Arthroscopic Capsulodesis for the Treatment of Dynamic Scapholunate Dissociations.

Management of scapholunate dissociations remains a significant challenge. Open approaches suffer from a disadvantageous further impairment of the stabilizing local structures. The minimally invasive arthroscopic technique described provides reliable stability of the scapholunate interosseous ligament complex in dynamic lesions. The anatomic key structure is the dorsal capsuloligamentous scapholunate septum, which provides a mechanical connection between the scaphoid, lunate, and dorsal capsule of the wrist. Arthroscopic capsuloplasty aims to tighten and stabilize this complex structure in long term. This approach preserves the adjacent structures, namely the secondary wrist stabilizers and their neuromuscular feedback loops. With a certain degree of experience in wrist arthroscopy, the technique is reliably adaptable and reproducible.

Evaluation of a Novel Thiol-Norbornene-Functionalized Gelatin Hydrogel for Bioprinting of Mesenchymal Stem Cells.

Introduction: Three-dimensional bioprinting can be considered as an advancement of the classical tissue engineering concept. For bioprinting, cells have to be dispersed in hydrogels. Recently, a novel semi-synthetic thiolene hydrogel system based on norbornene-functionalized gelatin (GelNB) and thiolated gelatin (GelS) was described that resulted in the photoclick hydrogel GelNB/GelS. In this study, we evaluated the printability and biocompatibility of this hydrogel system towards adipose-tissue-derived mesenchymal stem cells (ASCs). Methods: GelNB/GelS was synthesized with three different crosslinking densities (low, medium and high), resulting in different mechanical properties with moduli of elasticity between 206 Pa and 1383 Pa. These hydrogels were tested for their biocompatibility towards ASCs in terms of their viability, proliferation and differentiation. The extrusion-based bioprinting of ASCs in GelNB/GelS-high was performed to manufacture three-dimensional cubic constructs. Results: All three hydrogels supported the viability, proliferation and chondrogenic differentiation of ASCs to a similar extent. The adipogenic differentiation of ASCs was better supported by the softer hydrogel (GelNB/GelS-low), whereas the osteogenic differentiation was more pronounced in the harder hydrogel (GelNB/GelS-high), indicating that the differentiation fate of ASCs can be influenced via the adaption of the mechanical properties of the GelNB/GelS system. After the ex vivo chondrogenic differentiation and subcutaneous implantation of the bioprinted construct into immunocompromised mice, the production of negatively charged sulfated proteoglycans could be observed with only minimal inflammatory signs in the implanted material. Conclusions: Our results indicate that the GelNB/GelS hydrogels are very well suited for the bioprinting of ASCs and may represent attractive hydrogels for subsequent in vivo tissue engineering applications.

Ulnocarpal Impaction.

Ulnocarpal impaction syndrome is a common cause for ulnarsided wrist pain caused by an abutment between the ulnar head and the lunotriquetral complex. This pain is typically triggered by load bearing and rotation of the forearm. Radiographic examination is often associated with positive ulnar variance and cysts in the lunate, edema of the ulnoproximal lunate is shown in MRI. Operative treatment aims to reduce load on the lunate, either by open ulnar shortening osteotomy or arthroscopic wafer procedure.

Direct comparison of the immunogenicity of major histocompatibility complex-I and -II deficient mesenchymal stem cells in vivo.

Mesenchymal stem cells (MSCs) play an important role in tissue engineering applications aiming at the regeneration or substitution of damaged tissues. In this context, off-the-shelf allogeneic MSCs would represent an attractive universal cell source. However, immune rejection is a major limitation for the clinical use of allogeneic MSCs. Immune rejection is mediated by the expression of major histocompatibility complexes (MHC)-I and -II on the donor cells. In this study, we eliminated MHC-I and/or MHC-II expression in human MSCs by using the CRISPR/Cas9 technology and investigated the effect of the individual or combined knockout of MHC-I and MHC-II on MSC survival after transplantation into immunocompetent mice. Elimination of MHC-I and/or MHC-II expression did not affect mesenchymal marker gene expression, viability, proliferation and the differentiation potential of MSCs in vitro. However, cell survival of transplanted MSCs was significantly elevated in MHC-I and MHC-II deficient MSCs. A direct side-by-side comparison does not reveal any significant difference in the immunogenicity of MHC-I and MHC-II knockout MSCs. Moreover, double knockout of MHC-I and MHC-II did not further increase in vivo cell survival of transplanted MSCs. Our results demonstrate that knockout of MHC-I and/or MHC-II represents an effective strategy to prevent immune rejection of allogeneic MSCs.

In vivo evaluation of bioprinted prevascularized bone tissue.

Bioprinting can be considered as a progression of the classical tissue engineering approach, in which cells are randomly seeded into scaffolds. Bioprinting offers the advantage that cells can be placed with high spatial fidelity within three-dimensional tissue constructs. A decisive factor to be addressed for bioprinting approaches of artificial tissues is that almost all tissues of the human body depend on a functioning vascular system for the supply of oxygen and nutrients. In this study, we have generated cuboid prevascularized bone tissue constructs by bioprinting human adipose-derived mesenchymal stem cells (ASCs) and human umbilical vein endothelial cells (HUVECs) by extrusion-based bioprinting and drop-on-demand (DoD) bioprinting, respectively. The computer-generated print design could be verified in vitro after printing. After subcutaneous implantation of bioprinted constructs in immunodeficient mice, blood vessel formation with human microvessels of different calibers could be detected arising from bioprinted HUVECs and stabilization of human blood vessels by mouse pericytes was observed. In addition, bioprinted ASCs were able to synthesize a calcified bone matrix as an indicator of ectopic bone formation. These results indicate that the combined bioprinting of ASCs and HUVECs represents a promising strategy to produce prevascularized artificial bone tissue for prospective applications in the treatment of critical-sized bone defects.

Comparing the diagnostic performance of radiation dose-equivalent radiography, multi-detector computed tomography and cone beam computed tomography for finger fractures - A phantom study.

PURPOSE: To compare the diagnostic performance and raters´confidence of radiography, radiography equivalent dose multi-detector computed tomography (RED-MDCT) and radiography equivalent dose cone beam computed tomography (RED-CBCT) for finger fractures. METHODS: Fractures were inflicted artificially and randomly to 10 cadaveric hands of body donors. Radiography as well as RED-MDCT and RED-CBCT imaging were performed at dose settings equivalent to radiography. Images were de-identified and analyzed by three radiologists regarding finger fractures, joint involvement and confidence with their findings. Reference standard was consensus reading by two radiologists of the fracturing protocol and high-dose multi-detector computed tomography (MDCT) images. Sensitivity and specificity were calculated and compared with Cochrane´s Q and post hoc analysis. Rater´s confidence was calculated with Friedman Test and post hoc Nemenyi Test. RESULTS: Rater´s confidence, inter-rater correlation, specificity for fractures and joint involvement were higher in RED-MDCT and RED-CBCT compared to radiography. No differences between the modalities were found regarding sensitivity. CONCLUSION: In this phantom study, radiography equivalent dose computed tomography (RED-CT) demonstrates a partly higher diagnostic accuracy than radiography. Implementing RED-CT in the diagnostic work-up of finger fractures could improve diagnostics, support correct classification and adequate treatment. Clinical studies should be performed to confirm these preliminary results.

Calmodulin Regulated Spectrin Associated Protein 1 mRNA is Directly Regulated by miR-126 in Primary Human Osteoblasts.

Vascularization is essential for bone development, fracture healing, and bone tissue engineering. We have previously described that coculture of primary human osteoblasts (hOBs) and human umbilical vein endothelial cells (HUVECs) improves differentiation of both cell types. Investigating the role of microRNAs (miRNAs) in this system, we found that miR-126 is highly upregulated in hOBs following coculturing with HUVECs. In this study we performed miR-126 gain-of-function and loss-of-function experiments in hOBs followed by microarray analysis in order to identify targets of miR-126. The transcript cluster IDs were sieved by applying cut-off criteria and by selecting transcripts which were upregulated following miR-126 downregulation and vice versa. The calmodulin regulated spectrin associated protein 1 (CAMSAP1) mRNA was confirmed to be differentially regulated by miR-126. Using the luciferase reporter assay it was demonstrated that CAMSAP1 is directly targeted by miR-126. In this study, we show that miR-126 and CAMSAP1 directly interact in hOBs. This finding has potential implications for tissue engineering applications. J. Cell. Biochem. 118: 1756-1763, 2017. © 2016 Wiley Periodicals, Inc.

Comparison of Diagnostic Accuracy of Radiation Dose-Equivalent Radiography, Multidetector Computed Tomography and Cone Beam Computed Tomography for Fractures of Adult Cadaveric Wrists.

PURPOSE: To compare the diagnostic accuracy of radiography, to radiography equivalent dose multidetector computed tomography (RED-MDCT) and to radiography equivalent dose cone beam computed tomography (RED-CBCT) for wrist fractures. METHODS: As study subjects we obtained 10 cadaveric human hands from body donors. Distal radius, distal ulna and carpal bones (n = 100) were artificially fractured in random order in a controlled experimental setting. We performed radiation dose equivalent radiography (settings as in standard clinical care), RED-MDCT in a 320 row MDCT with single shot mode and RED-CBCT in a device dedicated to musculoskeletal imaging. Three raters independently evaluated the resulting images for fractures and the level of confidence for each finding. Gold standard was evaluated by consensus reading of a high-dose MDCT. RESULTS: Pooled sensitivity was higher in RED-MDCT with 0.89 and RED-MDCT with 0.81 compared to radiography with 0.54 (P = < .004). No significant differences were detected concerning the modalities' specificities (with values between P = .98). Raters' confidence was higher in RED-MDCT and RED-CBCT compared to radiography (P < .001). CONCLUSION: The diagnostic accuracy of RED-MDCT and RED-CBCT for wrist fractures proved to be similar and in some parts even higher compared to radiography. Readers are more confident in their reporting with the cross sectional modalities. Dose equivalent cross sectional computed tomography of the wrist could replace plain radiography for fracture diagnosis in the long run.

A self-made, low-cost infrared system for evaluating the sciatic functional index in mice.

The sciatic functional index (SFI) is a popular parameter for peripheral nerve evaluation that relies on footprints obtained with ink and paper. Drawbacks include smearing artefacts and a lack of dynamic information during measurement. Modern applications use digitized systems that can deliver results with less analytical effort and fewer mice. However, the systems are expensive (€40,000). This study aimed to evaluate the applicability and precision of a self-made, low-cost infrared system for evaluating SFI in mice. Mice were subjected to unilateral sciatic nerve crush injury (crush group; n = 7) and sham operation (sham group; n = 4). They were evaluated on the day before surgery, the 2(nd), 4(th) and 6(th) days after injury, and then every day up to the 23(rd) day after injury. We compared two SFI evaluation methods, i.e., conventional ink-and-paper SFI (C-SFI) and our infrared system (I-SFI). Our apparatus visualized footprints with totally internally reflected infrared light (950 nm) and a camera that can only detect this wavelength. Additionally we performed an analysis with the ladder beam walking test (LBWT) as a reference test. I-SFI assessment reduced the standard deviation by about 33 percent, from 11.6 to 7.8, and cut the variance around the baseline to 21 percent. The system thus requires fewer measurement repetitions and fewer animals, and cuts the cost of keeping the animals. The apparatus cost €321 to build. Our results show that the process of obtaining the SFI can be made more precise via digitization with a self-made, low-cost infrared system.

Overexpression of hypoxia-inducible factor-1 alpha improves vasculogenesis-related functions of endothelial progenitor cells.

Postnatal vasculogenesis is mediated by mobilization of endothelial progenitor cells (EPCs) from bone marrow and homing to ischemic tissues. This feature emphasizes this cell type for cell-based therapies aiming at the improvement of neovascularization in tissue engineering applications and regenerative medicine. In animal models, it was demonstrated that implantation of EPCs from cord blood (cbEPCs) led to the formation of a complex functional neovasculature, whereas EPCs isolated from adult peripheral blood (pbEPCs) showed a limited vasculogenic potential, which may be attributed to age-related dysfunction. Recently, it was demonstrated that activation of hypoxia-inducible factor-1α (Hif-1α) improves cell functions of progenitor cells of mesenchymal and endothelial origin. Thus, we hypothesized that overexpression of Hif-1α may improve the vasculogenesis-related phenotype of pbEPCs. In the present study, we overexpressed Hif-1α in pbEPCs and cbEPCs by using recombinant adenoviruses and investigated effects on stem cell- and vasculogenesis-related cell parameters. Overexpression of Hif-1α enhanced proliferation, invasion, cell survival and in vitro capillary sprout formation of both EPC populations. Migration was increased in cbEPCs upon Hif-1α overexpression, but not in pbEPCs. Cellular senescence was decreased in pbEPCs, while remained in cbEPCs, which showed, as expected, intrinsically a dramatically lower senescent phenotype in relation to pbEPCs. Similarly, the colony-formation capacity was much higher in cbEPCs in comparison to pbEPCs and was further increased by Hif-1α overexpression, whereas Hif-1α transduction exerted no significant influence on colony formation of pbEPCs. In summary, our experiments illustrated multifarious effects of Hif-1α overexpression on stem cell and vasculogenic parameters. Therefore, Hif-1α overexpression may represent a therapeutic option to improve cellular functions of adult as well as postnatal EPCs.

Transcriptomic Changes in Osteoblasts Following Endothelial Cell-Cocultivation Suggest a Role of Extracellular Matrix in Cellular Interaction.

Vascularization is important for bone development, fracture healing and engineering of artificial bone tissue. In the context of bone tissue engineering, it was shown that coimplantation of human primary umbilical vein endothelial cells (HUVECs) and human osteoblasts (hOBs) results in the formation of functional blood vessels and enhanced bone regeneration. Implanted endothelial cells do not only contribute to blood vessel formation, but also support proliferation, cell survival and osteogenic differentiation of coimplanted hOBs. These effects are partially mediated by direct heterotypic cell contacts. In a previous report we could show that cocultivated hOBs strongly increase the expression of genes involved in extracellular matrix (ECM) formation in HUVECs, suggesting that ECM may be involved in the intercellular communication between hOBs and HUVECs. The present study aimed at investigating whether comparable changes occur in hOBs. We therefore performed a microarray analysis of hOBs cultivated in direct contact with HUVECs, revealing 1,004 differentially expressed genes. The differentially expressed genes could be assigned to the functional clusters ECM, proliferation, apoptosis and osteogenic differentiation. The microarray data could be confirmed by performing quantitative real time RT-PCR on selected genes. Furthermore, we could show that the ECM produced by HUVECs increased the expression of the osteogenic differentiation marker alkaline phosphatase (ALP) in hOBs. In summary, our data demonstrate that HUVECs provoke complex changes in gene expression patterns in cocultivated hOBs and that ECM plays and important role in this interaction. J. Cell. Biochem. 117: 1869-1879, 2016. © 2016 Wiley Periodicals, Inc.

Comparison of Multidetector Computed Tomography and Flat-Panel Computed Tomography Regarding Visualization of Cortical Fractures, Cortical Defects, and Orthopedic Screws: A Phantom Study.

To compare the visualization of cortical fractures, cortical defects, and orthopedic screws in a dedicated extremity flat-panel computed tomography (FPCT) scanner and a multidetector computed tomography (MDCT) scanner.We used feet of European roe deer as phantoms for cortical fractures, cortical defects, and implanted orthopedic screws. FPCT and MDCT scans were performed with equivalent dose settings. Six observers rated the scans according to number of fragments, size of defects, size of defects opposite orthopedic screws, and the length of different screws. The image quality regarding depiction of the cortical bone was assessed. The gold standard (real number of fragments) was evaluated by autopsy.The correlation of reader assessment of fragments, cortical defects, and screws with the gold standard was similar for FPCT and MDCT. Three readers rated the subjective image quality of the MDCT to be higher, whereas the others showed no preferences.Although the image quality was rated higher in the MDCT than in the FPCT by 3 out of 6 observers, both modalities proved to be comparable regarding the visualization of cortical fractures, cortical defects, and orthopedic screws and of use to musculoskeletal radiology regarding fracture detection and postsurgical evaluation in our experimental setting.

Utilization of a genetically modified muscle flap for local BMP-2 production and its effects on bone healing: a histomorphometric and radiological study in a rat model.

AIM OF THE STUDY: We developed an experimental rat model to explore the possibility of enhancing the healing of critical-size bone defects. The aim of this study was to demonstrate the feasibility of this concept by achieving high local BMP-2 expression via a transduced muscle flap that would facilitate bony union while minimizing systemic sequelae. METHODS: The transduction potential of the adenoviral vector encoding for BMP-2 was tested in different cell lines in vitro. In vivo experiments consisted of harvesting a pedicled quadriceps femoris muscle flap with subsequent creation of a critical-size defect in the left femur in Sprague-Dawley rats. Next, the pedicled muscle flap was perfused with high titers of Ad.BMP-2 and Ad.GFP virus, respectively. Twelve animals were divided into three groups comparing the effects of Ad.BMP-2 transduction to Ad.GFP and placebo. Bone healing was monitored radiologically with subsequent histological analysis post-mortem. RESULTS: The feasibility of this concept was demonstrated by successful transduction in vitro and in vivo as evidenced by a marked increase of BMP-2 expression. The three examined groups only showed minor difference regarding bone regeneration; however, one complete bridging of the defect was observed in the Ad.BMP-2 group. No evidence of systemic viral contamination was noted. CONCLUSIONS: A marked increase of local BMP-2 expression (without untoward systemic sequelae) was detected. However, bone healing was not found to be significantly enhanced, possibly due to the small sample size of the study.

Cell-assisted lipotransfer.

BACKGROUND: Because of their easy accessibility and versatile biological properties, mesenchymal stem cells taken from fatty tissue (adipose-derived stem cells, ADSC) are attractive for various potential clinical uses. For example, ADSC can be added to fatty tissue before transplantation in the hope of improving the outcome of autologous lipotransfer: the modified procedure is called cell-assisted lipotransfer. The clinical use and commercial promotion of this novel stem-cell treatment (and others) are spreading rapidly, even though there is not yet any clear clinical evidence for its safety and efficacy. METHODS: In cooperation with the German Cochrane Center, we systematically searched the literature according to the PRISMA criteria. Eight major medical databases were searched. The retrieved publications were examined by two independent reviewers and assessed using objective criteria. RESULTS: After screening of the 3161 retrieved publications by title, abstract, and (where appropriate) full text, 78 were still considered relevant. 13 of these were reports of clinical studies; only 3 of the 13 met criteria for grade II or III evidence. The studies that were analyzed involved a total of 286 cell-assisted lipotransfer procedures with a longest follow-up time of 42 months. Oncological safety was not demonstrated. CONCLUSION: The studies published to date have not shown that cell-assisted lipotransfer is generally superior to conventional autologous lipotransfer. They dealt with safety aspects inappropriately or not at all. The case of cell-assisted lipotransfer illustrates the indispensability of high-quality clinical evidence before the introduction of novel stem-cell-based treatments.

Aesthetic surgery performed by plastic surgery residents: an analysis of safety and patient satisfaction.

BACKGROUND: Aesthetic surgery is an integral component of plastic surgery. Despite its importance, adequate training in aesthetic surgery is met with challenges. Although the educational benefit of resident clinics has been demonstrated, such clinics are rarely found outside the United States. The objective of the present study was to assess safety and patient satisfaction associated with aesthetic surgery procedures performed by plastic surgery residents at a German academic medical center. METHODS: The study had 2 components, namely, a retrospective chart review and an administration of a patient satisfaction survey. Only patients who underwent a surgical intervention by a plastic surgery resident between 2003 and 2011 were included in the study. Parameters of interest included age, sex, procedure performed, number of procedures, revenue (in &OV0556;), length of follow-up, revision rate, and postoperative complication rate. Patient satisfaction was assessed by the client satisfaction questionnaire-8. RESULTS: A total of 273 aesthetic procedures were performed in 206 patients with an increase in recent years. The median follow-up period was 49.5 months. The most frequently performed procedures were liposuction (n = 59), breast augmentation (n = 53), and upper eyelid blepharoplasty (n = 31). One hundred ninety-two (90.3%) patients had an uneventful postoperative course. The client satisfaction questionnaire-8 questionnaire was completed by 110 patients (response rate, 50.2%). The median value of 28 indicates a high degree of patient satisfaction. An association between occurrence of major complications and patient satisfaction was seen. CONCLUSIONS: Aesthetic surgery performed by plastic surgery residents under supervision by attending physicians is safe and provides for high levels of patient satisfaction postoperatively. Offering these services may be able to bridge the gap between providing high-quality aesthetic surgery training while yet recruiting an increasing number of patients who may appreciate the lower fees associated with these services.

Effects of endothelial cells on proliferation and survival of human mesenchymal stem cells and primary osteoblasts.

Angiogenesis is a fundamental process in bone formation, remodeling, and regeneration. Moreover, for the regeneration of bone in tissue engineering applications, it is essential to support neovascularization. This can be achieved by cell-based therapies using primary endothelial cells, which are able to form functional blood vessels upon implantation. In bone composite grafts, coimplanted endothelial cells do not only support neovascularization but also support osteogenic differentiation of osteoblasts and osteoprogenitor cells. In this study, we investigated the effect of endothelial cells on proliferation and cell survival of human primary osteoblasts (hOBs) and human mesenchymal stem cells (MSCs). Human umbilical vein endothelial cells (HUVECs) stimulated hOB and MSC proliferation, whereas proliferation of HUVECs was unaffected by cocultured hOBs or MSCs. The effect of HUVEC cocultivation on hOB and MSC proliferation was more pronounced in direct cocultures than in indirect cocultures, indicating that this effect is at least partially dependent on the formation of heterotypic cell contacts between HUVECs and hOBs or MSCs. Furthermore, HUVEC cocultivation reduced low-serum induced apotosis of hOBs and MSCs by a mechanism involving increased phosphorylation and inactivation of the proapoptotic protein Bad. In summary, our experiments have shown that cocultured HUVECs increase the proliferation and reduce low-serum induced apoptosis of hOBs and MSCs.

An integrated therapy concept for reduction of postoperative complications after resection of a panniculus morbidus.

BACKGROUND: Panniculus morbidus is characterized by an edematous, painful hanging abdominal mass, due to laxity and redundancy of the abdominal skin in morbid obesity, particularly after massive weight loss. Panniculectomy, by wedge resection, is a salvage procedure with high satisfaction rates though associated with high complication rates. Here we investigated the effects of perioperative complex decongestive physical therapy (CDP) on outcome and complication rates. METHODS: We retrospectively analyzed the clinical course and outcome of 24 patients receiving panniculectomy between 1998 and 2009 in our department of plastic surgery. Sixteen patients received perioperative CDP, and eight patients did not receive any form of decongestive treatment. We analyzed the incidence of complications, reoperation, blood transfusions, and length of hospital stay based on chart reviews. Complications were categorized as minor or major according to the necessity of readmission or reoperation. CDP was performed for 4-6 weeks preoperatively and 2 weeks postoperatively. RESULTS: The incidence of major complications (p = 0.001), the rate of postoperative blood transfusions (p = 0.028), wound healing disorders (p = 0.021), and the incidence for complications (p = 0.001), whether minor or major, were significantly reduced in the CDP group. In summary, 12 of 16 patients within the CDP group had an uneventful course, whereas all non-CDP patients had at least one complication. CONCLUSIONS: Adequate perioperative CDP treatment in a lymphological clinic may reduce the rate of early postoperative complications after resection of panniculus morbidus.

Up-regulation of alkaline phosphatase expression in human primary osteoblasts by cocultivation with primary endothelial cells is mediated by p38 mitogen-activated protein kinase-dependent mRNA stabilization.

For the regeneration of bone in tissue engineering applications, it is essential to provide cues that support neovascularization. This can be achieved by cell-based therapies using mature endothelial cells (ECs) or endothelial progenitor cells. In this context, ECs were used in various in vivo studies in combination with primary osteoblasts to enhance neovascularization of bone grafts. In a previous study, we have shown that cocultivation of human primary ECs and human primary osteoblasts (hOBs) leads to a cell contact-dependent up-regulation of alkaline phosphatase (ALP) expression in osteoblasts, indicating that cocultivated ECs may support osteogenic differentiation and osteoblastic cell functions. In the present study, we investigated this effect in more detail, revealing a time and cell number dependency of EC-mediated up-regulation of the early osteoblastic marker ALP, whereas osteocalcin, a late marker of osteogenesis, was down-regulated. The effect on ALP expression was bidirectional specific for both cell types. Functional inhibition of gap junctional communication between ECs and hOBs by 18alpha-glycyrrhetinic acid had only a weak suppressive effect on EC-mediated ALP up-regulation. In contrast, inhibition of p38 mitogen-activated protein kinase nearly completely prevented the EC-mediated stimulation of osteoblastic ALP expression. To investigate the molecular mechanism underlying the ALP up-regulation, we examined the effect of EC cocultivation on osteoblastic ALP promoter activity as well as mRNA stability. Cocultivation of ECs with hOBs significantly elevated the half-life of osteoblastic ALP mRNA without affecting its promoter activity. In summary, our data show that EC-mediated up-regulation of osteoblastic ALP expression is cell-type specific and is posttranscriptionally regulated via p38 mitogen-activated protein kinase-dependent mRNA turn-over.

"Turnover proteome" of human atrial trabeculae.

Most of the biologically relevant data on cardiomyocytes are derived from isolated cells under conditions that are, to some extent, altered compared to the natural milieu of the functional heart. The handling procedure of the dissection, isolation, and short-term culturing induces changes in the cells such that the subsequently measured parameters (among others, the protein synthesis) reflect the actual experimental conduct rather than the intrinsic properties of these terminally differentiated cells. Although it is known that the protein synthetic machinery of isolated cardiomyocytes is operational and functional, the biosynthetic yield of human cardiomyocytes in the natural milieu of the trabeculae remains to be established, with a special emphasis to clarify whether the protein synthesis includes just a limited set of polypeptides or it encompasses all cellular constituents. Knowledge on this issue is a prerequisite for achieving further advances in our understanding of heart remodeling related to hypertrophy in particular, and for attempting interventions leading to repair of damaged heart in general. The experimental system of "organ bath" enables simultaneous registration of contractile forces of portions of cardiac muscle tissue (and other myocyte-containing tissues) and biosynthetic labeling of newly synthesized cellular constituents. The organ bath methodology was adapted for this project such as enabling to measure molecular changes in response to in vitro applied stimuli. Instead of Krebs-Henseleit-solution, as used in classical protocols of organ bath studies, we utilized cell culture media suitable to experimental conditions related to metabolic labeling. Proteome patterns established by performing two-dimensional gel electrophoresis of the extracts from biosynthetically labeled trabeculae revealed that cardiomyocytes synthesize the full spectrum of cellular proteins. Proteomic silver-stain readout was used to obtain samples for spot excisions, as material suitable for mass spectrometric analysis. Protein spots were identified both from the excised spots and also by matching with the in-house- and www-databases (Swiss-Prot/High-Performance Heart). From our findings that protein synthesis in terminally differentiated cardiomyocytes is not confined just to the synthesis of those structures needed for the post-mitotic house-keeping functions, we conclude that this model might serve as a valid experimental system to study and elucidate the effects of various pharmacological compounds under conditions where physiology (contractile forces) and biochemistry (protein synthesis) of intact human heart tissue are monitored simultaneously.