Circ_0003764 Regulates the Osteogenic Differentiation of Periodontal Ligament Stem Cells.

INTRODUCTION AND AIMS: Specific circular RNAs (circRNAs) have been proven to play crucial roles in osteogenesis in vitro and in vivo. This study aims to identify a certain circRNA involved in the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and explore its regulatory role. METHODS: The expression of 5 candidate circRNAs (circ_0026344, circ_ACAP2, circ_0003764, circ_0008259, and circ_0060731) was detected by real-time quantitative reverse-transcription polymerase chain reaction (qRT-PCR) after PDLSCs were cultured in the osteogenic induction medium or medium supplemented with tumour necrosis factor-α (TNF-α, 10 ng/mL) for 3 and 7 days. The circRNA significantly decreased in both 3 and 7 days of osteogenic induction in PDLSCs and markedly increased in TNF-α-induced PDLSCs for 3 and 7 days screened. Identified circRNA was knocked down or overexpressed, and the effect on the osteogenic differentiation of PDLSCs was investigated by qRT-PCR, western blot, alkaline phosphatase (ALP) staining, and alizarin red S (ARS) staining. Cell counting kit-8 (CCK-8) assay and 5-ethynyl-2'-deoxyuridine (EdU) assay were applied to detect the effect of the circRNA on the proliferation of PDLSCs. RESULTS: qRT-PCR results showed that the expression of circ_0003764 was significantly decreased when PDLSCs were cultured in the osteogenic induction medium for 3 or 7 days, whereas it was dramatically increased in TNF-α-induced PDLSCs. Knockdown of circ_0003764 promoted the expression of the osteogenesis-related genes (RUNX2, ALP, OCN) and proteins (RUNX2, OCN), enhanced the ALP activity, and elevated the mineralization by PDLSCs, as shown by ARS staining. However, with the overexpression of circ_0003764, the osteogenic differentiation capacity of PDLSCs was significantly reduced. The CCK-8 and EdU results indicated that circ_0003764 could inhibit the proliferation of PDLSCs. CONCLUSION: Circ_0003764 is involved in the osteogenesis process and inhibits the osteogenic differentiation and proliferation of PDLSCs. CLINICAL RELEVANCE: This study indicates that circ_0003764 can serve as a diagnostic and therapeutic target in bone regeneration-related diseases treated by PDLSCs-based tissue engineering.

Advances in Regenerative Dentistry Approaches: An Update.

Regenerative dentistry is a rapidly evolving field in dentistry, which has been driven by advancements in biomedical engineering research and the rising treatment expectations and demands that exceed the scope of conventional approaches. Tissue engineering, the foundation of regenerative dentistry, mainly focuses on 3 key components: stem cells, bioactive molecules, and scaffolds. Dental tissue-derived stem cells are especially significant in this regard due to their remarkable properties. Regenerative techniques have provided novel approaches to many conventional treatment strategies in various disciplines of dentistry. For instance, regenerative endodontic procedures such as pulp revascularisation have provided an alternative approach to conventional root canal treatment. In addition, conventional surgical and nonsurgical periodontal treatment is being taken over by modified approaches of guided tissue regeneration with the aid of 3-dimensional bioprinting and computer-aided design, which has revolutionised oral and maxillofacial tissue engineering. This review presents a concise overview of the latest treatment strategies that have emerged into clinical practice, potential future technologies, and the role of dental tissue-derived stem cells in regenerative dentistry.

Emerging Roles of YAP/TAZ in Tooth and Surrounding: from Development to Regeneration.

Yes associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ) are ubiquitous transcriptional co-activators that control organ development, homeostasis, and tissue regeneration. Current in vivo evidence suggests that YAP/TAZ regulates enamel knot formation during murine tooth development, and is indispensable for dental progenitor cell renewal to support constant incisor growth. Being a critical sensor for cellular mechano-transduction, YAP/TAZ lays at the center of the complex molecular network that integrates mechanical cues from the dental pulp chamber and surrounding periodontal tissue into biochemical signals, dictating in vitro cell proliferation, differentiation, stemness maintenance, and migration of dental stem cells. Moreover, YAP/TAZ-mediated cell-microenvironment interactions also display essential regulatory roles during biomaterial-guided dental tissue repair and engineering in some animal models. Here, we review recent advances in YAP/TAZ functions in tooth development, dental pulp, and periodontal physiology, as well as dental tissue regeneration. We also highlight several promising strategies that harness YAP/TAZ activation for promoting dental tissue regeneration.

Long non-coding RNAs MALAT1 and NEAT1 in non-syndromic orofacial clefts.

Long non-coding RNAs (lncRNAs) are thought to play important roles in non-syndromic orofacial clefts (NSOFC). Clinical diagnosis was categorized as either non-syndromic cleft lip with or without cleft palate (NSCL/P), or non-syndromic cleft palate only (NSCPO). Tissues excised from the trimmed wound edge were reserved as experimental samples; adjacent normal control was used as a positive control, and tissue from healthy individuals was used as a blank control. Target lncRNAs in the collected tissues were identified using microarrays and quantitative reverse transcription PCR (RT-qPCR). Immunohistochemical (IHC) staining and RT-qPCR were used to verify the target mRNAs. Pathway, gene ontology (GO) enrichment, and TargetScan predictions were employed to construct competing endogenous RNA networks (ceRNA networks) and explore their potential functions. RNA-Seq revealed 24 upregulated and 43 downregulated lncRNAs; MALAT1 and NEAT1 were screened and validated using RT-qPCR. Common NSOFC risk factors were positively correlated with MALAT1 and NEAT1 expression. Bioinformatics predicted four ceRNA networks; GO enrichment focused on their potential functions. RT-qPCR and IHC data were consistent with respect to expression levels of proteins and the mRNAs that encode them. As MALAT1 and NEAT1 are associated with the severity of NSOFC, they represent potential therapeutic targets and prognostic biomarkers.

Sema4D-plexin-B1 signaling in recruiting dental stem cells for vascular stabilization on a microfluidic platform.

The recruitment of mural cells is critical for stabilization of nascent vessels. Stem cells from human exfoliated deciduous teeth (SHED) are considered to have mural cell-like properties. However, the signaling mechanisms that regulate the cross-talk between endothelial cells and SHED in recruiting them as mural cells is much less well understood. Herein, using a 3D biomimetic microfluidic device, for the first time, we unraveled the role of semaphorin 4D (Sema4D)-plexin-B1 signaling in the recruitment of SHED as mural cells during angiogenic sprouting and vasculature formation by endothelial cells (ECs) in a 3D fibrin matrix. The specific compartmentalized design of the microfluidic chip facilitated recreation of the multi-step dynamic process of angiogenesis in a time and space dependent manner. Enabled by the chip design, different morphogenic steps of angiogenesis including endothelial proliferation, migration & invasion, vascular sprout formation and recruitment of mural cells as well as functional aspects including perfusion and permeability were examined under various pharmacological and genetic manipulations. The results showed that Sema4D facilitates the interaction between endothelial cells and SHED and promotes the recruitment of SHED as mural cells in vascular stabilization. Our results further demonstrated that Sema4D exerts these effects by acting on endothelial-plexin-B1 by inducing expression of platelet-derived growth factor (PDGF)-BB, which is a major mural cell recruitment factor.

ORAL HEALTH AND VITAMIN D IN ADULT - A SYSTEMATIC REVIEW.

This systematic review aimed to provide a synthesis of the evidence relating to how the provision of Vitamin D supplements influences oral health status. An electronic database search was performed across six databases using a standardised search strategy. The PICO framework was used to define the review question. The screening and selection followed PRISMA process. The quality of reporting was assessed using CONSORT guidelines, and the bias was assessed using the revised Cochrane tool RoB2. A total of 1812 studies were retrieved. 1427 studies were excluded due to unmet inclusion criteria. Full texts of 75 potential studies were retrieved and ultimately six studies met the inclusion criteria. There were limitations in the quality of reporting of studies (between 49% and 73%). 70% of the risk of bias items were in the low risks category. Vitamin D interventions varied with respect to dosage and duration. Qualitative syntheses identified significantly better oral health outcomes. Heterogeneity of study design, intervention and outcomes precluded quantitative synthesis. Few clinical trials investigated the effect of Vitamin D supplementation on oral health. There is considerable heterogeneity among studies interventions and oral health outcomes. Quality of reporting of studies have limitations and there is evidence of study biases. Nonetheless, qualitative synthesis of the evidence suggest that Vitamin D supplements improve oral health outcomes, particularly periodontal health. Calcium may also play a significant role. Further high-quality trials are required of comparable Vitamin D supplements with similar oral health outcomes focus to inform quantitative synthesis of the evidence.

Regenerative Potential of Mesenchymal Stem Cell-Derived Extracellular Vesicles.

Mesenchymal stem cells (MSCs) are multipotent cells that can differentiate into diverse cell lineages. MSC based therapy has become a widely experimented treatment strategy in regenerative medicine with promising outcomes. Recent reports suggest that much of the therapeutic effects of MSCs are mediated by their secretome that is expressed through extracellular vesicles (EVs). EVs are lipid bilayer bound components that carry cellular proteins, mRNA, lncRNAs, and other molecules in order to mediate intercellular communication and signaling. In fact, MSC-derived EVs have been observed to implement the same therapeutic effects as MSCs with minimal adverse effects and could be used as an alternative treatment method to MSC-based therapy. The regenerative activity of MSC-EVs has been observed in relation to multiple cell/tissue lineages using various animal models. However, further research and clinical trials are essential for the advancement of this novel treatment strategy. This review provides an insight into the available literature on applications of MSC-EVs in relation to angiogenesis, neurogenesis, hepatic and kidney regeneration, and wound healing.

Indispensable Role of HIF-1α Signaling in Post-implantation Survival and Angio-/Vasculogenic Properties of SHED.

OBJECTIVES: Post-implantation survival and timely vascularization of stem-cell based constructs are critical factors in achieving successful outcomes in tissue regeneration approaches. Hypoxia inducible factor-1α (HIF-1α) is known to mediate adaptive functions to ischemic stress in many different cell types. The current study aimed to explore the role of HIF-1α in post-implantation survival and angio-/vasculogenesis of stem cells from human exfoliated deciduous teeth (SHED). METHODS: HIF-1α in SHED was suppressed using siRNA or chemical inhibitor (YC-1) and used in Matrigel plug assay conducted on severe combined immunodeficient mice. The plugs were retrieved on day 3 or 7 post-injection and analyzed for hypoxia status, ki67 expression, DNA fragmentation (TUNEL), cellularity, and vascularization by histology and immunohistochemistry for CD31, HIF-1α, pyruvate dehydrogenase kinase-1 (PDK1), hexokinase 2 (HK2), and glucose transporter 1 (Glut1). Cell viability of HIF-1α silenced SHED under different stress conditions (hypoxia, H2O2, and low glucose) in vitro was measured by CCK-8 assay. CM-H2DCFDA and MitoSOX Red were used to detect cellular and mitochondrial reactive oxygen species (ROS) levels, respectively. PDK1, HK2, and Glut1 expression were measured by western blotting and immunofluorescence. Secretory protein levels of vascular endothelial growth factor (VEGF) and the respective paracrine effects on endothelial cell proliferation and migration were detected by ELISA, CCK-8 assay, and trans-well assay, respectively. RESULTS: Histological analysis of Matrigel plugs showed significantly reduced cell survival in HIF-1α silenced or chemically inhibited SHED groups, which could be attributed to diminished metabolic adaptations as shown by decreased PDK1, HK2, and Glut1 expression. HIF-1α inhibition in SHED also resulted in significantly low blood vessel formation as observed by a low number of perfused and non-perfused vessels of human or mouse CD31 origin. The viability of HIF-1α silenced SHED was significantly affected under hypoxia, H2O2, and low-glucose conditions in vitro, which was reflected in increased cytoplasmic and mitochondrial ROS levels. Significantly reduced levels of VEGF in HIF-1α silenced SHED resulted in decreased paracrine angiogenic effects as shown by low proliferation and migration of endothelial cells. CONCLUSION: HIF-1α plays an indispensable role in post-implantation survival and angio-/vasculogenic properties of SHED by maintaining ROS homeostasis, inducing metabolic adaptations, and VEGF secretion.

2D DNA nanoporous scaffold promotes osteogenic differentiation of pre-osteoblasts.

Biofunctional materials with nanomechanical parameters similar to bone tissue may promote the adherence, migration, proliferation, and differentiation of pre-osteoblasts. In this study, deoxyribonucleic acid (DNA) nanoporous scaffold (DNA-NPS) was synthesized by the polymerization of rectangular and double-crossover (DX) DNA tiles. The diagonally precise polymerization of nanometer-sized DNA tiles (A + B) through sticky end cohesion gave rise to a micrometer-sized porous giant-sheet material. The synthesized DNA-NPS exhibited a uniformly distributed porosity with a size of 25 ± 20 nm. The morphology, dimensions, sectional profiles, 2-dimensional (2D) layer height, texture, topology, pore size, and mechanical parameters of DNA-NPS have been characterized by atomic force microscopy (AFM). The size and zeta potential of DNA-NPS have been characterized by the zeta sizer. Cell biocompatibility, proliferation, and apoptosis have been evaluated by flow cytometry. The AFM results confirmed that the fabricated DNA-NPS was interconnected and uniformly porous, with a surface roughness of 0.125 ± 0.08035 nm. The elastic modulus of the DNA-NPS was 22.45 ± 8.65 GPa, which was comparable to that of native bone tissue. DNA-NPS facilitated pre-osteoblast adhesion, proliferation, and osteogenic differentiation. These findings indicated the potential of 2D DNA-NPS in promoting bone tissue regeneration.

Conversion of stem cells from apical papilla into endothelial cells by small molecules and growth factors.

OBJECTIVES: Recently, a new strategy has been developed to directly reprogram one cell type towards another targeted cell type using small molecule compounds. Human fibroblasts have been chemically reprogrammed into neuronal cells, Schwann cells and cardiomyocyte-like cells by different small molecule combinations. This study aimed to explore whether stem cells from apical papilla (SCAP) could be reprogrammed into endothelial cells (ECs) using the same strategy. MATERIALS AND METHODS: The expression level of endothelial-specific genes and proteins after chemical induction of SCAP was assessed by RT-PCR, western blotting, flow cytometry and immunofluorescence. The in vitro functions of SCAP-derived chemical-induced endothelial cells (SCAP-ECs) were evaluated by tube-like structure formation assay, acetylated low-density lipoprotein (ac-LDL) uptake and NO secretion detection. The proliferation and the migration ability of SCAP-ECs were evaluated by CCK-8 and Transwell assay. LPS stimulation was used to mimic the inflammatory environment in demonstrating the ability of SCAP-ECs to express adhesion molecules. The in vivo Matrigel plug angiogenesis assay was performed to assess the function of SCAP-ECs in generating vascular structures using the immune-deficient mouse model. RESULTS: SCAP-ECs expressed upregulated endothelial-specific genes and proteins; displayed endothelial transcriptional networks; exhibited the ability to form functional tubular-like structures, uptake ac-LDL and secrete NO in vitro; and contributed to generate blood vessels in vivo. The SCAP-ECs could also express adhesion molecules in the pro-inflammatory environment and have a similar migration and proliferation ability as HUVECs. CONCLUSIONS: Our study demonstrates that the set of small molecules and growth factors could significantly promote endothelial transdifferentiation of SCAP, which provides a promising candidate cell source for vascular engineering and treatment of ischemic diseases.

DPSCs treated by TGF-ß1 regulate angiogenic sprouting of three-dimensionally co-cultured HUVECs and DPSCs through VEGF-Ang-Tie2 signaling.

BACKGROUND: Maintaining the stability and maturation of blood vessels is of paramount importance for the vessels to carry out their physiological function. Smooth muscle cells (SMCs), pericytes, and mesenchymal stem cells (MSCs) are involved in the maturation process of the newly formed vessels. The aim of this study was to investigate whether transforming growth factor beta 1 (TGF-ß1) treatment could enhance pericyte-like properties of dental pulp stem cells (DPSCs) and how TGF-ß1-treated DPSCs for 7 days (T-DPSCs) stabilize the newly formed blood vessels. METHODS: We utilized TGF-ß1 to treat DPSCs for 1, 3, 5, and 7 days. Western blotting and immunofluorescence were used to analyze the expression of SMC markers. Functional contraction assay was conducted to assess the contractility of T-DPSCs. The effects of T-DPSC-conditioned media (T-DPSC-CM) on human umbilical vein endothelial cell (HUVEC) proliferation and migration were examined by MTT, wound healing, and trans-well migration assay. Most importantly, in vitro 3D co-culture spheroidal sprouting assay was used to investigate the regulating role of vascular endothelial growth factor (VEGF)-angiopoietin (Ang)-Tie2 signaling on angiogenic sprouting in 3D co-cultured spheroids of HUVECs and T-DPSCs. Angiopoietin 2 (Ang2) and VEGF were used to treat the co-cultured spheroids to explore their roles in angiogenic sprouting. Inhibitors for Tie2 and VEGFR2 were used to block Ang1/Tie2 and VFGF/VEGFR2 signaling. RESULTS: Western blotting and immunofluorescence showed that the expression of SMC-specific markers (α-SMA and SM22α) were significantly increased after treatment with TGF-ß1. Contractility of T-DPSCs was greater compared with that of DPSCs. T-DPSC-CM inhibited HUVEC migration. In vitro sprouting assay demonstrated that T-DPSCs enclosed HUVECs, resembling pericyte-like cells. Compared to co-culture with DPSCs, a smaller number of HUVEC sprouting was observed when co-cultured with T-DPSCs. VEGF and Ang2 co-stimulation significantly enhanced sprouting in HUVEC and T-DPSC co-culture spheroids, whereas VEGF or Ang2 alone exerted insignificant effects on HUVEC sprouting. Blocking Tie2 signaling reversed the sprouting inhibition by T-DPSCs, while blocking VEGF receptor (VEGFR) signaling boosted the sprouting inhibition by T-DPSCs. CONCLUSIONS: This study revealed that TGF-ß1 can induce DPSC differentiation into functional pericyte-like cells. T-DPSCs maintain vessel stability through Ang1/Tie2 and VEGF/VEGFR2 signaling.

Fabrication of Tapered Fluidic Microchannels Conducive to Angiogenic Sprouting within Gelatin Methacryloyl Hydrogels.

INTRODUCTION: The transplantation of stem cells/tissue constructs into root canal space is a promising strategy for regenerating lost pulp tissue. However, the root canal system, which is cone shaped with a taper from the larger coronal end to the smaller apical end, limits the vascular supply and, therefore, the regenerative capacity. The current study aimed to fabricate built-in microchannels with different tapers to explore various approaches to endothelialize these microchannels. METHODS: The fluidic microchannels with varying tapers (parallel, 0.04, and 0.06) were fabricated within gelatin methacryloyl (GelMA) hydrogel (with or without stem cell from the apical papilla [SCAP] encapsulation) of different concentrations (5%, 7.5%, and 10% [w/v]). Green fluorescent protein-expressing human umbilical vein endothelial cells (HUVECs-GFP) were seeded alone or with SCAPs in coculture into these microchannels. Angiogenic sprouting was assessed by fluorescence and a confocal microscope and ImageJ software (National Institutes of Health, Bethesda, MD). Immunostaining was conducted to illustrate monolayer formation. Data were statistically analyzed by 1-way/2-way analysis of variance. RESULTS: HUVEC-only inoculation formed an endothelial monolayer inside the microchannel without angiogenic sprouting. HUVECs-GFP/SCAPs cocultured at a 1:1 ratio produced the longest sprouting compared with the other 3 ratios. The average length of the sprouting in the 0.04 taper microchannel was significantly longer compared with that in the parallel and 0.06 taper microchannels. Significant differences in HUVEC-GFP sprouting were observed in 5% GelMA hydrogel. Encapsulation of SCAPs within hydrogel further stimulated the sprouting of HUVECs. CONCLUSIONS: The coculture of SCAPs and HUVECs-GFP at a ratio of 1:1 in 0.04 taper fluidic microchannels fabricated with 5% (w/v) GelMA hydrogel with SCAPs encapsulated was found to be the optimal condition to enhance angiogenesis inside tapered microchannels.

Growth Factors and Small-molecule Compounds in Derivation of Endothelial Lineages from Dental Stem Cells.

INTRODUCTION: Incorporating fully assembled microvascular networks into bioengineered dental pulp constructs can significantly enhance functional blood flow and tissue survival upon transplantation. Endothelial cells (ECs), cellular building blocks of vascular tissue, play an essential role in the process of prevascularization. However, obtaining sufficient ECs from a suitable source for translational application is challenging. Dental stem cells (DSCs), which exhibit a robust proliferative ability and immunocompatibility because of their autologous origin, could be a promising alternative cell source for the derivation of endothelial lineages. Under specific culture conditions, DSCs differentiate into osteo/odontogenic, adipogenic, chondrogenic, and neurogenic cell lineages. METHODS: Recently, a new approach has been developed to directly reprogram cells using chemical cocktails and growth factors. Compared with the traditional reprogramming approach based on the forced expression of exogenous transcription factors, the chemical strategy avoids the risk associated with lentiviral transduction while offering a more viable methodology to drive cell lineage switch. The aim of this review was to unveil the concept of the use of small-molecule compounds and growth factors modulating key signaling pathways to derive ECs from DSCs. RESULTS: In addition, our preliminary study showed that stem cells from the apical papilla could be induced into EC-like cells using small-molecule compounds and growth factors. These EC-like cells expressed endothelial specific genes (CD31 and VEGFR2) and proteins (CD31, VEGF receptor 2, and vascular endothelial cadherin) as well as gave rise to vessel-like tubular structures in vitro. CONCLUSIONS: Our preliminary results suggest that chemical reprogramming might offer a novel way to generate EC-like cells from dental stem cells.

Scaffold-based and Scaffold-free Strategies in Dental Pulp Regeneration.

Regenerative dentistry has come a long way from pulp capping to pulp regeneration research, which aims to regenerate the pulp-dentin complex and restore its functions compromised by pulp injury and/or inflammation. Because of unique anatomic limitations of the tooth structure, engineering a suitable microenvironment that facilitates angio/vasculogenesis and innervation is a challenging task. Cell-based tissue engineering approaches have shown great potential in achieving this goal. Biomedical approaches in creating a regenerative microenvironment are mainly represented by either scaffold-based or scaffold-free strategies. The scaffold-based strategy mainly relies on the use of biomaterials to create a structural base that supports cells throughout the process of tissue formation. The scaffold could be a classic 3-dimensional construct with interconnected pores, a hydrogel with cells embedded in it, or a combination of these 2. The scaffold-free approach has been considered a bottom-up strategy that uses cell sheets, spheroids, or tissue strands as building blocks. The outcome of this strategy relies on the capacity of these building blocks to secrete a favorable extracellular matrix and to fuse into larger tissue constructs. Both the scaffold-free and scaffold-based systems are required as complementary, rather than competing, approaches for pulp regeneration. A combined synergetic strategy, through which multicellular building blocks could be integrated with robust 3-dimensional scaffolds, might represent an optimal solution to circumvent some of the major drawbacks of the current methods in pulp regeneration while concurrently fostering their advantages.

Sema4D/PlexinB1 promotes endothelial differentiation of dental pulp stem cells via activation of AKT and ERK1/2 signaling.

Inducing of dental pulp stem cells (DPSCs) into endothelial cells (ECs) to prevascularize pulp tissue constructs may offer a novel and viable approach for enhancing pulp regeneration. However, there are numerous challenges in current methods for the acquisition of sufficient translational ECs. It was known that Sema4D/PlexinB1 signaling exerts profound effects on enhancing vascular endothelial growth factor (VEGF) secretion and angiogenesis. Whether Sema4D/PlexinB1 could regulate endothelial differentiation of DPSCs is not yet investigated. In this study, when DPSCs were treated with Sema4D (2 µg/mL), ECs-specific (VEGFR1, VEGFR2, CD31, and vWF), and angiogenic genes and proteins were significantly upregulated. The induced ECs exhibited similar endothelial vessel formation ability to that of human umbilical vein endothelial cells (HUVECs). Furthermore, phosphorylation of AKT increased dramatically within 5 minutes (from 0.93 to 21.8), while p-ERK1/2 was moderately elevated (from 0.94 to 2.65). In summary, our results demonstrated that Sema4D/PlexinB1 signaling induces endothelial differentiation of DPSCs. The interactions of Sema4D, VEGF, ANGPTL4, ANG1, and HIF-1α may play a crucial role in mediating the differentiation process.

Overexpression of ephrinB2 in stem cells from apical papilla accelerates angiogenesis.

OBJECTIVES: We aimed to accelerate angiogenesis in pulp regeneration by modulating ephrinB2 expression in stem cells from apical papilla (SCAPs). MATERIALS AND METHODS: Stem cells from apical papilla were transducted with ephrinB2-lentiviral expression vector (ephrinB2-SCAPs) in experimental group and green fluorescent protein (GFP-SCAPs) in control group. The transduction efficiency was confirmed by real-time PCR and Western blot assays. MTT assay was performed to detect the proliferative capacity of SCAPs after transduction. In vitro Matrigel assay and in vivo Matrigel plug assay were carried out to evaluate the angiogenic capacity. RESULTS: Results showed that ephrinB2-SCAPs had significantly higher ephrinB2 expression than GFP-SCAPs. EphrinB2-SCAPs upregulated vascular endothelial growth factor (VEGF) secretion under hypoxia. In vitro Matrigel assay demonstrated that human umbilical vein endothelial cells (HUVECs) cocultured with ephrinB2-SCAPs under hypoxia formed vascular-like structures earlier than GFP-SCAPs. Animal experiments confirmed that SCAPs co-transplanted with HUVECs enabled to generate greater amount of blood vessels than SCAPs alone. EphrinB2-SCAPs produced increased number of blood vessels with references to GFP-SCAPs, and those co-transplanted with HUVECs generated vessels with larger and functional tubule volumes. CONCLUSIONS: Regulating ephrinB2 expression in SCAPs may act as a new avenue for enhancing angiogenesis in dental pulp regeneration.

Dental pulp stem cells overexpressing stromal-derived factor-1α and vascular endothelial growth factor in dental pulp regeneration.

OBJECTIVES: The current study aimed to investigate the effects of vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1α (SDF-1α) overexpressing dental pulp stem cells (DPSCs) in vascularized dental pulp regeneration in vivo. MATERIALS AND METHODS: Human DPSCs were transfected with VEGF or SDF-1α using premade lentiviral particles. Overexpression was verified by quantitative polymerase chain reaction (q-PCR), enzyme-linked immunosorbent assay (ELISA), and western blot analysis. Effects of SDF-1α and VEGF overexpressing DPSCs on their proliferation (CCK-8 and MTT assays) and endothelial vascular-tube formation (Matrigel assay) were investigated in vitro. Human tooth roots sectioned into 6-mm segments were injected with gene-modified DPSCs encapsulated in PuraMatrix hydrogel and implanted in the dorsum of severe-combined-immunodeficient (SCID) mice. Implants were retrieved after 4 weeks and examined for regenerated pulp-like tissue and vascularization using histology and immunohistochemistry. p < 0.05 was considered statistically significant. RESULTS: Gene-modified DPSCs expressed significantly high levels (p < 0.05) of SDF-1α and VEGF mRNA and proteins, respectively. Transfected DPSCs showed a significantly higher cell proliferation compared to that of wild-type DPSCs. Furthermore, they enhanced endothelial cell migration and vascular-tube formation on Matrigel in vitro. When injected into tooth root canals and implanted in vivo, DPSCs/SDF-1α + DPSCs/VEGF-mixed group resulted in significantly increased length of regenerated pulp-like tissue within the root canals compared to that of wild-type DPSCs/VEGF and DPSCs/SDF-1α groups. Vessel area density was significantly higher in DPSCs/SDF-1α and mixed DPSCs/SDF-1α + DPSCs/VEGF groups than in DPSCs-VEGF alone or wild-type DPSCs groups. CONCLUSION: A combination of VEGF-overexpressing and SDF-1α-overexpressing DPSCs could enhance the area of vascularized dental pulp regeneration in vivo. CLINICAL RELEVANCE: Enhancing vascularization in pulp regeneration is crucial to overcome the clinical limitation of the limited blood supply to the root canals via a small apical foramen enclosed by hard dentin.

The Role of Vasculature Engineering in Dental Pulp Regeneration.

Creating an optimal microenvironment that supports angiogenesis, cell-cell cross talk, cell migration, and differentiation is crucial for pulp/dentin regeneration. It was shown that dental stem cells being seeded onto a scaffold and transplanted in vivo could give rise to a new tissue similar to that of the native pulp. However, the unique structure of the tooth with a pulp space encased within hard dentin allows only a single blood supply from a small apical opening located at the apex of the root canals. Therefore, a further strategy that can address this limitation such as the incorporation of endothelial/endothelial progenitor cells or cells with high angiogenic potential into the transplant is required so that the added cells can contribute to the vascularization within the implant. However, the placement of 2 or more different cell types inside 3-dimensional porous scaffolds is technologically challenging. In contrast to the conventional scaffolding approach, self-assembly of monodispersed cells into 3-dimensional tissue mimics permits true physiological interactions between and among different types of cells without any influence from a secondary material. In this review, we discuss potential strategies that can be used in vasculature engineering in dental pulp regeneration with a specific emphasis on combining prevascularization and scaffold-based or scaffold-free approaches.

TGF-ß1-induced differentiation of SHED into functional smooth muscle cells.

BACKGROUND: Adequate vascularization is crucial for supplying nutrition and discharging metabolic waste in freshly transplanted tissue-engineered constructs. Obtaining the appropriate building blocks for vascular tissue engineering (i.e. endothelial and mural cells) is a challenging task for tissue neovascularization. Hence, we investigated whether stem cells from human exfoliated deciduous teeth (SHED) could be induced to differentiate into functional vascular smooth muscle cells (vSMCs). METHODS: We utilized two cytokines of the TGF-ß family, transforming growth factor beta 1 (TGF-ß1) and bone morphogenetic protein 4 (BMP4), to induce SHED differentiation into SMCs. Quantitative real-time polymerase chain reaction (RT-qPCR) was used to assess mRNA expression, and protein expression was analyzed using flow cytometry, western blot and immunostaining. Additionally, to examine whether these SHED-derived SMCs possess the same function as primary SMCs, in vitro Matrigel angiogenesis assay, fibrin gel bead assay, and functional contraction study were used here. RESULTS: By analyzing the expression of specific markers of SMCs (α-SMA, SM22α, Calponin, and SM-MHC), we confirmed that TGF-ß1, and not BMP4, could induce SHED differentiation into SMCs. The differentiation efficiency was relatively high (α-SMA+ 86.1%, SM22α+ 93.9%, Calponin+ 56.8%, and SM-MHC+ 88.2%) as assessed by flow cytometry. In vitro Matrigel angiogenesis assay showed that the vascular structures generated by SHED-derived SMCs and human umbilical vein endothelial cells (HUVECs) were comparable to primary SMCs and HUVECs in terms of vessel stability. Fibrin gel bead assay showed that SHED-derived SMCs had a stronger capacity for promoting vessel formation compared with primary SMCs. Further analyses of protein expression in fibrin gel showed that cultures containing SHED-derived SMCs exhibited higher expression levels of Fibronectin than the primary SMCs group. Additionally, it was also confirmed that SHED-derived SMCs exhibited functional contractility. When SB-431542, a specific inhibitor of ALK5 was administered, TGF-ß1 stimulation could not induce SHED into SMCs, indicating that the differentiation of SHED into SMCs is somehow related to the TGF-ß1-ALK5 signaling pathway. CONCLUSIONS: SHED could be successfully induced into functional SMCs for vascular tissue engineering, and this course could be regulated through the ALK5 signaling pathway. Hence, SHED appear to be a promising candidate cell type for vascular tissue engineering.