Characterization of cells in blood evoked from periapical tissues in immature teeth with pulp necrosis and their potential for autologous cell therapy in Regenerative Endodontics.

OBJECTIVE: The objectives of this study were to isolate, characterize progenitor cells from blood in the root canals of necrotic immature permanent teeth evoked from periapical tissues and evaluate the applicable potential of these isolated cells in Regenerative Endodontics. DESIGN: Ten necrotic immature permanent teeth from seven patients were included. Evoked bleeding from periapical tissues was induced after chemical instrumentation of the root canals. Cells were isolated from the canal blood and evaluated for cell surface marker expression, multilineage differentiation potential, proliferation ability, and target protein expression. Cell sheets formed from these cells were transferred into human root segments, and then transplanted into nude mice. Histological examination was performed after eight weeks. Data analysis was conducted using one-way ANOVA followed by Tukey's post-hoc comparison, considering p < 0.05 as statistically significant. RESULTS: The isolated cells exhibited characteristics typical of fibroblastic cells with colony-forming efficiency, and displayed Ki67 positivity and robust proliferation. Flow cytometry data demonstrated that at passage 3, these cells were positive for CD73, CD90, CD105, CD146, and negative for CD34 and CD45. Vimentin expression indicated a mesenchymal origin. Under differentiation media specific differentiation media, the cells demonstrated osteogenic, adipogenic, and chondrogenic differentiation potential. Subcutaneous root canals with cell sheets of isolated cells in nude mice showed the formation of pulp-like tissues. CONCLUSIONS: This study confirmed the presence of progenitor cells in root canals following evoked bleeding from periapical tissues of necrotic immature teeth. Isolated cells exhibited similar immunophenotype and regenerative potential with dental mesenchymal stromal cells in regenerative endodontic therapy.

Human Digested Dentin Matrix for Dentin Regeneration and the Applicative Potential in Vital Pulp Therapy.

INTRODUCTION: Human dentin is a natural acellular matrix with excellent reported biocompatibility. The aim was to fabricate a novel dentin matrix material from human dentin and investigate its applicative potential for vital pulp therapy. METHODS: Digested dentin matrix extract (DDME) was fabricated using controlled enzymatic digestion under acidic conditions. The surfaces and biocompatibility of DDME were then investigated, with its effects on the odontogenic differentiation of human dental pulp cells (hDPCs) also studied. The ability of DDME to induce mineralization was assessed in a nude mouse model. The performance of DDME as a pulp capping agent was evaluated in an in situ rat model. The molecular mechanism was verified by mRNA sequencing. RESULTS: A novel type of dentin matrix material with a uniform size of 8 µm was fabricated. DDME had a similar band compared with grinded dentin matrix, with a smaller size, and more uneven surface, as detected by Fourier-transform infrared spectrometer and X-ray photoelectron spectroscopy. DDME at low concentrations did not affect hDPC viability or proliferation, but enhanced runt-related transcription factor 2, dentin matrix acidic phosphoprotein 1, and COL1A1 (collagen type I alpha 1 chain) expression in hDPCs in vitro. DDME was superior to HA-TCP (hydroxyapatite-tricalcium phosphate) in dentin-like mineralized tissue formation after subcutaneous transplantation. In the rat model of pulpotomy, DDME showed visible curative effects. The underlying mechanism may be the inhibition of Hippo signaling following DDME treatment. DDME promoted Yes-associated protein (YAP) 1 nuclear influx, thereby enhancing the expression of DMP-1 (dentin matrix acidic phosphoprotein 1), which was reversed by YAP inhibitor treatment. CONCLUSIONS: Human DDME can be used as a biomaterial for dentin regeneration. The combined application of DDME and current pulp capping agents is a potential choice for vital pulp therapy.

Effect of conventional needle irrigation, passive ultrasonic irrigation, sonic irrigation and XP-endo finisher in removing intracanal Vitapex paste.

To compare the efficiency of conventional needle irrigation, passive ultrasonic irrigation, sonic irrigation and XP-endo Finisher in removing Vitapex paste from root canals. The root canals of human single-rooted teeth were prepared and obturated with Vitapex paste. After 2 weeks, the 48 teeth were randomly allocated into four groups: conventional needle irrigation (CNI) group, passive ultrasonic irrigation (PUI) group, sonic irrigation (SI) group and XP-endo Finisher (XP) group. The specimens of four groups were scanned using a micro-computed tomography after the Vitapex pastes was removed. The results showed that more residual paste was left in the CNI group than in the other three groups (p < 0.05). There was no significant difference among the PUI, SI and XP group (p > 0.05). And, the majority of the residual Vitapex was found in the apical third, with a small amount in the middle third after using any of the three agitated irrigation techniques.

Cryopreservation of human dental roots using vitrification for autologous human tooth tissue banking.

This study aims to preliminarily evaluate the feasibility of autologous transplantation of tooth tissues cryopreserved with vitrification, by investigating the influence of cryopreservation with vitrification on human dental root, regarding the morphology, microhardness, cell apoptosis, proliferation and differentiation. Freshly extracted human permanent premolars were collected with crown removed. Dental roots were cryopreserved using a commercial vitrification medium (Kitazatousa). After six-month storage in liquid nitrogen, cryopreserved roots were thawed, and then evaluated using histological and immunohistochemical methods. Microhardness of dentine was measured with a Vickers indenter. Cells in periodontal ligament and dental pulp tissues were isolated and characterized. The proliferation, immunophenotype, apoptosis and differentiation ability of cells isolated from cryopreserved roots were evaluated. The data was analyzed using one-way analysis of variance (ANOVA) and Student's t-test. The gross and histological morphology of dental roots was not significantly changed after vitrification and thawing. A few tiny cracks were found in 3 of all 10 cryopreserved samples. No obvious changes were found in microstructure of dentine under SEM observation. Dental pulp cells and periodontal ligament cells were successfully isolated from tissues of cryopreserved human dental roots. There were also no significant differences of those periodontal ligament cells in the two groups regarding morphology, immunophenotype, viability, proliferation and apoptosis. The osteogenic and adipogenic differentiation capability of periodontal ligament cells was maintained by cryopreservation with vitrification. In the conditions of this study, cryopreservation with vitrification preserves cell survival, hardness and structural integrity of dental roots. Vitrification can be a potential way to preserve tooth tissue for future auto-transplantation and autologous cell therapy.

Functional properties of LRRK2 mutations in Taiwanese Parkinson disease.

BACKGROUND/PURPOSE: Leucine-rich repeat kinase 2 (LRRK2) is a large protein encoding multiple functional domains. Mutations within different LRRK2 domains have been considered to be involved in the development of Parkinson disease by different mechanisms. Our previous study found three LRRK2 mutations-p.R767H, p.S885N, and p.R1441H-in Taiwanese patients with Parkinson disease. METHODS: We evaluated the functional properties of LRRK2 p.R767H, p.S885N, and p.R1441H mutations by overexpressing them in human embryonic kidney 293 and neuroblastoma SK-N-SH cells. The common p.G2019S mutation in the kinase domain was included for comparison. RESULTS: In 293 cells, overexpressed p.R1441H-but not p.R767H, p.S885N, or p.G2019-increased GTP binding affinity to prolong the active state. Overexpressed p.R1441H and p.G2019S generated inclusions in 293 cells. In SK-N-SH cells, the α-synuclein was coexpressed with wild type as well as mutated p.R767H, p.S885N, p.R1441H, and p.G2019 LRRK2 proteins. Part of the perinuclear inclusions formed by p.R1441H and p.G2019S were colocalized with α-synuclein. Additionally, p.S885N and p.R1441H mutations caused reduced interaction between LRRK2 and ARHGEF7, a putative guanine nucleotide exchange factor for LRRK2, whereas this interaction was well preserved in p.R767H and p.G2019S mutations. CONCLUSION: Our study suggests that p.R1441H protein facilitates the formation of intracellular inclusions, compromises GTP hydrolysis by increasing its affinity for GTP, and reduces its interaction with ARHGEF7.

Genetic variants ofLRRK2 in Taiwanese Parkinson's disease.

Genetic variants of leucine-rich repeat kinase 2 (LRRK2) were reported to alter the risk for Parkinson's disease (PD). However, the genetic spectrum of LRRK2 variants has not been clearly disclosed yet in Taiwanese population. Herein, we sequenced LRRK2 coding region in 70 Taiwanese early onset PD patients (age at onset ≤ 50), and found six amino acid-changing single nucleotide polymorphisms (SNPs, N551K, R1398H, R1628P, S1647T, G2385R and M2397T), one reported (R1441H) and 2 novel missense (R767H and S885N) mutations. We examined the frequency of identified LRRK2 variants by genotyping 573 Taiwanese patients with PD and 503 age-matched control subjects. The results showed that PD patients demonstrated a higher frequency of G2385R A allele (4.6%) than control subjects (2.1%; odds ratio = 2.27, 95% confidence interval: 1.38-3.88, P = 0.0017). Fewer PD patients (27.7%) carried the 1647T-2397T haplotype as compared with the control subjects (33.0%; odds ratio = 0.80, 95% confidence interval: 0.65-0.97, P = 0.0215). However, the frequency of 1647T-2385R-2397T haplotype (4.3%) in PD patients was still higher than in control subjects (1.9%, odds ratio: 2.15, 95% confidence interval: 1.27-3.78, P = 0.0058). While no additional subject was found to carry R767H and R1441H, one more patient was observed to carry the S885N variant. Our results indicate a robust risk association regarding G2385R and a new possible protective haplotype (1647T-2397T). Gene-environmental interaction and a larger cohort study are warranted to validate our findings. Additionally, two new missense mutations (R767H and S885N) regarding LRRK2 in PD patients were identified. Functional studies are needed to elucidate the effects of these LRRK2 variants on protein function.

Maternal and perinatal outcome in cases of fulminant viral hepatitis in late pregnancy.

OBJECTIVE: To investigate maternal and perinatal outcomes in cases of fulminant viral hepatitis in late pregnancy (FVHILP). METHODS: A multicenter retrospective study was conducted. The records of 40 patients with FVHILP were retrieved from 3 hospitals in China. To analyze the influence of mode of delivery on maternal and perinatal outcomes, women were allocated to the cesarean delivery group or the spontaneous vaginal delivery (SVD) group. To study the relationship between maternal outcome and perinatal outcome, patients were allocated to the maternal survival group or the non-survival group. RESULTS: There were no significant differences between the cesarean group and the SVD group in clinical manifestations or laboratory indices before delivery, or in fatality rate (P>0.05 for all), whereas there were significant differences in newborn weight, 1-minute Apgar score, and incidence of severe perinatal asphyxia between the maternal survival group and the non-survival group (P<0.05 for all). CONCLUSION: Maternal and perinatal outcomes in cases of FVHILP were not influenced by mode of delivery, whereas perinatal outcome significantly correlated with maternal outcome.

Analysis of prognosis-associated factors in fulminant viral hepatitis during pregnancy in China.

OBJECTIVE: To analyze factors contributing to the prognosis of patients with fulminant viral hepatitis in pregnancy (FVHP). METHODS: A retrospective study was conducted among 90 patients with FVHP who were admitted between January 1994 and August 2008 in 9 Chinese hospitals with expertise in the treatment of FVHP. RESULTS: Different clinical types of FVHP presented with different fatality rates; the acute type showed the highest fatality rate and the chronic type showed the lowest fatality rate. Serum albumin (Alb), total cholesterol (Tch), total bilirubin (Tbil), prothrombin activity (PTA), creatinine (Scr), white blood cell count (WBC), the phenomenon of "bilirubin-transaminase separation", and intractable complications correlated with the prognosis of FVHP. The fatality rate increased with decreasing Tch and PTA, increasing WBC, the appearance of "bilirubin-transaminase separation", and complications such as hepatic encephalopathy, hepatorenal syndrome, hemorrhage, and infection. The above indices were all significantly different between survivor and non-survivor groups. CONCLUSION: The clinical type of fulminant hepatitis, Alb, Tch, PTA, TBIL, Scr WBC, phenomenon of "bilirubin-transaminase separation", and intractable complications are important factors associated with prognosis of patients with FVHP. Dynamic monitoring of these indicators and active treatment of complications are key to the improvement of outcomes in patients with FVHP.

[Clinical diagnosis and treatment of acute fatty liver of pregnancy].

OBJECTIVE: To study the clinical diagnosis and treatment of acute fatty liver of pregnancy (AFLP). METHOD: The clinical data of 36 cases of AFLP were reviewed retrospectively, who were treated during Jan 1988- July 2007 in the Department of Obstetrics and Gynecology, Public Health Clinical Center Affiliated to Fu Dan University, Shanghai. RESULTS: All 36 cases were hospitalized in late pregnancy with various clinical symptoms and physical signs. Lab examinations were also performed. In the case of natural delivery, the rate of postpartum hemorrhage, and maternal mortality were 42%, and 50%, and the perinatal mortality and asphyxia in newborns were 50% and 58% respectively. In the case of cesarean section, the rate was 42%, 8%, and 13%, 38% respectively. The comprehensive therapies of termination of pregnancy by cesarean section plus medical supportive treatment can reduce the rate of postpartum hemorrhage, maternal mortality, perinatal mortality, and asphyxia in newborns significantly. Compared with the natural delivery, cesarean section had the higher rate of postpartum hemorrhage and the rate of asphyxia in newborns (P > 0.05), higher rate of maternal mortality and perinatal mortality (P < 0.05). All the death cases occurred in patients who only began to receive treatment 7 days after presentation of the symptoms. Compared with B ultrasonography, of which diagnosis rate of AFLP was 57%, CT had a higher diagnosis rate of 73% (P < 0.05). CONCLUSION: Early diagnosis, termination of pregnancy by cesarean section as soon as possible and a comprehensive therapy are the key to improve the mother and newborn's prognosis.