Induction of cell death by sodium hexachloroplatinate (IV) in the HEI-OC1 cell line, primary rat spiral ganglion cells and rat organ of Corti explants.

Although cochlear implants have become a well-established method for patients with sensory neural hearing loss, clinical results indicate that in some cases, corrosion of electrode contacts leads to high impedance that interferes with successful stimulation of the auditory nerve. As it is unclear whether corrosion products induce cell damage, we focused on cell culture models of the organ of Corti cell line (HEI-OC1), rat spiral ganglion cells (SGC) and rat organ of Corti explant (OCex) cultivated from neonatal rat cochleae to characterize the cytotoxicity of sodium hexachloroplatinate (IV) (Na2(PtCl6)). The oxidative activity in HEI-OC1 cells decreased with increasing Na2(PtCl6) concentrations between 8 and 16 ng/µl, and live cell staining with Calcein acetoxymethyl/Ethidium homodimer III revealed an increasing number of cells with disrupted membranes. Ultrastructural evidence of mitophagy followed by necroptosis was detected. Additionally, exposure of the SGC to 15-35 ng/µl Na2(PtCl6) dose-dependently reduced neuronal survival and neuritogenesis, as determined by neurofilament antigen staining. In parallel, staining glial cells and fibroblasts with specific antibodies confirmed the dose-dependent induction of cell death by Na2(PtCl6). Exposure of the OCex to 25-45 ng/µl Na2(PtCl6) resulted in severe concentration-dependent hair cell loss. Our data show for the first time that Na2(PtCl6) induces cell death in a concentration-dependent manner in inner ear cell types and tissues.

Synaptopodin Regulates Denervation-Induced Plasticity at Hippocampal Mossy Fiber Synapses.

Neurological diseases can lead to the denervation of brain regions caused by demyelination, traumatic injury or cell death. The molecular and structural mechanisms underlying lesion-induced reorganization of denervated brain regions, however, are a matter of ongoing investigation. In order to address this issue, we performed an entorhinal cortex lesion (ECL) in mouse organotypic entorhino-hippocampal tissue cultures of both sexes and studied denervation-induced plasticity of mossy fiber synapses, which connect dentate granule cells (dGCs) with CA3 pyramidal cells (CA3-PCs) and play important roles in learning and memory formation. Partial denervation caused a strengthening of excitatory neurotransmission in dGCs, CA3-PCs and their direct synaptic connections, as revealed by paired recordings (dGC-to-CA3-PC). These functional changes were accompanied by ultrastructural reorganization of mossy fiber synapses, which regularly contain the plasticity-regulating protein synaptopodin and the spine apparatus organelle. We demonstrate that the spine apparatus organelle and synaptopodin are related to ribosomes in close proximity to synaptic sites and reveal a synaptopodin-related transcriptome. Notably, synaptopodin-deficient tissue preparations that lack the spine apparatus organelle failed to express lesion-induced synaptic adjustments. Hence, synaptopodin and the spine apparatus organelle play a crucial role in regulating lesion-induced synaptic plasticity at hippocampal mossy fiber synapses.

In vitro impact of platinum nanoparticles on inner ear related cell culture models.

So far, it was supposed that the increase of electrical impedance following cochlear implant (CI) insertion was due to technical defects of the electrode, inflammatory and/or formation of scar tissue along the electrode. However, it was recently reported that corrosion of the platinum electrode contacts may be the reason for high impedances. It could be shown that platinum particles were stripped from the electrode surfaces. Its potential cytotoxic effects within the inner ear remains to be examined. In this study in vitro cell culture models of the mouse organ of Corti cell line (HEI-OC1) and the spiral ganglion (SG) cells derived from the cochleae neonatal rats were used to investigate the effects of the polyvinylpyrrolidone coated platinum nanoparticles (Pt-NPPVP, 3 nm) on cell metabolism, neuronal survival and neurite outgrowth. Our data revealed no decrease of the metabolic activity of the HEI-OC1 cells at Pt-NPPVP concentrations between 50-150 µg/ml. Also, staining with Calcein AM/EthD demonstrated prevalent presence of vital cells. As shown by transmission electron microscopy no Pt-NPPVP could be found at the cell surface or in the cytosol of the HEI-OC1 cells. Similarly, the SG cells exposed to 20-100 µg/ml Pt-NPPVP did not show any reduced survival rate and neurite outgrowth following staining of the neurofilament antigen even at the highest Pt-NPPVP concentration. Although the SG cells were exposed to Pt-NPPVP for further 72 h and 96 h immunocytochemical staining of the glial cells and fibroblasts presented normal cell morphology and growth independently of the cultivation period. Our data indicates that the used Pt-NPPVP do not trigger the cellular uptake and, thus, presumable do not initiate apoptotic pathways in cells of the organ of Corti cell line or the auditory nerve. The protection mechanisms to the Pt-NPPVP interactions remain to be clarified.

Stabilizing A Vascularized Autologous Matrix with Flexible Magnesium Scaffolds to Reconstruct Dysfunctional Left Ventricular Myocardium in a Large-Animal Feasibility Study.

The surgical reconstruction of dysfunctional myocardium is necessary for patients with severe heart failure. Autologous biomaterials, such as vascularized patch materials, have a regenerative potential due to in vivo remodeling. However, additional temporary mechanical stabilization of the biomaterials is required to prevent aneurysms or rupture. Degradable magnesium scaffolds could prevent these life-threatening risks. A left ventricular transmural defect was reconstructed in minipigs with a piece of the autologous stomach. Geometrically adaptable and degradable scaffolds made of magnesium alloy LA63 were affixed on the epicardium to stabilize the stomach tissue. The degradation of the magnesium structures, their biocompatibility, physiological remodeling of the stomach, and the heart's function were examined six months after the procedure via MRI (Magnetic Resonance Imaging), angiography, µ-CT, and light microscopy. All animals survived the surgery. Stable physiological integration of the stomach patch could be detected. No ruptures of the grafts occurred. The magnesium scaffolds showed good biocompatibility. Regenerative surgical approaches for treating severe heart failure are a promising therapeutic alternative to the currently available, far from optimal options. The temporary mechanical stabilization of viable, vascularized grafts facilitates their applicability in clinical scenarios.

Left Ventricular Wall Reconstruction with Autologous Vascularized Tunica Muscularis of Stomach in a Porcine Pilot Model.

INTRODUCTION: Surgical replacement of dysfunctional cardiac muscle with regenerative tissue is an important option to combat heart failure. But, current available myocardial prostheses like a Dacron or a pericardium patch neither have a regenerative capacity nor do they actively contribute to the heart's pump function. This study aimed to show the feasibility of utilizing a vascularized stomach patch for transmural left ventricular wall reconstruction. METHODS: A left ventricular transmural myocardial defect was reconstructed by performing transdiaphragmatic autologous transplantation of a vascularized stomach segment in six Lewe minipigs. Three further animals received a conventional Dacron patch as a control treatment. The first 3 animals were followed up for 3 months until planned euthanasia, whereas the observation period for the remaining 3 animals was scheduled 6 months following surgery. Functional assessment of the grafts was carried out via cardiac magnetic resonance tomography and angiography. Physiological remodeling was evaluated histologically and immunohistochemically after heart explantation. RESULTS: Five out of six test animals and all control animals survived the complex surgery and completed the follow-up without clinical complications. One animal died intraoperatively due to excessive bleeding. No animal experienced rupture of the stomach graft. Functional integration of the heterotopically transplanted stomach into the surrounding myocardium was observed. Angiography showed development of connections between the gastric graft vasculature and the coronary system of the host cardiac tissue. CONCLUSIONS: The clinical results and the observed physiological integration of gastric grafts into the cardiac structure demonstrate the feasibility of vascularized stomach tissue as myocardial prosthesis. The physiological remodeling indicates a regenerative potential of the graft. Above all, the connection of the gastric vessels with the coronary system constitutes a rationale for the use of vascularized and, therefore, viable stomach tissue for versatile tissue engineering applications.

Is P-Glycoprotein Functionally Expressed in the Limiting Membrane of Endolysosomes? A Biochemical and Ultrastructural Study in the Rat Liver.

The drug efflux transporter P-glycoprotein (Pgp; ABCB1) plays an important role in drug absorption, disposition, and elimination. There is an ongoing debate whether, in addition to its localization at the plasma membrane, Pgp may also be expressed at the limiting membrane of endolysosomes (ELs), mediating active EL drug sequestration. If true, this would be an important mechanism to prevent drugs from reaching their intracellular targets. However, direct evidence demonstrating the functional expression of Pgp at the limiting membrane of ELs is lacking. This prompted us to perform a biochemical and ultrastructural study on the intracellular localization of Pgp in native rat liver. For this purpose, we established an improved subcellular fractionation procedure for the enrichment of ELs and employed different biochemical and ultrastructural methods to characterize the Pgp localization and function in the enriched EL fractions. Whereas the biochemical methods seemed to indicate that Pgp is functionally expressed at EL limiting membranes, transmission electron microscopy (TEM) indicated that this only occurs rarely, if at all. Instead, Pgp was found in the limiting membrane of early endosomes and intraluminal vesicles. In additional TEM experiments, using a Pgp-overexpressing brain microvessel endothelial cell line (hCMEC/D3-MDR1-EGFP), we examined whether Pgp is expressed at the limiting membrane of ELs when cells are exposed to high levels of the Pgp substrate doxorubicin. Pgp was seen in early endosomes but only rarely in endolysosomes, whereas Pgp immunogold labeling was detected in large autophagosomes. In summary, our data demonstrate the importance of combining biochemical and ultrastructural methods to investigate the relationship between Pgp localization and function.

Fucosylated lipid nanocarriers loaded with antibiotics efficiently inhibit mycobacterial propagation in human myeloid cells.

Antibiotic treatment of tuberculosis (TB) is complex, lengthy, and can be associated with various adverse effects. As a result, patient compliance often is poor, thus further enhancing the risk of selecting multi-drug resistant bacteria. Macrophage mannose receptor (MMR)-positive alveolar macrophages (AM) constitute a niche in which Mycobacterium tuberculosis replicates and survives. Therefore, we encapsulated levofloxacin in lipid nanocarriers functionalized with fucosyl residues that interact with the MMR. Indeed, such nanocarriers preferentially targeted MMR-positive myeloid cells, and in particular, AM. Intracellularly, fucosylated lipid nanocarriers favorably delivered their payload into endosomal compartments, where mycobacteria reside. In an in vitro setting using infected human primary macrophages as well as dendritic cells, the encapsulated antibiotic cleared the pathogen more efficiently than free levofloxacin. In conclusion, our results point towards carbohydrate-functionalized nanocarriers as a promising tool for improving TB treatment by targeted delivery of antibiotics.

Toward acellular xenogeneic heart valve prostheses: Histological and biomechanical characterization of decellularized and enzymatically deglycosylated porcine pulmonary heart valve matrices.

The use of decellularized xenogeneic heart valves might offer a solution to overcome the issue of human valve shortage. The aim of this study was to revise decellularization protocols in combination with enzymatic deglycosylation, in order to reduce the immunogenicity of porcine pulmonary heart valves, in means of cells, carbohydrates, and, primarily, Galα1-3Gal (α-Gal) epitope removal. In particular, the valves were decellularized with sodium dodecylsulfate/sodium deoxycholate (SDS/SD), Triton X-100 + SDS (Tx + SDS), or Trypsin + Triton X-100 (Tryp + Tx) followed by enzymatic digestion with PNGaseF, Endoglycosidase H, or O-glycosidase combined with Neuraminidase. Results showed that decellularization alone reduced carbohydrate structures only to a limited extent, and it did not result in an α-Gal free scaffold. Nevertheless, decellularization with Tryp + Tx represented the most effective decellularization protocol in means of carbohydrates reduction. Overall, carbohydrates and α-Gal removal could strongly be improved by applying PNGaseF, in particular in combination with Tryp + Tx treatment, contrary to Endoglycosidase H and O-glycosidase treatments. Furthermore, decellularization with PNGaseF did not affect biomechanical stability, in comparison with decellularization alone, as shown by burst pressure and uniaxial tensile tests. In conclusion, valves decellularized with Tryp + Tx and PNGaseF resulted in prostheses with potentially reduced immunogenicity and maintained mechanical stability.

LAMTOR2 (p14) Controls B Cell Differentiation by Orchestrating Endosomal BCR Trafficking.

B-cell development and function depend on stage-specific signaling through the B-cell antigen receptor (BCR). Signaling and intracellular trafficking of the BCR are connected, but the molecular mechanisms of this link are incompletely understood. Here, we investigated the role of the endosomal adaptor protein and member of the LAMTOR/Ragulator complex LAMTOR2 (p14) in B-cell development. Efficient conditional deletion of LAMTOR2 at the pre-B1 stage using mb1-Cre mice resulted in complete developmental arrest. Deletion of LAMTOR2 using Cd19-Cre mice permitted analysis of residual B cells at later developmental stages, revealing that LAMTOR2 was critical for the generation and activation of mature B lymphocytes. Loss of LAMTOR2 resulted in aberrant BCR signaling due to delayed receptor internalization and endosomal trafficking. In conclusion, we identify LAMTOR2 as critical regulator of BCR trafficking and signaling that is essential for early B-cell development in mice.

Deficiency of fibroblast growth factor 2 (FGF-2) leads to abnormal spermatogenesis and altered sperm physiology.

In previous studies, we described the presence of fibroblast growth factor 2 (FGF-2) and its receptors (FGFRs) in human testis and sperm, which are involved in spermatogenesis and in motility regulation. The aim of the present study was to analyze the role of FGF-2 in the maintenance of sperm physiology using FGF-2 knockout (KO) mice. Our results showed that in wild-type (WT) animals, FGF-2 is expressed in germ cells of the seminiferous epithelium, in epithelial cells of the epididymis, and in the flagellum and acrosomal region of epididymal sperm. In the FGF-2 KO mice, we found alterations in spermatogenesis kinetics, higher numbers of spermatids per testis, and enhanced daily sperm production compared with the WT males. No difference in the percentage of sperm motility was detected, but a significant increase in sperm concentration and in sperm head abnormalities was observed in FGF-2 KO animals. Sperm from KO mice depicted reduced phosphorylation on tyrosine residues (a phenomenon that was associated with sperm capacitation) and increased acrosomal loss after incubation under capacitating conditions. However, the FGF-2 KO males displayed no apparent fertility defects, since their mating with WT females showed no differences in the time to delivery, litter size, and pup weight in comparison with WT males. Overall, our findings suggest that FGF-2 exerts a role in mammalian spermatogenesis and that the lack of FGF-2 leads to dysregulated sperm production and altered sperm morphology and function. FGF-2-deficient mice constitute a model for the study of the complex mechanisms underlying mammalian spermatogenesis.

Platinum corrosion products from electrode contacts of human cochlear implants induce cell death in cell culture models.

Despite the technological progress made with cochlear implants (CI), impedances and their diagnosis remain a focus of interest. Increases in impedance have been related to technical defects of the electrode as well as inflammatory and/or fibrosis along the electrode. Recent studies have demonstrated highly increased impedances as the result of corroded platinum (Pt) electrode contacts. This in vitro study examined the effects of Pt ions and compounds generated by corrosion of the electrode contacts of a human CI on cell metabolism. Since traces of solid Pt in surrounding cochlear tissues have been reported, the impact of commercially available Pt nanoparticles (Pt-NP, size 3 nm) on the cell culture model was also determined. For this purpose, the electrode contacts were electrically stimulated in a 0.5% aqueous NaCl solution for four weeks and the mass fraction of the platinum dissolute (Pt-Diss) was determined by mass spectrometry (ICP-MS). Metabolic activity of the murine fibroblasts (NIH 3T3) and the human neuroblastoma (SH-SY5Y) cells was determined using the WST-1 assay following exposure to Pt-Diss and Pt-NP. It was found that 5-50 µg/ml of the Pt-NP did not affect the viability of both cell types. In contrast, 100 µg/ml of the nanoparticles caused significant loss in metabolic activity. Furthermore, transmission electron microscopy (TEM) revealed mitochondrial swelling in both cell types indicating cytotoxicity. Additionally, TEM demonstrated internalized Pt-NP in NIH 3T3 cells in a concentration dependent manner, whereas endocytosis in SH-SY5Y cells was virtually absent. In comparison with the Pt-NP, the corrosion products (Pt-Diss) with concentrations between 1.64 µg/ml and 8.2 µg/ml induced cell death in both cell lines in a concentration dependent manner. TEM imaging revealed both mitochondrial disintegration and swelling of the endoplasmic reticulum, suggesting that Pt ions trigger cytotoxicity in both NIH 3T3 and SH-SY5Y cell lines by interacting with the respiratory chain.

Metalloprotease-mediated cleavage of PlexinD1 and its sequestration to actin rods in the motoneuron disease spinal muscular atrophy (SMA).

Cytoskeletal rearrangement during axon growth is mediated by guidance receptors and their ligands which act either as repellent, attractant or both. Regulation of the actin cytoskeleton is disturbed in Spinal Muscular Atrophy (SMA), a devastating neurodegenerative disease affecting mainly motoneurons, but receptor-ligand interactions leading to the dysregulation causing SMA are poorly understood. In this study, we analysed the role of the guidance receptor PlexinD1 in SMA pathogenesis. We showed that PlexinD1 is cleaved by metalloproteases in SMA and that this cleavage switches its function from an attractant to repellent. Moreover, we found that the PlexinD1 cleavage product binds to actin rods, pathological aggregate-like structures which had so far been described for age-related neurodegenerative diseases. Our data suggest a novel disease mechanism for SMA involving formation of actin rods as a molecular sink for a cleaved PlexinD1 fragment leading to dysregulation of receptor signaling.

Innovative 3D Model of the Human Middle Ear in High Resolution with a Histological Microgrinding Method: A Feasibility Study and Comparison with µCT.

Conclusion. The development of a histological 3D model of the tympanic cavity visualizes the exact microanatomy of the sound conduction organ and is therefore essential for finite elements simulations and surgical training. Objectives. So far, no accurate histological 3D model of the sound conduction system existed in literature. For 3D reconstruction of the very fine structures inside and outside the auditory ossicles, a method based on histological slices allows a more differential analysis of both hard and soft tissues and could thus be superior to µCT. Method. A complete temporal bone was embedded in epoxy resin and microground in distances of about 34 µm. After photodocumentation of every plane, a 3D reconstruction was performed by using the Computer Aided Design (CAD) program Rhinoceros 5®. For comparison, a µCT of the same specimen resulted in a 3D model of the calcified structures in the middle ear. Results. The histological 3D model gives an excellent overview to all anatomical soft and bony tissues of the human auditory ossicles. Specifically the fine blood vessel system and the exact dimension of cartilage areas inside the ossicles can be illustrated much more precisely than with µCT data. The present technique also allows the evaluation of the fine connecting ligaments inside the tympanic cavity.

Photochemical coating of Kapton® with hydrophilic polymers for the improvement of neural implants.

The polyimide Kapton® was coated photochemically with hydrophilic polymers to prevent undesirable cell growth on the polyimide surface. The polymer coatings were generated using photochemically reactive polymers synthesized by a simple and modular strategy. Suitable polymers or previously synthesized copolymer precursors were functionalized with photoactive arylazide groups by a polymer analogous amide coupling reaction with 4-azidobenzoic acid. A photoactive chitosan derivative (chitosan-Az) and photochemically reactive copolymers containing DMAA, DEAA or MTA as primary monomers were synthesized using this method. The amount of arylazide groups in the polymers was adjusted to approximately 5%, 10% and 20%. As coating on Kapton® all polymers effect a significantly reduced water contact angle (WCA) and consequently a rise of the surface hydrophilicity compared to the untreated Kapton®. The presence of the polymer coatings was also proven by ATR-IR spectroscopy. Coatings with chitosan-Az and the DEAA copolymer cause a distinct inhibition of the growth of fibroblasts. In the case of the DMAA copolymer even a strong anti-adhesive behavior towards fibroblasts was verified. Biocompatibility of the polymer coatings was proven which enables their utilization in biomedical applications.

Biocompatibility of silver containing silica films on Bioverit® II middle ear prostheses in rabbits.

For several centuries silver is known for its antibacterial effects. The middle ear is an interesting new scope for silver application since chronic inflammations combined with bacterial infection cause complete destruction of the fragile ossicle chain and tympanic membrane. The resulting conductive deafness requires tympanoplasty for reconstruction. Strategies to prevent bacterial growth on middle ear prostheses are highly recommended. In this study, rabbits were implanted with Bioverit® II middle ear prostheses functionalized with silver containing dense and nanoporous silica films which were compared with pure silica coatings as well as silver sulfadiazine cream applied on nanoporous silica coating. The health status of animals was continuously monitored; blood was examined before and after implantation. After 21 days, the middle ears were inspected; implants and mucosal samples were processed for electron microscopy. Autopsies were performed and systemic spreading of silver was chemically analyzed exemplarily in liver and kidneys. For verification of direct cytotoxicity, NIH 3T3 cells were cultured on similar silver containing silica coatings on glass up to 3 days. In vitro a reduced viability of fibroblasts adhering directly on the samples was detected compared to cells growing on the surrounding plastic of the same culture dish. In transmission electron microscopy, phagocytosed silver silica fragments, silver sulfadiazine cream as well as silver nanoparticles were noticed inside endosomes. In vivo, clinical and post mortem examinations were inconspicuous. Chemical analyses showed no increased silver content compared to controls. Mucosal coverages on almost all prostheses were found. But reduction of granulation tissue was only obvious around silver-coated implants. Single necroses and apoptosis in the mucosa were correlated by intracellular accumulation of metallic silver. For confirming supportive healing effects of middle ear implants, silver ion aggregates need to be tested in the future to optimize biocompatibility while assuring bactericidal effects in the middle ear.

Redundant role of protein kinase C delta and epsilon during mouse embryonic development.

Protein Kinase C delta and epsilon are mediators of important cellular events, such as cell proliferation, migration or apoptosis. The formation of blood vessels, i.e., vasculo- and angiogenesis, is a process where these isoforms have also been shown to participate. However, mice deficient in either Protein Kinase C delta or epsilon are viable and therefore their individual contribution to the formation of the vasculature appeared so far dispensable. In this study, we show that double null mutation of Protein Kinase C delta and epsilon causes embryonic lethality at approximately E9.5. At this stage, whole mount staining of the endothelial marker CD31 in double null embryos revealed defective blood vessel formation. Moreover, culture of double deficient mouse allantois showed impaired endothelial cell organization, and analyses of double deficient embryo sections showed dilated vessels, decreased endothelial-specific adherent junctions, and decreased contact of endothelial cells with mural cells. Protein kinase C delta and epsilon also appeared essential for vascular smooth muscle cell differentiation, since α-smooth muscle actin, a classical marker for vascular smooth muscle cells, was almost undetectable in double deficient embryonic aorta at E9.5. Subsequent qPCR analyses showed decreased VE-cadherin, Vegfr2, Cd31, Cdh2, Ets1, and Fli-1, among other angiogenesis related transcripts in double deficient embryos. Taken together, these data suggest for the first time an in vivo redundant role between members of the novel Protein Kinase C subfamily that allows for mutual compensation during mouse embryonic development, with vasculogenesis/angiogenesis as an obvious common function of these two Protein Kinase Cs. Protein Kinase C delta and epsilon might therefore be useful targets for inhibiting vasculo- and/or angiogenesis.

JAGN1 deficiency causes aberrant myeloid cell homeostasis and congenital neutropenia.

The analysis of individuals with severe congenital neutropenia (SCN) may shed light on the delicate balance of factors controlling the differentiation, maintenance and decay of neutrophils. We identify 9 distinct homozygous mutations in the JAGN1 gene encoding Jagunal homolog 1 in 14 individuals with SCN. JAGN1-mutant granulocytes are characterized by ultrastructural defects, a paucity of granules, aberrant N-glycosylation of multiple proteins and increased incidence of apoptosis. JAGN1 participates in the secretory pathway and is required for granulocyte colony-stimulating factor receptor-mediated signaling. JAGN1 emerges as a factor that is necessary in the differentiation and survival of neutrophils.

Tracheal cartilage - evaluating the potential of a novel biomaterial for reconstructive cardiovascular procedures.

Modern cardiovascular medicine aims for procedures that preferably involve biological materials and, ideally, living implants. Thereby, the regenerative capacity of the target organ may be preserved or even supported, with a potential implant growth capacity during the following time. In the current study we sought to evaluate the integrative capacity of vital and non-vital tracheal cartilage rings (TCRs) of allogenic or xenogenic origin (allo-/xeno-vTCR; allo-/xeno-nvTCR) as biomaterials under the in vivo functional load of the circulatory system. Ovine and porcine vTCRs and nvTCRs were implanted in the mitral valve (MV) position for 3 and 9 months (n = 3 each), respectively, in lambs. MV function and TCR position were analysed by echocardiography. Tissue morphology (planimetry), vitality (live/dead-assay) and implant endothelialization (scanning electron microscopy) were analysed. No functional impairment or significant MV insufficiency or stenosis was observed in any group. TCR shrinkage was observed in all xeno-TCRs and allo-nvTCRs at 3 months. Only TCRs of allogenic groups at 9 months and allo-vTCRs at 3 months showed a ring area comparable to its size at implantation. Moreover, allogenic vital cartilage showed superior tissue integration, greater endothelialization, less inflammation and calcification. Interestingly, in this group viable cartilage cells were found up to 9 months after implantation. Allogenic viable cartilage may represent a well-suited living material for reconstructive cardiovascular procedures, and further studies are warranted to elucidate the benefits of this novel material, particularly as a structurally supportive component in growing recipients.

Highly specific detection of myostatin prodomain by an immunoradiometric sandwich assay in serum of healthy individuals and patients.

BACKGROUND: Myostatin is a muscle derived factor that functions as a negative regulator of skeletal muscle growth. Induction of myostatin expression was observed in rodent models of muscle wasting and in cachectic patients with cancer or pulmonary disease. Therefore, there is an increasing interest to use serum myostatin as a biomarker. METHODS: We established an immunoradiometric sandwich assay (IRMA), which uses a commercially available chicken polyclonal, affinity purified antibody directed against human myostatin prodomain. We determined the serum concentrations of myostatin prodomain in 249 healthy individuals as well as 169 patients with heart failure, 53 patients with cancer and 44 patients with chronic pulmonary disease. RESULTS: The IRMA had a detection limit of 0.7ng/ml, an intraassay imprecision of ≤14.1% and an interassay imprecision of ≤ 18.9%. The specificity of our assay was demonstrated by size exclusion chromatography, detection of myostatin by Western-blotting and a SMAD-dependent transcriptional-reporter assay in the signal-rich serum fractions, as well as lack of interference by unspecific substances like albumin, hemoglobin or lipids. Myostatin prodomain was stable at room temperature and resistant to freeze-thaw cycles. Apparently healthy individuals over the age of 55 had a median myostatin prodomain serum concentration of 3.9ng/ml (25(th)-75(th) percentiles, 2-7ng/ml) and we could not detect increased levels in patients with stable chronic heart failure or cancer related weight loss. In contrast, we found strongly elevated concentrations of myostatin prodomain (median 26.9ng/ml, 25(th)-75(th) percentiles, 7-100ng/ml) in the serum of underweight patients with chronic pulmonary disease. CONCLUSIONS: We established a highly specific IRMA for the quantification of myostatin prodomain concentration in human serum. Our assay could be useful to study myostatin as a biomarker for example in patients with chronic pulmonary disease, as we detected highly elevated myostatin prodomain serum levels in underweight individuals of this group.

In vivo degradation of magnesium alloy LA63 scaffolds for temporary stabilization of biological myocardial grafts in a swine model.

Synthetic or biological patch materials used for surgical myocardial reconstruction are often fragile. Therefore, a transient support by degradable magnesium scaffolds can reduce the risk of dilation or rupture of the patch until physiological remodeling has led to a sufficient mechanical durability. However, there is evidence that magnesium implants can influence the growth and physiological behavior of the host's cells and tissue. Hence, we epicardially implanted scaffolds of the magnesium fluoride-coated magnesium alloy LA63 in a swine model to assess biocompatibility and degradation kinetics. Chemical analysis of the pigs' organs revealed no toxic accumulation of magnesium ions in the skeletal muscle, myocardium, liver, kidney, and bone of the pigs 1, 3, and 6 months postimplantation. The implants were surrounded by a fibrous granulation tissue, but no signs of necrosis were histologically evaluable. A sufficiently slow degradation rate of the magnesium alloy scaffold can be demonstrated via micro-computed tomography investigation. We conclude that stabilizing scaffolds of the magnesium fluoride-coated magnesium alloy LA63 can be used for epicardial application because no significant adverse effects to myocardial tissue were noted. Thus, degradable stabilizing scaffolds of this magnesium alloy with a slow degradation rate can extend the indication of innovative biological and synthetic patch materials.