Mesoporous Bioactive Glass-Incorporated Injectable Strontium-Containing Calcium Phosphate Cement Enhanced Osteoconductivity in a Critical-Sized Metaphyseal Defect in Osteoporotic Rats.

In this study, the in vitro and in vivo bone formation behavior of mesoporous bioactive glass (MBG) particles incorporated in a pasty strontium-containing calcium phosphate bone cement (pS100G10) was studied in a metaphyseal fracture-defect model in ovariectomized rats and compared to a plain pasty strontium-containing calcium phosphate bone cement (pS100) and control (empty defect) group, respectively. In vitro testing showed good cytocompatibility on human preosteoblasts and ongoing dissolution of the MBG component. Neither the released strontium nor the BMG particles from the pS100G10 had a negative influence on cell viability. Forty-five female Sprague-Dawley rats were randomly assigned to three different treatment groups: (1) pS100 (n = 15), (2) pS100G10 (n = 15), and (3) empty defect (n = 15). Twelve 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 in all animals. The originated fracture-defect was substituted with pS100 or pS100G10 or left empty. After six weeks, histomorphometrical analysis revealed a statistically significant higher bone volume/tissue volume ratio in the pS100G10 group compared to the pS100 (p = 0.03) and empty defect groups (p = 0.0001), indicating enhanced osteoconductivity with the incorporation of MBG. Immunohistochemistry revealed a significant decrease in the RANKL/OPG ratio for pS100 (p = 0.004) and pS100G10 (p = 0.003) compared to the empty defect group. pS100G10 showed a statistically higher expression of BMP-2. In addition, a statistically significant higher gene expression of alkaline phosphatase, osteoprotegerin, collagen1a1, collagen10a1 with a simultaneous decrease in RANKL, and carbonic anhydrase was seen in the pS100 and pS100G10 groups compared to the empty defect group. Mass spectrometric imaging by time-of-flight secondary ion mass spectrometry (ToF-SIMS) showed the release of Sr2+ ions from both pS100 and pS100G10, with a gradient into the interface region. ToF-SIMS imaging also revealed that resorption of the MBG particles allowed for new bone formation in cement pores. In summary, the current work shows better bone formation of the injectable pasty strontium-containing calcium phosphate bone cement with incorporated mesoporous bioactive glass compared to the bioactive-free bone cement and empty defects and can be considered for clinical application for osteopenic fracture defects in the future.

A new small animal model for simulating a two-stage-revision procedure in implant-related methicillin-resistant Staphylococcus aureus bone infection.

BACKGROUND: Implant-related bone infections with methicillin-resistant Staphylococcus aureus (MRSA) remain a challenge for orthopedic surgeons. This devasting complication may lead to functional impairment and loss of the affected limbs. High failure rates in treatment make improvement of surgical treatment necessary. Beside an already established demanding and costly large animal model, a small animal model of a two-stage revision does not exist, yet. Thus, the purpose of this study was to establish a preclinical small animal model to simulate a two-stage revision in implant-related MRSA infection. MATERIALS AND METHODS: In twelve rabbits Steel K-wires were implanted into the intramedullary canal of the left tibia, followed by inoculation with MRSA. Two different clinical isolates of MRSA-strains were used in two different concentrations (CFUs; 105 and 107 colony forming units (CFUs). This led to four groups of three rabbits each. Eleven rabbits survived the whole study period. After four weeks the inoculated K-wires were removed and replaced with vancomycin loaded PMMA-spacers (stage 1). Twenty-eight days later new K-wire implants were placed intramedullary (stage 2). After 84 days all animals were sacrificed. Tibiae were analyzed microbiologically, radiologically and histologically. RESULTS: In every rabbit K-wire associated infection could be established within the first four weeks. After irrigation and debridement at revision one (stage 1), infection could be eradicated in 67% of group I, in 50% of group II and in 33% of group III and IV. Recurrence of the infection could be determined in all animals of group I and IV at day 84. X-ray analysis and histology both demonstrated clear signs of osteomyelitis after twelve weeks. Survival, clinical observations and weight assessment confirmed the ethical justifiable stress of the animals during the experiment. CONCLUSION: The presented small animal model of a two-stage revision in implant-related infection is a promising preclinical set-up for assessment of new treatment strategies of implant-related infections. Both high survival as well as reinfection rates were possible by simulating the clinical gold standard of two-stage revision surgery in an MRSA implant-related infection model. Therefore, the model can be deemed suitable for further preclinical in vivo testing.

Nonsurgical Clinical Management of Periapical Lesions Using Calcium Hydroxide-Iodoform-Silicon-Oil Paste.

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.

High resolution imaging and 3D analysis of Ag nanoparticles in cells with ToF-SIMS and delayed extraction.

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.

Strontium and bisphosphonate coated iron foam scaffolds for osteoporotic fracture defect healing.

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, µCt 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.

A New Clinically Relevant T-Score Standard to Interpret Bone Status in a Sheep Model.

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 <-2.5) after 3 months of OXDS treatment into an osteopenic bone status (T-score <-1.5 to -2.4). CONCLUSIONS Using an independent baseline enhanced the statistical significance and showed the clinical relevance. Furthermore, an independent baseline is a reliable alternative to use of a new control group for future experiments and thus reduces the number of animals needed by eliminating the need for a control and corresponding to clinical practice.

Nicotinic Acetylcholine Receptor α9 and α10 Subunits Are Expressed in the Brain of Mice.

The α9 and α10 nicotinic acetylcholine receptor (nAChR) subunits are likely to be the evolutionary precursors to the entire cys-loop superfamily of ligand-gated ion channels, which includes acetylcholine, GABA, glycine and serotonin ionotropic receptors. nAChRs containing α9 and α10 subunits are found in the inner ear, dorsal root ganglia and many non-excitable tissues, but their expression in the central nervous system has not been definitely demonstrated. Here we show the presence of both α9 and α10 nAChR subunits in the mouse brain by RT-PCR and immunochemical approaches with a range of nAChR subunit-selective antibodies, which selectivity was demonstrated in the brain preparations of α7-/-, α9-/- and α10-/- mice. The α9 and α10 RNA transcripts were found in medulla oblongata (MO), cerebellum, midbrain (MB), thalamus and putamen (TP), somatosensory cortex (SC), frontal cortex (FC) and hippocampus. High α9-selective signal in ELISA was observed in the FC, SC, MO, TP and hippocampus and α10-selective signal was the highest in MO and FC. The α9 and α10 proteins were found in the brain mitochondria, while their presence on the plasma membrane has not been definitely confirmed The α7-, α9- and α10-selective antibodies stained mainly neurons and hypertrophied astrocytes, but not microglia. The α9- and α10-positive cells formed ordered structures or zones in cerebellum and superior olive (SO) and were randomly distributed among α7-positive cells in the FC; they were found in CA1, CA3 and CA4, but not in CA2 region of the hippocampus. The α9 and α10 subunits were up-regulated in α7-/- mice and both α7 and α9 subunits were down-regulated in α10-/- mice. We conclude that α9 and α10 nAChR subunits are expressed in distinct neurons of the mouse brain and in the brain mitochondria and are compensatory up-regulated in the absence of α7 subunits.

Osteocyte Regulation of Receptor Activator of NF-κB Ligand/Osteoprotegerin in a Sheep Model of Osteoporosis.

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.

TLR9 mediates S. aureus killing inside osteoblasts via induction of oxidative stress.

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.

Bone formation and degradation behavior of nanocrystalline hydroxyapatite with or without collagen-type 1 in osteoporotic bone defects - an experimental study in osteoporotic goats.

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.

Effects of macroporous, strontium loaded xerogel-scaffolds on new bone formation in critical-size metaphyseal fracture defects in ovariectomized rats.

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.

Neovascularization of osteoporotic metaphyseal bone defects: A morphometric micro-CT study.

PURPOSE: 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. MATERIALS AND METHODS: 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. RESULTS: 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 (p<0.001) in the critical size defect compared to the intact contralateral femur. Blood volume related vascular surface (vascular surface/volume) increased significantly (p<0.001). Connectivity density and tissue volume related vascular surface (vascular surface density) did not change significantly. CONCLUSIONS: 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.

Effects of dendritic core-shell glycoarchitectures on primary mesenchymal stem cells and osteoblasts obtained from different human donors.

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.

Cholinergic activation of the murine trachealis muscle via non-vesicular acetylcholine release involving low-affinity choline transporters.

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.

Impaired extracellular matrix structure resulting from malnutrition in ovariectomized mature rats.

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.

Histological Comparison of New Biodegradable Magnesium-Based Implants for Maxillofacial Applications.

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.

Small changes in bone structure of female α7 nicotinic acetylcholine receptor knockout mice.

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 < 0.05) and cortical thickness (p < 0.05) compared to α7WT, whereas gene expression of osteoclast marker cathepsin K was declined. ToF-SIMS analysis detected a decrease in trabecular calcium content and an increase in C4H6N(+) (p < 0.05) and C4H8N(+) (p < 0.001) collagen fragments whereas a loss of osteoid was found by means of TEM. CONCLUSIONS: Our results on female α7KO bone identified differences in bone strength and composition. In addition, we could demonstrate that α7-nAChRs are involved in regulation of bone remodelling. In contrast to mAChR M3 and nAChR subunit α2 the α7-nAChR favours reduction of bone strength thereby showing similar effects as α7ß2-nAChR in male mice. nAChR are able to form heteropentameric receptors containing α- and ß-subunits as well as the subunits α7 can be arranged as homopentameric cation channel. The different effects of homopentameric and heteropentameric α7-nAChR on bone need to be analysed in future studies as well as gender effects of cholinergic receptors on bone homeostasis.

In vitro assessment of nanosilver-functionalized PMMA bone cement on primary human mesenchymal stem cells and osteoblasts.

Peri-prosthetic infections caused by multidrug resistant bacteria have become a serious problem in surgery and orthopedics. The aim is to introduce biomaterials that avoid implant-related infections caused by multiresistant bacteria. The efficacy of silver nanoparticles (AgNP) against a broad spectrum of bacteria and against multiresistant pathogens has been repeatedly described. In the present study polymethylmethacrylate (PMMA) bone cement functionalized with AgNP and/or gentamicin were tested regarding their biocompatibility with bone forming cells. Therefore, influences on viability, cell number and differentiation of primary human mesenchymal stem cells (MSCs) and MSCs cultured in osteogenic differentiation media (MSC-OM) caused by the implant materials were studied. Furthermore, the growth behavior and the morphology of the cells on the testing material were observed. Finally, we examined the induction of cell stress, regarding antioxidative defense and endoplasmatic reticulum stress. We demonstrated similar cytocompatibility of PMMA loaded with AgNP compared to plain PMMA or PMMA loaded with gentamicin. There was no decrease in cell number, viability and osteogenic differentiation and no induction of cell stress for all three PMMA variants after 21 days. Addition of gentamicin to AgNP-loaded PMMA led to a slight decrease in osteogenic differentiation. Also an increase in cell stress was detectable for PMMA loaded with gentamicin and AgNP. In conclusion, supplementation of PMMA bone cement with gentamicin, AgNP, and both results in bone implants with an antibacterial potency and suitable cytocompatibility in MSCs and MSC-OM.

Rifampicin-fosfomycin coating for cementless endoprostheses: antimicrobial effects against methicillin-sensitive Staphylococcus aureus (MSSA) and methicillin-resistant Staphylococcus aureus (MRSA).

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 28days 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.

Biomimetic PEG-catecholates for stabile antifouling coatings on metal surfaces: applications on TiO2 and stainless steel.

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.