Accepted Manuscript Title: Orthodontic Treatment Mediates Dental Pulp Microenvironment via IL17A Author: Wenjing Yu Yueling Zhang Chunmiao Jiang Wei He Yating Yi Jun Wang PII: DOI: Reference:
S0003-9969(16)30009-7 http://dx.doi.org/doi:10.1016/j.archoralbio.2016.01.009 AOB 3533
To appear in:
Archives of Oral Biology
Received date: Revised date: Accepted date:
5-7-2015 22-12-2015 19-1-2016
Please cite this article as: Yu Wenjing, Zhang Yueling, Jiang Chunmiao, He Wei, Yi Yating, Wang Jun.Orthodontic Treatment Mediates Dental Pulp Microenvironment via IL17A.Archives of Oral Biology http://dx.doi.org/10.1016/j.archoralbio.2016.01.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Microenvironment via IL17A
Wenjing Yu1,2, Yueling Zhang1, Chunmiao Jiang1, Wei He1, Yating Yi1, Jun Wang1*
State Key Laboratory of Oral Diseases, Department of Orthodontics, West China
Hospital and School of Stomatology, Sichuan University, Chengdu, Sichuan, China 2
Department of Anatomy and Cell Biology, University of Pennsylvania, School of Dental
Medicine, Philadelphia, PA, USA
Corresponding author: Jun Wang, DDS, PhD, State Key Laboratory of Oral Diseases,
Department of Orthodontics, West China Hospital and School of Stomatology, Sichuan University, No.14, 3rd section, Renmin South Road, Chengdu, Sichuan, 610041, China. Fax: +86 (28) 85582167 Email: [email protected]
Dental tissue, such as pulp, remodeling may be elevated during day 4 to 14 after orthodontic treatment.
Orthodontic tooth movement promoted self-reproductive and multidifferentiation capacities of DPSCs, especially at 7 days after orthodontic loading in rats.
Orthodontic-induced inflammation occurred in dental pulp via elevated IL17A secretion during orthodontic tooth movement.
IL17A treatment was able to promote potential of DPSCs differentiation and selfrenewal in vitro.
Abstract Objective: Orthodontic treatment induces dental tissue remodeling; however, dental pulp stem cell (DPSC)-mediated pulp micro-environmental alteration is still largely uncharacterized. In the present study, we identified elevated interleukin-17A (IL17A) in the dental pulp, which induced the osteogenesis of DPSCs after orthodontic force loading. Design: Tooth movement animal models were established in Sprague-Dawley rats, and samples were harvested at 1, 4, 7, 14, and 21 days after orthodontic treatment loading. DPSC self-renewal and differentiation at different time points were examined, as well as the alteration of the microenvironment of dental pulp tissue by histological analysis and the systemic serum IL17A expression level by an ELISA assay. In vitro recombinant IL17A treatment was used to confirm the effect of IL17A on the enhancement of DPSC self-renewal and differentiation. Results: Orthodontic treatment altered the dental pulp microenvironment by activation of the pro-inflammatory cytokine IL17A in vivo. Orthodontic loading significantly promoted the self-renewal and differentiation of DPSCs. Inflammation and elevated IL17A secretion occurred in the dental pulp during orthodontic tooth movement. Moreover, in vitro recombinant IL17A treatment mimicked the enhancement of the selfrenewal and differentiation of DPSCs.
Conclusions: Orthodontic treatment enhanced the differentiation and self-renewal of DPSCs, mediated by orthodontic-induced inflammation and subsequent elevation of IL17A level in the dental pulp microenvironment.
Keywords: Orthodontic tooth movement; Dental pulp microenvironment; Dental pulp stem cells; Interleukin 17A.
Introduction Orthodontic tooth movement is characterized by remodeling, generally considered the essence of orthodontic treatment, in dental and periodontal tissues, including the dental pulp, periodontal ligament (PDL), alveolar bone, and gingiva (1, 2). Large studies have focused on the PDL tissue and cell changes followed by orthodontic tooth movement (35); however, the underlying mechanism of tissue remodeling by orthodontic treatment is still largely unknown. In 2004, Seo et al. first established evidence to show that human PDL tissue contains periodontal ligament stem cells (PDLSCs), which provided an avenue to study the dental microenvironment by local stem cells in orthodontic treatment (6). Since then, the function of dental-tissue-derived mesenchymal stem cells (MSCs) has been carefully studied in periodontal tissue remodeling after orthodontic force loading (7, 8). Human dental pulp stem cells (DPSCs) were first isolated and identified as a novel population of post-natal somatic stem cells that are clonogenic, highly proliferative, and capable of pulp-like tissue regeneration (9, 10). In addition, DPSCs not only have similar colony forming ability, morphology, and immunomodulatory properties as bone marrow MSCs (BMMSCs), but are also capable of multiple types of differentiation, including odontogenesis, osteogenesis, chondrogenesis, adipogenesis and neuronal differentiation (11-14). In 2010, Alge et al. compared rat DPSCs with donor-matched BMMSCs, and revealed that DPSCs had an increased proliferation rate, a higher number of
stem/progenitor colonies, and an increased mineralization potential (15). Due to their easy accessibility in the clinic and unique stem cell properties, DPSCs have been considered as a promising application in regenerative medicine, including but not limited to techniques such as dental tissue repair (10, 16-19). Orthodontic tooth movement induces vasodilatation in the dental pulp (20). Additionally, several genetic markers in the dental pulp were identified using the GeneFishing technique during orthodontic treatment, suggesting that molecular signaling regulates pulp tissue remodeling at the cellular level (21). These studies indicated that dental pulp tissue continues to remodel during orthodontic tooth movement; however, the regulation of dental pulp tissue remodeling after orthodontic loading is still not fully understood. Interleukin-17A (IL17A), a pro-inflammatory cytokine, has been demonstrated to regulate downstream signaling in many cell types, including mesenchymal cells, epithelial cells, and macrophages. At the functional level, IL17A is associated with pathology in numerous autoimmune and inflammatory diseases, such as rheumatoid arthritis, multiple sclerosis, and systemic lupus erythematosus (22). Recently, IL17A secreting cells have been suggested to be involved in tooth movement and tooth root/mandibular bone resorption (23, 24). Since orthodontic tooth movement induces an inflammatory response in dental pulp tissue, we hypothesized that IL17A might be involved in pulp remodeling via the regulation of DPSC differentiation. Tooth movement in the rat using a close-coiled spring is a good model for examining force-induced remodeling (3, 25). We hypothesize that orthodontic force treatment will lead to alteration of DPSCs in vivo and that inflammation-induced IL17A may be involved in this process.
Materials and methods Animals. Female Sprague-Dawley rats were purchased from The Harlan Laboratory. The generation of orthodontic tooth-movement rats was performed as previously described (3, 25), and age-matched rats receiving sham operations served as controls. Briefly, the
bilateral maxillary first molars were moved mesially using closed coil springs. An undercut was made in the incisors, and the spring was ligated to the first maxillary molars and incisors, delivering a force of 50 g. All animal experiments were performed according to a protocol approved by the Medical Ethics Committee, West China Hospital of Stomatology, Sichuan University (SN: SKLODLL2013A161) ELISA. Peripheral blood serum was collected, and IL17A protein level was detected and analyzed using the Rat IL17A ELISA MAX™ Deluxe kit (BioLegend), Briefly, the captured antibody was incubated in a 96-well plate one day prior to running the ELISA assay. After washing and blockage of non-specific binding, samples and standards were added to a coated plate and incubated for 2 h, followed by one hour incubation with detection
Tetramethylbenzidine (TMB) substrate was reacted with HRP, and the reaction was stopped by 2 N sulfuric acid, after which the absorbance at 450 nm was noted. Isolation and culture of rat MSCs and human DPSCs (hDPSCs). For the isolation of bone marrow MSCs, bone marrow cells were flushed out of the bone cavity of rat femurs with 2% fetal bovine serum (FBS; EquitechBio,) in PBS. For isolation of rat dental pulp MSCs, the first molars of rats were carefully split apart and digested with 1 mg/mL collagenase type I (Worthington) and 2 mg/mL dispase (Roche) in PBS for 1 h at 37℃ to release the mesenchymal cells. To isolate hDPSCs, pulp tissue was gently separated from the crown and root of normal human third molars collected from adults in the clinic. The study was approved by the Institutional Review Board (IRB#HS-08-00281) at the University of Southern California (Los Angeles, CA, USA), and written informed consent was obtained from each participant. The pulp tissue was then digested in a solution of 3 mg/mL collagenase type I and 4 mg/mL dispase for 1 h at 37°C. A singlecell suspension of BM-derived all-nuclear cells (ANCs; 15×106) was seeded into 100 mm culture dishes (Corning) and cultured at 37℃ with 5% CO2. 48 h later, non-adherent cells were removed, and attached cells were cultured for 16 days in 𝛼-MEM (Invitrogen) supplemented with 20% FBS, 2 mM L-glutamine (Invitrogen), 55 μM 2-mercaptoethanol (Invitrogen), 100 U/mL penicillin and 100 μg/ml streptomycin (Invitrogen). The medium of the attached single colonies was changed frequently. The IL17A medium
concentration was 10 ng/mL, and the recombinant protein was purchased from eBioscience. Flow cytometric analysis was performed to confirm MSC surface markers. FITC Anti-rat CD29, CD45 and CD90 were purchased from BioLegend. Osteogenic and adipogenic differentiation assays of MSCs. For differentiation induction in vitro, rat MSCs were seeded into 6-well plates (Corning) and cultured in growth medium until the cells reached confluence. To induce osteogenic differentiation, the MSCs were cultured under osteogenic culture conditions, containing 2 mM βglycerophosphate (Sigma-Aldrich), 100 μM L-ascorbic acid phosphate (Wako), and 10 nM dexamethasone (Sigma-Aldrich) in the growth medium. After 3-4 weeks induction, 1% Alizarin Red S (Sigma-Aldrich) staining was performed to detect matrix mineralization (26). After staining, plates were scanned and images were converted to black and white mode. To retain Alizarin Red S, images were analyzed and quantiﬁed using NIH ImageJ software with a 50% threshold by determining the area positive for dye staining expressed as a fraction of the total area. For adipogenic induction, 500 nM isobutylmethylxanthine (Sigma-Aldrich), 60 μM indomethacin (Sigma-Aldrich), 500 nM hydrocortisone (Sigma-Aldrich), 10 μg/mL insulin (Sigma-Aldrich), and 100 nM Lascorbic acid phosphate were added to the growth medium. After 2-3 weeks, the induced cells were stained with Oil Red O (Sigma-Aldrich). The positive cells were quantified under microscopy and are shown relative to the total cell count. Western blot analysis. Cells were lysed in M-PER mammalian protein extraction reagent (Thermo Fisher Scientific) with protease and phosphatase inhibitors (Roche), and then protein content was quantified using the PierceTM BCA Protein Assay Kit (Thermo scientific). 30 μg of protein was separated on an SDS-PAGE gel and transferred to nitrocellulose membranes (Millipore). The membranes were blocked with 1% non-fat milk (Santa Cruz), 4% BSA, and 0.05% Tween-20 for 1 h and then incubated overnight with primary antibodies diluted in incubation buffer, according to the manufacturer’s instructions. Antibodies to rat and human alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), Peroxisome proliferator-activated receptor gamma (PPAR-γ), and lipoprotein lipase (LPL) were purchased from Santa Cruz Biotechnology, Inc. Antibody to rat β-actin was purchased from Sigma-Aldrich. The
membranes were incubated for 1 h in HRP-conjugated secondary antibody (Santa Cruz Biotechnology, Inc.) diluted at 1: 100,000 in incubation buffer. Immunoreactive proteins were then detected by SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific) and BioMax film (Kodak). BrdU staining. MSCs were seeded into 8-well chamber slides (Thermo scientific) at a concentration of 2×104 per well. After 4 h, BrdU labeling reagent (Invitrogen) was added to the medium, after which the cells were incubated at 37°C overnight. The cells were stained using the Streptavidin-Biotin System for the BrdU Staining kit (Invitrogen) (27), and the positive cells were quantified under microscopy and presented relative to the total number of cells. Histological analyses. Maxilla were fixed in 4% paraformaldehyde (Sigma-Aldrich) and decalcified with 10% EDTA (pH 7.4) followed by paraffin embedding. Paraffin sections (7μm) were stained with hematoxylin and eosin (H&E) and analyzed using ImageJ software. To perform immunofluorescent staining, the paraffin-embedded sections were incubated with primary anti-IL17A antibody (1:200; Santa Cruz Biotechnology, Inc.) at 4°C overnight with rabbit IgG antibody used as negative control, and then treated with Alexa Fluor 594-conjugated secondary antibody (1:200, Life technologies) for 30 min at room temperature. Finally, slides were mounted with VECTASHIELD mounting medium (Vector Laboratories). Statistics. Data were analyzed using SPSS 11.5 software and are expressed as mean ± SD values. Comparisons between two groups were analyzed by independent unpaired two-tailed Student’s t-tests, and comparisons between more than two groups were analyzed by one-way analysis of variance (ANOVA). A P-value < 0.05 was considered statistically significant.
Results Measurement of tooth movement To test our hypothesis that DPSCs may be involved in orthodontic treatment-induced dental pulp tissue remodeling, we first established and examined a rat model of orthodontic tooth movement. The distance of tooth movement gradually increased in a time-dependent manner after orthodontic loading, by which the speed of tooth movement was slightly increased from day 1 to day 14 and decreased after day 14 (Figure 1A). From day 4 to day 14, teeth moved at a higher speed, and 67% of total movement was completed throughout 21 days (Figure 1A). This result indicates that dental tissue remodeling for tissues such as pulp may be elevated between day 4 and 14, and DPSCs may also be activated during this timeframe after orthodontic treatment.
Isolation and identification of DPSCs To examine whether DPSCs are involved in dental pulp tissue remodeling during orthodontic treatment, we then intended to isolate DPSCs from the animal model at different time points. It is well documented that clonogenic populations of DPSCs can be isolated from human and rat dental pulp tissues (9, 28). Here, we demonstrated the clonogenesis process of primary DPSCs. Cells began to proliferate on day 2, and mature clones could be seen on day 10 in primary cultured DPSCs (Figure 1B). P1 cells were used for flow cytometry analysis to examine the mesenchymal stem cell surface markers of DPSCs. The results showed that DPSCs had positive expression of the MSC surface markers CD29 and CD90, but were negative for the hematopoietic stem cell marker CD45, which was similar to that seen in BMMSCs (Figure 1C), implying that rat DPSCs may have similar properties as BMMSCs.
The self-renewal and differentiation capacities of DPSCs after orthodontic force loading We next examined the self-reproductive and multidifferentiation capacities of DPSCs after orthodontic loading. DPSCs after orthodontic force loading showed higher selfrenewal compared to control cells, as assessed by 5-bromo-2’-deoxyuridine (BrdU) incorporation assays (Figure 2A, 2B). Using rat BMMSCs as positive controls, the highest self-renewal capacity among all time points occurred at 7 days post-orthodontic treatment in rat DPSCs, which was comparable to that seen in BMMSCs. This data indicates that orthodontic treatment stimulus promotes the DPSC self-reproductive capacity, and this orthodontically induced self-reproductive enhancement reached its peak after 7 days of force loading. Next, we examined the ability of rat DPSCs to differentiate towards the adipogenic and osteogenic lineages. Under adipogenic inductive conditions, the amount of differentiation of normal DPSCs was limited, as determined by Oil Red O staining (Figure 2C, 2D). After introduction of orthodontic treatment, the adipogenesis potential of DPSCs increased in a time-dependent manner until 14 days after treatment (Figure 2C, 2D). Western Blot analysis showed similar results, that orthodontic treatment increased the expression of the adipogenic proteins PPARγ and LPL; however, the protein levels reached their peak at 4 days after orthodontic loading and then began to decrease (Figure 2E). In addition, Alizarin Red staining result showed that DPSCs had a similar osteogenic capacity compared to BMMSCs, while the osteogenesis of DPSCs after orthodontic loading significantly increased when compared to the untreated group. Osteogenesis increased until 7 days after orthodontic loading, and slightly decreased at 14 days (Figure 2F, 2G). Likewise, Western Blot analysis further confirmed the orthodontically induced enhancement of osteogenic capacity by the increased expression levels of the osteogenic proteins RUNX2, OCN and ALP (Figure 2H). These findings suggest that orthodontic tooth movement promotes the self-reproductive and multidifferentiation capacities of DPSCs, especially at 7 days after orthodontic loading, in rats.
Orthodontic-induced dental pulp microenvironment alteration contributes to the enhancement of self-renewal and differentiation in DPSCs To further explore how orthodontic force leads to the enhancement of the selfreproductive and differentiation capacities in DPSCs, we performed histological analysis and found that the number and area of blood vessels immediately increased at day 1 in the dental pulp of the orthodontic-treated rat teeth after the orthodontic treatment load was applied. The blood vessels gradually increased in number and area, reaching a peak at day 14 after loading, a level that was maintained until day 21, as seen by Hematoxylin and Eosin (HE) staining (Figure 3A, 3B). This data suggests that orthodontic treatment induced aseptic inflammation through blood vessel dilation in dental pulp tissue. Since the pro-inflammatory cytokine IL-17A is involved in multiple types of cells for the pathophysiology of numerous autoimmune and inflammatory diseases (22), we then examined the level of IL17A in the orthodontic tooth movement model. Using an ELISA assay, the results demonstrated that the level of IL17A in rat serum significantly increased after orthodontic loading. The serum IL17A level increased dramatically on the first day of orthodontic loading and then began to drop slowly. On day 14 after treatment loading, the serum IL17A level returned a level comparable to that seen in the control group (Figure 3E). Then, we used immunofluorescent staining to detect IL17A expression level in the dental pulp tissue of teeth that underwent movement in the experimental rats. The results displayed that IL17A exists in the dental pulp tissue under normal conditions, whereas it increases immediately after orthodontic loading and then drops to the normal level at day 14 (Figure 3C, 3D). These results imply that orthodonticinduced inflammation occurs in dental pulp via elevated IL17A secretion during orthodontic tooth movement, in which alteration of the microenvironment of the dental pulp might be the key to promoting DPSC self-renewal and differentiation.
Recombinant IL17A enhances hDPSC self-renewal and differentiation In order to confirm the hypothesis that IL17A plays an important role in the orthodonticinduced alteration of DPSC differentiation, which may be utilized in human clinical 10
applications, we then applied 10 ng/mL recombinant human IL17A protein to hDPSCs and examined their differentiation and self-renewal in vitro. The osteogenic and adipogenic assays showed a remarkable upregulation of osteogenesis and adipogenesis by Alizarin Red staining and Oil Red O staining, respectively (Figure 4A, 4B). By Western Blot analysis, we then confirmed the elevated protein expression levels of osteogenic and adipogenic markers after IL17A treatment (Figure 4D, 4E). Additionally, BrdU staining indicated an increase of self-renewal capacity in the IL17A treatment group (Figure 4C). These data illustrate that increased IL17A expression promotes the differentiation and self-renewal of DPSCs.
Discussion In the present study, we found that orthodontic force treatment promoted the self-renewal and differentiation of DPSCs in vivo, related to force-induced inflammation in the dental pulp via IL17A. Specifically, treatment with recombinant IL17A stimulated differentiation of DPSCs in vitro. By focusing on PDL tissue and cell remodeling during orthodontic tooth movement, the functional alteration of PDLSCs after force loading has previously been studied. However, little is yet known about how DPSCs function during orthodontic loading, since dental pulp remodeling has also been attributed to orthodontic tooth movement. In this study, we show that the speed of orthodontic tooth movement is not static. Teeth moved slowly at the very beginning of force loading, which accelerated from day 4 until day 14 after treatment, and then slowed down again. Our findings show a similar pattern in the speed of rat orthodontic tooth movement as in previous publications (33, 34). This might suggest that the dental pulp microenvironment, as well as DPSCs, may be primed to facilitate tooth movement-mediated tissue remodeling from day 4 to day 14 after orthodontic loading. In this study, we show that the speed of orthodontic tooth movement is not static. Teeth moved slowly at the very beginning of force loading, which accelerated from day 4 until
day 14 after treatment, and then slowed down again. Our findings show a similar pattern in the speed of rat orthodontic tooth movement as in previous publications (33, 34). This might suggest that the dental pulp microenvironment, as well as DPSCs, may be primed to facilitate tooth movement-mediated tissue remodeling from day 4 to day 14 after orthodontic loading. Orthodontic-induced alteration of the dental pulp microenvironment has been reported in previous studies (21, 35), which might lead to some side effects of orthodontic treatment such as loss of tooth vitality and orthodontic-induced root resorption (36, 37). However, it remained unknown whether the alteration in dental pulp microenvironment is related to DPSCs. To answer this question, we isolated and studied DPSCs after orthodontic loading, and we show for the first time that DPSCs are in an active state during orthodontic tooth movement, with elevated self-renewal and differentiation after orthodontic loading. Inflammation and enhanced angiogenesis are characteristics of orthodontic-loaded teeth (25, 38). Our findings also showed a significant increase in the number and area of blood vessels in the dental pulp of the experimental rat teeth. IL17A, a pro-inflammatory cytokine, is able to induce an inflammatory reaction as well as the production of other inflammatory cytokines, such as IL6, TNFα, and IFNγ (39, 40), which are associated with the pathology of numerous autoimmune and inflammatory diseases (41, 42). In 2013, Ruili et al. claimed that IL17A could activate the NFkappaB pathway to downregulate TGF-β production in MSCs, resulting in the abolishment of MSC-based immunomodulation (43). T helper 17 (Th17) cells and macrophages, as the major source of IL17A production, have been reported as associating with orthodontic tooth remodeling (24, 44). The role of IL17A in osteogenic differentiation has been extensively studied; however, the findings are controversial, suggesting that IL17A may play different roles in certain conditions (45, 46). Based on these findings, we speculated that IL17A might associate with the orthodontic-induced alteration of the pulp microenvironment and the enhancement of DPSC lineage differentiation. We then examined the IL17A expression levels in systemic serum and local dental pulp tissue, and illustrated a significant increase of IL17A expression both systemically and locally, suggesting that IL17A might play a critical role in orthodontic-induced dental tissue 12
remodeling. In addition, IL17A markedly enhanced both osteogenic and adipogenic differentiation, as well as the self-renewal of DPSCs in in vitro culture, which indicated that orthodontic-induced pulp tissue remodeling is mediated by IL17A, mainly through DPSCs. These findings suggest that IL17A, as well as other cytokines and chemokines, may be involved in force-induced pulp remodeling by the interplay with DPSCs, and immune cells will be an important target in orthodontic force-induced alteration of the dental pulp microenvironment. Taken together, our results indicate that orthodontic treatment induced inflammation and altered the microenvironment of the dental pulp tissue via increased IL17A expression, which promoted DPSC differentiation and self-renewal.
Conclusions Orthodontic treatment can enhance DPSC differentiation and self-renewal, due to orthodontic-induced inflammation and microenvironment alteration in the dental pulp via elevated IL17A expression after orthodontic loading.
Acknowledgement This work was supported by grants from the National Natural Science Foundation of China (No. 81271718). The authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.
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Figure legends Figure 1. The isolation and identification of DPSCs. (A) The distance that teeth moved at different time points. During day 4 to day 14, the teeth had a higher speed, suggesting that DPSCs may be more active during that period. (B) The colony formation process of primary rat DPSC culture. The bar represents 500 μm. (C) The expression of cell surface markers determined by flow cytometry, showing that DPSCs have similar cell surface markers as BMMSCs. Figure 2. Orthodontic force enhanced the self-renewal and differentiation capacities of DPSCs. (A, B) BrdU staining of DPSCs at different time points of orthodontic loading. These results indicate that an orthodontic force stimulus increased the DPSC selfreproductive capacity, and this forced-induced self-reproductive enhancement reached its peak on day 7. (C, D) Oil Red O staining of DPSCs at different time points of orthodontic loading shows lipid accumulation. DPSCs lacked the ability of adipogenic differentiation compared to BMMSCs, but the orthodontic tooth movement force triggered adipogenesis, especially after 14 days. (E) Western Blot results showed that orthodontic force increased adipogenic protein expression. (F, G) Alizarin Red staining of DPSCs at different time points of orthodontic loading shows calcium nodule formation. DPSCs had a similar osteogenesis capacity compared to BMMSCs, and orthodontic force stimulated this osteogenic differentiation capacity better than in BMMSCs. (H) Western Blot results showed increased osteogenic protein expression in DPSCs after orthodontic force loading, especially after 7 days. The bar represents 50 μm. Error bars represent the SD. Statistical significance was determined with one-way ANOVA, *P < 0.05; **P < 0.01; ***P < 0.005; NS, not significant. Figure
microenvironment alteration. (A, B) HE Staining of maxillary first molar sections showed increased blood vessel number and area during orthodontic loading. (C, D) Immunofluorescence of the maxillary first molar sections. This revealed that in the dental pulp tissue, the level of IL17A expression increased immediately on day 1 of orthodontic force loading, and then returned to the normal level. (E) ELISA of the serum IL17A level from orthodontically treated rats at different time points. This data illustrates that on
the first day of orthodontic tooth movement, the serum IL-17A level increased dramatically, and then began to decline slowly. The bar represents 50 μm. Error bars represent the SD. Statistical significance was determined with one-way ANOVA, *P < 0.05; **P < 0.01; ***P < 0.005; NS, not significant.
Figure 4. IL17A enhanced DPSC differentiation in vitro. (A) Alizarin Red staining of hDPSCs. IL17A stimulated increased calcium nodule formation compared to the control group. (B) Oil Red O staining of hDPSCs. IL17A triggered lipid accumulation in hDPSCs. (C) BrdU staining of hDPSCs. IL17A stimulated replication of hDPSCs at a higher level than the control group. (D, E) Western Blot results show that IL17A treatment increased the expression of both osteogenic and adipogenic proteins in hDPSCs. The bar represents 50 μm. Error bars represented the SD. Statistical significance was determined with independent unpaired two-tailed Student's t-tests, *P < 0.05; **P < 0.01.