Comparative proteomic analysis of osteogenic differentiated human adipose tissue and bone marrow-derived stromal cells.

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.

TNF-α modulation via Etanercept restores bone regeneration of atrophic non-unions.

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-ß, 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.

Inflammatory processes and elevated osteoclast activity chaperon atrophic non-union establishment in a murine model.

BACKGROUND: Delayed 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. METHODS: In 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. RESULTS: Histological 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. CONCLUSION: Taken 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.

Bone allografts combined with adipose-derived stem cells in an optimized cell/volume ratio showed enhanced osteogenesis and angiogenesis in a murine femur defect model.

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. KEY MESSAGES: Bone tissue engineering. Mesenchymal stem cells derived from human adipose tissue (hASCs). Optimal cell/volume ratio of cell-seeded scaffolds. Increased osteogenesis and angiogenesis in vivo.

Adipose-Derived Stromal Cells Are Capable of Restoring Bone Regeneration After Post-Traumatic Osteomyelitis and Modulate B-Cell Response.

Bone infections are a frequent cause for large bony defects with a reduced healing capacity. In previous findings, we could already show diminished healing capacity after bone infections, despite the absence of the causing agent, Staphylococcus aureus. Moreover, these bony defects showed reduced osteoblastogenesis and increased osteoclastogenesis, meaning elevated bone resorption ongoing with an elevated B-cell activity. To overcome the negative effects of this postinfectious inflammatory state, we tried to use the regenerative capacity of mesenchymal stem cells derived from adipose tissue (adipose-derived stem cells [ASCs]) to improve bone regeneration and moreover were curious about immunomodulation of applicated stem cells in this setting. Therefore, we used our established murine animal model and applicated ASCs locally after sufficient debridement of infected bones. Bone regeneration and resorption as well as immunological markers were investigated via histology, immunohistochemistry, Western blot, and fluorescence-activated cell scanning (FACS) analysis and µ-computed tomography (CT) analysis. Interestingly, ASCs were able to restore bone healing via elevation of osteoblastogenesis and downregulation of osteoclasts. Surprisingly, stem cells showed an impact on the innate immune system, downregulating B-cell population. In summary, these data provide a fascinating new and innovative approach, supporting bone healing after bacterial infections and moreover gain insights into the complex ceremony of stem cell interaction in terms of bone infection and regeneration. Stem Cells Translational Medicine 2019;8:1084-1091.

Activin Receptor 2 Antagonization Impairs Adipogenic and Enhances Osteogenic Differentiation in Mouse Adipose-Derived Stem Cells and Mouse Bone Marrow-Derived Stem Cells In Vitro and In Vivo.

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.

Interaction with the GDF8/11 pathway reveals treatment options for adenocarcinoma of the breast.

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.