Osteogenic differentiation of human bone marrow-derived mesenchymal stem cells is enhanced by an aragonite scaffold.

Bone graft substitutes and bone void fillers are predominantly used to treat bone defects and bone fusion in orthopaedic surgery. Some aragonite-based scaffolds of coralline exoskeleton origin exhibit osteoconductive properties and are described as useful bone repair scaffolds. The purpose of this study was to evaluate the in vitro osteogenic potential of the bone phase of a novel aragonite-based bi-phasic osteochondral scaffold (Agili-C™, CartiHeal Ltd.) using adult human bone marrow-derived mesenchymal stem cells (MSCs). Analyses were performed at several time intervals: 3, 7, 14, 21, 28 and 42 days post-seeding. Osteogenic differentiation was assessed by morphological characterisation using light microscopy after Alizarin red and von Kossa staining, and scanning electron microscopy. The transcript levels of alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX2), bone gamma-carboxyglutamate (BGLAP), osteonectin (SPARC) and osteopontin (SPP1) were determined by quantitative PCR. Proliferation was assessed by a thymidine incorporation assay and proliferating cell nuclear antigen (PCNA) immunocytochemistry. Our results demonstrate that the bone phase of the bi-phasic aragonite-based scaffold supports osteogenic differentiation and enhanced proliferation of bone marrow-derived MSCs at both the molecular and histological levels. The scaffold was colonized by differentiating MSCs, suggesting its suitability for incorporation into bone voids to accelerate bone healing, remodelling and regeneration. The mechanism of osteogenic differentiation involves scaffold surface modification with de novo production of calcium phosphate deposits, as revealed by energy dispersive spectroscopy (EDS) analyses. This novel coral-based scaffold may promote the rapid formation of high quality bone during the repair of osteochondral lesions.

Long-term viability and function of transplanted islets macroencapsulated at high density are achieved by enhanced oxygen supply.

Transplantation of encapsulated islets can cure diabetes without immunosuppression, but oxygen supply limitations can cause failure. We investigated a retrievable macroencapsulation device wherein islets are encapsulated in a planar alginate slab and supplied with exogenous oxygen from a replenishable gas chamber. Translation to clinically-useful devices entails reduction of device size by increasing islet surface density, which requires increased gas chamber pO2. Here we show that islet surface density can be substantially increased safely by increasing gas chamber pO2 to a supraphysiological level that maintains all islets viable and functional. These levels were determined from measurements of pO2 profiles in islet-alginate slabs. Encapsulated islets implanted with surface density as high as 4,800 islet equivalents/cm3 in diabetic rats maintained normoglycemia for more than 7 months and provided near-normal intravenous glucose tolerance tests. Nearly 90% of the original viable tissue was recovered after device explantation. Damaged islets failed after progressively shorter times. The required values of gas chamber pO2 were predictable from a mathematical model of oxygen consumption and diffusion in the device. These results demonstrate feasibility of developing retrievable macroencapsulated devices small enough for clinical use and provide a firm basis for design of devices for testing in large animals and humans.

The efficacy of an immunoisolating membrane system for islet xenotransplantation in minipigs.

Developing a device that protects xenogeneic islets to allow treatment and potentially cure of diabetes in large mammals has been a major challenge in the past decade. Using xenogeneic islets for transplantation is required in light of donor shortage and the large number of diabetic patients that qualify for islet transplantation. Until now, however, host immunoreactivity against the xenogeneic graft has been a major drawback for the use of porcine islets. Our study demonstrates the applicability of a novel immunoprotective membrane that allows successful xenotransplantation of rat islets in diabetic minipigs without immunosuppressive therapy. Rat pancreatic islets were encapsulated in highly purified alginate and integrated into a plastic macrochamber covered by a poly-membrane for subcutaneous transplantation. Diabetic Sinclair pigs were transplanted and followed for up to 90 days. We demonstrated a persistent graft function and restoration of normoglycemia without the need for immunosuppressive therapy. This concept could potentially offer an attractive strategy for a more widespread islet replacement therapy that would restore endogenous insulin secretion in diabetic patients without the need for immunosuppressive drugs and may even open up an avenue for safe utilization of xenogeneic islet donors.

Enhanced oxygen supply improves islet viability in a new bioartificial pancreas.

The current epidemic of diabetes with its overwhelming burden on our healthcare system requires better therapeutic strategies. Here we present a promising novel approach for a curative strategy that may be accessible for all insulin-dependent diabetes patients. We designed a subcutaneous implantable bioartificial pancreas (BAP)-the "ß-Air"-that is able to overcome critical challenges in current clinical islet transplantation protocols: adequate oxygen supply to the graft and protection of donor islets against the host immune system. The system consists of islets of Langerhans immobilized in an alginate hydrogel, a gas chamber, a gas permeable membrane, an external membrane, and a mechanical support. The minimally invasive implantable device, refueled with oxygen via subdermally implanted access ports, completely normalized diabetic indicators of glycemic control (blood glucose intravenous glucose tolerance test and HbA1c) in streptozotocin-induced diabetic rats for periods up to 6 months. The functionality of the device was dependent on oxygen supply to the device as the grafts failed when oxygen supply was ceased. In addition, we showed that the device is immuno-protective as it allowed for survival of not only isografts but also of allografts. Histological examination of the explanted devices demonstrated morphologically and functionally intact islets; the surrounding tissue was without signs of inflammation and showed visual evidence of vasculature at the site of implantation. Further increase in islets loading density will justify the translation of the system to clinical trials, opening up the potential for a novel approach in diabetes therapy.

Improvement of islet function in a bioartificial pancreas by enhanced oxygen supply and growth hormone releasing hormone agonist.

Islet transplantation is a feasible therapeutic alternative for metabolically labile patients with type 1 diabetes. The primary therapeutic target is stable glycemic control and prevention of complications associated with diabetes by reconstitution of endogenous insulin secretion. However, critical shortage of donor organs, gradual loss in graft function over time, and chronic need for immunosuppression limit the indication for islet transplantation to a small group of patients. Here we present a promising approach to address these limitations by utilization of a macrochamber specially engineered for islet transplantation. The s.c. implantable device allows for controlled and adequate oxygen supply and provides immunological protection of donor islets against the host immune system. The minimally invasive implantable chamber normalized blood glucose in streptozotocin-induced diabetic rodents for up to 3 mo. Sufficient graft function depended on oxygen supply. Pretreatment with the growth hormone-releasing hormone (GHRH) agonist, JI-36, significantly enhanced graft function by improving glucose tolerance and increasing ß-cell insulin reserve in rats thereby allowing for a reduction of the islet mass required for metabolic control. As a result of hypervascularization of the tissue surrounding the device, no relevant delay in insulin response to glucose changes has been observed. Consequently, this system opens up a fundamental strategy for therapy of diabetes and may provide a promising avenue for future approaches to xenotransplantation.

Electrochemical lab on a chip for high-throughput analysis of anticancer drugs efficiency.

We describe a new method for rapid, sensitive, and high-throughput detection of colon cancer cells' response to differentiation therapy, using a novel electrochemical lab-on-a-chip system. Differentiation-inducing agents such as butyric acid and its derivatives were introduced to miniature colon cancer samples within the nanovolume chip chambers. The efficacy of each of the differentiation-inducing agents was evaluated by electrochemical detection of the cellular enzymatic activity level, whereas reappearance of normal enzymatic activity denotes effective therapy. The results demonstrate the ability to evaluate simultaneously multiplex drug effects on miniature tumor samples (approximately 15 cells) rapidly (5 minutes) and sensitively, with quantitative correlation between cancer cells' number and the induced current. The use of miniature analytical devices is of special interest in clinically relevant samples, in that it requires less tissue for diagnosis, and enables high-throughput analysis and comparison of various drug effects on one small tumor sample, while maintaining uniform biological and environmental conditions.

Genetically engineered pfabA pfabR bacteria: an electrochemical whole cell biosensor for detection of water toxicity.

We describe here a bacterial sensor for electrochemical detection of toxic chemicals. The sensor constitutes recombinant bacteria harboring plasmids encoding the fabA and fabR genes and has high-resolution amperometric response to membrane-damaging chemicals. For example, it can detect phenol at concentrations ranging between 1.6 and 16 ppm within 20 min. The high sensitivity is achieved by using the fabA promoter fused to a reporter gene-encoded beta-galactosidase on a low copy number plasmid, under the control of the FabR repressor. The use of electrochemical whole cell sensors enables sensitive, fast, easy to operate, and cost-effective detection of water toxicity threats.

Novel integrated electrochemical nano-biochip for toxicity detection in water.

An electrochemical nano-biochip for water toxicity detection is presented. We describe chip design, fabrication, and performance. Bacteria, which have been genetically engineered to respond to environmental stress, act as a sensor element and trigger a sequence of processes, which leads to generation of electrical current. This novel, portable and miniature device provides rapid and sensitive real-time electrochemical detection of acute toxicity in water. A clear signal is produced within less than 10 min of exposure to various concentrations of toxicants, or to stress conditions, with a direct correlation between the toxicant concentration and the induced current.

Electrochemical detection of xenoestrogenic and antiestrogenic compounds using a yeast two-hybrid-17-beta-estradiol system.

The goal of this study was to determine the effects of various compounds on the 17-beta-estradiol-induced dimerization of the human estrogen receptor alpha (hERalpha), a nuclear transcription factor. For this purpose, we used a modified yeast two-hybrid (YTH) bioassay designed to study protein-protein interactions, based on the electrochemical monitoring of hERalpha dimerization and detected as beta-D-galactosidase reporter gene activity in a synthetic substrate p-aminophenyl-beta-D-galactopyranoside (pAPG). Compared with 17-beta-estradiol activity, genistein, bisphenol-A (BPA), and naringenin induced dimerization to a lower extent by four, five and six magnitudes of orders of magnitude, respectively. In the presence of physiological concentrations of 17-beta-estradiol, both tamoxifen and the analgesic drug acetaminophen inhibited hER dimerization in an antiestrogenic manner.

Electrochemical phagemid assay for the specific detection of bacteria using Escherichia coli TG-1 and the M13KO7 phagemid in a model system.

We describe a reporter phagemid system for the specific amperometric detection of bacteria. We constructed a phagemid a bacteriophage containing a bacterial plasmid using the M13KO7 helper phage and a commercial plasmid, pFLAG-ATS-BAP, which contains a gene encoding for a reporter enzyme, alkaline phosphatase. In the bacteria, the enzyme reacts with the substrate, p-aminophenyl phosphate, in the periplamic space that separates the outer plasma membrane from the cell wall. Thus, the activity of the reporter enzyme can be measured directly in an electrochemical cell without further treatment. The product of the enzymatic activity, p-aminophenol, diffuses out and is oxidized at the working electrode with an applied potential of 220 mV vs the reference electrode Ag/AgCl. The lower detection limit was 1 cfu/mL E. coli TG1 in less than 3 h in a very specific manner. The use of plasmid alkaline phosphatase as the reporter increased the sensitivity by 10-fold over our earlier electrochemical lytic phage method. Such a system can be used for the rapid detection of any strain of bacteria using the appropriate bacteriophage and reporter gene.