Degree of early integration of titanium alloy implants into bone is an important predictor of long term implant success in arthroplasty. The correlation between observations on early cell adhesion and the ability of modified surfaces to affect osseointegration of implants in in vivo models is unclear. We hypothesised that observation of increased focal adhesion complexes in early cultures of osteoblasts would correlate with increased osseointegration of treated implants in an animal model. Longer term culture of rat osteoblasts for alkaline phosphatase activity indicated that cells cultured on the 9V treated surfaces were displaying greater alkaline phosphatase activity at 14 days. Bone nodule formation at 28 days demonstrated a trend towards smaller area of bone nodules on the surfaces treated at 9V then those treated at 3V and 5V. A rat model was employed for testing mechanical push-out strength of experimental implants and demonstrated a trend towards increased yield strength of the bone-implant interface for implants treated at 3V180s and 5V180s. Histomorphometry was performed and no statistically significant differences in percentage area of contact with mineralised bone matrix were seen, although there was a trend for greater mineralised matrix contact on the polished and 9V180s treated implants. Previous experiments demonstrated cells on the 9V treated surfaces were well spread and had significantly increased size and number of focal adhesions. This was regarded as indicating more successful cell adhesion. The above results demonstrate that this early trend disappeared in longer term culture did not persist in experiments in an animal model.

Background

Membrane type 1 matrix metalloproteinase (MT1-MMP) plays a role in the progression of several common solid cancers. Given that osteosarcoma features extensive local invasion and haematogenous metastases, we hypothesised that osteosarcoma cells utilise MT1-MMP to drive these processes. Moreover, since hypoxia regulates MT1-MMP expression in breast cancer we investigated the effects of hypoxia on MT1-MMP expression in osteosarcoma cells.

Background

Uncemented implants are an important part of the arthroplasty armamentarium. Risk of aseptic loosening and failure of these components is related to initial osseointegration - the formation of a seamless bone-implant interface without interposition of fibrous tissue.

We used an atomic layer deposition (ALD) approach to create titanium oxide nanolayers on ultra high molecular weight polyethylene (UHMWPE) surfaces. These materials were then characterised in terms of rat osteoblast adhesion, morphology and differentiation.

UHMWPE discs produced from a machined cylinder or impact moulded discs were coated with titanium oxide by ALD. Light, atomic force microscopy and scanning electron microscopy with EDX were used to characterise the coated surfaces. These approaches showed 1-1.5 micron tooling grooves with a periodicity of 40 microns on the machined discs whilst the moulded discs exhibited nanotopographical features. The titanium oxide coating was successfully deposited on discs from both sources but was not uniform across the surfaces, with vein-like ‘creases’ clearly visible. We believe that these features are due to the thermal expansion of the UHMWPE discs during the ALD process and their subsequent cooling.

Coated and uncoated discs were seeded with osteoblasts for 24 hours, then fixed. Immunofluorescence microscopy and computer-based image processing enabled determination of osteoblast numbers, size and shape. A trend of larger average cell area was associated with the coated discs and P<0.01 for an H0 of no difference in cell area between coated and uncoated grooved discs.

Osteoblasts were also cultured on the discs in osteogenic medium to promote bone nodule formation. After a few weeks, von Kossa staining and computer-based image processing allowed calculation of surface area covered with bone nodules for each of the discs. Based on results from three of each type of disc, a significantly greater proportion of the surface area of coated discs was covered with calcified deposits compared to uncoated discs (P<0.025 for grooved discs and P<0.005 for smooth discs). On average, the coated discs had bone nodules on 1.4 times the surface area as compared to their uncoated counterparts.

The hypothesis for our study was that TiO2 coating of a polymer might better promote osteoblast interaction with the biomaterial surface leading to enhanced osteogenesis. Our preliminary data support this view and suggest that this approach could likely be exploited in the fabrication of implant materials with tailored biological activity.

Clinical proteomics is an exciting new sub-discipline of proteomics that involves the application of proteomic technologies at the bedside to identify new biomarkers, associated with specific diseases. In this study to compare serum protein profiles between identical age-matched groups of fracture and non-fracture controls, we looked at the initial proteomic profile of 10 patients who had fractures and compared them to age-matched controls to see if there was any specific difference indicative of fracture.

One way to improve orthopaedic materials is to understand the exact architectural parameters that influence bone cell behaviour. In this study substrates with highly controlled surface features were created using photo-lithographic processes. These surfaces were contrasted for their ability to influence osteoblast activity and inter-cellular communication.

An etched silicon wafer was created by photolithography and used to hot-emboss grooved substrates (10-30micrometers wide/ 5-16micrometers deep) in poly-carbonate (PC). Smaller features were created on polydimethylsiloxane (PDMS) by casting over a photo-resist patterned silicon wafer. Rat osteoblasts were routinely cultured on flat or micro-fabricated substrates or in media supplemented with osteogenic stimuli for 35 days. Alkaline phosphatase activity was colourimetri-cally localised, and mineralised matrix visualised with Von Kossa staining. Connexin-43 was immunolocalised with a CY-2 conjugated antibody. Intracellular communication was studied using a dye (Lucifer yellow) transfer technique and fluorescence microscopy.

Osteoblasts were aligned on the grooved surface. In 10micrometers grooves, cells were in single rows while at 30micrometers the rows were two/three cells wide. Culture of osteoblasts on these surfaces under osteogenic conditions demonstrated alkaline phosphatase activity comparable to flat surfaces but after 28-35 days there was little evidence of bone-like nodules on the grooved substrates. We hypothesized that on grooved substrates cell:cell communication is compromised thus gap-junctions were studied. Image analysis showed that there was lower connexin-43 expression in cells on grooved substrates and fewer discrete gap junction complexes compared to flat surfaces (p< 0.05 ANOVA.). There were also differences between the grooves with con-nexin-43 most abundant on the widest (30micrometers) and deepest grooves (16micrometers). Analysis of dye transfer demonstrated that whilst cell:cell coupling was maintained within grooves it was reduced at the boundaries of the groove. A surface of asymmetric arrays of micro-columns (diameter 5micrometers) was fabricated to retain lateral interactions between osteoblasts whilst still aligning cells. Osteoblast differentiation now resulted in the formation of numerous bone-like nodules and matrix was aligned in the direction of the shortest column distances.

Maintaining appropriate cell:cell communication structures is pivotal in the process of osteoblast differentiation and the design of novel biomaterial surfaces should ensure that cells can maintain these critical interactions.

Introduction: MEPE was identified in patients with tumors and oncogenic hypophosphatemic osteomalacia (OHO), and therefore thought to inhibit osteoblast differentiation and proliferation. However when looking at the structure of MEPE in detail a number of important domains are observed, including a glycosamino-glycan-attachment site, and a RGD cell-attachment motif. The RGD motif is by far the best characterized peptide sequence for stimulating cell adhesion on synthetic surfaces. Glycosaminoglycan attached to MEPE also has the potential to interact with numerous growth factors, proteases and cell surface receptors. MEPE shares molecular similarities with several dentin-bone phosphoglycoproteins which exhibit an ASARM motif shown to potently inhibit calcium crystallization and crystal growth in the salivary duct system. More recently the ASARM peptide sequence has been shown to be a inhibitor of osteoblast mineralization.

Method: To test the hypothesis that MEPE has multiple functional sites, PCR Primers were designed to provide a truncated MEPE protein, which contained pro-osteogenic motifs and had the anti-osteogenic ASARM motif removed. PCR products were cloned using the pBAD TOPO® TA Expression Kit. MEPE was than expressed in E. coli and purified by HIS column chromatography. Expression of truncated MEPE was confirmed by coomassie staining, Western blot with an antibody to an epitope tag and sequence analysis. Truncated MEPE was passively absorbed overnight at 4 oC in a 96 well plate (0.3–50 micrograms) and Fibronectin was laid down (30 micrograms) as a positive control. Primary rat osteoblasts in serum free medium were seeded into the wells (10,000 cells/well) in triplicate and incubated at 37oC for 24 hours. MTT assay was used to estimate cell number, the coloured product absorbance was then determined at 490nm and adhesion was expressed relative to fibronectin. In addition we laid down truncated MEPE into three 8 well chamber slides as above. This was left overnight at 4 oC. Primary rat osteoblasts were then seeded into the wells (10,000 cells/well) in triplicate and incubated at 37oC for 4 hours in serum free medium. Cells were viewed and images captured with a phase contrast microscope.

Results: We have successfully expressed MEPE in E. Coli and devised a purification strategy for obtaining protein. This has been confirmed by coomassie, silver stain and Western blot analysis. The MTT assay showed a significant increase in cell adhesion and proliferation within wells coated with 50 micrograms (70% +/− 0.67(relative to fibronectin)), 30 micrograms (63% +/− 0.81), 3 micrograms (54% +/− 2.4) of MEPE when compared with TCP (32% +/− 0.56). Furthermore we have shown increased osteoblast spreading with increasing dose when compared to tissue culture plastic alone.

Conclusion: The data shows a dose dependent response of osteoblast to increasing concentrations of the novel MEPE protein. This provides evidence that MEPE without the ASARM domain increases osteoblast adhesion, cell anchorage and spreading. Further studies are currently been undertaken to establish its long term effects on osteoblast function and suitability for incorporation into orthopaedic biomaterials.

Introduction: Several recent studies have highlighted the influence of topographical features on the response of cells to biomaterial surfaces, both in terms of their adhesion, morphology and gene expression. Initial cell adhesion events are believed to be pivotal in dictating subsequent host response to implant materials and therefore understanding the mechansims that regulate these events is fundemental to the design and engineering of the next generation of biomaterials. In our studies we evaluated the adhesion associated events of osteoblasts on four orthopaedic metals, each produced to the same surface finnish. Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) were used to determine the nanometre scale topography and immunofluorescence microscopy and image analysis performed to evaluate cell morphology.

Methods: Vitallium, titanium grade 2 (Ti2), Ti6Al4V and TM2F discs were prepared by Stryker, machined and finished to 1 micron. SEM and AFM were then used to analyse surface topography. Rat primary osteoblasts were then seeded at low density onto the metal discs and allowed to adhere and spread for 24 hours. The cells where fixed and focal adhesions stained with an anti-vinculin Mab. The actin cytoskeleton was counterstained with TRITC phalloidin and nuclei stained with DAPI. Images where captured on both a standard epiflourescence microscope and a confocal microscope. Image analysis was performed using ScionImage^TM to determine cell area, major X/Y axis lengths and numbers of focal adhesions per cell.

Results: Gross observation of all samples revealed a perfectly smooth and flat surface. SEM and AFM analysis showed that at the nanometre scale each exhibited varying degrees of surface roughness. Vitallium was the smoothest with scratches a few nanometres deep running across the surface. In contrast Ti6Al4V, Ti2 and TM2F had increasing degrees of surface roughness, each with details that measured up to a few microns in height.

We measured 1: the area occupied by a cell and 2: the number of focal adhesions per cell. The largest values of osteoblastic cell area were seen with the smoother vitallium surface. In contrast, samples with more numerous and larger surface features resulted in the osteoblasts covering a smaller area and being confined by topographical elements (Ti2> TM2F> Ti6Al4V). In terms of adhesion, there were generally more focal adhesions per cell on rougher surfaces (Ti6Al4V> TM2F> Vitallium> Ti2).

Conclusions: The different nanometre scale features introduced through the manufacturing process of different orthopaedic implant materials influence the adhesion and cell morphology of osteoblast cells within the first 24 hours of contact. This may have consequences for later differentiation and function of these cells.

Introduction: Vascular Endothelial Growth Factor (VEGF) is a proangiogenic cytokine that is expressed highly by many solid tumours often correlating with poor prognosis. VEGF has also been shown to interact with osteoclasts and their precursors in organ cultures to increase differentiation and survival and VEGF receptors have been found on osteoclasts in vitro. In this work we aimed to investigate the expression of VEGF and its receptors in bone metastases from primary breast tumours and further characterise its effects on osteoclasts. We performed immunolocalisation of VEGF in bone metastases and using VEGF and VEGF receptor-specific ligands we assessed their role in osteoclastogenesis in vitro.

Methods: Seventeen specimens of breast cancer metastases to bone were immunohistochemically stained with antibodies to VEGF and its receptors VEGFR1 and 2, and the macrophage marker CD68.

To investigate osteoclastogenesis in vitro Peripheral Blood Mononuclear Cells (PBMC) were isolated from healthy volunteers and cultured over a two-week period under stimulation by cytokines (RANKL, M-CSF, VEGF, PlGF, a specific ligand for VEGFR 1 and VEGF-D, a specific ligand for VEGFR 2). RAW 264.7 cells (a mouse monocyte/macrophage cell line able to differentiate into osteoclast-like cells) were cultured for seven days under stimulation by cytokines (RANKL, VEGF and M-CSF). Osteoclasts were identified by staining for Tartrate Resistant Acid Phophatase (TRAP) and numbers of multinucleated cells counted per treatment. Culture on ivory slices was performed to measure resorption activity of the osteoclasts.

Results: The immunohistochemistry demonstrated that breast cancer metastases express VEGF strongly and that the osteoclasts surrounding metastases express both VEGFR1 (12 of 14 specimens) and VEGFR2 (14 of 14 specimens).

The PBMCs stimulated by VEGF and RANKL together differentiated into multinucleated TRAP positive cells in similar numbers (22±4.7) per field of view to the M-CSF and RANKL (27.3±7.2). Resorption of ivory was identified in these cultures. Stimulation with PlGF and RANKL resulted in increased osteoclastogenesis but VEGF-D with RANKL had little effect. Similar results were seen in triplicate experiments RAW 264.7 cells also differentiated into osteoclast-like cells after stimulation with VEGF and RANKL similar to M-CSF and RANKL.

Discussion and Conclusions: VEGF is able to induce the differentiation of human and mouse osteoclast-like cells from monocyte precursors in the presence of RANKL and this seems to be mediated by VEGFR1. This may lead to an increase in bone resorption in physiological and pathological situations where there is an increase in VEGF, such as in tumours, embryogenesis and fracture repair. VEGF signalling could be a therapeutic target for osteoclast inhibtion in these situations.

Introduction: The biological processes underlying osteolysis in aseptic loosening are not completely understood, but are believed to include factors such as hydrostatic pressure and wear debris. Characterisation of the pseudosynovial membrane from failed implants has revealed numerous cell types with well characterised roles in osteoclastogenesis and bone resorption. More recent work has demonstrated the presence of immunomodulatory cells, including T cells. IL-17 is a T cell product that is believed to be capable of inducing bone resorption. The aims of our study were to characterise the effects of IL-17 on the expression of RANKL and OPG by synovial fibroblasts and to evaluate its role in supporting osteoclastogenesis in vitro.

Materials and Methods: Synovial fibroblasts (SFB) were isolated from tissue obtained at joint replacement surgery. SFB were expanded in culture and used in experiments between passage 4 and 5. Human SFB, and for comparison the human osteosarcoma cell line MG63, were treated with IL-17 (5 and 50ng/ml) for up to 48 hours. The expression and production of RANKL and OPG at 6, 24 and 48hours was assessed by RT-PCR, quantiative real-time PCR, Northern blot and Western blot analyses. To investigate osteoclastogenesis, peripheral blood mononuclear cells (PBMCs) were cultured with IL-17 (5 and 50ng/ml) either alone or with M-CSF (25 ng/ml). After 14–21 days, cultures were fixed and stained for tartrate-resistant acid phosphatase (TRAP) and multinucleated, TRAP positive cells counted. Experiments were repeated on ivory slices and resorption evaluated.

Results: RT-PCR and QT-PCR analysis demonstrated that RANKL mRNA levels in SFB (4 of 5 patients) are enhanced by IL-17 in a biphasic manner. RANKL expression was elevated at 6 hours, returned to near control values at 24 hours before demonstrating increased levels at 48 hours. The expression of RANKL in MG63 cells was enhanced by IL-17 (5ng/ml) at 6 and 24 hours, and by IL-17 (50ng/ml) at 48 hours. The expression of OPG by SFB was upregulated by IL-17 (5 and 50ng/ml) at 6, 24 and 48 hours. The elevated expression of OPG in MG63 cells by IL-17 was time dependent, and this elevated expression was confirmed by Western blot. In cultures of PBMCs, IL-17 alone increased the numbers of TRAP+ve multinucleate cells dose-dependently. Similar levels of TRAP+ve cells were observed in the cultures treated with RANKL and M-CSF, but numbers of multinucleated cells were further increased when M-CSF was supplemented with IL-17. Resorption of ivory wafers was also observed in cultures treated with IL-17.

Conclusions: These results suggest that IL-17 induced osteoclast formation could contribute to the bone loss associated with a wide range of pathological states involving osteolysis and aseptic loosening.

Background: The next generation of biomaterial surfaces for use in orthopaedic surgery will be functionalised to promote osteogenesis. This will be achieved in part by the stable addition of functional bioactive molecules onto the biomaterial surface. Heparan sulphate is a complex glycosaminoglycan (GAG) that displays cell and tissue specific differences in size and levels of sulphation. It is this heterogeneity that underlies the numerous biological roles of heparan sulphate, including binding of growth factors and proteases. Findings by others have shown that the addition of heparan sulphate proteoglycans stimulate osteoblast differentiation in vitro.

Aims: To characterise heparan sulphate structures that support and enhance osteogenesis and have the potential for tissue engineering.

Experiment 1. In order to further investigate the role of heparan-sulphate proteoglycans (HSPGs) in osteogenesis we supplemented cultures of differentiating rat osteo-blasts with sodium chlorate (an inhibitor of the enzyme that sulphates GAG chains) or 4-methylumbelliferyl-b-D-xyloside, BDX (an artificial acceptor of GAG chain synthesis). Interestingly the addition of chlorate to our culture system significantly stimulated alkaline phosphatase levels and increased the area of Von Kossa stained bone-like nodules. Whereas, when BDX was added to differentiating rat osteoblasts there was no increase in alkaline phosphatase activity or nodule area.

Experiment 2. Further characterisation of the HSPGs in chlorate treated osteoblasts showed that whilst they were less sulphated than untreated cells (as shown by low salt elution from an anion exchange chromatography column) they were much more abundant. These observations led us to hypothesise that less sulphated forms of heparan sulphate may well stimulate osteo-blast differentiation.

Experiment 3. To test this hypothesis we took the fully sulphated form of heparan sulphate, heparin and selectively desulphated it using DMSO/methanol (9:1) at 97°C and specifically N-resulphated or N-acetylated. These partially desulphated heparins were then added to osteoblasts cultured under osteogenic conditions. Quantification of bone nodule formation showed that specifically desulphated heparin significantly increased mineralised areas compared to controls whilst the addition of heparin inhibited osteogenesis. How these modified heparan sulphates exert their effect on bone cells is unknown, but a well characterised role of heparan sulphate is the support of FGF signalling. In preliminary studies we have shown through the activation of p42/44 MAPK and proliferation assays that the modified heparan sulphates are able to support FGF signalling in bone cells.

Experiment 4. Currently were are attaching our desulphated heparin to biomaterial scaffolds and examining osteoblast attachment and migration/ingrowth in cell culture.

Conclusion: We have isolated heparan sulphate chains that demonstrate osteogenic properties and have the potential for enhancing biological interactions of orthopaedic implant materials.

Introduction and Aims: To investigate the expression of Vascular Endothelial Growth Factor (VEGF) and its receptors in bone metastases from primary breast tumors and further characterise its effects on osteoclasts in vitro.

Method and Results: Seventeen specimens of breast cancer metastases to bone were immunohistochemically stained for VEGF, its receptors VEGFR1 and 2, and the macrophage marker CD68. This demonstrated that breast cancer metastases express VEGF strongly and that surrounding osteoclasts express both VEGFR1 (12 of 14 specimens) and VEGFR2 (14 of 14 specimens).

To investigate osteoclastogenesis in vitro, Peripheral Blood Mononuclear Cells (PBMC) were isolated from healthy volunteers and cultured under stimulation by cytokines. Tartrate Resistant Acid Phophatase (TRAP) positive multinucleated cells were counted in duplicate per treatment and experiments repeated three times. VEGF and RANKL together induced differentiation of multinucleated TRAP-positive cells in similar numbers (22±4.7[SE]) per field of view to M-CSF and RANKL (27.3±7.2[SE]). Stimulation with PlGF (a specific ligand for VEGFR1) and RANKL induced osteoclastogenesis, but VEGF-D (a specific ligand for VEGFR2) with RANKL had little effect.

RAW 264.7 cells (mouse monocyte cell line) differentiated into osteoclast-like cells after stimulation with VEGF and RANKL similar to M-CSF and RANKL. Culture under the same conditions on ivory disks was performed and resorption of ivory by osteoclasts from both PBMC and RAW cells was identified.

Conclusion: VEGF, the angiogenic cytokine, is expressed highly by many solid tumors often correlating with poor prognosis. We have shown that VEGF induces monocytes to differentiate into osteoclast-like cells in the presence of RANKL and this seems to be mediated by VEGFR1. VEGF may therefore play a role in physiological bone resorption and in pathological situations, such as tumor osteolysis and consequently VEGF signalling may be a therapeutic target for osteoclast inhibition.

Enzymes that breakdown components of the extracellular matrix (ECM) are of fundamental importance, not only in normal bone physiology but also in pathological processes. For instance the temporal and spatial distribution of proteoglycans is not only critical for the mineralisation of bone but is also believed to be responsible for dictating the local bioavailability of glycosaminoglycan-binding growth factors. A sub-family of the ADAMs (a disintegrin and metalloproteinase) has been identified, that contains thrombospondin-like motifs (ADAMTS), and ADAMTS1, 4 and 5 have recently been shown to cleave the major proteoglycan of cartilage, aggrecan. We propose that ADAMTS family members play a novel role in regulating osteoblast function by determining the distribution of proteoglycan in bone.

RT-PCR and Northern blotting experiments have shown expression of ADAMTS1, 3, 4 and 5 in primary rat osteoblasts and in the osteosarcoma cell lines, MG63, TE85 and SaOS-2. ADAMTS1 transcript levels increased with time in primary rat osteoblasts driven by dexamethasone, beta-glycerophosphate and ascorbic acid phosphate to produce bone-like nodules in vitro. Whereas levels of ADAMTS4 that were initially raised in this culture system then became undetectable as mineralisation proceeded.

Since we are interested in the relationship between the osteoblast and matrix molecules, we plated TE85 cells onto an ECM synthesised by MG63 cells and isolated RNA at 1, 24 and 48 hours. Northern analysis showed a transient upregulation of mRNA for both ADAMTS1 and 5 at 1h that was reduced to control levels at 24 and 48h. Transcripts for ADAMTS1 and 3 were also upregulated in primary rat osteoblasts when seeded on ECM molecules like fibronectin and type I collagen for 48 hours. There was however no change in the expression levels of ADAMTS4 when plated on to any of the substrates at any of the time points tested.

These data suggests that cells of the osteoblast lineage express ADAMTS1, 3, 4 and 5 and that individual transcript levels can be regulated by ECM components. The focalised production of ADAMTS family members in response to matrix-derived and other cues may be an important part of bone formation and may have important implications for the way that cells of the osteoblast lineage interact with implant and other biomaterials.