Tissue engineering of sizeable cell-scaffold constructs is limited by gradients in tissue quality from the periphery toward the center. Because homogenous delivery of oxygen to three-dimensional (3D) cell cultures remains an unsolved challenge, we hypothesized that uneven oxygen supply may impede uniform cellular growth on scaffolds. In this study we challenged static and dynamic 3D culture systems designed for bone tissue engineering applications with a well-growing subclone of MC3T3-E1 preosteoblasts and continuously measured the oxygen concentrations in the center of cell-seeded scaffolds and in the surrounding medium. After as little as 5 days in static culture, central oxygen concentrations dropped to 0%. Subsequently, cells died in central regions of the scaffold but not in its periphery, where oxygen levels were approximately 4%. The use of perfusion bioreactors successfully prevented cell death, yet central oxygen concentrations did not rise above 4%. We conclude that 3D culture in vitro is associated with relevant oxygen gradients, which can be the cause of inhomogeneous tissue quality. Perfusion bioreactors prevent cell death but they do not entirely eliminate 3D culture-associated oxygen gradients. Therefore, we advise continuous oxygen monitoring of 3D culture systems to ensure tissue quality throughout engineered constructs.

DNA damage activates cell cycle checkpoints in yeast and human cells. In the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe checkpoint-deficient mutants have been characterized, and the corresponding genes have been cloned. Searches for human homologs of S. pombe rad1, rad9, and hus1 genes identified the potential human homologs hRad1, hRad9, and hHus1; however, little is known about the roles of these proteins in human cells. The present studies demonstrate that hRad1 and hHus1 associate in a complex that interacts with a highly modified form of hRad9, but hHus1 and hRad1 do not associate with hRad17. In addition to being a key participant in complex formation, hRad9 is phosphorylated in response to DNA damage. Together, these results suggest that hRad9, hRad1, and hHus1 are central components of a DNA damage-responsive protein complex in human cells.DNA damage triggers a variety of cellular responses in eukaryotic cells, including the induction of a regulatory signaling network that activates checkpoint controls (1-3). Checkpoint activation transiently blocks cell cycle progression by arresting cells in G 1 and G 2 /M and slowing progression through S phase. Genetic studies in the yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe have identified many of the relevant players in DNA damage-induced checkpoint activation (4, 5). A common theme that emerges from these studies is that checkpoint-deficient yeast are dramatically more sensitive to genotoxins than their wild-type counterparts (1-3), suggesting that the checkpoint proteins play critical roles in cellular responses to DNA damage.Recent studies suggest that key checkpoint regulators may be conserved between yeast and humans. Cloning of the human gene mutated in AT (ATM) revealed that the ATM gene exhibited significant homology with the S. pombe rad3 (sprad3) and S. cerevisiae MEC1 (scMEC1) checkpoint genes (6). The corresponding proteins scMec1, spRad3, and Atm are protein kinases that share large stretches of homology, including a phosphatidylinositol 3-kinase-related kinase (PIKK) 1 domain. The PIKKs are also functionally conserved as mutation of scMEC1, sprad3, or ATM disrupts ionizing radiation (IR)-induced checkpoints and sensitizes the organisms to IR (7).The PIKKs are integral players in a tentative DNA damageinducible signaling pathway that interfaces with the cell cycle machinery (2, 3, 8). In this model, an unidentified sensor recognizes damaged DNA. Potential sensor candidates include the checkpoint proteins, spRad1, spRad9, spRad17, and spHus1. The sensor relays a signal to PIKK family members, which, at least in the case of ATM, are activated by IR (9, 10). In S. pombe, a PIKK-dependent pathway is required for DNA damage-induced activation of spChk1 (11), a protein kinase that phosphorylates Cdc25 and blocks its ability to activate Cdc2, thereby preventing cell cycle transition through the G 2 /M boundary.The similarities between yeast and humans extend beyond these proteins, as human homologs of sprad1 (12-14), spra...

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Tenomodulin (Tnmd) is the best-known mature marker for tendon and ligament lineage cells. It is important for tendon maturation, running performance and has key implications for the resident tendon stem/progenitor cells (TSPCs). However, its exact functions during the tendon repair process still remain elusive. Here, we established an Achilles tendon injury model in a Tnmd knockout (Tnmd−/−) mouse line. Detailed analyses showed not only a very different scar organization with a clearly reduced cell proliferation and expression of certain tendon-related genes, but also increased cell apoptosis, adipocyte and blood vessel accumulation in the early phase of tendon healing compared with their wild-type (WT) littermates. In addition, Tnmd−/− tendon scar tissue contained augmented matrix deposition of biglycan, cartilage oligomeric matrix protein (Comp) and fibronectin, altered macrophage profile and reduced numbers of CD146-positive cells. In vitro analysis revealed that Tnmd−/− TSPCs exhibited significantly reduced migration and proliferation potential compared with that of WT TSPCs. Furthermore, Tnmd−/− TSPCs had accelerated adipogenic differentiation accompanied with significantly increased peroxisome proliferator-activated receptor gamma (Pparγ) and lipoprotein lipase (Lpl) mRNA levels. Thus, our results demonstrate that Tnmd is required for prevention of adipocyte accumulation and fibrovascular scar formation during early tendon healing.

Osteogenic differentiation of human mesenchymal stem cells (hMSCs) into osteoblasts is a prerequisite for subsequent bone formation. Numerous studies have explored osteogenic differentiation under standard tissue culture conditions, which usually employ 21% of oxygen. However, bone precursor cells such as hMSCs reside in stem cell niches of low-oxygen atmospheres. Furthermore, they are subjected to low oxygen concentrations when cultured on three-dimensional scaffolds in vitro, and even more so after transplantation when vascularization has yet to be established. Similarly, hMSCs are exposed to low oxygen in the fracture microenvironment following bony injury. Recent studies revealed that hypoxic preconditioning improves cellular engraftment and survival in low-oxygen atmospheres. In our study we investigated the osteogenic differentiation potential of hMSCs under 2% O(2) (hypoxia) in comparison to a standard tissue culture oxygen atmosphere of 21% (normoxia). We assessed the osteogenic differentiation of hMSCs following hypoxic preconditioning to address whether this pretreatment is beneficial for subsequent differentiation processes as well. To validate our findings we carefully characterized the extent of hypoxia exerted and its effect on cell survival and proliferation. We found that hMSCs proliferate better if cultured under 2% of oxygen. We confirmed that osteogenic differentiation of hMSCs is indeed inhibited if osteogenic induction is carried out under constant hypoxia. Finally, we showed for the first time that hypoxic preconditioning of hMSCs prior to osteogenic induction restores osteogenic differentiation of hMSCs under hypoxic conditions. Collectively, our results indicate that maintaining constant levels of oxygen improves the osteogenic potential of hMSCs and suggest that low oxygen concentrations may preserve the stemness of hMSCs. In addition, our data support the hypothesis that if low-oxygen atmospheres are expected at the site of implantation, hypoxic pretreatment may be beneficial for the cells' subsequent in vivo performance.

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