Richard Stange

Estrogen-dependent and C-C chemokine receptor-2-dependent pathways determine osteoclast behavior in osteoporosis

Understanding the mechanisms of osteoclastogenesis is crucial for developing new drugs to treat diseases associated with bone loss, such as osteoporosis. Here we report that the C-C chemokine receptor-2 (CCR2) is crucially involved in balancing bone mass. CCR2-knockout mice have high bone mass owing to a decrease in number, size and function of osteoclasts. In normal mice, activation of CCR2 in osteoclast progenitor cells results in both nuclear factor-κB (NF-κB) and extracellular signal–related kinase 1 and 2 (ERK1/2) signaling but not that of p38 mitogen-activated protein kinase or c-Jun N-terminal kinase. The induction of NF-κB and ERK1/2 signaling in turn leads to increased surface expression of receptor activator of NF-κB (RANK, encoded by Tnfrsf11a), making the progenitor cells more susceptible to RANK ligand-induced osteoclastogenesis. In ovariectomized mice, a model of postmenopausal osteoporosis, CCR2 is upregulated on wild-type preosteoclasts, thus increasing the surface expression of RANK on these cells and their osteoclastogenic potential, whereas CCR2-knockout mice are resistant to ovariectomy-induced bone loss. These data reveal a previously undescribed pathway by which RANK, osteoclasts and bone homeostasis are regulated in health and disease.

Early structural changes in cartilage and bone are required for the attachment and invasion of inflamed synovial tissue during destructive inflammatory arthritis

Objective To elucidate the mechanisms involved in cartilage damage in an experimental model of rheumatoid arthritis (RA) by specifically addressing the time course of extracellular matrix degradation and the contribution of cell–matrix interactions for initiation and perpetuation of this process. Methods The human tumour necrosis factor (TNF) transgenic (hTNFtg) mouse model of RA was used to analyse the time course of pannus attachment to the cartilage and cartilage destruction, respectively, and crossed hTNFtg mice with interleukin (IL)-1−/− animals were used to investigate the role of IL-1 on these TNF-induced mechanisms in vivo. In addition, an in vitro attachment assay using synovial fibroblasts (SFs) from hTNFtg mice and freshly isolated articular cartilage was used to determine the role of proteoglycan loss in attachment of SFs and the role of the transmembrane heparan sulfate proteoglycan syndecan-4. Results In vivo analyses of hTNFtg mice showed that proteoglycan loss induced by IL-1 precedes and constitutes an important prerequisite for these processes as, in hTNFtg mice, IL-1 deficiency protected from the loss of cartilage proteoglycans and almost completely prevented the attachment and subsequent invasion of inflamed synovial tissue into cartilage. In vitro studies confirmed that loss of cartilage proteoglycans is required for attachment of SFs and that syndecan-4 is prominently involved in SF attachment and activation. Conclusions The results of this study suggest that the loss of cartilage proteoglycans is an early event in the course of destructive arthritis that facilitates the attachment of the inflamed synovial membrane and also initiates matrix degradation and inflammation through cell–matrix interactions.

Quantification, Localization, and Expression of IGF-I and TGF-β1 During Growth Factor-Stimulated Fracture Healing

Because of the increasing interest on stimulating fracture healing, knowledge about the role and chronology of growth factors during the healing process is important. The purpose of this study was to quantify the protein concentration of IGF-I and TGF-β1 during rat tibial fracture healing 5, 10, and 15 days after fracture using ELISA methods and to analyze the distribution of the proteins and the related mRNA expression in the fracture callus by immunohistochemistry and in situ hybridization. The following three groups were analyzed: Fractured tibiae intramedullary stabilized with K-wires coated with IGF-I and TGF-β1 compared with fractures stabilized with uncoated K-wires and unfractured tibiae. The weight of the callus increased during the healing process in both experimental groups. The protein concentration of IGF-I and TGF-β1 in the fracture callus showed significant changes between the investigated time points and treatment groups compared with the unfractured tibia. IGF-I increased with healing time whereas TGF-β1 revealed a constantly elevated level at the investigated time points. Mesenchymal cells, osteoblasts, osteocytes, proliferating and immature chondrocytes, and osteoclasts expressed both growth factors. No differences in the expression and localization pattern of the growth factors were detectable among the groups. Using the different methods for quantification and visualization of the growth factors, no differences (except the increased IGF-I concentration at day 15 in the growth factor group) were seen between the normal and the growth factor-stimulated fracture healing as an indication for physiological healing after exogenous growth factor treatment.

Biomechanical comparison of two angular stable plate constructions for periprosthetic femur fracture fixation

PurposeFractures of the femur associated with total hip arthroplasty are a significant concern in orthopaedic and trauma surgery. However, little is known about the different biomechanical properties of internal fixation systems in combination with periprosthetic fractures. In this study two new internal fixation systems for periprosthetic fractures are investigated using a cadaver fracture model simulating a Vancouver B1 periprosthetic femur fracture.MethodsNine pairs of fresh-frozen cadaver femurs were scanned by dual X-ray absorptiometry. Cementless total hip prostheses were implanted and a periprosthetic femur fracture was simulated. Fractures were randomly fixed either with the fixed angle locking attachment plate (LAP®, Depuy Synthes®, Solothurn, Switzerland) or the variable angle non-contact bridging plate (NCB®, Zimmer GmbH, Winterthur, Switzerland). Each construct was cyclically loaded to failure in axial compression.ResultsAxial stiffness and cycles to failure were significantly higher in the NCB group. Both systems were able to be fixed well around the femoral stem.ConclusionThe two different internal fixation systems for periprosthetic fractures differed significantly in our setup. The non-contact bridging plate system revealed significantly higher failure load and may be the preferred option where high stability and load capacity is needed right after operation.

Impaired biomechanical properties correlate with neoangiogenesis as well as VEGF and MMP-3 expression during rat patellar tendon healing

Recent studies reveal an important role of vascular endothelial growth factor (VEGF)‐induced angiogenesis in degenerative tendon diseases. The way how VEGF influences mechanical properties of the tendons is not well understood yet. We here hypothesized that tendinopathy results in a hypoxia‐mediated stimulation of VEGF and that the mechanical stability of the tendon is impaired in an angiogenic process by VEGF‐induced matrix metalloproteinases (MMPs). A modified in situ freezing model of patellar tendon was used to create a tendinopathy. 0, 7, 14, and 28 days post‐surgical animals were sacrificed and patellar tendons were dissected for biomechanical and immunohistochemical analysis. Native tendons were used as controls. Immunohistochemical staining revealed a peak in HIF‐1α stabilization immediately after surgery. Both VEGF and MMP‐3 were increased 7 days after surgery. Angiogenesis was also abundant 7 days after surgery. In contrast, biomechanical stability of the tendon was decreased 7 days after surgery. The current results reveal a time‐dependent correlation of HIF‐1/VEGF‐induced and MMP‐3‐supported angiogenesis with decreased biomechanical properties during tendon healing. The therapeutical modulation of neoangiogenesis by influencing the level of VEGF and MMP‐3 might be a promising target for new approaches in degenerative tendon diseases.

Syndecan 4 supports bone fracture repair, but not fetal skeletal development, in mice

OBJECTIVE Syndecan 4, a heparan sulfate proteoglycan, has been associated with osteoarthritis. The present study was undertaken to analyze the functional role of syndecan 4 in endochondral ossification of mouse embryos and in adult fracture repair, which, like osteoarthritis, involves an inflammatory component. METHODS Sdc4 promoter activity was analyzed in Sdc4(-/-) lacZ-knockin mice, using β-galactosidase staining. Endochondral ossification in embryos from embryonic day 16.5 was assessed by histologic and immunohistologic staining. Bone fracture repair was analyzed in femora of adult mice on days 7 and 14 postfracture. To evaluate Sdc2 and Sdc4 gene expression with and without tumor necrosis factor α (TNFα) and Wnt-3a stimulation, quantitative real-time polymerase chain reaction was performed. RESULTS In Sdc4(-/-) lacZ-knockin animals, syndecan 4 promoter activity was detectable at all stages of chondrocyte differentiation, and Sdc4 deficiency inhibited chondrocyte proliferation. Aggrecan turnover in the uncalcified cartilage of the epiphysis was decreased transiently in vivo, but this did not lead to a growth phenotype at birth. In contrast, among adult mice, fracture healing was markedly delayed in Sdc4(-/-) animals and was accompanied by increased callus formation. Blocking of inflammation via anti-TNFα treatment during fracture healing reduced these changes in Sdc4(-/-) mice to levels observed in wild-type controls. We analyzed the differences between the mild embryonic and the severe adult phenotype, and found a compensatory up-regulation of syndecan 2 in the developing cartilage of Sdc4(-/-) mice that was absent in adult tissue. Stimulation of chondrocytes with Wnt-3a in vitro led to increased expression of syndecan 2, while stimulation with TNFα resulted in up-regulation of syndecan 4 but decreased expression of syndecan 2. TNFα stimulation reduced syndecan 2 expression and increased syndecan 4 expression even in the presence of Wnt-3a, suggesting that inflammation has a strong effect on the regulation of syndecan expression. CONCLUSION Our results demonstrate that syndecan 4 is functionally involved in endochondral ossification and that its loss impairs fracture healing, due to inhibition of compensatory mechanisms under inflammatory conditions.

Age-related bone deterioration is diminished by disrupted collagen sensing in integrin α2β1 deficient mice.

Collagen binding integrins are of essential importance in the crosstalk between cells and the extracellular matrix. Integrin α2β1 is a major receptor for collagen I, the most abundant protein in bone. In this study we show for the first time that integrin α2 deficiency is linked to collagen type I expression in bone. Investigating the femurs of wild type and integrin α2β1 deficient mice, we found that loss of integrin α2 results in altered bone properties. Histomorphometric analysis of integrin α2 long bones displayed more trabecular network compared to wild type bones. During age related bone loss the integrin α2β1 deficient bones retain trabecular structure even at old age. These findings were supported by functional, biomechanical testing, wherein the bones of integrin α2β1 deficient mice do not undergo age-related alteration of biomechanical properties. These results might be explained by the increased presence of collagen in integrin α2β1 deficient bone. Collagen type I could be detected in higher quantities in the integrin α2β1 deficient bones, forming abnormal, amorphous fibrils. This was linked to higher expression levels of collagen type I and other bone formation related proteins as alkaline phosphatase of integrin α2β1 deficient osteoblasts. Osteoclasts of integrin α2β1 deficient mice did not show any differences. Consequently these results indicate that the absence of integrin α2β1 alleviates the effects of age related bone degradation through over-expression of collagen type I and demonstrate a molecular mechanism how collagen binding integrins might directly impact bone aging.

Local Delivery of Growth Factors Using Coated Suture Material

The optimization of healing processes in a wide range of tissues represents a central point for surgical research. One approach is to stimulate healing processes with growth factors. These substances have a short half-life and therefore it seems useful to administer these substances locally rather than systemically. One possible method of local delivery is to incorporate growth factors into a bioabsorbable poly (D, L-lactide) suspension (PDLLA) and coat suture material. The aim of the present study was to establish a procedure for the local delivery of growth factors using coated suture material. Sutures coated with growth factors were tested in an animal model. Anastomoses of the colon were created in a rat model using monofilament sutures. These were either untreated or coated with PDLLA coating alone or coated with PDLLA incorporating insulin—like growth factor-I (IGF-I). The anastomoses were subjected to biomechanical, histological, and immunohistochemical examination. After 3 days the treated groups showed a significantly greater capacity to withstand biomechanical stress than the control groups. This finding was supported by the results of the histomorphometric. The results of the study indicate that it is possible to deliver bioactive growth factors locally using PDLLA coated suture material. Healing processes can thus be stimulated locally without subjecting the whole organism to potentially damaging high systemic doses.

Bioactive-Coated Implants in Trauma Surgery

Complications still occur in musculoskeletal surgery despite improvements in operating techniques and optimization of implants. Problems include delayed fracture healing, non-unions and extensive osseous infections. Growth factors for local application are in clinical use, but have not become widely accepted. Reasons may be that these proteins are expensive and of limited availability and considerable quantities have to be implanted locally. Coated implants incorporating active ingredients could release drugs locally and thereby generate a high concentration directly in the area of interest without systemic side effects. Compounds that could be used in this way include growth factors for the improvement of fracture healing and antibiotics for prophylaxis of implant-related infections. The biodegradable poly(D,L-lactide) coating of implants can facilitate the local controlled release of incorporated growth factors directly into the fracture and thus serves both as a fracture stabilization device and as a carrier for active components. This review presents different models (fracture healing; intervertebral fusion; infection model) demonstrating the efficiency of the coating technology. These findings seem to justify the transfer of this technology into clinical settings. In a preliminary study, gentamicin-coated intramedullary tibial nails were implanted in patients exhibiting fractures with severe soft tissue damage. The preliminary findings do not allow conclusions to be drawn in respect of therapy of fractures with severe soft tissue damage or revision surgery. However, the coating seems to be suitable as a “key technology” for the incorporation of active ingredients and might be helpful in revision arthroplasty.

Influence of Synovial Fluid on Human Osteoblasts: An In Vitro Study

Osseous graft healing at the tendon bone interface after anterior cruciate ligament (ACL) reconstruction is unsatisfactory in 10—25%, depending on the evaluation criteria or the kind of graft used for reconstruction. Mechanical as well as biological aspects are currently discussed. Since osteoblasts play an important role in the osseous integration of an ACL graft, we hypothesize that synovial fluid (SF), when entering the bone tunnel, has an inhibitory effect on osteoblasts. In order to verify this hypothesis, human osteoblasts (p3) were incubated in the presence of SF or partially purified SF. Proliferation was assayed using MTT or BrdU assay. Gene expression of osteoblast markers (alkaline phosphatase, collagen I, and osteocalcin) were determined by TaqMan analysis. In the control group, SF was exchanged by fetal calf serum (FCS). The results showed osteoblast proliferation in the presence of SF as well as in partially purified heat-pretreated synovial fluid. Native SF induced alkaline phosphatase and collagen I gene expression. No induction of the osteocalcin gene was observed in the experiment. These results were comparable to that obtained with FCS. These findings suggest that SF stimulated proliferation of osteoblasts in vitro. This effect is mediated, in part, by heat-stable components of SF. In addition, the expression of osteoblast marker genes alkaline phosphatase and collagen I, but not osteocalcin, was induced by SF. Therefore, problems associated with cruciate ligament reconstruction might be due to the inhibition of osteoblast differentiation. If so, this is not a specific attribute of SF, but also applies to serum.