The bioactive polyetheretherketone (PEEK) was fabricated by the combination of PEEK and CaO-SiO. 2. particles, which formed hydroxyapatite on its surfaces in simulated body fluid and showed good mechanical propeties. The study revealed osteoblast-like cell proliferation and gene expression on the bioactive PEEK. Materials and Methods. Peek and bioactive PEEK discs (24 mm in diameter and 2 mm in thickness) were prepared. Bioactive PEEk was produced by the combination of 80 vol% Peek powder and 20 vol% CaO-SiO. 2. particles (30CaO · 70SiO. 2. ). Discs were sterilized with ethylene oxide gas. The study was approved by the ethics committee in Chiba University. Human osteoblast-like cells were used in the study. The cells at passage 3–5 were used in the experiments. 2 × 10. 5. cells /disc were culture at 37°C in a humidified atmosphere with 5% CO. 2. , and the media was replaced every 3 days. At days 3, 7, 21, the culture media, cells and discs were collected respectively. Cell attachment assay was performed. Cells were seeded at a density of 4 × 10. 5. cells /well and incubated for 2 hours at 37 C in a humidified atmosphere with 5% CO. 2. The cells on the discs were evaluated by DNA content. The real-time PCR was performed with regard to type I collagen (COLI), osteocalcin (OC), osteonectin (ON), osteopontin (OPN), and GAPDH. The alkaline phosphatase activity (ALP) was measeured at 3, 7, and 21 days, which samples as used in the DNA-content assay. Alizalin Red Staining was performed at day 21 to quantify calcification deposits in discs. Results were analyzed using Student's t-test with at least three samples. The level of siginificance was set at p=0.05. Results. The content of DNA showed similar increases on PEEK and bioactive PEEK in the course of day 3, 7, 21. The cell attachment of bioactive PEEK was two times larger than that of PEEK. Real-time PCR results of human osteoblast-like cells cultured on PEEK and bioactive PEEK discs were shown in Fig. 1. There were no significant differences between cells on PEEK and bioactive PEEK with respect to COL I and ON mRNA expression. However, human osteoblast-like cells on bioactive PEEK presented higher expression of OPN and OCN mRNA at day 21. No significant differences were found in ALP activity of both discs. Calcification deposits were observed only on bioactive PEEK at day 21. Discussion. The bioactive PEEK, with the combination of 80 vol% Peek powder and 20 vol% CaO-SiO. 2. particles (30CaO · 70SiO. 2. ) showed 123.5 MPa and 6.43 GPa in bending strength and Young's modulus, respectively. The bioactive PEEK has the aggregated CaO-SiO. 2. oarticles between the PEEK particles on its surface, which causes hydroxyapatite formation in vivo. The mechanism is considered to enhance the osteoblast attachment ability, and induce OPN and OC mRNA expression, following the calcification of human osteobloast-like cells. Therefore, the study indicated that bioactive PEEK is the most promising for use as an orthopedic implant

Objectives. In order to ensure safety of the cell-based therapy for bone regeneration, we examined in vivo biodistribution of locally or systemically transplanted osteoblast-like cells generated from bone marrow (BM) derived mononuclear cells. Methods. BM cells obtained from a total of 13 Sprague-Dawley (SD) green fluorescent protein transgenic (GFP-Tg) rats were culture-expanded in an osteogenic differentiation medium for three weeks. Osteoblast-like cells were then locally transplanted with collagen scaffolds to the rat model of segmental bone defect. Donor cells were also intravenously infused to the normal Sprague-Dawley (SD) rats for systemic biodistribution. The flow cytometric and histological analyses were performed for cellular tracking after transplantation. Results. Locally transplanted donor cells remained within the vicinity of the transplantation site without migrating to other organs. Systemically administered large amounts of osteoblast-like cells were cleared from various organ tissues within three days of transplantation and did not show any adverse effects in the transplanted rats. Conclusions. We demonstrated a precise assessment of donor cell biodistribution that further augments prospective utility of regenerative cell therapy. Cite this article: Bone Joint Res 2014;3:76–81

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

Previously, we have demonstrated reduced biomechanical bone strength and matrix quality in Tachykinin (Tac)1-deficient mice lacking the sensory neuropeptide substance P (SP). A similar distortion of bone microarchitecture was described for α-calcitonin gene-related pepide (α-CGRP)-deficient mice. In previous studies we observed alterations in cell survival and differentiation capacity of bone cells isolated from wildtype mice when stimulated with SP and α-CGRP. We assume that changes in sensory neurotransmitter balance modulate bone cell metabolism thereby possibly contributing to inferior bone quality. In order to explore this hypothesis, we investigated and compared metabolic parameters in osteoblasts and osteoclasts isolated from SP- and α-CGRP-deficient mice and wildtype (WT) controls. Bone marrow-derived macrophages (BMMs) and osteoblast-like cells from female C57Bl/6J (WT-control), Tac1-deficient (Tac1-/−) and α-CGRP-deficient (α-CGRP-/−) mice were isolated and differentiated according to established protocols (Niedermair et al., 2014). Cell metabolism studies were performed for enzyme activity and cell survival. We observed reduced numbers of BMM from Tac1-/− and α-CGRP-/− mice after initial seeding compared to WT but no changes in viability. Osteoblast-like cells from Tac1-/− mice tend to migrate out faster from bone chips compared to WT-controls whereas migration of osteoblast-like cells from α-CGRP-/− mice was not affected. Osteoblasts and osteoclast/BMM cultures from WT mice endogenously synthesize and secrete SP as well as α-CGRP at a picomolar range. We found no changes regarding BMM or osteoblast proliferation from both, Tac1-/− and α-CGRP-/− mice when compared to WT-controls. Caspase 3/7-activity was reduced by trend in osteoclast/BMM cultures of α-CGRP-/− mice and significantly reduced in osteoclast/BMM cultures of Tac1-/− mice compared to WT-controls. We found significantly higher Caspase 3/7-activity in osteoblasts of Tac1-/− mice after 14 days of osteogenic culture conditions when compared to WT-controls whereas osteoblasts of α-CGRP-/− mice were unaffected. Cathepsin K enzyme activity was significantly reduced in osteoclast/BMM cultures of Tac1-/− and α-CGRP-/− mice compared to WT-controls. ALP activity of Tac1-/− osteoblasts was higher after 7 days and reduced after 21 days of osteogenic culture compared to WT-controls whereas ALP activity of osteoblasts of α-CGRP-/− mice was unchanged. Acccording to our in vitro observations, we suggest some reduction in bone resorption rate but concomitantly a reduction in bone formation rate in Tac1-/− mice compared to WT-controls resulting in a net bone loss in these mice as bone resorption is faster than bone formation. Furthermore, we assume that bone resorption rate is slightly reduced in α-CGRP-/− mice but bone formation rate seems to be unchanged. Therefore we hypothesize that additional conditions present in vivo might contribute to the inferior bone properties of α-CGRP-/− mice

The search for bone substitutes has stimulated the study of growth factors with osteoinductive properties. Bone morphogenetic proteins (BMPs) have been shown to have a central role in endochondral and intramembranous bone formation and are thought to promote normal bone healing. Recent studies demonstrated that platelet-rich plasma (PRP) can provide several growth factors and stimulate osteogenesis. The aim of the present study was to analyse the in vitro effects of rhBMP-7 and PRP on phenotype and proliferation of cells from the site of non-union and from non-affected bone. During the surgical treatment of seven cases of non-union, normal cancellous bone and tissue from the non-union site were harvested. Osteoblast-like cells and fibroblast-like cells were isolated and characterised. Mesenchymal cells were obtained from bone marrow of the same patients. Each cell type was incubated with rhBMP-7 and PRP at different concentrations. Proliferation rate and alkaline phosphatase activity were assessed at 3, 7, 15 and 30 days. Histochemical and immunohistochemical analyses were performed at 15 and 30 days. The proliferation rate of osteoblast-like cells and mesenchymal cells wasalways higher than that of fibroblast-like cells from the non-union site. Growth factors induced mesenchymal cells to express osteoblast phenotype markers. The results suggest that fibroblast-like cells from the site of non-union are poorly responsive to growth factors, even at highest stimulation. In surgical practice these data strongly suggest adding osteoblast-like cells and mesenchymal cells from non-affected sites at the non-union site to enhance the osteogenic response to growth factors

Autogenous cancellous bone is the most effective material in stimulating osteogenic response and the standard graft augmentation for patients with nonunions or bone defects. However it may not be available in sufficient quantity and bone harvesting may give rise to morbidity. Allograft does not have the osteogenic potential of autogenous bone and may be complicated by immunological reaction and transmission of infections. The search of bone substitutes has led to study several growth factors capable of inducing bone formation. Bone morphogenetic proteins (BMPs) have been shown to have a central role in endochondral and intramembranous bone formation and are thought to promote normal bone healing process. Recent studies demonstrated that platelet-rich plasma (PRP) provides several growth factors and stimulates osteogenesis. The aim of the study was the evaluation of rhBMP-7 (rhOP-1) and PRP effects on the different cells detected at the site of nonunion, such as osteoblast-like cells, fibroblast-like cells and mesenchymal cells. During the surgical treatment of seven nonunion cases, cancellous bone and nonunion tissue were harvested. Osteoblast-like cells and fibroblast-like cells were isolated and characterized. Mesenchymal cells were obtained from bone marrow samples of the same patients. Each cell type was incubated with rhBMP-7 and PRP at different concentrations. Proliferation rate and alkaline phosphatase (ALP) activity were assessed at 3, 7, 15, and 30 days. cytochemical and immunocytochemical analysis were performed at 15 and 30 days. Proliferation rate was higher in osteoblast-like cells and mesenchymal cells than in fibroblast-like cells. Growth factors induced mesenchymal cells to express osteoblast phenotype markers. The results show that fibroblast-like cells at the site of nonunion are responsive to growth factors stimulation, though their low osteoblastic differentiation rate, even at highest concentration of growth factors. These data suggest that the use of growth factors in nonunion treatment should be combined with autologous cancellous bone and/or bone marrow graft, sources of target cells, in order to enhance osteogenic response

Aims. Bone regeneration during treatment of staphylococcal bone infection is challenging due to the ability of Staphylococcus aureus to invade and persist within osteoblasts. Here, we sought to determine whether the metabolic and extracellular organic matrix formation and mineralization ability of S. aureus-infected human osteoblasts can be restored after rifampicin (RMP) therapy. Methods. The human osteoblast-like Saos-2 cells infected with S. aureus EDCC 5055 strain and treated with 8 µg/ml RMP underwent osteogenic stimulation for up to 21 days. Test groups were Saos-2 cells + S. aureus and Saos-2 cells + S. aureus + 8 µg/ml RMP, and control groups were uninfected untreated Saos-2 cells and uninfected Saos-2 cells + 8 µg/ml RMP. Results. The S. aureus-infected osteoblasts showed a significant number of intracellular bacteria colonies and an unusual higher metabolic activity (p < 0.005) compared to uninfected osteoblasts. Treatment with 8 µg/ml RMP significantly eradicated intracellular bacteria and the metabolic activity was comparable to uninfected groups. The RMP-treated infected osteoblasts revealed a significantly reduced amount of mineralized extracellular matrix (ECM) at seven days osteogenesis relative to uninfected untreated osteoblasts (p = 0.007). Prolonged osteogenesis and RMP treatment at 21 days significantly improved the ECM mineralization level. Ultrastructural images of the mineralized RMP-treated infected osteoblasts revealed viable osteoblasts and densely distributed calcium crystal deposits within the extracellular organic matrix. The expression levels of prominent bone formation genes were comparable to the RMP-treated uninfected osteoblasts. Conclusion. Intracellular S. aureus infection impaired osteoblast metabolism and function. However, treatment with low dosage of RMP eradicated the intracellular S. aureus, enabling extracellular organic matrix formation and mineralization of osteoblasts at later stage. Cite this article: Bone Joint Res 2022;11(5):327–341

Objectives. We have observed clinical cases where bone is formed in the overlaying muscle covering surgically created bone defects treated with a hydroxyapatite/calcium sulphate biomaterial. Our objective was to investigate the osteoinductive potential of the biomaterial and to determine if growth factors secreted from local bone cells induce osteoblastic differentiation of muscle cells. Materials and Methods. We seeded mouse skeletal muscle cells C2C12 on the hydroxyapatite/calcium sulphate biomaterial and the phenotype of the cells was analysed. To mimic surgical conditions with leakage of extra cellular matrix (ECM) proteins and growth factors, we cultured rat bone cells ROS 17/2.8 in a bioreactor and harvested the secreted proteins. The secretome was added to rat muscle cells L6. The phenotype of the muscle cells after treatment with the media was assessed using immunostaining and light microscopy. Results. C2C12 cells differentiated into osteoblast-like cells expressing prominent bone markers after seeding on the biomaterial. The conditioned media of the ROS 17/2.8 contained bone morphogenetic protein-2 (BMP-2 8.4 ng/mg, standard deviation (. sd. ) 0.8) and BMP-7 (50.6 ng/mg, . sd. 2.2). In vitro, this secretome induced differentiation of skeletal muscle cells L6 towards an osteogenic lineage. Conclusion. Extra cellular matrix proteins and growth factors leaking from a bone cavity, along with a ceramic biomaterial, can synergistically enhance the process of ectopic ossification. The overlaying muscle acts as an osteoinductive niche, and provides the required cells for bone formation. Cite this article: D. B. Raina, A. Gupta, M. M. Petersen, W. Hettwer, M. McNally, M. Tägil, M-H. Zheng, A. Kumar, L. Lidgren. Muscle as an osteoinductive niche for local bone formation with the use of a biphasic calcium sulphate/hydroxyapatite biomaterial. Bone Joint Res 2016;5:500–511. DOI: 10.1302/2046-3758.510.BJR-2016-0133.R1

Aims. This study aimed to examine the effects of tumour necrosis factor-alpha (TNF-α) on osteoblasts in metal wear-induced bone loss. Methods. TNF-α immunoexpression was examined in periprosthetic tissues of patients with failed metal-on-metal hip arthroplasties and also in myeloid MM6 cells after treatment with cobalt ions. Viability and function of human osteoblast-like SaOs-2 cells treated with recombinant TNF-α were studied by immunofluorescence, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay, western blotting, and enzyme-linked immunosorbent assay (ELISA). Results. Macrophages, lymphocytes, and endothelial cells displayed strong TNF-α immunoexpression in periprosthetic tissues containing metal wear debris. Colocalization of TNF-α with the macrophage marker CD68 and the pan-T cell marker CD3 confirmed TNF-α expression in these cells. Cobalt-treated MM6 cells secreted more TNF-α than control cells, reflecting the role of metal wear products in activating the TNF-α pathway in the myeloid cells. While TNF-α did not alter the immunoexpression of the TNF-receptor 1 (TNF-R1) in SaOs-2 cells, it increased the release of the soluble TNF-receptor 1 (sTNF-R1). There was also evidence for TNF-α-induced apoptosis. TNF-α further elicited the expression of the endoplasmic reticulum stress markers inositol-requiring enzyme (IRE)-1α, binding-immunoglobulin protein (BiP), and endoplasmic oxidoreductin1 (Ero1)-Lα. In addition, TNF-α decreased pro-collagen I α 1 secretion without diminishing its synthesis. TNF-α also induced an inflammatory response in SaOs-2 cells, as evidenced by the release of reactive oxygen and nitrogen species and the proinflammatory cytokine vascular endothelial growth factor. Conclusion. The results suggest a novel osteoblastic mechanism, which could be mediated by TNF-α and may be involved in metal wear debris-induced periprosthetic bone loss. Cite this article: Bone Joint Res 2020;9(11):827–839

Introduction and Objective. Total joint replacement (TJR) is indicated for patients with end-stage osteoarthritis (OA) where conservative treatment has failed. Approximately 1.3 million primary hip replacement surgeries have been recorded in the United Kingdom since 2003 and this number is set to rise due to an increase in obesity as well as an ageing population. Total hip replacement (THR) has a survival rate of 85% at 20 years; the most common reason for failure is aseptic loosening which often occurs secondary to osteolysis caused by immune-mediated inflammation responses to wear debris generated from the materials used in the THR implant. Therefore, by understanding the biological steps by which biomaterials cause immune-mediated reactions it should be possible to prevent them in the future thereby reducing the number of costly revision surgeries required. Materials and Methods. The human osteoblast-like cell line (MG-63) was seeded at a density of 100,000 cell per well of a 6-well plate and treated with and increasing doses (0.5, 5, and 50mm. 3. per cell) of cobalt-chromium (CoCr) particles generated on a six-station pin-on-plate wear generator or commercially available ceramic oxide nanopowders (Al. 2. O. 3. and ZrO. 2. ) for 24 hours. TNF-alpha was used as a positive control and untreated cells as a negative control. Cells were then analysed by transmission electron microscopy (TEM) to determine whether the osteoblasts were capable of phagocytosing these biomaterials. MG-63 cells were used in conjunction with trypan blue and the XTT Cell Proliferation II Kit to assess cytotoxicity of the biomaterials investigated. Cells supernatants were also collected and analysed by enzyme-linked immunosorbant assay (ELISA) to investigate changes in pro-inflammatory protein secretion. Protein extracted from lysed cells was used for western blotting analysis to investigate RANKL protein expression to determine changes to osteolytic activation. Lysed cells were also used for RNA extraction and subsequent cDNA synthesis for real-time quantitative polymerase chain reaction (RT-qPCR) in order to assess changes to pro-inflammatory gene expression. Results. There was no significant change to cellular viability or proliferation in the osteoblasts treated with CoCr, Al. 2. O. 3. or ZrO. 2. when compared to the untreated negative control. TEM images showed clear and distinct intracellular vesicles within the cell cytoplasm which contained CoCr, Al. 2. O. 3. and ZrO. 2. RANKL expression increased at 5 and 50mm. 3. per cell CoCr and 50mm. 3. per cell Al. 2. O. 3. and ZrO. 2. Pro-inflammatory protein secretion of CXCL10, IL-8, and IL-6 all significantly increased at 50mm. 3. per cell CoCr, Al. 2. O. 3. , and ZrO. 2. Similarly to the protein secretion, CXCL10, IL-8, and IL-6 gene expression was significantly upregulated at 50mm. 3. per cell CoCr, Al. 2. O. 3. , and ZrO. 2. Conclusions. Increased in vitro RANKL expression in response to CoCr, Al. 2. O. 3. , and ZrO. 2. may result in disruption of bone metabolism and lead to osteolysis which can contribute to aseptic loosening in vivo. Significant increases in IL-6 are particularly important because as well as being a pro-inflammatory cytokine, IL-6 is also secreted by osteoblasts in order to stimulate mature osteoclast formation to mediate bone breakdown. CXCL10 and IL-8 are chemotactic cytokines and increased secretion in response to implant biomaterials can contribute to ongoing pro-inflammatory responses through the recruitment of monocytes and neutrophils respectively. This is interesting as in vivo data demonstrates increased cellular infiltrate in patients experiencing responses to implant materials. Overall, these findings show clear immune activation as well as altered metabolism of MG-63 osteoblast cells in response to implant wear debris which is in agreement with in vivo clinical reports

Biomaterials used in regenerative medicine should be able to support and promote the growth and repair of natural tissues. Bioactive glasses (BGs) have a great potential for applications in bone tissue engineering [1, 2]. As it is well known BGs can bond to host bone and stimulate bone cells toward osteogenesis. Silicate BGs, e.g. 45S5 Bioglass® (composition in wt.%: 45 SiO. 2. , 6 P. 2. O. 5. , 24, 5 Na. 2. O and 24.5 CaO), exhibit positive characteristics for bone engineering applications considering that reactions on the material surface induce the release of critical concentrations of soluble Si, Ca, P and Na ions, which can lead to the up regulation of different genes in osteoblastic cells, which in turn promote rapid bone formation. BGs are also increasingly investigated for their angiogenic properties. This presentation is focused on cell behavior of osteoblast-like cells and osteoclast-like cells on BGs with varying sample geometry (including dense discs for material evaluation and coatings of highly porous Al. 2. O. 3. -scaffolds as an example of load-bearing implants). To obtain mechanically competent porous samples with trabecular architecture analogous to those of cancellous bone, in this study Al. 2. O. 3. scaffolds were fabricated by the well-known foam replication method and coated with Bioglass® by dip coating. The resulted geometry and porosity were proven by SEM and μCT. Originating from peripheral blood mononuclear cells formed multinucleated giant cells, i.e. osteoclast-like cells, after 3 weeks of stimulation with RANKL and M-CSF. Thus, the bioactive glass surface can be considered a promising material for bone healing, providing a surface for bone remodeling. Osteoblast-like cells and bone marrow stromal cells were seeded on dense bioactive glass substrates and coatings showing an initial inhibited cell attachment but later a strong osteogenic differentiation. Additionally, cell attachment and differentiation studies were carried out by staining cytoskeleton and measuring specific alkaline phosphatase activity. In this context, 45S5 bioactive glass surfaces can be considered a highly promising material for bone tissue regeneration, providing very fast kinetics for bone-like hydroxyapatite formation (mineralization). Our examinations revealed good results in vitro for cell seeding efficacy, cell attachment, viability, proliferation and cell penetration onto dense and porous Bioglass®-coated scaffolds. Recent in vivo investigations [3] have revealed also the angiogenic potential of bioactive glass both in particulate form and as 3D scaffolds confirming the high potential of BGs for bone regeneration strategies at different scales. Implant surfaces based on bioactive glasses offer new opportunities to develop these advanced biomaterials for the next generation of implantable devices and tissue scaffolds with desired tissue-implant interaction

The material most widely used in orthopaedics is hydroxyapatite (HA), anyway many differences are still present between synthetic HA and biological HA. The aim of this study was to compare adhesion, proliferation and differentiation of human osteoblast-like cells on hydroxyapatite discs with different porosity and on plastic cultures. Human osteoblast-like cells were isolated from 4 young patients (mean age 24.5 years old), treated with collagenase and maintained in Dulbecco’s modified essential medium-10% fetal calf serum. Cells were plated on hydroxyapatite discs with 3 different porosities (35%, 35–55% e 55%) and on plastic cultures used as control. The proliferation was determined by the MTT colorimetric method, and alkaline phosphatase (ALP) activity was measured by a spettrophotometric method. Type I collagen and osteonectin production were demonstrated with fluorescence microscopy and osteoblast adhesion was studied by scanning electron microscopic (SEM) analysis. Results were analysed by one-way analysis of variance (ANOVA). Osteoblast proliferation on HA was three- to six-fold lower then on plastic. At 28 days, 2141 (± 350) cells/well grew on the most porous disks, with highly significant differences from controls. The ALP production was 2–3 fold lower on HA than on plastic. In the most porous disks, the mean ALP activity was of 2.95 (± 0.07) UI/well after 28 days, higher than in the other two groups. The type-I collagen and the osteonectin fluorescence reaction evidenced a cytoplasmic and a matrix labeling on HA at different porosities. SEM analysis showed osteoblasts with a flattened morphology and only few of them were metabolic active. At 21 and 28 days, proliferation rate and ALP activity on the three HA cultures were significantly different (p< 0.05). A decrease in cell population and increased ALP activity were observed on the most porous material, and high proliferation and poor differentiation rates on the less porous disks

Introduction: The use of uncemented arthroplasty in joint replacement surgery requires osseointegration of the prosthesis to maximise function and longevity. It has been demonstrated that osteoblast-like cells will preferentially proliferate, differentiate and produce mineralised matrix in pits and grooves on non-biological surfaces, of similar dimensions to those of Howslip’s lacunae produced by osteoclasts in vitro. The hypotheses of this study were that a photochemically etched titanium alloy surface would 1) induce proliferation and differentiation in osteoblast-like cells; 2) induce osteoblastic differentiation of human mesenchymal stem cells and 3) induce greater bone to implant contact in a caprine model. Methods: Three microgrooved titanium alloy surfaces (fine, medium & coarse) were created by photochemical etching, with dimensions of 200 to 515 microns. Human Mesenchymal stem cells (MSC) and Human Osteosarcoma (HOS) cells (TE-85) were seeded onto these surfaces and cultured in standard media; in the case of MSC, with and without the addition of osteogenic supplements. At intervals of time each surface and cell type were assessed for proliferation by Alamar blue assay and osteoblastic differentiation by Alkaline Phosphatase expression. A polished titanium surface was used as a control. A plate of each surface dimension was placed into a femoral condyle of ten adult male goats. The animals were euthanased at 6 and 12 weeks post-implantation. The specimens were histologically processed and examined under light and backscattered electron microscopy to establish the percentage of bone to implant contact and the presence of new bone within the grooves. Results: In vitro, all cells showed an increase in proliferation with time, the greatest occurring on the coarse surface. Alkaline phosphatase expression showed a rise with time on all surfaces, the greatest being on the coarse surface seeded with HOS cells (p< 0.05). MSC could not be induced to differentiate to an osteogenic lineage by these surface textures alone. On addition of osteogenic supplements their results followed the trends of HOS cells. In vivo, histomorphometric analysis showed significantly greater bone implant contact on the coarse surface at both 6 and 12 weeks (p< 0.05). In a number of cases there were signs of osteogenesis occurring deep within the pits and grooves. Discussion: This study confirms that a photochemically etched surface topography mimicking that created by osteoclasts will increase the proliferation and differentiation of osteoblastic cells in vitro. The rate of differentiation of these cells increased significantly in relation to the size of the grooves. When implanted in vivo these same surfaces were shown to support osseointegration. This surface has the potential to improve the function of uncemented arthroplasties in the future

Introduction. We have developed a new synthetic hydrogel that can be injected directly into the intervertebral disc (IVD) without major surgery. Designed to improve fixation of joint prosthesis, support bone healing or improve spinal fusion, the liquid may support the differentiation of native IVD cells towards osteoblast-like cells cultured within the hydrogel. Here we investigate the potential of this gel system (Bgel) to induce bone formation within intervertebral disc tissue. Methods. IVD tissue obtained from patients undergoing discectomy, or cadaveric samples, were cultured within a novel explant device. The hydrogel was injected, with and without mesenchymal stem cells (MSCs), and cultured under hypoxia, to mimic the degenerate IVD environment, for 4 weeks. Explants were embedded to wax and native cellular migration into the hydrogel was investigated, together with cellular phenotype and matrix deposition. Results. Increased collagen deposition was seen in tissue explants injected with Bgel, with evidence of elevated native cell migration towards the hydrogel. Increased collagen staining was seen in explants injected with Bgel together with MSCs. Alizarin red staining was utilised to investigate calcium deposition. Tissue explants, in the absence of Bgel, showed limited calcium deposition. This was increased in hydrogel-treated samples, with large clumping regions in the tissue that was injected with Bgel and MSCs. Conclusion. The injection of our synthetic hydrogel into disc tissue explants increased the amount of collagen and calcium deposition. This was further enhanced by the incorporation of MSCs, suggesting the promotion of bone formation. Current work is investigating phenotypic markers for bone formation within these tissues. CS and CLM have a patent on the hydrogel system described in this abstract. Funded by EPSRC and Grow MedTech

Uncemented implants combining antimicrobial properties with osteoconductivity would be highly desirable in revision surgery due to periprosthetic joint infection (PJI). Silver coatings convey antibacterial properties, however, at the cost of toxicity towards osteoblasts. On the other hand, topological modifications such as increased surface roughness or porosity support osseointregation but simultaneously lead to enhanced bacterial colonization. In this study, we investigated the antibacterial and osteoconductive properties of silver-coated porous titanium (Ti) alloys manufactured by electron beam melting, rendering a macrostructure that mimics trabecular bone. Trabecular implants with silver coating (TR-Ag) or without coating (TR) were compared to grit-blasted Ti6Al4V (GB) and glass cover slips as internal controls. Physicochemical characterization was performed by X-ray diffraction (XRD) and energy dispersive X-rays (EDX) together with morphological characterization through electron scanning microscopy (SEM). Bacterial adherence after incubation of samples with Staphylococcus (S.) aureus and S. epidermidis strains harvested from PJI patients was quantitatively assessed by viable count after detachment of adherent bacteria by collagenase/dispase treatment. Primary human osteoblasts (hOB) were used to investigate the osteoconductive potential by lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activity. Cell morphology was investigated by fluorescence microscopy after staining with carboxifluorescein diacetate succinimidyl ester (CFDA-SE) and 4′,6-diamidino-2-phenylindole (DAPI). The trabecular implants depicted a porosity of 70% with pore sizes of 600µm. The amount of silver analyzed by EDX accounted for 35%wt in TR-Ag but nil in TR. Silver-coated TR-Ag implants had 24% lower S. aureus viable counts compared to non-coated TR analogues, and 9% lower compared to GB controls. Despite trabecular implants, both with and without silver, had higher viable counts than GB, the viable count of S. epidermidis was 42% lower on TR-Ag compared to TR. The percentage of viable hOB, measured by LDH and normalized to controls and area at 1 day, was lower on both TR-Ag (18%) and on TR (13%) when compared with GB (89%). However, after 1 week, cell proliferation increased more markedly on trabecular implants, with a 5-fold increase on TR-Ag, a 3.4-fold increase on TR, and a 1.7-fold increase on GB. Furthermore, after 2 weeks of hOB culture, proliferation increased 20-fold on TR-Ag, 29-fold on TR, and 3.9-fold for GB, compared to 1 day. The osteoconductive potential measured by ALP illustrated slightly higher values for TR-Ag compared to TR at 1 day and 2 weeks, however below those of GB samples. Cell morphology assessed by microscopy showed abundant growth of osteoblast-like cells confined to the pores of TR-Ag and TR. Overall, our findings indicate that the silver coating of trabecular titanium exerts limited cytotoxic effects on osteoblasts and confers antimicrobial effects on two PJI-relevant bacterial strains. We conclude that improving material design by mimicking the porosity and architecture of cancellous bone can enhance osteoconductivity while the deposition of silver confers potent antimicrobial properties

Despite the increasing availability of bone grafting materials, the regeneration of large bone defects remains a challenge. Especially infection prevention while fostering regeneration is a crucial issue. Therefore, loading of grafting material with antibiotics for direct delivery to the site of need is desired. This study evaluates the concept of local delivery using in vitro and in vivo investigations. We aim at verifying safety and reliability of a perioperative enrichment procedure of demineralized bone matrix (DBM) with gentamicin. DBM (DBMputty, DIZG, Germany) was mixed with antibiotic using a syringe with an integrated mixing propeller (Medmix Systems, Switzerland). Gentamicin, as powder or solution, was mixed with DBM at different concentrations (25 −100 mg/g DBM), release and cytotoxicity was analyzed. For in vivo analysis, sterile drill hole defects (diameter: 6 mm, depth: 15 mm) were created in diaphyseal and metaphyseal bones of sheep (Pobloth et al. 2016). Defects (6 – 8 per group and time point) were filled with DBM or DBM enriched with gentamicin (50 mg/g DBM) or left untreated. After three and nine weeks, defect regeneration was analyzed by µCT and histology. The release experiments revealed a burst release of gentamicin from DBM independent of the used amount, the sampling strategy, or the formulation (powder or solution). Gentamicin was almost completely released after three days in all set-ups. Eluates showed an antimicrobial activity against S. aureus over at least three days. Eluates had no negative effect on viability and alkaline phosphatase activity of osteoblast-like cells (partially published Bormann et al. 2014). µCT and histology of the drill hole defects revealed a reduced bone formation with gentamicin loaded DBM. After nine weeks significantly less mineralized tissue was detectable in metaphyseal defects of the gentamicin group. Histological evaluation revealed new bone formation starting at the edges of the drill holes and growing into the center over time. The amount of DBM decreased over time due to the active removal by osteoclasts while osteoblasts formed new bone. Using this mixing procedure, loading of DBM was fast, reliable and possible during surgical setting. In vitro experiments revealed a burst and almost complete release after three days, antimicrobial activity and good biocompatibility of the eluates. Gentamicin/DBM concentration was in the range of clinically used antibiotic-loaded-cement for prophylaxis and treatment in joint replacement (Jiranek et al. 2006). The delayed healing seen in vivo was unexpected due to the good biocompatibility found in vitro. A reduced healing was also seen in spinal fusion where DBM was mixed with vancomycin (Shields et al. 2017), whereas DBM with gentamicin or DBM/bioactive glass with tobramycin had no negative effect on osteoinductivity or femur defect healing, respectively (Lewis et al. 2010, Shields et al. 2016). In conclusion, loading of DBM with gentamicin showed a proper antibiotic delivery over several days, covering the critical phase shortly after surgery. Due to the faster and complete release of the antibiotic compared to antibiotic loaded cement, the amount of antibiotic should be much lower in the DBM compared to cement

Introduction Efficient control of osteoblast metabolism is crucial for the development of methods for enhancement of bone fracture repair and in the treatment of osteoporosis. If extracellular matrix elaboration by osteoblast could be controlled on the cellular level, new theurapeutical means might be developed. The current methods for osteoblast metabolic manipulation include mechanical, electromagnetic, hormonal and biochemical, i.e. growth factors and cytokines, means. All this methods have different degrees of therapeutic success. Finding of additional pathways of metabolic stimulation of osteoblast will provide an important insight for the understanding of human bone mass maintenance. The recent report of the existence of peripheral ben-zodiazepine receptor(PBR) in mammalian fibroblast arises the possibility of the existence of an unknown cellular pathway for mesenchymal cells metabolic regulation through this receptor. The PBR is a part of the mitochondrial permeability transition complex with important role in cell proliferation, differentiation, steroidogenesis, immunity and apoptosis, i.e. this complex is involved in most of the cellular metabolic activities. The PBR was identified in various organs, especially with enhanced steroidogenetic activity, but never has been investigated in bone. Therefore PBR’s identification in the human osteoblast may reveal a new cellular pathway of its metabolism. Methods Cultures of confluent layers of osteoblast-like cells originated from human cancellous bone from distal femur. The samples were taken during osteoarthritic knee replacements. Chips of cancellous bone, 2 – 3 grams in total, were incubated in DMEM with heat-inactivated fetal calf serum (10%), 20mM HEPES buffer, 2mM L-glutamine, 100 μ M ascorbate-2-phosphate, 10nM dexam-etasone, 50 U ml-ml penicillin, 150μml-ml streptomicin at 37°C in humidified atmospheric environment of 95% air with 5% CO. 2. ( v:v ) for 30 days. Human osteoblast-like cells grew out from the chips as adherent to the plastic culture plates until confluency. The human bone cell cultures obtained by this method have been shown previously to express osteoblast-like characteristics. The PBR in the homogenized osteblast-like cells was identified by using its selective ligand PK11195. The affinity and density of the PBR was estimated by the scatchard analysis. Results We found that binding of the ligand [. 3. H]PK11195 to the human osteoblast PBR is saturable with a single population of binding sites (r=0.92 – 0.95). The equilibrium dissociation constant (K. d. ) equaled 9.15-9.34 nM and density of receptors (B. max. ) was 7,672–7,691 fmol/mg protein. Discussion The PBR receptor was identified in the human osteoblast with affinity to the PK11195 in the same magnitude as previously found in other tissues. The density of the PBR in the osteoblast appeared higher comparing to uterus, kidney, brain and placenta from different mammalian origin. PBR’s density in osteoblast is comparable only to the adrenal tissue, that is known to have its highest values. PBR density in the human osteoblast is also higher than in the rat’s skeletal fibroblast, and although this may suggest a higher differrention of the osteoblast, the interspecies comparison might be misleading. These data suggest that the human osteoblast is one of the important sites rich with PBR. The exact role of the PBR in the human osteoblast metabolism is not known yet and will be further investigated

The steric and electrostatic complementarity of natural proteins and other macromolecules are a result of evolutionary processes. The role of such complementarity is well established in protein-protein interactions, accounting for the known protein complexes. To our knowledge, non-biological systems have not been a part of such evolutionary processes. Therefore, it is desirable to design and develop nonbiological surfaces, such as implant devices (e.g. bone growth for non-cemented fixation), that exhibit such complementarity effects with the natural proteins. Cell attachment and spreading in vitro is generally mediated by adhesive proteins such as fibronectin and vitronectin [. 1. ]. The primary interaction between cells and adhesive proteins occurs through integrin and an RGD amino acid sequence. The adsorption of adhesive proteins plays an important role in cell adhesion and bone formation to an implant surface [. 1. ]. The ability of the implant surface to adsorb these proteins determines its aptitude to support cell adhesion and spreading and its biocompatibility. For example, the enhancement of osteoblast precursor attachment on hydroxyapatite (HA) as compared to titanium and stainless steel was related to increased fibronectin and vitronectin absorption [. 2. ]. The role of surface characteristics, such as topography, has been studied in recent years without the emergence of a comprehensive and consistent model [. 1. ]. For example, while no statistically significant influence of surface roughness on osteoblast proliferation and cell viability was detected in the study of metallic titanium surfaces [. 3. ], the TiO2 film enhances osteoblast adhesion, proliferation and differentiation upon an increase in roughness [. 4. ]. We designed and produced ceramic [. 5. ] and metallic coatings via an ion beam assisted deposition process with spatial dispersion (roughness) comparable to the size of proteins (3–20nm). Our ceramic and cobaltchrome (CoCr) coatings exhibit high hardness and contact angles with serum of 0° and 40° to 50°, respectively. Furthermore, our theoretical calculations and quantum-mechanical modeling clearly indicate that the spatial electric potential variation across our designed ceramic surfaces is comparable to the electrostatic potential variation of proteins such as fibronectin, promoting increased absorption on these surfaces. Therefore, an increase in the concentration of adhesive proteins on the designed surfaces results in the enhancement of the focal adhesion of cells. Our experimental results of the adhesion and proliferation of osteoblast-like stromal cells from mouse bone marrow indicate that our nanostructured coatings are three to five times better than growing on HA and orthopaedic grades of titanium and CoCr. Our results are consistent with the steric and electrostatic complementarity of nanostructured surfaces and adhesive proteins. This paper presents the adhesion and proliferation of osteoblast-like cells on micro-and nanostructured surfaces and provides new models describing the mechanism responsible for the enhancement of cell adhesion on nanostructured ceramic and metallic surfaces compared with orthopaedic materials

Aim. Toxin-antitoxin (TA) systems are small genetics elements found in the majority of bacteria which encode a toxin causing bacterial growth arrest and an antitoxin counteracting the toxic effect. In Salmonella and E. coli, TA systems were shown to be involved in the formation of persisters. Persisters are a bacterial subpopulation with low growth rate and high tolerance to antibiotics. They could be responsible for antibiotic treatment failure in chronic infections and relapses, notably in bone and joint infections (BJI) caused by Staphylococcus aureus. Currently, two type II TA system families were described in S. aureus, mazEF and axe/txe, but their physiological roles are not well described. In this work, we studied the importance of mazEF in the intracellular survival of S. aureus inside osteoblasts, one of the mechanisms considered in the chronicity of S. aureus BJI. Methods. Using an ex vivo model of intracellular infection of human osteoblast-like cells (MG-63), two strains of S. aureus HG003 wild type and its isogenic mutant HG003 ΔmazEF were compared in terms of : i) internalization and intracellular survival by lysostaphin protective assay and ii) cytotoxicity by quantifying LDH in the culture supernatant, 24h and 48h after infection. Results. The comparison of the two strains revealed that HG003 ΔmazEF had a lower capacity to be internalized by osteoblasts compared to the wild type (p=0.02). However, intracellular survival was greater for HG003 ΔmazEF compared to the wild type 24h and 48h post-infection (p=0.02 and 0.001 respectively). Concerning the bacteria-induced cell death, HG003 ΔmazEF appeared to be less cytotoxic than the wild type strain at 24h post infection (p=0.007) whereas no more differences could be observed after 48h. This delayed cytotoxicity with HG003 ΔmazEF was also observed after incubation of culture supernatants with osteoblasts during 8 hours, suggesting that the differences observed could be caused by a secreted molecule. Conclusions. Our results suggest that the mazEF system could be involved in S. aureus BJI physiopathology regulating cytotoxicity and persistence in osteoblasts. Our prospect is to identify the target of the mazF toxin which could be a therapeutic target

The increased incidence of type 2 Diabetes Mellitus is associated with an impaired skeletal structure and a higher prevalence of bone fractures. Sclerostin is a negative regulator of bone formation produced by osteocytes and there is recent evidence that its expression in serum is elevated in diabetic patients compared to control subjects. In this study, we test whether hyperglycemia affects serum and bone sclerostin levels in a rat model of type 2 Diabetes as well as sclerostin production by osteoblasts in culture. We used Zucker diabetic fatty (ZDF) male rats (n=6) that spontaneously develop obesity and frank diabetes around 8–9 weeks of age and Zucker lean rats as controls (n=6) to examine sclerostin expression in serum at 9, 11 and 13 weeks using a specific ELISA. Sclerostin expression in bone tibiae was examined at 12 weeks using immunocytochemistry. Rat osteoblast-like cells UMR-106 were cultured in the presence of increasing concentrations of glucose (5, 11, 22 and 44 mM) during 48 hours and sclerostin mRNA expression and release in the supernatant determined by quantitative PCR and ELISA, respectively. Our results show that serum sclerostin levels are higher in the diabetic rats compared to lean rats at 9 weeks (+ 140%, p<0.01). Our preliminary results using immunocytochemistry for sclerostin did not show any major difference in sclerostin expression in tibiae of diabetic rats compared to lean ones, although we observed many osteocytic empty lacunae in cortical bone from diabetic rats. Glucose dose-dependent stimulated sclerostin mRNA and protein production in mature UMR106 cells while it had no effect on osteocalcin expression. Altogether, our data suggest that sclerostin production by mature osteoblasts is increased by hyperglycemia in vitro and enhanced in serum of diabetic rats. Furthers studies are required to determine whether sclerostin could contribute to the deleterious effect of Diabetes on bone