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Mesenchymal stromal cells are promising candidates for regenerative applications upon treatment of bone defects. Bone marrow‐derived stromal cells (BMSCs) are limited by yield and donor morbidity but show superior osteogenic capacity compared to adipose‐derived stromal cells (ASCs), which are highly abundant and easy to harvest. The underlying reasons for this difference on a proteomic level have not been studied yet. Human ASCs and BMSCs were characterized by FACS analysis and tri‐lineage differentiation, followed by an intraindividual comparative proteomic analysis upon osteogenic differentiation. Results of the proteomic analysis were followed by functional pathway analysis. 29 patients were included with a total of 58 specimen analysed. In these, out of 5148 identified proteins 2095 could be quantified in >80% of samples of both cell types, 427 in >80% of ASCs only and 102 in >80% of BMSCs only. 281 proteins were differentially regulated with a fold change of >1.5 of which 204 were higher abundant in BMSCs and 77 in ASCs. Integrin cell surface interactions were the most overrepresented pathway with 5 integrins being among the proteins with highest fold change. Integrin 11a, a known key protein for osteogenesis, could be identified as strongly up‐regulated in BMSC confirmed by Western blotting. The integrin expression profile is one of the key distinctive features of osteogenic differentiated BMSCs and ASCs. Thus, they represent a promising target for modifications of ASCs aiming to improve their osteogenic capacity and approximate them to that of BMSCs.

Abstract Impaired bone homeostasis caused by osteomyelitis provokes serious variations in the bone remodeling process, thereby involving multiple inflammatory cytokines to activate bone healing. We have previously established a mouse model for post-traumatic osteomyelitis and studied bone regeneration after sufficient debridement. Moreover, we could further characterize the postinfectious inflammatory state of bony defects after debridement with elevated osteoclasts and decreased bone formation despite the absence of bacteria. In this study, we investigated the positive effects of Wnt-pathway modulation on bone regeneration in our previous established mouse model. This was achieved by local application of Wnt3a, a recombinant activator of the canonical Wnt-pathway. Application of Wnt3a could enhance new bone formation, which was verified by histological and μ-CT analysis. Moreover, histology and western blots revealed enhanced osteoblastogenesis and downregulated osteoclasts in a RANKL-dependent manner. Further analysis of Wnt-pathway showed downregulation after bone infections were reconstituted by application of Wnt3a. Interestingly, Wnt-inhibitory proteins Dickkopf 1 (DKK1), sclerostin, and secreted frizzled protein 1 (sFRP1) were upregulated simultaneously to Wnt-pathway activation, indicating a negative feedback for active form of Beta-catenin. In this study, we could demonstrate enhanced bone formation in defects caused by post-traumatic osteomyelitis after Wnt3a application. Key messages Osteomyelitis decreases bone regeneration Wnt3a restores bone healing after infection Canonical Wnt-pathway activation with negative feedback

Background: The optimal therapy for deep burn wounds is based on the early debridement of necrotic tissue followed by wound coverage to avoid a systemic inflammatory response and optimize scar-free healing. The outcomes are affected by available resources and underlying patient factors, which represent challenges in burn care and suboptimal outcomes. In this study, we aimed to determine optimal burn-wound management using enzymatic debridement (NexoBrid™, MediWound Germany GmbH, Rüsselsheim, Germany) and intact fish skin (Kerecis® Omega3 Wound, Isafjordur, Iceland). Methods: In this retrospective case series, 12 patients with superficial or deep dermal burn wounds were treated with enzymatic debridement followed by fish skin, Suprathel® (PolyMedics Innovations GmbH, Denkendorf, Germany), or a split-thickness skin graft (STSG). Patients’ outcomes regarding healing and scar quality were collected objectively and subjectively for 12 months after the burn injury. Results: Wounds treated with fish skin demonstrated accelerated wound healing, a significantly higher water-storage capacity, and better pain relief. Furthermore, improved functional and cosmetic outcomes, such as elasticity, skin thickness, and pigmentation, were demonstrated. The pain and itch expressed as POSAS scores (Patient and Observer Scar Assessment Scale) for fish skin decreased compared to those for wounds managed with an STSG or Suprathel. Importantly, fish skin-treated wounds had significantly improved sebum production and skin elasticity than those treated with Suprathel but showed no significant superiority compared to STSG-treated wounds. Conclusions: Enzymatic debridement in combination with intact fish skin grafts resulted in the faster healing of burn wounds and better functional and aesthetic outcomes than split-thickness skin grafts and Suprathel treatment.

Ischemia reperfusion (IR) injury remains an important topic in clinical medicine. While a multitude of prophylactic and therapeutic strategies have been proposed, recent studies have illuminated protective effects of myostatin inhibition. This study aims to elaborate on the intracellular pathways involved in myostatin signaling and to explore key proteins that convey protective effects in IR injury. We used CRISPR/Cas9 gene editing to introduce a myostatin (Mstn) deletion into a C2C12 cell line. In subsequent experiments, we evaluated overall cell death, activation of apoptotic pathways, ROS generation, lipid peroxidation, intracellular signaling via mitogen-activated protein kinases (MAPKs), cell migration, and cell proliferation under hypoxic conditions followed by reoxygenation to simulate an IR situation in vitro (hypoxia reoxygenation). It was found that mitogen-activated protein kinase kinase 3/6, also known as MAPK/ERK Kinase 3/6 (MEK3/6), and subsequent p38 MAPK activation were blunted in C2C12-Mstn−/− cells in response to hypoxia reoxygenation (HR). Similarly, c-Jun N-terminal kinase (JNK) activation was negated. We also found the intrinsic activation of apoptosis to be more important in comparison with the extrinsic activation. Additionally, intercepting myostatin signaling mitigated apoptosis activation. Ultimately, this research validated protective effects of myostatin inhibition in HR and identified potential mediators worth further investigation. Intercepting myostatin signaling did not inhibit ROS generation overall but mitigated cellular injury. In particular, intrinsic activation of apoptosis origination from mitochondria was alleviated. This was presumably mediated by decreased activation of p38 caused by the diminished kinase activity increase of MEK3/6. Overall, this work provides important insights into HR signaling in C2C12-Mstn−/− cells and could serve as basis for further research.

Critical sized defects, especially in long bones, pose one of the biggest problems in orthopedic surgery. By definition, these defects do not heal without further treatment. Different therapeutic options range from autologous bone grafts, for example, free vascularized bone grafts, to commercially available bone allografts. Disadvantages of these bone allografts are related to reduced osteogenesis, since they are solely composed of cell-free bone matrix. The purpose of this study was to investigate the cell seeding efficiency of human adipose-derived stem cells (hASCs) on human bone allografts in vitro and furthermore analyze these optimized seeded allografts in a critical sized defect model in vivo. Cancellous human bone allografts were colonized with human ASCs in vitro. Cell seeding efficiency was evaluated by Cell Counting Kit-8 assay. Thereafter, optimized hASC-seeded bone scaffolds were examined in a murine femur defect model, stabilized with the MouseExFix system. Subsequently, x-ray analysis and histology were performed. Examination of cell seeding efficiency revealed an optimum starting population of 84,600 cells per 100 mm3 scaffold. In addition, scaffolds seeded with hASCs showed increased osteogenesis compared with controls. Histological analysis revealed increased remodeling and elevated new bone formation within hASC-seeded scaffolds. Moreover, immunohistochemical stainings revealed increased proliferation, osteogenesis, and angiogenesis. In this study, we systemically optimized cell/volume ratio of two promising components of tissue engineering: hASCs and human bone allografts. These findings may serve as a basis for future translational studies.

Ischemia reperfusion (IR) injury plays a pivotal role in many diseases and leads to collateral damage during surgical interventions. While most studies focus on alleviating its severity in the context of brain, liver, kidney, and cardiac tissue, research as regards to skeletal muscle has not been conducted to the same extent. In the past, GDF8 (myostatin), primarily known for supressing muscle growth, has been implicated in inflammatory circuits, and research provided promising results for cardiac IR injury mitigation by inhibiting GDF8 cell surface receptor ACVR2B. This generated the question if interrupting GDF8 signaling could temper IR injury in skeletal muscle. Examining human specimens from free myocutaneous flap transfer demonstrated increased GDF8 signaling and tissue damage in terms of apoptotic activity, cell death, tissue edema, and lipid peroxidation. In subsequent in vivo MstnLn/Ln IR injury models, we identified potential mechanisms linking GDF8 deficiency to protective effects, among others, inhibition of p38 MAPK signaling and SERCA2a modulation. Furthermore, transcriptional profiling revealed a putative involvement of NK cells. Collectively, this work establishes a protective role of GDF8 deficiency in skeletal muscle IR injury.

BackgroundDelayed bone healing, especially in long bones poses one of the biggest problems in orthopeadic and reconstructive surgery and causes tremendous costs every year. There is a need for exploring the causes in order to find an adequate therapy. Earlier investigations of human scaphoid non-union revealed an elevated osteoclast activity, accompanied by upregulated levels of TGF-beta and RANKL. Interestingly, scaphoid non-union seemed to be well vascularized.MethodsIn the current study, we used a murine femur-defect model to study atrophic non unions over a time-course of 10 weeks. Different time points were chosen, to gather insights into the dynamic processes of non-union establishment.ResultsHistological analyses as well as western blots and qRT-PCR indicated enhanced osteoclast activity throughout the observation period, paralleled by elevated levels of TGF-beta, TNF-alpha, MMP9, MMP13 and RANKL, especially during the early phases of non-union establishment. Interestingly, elevated levels of these mediators decreased markedly over a period of 10 weeks, as inflammatory reaction during non-union establishment seemed to wear out. To our surprise, osteoblastogenesis seemed to be unaffected during early stages of non-union establishment.ConclusionTaken together, we gained first insights into the establishment process of atrophic non unions, in which inflammatory processes accompanied by highly elevated osteoclast activity seem to play a leading role.

Introduction The optimal therapy for deep burn wounds is based on the principle of rapid necrectomy and coverage in order to achieve healing that is as scar-free as possible. The available infrastructure and the patient’s condition represent limitations. With enzymatic debridement, selective bedside debridement can now be performed, however the optimal cover after enzymatic debridement has not been elucidated to date. In this study we compare superficial dermal and deep dermal wounds, which are either covered with lactic acid membrane, piscine skin, or split-thickness skin graft. To validate our approach the healed burn wounds were examined for objective (elasticity, water content, sebum, wound healing) and subjective skin quality as part of our standard follow-up care. Methods In this study, 12 patients who had received piscine skin, lactic acid membrane, or split-thickness skin graft after enzymatic debridement were retrospectively examined objectively and subjectively for scar quality as part of follow-up care 12 months after the accident. The wound healing process was also documented. Results Acceleration of wound healing was observed with the application of piscine skin vs split-thickness skin graft or lactic acid membrane. Skin elasticity was comparable to that of split-thickness skin graft but significantly better than lactic acid membrane. The sebum production in wounds treated with piscine skin was higher compared to lactic acid membrane covered wounds. The water storage capacity in the piscine skin treated wounds was also significantly higher than in lactic acid membrane or split-thickness skin graft treated wounds. Using the POSAS score, an improvement in elasticity, thickness, pigmentation, and relief was shown in piscine skin treated wounds, as well as a reduction in pain and itching, compared to split-thickness skin graft or lactic acid membrane. Conclusions The use of intact piscine skin immediately following enzymatic debridement in burn wounds results in faster wound healing and better patient outcomes compared to split-thickness skin graft or lactic acid membrane.

Impaired bone homeostasis caused by osteomyelitis provokes serious variations in the bone remodeling process, thereby involving multiple inflammatory cytokines to activate bone healing. We have previously established a mouse model for post-traumatic osteomyelitis and studied bone regeneration after sufficient debridement. Moreover, we could further characterize the postinfectious inflammatory state of bony defects after debridement with elevated osteoclasts and decreased bone formation despite the absence of bacteria. In this study, we investigated the positive effects of Wnt-pathway modulation on bone regeneration in our previous established mouse model. This was achieved by local application of Wnt3a, a recombinant activator of the canonical Wnt-pathway. Application of Wnt3a could enhance new bone formation, which was verified by histological and μ-CT analysis. Moreover, histology and western blots revealed enhanced osteoblastogenesis and downregulated osteoclasts in a RANKL-dependent manner. Further analysis of Wnt-pathway showed downregulation after bone infections were reconstituted by application of Wnt3a. Interestingly, Wnt-inhibitory proteins Dickkopf 1 (DKK1), sclerostin, and secreted frizzled protein 1 (sFRP1) were upregulated simultaneously to Wnt-pathway activation, indicating a negative feedback for active form of Beta-catenin. In this study, we could demonstrate enhanced bone formation in defects caused by post-traumatic osteomyelitis after Wnt3a application. KEY MESSAGES: Osteomyelitis decreases bone regeneration Wnt3a restores bone healing after infection Canonical Wnt-pathway activation with negative feedback.

Simple Summary Soft tissue sarcomas are relatively rare malignant diseases. Part of the diagnosis and follow-up includes medical imaging of the thorax for detection of lung metastases. A Python script was created and trained using a set of lung X-rays and concordant CT scans from a high-volume German-speaking sarcoma center. It is capable of detecting malignant metastasis in the lung with a precision of 71.2%, specificity of 90.5%, sensitivity of 94% and accuracy of 91.2%. Furthermore, the program was able to detect even small nodules with a size

Osteomyelitis is a frequent consequence of open fractures thus representing a common bone infection with subsequent alteration of bone regeneration. Impaired bone homeostasis provokes serious variations in the bone remodeling process, thereby involving multiple inflammatory cytokines to activate bone healing. Our previously established mouse model of posttraumatic osteomyelitis provides the chance to study regulation of selected cytokines after surgical debridement of osteomyelitis thus illustrating the course of initial infectious recovery. An inflammatory cytokine array revealed specifically upregulated cytokines in debrided animals after bone infection, that were verified by Western blot analysis, identifying increased levels of CCL2, CCL3, and CXCL2. Increased osteoclastogenesis after debridement of osteomyelitis was demonstrated by Calcitonin-receptor and RANKL detection via immunohistochemical and -fluorescence stainings. The substantial protein analysis was complemented by uncovering diminished osteogenesis and proliferation in debrided group, tracking Osteocalcin, RUNX2, and PCNA expression. Interestingly TNF-α expression seemed to have no effect on altered bone regeneration after bone infection. Additional flow cytometry analysis proved elevated B cell activity, subsequently increased osteoclast activity and accelerated bone resorption. Based on the variety of severely altered cytokines, we propose a RANKL-dependent osteoclastogenesis after debridement of osteomyelitis coinciding with elevated B cells and simultaneously decreased osteogenesis. A comprehensive understanding of these mechanisms provides new therapeutic options of osteomyelitis cure and is of great importance in prospective medical treatment. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2425-2434, 2017.

This article describes a mass spectrometry data set generated from osteogenic differentiated bone marrow stromal cells (BMSCs) and adipose tissue derived stromal cells (ASCs) of a 24-year old healthy donor. Before osteogenic differentiation and performing mass spectrometric measurements cells have been characterized as mesenchymal stromal cells via FACS-analysis positive for CD90 and CD105 and negative for CD14, CD34, CD45 and CD11b and tri-lineage differentiation. After osteogenic differentiation, both cell types were homogenized and then fractionated by SDS gel electrophoresis, resulting in 12 fractions. The proteins underwent an in-gel digestion, spiked with iRT peptides and analysed by nanoHPLC-ESI-MS/MS, resulting in 24 data files. The data files generated from the described workflow are hosted in the public repository ProteomeXchange with identifier PXD015026. The presented data set can be used as a spectral library for analysis of key proteins in the context of osteogenic differentiation of mesenchymal stromal cells for regenerative applications. Moreover, these data can be used to perform comparative proteomic analysis of different mesenchymal stromal cells or stem cells upon osteogenic differentiation. In addition, these data can also be used to determine the optimal settings for measuring proteins and peptides of interest. Keywords: Proteome, Spectral peptide library, Mesenchymal stromal cells, Adipose derived stromal cells, Bone marrow derived stromal cells, Osteogenic differentiation, Osteogenesis

Breast adenocarcinoma continues to be the most frequently diagnosed tumor entity. Despite established therapy options, mortality for breast cancer remains to be as high as 40,000 patients in the US annually. Thus, a need to develop a patient-oriented, targeted therapy exists. In this study, we investigated the interaction of breast adenocarcinoma with the ubiquitously present protein Follistatin and subsequently the GDF8/11 pathway. We analyzed primary histological samples from adenocarcinoma patients for expression of Follistatin and GDF8/11. Furthermore, expression levels of Follistatin and GDF8/11 in MCF7 were compared with MCF10a cells. From the resulting data, GDF8 and Follistatin were used as chemotherapeutic agents in MCF7 cells and their migratory, proliferative behavior and viability were measured. From the experiments, we were able to detect a significantly increased expression of Follistatin and GDF8/11 in the low malignant breast adenocarcinoma (G1) as compared to benign breast fibroadenoma. Interestingly, a decrease was demonstrated in higher grade malignancies. These findings were accompanied by the clinical observation that increased expression of Follistatin and GDF8 is associated with a higher overall survival rate of breasts cancer patients. Substitution of GDF8 and Follistatin reduces the viability of the MCF7 cells and disrupts the migrative and proliferative potential. In summary, MCF7 cells show high chemosensitivity to Follistatin and especially GDF8 and both proteins might serve as targets to improve systemic treatment in breast cancer. In contrast to most established chemotherapy regimens Follistatin and GDF8 show no cytotoxicity to other organs.

Metabolic diseases like diabetes mellitus cause bone healing deficiencies. We found significant impairment of bone regeneration, osteogenic differentiation and proliferation in diabetic bone. Moreover recent studies suggest a highly underestimated importance of GDF8 (Myostatin) in bone metabolism. Our goal was to analyze the role of GDF8 as a regulator of osteogenic differentiation, proliferation and bone regeneration. We used a murine tibial defect model in diabetic (Leprdb−/−) mice. Myostatin-Inhibitor Follistatin was administered in tibial bony defects of diabetic mice. By means of histology, immunohistochemistry and QRT-PC osteogenesis, differentiation and proliferation were analyzed. Application of Myostatin-inhibitor showed a significant improvement in diabetic bone regeneration compared to the control group (6.5 fold, p

Metabolic diseases like diabetes mellitus cause bone healing deficiencies. We found significant impairment of bone regeneration, osteogenic differentiation and proliferation in diabetic bone. Moreover recent studies suggest a highly underestimated importance of GDF8 (Myostatin) in bone metabolism. Our goal was to analyze the role of GDF8 as a regulator of osteogenic differentiation, proliferation and bone regeneration. We used a murine tibial defect model in diabetic (Leprdb−/−) mice. Myostatin-Inhibitor Follistatin was administered in tibial bony defects of diabetic mice. By means of histology, immunohistochemistry and QRT-PC osteogenesis, differentiation and proliferation were analyzed. Application of Myostatin-inhibitor showed a significant improvement in diabetic bone regeneration compared to the control group (6.5 fold, p

Soft tissue sarcomas (STS) are heterogeneous malignant tumors of mesenchymal origin. Due to low incidence and high number of different histological subtypes, their pathogenesis and thus potential targets for their therapy remain barely investigated. Several studies revealed significant higher EPHB4 expression in malignancies such as prostate and colorectal cancer showing survival advantages for these tumor cells. Therefore we studied the expression of EPHB4 in a total of 46 clinical human specimens of different STS and human fibroblasts. EPHB4 mRNA and protein expression were significantly increased in synovial sarcoma. After targeting EPHB4 in fibrosarcoma, synovial sarcoma, liposarcoma and MFH sarcoma cell lines by siRNA or by inhibition of autophosphorylation using the specific EPHB4 kinase inhibitor NVP-BHG712 a decreased proliferation rate/vitality of synovial- and fibrosarcoma cells was observed. Silencing of EPHB4 significantly reduced the transmigration of synovial sarcoma cells towards fibroblasts and endothelial cells. In addition, we assessed the anti-metastatic effect of EPHB4 inhibition in vivo by intraperitoneal administration of the EPHB4 inhibitor in an appropriate sarcoma lung metastasis xenograft model. As result 43% of NVP-BHG712 treated mice (n = 3/7) developed pulmonary metastases whereas all control mice (n = 5) revealed lung metastases. The residual 57% of mice (n = 4/7) showed only small local tumor cell spots. Size measurements of the Vimentin positive area explained significant decrease in lung metastasis formation (p

Host defense peptides, in particular LL-37, are emerging as potential therapeutics for promoting wound healing and inhibiting bacterial growth. However, effective delivery of the LL-37 peptide remains limiting. We hypothesized that skin-targeted electroporation of a plasmid encoding hCAP-18/LL-37 would promote the healing of wounds. The plasmid was efficiently delivered to full-thickness skin wounds by electroporation and it induced expression of LL-37 in the epithelium. It significantly accelerated reepithelialization of nondiabetic and diabetic wounds and caused a significant VEGFa and interleukin (IL)-6 induction. IL-6 was involved in LL-37-mediated keratinocyte migration in vitro and IL-6 neutralizing antibodies delivered to mice were able to suppress the wound healing activity of the hCAP-18/LL-37 plasmid. In a hindlimb ischemia model, electroporation of the hCAP-18/LL-37 plasmid increased blood perfusion, reduced muscular atrophy, and upregulated the angiogenic chemokines VEGFa and SDF-1a, and their receptors VEGF-R and CXCR-4. These findings demonstrate that a localized gene therapy with LL-37 is a promising approach for the treatment of wounds.

Disseminated soft tissue sarcomas (STS) present a therapeutic dilemma. The first-line cytostatic doxorubicin demonstrates a response rate of 30% and is not suitable for elderly patients with underlying cardiac disease, due to its cardiotoxicity. Well‑tolerated alternative treatment options, particularly in palliative situations, are rare. Therefore, the present study assessed the anti‑proliferative effects of the natural compounds epigallocatechin-3-gallate (EGCG), silibinin and noscapine on STS cells. A total of eight different human STS cell lines were used in the study: Fibrosarcoma (HT1080), liposarcoma (SW872, T778 and MLS‑402), synovial sarcoma (SW982, SYO1 and 1273) and pleomorphic sarcoma (U2197). Cell proliferation and viability were analysed by 5‑bromo-2'-deoxyuridine and MTT assays and real‑time cell analysis (RTCA). RTCA indicated that noscapine did not exhibit any inhibitory effects. By contrast, EGCG decreased proliferation and viability of all cell lines except for the 1273 synovial sarcoma cell line. Silibinin exhibited anti‑proliferative effects on all synovial sarcoma, liposarcoma and fibrosarcoma cell lines. Liposarcoma cell lines responded particularly well to EGCG while synovial sarcoma cell lines were more sensitive to silibinin. In conclusion, the green tea polyphenol EGCG and the natural flavonoid silibinin from milk thistle suppressed the proliferation and viability of liposarcoma, synovial sarcoma and fibrosarcoma cells. These compounds are therefore potential candidates as mild therapeutic options for patients that are not suitable for doxorubicin‑based chemotherapy and require palliative treatment. The findings from the present study provide evidence to support in vivo trials assessing the effect of these natural compounds on solid sarcomas.

The reasons for the distinct chemoresistance of liposarcomas and their high risk of local recurrence still remain unclear. Depending on the histological subtype of liposarcoma, first-line therapy with the cytostatic agent, doxorubicin, only achieves response rates of approximately 36%. Approximatley 70% of all local recurrences develop in spite of complete surgical resection of the primary tumor with microscopically negative margins. In this study, we aimed to assess the influence of tumor-associated fibroblasts (TAFs) obtained from surgically removed liposarcomas on the well-established human liposarcoma SW872 cell line. Intratumoral TAFs were isolated from intermediate- and high-grade liposarcoma samples. The human liposarcoma cell line, SW872, was co-cultured with the corresponding TAFs or with dermal fibroblasts as a control. The proliferation (by BrdU assay), cell viability (by MTT assay) and sensitivity to doxorubicin (using the iCELLigence system) of the co-cultured SW872 cells were examined. The SW872 cells exhibited a significant increase in proliferation and viability when co-cultured with the TAFs. As detected by real-time cell analysis, the SW872 cells co-cultured with the TAFs exhibited a diminished response towards doxorubicin. Notably, co-culture with TAFs obtained from high-grade liposarcoma samples resulted in higher proliferation and increased chemoresistance than co-culture with TAFs obtained from intermediate-grade liposarcoma samples. The findings of the present study thus indicate that TAFs from liposarcomas enhance the proliferation and decrease the chemosensitivity of SW872 liposarcoma cells significantly compared with normal fibroblasts from the dermis. TAFs from more malignant liposarcomas promoted tumor cell proliferation and chemoresistance more strikingly than TAFs from less malignant liposarcomas. These data provide evidence for the influence of the tumor microenvironment on liposarcoma and support for further investigations in patients with different liposarcoma subentities, assessing the influence of TAFs on tumor progression.

Sarcomas display a rare and heterogeneous group of tumors. Treatment options are limited. Host defense peptides (HDPs), effector molecules of the innate immune system, might provide a more effective treatment option. The aim of our study was to analyze the oncolytic activity and mode of action of a designer HDP. In vitro, the human liposarcoma cell line SW-872 and primary human fibroblasts as a control were exposed to [D]-K(3)H(3)L(9), a 15-mer D,L-amino acid designer peptide. Cell growth (MTT assay), proliferation (BrdU assay) and genotoxicity (TUNEL assay) were analyzed. The mode of action was examined via fluorescence-activated cell sorter (FACS) analysis and confocal laser scanning microscopy. In vivo, [D]-K(3)H(3)L(9) (n = 7) was administered intratumorally in a SW-872 xenograft mouse model (Foxn1nu/nu). Phosphate buffered saline served as a control (n = 5). After 4 weeks, tumor sections were histologically analyzed with respect to proliferation, cytotoxicity, vessel density and signs of apoptosis and necrosis, respectively. In vitro, [D]-K(3)H(3)L(9) highly significantly (p < 0.01) inhibited cell metabolism and proliferation. TUNEL assay revealed corresponding genotoxicity. FACS analysis suggested induction of necrosis as a cause of cell death. The mean tumor volume of the control group exponentially increased sevenfold, whereas the mean tumor growth was negligible in the treatment group. Macroscopically, [D]-K(3)H(3)L(9) induced full tumor remission in 43% of treated animals and partial remission in 43%. Vessel density was significantly reduced by 52%. Morphological analyses supported the hypothesis of cancer cell killing by necrosis. In summary, [D]-K(3)H(3)L(9) exerts very promising oncolytic activity on liposarcoma cells. Our study demonstrates the potential of HDPs as a novel therapeutic option in future soft tissue sarcoma therapy.

Wound infections with multi-drug resistant bacteria increase morbidity and mortality and have considerable socioeconomic impact. They can lead to impaired wound healing, resulting in rising treatment costs. The aim of this study was to investigate an ex vivo human wound infection model. Human full-thickness skin from the operating room (OR) was placed into the Bo-Drum and cultivated for 7 days in an air-liquid interphase. On day 8, the skin was inoculated with either (1) Pseudomonas aeruginosa, (2) Staphylococcus aureus (10(5) CFU, n = 3) or (3) carrier control. 1, 3 and 7 days after inoculation colony forming units in the tissue/media were determined and cytokine expression was quantified. A reliable and reproducible wound infection could be established for 7 days. At this time point, 1.8 x 10(8) CFU/g tissue of P. aeruginosa and 2 x 10(7) CFU/g tissue of S. aureus were detected. Immunohistochemical analysis demonstrated bacterial infection and epidermolysis in infected skin. RT-PCR analysis exhibited a significant induction of proinflammatory cytokines after infection. The BO-drum is a robust, easy-to-use, sterilizable and reusable ex vivo full-skin culture system. For investigation of wound infection, treatment and healing, the BO-drum presents a convenient model and may help to standardize wound research.

Malignant fibrous histiocytoma (MFH) or undifferentiated pleomorphic sarcoma (UPS) is the most common soft-tissue sarcoma of late adult life. Further advances in genetic characterization are warranted. The aim of this study was to search for numerical and structural chromosomal anomalies in UPS.We investigated five sarcoma-specific chromosomal translocations, five oncogene amplifications as well as the numerical karyotype of 19 UPS samples and one UPS/MFH cell line (U2197) using FISH probes on interphase nuclei.Our results demonstrate that chromosomal translocations involving CHOP, SYT, EWS, FUS and FKHR genes are absent. Furthermore, amplification of ERBB2 (10.5%) and MDM2 (10.5%) was observed whereas the EGFR, C-MYC and N-MYC genes were not amplified. Interestingly, predominant aneuploidies were found in eight chromosomes.The data demonstrate rarity of sarcoma-specific chromosomal breaks and oncogene amplifications in UPS, yet polysomic chromosomes appear more characteristically in this condition.