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PubMed2001

Stem cell bioengineering.

Tissue engineering and cellular therapies, either on their own or in combination with therapeutic gene delivery, have the potential to significantly impact medicine. Implementation of technologies based on these approaches requires a readily available source of cells for the generation of cells and tissues outside a living body. Because of their unique capacity to regenerate functional tissue for the lifetime of an organism, stem cells are an attractive "raw material" for multiple biotechnological applications. By definition they are self-renewing because on cell division they can generate daughter stem cells. They are also multipotent because they can differentiate into numerous specialized, functional cells. Recent findings have shown that stem cells exist in most, if not all, tissues, and that stem cell tissue specificity may be more flexible than originally thought. Although the potential for producing novel cell-based products from stem cells is large, currently there are no effective technologically relevant methodologies for culturing stem cells outside the body, or for reproducibly stimulating them to differentiate into functional cells. A mechanistic understanding of the parameters important in the control of stem cell self-renewal and lineage commitment is thus necessary to guide the development of bioprocesses for the ex vivo culture of stem cells and their derivates.

Adult↗
PubMed2005

Thin and strong! The bioengineering dilemma in the structural and functional design of the blood-gas barrier.

In gas exchangers, the tissue barrier, the partition that separates the respiratory media (water/air and hemolymph/blood), is exceptional for its remarkable thinness, striking strength, and vast surface area. These properties formed to meet conflicting roles: thinness was essential for efficient flux of oxygen by passive diffusion, and strength was crucial for maintaining structural integrity. What we have designated as "three-ply" or "laminated tripartite" architecture of the barrier appeared very early in the evolution of the vertebrate gas exchanger. The design is conspicuous in the water-blood barrier of the fish gills through the lungs of air-breathing vertebrates, where the plan first appeared in lungfishes (Dipnoi) some 400 million years ago. The similarity of the structural design of the barrier in respiratory organs of animals that remarkably differ phylogenetically, behaviorally, and ecologically shows that the construction has been highly conserved both vertically and horizontally, i.e., along and across the evolutionary continuum. It is conceivable that the blueprint may have been the only practical construction that could simultaneously grant satisfactory strength and promote gas exchange. In view of the very narrow allometric range of the thickness of the blood-gas barrier in the lungs of different-sized vertebrate groups, the measurement has seemingly been optimized. There is convincing, though indirect, evidence that the extracellular matrix and particularly the type IV collagen in the lamina densa of the basement membrane is the main stress-bearing component of the blood-gas barrier. Under extreme conditions of operation and in some disease states, the barrier fails with serious consequences. The lamina densa which in many parts of the blood-gas barrier is <50 nm thin is a lifeline in the true sense of the word.

Animals↗
PubMed2003

Development and use of an ELISA test to detect IgE antibody to Cry9c following possible exposure to bioengineered corn.

BACKGROUND: Starlink(TM), a variety of corn genetically engineered to contain the insecticidal protein Cry9c, had not been approved for human consumption because it possessed some characteristics associated with allergenic proteins. However, in the fall of 2000 CRY9C DNA was detected in several corn-containing products, suggesting that Starlink corn had entered the human food supply. Subsequently, consumers, following consumption of corn products, reported a number of adverse health events, possibly consistent with allergic reaction. METHODS: To investigate the possibility of allergic reactions due to Cry9c in these consumers an ELISA test was developed for the purpose of detecting IgE antibodies to Cry9c and blood samples were taken from a total of 18 people who self-reported allergic reactions. Sera collected prior to the 1996 development of Starlink were used as negative controls. RESULTS: None of the adverse event sera were found to be reactive with recombinant Cry9c antigen, based on comparison with normal controls. Although a known human positive control serum containing IgE specific for Cry9c was not available, other controls were incorporated into the ELISA protocol, including the use of sera from subjects allergic to other allergens and their homologous antigens (cat, grass, peanut) to validate the IgE detection reagents. CONCLUSIONS: While the results do not support the likely occurrence of allergic reactions to Cry9c, such reactions cannot be ruled out, nor can the possibility that sera might react with unique glycosylated epitopes of Cry9c that may be expressed in the corn plant/seed.

Bacillus thuringiensis Toxins↗
PubMed2006

The development of a serum-free derived bioengineered conjunctival epithelial equivalent using an ultrathin poly(epsilon-caprolactone) membrane substrate.

PURPOSE: To evaluate the use of an ultrathin poly(epsilon-caprolactone) (PCL) membrane as a substrate for the development of a serum-free-derived conjunctival epithelial equivalent. METHODS: Ultrathin PCL membranes 6 microm in thickness were prepared by solvent casting and biaxial stretching and analyzed by atomic force microscopy (AFM), scanning electron microscopy (SEM), tensile testing, and water-contact angle measurement. Rabbit conjunctival epithelial cells were cultivated on sodium hydroxide (NaOH)-treated PCL membranes and untreated PCL membranes in serum-free medium. The proliferative capacity of cultivated cells was analyzed with a bromodeoxyuridine (BrdU) ELISA proliferation assay. Conjunctival equivalents were xenografted into severe combined immune-deficient (SCID) mice. Immunostaining for tissue-specific and basement membrane-related proteins was performed. RESULTS: After biaxial stretching, the tensile strength of PCL membranes increased from 21 to 42 MPa, with a Young's modulus of 225 MPa. AFM and SEM showed that biaxially stretched PCL membranes consisted of closely packed microfibrils. PCL membranes supported the attachment and proliferation of conjunctival epithelial cells to form confluent stratified epithelial sheets. Surface modification with NaOH resulted in greater hydrophilicity and cellular proliferation than that of untreated membranes. Transplanted conjunctival equivalents underwent greater proliferation and stratification in vivo. Cultivated conjunctival cells expressed K4, K19, MUC5AC, and Ki67, whereas collagen IV and integrin beta4 were detected at the basement membrane junction. CONCLUSIONS: An ultrathin PCL membrane was shown to be biocompatible, mechanically strong enough to stand up to handling, and able to support conjunctival epithelial cell proliferation. This membrane may have potential for use as a scaffold matrix for tissue-engineered conjunctival equivalents.

Amnion↗
PubMed2006

Control of filtering bleb structure through tissue bioengineering: An animal model.

PURPOSE: To devise a means of providing controlled resistance between the anterior chamber and the subconjunctival space after trabeculectomy by implantation of a biodegradable, porous collagen matrix. METHODS: Matrices were implanted in the right eyes of 17 rabbits after trabeculectomy, while left eyes served as surgical controls. The scleral flap was sutured loosely, and the implant provided pressure on the scleral flap to reduce overfiltration. Trabeculectomy in the control eyes was performed with tight sutures using standard methodology. Intraocular pressure (IOP) was measured before surgery and on days 3, 7, 14, 21, and 28 after surgery. Masson trichrome and alpha-smooth muscle actin stains were used for histologic study of the filtering blebs. RESULTS: The initial postoperative IOP reduction was approximately equal, at 14% to 16%, for both groups. In the implanted group, the IOP continued to decrease to 55% below baseline at day 28 as the implant gradually degraded. In the control group, IOP had returned to the preoperative level by day 21. Histologic examination with Masson trichrome and alpha-smooth muscle actin stains showed a prominent bleb in the implanted group compared with scar formation and limited bleb formation in the control group. CONCLUSIONS: Implantation of a biodegradable, porous collagen matrix in the subconjunctival space offers the potential for a new means of avoiding early scar formation and maintaining long-term IOP control by creating a loosely structured filtering bleb.

Absorbable Implants↗