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CXC Chemokine Receptor 4 Is Expressed Paravascularly in Apical Papilla and Coordinates with Stromal Cell–derived Factor-1α during Transmigration of Stem Cells from Apical Papilla
Clinical Research
CXC Chemokine Receptor 4 Is Expressed Paravascularly in Apical Papilla and Coordinates with Stromal Cell–derived Factor-1a during Transmigration of Stem Cells from Apical Papilla Jing-Yi Liu, DDS,* Xue Chen, DDS,* Lin Yue, DDS, PhD,* George T.-J. Huang, DDS, MSD, DSc,† and Xiao-Ying Zou, DDS, MD* Abstract Introduction: Stem cells from the apical papilla (SCAPs) at the apex may be attracted into the root canal space as a cell source for pulp-dentin regeneration. To test this possibility, we used in vitro transmigration models to investigate whether SCAPs can be chemoattracted by the delivery of the chemotactic cytokine stromal cell–derived factor-1a (SDF-1a). Methods: We first examined the expression of CXC chemokine receptor 4 (CXCR4) for SDF-1a in the apical papilla and in cultured SCAPs using immunofluorescence, reverse-transcription polymerase chain reaction (RT-PCR), and flow cytometric analyses. A standard Transwell migration assay and a 3-dimensional cell migration assay were used to analyze transmigration of SCAPs via the SDF-1a/CXCR4 axis. Results: CXCR4 was expressed in the paravascular region of the apical papilla and detected in SCAP cultures. Most cultured SCAPs harbored intracellular CXCR4 (58%–99%, n = 4), whereas only a few cells had detectable CXCR4 on the cell surface (0.3%–2.34%, n = 4). Although SDF-1a had no significant effect on SCAP proliferation, it significantly promoted a higher number of migrated cells; this effect was abolished by antiCXCR4 antibodies. Interestingly, cell surface CXCR4 on SCAPs was not detectable until after transmigration. The 3-dimensional migration assay revealed that SDF1a significantly enhanced SCAP migration in the collagen gel. Conclusions: SCAPs can be chemoattracted via the SDF-1a/CXCR4 axis, suggesting that SDF-1a may be used clinically to induce CXCR4expressing SCAPs in the apical papilla to transmigrate into the root canal space as an endogenous cell source for pulp regeneration. (J Endod 2015;41:1430–1436)
Key Words 3-dimensional transmigration assay, cell homing, CXC chemokine receptor 4, stem cells from the apical papilla, stromal cell–derived factor-1a
R
egenerative endodontics has gained attention in the treatment of endodontically involved teeth, particularly immature permanent teeth. Successful de novo regeneration of pulp- and dentinlike tissues using animal models has been shown using cell transplantation into the root canal space (1–3). However, such cell-based regeneration approaches face hurdles such as complex cell processing procedures that require a specific facility—good manufacturing practice (4). A recently developed clinical treatment protocol termed revitalization or revascularization is a cell-free approach that allows the growth of additional root structure (ie, apical maturation and thickening of the root) (5). Although revitalization procedures have shown some clinical success evidenced by many clinical case reports and clinical research (6, 7), histologic studies have shown little evidence of actual pulp regeneration in human cases (8) or animal studies (9). The concept of cell homing is to use the mobilized endogenous cells in the host to regenerate the lost tissue (4, 10, 11). Stromal cell–derived factor-1a (SDF-1a), a member of the CXC cytokine subfamily, is a widely expressed chemotactic cytokine that mediates cell migration through its binding with CXC chemokine receptor 4 (CXCR4) (12, 13). The primary role of the SDF-1a/CXCR4 axis is to mobilize CD34+ hematopoietic progenitor cells to different organs during development and different niches within the bone marrow during differentiation and maturation of hematopoietic progenitor cells (14). The SDF-1a/CXCR4 axis is necessary for tissue repair and/or regeneration under multiple pathological conditions, such as myocardial infarction (15, 16) and acute kidney injury (17). Regarding pulp injury and regeneration, the expression of CXCR4 and SDF-1a was found to promote the transmigration of dental pulp cells (18). In immature teeth with necrotic pulp, endogenous dental pulp stem cells (DPSCs) are often no longer present, whereas stem cells from the apical papilla (SCAPs), if present, may be attracted into the canal space for pulp-dentin regeneration (19, 20). SCAPs are highly proliferative in cultures and have a strong odontogenic differentiation capacity (13, 21). They have been used as an exogenous cell source to regenerate pulp- and dentin-like tissues in root canal space and to form bioroots in animal models (2, 20). When in situ, because of their proximity to the vascular-rich periapical
From the *Department of Cariology, Endodontology and Operative Dentistry, School and Hospital of Stomatology, Peking University, Beijing, People’s Republic of China; and †Department of Bioscience Research, College of Dentistry, University of Tennessee Health Science Center, Memphis, Tennessee. Address requests for reprints to Dr Xiao-Ying Zou, Department of Cariology, Endodontology and Operative Dentistry, Peking University School and Hospital of Stomatology, 22 South Zhongguancun Avenue, Haidian District, Beijing 100081, PR China. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2015.04.006
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Clinical Research tissues, SCAPs in the apical papilla may survive endodontic infection (19, 22); therefore, they may be attracted into the canal space for pulp regeneration. In the present study, we hypothesized that SCAPs can be chemoattracted to migrate via the SDF-1a/CXCR4 axis. We investigated the expression of CXCR4 in both apical papilla and cultured SCAPs, and in vitro transmigration models were used to determine the chemotactic effect of SDF-1a on SCAPs.
Materials and Methods Sample Collection Human impacted third molars (n = 8) with an open apex were collected from healthy patients (8 donors aged 13–24 years) in the Oral and Maxillofacial Surgery Department at Peking University School of Stomatology with a protocol approved by the Ethical Committee of Peking University Health Science Center. Immediately upon extraction, the apical papilla was carefully cut off from the root apex. Each tooth was split cross-sectionally, and the dental pulp was then carefully extracted by tweezers, with the apical 3 mm cut off to avoid including the apical papilla. Cell Culture SCAPs and DPSCs were isolated as described previously (2). Briefly, tissues were minced and digested in a solution of 3 mg/mL collagenase type I and 4 mg/mL dispase for 30 to 60 minutes at 37 C. The isolated cells were seeded and cultured with alpha modification of Eagle medium (aMEM; Gibco, Grand Island, NY) supplemented with 15% fetal bovine serum (FBS; Gibco), 2 mmol/L L-glutamine, 100 U/mL penicillin G, and 100 mg/mL streptomycin and maintained in 5% CO2 at 37 C. Colony formation units of fibroblastic cells were normally observed within 1 to 2 weeks. These heterogeneous populations of adherent, clonogenic dental stem/progenitor cells were tested for their cell surface marker expression by flow cytometric analysis; they were positive for STRO-1, CD146, CD90 and CD105 and negative for CD45 (13). Cells at passage (p) 2 or p3 were used for experiments. Cells isolated from each tooth/donor were grown, maintained, and used separately for each independent experiment. Immunofluorescence Frozen apical papilla and pulp tissues were cryosectioned at 5 mm and double stained for CXCR4/vWF or CXCR4/STRO-1. The sections were permeabilized by 0.5% Triton X-100 (Sigma-Aldrich, St Louis, MO) and blocked with 10% goat serum albumin. The sections were then incubated with primary mouse monoclonal antihuman CXCR4 (1:100; R&D Systems, Abington, UK) and rabbit polyclonal antihuman von Willebrand factor (vWF) (1:4000; Dako, Copenhagen, Denmark) antibodies or with CXCR4 and mouse immunoglobulin (Ig)M antihuman STRO-1 antibodies (1:200; eBioscience, San Diego, CA) followed by secondary antibodies including fluorescein isothiocyanate (FITC) conjugated goat antimouse IgG (ZSGB-Bio, Beijing, China) and phycoerythrin (PE)-conjugated goat antirabbit IgG (Life Technologies, Carlsbad, CA) or goat antimouse IgG and PE-conjugated goat antimouse IgM (eBioscience). Sections were then counterstained with 40 ,6-diamidino-2-phenylindole (DAPI, ZSGB-Bio) and mounted for images analysis under a confocal laser scanning microscope at magnifications of 40 and 63 (488-nm laser diode for FITC, 405-nm laser diode for DAPI/dsDNA [LSM5; Carl Zeiss, Jena, Germany]). For immunocytofluorescence, 5 103 cells (p2)/coverslip were fixed in 4% paraformaldehyde, permeabilized by 0.5% Triton X-100 or without permeabilization, blocked by 10% goat serum for 30 minutes, and followed by incubation with primary mouse monoclonal antihuman JOE — Volume 41, Number 9, September 2015
CXCR4 (1:100; R&D Systems) followed by secondary antibodies FITCconjugated goat antimouse IgG (ZSGB-Bio); the cell nuclei were stained with DAPI.
Reverse-transcription Polymerase Chain Reaction Total RNA was isolated from tissues or cultured cells using TRIzol (Invitrogen, Carlsbad, CA). Extracted RNA was reverse transcribed to complementary DNA using the RT Reagent Kit (Fermentas, Burlington, ON, Canada). Polymerase chain reaction (PCR) amplification was performed using a PCR Kit (Fermentas) with the following amplification steps: initial denaturation at 95 C for 3 minutes followed by 35 cycles of 95 C for 30 seconds, 53 C for 30 seconds (CXCR4) or 50 C for 30 seconds (glyceraldehyde phosphate dehydrogenase, GAPDH), 72 C for 30 seconds, and a final extension at 72 C for 10 minutes. The primers were as follows: CXCR4: 50 -CCGTGGCAAACTGGTACTTT-30 (forward), 50 -GACGCCAACATAGACCACCT-30 (reverse) and GAPDH: 50 -CAAGGCTGAGAACGGGAAGC-30 (forward), 50 -AGGGGGCAGAGATGATGACC-30 (reverse). The PCR products were examined by 1.5% agarose gel electrophoresis and detected by a VILBER Fusion Fx5 (Vilber Lourmat, Collegien, France). Flow Cytometry SCAPs (1 106, p3) were resuspended in 0.5% bovine serum albumin (BSA). For the detection of cell surface CXCR4, cells were stained with PE-conjugated monoclonal antihuman CXCR4 antibody (R&D Systems). For intracellular CXCR4 staining, cells were first blocked with nonconjugated antihuman CXCR4 monoclonal antibody (clone 12G5, R&D Systems), fixed with 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) followed by incubation with PE-conjugated monoclonal antihuman CXCR4 antibody. Cells were then analyzed on a FACSCalibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ) with Cell Quest Pro software (BD Bioscience, Bedford, MA). Cell Counting Kit-8 Assay for Cell Proliferation SCAPs (p2) were seeded into 96-well plates at 4 103 cells/well. Cells were divided into 5 groups: the negative control group and 4 experimental groups each treated with a different concentration of SDF-1a (ie, 25, 50, 100, and 200 ng/mL) (R&D Systems). After incubation with the presence of SDF-1a for 1, 3, 5, 7, and 9 days, cell viability was determined using the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. The optical density (OD) was measured with an Elx808 enzyme-linked immunosorbent assay reader (BioTek, Winooski, VT) at 450 nm (OD450). Cell Transmigration Assay SCAPs (p3) were loaded into the upper chambers of 24-well Transwell inserts (8-mm pore; Corning, Corning, NY) at 5 103 cells/well. There were 3 experimental groups: 1. The negative control group: The lower chamber contained only culture medium. 2. The SDF-1a group: The lower chamber medium was supplemented with various concentrations (ie, 25, 50, 100, and 200 ng/mL) of SDF-1a. 3. The neutralized receptor group: SCAPs were incubated in medium supplemented with 10 mg/mL anti-CXCR4 monoclonal antibody (clone 12G5, R&D Systems) in its upper chamber and the lower chamber medium was supplemented with 100 ng/mL SDF-1a (18, 23).
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Clinical Research The Transwell cultures were incubated for 24 hours. The nontransmigrated cells on the upper side of the membrane were wiped out, and cells transmigrated to the lower side of the membranes were fixed with 95% ethanol and stained with 0.1% crystal violet for evaluation under a microscope. Nine randomly selected fields from each membrane were selected for cell counting.
To determine whether the cell surface CXCR4 expression changed during the transmigration process, Transwell inserts were also collected for immunofluorescence staining after the 24-hour Transwell assay. For the nonmigrated cell staining (upper side of the membrane), we wiped out the cells on the lower side of the membrane. Conversely, for staining of the transmigrated cells, cells on the upper side of the membrane were
Figure 1. CXCR4 expression in apical papilla and pulp. (A) RT-PCR analysis of CXCR4 expression in the apical papilla and dental pulp. Numbers represent different samples (#1: 22-year-old woman, #2: 20-year-old woman, and #3: 21-year-old woman, all third molars). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control. (): Milli-Q water (EMD Millipore, Billerica, MA) served as the negative control. (B) Immunofluorescence double staining of the apical papilla and dental pulp showing CXCR4 (green) coexpressed with vWF (red). CXCR4 in the apical papilla was expressed in the paravascular region where vWF-positive endothelial cells were also present (sample #4: 21-year-old man, third molar). (C) Immunofluorescence analysis of CXCR4 (green) and STRO-1 (red) in apical papilla and dental pulp. CXCR4-expressing cells in the apical papilla were also immunoreactive to anti–STRO-1 antibodies (sample #4). Cell nuclei exhibited blue fluorescence by DAPI counterstaining. Images are representative data of independent experiments with consistent results. Scale bar = (B) 20 mm and (C) 50 mm. Apical, apical papilla; Pulp, dental pulp tissue.
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Clinical Research wiped out. The immunofluorescence staining followed the same procedures described previously but without permeabilization.
3-dimensional Cell Migration Assay A 3-dimensional (3D) collagen gel was made as described previously (24). Briefly, after neutralizing with 1-N NaOH, rat-tail collagen type I solution (3 mg/mL) (BD Bioscience) was dispensed into the upper chambers of 24-well Transwell inserts (0.4-mm pore, Corning) and allowed gelation. SCAPs (p3) resuspended in culture medium were seeded on the surfaces of the gel at 5 103 cells/well and incubated with or without 100 ng/mL SDF-1a in the lower chamber. After a 7-day incubation, gels
were cut out and fixed in 10% formalin. After cell nuclear staining by DAPI, SCAPs migrated into the gel were analyzed by a confocal laser scanning microscope (speed at 400 Hz, objective magnification 20; diode 405, SP8; Leica, Wetzlar, Germany). One randomly selected central field from the surface of each gel was chosen for 3D reconstruction with the LAS AF 3D Visualization software (Leica). According to the reconstructed images (600 mm 600 mm 300 mm), cell proliferation was evaluated on x-y axial planes, whereas y-z axial planes reflected the cell migration depth. By dividing the y-z axial plane into 3 migration depth levels (ie, 0–100 mm, 100–200 mm, 200–300 mm), the numbers of migrated cells were counted according to the successive layers of the 3D reconstructed images.
Figure 2. CXCR4 expression in SCAPs. (A) RT-PCR analysis of CXCR4 expression in SCAPs and DPSCs. Numbers represent different samples (#5: 18-year-old woman, #6: 23-year-old woman, #7: 22-year-old woman, and #8: 13-year-old male; all from p2, third molars). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as internal control genes. (): Milli-Q water served as the negative control. (B) Immunofluorescence staining of SCAPs expressing CXCR4 (green) after permeabilization using Triton X-100, whereas CXCR4 was hardly expressed by nonpermeabilized SCAPs. Blue fluorescence revealed the cell nucleus (sample #7, p2). Images are representative data of independent experiments with consistent results. Scale bar = 50 mm. (C) Flow cytometric analysis of CXCR4 expression in SCAPs. SCAPs (1 106) at p3 were incubated with PE-conjugated monoclonal anti-CXCR4 antibodies for cell surface staining. Prior blockage of cell surface CXCR4 and cell permeabilization was performed before intracellular CXCR4 staining (samples #5–#8, p3). Green areas indicate positive signals; dashed lines are the isotype control. (D) After coincubation with 100 ng/mL SDF-1a in the medium for 24, 36, or 48 hours, there was no significant change of cell surface CXCR4 expression (sample #6, p3). (E) Cell Counting Kit-8 assay of SCAP proliferation. The OD450 values represent relative numbers of cells (mean standard deviation). After incubation with SDF-1a (25, 50, 100, or 200 ng/mL) for 1, 3, 5, 7 and 9 days, there was no significant difference between the negative control (nontreated) and SDF-1a–treated groups (P > .05) (sample #6, p3). CTRL, control.
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Clinical Research Data Analysis All experiments were performed using cells cultured from at least 3 different donors and assayed at least in triplicate. All values are presented as mean standard deviation. Statistical analysis was performed with the SPSS 22.0 software (IBM SPSS Statistics, Armonk, NY) using the Student t test when only 2 groups were compared. One-way analysis of variance was used followed by the least significant difference test when comparing 3 groups or more. Values of P < .05 were considered statistically significant.
Results The SDF-1a Receptor CXCR4 Was Expressed in the Apical Papilla CXCR4 expression was detected by reverse-transcription polymerase chain reaction (RT-PCR) in both the apical papilla and pulp tissue as shown in Figure 1A. Using immunofluorescence staining, we localized CXCR4 in the apical papilla to be in the paravascular region where vWFpositive endothelial cells were also present. In addition, CXCR4expressing cells in the apical papilla were also immunoreactive to anti–STRO-1 antibodies. A similar observation was made in the pulp tissue as indicated in Figure 1B and C.
Isolated SCAP in Cultures Expressed CXCR4 The expression of CXCR4 was detected by RT-PCR in both SCAPs and DPSCs in cultures (Fig. 2A). In the immunocytofluorescence study, no CXCR4 staining was detected when cells were not permeabilized, indicating a lack of cell surface CXCR4 expression. We then examined intracellular expression of CXCR4 and found that 77.55% (38 cells/49 cells from 14 randomly selected fields) of SCAPs expressed intracellular CXCR4 (Fig. 2B). Flow cytometric analysis showed that only 0.3%–2.34% (n = 4) of SCAPs expressed cell surface CXCR4 when cells were stained without permeabilization (Fig. 2C). Up to 57.49%–98.48% (n = 4) of SCAPs stained positively for intracellular CXCR4 expression, indicating that most cells expressed intracellular CXCR4, whereas only a low percentage of them expressed cell surface CXCR4. SDF-1a Had Little to No Effect on CXCR4 Expression or Proliferation of SCAPs We tested the effect of SDF-1a on SCAP CXCR4 surface expression and found no significant induction of cell surface CXCR4 expression after incubation with 100 ng/mL SDF-1a for 24, 36, or 48 hours compared with controls (Fig. 2D). We also determined SCAP proliferation using Cell Counting Kit-8 assays after cells were treated with different concentrations of SDF-
Figure 3. Effects of SDF-1a on the chemotactic capability of SCAPs. (A) A schematic diagram of a Transwell insert (8-mm pore). (B) Transmembrane migration of SCAPs under microscopy with 0.1% crystal violet staining. (C) Numbers of migrated cells in each group. There were significantly more cells transmigrated in the SDF1a groups compared with the negative control in a dose-dependent manner, except for the 25 ng/mL SDF-1a group. The neutralized receptor group has no significant difference with the negative control (*P < .05 compared with the negative control). (D) Immunofluorescence analysis of CXCR4 surface expression in SCAPs before and after 24-hour cell transmigration. CXCR4 was hardly detected in the pool SCAPs or nontransmigrated cells, whereas most of the SCAPs became surface CXCR4+ (green) after transmigration. Cell nuclei were counterstained with DAPI (blue). SCAPs were from sample #6 at p3. Images are representative data of independent experiments with consistent results. Scale bar = (B and D) 50 mm. NEG, negative control group; NEU, neutralized receptor group; PC, polycarbonate.
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Clinical Research 1a. There was no significant difference between the negative control and those SDF-1a–treated groups (P > .05) (Fig. 2E).
SDF-1a/CXCR4 Axis Mediated SCAP Migration In Vitro The Transwell migration assay (Fig. 3A) showed that more SCAPs were seen on the lower side of the membrane in the SDF-1a groups than that in the control group after 24 hours of treatment in a dosedependent manner (F = 26.003, P < .001), except for the 25 ng/mL SDF-1a group (P = .205 > .05) (Fig. 3B and C). The number of migrated cells reached the peak at 100 ng/mL SDF-1a. There was no significant difference between 100 ng/mL and 200 ng/mL SDF-1a groups (P = .136 > .05). When cells were pretreated with antiCXCR4 antibodies and then treated with 100 ng/mL SDF-1a, the transmigrated cell number significantly decreased compared with the 100 ng/mL SDF-1a group without anti-CXCR4 antibody in its upper chamber (P < .001) (Fig. 3C). In addition, by immunofluorescence staining, we did not detect cells expressing surface CXCR4 before their transmigration. We only observed a low percentage of the nonmigrated cells expressing CXCR4 (3.23%, 1 cell/31 cells, from 6 randomly selected fields). However, cell surface CXCR4 was detected in most of the transmigrated cells (88.89%, 24 cells/27 cells, from 6 randomly selected fields) (Fig. 3D), indicating that SCAPs began expressing surface CXCR4 after transmigration. SCAP Migrated into the 3D Collagen Gel toward SDF-1a Chemoattraction Using a 3D collagen gel Transwell model (Fig. 4A), we were able to observe the migration of SCAPs from the gel surface into the gel as the
result of chemoattraction by SDF-1a. SCAPs were sparsely seeded onto the 3D cylindrical collagen gel surface on day 0 (Fig. 4Ba). After 7 days, cells migrated into the gel at different levels. More cells migrated into deeper levels in the SDF-1a group than those in the control group (Fig. 4Bb). At the upper level (0–100 mm below the gel surface), the control group had significantly more cells than the SDF-1a–treated group (t = 6.72, P = .003 < .05). At the deeper level (100–200 mm below the gel surface), SDF-1a–treated group recruited significantly more cells than the control (t = 16.063, P < .001). As for the level of 200–300 mm below the gel surface, less migrated cells were found in both groups and no difference can be detected between the 2 groups although the mean value is higher for the SDF-1a group (t = 2.744, P = .052 > .05) (Fig. 4C).
Discussion The present study shows for the first time the in situ expression of SDF-1a receptor CXCR4 in the apical papilla. It is mainly located paravascularly, coexpressed with STRO-1. Its expression was also detected in cultured SCAPs. In our Transwell migration studies including the use of a 3D gel model, we found that SDF-1a can significantly promote the migration of SCAPs. Specifically, this migration could be blocked by the use of anti-CXCR4 antibodies, indicating that SDF-1a/CXCR4 axis was mediating the SCAP chemotaxis and transmigration. This finding has significant implications in that SCAPs residing in the apical papilla of an immature permanent tooth may be chemoattracted into the pulp space by SDF-1a. This phenomenon may be used as a strategy for pulp regeneration.
Figure 4. Chemotactic recruitment of SCAPs into a 3D collagen gel Transwell model. (A) A schematic diagram of a 3D collagen gel Transwell model (0.4-mm pore). (Ba) x-y axial planes of 3D reconstructed images. SCAPs were sparsely seeded onto the 3D cylindrical collagen gel surface on day 0. By day 7, cells underwent significant proliferation. (Bb) Cell migration depth in collagen gel. More cells migrated into deeper levels in the SDF-1a group (100 ng/mL) than those in the control group (nontreated). Cell nuclei were counterstained by DAPI (blue fluorescence). Images are representative data of independent experiments with consistent results. (C) Quantitative analysis of migration depth. The delivery of SDF-1a recruited significantly more cells into much deeper levels (mainly 100–200 mm) in collagen gel than the negative control (*P < .05 compared with negative control). Scale bar = (Ba) 100 mm, (Bb) 50 mm (sample #7, p3). PC, polycarbonate. JOE — Volume 41, Number 9, September 2015
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Clinical Research Our immunofluorescence studies showed that CXCR4 in healthy dental apical papilla colocalized with vWF-positive endothelial cells but appeared to be expressed by different populations as indicated by the merged images of the 2 different fluorescence wavelengths (Fig. 1B). By double staining with STRO-1, we found that this CXCR4+ paravascular niche appeared to coincide with the STRO-1+ populations (Fig. 1C). Our RT-PCR results indicated that isolated SCAPs also expressed CXCR4, suggesting that the CXCR4+ cells in the paravascular niche, or at least some of them, may be the isolated SCAPs. Our flow cytometric data revealed that the CXCR4 was mainly expressed intracellularly, not on the cell surface. This finding is similar to that found in bone marrow–derived mesenchymal stem cells (BMMSCs) (23, 25, 26) in which the majority of CXCR4 is localized in endosomal compartments, and it cycles continuously to the cell surface and re-enters into the cells via endocytosis involving clathrin-coated pits (25, 27). Our Transwell transmigration assays showed that SDF-1a promoted the transmigration of SCAPs in a dose-dependent manner in vitro. The 3D Transwell model in our studies that simulates the in vivo 3D space further showed that SCAPs could be recruited and migrated to a significant distance in responding to SDF-1a treatment. Although our flow cytometric results suggested that CXCR4 was rarely expressed on the cell surface, the SDF-1a gradient may induce translocation of cytoplasmic CXCR4 to the membrane, increasing the CXCR4expressing population of SCAPs. This phenomenon was observed in the studies of the SDF-1a/CXCR4 axis during the chemoattraction in MSCs (28). It appears that the spatiotemporal gradient of SDF-1a affects cellular CXCR4 production and its surface expression (28). It should be noted that SDF-1a did not significantly change the cell surface CXCR4 expression after coincubation. However, when SDF-1a was added in the lower chamber of the Transwells, transmigrated SCAPs expressed cell surface CXCR4. In our study, no gradient existed when cells were just incubated in the medium with SDF-1a, whereas SDF-1a gradient was established in the Transwell model. The anti-CXCR4 antibody inhibited the cell transmigration, indicating that SDF-1a/CXCR4 axis was mediating this process. To verify that this cell migration was attributed to the cell recruitment via the SDF-1a/CXCR4 axis rather than a potential cell proliferation effect exerted by SDF-1a, we verified that SDF-1a did not affect the proliferation of SCAPs (Fig. 2E). This is consistent with results from studies of other MSCs, such as DPSCs (18, 24) and BMMSCs (29, 30), although SDF-1a can enhance the proliferation of B-cell progenitors (31) and other cell types (32, 33). In conclusion, our present study is the first step toward testing the possibility of chemoattracting endogenous SCAPs in the apical papilla into the root canal space for pulp regeneration via the SDF-1a/ CXCR4 axis mechanism.
Acknowledgments Supported by grants from the National Natural Science Foundation of China 81200773 (X.Y.Z.) and the National Institutes of Health R01 DE019156 (G.T.-J.H.). The authors deny any conflicts of interest related to this study.
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Dentin and dental pulp regeneration by the patient’s endogenous cells. Endod Topics 2013;28:106–17. 11. Lee CH, Cook JL, Mendelson A, et al. Regeneration of the articular surface of the rabbit synovial joint by cell homing: a proof of concept study. Lancet 2010;376:440–8. 12. Bleul CC, Fuhlbrigge RC, Casasnovas JM, et al. A highly efficacious lymphocyte chemoattractant, stromal cell-derived factor 1 (SDF-1). J Exp Med 1996;184:1101–9. 13. Huang GT, Gronthos S, Shi S. Mesenchymal stem cells derived from dental tissues vs. those from other sources: their biology and role in regenerative medicine. J Dent Res 2009;88:792–806. 14. Aiuti A, Webb IJ, Bleul C, et al. The chemokine SDF-1 is a chemoattractant for human CD34+ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ progenitors to peripheral blood. J Exp Med 1997;185:111–20. 15. Yu J, Li M, Qu Z, et al. SDF-1/CXCR4-mediated migration of transplanted bone marrow stromal cells toward areas of heart myocardial infarction through activation of PI3K/Akt. J Cardiovasc Pharmacol 2010;55:496–505. 16. Zhang G, Nakamura Y, Wang X, et al. Controlled release of stromal cell-derived factor-1 alpha in situ increases c-kit+ cell homing to the infarcted heart. Tissue Eng 2007;13:2063–71. 17. Togel F, Isaac J, Hu Z, et al. Renal SDF-1 signals mobilization and homing of CXCR4positive cells to the kidney after ischemic injury. Kidney Int 2005;67:1772–84. 18. Jiang L, Zhu YQ, Du R, et al. The expression and role of stromal cell-derived factor1alpha-CXCR4 axis in human dental pulp. J Endod 2008;34:939–44. 19. Huang GT, Sonoyama W, Liu Y, et al. The hidden treasure in apical papilla: the potential role in pulp/dentin regeneration and bioroot engineering. J Endod 2008;34:645–51. 20. Sonoyama W, Liu Y, Fang D, et al. Mesenchymal stem cell-mediated functional tooth regeneration in swine. PLoS One 2006;1:e79. 21. Sonoyama W, Liu Y, Yamaza T, et al. Characterization of the apical papilla and its residing stem cells from human immature permanent teeth: a pilot study. J Endod 2008;34:166–71. 22. Diogenes A, Henry MA, Teixeira FB, et al. An update on clinical regenerative endodontics. Endod Topics 2013;28:2–23. 23. Wynn RF, Hart CA, Corradi-Perini C, et al. A small proportion of mesenchymal stem cells strongly expresses functionally active CXCR4 receptor capable of promoting migration to bone marrow. Blood 2004;104:2643–5. 24. Suzuki T, Lee CH, Chen M, et al. Induced migration of dental pulp stem cells for in vivo pulp regeneration. J Dent Res 2011;90:1013–8. 25. Pelekanos RA, Ting MJ, Sardesai VS, et al. Intracellular trafficking and endocytosis of CXCR4 in fetal mesenchymal stem/stromal cells. BMC Cell Biol 2014;15:15. 26. Son BR, Marquez-Curtis LA, Kucia M, et al. Migration of bone marrow and cord blood mesenchymal stem cells in vitro is regulated by stromal-derived factor-1CXCR4 and hepatocyte growth factor-c-met axes and involves matrix metalloproteinases. Stem Cells 2006;24:1254–64. 27. Zhang Y, Foudi A, Geay JF, et al. Intracellular localization and constitutive endocytosis of CXCR4 in human CD34+ hematopoietic progenitor cells. Stem Cells 2004;22:1015–29. 28. Iannone M, Ventre M, Pagano G, et al. Defining an optimal stromal derived factor-1 presentation for effective recruitment of mesenchymal stem cells in 3D. Biotechnol Bioeng 2014;111:2303–16. 29. Ji W, Yang F, Ma J, et al. Incorporation of stromal cell-derived factor-1alpha in PCL/ gelatin electrospun membranes for guided bone regeneration. Biomaterials 2013; 34:735–45. 30. Liu YS, Ou ME, Liu H, et al. The effect of simvastatin on chemotactic capability of SDF-1alpha and the promotion of bone regeneration. Biomaterials 2014;35: 4489–98. 31. Nagasawa T, Hirota S, Tachibana K, et al. Defects of B-cell lymphopoiesis and bonemarrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature 1996; 382:635–8. 32. Imitola J, Raddassi K, Park KI, et al. Directed migration of neural stem cells to sites of CNS injury by the stromal cell-derived factor 1alpha/CXC chemokine receptor 4 pathway. Proc Natl Acad Sci U S A 2004;101:18117–22. 33. Shen B, Zheng MQ, Lu JW, et al. CXCL12-CXCR4 promotes proliferation and invasion of pancreatic cancer cells. Asian Pac J Cancer Prev 2013;14:5403–8.
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CXC Chemokine Receptor 4 Is Expressed Paravascularly in Apical Papilla and Coordinates with Stromal Cell–derived Factor-1a during Transmigration of Stem Cells from Apical Papilla Jing-Yi Liu, DDS,* Xue Chen, DDS,* Lin Yue, DDS, PhD,* George T.-J. Huang, DDS, MSD, DSc,† and Xiao-Ying Zou, DDS, MD* Abstract Introduction: Stem cells from the apical papilla (SCAPs) at the apex may be attracted into the root canal space as a cell source for pulp-dentin regeneration. To test this possibility, we used in vitro transmigration models to investigate whether SCAPs can be chemoattracted by the delivery of the chemotactic cytokine stromal cell–derived factor-1a (SDF-1a). Methods: We first examined the expression of CXC chemokine receptor 4 (CXCR4) for SDF-1a in the apical papilla and in cultured SCAPs using immunofluorescence, reverse-transcription polymerase chain reaction (RT-PCR), and flow cytometric analyses. A standard Transwell migration assay and a 3-dimensional cell migration assay were used to analyze transmigration of SCAPs via the SDF-1a/CXCR4 axis. Results: CXCR4 was expressed in the paravascular region of the apical papilla and detected in SCAP cultures. Most cultured SCAPs harbored intracellular CXCR4 (58%–99%, n = 4), whereas only a few cells had detectable CXCR4 on the cell surface (0.3%–2.34%, n = 4). Although SDF-1a had no significant effect on SCAP proliferation, it significantly promoted a higher number of migrated cells; this effect was abolished by antiCXCR4 antibodies. Interestingly, cell surface CXCR4 on SCAPs was not detectable until after transmigration. The 3-dimensional migration assay revealed that SDF1a significantly enhanced SCAP migration in the collagen gel. Conclusions: SCAPs can be chemoattracted via the SDF-1a/CXCR4 axis, suggesting that SDF-1a may be used clinically to induce CXCR4expressing SCAPs in the apical papilla to transmigrate into the root canal space as an endogenous cell source for pulp regeneration. (J Endod 2015;41:1430–1436)
Key Words 3-dimensional transmigration assay, cell homing, CXC chemokine receptor 4, stem cells from the apical papilla, stromal cell–derived factor-1a
R
egenerative endodontics has gained attention in the treatment of endodontically involved teeth, particularly immature permanent teeth. Successful de novo regeneration of pulp- and dentinlike tissues using animal models has been shown using cell transplantation into the root canal space (1–3). However, such cell-based regeneration approaches face hurdles such as complex cell processing procedures that require a specific facility—good manufacturing practice (4). A recently developed clinical treatment protocol termed revitalization or revascularization is a cell-free approach that allows the growth of additional root structure (ie, apical maturation and thickening of the root) (5). Although revitalization procedures have shown some clinical success evidenced by many clinical case reports and clinical research (6, 7), histologic studies have shown little evidence of actual pulp regeneration in human cases (8) or animal studies (9). The concept of cell homing is to use the mobilized endogenous cells in the host to regenerate the lost tissue (4, 10, 11). Stromal cell–derived factor-1a (SDF-1a), a member of the CXC cytokine subfamily, is a widely expressed chemotactic cytokine that mediates cell migration through its binding with CXC chemokine receptor 4 (CXCR4) (12, 13). The primary role of the SDF-1a/CXCR4 axis is to mobilize CD34+ hematopoietic progenitor cells to different organs during development and different niches within the bone marrow during differentiation and maturation of hematopoietic progenitor cells (14). The SDF-1a/CXCR4 axis is necessary for tissue repair and/or regeneration under multiple pathological conditions, such as myocardial infarction (15, 16) and acute kidney injury (17). Regarding pulp injury and regeneration, the expression of CXCR4 and SDF-1a was found to promote the transmigration of dental pulp cells (18). In immature teeth with necrotic pulp, endogenous dental pulp stem cells (DPSCs) are often no longer present, whereas stem cells from the apical papilla (SCAPs), if present, may be attracted into the canal space for pulp-dentin regeneration (19, 20). SCAPs are highly proliferative in cultures and have a strong odontogenic differentiation capacity (13, 21). They have been used as an exogenous cell source to regenerate pulp- and dentin-like tissues in root canal space and to form bioroots in animal models (2, 20). When in situ, because of their proximity to the vascular-rich periapical
From the *Department of Cariology, Endodontology and Operative Dentistry, School and Hospital of Stomatology, Peking University, Beijing, People’s Republic of China; and †Department of Bioscience Research, College of Dentistry, University of Tennessee Health Science Center, Memphis, Tennessee. Address requests for reprints to Dr Xiao-Ying Zou, Department of Cariology, Endodontology and Operative Dentistry, Peking University School and Hospital of Stomatology, 22 South Zhongguancun Avenue, Haidian District, Beijing 100081, PR China. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2015 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2015.04.006
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Clinical Research tissues, SCAPs in the apical papilla may survive endodontic infection (19, 22); therefore, they may be attracted into the canal space for pulp regeneration. In the present study, we hypothesized that SCAPs can be chemoattracted to migrate via the SDF-1a/CXCR4 axis. We investigated the expression of CXCR4 in both apical papilla and cultured SCAPs, and in vitro transmigration models were used to determine the chemotactic effect of SDF-1a on SCAPs.
Materials and Methods Sample Collection Human impacted third molars (n = 8) with an open apex were collected from healthy patients (8 donors aged 13–24 years) in the Oral and Maxillofacial Surgery Department at Peking University School of Stomatology with a protocol approved by the Ethical Committee of Peking University Health Science Center. Immediately upon extraction, the apical papilla was carefully cut off from the root apex. Each tooth was split cross-sectionally, and the dental pulp was then carefully extracted by tweezers, with the apical 3 mm cut off to avoid including the apical papilla. Cell Culture SCAPs and DPSCs were isolated as described previously (2). Briefly, tissues were minced and digested in a solution of 3 mg/mL collagenase type I and 4 mg/mL dispase for 30 to 60 minutes at 37 C. The isolated cells were seeded and cultured with alpha modification of Eagle medium (aMEM; Gibco, Grand Island, NY) supplemented with 15% fetal bovine serum (FBS; Gibco), 2 mmol/L L-glutamine, 100 U/mL penicillin G, and 100 mg/mL streptomycin and maintained in 5% CO2 at 37 C. Colony formation units of fibroblastic cells were normally observed within 1 to 2 weeks. These heterogeneous populations of adherent, clonogenic dental stem/progenitor cells were tested for their cell surface marker expression by flow cytometric analysis; they were positive for STRO-1, CD146, CD90 and CD105 and negative for CD45 (13). Cells at passage (p) 2 or p3 were used for experiments. Cells isolated from each tooth/donor were grown, maintained, and used separately for each independent experiment. Immunofluorescence Frozen apical papilla and pulp tissues were cryosectioned at 5 mm and double stained for CXCR4/vWF or CXCR4/STRO-1. The sections were permeabilized by 0.5% Triton X-100 (Sigma-Aldrich, St Louis, MO) and blocked with 10% goat serum albumin. The sections were then incubated with primary mouse monoclonal antihuman CXCR4 (1:100; R&D Systems, Abington, UK) and rabbit polyclonal antihuman von Willebrand factor (vWF) (1:4000; Dako, Copenhagen, Denmark) antibodies or with CXCR4 and mouse immunoglobulin (Ig)M antihuman STRO-1 antibodies (1:200; eBioscience, San Diego, CA) followed by secondary antibodies including fluorescein isothiocyanate (FITC) conjugated goat antimouse IgG (ZSGB-Bio, Beijing, China) and phycoerythrin (PE)-conjugated goat antirabbit IgG (Life Technologies, Carlsbad, CA) or goat antimouse IgG and PE-conjugated goat antimouse IgM (eBioscience). Sections were then counterstained with 40 ,6-diamidino-2-phenylindole (DAPI, ZSGB-Bio) and mounted for images analysis under a confocal laser scanning microscope at magnifications of 40 and 63 (488-nm laser diode for FITC, 405-nm laser diode for DAPI/dsDNA [LSM5; Carl Zeiss, Jena, Germany]). For immunocytofluorescence, 5 103 cells (p2)/coverslip were fixed in 4% paraformaldehyde, permeabilized by 0.5% Triton X-100 or without permeabilization, blocked by 10% goat serum for 30 minutes, and followed by incubation with primary mouse monoclonal antihuman JOE — Volume 41, Number 9, September 2015
CXCR4 (1:100; R&D Systems) followed by secondary antibodies FITCconjugated goat antimouse IgG (ZSGB-Bio); the cell nuclei were stained with DAPI.
Reverse-transcription Polymerase Chain Reaction Total RNA was isolated from tissues or cultured cells using TRIzol (Invitrogen, Carlsbad, CA). Extracted RNA was reverse transcribed to complementary DNA using the RT Reagent Kit (Fermentas, Burlington, ON, Canada). Polymerase chain reaction (PCR) amplification was performed using a PCR Kit (Fermentas) with the following amplification steps: initial denaturation at 95 C for 3 minutes followed by 35 cycles of 95 C for 30 seconds, 53 C for 30 seconds (CXCR4) or 50 C for 30 seconds (glyceraldehyde phosphate dehydrogenase, GAPDH), 72 C for 30 seconds, and a final extension at 72 C for 10 minutes. The primers were as follows: CXCR4: 50 -CCGTGGCAAACTGGTACTTT-30 (forward), 50 -GACGCCAACATAGACCACCT-30 (reverse) and GAPDH: 50 -CAAGGCTGAGAACGGGAAGC-30 (forward), 50 -AGGGGGCAGAGATGATGACC-30 (reverse). The PCR products were examined by 1.5% agarose gel electrophoresis and detected by a VILBER Fusion Fx5 (Vilber Lourmat, Collegien, France). Flow Cytometry SCAPs (1 106, p3) were resuspended in 0.5% bovine serum albumin (BSA). For the detection of cell surface CXCR4, cells were stained with PE-conjugated monoclonal antihuman CXCR4 antibody (R&D Systems). For intracellular CXCR4 staining, cells were first blocked with nonconjugated antihuman CXCR4 monoclonal antibody (clone 12G5, R&D Systems), fixed with 4% paraformaldehyde, and permeabilized with 0.5% Triton X-100 (Sigma-Aldrich) followed by incubation with PE-conjugated monoclonal antihuman CXCR4 antibody. Cells were then analyzed on a FACSCalibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ) with Cell Quest Pro software (BD Bioscience, Bedford, MA). Cell Counting Kit-8 Assay for Cell Proliferation SCAPs (p2) were seeded into 96-well plates at 4 103 cells/well. Cells were divided into 5 groups: the negative control group and 4 experimental groups each treated with a different concentration of SDF-1a (ie, 25, 50, 100, and 200 ng/mL) (R&D Systems). After incubation with the presence of SDF-1a for 1, 3, 5, 7, and 9 days, cell viability was determined using the Cell Counting Kit-8 (CCK-8; Dojindo Laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. The optical density (OD) was measured with an Elx808 enzyme-linked immunosorbent assay reader (BioTek, Winooski, VT) at 450 nm (OD450). Cell Transmigration Assay SCAPs (p3) were loaded into the upper chambers of 24-well Transwell inserts (8-mm pore; Corning, Corning, NY) at 5 103 cells/well. There were 3 experimental groups: 1. The negative control group: The lower chamber contained only culture medium. 2. The SDF-1a group: The lower chamber medium was supplemented with various concentrations (ie, 25, 50, 100, and 200 ng/mL) of SDF-1a. 3. The neutralized receptor group: SCAPs were incubated in medium supplemented with 10 mg/mL anti-CXCR4 monoclonal antibody (clone 12G5, R&D Systems) in its upper chamber and the lower chamber medium was supplemented with 100 ng/mL SDF-1a (18, 23).
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Clinical Research The Transwell cultures were incubated for 24 hours. The nontransmigrated cells on the upper side of the membrane were wiped out, and cells transmigrated to the lower side of the membranes were fixed with 95% ethanol and stained with 0.1% crystal violet for evaluation under a microscope. Nine randomly selected fields from each membrane were selected for cell counting.
To determine whether the cell surface CXCR4 expression changed during the transmigration process, Transwell inserts were also collected for immunofluorescence staining after the 24-hour Transwell assay. For the nonmigrated cell staining (upper side of the membrane), we wiped out the cells on the lower side of the membrane. Conversely, for staining of the transmigrated cells, cells on the upper side of the membrane were
Figure 1. CXCR4 expression in apical papilla and pulp. (A) RT-PCR analysis of CXCR4 expression in the apical papilla and dental pulp. Numbers represent different samples (#1: 22-year-old woman, #2: 20-year-old woman, and #3: 21-year-old woman, all third molars). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control. (): Milli-Q water (EMD Millipore, Billerica, MA) served as the negative control. (B) Immunofluorescence double staining of the apical papilla and dental pulp showing CXCR4 (green) coexpressed with vWF (red). CXCR4 in the apical papilla was expressed in the paravascular region where vWF-positive endothelial cells were also present (sample #4: 21-year-old man, third molar). (C) Immunofluorescence analysis of CXCR4 (green) and STRO-1 (red) in apical papilla and dental pulp. CXCR4-expressing cells in the apical papilla were also immunoreactive to anti–STRO-1 antibodies (sample #4). Cell nuclei exhibited blue fluorescence by DAPI counterstaining. Images are representative data of independent experiments with consistent results. Scale bar = (B) 20 mm and (C) 50 mm. Apical, apical papilla; Pulp, dental pulp tissue.
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Clinical Research wiped out. The immunofluorescence staining followed the same procedures described previously but without permeabilization.
3-dimensional Cell Migration Assay A 3-dimensional (3D) collagen gel was made as described previously (24). Briefly, after neutralizing with 1-N NaOH, rat-tail collagen type I solution (3 mg/mL) (BD Bioscience) was dispensed into the upper chambers of 24-well Transwell inserts (0.4-mm pore, Corning) and allowed gelation. SCAPs (p3) resuspended in culture medium were seeded on the surfaces of the gel at 5 103 cells/well and incubated with or without 100 ng/mL SDF-1a in the lower chamber. After a 7-day incubation, gels
were cut out and fixed in 10% formalin. After cell nuclear staining by DAPI, SCAPs migrated into the gel were analyzed by a confocal laser scanning microscope (speed at 400 Hz, objective magnification 20; diode 405, SP8; Leica, Wetzlar, Germany). One randomly selected central field from the surface of each gel was chosen for 3D reconstruction with the LAS AF 3D Visualization software (Leica). According to the reconstructed images (600 mm 600 mm 300 mm), cell proliferation was evaluated on x-y axial planes, whereas y-z axial planes reflected the cell migration depth. By dividing the y-z axial plane into 3 migration depth levels (ie, 0–100 mm, 100–200 mm, 200–300 mm), the numbers of migrated cells were counted according to the successive layers of the 3D reconstructed images.
Figure 2. CXCR4 expression in SCAPs. (A) RT-PCR analysis of CXCR4 expression in SCAPs and DPSCs. Numbers represent different samples (#5: 18-year-old woman, #6: 23-year-old woman, #7: 22-year-old woman, and #8: 13-year-old male; all from p2, third molars). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as internal control genes. (): Milli-Q water served as the negative control. (B) Immunofluorescence staining of SCAPs expressing CXCR4 (green) after permeabilization using Triton X-100, whereas CXCR4 was hardly expressed by nonpermeabilized SCAPs. Blue fluorescence revealed the cell nucleus (sample #7, p2). Images are representative data of independent experiments with consistent results. Scale bar = 50 mm. (C) Flow cytometric analysis of CXCR4 expression in SCAPs. SCAPs (1 106) at p3 were incubated with PE-conjugated monoclonal anti-CXCR4 antibodies for cell surface staining. Prior blockage of cell surface CXCR4 and cell permeabilization was performed before intracellular CXCR4 staining (samples #5–#8, p3). Green areas indicate positive signals; dashed lines are the isotype control. (D) After coincubation with 100 ng/mL SDF-1a in the medium for 24, 36, or 48 hours, there was no significant change of cell surface CXCR4 expression (sample #6, p3). (E) Cell Counting Kit-8 assay of SCAP proliferation. The OD450 values represent relative numbers of cells (mean standard deviation). After incubation with SDF-1a (25, 50, 100, or 200 ng/mL) for 1, 3, 5, 7 and 9 days, there was no significant difference between the negative control (nontreated) and SDF-1a–treated groups (P > .05) (sample #6, p3). CTRL, control.
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Clinical Research Data Analysis All experiments were performed using cells cultured from at least 3 different donors and assayed at least in triplicate. All values are presented as mean standard deviation. Statistical analysis was performed with the SPSS 22.0 software (IBM SPSS Statistics, Armonk, NY) using the Student t test when only 2 groups were compared. One-way analysis of variance was used followed by the least significant difference test when comparing 3 groups or more. Values of P < .05 were considered statistically significant.
Results The SDF-1a Receptor CXCR4 Was Expressed in the Apical Papilla CXCR4 expression was detected by reverse-transcription polymerase chain reaction (RT-PCR) in both the apical papilla and pulp tissue as shown in Figure 1A. Using immunofluorescence staining, we localized CXCR4 in the apical papilla to be in the paravascular region where vWFpositive endothelial cells were also present. In addition, CXCR4expressing cells in the apical papilla were also immunoreactive to anti–STRO-1 antibodies. A similar observation was made in the pulp tissue as indicated in Figure 1B and C.
Isolated SCAP in Cultures Expressed CXCR4 The expression of CXCR4 was detected by RT-PCR in both SCAPs and DPSCs in cultures (Fig. 2A). In the immunocytofluorescence study, no CXCR4 staining was detected when cells were not permeabilized, indicating a lack of cell surface CXCR4 expression. We then examined intracellular expression of CXCR4 and found that 77.55% (38 cells/49 cells from 14 randomly selected fields) of SCAPs expressed intracellular CXCR4 (Fig. 2B). Flow cytometric analysis showed that only 0.3%–2.34% (n = 4) of SCAPs expressed cell surface CXCR4 when cells were stained without permeabilization (Fig. 2C). Up to 57.49%–98.48% (n = 4) of SCAPs stained positively for intracellular CXCR4 expression, indicating that most cells expressed intracellular CXCR4, whereas only a low percentage of them expressed cell surface CXCR4. SDF-1a Had Little to No Effect on CXCR4 Expression or Proliferation of SCAPs We tested the effect of SDF-1a on SCAP CXCR4 surface expression and found no significant induction of cell surface CXCR4 expression after incubation with 100 ng/mL SDF-1a for 24, 36, or 48 hours compared with controls (Fig. 2D). We also determined SCAP proliferation using Cell Counting Kit-8 assays after cells were treated with different concentrations of SDF-
Figure 3. Effects of SDF-1a on the chemotactic capability of SCAPs. (A) A schematic diagram of a Transwell insert (8-mm pore). (B) Transmembrane migration of SCAPs under microscopy with 0.1% crystal violet staining. (C) Numbers of migrated cells in each group. There were significantly more cells transmigrated in the SDF1a groups compared with the negative control in a dose-dependent manner, except for the 25 ng/mL SDF-1a group. The neutralized receptor group has no significant difference with the negative control (*P < .05 compared with the negative control). (D) Immunofluorescence analysis of CXCR4 surface expression in SCAPs before and after 24-hour cell transmigration. CXCR4 was hardly detected in the pool SCAPs or nontransmigrated cells, whereas most of the SCAPs became surface CXCR4+ (green) after transmigration. Cell nuclei were counterstained with DAPI (blue). SCAPs were from sample #6 at p3. Images are representative data of independent experiments with consistent results. Scale bar = (B and D) 50 mm. NEG, negative control group; NEU, neutralized receptor group; PC, polycarbonate.
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Clinical Research 1a. There was no significant difference between the negative control and those SDF-1a–treated groups (P > .05) (Fig. 2E).
SDF-1a/CXCR4 Axis Mediated SCAP Migration In Vitro The Transwell migration assay (Fig. 3A) showed that more SCAPs were seen on the lower side of the membrane in the SDF-1a groups than that in the control group after 24 hours of treatment in a dosedependent manner (F = 26.003, P < .001), except for the 25 ng/mL SDF-1a group (P = .205 > .05) (Fig. 3B and C). The number of migrated cells reached the peak at 100 ng/mL SDF-1a. There was no significant difference between 100 ng/mL and 200 ng/mL SDF-1a groups (P = .136 > .05). When cells were pretreated with antiCXCR4 antibodies and then treated with 100 ng/mL SDF-1a, the transmigrated cell number significantly decreased compared with the 100 ng/mL SDF-1a group without anti-CXCR4 antibody in its upper chamber (P < .001) (Fig. 3C). In addition, by immunofluorescence staining, we did not detect cells expressing surface CXCR4 before their transmigration. We only observed a low percentage of the nonmigrated cells expressing CXCR4 (3.23%, 1 cell/31 cells, from 6 randomly selected fields). However, cell surface CXCR4 was detected in most of the transmigrated cells (88.89%, 24 cells/27 cells, from 6 randomly selected fields) (Fig. 3D), indicating that SCAPs began expressing surface CXCR4 after transmigration. SCAP Migrated into the 3D Collagen Gel toward SDF-1a Chemoattraction Using a 3D collagen gel Transwell model (Fig. 4A), we were able to observe the migration of SCAPs from the gel surface into the gel as the
result of chemoattraction by SDF-1a. SCAPs were sparsely seeded onto the 3D cylindrical collagen gel surface on day 0 (Fig. 4Ba). After 7 days, cells migrated into the gel at different levels. More cells migrated into deeper levels in the SDF-1a group than those in the control group (Fig. 4Bb). At the upper level (0–100 mm below the gel surface), the control group had significantly more cells than the SDF-1a–treated group (t = 6.72, P = .003 < .05). At the deeper level (100–200 mm below the gel surface), SDF-1a–treated group recruited significantly more cells than the control (t = 16.063, P < .001). As for the level of 200–300 mm below the gel surface, less migrated cells were found in both groups and no difference can be detected between the 2 groups although the mean value is higher for the SDF-1a group (t = 2.744, P = .052 > .05) (Fig. 4C).
Discussion The present study shows for the first time the in situ expression of SDF-1a receptor CXCR4 in the apical papilla. It is mainly located paravascularly, coexpressed with STRO-1. Its expression was also detected in cultured SCAPs. In our Transwell migration studies including the use of a 3D gel model, we found that SDF-1a can significantly promote the migration of SCAPs. Specifically, this migration could be blocked by the use of anti-CXCR4 antibodies, indicating that SDF-1a/CXCR4 axis was mediating the SCAP chemotaxis and transmigration. This finding has significant implications in that SCAPs residing in the apical papilla of an immature permanent tooth may be chemoattracted into the pulp space by SDF-1a. This phenomenon may be used as a strategy for pulp regeneration.
Figure 4. Chemotactic recruitment of SCAPs into a 3D collagen gel Transwell model. (A) A schematic diagram of a 3D collagen gel Transwell model (0.4-mm pore). (Ba) x-y axial planes of 3D reconstructed images. SCAPs were sparsely seeded onto the 3D cylindrical collagen gel surface on day 0. By day 7, cells underwent significant proliferation. (Bb) Cell migration depth in collagen gel. More cells migrated into deeper levels in the SDF-1a group (100 ng/mL) than those in the control group (nontreated). Cell nuclei were counterstained by DAPI (blue fluorescence). Images are representative data of independent experiments with consistent results. (C) Quantitative analysis of migration depth. The delivery of SDF-1a recruited significantly more cells into much deeper levels (mainly 100–200 mm) in collagen gel than the negative control (*P < .05 compared with negative control). Scale bar = (Ba) 100 mm, (Bb) 50 mm (sample #7, p3). PC, polycarbonate. JOE — Volume 41, Number 9, September 2015
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Clinical Research Our immunofluorescence studies showed that CXCR4 in healthy dental apical papilla colocalized with vWF-positive endothelial cells but appeared to be expressed by different populations as indicated by the merged images of the 2 different fluorescence wavelengths (Fig. 1B). By double staining with STRO-1, we found that this CXCR4+ paravascular niche appeared to coincide with the STRO-1+ populations (Fig. 1C). Our RT-PCR results indicated that isolated SCAPs also expressed CXCR4, suggesting that the CXCR4+ cells in the paravascular niche, or at least some of them, may be the isolated SCAPs. Our flow cytometric data revealed that the CXCR4 was mainly expressed intracellularly, not on the cell surface. This finding is similar to that found in bone marrow–derived mesenchymal stem cells (BMMSCs) (23, 25, 26) in which the majority of CXCR4 is localized in endosomal compartments, and it cycles continuously to the cell surface and re-enters into the cells via endocytosis involving clathrin-coated pits (25, 27). Our Transwell transmigration assays showed that SDF-1a promoted the transmigration of SCAPs in a dose-dependent manner in vitro. The 3D Transwell model in our studies that simulates the in vivo 3D space further showed that SCAPs could be recruited and migrated to a significant distance in responding to SDF-1a treatment. Although our flow cytometric results suggested that CXCR4 was rarely expressed on the cell surface, the SDF-1a gradient may induce translocation of cytoplasmic CXCR4 to the membrane, increasing the CXCR4expressing population of SCAPs. This phenomenon was observed in the studies of the SDF-1a/CXCR4 axis during the chemoattraction in MSCs (28). It appears that the spatiotemporal gradient of SDF-1a affects cellular CXCR4 production and its surface expression (28). It should be noted that SDF-1a did not significantly change the cell surface CXCR4 expression after coincubation. However, when SDF-1a was added in the lower chamber of the Transwells, transmigrated SCAPs expressed cell surface CXCR4. In our study, no gradient existed when cells were just incubated in the medium with SDF-1a, whereas SDF-1a gradient was established in the Transwell model. The anti-CXCR4 antibody inhibited the cell transmigration, indicating that SDF-1a/CXCR4 axis was mediating this process. To verify that this cell migration was attributed to the cell recruitment via the SDF-1a/CXCR4 axis rather than a potential cell proliferation effect exerted by SDF-1a, we verified that SDF-1a did not affect the proliferation of SCAPs (Fig. 2E). This is consistent with results from studies of other MSCs, such as DPSCs (18, 24) and BMMSCs (29, 30), although SDF-1a can enhance the proliferation of B-cell progenitors (31) and other cell types (32, 33). In conclusion, our present study is the first step toward testing the possibility of chemoattracting endogenous SCAPs in the apical papilla into the root canal space for pulp regeneration via the SDF-1a/ CXCR4 axis mechanism.
Acknowledgments Supported by grants from the National Natural Science Foundation of China 81200773 (X.Y.Z.) and the National Institutes of Health R01 DE019156 (G.T.-J.H.). The authors deny any conflicts of interest related to this study.
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JOE — Volume 41, Number 9, September 2015