George A. Thouas

Simplified technique for differential staining of inner cell mass and trophectoderm cells of mouse and bovine blastocysts.

Histological staining and counting of blastocyst inner cell mass (ICM) and trophectoderm (TE) cells differentially with chromatin-specific dyes is a more accurate indicator of cultured blastocyst quality and normality than total cell number assessment. The aim of this study was to test the effectiveness of a simplified method of chemically-defined differential blastocyst staining. The TE of cultured mouse and bovine blastocysts of different developmental stages was stained when blastocysts were treated with a permeabilizing solution containing the ionic detergent Triton X-100 and the fluorochrome propidium iodide. Blastocysts were then incubated in a second solution containing 100% ethanol (for fixation) and the secondary fluorochrome bisbenzimide. Fixed and stained whole blastocysts were mounted and assessed for cell number using ultraviolet fluorescent microscopy. Using this method, in-vitro cultured mouse blastocysts (day 4.5) were shown to have an ICM:TE ratio of 1:2.63 with an average total cell count of 75.3 +/- 3. While day 7 and 8 in-vitro produced bovine blastocysts were shown to have an ICM:TE ratio of 1:3.42 and 1:3.36 with an average total cell count of 151.3 +/- 5.48 and 217.8 +/- 8.75 respectively. Blastocyst staining patterns indicate that this modified technique represents a simple and reliable alternative to current bichromatic blastocyst staining techniques for the differential assessment of cell numbers and may be useful for the assessment of blastocysts derived from in-vitro maturation, novel culture systems and advanced reproductive technologies such as cloning.

Somatic cell cloning without micromanipulators.

Until now, micromanipulators have been regarded as indispensable for somatic cell nuclear transfer. This paper describes an improved zona-free nuclear transfer procedure with manual bisection of oocytes, selection of cytoplasts by Hoechst staining, and two-step fusion of somatic cells from primary granulosa cell cultures with two cytoplasts. Blastocyst rates in the three systems tested for zona-free embryo culture were 0%, 18%, and 36% for microdrops, well of the wells (WOW system), and microcapillaries (GO system), respectively. This simple, rapid, and inexpensive procedure may become a useful alternative to the existing techniques for somatic cell nuclear transfer for large-scale application of the technology.

Paper Diagnostic for Instantaneous Blood Typing

Agglutinated blood transports differently onto paper than stable blood with well dispersed red cells. This difference was investigated to develop instantaneous blood typing tests using specific antibody-antigen interactions to trigger blood agglutination. Two series of experiments were performed. The first related the level of agglutination and the fluidic properties of blood on its transport in paper. Blood samples were mixed at different ratios with specific and nonspecific antibodies; a droplet of each mixture was deposited onto a filter paper strip, and the kinetics of wicking and red cell separation were measured. Agglutinated blood phase separated, with the red blood cells (RBC) forming a distinct spot upon contact with paper while the plasma wicked; in contrast, stable blood suspensions wicked uniformly. The second study analyzed the wicking and the chromatographic separation of droplets of blood deposited onto paper strips pretreated with specific and nonspecific antibodies. Drastic differences in transport occurred. Blood agglutinated by interaction with one of its specific antibodies phase separated, causing a chromatographic separation. The red cells wicked very little while the plasma wicked at a faster rate than the original blood sample. Blood agglutination and wicking in paper followed the concepts of colloids chemistry. The immunoglobin M antibodies agglutinated the red blood cells by polymer bridging, upon selective adsorption on the specific antigen at their surface. The transport kinetics was viscosity controlled, with the viscosity of red cells drastically increasing upon blood agglutination. Three arm prototypes were investigated for single-step blood typing.

Mitochondrial Dysfunction in Mouse Oocytes Results in Preimplantation Embryo Arrest In Vitro

Abstract Oocyte mitochondrial dysfunction has been proposed as a cause of high levels of developmental retardation and arrest that occur in human preimplantation embryos generated using assisted reproductive technology in the treatment of some causes of female infertility. To investigate this, a model of mitochondrial dysfunction was developed in mouse oocytes using a method of photosensitization of the mitochondrion-specific dye, rhodamine-123. After in vitro fertilization, dye-loaded and photosensitized oocytes showed developmental arrest in proportion to irradiation time. Morphological and metabolic assessments of zygotes indicated an increase in mitochondrial permeability that subsequently resulted in apoptotic degeneration. Development was partially restored by inhibition of mitochondrial permeability transition pore formation by oocyte pretreatment with cyclosporin A. Oocyte mitochondria are therefore physiological regulators of early embryo development and potential sites of pathological insult that may perturb oocyte and subsequent preimplantation embryo viability. These findings have important implications for the treatment of clinically infertile women using assisted reproductive technologies.

An elastomeric patch derived from poly(glycerol sebacate) for delivery of embryonic stem cells to the heart.

We hypothesize that a combinatorial approach of ventricle constraint and stem cell therapy would offer a greater benefit for the treatment of heart failure than either strategy alone. A heart patch would serve two therapeutic purposes: biomechanical support and cell delivery. In this study, we describe a hybrid heart patch engineered from a synthetic elastomer, poly(glycerol sebacate) (PGS), supplemented with cardiomyocytes differentiated from human embryonic stem cells (hESCs). In line with two therapeutically relevant considerations, i.e. biocompatibility and cell delivery efficiency, the PGS was (a) pre-conditioned in culture medium for 6 days, and (b) prepared without gelatin coatings to facilitate detachment and delivery of cardiomyocytes following patch implantation. Following pre-conditioning under physiological conditions, the PGS patch material without gelatin coating was found to satisfactorily support cardiomyocyte viability and attachment, with active cell beating for periods of longer than 3 months until interrupted. Dynamic culture studies revealed that cells detached more efficiently from the uncoated surface of PGS than from gelatin-coated PGS. No significant differences were detected between the beating rates of human embryonic stem cell-derived cardiomyocytes on tissue culture plate and the pre-conditioned and gelatin-uncoated PGS. PGS patches sutured over the left ventricle of rats in vivo remained intact over a 2 week period without any deleterious effects on ventricular function. We conclude that PGS is a suitable biomaterial for stem cell-based regeneration strategies to restore cardiomyocyte function, and the hybrid heart patch engineered under optimal conditions would be a promising support device for the cardiac repair.

The mechanical characteristics and in vitro biocompatibility of poly(glycerol sebacate)-Bioglass® elastomeric composites

Biodegradable elastomeric materials have gained much recent attention in the field of soft tissue engineering. Poly(glycerol sebacate) (PGS) is one of a new family of elastomers which are promising candidates used for soft tissue engineering. However, PGS has a limited range of mechanical properties and has drawbacks, such as cytotoxicity caused by the acidic degradation products of very soft PGS and degradation kinetics that are too fast in vivo to provide sufficient mechanical support to the tissue. However, the development of PGS/based elastomeric composites containing alkaline bioactive fillers could be a method for addressing these drawbacks and thus may pave the way towards wide clinical applications. In this study, we synthesized a new PGS composite system consisting of a micron-sized Bioglass filler. In addition to much improved cytocompatibility, the PGS/Bioglass composites demonstrated three remarkable mechanical properties. First, contrary to previous reports, the addition of microsized Bioglass increases the elongation at break from 160 to 550%, while enhancing the Youngs modulus of the composites by up to a factor of four. Second, the modulus of the PGS/Bioglass composites drops abruptly in a physiological environment (culture medium), and the level of drop can be tuned such that the addition of Bioglass does not harden the composite in vivo and thus the desired compliance required for soft tissue engineering are maintained. Third, after the abrupt drop in modulus, the composites exhibited mechanical stability over an extended period. This latter observation is an important feature of the new composites, because they can provide reliable mechanical support to damaged tissues during the lag phase of the healing process. These mechanical properties, together with improved biocompatibility, make this family of composites better candidates than plastic and related composite biomaterials for the applications of tissue engineering.

Progress and challenges in biomaterials used for bone tissue engineering: bioactive glasses and elastomeric composites

Driven by the increasing economic burden associated with bone injury and disease, biomaterial development for bone repair represents the most active research area in the field of tissue engineering. This article provides an update on recent advances in the development of bioactive biomaterials for bone regeneration. Special attention is paid to the recent developments of sintered Na-containing bioactive glasses, borate-based bioactive glasses, those doped with trace elements (such as Cu, Zn, and Sr), and novel elastomeric composites. Although bioactive glasses are not new to bone tissue engineering, their tunable mechanical properties, biodegradation rates, and ability to support bone and vascular tissue regeneration, as well as osteoblast differentiation from stem and progenitor cells, are superior to other bioceramics. Recent progresses on the development of borate bioactive glasses and trace element-doped bioactive glasses expand the repertoire of bioactive glasses. Although boride and other trace elements have beneficial effects on bone remodeling and/or associated angiogenesis, the risk of toxicity at high levels must be highly regarded in the design of new composition of bioactive biomaterials so that the release of these elements must be satisfactorily lower than their biologically safe levels. Elastomeric composites are superior to the more commonly used thermoplastic-matrix composites, owing to the well-defined elastic properties of elastomers which are ideal for the replacement of collagen, a key elastic protein within the bone tissue. Artificial bone matrix made from elastomeric composites can, therefore, offer both sound mechanical integrity and flexibility in the dynamic environment of injured bone.

Sex-related physiology of the preimplantation embryo

Male and female preimplantation mammalian embryos differ not only in their chromosomal complement, but in their proteome and subsequent metabolome. This phenomenon is due to a finite period during preimplantation development when both X chromosomes are active, between embryonic genome activation and X chromosome inactivation, around the blastocyst stage. Consequently, prior to implantation male and female embryos exhibit differences in their cellular phenotype. Manifestations of such differences include altered total activity of specific X-linked enzymes and the metabolic pathways they regulate. Subsequently, one would expect to be able to determine differences in the rate of consumption and utilization of specific nutrients between male and female embryos. Data to date on animal models support this, with sex-specific differences in glucose and amino acid utilization being reported for the mouse and cow blastocysts. Such differences in metabolic phenotype may logically be involved in the reported differences in growth rates between preimplantation embryos of different sex. As the fields of proteomics and metabolomics are being increasingly applied to human assisted conception it is prudent to consider how such technologies may be applied to identify sex differences in the human embryo. Such data would have implications far beyond current invasive technologies used to identify the sex of an embryo conceived in vitro for the diagnosis of X-linked diseases.

Fabrication and characterization of sol–gel derived 45S5 Bioglass®–ceramic scaffolds

Although Bioglass® has existed for nearly half a century its ability to trigger bone formation and tuneable degradability is vastly superior to other bioceramics, such as SiO(2)-CaO bioactive glasses. The sol-gel process of producing glass foams is well established for SiO(2)-CaO compositions, but not yet established for 45S5 composites containing Na(2)O. In this work the sol-gel derived 45S5 Bioglass® has for the first time been foamed into highly porous three-dimensional scaffolds using a surfactant, combined with vigorous mechanical stirring and subsequent sintering at 1000°C for 2 h. It was found that the mechanical strength of the sintered sol-gel derived Bioglass® scaffolds was significantly improved, attributable to the small fraction of material on the pore walls. More importantly, the compressive strength of the three-dimensional scaffolds produced by this surfactant foaming method could be predicted using Gibson and Ashbys closed cell model of porous networks. A comparative experiment revealed that ion release from the sol-gel derived Bioglass® foams was faster than that of counterparts produced by the replication technique. In vitro evaluation using osteoblast-like cells demonstrated that the sol-gel derived 45S5 Bioglass foams supported the proliferation of viable cell populations on the surface of the scaffolds, although few cells were observed to migrate into the virtually closed pores within the foams. Further work should be focused on modifications of the reaction conditions or alternative foaming techniques to improve pore interconnection.

Bone tissue engineering scaffolding: computer-aided scaffolding techniques

Tissue engineering is essentially a technique for imitating nature. Natural tissues consist of three components: cells, signalling systems (e.g. growth factors) and extracellular matrix (ECM). The ECM forms a scaffold for its cells. Hence, the engineered tissue construct is an artificial scaffold populated with living cells and signalling molecules. A huge effort has been invested in bone tissue engineering, in which a highly porous scaffold plays a critical role in guiding bone and vascular tissue growth and regeneration in three dimensions. In the last two decades, numerous scaffolding techniques have been developed to fabricate highly interconnective, porous scaffolds for bone tissue engineering applications. This review provides an update on the progress of foaming technology of biomaterials, with a special attention being focused on computer-aided manufacturing (Andrade et al. 2002) techniques. This article starts with a brief introduction of tissue engineering (Bone tissue engineering and scaffolds) and scaffolding materials (Biomaterials used in bone tissue engineering). After a brief reviews on conventional scaffolding techniques (Conventional scaffolding techniques), a number of CAM techniques are reviewed in great detail. For each technique, the structure and mechanical integrity of fabricated scaffolds are discussed in detail. Finally, the advantaged and disadvantage of these techniques are compared (Comparison of scaffolding techniques) and summarised (Summary).