Osteoporosis accounts for a leading cause of degenerative skeletal disease in the elderly. Osteoblast dysfunction is a prominent feature of age-induced bone loss. While microRNAs regulate osteogenic cell behavior and bone mineral acquisition, however, their function to osteoblast senescence during age-mediated osteoporosis remains elusive. This study aims to utilize osteoblast-specific microRNA-29a (miR-29a) transgenic mice to characterize its role in bone cell aging and bone mass. Young (3 months old) and aged (9 months old) transgenic mice overexpressing miR-29a (miR-29aTg) driven by osteocalcin promoter and wild-type (WT) mice were bred for study. Bone mineral density, trabecular morphometry, and biomechanical properties were quantified using μCT imaging, material testing system and histomorphometry. Aged osteoblasts and senescence markers were probed using immunofluorescence, flow cytometry for apoptotic maker annexin V, and RT-PCR. Significantly decreased bone mineral density, sparse trabecular morphometry (trabecular volume, thickness, and number), and poor biomechanical properties (maximum force and breaking force) along with low miR-29a expression occurred in aged WT mice. Aging significantly upregulated the expression of senescence markers p16INK4a, p21Waf/Cip1, and p53 in osteoporotic bone in WT mice. Of note, the severity of bone mass and biomechanical strength loss, as well as bone cell senescence, was remarkably compromised in aged miR-29aTg mice. In vitro, knocking down miR-29a accelerated senescent (β-galactosidase activity and senescence markers) and apoptotic reactions (capsas3 activation and TUNEL staining), but reduced mineralized matrix accumulation in osteoblasts. Forced miR-29a expression attenuated inflammatory cytokine-induced aging process and retained osteogenic differentiation capacity. Mechanistically, miR-29a dragged osteoblast senescence through targeting 3′-untranslated region of anti-aging regulator FoxO3 to upregulate that of expression as evident from luciferase activity assessment. Low miR-29a signaling speeds up aging-induced osteoblast dysfunction and osteoporosis development. Gain of miR-29a function interrupts osteoblast senescence and shields bone tissue from age-induced osteoporosis. The robust analysis sheds light to the protective actions of miR-29a to skeletal metabolism and conveys a perspective of miR-29a signaling enhancement beneficial for aged skeletons

Peri-prosthetic osteolysis and subsequent aseptic loosening is the most common reason for revising total hip replacements. Wear particles originating from the prosthetic components interact with multiple cell types in the peri-prosthetic region resulting in an inflammatory process that ultimately leads to peri-prosthetic bone loss. These cells include macrophages, osteoclasts, osteoblasts and fibroblasts. The majority of research in peri-prosthetic osteolysis has concentrated on the role played by osteoclasts and macrophages. The purpose of this review is to assess the role of the osteoblast in peri-prosthetic osteolysis. In peri-prosthetic osteolysis, wear particles may affect osteoblasts and contribute to the osteolytic process by two mechanisms. First, particles and metallic ions have been shown to inhibit the osteoblast in terms of its ability to secrete mineralised bone matrix, by reducing calcium deposition, alkaline phosphatase activity and its ability to proliferate. Secondly, particles and metallic ions have been shown to stimulate osteoblasts to produce pro inflammatory mediators in vitro. In vivo, these mediators have the potential to attract pro-inflammatory cells to the peri-prosthetic area and stimulate osteoclasts to absorb bone. Further research is needed to fully define the role of the osteoblast in peri-prosthetic osteolysis and to explore its potential role as a therapeutic target in this condition. Cite this article: Bone Joint J 2013;95-B:1021–5

The T-lymphocyte secreted pro-inflammatory cytokine, interleukin-17F (IL-17F), was found to be a key mediator in the cellular response of the immune system in the early phase of fracture repair but its intracellular signaling processes are currently not known in osteoblasts. The objective of this study was to identify the signaling proteins and crucial gene targets involved in osteoblast activation via IL-17F. It was hypothesised that IL-17F stimulated osteoblast maturation through a novel GSK3beta / beta-catenin independent pathway. Mouse pre-osteoblast cell line (MC3T3-E1) was used for IL-17F or Wnt3a treatment. Desired proteins were detected using western blot analysis (antibodies: Phospho-GSK-3beta (Tyr 216), Phospho-GSK-3beta (Ser9), Runx2/cbfa1, TRAF6, Act1, p-ERK2, p-JNK and p-MAPK, C/EBP-beta and & delta). Gene-specific siRNAs of mouse IL-17Ra, IL-17Rc and a non-targeting siRNA (control) were utilised. MC3T3-E1 were transfected with IL-17Ra, IL-17Rc or Negative Control and treated with IL-17F. Chromatin Immunoprecipitation (ChIP-qPCR) was used to evaluate the mouse Runx2 P1 promoter region. IL-17F increased expression of Col1, BSP, Runx2/cbfa1 and osteocalcin in MC3T3-E1 cells. Western blot analysis confirmed expression of known Wnt signaling proteins TRAF6, Act1, p-ERK2, p-JNK and p-MAPK in both IL-17F and Wnt3a treated cultures, including up-regulation of Runx2/cbfa1, a key transcription factor associated with osteoblast differentiation. IL-17F up-regulation of Runx2/cbfa1 appears independent of the Wnt/beta-catenin pathway as phosphorylated GSK-3beta at the Ser9 site was not detected with IL-17F treatment. Despite this, IL-17F treatment still increased expression of Runx2/cbfa1 downstream, lending evidence for a GSK3beta/beta-catenin independent manner of IL-17F stimulated osteogenesis. While IL-17F and Wnt3a both induced expression of C/EBP-delta, only IL-17F treatment induced expression of C/EBP-beta, an upstream transcription factor of Runx2/cbfa1. Further, siRNA knock down of the IL-17 receptors directly decreased Act1, C/EBP-beta and Runx2/cfba1 expression. By ChIP analysis, IL-17F was shown to upregulate C/EBP-beta expression and stimulated its binding to the P1 Promoter of the Runx2/cbfa1 gene. The C/EBP-beta transcription factor was shown to be a key regulator of early osteogenesis. C/EBP-beta up-regulates Runx2/cbfa1 expression by directly binding to the Runx2/cbfa1 P1 promoter in osteoblasts. C/EBP-beta was activated in the osteoblast by IL-17F but not by Wnt3a adding further support to a novel GSK3beta/beta-catenin independent pathway. Our data shows that IL-17F, a cytokine secreted by T-lymphocytes, stimulates osteoblast maturation through a novel GSK3beta/beta-catenin independent pathway and reveals a crucial interaction between C/EBP-beta and the Runx2/cbfa1 P1 promoter not previously been shown in osteogenesis signaling further

Titanium (Ti) is well known in orthopedic implant materials such as total hip replacement arthroplasty. Osseointegration of orthopedic implants is defined as the formation of a direct interface between the implant and the bone without intervening soft tissue. Unmodified Ti is not sufficient to complete adhesion between Ti surface and host bone with subsequent implant loosening over time and ultimately implant failure. An effective approach to enhance the biological activity of orthopedic implants and improve post-implantation healing is to modify the implant surface. The aim of this study was to investigate the effect of functionalized titanium (Ti) with alendronate (Aln) and bone morphogenic protein-2 (BMP-2) for enhancement of osteoblast activity in vitro. Aln and/or BMP-2 were sequentially immobilized to the heparinized-Ti (Hep-Ti) surface. The compositions of pristine Ti and Hep-Ti with or without Aln and/or BMP-2 were characterized by scanning electron microscope (SEM) and X-ray photoelectron spectroscopy (XPS). Osteoblast activities on all Ti substrates were investigated by cell proliferation assays, alkaline phosphate (ALP) activity, calcium deposition, gene expressions of osteocalcin and osteopontin. The modified Ti surface with heparin, Aln, BMP-2 and Aln/BMP-2 showed similar morphologies compared to that of pristine Ti on scanning electron microscope (SEM) and X-ray photoelectron spectroscopy (XPS). Aln or BMP-2 from Aln/Hep-Ti, BMP-2/Hep-Ti or Aln/BMP-2/ Hep-Ti substrates exhibited sustained release profiles up to 4 weeks. No significant cytotoxic effects were observed for incubation periods for up to 48 h. the ALP activity of MG-63 cells cultured on Hep-Ti was not significantly different compared to those cultured on pristine Ti for 7, 14, and 21 days. Alkaline phosphatase(ALP) activities of osteoblasts cultured on Ti groups immobilized with Aln, BMP-2, or Aln/BMP-2 were significantly increased when compared to pristine Ti(p < 0.05). Calcium deposition was markedly increased in Aln/BMP-2/Hep-Ti compared to Aln/Hep-Ti or BMP-2/Hep-Ti, respectively (p < 0.05). mRNA expressions of osteocalcin(OCN) and osteopontin(OPN) of osteoblasts grown on Aln/Hep-Ti, BMP-2/Hep-Ti, and Aln/BMP-2/Hep-Ti were significantly higher than of those grown on pristine Ti (p < 0.05). Based on the results of the in vitro studies, we showed that co-delivery of alendronate and BMP-2 had an additive effect on osteoblast activity and mineralization when compared with pristine Ti as well as alendronate or BMP-2 alone. Functionalized Ti systems with alendronate and BMP-2 can give a good solution to solve the most common problems associated with orthopedic and dental implants. Furthermore, in vivo studies required to determine the optimal doses of alendronate and BMP-2 for clinical application

Orthopaedic surgeons are astounded with the strength of bone found in Polynesians. Furthermore the rate at which new Polynesian bone over-grows metal fixation of a recent fracture is impressive. Studies demonstrate that Polynesians have a higher Bone Mineral Density (BMD) than age and weight matched Europeans in NZ (1, 2). In addition, Polynesians have a lower incidence of hip fractures when compared to other ethnic groups (3). This suggests that the higher BMD or other inherent differences must account for the lower incidence of hip fractures in Polynesians. The aim of this study was to identify (if any) a difference in osteoblast mitosis between European and Polynesian bone. Samples were collected from 13 patients that had joint replacements in accordance with the MCNZ ethics approval. The bone is processed and osteoblasts cultured in the lab to 50% confluence. The cells are then tagged with Propidium Iodide. Using Fluorescence-Activated Cell Sorting (or FACS) the number of osteoblasts in the different phases of the cell cycle are counted. The percentage of cells in G0/G1, S and G2/M phase can be determined by entering the FACS data into a program called mod-fit. This study shows that Polynesians have a greater proportion of cells undergoing replication (i.e S-phase) than their European counterparts. Incidentally we have also shown that the proportion of cells undergoing mitosis lowers with age irrespective of ethnicity

Bacterial contamination of endoprostheses especially in revision surgery is an upcoming problem according to increasing number of joint replacements. Early adherence of bacteria producing a biofilm is difficult to treat. Silver coating of implants offers the opportunity to avoid bacterial adhesions acting against all relevant bacteria causing infections on the implant. We developed a new technique of nano-silver coating using elemental silver covered with SiOxCy whose thickness can be varied determing duration of the coating on the implant. The SiOxCy and silver is completely soluble at least at 3 months. The silver coatings used so far are measuring at least 10um and they are not soluble making a cementless implantation of the endoprostheses impossible. The aim of this study was to test the compatibility of the new combined coating with human osteoblastic cells. The test was carried out with fHOB 1.19 (ATCCR CRL-11372TM). The cells were cultivated in 1:1 mixture of DMEM/Ham's F12 with usual supplements. The protein content was measured colourimetrically using BCA reagents and staining of the cells was done with XTT-reagent (Roche). The cells were incubated on Titanium and PEEK with and without coating for 2,6,16 and 48 hours. No adverse effects of the silver coating on the early cell adhesion at 2 and 6 hours and the further proliferation at 16 and 48 hours were observed. The adhesion on Titanium showed no significant difference against coated Titanium but an improvement of cell adhesion was seen on coated PEEK. This soluble silver coating did not negatively influence human osteoblastic cells. As the complete surfacing is soluble it might be possible to combine early protection against bacteria and osseous integration. An animal study is in progress verifying the in vitro results. It should investigate the maximum duration of the coating on the implant not disturbing osseous integration

Aim. Bone regeneration following the treatment of Staphylococcal bone infection or osteomyelitis is challenging due to the ability of Staphylococcus aureus to invade and persist within bone cells, which could possibly lead to antimicrobial tolerance and incessant bone destruction. Here, we investigated the influence of Staphylococcal bone infection on osteoblasts metabolism and function, with the underlying goal of determining whether Staphylococcus aureus-infected osteoblasts retain their ability to produce extracellular mineralized organic matrix after antibiotic treatment. Method. Using our in vitro infection model, human osteoblasts-like Saos-2 cells were infected with high-grade Staphylococcus aureus EDCC 5055 strain, and then treated with 8 µg/ml rifampicin and osteogenic stimulators up to 21-days. Results. Immunofluorescence and transmission electron microscopic (TEM) imaging demonstrated the presence of intracellular bacteria within the infected osteoblasts as early as 2 hours post-infection. TEM micrographs revealed intact intracellular bacteria with dividing septa indicative of active replication. The infected osteoblasts showed significant amounts of intracellular bacteria colonies and alteration in metabolic activity compared to the uninfected osteoblasts (p≤0.001). Treatment of S. aureus-infected osteoblasts with a single dose of 8 µg/ml rifampicin sufficiently restored the metabolic activity comparative to the uninfected groups. Alizarin red staining and quantification of the rifampicin-treated infected osteoblasts revealed significantly lower amount of mineralized extracellular matrix after 7-days osteogenesis (p<0.05). Interestingly, prolonged osteogenic stimulation and rifampicin-treatment up to 21 days improved the extracellular matrix mineralization level comparable to the rifampicin-treated uninfected group. However, the untreated (native) osteoblasts showed significantly more quantity of mineral deposits (p≤0.001). Ultrastructural analysis of the rifampicin-treated infected osteoblasts at 21-days osteogenesis revealed active osteoblasts and newly differentiated osteocytes, with densely distributed calcium crystal deposits within the extracellular organic matrix. Moreover, residual colony of dead bacteria bodies and empty vacuoles of the fully degraded bacteria embedded within the mineralized extracellular matrix. Gene expression level of prominent bone formation markers, namely RUNX2, COL1A1, ALPL, BMP-2, SPARC, BGLAP, OPG/RANKL showed no significant difference between the infected and uninfected osteoblast at 21-days of osteogenesis. Conclusions. Staphylococcus aureus bone infection can drastically impair osteoblasts metabolism and function. However, treatment with potent intracellular penetrating antibiotics, namely rifampicin restored the metabolic and bone formation activity of surviving osteoblasts. Delay in early osteogenesis caused by the bacterial infection was significantly improved over time after successful intracellular bacteria eradication

Aim. Periprosthetic joint infections follow 1-3% of arthroplasty surgeries, with the biofilm nature of these infections presenting a significant treatment challenge. 1. Prevention strategies include antibiotic-loaded bone cement; however, increases in cementless procedures means there is an urgent need for alternative local antimicrobial delivery methods. 2. A novel, ultrathin, silica-based sol-gel technology is evaluated in this research as an anti-infective coating for orthopaedic prosthetic devices, providing local antibiotic release following surgery. Method. Reduction in clinically relevant microbial activity and biofilm reduction by antimicrobial sol-gel coatings, containing a selection of antibiotics, were assessed via disc diffusion and microdilution culture assays using the Calgary biofilm device. 3. Proliferation, morphology, collagen, and calcium production by primary bovine osteoblasts cultured upon antibiotic sol-gel surfaces were examined, and cytotoxicity evaluated using Alamar blue staining and lactate dehydrogenase assays. Concentrations of silica, calcium and phosphorus compounds within the cell layer cultured on sol-gel coatings and concentrations eluted into media, were quantified using ICP-OES. Furthermore, cellular phenotype was assessed using alkaline phosphatase activity with time in culture. Results. Low antibiotic concentrations within sol-gel had an inhibitory effect on clinically relevant biofilm growth, for example 0.8 mg ml. -1. tobramycin inhibited clinically isolated S. aureus (MRSA) growth with an 8-log reduction in viable colony forming units. There was no significant difference in metabolic activity between untreated and sol-gel exposed primary bovine osteoblasts in elution-based assays. Reduction (2-fold) in metabolic activity in direct contact assays after 48 hours exposure was likely to be due to increased osteoinduction, whereas no impact upon cell proliferation were observed (p=0.92 at 14 days culture). The morphology of primary osteoblasts was unaffected by culture on sol-gel coatings and collagen production was maintained. Calcium containing nodule production within bovine osteoblastic cells was increased 16-fold after 14 days culture upon sol-gel. Conclusions. The ultrathin sol-gel coating showed low cytotoxicity, strong biofilm reducing activity and antimicrobial activity, which was comparable to antibiotics alone, demonstrating that sol-gel delivery of antibiotics could provide local antimicrobial effects to inhibit PJI growth without the need for bone cement. Future work will develop and evaluate sol-gel performance in an ex vivo explant bone infection model which will reduce the need for animal experimentation

Aim. Here, we are aimed to evaluate bacteriophage (191219) to treat S. aureus implant-associated bone infections by means of testing against S. aureus during its planktonic, biofilm and intracellular growth phases and finally assessing antimicrobial effect on in vivo biofilm formed on metal K-wire in an alternative insect model Galleria mellonella. Method. The bacteriophages (191219) were provided from D&D Pharma GmbH. These bacteriophages were tested against S. aureus EDCC 5055 (MSSA) and S. aureus DSM 21979 (MRSA) strains. To assess the activity of bacteriophages against planktonic growth phase, bacteriophages, and S. aureus EDCC 5055(1×10. 7. CFU/ml) were co-cultured in LB media as multiplicity of infection (MOI) of 10, 1, 0.1, and 0.01 for 24 hours at 37. o. C and finally plated out on the LB agar plates to estimate the bacterial growth. The antimicrobial activity of bacteriophages on biofilms in vitro was measured by analysing the incubating the several fold dilutions of bacteriophages in LB media with biofilms formed on 96-well plate. The eradication of biofilm was analysed with crystal violet as well as CFU analysis methods. Later, the effect of bacteriophages on intracellular growth of S. aureus in side osteoblast was tested by treating the S. aureus infected osteoblasts at 2h, 4h and 24h time points of post treatment. In addition, we have analysed synergistic effect with gentamicin and rifampicin antibiotics to clear intracellular S. aureus. Finally, experiments are performed to prove the effect of bacteriophages to clear in vivo biofilm using alternative insect model G. mellonella as well as to detect the presence of bacteriophages inside the osteoblasts through transmission electron microscopy (TEM) analysis. Results. Our results demonstrate the in vitro efficacy of bacteriophages against planktonic S. aureus. Transmission electron microscopy (TEM) experiments revealed severe infection of bacteria by bacteriophages. Bacteriophages also eradicated in a dose-dependent manner in vitro S. aureus biofilm formation and were active against intracellular S. aureus in an osteoblastic cell line. TEM analysis visualized the effect of the bacteriophages on S. aureus inside the osteoblasts with the destruction of the intracellular bacteria and formation of new bacteriophages. For the Galleria infection model, single administration of phages failed to show improvement in survival rates, but exhibited some synergistic effects with gentamicin or rifampicin, which was not statistically significant. Conclusions. In summary, bacteriophages could be a potential adjuvant treatment strategy for patients with implant-associated biofilm infections. Further preclinical and clinical trials are required to establish adequate treatment protocols

One out of nine Canadian males would suffer prostate cancer (PC) during his lifetime. Life expectancy of males with PC has increased with modern therapy and 90% live >10 years. However, 20% of PC-affected males would develop incurable metastatic diseases. Bone metastases (BM) are present in ~80% of metastatic PC patients, and are the most severe complication of PC, generating severe pain, fractures, spinal cord compression, and death. Interestingly, PC-BMs are mostly osteoblastic. However, the structure of this newly formed bone and how it relates to pain and fracture are unknown. Due to androgen antagonist treatment, different PC phenotypes develop with differential dependency on androgen receptor (AR) signaling: androgen-dependent (AR+), double negative (AR-) and neuroendocrine. How these phenotypes are related to changes in bone structure has not been studied. Here we show a state-of-the-art structural characterization of PCBM and how PC phenotypes are associated to abnormal bone formation in PCBM. Cadaveric samples (n=14) obtained from metastases of PC in thoracic or lumbar vertebrae (mean age 74yo) were used to analyze bone structure. We used micro-computed tomography (mCT) to analyze the three-dimensional structure of the bone samples. After imaging, the samples were sectioned and one 3mm thick section was embedded in epoxy-resin, ground and polished. Scanning electron microscopy (SEM)/energy-dispersive X-ray spectroscopy (EDS) and quantitative backscattering electron (qBSE) imaging were used to determine mineral morphology and composition. Another section was used for histological analysis of the PC-affected bone. Collagen structure, fibril orientation and extracellular matrix composition were characterized using histochemistry. Additionally, we obtained biopsies of 3 PCBM patients undergoing emergency decompression surgery following vertebral fracture and used them for immunohistological characterization. By using mCT, we observed three dysmorphic bone patterns: osteolytic pattern with thinned trabecula of otherwise well-organized structures, osteoblastic pattern defined as accumulation of disorganized matrix deposited on pre-existing trabecula, and osteoblastic pattern with minimum residual trabecula and bone space dominated by accumulation of disorganized mineralized matrix. Comparing mCT data with patho/clinical parameters revealed a trend for higher bone density in males with larger PSA increase. Through histological sections, we observed that PC-affected bone, lacks collagen alignment structure, have a higher number of lacunae and increased amount of proteoglycans as decorin. Immunohistochemistry of biopsies revealed that PC-cells inside bone organize into two manners: i) glandular-like structures where cells maintain their polarization in the expression of prostate markers, ii) diffuse infiltrate that spreads along bone surfaces, with loss of cell polarity. These cells take direct contact with osteoblasts in the surface of trabecula. We define that PCBM are mostly composed by AR+ with some double negative cells. We did not observe neuroendocrine phenotype cells. PCBMs generate predominantly osteoblastic lesions that are characterized by high lacunar density, lack of collagen organization and elevated proteoglycan content. These structural changes are associated with the infiltration of PC cells that are mostly androgen-dependent but have lost their polarization and contact directly with osteoblasts, perhaps altering their function. These changes could be associated with lower mechanical properties that led to fracture and weakness of the PCBM affected bone

Previous studies have described an age-dependent distortion of bone microarchitecture for α-CGRP-deficient mice (3). In addition, we observed changes in cell survival and activity of osteoblasts and osteoclasts isolated from young wildtype (WT) mice when stimulated with α-CGRP whereas loss of α-CGRP showed only little effects on bone cell metabolism of cells isolated from young α-CGRP-deficient mice. We assume that aging processes differently affect bone cell metabolism in the absence and presence of α-CGRP. To further explore this hypothesis, we investigated and compared cell metabolism of osteoblasts and bone marrow derived macrophages (BMM)/osteoclast cultures isolated from young (8–12 weeks) and old (9 month) α-CGRP-deficient mice and age matched WT controls. Isolation/differentiation of bone marrow macrophages (BMM, for 5 days) to osteoclasts and osteoblast-like cells (for 7/14/21 days) from young (8–12 weeks) and old (9 month) female α-CGRP−/− and WT control (both C57Bl/6J) mice according to established protocols. We analyzed cell migration of osteoblast-like cells out of femoral bone chips (crystal violet staining), proliferation (BrdU incorporation) and caspase 3/7-activity (apoptosis rate). Alkaline phosphatase (ALP) activity reflects osteoblast bone formation activity and counting of multinucleated (≥ 3 nuclei), TRAP (tartrate resistant acid phosphatase) stained osteoclasts reflects osteoclast differentiation capacity. We counted reduced numbers of BMM from young α-CGRP−/− mice after initial seeding compared to young WT controls but we found no differences between old α-CGRP−/− mice and age-matched controls. Total BMM number was higher in old compared to young animals. Migration of osteoblast-like cells out of bone chips was comparable in both, young and old α-CGRP−/− and WT mice, but number of osteoblast-like cells was lower in old compared to young animals. Proliferation of old α-CGRP−/− BMM was higher when compared to age-matched WT whereas proliferation of old α-CGRP−/− osteoblasts after 21 days of osteogenic differentiation was lower. No differences in bone cell proliferation was detected between young α-CGRP−/− and age-machted WT mice. Caspase 3/7 activity of bone cells from young as well as old α-CGRP−/− mice was comparable to age-matched controls. Number of TRAP-positive multinucleated osteoclasts from young α-CGRP−/− mice was by trend higher compared to age-matched WT whereas no difference was observed in osteoclast cultures from old α-CGRP−/− mice and old WT. ALP activity, as a marker for bone formation activity, was comparable in young WT and α-CGRP−/− osteoblasts throughout all time points whereas ALP activity was strongly reduced in old α-CGRP−/− osteoblasts after 21 days of osteogenic differentiation compared to age-matched WT. Our data indicate that loss of α-CGRP results in a reduction of bone formation rate in older individuals caused by lower proliferation and reduced activity of osteogenic cells but has no profound effects on bone resorption rate. We suggest that the osteopenic bone phenotype described in aged α-CGRP-deficient mice could be due to an increase of dysfunctional matured osteoblasts during aging resulting in impaired bone formation

Aim. Staphylococcus aureus is the first causative agent of bone and joints infections (BJI). It causes difficult-to-treat infections because of its ability to form biofilms, and to be internalized and persist inside osteoblastic cells. Recently, phage therapy has emerged as a promising therapy to improve the management of chronic BJI. In the present study, we evaluated the efficacy of an assembly of three bacteriophages previously used in a clinical case report (Ferry, 2018) against S. aureus in in vitro models of biofilm and intracellular osteoblast infection. Methods. Using HG001 S. aureus, the bactericidal activities of the assembly of the three bacteriophages (Pherecydes Pharma) used alone or in association with vancomycin or rifampicin were compared by quantifying the number of viable bacteria in mature biofilms and infected osteoblasts after 24h of exposure. Results. The activity of bacteriophages against biofilm-embedded S. aureus was dose-dependent. Synergistic effects were observed when bacteriophages were combined to antibiotics at the lowest concentrations, with no significant bactericidal activity in monotherapy. In the human osteoblast infection model, we were able to show that phage penetration into osteoblasts was only possible when the cells were infected, suggesting a S. aureus dependent Trojan horse mechanism. The intracellular inoculum in osteoblasts treated with bacteriophages or vancomycin was significantly higher than in cells treated with lysostaphin, used as control condition of rapid killing of bacteria released in the extracellular media after death of infected cells and absence of intracellular activity. These results suggest that bacteriophages are probably both i) inactive in the intracellular compartment and ii) unable to kill all bacteria released after cell lysis into the extracellular medium fast enough to prevent them from reinfecting other osteoblasts. Conversely, the intracellular inoculum recovered from cells treated with vancomycin+bacteriophages was significantly lower than the one inside cells treated with vancomycin or bacteriophages alone, suggesting that this combination allowed a better control of released bacteria in the extracellular media. Finally, bacteriophages did not increase the activity of rifampicin in this model. Conclusion. In conclusion, we showed that the bacteriophages tested were highly active against S. aureus in mature biofilm but had no activity against bacteria internalized in osteoblasts. Additional studies using animal models of BJI and well-conducted clinical trials are needed to further evaluate phage therapy and its positioning in the management of these infections

Fracture non-union can be as high as 20% in certain clinical scenarios and has a high associated socioeconomic burden. Boron has been shown to regulate the Wnt/β-catenin pathway in other bodily processes. However, this pathway is also critical for bone healing. Here we aim to demonstrate that the local delivery of boric acid can accelerate bone healing, as well as to elucidate how boric acid, via the regulationtheWnt/β-catenin pathway, impacts theosteogenic response of bone-derived osteoclasts and osteoblasts during each phase of bone repair. Bilateral femoral cortical defects were created in 32 skeletally mature C57 mice. On the experimental side, boric acid (8mg/kg concentration) was injected locally at the defect site whereas on the control side, saline was used. Mice were euthanized at 7, 14, and 28 days. MicroCT was used to quantify bone regeneration at the defect. Histological staining for ALP and TRAP was used to quantify osteoblast and osteoclast activity respectively. Immunohistochemical antibodies, β-catenin and CD34 were used to quantify active β-catenin levels and angiogenesis respectively. Sclerostin and GSK3β were also quantified and are both inhibitors of the wnt signaling pathway via degradation and inactivation of β-catenin. The boron group exhibited higher bone volume and trabecular thickness at the defect site by 28 days on microCT. ALP activity was significantly higher in boron group at 7 days whereas boron had no effect on TRAP activity. Additionally, CD34 staining revealed increased angiogenesis at 14 days in boron treated groups. β-catenin activity on immunohistochemistry was significantly higher in the boron group at 7 days, GSK3β was significantly higher in the boron group at 14 days and Sclerostin was significantly higher in the boron group at 28 days. Boron appears to increase osteoblast activity at the earlier phases of healing. The corresponding early increase in β-catenin along with ALP likely supports that boron increases osteoblast activity via the wnt/β-catenin pathway. Increased angiogenesis at 14 days could be a separate mechanism increasing bone formation independent of wnt/β-catenin activation. Neither GSK3β or Sclerostin levels correlated with β-catenin activity therefore boron likely increases β-catenin through a mechanism independent of both GSK3β and Sclerostin. The addition of this inexpensive and widely available ion could potentially become a non-invasive, cost-effective treatment modality to augment fracture healing and decrease non-union rates in high risk patients

Hip and knee arthroplasty (HKA) are two of the most successful orthopaedic procedures. However, one major complication necessitating revision surgery is osteolysis causing aseptic loosening of the prosthesis. JAK-STAT has been demonstrated to influence bone metabolism and can be regulated by microRNA (miRNA). Adult patients with osteolysis or aseptic loosening undergoing revision HKA were recruited. Age and gender matched patients undergoing primary hip or knee arthroplasty were our controls. Samples of bone, tissue and blood were collected and RNA isolation was performed. The best quality samples were used for RNA-sequencing. Data analysis was performed using RStudio and Galaxy to identify differentially expressed genes. Western blotting of IL6 was used to confirm protein expression. Five circulating miRNA were identified which had 10 differentially expressed genes in bone and 11 differentially expressed genes in tissue related to the JAK-STAT pathway. IL6 in bone and EpoR in bone were highly significant and IL6 in tissue, MPL in bone, SOCS3 in tissue, JAK3 in bone and SPRED1 in bone were borderline significant. Western blot results demonstrated up-expression of IL6 in bone tissue of revision patients. Periprosthetic osteolysis and aseptic loosening can be attributed to miRNA regulation of the JAK-STAT pathway in osteoblasts and osteoclasts, leading to increased bone resorption. These findings can be used for further experiments to determine utility in the clinical setting for identifying diagnostic markers or therapeutic targets

Bone regeneration includes a well-orchestrated series of biological events of bone induction and conduction. Among them, the Wnt/β-catenin signaling pathway is critical for bone regeneration. Being involved in several developmental processes, Wnt/β-catenin signaling must be safely targeted. There are currently only few specific therapeutic agents which are FDA-approved and already entered clinical trials. A published work has shown that Tideglusib, a selective and irreversible small molecule non-ATP-competitive glycogen synthase kinase 3-β(GSK-3β) inhibitor currently in trial for Alzheimer's patients, can promote tooth growth and repair cavities. [1]Despite some differences, they are some similarities between bone and tooth formation and we hypothesise that this new drug could represent a new avenue to stimulate bone healing. In this work, we locally delivered Tideglusib (GSK3β inhibitor) in the repair of femoral cortical window defects and investigated bone regeneration. A biodegradable FDA-approved collagen sponge was soaked in GSK-3βinhibitor solution or vehicle only (DMSO) and was implanted in 1 × 2 mm unicortical defects created in femora of 35 adult wild-type male mice. Bone defect repair on control and experimental (GSK-3βinhibitor) groups was assessed after 1 week (n=22), 2 weeks (n=24) and 4 weeks (n=24) with microCT and histological analysis foralkaline phosphatase (ALP, osteoblast activity), tartrate resistant acid phosphatase (TRAP, osteoclasts), and immunohistochemistry to confirm the activation of the Wnt/β-catenin pathway. Our results showed that Tideglusib significantly enhanced cortical bone bridging (20.6 ±2.3) when compared with the control (12.7 ±1.9, p=0.001). Activity of GSK-3β was effectively downregulated at day 7 and 14 resulting in a higher accumulation of active β-catenin at day 14 in experimental group (2.5±0.3) compared to the control (1.1±0.2, p=0.03). Furthermore, the onset of ALP activity appears earlier in the experimental group (day 14, 1.79±0.28), a level of activity never reached at any end-point by the control defects. At 4 weeks treatment, we observed a significant drop in ALP in the experimental group (0.47±0.05) compared to the control (1.01±0.19, p=0.02) and a decrease in osteoclast (experimental=1.32±0.36, control=2.23±0.67, p=0.04). Local downregulation of GSK-3β by tideglusib during bone defect repair resulted in significant increase in amount of new bone formation. The early upregulation of osteoblast activity is one explanation of bone healing augmentation. This is likely the effect of upregulation of β-catenin following pharmaceutical inhibition of GSK-3β since β-catenin activation is known to positively regulate osteoblasts, once committed to the osteoblast lineage. As a GSK-3β inhibitor, Tideglusib demonstrates a different mechanism of action compared with other GSK-3β antagonists as treatment was started immediately upon injury and did not interfere with precursor cells recruitment and commitment. This indicates that tideglusib could be used at the fracture site during the initial intraoperative internal fixation without the need for further surgery. This safe and FDA-approved drug could be used in prevention of non-union in patients presenting with high risk for fracture-healing complications

Aim. The osteolytic process of osteomyelitis is, according to textbooks, caused by increased osteoclast activity due to RANKL production by osteoblasts. However, recent findings contradict this theory. Therefore, the aim was to investigate, in a porcine osteomyelitis model, how osteolysis is affected by massive inflammation and RANKL blocking, respectively. In parallel, patients with chronic osteomyelitis, diabetes, foot osteomyelitis, and fracture related infections (FRI) were included for advanced histological analysis of osteolysis. Methods. In pigs, a tibial implant cavity was created and inoculated with 10. 4. CFU of Staphylococcus aureus: Group A (n=7). Group B (n=7); + 1cm. 3. spongostan into the cavity. Group C (n=4); + systemic Denosumab treatment. Spongostan was used as an avascular material to support bacterial growth and thus increase the inflammatory response. Denosumab treatment was administrated to suppress osteoclast activity by RANKL inhibition (as in osteoporotic patients). The volume of osteolysis was accessed by CT scans. Immunohistochemistry with antibodies towards Cathepsin K was used to identify osteoclasts within the bone lesions. Briefly, the number of Cathepsin K positive cells, i.e., both precursors and bone resorbing osteoclasts, respectively, were counted in 10 high power fields (400x). In total, 50 bone infection patients were included (Herlev Hospital). From each patient five parried samples were taken for histology and microbiology, respectively. Histopathology, CT osteolysis volume estimation, and molecular expression of osteoclasts and inflammatory markers are ongoing. One FRI patient was osteoporotic and treated with Denosumab for 6 years. Results. All pigs were confirmed infected in the implant cavity. The volume (2.41 ± 1.29cm. 3. ) of osteolysis was significantly increased in the spongostan group in comparison to Group A (1.24 ± 0.59 cm. 3. ) (p=0.04). Thereby, the spongostan group had bacteria deeper into the bone from the inoculation point. Sufficient Denosumab treatment, i.e. reduced serum Ca was seen in 3 pigs. None of the Denosumab treated pigs showed reduced osteolysis in comparison to Group A (1.42 ± 0.63 cm. 3. ). The Cathepsin K score of Group C was 17 (15-23 IQR) of precursor osteoclasts and 2 (0-2 IQR) of osteoclasts in Howship lacunae. The Denosumab treated patient showed substantial osteolysis and histological analysis confirmed acute inflammatory. Conclusions. Application of spongostan, i.e., bacterial host optimization and massive inflammation promotes osteolysis and local bacterial dissemination. Osteoclast blocking with Denosumab showed no impact on osteolysis. Elucidation of the pathophysiology causing bone loss in osteomyelitis is fundamental. However, the widely accepted osteoclast-based theory might not be the only relevant

Aim. Biomaterial-associated infections (BAI) present a formidable clinical challenge. Bioactive glasses (BG) have proven highly successful in diverse clinical applications, especially in dentistry and orthopaedics. In this study, we aimed to determine the effect of three commonly used BG composition and particle sizes on cell and bacterial attachment and growth. Our focus is on understanding the changes in pH and osmotic pressure in the surrounding environment during glass degradation. Method. First, three different melt-derived glasses were characterized by analyzing particle size and glass network structure using Raman and NMR. The different glasses were then tested in vitro by seeding 4x 10. 4. cells/well (SaOS Cell line) in a 48 well plate. After a pre-incubation period of 72 hours, the different BGs and particle sizes were added to the cells and the pH value, ion release and live/dead staining was measured every hour. The effect of BG against bacteria (S. epidermidis) was analyzed after 24 and 72 hours of treatment by using XTT viability assay and CFU counting by plating out the treated aliquot agar to estimate the viable bacteria cells. Results. All three BG compositions tested showed a significant increase in pH, which was highest in BG composition 45S5 with a value of 11 compared to the other BG compositions 10 and 9 in S53P4 and 13-93 respectively. This strong increase in the pH in all BG samples tested results in a strongly reduced cell viability rate of more than 75% compared to the untreated control and 6-fold reduction in bacterial viability compared to the untreated control. The live/ dead assay also showed an increased cell viability with increasing glass particle size (i. e smallest glass particle < 25% viable cell and largest glass particle> 65% viable cell). The ion release concentration over 50 h showed an increase in sodium ions to 0.25 mol/L, calcium to 0.003 mol/L and a decrease in phosphorus. Conclusions. These results show that the composition of the bioactive glass and the choice of particle size have a major influence on subsequent applications. In addition to the different compositions of the BG, particle size and additional medium change also influence the pH and ion release, and therefore also on cells or bacteria viability. The sizes of the bioactive glass particle are inversely proportional to it. Further tests are necessary to develop custom design BG compositions, which simultaneously stimulate osteoblasts proliferation and prevent microbial adhesion

The inflammatory cascade associated with prosthetic implant wear debris, in addition to diseases such as rheumatoid arthritis and periodontitis, it is shown to drastically influence bone turnover in the local environment. Ultimately, this leads to enhanced osteoclastic resorption and the suppression of bone formation by osteoblasts causing implant failure, joint failure, and tooth loosening in the respective conditions if untreated. Regulation of this pathogenic bone metabolism can enhance bone integrity and the treatment bone loss. The current study used novel compounds that target a group of enzymes involved with the epigenetic regulation of gene expression and protein function, histone deacetylases (HDAC), to reduce the catabolism and improve the anabolism of bone material in vitro. Human osteoclasts were differentiated from peripheral blood monocytes and cultured over a 17 day period. In separate experiments, human osteoblasts were differentiated from human mesenchymal stem cells isolated from bone chips collected during bone marrow donations, and cultured over 21 days. In these assays, cells were exposed to the key inflammatory cytokine involved with the cascade of the abovementioned conditions, tumour necrosis factor-α (TNFα), to represent an inflammatory environment in vitro. Cells were then treated with HDAC inhibitors (HDACi) that target the individual isoforms previously shown to be altered in pathological bone loss conditions, HDAC-1, −2, −5 and −7. Analysis of bone turnover through dentine resorptive measurements and bone mineral deposition analyses were used to quantify the activity of bone cells. Immunohistochemistry of tartrate resistant acid phosphatase (TRAP), WST-assay and automated cell counting was used to assess cell formation, viability and proliferation rates. Real-time quantitative PCR was conducted to identify alterations in the expression of anti- and pro-inflammatory chemokines and cytokines, osteoclastic and osteoblastic factors, in addition to multiplex assays for the quantification of cytokine/chemokine release in cell supernatant in response to HDACi treatments in the presence or absence of TNFα. TNFα stimulated robust production of pro-inflammatory cytokines and chemokines by PBMCs (IL-1β, TNFα, MCP1 and MIP-1α) both at the mRNA and protein level (p < 0 .05). HDACi that target the isoforms HDAC-1 and −2 in combination significantly suppressed the expression or production of these inflammatory factors with greater efficacy than targeting these HDAC isoforms individually. Suppression of HDAC-5 and −7 had no effect on the inflammatory cascade induced by TNFα in monocytes. During osteoclastic differentiation, TNFα stimulated the size and number of active cells, increasing the bone destruction observed on dentine slices (p < 0 .05). Targeting HDAC-1 and −2 significantly reduced bone resorption through modulation of the expression of RANKL signalling factors (NFATc1, TRAF6, CatK, TRAP, and CTR) and fusion factors (DC-STAMP and β3-integerin). Conversely, the anabolic activity of osteoblasts was preserved with HDACi targeting HDAC-5 and −7, significantly increasing their mineralising capacity in the presence of TNFαthrough enhanced RUNX2, OCN and Coll-1a expression. These results identify the therapeutic potential of HDACi through epigenetic regulation of cell activity, critical to the processes of inflammatory bone destruction

In order to improve fast osseointegration, to modulate inflammatory response and to avoid biofilm formation, several attempts of surface modifications of titanium alloy in term of surface topography and chemistry have been performed over years, but this is still an open issue. In our research work, a patented chemical treatment was developed and tailored to improve fast osseointegration and to allow further surface functionalization in order to get a multifunctional surface. After the chemical treatment, Ti6Al4V shows a micro and nano-textured surface oxide layer with high density of hydroxyls groups, as summarized Figure 1: it is able to induce apatite precipitation (during soaking in Simulated Body Fluid), high wettability by blood, specific protein adsorption, positive osteoblast response and surface mechanical resistance to implantation friction. Hydroxyl groups exposed by the treated surface also allow binding natural biomolecules such as polyphenols, which can further improve the rate and quality of osseointegration by adding anti-inflammatory, antibacterial and antitumoral effects suitable for implants in critical situations. Polyphenols have the further added value of being a low cost and eco-sustainable product, extractable from byproducts of wine and food industry. On the chemically treated and functionalized samples, the surface characterization was performed using Folin&Ciocalteu test, fluorescence microscopy and XPS analysis in order to check the presence and activity of the grafted biomolecules (polyphenols from red grape pomace and green tea leaves). Cell tests were performed with Kusa A-1 cells highlighting the ability of polyphenols to improve osteoblasts differentiation and deposition of mineralized extracellular matrix. Surface functionalization can also be performed with chitin derived biomolecules to reduce inflammation. With the purpose of obtaining the antibacterial effect, during the chemical treatment a silver precursor can also be added to obtain in situ reduced silver nanoparticles embedded in the nano-structured oxide layer. The samples containing nanoparticles on the surface were characterized by means of TEM and FESEM observation highlighting the presence of well distributed and small-sized nanoparticles on the surface and through the thickness of the oxide layer. A long-lasting release in water was observed up to 14 days and antibacterial tests on Staphylococcus aureus showed the ability of the surface to reduce bacteria viability avoiding biofilm formation. The results showed that the patented chemical treatment can improve the response of osteoblasts to titanium alloy implants, but is also a promising way to obtain multifunctional surfaces with antibacterial, antioxidant, anti-inflammatory and antitumoral properties that can be the future of orthopedic implants

Osteogenesis Imperfecta (OI) is a heritable bone disorder characterized by bone fragility and often caused by mutations in the Type I collagen-encoding genes COL1A1 and COL1A2. The pathophysiology of OI, particularly at the cellular level, is still not well understood. This contributes to the lack of a cure for this disorder as well as an effective preventive or management options of its complications. In the bone environment, mesenchymal stem cells (MSCs) and osteoblasts (Ob) exert their function, at least partially, through the secretion of extracellular vesicles (EV). EV is a heterogeneous group of nanosized membrane-enclosed vesicles that carry/transfer a cargo of proteins, lipid and nucleic acids from the secreting cell to its target cells. Our objective is to characterize EVs secreted by human control (HC)- and OI-MSCs and their derived Obs, with focus on their protein content. We hypothesize that there will be differences in the protein content of EVs secreted by OI-Obs compared to HC-Ob, which may indicate a deviation from healthy Ob behavior and, thus, a role in OI pathophysiology. MSCs were harvested from the adipose tissue of four COL1A1-OI and two HC patients. They were proliferated in an EV-depleted media, then induced to differentiate to extracellular matrix (ECM)-producing osteoblasts, which then gets mineralized. EVs secreted by MSCs (MSC-EV) and Obs (Ob-EV) were then purified and concentrated. Using liquid chromatography- tandem mass spectrometry, proteomic analysis of the EV groups was done. A total of 384 unique proteins were identified in all EVs, 373 were found in Vesiclepedia indicating a good enrichment of our samples with EV proteins. 67 proteins of the total 384 were exclusively or significantly upregulated (p-value < 0 .05) in OI-Ob-EV and 28 proteins in the HC-Ob-EVs, relative to each other. These two groups of differentially expressed proteins were compared by Gene Ontology (GO) analysis of their cellular compartment, molecular functions and biological processes. We observed that there were differences in the cellular origin of EV-proteins, which may indicate heterogeneity of the isolated EVs. Molecular function and biological process analyses of the HC-Ob-EV proteins showed, as expected, predominantly calcium-related activities such as extracellular matrix (ECM) mineralization. OI-Ob-EV proteins were still predominantly exhibiting ECM organization and formation functions. Annexins A1,2,4,5 and 6 were differentially and significantly upregulated by the HC-Ob-EVs. Fibronectin (FN), Fibulin-1 and −2, and Laminins (α4 & γ1), which are amongst the early non-collagenous proteins to form the ECM, were differentially and significantly upregulated in the OI-Ob-EVs. We concluded that the persistent expression of Fibronectin (FN), Fibulin-1 and −2, and Laminins in OI-Ob-EVs might indicate the presence of an immature ECM that the OI-Obs are trying to organize. ECM mineralization is largely dependent on the presence of an organized mature ECM, and this being compromised in OI bone environment, may be a contributor to the bone fragility seen in these patients. Annexins, which are calcium-binders that are vital for ECM mineralization, were significantly downregulated in the OI-Ob-EVs and this may be a further contributor to ECM mineralization impairment and bone fragility