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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↗

Bioengineering education, 1986--Part III.

This paper presents an overview of the Bioengineering programs available today in the northern midwest states of Illinois, Iowa, South Dakota, Minnesota, and Wisconsin. Two-year and four-year undergraduate programs and graduate programs are described, including contact information and course requirements. As Part III of the Journal's continuing series on college-level education for the fields of Biomedical and Clinical Engineering, the purpose of the paper is not to evaluate each program, but to illustrate the breadth of programs offered.

Biomedical Engineering↗

Bioengineering education, 1986--Part VI.

This paper constitutes the sixth part of the Journal's series on college-level Bioengineering programs. Contact information and descriptions of programs at 16 colleges or universities in the states of Kentucky, Maryland, Pennsylvania, Virginia, and West Virginia, and the District of Columbia, are presented herein. It is not the intent of this paper to evaluate each program, but to illustrate the breadth of biomedical and related engineering educational opportunities in this area of the United States.

Biomedical Engineering↗

Bioengineering education, 1986--Part VII.

This paper concludes the Journal's series on Bioengineering Education in the United States. In this issue, college-level programs in the states of Alabama, Florida, Georgia, Louisiana, Mississippi, North Carolina, South Carolina, and Tennessee are described. These programs range from two-year bachelor degree, master's degree, doctoral degree, and M.D./Ph.D. combined programs. The intent of this paper is not to evaluate the various programs, but to illustrate the breadth of biomedical and related engineering educational opportunities in this region.

Biomedical Engineering↗

Regeneration of intraoral defects after tumor resection with a bioengineered human dermal replacement (Dermagraft).

The experiences of seven patients with squamous cell carcinomas of the oral cavity who underwent reconstruction with a bioengineered human dermal replacement (Dermagraft) are examined. The human dermal replacement consists of fibroblasts seeded onto a three-dimensional polymer scaffold to create a living dermal structure. In this setting, the fibroblasts secrete a mixture of growth factors and matrix proteins in physiological concentration that is essential for wound healing and epithelization. The fibroblast tissue remains metabolically active after cryopreservation and can be used as an off-the-shelf tissue to cover medium-sized defects and avoid donor-site morbidity. In the first series of patients treated with this tissue, defect closure was achieved without functional problems, allowing optimal postoperative monitoring for tumor recurrence.

Aged↗

Bioengineered implantation of megalin-expressing cells: a potential intracorporeal therapeutic model for uremic toxin protein clearance in renal failure.

Patients who have renal failure and are on dialysis therapy experience serious complications caused by low-molecular-weight uremic toxin proteins normally filtered by glomeruli and metabolized by proximal tubule cells (PTC). Dialysis-related amyloidosis is one such complication induced by systemic deposition of amyloid proteins derived from 12-kD beta(2)-microglobulin (beta(2)-m). Despite the use of high-flux membrane hemodialysis devices and direct absorbent columns, the removal of beta(2)-m is suboptimal, because the effects are transient and insufficient. Megalin is expressed in the apical membranes of PTC and recognized as a multiligand endocytic receptor that binds numerous low-molecular-weight proteins, including beta(2)-m. This study tested the feasibility of an intracorporeal therapeutic model of continuous beta(2)-m removal using megalin-expressing cell implantation. By cell association and degradation assays, rat yolk sac-derived L2 cells were identified to internalize and degrade beta(2)-m via megalin. The cells were effectively implanted within the subcutaneous tissues of nude mice using a type I collagen scaffold and a method inducing local angiogenesis. After nephrectomy and intraperitoneal injection with (125)I-beta(2)-m, it was found that the implanted cells took up the labeled ligand, efficiently removing it from the blood. Bioengineered implantation of megalin-expressing cells may represent a new supportive therapy for dialysis patients to compensate for the loss of renal protein metabolism and remove uremic toxin proteins.

Animals↗

A 1-year study of osteoinduction in hydroxyapatite-derived biomaterials in an adult sheep model: part II. Bioengineering implants to optimize bone replacement in reconstruction of cranial defects.

The present study investigated hydroxyapatite biomaterials implanted in critical-size defects in the calvaria of adult sheep to determine the optimal bioengineering of hydroxyapatite composites to facilitate bone ingrowth into these materials. Five calvarial defects measuring 16.8 mm in diameter were made in each of 10 adult sheep. Three defects were filled with cement paste composites of hydroxyapatite and beta-tricalcium phosphate as follows: (1) 100 percent hydroxyapatite-cement paste, (2) 60 percent hydroxyapatite-cement paste, and (3) 20 percent hydroxyapatite-cement paste. One defect was filled with a ceramic composite containing 60 percent hydroxyapatite-ceramic, and the fifth defect remained unfilled. One year after implantation, the volume of all biomaterials was determined by computed tomography, and porosity and bone replacement were determined using backscatter electron microscopy. Computed tomography-based volumetric assessment 1 year after implantation demonstrated that none of the unfilled cranial defects closed over the 1-year period, confirming that these were critical-size defects. There was a significant increase in volume in both the cement paste and ceramic implants containing 60 percent hydroxyapatite (p < 0.01). There was no significant change in volume of the remaining cement paste biomaterials. Analysis of specimens by backscatter electron microscopy demonstrated mean bone replacement of 4.8 +/- 1.4 percent (mean +/- SEM) in 100 percent hydroxyapatite-cement paste, 11.2 +/- 2.3 percent in 60 percent hydroxyapatite-cement paste, and 28.5 +/- 4.5 percent in 20 percent hydroxyapatite-cement paste. There was an inverse correlation between the concentration of hydroxyapatite and the amount of bone replacement in the cement paste for each composite tested (p < 0.01). Bone replacement in 60 percent hydroxyapatite-ceramic composite (13.6 +/- 2.0 percent) was not significantly different from that in 60 percent hydroxyapatite-cement paste. Of note is that the ceramic composite contained macropores (200 to 300 microm) that did not change in size over the 1-year period. All cement paste composites initially contained micropores (3 to 5 nm), which remained unchanged in 100 percent hydroxyapatite-cement paste. Cement paste implants containing increased tricalcium phosphate demonstrated a corresponding increase in macropores following resorption of the tricalcium phosphate component. Bone replacement occurred within the macropores of these implants. In conclusion, there was no significant bone ingrowth into pure hydroxyapatite-cement paste (Bone Source, Stryker-Leibinger Inc., Dallas, Texas) in the present study. The introduction of macropores in a biomaterial can optimize bone ingrowth for reconstruction of critical-size defects in calvaria. This was demonstrated in both the ceramic composite of hydroxyapatite tested and the cement paste composites of hydroxyapatite by increasing the composition of a rapidly resorbing component such as beta-tricalcium phosphate.

Animals↗

Bioengineering lactic acid bacteria to secrete the HIV-1 virucide cyanovirin.

An urgent need exists to prevent the sexual transmission of HIV-1. With prevalence rates exceeding 35% in parts of sub-Saharan Africa, increasing attention has been placed on developing and testing microbicidal agents capable of preventing virus transmission at mucosal sites. HIV-1 microbicides must meet several requirements before their widespread use. The drugs must be able to neutralize a diversity of HIV-1 strains, not induce mucosal inflammation, be associated with minimal side effects, and be effective for a prolonged period after a single application. Recent work has demonstrated the utility of recombinant lactic acid bacteria (LAB) as agents of mucosal drug delivery. Here, we describe the bioengineering of strains of LAB to secrete the prototypic virucidal compound cyanovirin (CV-N) and demonstrate the anti-HIV-1 activity of secreted CV-N. Our results suggest that recombinant LAB may serve as effective microbicidal compounds and deserve in vivo testing in simian immunodeficiency virus models of mucosal virus transmission.

Bacterial Proteins↗

Functional bioengineered corneal epithelial sheet grafts from corneal stem cells expanded ex vivo on a temperature-responsive cell culture surface.

BACKGROUND: Limbal stem-cell deficiency by ocular trauma or diseases causes corneal opacification and visual loss. Recent attempts have been made to fabricate corneal epithelial graft constructs, but the technology is still evolving. We have developed a novel cell-sheet manipulation technology using temperature-responsive culture surfaces to generate functional, cultivated corneal epithelial cell sheet grafts. METHODS: Human or rabbit limbal stem cells were cocultured with mitomycin C-treated 3T3 feeder layers on temperature-responsive culture dishes at 37 degrees C. Cell sheets were harvested from the dishes after 2 weeks by reducing temperature to 20 degrees C. Histologic analyses, immunoblotting, and colony-forming assay were performed to characterize the cell sheets. Autologous transplantation was undertaken to reconstruct the corneal surfaces of rabbits with experimentally induced limbal stem cell deficiencies. RESULTS: Multilayered corneal epithelial sheets were harvested intact simply by reducing the temperature, without the use of proteases. Cell-cell junctions and extracellular matrix on the basal side of the sheet, critical to sheet integrity and function, remained intact. A viable population of corneal progenitor cells, close in number to that originally seeded, was found in the sheets. Harvested sheets were easily manipulated, transplantable without any carriers, and readily adhesive to corneal stroma so that suturing was not required. Corneal surface reconstruction in rabbits was highly successful. CONCLUSIONS: Cell sheet engineering technology allows us to create intact, transplantable corneal epithelial cell sheets that retain stem cells from limbal stem cells expanded ex vivo. Our research indicates highly promising clinical capabilities for our bioengineered corneal epithelial sheet.

3T3 Cells↗

Restoration of cardio-circulatory regulation by rate-adaptive pacemaker systems: the bioengineering view of a clinical problem.

In the past, the development of rate-adaptive (sensor-controlled) pacemaker systems seems to have been determined primarily by the availability, compatibility and other properties of the technical sensor. This paper, however, focuses on the system-physiological aspect in an attempt to answer the question to what extent physiological cardiovascular control is restored by the pacemaker system. This is a question which should be asked before attempting to design a sensor-controlled system and especially before designing multisensor systems with infinite combinations. Four categories are defined: direct bridging ("shunting"), open loop systems, closed systems using cardiorespiratory or metabolic coupling and those using cardiac signals. Further subdivisions are shown. From the bioengineering as well as from the physiological viewpoint a system should preferably not combine sensors from one and the same of these categories. At present direct bridging is available only for the atrioventricular (AV)-block, so that for sick-sinus-syndrome (SSS) patients feedback control via cardiac signals ("inotropic" pacemaker) comes nearest the goal without, however, ideally bridging the gap. Open-loop systems should no longer be developed as single-sensor systems. A well developed activity sensor, however, which quickly pinpoints the most prominent stressor of cardiovascular control is best suited to complement another sensory system achieving closed-loop control. New and promising concepts orientated toward direct bridging are the analysis of monophasic action potentials and the "dromotropic" concept, both of which seek direct correlation with the "chronotropic" information not available in SSS patients.

Adaptation, Physiological↗

Perspectives on MEMS in bioengineering: a novel capacitive position microsensor.

We describe a novel capacitive position sensor using micromachining to achieve high sensitivity and large range of motion. These sensors require a new theoretical framework to describe and optimize their performance. Employing a complete description of the electrical fields, the sensor should deviate from the standard geometries used for capacitive sensors. By this optimization, the sensor gains a twofold increase in sensitivity. Results on a PC board 10x model imply that the micromachined sensor should achieve a sensitivity of less than 10 nm over 500-micron range of travel. Some bioengineering applications are addressed, including positioning of micromirrors for laser surgery and dose control for implantable drug delivery systems.

Drug Delivery Systems↗

Moisturizing effect of cosmetic formulations containing Aloe vera extract in different concentrations assessed by skin bioengineering techniques.

BACKGROUND/PURPOSE: The polysaccharide-rich composition of Aloe vera extracts (Aloe barbadensis Miller), often used in cosmetic formulations, may impart moisturizing properties to the product. The aim of this study was to evaluate the effect of cosmetic formulations containing different concentrations of freeze-dried Aloe vera extract on skin hydration, after a single and a 1- and 2-week period of application, by using skin bioengineering techniques. METHODS: Stable formulations containing 5% (w/w) of a trilaureth-4 phosphate-based blend were supplemented with 0.10%, 0.25% or 0.50% (w/w) of freeze-dried Aloe vera extract and applied to the volar forearm of 20 female subjects. Skin conditions in terms of the water content of the stratum corneum and of transepidermal water loss (TEWL) (Corneometer CM 825 and Tewameter TM 210) were analysed before and after a single and 1- and 2-week period of daily application. RESULTS: After a single application, only formulations supplemented with 0.25% and 0.50% (w/w) of Aloe vera extract increased the water content of the stratum corneum, while after the 2-week period application, all formulations containing the extract (0.10%, 0.25% and 0.50%) had the same effect, in both cases as compared with the vehicle. TEWL was not modified after a single and after 1- and 2-week period of application, when compared with the vehicle. CONCLUSION: Our results show that freeze-dried Aloe vera extract is a natural effective ingredient for improving skin hydration, possibly through a humectant mechanism. Consequently, it may be used in moisturizing cosmetic formulations and also as a complement in the treatment of dry skin.

Adult↗

Electrical impedance compared with other non-invasive bioengineering techniques and visual scoring for detection of irritation in human skin.

A new, non-invasive device, which enables local measurements of electrical impedance to a controlled depth, has been used to evaluate the degree of irritation in human skin. The results have been compared with those obtained using other non-invasive techniques, i.e. with transepidermal water loss (TEWL), electrical capacitance moist measurement (ECM), laser-Doppler flowmetry (LDF), and visual scoring. Sodium lauryl sulphate solutions (0.1-5.0%), and a blank, were applied in Finn Chambers for 24 h on the volar forearm of 10 healthy volunteers. Values were recorded before application of the test chambers, and at 1 h, 24 h, 1 week and 2 weeks after removal. Thus, both the degree of initial damage and the healing process were monitored. There was close agreement among values obtained using electrical impedance, TEWL and visual readings. Results obtained using ECM and LDF were not consistent with the other three methods. In evaluating irritation of the skin, the practical situation may influence the choice of bioengineering tools. In most circumstances, a combination of methods would be preferable.

Adult↗

Fungal tyrosinases: new prospects in molecular characteristics, bioengineering and biotechnological applications.

Tyrosinases are type-3 copper proteins involved in the initial step of melanin synthesis. These enzymes catalyse both the o-hydroxylation of monophenols and the subsequent oxidation of the resulting o-diphenols into reactive o-quinones, which evolve spontaneously to produce intermediates, which associate in dark brown pigments. In fungi, tyrosinases are generally associated with the formation and stability of spores, in defence and virulence mechanisms, and in browning and pigmentation. First characterized from the edible mushroom Agaricus bisporus because of undesirable enzymatic browning problems during postharvest storage, tyrosinases were found, more recently, in several other fungi with relevant insights into molecular and genetic characteristics and into reaction mechanisms, highlighting their very promising properties for biotechnological applications. The limit of these applications remains in the fact that native fungal tyrosinases are generally intracellular and produced in low quantity. This review compiles the recent data on biochemical and molecular properties of fungal tyrosinases, underlining their importance in the biotechnological use of these enzymes. Next, their most promising applications in food, pharmaceutical and environmental fields are presented and the bioengineering approaches used for the development of tyrosinase-overproducing fungal strains are discussed.

Amino Acid Sequence↗

Evaluation of natural change of skin function in split-thickness skin grafts by noninvasive bioengineering methods.

BACKGROUND: Autologous split-thickness skin grafts (STSGs) are considered the mainstay for the treatment of large full-thickness wounds. There have been few studies reporting the natural change of the skin function in STSGs after procedure, however. OBJECTIVE: The objective was to evaluate the natural change of the skin function in STSG using noninvasive bioengineering methods. METHODS: Eighteen patients were eligible for the study. The skin functions of the graft and the control site were evaluated by an evaporimeter, corneometer, mexameter, and cutometer at Postoperation Days 0.5, 1, 2, 3, 6, 9, and 12 months. RESULTS: Transepidermal water loss (TEWL) of the graft was maintained around that of the normal skin. The values of the skin hydration testing generally decreased during the follow-up period. Erythema was highly maintained for the whole period. For the pigmentation, the ratio tended to increase after 6 months. The skin pliability of the graft was abruptly decreased at 0.5 month, and it recovered from 3 to 12 months. The value did not reach that of the normal skin, however. CONCLUSION: Our results showed that the STSGs had changed within the frame of the skin function, including the TEWL, epidermal hydration, color, and pliability, throughout 1 year after surgery. The authors have indicated no significant interest with commercial supporters.

Adolescent↗