Expansion and homing of umbilical cord blood hematopoietic stem and progenitor cells for clinical transplantation.

The successful expansion of hematopoietic stem and progenitor cells (HSPCs) from umbilical cord blood (UCB) for transplantation could revolutionize clinical practice by improving transplantation-related outcomes and making available UCB units that have suboptimal cell doses for transplantation. New cytokine combinations appear able to promote HSPC growth with minimal differentiation into mature precursors and new agents, such as insulin-like growth factor-binding protein 2, are being used in clinical trials. Molecules that simulate the HSPC niche, such as Notch ligand, have also shown promise. Further improvements have been made with the use of mesenchymal stromal cells, which have made possible UCB expansion without a potentially deleterious prior CD34/CD133 cell selection step. Chemical molecules, such as copper chelators, nicotinamide, and aryl hydrocarbon antagonists, have shown excellent outcomes in clinical studies. The use of bioreactors could further add to HSPC studies in future. Drugs that could improve HSPC homing also appear to have potential in improving engraftment times in UCB transplantation. Technologies to expand HSPC from UCB and to enhance the homing of these cells appear to have attained the goal of accelerating hematopoietic recovery. Further discoveries and clinical studies are likely to make the goal of true HSPC expansion a reality for many applications in future.

Retinal vascular fractal dimension is associated with cognitive dysfunction.

Fractal analysis is a method used to quantify the geometric branching complexity and density of retinal vessels. This study examined the relationship of retinal vascular fractal dimension and other retinal vascular parameters with cognitive dysfunction in an older Asian population. Subjects aged 60 years and older from the Singapore Malay Eye Study were selected for analysis. Retinal vascular fractal dimension (Df) and other quantitative retinal vascular parameters (branching angle, tortuosity, and caliber) were measured based on a standardized grading protocol from photographs of the retinal fundus using a computer-assisted program. Qualitative retinal signs were also assessed from photographs. Cognitive dysfunction was defined as a locally validated Abbreviated Mental Test (AMT) score ≤6/10 in participants with 0-6 years of formal education and an AMT score ≤8/10 in those with more than 6 years of formal education. Cognitive dysfunction was identified in 262 of the 1202 participants (21.8%). Decreased retinal vascular Df was significantly associated with lower AMT score (P = .019). In multivariate logistic regression analysis, participants with lower retinal vascular Df values were more likely to have cognitive dysfunction (odds ratio, 1.71; 95% confidence interval, 1.03-2.82, comparing the lowest and highest Df quintiles). In subgroup analysis stratified for cardiovascular risk factors, this association was present in participants with hypertension and current smokers. Other retinal vascular signs were not associated with cognitive dysfunction. Decreased retinal vascular Df is associated with cognitive dysfunction in older persons. Rarefaction of the retinal vasculature may reflect similar changes in the cerebral microvasculature that may contribute to cognitive deterioration.

Mesenchymal stromal cell supported umbilical cord blood ex vivo expansion enhances regulatory T cells and reduces graft versus host disease.

BACKGROUND AIMS: Double cord blood transplantation (DCBT) may shorten neutrophil and platelet recovery times compared with standard umbilical cord blood transplantation. However, DCBT may be associated with a higher incidence of graft versus host disease (GVHD). In this study, we explored the effect of ex vivo expansion of a single cord blood unit (CBU) in a DCBT setting on GVHD and engraftment. METHODS: Post-thaw cryopreserved CBUs from cord blood banks, hereinafter termed "banked" CBUs, were co-cultured with confluent bone marrow mesenchymal stromal cells (MSCs) supplemented with a cytokine cocktail comprising 100 ng/mL stem cell factor, 50 ng/mL flt3-ligand, 100 ng/mL thrombopoietin and 20 ng/mL insulin-like growth factor binding protein 2 for 12 days. RESULTS: When DCBT of one unexpanded and one expanded CBU was performed in non-obese diabetic/severe combined immunodeficient-IL2Rgamma(null) (NOD/SCID-IL2γ(-/-), NSG) mice, the expanded CBU significantly boosted in vivo hematopoiesis of the unexpanded CBU. The median survival of NSG mice was significantly improved from 63.4% (range, 60.0-66.7%) for mice receiving only unexpanded units to 86.5% (range, 80.0-92.9%) for mice receiving an expanded unit (P < 0.001). The difference in survival appeared to be due to a lower incidence of GVHD in the mice receiving expanded cells. This effect on GVHD was mediated by a significant increase in regulatory T cells seen in the presence of MSC co-culture. CONCLUSIONS: MSC-supported ex vivo expansion of "banked" CBU boosted unexpanded CBU hematopoiesis in vivo, increased regulatory T cell content and decreased the incidence of GVHD.

Development of a chitosan-based wound dressing with improved hemostatic and antimicrobial properties.

Hemorrhage remains a leading cause of early death after trauma, and infectious complications in combat wounds continue to challenge caregivers. Although chitosan dressings have been developed to address these problems, they are not always effective in controlling bleeding or killing bacteria. We aimed to refine the chitosan dressing by incorporating a procoagulant (polyphosphate) and an antimicrobial (silver). Chitosan containing different amounts and types of polyphosphate polymers was fabricated, and their hemostatic efficacies evaluated in vitro. The optimal chitosan-polyphosphate formulation (ChiPP) accelerated blood clotting (p = 0.011), increased platelet adhesion (p=0.002), generated thrombin faster (p = 0.002), and absorbed more blood than chitosan (p < 0.001). Silver-loaded ChiPP exhibited significantly greater bactericidal activity than ChiPP in vitro, achieving a complete kill of Pseudomonas aeruginosa and a > 99.99% kill of Staphylococcus aureus consistently. The silver dressing also significantly reduced mortality from 90% to 14.3% in a P. aeruginosa wound infection model in mice. Although the dressing exerted severe cytotoxicity against cultured fibroblasts, wound healing was not inhibited. This study demonstrated for the first time, the application of polyphosphate as a hemostatic adjuvant, and developed a new chitosan-based composite with potent hemostatic and antimicrobial properties.

Inducing hepatic differentiation of human mesenchymal stem cells in pellet culture.

Extensive cell-cell or cell-matrix interaction in three-dimensional (3D) culture is important for the maintenance of adult hepatocyte function and the maturation of hepatic progenitors. However, although there is significant interest in inducing the transdifferentiation of adult stem cells into the hepatic lineage, very few studies have been conducted in a 3D culture configuration. The aim of this study is to investigate the differentiation of mesenchymal stem cells (MSC) into hepatocytes in a pellet configuration, with or without the presence of small intestinal submucosa (SIS). After 4 weeks of differentiation with growth factors bFGF, HGF, and OsM, we obtained hepatocyte-like cells that expressed a subset of hepatic genes, secreted albumin and urea, stored glycogen, and showed inducible CYP3A4 mRNA levels. When these cells were implanted into livers of hepatectomized rats, they secreted human albumin into the bloodstream. The hepatic differentiation of MSC was faster in cell pellets without SIS. The plausible explanations for this finding may be related to the mass transport issues of the two different pellets and the role of cell-cell contact over cell-matrix interactions. The findings of this study should help in the design of optimal culture configurations for efficient hepatic differentiation of adult stem cells.

Hepatic differentiation potential of commercially available human mesenchymal stem cells.

The ready availability and low immunogenicity of commercially available mesenchymal stem cells (MSC) render them a potential cell source for the development of therapeutic products. With cell source a major bottleneck in hepatic tissue engineering, we investigated whether commercially available human MSC (hMSC) can transdifferentiate into the hepatic lineage. Based on previous studies that find rapid gain of hepatic genes in bone marrow-derived stem cells cocultured with liver tissue, we used a similar approach to drive hepatic differentiation by coculturing the hMSC with rat livers treated or untreated with gadolinium chloride (GdCl(3)). After a 24-hour coculture period with liver tissue injured by GdCl(3) in a Transwell configuration, approximately 34% of the cells differentiated into albumin-expressing cells. Cocultured cells were subsequently maintained with growth factors to complete the hepatic differentiation. Cocultured cells expressed more hepatic gene markers, and had higher metabolic functions and P450 activity than cells that were only differentiated with growth factors. In conclusion, commercially available hMSC do show hepatic differentiation potential, and a liver microenvironment in culture can provide potent cues to accelerate and deepen the differentiation. The ability to generate hepatocyte-like cells from a commercially available cell source would find interesting applications in liver tissue engineering.

Use of Valacyclovir for the treatment of cytomegalovirus antigenemia after hematopoietic stem cell transplantation.

BACKGROUND: Valacyclovir has been used for prophylaxis against cytomegalovirus (CMV) infection after hematopoietic stem cell transplantation (HSCT). We investigated the efficacy and safety of high-dose Valacyclovir as pre-emptive therapy in patients with CMV antigenemia after HSCT. METHODS: In a retrospective single center study of 61 patients, we compared the rates of viral clearance, recurrent antigenemia and adverse events in patients with pp65 CMV antigenemia who received high dose Valacyclovir (n = 15), Valganciclovir (n = 16), and Foscarnet (n = 30). RESULTS: Overall, 60/61 (98 %) of cases achieved CMV antigenemia clearance by day 28, and no patient developed CMV disease. After adjusting for age, sex, diagnosis, CMV serological status, donor type, CMV antigen level, graft-versus-host disease (GVHD) therapy, and conditioning regimen, there were no significant differences in the rates of viral clearance at day 14 in patients who received Valganciclovir (0.18, 95 % confidence interval (CI) 0.01 to 2.15, p = 0.17) and Foscarnet (OR 0.22, 95 % CI 0.03 to 2.40, p = 0.22), compared with Valacyclovir (assigned OR = 1.00). Recurrent antigenemia by day 180 after clearance of the initial CMV episode occurred in 34/61 (56 %) of patients. Using the multivariate model adjusting for the same covariates, there were also no significant differences in secondary episodes of CMV between treatment groups. With regards to adverse effect monitoring, Foscarnet led to significantly increased creatinine levels (P = 0.009), while Valganciclovir led to significant decrease in neutrophil counts (P = 0.012). CONCLUSION: High dose Valacyclovir is a potential alternative to Valganciclovir and Foscarnet in the stable post-HSCT patient who has cytopenia and is not keen for inpatient treatment of CMV antigenemia.

Measurement of macular fractal dimension using a computer-assisted program.

PURPOSE: Macular diseases may be associated with an altered retinal vasculature. We describe and test new software for the measurement of retinal vascular fractal dimension to quantify the complexity of retinal vasculature at the macula (D mac) and to compare this with fractal dimension measured around the optic disc (D disc). METHODS: A total of 342 macular-centered and optic disc-centered digital retinal photographs from 171 subjects was selected randomly from a population-based study. Retinal vascular fractional dimension (Df) was measured by two trained graders using a computer-assisted program (SIVA-FA, software version 1.0, National University of Singapore) on macula-centered (D mac) and optic disc-centered (D disc) photographs, to assess intergrader reliability. Measurements were repeated after two weeks to determine intragrader reliability. A separate 50 pairs of consecutively repeated images were selected and measured using SIVA-FA to assess intrasession reliability. Reliability analyses were conducted using intraclass correlation coefficients (ICC), and multiple linear regression analyses were performed to compare factors associated with D mac and D disc measurements. RESULTS: The mean (SD) D mac and D disc values were 1.453 (0.060) and 1.484 (0.043), respectively, and were highly correlated (r = 0.70, P < 0.001). Intragrader, intergrader, and intrasession reliability for both Df measures was high (ICCs ranging from 0.88-0.99). In multiple regression analyses, age (both ß = -0.03, P < 0.001) and hypertension (ß = -0.02, P = 0.011; ß = -0.02, P = 0.021, respectively) were independently associated with D mac and D disc. CONCLUSIONS: The complexity of the retinal vasculature in the macula can be measured reliably and may be a useful tool to study parafoveal vascular networks in macula diseases, such as diabetic maculopathy.

Myopia and cognitive dysfunction: the singapore malay eye study.

PURPOSE: To investigate a possible relationship between refractive error and cognitive function. METHODS: This population-based, cross-sectional study included 1032 persons aged 60 to 79 who participated in the Singapore Malay Eye Study. Refraction (sphere, cylinder, and axis) was measured using an autorefractor, and spherical equivalent was defined as sphere plus half negative cylinder. Refractive errors were defined as myopia (spherical equivalent < -0.5), emmetropia (-0.5 diopter [D] ≤ spherical equivalent ≤ 0.5 D), and hyperopia (spherical equivalent > 0.5 D). Visual acuity was measured with a logMAR chart. Cognitive dysfunction, assessed using the Abbreviated Mental Test, was defined based on education-specific cutoff values. RESULTS: Compared with individuals with emmetropia, persons with myopia were almost twice as likely to have cognitive dysfunction (odds ratio 1.82; 95% confidence interval 1.05-3.15), after adjusting for age, sex, body mass index, income, education, and hours of reading and writing per day. Hyperopia was not associated with cognitive dysfunction. The association remained significant after further adjustment for uncorrected refractive errors or best-corrected visual acuity. CONCLUSIONS: Our results provide evidence on a novel association between myopia and cognitive dysfunction.

Polycaprolactone-based fused deposition modeled mesh for delivery of antibacterial agents to infected wounds.

Infections represent a significant source of site morbidity following tissue trauma. Scarring and tissue adhesion remain the challenging issues yet to be solved. Prolonged inflammation and morphology of the re-epithelisated layer are important considerations. We hypothesized that the solution lies not only in the biochemistry of biomaterial but also the micro-architecture of the scaffold used as the matrix for wound healing. Targeted delivery of antibiotics may provide an efficacious means of infection control through adequate release. Here, we study the use of 3-dimensional polycaprolactone-tricalcium phosphate (PCL-TCP) mesh for the delivery of gentamicin sulphate (GS) fabricated using a solvent-free method. PCL-TCP meshes incorporated with varying loads of GS were evaluated in vitro for elution profile, antimicrobial efficacy and cytotoxicity. Results showed that PCL-TCP meshes incorporated with 15 wt% GS (PT15) efficiently eliminate bacteria within 2 h and demonstrate low cytotoxicity. Subsequently, PT15 meshes were evaluated using an infected full thickness wound mice model, and observed to eliminate bacteria in the wounds effectively. Additionally, mice from the PT15 treatment group (TG) showed no observable signs of overall infection through neutrophil count by day 7 and displayed efficient wound healing (94.2% wound area reduction) by day 14. Histology also showed significantly faster healing in TG through neo-collagen deposition and wound re-epithelisation. The meshes from TG were also observed to be expelled from wounds while gauze fibers from CG were integrated into wounds during healing.