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Angela Quispe-Salcedo, [Tomohiko Yamazaki](https://orcid.org/0000-0003-2136-8042), Hayato Ohshima

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[Effects of Synthetic Toll-Like Receptor 9 Ligand Molecules on Pulpal Immunomodulatory Response and Repair after Injuries](https://mdr.nims.go.jp/datasets/6bcb73d3-df2b-4c97-b074-9bcc47e64e4c)

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Effects of Synthetic Toll-Like Receptor 9 Ligand Molecules on Pulpal Immunomodulatory Response and Repair after InjuriesCitation: Quispe-Salcedo, A.;Yamazaki, T.; Ohshima, H. Effects ofSynthetic Toll-Like Receptor 9 LigandMolecules on Pulpal Immunomodulatory Response and Repair afterInjuries. Biomolecules 2024, 14, 931.https://doi.org/10.3390/biom14080931Academic Editor: Myron R.SzewczukReceived: 22 June 2024Revised: 26 July 2024Accepted: 29 July 2024Published: 1 August 2024Copyright: © 2024 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).biomoleculesArticleEffects of Synthetic Toll-Like Receptor 9 Ligand Molecules onPulpal Immunomodulatory Response and Repair after InjuriesAngela Quispe-Salcedo 1 , Tomohiko Yamazaki 2 and Hayato Ohshima 1,*1 Division of Anatomy and Cell Biology of the Hard Tissue, Niigata University Graduate School of Medical andDental Science, Niigata 951-8514, Japan; aquispesa@dent.niigata-u.ac.jp2 Research Center for Macromolecules and Biomaterials, National Institute of Material Sciences (NIMS),Tsukuba 305-0047, Japan; yamazaki.tomohiko@nims.go.jp* Correspondence: histoman@dent.niigata-u.ac.jp; Tel.: +81-25-227-2812Abstract: Synthetic oligodeoxynucleotides (ODNs) containing unmethylated cytosine–phosphate–guanine (CpG) motifs (CpG-ODNs) are ligand molecules for Toll-like receptor 9 (TLR9), whichis expressed by odontoblasts in vitro and dental pulp cells. This study determined the effectsof CpG-ODNs on pulpal immunomodulatory response and repair following injury. Briefly, theupper right first molars of three-week-old mice were extracted, immersed in Type A (D35) or B(K3) CpG-ODN solutions (0.1 or 0.8 mM) for 30 min, and then replanted. Pulpal healing andimmunomodulatory activity were assessed by hematoxylin–eosin and AZAN staining, as well asimmunohistochemistry. One week following the operation, inflammatory reactions occurred in all ofthe experimental groups; however, re-revascularization and newly formed hard tissue depositionwere observed in the pulp chamber of all groups at week 2. A positive trend in the expression ofimmune cell markers was observed toward the CpG-ODN groups at 0.1 mM. Our data suggest thatsynthetic CpG-ODN solutions at low concentrations may evoke a long-lasting macrophage–TLR9-mediated pro-inflammatory, rather than anti-inflammatory, response in the dental pulp to modulatethe repair process and hard tissue formation. Further studies are needed to determine the effects ofcurrent immunomodulatory agents in vitro and in vivo and develop treatment strategies for dentaltissue regeneration.Keywords: dental pulp; CpG oligonucleotide; Toll-like receptor 9; mice; odontoblasts; tooth replantation;tooth injuries1. IntroductionSince their discovery in the early 90s, synthetic oligodeoxynucleotides (ODNs) con-taining unmethylated cytosine–phosphate–guanine (CpG) motifs (CpG-ODNs) have beenevaluated because of their high immunostimulatory activity. Comparable to bacterial DNA,synthetic CpG-ODNs trigger an immunostimulatory effect that leads to the maturation,differentiation, and proliferation of immune cells, such as B- and T lymphocytes, naturalkiller (NK) cells, monocytes, macrophages, and dendritic cells (DCs) [1,2]. Moreover, therecognition of CpG motifs is associated with Toll-like receptor (TLR) signaling. Earlystudies demonstrated that macrophages from TLR9 knockout mice did not respond toCpG DNA but to ligands for TLR2 and TLR4. Moreover, TLR9 expression in immune cellscorrelated with responsiveness to CpG DNA, inducing an innate immune response thattriggers alterations in the cellular reduction–oxidation balance and the induction of cellsignaling pathways, including the mitogen-activated protein kinases and necrosis factorkappa beta (NFκB) [3–5]. Regarding the structural and functional differences betweenboth types, Type A CpG-ODNs contain a central palindromic CpG motif and form higher-order structures. They are primarily used for their strong interferon response, targetingplasmacytoid dendritic cells (pDCs) and eliciting systemic immunity. Type A CpG-ODNsBiomolecules 2024, 14, 931. https://doi.org/10.3390/biom14080931 https://www.mdpi.com/journal/biomoleculeshttps://doi.org/10.3390/biom14080931https://doi.org/10.3390/biom14080931https://creativecommons.org/https://creativecommons.org/licenses/by/4.0/https://creativecommons.org/licenses/by/4.0/https://www.mdpi.com/journal/biomoleculeshttps://www.mdpi.comhttps://orcid.org/0000-0002-6449-4631https://orcid.org/0000-0003-2136-8042https://orcid.org/0000-0001-8571-9900https://doi.org/10.3390/biom14080931https://www.mdpi.com/journal/biomoleculeshttps://www.mdpi.com/article/10.3390/biom14080931?type=check_update&version=1Biomolecules 2024, 14, 931 2 of 16also induce small amounts of inflammatory cytokines, such as IL-6. In contrast, Type BCpG-ODNs form linear structures and induce IL-6 secretion from TLR9-expressing cells,mainly B- and macrophage cells in mice. This results in the activation of B cells, promotingtheir proliferation and differentiation [1,2]. Current therapeutic applications of CpG-ODNsinclude their role as activators of innate immune defense against infections and potentagents for immunotherapy against cancer, allergies, asthma, and vaccine adjuvants [6,7].Despite multiple applications from basic research to clinical medicine, their potential use inthe dental field has not yet been evaluated.Dental trauma, such as luxation injuries or avulsion, and replantation directly affectthe dental pulp, periodontium, and surrounding bone, resulting in total severance of theneurovascular supply [8]. The outcome of pulpal severance will be either total pulpalre-vascularization or the development of partial or total pulp necrosis, which is dependenton the race between cellular ingrowth and bacterial invasion [8,9]. Following injury,an innate immune response occurs in the pulpal tissue, in which vascular alterationsand inflammatory cell infiltration are activated to eliminate the irritating molecules [9].Odontoblasts are located at the outermost cell layer of the dental pulp and are consideredthe first line of defense, as they are involved in the innate and adaptive immunity ofthe dental pulp against invading bacteria and noxious stimuli from the microflora ofthe oral cavity [10,11]. For example, when bacteria and their products invade deeplyinto dentinal tubules, odontoblasts are the first pulpal cells encountered by these dentin-invading microorganisms. They sense pathogen-associated molecular patterns (PAMPs)shared by microorganisms through specialized pattern recognition receptors (PRPs) atthe dentin–pulp interface [12–14]. In vitro and in vivo studies have demonstrated thatodontoblasts from humans and rodents express several PRRs, including members of theTLR family from 1–10 and nucleotide oligomerization binding domains. Interestingly,differentiated odontoblasts from mice, rats, and humans constitutively express TLR9 genesin vitro [11,12,15–19], which recognize CpG motifs in bacterial DNA and synthetic CpG-ODNs to elicit a tissular innate immune response. Exposure to CpG DNA induces a strongpro-inflammatory response in cultured mouse odontoblasts through the TLR9/MyD88/NF-κB signaling pathway. Similarly, exposure to CpG-ODNs induces a significant increase ininterleukin (IL)-8 and matrix metalloproteinase (MMP)-13 expression through the TLR9,MyD88, NF-κB, and ERK pathways in odontoblast-like cells in mice [15,18,19]. Althoughinformative, the available in vitro data on the immunostimulatory effects of CpG-ODNs inodontoblast-like cells should be analyzed in living organisms to determine the biologicalresponse to synthetic CpG-ODNs in pulpal tissue, particularly following injury.We established a tooth replantation model using mice [20–23] to assess the chrono-logical changes and cellular events that occur in dental pulp following injury. This modelmimics the clinical situation of avulsion, in which a tooth is completely displaced fromthe alveolar socket, thus making it a reliable experimental design to evaluate the effects ofexperimental compounds on the pulpal tissue of extracted teeth following replantation. Inthis study, we analyzed for the first time the effects of synthetic CpG-ODNs, TLR 9 ligandmolecules, on the pulpal immunomodulatory response and repair processes after injuryusing a mouse model for intentionally delayed tooth replantation.2. Materials and Methods2.1. In Vivo Experimental Procedures2.1.1. AnimalsAll animal experiments were conducted following the ARRIVE guidelines [24], and theprotocol was reviewed by the Institutional Animal Care and Use Committee and approvedby the President of Niigata University (approval #: SA00298 and SA01017).Ninety-two male Crlj:CD1 Institute of Cancer Research (ICR) mice (three weeks old)were obtained from Charles River Laboratories Japan (Yokohama, Japan). The animals weredivided into six experimental groups as follows: Distilled water (DW), Hank’s Balanced SaltSolution (HBSS, Fujifilm Wako Pure Chemical Industries, Ltd., Osaka, Japan), and CpG-ODNBiomolecules 2024, 14, 931 3 of 16A and B groups at 0.1 mM and 0.8 mM concentrations (see Supplementary Table S1). Thecharacteristics of the synthetic CpG-ODN solutions were as follows:CpG-ODN A: Class A CpG oligonucleotide (human and mouse), endotoxin-freeD35, partially phosphorothioated backbone, and the DNA sequence 5′-GGT GCA TCGATG CAG GGG GG-3 (Catalog #: 65001; GeneDesign Inc., Osaka, Japan) tested at finalconcentrations of 0.63 mg/mL (0.1 mM) and 5 mg/mL (0.8 mM) in DW.CpG-ODN B: Class B CpG oligonucleotide (human and mouse), endotoxin-free K3,phosphorothioated backbone, and the DNA sequence 5′-ATC GAC TCT CGA GCG TTCTC-3′, (Catalog #: 65003; GeneDesign) tested at final concentrations of 0.63 mg/mL (0.1 mM)and 5 mg/mL (0.8 mM) in DW.2.1.2. Tooth ReplantationThe intentionally delayed tooth replantation injury model was performed, as describedin our previous studies [20,21]. Briefly, the upper left and right first molars of each animal(one mouse, n = two teeth) were consecutively extracted under deep anesthesia followingan intraperitoneal injection of a mixed solution (0.05–0.1 mL/10 g) comprising 1.875 mLof Domitor® (Nippon Zenyaku Kogyo Co., Ltd., Koriyama, Japan), 2 mL of midazolam(Sandoz KK, Tokyo, Japan), 2.5 mL of Vetorphale® (Meiji Seika Pharma Co., Ltd., Tokyo,Japan), and 18.625 mL of physiological saline using a pair of modified dental forceps.The extracted teeth were immersed for 30 min in the experimental solutions and thenrepositioned in their original sockets without further treatment, such as the fixation of teethor relief of occlusion.2.2. Tissue PreparationSamples were collected from groups of 5–11 animals on weeks 1 and 2 following theoperation. At each observation point, the mice were transcardially perfused with physiolog-ical saline, followed by 4% paraformaldehyde (PFA; Sigma-Aldrich, St. Louis, MO, USA) in0.1 M of phosphate buffer (pH 7.4) under deep anesthesia with an intraperitoneal injectionof a mixed solution of Domitor®, midazolam, Vetorphale®, and physiological saline. Themaxillae were removed en bloc and immersed in 4% PFA for another 24 h at 4 ◦C. Afterdecalcification in Morse’s solution (10% sodium citrate and 22.5% formic acid) for 4–5 daysat 4 ◦C, the samples were dehydrated through a series of ethanol grades, embedded inparaffin, and sagittally cut at a thickness of 4 µm. The sections were then processed forhematoxylin–eosin (H&E) staining, AZAN staining, and immunohistochemistry.2.3. Immunohistochemical ProceduresSections were processed using the Envision + Horseradish Peroxidase System (DakoJapan, Tokyo, Japan; catalog #: K5027) and a mouse anti-nestin monoclonal antibodydiluted to 1:200 (Millipore, Temecula, CA, USA; catalog #: MAB353) and a rabbit poly-clonal anti-mannose receptor (CD206) antibody diluted to 1:1000 (Abcam, Cambridge,MA, USA; catalog #: ab64693). The avidin–biotin–peroxidase complex (Vectastain ABCKit, Vector Laboratories, Burlingame, CA, USA) method was performed using a mousemonoclonal anti-Ki67 antibody diluted to 1:100 (Dako Japan; catalog #: M7249), a ratmonoclonal anti-F4/80 antibody diluted to 1:250 (Novus Biologicals USA, Centennial,CO, USA; catalog #: NB600-404), and a mouse monoclonal anti-TLR9 antibody dilutedto 1:200 (Abcam; catalog #: ab12121), along with a biotinylated anti-mouse IgG (H+L)diluted to 1:100 (Vector Laboratories; catalog #: BA-2001) for Ki-67 and TLR9, and a bi-otinylated anti-rat IgG (H+L) diluted to 1:100 (Vector Laboratories; catalog #: BA-4000)for F4/80. To visualize the sections, 0.05 M of Tris-HCl buffer (pH 7.6) containing 0.04%3-3′-diaminobenzidine tetrahydrochloride and 30% H2O2 was used. The sections werecounterstained with hematoxylin.Biomolecules 2024, 14, 931 4 of 162.4. Cell Counting and Statistical AnalysisData analyses from H&E staining and immunohistochemistry at weeks 1 and 2 wereanalyzed using Image J software (Image J 1.53k, National Institutes of Health, Bethesda,MD, USA). The percentage of nestin-positive perimeters was calculated relative to the totalperimeter of the pulp–dentin border. Similarly, the rate of newly formed hard tissue areaswas calculated relative to the total area of the dental pulp. The percentage of cell density forKi-67- and CD206-positive cells in the pulpal area of each specimen was obtained using amine-squared grid of 2600 µm2 (total counting area: 23,400 µm2). For the analysis of F4/80and TLR9 immunostaining, the percentage of the area fraction or the stained area relativeto the total area of the pulp chamber was quantitated by adjusting the threshold using theplug-in Color Deconvolution 2 for Image J. All data are presented as the mean and standarddeviation for each group. Statistical analysis was performed using IBM® SPSS® software(Ver 21, IBM, Tokyo, Japan). Data normality was analyzed with the Shapiro–Wilk test. Forcomparisons between groups, Bonferroni’s test for multiple comparisons was used afterthe confirmation of data normality and homogeneity of variance. The samples showing nonormal distribution were compared by the Kruskal–Wallis test for more than three groupsor the Mann–Whitney U test for two groups.3. Results3.1. Histological Evaluation of the Dental Pulp Healing Process by H&E and AZAN Staining andNestin ImmunohistochemistryH&E and AZAN staining were performed to evaluate the morphological changesin the pulpal tissue and the presence of collagen-related matrices in the dental pulp,respectively. Furthermore, nestin was used as a marker to identify surviving odontoblastsand/or newly differentiated odontoblast-like cells. The progression of pulpal healing wasdetermined by the percentage of the nestin-positive perimeter at the pulp–dentin border.In addition, the newly formed hard tissue, such as tertiary dentin or bone-like tissue inthe dental pulp, was determined based on nestin-positive or -negative expression aroundthese areas. One week after replantation, the pulpal tissue of the replanted teeth exhibitedinflammatory features based on the type and concentration of each experimental solution(Figure 1A,D,G,J,M). Severe inflammatory reactions, including hemorrhagic areas, wereobserved in the whole dental pulp of replanted teeth treated with CpG-ODN A at 0.8 mM(asterisk in Figure 1G). Re-vascularization did not occur in the coronal pulp of the CpG-ODN B groups at 0.1 mM and 0.8 mM, whereas clear blood lumens were distinguished inthe pulpal floor of the DW and CpG-ODN A 0.1 mM groups (double arrows in Figure 1A,D),and the HBSS group (double arrows in Supplementary Figure S1A). Collagen-related bluematrices were faintly observed by AZAN staining in the dental pulp of the DW (arrowin Figure 1B) and HBSS groups (arrow in Supplementary Figure S1B) but barely detectedin the CpG-ODN groups (Figure 1E,H,K,N). Immunohistochemistry for nestin revealedthe presence of surviving odontoblasts and/or odontoblast-like cells (arrows) aligningunder the pulp–dentin border of the replanted teeth, as well as numerous nestin-positivefilamentous structures (arrowheads), occupying the pulpal areas where the healing processwas occurring (Figure 1C,F,I,L,O and Figure S1C). Most of the cells located under thetip of the pulpal horns lacked nestin immunoreactivity in the DW (Figure 1C), HBSS(Supplementary Figure S1C), CpG-ODN A 0.1 mM, and CpG-ODN B 0.1 mM and 0.8 mMgroups (Figure 1F,L,O), whereas only scattered nestin-positive cells were detected in thecoronal pulp of replanted teeth of the CpG-ODN A 0.8 mM group (Figure 1I). Significantdifferences in the rate of nestin-positive perimeters over the total perimeter of the dentalpulp of replanted teeth were observed between the HBSS and CpG-ODN groups and DW(p < 0.05 or p = 0.01), particularly those treated with a concentration of 0.8 mM (p < 0.01)(Figure 2A).Biomolecules 2024, 14, 931 5 of 16Biomolecules 2024, 14, x FOR PEER REVIEW 5 of 17   Figure 1. Hematoxylin–eosin (H&E) staining (A,D,G,J,M), AZAN staining (B,E,H,K,N), and nestin immunoreactivity (C,F,I,L,O) in samples of extracted teeth treated with DW (A–C), CpG-ODN A at 0.1 mM (D–F), CpG-ODN A at 0.8 mM (G–I), CpG-ODN B at 0.1 mM (J–L), and CpG-ODN B at 0.8 mM (M–O) one week after replantation. (A,D,G,J,M) The pulpal tissue of all replanted teeth shows inflammatory features. Hemorrhagic areas are observed in CpG-ODN A at 0.8 mM (asterisk in G). Clear blood lumens are distinguished in the pulpal floor of the DW and CpG-ODN A 0.1 mM groups (double arrows in A,D). (B,E,H,K,N) Collagen-related matrices are faintly observed in blue color in the DW group (arrow in B) but barely in the CpG-ODN groups. (C,F,I,L,O) Surviving odontoblasts and/or odontoblast-like cells (arrows) align under the pulp–dentin border of replanted teeth, as well as in numerous nestin-positive filamentous structures (arrowheads). AB: Alveolar bone, DP: Dental pulp. Scale bars: 250 µm (A,D,G,J,M) and 100 µm (B,C,E,F,H,I,K,L,N,O). Figure 1. Hematoxylin–eosin (H&E) staining (A,D,G,J,M), AZAN staining (B,E,H,K,N), and nestinimmunoreactivity (C,F,I,L,O) in samples of extracted teeth treated with DW (A–C), CpG-ODN Aat 0.1 mM (D–F), CpG-ODN A at 0.8 mM (G–I), CpG-ODN B at 0.1 mM (J–L), and CpG-ODN B at0.8 mM (M–O) one week after replantation. (A,D,G,J,M) The pulpal tissue of all replanted teethshows inflammatory features. Hemorrhagic areas are observed in CpG-ODN A at 0.8 mM (asterisk in(G)). Clear blood lumens are distinguished in the pulpal floor of the DW and CpG-ODN A 0.1 mMgroups (double arrows in (A,D)). (B,E,H,K,N) Collagen-related matrices are faintly observed inblue color in the DW group (arrow in B) but barely in the CpG-ODN groups. (C,F,I,L,O) Survivingodontoblasts and/or odontoblast-like cells (arrows) align under the pulp–dentin border of replantedteeth, as well as in numerous nestin-positive filamentous structures (arrowheads). AB: Alveolar bone,DP: Dental pulp. Scale bars: 250 µm (A,D,G,J,M) and 100 µm (B,C,E,F,H,I,K,L,N,O).At week 2, all groups exhibited newly formed hard tissue in the dental pulp, particu-larly in the root area (asterisks in Figure 3A,E,I,M,Q and Figure S2A). Despite the presenceof pulpal inflammatory reactions previously observed one week after replantation, thedental pulp of the replanted teeth treated with the CpG-ODN solutions recovered its charac-teristic features (Figure 3E,I,M,Q), including re-vascularization, which was comparable withthose treated with DW (Figure 3A) and HBSS (Supplementary Figure S2A). However, mostBiomolecules 2024, 14, 931 6 of 16samples in the DW and CpG-ODN groups and some in the HBSS group showed some rootankylosis at this stage (Figure 3A,E,I,M,Q and Supplementary Figure S2A). AZAN stainingrevealed that the blue areas related to collagen deposition in the newly formed hard tissuewere similar in all experimental groups (Figure 3B,F,J,N,R and Figure S2B). A slight positivetendency for the occurrence of newly formed hard tissue in the dental pulp of replantedteeth was observed for the HBSS group and CpG-ODN A and B groups at a concentrationof 0.1 mM; however, no significant differences were observed among the groups (Figure 2C).Nestin immunostaining revealed the presence of different healing patterns in the repairedpulpal tissue (Figure 3C,D,G,H,K,L,O,P,S,T and Figure S2C). Tertiary dentin, including pulpstones (Figure 3C,D,G,H,O,P) and nestin-negative bone-like tissue (arrows) coexisted in thedental pulp (Figure 3K,L,S,T). Nestin-positive newly differentiated odontoblast-like cellswere arranged beneath the tertiary dentin or surrounding the pulp stones. No significantdifferences were observed in the rate of nestin-positive perimeters relative to the totalperimeter of the dental pulp of replanted teeth among the groups; however, the CpG-ODNB 0.8 mM group exhibited the lowest rate compared with the other groups (Figure 2B). Inaddition, we observed that both CpG-ODN A and B at 0.1 mM concentration, as well as theDW and HBSS groups, tended to favor the deposition of tertiary dentin, which reduced therate of bone-like tissue formation in the pulpal tissue; however, no significant differenceswere observed among the groups (Figure 2D). Based on our observations, exposure to lowconcentrations (0.1 mM) of synthetic CPG-ODN Type A or B solutions appears to causeless damage to pulpal tissue in terms of inflammatory responses and, therefore, a higheroccurrence rate of hard tissue formation in the dental pulp.Biomolecules 2024, 14, x FOR PEER REVIEW 6 of 17   Figure 2. Quantitative analysis of H&E staining and nestin immunohistochemistry. (A) The percent-age (%) of the nestin-positive perimeter one week after replantation. Significant differences are evi-dent between the HBSS and CpG-ODN groups and DW. (B) During week 2, there are no significant differences in the rate of nestin-positive perimeters among the groups. (C) The occurrence rate (%) of hard tissue formation in the dental pulp during week 2 shows no significant differences among groups but a slight positive tendency toward the HBSS group and CpG-ODN A and B groups at 0.1 mM. (D) There are no significant differences in the percentage (%) of nestin-positive and -negative areas in the dental pulp at week 2 after replantation. At week 2, all groups exhibited newly formed hard tissue in the dental pulp, partic-ularly in the root area (asterisks in Figures 3A,E,I,M,Q and S2A). Despite the presence of pulpal inflammatory reactions previously observed one week after replantation, the den-tal pulp of the replanted teeth treated with the CpG-ODN solutions recovered its charac-teristic features (Figure 3E,I,M,Q), including re-vascularization, which was comparable with those treated with DW (Figure 3A) and HBSS (Supplementary Figure S2A). How-ever, most samples in the DW and CpG-ODN groups and some in the HBSS group showed some root ankylosis at this stage (Figure 3A,E,I,M,Q and Supplementary Figure S2A). AZAN staining revealed that the blue areas related to collagen deposition in the newly formed hard tissue were similar in all experimental groups (Figures 3B,F,J,N,R and S2B). A slight positive tendency for the occurrence of newly formed hard tissue in the dental pulp of replanted teeth was observed for the HBSS group and CpG-ODN A and B groups at a concentration of 0.1 mM; however, no significant differences were observed among the groups (Figure 2C). Nestin immunostaining revealed the presence of different healing patterns in the repaired pulpal tissue (Figures 3C,D,G,H,K,L,O,P,S,T and S2C). Tertiary dentin, including pulp stones (Figure 3C,D,G,H,O,P) and nestin-negative bone-like tissue (arrows) coexisted in the dental pulp (Figure 3K,L,S,T). Nestin-positive newly differentiated odontoblast-like cells were arranged beneath the tertiary dentin or sur-rounding the pulp stones. No significant differences were observed in the rate of nestin-positive perimeters relative to the total perimeter of the dental pulp of replanted teeth Figure 2. Quantitative analysis of H&E staining and nestin immunohistochemistry. (A) The per-centage (%) of the nestin-positive perimeter one week after replantation. Significant differences areevident between the HBSS and CpG-ODN groups and DW. (B) During week 2, there are no significantdifferences in the rate of nestin-positive perimeters among the groups. (C) The occurrence rate (%)of hard tissue formation in the dental pulp during week 2 shows no significant differences amonggroups but a slight positive tendency toward the HBSS group and CpG-ODN A and B groups at0.1 mM. (D) There are no significant differences in the percentage (%) of nestin-positive and -negativeareas in the dental pulp at week 2 after replantation.Biomolecules 2024, 14, 931 7 of 16Biomolecules 2024, 14, x FOR PEER REVIEW 7 of 17  among the groups; however, the CpG-ODN B 0.8 mM group exhibited the lowest rate compared with the other groups (Figure 2B). In addition, we observed that both CpG-ODN A and B at 0.1 mM concentration, as well as the DW and HBSS groups, tended to favor the deposition of tertiary dentin, which reduced the rate of bone-like tissue for-mation in the pulpal tissue; however, no significant differences were observed among the groups (Figure 2D). Based on our observations, exposure to low concentrations (0.1 mM) of synthetic CPG-ODN Type A or B solutions appears to cause less damage to pulpal tis-sue in terms of inflammatory responses and, therefore, a higher occurrence rate of hard tissue formation in the dental pulp.   Figure 3. H&E staining (A,E,I,M,Q), AZAN staining (B,F,J,N,R), and nestin immunoreactivity (C,D,G,H,K,L,O,P,S,T) in samples of extracted teeth treated with DW (A–D), CpG-ODN A—0.1 mM (E–H), CpG-ODN A at 0.8 mM (I–L), CpG-ODN B at 0.1 mM (M–P), and CpG-ODN B at 0.8 mM (Q–T) two weeks after replantation. (A,E,I,M,Q) The dental pulp of the DW and CpG-ODN groups shows re-vascularization and newly formed hard tissue areas (asterisks). (B,F,J,N,R) The AZAN-stained blue areas are similar for all experimental groups. (C,D,G,H,O,P) Nestin im-munostaining reveals the presence of tertiary dentin, including pulp stones. (K,L,S,T) Nestin-nega-tive bone-like tissue (arrows) coexists in the dental pulp. (D,H,L,P,T) Higher magnified views of the boxes in (C,G,K,O,S), respectively. AB: Alveolar bone, B: Bone-like tissue, DP: Dental pulp, OLC: Figure 3. H&E staining (A,E,I,M,Q), AZAN staining (B,F,J,N,R), and nestin immunoreactivity(C,D,G,H,K,L,O,P,S,T) in samples of extracted teeth treated with DW (A–D), CpG-ODN A—0.1 mM(E–H), CpG-ODN A at 0.8 mM (I–L), CpG-ODN B at 0.1 mM (M–P), and CpG-ODN B at 0.8 mM(Q–T) two weeks after replantation. (A,E,I,M,Q) The dental pulp of the DW and CpG-ODN groupsshows re-vascularization and newly formed hard tissue areas (asterisks). (B,F,J,N,R) The AZAN-stained blue areas are similar for all experimental groups. (C,D,G,H,O,P) Nestin immunostainingreveals the presence of tertiary dentin, including pulp stones. (K,L,S,T) Nestin-negative bone-liketissue (arrows) coexists in the dental pulp. (D,H,L,P,T) Higher magnified views of the boxes in(C,G,K,O,S), respectively. AB: Alveolar bone, B: Bone-like tissue, DP: Dental pulp, OLC: Odontoblast-like cells, TD: Tertiary dentin. Scale bars: 250 µm (A–C,E–G,I–K,M–O,Q–S) and 50 µm (D,H,L,P,T).3.2. Analysis of Cell Proliferation in the Pulpal Tissue by Ki-67 ImmunohistochemistryCell proliferation is a biological event that precedes the process of cell differentiationin tissues undergoing repair/regeneration processes. Ki-67 immunohistochemistry wasperformed to evaluate the proliferative activity in the dental pulp of replanted teeth exposedto DW, HBSS, and synthetic CpG-ODN solutions. The analysis of Ki-67-positive cellsindicates that the inflammatory reactions associated with the replantation procedures, inparticular, the use of CpG-ODN solutions at high concentrations (0.8 mM), affected cellBiomolecules 2024, 14, 931 8 of 16proliferation during the initial stages of the pulpal healing process. A positive tendencyfor cell proliferation was observed toward the DW and HBSS groups one week afterreplantation, in which a significant difference (p < 0.05) was observed in the coronaland root dental pulp between the DW and CpG-ODN B 0.8 mM groups at this stage(Figure 4A,B). At week 2, the number of proliferative cells in the CpG-ODN groups wasrestored to the level of the DW and HBSS groups, along with the establishment of healingpatterns in the dental pulp. No significant differences were observed among the groups atthis stage (Figure 4C,D). Thus, the data suggest that the inhibition of proliferative activityinduced by the synthetic CpG-ODN solutions at week 1 ceased until week 2, allowing thedifferentiation process of hard-tissue-forming cells at week 2.Biomolecules 2024, 14, x FOR PEER REVIEW 8 of 17  Odontoblast-like cells, TD: Tertiary dentin. Scale bars: 250 µm (A–C,E–G,I–K,M–O,Q–S) and 50 µm (D,H,L,P,T). 3.2. Analysis of Cell Proliferation in the Pulpal Tissue by Ki-67 Immunohistochemistry Cell proliferation is a biological event that precedes the process of cell differentiation in tissues undergoing repair/regeneration processes. Ki-67 immunohistochemistry was performed to evaluate the proliferative activity in the dental pulp of replanted teeth ex-posed to DW, HBSS, and synthetic CpG-ODN solutions. The analysis of Ki-67-positive cells indicates that the inflammatory reactions associated with the replantation proce-dures, in particular, the use of CpG-ODN solutions at high concentrations (0.8 mM), af-fected cell proliferation during the initial stages of the pulpal healing process. A positive tendency for cell proliferation was observed toward the DW and HBSS groups one week after replantation, in which a significant difference (p < 0.05) was observed in the coronal and root dental pulp between the DW and CpG-ODN B 0.8 mM groups at this stage (Fig-ure 4A,B). At week 2, the number of proliferative cells in the CpG-ODN groups was re-stored to the level of the DW and HBSS groups, along with the establishment of healing patterns in the dental pulp. No significant differences were observed among the groups at this stage (Figure 4C,D). Thus, the data suggest that the inhibition of proliferative ac-tivity induced by the synthetic CpG-ODN solutions at week 1 ceased until week 2, allow-ing the differentiation process of hard-tissue-forming cells at week 2.  Figure 4. The percentage (%) of Ki-67-positive cell density in the coronal (A,C) and pulpal (B,D) root area at weeks 1 (A,B) and 2 (C,D) after replantation in the DW, HBSS, and CpG-ODN A and B 0.1 mM groups. (A,B) The number of Ki-67-positive cells decreases particularly in the CpG-ODN groups one week after replantation. A significant difference is noted between DW and CpG-ODN B at 0.8 mM in the coronal and root pulp. (C,D) At week 2, the number of proliferating cells in the CpG-ODN groups reaches the level of the DW and HBSS groups. No significant differences are observed among the groups.   Figure 4. The percentage (%) of Ki-67-positive cell density in the coronal (A,C) and pulpal (B,D) rootarea at weeks 1 (A,B) and 2 (C,D) after replantation in the DW, HBSS, and CpG-ODN A and B 0.1 mMgroups. (A,B) The number of Ki-67-positive cells decreases particularly in the CpG-ODN groups oneweek after replantation. A significant difference is noted between DW and CpG-ODN B at 0.8 mMin the coronal and root pulp. (C,D) At week 2, the number of proliferating cells in the CpG-ODNgroups reaches the level of the DW and HBSS groups. No significant differences are observed amongthe groups.3.3. Assessment of Macrophage Activity in the Dental Pulp by F4/80 and CD206ImmunohistochemistryImmunohistochemistry for F4/80 and CD206 was performed to evaluate macrophageactivity. F4/80 is considered a pan macrophage marker, whereas CD206 is a marker forthe M2 subset, macrophages associated with anti-inflammatory activity and tissue repair.One week after intentionally delaying tooth replantation, most samples exhibited a mild tomoderate reaction to the F4/80 antibody (Figure 5A,E,I and Supplementary Figure S3A).F4/80 exhibited a dendritic cell-like pattern of expression in the dental pulp. At week 1,F4/80 was expressed by cells located in the central pulp and surrounding cell debris. F4/80-negative areas corresponded with those experiencing inflammatory conditions, wherepulpal degeneration had occurred, such as in the pulpal horns. There was no significantBiomolecules 2024, 14, 931 9 of 16difference in the percentage of F4/80-positive areas among all groups in the coronal orroot pulp; however, a positive trend was noted toward the CpG-ODN B 0.1 mM group(Supplementary Figure S4A,B). Interestingly, the presence of M2 macrophages (CD206-positive cells) was scarce at this observation point, particularly in the areas where F4/80-positive cells appeared (Figure 5B,F,J and Figure S3B). No significant differences wereobserved in the percentage of CD-206-positive cells among the groups at week 1, althougha positive tendency was observed toward the CpG-ODN A and B 0.1 mM groups in thecoronal and root pulp (Figure 6A,B). At week 2, the immunoreaction of F4/80 increasedin intensity in the CpG-ODN groups despite the onset of healing in the dental pulp (Fig-ure 5C,G,K and Figure S3C), which was primarily located in the subodontoblastic layer,central pulp, and surrounding blood capillaries and pulpal stones (Figure 5G,K), whereasonly a few F4/80-positive cells were observed beneath the predentin. There were no sig-nificant differences in the F4/80-positive area in the coronal and root dental pulp amongthe groups at this stage (Supplementary Figure S4C,D). Regarding the activity of M2macrophages, a small increase in the rate of CD206-positive cell density was noted at thisstage. CD206-positive cells increased in number in the subodontoblast layer and centralpulp, even in the areas where F4/80-positive cells were observed (Figure 5D,H,L and Fig-ure S3D). Although no significant differences were evident among the groups in the coronalpulp (Figure 6C), a significant difference in CD206-positive cell density was observed inthe root pulp between DW and HBSS, and the CpG-ODN A 0.1 mM group, respectively(p < 0.05) (Figure 6D). Histological and quantitative data from immunohistochemistry forF4/80 and CD206 suggest that synthetic CPG-ODN solutions appeared to increase theactivity of dental pulp macrophages without significant M2 subset commitment in bothobservation periods because intense expression for F4/80 was still observed at week 2.Biomolecules 2024, 14, x FOR PEER REVIEW 9 of 17  3.3. Assessment of Macrophage Activity in the Dental Pulp by F4/80 and CD206 Immunohisto-chemistry Immunohistochemistry for F4/80 and CD206 was performed to evaluate macrophage activity. F4/80 is considered a pan macrophage marker, whereas CD206 is a marker for the M2 subset, macrophages associated with anti-inflammatory activity and tissue repair. One week after intentionally delaying tooth replantation, most samples exhibited a mild to moderate reaction to the F4/80 antibody (Figure 5A,E,I and Supplementary Figure S3A). F4/80 exhibited a dendritic cell-like pattern of expression in the dental pulp. At week 1, F4/80 was expressed by cells located in the central pulp and surrounding cell debris. F4/80-negative areas corresponded with those experiencing inflammatory conditions, where pulpal degeneration had occurred, such as in the pulpal horns. There was no significant difference in the percentage of F4/80-positive areas among all groups in the coronal or root pulp; however, a positive trend was noted toward the CpG-ODN B 0.1 mM group (Supplementary Figure S4A,B). Interestingly, the presence of M2 macrophages (CD206-positive cells) was scarce at this observation point, particularly in the areas where F4/80-positive cells appeared (Figures 5B,F,J and S3B). No significant differences were observed in the percentage of CD-206-positive cells among the groups at week 1, although a positive tendency was observed toward the CpG-ODN A and B 0.1 mM groups in the coronal and root pulp (Figure 6A,B). At week 2, the immunoreaction of F4/80 increased in intensity in the CpG-ODN groups despite the onset of healing in the dental pulp (Figures 5C,G,K and S3C), which was primarily located in the subodontoblastic layer, central pulp, and sur-rounding blood capillaries and pulpal stones (Figure 5G,K), whereas only a few F4/80-positive cells were observed beneath the predentin. There were no significant differences in the F4/80-positive area in the coronal and root dental pulp among the groups at this stage (Supplementary Figure S4C,D). Regarding the activity of M2 macrophages, a small increase in the rate of CD206-positive cell density was noted at this stage. CD206-positive cells increased in number in the subodontoblast layer and central pulp, even in the areas where F4/80-positive cells were observed (Figures 5D,H,L and S3D). Although no signifi-cant differences were evident among the groups in the coronal pulp (Figure 6C), a signif-icant difference in CD206-positive cell density was observed in the root pulp between DW and HBSS, and the CpG-ODN A 0.1 mM group, respectively (p < 0.05) (Figure 6D). Histo-logical and quantitative data from immunohistochemistry for F4/80 and CD206 suggest that synthetic CPG-ODN solutions appeared to increase the activity of dental pulp mac-rophages without significant M2 subset commitment in both observation periods because intense expression for F4/80 was still observed at week 2.  Figure 5. Immunohistochemical evaluation of macrophage activity in dental pulp. F4/80(A,C,E,G,I,K) and CD206 (B,D,F,H,J,L) immunohistochemistry in the HBSS (A,B) and DW(C,D) groups and the CpG-ODN A (E–H) and CpG-ODN B (I–L) 0.1 mM groups at weeks 1(A,B,E,F,I,J) and 2 (C,D,G,H,K,L) following replantation. (A,E,I) Most samples exhibit a mild tomoderate reaction to the F4/80 antibody, with negative areas corresponding to those under inflamma-tory conditions one week after replantation. (C,G,K) At week 2, F4/80 immunoreactivity increasesin intensity in the pulpal tissue, particularly in the subodontoblast layer, central pulp, and areassurrounding the blood capillaries and pulpal stones in the CpG-ODN A and B 0.1 mM groups.(B,F,J) CD206-positive cells are scarce at week 1, particularly in the areas where F4/80-positive cellsare observed. (D,H,L) At week 2, CD206-positive cells increase in number in the subodontoblast layerand central pulp, even in the areas where F4/80-positive cells are observed. DP: Dental pulp. Scalebar: 50 µm (A–L).Biomolecules 2024, 14, 931 10 of 16Biomolecules 2024, 14, x FOR PEER REVIEW 10 of 17  Figure 5. Immunohistochemical evaluation of macrophage activity in dental pulp. F4/80 (A,C,E,G,I,K) and CD206 (B,D,F,H,J,L) immunohistochemistry in the HBSS (A,B) and DW (C,D) groups and the CpG-ODN A (E–H) and CpG-ODN B (I–L) 0.1 mM groups at weeks 1 (A,B,E,F,I,J) and 2 (C,D,G,H,K,L) following replantation. (A,E,I) Most samples exhibit a mild to moderate reac-tion to the F4/80 antibody, with negative areas corresponding to those under inflammatory condi-tions one week after replantation. (C,G,K) At week 2, F4/80 immunoreactivity increases in intensity in the pulpal tissue, particularly in the subodontoblast layer, central pulp, and areas surrounding the blood capillaries and pulpal stones in the CpG-ODN A and B 0.1 mM groups. (B,F,J) CD206-positive cells are scarce at week 1, particularly in the areas where F4/80-positive cells are observed. (D,H,L) At week 2, CD206-positive cells increase in number in the subodontoblast layer and central pulp, even in the areas where F4/80-positive cells are observed. DP: Dental pulp. Scale bar: 50 µm (A–L).  Figure 6. The percentage (%) of CD206-positive cells in the coronal (A,C) and pulpal (B,D) root area at weeks 1 (A,B) and 2 (C,D) after replantation in the DW and HBSS groups and the CpG-ODN A and B 0.1 mM groups. (A,B) There are no significant differences among the groups at week 1, alt-hough a positive tendency is evident toward the CpG-ODN A and B 0.1 mM groups in the coronal and root pulp. (C,D) At week 2, there is no significant difference among the groups in the coronal pulp, whereas a significant difference is found in the root pulp between the DW and HBSS groups and CpG-ODN A 0.1 mM group, respectively. 3.4. TLR9 Immunoexpression in the Afflicted Dental Pulp Synthetic CpG-ODNs are ligand molecules of TLR9. The ligand–receptor activity in-duced by the experimental solutions in the different cell populations of the pulpal tissue was evaluated. One week after the operation, TLR9 immunoreactivity was faintly de-tected in some pulpal cells, the surrounding cell debris matrix, and around the blood ca-pillaries in samples from the DW and HBSS groups (arrows in Figure 7A,D), whereas scattered TLR9-positive cells were observed around the pulpal stones and central pulp at week 2 (Figure 7J and arrows in Figure 7M). Nestin-positive filaments and cells were not correlated with TLR9-positive cells (arrowheads in Figure 7G, and Figure 7P). Some sam-ples that sustained pulpal inflammatory reactions in the CpG-ODN A and B 0.1 mM groups showed clear TLR9 immunoreactivity one week after the operation (Figure Figure 6. The percentage (%) of CD206-positive cells in the coronal (A,C) and pulpal (B,D) root areaat weeks 1 (A,B) and 2 (C,D) after replantation in the DW and HBSS groups and the CpG-ODN A andB 0.1 mM groups. (A,B) There are no significant differences among the groups at week 1, althougha positive tendency is evident toward the CpG-ODN A and B 0.1 mM groups in the coronal androot pulp. (C,D) At week 2, there is no significant difference among the groups in the coronal pulp,whereas a significant difference is found in the root pulp between the DW and HBSS groups andCpG-ODN A 0.1 mM group, respectively.3.4. TLR9 Immunoexpression in the Afflicted Dental PulpSynthetic CpG-ODNs are ligand molecules of TLR9. The ligand–receptor activityinduced by the experimental solutions in the different cell populations of the pulpal tissuewas evaluated. One week after the operation, TLR9 immunoreactivity was faintly detectedin some pulpal cells, the surrounding cell debris matrix, and around the blood capillariesin samples from the DW and HBSS groups (arrows in Figure 7A,D), whereas scatteredTLR9-positive cells were observed around the pulpal stones and central pulp at week 2(Figure 7J and arrows in Figure 7M). Nestin-positive filaments and cells were not correlatedwith TLR9-positive cells (arrowheads in Figure 7G, and Figure 7P). Some samples thatsustained pulpal inflammatory reactions in the CpG-ODN A and B 0.1 mM groups showedclear TLR9 immunoreactivity one week after the operation (Figure 7B,C,E,F). Afflicted cellslocated under the pulpal horns beneath the pulp–dentin border showed a positive reactionfor TLR9 (arrows) and a negative immunoreaction for nestin (Figure 7H, and arrowheadsin Figure 7I). At week 2, a less intense TLR9 immunoreactivity was observed in the pulpalcells surrounding the newly formed hard tissue and central pulpal tissue (arrows) ofsome samples in the CpG-ODN groups (Figure 7K,L,N,O). Some of these cells correlatedwith nestin-positive cells surrounding the newly formed hard tissue areas (arrowheadsin Figure 7Q,R). The quantitative analysis of the percentage of TLR9-cell positive areas inthe dental pulp showed no significant differences among groups during both observationperiods (Supplementary Figure S5A,B). Therefore, these novel data suggest that syntheticCpG-ODNs may activate TLR9 in specific cell populations of non-odontoblastic lineage andpossibly in dental pulp progenitor cells at week 1. Consequently, some TLR9-positive cellsseemed to coincide with the newly differentiated hard-tissue-forming cells surroundingtertiary dentin and/or bone-like tissue areas in the dental pulp at week 2.Biomolecules 2024, 14, 931 11 of 16Biomolecules 2024, 14, x FOR PEER REVIEW 12 of 17   Figure 7. The immunoexpression of TLR9 (A–F,J–O) and nestin immunohistochemistry (G–I,P–R) in the dental pulp of replanted teeth treated with HBSS (A,D,G,J,M,P) and CpG-ODN A (B,E,H,K,N,Q) and B (C,F,I,L,O,R) at 0.1 mM at weeks 1 (A–I) and 2 (J–R), following replantation. (Arrows in A,D) TLR9 immunoreactivity is faintly detected in samples from the HBSS group at week 1. (B,C,E,F) TLR9 immunoreactivity is observed in some samples that sustained pulpal inflam-matory reactions in the CpG-ODN groups at this stage (arrows in E,F). (G–I) Nestin-positive fila-ments (arrowheads) and cells do not correlate with TLR9-positive cells. (J,M) Scattered TLR9-posi-tive cells (arrows in M) are observed around pulpal stones in the HBSS group at week 2. (K,L,N,O) TLR9 immunoreactivity is observed in cells surrounding pulpal stones of some samples in the CpG-ODN groups (arrows in N,O). (P–R) TLR9-positive cells partially correlate with nestin-positive cells at this stage (arrowheads in Q,R). (D–I,M–O) Magnified views of the boxed areas in (A) and (J–L), respectively. B: Bone-like tissue, DP: Dental pulp, OLC: Odontoblast-like cells, TD: Tertiary dentin. Scale bars: 100 µm (A–C,J–L) and 25 µm (D–I,M–R). Figure 7. The immunoexpression of TLR9 (A–F,J–O) and nestin immunohistochemistry(G–I,P–R) in the dental pulp of replanted teeth treated with HBSS (A,D,G,J,M,P) and CpG-ODN A(B,E,H,K,N,Q) and B (C,F,I,L,O,R) at 0.1 mM at weeks 1 (A–I) and 2 (J–R), following replantation.(Arrows in (A,D)) TLR9 immunoreactivity is faintly detected in samples from the HBSS group at week1. (B,C,E,F) TLR9 immunoreactivity is observed in some samples that sustained pulpal inflammatoryreactions in the CpG-ODN groups at this stage (arrows in (E,F)). (G–I) Nestin-positive filaments(arrowheads) and cells do not correlate with TLR9-positive cells. (J,M) Scattered TLR9-positive cells(arrows in (M)) are observed around pulpal stones in the HBSS group at week 2. (K,L,N,O) TLR9immunoreactivity is observed in cells surrounding pulpal stones of some samples in the CpG-ODNgroups (arrows in (N,O)). (P–R) TLR9-positive cells partially correlate with nestin-positive cells atthis stage (arrowheads in (Q,R)). (D–I,M–O) Magnified views of the boxed areas in and (A, J–L),respectively. B: Bone-like tissue, DP: Dental pulp, OLC: Odontoblast-like cells, TD: Tertiary dentin.Scale bars: 100 µm (A–C,J–L) and 25 µm (D–I,M–R).Biomolecules 2024, 14, 931 12 of 164. DiscussionTo our knowledge, this is the first study describing the effects of synthetic CpG-ODNs on the immunomodulatory response and dental pulp repair using in vivo murinetooth injury models, such as intentionally delayed tooth replantation. The properties,molecular mechanisms, and therapeutic uses of synthetic CpG-ODNs have been extensivelyreported [1–7,25–30]; however, only a few studies have described their potential use indentistry [31–33]. In the present study, the topical use of synthetic CpG-ODNs followingtooth injury elicited an inflammatory reaction and a low rate of cell proliferation in thedental pulp during the first week after treatment. This led to the establishment of twodifferent healing patterns: tertiary dentin and a mixed form of tertiary dentin and bone-like tissue in the dental pulp at week 2 following the operation. However, we did notobserve significant differences among the groups because the use of synthetic CpG-ODNsdid not show any clear advantages at different stages of the dental pulp repair process,either from a cellular or morphological point of view. Therefore, the concentration ofsynthetic CpG-ODN solutions and the immersion time may be important factors in thetreatment of the afflicted pulp. Our unpublished preliminary data show that treatment withsynthetic CpG-ODN A and B at a concentration of 1.57 mM resulted in the strongest pulpalinflammatory reactions among the tested concentrations on week 1 after replantation andthe lowest occurrence of hard tissue formation in the dental pulp at week 2, suggesting thatexposure to synthetic CpG-ODN solutions at high concentrations worsens the prognosisof the afflicted pulpal tissue. These results are consistent with those of a previous studythat analyzed the effects of different concentrations and immersion times of extractedteeth of mice in an experimental triple antibiotic (3Mix) solution before replantation. Theimmersion of the extracted teeth in a phosphate-buffered saline (PBS) solution, followedby short immersion (5 to 15 min) in the 3Mix solution at a standard (low) concentration,improved both pulpal and periodontal healing [20]. In addition, our preliminary data provethat immersion for 30 min is sufficient to ensure the penetration of CpG-ODNs into thepulpal tissue. Under the same experimental conditions, the extracted teeth of the mice wereimmersed into a GFP-conjugated CpG-ODN solution and then replanted. One hour afterreplantation, a GFP signal was detected in the coronal pulp, which remained invariableduring the following 24 h (Supplementary Figure S6). This suggests that CpG-ODNs mayhave a long-lasting or residual effect on the exposed tissues, which elicits a continuedimmune response in the pulpal cells. CpG-ODNs with a phosphorothioate backbone,the ones used in this study, are less susceptible to DNase digestion, resulting in longerin vivo half-lives compared with ODNs with a phosphodiester backbone [5]. Based onthese structural characteristics, they are optimal immune adjuvants, as they can extend theeffect of vaccines [34,35]. Thus, further in vivo studies on the effect of synthetic CpG-ODNson dental tissue should consider these variables under various conditions, such as shorterimmersion times (<30 min), lower concentrations, or the use of transfer solutions (HBSS orDW) before or after immersion, to find the ideal scenario to promote the acceleration ofpulpal healing following injury.This study shows the presence of TLR9-positive cells in the dental pulp of somesamples from the HBSS and CpG-ODN groups at weeks 1 or 2 after replantation. Todate, TLR9 mRNA expression has been confirmed in human- and murine-differentiatedodontoblasts in vitro [11,12,15–19]. Nestin immunostaining confirmed that the TLR9-positive cells observed one week after replantation in the CpG-ODN groups are not ofodontoblast lineage but leukocytes or antigen-presenting cells, such as pulpal macrophagesor dendritic cells. As CpG-ODNs can directly activate B cells, macrophages, and dendriticcells [1–5,7,36,37], we hypothesize that CpG-ODNs may exert a direct immunostimulatoryeffect on resident immune cells of the dental pulp following their exposure. Moreover,pulpal progenitors may also be stimulated by direct exposure to CpG-ODNs through theprolonged activation of TLR9. The latter could explain why some TLR9-positive cells wereobserved around pulpal stones in some samples of the CpG-ODN A 0.1 mM group atweek 2 when the healing patterns were already established in the dental pulp of all groups.Biomolecules 2024, 14, 931 13 of 16In the absence of comparable in vivo data, further studies are needed to determine theexpression of TLR9 in dental pulp cells under normal and pathological conditions and howCpG-ODN activates dental pulp antigen-presenting cells and/or progenitor cells followingdental injuries.Similarly, this study shows that exposure to synthetic CpG-ODNs tends to stimulatemacrophage activity in the coronal and root dental pulp after replantation compared withHBSS and DW. Several recent studies have highlighted the importance of macrophagepolarization in the healing of pulpal tissue [38–42]. Macrophages present in different tis-sues, including the dental pulp, can polarize in response to changes in their environment.For example, M1 macrophages are primarily involved in pro-inflammatory responses bysecreting cytokines, such as interleukin 1β (1L-1β), tumor necrosis factor-alpha (TNF-α),IL-6, reactive oxygen species (ROS), and nitric oxide (NO) [41], whereas M2 macrophagescan induce an anti-inflammatory response and tissue repair [43,44]. Although we did notanalyze the presence of M1 macrophages, we presume that exposure to CpG-ODNs at0.1 mM may activate the M1 subset because the quantitative analysis of F4/80 immunohis-tochemistry revealed a slight trend toward CpG-ODNs compared with that in the HBSS andDW groups at week 2. Moreover, the percentage of M2 macrophages in the coronal and rootpulp decreased at the same stage, particularly after treatment with CpG-ODNs-A 0.1 mMcompared with HBSS and DW. The pro-inflammatory immune response observed in thepresent study was previously reported by He et al. [15,18,19], albeit using an in vitro ap-proach with murine-differentiated odontoblasts. Based on the premise that CpG-ODN canstimulate inflammation and induce the M1 macrophage phenotype that restricts angiogen-esis [45], recent studies have demonstrated that novel delivery approaches of CpG-ODNscan effectively regulate a tumor-associated M2 macrophage transformation into the M1phenotype [46–48], thus facilitating the development of promising cancer therapeutics. Incontrast, M2 macrophages were shown to be key to reversing the early stages of pulpalinflammation and achieving wound healing in a rat model for cavity preparation andorthodontic tooth movement [38,40]. Therefore, exposure to synthetic CpG-ODNs mayalter the interactions of M1/M2 macrophages with other cell populations, such as resi-dent dendritic cells and dental pulp progenitor cells, which are essential for odontoblastdifferentiation under pathological conditions [49,50].Overall, our findings suggest that treatment with low-concentration synthetic CpG-ODN solutions, either Type A or B at 0.1 mM, may evoke a long-lasting macrophage–TLR9-mediated pro-inflammatory, rather than anti-inflammatory, response in the dentalpulp. Both types of synthetic CpG-ODNs primarily enhanced the immune responses byactivating TLR9 signaling pathways in the dental pulp. Our data show a positive trendtoward the use of synthetic CpG-ODN solutions at low concentrations (0.1 mM) to promotehealing of the afflicted pulpal tissue better than the 0.8 mM concentration during bothobservation periods. However, when comparing both CpG-ODN types at 0.8 mM, TypeB seems to provide a slightly better situation for pulpal healing than Type A at week 1,especially in the coronal pulp. Based on this observation, it is possible to assume that TypeB may have a less adverse effect on the dental pulp than Type A during the initial stagesafter tooth replantation and, therefore, tends to result in increased hard tissue depositionat week 2. The evidence that Type A CpG-ODN can target pDCs and enhance systemicimmunity, while Type B CpG-ODN can induce proinflammatory cytokines from B cellsand macrophages, including those present in inflamed pulpal tissue [51,52], supportsthe tentative assumption that Type B is superior to Type A for the healing of the injureddental pulp, although this claim needs to be further confirmed by additional studies.Despite the disadvantages that may have arisen from our experimental design, such asthe prolonged immersion time, we conclude that immersion for 30 min in CpG-ODNsolutions may have a detrimental effect on the process of pulpal repair. This might resultin the overactivation of the M1 macrophage subset and/or the M2-to-M1 macrophagetransformation in the afflicted dental pulp of replanted teeth. Although this study did notprovide evidence on this subject, the concerns regarding M1 macrophages must be furtherBiomolecules 2024, 14, 931 14 of 16analyzed in detail to support our current hypothesis. Shorter immersion times in syntheticCpG-ODNs may effectively activate the M1 macrophage subset, which is needed duringthe initial inflammatory response for the removal of microbial-derived noxious productsor cell debris. The timing in which the activity of M1 macrophages should give way toM2 macrophages to initiate anti-inflammatory activity and the proper balance betweenM1/M2 polarized macrophages in order to promote tissue repair in the afflicted dentalpulp also need to be elucidated. In addition, the mechanisms behind the differentiation ofhard-tissue-forming cells and the subsequent deposition of tertiary dentin and/or bone-liketissue, while sustained macrophagic activity continues to occur within the dental pulp,remain to be determined. Further studies analyzing the effects of immunomodulatoryagents in vitro and in vivo are needed to develop therapeutic strategies for dental tissueregeneration.Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/biom14080931/s1, Figure S1: Histological evaluation of samplestreated with HBSS at week 1; Figure S2: Histological evaluation of samples treated with HBSS at week2; Figure S3: F4/80 and CD206 immunohistochemistry in samples of DW and HBSS groups at weeks 1and 2; Figure S4: The percentage of F4/80-positive area in the dental pulp; Figure S5: The percentageof TLR9-positive area in the dental pulp; Figure S6: The penetration of GFP-conjugated syntheticCpG-ODNs in the pulpal tissue; Table S1: The distribution of animals per experimental group.Author Contributions: Conceptualization, A.Q.-S., T.Y. and H.O.; methodology, H.O.; validation,A.Q.-S., T.Y. and H.O.; formal analysis, A.Q.-S. and H.O.; investigation, A.Q.-S. and H.O.; data cura-tion, A.Q.-S. and H.O.; writing—original draft preparation, A.Q.-S. and H.O.; writing—review andediting, A.Q.-S., T.Y. and H.O.; visualization, A.Q.-S. and H.O.; supervision, H.O.; funding acquisition,A.Q.-S. and H.O. All authors have read and agreed to the published version of the manuscript.Funding: This research was funded by the Japan Society for the Promotion of Science (JSPS), “FY 2020JSPS Postdoctoral Fellowship for Overseas Researchers (Grant number P20412)”, and JSPS KAKENHI(Grant Numbers 22K21011 and 23H03078).Institutional Review Board Statement: The animal study protocol was approved by the InstitutionalAnimal Care and Use Committee and approved by the President of Niigata University (approvalnumbers SA00298 and SA01017).Informed Consent Statement: Not applicable.Data Availability Statement: The original data presented in the study are included in the arti-cle/Supplementary Materials. Further inquiries can be directed to the corresponding author.Acknowledgments: The authors cordially thank H. Ida-Yonemochi, Y. Seino, K. Saito, and M. Kawachifor their technical assistance and animal care.Conflicts of Interest: The authors declare no conflicts of interest.References1. Krieg, A.M. 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