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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.

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.

The intention of the current work is to assess new bone formation and degradation behavior of nanocrystalline hydroxyapatite with (HA/col-1) or without collagen-type I (HA) in osteoporotic metaphyseal bone defects in goats. After ovariectomy and special low-calcium diet for three months, 3 drill hole defects in the vertebrae of L3, L4, L5, 4 drill hole defects in the right and left iliac crest and 1 drill hole defect at the distal femur were created in three Chinese mountain goats with a total of 24 defects. The defects were either filled with one of the biomaterials or left empty (empty defect control group). After 42 days, the animals were euthanized and the samples were assessed for new bone formation using high-resolution peripheral quantitative computed tomography (HR-pQCT) and histomorphometry with 2 regions of interest. Detail histology, enzymehistochemistry and immunohistochemistry as well as connexin-43 in situ hybridization and transmission electron microscopy were carried out for evaluation of degradation behavior of the materials and cellular responses of the surrounding tissue in respect to the implants. HR-pQCT showed the highest BV/TV ratio (p = 0.008) and smallest trabecular spacing (p = 0.005) for HA compared to the other groups in the region of interest at the interface with 1mm distance to the initially created defect. The HA/col-1 yielded the highest connectivity density (Conn.D) (p = 0.034) and the highest number of trabeculae (Tb.N) (p = 0.002) compared to the HA and the control group. Histomorphometric analysis for the core region of the initially created defect revealed a statistically higher new bone formation in the HA (p = 0.001) and HA/col-1 group (p = 0.001) compared to the empty defect group including all defect sites. This result was confirmed for site specific analysis with significant higher new bone formation for the HA group for vertebral defects compared to the empty defect group (p = 0.029). For the interface region, no statistically significant differences were found between the three groups (p = 0.08). Histology revealed a good biocompatibility without inflammatory reaction for the HA- and HA/col-1 implants with a higher fragmentation of the HA-implant compared to the HA/col-1 biomaterial and formation of new bone in the region between the biomaterial fragments by osteoblasts. Fragmentation was shown by transmission electron microscopy to be caused by multinuclear osteoclast-like cells with degradation of the implant via intracellular incorporation of degraded implant material particles. In conclusion, both nanoparticulate HA with and without collagen type-1 showed better new bone formation compared to untreated drill hole defects in metaphyseal regions of this osteoporotic Chinese mountain goat model with good biocompatibility.

Acetylcholine (ACh) is an important signaling molecule in non-neuronal systems where it is involved in regulation of viability, proliferation, differentiation and migration of mesenchymal stem cells (MSC) that are capable to differentiate into osteoblasts, chondrocytes and adipocytes. Patients with the systemic disease osteoporosis show altered MSC properties, reduced bone formation and mineral density followed by increased bone fragility and high fracture incidence. Here we asked whether nicotinic and muscarinic acetylcholine receptors (AChR) are expressed in osteoblasts, adipocytes and chondrocytes differentiated from bone MSC extracted from human reaming debris (RDMSC) that was harvested during surgery of long bone diaphyseal fractures. Using RT-PCR, AChR were detected in RDMSC, osteoblasts, chondrocytes and adipocytes of male and female bone-healthy and of female osteoporotic donors. An up-regulation in multiplicity and occurrence of AChR subtypes was found in female compared to male donors and in osteoblast of male donors compared to adipocytes. Real-time RT-PCR analysis resulted in a significant increase in relative expression of nAChR α9 in chondrocytes compared to adipocytes of healthy female donors. The nAChR subunit α10 was significantly up-regulated in osteoblasts of healthy compared to osteoporotic donors as well as the mAChR M5 that is additionally decreased in osteoporotic osteoblasts compared to MSC and chondrocytes of osteoporotic donors. In summary, the gene expression of AChR during differentiation of RDMSC and its regulation in cells of osteoporotic donors lead to the assumption that AChR signaling is involved in bone formation and might be utilized to stimulate bone remodeling processes.

High frequency of osteoporosis is found in postmenopausal women where several molecular components were identified to be involved in bone loss that subsequently leads to an increased fracture risk. Bone loss has already been determined in male mice with gene deficiency of muscarinic acetylcholine receptor M3 (M3R-KO). Here we asked whether bone properties of female 16-week old M3R-KO present similarities to osteoporotic bone loss by means of biomechanical, radiological, electron microscopic, cell- and molecular biological methods. Reduced biomechanical strength of M3R-KO correlated with cortical thickness and decreased bone mineral density (BMD). Femur and vertebrae of M3R-KO demonstrated a declined trabecular bone volume, surface, and a higher trabecular pattern factor and structure model index (SMI) compared to wild type (WT) mice. In M3R-KO, the number of osteoclasts as well as the cathepsin K mRNA expression was increased. Osteoclasts of M3R-KO showed an estimated increase in cytoplasmic vesicles. Further, histomorphometrical analysis revealed up-regulation of alkaline phosphatase. Osteoblasts and osteocytes showed a swollen cytoplasm with an estimated increase in the amount of rough endoplasmatic reticulum and in case of osteocytes a reduced pericellular space. Thus, current results on bone properties of 16-week old female M3R-KO are related to postmenopausal osteoporotic phenotype. Stimulation and up-regulation of muscarinic acetylcholine receptor subtype M3 expression in osteoblasts might be a possible new option for prevention and therapy of osteoporotic fractures. Pharmacological interventions and the risk of side effects have to be determined in upcoming studies.

Butyrylcholinesterase (BChE) degrades acetylcholine in addition to acetylcholinesterase (AChE) which is involved in embryonic development of limbs. Since BChE is expressed by osteoblast-like cells we asked whether it is functional in adult bone remodeling. We addressed this issue by analyzing BChE gene-deficient mice (BChE-KO). Bones were extracted from 16-week old female BChE-KO and corresponding wild type mice (WT). Femoral bones were used for biomechanical testing and μCT evaluation of cancellous and cortical bone. Also vertebrae Th12 and L1 were investigated with μCT while L3 was used for tartrate-resistant acidic phosphatase (TRAP) histomorphometry and Th10 for gene expression analysis by means of real-time RT-PCR. BChE-KO did not reveal significant differences in biomechanical bone strength and bone mineral density determined by μCT. Microarchitecture of cancellous and cortical bone showed an increase in μCT parameters like trabecular thickness, trabecular separation, and relative cortical bone area of femoral BChE-KO bone compared to WT. In vertebrae no changes of microstructure and mRNA expression were detected. However, osteoclast histomorphometry with TRAP stained sections demonstrated a significant increase in relative osteoclast number. In conclusion, in adult murine bone the role of BChE is limited to bone specific changes in microarchitecture and to an increase in relative number of bone resorbing osteoclasts whereas the main collagen resorbing enzyme Cathepsin-K (CtsK) was stably expressed. Besides, AChE might be able to compensate the lack of BChE. Thus, further analyses using bone tissue specific AChE BChE cre–lox double knockout mice would be helpful.

BACKGROUND Anterior knee pain is the most common complication after intramedullary tibial nailing. The cause is often multifactorial and varies among individuals. Violation of the anterior intermeniscal ligament (AIL) during intramedullary tibial nailing might be a possible source of postsurgical anterior knee pain. Although there is a certain ambiguity regarding the importance and function of the AIL, neural structures in the AIL tissue might play a significant role with respect to functional purposes and pain perception. METHODS We subjected 6 AIL specimens to histologic examination to identify the neural structures that are a mandatory requirement as a source of anterior knee pain. Specifically, we performed three-dimensional immunohistochemical investigation of subtyping, orientation, and detailed characterization of neural structures within the AIL tissue. RESULTS Histologic and three-dimensional immunohistochemical examinations confirmed the presence of neural structures in all 6 AIL specimens. We identified myelinated and unmyelinated nerve fibers, as well as all types of mechanoreceptors. CONCLUSIONS Free nerve endings are a mandatory requirement for pain perception as a result of AIL violation during tibial nailing. Our verification of all different types of mechanoreceptors in the AIL tissue makes a role of the ligament in knee joint function and proprioception highly probable. Further investigations are necessary to clarify possible correlations between neural supply and function of the AIL. Violation of the ligament during operative procedures should be avoided, although the significance of the AIL is still debated.

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.

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.

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.

Discrepancies in bone healing between osteoporotic and non-osteoporotic bone remain uncertain. The focus of the current work is to evaluate potential healing discrepancies in a metaphyseal defect model in rat femora. Female Sprague-Dawley rats were either ovariectomized (OVX, n=14) and combined with a calcium-, phosphorus- and vitamin D3-, soy- and phytoestrogen-free diet or received SHAM operation with standard diet rat (SHAM, n=14). Three months post-ovariectomy, DEXA measurement showed a reduction of bone mineral density reflecting an osteoporotic bone status in OVX rats. Rats then underwent a 3 mm wedge-shaped osteotomy at the distal metaphyseal area of the left femur stabilized with a T-shaped mini-plate and allowed to heal for 6 weeks. Biomechanical competence by means of a non-destructive three-point bending test showed significant lower flexural rigidity in the OVX rats at 3 mm lever span compared to SHAM animals (p=0.048) but no differences at 10 mm lever span. Microcomputer tomography (μCT) showed bridging cortices and consolidation of the defect in both groups, however, no measurable differences were found in either total ossified tissue or vascular volume fraction. Furthermore, histology showed healing discrepancies that were characterized by cartilaginous remnant and more unmineralized tissue presence in the OVX rats compared to more mature consolidation appearance in the SHAM group. In summary, bone defect healing in metaphyseal bone slightly differs between osteoporotic and non-osteoporotic bone in the current 3 mm defect model in both 3mm lever span biomechanical testing and histology.

Background A scaffold for treatment of deep osteochondral defects should be stable, integrate well, and provide a surface for chondrocytes. To meet these demands, a biphasic scaffold of allogenous sterilized bone with a collagen surface was developed. Integration was tested in the sheep model. Material and methods Cartilage chips were taken from the nonweight-bearing area of the left knee of 12 sheep and cultured. After 4 wk a second procedure followed and defects of 9.4-mm diameter at the weight-bearing area of the medial femoral condyle of the right knee were created. The sterilized scaffold was inserted and the cultured autologous chondrocytes were dripped onto the surface. After 6 wk, 3 mo, and 6 mo the animals were sacrificed; the explanted femoral condyles were evaluated macroscopically and using histologic, immunohistochemical, and electronmicroscopic methods. Results After 6 wk the level of the surface was well preserved, after 3 mo parts of the scaffold were sintered but after 6 mo the surface was continuous. Full integration of the allogenous bone could be observed after 6 mo. The surface of the scaffold after 6 wk consisted of bone, but after 3 mo some chondrocytes and after 6 mo a continuous chondral layer could be detected. Conclusions The biphasic scaffold of allogenous bone and collagen proved to be stable and sufficiently integrated in the short- and midterm interval. Whether the chondrocytes on the surface had been derived from implanted chondrocytes or the scaffold with its surface was sufficiently chondroconductive must be answered in further investigations.

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.

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.

Aims Functional nicotinic acetylcholine receptors (nAChR) have been identified in airway epithelia and their location in the apical and basolateral membrane makes them targets for acetylcholine released from neuronal and non-neuronal sources. One function of nAChR in airway epithelia is their involvement in the regulation of transepithelial ion transport by activation of chloride and potassium channels. However, the mechanisms underlying this nicotine-induced activation of ion transport are not fully elucidated. Thus, the aim of this study was to investigate the involvement of adenylyl cyclases in the nicotine-induced ion current in mouse tracheal epithelium. Main methods To evaluate the nicotine-mediated changes of transepithelial ion transport processes electrophysiological Ussing chamber measurements were applied and nicotine-induced ion currents were recorded in the absence and presence of adenylyl cyclase inhibitors. Key findings The ion current changes induced by nicotine (100 μM, apical) were not altered in the presence of high doses of atropine (25 μM, apical and basolateral), underlining the involvement of nAChR. Experiments with the transmembrane adenylyl cyclase inhibitor 2'5'-dideoxyadenosine (50 μM, apical and basolateral) and the soluble adenylyl cyclase inhibitor KH7 (10 μM, apical and basolateral) both reduced the nicotine-mediated ion current to a similar extent. Yet, a statistically significant reduction was obtained only in the experiments with KH7. Significance This study indicates that nicotine binding to nAChR in mouse tracheal epithelium activates transepithelial ion transport involving adenylyl cyclase activity. This might be important for novel therapeutic strategies targeting epithelial ion transport mediated by the non-neuronal cholinergic system.

Bone loss varies according to disease and age and these variations affect bone cells and extracellular matrix. Osteoporosis rat models are widely investigated to assess mechanical and structural properties of bone; however, bone matrix proteins and their discrepant regulation of diseased and aged bone are often overlooked. The current study considered the spine matrix properties of ovariectomized rats (OVX) against control rats (Sham) at 16 months of age. Diseased bone showed less compact structure with inhomogeneous distribution of type 1 collagen (Col1) and changes in osteocyte morphology. Intriguingly, demineralization patches were noticed in the vicinity of blood vessels in the OVX spine. The organic matrix structure was investigated using computational segmentation of collagen fibril properties. In contrast to the aged bone, diseased bone showed longer fibrils and smaller orientation angles. The study shows the potential of quantifying transmission electron microscopy images to predict the mechanical properties of bone tissue.

The bladder urothelium not only provides a diffusion barrier but it also serves a sensor function and releases signalling molecules that are considered to act in a paracrine and autocrine fashion, e.g. by acetylcholine. Its actions are conferred by two classes of receptors, i.e. G-protein-coupled muscarinic receptors (MR) and ionotropic nicotinic receptors (nAChR). In this study we set out to determine the expression and distribution of all MR subtypes (M1R-M5R) and nAChR alpha-subunits 7, 9 and 10 in the human urothelium by means of RT-PCR and immunohistochemistry, respectively. Real-time RT-PCR revealed a rank order of MR subtype expression of M2R>>M3R=M5R>M4R=M1R. Immunohistochemistry demonstrated differential distribution patterns with M1R being restricted to basal cells, M2R nearly exclusively found in umbrella cells, whereas M3R and M4R were homogenously distributed and M5R was seen in a decreasing gradient from luminal to basal. As for nAChR alpha-subunits, rank order of expression is alpha7>>alpha10>alpha9, and they were observed throughout the urothelium with a gradient decreasing from luminal to basal in intensity. In conclusion, the human urothelium carries multiple cholinergic receptor subtypes, with predominant expression of M2R, M3R and alpha7-nAChR. Their distribution as well as that of the less expressed subtypes is layer-specific in the urothelium. In view of the multiplicity of pathways to which different cholinergic receptor subtypes are coupled, we propose that this layer-specific distribution serves to stratify cholinergic regulation of human urothelial function.

Acetylcholine (ACh), derived both from nerve fibres and from non-neuronal sources such as epithelial cells, is a major regulator of airway function. There is evidence that dysfunction of the neuronal cholinergic system is involved in the pathogenesis of asthma. Here, we asked whether the pulmonary non-neuronal ACh-synthesis and release machinery is altered in a rat and a mouse model of allergic airway disease. Animals were sensitized against ovalbumin, challenged by allergen inhalation, and sacrificed 24 or 48 h later. Targets of investigation were the high-affinity choline transporter-1 (CHT1), that mediates cellular uptake of choline, the ACh-synthesizing enzyme choline acetyltransferase (ChAT), the vesicular ACh transporter (VAChT), and the polyspecific organic cation transporters (OCT1-3), which are able to translocate choline and ACh across the plasma membrane. With cell-type specific distribution patterns, immunohistochemistry identified these proteins in airway epithelial cells and alveolar macrophages. Real-time RT-PCR revealed significant decreases in ChAT-, CHT1-, VAChT-, OCT-mRNA in the lung of sensitized and allergen challenged animals. These data were supported by immunohistochemistry, demonstrating reduced labeling intensity of airway epithelial cells. ChAT-, CHT1-, VAChT-, and OCT1-mRNA were also significantly reduced in cells recovered by bronchoalveolar lavage from sensitized and challenged rats. In conclusion, the pulmonary non-neuronal cholinergic system is down-regulated in acute allergic airway inflammation. In view of the role of ACh in maintenance of cell-cell-contacts, stimulation of fluid-secretion and of ciliary beat frequency, this down-regulation may contribute to epithelial shedding and ciliated cell dysfunction that occur in this pathological condition.

Neovascularization is essential for bone regeneration in fractures. This study aimed to investigate the microvascular morphology and distribution in the non-injured femur and the neovascularization of the metaphyseal critical size defect in a small animal model of osteoporosis.Female rats (n=7) were ovariectomized (OVX) and received a multideficiency diet. Three months after OVX, a 5mm wedge shaped critical size defect was cut at the distal femoral metaphysis and stabilized with a T-shaped mini-plate. After six weeks, the animals were euthanized, and femora were removed and decalcified for micro-CT measurement of fracture neovascularization.No fracture healing was observed along the critical size defects. In the non-injured bone, micro-vessel distribution showed a specific pattern, thereby enabling a differentiation between epi-, meta- and diaphysis. Micro-CT based morphometry revealed a significant reduction of the vascular volume fraction as well as the vascular thickness (p0.001) in the critical size defect compared to the intact contralateral femur. Blood volume related vascular surface (vascular surface/volume) increased significantly (p0.001). Connectivity density and tissue volume related vascular surface (vascular surface density) did not change significantly.Micro-CT based vascular morphometry demonstrated differences between epi-, meta- and diaphysis in the non-injured bone as well as differences between the critical size defect and the non-injured metaphysis. As angiogenesis is a crucial prerequisite that precedes osteogenesis, our results may influence further evaluation of osteoconductive or osteogenic biomaterials in this small animal model of osteoporosis.

Bone loss is a symptom related to disease and age, which reflects on bone cells and ECM. Discrepant regulation affects cell proliferation and ECM localization. Rat model of osteoporosis (OVX) was investigated against control rats (Sham) at young and old ages. Biophysical, histological and molecular techniques were implemented to examine the underlying cellular and extracellular matrix changes and to assess the mechanisms contributing to bone loss in the context of aging and the widely used osteoporotic models in rats. Bone loss exhibited a compromised function of bone cells and infiltration of adipocytes into bone marrow. However, the expression of genes regulating collagen catabolic process and adipogenesis was chronologically shifted in diseased bone in comparison with aged bone. The data showed the involvement of Wnt signaling inhibition in adipogenesis and bone loss due to over-expression of SOST in both diseased and aged bone. Further, in the OVX animals, an integrin-mediated ERK activation indicated the role of MAPK in osteoblastogenesis and adipogenesis. The increased PTH levels due to calcium and estrogen deficiency activated osteoblastogenesis. Thusly, RANKL-mediated osteoclastogenesis was initiated. Interestingly, the data show the role of MEPE regulating osteoclast-mediated resorption at late stages in osteoporotic bone. The interplay between ECM and bone cells change tissue microstructure and properties. The involvement of Wnt and MAPK pathways in activating cell proliferation has intriguing similarities to oncogenesis and myeloma. The study indicates the importance of targeting both pathways simultaneously to remedy metabolic bone diseases and age-related bone loss.

In addition to quantal, vesicular release of acetylcholine (ACh), there is also non-quantal release at the motor endplate which is insufficient to evoke postsynaptic responses unless acetylcholinesterase (AChE) is inhibited. We here addressed potential non-quantal release in the mouse trachea by organ bath experiments and (immuno)histochemical methods. Electrical field stimulation (EFS) of nerve terminals elicited tracheal constriction that is largely due to ACh release. Classical enzyme histochemistry demonstrated acetylcholinesterase (AChE) activity in nerve fibers in the muscle and butyrylcholinesterase (BChE) activity in the smooth muscle cells. Acute inhibition of both esterases by eserine significantly raised tracheal tone which was fully sensitive to atropine. This effect was reduced, but not abolished, in AChE, but not in BChE gene-deficient mice. The eserine-induced increase in tracheal tone was unaffected by vesamicol (10(-5)M), an inhibitor of the vesicular acetylcholine transporter, and by corticosterone (10(-4)M), an inhibitor of organic cation transporters. Hemicholinium-3, in low concentrations an inhibitor of the high-affinity choline transporter-1 (CHT1), completely abrogated the eserine effects when applied in high concentrations (10(-4)M) pointing towards an involvement of low-affinity choline transporters. To evaluate the cellular sources of non-quantal ACh release in the trachea, expression of low-affinity choline transporter-like family (CTL1-5) was evaluated by RT-PCR analysis. Even though these transporters were largely abundant in the epithelium, denudation of airway epithelial cells had no effect on eserine-induced tracheal contraction, indicating a non-quantal release of ACh from non-epithelial sources in the airways. These data provide evidence for an epithelium-independent non-vesicular, non-quantal ACh release in the mouse trachea involving low-affinity choline transporters.

Acetylcholinesterase (AChE) hydrolyzes acetylcholine (ACh) to acetate and choline and thereby terminates nerve impulse transmission. ACh is also expressed in bone tissue and enhances here proliferation and differentiation of osteoblasts, which makes it interesting to investigate effects of AChE deficiency on bone. To our knowledge, this is the first study that analyzed bone of heterozygous acetylcholinesterase-knockout (AChE-KO) mice. Tibia, femur, thoracic and lumbar vertebrae of 16-week-old female heterozygous AChE-KO mice and their corresponding wildtypes (WT) were analyzed using real-time RT-PCR, dual-energy X-ray absorptiometry, biomechanics, micro-computed tomography, histology and histomorphometry. Our data revealed that heterozygous AChE-KO did not cause negative effects upon bone parameters analyzed. In contrast, the number of osteoclasts per perimeter was significantly reduced in lumbar vertebrae. In addition, we found a significant decrease in trabecular perimeter of lumbar vertebrae and cortical area fraction (Ct.Ar/Tt.Ar) in the mid-diaphysis of femurs of AChE-KO mice compared to their WT. Therefore, presumably a local homozygous knockout of AChE or AChE-inhibitor administration might be beneficial for bone formation due to ACh accumulation. However, many other bone parameters analyzed did not differ statistically significantly between AChE-KO and WT mice. That might be reasoned by the compensating effect of butyrylcholinesterase (BChE).

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.

Neuronal nicotinic acetylcholine receptors (nAChR) are ligand-gated ion channels that consist of various subunits. During ontogeny, muscular and neuronal nAChR undergo changes in the distribution and subunit composition in skeletal muscle and brain, respectively. Here, we have investigated the occurrence of the ligand-binding alpha-subunits of neuronal nAChR by means of reverse transcription/polymerase chain reaction and immunohistochemistry in the rat heart during prenatal and postnatal development and after capsaicin-induced sensory denervation. mRNAs coding for the alpha4, alpha5, alpha7 and alpha10 subunits were detected throughout all developmental stages. Messenger coding for the alpha2 subunit was first detectable at developmental stage E20; alpha3 subunit mRNA was expressed throughout all prenatal developmental stages, whereas it was restricted postnatally to the atria. mRNA for alpha6 was observed at E14-P8 but was absent thereafter. At no developmental stage could an unequivocal signal for alpha9 nAChR subunit mRNA be obtained. The expression pattern was unchanged by capsaicin treatment. Immunohistochemistry demonstrated alpha7 subunits on cardiac neurons, fibroblasts and cardiomyocytes and alpha2/4 subunits on cardiomyocytes with a postnatal redistribution to intercalated discs, as shown by cryo-immunoelectron microscopy. Our results indicate an additional non-neuronal expression of nAChR subunits in the rat heart that, as in skeletal muscle, precedes functional innervation and then undergoes changes in its distribution on the surface of cells.

Stimulation of nicotinic acetylcholine receptors (nAChR) excites peripheral sensory nerve fibres, but also exert antinociceptive effects. The differences in these nAChR-mediated effects could be related to the expression of different nAChR subtypes located on nociceptive neurons. In the present study, we focused on the recently described alpha 10-nAChR subunit, and on alpha 4 and alpha 7 subunits, which are the most abundant subunits in the central nervous system. In nociceptive neurons from thoracic and lumbar dorsal root ganglia (DRG), nAChR subunits were found at transcriptional (RT-PCR), translational (immunohistochemistry) and functional levels. Cultured DRG neurons express mRNA for the subunits alpha 2-7 and alpha 10. The alpha-subunit proteins 4, 7 and 10 were colocalised in virtually all nociceptive neurons that were identified by immunoreactivity for the vanilloid receptor TRPV-1. These findings were corroborated by current recordings and calcium measurements, which revealed excitatory inward currents and calcium responses in capsaicin sensitive neurons.

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.

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.

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.

Aims In the oviduct, muscarinic acetylcholine receptors (MR) are linked with motility regulation and nicotinic receptors (nAChR) with ectopic pregnancy. We here aimed to determine the repertoire of cholinergic receptor expression in the murine oviduct and their functional coupling to regulation of intracellular calcium concentration ([Ca 2+ ] i ). Main methods Cholinergic receptor transcripts were assessed by RT-PCR in oviductal segments (ampulla, isthmus, uterotubar junction) in all cyclic stages and pregnancy, and in laser-microdissected samples of epithelium and smooth muscle, nAChR subunit α3 distribution in tissue sections using an appropriate genetic reporter mouse strain. [Ca 2+ ] i responses were monitored in ciliated and non-ciliated oviductal cells isolated from wild-type and MR subtypes 1 and 3 gene deficient mice. Key findings Transcripts for all MR subtypes (M1–M5) are constantly expressed whereas there is some variability in nAChR expression from individual to individual. The qualitative expression pattern is independent from the hormonal status of the animal, except for nAChR α7, which is less present during pregnancy. The epithelium expresses M1, M3, nAChR α7 (data from laser-assisted microdissection) and nAChR α3 (ultrastructural investigation of reporter mice). MR dominate over nAChR in increasing [Ca 2+ ] i with being M3 the major, but not sole subtype driving this effect. The general nAChR inhibitor mecamylamine enhances muscarinic and purinergic responses. Significance In conclusion, the murine oviduct is endowed with a multiplicity of muscarinic and nicotinic receptors subtypes that, with respect to regulation of [Ca 2+ ] i , are inversely linked to each other. The major, but not sole, cholinergic receptor driving increase in [Ca 2+ ] i is M3.

The purpose of this study is to assess the mechanical efficacy of an alkylene bis(dilactoyl)-methacrylate-based degradable bone adhesive in 36 sheep. Bone segmentation with osteotomies of the metaphyseal ulna was performed and adhesive was applied into the osteotomy gaps in 18 sheep. The remaining 18 animals served as controls. The segment was subsequently stabilized without any osteosynthesis in all sheep. Six animals of the adhesive group and 6 controls were killed after 21, 42 and 84 days, respectively. Bond strength of the adhesive and quality of fracture healing was studied using biomechanical, histological and radiological methods. There were no significant differences in biomechanical analysis between both groups at any time. However, an increase of in vivo bond strength with the highest stiffness of 102.83 N/mm(2) was observed in the adhesive group after 84 days. In vitro analysis showed non-significant differences in bond strength during polymerization time. Histomorphometric investigations revealed significant differences in osteotomy cross-section area after 84 days, with higher areas of callus in the control. After 84 days the X-ray examinations showed completely bridged gaps in four of six animals in the adhesive and in five animals in the control group. This bone adhesive exhibited good in vivo and in vitro bond strength and mechanical efficiency in both the short and long term without impairment of physiological fracture healing.

In complex tissues where multiple subtypes of nicotinic acetylcholine receptors (nAChRs) are expressed, immunohistochemistry has been the most popular tool for investigation of nAChR subunit distribution. However, recent studies with nAChR subunit knockout mice demonstrated that a large panel of antibodies is unsuitable. Thus, we aimed to develop a histochemical method for selective labeling of alpha7 nAChR with neurotoxins, utilizing alpha7 nAChR-transfected cells, dorsal root ganglia (DRG) and spinal cord from wild-type and knockout mouse. The specificity of Alexa Fluor 488-conjugated alpha-bungarotoxin (Alexa-alphaBgt) was demonstrated in binding to alpha7-transfected cells inhibited by long-chain alpha-cobratoxin (CTX), but not short-chain alpha-neurotoxin II (NTII). In contrast, binding to Torpedo muscle-type nAChRs and to motor end plates in mouse tongue sections was prevented by both CTX and NTII. In tissue sections of DRG, expressing all neuronal nAChR subunits, only CTX precluded Alexa-alphaBgt labeling of neurons, with no staining for alpha7 nAChR knockout tissue. It proved that alpha7 nAChRs are the major alphaBgt-binding sites in mouse DRG. Corresponding results were obtained for terminals in the spinal cord. Thus, we present a protocol utilizing Alexa-alphaBgt and non-labeled CTX/NTII that allows specific histochemical detection of alpha7 nAChR with a spatial resolution at the level of single axon terminals.

High amounts of acetylcholine (ACh) and its synthesising enzyme choline acetyltransferase (ChAT) have been detected in the placenta. Since the placenta is not innervated by extrinsic or intrinsic cholinergic neurons, placental ACh and ChAT originate from non-neuronal sources. In neurons, cytoplasmic ACh is imported into synaptic vesicles by the vesicular acetylcholine transporter (VAChT), and released through vesicular exocytosis. In view of the coordinate expression of VAChT and ChAT from the "cholinergic gene locus" in neurons, we asked whether VAChT is coexpressed with ChAT in rat placenta, and investigated this issue by means of RT-PCR, in situ hybridisation, western blot and immunohistochemistry. Messenger RNA and protein of the common type of ChAT (cChAT), its splice variant peripheral ChAT (pChAT), and VAChT were detected in rat placenta with RT-PCR and western blot. ChAT in situ hybridisation signal and immunoreactivity for cChAT and pChAT were observed in nearly all placental cell types, while VAChT mRNA and immunolabelling were detected in the trophoblast, mesenchymal cells and the visceral yolk sac epithelial cells. While ChAT is nearly ubiquitously expressed in rat placenta, VAChT immunoreactivity is localised cell type specifically, implying that both vesicular and non-vesicular ACh release machineries prevail in placental cell types.

Smoking during pregnancy causes low birth weight, premature delivery, neonatal morbidity, and mortality. Nicotine is a main pathogenic compound of cigarette smoke, and depresses active amino-acid uptake by human placental villi. It binds to the acetylcholine binding site of the alpha-subunits of nicotinic acetylcholine receptors (nAChR). Eight different neuronal nAChR alpha-subunits have been identified in mammals. Here, we investigated their localisation and distribution in the human and rat placenta by RT-PCR and immunofluorescence. The mRNAs of all alpha-subunits are expressed in the human and rat placenta. Immunohistochemically, subunits alpha2-5, alpha7, alpha9 and alpha10 are localised in different combinations in rat cytotrophoblast, human and rat syncytiotrophoblast, vascular smooth muscle cells, endothelial cells, Hofbauer cells, human amnion epithelium and rat visceral yolk sac epithelium. Thus, all human and rat placental cell types exhibit receptor subunits with binding sites for the endogenous ligand ACh and nicotine. ACh is suggested to be an important placental signalling molecule that, through stimulation of nAChR, controls the uptake of nutrients, blood flow and fluid volume in placental vessels, and the vascularisation during placental development. Chronic stimulation of nAChR by nicotine might result in unbalanced receptor activation or functional desensitisation followed by the known pathological effects of smoking.

The contribution of nicotinic acetylcholine receptors (nAChRs) to stimulation of NO-production was investigated in isolated rat dorsal root ganglion (DRG) neurons utilizing an NO-sensitive fluorescent indicator 4,5-diaminofluorescein-diacetate (DAF-2DA) and appropriate channel blockers. RT-PCR and immunohistochemical analysis of NOS isoforms in cultured neurons revealed the expression of eNOS in the vast majority of neurons, nNOS in about 5–10%, and iNOS only exceptionally. Application of nicotine resulted in an abrupt increase in DAF-2T fluorescence in 65% of neuronal cell bodies that was fully sensitive to the general nAChR antagonist mecamylamine. Methyllycaconitine reduced the number of nicotine-sensitive neurons and the extent of NO-generation. Thus, α7- and/or α9/10-nAChRs are required for nicotine-induced NO-production in a subpopulation of DRG neurons, and appear to be partially involved in the remaining, larger subpopulation.

We investigated the occurrence and distribution of the ligand-binding alpha-subunits of nicotinic acetylcholine receptors in the rat arterial system in situ by means of RT-PCR and immunohistochemistry. Except the alpha9-subunit, all other mammalian non-muscular alpha-subunits were expressed in the arterial wall--either in endothelial or in smooth muscle cells--suggesting it as a direct target of nicotine and endogenous acetylcholine. The distribution pattern of alpha-subunits found in smooth muscle cells varied considerably among the individual elastic, muscular and intraparenchymal arteries investigated, suggesting that non-neuronal cholinergic signalling via nicotinic receptors in the vascular wall includes components that are highly specific for individual arteries.

Uptake of choline by the high-affinity choline transporter CHT1 is the rate-limiting step in neuronal acetylcholine (ACh) synthesis. Here, we investigated by RT-PCR, in-situ hybridisation, immunohistochemistry, and Western blotting whether CHT1 is also expressed in cholinergic epithelia. CHT1-mRNA and -protein were detected in keratinocytes of human skin, rat skin and tongue, the human keratinocyte cell line HaCaT, and the ciliated cells of the rat tracheal epithelium. Immunohistochemically, CHT1 was predominantly localized to the epithelial cell membranes, in case of ciliated tracheal cells it was restricted to the apical membrane. This is the first study to demonstrate the expression of CHT1 in non-neuronal cells. The close apposition of CHT1 to reported sites of localization of choline acetyltransferase in these cells is strongly in favour of ACh synthesis being fuelled by choline uptake via CHT1 in these epithelia.

Previous binding studies have suggested the presence of a so far unknown nicotinic acetylcholine receptor subunit in dorsal root ganglia (Pugh et al., 1995). Here, we investigated whether the most recently identified subunit, alpha10, and its potential interaction partner, alpha9 (Elgoyhen et al., 2001), are expressed in these ganglia. All neurons of rat dorsal root ganglia, but no glial cells, expressed both alpha9 and alpha10 mRNA in in situ hybridization, and exhibited alpha10 immunoreactivity using a newly raised antibody. These findings were confirmed by RT-PCR and western blotting. The data show that dorsal root ganglion neurons coexpress alpha9 and alpha10 nicotinic receptor subunits, thereby providing the first example of neuronal expression of this receptor subunit pair.

The rate-limiting step in neuronal acetylcholine (ACh) synthesis is the uptake of choline via a high-affinity transporter. We have generated antisera against the recently identified transporter CHT1 to investigate its distribution in rat motor neurons and skeletal muscle and have used these antisera in combination with (1) antisera against the vesicular acetylcholine transporter (VAChT) to identify cholinergic synapses and (2) Alexa-488-labelled alpha-bungarotoxin to identify motor endplates. In the motor unit, immunohistochemistry and RT-PCR have demonstrated that CHT1 is restricted to motoneurons and absent from the non-neuronal ACh-synthesizing elements, e.g. skeletal muscle fibres. In addition, CHT1 is also present in parasympathetic neurons of the tongue, as evidenced by immunohistochemistry and RT-PCR. CHT1 immunoreativity is principally found at all segments (perikaryon, dendrites, axon) of the motoneuron but is enriched at neuro-neuronal and neuro-muscular synapses. This preferential localisation matches well with its anticipated pivotal role in synaptic transmitter recycling and synthesis.

Background Nicotinic acetylcholine receptors (nAChR) have been identified on a variety of cells of the immune system and are generally considered to trigger anti-inflammatory events. In the present study, we determine the nAChR inventory of rat alveolar macrophages (AM), and investigate the cellular events evoked by stimulation with nicotine. Methods Rat AM were isolated freshly by bronchoalveolar lavage. The expression of nAChR subunits was analyzed by RT-PCR, immunohistochemistry, and Western blotting. To evaluate function of nAChR subunits, electrophysiological recordings and measurements of intracellular calcium concentration ([Ca2+]i) were conducted. Results Positive RT-PCR results were obtained for nAChR subunits α3, α5, α9, α10, β1, and β2, with most stable expression being noted for subunits α9, α10, β1, and β2. Notably, mRNA coding for subunit α7 which is proposed to convey the nicotinic anti-inflammatory response of macrophages from other sources than the lung was not detected. RT-PCR data were supported by immunohistochemistry on AM isolated by lavage, as well as in lung tissue sections and by Western blotting. Neither whole-cell patch clamp recordings nor measurements of [Ca2+]i revealed changes in membrane current in response to ACh and in [Ca2+]i in response to nicotine, respectively. However, nicotine (100 μM), given 2 min prior to ATP, significantly reduced the ATP-induced rise in [Ca2+]i by 30%. This effect was blocked by α-bungarotoxin and did not depend on the presence of extracellular calcium. Conclusions Rat AM are equipped with modulatory nAChR with properties distinct from ionotropic nAChR mediating synaptic transmission in the nervous system. Their stimulation with nicotine dampens ATP-induced Ca2+-release from intracellular stores. Thus, the present study identifies the first acute receptor-mediated nicotinic effect on AM with anti-inflammatory potential.

BACKGROUND Osteoporosis is diagnosed by bone loss using a radiological parameter called T-score. Preclinical studies use DXA to evaluate bone status were the T-score is referenced on bone mineral density (BMD) values of the same animals before treatment. Clinically, the reference BMD represents values of an independent group of healthy patients around 30 years old. The present study established a clinically similar T-score standard to diagnose osteoporosis in a sheep model. MATERIAL AND METHODS We used 31 female merino land sheep (average 5.5 years old) to study osteoporosis. The following groups were compared using DXA measurement: 1) control; 2) ovariectomized (OVX); 3) OVX combined with a deficient diet (OVXD); and 4) OVXD combined with methylprednisolone administration (OVXDS). Further, an independent group of 32 healthy sheep (4-6 years old) were measured as an independent baseline. BMD was measured at 0 months, 3 months, and 8 months after treatment. RESULTS The same significance pattern between the treated groups and either baseline groups was seen. However, using an independent baseline changed the "clinical" interpretation of the data from an osteoporotic bone status (T-score

New bone formation was studied in a metaphyseal fracture-defect in ovariectomized rats stimulated by a plain and a strontium-enriched macroporous silica/collagen scaffold (ScB30 and ScB30Sr20) and a compact silica/collagen xerogel (B30). 45 female Sprague-Dawley rats were randomly assigned to three different treatment groups: (1) ScB30 (n=15), (2) ScB30Sr20 (n=15), and (3) B30 (n=15). 12 weeks after bilateral ovariectomy and multi-deficient diet, a 4 mm wedge-shaped fracture-defect was created at the metaphyseal area of the left femur. A 7-hole T-shaped plate at the lateral aspect of the femur stabilized the bone and the defect was filled with ScB30, ScB30Sr20 or B30 subsequently. After six weeks, histomorphometrical analysis revealed a statistically significant higher bone volume/tissue volume ratio in the ScB30Sr20 group compared to ScB30 (p=0.043) and B30 (p=0.0001) indicating an improved formation of new bone by the strontium-enriched macroporous silica/collagen scaffold. Furthermore, immunohistochemical results showed increased expression of BMP2 and OPG and a decreased RANKL expression in the ScB30Sr20 group. This was further confirmed with the gene expression analysis where an increase in prominent bone formation markers (ALP, OCN, Runx2, Col1a1 and Col10a1) was seen. No material remnants were found in the scaffold group indicating an almost complete degradation process of the biomaterials. This is confirmed by ToF-SIMS analysis that did not detect any strontium in the ScB30Sr20 group neither in the defect nor in the surrounding tissue. Taken together, this study shows the stimulating effects of strontium through increased bone formation by up regulation of osteoanabolic markers. This work also indicates the importance of material porosity, geometry and biodegradability in bone healing.

The arterial vascular wall contains a non-neuronal intrinsic cholinergic system. The rate-limiting step in acetylcholine (ACh) synthesis is choline uptake. A high-affinity choline transporter, CHT1, has recently been cloned from neural tissue and has been identified in epithelial cholinergic cells. Here we investigated its presence in rat and human arteries and in primary cell cultures of rat vascular cells (endothelial cells, smooth muscle cells, fibroblasts). CHT1-mRNA was detected in the arterial wall and in all isolated cell types by RT-PCR using five different CHT1-specific primer pairs. Antisera raised against amino acids 29-40 of the rat sequence labeled a single band (50 kD) in Western blots of rat aorta, and an additional higher molecular weight band appeared in the hippocampus. Immunohistochemistry demonstrated CHT1 immunoreactivity in endothelial and smooth muscle cells in situ and in all cultured cell types. A high-affinity [3H]-choline uptake mechanism sharing characteristics with neuronal high-affinity choline uptake, i.e., sensitivity to hemicholinium-3 and dependence on sodium, was demonstrated in rat thoracic aortic segments by microimager autoradiography. Expression of the high-affinity choline transporter CHT1 is a novel component of the intrinsic non-neuronal cholinergic system of the arterial vascular wall, predominantly in the intimal and medial layers.