Adipose tissue-derived stromal/stem cells + cholecalciferol: a pilot study in recent-onset type 1 diabetes patients

ABSTRACT Objective: Adipose tissue-derived stromal/stem cells (ASCs) and vitamin D have immunomodulatory actions that could be useful for type 1 diabetes (T1D). We aimed in this study to investigate the safety and efficacy of ASCs + daily cholecalciferol (VIT D) for 6 months in patients with recent-onset T1D. Materials and methods: In this prospective, dual-center, open trial, patients with recent onset T1D received one dose of allogenic ASC (1 x 106 cells/kg) and cholecalciferol 2,000 UI/day for 6 months (group 1). They were compared to patients who received chol-ecalciferol (group 2) and standard treatment (group 3). Adverse events were recorded; C-peptide (CP), insulin dose and HbA1c were measured at baseline (T0), after 3 (T3) and 6 months (T6). Results: In group 1 (n = 7), adverse events included transient headache (all), mild local reactions (all), tachycardia (n = 4), abdominal cramps (n = 1), thrombophlebitis (n = 4), scotomas (n = 2), and central retinal vein occlusion at T3 (n = 1, resolution at T6). Group 1 had an increase in basal CP (p = 0.018; mean: 40.41+/-40.79 %), without changes in stimulated CP after mixed meal (p = 0.62), from T0 to T6. Basal CP remained stable in groups 2 and 3 (p = 0.58 and p = 0.116, respectively). Group 1 had small insulin requirements (0.31+/- 0.26 UI/kg) without changes at T6 (p = 0.44) and HbA1c decline (p = 0.01). At T6, all patients (100%; n = 7) in group 1 were in honeymoon vs 75% (n = 3/4) and 50% (n = 3/6) in groups 2 and 3, p = 0.01. Conclusions: Allogenic ASC + VIT D without immunosuppression was safe and might have a role in the preservation of β-cells in patients with recent-onset T1D. ClinicalTrials.gov: NCT03920397.

Long non-coding RNA potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 regulates the proliferation and osteogenic differentiation of human periodontal ligament stem cells by targeting miR-24-3p / 华西口腔医学杂志

OBJECTIVES@#This study aims to explore the effect and molecular mechanism of long non-coding RNA (lncRNA) potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (KCNQ1OT1) on proliferation and osteogenic differentiation in human periodontal ligament stem cells (hPDLSCs).@*METHODS@#The hPDLSCs of normal periodontal tissues were isolated and cultured. The mineralized solution induced the osteoblast differentiation of hPDLSCs. The down-regulation of lncRNA KCNQ1OT1, the overexpression of anti-miR-24-3p on the proliferation and the levels of osteocalcin (OCN), osteopontin (OPN) and alkaline phosphatase (ALP) of hPDLSCs were investigated. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the levels of lncRNA KCNQ1OT1, miR-24-3p, OCN, OPN, and ALP. Methyl thiazolyl tetrazolium (MTT) method was used to detect cell viability and activity. Cell proliferation was evaluated by MTT. Western blot was used to detect protein expression. The targeted relationship between lncRNA KCNQ1OT1 and miR-24-3p was detected by double-luciferase experiment.@*RESULTS@#The expression level of lncRNA KCNQ1OT1 increased, and that of miR-24-3p decreased during the osteogenesis of hPDLSCs (@*CONCLUSIONS@#Down-regulation of lncRNA KCNQ1OT1 inhibited the proliferation and osteogenic differentiation of hPDLSCs by targeting the up-regulated expression of miR-24-3p.

Phenotype changes of oral epithelial stem cells after in vitro culture

Abstract The aim of our study was to isolate populations of keratinocyte stem cells based on the expression of cell surface markers and to investigate whether the culture could affect their phenotype. keratinocytes from human oral mucosa were sorted based on the expression of the epithelial stem cell markers p75NTR and CD71. We also examined the co-expression of other epithelial stem markers such as integrins β1 and α6 and their stem cell-like proprieties in in vitro assays. Three passages after being sorted by MACS, more than 93% of the p75NTR+ve cells lost the expression of p75NTR, while 5.46% of the p75NTR-ve gained it. Within the small population of the p75NTR+ve cells, 88% co-expressed other epithelial stem cell markers such as integrins β1 and α6, while only 28% of p75NTR-ve cells co-expressed these markers. These results were confirmed by sorting cells by FACS. Additionally, when double staining was used for sorting cells, 99% of the p75NTR+veCD71-ve and 33% of the p75NTR-veCD71+ve cells expressed both integrins, but just one week after culture, only 1.74% of the p75NTR+veCD71-ve cells still expressed p75NTR and only 0.32% still expressed CD71. Similar results were obtained when co-culturing p75NTR+ve and p75NTR-ve populations before analysis. Our results suggest that phenotype changes may be part of an intrinsic cellular mechanism to conserve levels of protein expression as they may found in the human body. In addition, in vitro culture may not offer ideal conditions for epithelial stem cell maintenance due to phenotype changes under standard culture conditions.

Characteristics of human natal stem cells cultured in allogeneic medium

Abstract Recently, human natal dental pulp stem cells (hNDP-SCs) have been characterized in vitro and it has been shown that they satisfy criteria defining human mesenchymal stromal cells (MSCs), as proposed by the International Society for Cellular Therapy. However, these results were reached in the presence of xenogeneic expansion medium, which has the potential to alter the cells' functional capacity. To determine the validity of the previously reported hNDP-SCs characteristics for human cell therapy, we have cultured hNDP-SCs in allogeneic expansion medium. Two hNDP-SC lineages were isolated from vital natal teeth, donated by a healthy newborn female and cultured in 2% platelet rich plasma (PRP). Analysis of the phenotypic expressions, proliferation rates, viability, telomerase length and in vitro adipogenic, osteogenic and chondrogenic differentiation potentials of two hNDP-SCs lineages (Zn001 and Zn002) were performed. Both lineages displayed similar morphology, proliferation rates, adipogenic, chondrogenic and osteogenic differentiation potential. Telomere shortening by 41.0% and 13.49% occurred from 3rd till 14th passage for lineages Zn001 and Zn002 respectively. Viability of both lineages was higher than 90%. Flow cytometry demonstrated that both lineages were positive to the majority of tested markers, including markers, which were negatively, expressed when hNDP-SCs were cultured previously in xenogeneic medium. Using immune-cytochemistry the cells were shown to express beta III-tubulin, nestin, neurofilaments and Nanog. PRP used as allogeneic medium is suitable for cultivation of hNDP-SCs.

Resumo Recentemente, células-tronco da polpa dental humana (hNDP-SCs) foram caracterizadas in vitro e foi demonstrado que elas satisfazem critérios que definem células mesenquimais do estroma humana (MSCs), tal como proposto pela Sociedade Internacional para Terapia Celular. No entanto, esses resultados foram alcançados na presença de meio de expansão xenogênico, que tem o potencial de alterar a capacidade funcional das células. Para determinar a validade das características das hNDP-SCs anteriormente relatadas para a terapia celular humana, cultivamos hNDP-SCs em meio de expansão alogênico. Duas linhagens hNDP-SC foram isoladas de dentes natais vitais, doadas por uma recém-nascida saudável, e cultivadas em plasma rico em plaquetas a 2% (PRP). Análises das expressões fenotípicas, taxas de proliferação, viabilidade, comprimento de telomerase e potenciais de diferenciação adipogênica, osteogênica e condrogênica in vitro das duas linhagens hNDP-SC (Zn001 e Zn002) foram realizadas. Ambas as linhagens apresentaram morfologia, taxas de proliferação, potencial de diferenciação adipogênico, condrogênico e osteogênico semelhantes. O encurtamento dos telômeros em 41,0% e 13,49% ocorreu da 3ª até a 14ª passagem para as linhagens Zn001 e Zn002, respectivamente. A viabilidade de ambas as linhagens foi superior a 90%. A citometria de fluxo demonstrou que ambas as linhagens foram positivas para a maioria dos marcadores testados, incluindo marcadores, que foram negativamente expressados quando hNDP-SCs foram previamente cultivadas em meio xenogênico. Usando análise imunocitoquímica, as células mostraram expressar a beta III-tubulina, nestina, neurofilamentos e Nanog. O PRP usado como meio alogênico mostrou-se adequado para o cultivo de hNDP-SCs.

Human dental pulp stem cell adhesion and detachment in polycaprolactone electrospun scaffolds under direct perfusion

Cell adhesion in three-dimensional scaffolds plays a key role in tissue development. However, stem cell behavior in electrospun scaffolds under perfusion is not fully understood. Thus, an investigation was made on the effect of flow rate and shear stress, adhesion time, and seeding density under direct perfusion in polycaprolactone electrospun scaffolds on human dental pulp stem cell detachment. Polycaprolactone scaffolds were electrospun using a solvent mixture of chloroform and methanol. The viable cell number was determined at each tested condition. Cell morphology was analyzed by confocal microscopy after various incubation times for static cell adhesion with a high seeding density. Scanning electron microscopy images were obtained before and after perfusion for the highest flow rate tested. The wall pore shear stress was calculated for all tested flow rates (0.005-3 mL/min). An inversely proportional relationship between adhesion time with cell detachment under perfusion was observed. Lower flow rates and lower seeding densities reduced the drag of cells by shear stress. However, there was an operational limit for the lowest flow rate that can be used without compromising cell viability, indicating that a flow rate of 0.05 mL/min might be more suitable for the tested cell culture in electrospun scaffolds under direct perfusion.

Células madre: buscando marcadores de superficie celular que predispongan compromiso de diferenciación cardiaca / Stem cells: searching predisposition to cardiac commitment by surface markers expression

Resumen Actualmente las enfermedades cardiovasculares se han convertido en un serio problema para los sistemas de salud de todo el mundo, ya que son la principal causa de muerte y representan una enorme carga económica. Este problema ha sido abordado con diferentes estrategias, entre ellas con la ayuda de terapia celular, aunque sin resultados contundentes. Durante más de 20 años, se ha utilizado una gran variedad de células madre en diferentes modelos de infarto del miocardio. El uso de células madre cardiacas (CSC) parece ser la mejor opción, pero la inaccesibilidad y la escasez de estas células hacen que su uso sea muy limitado. Además, existe un riesgo elevado pues tienen que obtenerse directamente del corazón del paciente. A diferencia de las CSC, las células madre adultas derivadas de médula ósea o tejido adiposo, entre otras, representan una opción atractiva debido a su fácil accesibilidad y abundancia, pero sobre todo a la probable existencia de progenitores cardiacos entre sus diferentes subpoblaciones. En esta revisión hacemos un análisis de los marcadores de superficie presentes en CSC en comparación con otras células madre adultas, y sugerimos la preexistencia de células que comparten marcadores de superficie específicos con CSC, la presencia de un inmunofenotipo predecible, aunque en proporciones bajas, pero con un potencial de diferenciación cardiaca similar a las CSC, lo cual podría aumentar su valor terapéutico. Este estudio revela las nuevas perspectivas con respecto a la presencia de dichos marcadores, los cuales comprometerían algunas de estas subpoblaciones a diferenciarse a tejido cardiaco.

Abstract It is well-known that cardiovascular diseases are the leading cause of death world- wide, and represent an important economic burden to health systems. In an attempt to solve this problem, stem cell therapy has emerged as a therapeutic option. Within the last 20 years, a great variety of stem cells have been used in different myocardial infarction models. Up until now, the use of cardiac stem cells (CSCs) has seemed to be the best option, but the inaccessibility and scarcity of these cells make their use unreliable. Additionally, there is a high risk as they have to be obtained directly from the heart of the patient. Unlike CSCs, adult stem cells originating from bone marrow or adipose tissue, among others, appear to be an attractive option due to their easier accessibility and abundance, but particularly due to the probable existence of cardiac progenitors among their different sub-populations. In this review an analysis is made of the surface markers present in CSCs compared with other adult stem cells. This suggested the pre-existence of cells sharing specific surface markers with CSCs, a predictable immunophenotype present in some cells, although in low proportions, and with a potential of cardiac differentiation that could be similar to CSCs, thus increasing their therapeutic value. This study highlights new perspectives regarding MSCs that would enable some of these sub-populations to be differentiated at cardiac tissue level.

Evidence of progenitor cells in the adult human cochlea: sphere formation and identification of ABCG2

OBJECTIVES: The aim of this study was to search for evidence of stem or progenitor cells in the adult human cochlea by testing for sphere formation capacity and the presence of the stem cell marker ABCG2. METHODS: Cochleas removed from patients undergoing vestibular schwannoma resection (n=2) and from brain-dead organ donors (n=4) were dissociated for either flow cytometry analysis for the stem cell marker ABCG2 or a sphere formation assay that is widely used to test the sphere-forming capacity of cells from mouse inner ear tissue. RESULTS: Spheres were identified after 2-5 days in vitro, and the stem cell marker ABCG2 was detected using flow cytometric analysis after cochlear dissociation. CONCLUSIONS: Evidence suggests that there may be progenitor cells in the adult human cochlea, although further studies are required.

Aldefluor protocol to sort keratinocytes stem cells from skin

Abstract Purpose: To investigate the use Aldefluor® and N, N - Dimethylaminobenzaldehyde (DEAB) to design a protocol to sort keratinocyte stem cells from cultured keratinocytes from burned patients. Methods: Activated Aldefluor® aliquots were prepared and maintained at temperature between 2 to 8°C, or stored at -20°C. Next, the cells were collected following the standard protocol of sample preparation. Results: Best results were obtained with Aldefluor® 1.5µl and DEAB 15 µl for 1 x 106 cells, incubated at 37°C for 15 minutes. Flow cytometer range for keratinocyte stem cells separation was evaluated. There were 14.8% of stem cells separated in one sample of keratinocyte culture used to pattern the protocol. After being defined the ideal concentration, the same test pattern was performed in other keratinocyte samples. We observed a final mean of 10.8%. Conclusion: Aldefluor® has been shown as a favorable marking of epidermal keratinocyte stem cells for subsequent separation on a flow cytometer, with detection of 10.8% of epidermal keratinocyte stem cells, in this protocol.

Low-level laser irradiation promotes proliferation of cryopreserved adipose-derived stem cells / Laser de baixa intensidade promove proliferação de células-tronco derivadas de tecido adiposo criopreservadas

ABSTRACT Objective To evaluate the effect of low-level laser irradiation on proliferation and viability of murine adipose-derived stem cells previously submitted to cryopreservation. Methods Adipose-derived stem cells were isolated from inguinal fat pads of three mice, submitted to cryopreservation in fetal bovine serum with 10% dimethylsulfoxide for 30 days and then thawed and maintained in normal culture conditions. Culture cells were either irradiated or not (control) with an InGaAIP diode laser at zero and 48 hours, using two different energy densities (0.5 and 1.0J/cm2). Cell proliferation was evaluated by trypan blue exclusion method and MTT assay at intervals of zero, 24, 48, and 72 hours after the first laser application. Cell viability and apoptosis of previously cryopreserved cells submitted to laser therapy were evaluated by flow cytometry. Results The Irradiated Groups (0.5 and 1.0J/cm2) showed an increased cell proliferation (p<0.05) when compared to the Control Group, however no significant difference between the two energy densities was observed. Flow cytometry revealed a percentage of viable cells higher than 99% in all groups. Conclusion Low-level laser irradiation has stimulatory effects on the proliferation of adipose-derived stem cells previously submitted to cryopreservation.

RESUMO Objetivo Avaliar o efeito do laser de baixa intensidade na proliferação e na viabilidade de células-tronco derivadas de tecido adiposo murinas previamente submetidas à criopreservação. Métodos Células-tronco derivadas de tecido adiposo foram isoladas da região inguinal de três camundongos, submetidas à criopreservação em soro fetal bovino com 10% de dimetilsulfóxido por 30 dias e, depois, descongeladas e mantidas em condições normais de cultivo. As células cultivadas foram irradiadas ou não (controle) com um laser de diodo InGaAIP nos intervalos de zero e 48 horas, utilizando duas densidades de energia diferentes (0,5 e 1,0J/cm2). A proliferação celular foi avaliada pelo método de exclusão de azul de tripan e ensaio MTT, nos intervalos de zero, 24, 48 e 72 horas após a primeira aplicação do laser. A viabilidade celular e a apoptose das células previamente criopreservadas submetidas à laserterapia foram avaliadas por citometria de fluxo. Resultados Os Grupos Irradiados (0,5 e 1,0J/cm2) apresentaram aumento da proliferação celular (p<0,05) quando comparados ao Grupos Controle, porém não foi observada diferença significativa entre as duas densidades de energia. A citometria de fluxo revelou percentagem de células viáveis superior a 99% em todos os grupos. Conclusão O laser de baixa intensidade tem efeitos estimuladores sobre a proliferação de células-tronco derivadas de tecido adiposo previamente submetidas à criopreservação.

How has dental pulp stem cells isolation been conducted? A scoping review

Abstract: The objective of this study was to realize a scoping review the literature in order to identify the profile of DPSCs isolation and analyze the possible risk factors that could change the native behavior of these cells. An initial search was conducted using the following MeSH terms: "(dental pulp stem cell [MeSH])"; "(dental pulp [MeSH])" AND "(stem cell [MeSH])"; "("dental pulp stem cell" [MeSH]")". The electronic search was done without date restriction up to and including April 2014, in PubMed, Scopus, Scielo and ISI Web of Knowledge databases. Studies were submitted to inclusion and exclusion criteria and 222 articles were included. Data showed that over the past 15 years many studies have been conducted using DPSCs. However this is the first systematic review regarding the isolation of stem cell, and more specifically of dental pulp stem cells. The isolation of dental pulp stem cells showed great variability, hampering the development of standard protocols to achieve in vitro dental pulp stem cells with similar characteristics. This scoping review combined, for the first time, the methodologies used for dental pulp stem isolation, highlighting the most frequently used.

Medium modification with bone morphogenetic protein 2 addition for odontogenic differentiation

Abstract The aim of this study was to evaluate whether medium modification improves the odontogenic differentiation of human dental pulp stem cells (DPSC) in vitro and in vivo. DPSC isolated from human impacted third molar teeth were analysed for clusters of differentiation with flow cytometry. Odontogenic differentiation was stimulated by medium modification with the addition of bone morphogenetic protein 2 (BMP2). The expression of dentin sialophosphoprotein, dentin matrix protein 1, enamelysin/matrix metalloproteinase 20 and the phosphate-regulating gene with homologies to endopeptidases on the X chromosome of the cells were analysed with RT-PCR at 7, 14 and 21 days. Then, DPSC were transplanted on the back of immunocompromised mice via a hydroxyapatite tricalcium phosphate scaffold, and the structure of the formed tissue was investigated. The cells were identified as mesenchymal stem cells with a 98.3% CD73 and CD90 double-positive cell rate. The increase in mineralization capacity and expression of human enamel-dentin specific transcripts proportional to the culture period were determined after differentiation. Six weeks after transplantation, an osteo-dentin matrix was formed in the group in which odontogenic differentiation was stimulated, and the odontogenic characteristics of the matrix were confirmed by histological examination and RT-PCR analysis. Odontogenic differentiation of the isolated and characterized human DPSC was improved with medium modification by the addition of BMP2 in vitro and in vivo. The defined medium and applied technique have a potential use for forming reparative dentin in the future, but the effects of the method should be investigated in long-term studies.

Differentiation of Human Dental Pulp Stem Cells into Dopaminergic Neuron-like Cells in Vitro

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.

Differentiation of Human Dental Pulp Stem Cells into Dopaminergic Neuron-like Cells in Vitro

We investigated the potential of human dental pulp stem cells (hDPSCs) to differentiate into dopaminergic neurons in vitro as an autologous stem cell source for Parkinson's disease treatment. The hDPSCs were expanded in knockout-embryonic stem cell (KO-ES) medium containing leukemia inhibitory factor (LIF) on gelatin-coated plates for 3-4 days. Then, the medium was replaced with KO-ES medium without LIF to allow the formation of the neurosphere for 4 days. The neurosphere was transferred into ITS medium, containing ITS (human insulin-transferrin-sodium) and fibronectin, to select for Nestin-positive cells for 6-8 days. The cells were then cultured in N-2 medium containing basic fibroblast growth factor (FGF), FGF-8b, sonic hedgehog-N, and ascorbic acid on poly-l-ornithine/fibronectin-coated plates to expand the Nestin-positive cells for up to 2 weeks. Finally, the cells were transferred into N-2/ascorbic acid medium to allow for their differentiation into dopaminergic neurons for 10-15 days. The differentiation stages were confirmed by morphological, immunocytochemical, flow cytometric, real-time PCR, and ELISA analyses. The expressions of mesenchymal stem cell markers were observed at the early stages. The expressions of early neuronal markers were maintained throughout the differentiation stages. The mature neural markers showed increased expression from stage 3 onwards. The percentage of cells positive for tyrosine hydroxylase was 14.49%, and the amount was 0.526 ± 0.033 ng/mL at the last stage. hDPSCs can differentiate into dopaminergic neural cells under experimental cell differentiation conditions, showing potential as an autologous cell source for the treatment of Parkinson's disease.

Small activating RNA induces myogenic differentiation of rat adipose-derived stem cells by upregulating MyoD

ABSTRACTPurpose:RNA activation (RNAa) is a mechanism of gene activation triggered by promoter-targeted small double stranded RNAs (dsRNAs), also known as small activating RNAs (saRNAs). Myogenic regulatory factor MyoD is regarded as the master activator of myogenic differentiation cascade by binding to enhancer of muscle specific genes. Stress urinary incontinence (SUI) is a condition primarily resulted from urethral sphincter deficiency. It is thus expected that by promoting differentiation of adipose-derived stem cells (ADSCs) into myoblasts by activating MyoD gene through RNAa may offer benefits to SUI.Materials and Methods:Rats ADSCs were isolated, proliferated in vitro, and identified by flow cytometry. Purified ADSCs were then transfected with a MyoD saRNA or control transfected. Real-time polymerase chain reaction (RT-PCR) and western blotting were used to detect MyoD mRNA and protein expression, respectively. Immunocytochemical staining was applied to determine the expression of desmin protein in transfected cells. Cell viability was measured by using CellTiter 96® AQueous One Solution Cell Proliferation Assay kit.Results:Transfection of a MyoD saRNA (dsMyoD) into ADSCs significantly induced the expression of MyoD at both the mRNA and protein levels, and inhibited cell proliferation. Desmin protein expression was detected in dsMyoD treated ADSCs 2 weeks later.Conclusion:Our findings show that RNAa mediated overexpression of MyoD can promote transdifferentiation of ADSCs into myoblasts and may help treat stress urinary incontinence (SUI)–a condition primarily resulted from urethral sphincter deficiency.

Characterization of victims of aggression and transportation accidents treated at the Forensic Medicine and Dentistry Institute - Campina Grande, Paraíba, Brazil - 2010 / Caracterização de vítimas de agressão e de acidentes de transporte atendidas no Instituto de Medicina e Odontologia Forense - Campina Grande, Paraíba, Brasil - 2010

The objective of this cross-sectional census study was to characterize agression and land-based transport accidents in a city in the Northeast of Brazil. Data was analyzed from live victims who were treated at a forensic service (N = 2.379). In the descriptive analysis, the majority of events were represented by aggression (71.6%); which occurred on weekdays (65%), with 35.1% at night. Trauma occurred to the whole body (63.6%) and to soft tissue (74.2%). On the basis of multiple correspondence analysis, two dimensions were formed: the first dimension (internal reliability = 0.654) was formed by the cause of the event, the trauma and the age group and the second dimension (reliability = 0.514), by age group, occupation and civil status. Three groups with distinct profiles were formed for accidents and aggression: young women who suffered aggression, with trauma to the face and soft tissues during the evening and at weekends; adult men who suffered car accidents, in the morning and on work days; and retired elderly widowers, who were run over.

O objetivo deste estudo tranversal censitário foi caracterizar a agressão e os acidentes de transporte terrestre em uma cidade do nordeste do Brasil. Foram analisados os dados de vítimas vivas que foram atendidas em um serviço forense (N = 2.379). Na análise descritiva, a maioria dos eventos foi a agressão (71,6%); que ocorreu nos dias úteis (65%), sendo 35,1% no período noturno. Os traumas ocorreram no corpo todo (63,6%) e envolveram o tecido mole (74,2%). A partir da análise de correspondência múltipla formaram-se duas dimensões: a primeira dimensão (confiabilidade interna = 0,654) foi formada pela causa do evento, o trauma e a faixa etária e, a segunda dimensão (confiabilidade interna = 0,514), pela faixa etária, a ocupação e o estado civil. Formaram-se três grupos com perfis distintos para os acidentes e agressão. Mulheres jovens que sofreram agressões com traumas faciais, em tecidos moles, no período da tarde e durante os finais de semana. Homens, adultos que sofreram acidentes automobilísticos, pela manhã e em dias úteis, e idosos, viú vos, aposentados e que sofreram atropelamento. Há um elevado número de vítimas de agressão interpessoal, seguido por acidentes de moto e os tipos de acidentes estão associados a grupos populacionais.

Stem Cell Therapy in Dentistry: An Overview.

Cells with a unique capacity for self-renewal and potency are called stem cells. With appropriate biochemical signals stem, cells can be transformed into desirable cells. Regeneration of oral and maxillofacial structures is earned out by using stem cell therapy, and this has gained momentum in recent days. Future tissues like tissue engineered bone grafts, engineered joints and cranial sutures can be developed with stem cell therapy. We have described the properties, types and advantages of dental stem cells. Emphasis is been given to the possibilities of stem cell therapy in the oral and maxillofacial region including regeneration of tooth and craniofacial defects.

Diferenciação odonto/osteogênica de células-tronco mesenquimais fotomoduladas em hidrogel com incorporação de proteína morfogenética óssea 4 / Odonto/osteogenic differentiation of photomodulated mesenchymal stem cells in BMP4-loaded hydrogel

Este estudo avaliou a influência da fototerapia a laser (FTL) na proliferação e diferenciação de células-tronco da polpa dentária humana (DPSCs; do inglês, Dental Pulp Stem Cells ) encapsuladas em carreador injetável e termoresponsivo (PL; Pluronic® F-127, Sigma-Aldrich, MO, EUA) com incorporação de proteína morfogenética óssea 4 recombinante humana (rhBMP4) (sistema PL/rhBMP4). O biomaterial foi caracterizado de acordo com seus perfis de embebição e dissolução, liberação de rhBMP4 e sua estrutura morfológica. DPSCs foram isoladas, caracterizadas e encapsuladas em PL para confirmar sua viabilidade e seu potencial de diferenciação (adipo e osteogênico) em comparação com células-tronco mesenquimais de medula óssea (BMMSCs; do inglês, Bone Marrow Mesenchymal Stem Cells). Quando encapsuladas no sistema PL/rhBMP4, DPSCs foram irradiadas com duas densidades de energia diferentes utilizando laser de diodo de fosfeto de índio-gálio-alumínio (InGaAlP), modos contínuo, pontual e em contato [660 nm, 0,028 cm2, 20 mW, 0,71 W/cm2, 3 J/cm2 (4 s) ou 5 J/cm2 (7 s)]. Os ensaios de PKH26 (do inglês, Red Fluorescent Cell Linker), CFU-F (do inglês, Coloning Forming Units - Fibroblastic), e MTT (do inglês, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide)) foram utilizados para avaliar adesão/proliferação, diferenças na capacidade formadora de colônias e viabilidade das DPSCs (neste último caso sob estresse nutricional), respectivamente. Finalmente, a diferenciação odonto/osteogênica foi analisada por qRT-PCR e confirmada por ensaio de vermelho de alizarina. O biomaterial embebeu e dissolveu rapidamente; densa rede tubular e reticular com poros interconectados foi observada.

DPSCs e BMMSCs apresentaram alta viabilidade celular quando encapsuladas em PL. Ambas as linhagens celulares tiveram êxito em se diferenciar em tecidos adiposo e ósseo. De acordo com o PKH26, DPSCs puderam aderir e proliferar no sistema PL/rhBMP4. DPSCs irradiadas encapsuladas tanto em PL como em PL/rhBMP4 formaram mais CFU-F que os controles não irradiados. Sob estresse nutricional, DPSCs semeadas no PL e irradiadas com 5 J/cm2 exibiram maior taxa de viabilidade celular em relação aos grupos não irradiados e irradiados com 3 J/cm2. Na presença de rhBMP4, os grupos irradiados tanto com 3 J/cm2 quanto com 5 J/cm2 apresentaram deposição mineral precoce quando comparados aos grupos não irradiados. Ainda, após 21 dias de diferenciação odonto/osteogênica, DPSCs irradiadas produziram maior quantidade de nódulos mineralizados. A irradiação com 5 J/cm2 levou ao aumento significativo da expressão de genes envolvidos na diferenciação odonto/osteogênica, como colágeno tipo I (COL1A1), osteocalcina (OCN), proteína da matriz dentinária 1 (DMP1), sialofosfoproteina dentinária (DSPP) e proteína heat shock 27 kDa (HSPB1). A associação entre rhBMP4 e FTL promove proliferação e diferenciação odonto/osteogênica de DPSCs acelerando e aumentando notavelmente a formação de tecido mineralizado, em especial quando a densidade de energia de 5 J/cm2 é aplicada.

This study evaluated the influence of laser phototherapy (LPT) on dental pulp stem cells (DPSCs) proliferation and differentiation upon encapsulation in an injectable and thermo-responsive cell carrier (PL; Pluronic® F-127, Sigma-Aldrich, MO, USA) loaded with human recombinant bone morphogenetic protein 4 (rhBMP4)(PL/rhBMP4 system). The biomaterial was characterized according to its swelling and dissolution profiles, release of rhBMP4 and morphological structure. DPSCs were isolated, characterized and encapsulated in PL to confirm their viability and multilineage differentiation potential (adipo and osteogenic) in comparison to bone marrow mesenchymal stem cells (BMMSCs). When encapsulated in the PL/rhBMP4 system, DPSCs were irradiated with two different energy densities using a continuous-wave indium-gallium-aluminum-phosphide (InGaAlP) diode laser [660 nm, 0.028 cm2, 20 mW, 0.71 W/cm2, 3 J/cm2 (4 s) or 5 J/cm2 (7 s)] in punctual and contact modes. The PKH26 (Red Fluorescent Cell Linker), the CFU-F (Coloning Forming Units - Fibroblastic), and the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assays were used to assess differences in cell adhesion/proliferation, colony forming units formation ability, and cell viability of DPSCs (in this case under nutritional stress), respectively. Then, alizarin red and qRT-PCR analyzes were used to evaluate odonto/osteogenic differentiation. The biomaterial swelled and dissolved rapidly; dense tubular and reticular network morphology with well-interconnected pores was observed. DPSCs and BMMSCs presented high cell viability when encapsulated in PL. Both cell lineages successfully differentiated into bone or adipose tissues. According to PKH26,

DPSCs were able to adhere and proliferate in the PL/rhBMP4 system. Irradiated DPSCs encapsulated in either PL or PL/rhBMP4 system formed more CFU-F than non-irradiated controls. Under nutritional stress, DPSCs encapsulated in the hydrogels with no rhBMP4 and irradiated at 5 J/cm2 exhibited higher cell viability than the other groups. In the presence of rhBMP4, the groups irradiated both at 3 and 5 J/cm2 energy densities displayed earlier mineral deposition than the non-irradiated groups. Moreover, after 21 days of odonto/osteogenic differentiation, irradiated DPSCs produced greater nodule formation than the control groups. At the energy density of 5 J/cm2, there were significant upregulation of genes involved in odonto/osteoblast differentiation, such as type I collagen (COL1A1), osteocalcin (OCN), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP) and heat shock protein 27 kDa (HSPB1). The association between rhBMP4 and LPT promotes cell proliferation and odonto/osteogenic differentiation of DPSCs accelerating and increasing the formation of mineralized tissue, in particular when the energy density of 5 J/cm2 is applied.

Diferenciação odonto/osteogênica de células-tronco mesenquimais fotomoduladas em hidrogel com incorporação de proteína morfogenética óssea 4 / Odonto/osteogenic differentiation of photomodulated mesenchymal stem cells in BMP4-loaded hydrogel

Este estudo avaliou a influência da fototerapia a laser (FTL) na proliferação e diferenciação de células-tronco da polpa dentária humana (DPSCs; do inglês, Dental Pulp Stem Cells ) encapsuladas em carreador injetável e termoresponsivo (PL; Pluronic® F-127, Sigma-Aldrich, MO, EUA) com incorporação de proteína morfogenética óssea 4 recombinante humana (rhBMP4) (sistema PL/rhBMP4). O biomaterial foi caracterizado de acordo com seus perfis de embebição e dissolução, liberação de rhBMP4 e sua estrutura morfológica. DPSCs foram isoladas, caracterizadas e encapsuladas em PL para confirmar sua viabilidade e seu potencial de diferenciação (adipo e osteogênico) em comparação com células-tronco mesenquimais de medula óssea (BMMSCs; do inglês, Bone Marrow Mesenchymal Stem Cells). Quando encapsuladas no sistema PL/rhBMP4, DPSCs foram irradiadas com duas densidades de energia diferentes utilizando laser de diodo de fosfeto de índio-gálio-alumínio (InGaAlP), modos contínuo, pontual e em contato [660 nm, 0,028 cm2, 20 mW, 0,71 W/cm2, 3 J/cm2 (4 s) ou 5 J/cm2 (7 s)]. Os ensaios de PKH26 (do inglês, Red Fluorescent Cell Linker), CFU-F (do inglês, Coloning Forming Units - Fibroblastic), e MTT (do inglês, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide)) foram utilizados para avaliar adesão/proliferação, diferenças na capacidade formadora de colônias e viabilidade das DPSCs (neste último caso sob estresse nutricional), respectivamente. Finalmente, a diferenciação odonto/osteogênica foi analisada por qRT-PCR e confirmada por ensaio de vermelho de alizarina. O biomaterial embebeu e dissolveu rapidamente; densa rede tubular e reticular com poros interconectados foi observada. DPSCs e BMMSCs apresentaram alta viabilidade celular quando encapsuladas em PL. Ambas as linhagens celulares tiveram êxito em se diferenciar em tecidos adiposo e ósseo. De acordo com o PKH26, DPSCs puderam aderir e proliferar no sistema PL/rhBMP4. DPSCs irradiadas encapsuladas tanto em PL como em PL/rhBMP4 formaram mais CFU-F que os controles não irradiados. Sob estresse nutricional, DPSCs semeadas no PL e irradiadas com 5 J/cm2 exibiram maior taxa de viabilidade celular em relação aos grupos não irradiados e irradiados com 3 J/cm2. Na presença de rhBMP4, os grupos irradiados tanto com 3 J/cm2 quanto com 5 J/cm2 apresentaram deposição mineral precoce quando comparados aos grupos não irradiados. Ainda, após 21 dias de diferenciação odonto/osteogênica, DPSCs irradiadas produziram maior quantidade de nódulos mineralizados. A irradiação com 5 J/cm2 levou ao aumento significativo da expressão de genes envolvidos na diferenciação odonto/osteogênica, como colágeno tipo I (COL1A1), osteocalcina (OCN), proteína da matriz dentinária 1 (DMP1), sialofosfoproteina dentinária (DSPP) e proteína heat shock 27 kDa (HSPB1). A associação entre rhBMP4 e FTL promove proliferação e diferenciação odonto/osteogênica de DPSCs acelerando e aumentando notavelmente a formação de tecido mineralizado, em especial quando a densidade de energia de 5 J/cm2 é aplicada.

This study evaluated the influence of laser phototherapy (LPT) on dental pulp stem cells (DPSCs) proliferation and differentiation upon encapsulation in an injectable and thermo-responsive cell carrier (PL; Pluronic® F-127, Sigma-Aldrich, MO, USA) loaded with human recombinant bone morphogenetic protein 4 (rhBMP4)(PL/rhBMP4 system). The biomaterial was characterized according to its swelling and dissolution profiles, release of rhBMP4 and morphological structure. DPSCs were isolated, characterized and encapsulated in PL to confirm their viability and multilineage differentiation potential (adipo and osteogenic) in comparison to bone marrow mesenchymal stem cells (BMMSCs). When encapsulated in the PL/rhBMP4 system, DPSCs were irradiated with two different energy densities using a continuous-wave indium-gallium-aluminum-phosphide (InGaAlP) diode laser [660 nm, 0.028 cm2, 20 mW, 0.71 W/cm2, 3 J/cm2 (4 s) or 5 J/cm2 (7 s)] in punctual and contact modes. The PKH26 (Red Fluorescent Cell Linker), the CFU-F (Coloning Forming Units - Fibroblastic), and the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] assays were used to assess differences in cell adhesion/proliferation, colony forming units formation ability, and cell viability of DPSCs (in this case under nutritional stress), respectively. Then, alizarin red and qRT-PCR analyzes were used to evaluate odonto/osteogenic differentiation. The biomaterial swelled and dissolved rapidly; dense tubular and reticular network morphology with well-interconnected pores was observed. DPSCs and BMMSCs presented high cell viability when encapsulated in PL. Both cell lineages successfully differentiated into bone or adipose tissues. According to PKH26, DPSCs were able to adhere and proliferate in the PL/rhBMP4 system. Irradiated DPSCs encapsulated in either PL or PL/rhBMP4 system formed more CFU-F than non-irradiated controls. Under nutritional stress, DPSCs encapsulated in the hydrogels with no rhBMP4 and irradiated at 5 J/cm2 exhibited higher cell viability than the other groups. In the presence of rhBMP4, the groups irradiated both at 3 and 5 J/cm2 energy densities displayed earlier mineral deposition than the non-irradiated groups. Moreover, after 21 days of odonto/osteogenic differentiation, irradiated DPSCs produced greater nodule formation than the control groups. At the energy density of 5 J/cm2, there were significant upregulation of genes involved in odonto/osteoblast differentiation, such as type I collagen (COL1A1), osteocalcin (OCN), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP) and heat shock protein 27 kDa (HSPB1). The association between rhBMP4 and LPT promotes cell proliferation and odonto/osteogenic differentiation of DPSCs accelerating and increasing the formation of mineralized tissue, in particular when the energy density of 5 J/cm2 is applied.

Clinical implications of advances in liver regeneration

Remarkable advances have been made recently in the area of liver regeneration. Even though liver regeneration after liver resection has been widely researched, new clinical applications have provided a better understanding of the process. Hepatic damage induces a process of regeneration that rarely occurs in normal undamaged liver. Many studies have concentrated on the mechanism of hepatocyte regeneration following liver damage. High mortality is usual in patients with terminal liver failure. Patients die when the regenerative process is unable to balance loss due to liver damage. During disease progression, cellular adaptations take place and the organ microenvironment changes. Portal vein embolization and the associating liver partition and portal vein ligation for staged hepatectomy are relatively recent techniques exploiting the remarkable progress in understanding liver regeneration. Living donor liver transplantation is one of the most significant clinical outcomes of research on liver regeneration. Another major clinical field involving liver regeneration is cell therapy using adult stem cells. The aim of this article is to provide an outline of the clinical approaches being undertaken to examine regeneration in liver diseases.