The scarcity of transplant allografts for diseased organs has prompted efforts at tissue regeneration using seeded scaffolds, an approach hampered by the enormity of cell types and complex architectures. Our goal was to decellularize intact organs in a manner that retained the matrix signal for differentiating pluripotent cells. We decellularized intact rat kidneys in a manner that preserved the intricate architecture and seeded them with pluripotent murine embryonic stem cells antegrade through the artery or retrograde through the ureter. Primitive precursor cells populated and proliferated within the glomerular, vascular, and tubular structures. Cells lost their embryonic appearance and expressed immunohistochemical markers for differentiation. Cells not in contact with the basement membrane matrix became apoptotic, thereby forming lumens. These observations suggest that the extracellular matrix can direct regeneration of the kidney, and studies using seeded scaffolds may help define differentiation pathways.

A major hurdle in most current gene therapy modalities is the ability to transduce target tissues at very high efficiencies that ultimately lead to therapeutic levels of transgene expression. We have developed a novel method of recombinant adeno-associated virus 2 (rAAV) delivery that results in increased vector transduction efficiencies using microspheres reversibly conjugated to rAAV vectors. We hypothesize that conjugation to microspheres should result in a higher effective concentration of vector as well as longer relative exposure time of vector to target cells as it moves through the tissue vasculature. In vitro experiments demonstrate that the same level of transduction seen with free vector can be achieved using 1% of vector when conjugated to microspheres. In addition, using magnetic microspheres, the region of infection can be targeted. In vivo, we demonstrate that microsphere-mediated delivery of rAAV vector results in higher transduction efficiencies than delivery with free vector alone when administered either intramuscularly or intravenously. Furthermore, we demonstrate targeting of transgene expression to specific tissues by retention of microsphere-bound vector in the capillary bed. These studies demonstrate a novel method to deliver rAAV vectors more effectively that could prove to be a successful alternative mode of virus-mediated human gene therapy.

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Conclusion: We provide new evidence for matrix-to-cell signaling in acellular whole organ scaffolds that induces differentiation of pluripotent precursor cells to endothelial lineage. Production of mouse basement membrane supports remodeling of host (rat)-derived scaffolds and thereby warrants further investigation as a promising approach for xenotransplantation.Methods: We previously showed that murine embryonic stem cells arterially seeded into acellular rat whole kidney scaffolds multiply and demonstrate morphologic, immunohistochemical and gene expression evidence for differentiation. Vascular cell endothelialization was now further tested by endothelial specific BsLB4 lectin and anti-VEGFR2 (Flk1) antibodies. Remodeling of the matrix basement membranes from rat to mouse ("murinization") was assessed by a monoclonal antibody specific for mouse laminin b1 chain.

The present study evaluated the potential for reestablishment of connective tissue attachment after exclusion of the gingival connective tissue and epithelium during healing after periodontal surgery in a dog model. Buccal mucoperiosteal flaps were elevated in all four premolar regions in two dogs. The buccal bone was reduced to approximately 25% of its original level on 24 roots. Before the elevated flaps were replaced and sutured, membranes were adjusted to cover the exposed root surfaces. On eight surfaces Millipore filters were used and on eight surfaces biodegradable polylactic acid membranes were used. No membranes were placed over the remaining eight root surfaces which served as controls. After two months of healing, the animals were sacrificed and all the roots were subjected to histological analysis. In all of the roots covered with the polylactic acid membrane, newly formed cementum with inserting collagen fibers was observed and covered on the average 46% of the initial defect. Newly formed bone covered 39% of the initial defect. The roots covered by Millipore filters were characterized by significantly less new attachment and bone. In the control roots only minor new attachment was found in the bottom of the defect in some roots. The results suggest that the establishment of a connective tissue attachment is clearly favored by the placement of a polylactic acid membrane to exclude the epithelium and gingival connective tissue during healing.