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Despite the high incidence of metaphyseal bone fractures in patients, the mechanisms underlying the healing processes are poorly understood due to the lack of suitable experimental animal models. Hence, the present study was conducted to establish and characterise a clinically relevant large-animal model for metaphyseal bone healing. Six female adult Merino sheep underwent full wedge-shaped osteotomy at the distal left femur metaphysis. The osteotomy was stabilised internally with a customised anatomical locking titanium plate that allowed immediate post-operative full-weight bearing. Bone healing was evaluated at 12 weeks post-fracture relative to the untouched right femur. Histological and quantitative micro-computed tomography results revealed an increased mineralised bone mass with a rich bone microarchitecture. New trabeculae healed by direct intramembranous ossification, without callus and cartilaginous tissue formation. Stiffness at the cortical and trabecular regions was comparable in both groups. Functional morphological analysis of the osteocyte lacunae revealed regularly arranged spherically shaped lacunae along with the canalicular network. Bone surface biochemical analysis using time-of-flight secondary-ion mass spectrometry showed high and homogeneously distributed levels of calcium and collagenous components. Ultrastructure imaging of the new trabeculae revealed a characteristic parallel arrangement of the collagen fibrils, evenly mineralised by the dense mineral substance. The specialised bone cells were also characterised by their unique structural features. Bone remodelling in the fractured femur was evident in the higher expression levels of prominent bone formation and resorption genes. In conclusion, the novel metaphyseal fracture model is beneficial for studying healing and treatment options for the enhancement of metaphyseal bone defects.

Allogeneic bone derived from living donors being necessary to match demand for bone transplantation and thermodisinfection of femoral heads is an established sterilization method. During the thermodisinfection the peripheral bone is exposed to maximum 86 °C for 94 min providing 82.5 °C within the center of the femoral head for at least 15 min. This study examined the compression force of the central and representative peripheral regions of native and thermodisinfected human femoral heads to observe wether different duration and intensity of heat exposure might alter mechanic behaviour. Slices from the equatorial region of human femoral heads were taken from each 14 native and thermodisinfected human femoral heads. The central area revealed a significantly higher compression force for native (p ≤ 0.001) and for thermodisinfected bone (p = 0.002 and p = 0.005) compared with peripheral regions since no relevant differences were found between the peripheral and intermediate areas themselves. A small reduction of compression force for thermodisinfected bone was shown since this did not appear significant due to the small number of specimens. The heat exposure did not alter the pre-existing anatomical changes of the microarchitecture of the native femoral heads from the center towards the peripheral regions. The heterogeneity of microstructure of the femoral head might be of interest concerning clinical applications of bone grafts since the difference between native and thermodisinfected bone appears moderate as shown previously. The different quantity of heat exposure did not reveal any significant influence on compression force which might enable thermodisinfection of preformed bone pieces for surgical indications.

The development of new and better implant materials adapted to osteoporotic bone is still urgently required. Therefore, osteoporotic muscarinic acetylcholine receptor M3 (M3 mAChR) knockout (KO) and corresponding wild type (WT) mice underwent osteotomy in the distal femoral metaphysis. Fracture gaps were filled with a pasty &alpha, tricalcium phosphate (&alpha, TCP)-based hydroxyapatite (HA)-forming bone cement containing mesoporous bioactive CaP-SiO2 glass particles (cement/MBG composite) with or without Brain-Derived Neurotrophic Factor (BDNF) and healing analyzed after 35 days. Histologically, bone formation was significantly increased in WT mice that received the BDNF-functionalized cement/MBG composite compared to control WT mice without BDNF. Cement/MBG composite without BDNF increased bone formation in M3 mAChR KO mice compared to equally treated WT mice. Mass spectrometric imaging showed that the BDNF-functionalized cement/MBG composite implanted in M3 mAChR KO mice was infiltrated by newly formed tissue. Leukocyte numbers were significantly lower in M3 mAChR KO mice treated with BDNF-functionalized cement/MBG composite compared to controls without BDNF. C-reactive protein (CRP) concentrations were significantly lower in M3 mAChR KO mice that received the cement/MBG composite without BDNF when compared to WT mice treated the same. Whereas alkaline phosphatase (ALP) concentrations in callus were significantly increased in M3 mAChR KO mice, ALP activity was significantly higher in WT mice. Due to a stronger effect of BDNF in non osteoporotic mice, higher BDNF concentrations might be needed for osteoporotic fracture healing. Nevertheless, the BDNF-functionalized cement/MBG composite promoted fracture healing in non osteoporotic bone.

Bone substitute materials loaded with mediators that stimulate fracture healing are demanded in the clinical treatment in trauma surgery and orthopedics. Brain-derived neurotrophic factor (BDNF) enhances the proliferation and differentiation of mesenchymal stem cells into osteoblast. To load the implants with BDNF, a drug delivery system that allows the release of BDNF under spatiotemporal control would improve functionality. Polyelectrolyte complex nanoparticles (PECNP) have been reported as a suitable drug delivery system. The suitability of PECNP in contact with osteocytes as the main cell type of bone is not known so far. Thus, we aimed to verify that BDNF and PECNP loaded with BDNF (PECNP+BDNF) as well as pure PECNP have no negative effects on osteocytes in vitro. Therefore, the murine osteocyte cell line MLO-Y4 was treated with BDNF and PECNP+BDNF. The effects on proliferation were analyzed by the BrdU test (n = 5). The results demonstrated a significant increase in proliferation 24 h after BDNF application, whereas PECNP+BDNF did not lead to significant changes. Thus, we conclude that BDNF is an appropriate mediator to stimulate osteocytes. Since the addition of PECNP did not affect the viability of osteocytes, we conclude that PECNP are a suitable drug delivery system for bone implants.

Alterations in bone strength and structure were found in knockout (KO) mouse strains with deletion of several acetylcholine receptors. Interestingly, the expression of the nicotinic acetylcholine receptors (nAChR) subunit α10 was down-regulated in osteogenic differentiated mesenchymal stem cells of patients with osteoporosis whereas the expression of subunit α9 was not altered. Since nAChR subunits α9 and α10 are often combined in a functional receptor, we analyzed here the bone of adult female KO mice with single deletion of either nAChR alpha9 (α9KO) or alpha10 (α10KO). Biomechanical testing showed a significant decrease of bending stiffness and maximal breaking force in α9KO compared to their corresponding wild type mice. Furthermore, an increase in trabecular pattern factor (Tb.Pf) and structure model index (SMI) was detected by μCT in α9KO indicating reduced bone mass. On the mRNA level a decrease of Collagen 1α1 and Connexin-43 was measured by real-time RT-PCR in α9KO while no alteration of osteoclast markers was detected in either mouse strain. Using electron microcopy we observed an increase in the number of osteocytes that showed signs of degeneration and cell death in the α9KO compared to their wild type mice, while α10KO showed no differences. In conclusion, we demonstrate alterations in bone strength, structure and bio-marker expression in α9KO mice which imply the induction of osteocyte degeneration. Thus, our data suggest that nAChR containing the α9 subunit might be involved in the homeostasis of osteocytes and therefore in bone mass regulation.

Staphylococcus aureus can cause wide range of infections from simple soft skin infections to severe endocarditis, bacteremia, osteomyelitis and implant associated bone infections (IABI). The focus of the present investigation was to study virulence properties of S. aureus isolates from acute and chronic IABI by means of their in vivo lethality, in vitro osteoblasts invasion, biofilm formation and subsequently whole genome comparison between high and low virulent strains. Application of insect infection model Galleria mellonella revealed high, intermediate and low virulence phenotypes of these clinical isolates, which showed good correlation with osteoblast invasion and biofilm formation assays. Comparative genomics of selected high (EDCC 5458) and low (EDCC 5464) virulent strains enabled the identification of molecular factors responsible for the development of acute and chronic IABI. Accordingly, the low virulent strain EDCC 5464 harbored point mutations resulting in frame shift mutations in agrC (histidine kinase in agr system), graS (histidine kinase in graSR, a two component system) and efeB (peroxidase in efeOBU operon, an iron acquisition system) genes. Additionally, we found a mobile element (present 11 copies in EDCC 5464) inserted at the end of β-hemolysin (hlb) and sarU genes, which are involved in the pathogenesis and regulation of virulence gene expression in coordination with quorum sensing system. All these results are in good support with the low virulence behavior of EDCC 5464. From the previous literature, it is well known that agr defective S. aureus clinical strains are isolated from the chronic infections. Similarly, low virulent EDCC 5464 was isolated from chronic implant-associated bone infections infection whereas EDCC 5458 was obtained from acute implant-associated bone infections. Laboratory based in vitro and in vivo results and insights from comparative genomic analysis could be correlated with the clinical conclusion of IABIs and allows evidence-based treatment strategies based on the pathogenesis of the strain to cure life devastating implant-associated infections.

Background Use of magnesium for resorbable metal implants is a new concept in orthopaedic and dental medicine. The majority of studies on magnesium’s biocompatibility in vitro have assessed the short-term effect of magnesium extract on cells. The aim of this study was to evaluate the influence of direct exposure to magnesium alloys on the bioactivity of primary human reaming debris-derived (HRD) cells. Materials and methods Pure Mg, Mg2Ag, WE43 and Mg10Gd were tested for biocompatibility. The study consisted of assessment of cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) test, evaluation of alkaline phosphatase (ALP) content, and study of cell morphology under light microscopy, scanning electron microscopy (SEM) and transmission electron microscopy (TEM), along with determination of calcification and pH changes induced by magnesium. Results The number of viable cells in the presence of Mg2Ag was high over the entire observation period. Inhibition of ALP content in osteogenic differentiating HRD was caused by pure Mg at day 14 and 28. All other magnesium alloys did not affect the ALP content. Exposure of HRD to magnesium increased the amount of lysosomes and endocytotic vesicles. Cellular attachment was generally the best for those crystals that formed on the surface of all materials. A decrease was observed in Ca2+ in the medium from day 1 to day 14. Conclusions In terms of cell morphology, cell viability and differentiation, cell density and the effect on the surrounding pH, Mg2Ag showed the most promising results. All magnesium materials induced calcification, which is beneficial for orthopaedic and dental applications.

Even though Ti-based implants are the most used materials for hard tissue replacement, they may present lack of osseointegration on the long term, due to their inertness. Hydrothermal treatment (HT) is a useful technique for the synthesis of firmly attached, highly crystalline coatings made of anatase titanium dioxide (TiO2), providing favorable nanoroughness and higher exposed surface area, as well as greater hydrophilicity, compared to the native amorphous oxide on pristine titanium. The hydrophilicity drops even more by photofunctionalization of the nanostructured TiO2-anatase coatings under UV light. Human mesenchymal stem cells exhibited a good response to the combination of the positive surface characteristics, especially in respect to the UVB pre-irradiation. The results showed that the cells were not harmed in terms of viability; even more, they were encouraged to differentiate in osteoblasts and to become osteogenically active, as confirmed by the calcium ion uptake and the formation of well-mineralized, bone-like nodule structures. In addition, the enrichment of hydroxyl groups on the HT-surfaces by UVB photofunctionalization accelerated the cell differentiation process and greatly improved the osteogenesis in comparison with the nonirradiated samples. The optimal surface characteristics of the HT-anatase coatings as well as the high potentiality of the photo-induced hydrophilicity, which was reached during a relatively short pre-irradiation time (5 h) with UVB light, can be correlated with better osseointegration ability in vivo; among the samples, the superior biological behavior of the roughest and most hydrophilic HT coating makes it a good candidate for further studies and applications.

Increasing evidence is showing that the non-neuronal cholinergic system plays an important role in the pathology of rheumatoid arthritis (RA). Choline transport into the cell is the rate-limiting step for the synthesis of acetylcholine (ACh), which can be released directly or in vesicles from the cell. However, in the human joint little is known about choline import or the release of ACh from the cell. Thus, we analyze the expression of members of the organic cation transporter (OCT), of the newly discovered choline transporter-like (CTL) family and of classical neuronal components such as the high-affinity choline transporter (CHT1) and the vesicular ACh transporter (VAChT) in the synovium and cartilage of the human hip joint from patients with osteoarthritis (OA) and RA. OCT1, OCT3 and OCTN1 and all members of the CTL family were expressed in synovial and cartilage samples. The expression of CTL1 and CTL2 was localized in synovial macrophages and fibroblasts. CHT1 mRNA expression was detectable only in the synovium, whereas VAChT was completely absent in all samples. Therefore, in the human joint, choline transport into the cell and the release of ACh seems to be mediated mainly by members of the OCT and CTL family. Expression of transporters appears not to be influenced by the pathological state, as no differences have been detected between joints from OA or RA patients. Importantly, however, all necessary components for choline import and the release of non-neuronal ACh are present in the human joint. Electronic supplementary material The online version of this article (doi:10.1007/s00441-014-2036-0) contains supplementary material, which is available to authorized users.

Background. The study aim is to avoid tooth extraction by nonsurgical treatment of periapical lesion. It assesses healing progress in response to calcium hydroxide-iodoform-silicon oil paste (CHISP). Numeric Pain Rating Scale was used to validate the approach. Furthermore, CHISP was used to treat cystic lesions secondary to posttraumatic avulsion of permanent teeth. Materials and Methods. Over 200 patients with radicular cysts were treated with CHISP through the root canal. Radiographs were used to verify lesion size and position, ensure correct delivery to the site, and monitor the progress of bone healing in the lesion area. Ten males and 10 females were randomly selected for statistical assessment. Results. No severe pain, complications, or failure in cyst healing was reported. Complete healing was achieved in an average of 75 days. Furthermore, healing of radicular cyst secondary to posttraumatic tooth avulsion was successful. Conclusion. CHISP indicated an antiseptic effect, which enhanced and shortened healing time of periapical lesions. The less invasive procedure avoids tooth extraction and reduces bone resorption. Cyst management with CHISP can remedy failed root canal treatments. The results show a bone regenerative capacity of CHISP suggested in first rapid phase and a second slow phase.

Osteoporosis induction in a sheep model by steroid administration combined with ovariectomy recapitulates decreased bone formation and substandard matrix mineralization in patients. Recently, the role of osteocytes has been frequently addressed, with focus on their role in osteoclastogenesis. However, the quantification of receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) signaling in osteocytes was not studied in sheep. The current study reproduced the sheep model of osteoporosis to study the RANKL/OPG ratio correlation to the method of osteoporosis induction. We investigated the induction of osteoporosis after 8 months using 31 female merino land sheep divided into four groups: control, ovariectomy, ovariectomy with dietary limitation, and ovariectomy with dietary limitation and steroid injection. In accordance to previous reports, the present study showed trabecular thinning, higher numbers of apoptotic osteocytes, and imbalanced metabolism, leading to defective mineralization. The global RANKL/OPG ratio in the spine after 8 months of steroid and dietary treatment was not different from that of the control. Interestingly, assessment of the osteocyte-specific RANKL/OPG ratio showed that the steroid-induced osteoporosis in its late progressive phase stimulates RANKL expression in osteocytes. Sclerostin is suggested to induce RANKL expression in osteocytes. The findings of this study can contribute to further explain the success of sclerostin antibodies in treating osteoporotic patients despite increased osteocyte-expressed RANKL.

The purpose of this work was to investigate new bone formation in macroporous iron foams coated with strontium (FeSr) or bisphosphonate (FeBiP) compared to plain iron foam (Fe) and empty defect in a critical size metaphyseal bone defect model in ovariectomized rats. 60 female rats were subjected to bilateral ovariectomy and multi-deficient diet for 3 months. A 4 mm wedge shaped metaphyseal osteotomy was created, fixed with a mini-plate and subsequently filled with Fe, FeSr, FeBiP or left empty. After 6 weeks, mCt analysis revealed a statistically significant increased bone formation at the implant interface in FeSr compared to FeBiP (p = 0.035) and Fe (p = 0.002), respectively. Increased mineralized tissue was also seen within the pores in FeSr (p = 0.023) compared to Fe. Histomorphometry revealed significantly increased bone formation at the implant interface in FeSr (p < 0.001) and FeBiP (p = 0.006) compared to plain Fe with increased osteoblast and decreased osteoclast activity in combination with increased BMP2 and decreased RANKL/OPG in immunohistochemistry. ToF-SIMS analysis showed overlapping Ca signals with Fe for both FeSr and FeBiP thereby indicating tissue in-growth into the scaffolds. In conclusion, iron foam with strontium or bisphosphonate coating are of further interest in metaphyseal fracture defects in osteopenic bone.

Staphylococcus aureus is the most clinically relevant pathogen regarding implant-associated bone infection and its capability to invade osteoblasts is well known. The aim of this study was to investigate firstly whether S. aureus is not only able to invade but also to proliferate within osteoblasts, secondly to delineate the mechanism of invasion and thirdly to clarify whether rifampicin or gentamicin can inhibit intracellular proliferation and survival of S. aureus. The SAOS-2 osteoblast-like cell line and human primary osteoblasts were infected with S. aureus EDCC5055 and S. aureus Rosenbach 1884. Both S. aureus strains were able to invade efficiently and to proliferate within human osteoblasts. Immunofluorescence microscopy showed intracellular invasion of S. aureus and transmission electron microscopy images could demonstrate bacterial division as a sign of intracellular proliferation as well as cytosolic bacterial persistence. Cytochalasin D, the major actin depolymerisation agent, was able to significantly reduce S. aureus invasion, suggesting that invasion was enabled by promoting actin rearrangement at the cell surface. 7.5 μg/mL of rifampicin was able to inhibit bacterial survival in SAOS-2 cells with almost complete elimination of bacteria after 4 h. Gentamicin could also kill intracellular S. aureus in a dose-dependent manner, an effect that was significantly lower than that observed using rifampicin. In conclusion, S. aureus is not only able to invade but also to proliferate in osteoblasts. Invasion seems to be associated with actin rearrangement at the cell surface. Rifampicin is effective in intracellular eradication of S. aureus whereas gentamicin only poorly eliminates intracellularly replicating bacteria.

In estrogen-deficient, postmenopausal women, vitamin D and calcium deficiency increase osteoporotic fracture risk. Therefore, a new rat model of combined ovariectomy and multiple-deficient diet was established to mimic human postmenopausal osteoporotic conditions under nutrient deficiency. Sprague-Dawley rats were untreated (control), laparatomized (sham), or ovariectomized and received a deficient diet (OVX-Diet). Multiple analyses involving structure (micro-computed tomography and biomechanics), cellularity (osteoblasts and osteoclasts), bone matrix (mRNA expression and IHC), and mineralization were investigated for a detailed characterization of osteoporosis. The study involved long-term observation up to 14 months (M14) after laparotomy or after OVX-Diet, with intermediate time points at M3 and M12. OVX-Diet rats showed enhanced osteoblastogenesis and osteoclastogenesis. Bone matrix markers (biglycan, COL1A1, tenascin C, and fibronectin) and low-density lipoprotein-5 (bone mass marker) were down-regulated at M12 in OVX-Diet rats. However, up-regulation of matrix markers and existence of unmineralized osteoid were seen at M3 and M14. Osteoclast markers (matrix metallopeptidase 9 and cathepsin K) were up-regulated at M14. Micro-computed tomography and biomechanics confirmed bone fragility of OVX-Diet rats, and quantitative RT-PCR revealed a higher turnover rate in the humerus than in lumbar vertebrae, suggesting enhanced bone formation and resorption in OVX-Diet rats. Such bone remodeling caused disturbed bone mineralization and severe bone loss, as reported in patients with high-turnover, postmenopausal osteoporosis. Therefore, this rat model may serve as a suitable tool to evaluate osteoporotic drugs and new biomaterials or fracture implants.

Fracture treatment in osteoporotic patients is still challenging. Osteoporosis emerges when there is an imbalance between bone formation and resorption in favor of resorption by osteoclasts. Thus, new implant materials for osteoporotic fracture treatment should promote bone formation and reduce bone resorption. Nanoparticles can serve as drug delivery systems for growth factors like Brain-Derived Neurotrophic Factor (BDNF), which stimulated osteoblast differentiation. Therefore, polyelectrolyte complex nanoparticles (PEC-NPs) consisting of poly(l-lysine) (PLL) and cellulose sulfate (CS), with or without addition of BDNF, were used to analyze their effect on osteoclasts in vitro. Live cell images showed that osteoclast numbers decreased after application of high PLL/CS PEC-NPs concentrations independent of whether BDNF was added or not. Real-time RT-PCR revealed that relative mRNA expression of cathepsin K and calcitonin receptor significantly declined after incubation of osteoclasts with high concentrations of PLL/CS PEC-NPs. Furthermore, Enzyme-Linked Immunosorbent Assay indicated that tartrate-resistant acidic phosphatase 5b activity was significantly reduced in the presence of high PLL/CS PEC-NPs concentrations. Consistent with these results, the pit formation analysis showed that less hydroxyapatite was resorbed by osteoclasts after incubation with high concentrations of PLL/CS PEC-NPs. BDNF had no influence on osteoclasts. We conclude that highly concentrated PLL/CS PEC-NPs dosages decreased osteoclastogenesis and osteoclasts activity. Moreover, BDNF might be a promising growth factor for osteoporotic fracture treatment since it did not increase osteoclast activity.

The first objective was to investigate new bone formation in a critical-size metaphyseal defect in the femur of ovariectomized rats filled with a strontium modified calcium phosphate cement (SrCPC) compared to calcium phosphate cement (CPC) and empty defects. Second, detection of strontium release from the materials as well as calcium and collagen mass distribution in the fracture defect should be targeted by time of flight secondary ion mass spectrometry (TOF-SIMS). 45 female Sprague Dawley rats were randomly assigned to three different treatment groups: (1) SrCPC (n = 15), (2) CPC (n = 15), and (3) empty defect (n = 15). Bilateral ovariectomy was performed and three months after multi-deficient diet, the left femur of all animals underwent a 4 mm wedge-shaped metaphyseal osteotomy that was internally fixed with a T-shaped plate. The defect was then either filled with SrCPC or CPC or was left empty. After 6 weeks, histomorphometric analysis showed a statistically significant increase in bone formation of SrCPC compared to CPC (p = 0.005) and the empty defect (p = 0.002) in the former fracture defect zone. Furthermore, there was a statistically significant higher bone formation at the tissue implant interface in the SrCPC group compared to the CPC group (p < 0.0001). These data were confirmed by immunohistochemistry revealing an increase in bone-morphogenic protein 2, osteocalcin and osteoprotegerin expression and a statistically significant higher gene expression of alkaline phosphatase, collagen10a1 and osteocalcin in the SrCPC group compared to CPC. TOF-SIMS analysis showed a high release of Sr from the SrCPC into the interface region in this area compared to CPC suggesting that improved bone formation is attributable to the released Sr from the SrCPC. (C) 2013 The Authors. Published by Elsevier Ltd. All rights reserved.

Acetylcholine (ACh) is not only a neurotransmitter but is an ancient molecule that can be released by and act on non-neuronal cells. In these cells the system of ACh-synthesizing enzymes, transporters, receptors and degrading enzymes is termed the non-neuronal cholinergic system (NNCS). There is increasing evidence that the NNCS is dysregulated in various diseases and can have an influence on their pathology. However, for many organ systems not much is known about the expression and function of the NNCS. Thus, this review focusses on the role of the NNCS in different organ systems in health and disease. Dysregulation of ACh synthesis and release, mutations or polymorphisms in genes encoding NNCS components, and auto-antibodies against NNCS components are common factors influencing disease progression. Pharmacological agents targeting the NNCS are already successfully in clinical use for some disorders, indicating that interfering with this system is very promising and more research is needed to elucidate the role of the NNCS in different tissues and pathological states.

Background Osteoporosis is a multi-factorial, chronic, skeletal disease highly prevalent in post-menopausal women and is influenced by hormonal and dietary factors. Because animal models are imperative for disease diagnostics, the present study establishes and evaluates enhanced osteoporosis obtained through combined ovariectomy and deficient diet by DEXA (dual-energy X-ray absorptiometry) for a prolonged time period. Material/methods Sprague-Dawley rats were randomly divided into sham (laparotomized) and OVX-diet (ovariectomized and fed with deficient diet) groups. Different skeletal sites were scanned by DEXA at the following time points: M0 (baseline), M12 (12 months post-surgery), and M14 (14 months post-surgery). Parameters analyzed included BMD (bone mineral density), BMC (bone mineral content), bone area, and fat (%). Regression analysis was performed to determine the interrelationships between BMC, BMD, and bone area from M0 to M14. Results BMD and BMC were significantly lower in OVX-diet rats at M12 and M14 compared to sham rats. The Z-scores were below -5 in OVX-diet rats at M12, but still decreased at M14 in OVX-diet rats. Bone area and percent fat were significantly lower in OVX-diet rats at M14 compared to sham rats. The regression coefficients for BMD vs. bone area, BMC vs. bone area, and BMC vs. BMD of OVX-diet rats increased with time. This is explained by differential percent change in BMD, BMC, and bone area with respect to time and disease progression. Conclusions Combined ovariectomy and deficient diet in rats caused significant reduction of BMD, BMC, and bone area, with nearly 40% bone loss after 14 months, indicating the development of severe osteoporosis. An increasing regression coefficient of BMD vs. bone area with disease progression emphasizes bone area as an important parameter, along with BMD and BMC, for prediction of fracture risk.

Background Staphylococcus aureus is the principle causative pathogen of osteomyelitis and implant-associated bone infections. It is able to invade and to proliferate inside osteoblasts thus avoiding antibiotic therapy and the host immune system. Therefore, development of alternative approaches to stimulate host innate immune responses could be beneficial in prophylaxis against S. aureus infection. TLR9 is the intracellular receptor which recognizes unmethylated bacterial CpG-DNA and activates immune cells. Synthetic CpG-motifs containing oligodeoxynucleotide (CpG-ODNs) mimics the stimulatory effect of bacterial DNA. Results Osteoblast-like SAOS-2 cells were pretreated with CpG-ODN type-A 2216, type-B 2006, or negative CpG-ODN 2243 (negative control) 4 h before infection with S. aureus isolate EDCC 5055 (=DSM 28763). Intracellular bacteria were streaked on BHI plates 4 h and 20 h after infection. ODN2216 as well as ODN2006 but not ODN2243 were able to significantly inhibit the intracellular bacterial growth because about 31 % as well as 43 % of intracellular S. aureus could survive the pretreatment of SAOS-2 cells with ODN2216 or ODN2006 respectively 4 h and 20 h post-infection. RT-PCR analysis of cDNAs from SAOS-2 cells showed that pretreatment with ODN2216 or ODN2006 stimulated the expression of TLR9. Pretreatment of SAOS-2 cells with ODN2216 or ODN2006 but not ODN2243 managed to induce reactive oxygen species (ROS) production inside osteoblasts as measured by flow cytometry analysis. Moreover, treating SAOS-2 cells with the antioxidant Diphenyleneiodonium (DPI) obviously reduced S. aureus killing ability of TLR9 agonists mediated by oxidative stress. Conclusions In this work we demonstrated for the first time that CPG-ODNs have inhibitory effects on S. aureus survival inside SAOS-2 osteoblast-like cell line. This effect was attributed to stimulation of TLR9 and subsequent induction of oxidative stress. Pretreatment of infected SAOS-2 cells with ROS inhibitors resulted in the abolishment of the CPG-ODNs killing effects.

Summary Although bone minerals have been widely studied by various techniques in previous studies, crystal structures, morphology of bone minerals and its building pathway remained still controversy. In this work, the ultrastructure of the mineralization front of rabbit femur has been studied by conventional and high-resolution (HR) transmission electron microscopy (TEM). In order to induce a healing and demineralization process the animals were subjected to a standardized osteotomy stabilized with titan screws and sonic pins. After 84 days follow-up time the newly build bone was investigated. The mineralization front of rabbit femur osteotomy contains partly mineralized collagen fibrils with a pronounced striped pattern together with a large number of agglomerated apatite platelets. The striation is caused by mineralization in the hole zones of the collagen fibrils, corresponding to the early stage of mineralization. In the TEM micrographs, the mineralization zone appears denser and compact when compared with fully mineralized bone, although most of the collagen fibrils are completely mineralized in the latter (higher concentration of interfibrillar apatite platelets within the mineralization zone). In bone some partly mineralized collagen fibrils are also observed, revealing the same arrangement, regular shape, and size of apatite platelets as collagen fibrils in the mineralization zone. Apatite platelets with irregular shapes are observed at the vortex-shaped outer boundary of the mineralization zone, i.e. at the interfaces with nonmineralized collagen or osteoblasts. HR TEM micrographs reveal that the platelets are assumably semicrystalline and that within the platelet nanocrystalline domains of apatite are embedded in an amorphous calciumphosphate matrix. SCANNING 35: 169-182, 2013. © 2012 Wiley Periodicals, Inc.

BACKGROUND AND PURPOSE Recent studies detected the expression of proteins involved in cholinergic metabolism in airway epithelial cells, although the function of this non-neuronal cholinergic system is not known in detail. Thus, this study focused on the effect of luminal ACh as a regulator of transepithelial ion transport in epithelial cells. EXPERIMENTAL APPROACH RT-PCR experiments were performed using mouse tracheal epithelial cells for ChAT and organic cation transporter (OCT) transcripts. Components of tracheal airway lining fluid were analysed with desorption electrospray ionization (DESI) MS. Effects of nicotine on mouse tracheal epithelial ion transport were examined with Ussing-chamber experiments. KEY RESULTS Transcripts encoding ChAT and OCT1–3 were detected in mouse tracheal epithelial cells. The DESI experiments identified ACh in the airway lining fluid. Luminal ACh induced an immediate, dose-dependent increase in the transepithelial ion current (EC50: 23.3 µM), characterized by a transient peak and sustained plateau current. This response was not affected by the Na+-channel inhibitor amiloride. The Cl--channel inhibitor niflumic acid or the K+-channel blocker Ba2+ attenuated the ACh effect. The calcium ionophore A23187 mimicked the ACh effect. Luminal nicotine or muscarine increased the ion current. Experiments with receptor gene-deficient animals revealed the participation of muscarinic receptor subtypes M1 and M3. CONCLUSIONS AND IMPLICATIONS The presence of luminal ACh and activation of transepithelial ion currents by luminal ACh receptors identifies a novel non-neuronal cholinergic pathway in the airway lining fluid. This pathway could represent a novel drug target in the airways.

Aims Alveolar hypoxia acutely elicits contraction of pulmonary arteries, leading to a rise in pulmonary arterial pressure (PAP) and shifting blood to better ventilated areas of the lung. The molecular mechanisms underlying this hypoxic pulmonary vasoconstriction (HPV) are still incompletely understood. Here, we investigated the role of succinate dehydrogenase (SDH; synonymous to mitochondrial complex II) in HPV, with particular emphasis on regional differences along the vascular bed and consequences for PAP and perfusion-to-ventilation matching, using mutant mice heterozygous for the SDHD subunit of complex II ( SDHD +/−). Methods and results Western blots revealed reduced protein content of complex II subunits SDHA, SDHB, and SDHC in lungs of SDHD +/− mice, despite unaffected mRNA content as determined by real-time PCR. Hypoxic pulmonary vasoconstriction of small (20–50 µm) intra-acinar and larger (51–100 µm) pre-acinar arteries was evaluated by videomorphometric analysis of precision-cut lung slices. The hypoxic response was detectable in pre-acinar arteries but absent from intra-acinar arteries of SDHD +/− mice. In isolated perfused lungs, basal PAP and its hypoxia-induced increase were indistinguishable between both mouse strains. Arterial oxygenation was measured after provocation of regional ventilatory failure by tracheal fluid instillation in anaesthetized mice, and it declined more in SDHD +/− than in wild-type mice. Conclusion SDHD is required for the formation of a stable mitochondrial complex II and it is selectively important for HPV of intra-acinar vessels. This specialized vascular segment participates in perfusion-to-ventilation matching but does not significantly contribute to the acute hypoxic rise in PAP that results from more proximal vasoconstriction.

The cholinergic system consists of acetylcholine (ACh), its synthesising enzyme, choline acetyltransferase (CHAT), transporters such as the high-affinity choline transporter (SLC5A7; also known as ChT1), vesicular ACh transporter (SLC18A3; also known as VAChT), organic cation transporters (SLC22s; also known as OCTs), the nicotinic ACh receptors (CHRN; also known as nAChR) and muscarinic ACh receptors. The cholinergic system is not restricted to neurons but plays an important role in the structure and function of non-neuronal tissues such as epithelia and the immune system. Using molecular and immunohistochemical techniques, we show in this study that non-neuronal cells in the parenchyma of rat testis express mRNAs forChat,Slc18a3,Slc5a7andSlc22a2as well as for the CHRN subunits in locations completely lacking any form of innervation, as demonstrated by the absence of protein gene product 9.5 labelling. We found differentially expressed mRNAs for eight α and three β subunits of CHRN in testis. Expression of the α7-subunit of CHRN was widespread in spermatogonia, spermatocytes within seminiferous tubules as well as within Sertoli cells. Spermatogonia and spermatocytes also expressed the α4-subunit of CHRN. The presence of ACh in testicular parenchyma (TP), capsule and isolated germ cells could be demonstrated by HPLC. Taken together, our results reveal the presence of a non-neuronal cholinergic system in rat TP suggesting a potentially important role for non-neuronal ACh and its receptors in germ cell differentiation.

Within this study, the authors use human mesenchymal stem cells incubated with silver nanoparticles (AgNPs) as a model system to systematically investigate the advantages and drawbacks of the fast imaging delayed extraction mode for two-dimensional and three-dimensional (3D) analyses at the cellular level. The authors compare the delayed extraction mode with commonly employed measurement modes in terms of mass and lateral resolution, intensity, and dose density. Using the delayed extraction mode for single cell analysis, a high mass resolution up to 4000 at m/z = 184.08 combined with a lateral resolution up to 360 nm is achieved. Furthermore, the authors perform 3D analyses with Ar-clusters (10 keV) and O2+ (500 eV) as sputter species, combined with Bi3+ and delayed extraction for analysis. Cell compartments like the nucleus are visualized in 3D, whereas no realistic 3D reconstruction of intracellular AgNP is possible due to the different sputter rates of inorganic and organic cell materials. Furthermore, the authors show that the sputter yield of Ag increases with the decreasing Ar-cluster size, which might be an approach to converge the different sputter rates.

Acetylcholine (ACh), a classical transmitter of parasympathetic nerve fibres in the airways, is also synthesized by a large number of non-neuronal cells, including airway surface epithelial cells. Strongest expression of cholinergic traits is observed in neuroendocrine and brush cells but other epithelial cell types--ciliated, basal and secretory--are cholinergic as well. There is cell type-specific expression of the molecular pathways of ACh release, including both the vesicular storage and exocytotic release known from neurons, and transmembrane release from the cytosol via organic cation transporters. The subcellular distribution of the ACh release machineries suggests luminal release from ciliated and secretory cells, and basolateral release from neuroendocrine cells. The scenario as known so far strongly suggests a local auto-/paracrine role of epithelial ACh in regulating various aspects on the innate mucosal defence mechanisms, including mucociliary clearance, regulation of macrophage function and modulation of sensory nerve fibre activity. The proliferative effects of ACh gain importance in recently identified ACh receptor disorders conferring susceptibility to lung cancer. The cell type-specific molecular diversity of the epithelial ACh synthesis and release machinery implies that it is differently regulated than neuronal ACh release and can be specifically targeted by appropriate drugs.

Background: A variety of materials have been used for bone augmentation, distraction osteotomy, and in post-cancer patients following tumor removal. However, a temporary metal implant that would resorb after successful treatment is a new concept. Magnesium was suggested as a suitable material for these purposes because it is biocompatible, has better mechanical properties than titanium, and stimulates new bone formation. This study evaluates histological appearance of magnesium-based implants and the surrounding bone. Materials and Methods: Three magnesium-based biomaterials were tested in a rabbit bone defect model: magnesium–hydroxyapatite (Mg–HA), W4 (96 % magnesium, 4 % yttrium), and pure magnesium (pure Mg). Animals were sacrificed after 6 and 12 weeks and the samples were analyzed histologically and histomorphometrically. Results: Mg–HA had the highest mean amount of tartrate-resistant acid phosphatase (TRAP) positive cells at the implantation site of all groups. It had shown the fastest degradation rate already at 6 weeks but the least amount of new bone formation. New bone was seen forming in direct contact with pure Mg and W4. The mean gas volume was highest in W4 compared to pure Mg and Mg–HA but this difference was not statistically significant. W4 had the lowest mean number of TRAP-positive cells of all materials. Conclusion: Pure Mg and W4 were shown to be the most promising materials in this study in respect to the bone response to the implant material. They could be used for screws and plates in bone augmentation procedures.

The biological impact of novel nano-scaled drug delivery vehicles in highly topical therapies of bone diseases have to be investigated in vitro before starting in vivo trials. Highly desired features for these materials are a good cellular uptake, large transport capacity for drugs and a good bio-compatibility. Essentially the latter has to be addressed as first point on the agenda. We present a study on the biological interaction of maltose-modified poly(ethyleneimine) (PEI-Mal) on primary human mesenchymal stem cell, harvested from reaming debris (rdMSC) and osteoblasts obtained from four different male donors. PEI-Mal-nanoparticles with two different molecular weights of the PEI core (5000 g/mol for PEI-5k-Mal-B and 25,000 g/mol for PEI-25k-Mal-B) have been administered to both cell lines. As well dose as incubation-time dependent effects and interactions have been researched for concentrations between 1 μg/ml to 1 mg/ml and periods of 24 h up to 28 days. Studies conducted by different methods of microscopy as light microscopy, fluorescence microscopy, transmission-electron-microscopy and quantitative assays (LDH and DC-protein) indicate as well a good cellular uptake of the nanoparticles as a particle- and concentration-dependent impact on the cellular macro- and micro-structure of the rdMSC samples. In all experiments PEI-5k-Mal-B exhibits a superior biocompatibility compared to PEI-25k-Mal-B. At higher concentrations PEI-25k-Mal-B is toxic and induces a directly observable mitochondrial damage. The alkaline phosphatase assay (ALP), has been conducted to check on the possible influence of nanoparticles on the differentiation capabilities of rdMSC to osteoblasts. In addition the production of mineralized matrix has been shown by von-Kossa stained samples. No influence of the nanoparticles on the ALP per cell has been detected. Additionally, for all experiments, results are strongly influenced by a large donor-to-donor variability of the four different rdMSC samples. To summarize, while featuring a good cellular uptake, PEI-5k-Mal-B induces only minimal adverse effects and features clearly superior biocompatibility compared to the larger PEI-25k-Mal-B. Electronic supplementary material The online version of this article (doi:10.1186/s12951-015-0128-y) contains supplementary material, which is available to authorized users.

Screws for ligament reconstruction are nowadays mostly made of poly-L-lactide (PLLA). However, magnesium-based biomaterials are gathering increased interest in this research field because of their good mechanical property and osteoanabolic influence on bone metabolism. The aim of this pilot study was to evaluate the biocompatibility of an interference screw for ligament reconstruction made of magnesium alloy W4 by diecasting and milling and using different PEO-coatings with calcium phosphates. PLLA and titanium screws were used as control samples. The screws were implanted in the femur condyle of the hind leg of a merino sheep. The observation period was six and twelve weeks and one year. Histomorphometric, immunohistochemical, immunofluorescence, and molecular biological evaluation were conducted. Further TEM analysis was done. In all magnesium screws a clinically relevant gas formation in the vicinity of the biomaterial was observed. Except for the PLLA and titanium control samples, no screw was fully integrated in the surrounding bone tissue. Regarding the fabrication process, milling seems to produce less gas liberation and has a better influence on bone metabolism than diecasting. Coating by PEO with calcium phosphates could not reduce the initial gas liberation but rather reduced the bone metabolism in the vicinity of the biomaterial.

In airway epithelia, non-neuronal cholinergic regulations have been described; however, the route for acetylcholine (ACh) release has not been verified. To investigate whether organic cation transporters (OCTs) serve this function, we studied the expression of OCTs in airway epithelia and their capability to translocate ACh. Using immunohistochemistry in rats and humans, OCT1, OCT2, and OCT3 were localized to the luminal membrane of ciliated epithelial cells. In humans, OCT2 showed the strongest expression in the luminal membrane. We expressed the OCT isoforms in oocytes of Xenopus laevis and measured uptake and efflux of ACh. Tracer flux measurements showed that ACh is transported by OCT1 and OCT2 but not by OCT3. Two-electrode-voltage-clamp measurements revealed that OCT2 mediates electrogenic uptake and efflux of ACh. For ACh uptake by human OCT2, a K(M) value of approximately 0.15 mM was determined. At -50 mV, ACh efflux by human OCT2 was trans-inhibited by micromolar concentrations of the inhalational glucocorticoid budesonide, which is used in treatment of asthma (K(i) approximately 2.7 microM). The data show that OCT1 and OCT2 mediate luminal ACh release in human airways and suggest that ACh release is blocked after inhalation of budesonide.

Choline is an essential component in acetylcholine biosynthesis, and is involved in cell signaling. It is unable to permeate the cell membrane and requires a transporter to enter the cell. Neurons that synthesize acetylcholine take up choline by a recently cloned high-affinity choline transporter (choline transporter 1) that is Na+-dependent and can be blocked by hemicholinium-3. The aim of this study was to determine the expression and to analyze the distribution of choline transporter 1 in human and rat skin. The mRNA for choline transporter 1 was detected in rat and human skin and in the human keratinocyte cell line HaCaT. A polyclonal anti-serum was developed against the N-terminal region of the human and rat protein. In rat and human skin, choline transporter 1 immunoreactivity was present in nerve fibers. In addition, keratinocytes, HaCaT cells and cells of the internal root sheath of the hair follicle contained choline transporter 1 immunoreactivity. The labeling patterns of nonconfluent vs confluent cultured cells and the distribution of choline transporter 1 along the epidermal layer suggest an association of choline transporter 1 with keratinocyte differentiation. In conclusion, this study shows the presence of the high-affinity choline transporter choline transporter 1 in nerve fibers and epithelial cells in the human and rat skin supporting the pivotal role of this transporter in both the neuronal and non-neuronal cholinergic system of the skin.

Background Recently, analysis of bone from knockout mice identified muscarinic acetylcholine receptor subtype M3 (mAChR M3) and nicotinic acetylcholine receptor (nAChR) subunit α2 as positive regulator of bone mass accrual whereas of male mice deficient for α7-nAChR (α7KO) did not reveal impact in regulation of bone remodeling. Since female sex hormones are involved in fair coordination of osteoblast bone formation and osteoclast bone degradation we assigned the current study to analyze bone strength, composition and microarchitecture of female α7KO compared to their corresponding wild-type mice (α7WT). Methods Vertebrae and long bones of female 16-week-old α7KO (n = 10) and α7WT (n = 8) were extracted and analyzed by means of histological, radiological, biomechanical, cell- and molecular methods as well as time of flight secondary ion mass spectrometry (ToF-SIMS) and transmission electron microscopy (TEM). Results Bone of female α7KO revealed a significant increase in bending stiffness (p

Background Cholinergic signaling via muscarinic acetylcholine receptors (mAChR) is known to influence various physiological functions. In bone, M3 mAChR and M5 mAChR were identified on the membrane of osteoblast-like cells. M3 mAChR seems to be particularly relevant for bone physiology, as signaling via this receptor was reported to increase bone formation and decrease bone resorption. Thus, in the present study we investigated the relative mRNA expression of M3 and M5 mAChR in bones of a rat osteoporosis model. Material and methods Osteoporosis was induced in Sprague-Dawley rats by bilateral ovariectomy and additional feeding of a diet deficient in calcium, vitamins C, D2, D3, and phosphorus, and free of soy and phytoestrogen. After a period of 3, 12, and 14 months, relative mRNA expression of M3 mAChR and M5 mAChR was analyzed in the 11th thoracic vertebra by real-time RT-PCR. Results Relative mRNA expression of M3 mAChR was significantly reduced in bones of osteoporotic rats compared to sham operated animals that served as controls. Further, M3 mAChR mRNA expression was significantly down-regulated when comparing 14-month osteoporotic rats to 3-month osteoporotic rats. Relative M5 mAChR mRNA was expressed to a lesser extent than M3 mAChR and did not show significant differences in mRNA expression level between the experimental groups. Conclusions M3 mAChR mRNA expression was reduced upon induction of osteoporosis and progression of disease was associated with further decrease of this receptor, indicating that M3 mAChR is involved in the development and regulation of osteoporosis.

New strategies to decrease infection rates in cementless arthroplasty are needed, especially in the context of the growing incidence of methicillin-resistant Staphylococcus aureus (MRSA) infections. The purpose of this study was to investigate the antimicrobial activity of a rifampicin–fosfomycin coating against methicillin-sensitive Staphylococcus aureus (MSSA) and MRSA in a rabbit infection prophylaxis model. Uncoated or rifampicin–fosfomycin-coated K-wires were inserted into the intramedullary canal of the tibia in rabbits and contaminated with an inoculation dose of 10 5 or 10 6 colony-forming units of MSSA EDCC 5055 in study 1 and MRSA T6625930 in study 2, respectively. After 28 days the animals were killed and clinical, histological and microbiological assessment, including pulse-field gel electrophoresis, was conducted. Positive culture growth in agar plate testing and/or clinical signs and/or histological signs were defined positive for infection. Statistical evaluation was performed using Fisher’s exact test. Both studies showed a statistically significant reduction of infection rates for rifampicin–fosfomycin-coated implants compared to uncoated K-wires ( P = 0.015). In both studies none of the 12 animals that were treated with a rifampicin–fosfomycin-coated implant showed clinical signs of infection or a positive agar plate testing result. In both studies, one animal of the coating group showed the presence of sporadic bacteria with concomitant inflammatory signs in histology. The control groups in both studies exhibited an infection rate of 100% with clear clinical signs of infection and positive culture growth in all animals. In summary, the rifampicin–fosfomycin-coating showed excellent antimicrobial activity against both MSSA and MRSA, and therefore warrants further clinical testing.

A correlation between obesity and bone metabolism is strongly assumed because adipocytes and osteoblasts originate from the same precursor cells and their differentiation is conversely regulated by the same factors. It is controversially discussed if obesity protects bone or leads to loss of bone mass. Thus, the aim of the present study was to investigate the influence of diet-induced mild obesity (11% increased body weight compared to control) on bone microstructure in mice. Four-week-old male C57BL/6J mice received a high-fat diet (HFD, 60% kcal from fat) and were analyzed by means of dual X-ray absorptiometry, histological methods, real-time RT-PCR, and transmission electron microscopy in comparison to control animals (10% kcal from fat). The cancellous bone mass, collagen 1α1 expression, amount of osteoid, and cohesion of cells via cell-to-cell contacts decreased in HFD mice whereas the bone mineral density and the amount of osteoblasts and osteoclasts were not modified. The amount of apoptotic osteocytes was increased in HFD mice in comparison to controls. We conclude that moderately increased body weight does not protect bone architecture from age-dependent degeneration. By contrast, bone microstructure is negatively affected and reduced maintenance of cell-cell contacts may be one of the underlying mechanisms.

Trimeric catecholates have been designed for the stable immobilization of effector molecules on metal surfaces. The design of these catecholates followed a biomimetic approach and was inspired by natural multivalent metal binders, such as mussel adhesion proteins (MAPs) and siderophores. Three catecholates have been conjugated to central scaffolds based on adamantyl or trisalkylmethyl core structures. The resulting triscatecholates have been immobilized on TiO2 and stainless steel. In a proof of concept study we have demonstrated the high stability of the resulting nanolayers at neutral and slightly acidic pH. Furthermore, polyethylene glycol (PEG) conjugates of our triscatecholates have been synthesized and were immobilized on TiO2 and stainless steel. The PEG coated surfaces showed excellent antifouling properties upon exposure to human blood and bacteria as demonstrated by fluorescence microscopy, ellipsometry and a bacterial assay with Staphylococcus epidermidis. In addition, our PEG-triscatecholates showed no cytotoxicity against bone-marrow stem cells on TiO2.

The prevention of implant-related infections is an important issue in medical research. The aim is to exploit the strong antimicrobial effect of silver nanoparticles (AgNP) to develop new antibacterial coatings for implants. However, there is still a serious lack of information on the influence of AgNP on bone metabolism. In the present study we have evaluated the influence of AgNP on cell stress, viability, proliferation and differentiation of primary human mesenchymal stem cells (MSC) and osteoblasts (OB). Finally, cellular uptake of the AgNP was examined. After 21 days impairment of cell viability of MSC and OB occurred at a concentration of 10 μg/g of AgNP. Cytotoxicity and inhibition of proliferation was highly time and dose dependent. No influence on cell differentiation, but an increase in cell stress, was observed. Uptake of AgNP into MSC and OB could be confirmed. In summary, these results demonstrate AgNP-mediated cytotoxicity at higher concentrations. Therefore, a therapeutical window for the application of AgNP in medical products might exist. However, the antibacterial benefits and potential health risks of AgNP need to be weighed in further studies.

Insertion of bone substitution materials accelerates healing of osteoporotic fractures. Biodegradable materials are preferred for application in osteoporotic patients to avoid a second surgery for implant replacement. Degraded implant fragments are often absorbed by macrophages that are removed from the fracture side via passage through veins or lymphatic vessels. We investigated if lymphatic vessels occur in osteoporotic bone defects and whether they are regulated by the use of different materials. To address this issue osteoporosis was induced in rats using the classical method of bilateral ovariectomy and additional calcium and vitamin deficient diet. In addition, wedge-shaped defects of 3, 4, or 5 mm were generated in the distal metaphyseal area of femur via osteotomy. The 4 mm defects were subsequently used for implantation studies where bone substitution materials of calcium phosphate cement, composites of collagen and silica, and iron foams with interconnecting pores were inserted. Different materials were partly additionally functionalized by strontium or bisphosphonate whose positive effects in osteoporosis treatment are well known. The lymphatic vessels were identified by immunohistochemistry using an antibody against podoplanin. Podoplanin immunopositive lymphatic vessels were detected in the granulation tissue filling the fracture gap, surrounding the implant and growing into the iron foam through its interconnected pores. Significant more lymphatic capillaries were counted at the implant interface of composite, strontium and bisphosphonate functionalized iron foam. A significant increase was also observed in the number of lymphatics situated in the pores of strontium coated iron foam. In conclusion, our results indicate the occurrence of lymphatic vessels in osteoporotic bone. Our results show that lymphatic vessels are localized at the implant interface and in the fracture gap where they might be involved in the removal of lymphocytes, macrophages, debris and the implants degradation products. Therefore the lymphatic vessels are involved in implant integration and fracture healing.

An improved interfacial drug delivery system (DDS) based on polyelectrolyte complex (PEC) coatings with controlled drug loading and improved release performance was elaborated. The cationic homopolypeptide poly(l-lysine) (PLL) was complexed with a mixture of two cellulose sulfates (CS) of low and high degree of substitution, so that the CS and PLL solution have around equal molar charged units. As drugs the antibiotic rifampicin (RIF) and the bisphosphonate risedronate (RIS) were integrated. As an important advantage over previous PEC systems this one can be centrifuged, the supernatant discarded, the dense pellet phase (coacervate) separated, and again redispersed in fresh water phase. This behavior has three benefits: (i) Access to the loading capacity of the drug, since the concentration of the free drug can be measured by spectroscopy; (ii) lower initial burst and higher residual amount of drug due to removal of unbound drug and (iii) complete adhesive stability due to the removal of polyelectrolytes (PEL) excess component. It was found that the pH value and ionic strength strongly affected drug content and release of RIS and RIF. At the clinically relevant implant material (Ti40Nb) similar PEC adhesive and drug release properties compared to the model substrate were found. Unloaded PEC coatings at Ti40Nb showed a similar number and morphology of above cultivated human mesenchymal stem cells (hMSC) compared to uncoated Ti40Nb and resulted in considerable production of bone mineral. RIS loaded PEC coatings showed similar effects after 24 h but resulted in reduced number and unhealthy appearance of hMSC after 48 h due to cell toxicity of RIS.

The intention of this study was to establish a new critical size animal model that represents clinically relevant situations with osteoporotic bone status and internally fixated metaphyseal defect fractures in which biomaterials for the enhancement of fracture healing in osteoporotic fracture defects can be studied. Twenty-eight rats were ovariectomized (OVX) and treated with a calcium-, phosphorus-, vitamin D3-, soy- and phytoestrogen-free diet. After 3months Dual-energy X-ray absorptiometry measurements showed statistically significant reductions in bone mineral density of the spine of -25.9% and of the femur of -21.3% of the OVX rats compared with controls, confirming osteoporosis in the OVX rats. The OVX rats then underwent either 3 or 5mm wedge-shaped osteotomy of the distal metaphyseal area of the femur that was internally stabilized with a T-shaped mini-plate. After 42days biomechanical testing yielded completely unstable conditions in the 5mm defect femora (bending stiffness 0Nmm(-2)) and a bending stiffness of 12,500Nmm(-2) in the 3mm defects, which showed the beginning of fracture consolidation. Micro-computed tomography showed statistically significant more new bone formation in the 3mm defects (4.83±0.37mm(2)), with bridging of the initial fracture defect area, compared with the 5mm defects (2.68±0.34mm(2)), in which no bridging of the initial defect was found. These results were confirmed by histology. In conclusion, the 5mm defect can be considered as a critical size defect model in which biomaterials can be tested.

Summary Background As women are the population most affected by multifactorial osteoporosis, research is focused on unraveling the underlying mechanism of osteoporosis induction in rats by combining ovariectomy (OVX) either with calcium, phosphorus, vitamin C and vitamin D2/D3 deficiency, or by administration of glucocorticoid (dexamethasone). Material/Methods Different skeletal sites of sham, OVX-Diet and OVX-Steroid rats were analyzed by Dual Energy X-ray Absorptiometry (DEXA) at varied time points of 0, 4 and 12 weeks to determine and compare the osteoporotic factors such as bone mineral density (BMD), bone mineral content (BMC), area, body weight and percent fat among different groups and time points. Comparative analysis and interrelationships among osteoporotic determinants by regression analysis were also determined. Results T scores were below-2.5 in OVX-Diet rats at 4 and 12 weeks post-OVX. OVX-diet rats revealed pronounced osteoporotic status with reduced BMD and BMC than the steroid counterparts, with the spine and pelvis as the most affected skeletal sites. Increase in percent fat was observed irrespective of the osteoporosis inducers applied. Comparative analysis and interrelationships between osteoporotic determinants that are rarely studied in animals indicate the necessity to analyze BMC and area along with BMD in obtaining meaningful information leading to proper prediction of probability of osteoporotic fractures. Conclusions Enhanced osteoporotic effect observed in OVX-Diet rats indicates that estrogen dysregulation combined with diet treatment induces and enhances osteoporosis with time when compared to the steroid group. Comparative and regression analysis indicates the need to determine BMC along with BMD and area in osteoporotic determination.

Although bone minerals have been widely studied by various techniques in previous studies, crystal structures, morphology of bone minerals and its building pathway remained still controversy. In this work, the ultrastructure of the mineralization front of rabbit femur has been studied by conventional and high-resolution (HR) transmission electron microscopy (TEM). In order to induce a healing and demineralization process the animals were subjected to a standardized osteotomy stabilized with titan screws and sonic pins. After 84 days follow-up time the newly build bone was investigated. The mineralization front of rabbit femur osteotomy contains partly mineralized collagen fibrils with a pronounced striped pattern together with a large number of agglomerated apatite platelets. The striation is caused by mineralization in the hole zones of the collagen fibrils, corresponding to the early stage of mineralization. In the TEM micrographs, the mineralization zone appears denser and compact when compared with fully mineralized bone, although most of the collagen fibrils are completely mineralized in the latter (higher concentration of interfibrillar apatite platelets within the mineralization zone). In bone some partly mineralized collagen fibrils are also observed, revealing the same arrangement, regular shape, and size of apatite platelets as collagen fibrils in the mineralization zone. Apatite platelets with irregular shapes are observed at the vortex-shaped outer boundary of the mineralization zone, i.e. at the interfaces with nonmineralized collagen or osteoblasts. HR TEM micrographs reveal that the platelets are assumably semicrystalline and that within the platelet nanocrystalline domains of apatite are embedded in an amorphous calciumphosphate matrix.

The present study focused on the influence of nicotinic acetylcholine receptors (nAChR) on ion transport processes in mouse tracheal epithelium. RT-PCR experiments revealed expression of the α3, α4, α5, α7, α9, α10, β2, and β4 nAChR subunits in mouse tracheal epithelium. In Ussing chamber recordings of mouse tracheae, apically applied nicotine (100 μM) induced a dose-dependent increase of the transepithelial short-circuit current (EC(50): 14.6 μM). The nicotine-induced effect (I(NIC)) was attenuated by mecamylamine (25 μM, apical) and methyllycaconitine (1 μM, apical). The nAChR agonist 1.1-dimethyl-4-phenylpiperatinium iodide (DMPP) (100 μM) revealed apical and basolateral location of the receptors. I(NIC) was not affected by the sodium channel inhibitor amiloride (10 μM, apical) or the cystic fibrosis transmembrane conductance regulator inhibitor CFTR(inh)-172 (20 μM, apical) but was reduced by the chloride channel inhibitor 5-nitro-2-(3-phenylpropylamino)benzoic acid (100 μM, apical), the Na(+)/K(+)/2Cl(-) cotransporter inhibitor bumetanide (200 μM, basolateral), the potassium channel inhibitor Ba(2+) (5 mM, basolateral), and 4.4'-diisothiocyanatostilbene-2.2'-disulfonate (100 μM, apical), indicating a contribution of Ca(2+)-activated chloride channels and potassium channels. Removal of extracellular Na(+) (apical) or Ca(2+) (apical) did not influence I(NIC) but reduced the DMPP effect. Experiments with the Ca(2+)-ionophore A23187, a mix of 3-isobutyl-1-methylxanthine and forskolin, or the inositol-1,4,5-triphospate (IP(3)) receptor inhibitor 2-aminoethyl-diphenyl-borinate (75 μM, apical) decreased I(NIC), indicating a nicotine-mediated increase of intracellular Ca(2+) and cAMP levels involving the IP(3) signaling pathway. These findings indicate the activity of Ca(2+)-permeable nAChRs and alternative metabotropic pathways by nAChR activation that mediate Cl(-) and K(+) transport in tracheal epithelium.

Bony integration of biomaterials is a complex process in which angiogenesis plays a crucial role. We evaluated micro- and nano-CT imaging to demonstrate and quantify neovascularization in bony integration of a biomaterial and to give an image based estimation for the needed resolution for imaging angiogenesis in an animal model of femora defect healing. In 8 rats 5 mm full-size defects were created at the left femur that was filled with silica-collagen bone substitute material and internally fixed with plate osteosynthesis. After 6 weeks the femora were infused in situ with Microfil, harvested and scanned for micro-CT (9 μm)3 and nano-CT (3 μm)3 imaging. Using those 3D images, the newly formed blood vessels in the area of the biomaterial were assessed and the total vascular volume fraction, the volume of the bone substitute material and the volume of the bone defect were quantitatively characterized. Results were complemented by histology. Differences were statistically assessed using (ANOVA). High-resolution nano-CT demonstrated new blood vessel formation surrounding the biomaterial in all animals at capillary level. Immunohistochemistry confirmed the newly formed blood vessels surrounding the bone substitute material. The mean vascular volume fraction (VVF) around the implant was calculated to be 3.01 ± 0.4%. The VVF was inversely correlated with the volume of the bone substitute material (r = 0.8) but not with the dimension of the fracture zone (r = 0.3). Nano-CT imaging is feasible for quantitative analysis of angiogenesis during bony integration of biomaterials and a promising tool in this context for the future.

A new role and source of the old mediator acetylcholine is described, which is produced by graft monocytes and attenuates monocytic ATP-signaling. During acute rejection, large numbers of leukocytes accumulate in the blood vessels of experimental renal allografts. About 70% of them are activated, cytotoxic monocytes that appear to be involved in allograft destruction. ACh exerts anti-inflammatory effects upon monocytes/macrophages and has been proposed to be a key player in neuroimmunological interactions. Its short half-life, however, makes it unlikely that neuronal ACh affects blood leukocytes. Renal transplantation was performed in the allogeneic DA to LEW and in the isogeneic LEW to LEW rat strain combination. Intravascular leukocytes were harvested after 4 days, and the expression of CHT1, cChAT, pChAT, and nAChR subunits was investigated by RT-PCR, immunoblotting, and immunohistochemistry. Monocytes were identified by double-labeling with ED1-antibody, directed to a CD68-like antigen. ACh content was measured by HPLC. [Ca2+]i was monitored by Fura-2. Intravascular graft leukocytes express CHT1 and cChAT mRNA and protein and pChAT protein. Their expression is strongly up-regulated in vivo during acute allograft rejection. Immunohistochemistry revealed CHT1, cChAT, and pChAT protein in ED1-positive monocytes. The ACh content of allograft intravascular leukocytes was sixfold higher than that of isografts. Intravascular leukocytes express nAChR subunits, and an ATP-induced increase in [Ca2+]i was augmented in vitro by a nAChR inhibitor in allograft but not isograft leukocytes. Intravascular graft leukocytes, among them monocytes, up-regulate non-neuronal ACh synthesis and develop auto-/paracrine cholinergic attenuation of ATP signaling during acute allograft rejection.