Kinetics of LYVE-1-positive M2-like macrophages in developing and repairing dental pulp in vivo and their pro-angiogenic activity in vitro

Tissue-resident macrophages expressing lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) are found in multiple tissues and organs. We aimed to evaluate the dynamics and biological functions of LYVE-1+ macrophages in dental pulp during post-injury tissue remodeling. Immunofluorescence staining of mouse embryos revealed that LYVE-1+ macrophages colonized dental pulp before birth. In mature rat molar dental pulp, LYVE-1+ macrophages were the main subset of macrophages expressing CD163, an M2 marker, and were distributed throughout the tissue. In response to dental pulp injury induced by cavity preparation, LYVE-1+ macrophages quickly disappeared from the affected area of the pulp and gradually repopulated during the wound healing process. RAW264.7 mouse macrophages cultured with a mixture of macrophage colony-stimulating factor, interleukin-4, and dexamethasone increased LYVE-1 expression, whereas lipopolysaccharide-stimulation decreased LYVE-1 expression. Enforced expression of Lyve1 in RAW264.7 cells resulted in increased mRNA expression of matrix metalloproteinase 2 (Mmp2), Mmp9, and vascular endothelial growth factor A (Vegfa). Lyve1-expressing macrophages promoted the migration and tube formation of human umbilical vein endothelial cells. In conclusion, LYVE-1+ tissue-resident M2-like macrophages in dental pulp showed dynamism in response to pulp injury, and possibly play an important role in angiogenesis during wound healing and tissue remodeling.

LYVE-1 + tissue-resident macrophages are seeded in the dental papilla before birth. Most tissue-resident macrophage populations are established during embryonic development 7 . To evaluate whether the dental papilla is populated by LYVE-1 + macrophages before birth, we examined mouse first molars at embryonic days (E) 12, 15 and 18. Immunostaining for LYVE-1 showed that LYVE-1 + cells were absent from dental papilla from the bud stage (E12) to the cap stage (E15), and started to be observed in the middle of the dental papilla at the bell stage (E18) of tooth development ( Fig. 2A). Double immunofluorescence staining for LYVE-1 and one of CD68, CD163, or MHC-II at E18 revealed that these LYVE-1 + cells were co-immunoreactive for both CD68 and CD163 (Fig. 2B,C) but negative for MHC-II (Fig. 2D). Additionally, at this stage, LYVE-1 − /CD68 + , LYVE-1 − /CD163 + , and LYVE-1 − /MHC-II + cells were scattered in the subodontoblastic layer ( Fig. 2B-D). Collectively, these findings revealed that embryonic-derived LYVE-1 + macrophages were colonized in the dental papilla from the bell-stage of tooth development and exhibited an identical phenotype to LYVE-1 + macrophages found in the adult dental pulp. LYVE-1 + macrophages initially disappear and latterly recolonize in the dental pulp in response to dentinal cavity preparation. Inflammation is one of the main factors to modulate the properties of tissue-resident macrophages by making them motile and changing the niche in which they reside 5 . To elucidate whether the inflammation status of the dental pulp impacts the distribution of the LYVE-1 + population, we performed cavity preparation without pulp exposure on rat molars, which can cause damage to the tissue beneath the cavity and subsequently trigger reversible inflammation. The inflammation status and wound-healing stages of the dental pulp after cavity preparation were histologically examined by hematoxylin and eosin (H&E) staining ( Fig. 3Aa-e). On day 1 post-cavity preparation, the pulp tissue just beneath the cavity showed a necrotic area with sparse cellular components and an accumulation of infiltrated inflammatory cells along the border between the necrotic and vital pulp (Fig. 3Ab). From day 3 to day 7 after cavity preparation, inflammatory cells decreased their number in the wounded pulp tissue (Fig. 3Ac-e). Coinciding with the resolution of inflammation, dental pulp cells were repopulated in the wounded area on day 3 post-cavity preparation, suggesting the early stage of wound healing (Fig. 3Ac). From day 5 to day 7 post-cavity preparation, newly differentiated odontoblast-like cells lined the dentin wall and produced a thin layer of reparative dentin beneath the cavity (Fig. 3Ad,e) (Supplementary Fig. 2).
We next examined the distribution of LVYE-1 + macrophages in the injured pulp after cavity preparation by immunofluorescence staining. At steady state, LYVE-1 + macrophages were widely distributed throughout the dental pulp (Fig. 3Ba). On the first day after cavity preparation, a drastic loss of LYVE-1 + cells was observed in the affected pulp tissue (Fig. 3Bb). Notably, despite an almost complete loss in the affected area of the pulp at 1 day post-cavity preparation, LYVE-1 + cells could be detected in the pulp tissue distant from the cavity (Supplemental Fig. 1 www.nature.com/scientificreports/ central coronal pulp, they were still absent from the tissue beneath the cavity (Fig. 3Bc). On day 5 post-cavity preparation, many LYVE-1 + cells were observed in the central coronal pulp, and a small number of these cells were detected near the cavity (Fig. 3Bd). At 7 days post-cavity preparation, recovery of the LYVE-1 + cell population was evident by their extensive presence in the dental pulp, including in the tissue adjacent to the cavity (Fig. 3Be). Quantitative analysis of LYVE-1 + cells confirmed that the number of these cells was significantly decreased on day 1 and gradually increased to a level comparable to the control on day 7 post-cavity preparation (Fig. 3C). Double immunofluorescence staining for LYVE-1 and another macrophage marker revealed that, despite the disappearance of LYVE-1 + /CD68 + and LYVE-1 + /CD163 + macrophages, many LYVE-1 − /CD68 + and LYVE-1 − /CD163 + macrophages were still present in the tissue beneath the cavity on day 1 post-cavity preparation (Fig. 3D,E). Collectively, the distribution and density of the LYVE-1 + population in the dental pulp highly depends on the inflammation status: they disappeared at the acute inflammatory phase and recovered at the healing phase when the tissue remodeling progressed.  www.nature.com/scientificreports/  (2) show high magnification of LYVE-1 + MHC − macrophages and LYVE-1 − CDMHC + macrophages, respectively. Images are representative of at least 3-4 different samples for each condition examined. De dental epithelium, dm dental mesenchyme, eo enamel organ, od odontoblast layer, dp dental papilla. Scale bar 100 µm. www.nature.com/scientificreports/  www.nature.com/scientificreports/

Pro-/anti-inflammatory stimuli alter the expression of LYVE-1 in RAW264.7 cells. In response
to cavity preparation, the number of LYVE-1 + macrophages significantly decreased during the acute phase of inflammation. Therefore, we hypothesized that pro-inflammatory stimuli abolish the expression of LYVE-1. To test this hypothesis, the murine monocytic cell line RAW264.7 was transfected with a LYVE-1 expression vector (pcDNA.3.1-DYK.LYVE-1) to generate Lyve1-expressing macrophages followed by lipopolysaccharide (LPS; 100 ng/ml) stimulation for 4 h. Expression of Lyve1 mRNA and protein was increased significantly in Lyve1expressing macrophages (Fig. 4A,B). Additionally, Lyve1-expressing macrophages showed strong M2 polarization, as revealed by their high mRNA expression of typical M2 macrophages markers arginase 1 (Agr1) and mannose receptor C type 1 (Mrc1) (Fig. 4C). Immunofluorescence staining revealed that LYVE-1-positive cells disappeared after LPS stimulation (Fig. 4D). As a transmembrane glycoprotein, the ectodomain of LYVE-1 can undergo proteolytic cleavage that produces soluble LYVE-1 (sLYVE-1) in response to different stimuli 28,29 . We considered the possibility that LPS induced ectodomain shedding of LYVE-1, which led to the loss of cell surface LYVE-1 and the accumulation of sLYVE-1 in the culture medium. Western blotting of cell lysates revealed decreased LYVE-1 protein levels upon LPS stimulation, whereas western blotting of culture media collected from LPS-treated cells showed a detectable band of sLYVE-1, which was shorter (55 kDa) compared with fulllength LYVE-1 (70 kDa) (Fig. 4E).
As we demonstrated, LYVE-1 + cells in the dental pulp expressed M2 macrophage markers and reappeared during the healing phase when the inflammation subsided. We next investigated the possibility that M2 polarization induces upregulation of LYVE-1. To induce M2 polarization, RAW264.7 cells were treated with a cocktail of macrophage-colony stimulating factor (M-CSF), interleukin-4 (IL-4), and synthetic glucocorticoid dexamethasone (MID) for 7 days. MID stimulation increased the mRNA levels of M2-markers Arg1 and Mrc1, along with Lyve1 (Fig. 4F). Western blotting and immunofluorescence staining further confirmed the increased protein levels of LYVE-1 in MID-treated RAW264.7 cells (Fig. 4G,H). Taken together, these results revealed that pro-inflammatory signals abolish the expression of LYVE-1, whereas anti-inflammatory signals activated RAW264.7 cells to the M2 direction, resulting in the upregulation of LYVE-1. We therefore suggest that the expression of LYVE-1 highly depends on the pro-/anti-inflammatory stimuli in the local environment where the macrophages reside.

Lyve1-expressing macrophages promote the migration and tube formation of human umbilical vein endothelial cells.
As a dominant population of tissue M2 macrophages that repopulates dental pulp in the healing phase after injury, LYVE-1 + macrophages potentially play an important role in wound healing. To examine this hypothesis, Lyve1-expressing macrophages were generated from RAW264.7 cells as described above. Overexpression of LYVE-1 resulted in a significant increase in matrix metalloproteinase 2 (Mmp2), MMP-9 (Mmp9), and vascular endothelial growth factor A (Vegfa) mRNA expression (Fig. 5A). In addition, higher VEGF-A protein production was detected in Lyve1-expressing macrophages compared with the control (Fig. 5B).
To further examine the role of Lyve1-expressing macrophages in wound healing, scratch wound healing and tube formation assays were conducted using conditioned media from Lyve1-expressing macrophages (CM-L) and RAW264.7 cells (CM-R). Endothelial cell growth basal medium-2 (EBM-2; Lonza, Walkersville, MD, Japan), with or without EGM-2 (endothelial cell growth medium-2; SingleQuot kit; Lonza, Walkersville, MD, USA) and endothelial cell growth factor component, were used as positive and negative controls, respectively. The results of the scratch wound healing assay showed that CM-L promoted the migration of human umbilical vein endothelial cells (HUVECs) to the scratched area faster than EBM and CM-R, whereas EBM supplemented with EGM (EBM/EGM) showed the greatest improvement in the HUVEC migration (Fig. 5C). At twenty-four hours after the initial scratch, HUVECs treated with CM-L closed approximately 71% of the original scratched areas compared with 57% and 48% in CM-L-and EBM-treated cells, respectively (Fig. 5D). The highest closure of the scratched area was observed in EBM-/EGM-treated cells used as the positive control (Fig. 5E). After 36 h, CM-L was comparable to EBM/EGM by promoting complete closure, while the scratched area still remained in the EBM (55%) or CM-R (84%) groups (Fig. 5E). The results of the tube formation assay revealed that treatment with CM-L promoted the physical organization of HUVECs into tube-like structures after 6 h (Fig. 5F). HUVECs treated with EBM/EGM showed the highest number of nodes and junctions and total vessel length compared with all experimental groups, whereas treatment with CM-L led to a significantly higher number of  www.nature.com/scientificreports/ nodes and junctions and total vessel length compared with CM-R and EBM (Fig. 5G). Thus, Lyve1-expressing macrophages promoted the migration and tube formation of HUVECs. These findings suggest the potential role of LYVE-1 + macrophages in the wound healing process by providing appropriate angiogenic factors such as matrix metalloproteinases and growth factors.

Discussion
In the present study, we identified a population of tissue-resident M2 macrophages expressing LYVE-1 in dental pulp. During embryogenesis, their initial colonization was detected in the dental papilla around the bell stage of tooth development. As the main tissue resident macrophage population of the dental pulp, they exhibited a certain degree of dynamism in response to pulpal inflammation induced by cavity preparation. In vitro data supported our hypothesis that the expression of Lyve1 highly depends on pro-/anti-inflammation signals. We further found that Lyve1-expressing macrophages promoted the migration and tube formation of HUVECs. On the basis of these findings, we propose that LYVE-1 + macrophages in the dental pulp rapidly respond to microenvironment changes and play an important role in supporting wound healing processes. Under homeostatic conditions, LYVE-1 + macrophages are found in many different tissues, including sclera, lung, skin, heart mesentery, synovial membrane, kidney, adipose tissue, thymus, lymph node, and aorta 13,14,30 The phenotype of LYVE-1 + macrophages has been reported so far. LYVE-1 + cells expressing M2-associated markers CD163 and CD206 are a predominant population of tissue-resident macrophages in the artery 14 . Similarly, LYVE-1 + macrophages found in the mammary gland are CD206-positive 31 . A recent study also demonstrated the presence of LYVE-1 + macrophage populations that are located in close proximity to capillaries in the lung, skin, and heart, and that express a high level of CD206 but are negative for MHC-II 13 . Consistent with these findings, LYVE-1 + macrophages in the normal dental pulp preferentially colonized tissue adjacent to capillaries, expressed CD163, and were distinct from MHC-II + antigen-presenting cells. The unique functions of LYVE-1 + macrophages in maintaining tissue homeostasis under steady-state conditions have been reported. For example, LYVE-1 + macrophages prevent arterial stiffness and collagen deposition 16 , restrain tissue fibrosis 13 , and modulate extracellular matrix turnover 31 . It is plausible that pulpal LYVE-1 + macrophages also play an important role in supporting dental pulp homeostasis because these cells were distributed throughout the dental pulp tissue under steady-state conditions and shared a similar phenotype to other LYVE-1 + macrophage populations found in different tissues.
Most tissue-resident macrophage populations are derived from embryonic progenitors that persist in the tissues and maintain themselves locally throughout adulthood 4,7,32,33 . The existence of embryonic-derived LYVE-1 + macrophages has been observed in the early stage of tissue development. For example, during mouse embryogenesis, CD68 + /LYVE-1 + macrophages invade the fetal heart around E14.5 and give rise to resident macrophages of the adult heart 32,34 . In line with these findings, we showed here that LYVE-1 + macrophages started to colonize the dental papilla around E18 and were phonetically identical to LYVE-1 + macrophages found in the mature pulp tissue. Therefore, we propose the concept that embryonic-derived LYVE-1 + macrophages potentially persist in dental pulp prior to birth and contribute to the LYVE-1 + macrophage population in adulthood.
Tissue-resident macrophages are highly plastic immune cells due to their ability to adapt to extrinsic factors derived from the tissue microenvironment 35 . Inflammation stimuli often result in the partial depletion of tissue-resident macrophage populations, which has been described as the macrophage disappearance reaction 9,36 . When the inflammation subsides, tissue niches return to steady-state conditions along with the recovery of tissue-resident macrophage populations 9 . For example, the number of LYVE-1 + -resident arterial macrophages decreases immediately after LPS exposure and subsequently rebounds to the level observed during steady-state conditions after 1 week 18 . In line with these findings, we observed the disappearance of LYVE-1 + cells in the dental pulp 1 day after cavity preparation and their recovery to normal levels within a week. The disappearance reaction of tissue-resident macrophages is possibly associated with their immigration to draining lymphatics for antigens processing, necroptotic cell death as the result of tissue damage, diminishment of LYVE-1 protein expression, and/or phenotypical and functional switching in response to changes in tissue niches 2,9,37,38 . However, functional enrichment analyses of gene expression profiling have revealed that genes upregulated in LYVE-1 + macrophages are not enriched for term-related antigen processing compared with LYVE-1 − macrophages in fat tissue 14 . Notably, cavity preparation in our model caused a necrotic wound that was limited to the peripheral area of the pulp tissue beneath the cavity and that exhibited a severe loss of LYVE-1 + macrophages. Hence, the disappearance of LYVE-1 + macrophages in the dental pulp after cavity preparation is attributed not to their migration to the lymph nodes but rather to cell death, diminishment of LYVE-1 protein expression, or their character switching in response to the signals in the local tissue microenvironment. Evidence supporting this notion includes the fact that LYVE-1 is a type I transmembrane glycoprotein that can undergo ectodomain shedding to form sLYVE-1 in response to certain signals including inflammation 28,29 . Shedding of LYVE-1 is observed in inflamed lymphatic vessels 28 . Moreover, our in vitro findings revealed the loss of cell surface LYVE-1 expression and the accumulation of sLYVE-1 in the culture medium after 4 h of LPS stimulation. Therefore, we propose the possibility that LYVE-1 + macrophages in inflamed pulp induced by cavity preparation can undergo shedding of its ectodomain and become LYVE-1 − macrophages.
The mechanism behind the renewal of LYVE-1 + macrophages in the dental pulp after cavity preparation remains unclear. It may be due to self-renewal by local proliferation of surviving macrophage subsets or the replenishment of recruited monocytes 39 . Indeed, we detected the remaining population of LYVE-1 + macrophages in the dental pulp after cavity preparation, distant from the cavities, suggesting that these cells can migrate toward the injured pulp tissue and give rise to the recovered population by local proliferation. Moreover, on recovery from the inflammatory phases, monocytes recruited to the tissue could identify homeostatic conditions for re-programming to an M2-like phenotype by Th2 cytokines (IL-4, IL-13 www.nature.com/scientificreports/ macrophages over time [40][41][42] . Our data also indicated that M2 macrophages activated by IL-4 upregulated LYVE-1 expression. We suggest that when inflammation is resolved, recruited monocytes can undergo reprogramming in response to anti-inflammatory cytokines in the local environment and replace the loss of original LYVE-1 + macrophages. Macrophages are critical in all phases of the wound healing process, which occurs in three overlapping stages: (1) inflammation; (2) resolution of inflammation; and (3) tissue vascularization and regeneration 35,43 . Most tissue-resident macrophages show strong M2 polarization 44 , which are critical players in the final stages of wound healing 43,45 . While inflammatory M1 macrophages initially infiltrate the wound in an attempt to phagocytose and kill the invaders, anti-inflammatory M2 macrophages subsequently provide growth factors to support the healing process after the dangerous pathogens have been eliminated 11,46,47 . It has been reported that cavity preparation in rat molars resulted in the destruction of capillary networks in the subodontoblastic layer immediately after the operation, and subsequently, the density and thickness of blood vessels began to increase in the wounded pulp and reached a peak on postoperative day 3 to day 5 48 . Our data revealed that LYVE-1 + macrophages expressing M2 macrophage markers repopulate dental pulp after 3 days post-injury, which coincides with the vascularization and regeneration phases of the wound healing process. Therefore, it is plausible that LYVE-1 + macrophages may participate in promoting wound healing. Previously, LYVE-1 + macrophages have been reported to participate in the angiogenesis of adipose tissue by secreting MMP-7, MMP-9, and MMP-12 17 . We found that Lyve1-expressing macrophages generated from RAW264.7 cells showed upregulation of angiogenesis-related genes and produced a high level of VEGF-A. Furthermore, conditioned media from Lyve1-expressing macrophages promoted the migration and tube formation of HUVECs. Collectively, these findings suggest that LYVE-1 + macrophages orchestrate tissue repair during the healing phases after injury of dental pulp by promoting angiogenesis via the secretion of pro-angiogenic factors such as MMPs and VEGF-A.
A limitation of the present study is that we evaluated the biological function using Lyve1-expressing RAW264.7 cells, and not LYVE-1 + macrophages sorted from dental pulp tissue. Strictly speaking, these cells have different properties. However, Lyve1-expressing RAW264.7 cells also expressed M2 markers such as Arg1 and Mrc1, suggesting that they share some similarities with LYVE-1 + macrophages existing in the dental pulp. Our findings contribute to the current understanding of LYVE-1 + macrophages, which are the predominant tissue-resident macrophage population in the dental pulp. However, their precise roles in the dental pulp during physiological and pathological conditions require further investigation. Recently, attention has been focused on pulp regeneration therapy in the field of endodontic treatment. In particular, induction of angiogenesis in newly formed tissue is critical 49 . Because we demonstrated the potentially pro-angiogenic ability of LYVE-1 + macrophages, targeting LYVE-1 + macrophages could be a promising therapeutic strategy for dental pulp regeneration.

Conclusion
We revealed that LYVE-1 + macrophages in dental pulp mainly comprised M2-like tissue resident macrophages. After pulp injury induced by cavity preparation, they drastically disappeared at the initial inflammatory phase and repopulated the area at the resolution phase. In vitro, Lyve1-expressing macrophages generated from RAW264.7 cells increased the mRNA expression of angiogenic MMPs and secreted VEGF-A and promoted the migration and tube formation of HUVEC cells. These findings suggest that Lyve1 + macrophages play an important role in angiogenesis during wound healing and tissue remodeling of injured dental pulp tissue.

Animal experiments and tissue preparation. All experiments were approved by the Animal Care and
Use Committee of Tokyo Medical & Dental University (A2017-155A) and were conducted in accordance with relevant ethical guidelines and regulations. All animal experiments are reported in compliance with the ARRIVE guidelines 50 . Male Sprague Dawley rats (8 weeks old, n = 30) and pregnant C57BL/6J mice (n = 4) were obtained from CLEA Japan (Tokyo, Japan) and housed in standard conditions (22 °C, 55% relative humidity, artificial illumination). To generate the pulpal injury model, rats were anesthetized with an intraperitoneal injection of ketamine hydrochloride (50 mg/kg, Ketalar; Sankyo, Tokyo, Japan) and xylazine hydrochloride (20 mg/kg, Selactar; Bayer Yakuhin, Osaka, Japan). Cavities without pulp exposure were prepared on the mesial surface of the upper first molars of both sides with #1/2 round burs using a dental handpiece motor under a stereoscopic microscope (Dental Microscope Z; Mani, Tochigi, Japan). The thickness of remaining dentin was approximately 200 µm. On days 1, 3, 5, and 7 after cavity preparation, the rats were killed under carbon dioxide euthanasia. The maxilla was collected and fixed with 4% paraformaldehyde overnight. The samples were demineralized with 17% EDTA for 3 weeks and embedded in an embedding medium (Tissue-Tek OCT compound; Sakura Finetek, Torrance, CA, USA) for frozen sections. To obtain embryos, the pregnant mice were killed under carbon dioxide euthanasia. The heads of fetal mice were harvested at the desired developmental stages (E13, E15, and E18) and immediately embedded in OCT compound to make fresh frozen samples. Cryostat sections (10 µm) from rat maxilla and mouse embryos were prepared for histological evaluation. For cultured cells, 35-mm imaging dishes with a polymer coverslip bottom (Asahi Techno Glass, Shizuoka, Japan) were used for cell seeding. The cells were fixed with 4% paraformaldehyde and immunofluorescence staining was performed as described above. Histological analyses were performed using a confocal laser scanning microscope (Leica TCS-SP8, Leica Microsystems; Wetzlar, Germany) and LAS AF confocal software (Version 1.8.3, Leica Microsystems).
For immunoperoxidase staining, sections were fixed with 4% paraformaldehyde for 10 min at 4 °C. Endogenous peroxidase activity was blocked by incubating the sections in 0.3% H 2 O 2 solution in PBS at room temperature for 10 min. The sections were incubated with an anti-LYVE-1 antibody (11-036; AngioBio) overnight at 4 °C, followed by 30-min incubation with biotinylated anti-rabbit IgG antibody (BA-1000; Vector Laboratories, Burlingame, CA, USA) and the avidin-biotin-peroxidase complex (R.T.U. Vectastain Universal ABC Kit, PK-7200; Vector Laboratories). The color reaction was performed using DAB substrate solution (ImmPACT DAB; Vector Laboratories). The sections were counterstained using methyl green (MUTO, Tokyo, Japan) and mounted using mounting medium (VectaMount; Vector Laboratories). Histology was observed under light microscopy (Axio Vert; Zeiss).
Preparation of conditioned media. RAW246.7 cells (10 5 cells/ml) were seeded on 60-mm culture plates and transfected with Lyve1 overexpression vector as described above. One day after transfection, the cells were washed thoroughly using PBS and replenished with serum-free EBM-2 for 24 h under standard conditions (37 °C, 5% CO 2 ). Conditioned media from Lyve1-expressing macrophages (CM-L) or RAW246.7 cells (CM-R) were collected, centrifuged at 3000 rpm for 5 min at 4 °C to remove cell debris and stored at − 30 °C.
Enzyme-linked immunosorbent assay. The protein level of VEGF-A in conditioned media was determined using DuoSet ELISA mouse VEGF (DY493; R&D Systems) in accordance with the manufacturer's instructions.
Wound healing assay. HUVECs (3.0 × 10 5 cells/ml) were seeded in 24-well plates in endothelial cell growth basal medium-2 (EBM-2; Lonza) containing EGM-2 (SingleQuot kit; Lonza). When HUVECs had formed a confluent cell monolayer, cells were starved in serum-free EMB-2 for 24 h. Cell monolayers were scratched with 200-μl pipette tips and carefully rinsed with PBS to create uncovered areas in the center of the cultured wells. Conditioned media (CM-L, CM-R), EBM-2 supplemented with EGM-2 (positive control), and serum-free EBM-2 (negative control) were added to culture plates. Images were captured immediately following media replacement and at 12 h and 36 h with a microscope (Axio Vert; Zeiss). The images were analyzed, and wound areas were measured using an optimized plugin for ImageJ to automatically recognize the wound healing size. The percentages of wound closure were calculated using the following equation: A T=0 is the initial wound area (μm 2 ) and A T=Δt is the wound area after 24 h or 36 h of the initial scratch (μm 2 ).
Tube formation assay. Fifty microliters ECM gel solution (Cell Biolabs, San Diego, CA, USA) was coated on 96-well plates followed by 30-min incubation at 37 °C for solidification. HUVECs were harvested and resuspended in conditioned media (CM-R, CM-L), EBM-2 supplemented with EGM-2 (positive control), and serumfree EBM-2 (negative control) at 10 5 cells/ml. One hundred fifty microliters cell suspension was seeded onto the solidified ECM gel and incubated for 6 h. Endothelial tubes were labeled with Calcein AM (Cell Biolabs) and examined under a fluorescence microscope (Axio Vert; Zeiss). Several randomized images per well were captured. The numbers of nodes and junctions and total tube length were quantified by ImageJ (version 1.8.0; National Institute of Health).
Cell quantification. Quantification of cell number was performed by manual cell counting of histological sections. The images used for cell counting were randomly captured at the mesial pulp horn area and mesial coronal pulp portion with a 40× objective lens and an acquisition resolution of 1024 × 1024 pixels. For each analysis, representative images (n ≥ 4) of each experimental group were used.