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Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disease resulting from inactivating mutations in the gene encoding the protein neurofibromin. NF1 manifests as a heritable susceptibility to tumours of neural tissue mainly located in the skin (neurofibromas) and pigmented skin lesions. Besides these more common clinical manifestations, many NF1 patients (50%) have abnormalities of the skeleton. Long bones are often affected (usually the tibia) and the clinical signs range from bowing to spontaneous fractures and non-unions. Here we present the analysis of bone fracture healing in the Nf1(Prx1)-knock-out mouse, a model of NF1 long bone dysplasia. In line with previously reported cortical bone injury results, fracture healing was impaired in Nf1(Prx1) mice. We showed that the defective fracture healing in Nf1(Prx1) mice is characterized by diminished cartilaginous callus formation and a thickening of the periosteal bone. These changes are paralleled by fibrous tissue accumulation within the fracture site. We identify a population of fibrous tissue cells within the Nf1 deficient fracture as alpha-smooth muscle actin positive myofibroblasts. Additionally, histological and in-situ hybridization analysis reveal a direct contact of the fracture site with muscle fascia, suggesting a possible involvement of muscle derived cells in the fracture deterioration.

Adequate blood supply and sufficient mechanical stability are necessary for timely fracture healing. Damage to vessels impairs blood supply; hindering the transport of oxygen which is an essential metabolite for cells involved in repair. The degree of mechanical stability determines the mechanical conditions in the healing tissues. The mechanical conditions can influence tissue differentiation and may also inhibit revascularization. Knowledge of the actual conditions in a healing fracture in vivo is extremely limited. This study aimed to quantify the pressure, oxygen tension and temperature in the external callus during the early phase of bone healing. Six Merino-mix sheep underwent a tibial osteotomy. The tibia was stabilized with a standard mono-lateral external fixator. A multi-parameter catheter was placed adjacent to the osteotomy gap on the medial aspect of the tibia. Measurements of oxygen tension and temperature were performed for ten days post-op. Measurements of pressure were performed during gait on days three and seven. The ground reaction force and the interfragmentary movements were measured simultaneously. The maximum pressure during gait increased (p=0.028) from three (41.3 [29.2-44.1] mm Hg) to seven days (71.8 [61.8-84.8] mm Hg). During the same interval, there was no change (p=0.92) in the peak ground reaction force or in the interfragmentary movement (compression: p=0.59 and axial rotation: p=0.11). Oxygen tension in the haematoma (74.1 mm Hg [68.6-78.5]) was initially high post-op and decreased steadily over the first five days. The temperature increased over the first four days before reaching a plateau at approximately 38.5 degrees C on day four. This study is the first to report pressure, oxygen tension and temperature in the early callus tissues. The magnitude of pressure increased even though weight bearing and IFM remained unchanged. Oxygen tensions were initially high in the haematoma and fell gradually with a low oxygen environment first established after four to five days. This study illustrates that in bone healing the local environment for cells may not be considered constant with regard to oxygen tension, pressure and temperature.

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.

After bone fracture, various cellular activities lead to the formation of different tissue types, which form the basis for the process of secondary bone healing. Although these tissues have been quantified by histology, their material properties are not well understood. Thus, the aim of this study is to correlate the spatial and temporal variations in the mineral content and the nanoindentation modulus of the callus formed via intramembranous ossification over the course of bone healing. Midshaft tibial samples from a sheep osteotomy model at time points of 2, 3, 6 and 9 weeks were employed. PMMA embedded blocks were used for quantitative back scattered electron imaging and nanoindentation of the newly formed periosteal callus near the cortex. The resulting indentation modulus maps show the heterogeneity in the modulus in the selected regions of the callus. The indentation modulus of the embedded callus is about 6 GPa at the early stage. At later stages of mineralization, the average indentation modulus reaches 14 GPa. There is a slight decrease in average indentation modulus in regions distant to the cortex, probably due to remodelling of the peripheral callus. The spatial and temporal distribution of mineral content in the callus tissue also illustrates the ongoing remodelling process observed from histological analysis. Most interestingly the average indentation modulus, even at 9 weeks, remains as low as 13 GPa, which is roughly 60% of that for cortical sheep bone. The decreased indentation modulus in the callus compared to cortex is due to the lower average mineral content and may be perhaps also due to the properties of the organic matrix which might be different from normal bone.

The AP-1 transcription factor family is a central regulator of skin and bone homeostasis. We have previously shown that specific deletion of JunB/AP-1 in epidermis (JunBmice) results in skin inflammation,myeloproliferative disease, lupus-like disease and osteopenia. While upregulation of serum IL-6 and G-CSF are observed in this model, genetic deletion of these cytokines does not rescue osteopenia in JunB mice. Thus, we carried out a screen for other cytokines that are regulated by the loss of JunB in the epidermis. We have identified IL-17A as a cytokine expressed in JunB epidermis in vivo, and hypothesize that IL-17A leads to osteopenia in JunBmice. To test this,we carried out osteoblast and osteoclast differentiation assays in the presence of recombinant IL-17A or serum from JunB mice. Although there were no changes in osteoclast differentiation under these conditions, osteoblast differentiation, as visualized by Alizarin Red staining, was inhibited. Osteoblast differentiation markers were also downregulated in cultures in the presence of recombinant IL-17A or serum from JunB mice. To understand the mechanism by which IL-17A leads to inhibition of osteoblast differentiation, we stimulated osteoblasts with IL-17A in vitro, and observed upregulation in phospho-STAT3, phospho-NFkB andphospho-p38 levels. To determine if IL-17Aupregulation in the epidermis is sufficient to induce bone loss, we analyzed bones of mice ectopically expressing IL-17A in the epidermis. Thesemice displayed bone loss and had decreased levels of serumosteocalcin aswell as decreased osteoblast differentiationmarker expression in vivo. Therefore, thesedata suggest thatepidermal IL002D17A is able to inducebone loss through its action on osteoblast differentiation. These findings will likely be relevant for the treatment of patients with inflammatory diseases and skeletal involvement, such as psoriatic arthritis. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.078 OC08 Depletion of cytotoxic T cells at the time of bone injury enhances bone healing outcome K. Schmidt-Bleek⁎, I. Konnecke, A. Ellinghaus, H.-D. Volk, G.N. Duda, H. Schell Julius Wolff Institut, Charite-Universitatsmedizin Berlin, Berlin, Germany Institut for Medical Immunology, Charite-Universitatsmedizin Berlin

Introduction: The fracture hematoma plays an important role in fracture healing. It contains different factors regulating cell proliferation, differentiation and matrix synthesis. Nevertheless, fracture hematoma is sometimes removed in clinical routine, e.g. in order to improve sight. Hypothesis:We tested the hypothesis, that removal of the fracture hematoma several days post osteotomy delays early fracture healing due to removal of biological potential. Material and methods: Twenty Merinomix sheep underwent a tibial osteotomy stabilized with a rigid external fixator. Animals were divided into three groups. In the D4 and D7 groups the fracture hematoma were removed 4 and 7 days post osteotomy respectively. The healing in the C group was left untreated. All animals were sacrificed 2 weeks post osteotomy. Sections through the osteotomy region were analyzed histologically and histomorphometrically. Results: In the C group the periosteally newly formed woven bone reached the gap. Hematoma was barely present. In the D4 and D7 groups hematoma dominated in the periosteal and endosteal areas. Small amounts of woven bone developed at a distance from the gap and from the hematoma. The D4 and D7 groups showed a significantly smaller (p=0.001) fraction of newly formed woven bone and a significantly higher (p=0.001) fraction of hematoma in the periosteal callus in comparison to the C group. Close to the hematoma, no bone or cartilage forming cells were present. Discussion and conclusion: Our study showed in accordance with the literature, that removal of fracture hematoma 4 or 7 days post osteotomy delays early fracture healing. This is generally explained by the removal of essential factors and cells. Interestingly, in our study we found evidence, that the secondary bleeding might as well hinder bone formation and healing, since there were no bone or cartilage forming cells present close to the new hematoma. This mechanism needs to be clarified in further analyses. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared. doi:10.1016/j.bone.2012.02.353 PP165 Bone formation in TiO2 bone scaffolds in extraction sockets of minipigs H. Tiainen, J.C. Wohlfahrt, A. Verket, S.P. Lyngstadaas, H.J. Haugen Department of Biomaterials, University of Oslo, Oslo, Norway Abstract: Objective: The aim of the present study was to demonstrate the in vivo performance and osteoconductive capacity of the porous TiO2 scaffolds in a large animal model, and to evaluate new bone formation within the TiO2 scaffolds following their placement into extraction sockets in minipig mandibles. Materials and methods: The porous TiO2 scaffolds were prepared by polymer sponge replication as previously described [1] and implanted into fresh extraction sockets in minipigs (n=15). Non-critical size defects were used in this study in order to ensure sufficient bone regeneration for the evaluation of bone ingrowth to the porous scaffold structure, and sham sites were used as positive control. Micro-CT and histology were used in the evaluation of bone ingrowth, bone quality, and implant integration six weeks after implantation. Results and discussion: The produced scaffolds had a mean pore size of ~400 μm and overall porosity above 83%. The pore Objective: The aim of the present study was to demonstrate the in vivo performance and osteoconductive capacity of the porous TiO2 scaffolds in a large animal model, and to evaluate new bone formation within the TiO2 scaffolds following their placement into extraction sockets in minipig mandibles. Materials and methods: The porous TiO2 scaffolds were prepared by polymer sponge replication as previously described [1] and implanted into fresh extraction sockets in minipigs (n=15). Non-critical size defects were used in this study in order to ensure sufficient bone regeneration for the evaluation of bone ingrowth to the porous scaffold structure, and sham sites were used as positive control. Micro-CT and histology were used in the evaluation of bone ingrowth, bone quality, and implant integration six weeks after implantation. Results and discussion: The produced scaffolds had a mean pore size of ~400 μm and overall porosity above 83%. The pore network was shown to be highly interconnected as the open porosity constituted over 99.9% of the total porosity and the interconnectivity of the pore space exceeded 90% through interconnects up to 250 μm in diameter. After six weeks of healing, newly formed bone trabeculae were found throughout the scaffold volume in relatively large quantities, indicating that the highly reticulated scaffold structure permits excellent bone tissue penetration to the entire pore volume in vivo. The newly formed bone tissue was found to occupy 74±11 % of the available pore space within the scaffold structure, and together the scaffold material and regenerated bone filled 84±10 % of total defect volume (Fig. 1). Furthermore, the regenerated bone tissue found in the interior regions of the TiO2 scaffolds showed evidence for vascularisation suggesting that the open pore network provides favourable condition for viable bone formation within the scaffold structure. In addition, mineralised bone tissue was found in direct contact with 50±22 % of the TiO2 struts, which together with the extensive bone ingrowth gives good mechanical stability to the implant site. Conclusion: The presence of a large quantity of viable mineralised bone tissue and the evidence of vascularisation in the scaffold interior indicate that the highly interconnected pore structure of the TiO2 scaffolds has excellent osteoconductive capacity and provides a favourable environment for bone ingrowth. Fig. 1. This article is part of a Special Issue entitled ECTS 2012. Disclosure of interest: None declared.