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Cryoprotectant permeability parameters for cells used in a bioengineered human corneal equivalent and applications for cryopreservation.

A human corneal equivalent is being developed with applications in pharmaceutical testing and biomedical research, but the distribution of this engineered tissue, depends on successful cryopreservation. Cryopreservation of tissues depends on the presence of cryoprotectants, their addition and removal, and exposure to conditions during freezing and thawing, all of which depend on cellular membrane permeabilities to water and cryoprotectant. This study defines the permeability properties that define the rate of water and cryoprotectant movement across the plasma membrane of isolated human corneal endothelial, keratocyte, and epithelial cells. Cells were transferred from isotonic conditions (300 mosm/kg) to 0.5, 1, or 2 M dimethyl sulfoxide and propylene glycol solutions at constant temperature, and cell volumes monitored using an electronic particle counter. Histograms describing cell volume changes over time after cryoprotectant exposure allowed calculation of hydraulic conductivity (Lp), cryoprotectant permeability (Ps), and the reflection coefficient (sigma). Experimental values for Lp and Ps at 4, 13, 22, and 37 degrees C were used to determine the Arrhenius activation energy (Ea). Defining the permeability parameters and temperature dependencies allows simulation of responses of human corneal cells to addition and removal of cryoprotectants and to freezing conditions, allowing amount of supercooling, intracellular electrolyte concentration, and intracellular cryoprotectant concentration to be calculated. Simulations also show that the constituent cells in the bioengineered cornea respond differently to addition and removal of cryoprotectants and to freezing. This study has defined the requirements during cryopreservation for the corneal cells; future work will define the matrix requirements which will allow the development of a cryopreservation protocol.

Cell Membrane Permeability↗

Use of a bioengineered skin equivalent for the management of difficult skin defects after pediatric multivisceral transplantation.

BACKGROUND: Primary wound closure is not always possible after pediatric multi-visceral transplantation because of oversized donor organs and/or intestinal or graft edema. We report our experience evaluating the safety and efficacy of Graftskin (Apligraf, Organogenesis, Canton, Mass), a bioengineered bi-layered human skin equivalent, for the management of difficult skin abdominal defects after multivisceral transplantation in a pediatric population. METHODS: A retrospective chart review was performed of pediatric multivisceral transplantation patients who were treated with Graftskin. Adverse events, course of wound reepithelialization, and time for complete closure were recorded. RESULTS: Four patients, 7 to 29 months old, were treated with Graftskin. One patient died because of unrelated reasons. Stimulation of the granulation, reepithelialization, and rapid reduction of the wound surface and depth occurred in the other 3 patients. Complete reepithelialization occurred within 5 months. No adverse events were noted. CONCLUSION: Graftskin was a successful treatment for difficult abdominal skin defects after liver and multivisceral transplantation in children.

Biomedical Engineering↗

Biodegradable polymer with collagen microsponge serves as a new bioengineered cardiovascular prosthesis.

OBJECTIVE: Biodegradable materials with autologous cell seeding have attracted much interest as potential cardiovascular grafts. However, pretreatment of these materials requires a complicated and invasive procedure that carries the risk of infection. To avoid these problems, we sought to develop a biodegradable graft material containing collagen microsponge that would permit the regeneration of autologous vessel tissue. The ability of this material to accelerate in situ cellularization with autologous endothelial and smooth muscle cells was tested with and without precellularization. METHODS: Poly(lactic-co-glycolic acid) as a biodegradable scaffold was compounded with collagen microsponge to form a vascular patch material. These poly(lactic-co-glycolic acid)-collagen patches with (n = 10) or without (n = 10) autologous vessel cellularization were used to patch the canine pulmonary artery trunk. Histologic and biochemical assessments were performed 2 and 6 months after the implantation. RESULTS: There was no thrombus formation in either group, and the poly(lactic-co-glycolic acid) scaffold was almost completely absorbed in both groups. Histologic results showed the formation of an endothelial cell monolayer, a parallel alignment of smooth muscle cells, and reconstructed vessel wall with elastin and collagen fibers. The cellular and extracellular components in the patch had increased to levels similar to those in native tissue at 6 months. CONCLUSIONS: The poly(lactic-co-glycolic acid)-collagen microsponge patch with and without precellularization showed good histologic findings and durability. This patch shows promise as a bioengineered material for promoting in situ cellularization and the regeneration of autologous tissue in cardiovascular surgery.

Animals↗

Bioengineering and physicochemical optimization of ergothioneine production by Aspergillus oryzae.

Ergothioneine (EGT) is a bioactive, rare variant of histidine with many applications in the medical, pharmaceutical, and food fields. Therefore, we aimed to investigate in this study the impact of genomic and physicochemical factors on EGT production by the industrial filamentous fungus Aspergillus oryzae. Firstly, to facilitate efficient EGT production, we analyzed the subcellular localization of the three EGT biosynthetic enzymes present in A. oryzae. During screening for the most potent producer of EGT among bioengineered transformants, the strain EgtACO overexpressing both AoegtA and AoegtC showed promising EGT production in DPY medium. Five days of incubation was the optimum period, and CZYP medium was the optimum medium for EGT production. Co-cultivation with the nisin Z-producing Lactococcus lactis JCM 7638 yielded EGT production equivalent to that of the EgtACO strain alone. Having broad-spectrum antimicrobial activity without suppressing growth of the EgtACO strain suggested that bacteriocin may help reduce the risk of contamination during long-term cultivation. Moreover, supplementing the production medium with L-methionine or zinc sulfate improved EGT production (1468.5 or 1565 mg/L, respectively). Furthermore, repeated inoculation of the producer strain EgtACO and incubation in blue light were the optimum conditions for EGT production (1895 mg/L). Finally, we achieved cost-effective EGT production using A. oryzae strain EgtACO under the optimal culture conditions using agricultural wastes: potato peel and sweet potato peel (293 and 308 mg/L, respectively).

Aspergillus oryzae↗

Osmotic parameters of cells from a bioengineered human corneal equivalent and consequences for cryopreservation.

A human corneal equivalent is under development with potential applications in pharmaceutical testing, biomedical research, and transplantation, but the ability to distribute this engineered tissue, depends on successful cryopreservation. Tissue recovery after exposure to conditions during cryopreservation depends on the response of its constituent cells to the changing environment as ice forms and solutes concentrate. This study defines the osmotic properties that define the rate of water movement across the plasma membrane of isolated human corneal endothelial, stroma, and epithelial cells. Cells were transferred from an isotonic (300 mosm/kg) to an anisotonic (150-1500 mosm/kg) solution at constant temperature, and cell volumes monitored using an electronic particle counter. Histograms describing cell volume changes over time after anisosmotic exposure allowed calculation of hydraulic conductivity (L(p)) and osmotically inactive volume fraction (V(b)). Experimental values for L(p) at 4, 13, 22, and 37 degrees C were used to determine the Arrhenius activation energy (E(a)). The L(p) for endothelial, stroma, and epithelial cells at 37 degrees C was 1.98+/-0.32,1.50+/-0.30, and 1.19+/-0.14 microm/min/atm, and the V(b) was 0.28, 0.27, and 0.41, respectively. The E(a) for endothelial, stroma, and epithelial cells was 14.8, 12.0, and 14.1 kcal/mol, respectively, suggesting the absence of aqueous pores. These osmotic parameters and temperature dependencies allow simulation of osmotic responses of human corneal cells to cryopreservation conditions, allowing amount of supercooling to be calculated to indicate the likelihood of intracellular freezing. Simulations show that differences in the osmotic parameters for the constituent cells in the bioengineered cornea result in significant implications for cryopreservation of the engineered corneal equivalent.

Biomedical Engineering↗

A novel in vitro model for xenorejection and immune mechanisms using bioengineered living skin equivalents.

We hypothesized that an in vitro bioengineered skin (LSE) could be used for the study of xenogeneic inflammatory or immunosuppressive mechanisms. Murine fibroblasts (10(4)/mL) were mixed with type 1 rat-tail collagen to form a matrix (approximately 5 days) on which human keratinocytes (NHEK, 10(5)/75 microL) were seeded. The xeno-LSE was used as a rejection target organ in vitro. Rejection was achieved by the addition of human lymphocytes (10(6)/75 microL). At various intervals of growth, control LSEs without lymphocytes (CON) and rejecting LSEs with immunocytes (REJ) were analyzed. Slides were stained with hematoxylin & eosin and examined under light microscopy. REJ xenocomposite LSEs showed classic histologic signs of rejection. There was separation of the D-E junction with the presence of dyskeratotic and necrotic cells with a concomitant inflammatory infiltrate. The CON LSEs showed essentially normal dermis (collagen fibroblast matrix) and keratinocytes. A significant finding was the separation of the D-E junction in the absence of an inflammatory infiltrate. Previous studies have shown that human keratinocytes can be grown with human fibroblasts that are histologically similar to natural human skin. This discohesive nature of the murine fibroblasts and human keratinocytes may represent an important intercellular interaction associated with xeno-cellular interactions and is worthy of further study. Furthermore, the ability to grow xeno-composite tissues has profound implications in the face of organ donor shortages. They may represent an eventual in vitro replacement of skin and even solid organs.

Adult↗

The regulation of biologic products derived from bioengineered plants.

Recently, there has been a large increase in the number and types of biological products--from therapeutic antibodies to vaccines for the prevention of infectious diseases--that are produced in bioengineered plant systems. We anticipate that this technology will be used increasingly on a commercial scale for the manufacture of human and animal products. These production systems have the capacity to produce very large quantities of products at lower costs and with reduced risks compared with mammalian systems.

Animals↗

Reprogramming somatic cell differentiation and the Hayflick Limit: contrasting two modern molecular bioengineering aims and their impact on the future of mankind.

The molecular biology of human cloning and aging research depend on the closely related laboratory techniques supported by a thorough understanding of cell-signaling processes. Unfortunately, the link between these two research fields has received only marginal attention in the lay press. Cloning is possible when somatic cell differentiation is successfully reprogrammed, and clinical control of cellular senescence depends on a proper reconfiguration of the predetermined number of divisions permitted during the cell life-cycle (the so-called "Hayflick Limit"). In this paper, we discuss these two concepts and compare the impact likely to be associated with bioengineering studies that facilitate both human cloning and longevity therapy.

Aging↗

Multiple effects of GDF-5 deficiency on skeletal tissues: implications for therapeutic bioengineering.

The growth/differentiation factors (GDFs) are a subfamily of the highly conserved group of bone morphogenetic protein (BMP) signaling molecules known to play a diverse set of roles in the skeletal system. GDFs 5, 6, and 7 in particular have been grouped together on the basis of the high degree of amino acid sequence homology in the C-terminal signaling region of these proteins. The existence of several naturally occurring and engineered mouse models with functional null mutations in these GDFs has led to a variety of investigations into the effects of GDF deficiency on skeletal tissues and processes. The best characterized of these models to date is the GDF-5-deficient brachypod (bp) mouse. In this paper, a comprehensive review of the studies performed on the bp mouse is provided in an effort to elucidate implications for potential therapeutic bioengineering applications using GDF-5. On the basis of the available evidence to date, GDF-5 may hold promise as a possible therapeutic agent for applications involving tendon/ligament repair as well as perhaps intervertebral disk degeneration, cartilage repair, and bone augmentation, although further detailed interventional studies will be required to investigate these potential applications.

Animals↗

Which bioengineering assay is appropriate for irritant patch testing with sodium lauryl sulfate?

For testing with sodium lauryl sulphate (SLS), measurements of transepidermal water loss (TEWL) and cutaneous blood flow with laser Doppler (LD) are considered to be the most reliable methods. The aim of this study was to determine which method of measurement should be preferred when conducting SLS testing under varying conditions. Patch testing with SLS at different concentrations and exposure times was performed. TEWL values were compared with those of LD. TEWL values showed distinct changes at low SLS concentrations and short application periods. By contrast, higher SLS concentrations were necessary to increase LD values. Short application of patches changed TEWL rather than LD values. When evaluating SLS patch testing by bioengineering methods, TEWL measurement appears to be more suitable for a test procedure that provokes mild skin reactions (SLS concentration <1%), whereas LD measurement is more appropriate to evaluate pronounced skin reactions (SLS concentration >or=1%).

Dermatitis, Irritant↗

A bioengineering study on the efficacy of a skin protectant lotion in preventing SLS-induced dermatitis.

BACKGROUND/AIMS: This study evaluated the efficacy of a dimethicone skin protectant lotion against sodium lauryl sulfate (SLS)-induced irritant contact dermatitis (ICD) by clinical visual grading and bioengineering techniques in 12 healthy humans. METHODS: The flexor aspects of both forearms of the subjects were used as test sites. Each test was duplicated to diminish the variations of the test sites. In a random order and a double-blind manner, two test sites were pretreated either with the testing protectant lotion or with its vehicle control prior to contact with SLS. Thirty minutes later, 0.2 ml of 0.5% SLS in a polypropylene chamber was applied to each pretreated site. One additional test site served as a positive control (without lotion), receiving the irritant only. After 24 h of exposure to the irritant, the chambers were removed. The efficacy of protective effect was determined by four parameters: visual scoring (VS), transepidermal water loss (TEWL), skin color (a* value), and cutaneous blood flow volume (BFV). All test sites were assessed with the parameters daily for 5 days. RESULTS: The VS data showed a significant decrease on the site pretreated with protectant lotion in comparison with the SLS-only treated site (P<0.01) and with the site pretreated with control vehicle (P<0.05) (overall for 5 days). TEWL value was significantly decreased in comparison with the SLS-only treated site (P=0.02 at day 2; P=0.008 at day 4; P=0.014 at day 5) and with the site pretreated with the control vehicle (P<0.05) (day 2, 4 and 5). However, the BFV and a* values did not show a statistical difference between protectant lotion, vehicle, and SLS-only treated sites. CONCLUSIONS: This study demonstrated that appropriate dimethicone skin protection products may provide certain benefits from surfactant ICD. The skin protectant lotion may be used to prevent ICD in home or work environments, where skin irritants may induce dermatitis or eczema.

Journal Article↗

A bioengineering system for in situ bioremediation of contaminated groundwater.

Much of the past and current focus of bioremediation has been on laboratory studies of microbial processes. By necessity, early studies have ignored important field properties, parameters, and processes that control the ultimate success of in situ bioremediation of contaminated groundwater. This paper presents a bioengineering systems approach that examines the impact of some of these field variables on common bioremediation practices. Using simple systems, the niche of biostimulation is shown to be aquifers with high contaminant sorption. A novel gas-phase biostimulation filter and a novel resting-state bioaugmentation/biofilter approach which show promise for effective field implementation are discussed.

Bacteria↗

CosmoDerm/CosmoPlast (human bioengineered collagen) for the aging face.

Type 1 collagen loss in the dermis is one of the primary causes of wrinkles seen in aged skin. Dermal fillers using type 1 collagen derived from bioengineered skin are now being used to treat facial wrinkles. These fillers, known by the trade names of CosmoDerm and CosmoPlast, can be used alone or in combination with hyaluronic acid fillers. The benefit of collagen-based dermal fillers is decreased downtime, decreased bruising, decreased pain on injection, and the ability to return lost structural components to aged skin. Many aesthetic physicians are beginning to use collagen- and hyaluronic-containing fillers in combination to replace both of these natural components of the skin that are lost during the aging process.

Age Factors↗

Accelerated healing of pyoderma gangrenosum treated with bioengineered skin and concomitant immunosuppression.

Pyoderma gangrenosum is a rare, destructive, neutrophilic dermatosis, the origin of which remains largely obscure. The ulcerative variant of this inflammatory disorder causes painful, necrotic, rapidly enlarging ulcers. Because of pathergy, many clinicians avoid managing these nonhealing ulcers with aggressive surgical debridement and autologous grafts. This article proposes that the application of an allogeneic cultured human skin equivalent (Graftskin) not only circumvents this problem, but also hastens re-epithelialization of the ulcer bed. An added benefit of the possible improvement of the cosmetic appearance of the final scar by preventing severe wound contracture is also postulated. We report a newly diagnosed case of ulcerative pyoderma gangrenosum; the use of bioengineered skin as an adjunct to concurrent immunosuppressive therapy with cyclosporine hastened the healing and diminished pain in a rapidly enlarging leg ulcer. Within 2 weeks, the ulcer was 30% to 40% healed, achieving 100% re-epithelialization within 6 weeks.

Adult↗

High-level expression of a bioengineered, cysteine-free hepatocyte-stimulating factor (interleukin 6)-like protein.

Hepatocyte-stimulating factor, interferon-beta 2, B-cell stimulation factor 2, and hybridoma/plasmacytoma growth factor are identical proteins presently referred to as interleukin 6 (IL-6). Through the use of synthetic oligonucleotide technology, we have constructed a biologically active recombinant IL-6 (rIL-6) gene based on the sequence of a human IL-6 cDNA. The synthetic gene encodes a cysteine-free, bioengineered rIL-6 protein that is expressed at high levels in Escherichia coli as a tripartite fusion protein. Cleavage of the fusion protein with collagenase releases a 23-kDa rIL-6 protein that can be easily purified to homogeneity. We show that the rIL-6 protein displays a range of biological activities similar to those of natural human IL-6, as demonstrated by its ability to (i) protect cells from viral infection, (ii) stimulate the synthesis of fibrinogen in rat FAZA 967 cells, and (iii) induce the terminal differentiation of B cells, resulting in elevated secretion of immunoglobulin.

Amino Acid Sequence↗

Repeated open application tests (ROAT) in patients allergic to colophony--evaluated visually and with bioengineering techniques.

It is desirable to further evaluate the clinical relevance of a positive patch test. The repeated open application test (ROAT) has been suggested as such a supplementary method. To compare the results of patch testing with the outcome of ROATs, 13 colophony-sensitive subjects and 9 controls were patch-tested with colophony in a serial dilution test. Five microliters, of three concentrations of a colophony solution and the vehicle were then applied to small test areas on the lower arm, once daily for 2 weeks. Prior to each application, all test sites were examined visually and with bioengineering techniques. In the ROATs, 10/13 colophony-sensitive subjects--but no controls--reacted to a 20% colophony solution, 4 also 1%. A correlation was found between the threshold concentration at patch testing and the outcome of ROATs. There was great variation in the reactivity in the ROATs. Objective measures for evaluating the ROAT reactions gave no further information than visual assessment.

Adolescent↗

A bioengineering analysis of human muscle and joint forces in the lower limbs during running.

A two-dimensional, dynamic bioengineering model of the lower limbs was developed in order to estimate muscle and joint forces present during running at 4.5 m s-1. Data were collected from four subjects using a force platform and cine film. Individual X-rays and anthropometric data from the lower limbs were utilized to produce accurate bone models of the subjects' legs. Electromyographic verification of the model was undertaken while a runner was undergoing treadmill running at 4.5 m s-1. Results indicate that peak muscle forces of 22 times subject body weight (22 BW) could be present in the quadriceps muscle group and 7 BW in the gastrocnemius. The anterior shin muscles were found to be active for the first 9% of stance phase only, and compressive loads of 33 BW were found in the knee joint. The relationship between these high forces in the lower limbs and running related injuries is discussed.

Adult↗

Therapeutic neovascularization: contributions from bioengineering.

A number of pathological entities and surgical interventions could benefit from therapeutic stimulation of new blood vessel formation. Although strategies designed for promoting neovascularization have shown promise in preclinical models, translation to human application has met with limited success when angiogenesis is used as the single therapeutic mechanism. While clinical protocols continue to be optimized, a number of exciting new approaches are being developed. Bioengineering has played an important role in the progress of many of these innovative new strategies. In this review, we present a general outline of therapeutic neovascularization, with an emphasis on investigations using engineering principles to address this vexing clinical problem. In addition, we identify some limitations and suggest areas for future research.

Angiogenesis Inducing Agents↗