Immunolocalization of the mechanogated ion channels PIEZO1 and PIEZO2 in human and mouse dental pulp and periodontal ligament

PIEZO1 and PIEZO2 are essential components of mechanogated ion channels, which are required for mechanotransduction and biological processes associated with mechanical stimuli. There is evidence for the presence of PIEZO1 and PIEZO2 in teeth and periodontal ligaments, especially in cell lines and mice, but human studies are almost nonexistent. Decalcified permanent human teeth and mouse molars were processed for immunohistochemical detection of PIEZO1 and PIEZO2. Confocal laser microscopy was used to examine the co‐localization of PIEZO 1 and PIEZO2 with vimentin (a marker of differentiated odontoblasts) in human teeth. In the outer layer of the human dental pulp, abundant PIEZO1‐ and PIEZO2‐positive cells were found that had no odontoblast morphology and were vimentin‐negative. Based on their morphology, location, and the absence of vimentin positivity, they were identified as dental pulp stem cells or pre‐odontoblasts. However, in mice, PIEZO1 and PIEZO2 were ubiquitously detected and colocalized in odontoblasts. Intense immunoreactivity of PIEZO1 and PIEZO2 has been observed in human and murine periodontal ligaments. Our findings suggest that PIEZO1 and PIEZO2 may be mechanosensors/mechanotransducers in murine odontoblasts, as well as in the transmission of forces by the periodontal ligament in humans and mice.

mechanical forces on dental pulp using animal and in vitro models.Various forces (stretching and pressure) that act on teeth are transmitted to the dental pulp and can subsequently influence pulp cell biology (Cai et al., 2011;Hata et al., 2012;He et al., 2021;Marrelli et al., 2018;Miyashita et al., 2017;Yang et al., 2018).Nevertheless, the mechanisms by which dental pulp cells sense environmental forces are only partially known, even though is an essential step in determining how mechanical stimuli are converted into functions, including lineage specification.
Owing to the scarcity of studies on the occurrence of mechanoproteins in human teeth, the present study was designed to investigate the distribution of PIEZO1 and PIEZO2 in human dental pulp and the periodontal ligament to contribute to the knowledge of tooth mechanobiology.Additionally, the presence of PIEZO1 and PIEZO2 in mouse molars was examined to confirm their presence in odontoblasts.

| Human teeth
Teeth that showed no evident pathologies (five canines and six molars) were removed for various reasons from patients aged 25-48 years (males, n = 4; females, n = 7) in different private clinics.Informed consent was obtained from all patients with exodontia, and the study was approved by the Ethics Committee of the Facultad de Medicina y Ciencias de la Salud of the University of Oviedo, Spain (Ref. .The teeth were washed with tap water, followed by cold saline, and then placed in a solution containing 10% formalin, 15.4 M nitric acid, and distilled water (10:5:85 v/v) until decalcification was complete (7-10 days).After decalcification, the samples were washed with tap water for 12 h and embedded in paraffin.Longitudinal 10-μm-thick sections were then cut, mounted on gelatin-coated microscope slides, and processed for immunohistochemistry.

| Mice teeth
Thirty C57B1/6 mice at 30 days old and of both sexes were obtained from the Animalarium of the University of Oviedo, Spain.The animals were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) via intraperitoneal injection.Thereafter, the mice were decapitated when they no longer responded to the painful stimuli.The cephalic skin and muscles, as well as the tongue and eyes, were removed and the skull was placed in buffered 10% formalin for 12 h, then in a solution containing 10% formalin, 15.4 M nitric acid, and distilled water (10:5:85 v/v; changed daily) until decalcification was completed (7-10 days).After decalcification, the rostral portion of the skull containing the teeth was washed with tap water for 12 h and embedded in paraffin.Sections were cut at a thickness of 10 μm and mounted on gelatine-coated microscope slides.This study was approved by the Animal Welfare Committee of the University of Oviedo, Spain.

| Single immunohistochemistry analysis
Deparaffinized and rehydrated human and mouse tooth sections were processed for the detection of PIEZO1 and PIEZO2 using the EnVision antibody complex detection kit (Dako, Copenhagen, Denmark) following the supplier's instructions.Briefly, endogenous peroxidase activity was inhibited (3% H 2 O 2 for 15 min) and non-specific binding was blocked (10% bovine serum albumin for 20 min).The sections were then incubated overnight at 4 C with the primary antibodies.The polyclonal anti-PIEZO1 antibody was raised in rabbits against a synthetic peptide, (C)EDLKPQHRRHISIR, corresponding to amino acids 1863-1876 of rat PIEZO1 (Alomone, Jerusalem, Israel), and was diluted 1:200.The polyclonal anti-PIEZO2 antibody was raised in rabbits against a synthetic peptide of human PIEZO2 with the following sequence: VFGFWAFGKHSAA ADITSSLSEDQVPGPFLVMVLIQFGT MVVD RALYLRK (Sigma-Aldrich, St Louis, MO, USA), and was diluted 1:200.Subsequently, the sections were incubated with an antirabbit EnVision-system-labeled polymer (Dako Cytomation) for 30 min, washed in a buffer solution, and treated with peroxidase-blocking buffer (Dako Cytomation).Finally, the slides were washed with a buffer solution and the immunoreaction was visualized with diaminobenzidine as a chromogen.After washing and dehydrating the slides, they were mounted with Entellan ® (Merk, Darmstadt, Germany).To ascertain the structural details, the sections were counterstained with Mayer's hematoxylin.

| Double immunofluorescence staining
Human tooth sections were also processed for the simultaneous detection of PIEZO1 and PIEZO2 together with vimentin, a marker of odontoblasts, pulp fibroblasts, and fibroblasts of the periodontal ligament (Solé-Magdalena et al., 2011, 2018).Nonspecific binding was prevented by incubating the sections for 30 min with a solution of 25% calf bovine serum in Tris-buffered saline (TBS) at pH 7.4.The sections were incubated overnight at 4 C in a humid chamber with a 1:1 mixture of polyclonal antibodies against PIEZO1 or PIEZO2 (diluted 1:100) and a mouse monoclonal anti-vimentin antibody (clone 334; Boehringer-Mannheim, Mannheim, Germany; diluted 1:500).After rinsing with TBS, the sections were incubated for 1 h with CFL488-conjugated bovine anti-rabbit IgG (sc-362260; Santa Cruz Biotech, Dallas, TX, USA) diluted 1:200 in TBS, rinsed again, and incubated for another hour with a CyTM3-conjugated donkey anti-mouse antibody (Jackson ImmunoResearch, Baltimore, MD, USA) diluted 1:100 in TBS.Both incubations were performed at room temperature in a dark humid chamber.Finally, the sections were washed, and cell nuclei were stained with DAPI (10 ng/mL).Triple fluorescence was detected using a Leica DMR-XA automatic fluorescence microscope (Microscopía fot onica y proceso de im agen, servicios científico-técnicos, Universidad de Oviedo) coupled with Leica Confocal Software, version 2.5 (Leica Microsystems, Heidelberg GmbH, Germany), and the captured images were processed using Image J version 1.43 at the Master Biophotonics Facility, McMaster University, Ontario, Canada (www.macbiophotonics.ca).For control purposes, representative sections were processed in the same way as described above, using non-immune rabbit or mouse sera instead of primary antibodies or omitting the primary antibodies during incubation.When available, additional controls were performed using pre-absorbed antisera.No immunoreactivity was detected under these conditions (data not shown).

| RESULTS
The distribution of the mechanoproteins PIEZO1 and PIEZO2 was analyzed using immunohistochemistry and immunofluorescence (combined with confocal laser microscopy) in sections from paraffin-embedded and decalcified human and mouse teeth.The processing of the teeth specimens was identical, following well-established protocols; however, it is possible that the intensity of immunostaining may have been altered by the components used in the decalcification process.

| Humans
No differences were noted in the location of the immunoreaction between the canines and molars.PIEZO1 was detected in a subpopulation of pulp cells near the dentinpulp junction within the odontoblast layer.However, these cells did not show a morphology typical of odontoblasts, and PIEZO1 immunoreactivity was not detected within the dentinal ducts, and thus was not detected in the odontoblast processes (Figure 1a,b).Positive PIEZO2 cells were also found in the odontoblast layer, in cells displaying a morphology similar to that of PIEZO1-positive cells.PIEZO2 was not observed in the dentinal tubules (Figure 1c,d).Blood vessel walls of the dental pulp were positive for PIEZO2.Sparce cells displaying PIEZO1 and PIEZO2 immunoreactivity were also observed in the core of the dental pulp and occasionally in the capillary walls (data not shown).When performing experiments on the colocalization of PIEZO1 or PIEZO2 with vimentin (which is a specific marker of differentiated odontoblasts), it was observed that PIEZO1-and PIEZO2-positive cells and vimentinpositive cells represented two different, independent cell populations (Figures 2 and 3).Therefore, cells displaying PIEZO1 and PIEZO2 were not mature odontoblasts and presumably, corresponded to a subpopulation of dental pulp stem cells and/or pre-odontoblasts.
Strong immunostaining was detected for PIEZO1 (Figure 4a) and PIEZO2 (Figure 4b,c) in the periodontal ligament, partially colocalized with vimentin in fibroblasts at the junction of the periodontal ligament with the cementum (Figure 4d-f).

| Mice
In mouse teeth, immunoreactivity for PIEZO1 (Figure 5a,b) and PIEZO2 (Figure 5d,e) was observed in all odontoblasts, especially in the basal pole, without any differences among the cells.In serial sections, it was observed that immunoreactivity for PIEZO1 and PIEZO2 was co-localized in all odontoblasts.In contrast, periodontal ligament fibroblasts showed an intense immunoreaction for PIEZO1 and PIEZO2 (Figure 5c,f).
In the present study, the expression of PIEZO1 and PIEZO2 in adult human and mouse teeth was investigated using immunohistochemistry. PIEZO1 and PIEZO2 are present in teeth; however, their functions are poorly understood and remain to be definitively established (Lin et al., 2022;Pei et al., 2021).
In humans, both proteins have been detected in a cellular subpopulation of the dental pulp within the odontoblast layer and periodontal ligament.To the best of our knowledge, this is the first study to analyze the presence of PIEZO1 and PIEZO2 in human teeth.However, our data did not suggest the presence of PIEZO1 or PIEZO2 in the pulp nerve fibers, as previously reported (Cho et al., 2022;Han et al., 2022;Sato et al., 2018;Won et al., 2017).The results for the periodontal ligament were consistent with those previously observed in mice (see Lukacs et al., 2023).
In mice, both odontoblasts and fibroblasts of the periodontal ligament are immunoreactive for PIEZO1 and PIEZO2.This is in agreement with previous studies reporting the presence of PIEZO1 (Mousawi et al., 2020;Ohyama et al., 2022;Sun et al., 2022) and PIEZO2 (Khatibi Shahidi et al., 2015) in murine odontoblasts and the occurrence of PIEZO1 (Jiang et al., 2021; F I G U R E 5 Immunohistochemical detection of PIEZO1 (a-c) and PIEZO2 (d-f) in mouse teeth.Positive immunoreactivity for PIEZO1 (a, b) and PIEZO2 (d, e) was observed in odontoblasts, and the proteins were apparently colocalized.Fibroblasts of the periodontal ligament also expressed PIEZO1 (c) and PIEZO2 (f).al, ameloblast layer; dp, dental pulp; ol, odontoblast layer.Jin et al., 2015) and PIEZO2 (Gao et al., 2017) in the periodontal ligament.
Nevertheless, the results for human and murine dental pulp cells were clearly different.We believe that these discrepancies may be due to species-specific differences between humans and mice.Human teeth contain heterogeneous pulp cell populations (Krivanek et al., 2020) that are not uniformly distributed but are organized into four zones from the dentin to the pulp core: the odontoblast layer, cell-free zone, cell-rich zone, and pulp core (D'Aquino et al., 2008;Fox & Heeley, 1980;França et al., 2019;Lizier et al., 2012).The external layer, in which PIEZO1-and PIEZO2-positive cells are localized, comprises odontoblasts and dental pulp stem cells (DPSCs).Therefore, based on topographical, morphological, and immunohistochemical criteria, the PIEZO1-and PIEZO2-positive cells we observed in the present study were not mature odontoblasts, but presumably, pre-odontoblasts/DPSCs.We attempted to identify the cells expressing PIEZO1 and PIEZO2 in human teeth using various DPSC markers, including CD44, CD73, CD105, CD106, and nestin.None of these proteins were clearly colocalized with PIEZO proteins.The results obtained were unclear, probably due to technical issues such as the use of "bridge" secondary antibodies together with polyclonal antibodies.Therefore, the nature of cells that express PIEZO proteins in the dental pulp remains to be clarified and further research is needed to resolve this question.
DPSCs are mechanosensitive (Gao et al., 2017), and mechanical stimuli can significantly increase DPSC proliferation in vitro (Han et al., 2022;Hata et al., 2012;Yang et al., 2018), but reduce DPSC survival and adhesion (Yu et al., 2009).Furthermore, Miyazaki et al. (2019) observed that during odontoblast differentiation, PIEZO1 functions as a mechanotransducer that connects pressure signals to intracellular signaling; however, no data exist for PIEZO2 in pre-odontoblasts/DPSCs.If the PIEZO1-and PIEZO2-positive cells that we identified in human dental pulp are DPSCs, it can be hypothesized that the forces acting on PIEZO1 and/or PIEZO2 may participate in the differentiation of DPSCs/pre-odontoblasts to odontoblasts.Further studies are necessary using double immunolabeling for PIEZO proteins and DPSC markers (Crende et al., 2020;Sui et al., 2020) to clearly identify PIEZO1-and PIEZO2-positive cells in human dental pulp.
It is well known that periodontal ligament cells are mechanosensitive and play a pivotal role in periodontal tissue homeostasis (De Jong et al., 2017;Lin et al., 2021).PIEZO1 is differentially adjusted by pressure (Jin et al., 2015;Shen et al., 2020) and PIEZO2 (Gao et al., 2017).The presence of these proteins in the periodontal ligament may be important for orthodontic treatments (Lukacs et al., 2023), but further research is needed to establish the role of mechanoproteins, particularly PIEZO, in induced tooth movement.

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I G U R E 1 Immunohistochemical detection of PIEZO1 (a, b) and PIEZO2 (c, d) in human teeth.Positive immunoreactivity for both proteins was detected in a cell subpopulation present in the outer layer pulp.d: dentin; fdp: fibroblasts of the dental pulp; ol: odontoblast layer.F I G U R E 2 Double immunofluorescence showing no co-localization of PIEZO1 (green fluorescence) with vimentin (red fluorescence), suggesting that PIEZO1 is not present in mature odontoblasts.Lens 60/1.25 oil; pinhole resolution: 1. XY 156 nm, and resolution Z 334 nm.d: dentin; fdp: fibroblasts of the dental pulp; ol: odontoblast layer.

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I G U R E 4 Immunohistochemical detection of PIEZO1 (a) and PIEZO2 (b, c) in human periodontal ligament.Double immunofluorescence showing no localization of PIEZO2 (d, f) green fluorescence with vimentin (e) red fluorescence.Lens 60/1.25 oil; pinhole resolution: 1. XY 156 nm, and resolution Z 334 nm.Pol: periodontal ligament.