Temporospatial Expression of Neuropeptide Substance P in Dental Pulp Stem Cells During Odontoblastic Differentiation in Vitro and Reparative Dentinogenesis in Vivo.

INTRODUCTION: Substance P (SP) is a neuropeptide released from the nervous fibers in response to injury. In addition to its association with pain and reactions to anxiety and stress, SP exerts various physiological functions by binding to the neurokinin-1 receptor (NK1R). However, the expression and role of SP in reparative dentinogenesis remain elusive. Here, we explored whether SP is involved in odontoblastic differentiation during reparative dentinogenesis. METHODS: Dental pulp stem cells (DPSCs) were isolated from healthy human dental pulp tissues and subjected to odontoblastic differentiation. The expression of SP and NK1R during odontoblastic differentiation was investigated in vitro. The effects of SP on odontoblastic differentiation of DPSCs were evaluated using alizarin red staining, alkaline phosphatase staining, and real-time polymerase chain reaction. After direct pulp capping with mineral trioxide aggregate, the expression of SP and NK1R during reparative dentin formation in rats were identified using histological and immunohistochemical staining. RESULTS: SP and NK1R expression increased during the odontoblastic differentiation of DPSCs. SP translocated to the nucleus when DPSCs were exposed to differentiation medium. NK1R was always present in the nuclei of DPSCs and odontoblast-like cells. Additionally, we discovered that 10-8 M SP marginally enhanced the odontoblastic differentiation of DPSCs, and that these effects could be impaired by the NK1R antagonist. Furthermore, SP and NK1R were expressed in odontoblast-like and dental pulp cells during reparative dentin formation in vivo. CONCLUSIONS: SP contributes to odontoblastic differentiation during reparative dentin formation by binding to the NK1R.

The role of different macrophages-derived conditioned media in dental pulp tissue regeneration.

Macrophages have been reported to play important roles in tissue repair and regeneration. While it is known that macrophages are present in the dental pulp, their role in dental pulp regeneration is not fully understood. In the present study, we investigated the effects of different phenotype macrophages conditioned medium on the cellular behaviors of hDPSCs and their extracellular matrix (cell sheets) in vitro. Moreover, twenty-four root fragments inserted with cell sheets cultured with different conditioned media were placed into the back subcutaneous space of 6-8-week-old male BALB/c nude mouse. The regenerated tissues in the root fragments were assessed via histologic analysis after 8 weeks of transplantation. M2 macrophages could promote the proliferation, migration, and osteogenic differentiation of hDPSCs. Dental pulp-like tissue with an odontoblast-like layer lining the dentinal surface and well-arranged collagen fibers was harvested in root fragment combined with M2 conditioned medium cultured cell sheet, whereas a large amount of calcium salt deposition and disorganization of collagen fibers were observed in root fragments combined with M1 conditioned medium cultured cell sheet. Therefore, promoting the transformation of M1 into M2 macrophage in dental pulp tissue regeneration may be a potential way for dental pulp regeneration via functional healing.

The Role of Small Extracellular Vesicles Derived from Lipopolysaccharide-preconditioned Human Dental Pulp Stem Cells in Dental Pulp Regeneration.

INTRODUCTION: Regenerative endodontics has created a desirable shift in the treatment paradigm despite current limitations of regenerative outcomes. Mesenchymal stem cells (MSCs) facilitate tissue regeneration and repair in a mild inflammatory environment. Small extracellular vesicles (sEVs) derived from MSCs play an imperative role in the paracrine modulation of regenerative responses modulated by MSCs. However, it remains unknown whether MSCs enhance dental pulp regeneration or whether this enhancement is mediated by sEVs in a mild inflammatory environment. The present study aimed to elucidate the effects of sEVs originated from lipopolysaccharide (LPS)-preconditioned human dental pulp stem cells (hDPSCs) on dental pulp regeneration. METHODS: All sEVs were isolated from hDPSCs cultured with or without LPS (ie, N-sEVs and L-sEVs, respectively). The effect of N-sEVs and L-sEVs on proliferation, migration, angiogenesis, and differentiation of rat bone marrow MSCs was identified in vitro. Moreover, N-sEVs or L-sEVs were implanted into rat pulpless root canal models, and the regenerated tissue in root canals was assessed via hematoxylin-eosin staining, Masson staining, and immunohistochemistry after 30 days of transplantation. RESULTS: Both N-sEVs and L-sEVs could modulate BMSC proliferation, migration, angiogenesis, and differentiation. Both kinds of sEVs enhanced the structure of the regenerated tissue closer to that of a normal dental pulp in vivo. L-sEVs had a more significant effect than N-sEVs. CONCLUSIONS: sEVs released by hDPSCs in a mild inflammatory microenvironment are capable of facilitating the regeneration of dental pulp through functional healing instead of scar healing, which has potential applications in regenerative endodontics.

Quality of Root Filling after Obturation with Gutta-percha and 3 Different Sealers of Minimally Instrumented Root canals of the Maxillary First Molar.

INTRODUCTION: The aim of this study was to compare the quality of root fillings completed by a modified single-cone (MSC) technique with 3 different sealers after minimal instrumentation and multisonic cleaning of root canals of maxillary first molars. METHODS: Root canals of 18 maxillary first molars were instrumented to size 15/.04 taper using rotary files. Sodium hypochlorite 5.25% was used during instrumentation; the final cleaning was performed by the GentleWave System (Sonendo Inc, Laguna Hills, CA). The specimens were allocated into 3 groups and root filled by the MSC technique using a size fitted gutta-percha master cone and GuttaFlow Bioseal (Coltene Whaledent GmBH + Co KG, Langenau, Switzerland), GuttaFlow 2 (Coltene Whaledent GmBH + Co KG), and MTA Fillapex (Angelus Industria de Produtos Odontológicos S/A, Londrina, PR, Brazil) sealers. Micro-computed tomographic scans were obtained before and after instrumentation, post-GentleWave, and after obturation. Reconstructed images were analyzed for the volumetric percentage of filling materials. Mesiobuccal roots of the selected teeth were sectioned at 0.5-mm increments starting at the apex of the root. The cross sections were further examined using a light microscope. RESULTS: The 3 groups had 90%-99% of the canal space filled with the root filling material. The mean volume of the filling material was higher in the GuttaFlow Bioseal and GuttaFlow 2 groups than in the MTA Fillapex group (P < .05). There was no significant difference among the apical, middle, and coronal thirds. The cross-sectional images showed no obvious gaps or voids in the GuttaFlow groups. After instrumentation, 49 of the 189 canal thirds (25.9%) had hard tissue debris in the root canal system. After GentleWave cleaning, only 4 of 63 canals (6.3%) and 4 of the 189 canal thirds (2.1%) still had debris. CONCLUSIONS: The MSC method with GuttaFlow 2 and GuttaFlow Bioseal sealers after multisonic cleaning of minimally instrumented molar canals resulted in high-quality root fillings. Multisonic cleaning of minimally instrumented molars seems to be effective in debris removal.

High-temperature Requirement Protein A1 Regulates Odontoblastic Differentiation of Dental Pulp Cells via the Transforming Growth Factor Beta 1/Smad Signaling Pathway.

INTRODUCTION: Dentinogenesis includes odontoblast differentiation and extracellular matrix maturation as well as dentin mineralization. It is regulated by numerous molecules. High-temperature requirement protein A1 (HtrA1) plays crucial roles in bone mineralization and development and is closely associated with the transforming growth factor beta (TGF-ß) signal in osteogenesis differentiation. Simultaneously, the TGF-ß1/small mother against decapentaplegic (Smad) signaling pathway is an important signaling pathway in various physiological processes and as a downstream regulation factor of HtrA1. However, the role of HtrA1 and its relationship with the TGF-ß1/Smad signaling pathway in dentin mineralization is unknown. METHODS: We detected the role of HtrA1 and its relationship with the TGF-ß1/Smad signaling pathway in odontoblastic differentiation of human dental pulp cells (hDPCs) in this study. First, hDPCs were cultured in mineralized medium, and odontoblastic differentiation was confirmed by investigating mineralized nodule formation, alkaline phosphatase (ALP) activity, and the expression of mineral-associated genes, including ALP, collagen I, and dentin sialophosphoprotein. Then, the expression of HtrA1 and TGF-ß1/Smad in hDPCs was investigated in hDPCs during mineralized induction. After HtrA1 knockdown by lentivirus, the mineralized nodule formation, ALP activity, and expression of mineral-associated genes and TGF-ß1/Smad genes were investigated to confirm the effect of HtrA1 on odontoblastic differentiation and its relationship with the TGF-ß1/Smad signaling pathway. RESULTS: The expression of HtrA1 and TGF-ß1 was increased during odontoblastic differentiation of hDPCs along with the messenger RNA expression of downstream factors of the TGF-ß1/Smad signaling pathway. In addition, lentivirus-mediated HtrA1 knockdown inhibited the process of mineralization and the expression of HtrA1 and TGF-ß1/Smad genes. CONCLUSIONS: These findings suggest that HtrA1 might positively regulate odontoblastic differentiation of hDPCs through activation of the TGF-ß1/Smad signaling pathway.

A New Method to Develop Human Dental Pulp Cells and Platelet-rich Fibrin Complex.

INTRODUCTION: Platelet-rich fibrin (PRF) has been used as a scaffold material in various tissue regeneration studies. In the previous methods to combine seed cells with PRF, the structure of PRF was damaged, and the manipulation time in vitro was also increased. The objective of this in vitro study was to explore an appropriate method to develop a PRF-human dental pulp cell (hDPC) complex to maintain PRF structure integrity and to find out the most efficient part of PRF. METHODS: The PRF-hDPC complex was developed at 3 different time points during PRF preparation: (1) the before centrifugation (BC) group, the hDPC suspension was added to the venous blood before blood centrifugation; (2) the immediately after centrifugation (IAC) group, the hDPC suspension was added immediately after blood centrifugation; (3) the after centrifugation (AC) group, the hDPC suspension was added 10 minutes after blood centrifugation; and (4) the control group, PRF without hDPC suspension. The prepared PRF-hDPC complexes were cultured for 7 days. The samples were fixed for histologic, immunohistochemistry, and scanning electron microscopic evaluation. Real-time polymerase chain reaction was performed to evaluate messenger RNA expression of alkaline phosphatase and dentin sialophosphoprotein. Enzyme-linked immunosorbent assay quantification for growth factors was performed within the different parts of the PRF. RESULTS: Histologic, immunohistochemistry, and scanning electron microscopic results revealed that hDPCs were only found in the BC group and exhibited favorable proliferation. Real-time polymerase chain reaction revealed that alkaline phosphatase and dentin sialophosphoprotein expression increased in the cultured PRF-hDPC complex. The lower part of the PRF released the maximum quantity of growth factors. CONCLUSIONS: Our new method to develop a PRF-hDPCs complex maintained PRF structure integrity. The hDPCs were distributed in the buffy coat, which might be the most efficient part of PRF.

[Three-dimensional structure of platelet-rich fibrin gel and its effect on proliferation of human dental pulp cells in vitro].

PURPOSE: This study was designed to analyze the three-dimensional structure of platelet-rich fibrin (PRF) gel as scaffold for dental pulp regeneration, and to investigate the effect of PRF gel on the proliferation of human dental pulp cells (hDPCs) in vitro. METHODS: PRF gel prepared by Choukroun's protocols was evaluated by light microscope and scanning electron microscope (SEM). The extract of PRF gel was collected at the time point of 7th day. The experimental group was divided into 2 subgroups (25% and 75%) according to the volume fraction of PRF gel extract. The 2 subgroups were named 1PRF and 3PRF, respectively. The hDPCs were isolated and cultured in DMEM with 1PRF and 3PRF, respectively. Cell proliferation was detected using cell counting kit-8 (CCK-8) at 24, 48, 72, 96, 120, 144 and 168 h, respectively. The data was analyzed with SPSS16.0 software package. RESULTS: Platelet and leukocyte were mainly distributed at the junction between the red corpuscles and the PRF gel, which was called "buffy coat". "Buffy coat" was constructed by large and dense fibrin clusters. At 24, 48, 72 and 96 h, there was no significant difference (OD) value of optical density(OD) between experimental group and control group(P>0.05). At 120, 144 and 168 h, the OD value in the experimental group was significant higher than that of the control group (P<0.05); there was no significant difference of OD value between group 1PRF and 3PRF (P>0.05). CONCLUSIONS: The results confirm that the three-dimensional structure of PRF gel is constituted by fibrin network. A large amount of platelet and leukocyte which can release high quantities of growth factors are concentrated in the PRF gel. The extract of PRF gel can accelerate the proliferation of hDPCs in time-dependent manner. PRF gel may be a proper scaffold for pulp regeneration. Supported by National Natural Science Foundation of China (81160133).

[Comparison of apical sealing ability of two filling systems].

PURPOSE: To compare the apical sealing ability of the two filling materials: Resilon/Epiphany Self-Etch and Gutta-percha/AH plus. METHODS: Thirty-six recently extracted mandibular premolar roots were included, 6 for the positive and negative groups, 28 for the experimental groups (14 for the Resilon/Epiphany SE group, 14 for the Gutta-percha / AH plus group).The roots were prepared according to the same standards, then the root canals were filled respectively with Resilon/Epiphany SE and Gutta-percha/AH plus. The teeth were put into the India ink to stain, and the apical sealing ability of the two filling materials: Resilon/Epiphany Self-Etch and Gutta-percha/AH plus were compared with the methods of dye leakage. SPSS13.0 software package was used for data analysis. RESULTS: The sealer of the Resilon/Epiphany SE was bonding to the resin filling material and the root canal dentin, and became a structure named mono-block. Compared with the Gutta-percha /AH plus group, the Resilon/Epiphany SE group had significantly lower apical leakage(P<0.05). CONCLUSIONS: The results show that the Resilon/Epiphany SE filling system has better apical sealing ability than the traditional filling material of Gutta-percha/AH plus, and the sealer of Resilon/Epiphany SE is bonded to the resin filling materials and the root canal dentin, and become a structure named monoblock.

[Evaluation of the cavity cleaning of ultrasonic instruments and slow-speed handpiece in posterior teeth root-end preparation].

PURPOSE: To compare the cleanliness of root end preparations by using ultrasonic instrumentation and slow-speed handpiece. METHODS: Thirty-two mesial roots of the first mandibular molars with two canals and mature root apices were assigned randomly to 2 groups, each group had 16 teeth. The root-end preparations were made respectively using ultrasonic diamond tip Berutti and NiTi tip RE2 and slow-speed handpiece with No.2 round bur. Root end cavities were examined under scanning electron microscope for further evaluation of the superficial debris and smear layer of the root end preparations. SPSS 13.0 software package was used for Kruskal Wallis test. RESULTS: Ultrasonic preparation had significantly less superficial debris and smear layer than slow-speed handpiece preparation (P<0.05). CONCLUSIONS: Ultrasonic instrument creates cleaner surfaces for root end cavities than slow-speed handpiece preparation in posterior teeth root end preparation.

[Observation of the number of molar root canal with X-ray imaging and three-dimensional imaging].

PURPOSE: To find a convenient way to reconstruct the morphologic characters of the molar root canal with 3dsmax8.0 software in the laboratory and evaluate the difference of the number of molar root canal between the in vivo X-ray image and three-dimensional image. METHODS: Twelve recently extracted molar roots were studied. In vivo X-ray images were taken before serial section. Starting at the root apex, serial cross sections were taken from each specimen using a saw microtome, and each cross section was photographed under a stereoscopic microscope. All of the digital photos were inputted into the computer to be reconstructed by 3dsmax software. RESULTS: A three-dimensional image of the molar root canal was built successfully. There were 19 molar root canals observed via in vivo X-ray image and 47 molar root canals observed in the three-dimensional image. The number of molar root canal that observed via in vivo X-ray image was less than that from the three-dimensional image. CONCLUSION: Use of saw microtome and 3dsmax software can reconstruct the three-dimensional image of the tooth.

[Pulp revascularization of immature anterior teeth with apical periodontitis].

OBJECTIVE: To compare the therapeutic efficacy both apexification and revascularization in the immature anterior teeth of animal model with apical periodontitis, and observe the histological situation of revascularization in the root canal. METHODS: Six immature anterior teeth of one animal model (dog) aged approximately 4.5 months was selected. Afterwards, periapical periodontitis pattern were established, the samples were randomly divided into the experimental group (revascularization, 3 teeth) and the control group (apexification, 3 teeth). To compare the development of root and the healing of periapical inflammation, the involved teeth were respectively radiographed 1, 4, 8 weeks after surgery. The animals were sacrificed after 8 weeks, and the closure of apical foramen and the content of root canal were observed by hematine-eosine (HE) staining. RESULTS: The postoperative radiography after 1 week and 4 weeks, the apical foramen size and the periapical radiolucency of the samples was shown no perceptual change. After 8 weeks, the experimental group periapical radiolucency area was obviously more narrowing, and had a apical closure tendency whereas the thickness of the root canal walls had imperceptible changed. While the control group periapical radiolucency change varied. The granulation tissue could be seen within the lumen of the experimental group, which contained a large number of irregular calcification, the calcification was obvious in the apical and adjacent the root canal wall. A small quantity of hard tissue was deposited in the apical of the control group. CONCLUSION: Revascularization may increase the recovery of immature anterior teeth with chronic periapical inflammation, the vital regenerative tissue within root canal is the granulation tissue contained calcification.

[Effects of different instruments in root-end preparation: scanning electron microscopic evaluation].

PURPOSE: To evaluate the effects of retrograde preparation between ultrasonic retrotips and traditional slow-speed handpiece. METHODS: 27 maxillary second bicuspid teeth with two canals and one isthmus were divided randomly into 3 groups with 9 in each. The root-end preparations were made respectively using ultrasonic diamond tip Berutti and NiTi tip RE2, ultrasonic #40K file and slow-speed handpiece with NO.2 round bur. The postoperative epoxy resin replicas were examined under scanning electron microscope for further evaluating the number and type of the micro-cracks, the quality of cavity margin. SPSS13.0 software package was used for Kruskal-Wallis test. RESULTS: The number of micro-crack of ultrasonic instruments were significantly less than that of slow-speed handpiece(P=0.002).The type of micro-cracks has no significant difference between the two techniques(P=0.657). On the quality of cavity margin, the slow-speed handpiece group had significant inferior to the ultrasonic #40K file group(P=0.039); there was no significantly difference between the ultrasonic Berutti and RE2 group and the slow-speed handpiece group (P=0.136); there was no significant difference between the two ultrasonic groups (P=0.637). CONCLUSION: Ultrasonic instrument is feasible in the root-end preparation.