Spider silk enhanced tissue engineering of cartilage tissue: Approach of a novel bioreactor model using adipose derived stromal cells.

Human cartilage tissue remains a challenge for the development of therapeutic options due to its poor vascularization and reduced regenerative capacities. There are a variety of research approaches dealing with cartilage tissue engineering. In addition to different biomaterials, numerous cell populations have been investigated in bioreactor-supported experimental setups to improve cartilage tissue engineering. The concept of the present study was to investigate spider silk cocoons as scaffold seeded with adipose-derived stromal cells (ASC) in a custom-made bioreactor model using cyclic axial compression to engineer cartilage-like tissue. For chemical induction of differentiation, BMP-7 and TGF-ß2 were added and changes in cell morphology and de-novo tissue formation were investigated using histological staining to verify chondrogenic differentiation. By seeding spider silk cocoons with ASC, a high colonization density and cell proliferation could be achieved. Mechanical induction of differentiation using a newly established bioreactor model led to a more roundish cell phenotype and new extracellular matrix formation, indicating a chondrogenic differentiation. The addition of BMP-7 and TGF-ß2 enhanced the expression of cartilage specific markers in immunohistochemical staining. Overall, the present study can be seen as pilot study and valuable complementation to the published literature.

Organization of Hannover Skin Bank: Sterile culture and procurement protocols for viable cryopreserved allogeneic skin grafts of living donors.

Preserved allogeneic donor skin still represents one of the gold standard therapies in temporary wound coverage in severely burned patients or chronic wounds. Allogeneic skin grafts are currently commercially available as cryo- or glycerol-preserved allografts through skin tissue banks all over the world. Most of the skin tissue banks rely on human cadaveric skin donations. Due to the chronic shortage of human allogeneic transplants, such as skin, and increasing costs in the procurement of allografts from other skin tissue banks, Hannover Medical School has been building up its own skin tissue bank based on allogeneic skin grafts from living donors who underwent surgical treatment (i.e., body-contouring procedures, such as abdominioplasties). This article presents procedures and protocols for the procurement and processing of allogeneic skin grafts according to national legislation and European regulations and guidelines. Beside protocols, initial microbiological data regarding the sterility of the harvested grafts are presented. The results currently form the basis for further investigations as well as clinical applications. In summary, a microbiological testing and acceptance procedure is presented that ensures adequate patient safety and skin viability.

Influence of Tamoxifen on Different Biological Pathways in Tumorigenesis and Transformation in Adipose-Derived Stem Cells, Mammary Cells and Mammary Carcinoma Cell Lines-An In Vitro Study.

Breast carcinoma is one of the most common malignant tumors in women. In cases of hormone-sensitive cells, tamoxifen as an anti-estrogenic substance is a first line medication in the adjuvant setting. The spectrum of autologous breast reconstructions ranges from fat infiltrations to complex microsurgical procedures. The influence of adipose-derived stem cells (ASC) on the tumor bed and a possibly increased recurrence rate as a result are critically discussed. In addition, there is currently no conclusive recommendation regarding tamoxifen-treated patients and autologous fat infiltrations. The aim of the present study was to investigate the effect of tamoxifen on the gene expression of a variety of genes involved in tumorigenesis, cell growth and transformation. Mammary epithelial cell line and mammary carcinoma cell lines were treated with tamoxifen in vitro as well as co-cultured with ASC. Gene expression was quantified by PCR arrays and showed increased expression in the mammary carcinoma cell lines with increasing time of treatment and concentration of tamoxifen. The data presented can be considered as an addition to the controversial discussion on the relationship between ASC and breast carcinoma cells. Further studies are needed to quantify the in vivo interaction of ASC and mammary carcinoma cells and to conclusively assess the impact of tamoxifen in reconstructive cases with fat grafting.

A New Standardized Clinical Model for Evaluation of Nerve Guides: A Single-Blinded Randomized Controlled Prospective Study.

BACKGROUND: Autologous nerve transplantation is the accepted clinical standard for reconstruction of peripheral nerve defects. Bioengineered nerve guides as an alternative to autologous nerve transplantation have been described in previous studies and several different types are commercially available. Examination of the implementation of these devices in the clinical setting is an important step in determining their applicability in reconstructive nerve surgery. METHODS: In a single-blinded randomized controlled prospective study, the authors examined 34 patients undergoing diagnostic nerve biopsy (2 cm). The biopsy-induced nerve gap was interposed using a segment of the lesser saphenous vein in 16 patients. Eighteen patients had no nerve reconstruction and served as a control group. A further 10 participants were included as a healthy cohort. Nerve regeneration was assessed using von Frey filaments preoperatively and 1 day and 3, 6, 9, and 12 months postoperatively. RESULTS: Patients who received defect bridging of the sural nerve with a venous graft showed better regeneration of lateral foot sensitivity than patients without venous graft. CONCLUSIONS: The distal sural nerve biopsy can be used as a baseline model to evaluate peripheral nerve regeneration. Newly developed nerve guides could be tested in other unpredictable and challenging clinical peripheral nerve lesions. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, II.

Preliminary application of native Nephila edulis spider silk and fibrin implant causes granulomatous foreign body reaction in vivo in rat's spinal cord.

After spinal cord injury, gliomesenchymal scaring inhibits axonal regeneration as a physical barrier. In peripheral nerve injuries, native spider silk was shown to be an effective scaffold to facilitate axonal re-growth and nerve regeneration. This study tested a two-composite scaffold made of longitudinally oriented native spider silk containing a Haemocomplettan fibrin sheath to bridge lesions in the spinal cord and enhance axonal sprouting. In vitro cultivation of neuronal cells on spider silk and fibrin revealed no cytotoxicity of the scaffold components. When spinal cord tissue was cultured on spider silk that was reeled around a metal frame, migration of different cell types, including neurons and neural stem cells, was observed. The scaffold was implanted into spinal cord lesions of four Wistar rats to evaluate the physical stress caused on the animals and examine the bridging potential for axonal sprouting and spinal cord regeneration. However, the implantation in-vivo resulted in a granulomatous foreign body reaction. Spider silk might be responsible for the strong immune response. Thus, the immune response to native spider silk seems to be stronger in the central nervous system than it is known to be in the peripheral body complicating the application of native spider silk in spinal cord injury treatment.

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.

In-Vitro Application of Magnetic Hybrid Niosomes: Targeted siRNA-Delivery for Enhanced Breast Cancer Therapy.

Even though the administration of chemotherapeutic agents such as erlotinib is clinically established for the treatment of breast cancer, its efficiency and the therapy outcome can be greatly improved using RNA interference (RNAi) mechanisms for a combinational therapy. However, the cellular uptake of bare small interfering RNA (siRNA) is insufficient and its fast degradation in the bloodstream leads to a lacking delivery and no suitable accumulation of siRNA inside the target tissues. To address these problems, non-ionic surfactant vesicles (niosomes) were used as a nanocarrier platform to encapsulate Lifeguard (LFG)-specific siRNA inside the hydrophilic core. A preceding entrapment of superparamagnetic iron-oxide nanoparticles (FexOy-NPs) inside the niosomal bilayer structure was achieved in order to enhance the cellular uptake via an external magnetic manipulation. After verifying a highly effective entrapment of the siRNA, the resulting hybrid niosomes were administered to BT-474 cells in a combinational therapy with either erlotinib or trastuzumab and monitored regarding the induced apoptosis. The obtained results demonstrated that the nanocarrier successfully caused a downregulation of the LFG gene in BT-474 cells, which led to an increased efficacy of the chemotherapeutics compared to plainly added siRNA. Especially the application of an external magnetic field enhanced the internalization of siRNA, therefore increasing the activation of apoptotic signaling pathways. Considering the improved therapy outcome as well as the high encapsulation efficiency, the formulated hybrid niosomes meet the requirements for a cost-effective commercialization and can be considered as a promising candidate for future siRNA delivery agents.

A Short History of Skin Grafting in Burns: From the Gold Standard of Autologous Skin Grafting to the Possibilities of Allogeneic Skin Grafting with Immunomodulatory Approaches.

Due to groundbreaking and pioneering developments in the last century, significant improvements in the care of burn patients have been achieved. In addition to the still valid therapeutic standard of autologous split-thickness skin grafting, various commercially available skin substitutes are currently available. Significant progress in the field of tissue engineering has led to the development of promising therapeutic approaches. However, scientific advances in the field of allografting and transplant immunology are of great importance. The achievement of various milestones over the past decades has provided thought-provoking impulses in the field of skin allotransplantation. Thus, biologically viable skin allotransplantation is still not a part of the clinical routine. The purpose of this article is to review the achievements in burn surgery with regards to skin allotransplantation in recent years.

Influence of glucose and insulin in human adipogenic differentiation models with adipose-derived stem cells.

Autologous fat grafting represents an attractive source for tissue engineering applications in the field of reconstructive medicine. However, in adipogenic differentiation protocols for human adipose-derived stem cells, the concentration of glucose and insulin varies considerably. With the intent to gain maximum tissue augmentation, we focused on the late phase of adipogenesis. In this study, we modified the differentiation protocol for adipose-derived stem cells by prolongation of the induction period and the application highly concentrated glucose and insulin. Human adipose-derived stem cells were isolated from subcutaneous depots and differentiated in a standard induction medium for the first two weeks, followed by two weeks with varying glucose and insulin concentrations. Morphological changes assessed using Oil-Red-O staining were examined for corresponding alterations in the expression of the adipogenic markers peroxisome proliferator-activated receptor gamma (PPARγ) and lipoprotein lipase (LPL). Furthermore, glucose and lactate levels in conditioned media were monitored over the period of differentiation. We found high-glucose media increasing the level of lipid accumulation and the size of single droplets whereas insulin significantly showed a dose-dependent negative effect on fat storage. However, whereas high glucose stimulated PPARγ transcription, expression levels in insulin-treated cells remained constant. Results permit assumptions that a high-glucose medium intensifies the degree of differentiation in mature adipocytes providing conditions to promote graft volume while we have identified highly concentrated insulin treatment as an inhibitor of lipid storage in the late adipogenic differentiation.

Down-Regulation of MHC Class I Expression in Human Keratinocytes Using Viral Vectors Containing US11 Gene of Human Cytomegalovirus and Cultivation on Bovine Collagen-Elastin Matrix (Matriderm®): Potential Approach for an Immune-Privileged Skin Substitute.

Skin transplantation, especially in burn patients, is still challenging because surgeons are faced with limited disposability of autologous donor side material. The in vitro culture of keratinocytes has become an important reconstructive option. However, only non-immunogenic allogenic keratinocytes offer the opportunity to develop a skin graft that can overcome rejection. The purpose of the study was to develop targeted gene modification of keratinocytes in order to reduce immunogenicity for the use as allogenic transplantable skin graft by decreasing the expression of MHC class I. To reduce MHC class I expression, viral vectors containing the US11 gene of human cytomegalovirus were generated and tested on their functionality using Western blotting, indirect immunofluorescence staining, and flow cytometry. Transfected keratinocytes were seeded on commercially available bovine collagen-elastin matrices and further cultured for histological and cell survival assays. Results showed transient down-regulation of MHC class I after 24 h post-transfection, with recovery of MHC class I expression after 48 h. Histological assessments showed long-term cell survival as well as histological patterns comparable to epidermal layers of healthy human skin. The data postulates the potential application of US11 transfected keratinocytes as an approach towards an immune-privileged skin substitute. Nevertheless, further studies and data are needed.

Effect of Exosomes from Rat Adipose-Derived Mesenchymal Stem Cells on Neurite Outgrowth and Sciatic Nerve Regeneration After Crush Injury.

Peripheral nerve injury requires optimal conditions in both macro-environment and microenvironment for promotion of axonal regeneration. However, most repair strategies of traumatic peripheral nerve injury often lead to dissatisfying results in clinical outcome. Though various strategies have been carried out to improve the macro-environment, the underlying molecular mechanism of axon regeneration in the microenvironment provided by nerve conduit remains unclear. In this study, we evaluate the effects of from adipose-derived mesenchymal stem cells (adMSCs) originating exosomes with respect to sciatic nerve regeneration and neurite growth. Molecular and immunohistochemical techniques were used to investigate the presence of characteristic exosome markers. A co-culture system was established to determine the effect of exosomes on neurite elongation in vitro. The in vivo walking behaviour of rats was evaluated by footprint analysis, and the nerve regeneration was assessed by immunocytochemistry. adMSCs secrete nano-vesicles known as exosomes, which increase neurite outgrowth in vitro and enhance regeneration after sciatic nerve injury in vivo. Furthermore, we showed the presence of neural growth factors transcripts in adMSC exosomes for the first time. Our results demonstrate that exosomes, constitutively produced by adMSCs, are involved in peripheral nerve regeneration and have the potential to be utilised as a therapeutic tool for effective tissue-engineered nerves.

In vitro enhancement and functional characterization of neurite outgrowth by undifferentiated adipose-derived stem cells.

Adipose­derived stem cells (ASCs) can easily be obtained and expanded in vitro for use in autologous cell therapy. Via their production of cytokines and neurotrophic factors, transplanted ASCs provide neuroprotection, neovascularization and induction of axonal sprouting. However, the influencing mechanism of undifferentiated ASCs on nerve regeneration is currently only partially understood. In the present study, undifferentiated ASCs and cutaneous primary afferent dorsal root ganglion (DRG) neurons were co­cultured in order to investigate their interaction. ASCs were isolated from adult rat fat tissue. The presence of characteristic stem cell markers was determined by flow cytometry in three subsequent passages. Adipogenic, osteogenic, chondrogenic and glial differentiation was performed in order to evaluate their differentiation capacity. A direct co­culture system with DRG cells was established to determine the effect of undifferentiated pluripotent ASCs on neurite elongation. Neurite outgrowth, length and number was examined in the co­culture and compared with single­culture cells and cells stimulated with nerve growth factor (NGF). In ASC cultures, NGF expression was assessed by ELISA. The present results demonstrated that the specific mesenchymal stem cell surface markers CD44, CD73 and CD90 were detected in all three subsequent passages of the isolated ASCs. In accordance, ASC differentiation into adipogenic, osteogenic, chondrogenic and Schwann cell phenotype was conducted successfully. Neurite outgrowth of DRG neurons was enhanced following co­culture with ASCs, resulting in increased neurite length after 24 h of cultivation. Furthermore, neurite outgrowth of DRG neurons was directed towards the undifferentiated ASC and direct cell­to­cell contact was observed. In summary, the results of the present study revealed an interaction between the two cell types with guidance of neurite growth towards the undifferentiated ASC. These findings suggest that the use of undifferentiated ASC optimizing tissue­engineered constructs may be promising for peripheral nerve repair.

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.

Anti-apoptotic protein Lifeguard does not act as a tumor marker in breast cancer.

The aim of the present study was to use an enzyme-linked immunosorbent assay (ELISA) to determine the concentrations of Lifeguard (LFG) protein in the serum of 36 patients diagnosed with breast cancer and to compare these values with the concentrations of LFG protein in the serum of 7 healthy volunteers in order to detect a possible association between the expression of LFG in the serum and the degree of malignancy of the disease. Although there is no direct association between the LFG protein concentration in the serum and the degree of malignancy of breast cancer, a statistically significant distribution of the concentration in all investigated samples was observed. This indicated an association between the LFG protein concentration in human serum with a currently unknown factor.

Characterization and Schwann Cell Seeding of up to 15.0 cm Long Spider Silk Nerve Conduits for Reconstruction of Peripheral Nerve Defects.

Nerve reconstruction of extended nerve defect injuries still remains challenging with respect to therapeutic options. The gold standard in nerve surgery is the autologous nerve graft. Due to the limitation of adequate donor nerves, surgical alternatives are needed. Nerve grafts made out of either natural or artificial materials represent this alternative. Several biomaterials are being explored and preclinical and clinical applications are ongoing. Unfortunately, nerve conduits with successful enhancement of axonal regeneration for nerve defects measuring over 4.0 cm are sparse and no conduits are available for nerve defects extending to 10.0 cm. In this study, spider silk nerve conduits seeded with Schwann cells were investigated for in vitro regeneration on defects measuring 4.0 cm, 10.0 cm and 15.0 cm in length. Schwann cells (SCs) were isolated, cultured and purified. Cell purity was determined by immunofluorescence. Nerve grafts were constructed out of spider silk from Nephila edulis and decellularized ovine vessels. Finally, spider silk implants were seeded with purified Schwann cells. Cell attachment was observed within the first hour. After 7 and 21 days of culture, immunofluorescence for viability and determination of Schwann cell proliferation and migration throughout the conduits was performed. Analyses revealed that SCs maintained viable (>95%) throughout the conduits independent of construct length. SC proliferation on the spider silk was determined from day 7 to day 21 with a proliferation index of 49.42% arithmetically averaged over all conduits. This indicates that spider silk nerve conduits represent a favorable environment for SC attachment, proliferation and distribution over a distance of least 15.0 cm in vitro. Thus spider silk nerve implants are a highly adequate biomaterial for nerve reconstruction.

Conditioned umbilical cord tissue provides a natural three-dimensional storage compartment as in vitro stem cell niche for human mesenchymal stroma/stem cells.

BACKGROUND: The use of large amounts of human multipotent mesenchymal stroma/stem cells (MSC) for cell therapies represents a desirable property in tissue engineering and banking in the field of regenerative medicine. METHODS AND RESULTS: Whereas cryo-storage of umbilical cord (UC) tissue pieces in liquid nitrogen without ingredients was associated with predominant appearance of apoptotic cells after thawing and re-culture, progressive growth of MSC was observed following use of cryo-medium. Moreover, conditioning of UC tissue pieces by initial explant culture and subsequent cryo-storage with cryo-medium accelerated a further MSC culture after thawing. These findings suggested that conditioning of UC tissue pieces provides an in vitro stem cell niche by maintenance of a 3-dimensional natural microenvironment for continuous MSC outgrowth and expansion. Indeed, culture of GFP-labeled UC tissue pieces was accompanied by increased outgrowth of GFP-labeled cells which was accelerated in conditioned UC tissue after cryo-storage. Moreover, cryopreserved conditioned UC tissue pieces in cryo-medium after thawing and explant culture could be cryopreserved again demonstrating renewed MSC outgrowth after repeated thawing with similar population doublings compared to the initial explant culture. Flow cytometry analysis of outgrowing cells revealed expression of the typical MSC markers CD73, CD90, and CD105. Furthermore, these cells demonstrated little if any senescence and cultures revealed stem cell-like characteristics by differentiation along the adipogenic, chondrogenic and osteogenic lineages. CONCLUSIONS: Expression of MSC markers was maintained for at least 10 freeze/thaw/explant culture cycles demonstrating that repeated cryopreservation of conditioned UC tissue pieces provided a reproducible and enriched stem cell source.

TRIM21, a negative modulator of LFG in breast carcinoma MDA-MB-231 cells in vitro.

Lifeguard (LFG) is a transmembrane protein which is highly expressed in tissues of the hippocampus and the cerebellum, especially during postnatal development. This protein is responsible for the protection of neurons against Fas-induced apoptosis, and the same effect can be seen in tumor cells derived from mastocarcinoma. However, the molecular function of LFG and its regulation in the carcinogenesis of human breast cells remains to be elucidated. In the present study, we investigated the connection of the interaction of LFG within an array analysis of over 9,000 different proteins. Results showed an interaction between the proteins tripartite motif-containing 21 (TRIM21) and LFG and a negative regulatory effect of TRIM21 towards LFG on the protein level. Furthermore, Fas-induced apoptosis decreased upon the addition of TRIM21 to the cultured cells. These results revealed TRIM21 to be a negative modulator of LFG in cells of mastocarcinoma in vitro. For all analyses, MDA-MB-231 cells were used. The interaction of TRIM21 and LFG was analyzed by co-immunoprecipitation. To examine changes in regulatory processes, western blot analyses, real-time PCR, activity of apoptotic process and flow cytometric analyses were carried out.

Acquisition of new tumor cell properties by MSC-derived exosomes.

Interaction between multi-functional mesenchymal stroma/stem cells (MSC) and human tumor cells involves the exchange of biological material via extracellular vesicles including exosomes. Protein analysis of MSC-derived exosomes demonstrated the presence of MMP-2 and MSC-specific markers including CD90 and ecto-5'-nucleotidase (CD73). Incubation of tumor cells with these membranous particles revealed a rapid uptake of MSC-released microvesicles whereby breast cancer cells incorporated ~19% and SCCOHT-1 cells representing a rare type of small cell ovarian cancer assimilated ~28% of available exosomes within 24 h. This interaction was accompanied by functional alterations of tumor cell properties during integration of exosomal content from MSC. Indeed, exosome-associated MMP-2 exhibited functional enzyme activity and MCF-7 breast cancer cells with undetectable MMP-2 protein acquired expression of this enzyme and corresponding gelatinase functionality after stimulation with MSC-derived exosomes. Similar effects were observed in SCCOHT-1 cells during culture in the presence of MSC-derived exosomes which enabled new metabolic activities in this tumor cell type. Together, these findings demonstrated that the internalization of MSC-derived exosomes was associated with the acquisition of new tumor cell properties by altering cellular functionalities and providing the capability to re-organize the tumor microenvironment.

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.