The ␤-cells in the pancreatic islets of Langerhans are the targets of autoreactive T-cells and are destroyed in type 1 diabetes. Macrophage-derived interleukin-1␤ ( I L -1␤) is important in eliciting ␤-cell dysfunction and initiating ␤-cell damage in response to microenvironmental changes within islets. In particular, IL-1␤ c a n impair glucose-stimulated insulin production in ␤-c e l l s in vitro and can sensitize them to Fas (CD95)/FasLtriggered apoptosis. In this report, we have examined the ability to block the detrimental effects of IL-1␤ b y genetically modifying islets by adenoviral gene transfer to express the IL-1 receptor antagonist protein. We demonstrate that adenoviral gene delivery of the cDNA encoding the interleukin-1 receptor antagonist protein (IL-1Ra) to cultured islets results in protection of human islets in vitro against IL-1␤-induced nitric oxide formation, impairment in glucose-stimulated insulin production, and Fas-triggered apoptosis activation. Our results further support the hypothesis that I L -1␤ antagonism in in situ may prevent intra-islet proinsulitic inflammatory events and may allow for an in vivo gene therapy strategy to prevent insulitis and the consequent pathogenesis of diabetes. D i a b e t e s 48: [1730][1731][1732][1733][1734][1735][1736] 1999

The demands of tissue engineering have driven a tremendous amount of research effort in 3D tissue culture technology and, more recently, in 3D printing. The need to use 3D tissue culture techniques more broadly in all of cell biology is well-recognized, but the transition to 3D has been impeded by the convenience, effectiveness, and ubiquity of 2D culture materials, assays, and protocols, as well as the lack of 3D counterparts of these tools. Interestingly, progress and discoveries in 3D bioprinting research may provide the technical support needed to grow the practice of 3D culture. Here we investigate an integrated approach for 3D printing multicellular structures while using the same platform for 3D cell culture, experimentation, and assay development. We employ a liquid-like solid (LLS) material made from packed granular-scale microgels, which locally and temporarily fluidizes under the focused application of stress and spontaneously solidifies after the applied stress is removed. These rheological properties enable 3D printing of multicellular structures as well as the growth and expansion of cellular structures or dispersed cells. The transport properties of LLS allow molecular diffusion for the delivery of nutrients or small molecules for fluorescence-based assays. Here, we measure viability of 11 different cell types in the LLS medium, we 3D print numerous structures using several of these cell types, and we explore the transport properties in molecular time-release assays.

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The long-term goal of the present study is to develop a clinically applicable approach to enhance natural repair mechanisms within cartilage lesions by targeting bone marrow-derived cells for genetic modification. To determine if bone marrow-derived cells infiltrating osteochondral defects could be transduced in situ, we implanted collagen-glycosaminoglycan (CG) matrices preloaded with adenoviral vectors containing various marker genes into lesions surgically generated in rabbit femoral condyles. Analysis of the recovered implants showed transgenic expression up to 21 days; however, a considerable portion was found in the synovial lining, indicating leakage of the vector and/or transduced cells from the matrix. As an alternative medium for gene delivery, we investigated the feasibility of using coagulated bone marrow aspirates. Mixture of an adenoviral suspension with the fluid phase of freshly aspirated bone marrow resulted in uniform dispersion of the vector throughout, and levels of transgenic expression in direct proportion to the density of nucleated cells in the ensuing clot. Furthermore, cultures of mesenchymal progenitor cells, previously transduced ex vivo with recombinant adenovirus, were readily incorporated into the coagulate when mixed with fresh aspirate. These vector-seeded and cell-seeded bone marrow clots were found to maintain their structural integrity following extensive culture and maintained transgenic expression in this manner for several weeks. When used in place of the CG matrix as a gene delivery vehicle in vivo, genetically modified bone marrow clots were able to generate similarly high levels of transgenic expression in osteochondral defects with better containment of the vector within the defect. Our results suggest that coagulates formed from aspirated bone marrow may be useful as a means of gene delivery to cartilage and perhaps other musculoskeletal tissues. Cells within the fluid can be readily modified with an adenoviral vector, and the matrix formed from the clot is completely natural, native to the host and is the fundamental platform on which healing and repair of mesenchymal tissues is based.

With the long-term goal of developing a gene-based treatment for osteoarthritis (OA), we performed studies to evaluate the equine joint as a model for AAV-mediated gene transfer to large, weight-bearing human joints. A self-complementary AAV2 vector containing the coding regions for human interleukin-1 receptor antagonist (hIL-1Ra) or green fluorescent protein (GFP) was packaged in AAV capsid serotypes 1, 2, 5, 8 and 9. Following infection of human and equine synovial fibroblasts in culture, we found that both were only receptive to transduction with AAV1, 2 and 5. For these serotypes, however, transgene expression from the equine cells was consistently at least 10-fold higher. Analyses of AAV surface receptor molecules and intracellular trafficking of vector genomes implicate enhanced viral uptake by the equine cells. Following delivery of 1 × 1011 vector genomes of serotypes 2, 5 and 8 into the forelimb joints of the horse, all three enabled hIL-1Ra expression at biologically relevant levels and effectively transduced the same cell types, primarily synovial fibroblasts and, to a lesser degree, chondrocytes in articular cartilage. These results provide optimism that AAV vectors can be effectively adapted for gene delivery to large human joints affected by OA.

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This study evaluated the potential of gene induced synoviocyte expression of a combination of insulin-like growth factor-I (AdIGF-I) and interleukin-1 receptor antagonist protein (AdIL-1Ra) to control articular cartilage degradation in vitro. Cartilage explants and synovial membrane were harvested from young mature horses. Synovial monolayers were established and either (1) maintained as untransduced controls; (2) transduced with AdIGF-I at 200 MOI in 500 pl serum-free medium; (3) transduced with AdIL-1Ra at 100 MOI; or (4) transduced with a combination of AdIGF-I (200 MOI) and AdIL-1Ra (100 MOI). Following transduction, cartilage explants were exposed to the synovial monolayer medium using co-culture inserts. Cultures were maintained for 6days in either serum-free medium or medium containing 10 ng/ml recombinant human interleukin-1 p. At termination, synovial cell RNA was isolated for real-time PCR analysis, and cartilage explants were collected for H&E and toluidine blue staining, immunohistochemistry for type I1 collagen and IGF-I, in situ localization of IGF-I and type I1 collagen gene expression, and biochemical assays. Synovial monolayers were readily transduced with both AdIGF-I and AdIL-1 Ra. IGF-I and IL-1 Ra protein were secreted at beneficial levels throughout the experiment, having peak concentrations of 94.6 nglml and 33.0 ng/ml, respectively. Transduction with IGF-I promoted cartilage production of proteoglycan and type I1 collagen, suggesting a beneficial role for healing injured cartilage. Transduction with IL-1Ra decreased the synovial expression of IL-la and IL-1 p and matrix metalloproteinases, indicating a mechanism for prevention of matrix degradation. The beneficial effects of the combination of anabolic growth factors and catabolic blockers were evident in improved preservation of proteoglycan content of cartilage explants exposed to the depleting effects of IL-I . These results show that gene therapy combining anabolic growth factors to stimulate matrix synthesis and catabolic blockers to prevent matrix degradation by IL-1, protects and causes partial restoration of cartilage matrix, and suggest a potential benefit of combination gene therapy for cartilage healing.

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