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PubMed1991

Application of multilayer films to molecular sensors: some examples of bioengineering at the molecular level.

The Langmuir-Blodgett technique is now a well established method for producing ultra-thin organic films on solid surfaces. These layers could find wide ranging uses in electronic and optoelectronic devices, or even as the basis of artificial biological systems. In this paper the film deposition process is briefly reviewed. Examples of multilayers incorporating biological molecules are presented and possible applications for such films in sensing structures are briefly discussed.

Adsorption↗
PubMed1994

Bioengineered soluble HLA-B7. Genesis, characterization, and occurrence of dimerization.

A soluble, secreted form of HLA-B7 was engineered by replacing the exons encoding the transmembrane and cytoplasmic domains of the B7 gene with a CI. The modified gene, gsB7, transfected into J27.2 or C1R cell lines, produced a secreted protein, sB7, serologically recognized as B7. Size fractionation showed one species of sB7 at the approximately 55 kD expected for an sB7 alpha-chain-beta 2m heteroduplex, and another at approximately 120 kD which had the same constituent chains and was a dimer of the 55-kD species. Dimer formation appeared to be related to protein concentration but not to disulfide bridging. The sB7 heavy chain on SDS-PAGE showed a doublet at approximately 39 and approximately 42 kD; enzyme analysis indicated that the two bands differed only by a carboxyl terminal polypeptide. Analysis of gsB7 transfectants' mRNA by Northern blots and PCR revealed message fully spliced or with retained CI, accounting for the 39- and 42-kD bands, respectively, and apparently untranslated message with I3 retained. sB7 was not detectable on the surface of gsB7 transfectants by CTLs, nor did it inhibit those CTLs. Production of the sB7 protein provides a ready, consistent source of soluble class I antigen for further study, including test materials for tolerogenicity studies in animal models.

Amino Acid Sequence↗
PubMed2006

Bioengineered cardiac cell sheet grafts have intrinsic angiogenic potential.

Previously, we have demonstrated the long-term survival of myocardial cell sheet constructs in vivo, with microvascular network formation throughout the engineered tissues. The understanding and control of these vascularization processes are a key factor for creating thicker functional tissues. Here, we show that cardiac cell sheets express angiogenesis-related genes and form endothelial cell networks in culture. After non-invasive harvest and stacking of cell sheets using temperature-responsive culture dishes, these endothelial cell networks are maintained and result in neovascularization upon in vivo transplantation. Interestingly, we also discovered that all of the graft vessels are derived from the grafts themselves and these vessels migrate to connect with the host vasculature. Finally, blood vessel formation within the grafts can be controlled by changing the ratio of endothelial cells. In conclusion, myocardial tissue grafts engineered with cell sheet technology have their own inherent potential for the in vivo neovascularization that can be regulated in vitro.

Animals↗
PubMed2005

Structural characterization of bioengineered human corneal endothelial cell sheets fabricated on temperature-responsive culture dishes.

For the purpose of corneal regenerative medicine, we fabricated human corneal endothelial cell sheets on temperature-responsive dishes, which could be non-invasively harvested as intact, transplantable sheets by simply reducing the culture temperature. Cells demonstrated hexagonal cell shape with numerous microvilli and cilia, and also exhibited abundant cytoplasmic organelles similar to these cells in vivo. Immunofluorescence for type IV collagen and fibronectin revealed that abundant extracellular matrix (ECM) was deposited on the basal surface throughout culture, and the deposited ECM was harvested along with the cell sheets by reducing culture temperature to 20 degrees C. Faint ECM remnants were observed on the dish surfaces after cell sheet detachment. Immunofluorescence for ZO-1 showed that tight junctions were established between cells, and immunoblotting indicated that intact ZO-1 was maintained during cell sheet harvest, while conventional proteolytic cell harvest methods resulted in the degradation of ZO-1. These results suggest that these transplantable corneal endothelial cell sheets can be applied to treat patients with damaged corneas.

Cell Culture Techniques↗
PubMed2004

Histological assessment of bioengineered new bone in repairing osteoperiosteal mandibular defects in sheep using recombinant human bone morphogenetic protein-7.

UNLABELLED: Numerous experimental studies have been published about osteoinductive bone morphogenetic proteins (BMPs). However, to our knowledge there has been no detailed histological study of a mandibular defect in a large mammal, reconstructed using BMPs. We describe here the histological features of rhBMP-7-induced bone in mandibular defects in sheep. METHODS: A 35 mm osteoperiosteal defect was created at the parasymphyseal region of the mandible in six adult sheep. The continuity of the mandible was maintained using a bony plate, and rhBMP-7 was applied on a type I collagen carrier. Bone labels were injected at selected time intervals during the follow-up period. The animals were killed after 3 months and bone samples were examined histologically, histomorphometrically, and by fluorescence microscopy. RESULTS AND CONCLUSIONS: We found a mixture of woven and lamellar bone that contained many cells with large nuclei. This had not reorganised to form cortical bone and the rhBMP-7-induced bone was more porous than the native bone. The newly-formed bone restored both endosteal and periosteal layers. rhBMP-7-induced bone was biocompatible and induced no ossification of soft tissue or abnormal growth of nearby vital structures. The mineral apposition rate was 1.98 microm/day (range 0.62-5.63 microm/day), a value close to that reported in humans. This suggests that BMPs have a limited effect in accelerating the rate of mineralisation, but promote the pre-mineralisation processes, and perhaps the formation of woven bone.

Animals↗
PubMedDate not supplied

CD117-positive cells and mast cells in adult human cardiac valves--observations and implications for the creation of bioengineered grafts.

BACKGROUND: There is no report to date of stem cells in human cardiac valves. We examined their possible presence, number, and distribution in valves removed at cardiac surgery from patients with a variety of underlying valve pathologies. METHODS: Grossly normal aortic and mitral valves were obtained from live heart transplant patients. Surgically excised valves with rheumatic mitral stenosis, aortic valve age-related degeneration, aortic valve changes of aortoannular ectasia, and mitral valves with myxomatous degeneration were studied. Immunohistochemical and histochemical studies were performed on sequential valve sections, including hematoxylin and eosin, hematoxylin phloxine saffron, Movat pentachrome, toluidine blue, CD31, CD34, and CD117. RESULTS: There were small clusters of CD117-positive cells in the fibrosa and spongiosa of mitral and aortic valves from all groups of valves. Sequential sectioning and staining showed that almost all of these cells were mast cells. However, in the mitral myxomatous valves and the mitral rheumatic valves, there were rare CD117-positive cells that did not have corresponding toluidine blue staining and thus could be valve mesenchymal stem cells. CONCLUSIONS: Most of the CD117-positive cells in normal and diseased adult heart valves are mast cells. These valve cells could play a role in valve pathology and injury. A very small number of possible valve stem cells were also identified. It is unlikely that these valve stem cells are sufficient in number to allow isolation and expansion for tissue engineering purposes.

Bioprosthesis↗
PubMed2005

Ovine panel reactive antibody assay of HLA responsivity to allograft bioengineered vascular scaffolds.

BACKGROUND: Increasing evidence implicates immune response as a contributing factor in the failure of allograft valve transplants. Increases in panel reactive antibodies have been identified in human subjects. To correlate these responses with novel preimplantation processing methods to reduce cellularity, both a relevant panel reactive antibody assay and a chronic implantation animal model are necessary. We modified a human flow cytometric panel reactive antibody assay for ovine model use to detect antibody responses to residual antigen-loading decellularized scaffolds engineered from pulmonary artery tissue. METHODS: A clinical panel reactive antibody assay was modified with anti-sheep antibodies. Dimethyl sulfoxide cryopreserved (n = 4) and decellularized scaffolds (n = 8) fashioned as patches from pulmonary arteries were implanted for study. Fresh (nonprocessed) tissue implants were used as positive controls (n = 2), and sham-treated animals were used as negative controls (n = 2). Baseline, 10-week, and 20-week blood samples were assayed for panel reactive antibody levels. Immunohistochemistry with anti-major histocompatibility complex antibodies were performed on preimplantation scaffolds. RESULTS: Chronic implants of fresh tissue stimulated strong panel reactive antibody responses. Classically cryopreserved tissues provoked modest panel reactive antibody responses to major histocompatibility complex I antigen and no response to major histocompatibility complex II antigen. Decellularized tissue scaffolds provoked minimal to no panel reactive antibody responses to either major histocompatibility complex I or II antigen. Immunohistochemistry correlated with the panel reactive antibody results by identifying significant amounts of major histocompatibility complex I and II in fresh tissue, reduced antigen staining in cryopreserved control tissues, and minimal amounts in decellularized tissues. CONCLUSIONS: These studies with an ovine modified panel reactive antibody assay confirmed minimal immune allosensitization to transplanted decellularized tissue patches. Qualifying criteria for putative tissue-engineered scaffolds should include minimal recipient panel reactive antibody response.

Analysis of Variance↗
PubMed2005

Measuring contact area, force, and pressure for bioengineering applications: using Fuji Film and TekScan systems.

The goal of this study was to compare the TekScan I-Scan Pressure Measurement System with two methods of analysis involving the Fuji Film Prescale Pressure Measuring System in estimating area, force and pressure. Fuji Film and TekScan sensors were alternately placed between a cylindrical peg and a finely ground steel base plate, and compressed with known forces. All Fuji stains were digitally scanned and analyzed. The Erase method of Fuji Film analysis consisted of manually removing portions of the image judged by the user to be outside the perimeter of the stain. The second method of Fuji Film analysis, termed the Threshold method, used the threshold tool to analyze only those pixels that were stained from loading. The TekScan system utilized special matrix-based sensors interfaced with a Windows compatible desktop computer that was equipped with specialized data acquisition hardware and analysis software. The data from this study did not support the hypothesis that all three methods would have accuracies within +/-5% of a known value, when estimating area, force and pressure. Specifically, the TekScan system was found to be more accurate than either of the Fuji Film methods when estimating area and pressure.

Biomedical Engineering↗
PubMed2006

A novel human detoxification system based on nanoscale bioengineering and magnetic separation techniques.

We describe the conceptual approach, theoretical background and preliminary experimental data of a proposed platform technology for specific and rapid decorporation of blood-borne toxins from humans. The technology is designed for future emergent in-field or in-hospital detoxification of large numbers of biohazard-exposed victims; for example, after radiological attacks. The proposed systems is based on nanoscale technology employing biocompatible, superparamagnetic nanospheres, which are functionalized with target-specific antitoxin receptors, and freely circulate within the human blood stream after simple intravenous injection. Sequestration of the blood-borne toxins onto the nanosphere receptors generates circulating nanosphere-toxin complexes within a short time interval; mathematical modeling indicates prevailing of unbound nanosphere receptors over target toxin concentrations at most therapeutic injection dosages. After a toxin-specific time interval nanosphere-toxin complexes are generated within the blood stream and, after simple arterial or venous access, the blood is subsequently circulated via a small catheter through a portable high gradient magnetic separator device. In this device, the magnetic toxin complexes are retained by a high gradient magnetic field and the detoxified blood is then returned back to the blood circulation (extracorporeal circulation). Our preliminary in vitro experiments demonstrate >95% first pass capture efficiency of magnetic spheres within a prototype high gradient magnetic separation device. Further, based on the synthesis of novel hydrophobic magnetite nanophases with high magnetization ( approximately 55 emu/g), the first biodegradable magnetic nanospheres at a size range of approximately 280 nm and functionalized with PEG-maleimide surface groups for specific antibody attachment are described here. In future applications, we envision this technology to be suitable for emergent, in-field usage for acutely biohazard exposed victims as both the injectable toxin-binding magnetic spheres and the separator device are made to be portable, light-weight, zero-power, and self- or helper-employed. Details of the technology are presented and the state-of-knowledge and research is discussed.

Biocompatible Materials↗
PubMed2005

Bioengineering solutions for hearing loss and related disorders.

Advances in digital signal processing, microelectronics, and power technology have produced devices that have contributed significantly to the quality of life and communication abilities of individuals with hearing impairment and tinnitus. Future technological developments will expand the benefits of current devices and offer new treatments for otologic disorders.

Adult↗
PubMed2004

Sustained phenotypic correction of hemophilia a mice following oncoretroviral-mediated expression of a bioengineered human factor VIII gene in long-term hematopoietic repopulating cells.

Hematopoietic stem cells (HSCs) are an attractive target cell population for hemophilia A gene therapy because of their capacity to regenerate the hematolymphoid system permanently following transplantation. Here we transplanted bone marrow (BM) cells transduced with a splicing-optimized MSCV oncoretroviral vector expressing a secretion-improved human factor VIII gene into immunocompromised hemophilic mice that had received a reduced dose conditioning regimen. An enhanced green fluorescent protein (EGFP) reporter gene linked to an encephalomyocarditis virus internal ribosome entry site was incorporated into the vector to allow preselection of transduced cells and facile evaluation of engraftment. Sustained expression of EGFP was demonstrated in the peripheral blood, and therapeutic levels of factor VIII were detected in the plasma of the majority of the recipients for the duration of the observation period (up to 22 weeks). Coordinate expression of factor VIII and EGFP (up to 19 weeks) was transferred to secondary BM transplant recipients, indicating that long-term repopulating HSCs had been successfully gene modified. Notably, the hemophilic phenotype of all treated mice was corrected, thus demonstrating the potential of HSC-directed oncoretroviral-mediated factor VIII gene transfer as a curative therapeutic strategy for hemophilia A.

Animals↗