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. 2021 Apr 29;100(12):1351–1358. doi: 10.1177/00220345211007427

Fabrication of Vascularized DPSC Constructs for Efficient Pulp Regeneration

C Katata 1,2, JI Sasaki 1,✉, A Li 1, GL Abe 1, JE Nör 3, M Hayashi 2, S Imazato 1,4
PMCID: PMC9290113  PMID: 33913364

Abstract

Dental pulp regeneration is a promising approach to restore the vitality of necrotic teeth. We have previously reported the fabrication of scaffold-free cell constructs containing only dental pulp stem cells (DPSCs) and their ability to form pulp-like tissue in the pulpless tooth. However, the DPSC construct could not build pulp-like tissue with a full root length because it is difficult to induce blood vessels from a small root canal foramen. Therefore, we hypothesized that vascular structure could be preformed in the DPSC construct by employing endothelial differentiation capability of DPSCs, and vascularized constructs might facilitate dental pulp regeneration in the pulpless tooth. In this study, vascularized DPSC constructs were fabricated by inducing endothelial differentiation, and then we investigated the behavior of differentiated DPSCs, the internal structure of cell constructs, and their pulp regenerative ability in vivo. We observed that DPSCs positive for CD31 and von Willebrand factor were localized at the outer layer of constructs and formed a reticulated lumen structure. The cells constituting the outer layer of the construct expressed endothelial differentiation markers at higher levels than cells in the inner part. These results indicated that DPSCs in the outer layer differentiated into endothelial cells and formed vascular-like structures in the cell construct. Next, a vascularized DPSC construct was transplanted into the human pulpless tooth that was implanted into immunodeficient mice in the subcutaneous space. After 6 wk of implantation, the vascularized construct formed pulp-like tissues with higher density of human CD31-positive blood vessels when compared with specimens implanted with a DPSC construct without prevascularization. These results suggest that the vascular structure formed in the DPSC construct facilitated the blood supply and enhanced pulp regeneration. This study demonstrates that a vascularized DPSC construct is a prospective biomaterial as an implant for novel dental pulp regeneration.

Keywords: tissue engineering, biomaterials, vascular endothelial cells, cell differentiation, endodontics, regenerative medicine

Introduction

Dental pulp is a highly vascularized and innervated tissue that provides sensory, immunoprotective, and regenerative functions to teeth (Tirino et al. 2012). Dental pulp consists of a heterogeneous cell population, with each cell type contributing to different tissue functions. Macrophages and dendritic cells in the pulp counteract the invasion of pathogens via phagocytosis and immune cell recruitment (Farges et al. 2015). Nerve fibers are densely distributed and induce pain as a protective response against external stimuli (Couve et al. 2014). Dental pulp stem cells (DPSCs) form dentin, which acts as a physical protective barrier for the tissue (Kaneko et al. 2013; Shah et al. 2020). All of these cell types are essential to the normal function of pulp tissue and are involved in repair processes. Therefore, regeneration of dental pulp in a pulpless tooth leads to restitution of defense and repair functions that have great significance to extend the tooth life (Nakashima et al. 2017).

To date, numerous tissue engineering approaches have been applied to dental pulp regeneration (Ahmed et al. 2020). Dental stem cells have been implanted in combination with a biodegradable scaffold, which formed pulp-like tissue within a sliced tooth root (Huang et al. 2010). In addition, autologous DPSC transplantation with atelocollagen has regenerated well-vascularized tissue in a pulpotomized tooth of a dog model (Iohara et al. 2013; Kim et al. 2015). Thus, most approaches employ a 3-dimensional (3D) scaffold to maintain the implanted cells and encourage the formation of a tissue-specific shape. However, many concerns have been raised about the use of scaffolds, including safety considerations regarding scaffold origins, low biocompatibility of base materials, potential cytotoxicity of degradation by-products, and inability to control the biodegradation period (Yildirimer and Seifalian 2014; Chen and Liu 2016). To address these issues, attempts are being made to develop novel scaffolding materials and to avoid scaffolds altogether.

We previously reported that scaffold-free 3D cell constructs can be fabricated using a thermoresponsive hydrogel (Sasakiet al. 2010; Itoh et al. 2018). This technology allows control of the size and shape of cell aggregates in vitro, which is promising for tissue engineering. DPSCs constituting cell constructs have a self-organization ability and dentin-like matrices form within the constructs under the conditions of odontoblastic differentiation (Yamamoto et al. 2014). Furthermore, DPSC constructs regenerate pulp-like tissue to a pulpless tooth in vivo. However, the pulp regeneration of DPSC constructs is confined and forms a cavity in the root canal with high probability (Itoh et al. 2018). Therefore, it remains difficult to achieve pulp regeneration in the full root length.

DPSCs are classified as mesenchymal stem cells, which originate from the neural crest and can differentiate into the vascular endothelial lineage as well as odontoblasts, osteoblasts, adipocytes, and chondrocytes (Aydin and Şahin 2019; Yianni and Sharpe 2019). They express vascular endothelial growth factor (VEGF) receptor 1 in an undifferentiated state and are therefore easier to differentiate into endothelial cells than mesenchymal stem cells derived from other tissues, such as bone marrow and adipose tissue (Janebodin et al. 2013; Yuan et al. 2015). We have also reported that endothelial differentiated DPSCs express VE-cadherin that contributes to anastomosis of DPSC-derived microvessels to host vasculature (Sasaki et al. 2020).

Here, we hypothesize that prevascularized 3D cell constructs can be fabricated by employing DPSC vasculogenesis ability in vitro to enhance dental pulp regeneration in the pulpless tooth in vivo. In this study, we formed a vascular network within a scaffold-free cell construct by inducing endothelial differentiation of DPSCs constituting the construct. Then, we evaluated the pulp regeneration efficiency of the novel vascularized cell constructs.

Materials and Methods

Endothelial Differentiation of DPSC Constructs

DPSCs isolated from human adult third molars (Lonza) were cultured in Dulbecco’s modified Eagle’s medium (Wako) containing 20% fetal bovine serum (Invitrogen) and 1% penicillin-streptomycin (Sigma-Aldrich). This medium was designated as growth medium. Rod-shaped 3D constructs consisting only of DPSCs were prepared in accordance with previously published protocols (Itoh et al. 2018). Briefly, DPSCs at passages 3 to 5 were cultured in a 100-mm dish until confluency was achieved. Confluent DPSCs were collected as a sheet-like structure using a cell scraper. Then, the sheet-like DPSC constructs were packed into the semicircular columnar grooves (length: 12 mm; width: 3 mm; depth 3 mm) of a thermoresponsive poly-N-isopropylacrylamide (pNIPAAm) gel mold. After 2 d of culture, DPSC constructs were harvested by expansion of the pNIPAAm gel at room temperature. The pNIPAAm gel molds were discarded, and only the resulting DPSC constructs were used for further experiments. The day that DPSC constructs were obtained from the gel mold was designated as day 0. DPSCs and cell constructs were maintained in a humidified incubator at 37°C with 5% CO2.

Endothelial differentiated cell constructs (E-CCs) were prepared by culturing DPSC constructs in endothelial differentiation medium (EGM2-MV [Lonza] supplemented with 50 ng/mL rhVEGF165 [R&D Systems]) (Sasaki et al. 2020). Stereoscopic images of the E-CCs were obtained, and the lengths of long axes were quantified with image analysis software (ImageJ; National Institutes of Health) for 20 d. DPSC constructs cultured in growth medium were used as a control.

Live/Dead Staining

Cell viability within E-CCs was assessed at 0, 5, 10, and 20 d of culture. Samples were washed with phosphate-buffered saline (PBS), and the central part of the construct was cut out into 0.5 mm in parallel with the long axis of the construct using a surgical scalpel. Sliced E-CCs were mounted on glass slides and then stained with Calcein AM or Ethidium homodimer 1 (Invitrogen). The stained samples were observed under a fluorescence microscope (TE2000; Nikon) at a wavelength of 475 nm for live cells and a wavelength of 559 nm for dead cells. The area occupied by living cells was semiquantitatively determined by ImageJ.

Histological Evaluation of E-CCs

E-CCs were harvested at 0, 5, 10, and 20 d of culture; fixed in 4% paraformaldehyde; and then embedded in paraffin. Hematoxylin-eosin (HE) staining was carried out on sections (4 μm thick) cut in parallel with the long axis of the cell construct. Stained sections were observed under a light microscope (ECLIPSE Ci-L; Nikon). Immunofluorescence staining was then performed to investigate the localization of endothelial differentiated cells, which were positive for CD31 and von Willebrand factor (vWF) (Nakashima et al. 2009), and undifferentiated cells expressing STRO-1 (Yu et al. 2010). Sections were deparaffinized, rehydrated, and then incubated with mouse monoclonal antibodies against human CD31 (Dako), vWF (Proteintech), STRO-1 (Santa Cruz Biotechnology), and mouse IgG isotype control (Abcam). Immunoreactive proteins and nuclei were visualized with Alexa Fluor 488 goat anti-mouse IgG and Hoechst 33342 (Invitrogen), respectively. Stained sections were observed under the fluorescence microscope.

Fluorescence imaging was also conducted using FITC-labeled microparticles to visualize the vasculature formation within E-CCs. Cell constructs at up to 20 d of culture were fixed and then immersed in PBS containing FITC-dextran beads (150 kDa; Sigma-Aldrich) for 24 h. The specimens were washed, and then the center part was cut out with a scalpel and observed at a wavelength of 490 nm (TE2000).

Real-Time Reverse Transcription Polymerase Chain Reaction

Expression of angiogenic and stemness marker genes in DPSCs constituting cell constructs was evaluated by real-time polymerase chain reaction (PCR) as described previously (Itoh et al. 2018). Briefly, at 5, 10, and 20 d of culture, E-CCs were cut into 1-mm layers from the outermost portion of the construct (outer layer) to the innermost portion of the construct (inner layer). Messenger RNA (mRNA) expression of VEGFA, C-X-C motif chemokine ligand 1 (CXCL1), Nanog, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Applied Biosystems) in each DPSC layer was evaluated by a TaqMan Gene Expression Cells-to-Ct Kit (Ambion). The expression levels of VEGFA, CXCL1, and Nanog were normalized to those of GAPDH using the ΔΔCt method.

In Vivo Pulp Regeneration Model

All animal surgeries followed ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines and a strict protocol that was approved by the Institutional Animal Care and Use Committee of Osaka University Graduate School of Dentistry (approval number: 26-021-0).

Extracted human single-rooted teeth were obtained from patients at Osaka University Dental Hospital under a protocol approved by the Ethics Committee of the Osaka University Graduate School of Dentistry (IRB number: H25-E23). The human teeth were processed as previously described (Itoh et al. 2018). Briefly, a human tooth root was cut with a low-speed cutter (IsoMet; Buehler) to obtain a 12-mm-long root segment, and the root canal was enlarged using K-files up to #80. An E-CC cultured for 10 d was placed in the treated root canal until the apical end was filled, the coronal side was sealed with calcium silicate cement (ProRoot MTA; Dentsply), and then the tooth/DPSC construct was implanted into the dorsal subcutaneous space of immunodeficient mice (CB.17.SCID; Clea Japan). Tooth roots with DPSC constructs cultured in growth medium were used as a control. Animals were kept in a specific pathogen-free environment with barriers and a controlled light cycle. They were provided with sterile food and water ad libitum. Six male mice aged 6 wk weighing 18 to 22 g were used in the experiments. Two midsagittal incisions were made on the dorsa, and 6 samples were implanted for each group.

Pulp Regeneration of E-CCs

After 6 wk of implantation, mice were euthanized using carbon dioxide, and then tooth roots were harvested and fixed in 10% neutral buffered formalin phosphate. Regenerated pulp-like tissue was observed by micro–computed tomography (micro-CT; R_mCT2, Rigaku). The root canal–to–tissue ratio was determined using 3D image analysis software (TRI/3D-BON; Ratoc) (Oshima et al. 2011; Kazmi et al. 2019).

Specimens were decalcified with Morse solution (Wako) and prepared for HE staining. For immunofluorescence staining, sections were incubated with monoclonal antibodies against human CD31, STRO-1, and dentin sialophosphoprotein (DSPP; Santa Cruz Biotechnology). Microscopic observation was performed as described above. Blood vessels formed by human CD31-positive cells were counted per unit area in the obtained images.

Statistical Analysis

One-way analysis of variance with Tukey’s honestly significant difference (HSD) test was used for comparisons of more than 2 groups. Student’s t test was used for in vitro comparisons of 2 groups. Statistical analysis of in vivo experiments was performed using the nonparametric Mann-Whitney U test. P < 0.05 was considered significantly different.

Results

Structure of the E-CC

Stereoscopic images and size alterations of cell constructs are shown in Figure 1A, B. DPSC constructs had an approximately 7-mm long axis and 3-mm short axis when obtained from the gel mold, which decreased in size by 10 d. Throughout the culture period, no significant difference in sizes was found between the E-CC and control.

Figure 1.

Figure 1.

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Characterization of dental pulp stem cell (DPSC) constructs cultured in an environment of endothelial differentiation (E-CC). (A) Stereoscopic images of E-CCs at 0, 5, 10, and 20 d of culture. (B) Size alterations of E-CC and DPSC constructs without endothelial differentiation (control). The size of these constructs was measured in stereoscopic images. The initial size of the cell construct (day 0) was defined as 100%, and their size alterations were compared between E-CCs and controls. At 7 d, the size decreased to 65.3% ± 3.1% in the E-CC group and 64.7% ± 6.1% in the control group. Similarly, no significant differences were found between the groups over the culture period (P < 0.05). Each point represents the mean ± SD. n = 5. (C) Live/dead staining of E-CCs at 0, 5, 10, and 20 d of culture. Images taken by fluorescent microscope were connected using the “Auto-Blend Layers” function of graphics software (Photoshop, Adobe Inc.). Day 0 panel was arranged manually. (D) Proportions of live cells were semiquantified in live/dead staining images. Different letters indicate significant differences among the days (P < 0.05). Each point represents the mean ± SD. n = 4. Scale bars: 1 mm.

Live/dead staining revealed that the E-CCs mainly consisted of living cells, and dead cells were localized only at the marginal part of the cell construct (Fig. 1C). There were 98.7% ± 1.2% living cells in the construct at day 0, and no significant difference was observed up to 10 d of culture. However, there were 95.1% ± 2.2% living cells after 20 d, which were significantly lower than those on days 0 and 5 (Fig. 1D).

The results of HE staining are shown in Figure 2A, B. The outer layer of the DPSC construct consisted of dense cells throughout the culture period. In the inner part of the constructs, cell density in a sparse structure at day 0 increased gradually, which compacted into a dense structure after 20 d of culture.

Figure 2.

Figure 2.

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Histological observation of endothelial differentiated cell constructs (E-CCs) cultured for up to 20 d. (A) Hematoxylin-eosin staining images of a whole E-CC and (B) magnified images of boxed areas shown in (A). The following panels show immunofluorescence staining of (C) von Willebrand factor (vWF), (D) CD31, and (E) STRO-1 and (F) fluorescence imaging using FITC-labeled microparticles. Arrows indicate dental pulp stem cells forming the vessel-like structure. Dotted lines indicate the outermost surface of the cell construct. Scale bars: (A–E) 100 µm; (F) 200 µm.

Vascular-Like Structure in the E-CC

Expression of vWF was rarely observed in the construct at day 0 and increased in the outer layer throughout the culture period (Fig. 2C). Notably, at day 20, a vascular-like reticular structure consisting of vWF-positive cells was formed at the outer layer of E-CCs. Similar to the results of vWF, CD31-positive cells were rarely observed at day 0, which gradually increased thereafter and built a luminal structure after 10 and 20 d (Fig. 2D, Appendix Fig. 1). Conversely, expression of STRO-1 was observed throughout the DPSC constructs during the early culture period but disappeared in the outer layer of constructs after 10 d (Fig. 2E). Immunostaining with IgG isotype control showed little nonspecific binding with the specimens (Appendix Fig. 2).

Fluorescence imaging revealed that FITC-labeled microparticles were distributed evenly over the DPSC construct at days 0 and 5 (Fig. 2F). Subsequently, vessel-like reticular formation was visualized by accumulation of fluorescence particles in the outermost layer at day 10. Furthermore, vessel-like hollows were structured after 20 d of culture. However, there was neither particle accumulation nor a vessel-like structure in DPSC constructs without endothelial differentiation (Appendix Fig. 3).

Endothelial Differentiation in DPSC Constructs

The results of real-time PCR are shown in Figure 3. The expression levels of the angiogenic markers VEGFA and CXCL1 were significantly higher in the outer layer of E-CCs than in the inner layer throughout the culture period (Fig. 3A, B). At day 5, fold-changes in VEGFA and CXCL1 expression within the outer layer of the constructs were 0.22 ± 0.05 and 0.026 ± 0.001, respectively, compared with that in the inner layer of constructs. Conversely, expression of Nanog in DPSCs was significantly higher in the inner layer of constructs than in the outer layer at days 5 and 10 (Fig. 3C). However, the fold-change of Nanog expression in the inner layer was 1.17 ± 0.16 on day 20 of culture, and no significant difference was found between cells in the inner and outer layers.

Figure 3.

Figure 3.

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Expression of endothelial differentiation and stemness marker genes in the inner and outer layers of endothelial differentiated cell constructs (E-CCs). Expression of (A) vascular endothelial growth factor A (VEGFA), (B) C-X-C motif chemokine ligand 1 (CXCL1), and (C) Nanog after 5, 10, and 20 d of culture was determined by quantitative real-time reverse transcription polymerase chain reaction. Gene expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. Data represent the mean ± SD. n = 4. *P < 0.05.

Enhanced Pulp Regeneration Using an E-CC

All animals tolerated the implantation without adverse events. There was no evidence of inflammation or fibrous capsule formation surrounding the implanted tooth. Stereoscopic images showed that teeth with an E-CC indicated the presence of blood components at the root apex. However, teeth with DPSC constructs without endothelial differentiation (control) did not exhibit any color changes after 6 wk of in vivo implantation (Fig. 4A). Micro–computed tomography (CT) observation showed that the root canal was filled with tissue in the E-CC–transplanted tooth, but cavities were observed in the regenerated tissue of the control group (Fig. 4B, Appendix Video). Quantitative analysis demonstrated that the ratio of tissue in the E-CC–transplanted tooth was 92.4% ± 6.2%, which was significantly higher compared with the control group (74.7% ± 12.5%) (Fig. 4C).

Figure 4.

Figure 4.

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Pulp regeneration with endothelial differentiated cell constructs (E-CCs) in vivo. Dental pulp stem cell construct-transplanted human tooth roots were implanted in the subcutaneous space of immunodeficient mice. After 6 wk, the implanted tooth was harvested and observed by (A) stereomicroscopy and (B) micro–computed tomography (micro-CT). The region enclosed by the white-dotted line indicates blood components in the transplanted E-CC at the apical part of the tooth. The black-dotted ellipse indicates the cavity formed in the control group. (C) Ratio of regenerated pulp-like tissue to the root canal was assessed quantitatively from micro-CT using 3-dimensional image analysis software. The median and interquartile range values are represented by vertical lines. The statistical difference between the groups was analyzed using Mann-Whitney’s U test. n = 6. *P < 0.05. Scale bars: 5 mm.

Histological evaluation revealed that pulp-like fibrous connective tissue and blood vessels had formed in both E-CC and control groups (Fig. 5A). Immunofluorescence staining showed that regenerated tissues in both groups contained blood vessels consisting of human CD31-positive cells (Fig. 5B). These blood vessels had blood cells inside, which indicated anastomosis between newly formed vessels to host vessels (Appendix Fig. 4A). In addition, the number of microvessels consisting of CD31-positive cells was significantly higher in the E-CC–transplanted root canal (1,101.1 ± 77.3/mm2) than the control group (906.2 ± 134.4/mm2) (Appendix Fig. 4B). In both groups, DSPP-positive cells were localized close to the dentin wall and capable of penetrating to the dentinal tubules (Fig. 5B, Appendix Fig. 4C). Cells positive for STRO-1 were observed only in the central part of the regenerated tissue, whereas DSPP- or STRO-1–positive cells showed similar distributions between E-CC and control groups (Fig. 5B).

Figure 5.

Figure 5.

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Histological observation of regenerated pulp-like tissue. (A) Hematoxylin-eosin staining of endothelial differentiated cell constructs (E-CCs) (right) and undifferentiated cell construct (left) transplanted human tooth root. Lower panels show magnified images of the box-enclosed area in the transplanted specimen. Black arrows indicate blood vessels formed in the regenerated tissue. Scale bars: 2 mm (50 μm in the magnified image). (B) Immunofluorescence staining of CD31, dentin sialophosphoprotein (DSPP), and STRO-1 was performed in the cell construct-transplanted tooth root. White arrowheads indicate blood vessels consisting of human CD31-positive cells. Dotted lines indicate the dentin surface of the tooth root canal. Scale bars: 50 μm for CD31; 100 µm for DSPP and STRO-1.

Discussion

Dental pulp regeneration is considered challenging in the field of regenerative medicine because the blood supply providing nourishment and oxygen to the tissue is received only from the apical end. Therefore, it is difficult to maintain cell survival near the crown (Li et al. 2016; Duncan et al. 2018). Moreover, the apical opening of the human matured tooth is too small (less than 1 mm) to induce enough vasculature for retention of pulp tissue in the full root length of 11 to 13 mm (Kim et al. 2013; Dissanayaka et al. 2015). Therefore, promotion of rapid vascularization is an efficient and effective approach to achieve dental pulp regeneration.

Structural analysis of E-CCs revealed that a vascular-like reticular structure was formed by differentiated DPSCs positive for endothelial marker proteins after 10 d of culture. In addition, a lumen structure was confirmed by fluorescence imaging at day 20. In this study, dextran beads with an average diameter of 85 Å (Jain et al. 2013; Chetprayoon et al. 2015), which were used as FITC carriers, were able to infiltrate the E-CC and accumulated easily at sparsely populated areas of the construct compared with dense areas. Therefore, the vascular-like structure was visualized using FITC-labeled dextran microparticles.

Cell viability is crucial for implantation of cell-based biomaterials (Jeyaraj et al. 2015; Jahani et al. 2020). In this study, >95% of DPSCs had survived in the E-CC even after 20 d of culture. Our previous study showed that DPSC constructs without endothelial differentiation contained 15% dead cells (Itoh et al. 2018). The high cell viability in E-CCs may be attributed to the environment that formed the vascular structure, which facilitated penetration of medium into the construct.

DPSCs in the outer layer expressed the endothelial differentiation markers VEGFA and CXCL1 (Yadlapati et al. 2017) at significantly higher levels than cells in the inner part of the E-CC throughout the culture period. Moreover, no significant difference in the expression of the stemness marker Nanog (Itoh et al. 2018) was observed at 20 d of culture. Immunofluorescence staining also showed degression of STRO-1 from the outer layer over the culture period. These results indicated that the stemness of DPSCs was lost at the center part of the E-CC after 20 d of endothelial induction. To regenerate the complex structures of dental pulp, a potential source of fibroblasts, nerve cells, and odontoblasts is indispensable (Zhai et al. 2019). Therefore, E-CCs containing undifferentiated DPSCs are suitable for implantation. Thus, E-CCs at 10 d of culture were used as implant materials for in vivo experiments.

In the E-CC–implanted group, the amount of pulp-like tissues was significantly increased compared with the control. Blood vessels consisting of CD31-positive cells also showed higher numbers in the E-CC, which contained host blood cells. These results suggested that the vascular structure had formed in advance, and differentiated DPSCs facilitated vasculogenesis and promoted the regeneration of pulp-like tissues. However, in the control group, host blood had difficulty circulating to the transplanted construct due to the lack of vessel-like structures, resulting in incomplete tissue regeneration (Unger et al. 2010; Khayat et al. 2017).

In regenerative endodontics, dentin and pulp form an inseparable unit because they are closely related functionally as well as embryologically and histologically (Rosa et al. 2013; Qu et al. 2015). Therefore, reassembling of odontoblasts is essential for successful pulp regeneration. In this study, the E-CC in contact with the dentin wall was stained for an odontoblast marker, and odontoblast-like cells had penetrated dentinal tubes. Moreover, there were stemness-maintaining DPSCs (STRO-1 positive) in the center of the regenerated tissue even at 6 wk after implantation. Bone morphogenetic protein 2 and transforming growth factor β1 in dentin play important roles in odontoblast differentiation of DPSCs (Galler et al. 2016; Miyashita et al. 2017). Thus, DPSCs in contact with the dentin wall may have differentiated into odontoblasts under the influence of these growth factors.

Overall, we succeeded in fabricating prevascularized cell constructs in vitro by inducing differentiation of DPSCs into vascular endothelial cells, which supported pulp-like tissue regeneration in vivo. This is the first study to report on the induction of vascular differentiation of 3D cell constructs in vitro prior to in vivo implantation as a method to improve blood perfusion to the implanted tissue. These DPSC constructs with a vascular-like network structure are applicable as implants that enhance dental pulp regeneration.

Author Contributions

C. Katata, J.I. Sasaki, contributed to conception, design, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript; A. Li, G.L. Abe, contributed to data acquisition and analysis, critically revised the manuscript; J.E. Nör, M. Hayashi, contributed to conception, drafted and critically revised the manuscript; S. Imazato, contributed to conception, design, data analysis and interpretation, drafted and critically revised the manuscript. All authors gave final approval and agree to be accountable for all aspects of the work.

Supplemental Material

sj-pdf-1-jdr-10.1177_00220345211007427 – Supplemental material for Fabrication of Vascularized DPSC Constructs for Efficient Pulp Regeneration
Click here for additional data file. (502.5KB, pdf)

Supplemental material, sj-pdf-1-jdr-10.1177_00220345211007427 for Fabrication of Vascularized DPSC Constructs for Efficient Pulp Regeneration by C. Katata, J.I. Sasaki, A. Li, G.L. Abe, J.E. Nör, M. Hayashi and S. Imazato in Journal of Dental Research

Acknowledgments

We thank Dr. Satoshi Yamaguchi for providing technical assistance with the computer design software and 3D printer.

Footnotes

A supplemental appendix to this article is available online.

Declaration of Conflicting Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding: The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by Grants-in-Aid for Scientific Research (17K11778 to J.I. Sasaki and 17H04383 to S. Imazato) from the Japan Society for the Promotion of Science.

Data Sharing Statement: The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Supplementary Materials

sj-pdf-1-jdr-10.1177_00220345211007427 – Supplemental material for Fabrication of Vascularized DPSC Constructs for Efficient Pulp Regeneration
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Supplemental material, sj-pdf-1-jdr-10.1177_00220345211007427 for Fabrication of Vascularized DPSC Constructs for Efficient Pulp Regeneration by C. Katata, J.I. Sasaki, A. Li, G.L. Abe, J.E. Nör, M. Hayashi and S. Imazato in Journal of Dental Research

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