Objectives This is to compare the volumes of irrigant apically extruded by five irrigation system... more Objectives This is to compare the volumes of irrigant apically extruded by five irrigation systems in an artificial socket model simulating clinical conditions. Materials and methods Twenty extracted human singlerooted teeth were enlarged to size 40/04 and then embedded in silicone impression material. The root canal space was irrigated with nominal 3% sodium hypochlorite (NaOCl) using standard needle irrigation (SNI) with a 30-gauge notched needle, EndoActivator (EA), XP Endo Finisher (XP Endo), EndoVac (EV), and photon-induced photoacoustic streaming (PIPS). Extruded NaOCl was collected, reacted with taurine to form taurine-monochloramine, and absorbance of taurinemonochloramine was measured at 252 nm using a spectrophotometer. The five irrigation systems were compared with repeated measures ANOVA and pairwise comparisons. R e s u l t s T h e E V g r o u p h a d v e r y l o w e x t r u s i o n (mean ± SD = 0.12 ± 0.2 μL) and differed significantly from the other four groups (P ≤ 0.001). Larger volumes of irrigant were extruded in the other irrigation groups. There were no significant differences in the extruded volumes among the SNI (7.4 ± 3.4 μL), EA (7.0 ± 6.1 μL), and XP Endo (7.8 ± 4.1 μL) groups (P = 1). The PIPS group had the highest mean extruded volume (12.9 ± 6.8 μL) and differed significantly from SNI (P = 0.030), EV (P < 0.0005), and EA (P = 0.02), but not XP Endo (P = 0.154). Conclusion Under the in vitro conditions of this study, irrigant extrusion appears unavoidable unless negative pressure irrigation such as EV is used. PIPS extrudes more irrigant than other systems, while SNI, EA, and XP Endo extrude similar volumes of irrigant. Clinical relevance The findings help clinicians select the optimal irrigation system to avoid irrigant extrusion.
To determine factors that may influence treatment outcome and healing time following root canal t... more To determine factors that may influence treatment outcome and healing time following root canal treatment. Root filled and restored teeth by pre-doctoral students were included in this study. Teeth/roots were followed-up regularly, and treatment outcome was evaluated at every follow-up appointment (healed, healing, uncertain or unsatisfactory). Host (age, immune condition, pulp/periapical diagnosis, tooth/root type, location and anatomy) and treatment factors (master apical file size, apical extension, voids and density of root filling) were recorded from patient dental records. Univariate, bivariate and multivariate analyses were performed to determine the impact of the factors on treatment outcomes and healing times. A total of 422 roots from 291 teeth met the inclusion criteria with a mean follow-up period of 2 years. The preoperative pulp condition, procedural errors during treatment, apical extension and density of root fillings significantly affected the treatment outcome. The average time required for a periapical lesion to heal was 11.78 months. The healing time increased in patients with compromised healing, patients older than 40 years, roots with Weine type II root canal systems, root canal systems prepared to a master apical file size &lt;35, and roots with overextended fillings (P &lt; 0.1). Multiple host and treatment factors affected the healing time and outcome of root canal treatment. Follow-up protocols should consider these factors before concluding the treatment outcome: patient&#39;s age, immune condition, as well as roots with overextended fillings, root canal systems with smaller apical preparations (size &lt;35) or roots with complex canal systems. Intervention may be recommended if the treatment quality was inadequate or if patients became symptomatic.
Introduction-The aim of this study was to determine the efficiency of 4 irrigation systems in eli... more Introduction-The aim of this study was to determine the efficiency of 4 irrigation systems in eliminating bacteria in root canals, particularly in dentinal tubules. Methods-Roots of human teeth were prepared to 25/04, autoclaved, and inoculated with Enterococcus faecalis for 3 weeks. Canals were then disinfected by (1) standard needle irrigation, (2) sonically agitating with EndoActivator, (3) XP Endo finisher, or (4) erbium:yttrium aluminum garnet laser (PIPS) (15 roots/group). The bacterial reduction in the canal was determined by MTT assays. For measuring live versus dead bacteria in the dentinal tubules (4 teeth/group), teeth were split open and stained with LIVE/DEAD BackLight. Coronal, middle, and apical thirds of the canal dentin were scanned by using a confocal laser scanning microscope (CLSM) to determine the ratio of dead/total bacteria in the dentinal tubules at various depths. Results-All 4 irrigation protocols significantly eliminated bacteria in the canal, ranging from 89.6% to 98.2% reduction (P < .001). XP Endo had the greatest bacterial reduction compared with other 3 techniques (P < .05). CLSM analysis showed that XP Endo had the highest level of dead bacteria in the coronal, middle, and apical segments at 50-μm depth. On the other hand, PIPS had the greatest bacterial killing efficiency at the 150-μm depth in all 3 root segments. Conclusions-XP Endo appears to be more efficient than other 3 techniques in disinfecting the main canal space and up to 50 μm deep into the dentinal tubules. PIPS appears to be most effective in killing the bacteria deep in the dentinal tubules.
Management of avulsed teeth after replantation often leads to an unfavorable outcome. Damage to t... more Management of avulsed teeth after replantation often leads to an unfavorable outcome. Damage to the thin and vulnerable periodontal ligament is the key reason for failure. Cell-or stem cell-based regenerative medicine has emerged in the past two decades as a promising clinical treatment modality to improve treatment outcomes. This concept has also been tested for the management of avulsed teeth in animal models. This review focuses on the discussion of limitation of current management protocols for avulsed teeth, cell-based therapy for periodontal ligament (PDL) regeneration in small and large animals, the challenges of de novo regeneration of PDL on denuded root in the edentulous region using a mini-swine model, and establishing a prospective new clinical protocol to manage avulsed teeth based on the current progress of cell-based PDL regeneration studies.
The potential of human mesenchymal stromal/stem cells (MSCs) including oral stem cells (OSCs) as ... more The potential of human mesenchymal stromal/stem cells (MSCs) including oral stem cells (OSCs) as a cell source to derive functional neurons has been inconclusive. Here we tested a number of human OSCs for their neurogenic potential compared to non-OSCs and employed various neurogenic induction methods. OSCs including dental pulp stem cells (DPSCs), gingiva-derived mesenchymal stem cells (GMSCs), stem cells from apical papilla and non-OSCs including bone marrow MSCs (BMMSCs), foreskin fibroblasts and dermal fibroblasts using non-neurosphere-mediated or neurosphere-mediated methods to guide them toward neuronal lineages. Cells were subjected to RT-qPCR, immunocytofluorescence to detect the expression of neurogenic genes or electrophysiological analysis at final stage of maturation. We found that induced DPSCs and GMSCs overall appeared to be more neurogenic compared to other cells either morphologically or levels of neurogenic gene expression. Nonetheless, of all the neural induction ...
Introduction-The aim was to quantify vascular network formation capacity after angiogenic inducti... more Introduction-The aim was to quantify vascular network formation capacity after angiogenic induction of human and swine dental pulp stem cells (DPSCs) in comparison to endothelial cells. Methods-Primary human (h) DPSCs or swine (s) DPSCs were induced in endothelial growth medium (EGM) for 7 days. The expression of endothelial marker, von Willebrand Factor (vWF) was determined by immunostaining. Induced (iDPSCs) and non-induced (n-iDPSCs) were seeded at different cell numbers onto Matrigel for vascular network formation assays and analyzed after 4, 8, 12, and 18 h in comparison to human endothelial cells (hMECs). Quantitative analysis of vascular tubule formation was performed using ImageJ. The vascular network formation was also conducted by co-culturing of n-iDPSCs and iDPSCs. Results-vWF was detected by immunofluorescence in both n-iDPSCs and iDPSCs (human and swine). Time-lapse microscopic observation showed that the vascular network was formed by iDPSCs, but not n-iDPSCs. After 4 h, iDPSCs showed vascular network formation while FDPSCs started to aggregate and formed clusters. ihDPSCs displayed a similar capacity to form vascular networks in Matrigel compared to hMECs based on quantitative analysis. isDPSCs had a higher capacity compared to ihDPSCs or hMECs (p<0.05) in forming the network structures including segments, nodes and mesh. isDPSCs than ihDPSCs and hMECs (p<0.05). Co-culture experiment showed that n-ihDPSCs co-localized on the angiogenic tubules and vascular networks formed by ihDPSCs.
Regeneration of periodontal tissues, particularly cementum, is key to regaining periodontal attac... more Regeneration of periodontal tissues, particularly cementum, is key to regaining periodontal attachment and health. Human periodontal ligament stem cells (hPDLSCs) have been shown to be a good cell source to regenerate periodontal tissues. However, their subpopulations and the differentiation induction in relation to cementogenic lineages is unclear. Thus, we aim to examine the expression of cementum-associated genes in PDLSC subpopulations and determine the effect of broadly used osteogenic stimulus or vitamin C (VC) on the expression of cementogenic and osteogenic genes in PDLSCs. Our real-time quantitative polymerase chain reaction (qPCR) analysis showed that cementogenic marker cementum attachment protein (CAP) expressed only slightly higher in STRO-1(+)/CD146(+), STRO-1(-)/CD146(+) and STRO-1(-)/CD146(-) subpopulations than in the original cell pool, while cementum protein 1 (CEMP1) expression in these subpopulations was not different from the original pool. Notably, under the s...
Characterizing subpopulations of stem cells is important to understand stem cell properties. Tiss... more Characterizing subpopulations of stem cells is important to understand stem cell properties. Tissue-nonspecific alkaline phosphatase (ALP) is associated with mineral tissue forming cells as well as stem cells. Information regarding ALP subpopulation of human periodontal ligament stem cells (hPDLSCs) is limited. In the present study, we examined ALP(+) and ALP(-) hPDLSC subpopulations, their surface markers STRO-1 and CD146, and the expression of stemness genes at various cell passages. We found that ALP(+) subpopulation had higher levels of STRO-1 (30.6 ± 5.6%) and CD146 (90.4 ± 3.3%) compared to ALP(-) (STRO-1: 0.5 ± 0.1%; CD146: 75.3 ± 7.2%). ALP(+) cells expressed significantly higher levels of stemness associated genes, NANOG, OCT4 and SOX than ALP(-) cells at low cell passages of 2-3 (p<0.05). ALP(+) and ALP(-) cells had similar osteogenic, chondrogenic and neurogenic potential while ALP(-), not ALP(+) cells, lacked adipogenic potential. Upon continuous culturing and passagi...
Polymeric scaffolds, which release growth factors in a temporally controlled manner, have success... more Polymeric scaffolds, which release growth factors in a temporally controlled manner, have successfully directed the differentiation of stem cells into monolithic tissues of a single lineage. However, engineering precise boundaries in multilineage functional tissues, such as the juxtaposed cartilaginous and osseous tissue present in articulated joints, often remains a challenge. This work demonstrates a precise materials system for in vitro reconstruction of the three-dimensional architecture of these types of human tissues. Multilayer poly(lactide-coglycolide) (PLG) scaffolds were used to produce spatiotemporal gradients to direct the differentiation of an initially uniform population of mesenchymal stem cells (MSCs) into juxtaposed cartilage and bone. Specifically, growth factors (chondrogenic transforming growth factor-b3 and osteogenic bone morphogenetic protein-4) and their neutralizing antibodies were incorporated within distinct layers of the PLG scaffolds to create spatially segregated morphogen fields within the scaffold volume. The multilayer PLG scaffold designs were optimized by mathematical modeling, and generation of spatially segregated morphogen gradients was validated by assessing activity of luciferase reporter cell lines responsive to each growth factor. Scaffolds seeded with MSCs demonstrated production of juxtaposed cartilage and bone, as evaluated by biochemical staining and western blotting for tissue-specific matrix proteins. This work demonstrates a significant advance for the engineering of implantable constructs comprising tissues of multiple lineages, with potential applications in orthopedic regenerative medicine.
Induced pluripotent stem cells (iPSCs) are a potent cell source for neurogenesis. Previously we h... more Induced pluripotent stem cells (iPSCs) are a potent cell source for neurogenesis. Previously we have generated iPSCs from human dental stem cells carrying transgene vectors. These exogenous transgenes may affect iPSC behaviors and limit their clinical applications. The purpose of this study was to establish transgene-free iPSCs (TF-iPSCs) reprogrammed from human stem cells of apical papilla (SCAP) and determine their neurogenic potential. A single lentiviral 'stem cell cassette' flanked by the loxP site (hSTEMCCA-loxP), encoding four human reprogramming factors, OCT4, SOX2, KLF4, and c-MYC, was used to reprogram human SCAP into iPSCs. Generated iPSCs were transfected with plasmid pHAGE2-EF1α-Cre-IRES-PuroR and selected with puromycin for the TF-iPSC subclones. PCR was performed to confirm the excision of hSTEMCCA. TF-iPSC clones did not resist to puromycin treatment indicating no pHAGE2-EF1α-Cre-IRES-PuroR integration into the genome. In vitro and in vivo analyses of their p...
Rapid advancements in the field of stem cell biology have led to many current efforts to exploit ... more Rapid advancements in the field of stem cell biology have led to many current efforts to exploit stem cells as therapeutic agents in regenerative medicine. However, current ex vivo cell manipulations common to most regenerative approaches create a variety of technical and regulatory hurdles to their clinical translation, and even simpler approaches that use exogenous factors to differentiate tissue-resident stem cells carry significant off-target side effects. We show that non-ionizing, low-power laser (LPL) treatment can instead be used as a minimally invasive tool to activate an endogenous latent growth factor complex, transforming growth factor-β1 (TGF-β1), that subsequently differentiates host stem cells to promote tissue regeneration. LPL treatment induced reactive oxygen species (ROS) in a dose-dependent manner, which, in turn, activated latent TGF-β1 (LTGF-β1) via a specific methionine residue (at position 253 on LAP). Laser-activated TGF-β1 was capable of differentiating hum...
Introduction: The loss of dental pulp may weaken teeth, rendering them susceptible to reinfection... more Introduction: The loss of dental pulp may weaken teeth, rendering them susceptible to reinfection, fracture, and subsequent tooth loss. Therefore, regeneration of pulp is considered an ideal treatment to preserve teeth. The aim of this study was to explore the capacity of dental pulp stem cells (DPSCs) and platelet-rich plasma (PRP) to regenerate dental pulp in canine mature permanent teeth. Methods: Pulpectomy with apical foramen enlarged to a #80 file was performed in 16 upper premolars of 4 beagle dogs. Four experimental groups were randomly established: (1) the blood clot group, (2) the autologous DPSCs group, (3) the PRP group, and (4) the DP + PRP group (a mixture of DPSCs and PRP). Four lower premolars without any further treatment after pulpectomy were used as the control group. All teeth were sealed with mineral trioxide aggregate and composite. Twelve weeks after transplantation, the teeth were subjected to radiographic and histologic examination. Results: Twenty-four of 32 experimental root canals gained newly formed tissues. All canals with an introduction of a blood clot showed histologic evidence of vital tissue formation. Cementum-like and periodontal ligament-like tissues along the internal root canal walls were typical structures in most cases. There is no significant difference between groups with or without autologous DPSC transplantation (exact chisquare test, P < .05). Conclusions: New vital tissues can be regenerated in permanent canine teeth after pulpectomy and enlargement of the apical foramen. Histologically, transplantation of DPSCs and/or PRP into root canals showed no enhancement in new tissue formation compared with inducement of a blood clot into the root canals alone.
AimTo investigate the new tissues growing into the pulp space of immature dog teeth that were inf... more AimTo investigate the new tissues growing into the pulp space of immature dog teeth that were infected, disinfected and filled with blood clot (BC), dental pulp cells (DPCs), platelet‐rich plasma (PRP) or a combination of DPCs and PRP in immature dog teeth with apical periodontitis.MethodologyFifty‐six immature roots from mandibular premolars of four beagles were divided into four experimental groups (n = 40) and two control groups. After the induction of apical periodontitis, the root canals of experimental groups were disinfected with NaOCl irrigation and a tri‐antibiotic paste medication. The canals were then filled with different materials according to the experimental group: BC group, DPCs group, PRP group or DPCs + PRP group. Access cavities were sealed with MTA and composite. Radiographs were taken after 90 days, and the jaws including the teeth were processed for histologic analysis. The data were statistically analysed using chi‐square evaluation and Student's t‐test.Re...
Stem cell-mediated regenerative endodontics has reached the human clinical trial phase; however, ... more Stem cell-mediated regenerative endodontics has reached the human clinical trial phase; however, many issues still exist that prevent such technology to be a widely used clinical practice. These issues are not straightforward and are complicated. They should be because pulp regeneration is dealing with a small dead-end space. In addition, when regeneration is needed, the space is often heavily infected. The true standard of pulp regeneration should be everything except generation of some fibrous connective tissue and amorphous mineral deposit. As of now, we are still far short of reaching the standard of complete vascularized and innervated pulp regeneration with newly formed tubular dentin in all types of teeth. Thus, we need to go back to the bench and use established animal models or create new animal models to tackle those issues. This article will address several key issues including the possibility of pulp regeneration in small canals of molar teeth by enhancing the neovascularization, and whether the organized tubular dentin can be generated on the canal walls. Data from our semi-orthotopic tooth fragment mouse model have shown that complete pulp regeneration using dental pulp stem cells (DPSCs) in small canal has been inconsistent because of limited blood supply. This inconsistency is similar in our orthotopic miniature swine model, although in some cases vascularized pulp-like tissue can be formed throughout the canal space after DPSC transplantation. Furthermore, no tubular dentin was observed in the orthotopic pulp regeneration, despite the fact that DPSCs have the capacity to generate some tubular dentin-like structure in the hydroxyapatite/tricalcium phosphate-mediated ectopic pulp/dentin formation model in mice. Potential strategies to be tested to address these regeneration issues are discussed herein.
International journal of oral biology : official journal of the Korean Academy of Oral Biology and the UCLA Dental Research Institute, 2004
The objective of this study was to determine whether cells from human pulp can be transduced to e... more The objective of this study was to determine whether cells from human pulp can be transduced to express the antimicrobial peptide--human β-defensin-2 (HBD-2). Primary human pulp cells and gingival fibroblasts from normal tissue, as well as two mouse cell lines (NIH 3T3 and AT-84) and a human cell line SCC-9 were transduced with a retroviral vector carrying HBD-2 cDNA. ELISA and Northern blot analyses were performed to detect HBD-2 expression by these transduced cells. Antimicrobail assays using recombinant HBD-2 were performed on two caries-associated bacteria Streptococcus mutnas and Lactobacillus acidophilus. The results showed that transduced pulp cells secreted 62.4 ± 27.2 ng/3 days of HBD-2, which was comparable to that by NIH 3T3 (78.0 ± 14.1 ng/4 days), and higher than those by gingival fibroblasts (17.9 ± 7.9 ng/3 days), AT-84 (2.6 ± 1.0 ng/3 days), and SCC-9 (47.6 ± 9.9 ng/3 days). Northern blot analysis showed that the levels of HBD-2 mRNA expression correlated with their ...
Mesenchymal stem cells (MSCs) have been isolated from the pulp tissue of permanent teeth (dental ... more Mesenchymal stem cells (MSCs) have been isolated from the pulp tissue of permanent teeth (dental pulp stem cells or DPSCs) and deciduous teeth (stem cells from human exfoliated deciduous teeth). We recently discovered another type of MSCs in the apical papilla of human immature permanent teeth termed stem cells from the apical papilla (SCAP). Here, we further characterized the apical papilla tissue and stem cell properties of SCAP using histologic, immunohistochemical, and immunocytofluorescent analyses. We found that the apical papilla is distinctive to the pulp in terms of containing less cellular and vascular components than those in the pulp. Cells in the apical papilla proliferated 2- to 3-fold greater than those in the pulp in organ cultures. Both SCAP and DPSCs were as potent in osteo/dentinogenic differentiation as MSCs from bone marrows, whereas they were weaker in adipogenic potential. The immunophenotype of SCAP is similar to that of DPSCs on the osteo/dentinogenic and gr...
Background: Potent stem/progenitor cells have been isolated from normal human dental pulps termed... more Background: Potent stem/progenitor cells have been isolated from normal human dental pulps termed dental pulp stem cells (DPSCs). However, it is unknown whether these cells exist in inflamed pulps (IPs). Aims: To determine whether DPSCs can be identified and isolated from IPs; and if they can be successfully cultured, whether they retain tissue regeneration potential in vivo. Materials & methods: DPSCs from freshly collected normal pulps (NPs) and IPs were characterized in vitro and their tissue regeneration potential tested using an in vivo study model. Results: The immunohistochemical analysis showed that IPs expressed higher levels of mesenchymal stem cell markers STRO-1, CD90, CD105 and CD146 compared with NPs (p < 0.05). Flow cytometry analysis showed that DPSCs from both NPs and IPs expressed moderate to high levels of CD146, stage-specific embryonic antigen-4, CD73 and CD166. Total population doubling of DPSCs-IPs (44.6 ± 2.9) was lower than that of DPSCs-NPs (58.9 ± 2.5) ...
The ultimate goal of this study is to regenerate lost dental pulp and dentin via stem=progenitor ... more The ultimate goal of this study is to regenerate lost dental pulp and dentin via stem=progenitor cell-based approaches and tissue engineering technologies. In this study, we tested the possibility of regenerating vascularized human dental pulp in emptied root canal space and producing new dentin on existing dentinal walls using a stem=progenitor cell-mediated approach with a human root fragment and an immunocompromised mouse model. Stem=progenitor cells from apical papilla and dental pulp stem cells were isolated, characterized, seeded onto synthetic scaffolds consisting of poly-D,L-lactide=glycolide, inserted into the tooth fragments, and transplanted into mice. Our results showed that the root canal space was filled entirely by a pulp-like tissue with well-established vascularity. In addition, a continuous layer of dentin-like tissue was deposited onto the canal dentinal wall. This dentin-like structure appeared to be produced by a layer of newly formed odontoblast-like cells expressing dentin sialophosphoprotein, bone sialoprotein, alkaline phosphatase, and CD105. The cells in regenerated pulp-like tissue reacted positively to anti-human mitochondria antibodies, indicating their human origin. This study provides the first evidence showing that pulp-like tissue can be regenerated de novo in emptied root canal space by stem cells from apical papilla and dental pulp stem cells that give rise to odontoblast-like cells producing dentin-like tissue on existing dentinal walls.
Mesenchymal progenitor cells (MPCs) are multipotent cells, derived from various adult tissues, th... more Mesenchymal progenitor cells (MPCs) are multipotent cells, derived from various adult tissues, that are capable of differentiating into several mesenchymal lineages, including osteoblasts, chondroblasts, and adipocytes. A large body of data suggested MPCs as a promising candidate cell type applicable for repair and regeneration of a variety of mesenchymal tissues such as bone, carti lage, and muscle [1,2]. MPCs were initially identified and isolated from bone marrow (BM) and are characterized by the expression of a number of cell surface markers [3-5]. Based on their clonogenic and multipotent differen tiation activities, to date, MPCs have been isolated from a number of adult tissues, including trabecular bone [6], fat [7,8], synovium [9,10], skin [11], thymus [11,12], periodontal ligament [13], as well as prenatal and perinatal sources such as umbilical cord blood [14], umbilical cord [15], palatine tonsil [16], and placenta [17]. e diversity of sources facilitates MPC accessibility, but also raises questions about possible phenotypic and functional discrepancies that must be addressed for their clinical use. e transforming growth factor-β (TGF-β) superfamily of secreted factors includes TGF-β, activins, Nodal, and bone morphogenetic proteins (BMPs). e activation of the TGF-β/activin/Nodal signaling pathway through SMAD2/3 is associated with the pluripotency of human embryonic stem cells (hESCs) and is required for the maintenance of their undifferentiated state [18]. rough the induction of Oct4, Nanog, Nodal, Wnt3, basic fibroblast growth factor (FGF-2), and FGF-8, Activin A was shown to be a key regulator for the "stemness" maintenance of hESCs [19]. Activin A, like other members of the TGF-β superfamily, has also been described to affect
Objectives This is to compare the volumes of irrigant apically extruded by five irrigation system... more Objectives This is to compare the volumes of irrigant apically extruded by five irrigation systems in an artificial socket model simulating clinical conditions. Materials and methods Twenty extracted human singlerooted teeth were enlarged to size 40/04 and then embedded in silicone impression material. The root canal space was irrigated with nominal 3% sodium hypochlorite (NaOCl) using standard needle irrigation (SNI) with a 30-gauge notched needle, EndoActivator (EA), XP Endo Finisher (XP Endo), EndoVac (EV), and photon-induced photoacoustic streaming (PIPS). Extruded NaOCl was collected, reacted with taurine to form taurine-monochloramine, and absorbance of taurinemonochloramine was measured at 252 nm using a spectrophotometer. The five irrigation systems were compared with repeated measures ANOVA and pairwise comparisons. R e s u l t s T h e E V g r o u p h a d v e r y l o w e x t r u s i o n (mean ± SD = 0.12 ± 0.2 μL) and differed significantly from the other four groups (P ≤ 0.001). Larger volumes of irrigant were extruded in the other irrigation groups. There were no significant differences in the extruded volumes among the SNI (7.4 ± 3.4 μL), EA (7.0 ± 6.1 μL), and XP Endo (7.8 ± 4.1 μL) groups (P = 1). The PIPS group had the highest mean extruded volume (12.9 ± 6.8 μL) and differed significantly from SNI (P = 0.030), EV (P < 0.0005), and EA (P = 0.02), but not XP Endo (P = 0.154). Conclusion Under the in vitro conditions of this study, irrigant extrusion appears unavoidable unless negative pressure irrigation such as EV is used. PIPS extrudes more irrigant than other systems, while SNI, EA, and XP Endo extrude similar volumes of irrigant. Clinical relevance The findings help clinicians select the optimal irrigation system to avoid irrigant extrusion.
To determine factors that may influence treatment outcome and healing time following root canal t... more To determine factors that may influence treatment outcome and healing time following root canal treatment. Root filled and restored teeth by pre-doctoral students were included in this study. Teeth/roots were followed-up regularly, and treatment outcome was evaluated at every follow-up appointment (healed, healing, uncertain or unsatisfactory). Host (age, immune condition, pulp/periapical diagnosis, tooth/root type, location and anatomy) and treatment factors (master apical file size, apical extension, voids and density of root filling) were recorded from patient dental records. Univariate, bivariate and multivariate analyses were performed to determine the impact of the factors on treatment outcomes and healing times. A total of 422 roots from 291 teeth met the inclusion criteria with a mean follow-up period of 2 years. The preoperative pulp condition, procedural errors during treatment, apical extension and density of root fillings significantly affected the treatment outcome. The average time required for a periapical lesion to heal was 11.78 months. The healing time increased in patients with compromised healing, patients older than 40 years, roots with Weine type II root canal systems, root canal systems prepared to a master apical file size &lt;35, and roots with overextended fillings (P &lt; 0.1). Multiple host and treatment factors affected the healing time and outcome of root canal treatment. Follow-up protocols should consider these factors before concluding the treatment outcome: patient&#39;s age, immune condition, as well as roots with overextended fillings, root canal systems with smaller apical preparations (size &lt;35) or roots with complex canal systems. Intervention may be recommended if the treatment quality was inadequate or if patients became symptomatic.
Introduction-The aim of this study was to determine the efficiency of 4 irrigation systems in eli... more Introduction-The aim of this study was to determine the efficiency of 4 irrigation systems in eliminating bacteria in root canals, particularly in dentinal tubules. Methods-Roots of human teeth were prepared to 25/04, autoclaved, and inoculated with Enterococcus faecalis for 3 weeks. Canals were then disinfected by (1) standard needle irrigation, (2) sonically agitating with EndoActivator, (3) XP Endo finisher, or (4) erbium:yttrium aluminum garnet laser (PIPS) (15 roots/group). The bacterial reduction in the canal was determined by MTT assays. For measuring live versus dead bacteria in the dentinal tubules (4 teeth/group), teeth were split open and stained with LIVE/DEAD BackLight. Coronal, middle, and apical thirds of the canal dentin were scanned by using a confocal laser scanning microscope (CLSM) to determine the ratio of dead/total bacteria in the dentinal tubules at various depths. Results-All 4 irrigation protocols significantly eliminated bacteria in the canal, ranging from 89.6% to 98.2% reduction (P < .001). XP Endo had the greatest bacterial reduction compared with other 3 techniques (P < .05). CLSM analysis showed that XP Endo had the highest level of dead bacteria in the coronal, middle, and apical segments at 50-μm depth. On the other hand, PIPS had the greatest bacterial killing efficiency at the 150-μm depth in all 3 root segments. Conclusions-XP Endo appears to be more efficient than other 3 techniques in disinfecting the main canal space and up to 50 μm deep into the dentinal tubules. PIPS appears to be most effective in killing the bacteria deep in the dentinal tubules.
Management of avulsed teeth after replantation often leads to an unfavorable outcome. Damage to t... more Management of avulsed teeth after replantation often leads to an unfavorable outcome. Damage to the thin and vulnerable periodontal ligament is the key reason for failure. Cell-or stem cell-based regenerative medicine has emerged in the past two decades as a promising clinical treatment modality to improve treatment outcomes. This concept has also been tested for the management of avulsed teeth in animal models. This review focuses on the discussion of limitation of current management protocols for avulsed teeth, cell-based therapy for periodontal ligament (PDL) regeneration in small and large animals, the challenges of de novo regeneration of PDL on denuded root in the edentulous region using a mini-swine model, and establishing a prospective new clinical protocol to manage avulsed teeth based on the current progress of cell-based PDL regeneration studies.
The potential of human mesenchymal stromal/stem cells (MSCs) including oral stem cells (OSCs) as ... more The potential of human mesenchymal stromal/stem cells (MSCs) including oral stem cells (OSCs) as a cell source to derive functional neurons has been inconclusive. Here we tested a number of human OSCs for their neurogenic potential compared to non-OSCs and employed various neurogenic induction methods. OSCs including dental pulp stem cells (DPSCs), gingiva-derived mesenchymal stem cells (GMSCs), stem cells from apical papilla and non-OSCs including bone marrow MSCs (BMMSCs), foreskin fibroblasts and dermal fibroblasts using non-neurosphere-mediated or neurosphere-mediated methods to guide them toward neuronal lineages. Cells were subjected to RT-qPCR, immunocytofluorescence to detect the expression of neurogenic genes or electrophysiological analysis at final stage of maturation. We found that induced DPSCs and GMSCs overall appeared to be more neurogenic compared to other cells either morphologically or levels of neurogenic gene expression. Nonetheless, of all the neural induction ...
Introduction-The aim was to quantify vascular network formation capacity after angiogenic inducti... more Introduction-The aim was to quantify vascular network formation capacity after angiogenic induction of human and swine dental pulp stem cells (DPSCs) in comparison to endothelial cells. Methods-Primary human (h) DPSCs or swine (s) DPSCs were induced in endothelial growth medium (EGM) for 7 days. The expression of endothelial marker, von Willebrand Factor (vWF) was determined by immunostaining. Induced (iDPSCs) and non-induced (n-iDPSCs) were seeded at different cell numbers onto Matrigel for vascular network formation assays and analyzed after 4, 8, 12, and 18 h in comparison to human endothelial cells (hMECs). Quantitative analysis of vascular tubule formation was performed using ImageJ. The vascular network formation was also conducted by co-culturing of n-iDPSCs and iDPSCs. Results-vWF was detected by immunofluorescence in both n-iDPSCs and iDPSCs (human and swine). Time-lapse microscopic observation showed that the vascular network was formed by iDPSCs, but not n-iDPSCs. After 4 h, iDPSCs showed vascular network formation while FDPSCs started to aggregate and formed clusters. ihDPSCs displayed a similar capacity to form vascular networks in Matrigel compared to hMECs based on quantitative analysis. isDPSCs had a higher capacity compared to ihDPSCs or hMECs (p<0.05) in forming the network structures including segments, nodes and mesh. isDPSCs than ihDPSCs and hMECs (p<0.05). Co-culture experiment showed that n-ihDPSCs co-localized on the angiogenic tubules and vascular networks formed by ihDPSCs.
Regeneration of periodontal tissues, particularly cementum, is key to regaining periodontal attac... more Regeneration of periodontal tissues, particularly cementum, is key to regaining periodontal attachment and health. Human periodontal ligament stem cells (hPDLSCs) have been shown to be a good cell source to regenerate periodontal tissues. However, their subpopulations and the differentiation induction in relation to cementogenic lineages is unclear. Thus, we aim to examine the expression of cementum-associated genes in PDLSC subpopulations and determine the effect of broadly used osteogenic stimulus or vitamin C (VC) on the expression of cementogenic and osteogenic genes in PDLSCs. Our real-time quantitative polymerase chain reaction (qPCR) analysis showed that cementogenic marker cementum attachment protein (CAP) expressed only slightly higher in STRO-1(+)/CD146(+), STRO-1(-)/CD146(+) and STRO-1(-)/CD146(-) subpopulations than in the original cell pool, while cementum protein 1 (CEMP1) expression in these subpopulations was not different from the original pool. Notably, under the s...
Characterizing subpopulations of stem cells is important to understand stem cell properties. Tiss... more Characterizing subpopulations of stem cells is important to understand stem cell properties. Tissue-nonspecific alkaline phosphatase (ALP) is associated with mineral tissue forming cells as well as stem cells. Information regarding ALP subpopulation of human periodontal ligament stem cells (hPDLSCs) is limited. In the present study, we examined ALP(+) and ALP(-) hPDLSC subpopulations, their surface markers STRO-1 and CD146, and the expression of stemness genes at various cell passages. We found that ALP(+) subpopulation had higher levels of STRO-1 (30.6 ± 5.6%) and CD146 (90.4 ± 3.3%) compared to ALP(-) (STRO-1: 0.5 ± 0.1%; CD146: 75.3 ± 7.2%). ALP(+) cells expressed significantly higher levels of stemness associated genes, NANOG, OCT4 and SOX than ALP(-) cells at low cell passages of 2-3 (p<0.05). ALP(+) and ALP(-) cells had similar osteogenic, chondrogenic and neurogenic potential while ALP(-), not ALP(+) cells, lacked adipogenic potential. Upon continuous culturing and passagi...
Polymeric scaffolds, which release growth factors in a temporally controlled manner, have success... more Polymeric scaffolds, which release growth factors in a temporally controlled manner, have successfully directed the differentiation of stem cells into monolithic tissues of a single lineage. However, engineering precise boundaries in multilineage functional tissues, such as the juxtaposed cartilaginous and osseous tissue present in articulated joints, often remains a challenge. This work demonstrates a precise materials system for in vitro reconstruction of the three-dimensional architecture of these types of human tissues. Multilayer poly(lactide-coglycolide) (PLG) scaffolds were used to produce spatiotemporal gradients to direct the differentiation of an initially uniform population of mesenchymal stem cells (MSCs) into juxtaposed cartilage and bone. Specifically, growth factors (chondrogenic transforming growth factor-b3 and osteogenic bone morphogenetic protein-4) and their neutralizing antibodies were incorporated within distinct layers of the PLG scaffolds to create spatially segregated morphogen fields within the scaffold volume. The multilayer PLG scaffold designs were optimized by mathematical modeling, and generation of spatially segregated morphogen gradients was validated by assessing activity of luciferase reporter cell lines responsive to each growth factor. Scaffolds seeded with MSCs demonstrated production of juxtaposed cartilage and bone, as evaluated by biochemical staining and western blotting for tissue-specific matrix proteins. This work demonstrates a significant advance for the engineering of implantable constructs comprising tissues of multiple lineages, with potential applications in orthopedic regenerative medicine.
Induced pluripotent stem cells (iPSCs) are a potent cell source for neurogenesis. Previously we h... more Induced pluripotent stem cells (iPSCs) are a potent cell source for neurogenesis. Previously we have generated iPSCs from human dental stem cells carrying transgene vectors. These exogenous transgenes may affect iPSC behaviors and limit their clinical applications. The purpose of this study was to establish transgene-free iPSCs (TF-iPSCs) reprogrammed from human stem cells of apical papilla (SCAP) and determine their neurogenic potential. A single lentiviral 'stem cell cassette' flanked by the loxP site (hSTEMCCA-loxP), encoding four human reprogramming factors, OCT4, SOX2, KLF4, and c-MYC, was used to reprogram human SCAP into iPSCs. Generated iPSCs were transfected with plasmid pHAGE2-EF1α-Cre-IRES-PuroR and selected with puromycin for the TF-iPSC subclones. PCR was performed to confirm the excision of hSTEMCCA. TF-iPSC clones did not resist to puromycin treatment indicating no pHAGE2-EF1α-Cre-IRES-PuroR integration into the genome. In vitro and in vivo analyses of their p...
Rapid advancements in the field of stem cell biology have led to many current efforts to exploit ... more Rapid advancements in the field of stem cell biology have led to many current efforts to exploit stem cells as therapeutic agents in regenerative medicine. However, current ex vivo cell manipulations common to most regenerative approaches create a variety of technical and regulatory hurdles to their clinical translation, and even simpler approaches that use exogenous factors to differentiate tissue-resident stem cells carry significant off-target side effects. We show that non-ionizing, low-power laser (LPL) treatment can instead be used as a minimally invasive tool to activate an endogenous latent growth factor complex, transforming growth factor-β1 (TGF-β1), that subsequently differentiates host stem cells to promote tissue regeneration. LPL treatment induced reactive oxygen species (ROS) in a dose-dependent manner, which, in turn, activated latent TGF-β1 (LTGF-β1) via a specific methionine residue (at position 253 on LAP). Laser-activated TGF-β1 was capable of differentiating hum...
Introduction: The loss of dental pulp may weaken teeth, rendering them susceptible to reinfection... more Introduction: The loss of dental pulp may weaken teeth, rendering them susceptible to reinfection, fracture, and subsequent tooth loss. Therefore, regeneration of pulp is considered an ideal treatment to preserve teeth. The aim of this study was to explore the capacity of dental pulp stem cells (DPSCs) and platelet-rich plasma (PRP) to regenerate dental pulp in canine mature permanent teeth. Methods: Pulpectomy with apical foramen enlarged to a #80 file was performed in 16 upper premolars of 4 beagle dogs. Four experimental groups were randomly established: (1) the blood clot group, (2) the autologous DPSCs group, (3) the PRP group, and (4) the DP + PRP group (a mixture of DPSCs and PRP). Four lower premolars without any further treatment after pulpectomy were used as the control group. All teeth were sealed with mineral trioxide aggregate and composite. Twelve weeks after transplantation, the teeth were subjected to radiographic and histologic examination. Results: Twenty-four of 32 experimental root canals gained newly formed tissues. All canals with an introduction of a blood clot showed histologic evidence of vital tissue formation. Cementum-like and periodontal ligament-like tissues along the internal root canal walls were typical structures in most cases. There is no significant difference between groups with or without autologous DPSC transplantation (exact chisquare test, P < .05). Conclusions: New vital tissues can be regenerated in permanent canine teeth after pulpectomy and enlargement of the apical foramen. Histologically, transplantation of DPSCs and/or PRP into root canals showed no enhancement in new tissue formation compared with inducement of a blood clot into the root canals alone.
AimTo investigate the new tissues growing into the pulp space of immature dog teeth that were inf... more AimTo investigate the new tissues growing into the pulp space of immature dog teeth that were infected, disinfected and filled with blood clot (BC), dental pulp cells (DPCs), platelet‐rich plasma (PRP) or a combination of DPCs and PRP in immature dog teeth with apical periodontitis.MethodologyFifty‐six immature roots from mandibular premolars of four beagles were divided into four experimental groups (n = 40) and two control groups. After the induction of apical periodontitis, the root canals of experimental groups were disinfected with NaOCl irrigation and a tri‐antibiotic paste medication. The canals were then filled with different materials according to the experimental group: BC group, DPCs group, PRP group or DPCs + PRP group. Access cavities were sealed with MTA and composite. Radiographs were taken after 90 days, and the jaws including the teeth were processed for histologic analysis. The data were statistically analysed using chi‐square evaluation and Student's t‐test.Re...
Stem cell-mediated regenerative endodontics has reached the human clinical trial phase; however, ... more Stem cell-mediated regenerative endodontics has reached the human clinical trial phase; however, many issues still exist that prevent such technology to be a widely used clinical practice. These issues are not straightforward and are complicated. They should be because pulp regeneration is dealing with a small dead-end space. In addition, when regeneration is needed, the space is often heavily infected. The true standard of pulp regeneration should be everything except generation of some fibrous connective tissue and amorphous mineral deposit. As of now, we are still far short of reaching the standard of complete vascularized and innervated pulp regeneration with newly formed tubular dentin in all types of teeth. Thus, we need to go back to the bench and use established animal models or create new animal models to tackle those issues. This article will address several key issues including the possibility of pulp regeneration in small canals of molar teeth by enhancing the neovascularization, and whether the organized tubular dentin can be generated on the canal walls. Data from our semi-orthotopic tooth fragment mouse model have shown that complete pulp regeneration using dental pulp stem cells (DPSCs) in small canal has been inconsistent because of limited blood supply. This inconsistency is similar in our orthotopic miniature swine model, although in some cases vascularized pulp-like tissue can be formed throughout the canal space after DPSC transplantation. Furthermore, no tubular dentin was observed in the orthotopic pulp regeneration, despite the fact that DPSCs have the capacity to generate some tubular dentin-like structure in the hydroxyapatite/tricalcium phosphate-mediated ectopic pulp/dentin formation model in mice. Potential strategies to be tested to address these regeneration issues are discussed herein.
International journal of oral biology : official journal of the Korean Academy of Oral Biology and the UCLA Dental Research Institute, 2004
The objective of this study was to determine whether cells from human pulp can be transduced to e... more The objective of this study was to determine whether cells from human pulp can be transduced to express the antimicrobial peptide--human β-defensin-2 (HBD-2). Primary human pulp cells and gingival fibroblasts from normal tissue, as well as two mouse cell lines (NIH 3T3 and AT-84) and a human cell line SCC-9 were transduced with a retroviral vector carrying HBD-2 cDNA. ELISA and Northern blot analyses were performed to detect HBD-2 expression by these transduced cells. Antimicrobail assays using recombinant HBD-2 were performed on two caries-associated bacteria Streptococcus mutnas and Lactobacillus acidophilus. The results showed that transduced pulp cells secreted 62.4 ± 27.2 ng/3 days of HBD-2, which was comparable to that by NIH 3T3 (78.0 ± 14.1 ng/4 days), and higher than those by gingival fibroblasts (17.9 ± 7.9 ng/3 days), AT-84 (2.6 ± 1.0 ng/3 days), and SCC-9 (47.6 ± 9.9 ng/3 days). Northern blot analysis showed that the levels of HBD-2 mRNA expression correlated with their ...
Mesenchymal stem cells (MSCs) have been isolated from the pulp tissue of permanent teeth (dental ... more Mesenchymal stem cells (MSCs) have been isolated from the pulp tissue of permanent teeth (dental pulp stem cells or DPSCs) and deciduous teeth (stem cells from human exfoliated deciduous teeth). We recently discovered another type of MSCs in the apical papilla of human immature permanent teeth termed stem cells from the apical papilla (SCAP). Here, we further characterized the apical papilla tissue and stem cell properties of SCAP using histologic, immunohistochemical, and immunocytofluorescent analyses. We found that the apical papilla is distinctive to the pulp in terms of containing less cellular and vascular components than those in the pulp. Cells in the apical papilla proliferated 2- to 3-fold greater than those in the pulp in organ cultures. Both SCAP and DPSCs were as potent in osteo/dentinogenic differentiation as MSCs from bone marrows, whereas they were weaker in adipogenic potential. The immunophenotype of SCAP is similar to that of DPSCs on the osteo/dentinogenic and gr...
Background: Potent stem/progenitor cells have been isolated from normal human dental pulps termed... more Background: Potent stem/progenitor cells have been isolated from normal human dental pulps termed dental pulp stem cells (DPSCs). However, it is unknown whether these cells exist in inflamed pulps (IPs). Aims: To determine whether DPSCs can be identified and isolated from IPs; and if they can be successfully cultured, whether they retain tissue regeneration potential in vivo. Materials & methods: DPSCs from freshly collected normal pulps (NPs) and IPs were characterized in vitro and their tissue regeneration potential tested using an in vivo study model. Results: The immunohistochemical analysis showed that IPs expressed higher levels of mesenchymal stem cell markers STRO-1, CD90, CD105 and CD146 compared with NPs (p < 0.05). Flow cytometry analysis showed that DPSCs from both NPs and IPs expressed moderate to high levels of CD146, stage-specific embryonic antigen-4, CD73 and CD166. Total population doubling of DPSCs-IPs (44.6 ± 2.9) was lower than that of DPSCs-NPs (58.9 ± 2.5) ...
The ultimate goal of this study is to regenerate lost dental pulp and dentin via stem=progenitor ... more The ultimate goal of this study is to regenerate lost dental pulp and dentin via stem=progenitor cell-based approaches and tissue engineering technologies. In this study, we tested the possibility of regenerating vascularized human dental pulp in emptied root canal space and producing new dentin on existing dentinal walls using a stem=progenitor cell-mediated approach with a human root fragment and an immunocompromised mouse model. Stem=progenitor cells from apical papilla and dental pulp stem cells were isolated, characterized, seeded onto synthetic scaffolds consisting of poly-D,L-lactide=glycolide, inserted into the tooth fragments, and transplanted into mice. Our results showed that the root canal space was filled entirely by a pulp-like tissue with well-established vascularity. In addition, a continuous layer of dentin-like tissue was deposited onto the canal dentinal wall. This dentin-like structure appeared to be produced by a layer of newly formed odontoblast-like cells expressing dentin sialophosphoprotein, bone sialoprotein, alkaline phosphatase, and CD105. The cells in regenerated pulp-like tissue reacted positively to anti-human mitochondria antibodies, indicating their human origin. This study provides the first evidence showing that pulp-like tissue can be regenerated de novo in emptied root canal space by stem cells from apical papilla and dental pulp stem cells that give rise to odontoblast-like cells producing dentin-like tissue on existing dentinal walls.
Mesenchymal progenitor cells (MPCs) are multipotent cells, derived from various adult tissues, th... more Mesenchymal progenitor cells (MPCs) are multipotent cells, derived from various adult tissues, that are capable of differentiating into several mesenchymal lineages, including osteoblasts, chondroblasts, and adipocytes. A large body of data suggested MPCs as a promising candidate cell type applicable for repair and regeneration of a variety of mesenchymal tissues such as bone, carti lage, and muscle [1,2]. MPCs were initially identified and isolated from bone marrow (BM) and are characterized by the expression of a number of cell surface markers [3-5]. Based on their clonogenic and multipotent differen tiation activities, to date, MPCs have been isolated from a number of adult tissues, including trabecular bone [6], fat [7,8], synovium [9,10], skin [11], thymus [11,12], periodontal ligament [13], as well as prenatal and perinatal sources such as umbilical cord blood [14], umbilical cord [15], palatine tonsil [16], and placenta [17]. e diversity of sources facilitates MPC accessibility, but also raises questions about possible phenotypic and functional discrepancies that must be addressed for their clinical use. e transforming growth factor-β (TGF-β) superfamily of secreted factors includes TGF-β, activins, Nodal, and bone morphogenetic proteins (BMPs). e activation of the TGF-β/activin/Nodal signaling pathway through SMAD2/3 is associated with the pluripotency of human embryonic stem cells (hESCs) and is required for the maintenance of their undifferentiated state [18]. rough the induction of Oct4, Nanog, Nodal, Wnt3, basic fibroblast growth factor (FGF-2), and FGF-8, Activin A was shown to be a key regulator for the "stemness" maintenance of hESCs [19]. Activin A, like other members of the TGF-β superfamily, has also been described to affect
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Papers by George Huang