RNASEH2C c.194G>A is a Chinese-specific founder mutation in three unrelated patients with Aicardi-Goutières syndrome 3.

Biallelic pathogenic variants in RNASEH2C cause Aicardi-Goutières syndrome 3 (AGS3, MIM #610329), a rare early-onset encephalopathy characterized by intermittent unexplained fever, chilblains, irritability, progressive microcephaly, dystonia, spasticity, severe psychomotor retardation and abnormal brain imaging. Currently, approximately 50 individuals with AGS3 and 19 variants in RNASEH2C have been revealed. Here, we reported the novel clinical manifestations and genotypic information of three unrelated Chinese patients with AGS3 caused by pathogenic variants in RNASEH2C. In addition to three novel missense variants (c.101G>A, p.Cys34Tyr; c.401T>A, p.Leu134Gln and c.434G>T, p.Arg145Leu), one missense variant (c.194G>A, p.Gly65Asp) reoccurred in all patients but was completely absent in South Asian and other ethnicities. Our study expanded the variant spectrum of RNASEH2C and identified RNASEH2C c.194G>A as a Chinese-specific founder mutation. The novel phenotypes, including mouth ulcers, hip dysplasia, retarded dentition and hypogonadism, observed in our patients greatly enriched the clinical characteristics of AGS3.

The fate and risk of microplastic and antibiotic sulfamethoxazole coexisting in the environment.

With huge amount of plastic entering to the environment, microplastic pollution has become a great concern. Microplastic behavior in the environment is important to evaluate its harm to ecosystem and human beings. It has been found that microplastic can be used as a carrier to adsorb and enrich heavy metals or organic pollutants in water or soil. With the development of industry and medicine, antibiotics are improperly used in many countries and most of them end up in wastewater. This study investigates the adsorption behavior of sulfamethoxazole (SMX) antibiotic onto virgin and aged polyamide 6 (PA6) microplastics. The maximum adsorption amount was 0.089 mg SMX/g PA6 at 25 °C and pH 7 with initial SMX concentration of 2 mg/L. Results reveal that the adsorption was mainly due to the chemical bounding. The impact of pH, salinity, and humic acid on the adsorption have been studied, and it was found that the pH has significant impact on the adsorption. At pH 5, the adsorption amount was 0.27 mg/g which is two times higher than that at pH 7. The SMX adsorbed on PA6 tends to be more likely desorbed in reservoir water than in ultrapure water. For instance, the desorption amount of SMX from virgin PA6 was 0.15 mg/L in reservoir water but 0.10 mg/L in ultrapure water. The study indicates that microplastics have great threat to environment.

Research on Graphene and Its Derivatives in Oral Disease Treatment.

Oral diseases present a global public health problem that imposes heavy financial burdens on individuals and health-care systems. Most oral health conditions can be treated in their early stage. Even if the early symptoms of oral diseases do not seem to cause significant discomfort, prompt treatment is essential for preventing their progression. Biomaterials with superior properties enable dental therapies with applications in restoration, therapeutic drug/protein delivery, and tissue regeneration. Graphene nanomaterials have many unique mechanical and physiochemical properties and can respond to the complex oral microenvironment, which includes oral microbiota colonization and high masticatory force. Research on graphene nanomaterials in dentistry, especially in caries, periodontitis therapy, and implant coatings, is progressing rapidly. Here, we review the development of graphene and its derivatives for dental disease therapy.

Impacts of ridge-furrow rainfall concentration systems and mulches on corn growth and yield in the semiarid region of China.

BACKGROUND: Plastic-covered ridge-furrow farming systems for rainfall concentration (RC) improve the water availability for crops and increase the water use efficiency (WUE), thereby stabilizing high yields. In this study, we optimized the mulching patterns for RC planting to mitigate the risks of drought during crop production in semiarid agricultural areas. We conducted a 4-year field study to determine the RC effects on corn production of mulching in furrows with 8% biodegradable films (RCSB ), liquid film (RCSL ), bare furrow (RCSN ) and conventional flat (CF) farming. RESULTS: We found that RC significantly (P > 0.05) increased the soil moisture in the top 0-100 cm layer and the topsoil temperature (0-20 cm) during the corn-growing period. Mulching with different materials in planting furrows further improved the rain-harvesting, moisture-retaining and yield-increasing effects of RC planting. Compared with CF, the 4-year average total dry matter amount per plant for RCSB , RCSL and RCSN treatments increased by 42.1%, 30.8% and 17.2%, respectively. The grain yield increased by 59.7%, 53.4% and 32.6%, respectively. CONCLUSION: Plastic-covered ridge and furrow mulched with biodegradable film and liquid film is recommended for use in the semiarid Loess Plateau of China to alleviate the effects of drought on crop production. © 2015 Society of Chemical Industry.

Smoking and perceived stress in relation to short salivary telomere length among caregivers of children with disabilities.

Telomere length (TL), the length of repeated DNA sequence that forms protective caps at the end of chromosomes, has emerged as a novel biomarker of cell aging and oxidative stress. There is increasing research exploring the associations of smoking and perceived stress with TL, and the results are inconsistent. This study aimed to examine whether smoking and perceived stress were associated with shortened salivary TL among primary caregivers of children with disabilities. Using a quantitative polymerase chain reaction method, salivary TL was assessed among 89 caregivers aged 19-69 years (87% were women) who took care of disabled children in the Patagonia Region, Chile. Interviewer-administered questionnaires were used to collect information on sociodemographic and lifestyle factors. The 14-item Perceived Stress Scale was used to assess perceived stress. Mean relative TL was 0.92 (standard error = 0.03). Smokers had age-adjusted mean TL that was 0.07 units lower (ß = -0.07, standard error = 0.03; p = 0.012) than non-smokers. Smokers were 2.17 times more likely to have shorter TL ( < 0.73, the lowest quartile of TL) than non-smokers (odds ratio = 3.17; 95% confidence interval = 1.05-9.52) with adjustment for age and perceived stress. Caregivers with higher perceived stress were 2.13 times more likely to have shorter TL (odds ratio = 3.13; 95% confidence interval = 1.03-9.55) than caregivers with lower perceived stress after adjustment for age and smoking. This study provides the first evidence of strong associations between smoking and perceived stress and shortened salivary TL among caregivers of children with disabilities. Larger studies with detailed information on smoking status are warranted to confirm our findings.

High-viscosity dietary fibers reduce adiposity and decrease hepatic steatosis in rats fed a high-fat diet.

Viscous dietary fiber consumption lowers the postprandial glucose curve and may decrease obesity and associated comorbidities such as insulin resistance and fatty liver. We determined the effect of 2 viscous fibers, one fermentable and one not, on the development of adiposity, fatty liver, and metabolic flexibility in a model of diet-induced obesity. Rats were fed a normal-fat (NF) diet (26% energy from fat), a high-fat diet (60% energy from fat), each containing 5% fiber as cellulose (CL; nonviscous and nonfermentable), or 5% of 1 of 2 highly viscous fibers-hydroxypropyl methylcellulose (HPMC; nonfermentable) or guar gum (GG; fermentable). After 10 wk, fat mass percentage in the NF (18.0%; P = 0.03) and GG groups (17.0%; P < 0.01) was lower than the CL group (20.7%). The epididymal fat pad weight of the NF (3.9 g; P = 0.04), HPMC (3.9 g; P = 0.03), and GG groups (3.6 g; P < 0.01) was also lower than the CL group (5.0 g). The HPMC (0.11 g/g liver) and GG (0.092 g/g liver) groups had lower liver lipid concentrations compared with the CL group (0.14 g/g liver). Fat mass percentage, epididymal fat pad weight, and liver lipid concentration were not different among the NF, HPMC, and GG groups. The respiratory quotient was higher during the transition from the diet-deprived to fed state in the GG group (P = 0.002) and tended to be higher in the HPMC group (P = 0.06) compared with the CL group, suggesting a quicker shift from fatty acid (FA) to carbohydrate oxidation. The HPMC group [15.1 nmol/(mg ⋅ h)] had higher ex vivo palmitate oxidation in muscle compared with the GG [11.7 nmol/(mg ⋅ h); P = 0.04] and CL groups [10.8 nmol/(mg ⋅ h); P < 0.01], implying a higher capacity to oxidize FAs. Viscous fibers can reduce the adiposity and hepatic steatosis that accompany a high-fat diet, and increase metabolic flexibility, regardless of fermentability.

Thermoresponsive worms for expansion and release of human embryonic stem cells.

The development of robust suspension cultures of human embryonic stem cells (hESCs) without the use of cell membrane disrupting enzymes or inhibitors is critical for future clinical applications in regenerative medicine. We have achieved this by using long, flexible, and thermoresponsive polymer worms decorated with a recombinant vitronectin subdomain that bridge hESCs, aiding in hESC's natural ability to form embryoid bodies (EBs) and satisfying their inherent requirement for cell-cell and cell-extracellular matrix contact. When the EBs reached an optimal upper size where cytokine and nutrient penetration becomes limiting, these long and flexible polymer worms facilitated EB breakdown via a temperature shift from 37 to 25 °C. The thermoresponsive nature of the worms enabled a cyclical dissociation and propagation of the cells. Repeating the process for three cycles (over eighteen days) provided a >30-fold expansion in cell number while maintaining pluripotency, thereby providing a simple, nondestructive process for the 3D expansion of hESC.

First insight of the intergenerational effects of tri-n-butyl phosphate and polystyrene microplastics to Daphnia magna.

As an emerging organic pollutant, tributyl phosphate (TnBP) can be easily adsorbed by microplastics, resulting in compound toxic effects. In the present work, the effects of polystyrene microplastics (PS-MPs) and TnBP on the survival, growth, reproduction and oxidative stress of Daphnia magna (D. magna) have been evaluated through multigenerational test. Compared with the alone exposure groups, the somatic growth rate and the expression values of growth related genes rpa1, mre11, rnha, and rfc3_5 in the F1 generation of the combined exposure groups were significantly lower (p < 0.05), indicating synergistic effect of PS-MPs and TnBP on the growth toxicity and transgenerational effects. In addition, compared with the PS-MPs groups, significantly lower average number of offspring and expression values of reproduction related genes ccnb, mcm2, sgrap, and ptch1 were observed in the combined exposure group and TnBP group (p < 0.05), indicating TnBP might be the major factor causing reproductive toxicity to D. magna. Although PS-MPs and TnBP alone or in combination also had toxic impacts on the growth, survival and reproduction of D. magna in generations F0 and F2, the effects were less than F1 generation. Regarding oxidative stress, the activity of SOD, CAT and GSH-Px and MDA content in the generations F0 and F1 of combined exposure groups were higher than the TnBP group but lower than the PS-MPs groups, suggesting that PS-MPs might be the dominant cause of the oxidative damage in D. magna and the presence of TnBP would alleviate oxidative stress by reducing the bioaccumulation of PS-MPs. The present work will provide a theoretical basis for further understanding of the toxic effects and ecological risks of combined TnBP and microplastic pollution on aquatic organisms.

Synergistic antibacterial and antifouling wound dressings: Integration of photothermal-activated no release and zwitterionic surface modification.

Addressing the pervasive issue of bacteria and biofilm infections is crucial in the development of advanced antifouling wound dressings. In this study, a novel wound healing treatment using sulfobetaine (SBMA) decorated electrospun fibrous membrane based on polycaprolactone (PCL)/nitric oxide (NO) donors was developed. The fabrication involved a dual strategy, first integrating NO donors into mesoporous polydopamine (MPDA) and complexed with PCL/PEI to electrospin nanofibers. The fibrous membrane exhibited a potent antibacterial response upon irradiation at 808 nm, owing to a combination of NO and photothermal effect that effectively targets bacteria and disrupts biofilms. Surface functionalization of the membrane with PEI allowed for the attachment of SBMA via Michael addition, fabricating a zwitterionic surface, which significantly hinders protein adsorption and reduces biofilm formation on the wound dressing. In vitro and in vivo assessments confirmed the rapid bactericidal capabilities and its efficacy in biofilm eradication. Combining photothermal activity, targeted NO release and antifouling surface, this multifaceted wound dressing addresses key challenges in bacterial infection management and biofilm eradication, promoting efficient wound healing.

Analysis, environmental occurrence, fate and potential toxicity of tire wear compounds 6PPD and 6PPD-quinone.

Tire wear compounds N-(1,3-dimethylbutyl)-N'-phenyl-p-phenylenediamine (6PPD) and its derivative 6PPD-quinone have been considered as emerging pollutants and attracted much attention recently. As an antioxidant and antiozonant widely used, 6PPD would be released during the production or use of rubber-related products. Because of the mass production and wide use of rubber-related products, 6PPD and 6PPD-quinone have been identified to be ubiquitous in the environment. In this study, we firstly reviewed the current available literature on the analytical procedures, concentrations and distribution of 6PPD and 6PPD-quinone, and then investigated the potential toxic effects of these two compounds on aquatic organisms. Current studies have been mainly focused on the occurrence of 6PPD and 6PPD-quinone in dust and water, while available information on atmosphere, soil, sediments and organisms is limited. The fate and distribution of 6PPD and 6PPD-quinone would be influenced by environmental factors such as temperature, illumination, and storm events, etc. Although 6PPD and 6PPD-quinone have potential adverse effects on aquatic organisms, and 6PPD-quinone has species-specific toxicity, toxicological mechanisms of these compounds are still unclear. Based on the review and analysis of current studies, some suggestions for future research of 6PPD and 6PPD-quinone are given.

Emergence of a non vaccine-cognate enterovirus A71 genotype C1 in mainland China.

BACKGROUND: EV-A71 is a common causative agent of hand foot and mouth disease. In mainland China, EV-A71 subgenotype C4 has been the sole circulating genotype since 2008, and was used in the production of multiple licensed vaccines. Here, we report the first detection EV-A71 C1 strains in China. METHODS: Full genomic sequence were obtained. The origin of the EV-A71 C1 strains were tracked down by Bayesian inferences. Recombination was analyzed using Simplot program. And the antigenicity were tested using the microneutralization test. RESULTS: The C1-GD2019 shared high identity with the C1-like lineage recently identified in Europe and was introduced into Guangdong in 2018-2019. Close genetic relatedness between the C1-GD2019 and Europe C1-like strains were observed except for the 3D-3'UTR region. The late showed high similarity with CVA genomes. Antigenic variance was found. The C1-GD2019 could not be effectively neutralized by EV-A71 C4a neutralizing antibody positive samples. CONCLUSION: This is the first report of EV-A71 subgenotype C1 isolated in China. It is a recombinant strain originating from C1-like strains recently identified in Europe and CVA strains. The different antigenicity between the C1 strains and C4a vaccine strains highlighted the importance on closely monitoring the EV-A71 C1 strains in China.

Molecular characterization and functional analysis of a novel WRKY transcription factor HbWRKY83 possibly involved in rubber production of Hevea brasiliensis.

WRKY transcription factors play important roles in plant growth and developmental processes and various stress responses, and are also associated with jasmonic acid (JA) signaling in the regulation of secondary metabolite biosynthesis in plants. The regulatory networks mediated by WRKY proteins in the latex production of Hevea brasiliensis (the Pará rubber tree) are poorly understood. In this study, one novel WRKY gene (designated HbWRKY83) was identified from the latex of H. brasiliensis, and its functions were characterized via gene expression analysis in both the latex and HbWRKY83-overexpressing transgenic Arabidopsis. HbWRKY83 gene contains an open reading frame (ORF) of 921 bp encoding a 306-amino-acid protein which is clustered with group IIc WRKY TF. HbWRKY83 is a nuclear-localized protein with transcriptional activity. Real-time quantitative PCR analysis demonstrated that the transcription level of HbWRKY83 was up-regulated by exogenous methyl jasmonate, Ethrel (ethylene releaser) stimulation, and bark tapping (mechanical wounding). Compared with the wild-type plants, overexpression of HbWRKY83 improved the tolerance of transgenic Arabidopsis lines to drought and salt stresses by enhancing the expression levels of ethylene-insensitive3 transcription factors (EIN3s) and several stress-responsive genes, including Cu/Zn superoxide dismutases CSD1 (Cu/Zn-SOD1) and CSD2 (Cu/Zn-SOD2), related to reactive oxygen species scavenging. Additionally, these genes were also significantly up-regulated by bark tapping. In combination, these results suggest that HbWRKY83 might act as a positive regulator of rubber production by activating the expression of JA-, ethylene-, and wound-responsive genes in the laticiferous cells of rubber trees.

Association between the number of teeth and frailty among Chinese older adults: a nationwide cross-sectional study.

OBJECTIVES: To explore the association between the number of teeth and frailty among older Chinese adults using a nationally representative sample. DESIGN: Cross-sectional analysis was carried out using the 2014 wave data from the Chinese Longitudinal Healthy Longevity Survey, which used a targeted random-sampling design. SETTING: This research was conducted in communities from nearly half of the counties and cities in 22 out of 31 provinces throughout China. PARTICIPANTS: Of the 6934 interviewees aged ≥65 years, the final analysis included 3635 older adults who had completed the 2014 wave survey on the variables included in the study. PRIMARY AND SECONDARY OUTCOME MEASURES: Outcome variables included frailty, measured by the Frailty Index, and number of teeth. Covariates included demographic characteristics (ie, age, sex, co-residence, marital status, years of education and financial support), body mass index (BMI) and health behaviours (ie, smoking, drinking and exercise). A univariate logistic regression was used to test the factors associated with frailty. A multiple logistic regression model was used, using the frailty score as the dependent variable and the number of teeth together with significant covariates as the independent variables. RESULTS: The prevalence of frailty was 27.68%. The mean number of teeth present was 9.23 (SD=10.03). The multiple logistic regression showed that older adults' demographic variables, health behaviours, BMI, tooth number and chewing pain were significantly associated with frailty. After adjusting for the covariates, older adults with fewer teeth had significantly higher odds of frailty than those with 20 or more teeth (no teeth: OR=2.07, 95% CI 1.53 to 2.80; 1 to 10 teeth: OR=1.77, 95% CI 1.31 to 2.38), except for older adults with 11 to 20 teeth (OR=1.30, 95% CI 0.93 to 1.82). CONCLUSIONS: The presence of fewer teeth is significantly associated with frailty status among older Chinese adults. Future studies are needed to explain the specific mechanisms underlying how oral health status is associated with frailty.

Methods for Expansion of Three-Dimensional Cultures of Human Embryonic Stem Cells Using a Thermoresponsive Polymer.

Human pluripotent stem cells (hPSCs) are viewed as promising candidates for applications in regenerative medicine and therapy due to their proliferative and pluripotent properties. However, obtaining clinically significant numbers of hPSCs remains a limiting factor and impedes their use in therapeutic applications. Conventionally, hPSCs are cultured on two-dimensional surfaces coated with a suitable substrate, such as Matrigel™. This method, however, requires a large surface area to generate sufficient cell numbers to meet clinical needs and is therefore impractical as a manufacturing platform for cell expansion. In addition, the use of enzymes for cell detachment and small molecule inhibitors to increase plating efficiency may impact future cell behavior when used for routine subculturing. In this study, we describe a protocol to generate and maintain hPSC aggregates in a three-dimensional suspension culture by utilizing thermoresponsive nanobridges. The property of the polymer used in the nanobridges enables passaging and expansion through a temperature change in combination with mechanically applied shear to dissociate aggregates; thus, we eliminate the need of enzymes or small molecules for cell dissociation and viability, respectively. Utilizing this platform, maintenance of human embryonic stem cells for three continuous passages demonstrated high expression levels in key pluripotent markers.

Micro-abrasion-corrosion behaviour of a biomedical Ti-25Nb-3Mo-3Zr-2Sn alloy in simulated physiological fluid.

The micro-abrasion-corrosion behaviour of the biomedical Ti-25Nb-3Mo-3Zr-2Sn alloy in Hank׳s solution with protein has been investigated using electrochemical measurements, tribological tests and scanning electron microscope (SEM) observations. The potentiodynamic polarization tests showed that the corrosion potential (Ecorr) exhibits the maximum value at the abrasive concentration of 0.05gcm(-3) despite of the load level. The tribological results indicated that the total material loss of the Ti-25Nb-3Mo-3Zr-2Sn alloy during micro-abrasion increased with the increasing abrasive concentration at a certain applied load. When the abrasive concentration is no more than 0.15gcm(-3), the total material loss increases with increasing load, while the total material loss exhibits the maximum value at a moderate load in case of higher abrasive concentration levels. This was ascribed to the three-body or two-body micro-abrasion-corrosion at different abrasive concentration levels. The wastage map, abrasion mode map and synergy map associated with the applied load and the abrasive concentration were constructed to evaluate the micro-abrasion-corrosion behaviour of the Ti-25Nb-3Mo-3Zr-2Sn alloy in potential biomedical applications.

[The Complete Sequence Analysis of 18 Strains of Coxsackievirus A6 in Guangdong Province of China].

In this study, we examined the complete genome of coxsackievirus A6 (CVA6) from hand, foot, and mouth disease in Guangdong Province from 2013,and explored the genetic similarities and differences in epidemic and non-epidemic stains of CVA6.Eighteen strains of CVA6 were included in complete genome sequencing, and the sequences were subject to phylogenetic analysis,sequence alignment analysis and genetic recombination analysis using the software DNASTAR6.0,MEGA5.2and SimPlot3.5.1.The results showed that the complete genome of 18 Guangdong CVA6strains ranged from 7390bp to 7392bp.No insertions or deletions were detected in the coding region. There were several insertions and deletions in 5'UTR and 3'UTR.Phylogenetic analysis indicated that the nucleotide and amino acid sequence identity between the 18 complete genomes were 90.5%-99.6% and 97.5%-99.9%,respectively.The strains isolated in2013 could be further divided into two clusters, III and IV, while the strains isolated in 2011 were only present in the IV cluster. Genetic recombination analysis revealed that the Guangdong representative strain of CVA6,GD870/2013,had gene recombination in the P2 and P3regions,while the GD839/2013 strain did not show obvious genetic recombination. Genome-wide analysis of CVA6 revealed that there are two possible transmitted chains, III and IV, in epidemic strains from Guangdong Province in 2013.The transmitted chain Ⅲ originated from the strain with genetic recombination in the P2 and P3regions,whichwas completely different from the chain IV. Transmission of chain IV of CVA6 was only observed in the nonepidemic 2011 strain.

[Preparation of interferon-alpha-loaded poly-L-lactic acid lamellar particles].

Lamellar particles(lamellae) were prepared by non-solvent precipitation from crystalic poly-L-lactic acid (PLLA). The PLLA lamellae exhibite a diamond or stepped irregular shape with a size range between 3-5 microns. Prepared without any surfactants and dispersing agents, the lamellar particles have clean surface, which is advantageous for the adsorption of proteins. The PLLA lamellar particles adsorbed protein cytokine interferon-alpha (IFN-alpha) with an adsorption efficiency of > or = 95%. The release of loaded IFN could continue for more than 10 days. The cell incubation experiments showed that the PLLA lamellar particles were easy to be phagocytosed by macrophages. The immunological experiments showed that the biological activity of IFN-alpha loaded on the PLLA lamellar particle was effectively retained.

Inhibition of ROCK-myosin II signaling pathway enables culturing of human pluripotent stem cells on microcarriers without extracellular matrix coating.

Large quantities of human pluripotent stem cells (hPSCs) needed for therapeutic applications can be grown in scalable suspended microcarrier cultures. These microcarriers are coated with animal or human extracellular matrix (ECM) proteins to promote cell growth and maintain pluripotency. However, the coating is costly for large-scale cultures and it presents safety risks. This study demonstrates that hPSCs can be propagated on noncoated positively charged cellulose microcarriers in a serum-free medium containing the ROCK inhibitor, (Y27632) or myosin inhibitor, Blebbistatin. In the presence of these two inhibitors, myosin phosphatase 1 and myosin light chain 2 were dephosphorylated suggesting that reduced myosin contractility is responsible for hPSC survival and growth on ECM coating-free microcarriers. Cells propagated on the noncoated microcarriers for 12 passages maintained their pluripotency and karyotype stability. Scalability was demonstrated by achieving a cell concentration of 2.3×106 cells/mL with 11.5-fold expansion (HES-3) in a 100-mL spinner flask. The differentiation capability of these cells toward three primary lineages is demonstrated via in vitro embryoid bodies and in vivo teratoma formations. Moreover, the directed differentiation to polysialylated neuronal cell adhesion molecule-positive (PSA-NCAM+) neural progenitors produced high cell concentrations (9.1±1.2×106 cells/mL) with a cell yield of 412±77 neural progenitor cells per seeded HES-3 and a PSA-NCAM expression level of 91±1.1%. This defined serum- and coating-free scalable microcarrier culturing system is a safer and less expensive method for generating large amounts of hPSCs for cell therapies.

[Maxillary sinus floor augmentation using gene-enhanced tissue engineered bone].

OBJECTIVE: To evaluate the effect of maxillary sinus elevation with gene-enhanced tissue engineering bone in dogs. METHODS: bone marrow stromal cells (BMSC) derived from dog marrow were cultured, and transduced with the adenovirus carrying bone morphogenetic protein-2 (BMP-2) gene (AdBMP-2), the adenovirus carrying green fluorescent protein (GFP) gene (AdGFP) in vitro. The bone formation ability of gene modified BMSC with scaffold was examined in nude mice and in elevated maxillary sinus of dog. Student's t-test was used for statistical analysis with SPSS 11.0 software package. RESULTS: Gene transfection efficiency reached up to (83.95 ± 2.43)% as demonstrated by GFP expression. Ectopic bone formation was detected in nude mice. As for maxillary sinus floor elevation in a dog model, new bone formation area in the AdBMP-2 gene transduced BMSC with Bio-Oss group was significantly higher than in BMSC with Bio-Oss group at 120 d (P < 0.05). CONCLUSIONS: AdBMP-2 gene transduced BMSC can stimulate ectopic bone formation in nude mice, and promote bone formation and maturation in the dog maxillary sinus.

Expansion of human embryonic stem cells on cellulose microcarriers.

This unit describes the routine maintenance and expansion of undifferentiated human embryonic stem cells (hESC) on cellulose microcarriers. Conventionally, hESCs have been maintained on feeder cells or extracellular matrix-coated two-dimensional tissue culture plates. The expansion of hESC on a tissue culture platform is limited by the available surface area and the requirement of repetitive subculturing to reach the required cell yield. Here, we show that expansion of hESC can be carried out in a three-dimensional suspension culture using Matrigel-coated cellulose microcarriers. hESCs from a tissue culture plate can be seeded directly onto the microcarriers; hESC microcarrier culture is passaged and expanded by mechanical dissociation of the cells without enzyme. Expansion of the culture in a 100-ml spinner flask is also described. Long-term culture of hESC on the microcarriers maintains typical pluripotent markers (OCT-4, Tra-1-60, and SSEA-4) and stable karyotype. Spontaneous differentiations of microcarrier-maintained hESCs in vitro (embryoid body formation) and in vivo (teratoma formation in SCID mouse) have demonstrated formation of the three germ layers. These protocols can also be applied equally well to human induced pluripotent stem cells.