Bone marrow-derived mesenchymal stem cells (MSCs) have the potential to differentiate along different mesenchymal lineages including those forming bone, cartilage, tendon, fat, muscle and marrow stroma that supports hematopoiesis. This differentiation potential makes MSCs candidates for cell-based therapeutic strategies for mesenchymal tissue injuries and for hematopoietic disorders by both local and systemic application. In the present study, rat marrow-derived MSCs were ex vivo culture-expanded, labeled with ¹¹¹In-oxine, and infused into syngeneic rats via intra-artery (i.a.), intravenous (i.v.) and intraperitoneal cavity (i.p.) infusions. In addition, for i.a. and i.v. infusions, a vasodilator, sodium nitroprusside, was administered prior to the cell infusion and examined for its effect on MSC circulation. The dynamic distribution of infused MSCs was monitored by real-time imaging using a gamma camera immediately after infusion and at 48 h postinfusion. After 48 h, radioactivity in excised organs, including liver, lungs, kidneys, spleen and long bones, was measured in a gamma well counter and expressed as a percentage of injected doses. After both i.a. and i.v. infusion, radioactivity associated with MSCs was detected primarily in the lungs and then secondarily in the liver and other organs. When sodium nitroprusside was used, more labeled MSCs cleared the lungs resulting in a larger proportion detected in the liver. Most importantly, the homing of labeled MSCs to the marrow of long bones was significantly increased by the pretreatment with vasodilator. These results indicate multiple homing sites for injected MSCs and that the distribution of MSCs can be influenced by administration of vasodilator.

Glutaredoxin (Grx) is a glutathione-dependent hydrogen donor for ribonucleotide reductase. Today glutaredoxins are known as a multifunctional family of GSHdisulfide-oxidoreductases belonging to the thioredoxin fold superfamily. In contrast to Escherichia coli and yeast, a single human glutaredoxin is known. We have identified and cloned a novel 18-kDa human dithiol glutaredoxin, named glutaredoxin-2 (Grx2), which is 34% identical to the previously known cytosolic 12-kDa human Grx1. The human Grx2 sequence contains three characteristic regions of the glutaredoxin family: the dithiol/disulfide active site, CSYC, the GSH binding site, and a hydrophobic surface area. The human Grx2 gene, located at chromosome 1q31.2-31.3, consisted of five exons that were transcribed to a 0.9-kilobase human Grx2 mRNA ubiquitously expressed in several tissues. Two alternatively spliced Grx2 mRNA isoforms that differed in their 5 region were identified. These corresponded to alternative proteins with a common 125-residue C-terminal Grx domain but with different N-terminal extensions of 39 and 40 residues, respectively. The 125-residue Grx domain and the two full-length variants were expressed in E. coli and exhibited GSH-dependent hydroxyethyl disulfide and dehydroascorbate reducing activities. Western blot analysis of subcellular fractions from Jurkat cells with a specific anti-Grx2 antibody showed that human Grx2 was predominantly located in the nucleus but also present in the mitochondria. We further showed that one of the mRNA isoforms corresponding to Grx2a encoded a functional N-terminal mitochondrial translocation signal.

Protein transduction domains (PTD), such as the HIV TAT and the herpes simplex virus VP22 proteins, are reported to translocate across the membranes of mammalian cells. The mechanism of PTD membrane translocation has largely remained elusive, but recent studies suggest that the reported PTD translocation is due to a fixation artifact. We have constructed and expressed the PTDs VP22, TAT, polyarginine, and polylysine fused to the green fluorescent protein to visualize these proteins in both living and fixed cells. The investigated PTDs strongly adhered to the surface of living cells and were internalized by constitutive endocytosis. No cytosolic or nuclear import of the proteins was detected. In contrast, the PTD-GFP fusion proteins were redistributed to the cytosol and nucleus directly after fixation. Our findings suggest that the PTDs only mediate cell surface adherence, a property shared with many other positively charged macromolecules. The cell surface adherence results in endocytosis and accumulation of proteins in endosomes. We suggest that the biological effects observed for PTD fusion proteins are due to cell surface interactions and internalization of the proteins into cells by classical endocytosis.

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Summary: Articular cartilage in adults has limited ability for self-repair. Some methods devised to augment the natural healing response stimulate some regeneration, but the repair is often incomplete and lacks durability. Hyaluronan-based polymers were tested for their ability to enhance the natural healing response. It is hypothesized that hyaluronan-based polymers recreate an embryonic-like milieu where host progenitor cells can rcgcncrate the damaged articular surface and underlying bone. Oskochondral dcfccts were made on the femoral condyles of 4-month-old rabbits and were left empty or filled with hyaluronan-based polymers. The polymers tested were ACP sponge. made of crosslinked hyaluronan, and HYAFF-11 sponge, made of benxylated hyaluronan. The rabbits were killed 4 and 12 weeks after surgery, and the condyles were processed for histology. All 12-week defects were scored with a 29-point scale, and the scores were compared with a Kruskall-Wallis analysis of variance on ranks. Untreated defects filled with bone tissue up to or beyond the tidemark, and the noncalcified surface layer varied from fibrous to hyaline-like tissue. Four weeks aftcr surgery, defects treated with ACP exhibited bone filling to the level of the tidemark and the surface layer was composed of hyaline-like cartilage well integrated with the adjacent cartilage. At 12 weeks, the specimens had bone beyond the tidemark that was covered with a thin layer of hyaline cartilage. Four weeks after surgery, defects treated with HYAFF-11 contained a rim of chondrogcnic cells at the interface of the implant and the host tissue. In general, the 12-week dcfccts exhibited good bone fill and the surface was mainly hyaline cartilage. Treated defects received significantly higher scores than untreated defects (p < 0.05), and ACP-trcatcd defects scored significantly higher than HYAFF-11-treated defects (p < 0.05). The introduction of these hyaluronan-based polymers into delects provides an appropriate scaffolding and favorable microenvironnient for the reparative process. Further work is required to lully assess thc long-term outcome of defects treated with these polymers.The successful regeneration of any tissue requires not oiily reparative cells with the potential to differentiate into the phenotypes required to restore the damaged site but also a microenvironment that supports the proliferation and differentiation of those cells (12). It is our hypothesis that these reparative cells recapitulate, a t least in part. the sequence of events that leads to the formation of differentiated tissues during embryonic development; therefore. materials that mimic an embryonic environment may be superior scaffolds for tissue-engineered repair of damaged tissues.Articular cartilage has no vascular supply or easy access to large quantities of progenitor cells: thus, cartilage repair is often unsuccessful. Some studies indicate that full-thickness lesions in young animals are

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