Search

Bone infections are one of the main reasons for impaired bone regeneration and non-union formation. In previous experimental animal studies we could already demonstrate that bone defects due to prior infections showed a markedly reduced healing capacity, which could effectively be enhanced via application of Wnt3a and Adipose-derived stromal cells (ASCs). For a more in-depth analysis, we investigated proliferation and mineralization of cultured osteoblasts infected with staph aureus and sought to investigate effects of Wnt3a and ASCs on infected osteoblasts. Primary murine osteoblasts were isolated from calvariae and infected with staph aureus. Infected osteoblasts received treatment via application of recombinant Wnt3a, ASC conditioned medium and were furthermore cocultured with ASCs. Osteoblasts were evaluated by Alamar blue assay for metabolic activity, TUNEL-assay for apoptosis, ALP and Alizarin Red staining for mineralization. In addition, immunoflourescent staining (IF) and qRT-PCR analyses were performed. Infected osteoblasts showed a markedly reduced ability for mineralization and increased apoptosis, which could be restored to physiological levels by Wnt3a and ASC treatment. Interestingly, metabolic activity of osteoblasts seemed to be unaffected by staph aureus infection. Additional analyses of Wnt-pathway activity revealed effective enhancement of canonical Wnt-pathway activity in Wnt3a-treated osteoblasts. In summary, we gained further osteoblast-related insights into pathomechanisms of reduced bone healing capacity upon infections.

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

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.

Treatment of atrophic non-unions, especially in long bones is a challenging problem in orthopedic surgery due to the high revision and failure rate after surgical intervention. Subsequently, there is a certain need for a supportive treatment option besides surgical treatment. In our previous study we gained first insights into the dynamic processes of atrophic non-union formation and observed a prolonged inflammatory reaction with upregulated TNF-α levels and bone resorption. In this study we aimed to improve bone regeneration of atrophic non-unions via TNF-α modulation in a previously established murine femoral segmental defect model. Animals that developed atrophic non-unions of the femur after 5 and 10 weeks were treated systemically for 10 and 5 weeks with Etanercept, a soluble TNF-α antibody. μCT scans and histology revealed bony bridging of the fracture gap in the treatment group, while bone formation in control animals without treatment was not evident. Moreover, osteoclasts were markedly decreased via modulation of the RANKL/OPG axis due to Etanercept treatment. Additionally, immunomodulatory effects via Etanercept could be observed as further inflammatory agents, such as TGF-β, IL6, MMP9 and 13 were decreased in both treatment groups. This study is the first showing beneficial effects of Etanercept treatment on bone regeneration of atrophic non-union formation. Moreover, the results of this study provide a new and promising therapeutic option which might reduce the failure rate of revision surgeries of atrophic non-unions.

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.

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.

Tumors, traumata, burn injuries or surgeries can lead to critical-sized bony defects which need to be reconstructed. Mesenchymal stem cells (MSCs) have the ability to differentiate into multiple cell lineages and thus present a promising alternative for use in tissue engineering and reconstruction. However, there is an ongoing debate whether all MSCs are equivalent in their differentiation and proliferation ability. The goal of this study was to assess osteogenic and adipogenic characteristic changes of adipose-derived stem cells (ASCs) and bone marrow-derived stem cells (BMSCs) upon Myostatin inhibition with Follistatin in vitro and in vivo. We harvested ASCs from mice inguinal fat pads and BMSCs from tibiae of mice. By means of histology, real-time cell analysis, immunohistochemistry, and PCR osteogenic and adipogenic proliferation and differentiation in the presence or absence of Follistatin were analyzed. In vivo, osteogenic capacity was investigated in a tibial defect model of wild-type (WT) mice treated with mASCs and mBMSCs of Myo-/- and WT origin. In vitro, we were able to show that inhibition of Myostatin leads to markedly reduced proliferative capacity in mBMSCs and mASCs in adipogenic differentiation and reduced proliferation in osteogenic differentiation in mASCs, whereas proliferation in mBMSCs in osteogenic differentiation was increased. Adipogenic differentiation was inhibited in mASCs and mBMSCs upon Follistatin treatment, whereas osteogenic differentiation was increased in both cell lineages. In vivo, we could demonstrate increased osteoid formation in WT mice treated with mASCs and mBMSCs of Myo-/- origin and enhanced osteogenic differentiation and proliferation of mASCs of Myo-/- origin. We could demonstrate that the osteogenic potential of mASCs could be raised to a level comparable to mBMSCs upon inhibition of Myostatin. Moreover, Follistatin treatment led to inhibition of adipogenesis in both lineages.

Introduction: Patients experiencing thermal injuries with an extent of over 20% of total body surface area suffer from systemic catabolic disease. The thermal trauma-induced loss of muscle mass causes a higher incidence for comorbidities and subsequently a higher mortality. In this study, we aimed to investigate the role of myostatin in the interplay with follistatin during muscle cachexia. Methods: Patients with burn injuries (>10% total body surface area) between the ages of 18 and 75 were prospectively included within the first 48 h after trauma to determine deviations of parameters connected to muscle catabolism. In the chronic state of burn injury (9–12 months after trauma), we re-evaluated myostatin and follistatin concentrations as well as muscle strength of the non-dominant forearm. Results: We were able to show a time-dependent alteration (9–12 months after burn injury) of myostatin with an initial decrease ( p Conclusion: With regard to the muscle metabolism after thermal trauma, our data suggest an acute anabolic response, presumably to spare muscle mass, which is converted to catabolic conditions accompanied by muscle strength reduction in the chronic phase. Myostatin plays a crucial role in this orchestration and initial myostatin concentration may predict the long-term muscle strength.

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.