The primary emphasis of tissue engineering is the design and fabrication of constructs for the replacement of nonfunctional tissue. Because tissue represents a highly organized interplay of cells and extracellular matrix, the fabrication of replacement tissue should mimic this spatial organization. This report details studies evaluating the use of a three-dimensional, direct-write cell deposition system to construct spatially organized viable structures. A direct-write bioassembly system was designed and fabricated to permit layer-by-layer placement of cells and extracellular matrix on a variety of material substrates. Human fibroblasts suspended in polyoxyethylene/polyoxypropylene were coextruded through a positive displacement pen delivery onto a polystyrene slide. After deposition, approximately 60% of the fibroblasts remained viable. Bovine aortic endothelial cells (BAECs) suspended in soluble collagen type I were coextruded via microdispense pen delivery onto the hydrophilic side of flat sheets of polyethylene terephthalate. After deposition with a 25-gauge tip, approximately 86% of the BAECs were viable. When maintained in culture for up to 35 days, the constructs remained viable and maintained their original spatial organization. These results indicate the potential for utilizing a direct-write, three-dimensional bioassembly tool to create viable, patterned tissue-engineered constructs.

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Development of optimal passive and active immunization strategies for cryptosporidiosis is dependent on the identification and characterization of functional parasite molecules which can be targeted. Each of a subset of five monoclonal antibodies (mAb) produced against immunoaffinity-isolated C. parvum antigens elicited distinct morphologic changes in sporozoites and merozoites, characterized by progressive formation and eventual release of membranous antigen-mAb precipitates from the sporozoite posterior m g g s MW, Stone AL, Arrowood MJ, Langer RC, Yount PA. Abstract # C46, Thud International Workshop on Pneumocystis, Toxoplarma, Cryptosporidium and Microsporidia (1994) Cleveland, OH; E g g s MW, Stone AL, Yount PA, Arrowood MJ, Langer RC, Bentley DL. Protective MQnQClOnd Antibodies Define a Ckcumspsrozoite-like Glycooproteh Exoantigen of Cryptosporidium parvum Sporozoites and Merozoites, submitted]. These changes were similar to, but more rapid in development and prominent than thoae elicited by therapeutic bovine colostral anti-C. parvum antibody we described previously [8], and resembled the malarial circumsporozoite precipitate (CSP) reaction [3]. Infectivity of sporozoites having undergone the CSP-like reaction was neutralized. Further, mAbs which elicited the CSP-like reaction provided marked passive protection against C. parvum oocyst challenge in neonatal BALB/c mice. The antigen mechanistically involved in the CSP-like reaction was released initially from the apical end of sporozoites. Cytoskeletal inactivation with cytochalasind did not inhibit apical release of the antigen, but did inhibit its posterior translocation. The CSP-like reaction was morphologically and antigenically distinct from P23 trail deposition [2] previously described for motile C. parvum sporozoites. The antigen recognized by mAbs which elicited the CSPlike reaction was localized to sporozoite apical complex organelles, including electrondense granules, by immunoelectron microscopy. In western blots of sporozoites or merozoites, the mAbs recognized multiple 46-230 kDa antigens and a high M, antigen, designated CSL, which variably migrated between 1200-1400 kDa in reducing SDS-

Islet transplantation for the purpose of treating insulin-sensitive diabetes is currently limited by several factors, including islet survival posttransplantation. In the current study, a tissue-engineered prevascularized pancreatic encapsulating device (PPED) was developed. Isolated islets were placed in collagen gels, and they exhibited fourfold more insulin release than islets not in collagen. The insulin released by beta-cells in islets encapsulated in collagen exhibited unobstructed diffusion within the collagen gels. Subsequent studies evaluated the ability to create a sandwich comprised of two layers of prevascularized collagen gels around a central collagen gel containing islets. In vitro characterization of the islets showed that islets are functional and responded to glucose stimulation. The PPEDs were implanted subcutaneously into severe combined immunodeficient mice. Islet survival was assessed after 7, 14, and 28 days. Immunohistochemical analysis was performed on the implants to detect insulin and the presence of intraislet endothelial cells. At all time points, insulin was localized in association with intact and partially dissociated islets. Moreover, cells that exhibited insulin staining were colocalized with intraislet endothelial cells. These data indicate that the PPED enhances islet survival by supporting islet viability and maintaining intraislet endothelial cell structures.

Optical coherence tomography (OCT) is an imaging modality capable of acquiring cross-sectional images of tissue using back-reflected light. Conventional OCT images have a resolution of 10-15 microm, and are thus best suited for visualizing tissue layers and structures. OCT images of collagen (with and without endothelial cells) have no resolvable features and may appear to simply show an exponential decrease in intensity with depth. However, examination of these images reveals that they display a characteristic repetitive structure due to speckle. The purpose of this study is to evaluate the application of statistical and spectral texture analysis techniques for differentiating living and non-living tissue phantoms containing various sizes and distributions of scatterers based on speckle content in OCT images. Statistically significant differences between texture parameters and excellent classification rates were obtained when comparing various endothelial cell concentrations ranging from 0 cells/ml to 25 million cells/ml. Statistically significant results and excellent classification rates were also obtained using various sizes of microspheres with concentrations ranging from 0 microspheres/ml to 500 million microspheres/ml. This study has shown that texture analysis of OCT images may be capable of differentiating tissue phantoms containing various sizes and distributions of scatterers.