In Vitro Differentiation of Myoblast Cell Lines on Spider Silk Scaffolds in a Rotating Bioreactor for Vascular Tissue Engineering.

Functional construction of tissue-engineered vessels as an alternative to autologous vascular grafts has been shown to be feasible, however the proliferation of seeded smooth-muscle cells remains a limiting factor. We employed a rotating bioreactor system to improve myoblast cell differentiation on a spider silk scaffold for tissue-engineered vessel construction. C2C12 myofibroblast cells were seeded on the surface of spider silk scaffold constructs and cultivated in a rotating bioreactor system with a continuous rotation speed (1 rpm). Cell function, cell growth and morphological structure and expression of biomarkers were analyzed using scanning electron microscopy, the LIVE/DEAD® assay, Western blot and quantitative real-time PCR analyses. A dense myofibroblast cell sheet could be developed which resembled native blood vessel muscular tissue in morphological structure and in function. Bioreactor perfusion positively affected cell morphology, and increased cell viability and cell differentiation. The expression of desmin, MYF5 and MEF2D surged as an indication of myoblast differentiation. Cell-seeded scaffolds showed a tear-down at 18 N when strained at a set speed (20 mm min-1). Spider silk scaffolds appear to offer a reliable basis for engineered vascular constructs and rotating bioreactor cultivation may be considered an effective alternative to complex bioreactor setups to improve cell viability and biology.

An Identification and Characterization of the Axolotl (Ambystoma mexicanum, Amex) Telomerase Reverse Transcriptase (Amex TERT).

The Mexican axolotl is one of the few vertebrates that is able to replace its lost body parts during lifespan. Due to its remarkable regenerative abilities, the axolotl emerged as a model organism especially for limb regeneration. Telomeres and the telomerase enzyme are crucial for regeneration and protection against aging processes and degenerating diseases. Despite its relevance for regeneration, the axolotl telomerase and telomere length have not yet been investigated. Therefore, in the present paper, we reveal the sequence of the axolotl telomerase reverse transcriptase gene (Tert) and protein (TERT). Multiple sequence alignment (MSA) showed the known conserved RT- and TERT-specific motifs and residues found in other TERTs. In addition, we establish methods to determine the Tert expression (RT-PCR) and telomerase activity (Q-TRAP) of adult axolotl and blastema tissues. We found that both differentiated forelimb tissue and regenerating blastema tissue express Tert and show telomerase activity. Furthermore, blastema tissue appears to exhibit a higher Tert expression and telomerase activity. The presence of active telomerase in adult somatic cells is a decisive difference to somatic cells of non-regenerating vertebrates, such as humans. These findings indicate that telomere biology may play a key role in the regenerative abilities of cells.

Identification of axolotl BH3-only proteins and expression in axolotl organs and apoptotic limb regeneration tissue.

Like other urodela amphibians, axolotls are able to regenerate lost appendages, even as adults, rendering them unique among higher vertebrates. In reaction to the severe trauma of a lost limb, apoptosis seems to be primarily implicated in the removal of injured cells and tissue homeostasis. Little, however, is known about apoptotic pathways and control mechanisms. Therefore, here we provide additional information regarding the mechanisms of tissue degradation. Expression patterns of Bcl-2 family members were analyzed using reverse transcriptase-PCR, western blotting and immunofluorescence. In our study, we identified ten putative axolotl orthologs of the Bcl-2 family. We demonstrated that BH3-only proteins are differentially expressed in some axolotl organs, while they are expressed broadly in tail composite tissue and limb regeneration blastema. The importance of Bcl-2 family members is also indicated by detecting the expression of proapoptotic protein Bak in spatial congruence to apoptosis in the early stages of limb regeneration, while Bcl-2 expression was slightly modified. In conclusion, we demonstrate that Bcl-2 family members are conserved in the axolotl and might be involved in the tissue degradation processes that occur during limb regeneration.

Preliminary investigations of spider silk in wounds in vivo - Implications for an innovative wound dressing.

The ideal wound dressing in particular for burn wounds has not been found yet. The aim of this study was to investigate native spider silk as a novel wound dressing. Release of inflammatory cytokines of macrophages and neutrophile granulocytes was determined via ELISA after exposure to spider silk. Migration of dermal cells as well as angiogenesis on spider silk was visualized with live video microscopy or chorioallantois membrane model, respectively. Native spider silk was placed in full-thickness skin wounds in a sheep in vivo-model and wounds were evaluated after 2, 4, 6, and 8weeks histologically as well as per quantitative real-time PCR. Minimal inflammatory cytokine release could be seen for spider silk. Ingrowth of single capillaries into bundles of spider silk and migration of keratinocytes as well as fibroblasts on spider silk fibres was proven. Macroscopically, a comparable wound closure could be seen in spider silk and in sham controls. In histological evaluation, a thicker epidermis was observed in spider silk treated wounds while collagen III/I expression ratio was comparable in both groups. As native spider silk has been described as highly biocompatible, it might represent an innovative alternative to common wound dressings.

Silks as scaffolds for skin reconstruction.

In this short review, we describe the use of high molecular weight proteins produced in the glands of several arthropods-commonly called silks-for the purpose to enhance human skin wound healing. To this end an extensive literature search has been performed, the publications have been categorized concerning silk preparation and application and summarized accordingly: Scaffolds to promote wound healing were prepared by processing the silks in different ways including solubilization of the protein fibers followed by casting or electrospinning. The silk scaffolds were additionally modified by coating or blending with the intention of further functionalization. In several approaches, the scaffolds were also vitalized with skin cells or stem cells. In vitro and in vivo models were implied to test for safety and efficiency. We conclude that silk scaffolds are characterized by an advantageous biocompatibility as well as an impressive versatility rendering them ideally suited for application in wounds. Nevertheless, further investigation is needed to exploit the full capacity of silk in different wound models and to achieve clinical transfer in time.

Identification of reference genes and validation for gene expression studies in diverse axolotl (Ambystoma mexicanum) tissues.

For the precise quantitative RT-PCR normalization a set of valid reference genes is obligatory. Moreover have to be taken into concern the experimental conditions as they bias the regulation of reference genes. Up till now, no reference targets have been described for the axolotl (Ambystoma mexicanum). In a search in the public database SalSite for genetic information of the axolotl we identified fourteen presumptive reference genes, eleven of which were further tested for their gene expression stability. This study characterizes the expressional patterns of 11 putative endogenous control genes during axolotl limb regeneration and in an axolotl tissue panel. All 11 reference genes showed variable expression. Strikingly, ACTB was to be found most stable expressed in all comparative tissue groups, so we reason it to be suitable for all different kinds of axolotl tissue-type investigations. Moreover do we suggest GAPDH and RPLP0 as suitable for certain axolotl tissue analysis. When it comes to axolotl limb regeneration, a validated pair of reference genes is ODC and RPLP0. With these findings, new insights into axolotl gene expression profiling might be gained.

Intravenous transplantation of mesenchymal stromal cells to enhance peripheral nerve regeneration.

Peripheral nerve injury is a common and devastating complication after trauma and can cause irreversible impairment or even complete functional loss of the affected limb. While peripheral nerve repair results in some axonal regeneration and functional recovery, the clinical outcome is not optimal and research continues to optimize functional recovery after nerve repair. Cell transplantation approaches are being used experimentally to enhance regeneration. Intravenous infusion of mesenchymal stromal cells (MSCs) into spinal cord injury and stroke was shown to improve functional outcome. However, the repair potential of intravenously transplanted MSCs in peripheral nerve injury has not been addressed yet. Here we describe the impact of intravenously infused MSCs on functional outcome in a peripheral nerve injury model. Rat sciatic nerves were transected followed, by intravenous MSCs transplantation. Footprint analysis was carried out and 21 days after transplantation, the nerves were removed for histology. Labelled MSCs were found in the sciatic nerve lesion site after intravenous injection and regeneration was improved. Intravenously infused MSCs after acute peripheral nerve target the lesion site and survive within the nerve and the MSC treated group showed greater functional improvement. The results of study suggest that nerve repair with cell transplantation could lead to greater functional outcome.