Preparation and characterization of methacryloylated hyaluronic acid/acellular Wharton's jelly composite hydrogel scaffold / 中国组织工程研究

BACKGROUND:As tissue engineering brings new hope to the worldwide problem of articular cartilage repair,the construction of light-curing 3D printed hydrogel scaffolds with biomimetic composition is of great significance for cartilage tissue engineering. OBJECTIVE:To construct a biomimetic methacryloylated hyaluronic acid/acellular Wharton's jelly composite hydrogel scaffold by digital light processing 3D printing technology,and to evaluate its biocompatibility. METHODS:Wharton's jelly was isolated and extracted from human umbilical cord,then decellulated,freeze-dried,ground into powder,and dissolved in PBS to prepare 50 g/L acellular Wharton's jelly solution.Methylallylated hyaluronic acid was prepared,lyophilized and dissolved in PBS to prepare 50 g/L methylallylated hyaluronic acid solution.Acellular Wharton's jelly solution was mixed with methacrylyacylated hyaluronic acid solution at a volume ratio of 1:1,and was used as bio-ink after adding photoinitiator.Methylacrylylated hyaluronic acid hydrogel scaffolds(labeled as HAMA hydrogel scaffolds)and methylacrylylated hyaluronic acid/acellular Wharton's jelly gel scaffolds(labeled as HAMA/WJ hydrogel scaffolds)were prepared by digital light processing 3D printing technology,and the microstructure,swelling performance,biocompatibility,and cartilage differentiation performance of the scaffolds were characterized. RESULTS AND CONCLUSION:(1)Under scanning electron microscope,the two groups of scaffolds showed a three-dimensional network structure,and the fiber connection of HAMA/WJ hydrogel scaffold was more uniform.Both groups achieved swelling equilibrium within 10 hours,and the equilibrium swelling ratio of HAMA/WJ hydrogel scaffold was lower than that of HAMA hydrogel scaffold(P＜0.05).(2)CCK-8 assay showed that HAMA/WJ hydrogel scaffold could promote the proliferation of bone marrow mesenchymal stem cells compared with HAMA hydrogel scaffold.Dead/live staining showed that bone marrow mesenchymal stem cells grew well on the two groups of scaffolds,and the cells on the HAMA/WJ hydrogel scaffolds were evenly distributed and more cells were found.Phalloidine staining showed better adhesion and spread of bone marrow mesenchymal stem cells in HAMA/WJ hydrogel scaffold than in HAMA.(3)Bone marrow mesenchymal stem cells were inoculated into the two groups for chondrogenic induction culture.The results of qRT-PCR showed that the mRNA expressions of agglutinoglycan,SOX9 and type Ⅱ collagen in the HAMA/WJ hydrogel scaffold group were higher than those in the HAMA hydrogel scaffold group(P＜0.05,P＜0.01).(4)These findings indicate that the digital light processing 3D bioprinting HAMA/WJ hydrogel scaffold can promote the proliferation,adhesion,and chondrogenic differentiation of bone marrow mesenchymal stem cells.

Potential role of dental pulp stem cells conditioned medium for odontoblastic differentiation

BACKGROUND: Functional bioengineered tooth regeneration using autologous or allogeneic alternative differentiated cells sources are thought to have a great potential in replacing conventional dentures. This study investigated the potential of dental pulp stem cells (DPSCs) conditioned medium for odontoblastic differentiation of Wharton's jelly mesenchymal stem cells (WJMSCs). The DPSCs derived from healthy adult permanent first molars were cultured at high confluence prior to conditioned medium collection. The WJMSCs were cultured in six different treatments, with varying ratios of culture media to DPSCs-conditioned medium. MTT assay was used to measure the rate of proliferation of WJMSCs, while immunocytochemistry staining was utilised to detect the expression of dental matrix protein 1 (DMP-1). The deposited calcium was detected and analysed via Alizarin-Red Staining (ARS). RESULTS: It was found that the proliferation of WJMSCs cultured under the mixture of complete medium and DPSCs conditioned medium showed significantly lower than the control; presumably the cells started to exit proliferative state prior differentiation. In 14 days of induction, the cells in all treatments showed osteoblastic-like morphology, calcium compound deposits were observed at day 7, 10 and 14 of differentiation suggested that DPSCs conditioned medium could lead to osteoblastic/odontoblastic differentiation. However, the DMP-1 protein can be seen only expressed minimally at day 14 of conditioned medium induction. CONCLUSIONS: In conclusion, DPSCs conditioned medium appeared as a potential odontoblastic induction approach for WJMSCs. To further investigate the stimulatory effects by DPSCs conditioned medium, specific signalling pathway need to be elucidated to enhance the differentiation efficiency.

Efecto del factor de crecimiento fibroblástico dos (FGF-2), en la reducción de la senescencia en células madre mesenquimales aisladas de gelatina de Wharton / Fibroblastic growth factor two (FGF-2), in reducing the senescent effect on mesenchymal stem cell isolated from Wharton ́s jelly

Resumen Introducción. Las células madre mesenquimales han generado interés en la ingeniería de tejidos, debido a sus propiedades proliferativas y capacidad de reparación de tejidos, sin embargo, para un trasplante exitoso, es necesario aumentar el número de células mediante un cultivo in-vitro. Durante este proceso la capacidad proliferativa disminuye, provocando cambios en la morfología y funcionalidad celular y afectando la viabilidad del cultivo, este estado se conoce como senescencia celular y como posibles causales, se ha considerado el estrés oxidativo y la falta de factores de crecimiento. Objetivos: Evaluar el efecto de FGF-2 sobre la senescencia de un cultivo de células madre mesenquimales aisladas de gelatina de Wharton y su papel en la regulación del estrés oxidativo. Metodología. Se añadieron dosis de 3,5 y 7,5 ng de FGF-2 al cultivo. Durante los pasajes 5 y 7, se estimó tanto la senescencia celular como la presencia de ROS (especies reactivas de oxígeno). Resultados.Se obtuvo en el pasaje 5, una diferencia significativa del 99,5% entre el control (+) con respecto a los tratamientos con FGF-2, sin embargo, en el pasaje 7 se observó un aumento en la producción de la enzima ß-galactosidasa y cambios morfológicos, confirmando un estado senescente en el cultivo en todos los tratamientos evaluados. Conclusión. Las dosis utilizadas en este estudio contribuyeron positivamente a disminuir el proceso senescente en el cultivo celular, además se determinó, que el FGF-2 puede prolongar el tiempo de cultivo, retardando parcialmente la concentración de especies reactivas de oxígeno

AbstractIntroduction. Mesenchymal stem cells have been generated interest in tissue engineering, due to their proliferative properties and tissue repair capacity, however, for a successful transplant process, it is necessary to increase the number of cells in a culture expansion process. During this process the proliferative capacity is limited, causing changes in cell morphology and functionality affecting the viability of the culture, this state is known as cell senescence. Oxidative stress and deregulation of growth factors are considered as reasons. Aims. To evaluate the effect of FGF-2 on the senescence of a mesenchymal stem cells culture isolated from Wharton ́s jelly and its role in the regulation of oxidative stress. Methodology: 3,5 and 7,5 ng doses of FGF-2 were added to the culture medium from passage 2, then the senescence of the culture was evaluated and the presence of reactive oxygen species was determined during passages 5 and 7. Results. We observed that in passage 5, there is a significant difference 99.5% between the control (+) concerning the FGF-2 treatments, however, in passage 7, an increase in the production of the enzyme ß-galactosidase was observed and changes in morphology such as: increase in size and elongated shape of the cell, confirming a senescent state on the culture in all the treatments evaluated. Conclusion. The doses used in this study contributed positively to decrease this process in a cell culture, also, the FGF- 2 can prolong the cultivation time, partially decreasing the concentration of reactive oxygen species

Absence of Wharton’s jelly: an association with feto-maternal morbidity

Umbilical cord contains two arteries and one vein connecting fetus to the placenta and is responsible for blood flow between the two. It is surrounded by Wharton’s jelly which is a gelatinous substance and functions as adventitia layer of umbilical vessels, thereby providing insulation and protection to the umbilical cord. Umbilical cord abnormalities are associated with poor perinatal outcomes. Very few cases of absent Wharton’s jelly are reported in literature. Ours might be the 8th one in which we did a lower segment caesarean section for meconium stained liquor but the baby died after 12 hours.

Elabela promotes differentiation of wharton’s jelly-derived mesenchymal stem cells into cardiomyocyte-like cells / 中国组织工程研究

BACKGROUND: Elabela is a new type of endogenous receptor of APJ discovered in recent years. It is widely distributed in the adult cardiovascular system and has a certain influence on cardiovascular diseases. However, the effect of Elabela on the differentiation of stem cells into cardiomyocytes and the expression of APJ in cardiomyocyte differentiation has not been studied yet. OBJECTIVE: To investigate the effect of Elabela on the differentiation of Wharton’s jelly-derived mesenchymal stem cells into cardiomyocytes. METHODS: The frozen mesenchymal stem cells were resuscitated. 5-Azacytidine was used to induce Wharton’s jelly-derived mesenchymal stem cells to differentiate into cardiomyocytes when the cell confluence reached 80%-90%. After 24 hours, the medium was replaced by low-glucose medium containing Elabela and 10% fetal bovine serum in the experimental group, and by low-glucose medium containing 10% fetal bovine serum in the control group. At 7, 14, 21, and 28 days after induction, cell morphology was observed. The total RNA and total protein of each group were collected. The myocardial specific markers Nkx2.5, cTnT and Connexin 43 mRNA and protein expression levels were detected by real-time fluorescent quantitative PCR and western blot assay. The expression of APJ in the induced cardiomyocytes was detected by real-time fluorescent quantitative PCR and flow cytometry. RESULTS AND CONCLUSION: (1) The expression levels of myocardial specific markers Nkx2.5, cTnT and Connexin 43 mRNA and protein were higher in the experimental group than in the control group in all stages of differentiation, and the expression of APJ was also higher in the experimental group than in the control group. (2) In summary, Elabela plays a certain promoting role in the differentiation of Wharton’s jelly-derived mesenchymal stem cells into oriented cardiomyocytes. Elabela, as another agonist of APJ, can promote the expression of APJ during the induced cell differentiation.

Unusual umbilical cord finding in a neonate

Umbilical cord cyst refers to any cystic lesion that are associated with the umbilical cord. They are classified as true cysts or pseudocysts. True cysts are small remnants of the allantois, whereas false cysts originate from liquefaction of Wharton Jelly. In present case, cyst was diagnosed at birth without any associated congenital anomalies and resolved spontaneously within a few days requiring nil surgical intervention. Umbilical cord cysts deserve special attention since 20% of them, regardless of type, are associated with structural or chromosomal anomalies. Because of this, fetal karyotyping and amniocentesis should be considered when cysts persist beyond the first trimester.

Histomorphometric Assessment Of Wharton’s Jelly Area In Normal And Pre-eclamptic Pregnancies And Its Correlation With Placental Weight

Introduction: Fetal capability to grow in utero depends on placental development and function. The goal of thisstudy was to appraise the effects of hypertension on placental weight and Wharton’s jelly area (WJA); andcorrelate them in normal and pre-eclamptic pregnancies.Material and Methods: Eighty placentae along with umbilical cord divided into forty each of normotensive andpre-eclamptic pregnancies were studied. The cross-sectional area of the umbilical cord and vessels area wasmeasured with the help of vernier scale and ocular micrometer respectively. WJA was calculated by deduction ofthe vascular area from the umbilical cord area. Placental weight was recorded by using a weighing machine andcorrelated with the WJA.Results: In the present study, mean placental weight was 445.45 ± 40.31 grams and WJA was 35.28 ± 8.42 mm2 inthe normal group. Whereas, in the pre-eclamptic group, mean placental weight was 408.95 ± 47.15 grams andWJA was 29.04 ± 8.09 mm2. Mean placental weight and WJA was significantly lower in the pre-eclamptic group.A significant positive correlation was found between WJA and placental weight (r = 0.710, p<0.0001) in normalgroup and (r = 0.764, p<0.0001) in pre-eclamptic group.Conclusion: Pre-eclampsia is associated with reducing placental weight and WJA. Low WJA may hamper the fetalgrowth. The current study shows a strong positive correlation between WJA and placental weight. So, the awarenessof this correlation will be helpful in the early observation of placental insufficiencies and provide sufficientinformation to take additional care in such conditions.

Morphometric Characterization Of Umbilical Cord Vessels And Neonatal Outcome

Morphologic and morphometric characterization of the umbilical cord and vessel components could greatlyassist in improving adverse maternal and neonatal outcomes. The aim of this study was to evaluate therelationship between morphometry of umbilical cord vessel components and neonatal outcome. A descriptivecross sectional study was conducted on 207 umbilical cords attached to placentae obtained from VictoryMaternity Home and Clinic in Kumasi (Ghana) between November, 2013 and October, 2014. Umbilical cordlength, diameter, and vessels’ diameter were measured with the umbilical cord still attached to the placenta.Neonatal anthropometries were recorded within 24 hours after delivery. The mean ± SD of vein diameter betweenneonates of normotensive 3.36 (±0.88) and hypertensive mothers 3.82 (± 0.50) showed a significant difference.The body length of neonates with short umbilical cord length was significantly lower (p < 0.05) than that of thosewith long cord lengths. Quantitative analysis indicated a positive linear relationship in umbilical cord and itsvessels components with neonatal anthropometry (p<0.05). In conclusion, the morphometry of the umbilicalcord and its vessels could predict maternal and neonatal outcome and therefore would be useful in earlydetection and management of neonatal abnormalities.

Comparison of Advanced Platelet Rich Fibrin (A-PRF) and Culture Media Conditioned Warton's Jelly (CMCWJ) on Fibroblast Cells Proliferation

Objective: To compare the potency of fibroblast cells proliferation in 12.5% and 25% Culture Media Conditioned Warton's Jelly (CMCWJ) and Advanced Platelet Rich Fibrin (A-PRF) cultured medium. Material and Methods: Fibroblast cells were divided into five groups: Group I (Control Group): serum-starved fibroblast without any treatment as a negative control; Group II: fibrolast that supplemented in 12.5% CMCWJ medium; Group III: fibrolast that supplemented 12.5% A-PRF medium; Group IV: fibrolast that supplemented 25% CMCWJ medium, and Group V: fibrolast that supplemented 25% A-PRF medium. The fibroblasts proliferation was counted by an automated cell counter. Statistical analysis was performed using One-way ANOVA and Post hoc Tamhane test was conducted to analyze the potential fibroblast proliferation differences in different concentration of CMCWJ and A-PRF group. Results: There were no significant differences in the fibroblast cell proliferation between GI and GIV, GII and GIV, GII and GIII, GII and GV, also GIV and GV. There were significant differences between GI and GII, GI and GIII, GI and GV, also GIII and GIV. Conclusion: The 12.5% CMCWJ group, 12.5% A-PRF group and 25% A-PRF group has excellent potential ability of fibroblast cells proliferation, meanwhile 25% CMCWJ group has the lowest mean potency of fibroblast cells proliferation compared to other groups. The 12.5% A-PRF Group has the highest mean of fibroblast cell proliferation amongst other groups.

Mesenchymal Stromal Cells from the Maternal Segment of Human Umbilical Cord is Ideal for Bone Regeneration in Allogenic Setting

Umbilical cord (UC) is a discarded product from the operating theatre and a ready source of mesenchymal stromal cells (MSCs). MSCs from UC express both embryonic and adult mesenchymal stem cell markers and are known to be hypoimmunogenic and non-tumorigenic and thus suitable for allogeneic cell transplantation. Our study aimed to determine the degree of immunotolerance and bone-forming capacity of osteodifferentiated human Wharton's jelly-derived mesenchymal stromal cells (hWJ-MSCs) from different segments of UC in an allogenic setting. UCs were obtained from healthy donors delivering a full-term infant by elective Caesarean section. hWJ-MSCs were isolated from 3 cm length segment from the maternal and foetal ends of UCs. Three-dimensional fibrin constructs were formed and implanted intramuscularly into immunocompetent mice. The mice were implanted with 1) fibrin construct with maternal hWJ-MSCs, 2) fibrin construct with foetal hWJ-MSCs, or 3) fibrin without cells; the control group received sham surgery. After 1 month, the lymphoid organs were analysed to determine the degree of immune rejection and bone constructs were analysed to determine the amount of bone formed. A pronounced immune reaction was noted in the fibrin group. The maternal segment constructs demonstrated greater osteogenesis than the foetal segment constructs. Both maternal and foetal segment constructs caused minimal immune reaction and thus appear to be safe for allogeneic bone transplant. The suppression of inflammation may be a result of increased anti-inflammatory cytokine production mediated by the hWJ-MSC. In summary, this study demonstrates the feasibility of using bone constructs derived from hWJ-MSCs in an allogenic setting.

Transplantation of Wharton's jelly alleviates inflammatory response and depression after traumatic brain injury in mice / 中华急诊医学杂志

Objective To investigate the influence of Wharton's jelly (W J) transplant on brain inflammation and mood status in traumatic brain injury (TBI) mouse model.Methods The WJ was isolated from human umbilical cord and cultured,and the cell phenotype of P3 human umbilical cord mesenchymal stem cells was identified by flow cytometry.The animal model was established by modified weight drop method.Experimental mice were randomly(random number) divided into Veh(normal saline)and WJ transplantation groups.After 3 days,water content of damaged brain was detected.Elisa kit was used to detect the expression of IL-1β and TNF-αt.The expression of GFAP,Ibal and CD68 were detected by immunofluorescence.Het(hydroethidine) staining was used to detect reactive oxygen species (ROS) production.Neurologic deficit score was used to evaluate the motor function,sucrose preference test,tail suspension test and forced swim test were used to detect the depression of mice.The data were expressed in ((-x)±s) and analysed by SPSS 21.0 software.Two-way ANOVA with repeated measures design was used to compare difference bewteen two groups at multiple interval.Results Compared with Veh group,the mice in the WJ group had a better performance in NDS scored test(d 1:14.8± 1.169 vs.15.2+ 1.472);3d:(11.0±1.414 vs.13.5+1.225);7d:(9.5±1.517 vs.12.0±1.549);14d:(7.7±0.816 vs.10.5±1.643);21d:(6.5±0.547 vs.9.0±1.265);28d:(5.3+0.816 vs.7.8±1.169),P＜0.05].After TBI,WJ tissue transplantation increased sucrose preference index from (54.49±1.505)％ to (64.56±2.279)％ (P=0.004),decreased immobility time using tail suspension test from (144.7±5.493)s to (115.7±4.660)s (P＜0.01),and decreased immobility time using forced swim test from (260.3±4.558)s to (215.8±5.003)s (P=0.002).After WJ transplantation,brain water content was reduced from (84.48±1.802)％ to (75.58+1.559)％ (P=0.004),the expression of IL-1β and TNF-α near injury area also decreased(P=0.000 6 and 0.000 3),as well as the expression of ROS(P=0.020).The fluorescence intensity of activated astrocytes decreased from (2 906±431.591)to (165 8±312.912) (P=0.041),and the number of microglias and activated microglias were both reduced(P=0.049 and P＜0.01) after TBI.Conclusions Wharton's jelly alleviated the inflammation and depression in traumatic brain injured mice.

Comparación entre Células Troncales Mesenquimales obtenidas de Médula Ósea, Tejido Adiposo y Gelatina de Wharton en base a los Criterios de la ISCT / Comparison between Mesenchymal Stem Cells obtained from Bone Marrow, Adipose Tissue and Wharton Gelatin according to the ISCT Criteria

RESUMEN: Las células troncales mesenquimales (CTM) representan una población heterogénea con capacidad para auto-renovarse y diferenciarse a distintos tipos celulares. Estas fueron descritas en un inicio en médula ósea (MO) a mediados del siglo pasado, desde entonces este tejido se ha convertido en el estándar de oro para la obtención y caracterización de CTM. Actualmente se sabe que este tipo de células se encuentran alojadas en nichos distribuidos por todo el organismo, donde contribuyen a los procesos de regeneración del tejido donde se localizan. No obstante, encontrar una fuente alterna de CTM con las mismas características que las de MO, pero que su extracción no suponga riesgo para el donador es fundamental para su utilización con fines terapéuticos. En este trabajo se aislaron células troncales de médula ósea, y se compararon con tejido adiposo y gelatina de Wharton y caracterizaron de acuerdo a los criterios de la Sociedad Internacional para la Terapia Celular (ISCT). Los resultados mostraron que la morfología, diferenciación osteogénica y adipogénica, así como la expresión de los antígenos de superficie CD90, CD73 y CD105 cumplen con los estándares, señalando a las provenientes de gelatina de Wharton como mejor opción.

ABSTRACT: Mesenchymal stem cells (MSC) represent a heterogeneous population with the capacity to self-renew and differentiate into different cell types. At the middle of the last century these cells initially were described in bone marrow (BM), thence this tissue has become the gold standard for obtaining and characterization of MSC. It is known that these cells are housed in specific areas called niches distributed throughout all body, where they contribute to tissue regeneration processes of self-tissue were they are located. However, finding an alternative source of CTM with the same characteristics that have showed in MO, but its obtention no represent a risk since the donor is essential to their use for therapeutic purposes. In this study we isolated mesenchymal stem cells from bone marrow, adipose tissue and Wharton's jelly and they were compared in their characteristics in according to the standards of the International Society for Cellular Therapy (ISCT). The results showed that the morphology as well as adipogenic and osteogenic differentiation and also the expression of surface antigens (CD90, CD73, and CD105) from all tissues accomplished the standards, although Wharton's jelly represented the best option.

Cardioprotective Effects of Wharton Jelly Derived Mesenchymal Stem Cell Transplantation in a Rodent Model of Myocardial Injury

BACKGROUND: Whartons jelly-derived mesenchymal stem cells are a valuable alternative source that possess multipotent properties, easy to obtain and available in large scale compared to BMMSCs. We investigated the possibility of cardiac function improvement post isoproterenol induced cardiac injury in a rat model following human WJMSCs transplantation. MATERIALS AND METHODS: MSCs were extracted and cultured from cord WJ, characterized by morphology, Immunophenotyping and differentiation to osteoblast and adipocytes. WJMSCs were labeled with PKH2 linker dye. Wistar rats were divided into control group, ISO group (injected with 2 doses of isoproterenol) to induce myocardial injury and ISO group transplanted with labelled WJMSCs. ECG, electrocardiographic patterns, cardiac marker enzymes, tracing of labeled MSCs and immunohistochemical analysis of myocardial cryosections were studied. RESULTS AND CONCLUSIONS: WJ derived MSCs were expanded for more than 14 passages while maintaining their un-differentiated state, were positive for MSC markers and were able to differentiate into adipocyte and osteoblast. We demonstrated that intravenously administered WJMSCs were capable of homing predominently in the ischemic myocardium. Cardiac markers were positively altered in stem cell treated group compared to ISO group. ECG and ECHO changes were improved with higher survival rate. WJMSCs could differentiate into cardiac-like cells (positive for cardiac specific proteins) in vivo. WJMSCs infusion promoted cardiac protection and reduced mortality, emphasizing a promising therapeutic role for myocardial insufficiency.

The osteogenesis ability of human umbilical cord Wharton's Jelly-derived mesenchymal stem cells and periodontal mesenchymal stem cells / 实用口腔医学杂志

Objective:To compare the osteogenesis ability between human umbilical cord Wharton's Jelly-derived mesenchymal stem cells(hUCWJMSCs) and human periodontal ligament mesenchymal stem cells (hPDLSCs) in vitro.Methods:hUCWJMSCs and hPDLSCs were in vitro cultured.The cell proliferation capacity was examined by MTT assay.After osteogenesis induction culture,ALP activity of the cells was determined,minerialization was observed by alizarin red staining,OPN and Runx2 mRNA expression was analyzed by Real-time PCR.Results:hUCWJMSCs grew faster than hPDLSCs.After osteogenic differentiation induction,hPDLSCs group showed higher ALP level,more mineralized nodule formation and higher Runx2 expression compared with hUCWJMSCs group (P ＜ 0.05);while the OPN expressed higher in hUCWJMSCs than in hPDLSCs (P ＜ 0.05).Conclusion:hUCWJMSCs and hPDLSCs have osteogenesis differentiation potential,hPDLSCs are more osteogenetic.

Attachment and Differentiation of Human Umbilical Cord Stem Cells on to the Tooth Root Surface with and without the Use of Fibroblast Growth Factor-An In Vitro Study

BACKGROUND AND OBJECTIVES: The purpose of this first of its kind study was to analyse the growth, development and attachment of cultured human umbilical cord stem cells alone or supplemented with basic Fibroblast Growth Factor (bFGF) on both healthy and periodontally diseased tooth surfaces in vitro. METHODS: Four groups of 12 root surface scaffolds each were classified as Group I- healthy root surfaces; Group II- periodontally diseased; Group III- Healthy with bFGF and Group IV- periodontally diseased root with bFGF. bFGF was applied in the concentration of 8 ng/ml on to the surface followed by incubation of cultured human umbilical cord stem cells (hUCMSCs) on the scaffolds. Scanning electron microscopy observations were made on 14th and 21st days to assess the proliferation and morphology of cells attached on the tooth surface. RESULTS: Cultured hUCMSCs demonstrated adhesion to tooth root scaffold. All the groups showed a significant increase in the number of cell attachment from 14th day to 21st day. The groups with bFGF showed a significant increase in attachment of cells when compared to the groups without bFGF. The cells showed an increase in number of flat cells from 14th day to 21st day in all the groups indicating an increased maturity of cells. Periodontally diseased groups had less maturity of cells than healthy groups. The groups supplemented with bFGF, had more mature cells than the groups without bFGF. CONCLUSIONS: hUCMSCs have the propensity to differentiate into cells that have the capacity to bind to root surfaces. hUCMSCs incubated with bFGF showed better proliferation and attachment to tooth root surfaces. The role of hUCMSCs can be further explored for periodontal regeneration.

Effect of human umbilical cord WJ-MHCs on TNF-α and NT-proBNP in the rats with heart failure of acute myocardial infarction / 重庆医学

Objective To observe the influence of human umbilical cord wharton′s jelly‐mesenchymal stem cells(WJ‐MHCs) on the tumor necrosis factorα (TNF‐α) and N‐terminal pro‐brain natriuretic peptide(NT‐proBNP) in rats with heart failure of a‐cute myocardial infarction .Methods Totally 80 male rat models of heart failure of acute myocardial infarction were made by isopre‐naline(ISO) 200 mg/kg injected subcutaneously twice at an interval of 24 hours .After one week ,24 survival rats were randomly di‐vided into WJ‐M HCs transplantation group and normal control group .Sham group was made of 12 health rats ,and then each of the three groups was subdivided into pre‐transplantation group and post‐transplantation group 4 weeks later .WJ‐MHCs transplantation group was transplanted with WJ‐MHCs with DAPI labeled after ISO injected one week .Sham group and normal group were un‐treated and normally bred .The left ventricular ejection fraction(LVEF) measured by before transplantation and post‐transplantation 4 weeks later .The injected cells and the expression of TNF‐αwas measured .Results Compared to pre‐transplantation group ,WJ‐M HCs transplantation group increased the LVEF(P<0 .05);compared to pre‐transplantation and normal control ,WJ‐M HCs trans‐plantation group reduced the TNF‐αand NT‐proBNP in the serum(P<0 .05)and the expression of TNF‐α from the heart tissue (P<0 .05);compared to normal transplantation ,WJ‐M HCs transplantation group reduced the mortality from 33 .3% to 16 .7% ;immunofluorescence demonstrated that transplanted cells were still found alive in the heart after transplantation 4 weeks later .Con‐clusion Transplantation of WJ‐MHCS down‐regulates TNF‐α and NT‐proBNP in the serum in the serum and the expression of TNF‐αfrom the heart tissue and up‐regulates the LVEF in rats with heart failure of acute myocardial infarction .

Optimization the methodology of isolating human ubilical cord mesenchymal stromal cells from Wharton's jelly and examination of their passage effect on immune phenotype using flow cytometry / 天津医药

Objective To observe the effects of different collagenase digestions on isolating human umbilical cord mesenchymal stromal cells (MSC) from Wharton’s jelly, to exam their differentiation ability and to investigate their passage effect on the immune phenotype. Methods Human umbilical cord samples were digested by collagenaseⅠorⅡorⅣfor 4-18 hours then were passed through sieves . Cells were collected by centrifugation then inoculated in DMEM/F12 medium at concentration within range of 4.8×103-1×104/cm2 to compare the effect of different digestions on MSC. Von kossa staining and tetracycline fluorescence was used to label the osteogenic differentiation capacity of MSC. Also RT-PCR was employed to identify the differentiate capacity of MSC into myocardial-like cells. The immunophenotype of MSCs were detected by flow cytometry after subculture. Results Using collagenaseⅠdigestion, the number of MSCs isolated from human umbilical cord in Wharton’s jelly and their vitality were much higher while the period to show cell extension and primary culture time were shorter than those using collagenaseⅡorⅣdigestions. The analysis of surface marker revealed that the expression of positive markers include CD29, CD44, CD73, CD90 and CD105 did not change with passages while the negative markers such as CD31, CD34 and HLA-DR increased significantly with passages;Differential experiments induced in vitro show that human umbilical cord MSC in wharton’s jelly had the ability to differentiate into osteoblasts and myocardial-like cells. Con?clusion The human umbilical cord MSC in Wharton’s jelly was successfully isolated by collagenaseⅠdigestion. This meth?od was simple with a high success rate while cell loss and damage were minimum. This makes large-scale cultivation possi? ble. Negative markers increased with cell passages. This phenomenon revealed that MSC showed directional differentiation.

Cryopreservation of Human Wharton's Jelly-derived Mesenchymal Stem Cells Following Controlled Rate Freezing Protocol Using Different Cryoprotectants; A Comparative Study

OBJECTIVES: To compare the effect of three different cryoprotectants on basic stem cell characteristics for the possibility of using well defined, dimethyl sulfoxide (DMSO) and serum free freezing solutions to cryopreserve human Wharton's jelly-derived mesenchymal stem cells (WJMSCs) following controlled rate freezing protocol. METHODS: The mesenchymal stem cells isolated from human Wharton's jelly were cryopreserved using 10% DMSO, 10% polyvinylpyrrolidone (PVP) and a cocktail solution comprising of 0.05 M glucose, 0.05 M sucrose and 1.5 M ethylene glycol following controlled rate freezing protocol. We investigated the post-thaw cell viability, morphology, proliferation capacity, basic stem cell characteristics, in vitro differentiation potential and apoptosis-related gene expression profile before and after cryopreservation. RESULTS: The cryoprotectant 10% DMSO has shown higher post-thaw cell viability of 81.2+/-0.58% whereas 10% PVP and cocktail solution have shown 62.87+/-0.35% and 72.2+/-0.23%, respectively at 0 h immediately thawing. The cell viability was further reduced in all the cryopreserved groups at 24 h later post-thaw culture. Further, the complete elimination of FBS in cryoprotectants has resulted in drastic reduction in cell viability. Cryopreservation did not alter the basic stem cell characteristics, plasticity and multipotency except proliferation rate. The expression of pro-apoptotic BAX and p53 genes were higher whilst p21 was lower in all the cryopreserved groups when compare to the control group of WJMSCs. CONCLUSION: Although 10% DMSO has shown higher post-thaw cell viability compare to 10% PVP and cocktail solution, the present study indicates the feasibility of developing a well-defined DMSO free cryosolution which can improve storage and future broad range applications of WJMSCs in regenerative medicine without losing their basic stem cell characteristics.

Absence of Wharton's jelly: case report / Ausência da geleia de Wharton: relato de caso

There are few reports in the literature of the absence of Wharton's Jelly. Here we report the seventh case in a primigravida, 22 years old, admitted after vaginal delivery of stillborn. The umbilical cord have a long segment with disruption of cord structures and the three blood vessels were completely separated from each other, with a minimum amount of Wharton's jelly remaining around each vessel. The absence of Wharton' jelly is associated with fetal distress, intrauterine growth restriction, and fetal death. Quantitative/qualitative studies of Wharton's jelly represent an open field of research for possible correlations with obstetric conditions and fetal deaths.

Na literatura, há poucos relatos sobre a ausência de geleia de Wharton. Relatamos o sétimo caso em uma primigesta de 22 anos, admitida após parto vaginal de feto natimorto. O cordão umbilical apresentava longo segmento com esfacelo da geleia e três vasos sanguíneos completamente separados uns dos outros, com mínima quantidade de geleia de Wharton remanescente ao redor de cada vaso. Ausência de geleia de Wharton associa-se a estresse, restrição de crescimento e óbitos fetais. Estudos quantitativos/ qualitativos sobre a geleia de Wharton representam campo de pesquisa aberto para possíveis correlações com condições e doenças obstétricas e óbitos fetais.

Avaliação do efeito de células-tronco mesenquimais humanas de várias origens na atividade de linhagem de células tumorais HepG-2 / Evaluation of the effect of human mesenchymal cells from several sources in the activity of tumor cells line

INTRODUÇÃO O câncer é uma das principais causas de morte no mundo, sendo responsável por cerca de 8 milhões de mortes por ano, segundo dados da OMS. As mortes por câncer são provocadas por tumores que se originam em órgãos como pulmão, fígado, estômago, intestino, mama e esôfago. As células-tronco mesenquimais (CTM) foram identificadas em vários órgãos e estudos da sua interação com células tumorais têm apresentado resultados indicando ação inibitória sobre alguns tipos de tumores. Para explorar essa questão foram analisados os efeitos sobre células tumorais de carcinoma hepatocelular humano (HepG-2) do meio condicionado (MC) obtido do cultivo de CTM isoladas de tecido adiposo (TA), líquido amniótico (LA) e geleia de Wharton (GW). MÉTODOS Os MCs foram coletados após 24 horas de incubação das CTM sub-confluentes com ?-MEM contendo 20% de soro fetal bovino (SFB). Os MCs foram centrifugados e passados através de filtros de 0,22 ?M e armazenadas a -20 °C. O MC da própria célula HepG-2 foi utilizado como controle. Os efeitos dos MCs sobre a proliferação de células HepG-2 foram testados por ensaio MTT, em várias concentrações após 24 h de incubação. O ciclo celular de células HepG-2 tratadas com MC a 25%, 50% ou 75% foi analisado por citometria de fluxo (coloração IP) utilizando o software Modfit LT. A expressão dos genes bcl-2, bcl-6, CCND1 foi analisada por RT-PCR. A proliferação celular foi avaliada pela expressão das proteínas survivina, Bcl-2, PCNA e Ki-67 e pela quantificação de mitocôndrias com corante MitoTracker, assim como pelo potencial de membrana mitocondrial por corante JC-1 Mitoscreen utilizando equipamento de high content analysis. RESULTADOS Os meios condicionados de células-tronco de tecido adiposo (MC-TA) não alteraram a proliferação de células tumorais HepG-2 e os meios condicionados de células-tronco de líquido amniótico (MC-LA) e de geleia de Wharton (MC-GW) provocam aumento da proliferação, confirmada pela contagem de células com núcleos...

INTRODUCTION Cancer is a leading cause of death worldwide, accounting for about 8 million deaths per year, according to WHO data. Cancer deaths are caused by tumors that originate in organs such as lung, liver, stomach, bowel, breast and esophagus. The mesenchymal stem cells (MSCs) have been identified in many organs and studies of their interaction with tumor cells have shown results indicating an inhibitory effect on some types of tumors. To explore this question the effects of the conditioned medium (CM) obtained from mesenchymal stem cell isolated from adipose tissue (AT), amniotic fluid (AF) and Wharton jelly (WJ) on tumor cells of human hepatocellular carcinoma (HepG-2) were analyzed. METHODS The MSC CM was collected after 24 hours incubation of sub confluent MSC with ?-MEM containing 20% fetal bovine serum (FBS). The MSC CM were centrifuged and passed through 0.22 ?M filter and stored at -20° C. The CM of HepG-2 cell itself was used as control. The effects of contrast media on proliferation of HepG-2 cells were tested by MTT assay at various concentrations after 24 h of incubation. The cell cycle HepG-2 cells treated with CM at 25%, 50% or 75% was analyzed by flow cytometry (PI staining) using Modfit software LT. The expression of the genes Bcl-2, Bcl-6, CCND1 was analyzed by RT-PCR. Cell proliferation was assessed by the expression of survivin, Bcl-2, Ki-67 and PCNA proteins, and the quantization of mitochondrial by MitoTracker dye, as well as the mitochondrial membrane potential by JC-1 Mitoscreen dye using high content analysis equipment. RESULTS The conditioned media of mesenchymal stem cells (MSC) from adipose tissue (AT-CM) did not alter the proliferation of tumor HepG-2 cells and conditioned media of MSC cells from amniotic fluid (AF-CM) and Wharton jelly (WJ-CM) caused increased proliferation, confirmed by counting cells with nuclei stained with Hoechst 33342. The cell cycle analysis showed that exposure of HepG-2 cells to AF-CM...