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Toben D.,Charite - Medical University of Berlin | Toben D.,Berlin Brandenburg Center for Regenerative Therapies | Schroeder I.,Charite - Medical University of Berlin | Schroeder I.,Berlin Brandenburg Center for Regenerative Therapies | And 18 more authors.
Journal of Bone and Mineral Research

Fracture healing is a unique biologic process starting with an initial inflammatory response. As in other regenerative processes, bone and the immune system interact closely during fracture healing. This project was aimed at further elucidating how the host immune system participates in fracture healing. A standard closed femoral fracture was created in wild-type (WT) and recombination activating gene 1 knockout (RAG1-/-) mice lacking the adaptive immune system. Healing was investigated using micro-computed tomography (ÂμCT), biomechanical testing, and histologic and mRNA expression analyses. Biomechanical testing demonstrated a significantly higher torsional moment on days 14 and 21 in the RAG1-/- mice compared to the WT group. ÂμCT evaluation of RAG1-/- specimens showed earlier mineralization and remodeling. Histologically, endochondral ossification and remodeling were accelerated in the RAG1-/- compared with the WT mice. Histomorphometric analysis on day 7 showed a significantly higher fraction of bone and a significantly lower fraction of cartilage in the callus of the RAG1-/- mice than in the WT mice. Endochondral ossification was accelerated in the RAG1-/- mice. Lymphocytes were present during the physiologic repair process, with high numbers in the hematoma on day 3 and during formation of the hard callus on day 14 in the WT mice. Expression of inflammatory cytokines was reduced in the RAG1-/- mice. In contrast, expression of anti-inflammatory interleukin 10 (IL-10) was strongly upregulated in RAG1-/- mice, indicating protective effects. This study revealed an unexpected phenotype of enhanced fracture healing in RAG1-/- mice, suggesting detrimental functions of lymphocytes on fracture healing. The shift from proinflammatory to anti-inflammatory cytokines suggests that immunomodulatory intervention strategies that maximise the regenerative and minimize the destructive effects of inflammation may lead to enhanced fracture repair. © 2011 American Society for Bone and Mineral Research. Source

Haupl T.,Charite - Medical University of Berlin | Stuhlmuller B.,Charite - Medical University of Berlin | Grutzkau A.,German Arthritis Research Center | Radbruch A.,German Arthritis Research Center | Burmester G.-R.,Charite - Medical University of Berlin
Annals of the Rheumatic Diseases

Transcription profiling has become a standard technology in research. It is mainly applied in the search for biomarkers to improve diagnostic and prognostic classification, to quantify disease activity and to predict or indicate response to therapy. This review will focus on rheumatoid arthritis and discuss considerations for sample selection, prerequisites for functional interpretation of data and the current status of information deduced in the field of biomarkers for the various clinical questions. In the next few years, prediction of response to treatment is the most important aim of biomarker research. With the growing number of new biological agents, there is increasing pressure to identify molecular parameters that will not only guide the therapeutic decision but also help to define the most important targets for which new biological agents should be tested in clinical studies. Source

Kolar P.,Charite University Hospital | Kolar P.,German Arthritis Research Center | Gaber T.,Charite University Hospital | Gaber T.,German Arthritis Research Center | And 8 more authors.
Clinical Orthopaedics and Related Research

Background: An effective immune system, especially during the inflammatory phase, putatively influences the quality and likelihood of bone healing. If and how this is reflected within the initial fracture hematoma is unclear. Questions/purposes: We therefore asked the following questions: (1) Does the local expression in fracture hematoma of genes involved in adaptation to hypoxia, migration, angiogenesis, and osteogenesis vary as compared to the peripheral blood? (2) Do these changes occur time dependently? (3) Is the gene expression during fracture hematoma formation altered by irradiation? Methods: Cells from fracture hematoma of 20 patients and hematomas formed in 40 patients after THA (20 without and 20 with preoperative radiation) were isolated and RNA was extracted to analyze the influence of oxygen deprivation during fracture healing on mRNA expression of genes (HIF1A, LDHA, and PGK1) involved in immunoregulation (IL6, IL8, CXCR4), angiogenesis (VEGF, IL8), and osteogenesis (SPP1, RUNX2) by quantitative PCR. Results: We observed locally increased LDHA gene expression in fracture hematoma cells (6-72 h post fracture) reflecting the adaptation to hypoxia. IL6, IL8, and VEGF upregulation indicated hypoxia-mediated inflammation and angiogenesis; increased CXCR4 expression reflected immigration of immune cells. Osteogenic differentiation was reflected in the increased expression of the SPP1 and RUNX2 genes. The increased expression of the LDHA, VEGF, IL8, SPP1 and RUNX2 genes occurred time dependently. Irradiation suppressed HIF1A, IL6, IL8, CXCR4, and RUNX2 gene expression. Conclusions: Our data suggest cells in the fracture hematoma (1) adapt to hypoxia and (2) promote inflammation in fracture healing at the mRNA level, indicating early involvement of the immune system. Clinical Relevance: The initial fracture hematoma is important for the onset of angiogenesis, chemotaxis, and osteogenesis. © 2011 The Association of Bone and Joint Surgeons®. Source

Hoff P.,Charite University Hospital | Hoff P.,German Arthritis Research Center | Buttgereit F.,Charite University Hospital | Buttgereit F.,German Arthritis Research Center | And 15 more authors.
International Orthopaedics

Purpose: Two of the most common joint diseases are rheumatoid arthritis (RA) and osteoarthritis (OA). Cartilage degradation and erosions are important pathogenetic mechanisms in both joint diseases and have presently gained increasing interest. The aim of the present study was to investigate the effects of the synovial fluid environment of OA patients in comparison with synovial fluids of RA patients on human chondrocytes in vitro. Methods: Primary human chondrocytes were incubated in synovial fluids gained from patients with OA or RA. The detection of vital cell numbers was determined by histology and by using the Casy Cell Counter System. Cytokine and chemokine secretion was determined by a multiplex suspension array. Results: Microscopic analysis showed altered cell morphology and cell shrinkage following incubation with synovial fluid of RA patients. Detection of vital cells showed a highly significant decrease of vital chondrocyte when treated with RA synovial fluids in comparison with OA synovial fluids. An active secretion of cytokines such as vascular endothelial growth factor (VEGF) of chondrocytes treated with OA synovial fluids was observed. Conclusions: Significantly increased levels of various cytokines in synovial fluids of RA, and surprisingly of OA, patients were shown. Activation of pro-inflammatory cytokines of human chondrocytes by synovial fluids of OA patient supports a pro-inflammatory process in the pathogenesis of OA. © 2012 Springer-Verlag Berlin Heidelberg. Source

Konnecke I.,Charite - Medical University of Berlin | Serra A.,German Arthritis Research Center | El Khassawna T.,Justus Liebig University | Schlundt C.,Charite - Medical University of Berlin | And 8 more authors.

Fracture healing is a regenerative process in which bone is restored without scar tissue formation. The healing cascade initiates with a cycle of inflammation, cell migration, proliferation and differentiation. Immune cells invade the fracture site immediately upon bone damage and contribute to the initial phase of the healing process by recruiting accessory cells to the injury site. However, little is known about the role of the immune system in the later stages of fracture repair, in particular, whether lymphocytes participate in soft and hard callus formation. In order to answer this question, we analyzed femoral fracture healing in mice by confocal microscopy. Surprisingly, after the initial inflammatory phase, when soft callus developed, T and B cells withdrew from the fracture site and were detectable predominantly at the femoral neck and knee. Thereafter lymphocytes massively infiltrated the callus region (around day 14 after injury), during callus mineralization. Interestingly, lymphocytes were not found within cartilaginous areas of the callus but only nearby the newly forming bone. During healing B cell numbers seemed to exceed those of T cells and B cells progressively underwent effector maturation. Both, osteoblasts and osteoclasts were found to have direct cell-cell contact with lymphocytes, strongly suggesting a regulatory role of the immune cells specifically in the later stages of fracture healing. © 2014 Elsevier Inc. Source

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