function.
Regarding the mechanisms by which ASA induces osteogenic diferentiation.Te results showed that the efect of ASA on odontoblastic diferentiation of hDPMSCs was likely related to the induction of JNK and p38 phosphorylation, which is consistent with the previous fndings [39,52,53].However, further study is required to determine the specifc mechanism involved in ASA-hDPMSCsmediated periodontal repair and regeneration by using signaling pathway blockers.
Stem cell research has been one of the most frequently studied felds in dental science and medicine.Recent tissue engineering techniques provide several approaches for regenerative medicine.Te major challenge with direct injection is the loss of cell numbers and the low level of integration.We are looking forward to applying ASA-hDPMSCs in combination with cellular scafold to improve the efciency of the cells after they have settled in target tissues.
Taken together, our study frst reports that ASApretreated hDPMSCs have a positive efect on reducing infammation and promoting regeneration of the alveolar bone in periodontitis.Tis technique promises an easy and noninvasive selective therapeutic strategy for oral diseases.

Conclusion
Te restoration of severe periodontal defects is still a complex and challenging feld for clinicians.Tis is the frst study to show that ASA has a substantial infuence on the osteogenic diferentiation of cells in vitro.Moreover, in experimental periodontitis rat models, ASA-pretreated hDPMSCs administration enhanced the OPG/RANKL ratio and inhibited bone resorption, indicating alveolar bone regeneration.Besides, to restore the microenvironmental imbalance, local infammatory mediators were reduced.Collectively, the current data suggest that combining ASA with hDPMSCs is a more efcient method of treating periodontitis.Enzyme-linked immunosorbent TNF-α:

Figure 3 :
Figure 3: Te in vivo dynamic fuorescence imaging showed distribution of hDPMSCs.Te black arrows indicate the sites of the local injection.All values represent the mean ± SD of triplicate experiments n � 3.