Hydrogen Sulfide Maintains Dental Pulp Stem Cell Function Via TRPV1

Yang et al. Cell Death Discovery (2018) 4:69 DOI 10.1038/s41420-018-0071-4 Cell Death Discovery

ARTICLE Open Access Hydrogen sulﬁde maintains dental pulp stem cell function via TRPV1-mediated calcium inﬂux Ruili Yang1,2,3,YiLiu4, Tingting Yu1,2,3, Dawei Liu1,2,3, Songtao Shi3, Yongsheng Zhou2,5 and Yanheng Zhou1,2

Abstract

Hydrogen sulfide (H2S), an endogenous gasotransmitter, mediated a variety of biological processes through multiple signaling pathways, and aberrant H2S metabolism has been associated with mesenchymal stem cell (MSC) dysfunction. Here we employed the small interfering RNA treatment for cystathionine β-synthase (CBS), cystathionine γ-lyase, the main enzymes to synthesize H2S, and CBS-knockout mice to analyze the effect of H2S on dental pulp homeostasis. We showed that H2Sdeficiency attenuated dental pulp stem cell (DPSC) osteogenic/dentinogenic differentiation in vitro and in vivo with enhanced cell proliferation. Mechanically, H2S facilitated the transient receptor potential action channel subfamily V member 1-mediated calcium (Ca2+) influx, which subsequently activated the β- 2+ catenin pathway. While H2Sdeficiency decreased Ca , resulting in glycogen synthase kinase-3β-mediated β-catenin degradation, which controls proliferation and differentiation of DPSCs. Consistently, H2S-deficient mice displayed disturbed pattern of dental pulp and less dentin formation. In this study, we identified a previously unknown mechanism by which H2S regulates DPSC lineage determination and dental pulp homeostasis. 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,;

Introduction stem cells (MSCs) to ensure bone homeostasis4,5. Our Hydrogen sulfide (H2S) could freely diffuse through cell previous studies showed that H2Sdeficiency resulted in membranes to elicit various cellular events. H2S is syn- bone marrow MSC impairment caused by H2Sdeficiency + thesized by three enzymes, namely cystathionine β- reduced sulfhydration of combined Ca2 transient synthase (CBS), cystathionine γ-lyase (CSE), and 3- receptor potential (TRP)-mediated (transient receptor mercaptopyruvate sulfurtransferase (MPST) from L- potential action channel subfamily V member 3 (TRPV3), 1 2+ 6 cysteine . Alternations of H2S metabolism have been TRPV6, and TRPM4) aberrant Ca influx .H2S donor linked to osteoporosis and T cell-related immune dis- was also reported to prevent trabecular bone loss induced 2,3 orders . Endogenous H2S is essential to maintain dif- by ovariectomy via increased Wnt ligands Wnt16, Wnt2b, ferentiation and proliferation of neural stem cells and Wnt6, and Wnt10b in the bone marrow7. human-induced pluripotent stem cells, and to restore the The oral cavity is abundant with a plethora of bacteria, function of endothelial progenitor cell and mesenchymal some of which are well known to produce H2S. When the dynamic ecological equilibrium in the biofilm is disturbed, bacteria may elicit oral diseases such as car- Correspondence: Yongsheng Zhou ([email protected])or ies, gingivitis, and periodontitis8. Several studies Yanheng Zhou ([email protected]) have indicated the role of H Sindentalstemcells9. 1Department of Orthodontics, Peking University School and Hospital of 2 Stomatology, 100081 Beijing, China Dental pulp stem cells (DPSCs), isolated from dental 2National Engineering Laboratory for Digital and Material Technology of pulp,werereportedtobehighlyproliferativeandcap- Stomatology, Beijing Key Laboratory of Digital Stomatology, 100081 Beijing, able of differentiating into a variety of cells including China Full list of author information is available at the end of the article odontoblasts, osteoblasts, adipocytes, and neural cells. Edited by A.E. Sayan

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Figure 1 Human dental pulp stem cells (DPSCs) produced H2S. a Western blot analysis showed that DPSCs expressed CBS, CSE, and MPST as BMMSCs. b, c DPSCs expressed CBS, CSE, and MPST assessed by flow cytometry (b) and immunofluorescence staining (c). d DPSCs produced H2Sin the culture supernatant. Knockdown of CBS or CSE, but not MPST, by siRNA decreased the production of H2S by DPSCs, while NaHS treatment could upregulate H2S concentration. **P < 0.01; scale bar: 20 μm. All experimental data were verified in at least three independent experiments

DPSCs could be induced to generate bone and dentin In this study, we showed that the gasotransmitter H2Sis and play an important role in maintaining dental pulp essential to maintain DPSC function via TRPV1-mediated + and dentin homeostasis10. Recently, studies showed Ca2 inﬂux-stimulated glycogen synthase kinase-3β 11,12 that H2S increased DPSC hepatic differentiation . (GSK3β)/β-catenin pathway. Exogenous H2S induced DPSC apoptosis by activating 13 mitochondrial pathway apoptosis .H2S may also Results be involved in periodontal ligament stem cell pro- DPSCs expressed CBS, CSE, and produced H2S liferation and differentiation and orthodontic tooth Aberrant H2S metabolism has been linked to MSC – movement14 17. However, it remains unclear as to how dysfunction; here we revealed that DPSCs expressed CBS, H2S molecularly maintains homeostasis of DPSCs and CSE, and ubiquitous enzyme MPST, the main catalyzed dental pulp. enzymes of H2S synthesis (Fig. 1a, c and Supplementary

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Figure 2 H2S was required to maintain homeostasis of DPSCs. a DPSC proliferation was increased after CBS or CSE siRNA treatment, as assessed by BrdU proliferation assay (a). b, c CBS or CSE siRNA treatment decreased DPSC osteogenic/dentiogenic differentiation, as assessed by Alizarin red staining, with decreased expression of RUNX2, ALP, and DSPP analyzed by qPCR. d When DPSCs were subcutaneously implanted into immunocompromised mice using HA/TCP as a carrier, bone/dentin-like hard tissue formation of DPSCs was decreased after CBS or CSE siRNA treatment. e ALP-positive and DSPP-positive cells were detected in the tissue formed by DPSC in vivo transplantation. CBS or CSE siRNA treatment decreased ALP-positive and DSPP-positive cells compared to the control ones. *P < 0.05, **P < 0.01; scale bar: 100 μm(a, b, d), 50 μm(e). HA HA/TCP, CT connect tissue, B/D bone/dentin. All experimental data were veriﬁed in at least three independent experiments

Figure 1). Meanwhile, DPSCs produced H2S in the cul- MPST siRNA treatment (Fig. 1d), and H2S con- ture supernatant, which was downregulated by CBS small centration was upregulated by H2S donor NaHS treat- interfering RNA (siRNA) or CSE siRNA treatment but not ment (Fig. 1d).

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2+ 2+ 2+ Figure 3 H2S activated TRPV1-mediated Ca influx. a NaHS treatment could induce Ca influx in DPSCs, as assessed by Ca levels using the ratio of emission in response to excitation at 340 and 380 nm on an Olympus Optical IX71 microscope. b NaHS treatment could induce Ca2+ influx in DPSCs as capsaicin treatment. c Capsaicin, but not NaHS, could induce Ca2+ influx in CBS siRNA-pre-treated DPSCs. d Capsaicin, but not NaHS, could induce Ca2+ influx in CSE siRNA-pre-treated DPSCs. e Both capsaicin-induced and NaHS-induced Ca2+ influx was blocked by capsazepine treatment. f Knockdown of TRPV1 by siRNA could partially block the Ca2+ influx induced by NaHS treatment. Caps capsaicin. All experimental data were verified in at least three independent experiments

H2S maintained DPSC function subcutaneously implanted into immunocompromised To analyze the role of H2S on DPSC function, we used mice using hydroxyapatite/tricalcium phosphate (HA/ CBS or CSE siRNA to treat DPSCs and found that DPSC TCP) as a carrier, bone/dentin-like hard tissue formation proliferation was increased after CBS or CSE siRNA were observed. After CBS or CSE siRNA treatment, bone/ treatment (Fig. 2a and Supplementary Figure 2a). CBS or dentin-like hard tissue formation of DPSCs was decreased CSE siRNA treatment inhibited DPSC osteogenic/denti- (Fig. 2d). Moreover, ALP-positive and DSPP-positive cells nogenic differentiation, accompanied by decreased were detected in the tissues formed by DPSC transplan- expression of osteogenesis-related genes RUNX2 and tation in vivo, while ALP-positive and DSPP-positive cells ALP, and dentin sialophosphoprotein (DSPP) (Fig. 2b, c). were decreased after CBS or CSE siRNA treatment CSE, but not CBS, siRNA treatment decreased the adi- (Fig. 2e). These data implied that DPSCs could differ- pogenic differentiation of DPSCs, along with decreased entiate into functional odontoblast-like cells and osteo- expression of adipogenesis-related genes LPL and PPARγ blast in vitro and in vivo, which was attenuated by H2S (Supplementary Figure 2b, 2c). When DPSCs were deﬁciency.

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Figure 4 TRPV1-mediated Ca2+ inﬂux was required to maintain DPSC function. a DPSC proliferation was increased after TRPV1 knockdown, as assessed by BrdU proliferation assay. b, c DPSC osteogenic/dentinogenic differentiation was decreased after TRPV1 knockdown, as assessed by Alizarin red staining, with decreased expression of RUNX2, ALP, and DSPP analyzed by qPCR. d Bone/dentin-like hard tissue formation of DPSCs in vivo decreased after TRPV1 knockdown by siRNA. e ALP-positive and DSPP-positive cells detected in tissues formed by DPSC transplantation were decreased after TRPV1 knockdown. f NaHS treatment upregulated p-GSK3β expression, and knockdown of TRPV1 by siRNA partially blocked NaHS-

induced GSK3β phosphorylation. g CBS or CSE siRNA treatment downregulated p-GSK3β expression. h H2S donor NaHS treatment activated β- catenin expression, which was partially blocked after TRPV1 knockdown. i NaHS treatment enhanced TOPflash activity in DPSCs, which was blocked after TRPV1 siRNA treatment, as assessed by luciferase activity. NaHS treatment failed to alter FOPflash activity. HA HA/TCP, CT connect tissue, B/D bone/dentin. *P < 0.05, **P < 0.01; scale bar: 100 μm(b, d), 50 μm(e). All experimental data were verified in at least three independent experiments

2+ H2S activated TRPV1-mediated Ca influx Our previous study revealed that combined TRPV3, 2+ 2+ Emerging evidences showed that H2S plays crucial role TRPV6, and TRPM4 Ca channels mediated Ca in maintaining calcium homeostasis in endothelial cells, influx-promoted osteogenic differentiation of BMMSCs6. 18 cardiomyocytes, and MSCs . Here we found that H2S First, we used TRPV3, TRPV6, and TRPM4 siRNAs + donor NaHS treatment induced Ca2 influx in DPSCs individually or in combination to treat DPSCs and found + + (Fig. 3a). The TRPV subfamily is Ca2 -permeable cation that inhibition of these Ca2 channels could not block + channel, which could be activated by different stimuli. NaHS-induced Ca2 influx (Supplementary Figure 3).

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Figure 5 β-Catenin signaling were decreased in Cbs−/− dental pulp. a Cbs−/− mice dental pulp showed more cells than control ones. b The average dentin thickness of mandible ﬁrst molar is less in Cbs−/− mice than control ones, as assessed by micro-CT. c DSPP+ cells decreased in Cbs−/− mice dental pulp compared to control ones. d PCNA+ cells increased in Cbs−/− mice dental pulp. e, f p-GSK3β+ and active-β-catenin cells decreased in Cbs−/− mice dental pulp. *P < 0.05, **P < 0.01, ***P < 0.001, scale bar: 200 μm left panel, 100 μm right panel (a), 1 mm (b), 20 μm(c–f). All experimental data were veriﬁed in at least three independent experiments

Studies found that TRPV1 was expressed in osteoblasts knockdown of TRPV1 by siRNA blocked NaHS-triggered 2+ and was important in maintaining osteoblast differentia- Ca influx in DPSCs (Fig. 3f), verifying that H2S stimu- + tion ability19. Here we revealed that capsaicin, which acts lates TRPV1 channel to trigger Ca2 influx in DPSCs. on the TRPV1 receptor in nociceptive neurons to activate + calcium influx, induced Ca2 influx in DPSCs. NaHS TRPV1-mediated Ca2+ influx was required to maintain + treatment induced similar Ca2 influx as capsaicin in DPSC function + DPSCs (Fig. 3b). When DPSCs were pretreated with CBS To verify the role of TRPV1-mediated Ca2 influx in or CSE siRNA treatment, attenuated NaHS, but not DPSCs, we analyzed DPSC function after TRPV1 knock- + capsaicin, triggered Ca2 influx (Fig. 3c, d). When DPSCs down and revealed that DPSC proliferation was increased were pretreated with capsazepine, a competitive antago- after TRPV1 knockdown (Fig. 4a). DPSC osteogenic/ + nist of TRPV1, the Ca2 influx induced both by NaHS and dentinogenic differentiation was decreased, along with capsaicin were blocked, which indicated that TRPV1 downregulated expression of RUNX2, ALP, and DPSS 2+ channels play a crucial role in mediated H2S-induced Ca after TRPV1 knockdown by siRNA (Fig. 4b, c). Moreover influx in DPSCs (Fig. 3e). Furthermore, we found that the in vivo bone/dentin-like hard tissue formed by DPSC

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+ transplantation also decreased after TRPV1 knockdown, proliferation was elevated. There were more PCNA cells −/− which was consistent with H2Sdeficiency. (Fig. 4d). infiltrated in Cbs mice dental pulp, indicating that H2S Consistently, less ALP-positive and DSPP-positive cells is required to restrict aberrant proliferation of stem cells, were detected in the tissues formed by DPSC transplan- which is consistent with previous reports15. Furthermore, − − tation after TRPV1 siRNA treatment (Fig. 4e). These we found that the dentin thickness in Cbs / mice + studies indicate that TRPV1-mediated Ca2 influx play decreased compared to the control ones, which was crucial roles in maintaining DPSC function. consistent with the decreased osteogenic and dentinogenic differentiation of H2Sdeficiency DPSCs in vitro and H2S activated β-catenin signaling in vivo. These results expand the physiological roles of Next, we analyzed the underlying mechanisms of how H2S in MSCs from bone to dental pulp and dentin 2+ Ca influx regulated DPSC function. We used H2S donor homeostasis. NaHS to treat DPSCs and found that NaHS treatment Calcium channels were one of main cellular events that 20,21 elevated phosphorylation of GSK3β, while knockdown of may contribute H2S-mediated molecular responses . + TRPV1 by siRNA blocked NaHS-induced phosphory- The L-type and T-type Ca2 channels and TRP channels lated-GSK3β (p-GSK3β) elevation (Fig. 4f). Consistently, are reported to be the alternative targets of H2S. TRP we revealed that CBS or CSE siRNA treatment down- channels were involved in diverse cellular functions such regulated p-GSK3β expression (Fig. 4g). β-Catenin sig- as gene expression, proliferation, differentiation, migra- naling plays crucial roles in determining stem cell lineage tion, and apoptosis. It is reported that H2S triggers TRPV1 1 differentiation . We found that H2S donor NaHS treat- to mediate chloride secretion in acute pancreatitis and 22,23 ment increased β-catenin and active-β-catenin expression, colon . In this study, we found that H2S donors which was blocked by TRPV1 knockdown (Fig. 4h). Next, mimicked the effect of capsaicin in the neurons, leading to 2+ 2+ we investigated whether H2S affected β-catenin tran- the Ca flux entry in DPSCs. Ca influx entry then scriptional activity by using luciferase assay analyzed by triggered GSK3β/β-catenin cascade to regulate DPSC the ratio of TOPflash and FOPflash. The results showed proliferation and differentiation. Furthermore, the study that H2S donor NaHS treatment increased pathway revealed that TRPV1 was essential to maintain DPSC activity. TRPV1 siRNA treatment attenuated NaHS- proliferation and differentiation. When TRPV1 was induced pathway activity elevation (Fig. 4i). These data knocked down by siRNA in DPSCs, the proliferation of implied that H2S gasotransmitter pathway activated DPSCs was elevated and osteogenic/dentinogenic differ- β-catenin signaling in DPSCs. entiation of DPSCs was inhibited, which firstly implied the role of TRPV1 in maintaining MSC capacity. Our pre- 2+ H2S-mediated β-catenin signaling contributed to dentin vious study reported that H2S-triggered Ca influx is formation required to maintain bone and MSC homeostasis. The In order to verify the role of β-catenin cascade in study revealed that combined TRPV3, TRPV6, and 2+ 2+ maintaining dental pulp homeostasis, we employed H2S- TRPM4 Ca channels mediated Ca influx to promote − − − − deficient (Cbs / ) mice and found that Cbs / mice osteogenic differentiation of BMMSCs6. In this study, we dental pulp showed disturbed pattern with increased cell found that the proliferation and differentiation capacity of infiltration than control ones (Fig. 5a). The average DPSCs could not be attenuated by TRPV3, TRPV6, and − − + thickness of dentin was decreased in Cbs / mice with TRPM4 Ca2 channels blockage. These differences may decreased DSPP-positive cells in the dental pulp com- attribute that DPSCs were derived from the neural crest pared to control ones (Fig. 5b, c). Furthermore, pro- and mesoderm during development, which were more + − − liferative PCNA cells increased in Cbs / mice dental accessible to differentiate into dentin-like tissue rather + + pulp (Fig. 5d), while p-GSK3β and active-β-catenin than the bone compared to BMMSCs. Functionally, − − cells decreased in Cbs / mice dental pulp (Fig. 5e, f), DPSCs also easily differentiated into neuronal and glial 24,25 which to some extent verifies that H2S-mediated β-cate- cells and acted as neuronal cells . These studies shed nin signaling is important in maintaining dentin forma- light on the different molecular target induced by H2Sto tion and DPSC function in vivo. illustrate the unique properties of MSCs derived from different tissues Discussion Variety of signal pathways may contribute to that H2S H2S regulates several physiological and pathological fine-tune its effects on different tissues and cells. H2S may processes via diverse mechanisms. In this study, we trigger the nuclear translocation of nuclear factor-κB (NF- showed that H2S is essential to maintain DPSC osteogenic κB) and affect the activity of numerous kinases including and dentinogenic differentiation. We used siRNA to p38 mitogen-activated protein kinase, extracellular signal- knockdown CBS and CSE, the main enzymes that cata- regulated kinase, and Akt signaling. H2S is able to pro- lyzed H2S production in DPSCs, and found that DPSC mote angiogenesis and vascular remodeling via the PI3K/

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Akt/survivin axis in endothelial cells by augmenting anti-GSK3β and anti-phospho-GSK3β antibodies were 20,21 phosphorylation of ERK and p38 .H2S deceases pro- purchased from Cell Signaling Inc. (San Francisco, CA, inflammatory genes involved in cardiac ischemic/reper- USA). Anti-β-actin antibody was purchased from Sigma- fusion injury via inhibiting the nuclear translocation of Aldrich Corporation (St. Louis, MO, USA). Unconjugated 26 2+ NF-κB . In BMMSCs, H2S-induced Ca influx pro- anti-CBS, CSE, DSPP (DSP) and TRPV1 were purchased moted β-catenin activity via the PKC/ERK pathway to from Abcam Inc. (Cambridge, MA, USA). 6 maintain bone homeostasis . However, we found that H2S treatment failed to alter ERK signaling in DPSCs (data + were not shown). We showed that TRPV1-mediated Ca2 Chemicals influx entry facilitates the phosphorylation of GSK3β to siRNA for CBS (hydroxylamine), CSE (DL-pro- disrupt the “destruction complex.” β-Catenin accumulates pargylglycine), TRPV1, TRPV3, TRPV6, and TRPM4 were in the cytoplasm and then translocates into the nucleus, purchased from Santa Cruz. H2S donor NaHS was pur- which promoted DPSC osteogenic differentiation. H2S chased from Sigma-Aldrich Corporation (St. Louis, MO, + deficiency decreased TRPV1-mediated Ca2 influx entry, USA). Capsaicin and capsazepine were purchased from leading to decreased phosphorylation of GSK3β. These EMD Millipore (Billerica, MA, USA). The concentrations data indicated that the response of different stem cells to of capsaicin and capsazepine used for the treatment H2S may be regulated by different signaling pathways. were 1 μM. Cytoplasmic β-catenin was degraded, resulting in DPSC − − osteogenic differentiation deficiency. In vivo, Cbs / mice Isolation and culture of human bone DPSCs dental pulp showed elevated cell infiltration and pro- Dental pulp from human extracted wisdom teeth were liferation and decreased GSK3β/β-catenin signaling. gently separated, minced, and digested with 2 mg/ml − − Moreover, the dentin formation of Cbs / mice was less collagenase type I (Worthington Biochemical, Freehold, than control ones. These data suggest that H2S-mediated NJ, USA) and 4 mg/ml dispase II (Roche Diagnostics, GSK3β/β-catenin cascade is important to maintain DPSC Indianapolis, IN, USA) in phosphate-buffered saline for 1 function and dental pulp homeostasis. These results shed h at 37 °C. Then, the cells were passed through a 70-μm light on the different biology and mechanism of H2S strainer (BD Biosciences, Franklin Lakes, NJ, USA) to get targeting on different MSCs and tissue homeostasis. single cells. The single-cell suspensions were cultured with α-minimum essential medium (α-MEM) supple- Conclusion mented with 15% fetal bovine serum (FBS), 100 mM L- Our study reveals that the levels of H2S are essential to ascorbic acid-2-phosphate, 2 mM L-glutamine, 100 U/ml + + maintain Ca2 homeostasis via TRPV1 Ca2 channel in penicillin, and 100 μg/ml streptomycin and passaged, as 2+ 27 DPSCs. H2Sdeficiency results in Ca influx/GSK3β/β- previously reported . catenin cascade response in DPSCs, leading to differentiation deficiency of DPSCs and dentin formation dis- Human bone marrow MSCs BMMSC culture orders (Supplementary Figure 4). Human bone marrow aspirates from healthy adult donors (20–35 years of age) were purchased from AllCells Materials and methods (Emeryville, CA, USA). Human bone marrow progenitor Mice cells were enriched using fresh human bone marrow by ® C57BL/6 (JAX #000664), B6.129P2-Cbstm1Unc/J (JAX RosetteSep Human Bone Marrow Progenitor Cell Pre- + − #002461) Cbs / mice were purchased from Jackson lab. Enrichment Cocktail (STEMCELL Technologies, Van- Female immunocompromised mice (beige nude/nude couver, BC, Canada) following the manufacturer’s XIDIII) were purchased from Harlan Laboratories. All instructions. The enriched single-cell suspensions were animal experiments were performed under institutionally cultured with α-MEM supplemented with 15% FBS, 100 approved protocols for the use of animal research at the mM L-ascorbic acid-2-phosphate, 2 mM L-glutamine, 100 University of Pennsylvania (IACUC# 805478) and Peking U/ml penicillin, and 100 μg/ml streptomycin and pas- University (#LA2012-65). saged, as previously reported27.

Antibodies and chemicals Cell proliferation assay Antibodies DPSCs were passaged to a 4-well chamber slide (Nunc, Anti-active-β-catenin and β-catenin antibodies were 1×104 per well) and incubated for 12 h at 37 °C in a 5% purchased from Millipore (Temecula, CA, USA). Anti- CO2 condition. Then, the cells were assayed for bromo- CD105-PE, anti-CD146-PE, anti-CD90-PE, anti-CD34- deoxyuridine (BrdU) incorporation, as per the manu- + PE, and anti-CD45-PE antibodies were purchased facturer’s instruction. BrdU and total cell numbers were + from BD Bioscience (San Jose, CA, USA). Unconjugated counted in ﬁve images per subject. The BrdU cells were

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+ indicated as a percentage of BrdU cells and the total cell excitation at 340 and 380 nm on an Olympus Optical number. IX71 microscope.

DPSC-mediated hard tissue formation in vivo Measurement of H2S Approximately 4.0 × 106 DPSCs were mixed with HA/ Cell culture supernatants were mixed with 0.25 ml Zn TCP ceramic particles (40 mg, Zimmer Inc., Warsaw, IN, acetate (1%) and 0.45 ml water for 10 min at room tem- USA) as a carrier and subcutaneously implanted into the perature. TCA (10%; 0.25 μl) was then added and cen- dorsal surface of 8- to 10-week-old immunocompromised trifuged (14,000 × g for 10 min at 4 °C). The supernatant mice (n = 4). At 8 weeks post-implantation, the implants was then collected and mixed with N,N-dimethyl-p-phe- were harvested, fixed in 4% paraformaldehyde, and then nylenediamine sulfate (20 μM) in 1.2 M HCl and FeCl3 decalcified with 5% EDTA, followed by paraffin embed- (30 μM) in 1.2 mol/l HCl. After 20 min, absorbance was ding. The 5 μm paraffin sections were stained with measured at 650 nm. hematoxylin and eosin and analyzed by NIH ImageJ. The newly formed mineralized tissue area in each field was In vitro transcription assays calculated and shown as a percentage of the total tissue β-Catenin-mediated transcriptional activity was mea- area. sured by the luciferase activity by using TOPflash and FOPflash described previously6. DPSCs (1 × 105) were Osteogenic/dentinogenic differentiation assay seeded onto a 24-well culture plate and cultured for 24 h. Osteogenic/dentinogenic differentiation is performed as TOPflash or FOPflash (10 ng/well) and renilla luciferase described in the Supplementary materials. (4 ng/well) were transfected into DPSCs using a Lipo- fectamine™ RNAiMAX kit (Invitrogen). After 15 h, the Adipogenic differentiation assay plates were assayed for firefly luciferase and renilla luci- ® Adipogenic differentiation is performed as described in ferase activities using the Dual-Luciferase Reporter Assay the Supplementary materials. System (Promega).

Immunofluorescent staining Micro computed tomography DPSCs were seeded on a 4-well chamber slide and The mandible of mice was scanned with a micro- incubated for 12 h at 37 °C in a 5% CO2 condition. The computed tomography (micro-CT) system (Inveon detailed method of immunofluorescent staining is MMCT, Berlin, Germany) at 80 kV, 500 µA, and an image described in the Supplementary materials. voxel size of 18 µm. The hemi-mandibles were excised (n = 6 per group). The hemi-mandible samples were direc- Immunohistochemical staining ted parallel to the occlusal plane and scanned for 2.70 Immunohistochemical staining is performed as descri- mm. The thickness of the first molar was measured with bed in the Supplementary materials. the DataViewer software (ver. 1.4.3; SkyScan).

Western blot Statistical analysis Western blot is performed as described in the Supple- P values were analyzed from two-tailed Student’s t test mentary materials. for the difference between two groups or one-way analysis of variance to compare the difference from more than two Quantitative PCR groups using the SPSS 18.0 software. P values <0.05 were For quantitative PCR (qPCR) analysis, total RNA was considered signiﬁcant. extracted with RNeasy Mini kit (Qiagen, Valencia, CA, USA), and complementary DNA was prepared using a Acknowledgements ® This work was supported by Supported by Beijing Natural Science Foundation SuperScript III Reverse Transcriptase (RT) kit (Invitro- (7182182), the Young Elite Scientist Sponsorship Program by CAST ® gen). qPCR was carried out using SYBR Green Supermix (2017QNRC001), International Science & Technology Cooperation Program of China No. 2015DFB30040 (to Y.Z.), the National Science Foundations of China, (Bio-Rad) on a Bio-Rad CFX96 Real Time System, as No. 81470717 (to Y.Z.), 81600865 (to R.Y.), and 81570953 (to Y.S. Z.). indicated by the manufacturer’s protocol. Author details 2+ 1Department of Orthodontics, Peking University School and Hospital of Measurement of Ca image 2 5 Stomatology, 100081 Beijing, China. National Engineering Laboratory for DPSCs (1 × 10 ) were seeded onto 60 mm culture dishes Digital and Material Technology of Stomatology, Beijing Key Laboratory of 3 and cultured for 24 h at 37 °C in 5% CO2. Then, the cells Digital Stomatology, 100081 Beijing, China. Department of Anatomy and Cell Biology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA were loaded with fura-2 AM (Invitrogen) and incubated 4 2+ 19104, USA. Laboratory of Tissue Regeneration and Immunology and for 1 h at 37 °C in 5% CO2 in the dark. Ca levels were Department of Periodontics, Beijing Key Laboratory of Tooth Regeneration and measured from the ratio of emission in response to Function Reconstruction, Capital Medical University School of Stomatology,

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100050 Beijing, China. 5Department of Prosthodontics, Peking University 8. Aas,J.A.,Paster,B.J.,Stokes,L.N.,Olsen,I.&Dewhirst,F.E.Defining the School and Hospital of Stomatology, 100081 Beijing, China normal bacterial flora of the oral cavity. J. Clin. Microbiol. 43,5721–5732 (2005). Authors' contributions 9. Zhang,J.H.,Dong,Z.&Chu,L.Hydrogensulfide induces apoptosis in human R.Y.: conception and design, collection and assembly of data, data analysis and periodontium cells. J. Periodontal Res. 45,71–78 (2010). interpretation, final approval of manuscript; Y.L., T.Y.: collection data and 10. Miura, M. et al. SHED: stem cells from human exfoliated deciduous teeth. Proc. interpretation, final approval of manuscript. Y.H.Z., Y.S. Z., S.S.: critically edited Natl. Acad. Sci. USA 100,5807–5812 (2003). the manuscript, financial support, final approval of manuscript. 11. Ishkitiev, N. et al. Hydrogen sulfide increases hepatic differentiation in tooth–pulp stem cells. J. 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