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.
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.
Examination of MT1-MMP expression in osteosarcoma biopsy tissue in relation to clinical outcome Assessment of MT1-MMP, together with hypoxia inducible factors HIF-1α and HIF-2α expression in a panel of osteosarcoma cell lines under normoxia and hypoxiaBackground
Aims
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. Modification of the surface properties of titanium alloy, to enhance suitability for early osseointegration.Background
Aim
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. 10 patients with single fractures of the long bones, wrist or ankle gave a blood sample upon presentation at the fracture clinic. 10 healthy, age-matched, non-fracture volunteers also donated blood. Plasma was isolated and the albumin and IgG fractions removed before loading equal amounts of each sample onto 2 dimensional polyacrylamide gels for analysis by isoelectric point in the first dimension and molecular mass in the second dimension. Protein profiles between fracture patients and non-fracture controls were contrasted using Phoretix 2D analysis software. Data analysis differentiated between the average gel of the patient group and the average gel of the control group. More than 300 protein spots were observed in both the control and patient group. Seven protein spots were identified which showed a statistically significant (p<0.05) difference between the control and patient samples. Of these, three spots (X, Y, Z) were clear, distinct and present in at least 80% of these gels. All the three spots were up regulated in the patient group as opposed to the control group. These proteins are currently being investigated further by MALDI-TOF TOF for specific protein identification. Proteomic analysis is already a powerful tool in the identification of disease markers. We aim to show here that there are differences seen in blood plasma profiles in fracture patients compared to non-fracture healthy controls. The differences seen may help us to understand the fracture repair process better.Materials and Methods
Discussion
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.
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).
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.
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.
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.
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.