Large-Volume Vascularized Muscle Grafts Engineered From Groin Adipose Tissue in Perfusion Bioreactor Culture.

BACKGROUND: Muscle tissue engineering still remains a major challenge. An axial vascular pedicle and a perfusion bioreactor are necessary for the development and maintenance of a large-volume engineered muscle tissue to provide circulation within the construct. This study aimed to determine whether large-volume vascularized muscle-like constructs could be made from rat groin adipose tissue in a perfusion bioreactor. METHODS: Epigastric adipofascial flaps based on the inferior superficial epigastric vessels were elevated bilaterally in male Lewis rats and connected to the bioreactor. The system was run using a cable pump and filled with myogenic differentiation medium in the perfusion bioreactor for 1, 3, 5, or 7 weeks. The resulting tissue constructs were characterized with respect to the morphology and muscle-related expression of genes and proteins. RESULTS: The histological examination demonstrated intact muscle-like tissue fibers; myogenesis was verified by the expression of myosin, MADS box transcription enhancer factor 2 D, desmin-a disintegrin and metalloproteinase domain (ADAM) 12-and M-cadherin using reverse transcription-polymerase chain reaction. Western blot analysis for desmin, MyoD1, N-cadherin, and ADAM12 was performed to verify the myogenic phenotype of the extracted differentiated tissue and prove the formation of muscle-like constructs. CONCLUSIONS: A large-volume vascularized muscle tissue could be engineered in a perfusion bioreactor. The resulting tissue had muscle-like histological features and expressed muscle-related genes and proteins, indicating that the trans-differentiation of adipose tissue into muscle tissue occurred.

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.

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.

What is the effect of nicotinic acetylcholine receptor stimulation on osteoarthritis in a rodent animal model?

OBJECTIVES: Despite the rising number of patients with osteoarthritis, no sufficient chondroprotective and prophylactic therapy for osteoarthritis has been established yet. The purpose of this study was to verify whether stimulation of the nicotinic acetylcholine receptor via nicotine has a beneficial effect on cartilage degeneration in the development of osteoarthritis and is capable of reducing the expression of proinflammatory cytokines and cartilage degrading enzymes in synovial membranes after osteoarthritis induction. METHODS: Experimental osteoarthritis was induced in Lewis rats using a standardized osteoarthritis model with monoiodoacetate. A total of 16 Lewis rats were randomized into four groups: control, sham + nicotine application, osteoarthritis, and osteoarthritis + nicotine application. Nicotine (0.625 mg/kg twice daily) was administered intraperitoneally for 42 days. We analyzed histological sections, radiological images and the expression of the proinflammatory cytokines, such as interleukin-1ß, tumor necrosis factor-α and interleukin-6, and of matrix metalloproteases 3, 9 and 13 and tissue inhibitors of metalloprotease-1 in synovial membranes via quantitative polymerase chain reaction. RESULTS: Histological and x-ray examination revealed cartilage degeneration in the osteoarthritis group compared to control or sham + nicotine groups (histological control vs osteoarthritis: p = 0.002 and x-ray control vs osteoarthritis: p = 0.004). Nicotine treatment reduced the cartilage degeneration without significant differences. Osteoarthritis induction led to a higher expression of proinflammatory cytokines and matrix metalloproteases as compared to control groups. This effect was attenuated after nicotine administration. The differences of proinflammatory cytokines and matrix metalloproteases did not reach statistical significance. CONCLUSION: With the present small-scale study, we could not prove a positive effect of nicotinic acetylcholine receptor stimulation on osteoarthritis due to a conservative statistical analysis and the consecutive lack of significant differences. Nevertheless, we found promising tendencies of relevant parameters that might prompt further experiments designed to evaluate the potency of stimulation of this receptor system as an additional treatment approach for osteoarthritis.

In Vitro Evaluation of Spider Silk Meshes as a Potential Biomaterial for Bladder Reconstruction.

Reconstruction of the bladder by means of both natural and synthetic materials remains a challenge due to severe adverse effects such as mechanical failure. Here we investigate the application of spider major ampullate gland-derived dragline silk from the Nephila edulis spider, a natural biomaterial with outstanding mechanical properties and a slow degradation rate, as a potential scaffold for bladder reconstruction by studying the cellular response of primary bladder cells to this biomaterial. We demonstrate that spider silk without any additional biological coating supports adhesion and growth of primary human urothelial cells (HUCs), which are multipotent bladder cells able to differentiate into the various epithelial layers of the bladder. HUCs cultured on spider silk did not show significant changes in the expression of various epithelial-to-mesenchymal transition and fibrosis associated genes, and demonstrated only slight reduction in the expression of adhesion and cellular differentiation genes. Furthermore, flow cytometric analysis showed that most of the silk-exposed HUCs maintain an undifferentiated immunophenotype. These results demonstrate that spider silk from the Nephila edulis spider supports adhesion, survival and growth of HUCs without significantly altering their cellular properties making this type of material a suitable candidate for being tested in pre-clinical models for bladder reconstruction.

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.

Three-Dimensional Modelling inside a Differential Pressure Laminar Flow Bioreactor Filled with Porous Media.

A three-dimensional computational fluid dynamics- (CFD-) model based on a differential pressure laminar flow bioreactor prototype was developed to further examine performance under changing culture conditions. Cell growth inside scaffolds was simulated by decreasing intrinsic permeability values and led to pressure build-up in the upper culture chamber. Pressure release by an integrated bypass system allowed continuation of culture. The specific shape of the bioreactor culture vessel supported a homogenous flow profile and mass flux at the scaffold level at various scaffold permeabilities. Experimental data showed an increase in oxygen concentration measured inside a collagen scaffold seeded with human mesenchymal stem cells when cultured in the perfusion bioreactor after 24 h compared to static culture in a Petri dish (dynamic: 11% O2 versus static: 3% O2). Computational fluid simulation can support design of bioreactor systems for tissue engineering application.

Noninvasive Oxygen Monitoring in Three-Dimensional Tissue Cultures Under Static and Dynamic Culture Conditions.

We present a new method for noninvasive real-time oxygen measurement inside three-dimensional tissue-engineered cell constructs in static and dynamic culture settings in a laminar flow bioreactor. The OPAL system (optical oxygen measurement system) determines the oxygen-dependent phosphorescence lifetime of spherical microprobes and uses a two-frequency phase-modulation technique, which fades out the interference of background fluorescence from the cell carrier and culture medium. Higher cell densities in the centrum of the scaffolds correlated with lower values of oxygen concentration obtained with the OPAL system. When scaffolds were placed in the bioreactor, higher oxygen values were measured compared to statically cultured scaffolds in a Petri dish, which were significantly different at day 1-3 of culture. This technique allows the use of signal-weak microprobes in biological environments and monitors the culture process inside a bioreactor.

Transdermal Nicotine Application Attenuates Cardiac Dysfunction after Severe Thermal Injury.

BACKGROUND: Severe burn trauma leads to an immediate and strong inflammatory response inciting cardiac dysfunction that is associated with high morbidity and mortality. The aim of this study was to determine whether transdermal application of nicotine could influence the burn-induced cardiac dysfunction via its known immunomodulatory effects. MATERIAL AND METHODS: A standardized rat burn model was used in 35 male Sprague Dawley rats. The experimental animals were divided into a control group, a burn trauma group, a burn trauma group with additional nicotine treatment, and a sham group with five experimental animals per group. The latter two groups received nicotine administration. Using microtip catheterization, functional parameters of the heart were assessed 12 or 24 hours after infliction of burn trauma. RESULTS: Burn trauma led to significantly decreased blood pressure (BP) values whereas nicotine administration normalized BP. As expected, burn trauma also induced a significant deterioration of myocardial contractility and relaxation parameters. After application of nicotine these adverse effects were attenuated. CONCLUSION: The present study showed that transdermal nicotine administration has normalizing effects on burn-induced myocardial dysfunction parameters. Further research is warranted to gain insight in molecular mechanisms and pathways and to evaluate potential treatment options in humans.

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.

In planta production of ELPylated spidroin-based proteins results in non-cytotoxic biopolymers.

BACKGROUND: Spider silk is a tear-resistant and elastic biopolymer that has outstanding mechanical properties. Additionally, exiguous immunogenicity is anticipated for spider silks. Therefore, spider silk represents a potential ideal biomaterial for medical applications. All known spider silk proteins, so-called spidroins, reveal a composite nature of silk-specific units, allowing the recombinant production of individual and combined segments. RESULTS: In this report, a miniaturized spidroin gene, named VSO1 that contains repetitive motifs of MaSp1 has been synthesized and combined to form multimers of distinct lengths, which were heterologously expressed as elastin-like peptide (ELP) fusion proteins in tobacco. The elastic penetration moduli of layered proteins were analyzed for different spidroin-based biopolymers. Moreover, we present the first immunological analysis of synthetic spidroin-based biopolymers. Characterization of the binding behavior of the sera after immunization by competitive ELISA suggested that the humoral immune response is mainly directed against the fusion partner ELP. In addition, cytocompatibility studies with murine embryonic fibroblasts indicated that recombinant spidroin-based biopolymers, in solution or as coated proteins, are well tolerated. CONCLUSION: The results show that spidroin-based biopolymers can induce humoral immune responses that are dependent on the fusion partner and the overall protein structure. Furthermore, cytocompatibility assays gave no indication of spidroin-derived cytotoxicity, suggesting that recombinant produced biopolymers composed of spider silk-like repetitive elements are suitable for biomedical applications.

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.

Successful nucleofection of rat adipose-derived stroma cells with Ambystoma mexicanum epidermal lipoxygenase (AmbLOXe).

INTRODUCTION: Adipose-derived stroma cells (ASCs) are attractive cells for cell-based gene therapy but are generally difficult to transfect. Nucleofection has proven to be an efficient method for transfection of primary cells. Therefore, we used this technique to transfect ASCs with a vector encoding for Ambystoma mexicanum epidermal lipoxygenase (AmbLOXe) which is a promising bioactive enzyme in regenerative processes. Thereby, we thought to even further increase the large regenerative potential of the ASCs. METHODS: ASCs were isolated from the inguinal fat pad of Lewis rats and were subsequently transfected in passage 1 using Nucleofector® 2b and the hMSC Nucleofector kit. Transfection efficiency was determined measuring co-transfected green fluorescent protein (GFP) in a flow cytometer and gene expression in transfected cells was detected by reverse transcription polymerase chain reaction (RT-PCR). Moreover, cell migration was assessed using a scratch assay and results were tested for statistical significance with ANOVA followed by Bonferroni's post hoc test. RESULTS: High initial transfection rates were achieved with an average of 79.8 ± 2.82% of GFP positive cells although longer cultivation periods reduced the number of positive cells to below 5% after four passages. Although successful production of AmbLOXe transcript could be proven the gene product had no measureable effect on cell migration. CONCLUSIONS: Our study demonstrates the feasibility of ASCs to serve as a vehicle of AmbLOXe transport for gene therapeutic purposes in regenerative medicine. One potential field of applications could be peripheral nerve injuries.

Observed changes in the morphology and phenotype of breast cancer cells in direct co-culture with adipose-derived stem cells.

BACKGROUND: Regarding aesthetics and long-term stability, cell-assisted lipotransfer is a promising method for breast reconstruction. Here, autologous fat grafts enriched with autologous adipose-derived stem cells are transferred. However, as adipose-derived stem cells secrete high amounts of growth factors, potential risks of tumor reactivation remain. In this study, influences of adipose-derived stem cells on inflammatory breast cancer cells were evaluated in a direct co-culture system. METHODS: Human adipose-derived stem cells were isolated and cultivated either alone or in a direct co-culture with the inflammatory breast carcinoma cell line T47D. At different time points, cell morphology was observed by scanning electron microscopy, cell membranes were stained by immunofluorescence, and gene expression was analyzed by real-time polymerase chain reaction. RESULTS: In co-cultures, T47D breast carcinoma cells showed tumorsphere-typical growth surrounded by a monolayer of adipose-derived stem cells. Direct cell-to-cell contacts could be observed between the two different cell types. Immunofluorescence revealed vesicular exchange and fusion between carcinoma cells and adipose-derived stem cells. Expression levels of transcriptional genes for typical malignancy markers were substantially higher in co-cultures compared with single cultures. CONCLUSIONS: Direct intercellular contact between carcinoma cells and adipose-derived stem cells by means of exosomal vesicular exchange was revealed. Breast cancer cells displayed a change towards a more malignant phenotype associated with higher rates of metastasis and worsened prognosis. As cell-assisted lipotransfer is often performed after breast cancer surgery, transfer of adipose-derived stem cells might lead to deterioration of prognosis in case of recurrence as it has been described for inflammatory breast cancer.

Murine embryonic fibroblast cell lines differentiate into three mesenchymal lineages to different extents: new models to investigate differentiation processes.

Various diseases, injuries, and congenital abnormalities may result in degeneration and loss of organs and tissues. Recently, tissue engineering has offered new treatment options for these common, severe, and costly problems in human health care. Its application is often based on the usage of differentiated stem cells. However, despite intensive research and growing knowledge, many questions remain unresolved in the process of cell differentiation. The aim of this study was to find standardized cell models for analyzing molecular mechanisms of cell differentiation. We investigated the multipotency of three standardized murine embryonic fibroblast cell cultures using histological staining, western blotting, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Our results demonstrated that NIH-3T3 and mouse embryonic fibroblast (MEF) cells were able to differentiate into adipogenic, chondrogenic, and osteogenic lineages expressing typical differentiation markers. Interestingly, Flp-In-3T3 cells did not differentiate into any of the three mesenchymal lineages, although this cell line is genetically closely related to NIH-3T3. The results were confirmed by histological staining. Flp-In-3T3, NIH-3T3, and MEF cells have usually been used for DNA transfections, recombinant protein expression, and as "feeder cells." Unlike mesenchymal stem cells (MSCs) and mesenchymal progenitor cells (MPCs), they are easy to obtain and to expand and are less prone to change their structure and morphology, even at higher passages. Our results suggest that Flp-In-3T3, MEF, and NIH-3T3 cells are highly suitable to be used as models to analyze molecular mechanisms of cell differentiation.

Role of lifeguard ß-isoform in the development of breast cancer.

In the last century there has been great progress in the treatment of breast cancer by improving drug and radiation therapy as well as surgical techniques. Despite this development, breast cancer remains a major cause of death among women in Europe and the US. The cause of breast cancer at the cellular level is still not fully understood. In the present study, we investigated the expression of the Lifeguard ß-isoform in breast cancer tissues. In contrast to Lifeguard, the ß­isoform has one transmembrane domain less, which is the last of seven (99 bp), and due to this we suspect that the Lifeguard ß-isoform exhibits a different function. We determined the expression and function of the ß-isoform of Lifeguard in breast cancer cell lines (MCF-7 and MDA-MB-231), a human breast epithelial cell line (MCF10A) and in breast tumour tissue sections. Western blotting, PCR arrays and immunofluorescence were used to investigate the expression of Lifeguard and its ß-isoform. Moreover, we investigated the ability of Lifeguard ß-isoform expression to inhibit apoptosis induced by Fas. Our results indicated that Lifeguard ß-isoform is strongly expressed in breast tumour tissues. More notably, we demonstrated that Fas sensitivity was reduced in the MCF10A breast cells expressing the Lifeguard ß-isoform. Taken together, our findings indicate the role of the Lifeguard ß-isoform as an anti­apoptotic protein and provide further evidence of the potential of the Lifeguard ß-isoform as a target for the development of novel therapeutic strategies.

Biomechanics and biocompatibility of woven spider silk meshes during remodeling in a rodent fascia replacement model.

OBJECTIVE: The aim of this study was to investigate biomechanical and immunogenic properties of spider silk meshes implanted as fascia replacement in a rat in vivo model. BACKGROUND: Meshes for hernia repair require optimal characteristics with regard to strength, elasticity, and cytocompatibility. Spider silk as a biomaterial with outstanding mechanical properties is potentially suitable for this application. METHODS: Commercially available meshes used for hernia repair (Surgisis and Ultrapro) were compared with handwoven meshes manufactured from native dragline silk of Nephila spp. All meshes were tied onto the paravertebral fascia, whereas sham-operated rats were sutured without mesh implantation. After 4 or 14 days, 4 weeks, and 4 or 8 months, tissue samples were analyzed concerning inflammation and biointegration both by histological and biochemical methods and by biomechanical stability tests. RESULTS: Histological sections revealed rapid cell migration into the spider silk meshes with increased numbers of giant cells compared with controls with initial decomposition of silk fibers after 4 weeks. Four months postoperatively, spider silk was completely degraded with the formation of a stable scar verified by constant tensile strength values. Surgisis elicited excessive stability loss from day 4 to day 14 (P < 0.001), with distinct inflammatory reaction demonstrated by lymphocyte and neutrophil invasion. Ultrapro also showed decreasing strength and poor elongation behavior, whereas spider silk samples had the highest relative elongation (P < 0.05). CONCLUSIONS: Hand-manufactured spider silk meshes with good biocompatibility and beneficial mechanical properties seem superior to standard biological and synthetic meshes, implying an innovative alternative to currently used meshes for hernia repair.