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.

Human mesenchymal stem cells (hMSCs) can be readily isolated from bone marrow and differentiate into multiple tissues, making them a promising target for future cell and gene therapy applications. The low frequency of hMSCs in bone marrow necessitates their isolation and expansion in vitro prior to clinical use, but due to senescence-associated growth arrest during culture, limited cell numbers can be generated. The lifespan of hMSCs has been extended by ectopic expression of human telomerase reverse transcriptase (hTERT) using retroviral vectors. Since malignant transformation was observed in hMSCs and retroviral vectors cause insertional mutagenesis, we ectopically expressed hTERT using lentiviral gene transfer. Single-cell-derived hMSC clones expressing hTERT did not show malignant transformation in vitro and in vivo after extended culture periods. There were no changes observed in the expression of tumour suppressor genes and karyotype. Cultured hMSCs lack telomerase activity, but it was significantly increased by ectopic expression of hTERT. HTERT expression prevented hMSC senescence and the cells showed significantly higher and unlimited proliferation capacity. Even after an extended culture period, hMSCs expressing hTERT preserved their stem cells character as shown by osteogenic, adipogenic and chon-drogenic differentiation. In summary, extending the lifespan of human mesenchymal stem cells by ectopic expression of hTERT using lentiviral gene transfer may be an attractive and safe way to generate appropriate cell numbers for cell and gene therapy applications.

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Almost 20 years after the invention of tissue engineering, autogenous bone grafting has remained the favored strategy for the treatment of bone defects. As an alternative, a vast variety of bone substitutes has been developed and is available for clinical use. The ongoing search for bone substitutes, however, reflects the limitations imposed to both autogenous and allogenous bone grafts as well as to bone substitute materials. The concept of tissue engineering holds great promise for the future treatment of osseous defects. Research in this interdisciplinary field is carried out to find a way of producing biologic substitutes as functional tissue replacement. For this, functionally active cells are applied on supporting scaffolds under controlled stimulation with growth factors. Scaffolds are temporary matrices for bone growth and provide a specific environment and architecture for tissue development. Ideally, scaffolds favor cellular attachment, growth and differentiation in vitro and in vivo. Especially ceramics and biodegradable polymers are widely used and have been tested in various animal studies. Yet, to allow for precise production of specific custom-made scaffolds, rapid prototyping (RP) techniques have recently drawn a lot of attention. Using these methods scaffolds with a predefined, well-controlled internal and external architecture mimicking the structure of natural bone can be generated. Although biocompatibility of the materials used in the process and the structural resolution that can be technically achieved so far limit the range of use, rapid manufacturing techniques do offer great opportunities to generate suitable scaffolds for bone tissue engineering in the near future.

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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(111)In-oxine labelling moderately impaired hMSC's functional integrity while preserving their stem cell character. Combined SPECT/CT imaging of (111)In-oxine-labelled hMSC opens the possibility for non-invasive sequential monitoring of therapeutic stem cells.

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