THE CHARACTERISATION OF MONOCYTE RESPONSES TO MEDIATORS RELEASED FROM OSTEOARTHRITIC AND PERIPROSTHETIC TISSUES

BACKGROUND: Aseptic loosening is the major cause of long-term failure of an arthroplasty. Pathophysiological periprosthetic osteolysis is driven by wear debris particles (WDP) accumulating in the surrounding joint tissues. Macrophages, professional phagocytic cells, internalise submicron WDP and upregulate production of inflammatory mediators, stimulating circulating monocytes to migrate into the inflamed joint tissues. These migrated monocytes/tissue macrophages fuse, forming foreign body giant cells (FBGC) and/or osteoclasts, instigating osteolysis at the bone-implant interface. However, the activation of monocytes while in circulation, subsequent migration and differentiation in response to WDP-induced inflammatory mediators are poorly understood. Thus, the alteration in functional surface receptors, gene expression and phagocytic commitments of monocytes needs evaluation. This study details how monocytes from three cohorts of orthopaedic patients respond to mediators derived from aseptic loosening periprosthetic synovium. METHODS: Two sets of patients were recruited for this study. First set; with cohorts of end stage OA (ES-OA, N=10), stable arthroplasty (N=10) and revision arthroplasty due to aseptic loosening (N=10), to isolate, characterise and test peripheral blood mononuclear cells (PBMNCs), from blood samples. A second set with two cohorts donated tissues debrided at surgery; Hip/Knee aseptic loosening revision synovium (N=13) and knee ES-OA synovium (N=25). These tissues were used for H&E staining and preparation of tissue conditioned media (TCM). Pooled revision synovial (RTCM) or ES-OA synovial (OACM) TCM was used for proteomic analysis and PBMNCs migration assays using a 3-μm pores transwell system. Serum free media (SFM) and monocyte chemoattractant protein-1 (MCP-1) were used as spontaneous and specific chemokine directed migration controls, respectively. Migrated cells were assessed using light microscopy. Characterisation by FACs included monocyte markers (CD14 and CD16), surface receptors for chemotaxis (CCR2 and CX3CR1), inflammation (CXCR2), cell adhesion (ITGAM), FBGC/osteoclast precursor formation (DCSTAMP) and monocyte-platelet aggregation (GP1BA). Functional commitment analysis utilised gene expression profiles and 1-μm fluorescence latex beads (LB) phagocytosis assay before and after migration. Parametric and/or non-parametric statistical analyses were undertaken and results presented as mean ± standard deviation. RESULTS: FBGC were identified in sections of revision synovial tissues. Proteomic analysis revealed proteins related to FBGC formation, osteoclast activity, inflammation, cytokine secretion and phagocytosis in RTCM but not in OACM. The PBMNCs monocyte percentage count (72.74±8.69%, p=0.001), cell adhesion receptor (ITGAM, p=0.024), FBGC formation gene expression (IL13RA1, p=0.041) and LB uptake/cell (8±4, p=0.001) were significantly increased in arthroplasty patients when compared with ES-OA patients. The RTCM migrated cell count, CD14+/ classical CD14++CD16- monocyte percentage count, cell adhesion receptor (ITGAM), inflammatory (TNF-α and IL-1β), survival (MAPK1), cell adhesion (C1qR1/CD93), FBGC formation (IL13RA1) genes expression and LB uptake/cell (7±5) were significantly increased (p<0.05) when compared with SFM. CONCLUSION: This study starts to unravel changes in monocytes in patients with an arthroplasty. Characteristic functional alterations in circulating monocytes, indicative of early commitment to the osteoclast lineage, could offer non-invasive, early warning of pathophysiological osteolysis or arthroplasty loosening. New targeted therapies could be developed to slow or prevent progression and reduce extensive revision surgery

In-vitro biocompatibility study of hydroxyapatite coated on Co-Cr-Mo with oxide interlayer

The effects of oxide interlayer on cobalt-chromium-molybdenum substrate were investigated in order to improve the quality of hydroxyapatite (HA) coating as well as enhance the cell responses. Substrates were oxidized at temperature of 850 °C and 1050 °C for 3 hours. Oxidized substrates were then coated with HA slurry using dip coating technique. Analysis of surface morphology, thickness and chemical composition of oxide interlayer prior to HA coating were performed using field-emission scanning electron microscopy, energy dispersive X-ray spectroscopy and grazing X-ray diffraction respectively. It seems that rough surface of oxide interlayer provides better mechanical interlocking of HA particles to the substrate surface with no visible micro-cracks. In addition, the HA coated substrates with oxide interlayer also demonstrate strong attachment and better proliferation of cells compared to HA coated substrates without oxide interlayer. The results also demonstrates that cells were spread out more actively as earlier as day 7 and have greater extensions of filopodium on HA coated substrates with oxide interlayer. It is concluded that the introduction of an intermediate oxide layer on Co-Cr-Mo substrate prior to HA coating has shown a positive effect in terms of improving the quality of HA coating as well as cell bioactivity performance

The physicochemical and biomechanical profile of forsterite and its osteogenic potential of mesenchymal stromal cells

It has been demonstrated that nanocrystalline forsterite powder synthesised using urea as a fuel in sol-gel combustion method had produced a pure forsterite (FU) and possessed superior bioactive characteristics such as bone apatite formation and antibacterial properties. In the present study, 3D-scaffold was fabricated using nanocrystalline forsterite powder in polymer sponge method. The FU scaffold was used in investigating the physicochemical, biomechanics, cell attachment, in vitro biocompatibility and osteogenic differentiation properties. For physicochemical characterisation, Fourier-transform infrared spectroscopy (FTIR), Energy dispersive X-ray (EDX), X-ray diffraction (XRD), Raman spectroscopy, Xray photoemission spectrometer (XPS) and Brunauer-Emmett-Teller (BET) were used. FTIR, EDX, XRD peaks and Raman spectroscopy demonstrated correlating to FU. The XPS confirmed the surface chemistry associating to FU. The BET revealed FU scaffold surface area of 12.67 m(2)/g and total pore size of 0.03 cm(3)/g. Compressive strength of the FU scaffold was found to be 27.18 +/- 13.4 MPa. The human bone marrow derived mesenchymal stromal cells (hBMSCs) characterisation prior to perform seeding on FU scaffold verified the stromal cell phenotypic and lineage commitments. SEM, confocal images and presto blue viability assay suggested good cell attachment and proliferation of hBMSCs on FU scaffold and comparable to a commercial bone substitutes (cBS). Osteogenic proteins and gene expression from day 7 onward indicated FU scaffold had a significant osteogenic potential (p < 0.05), when compared with day 1 as well as between FU and cBS. These findings suggest that FU scaffold has a greater potential for use in orthopaedic and/or orthodontic applications

In vitro evaluation of bioactivity of chemically deposited hydroxyapatite on polyether ether ketone

Polyether ether ketone (PEEK) is considered the best alternative material for titanium for spinal fusion cage implants due to its low elasticity modulus and radiolucent property. The main problem of PEEK is its bioinert properties. Coating with hydroxyapatite (HA) showed very good improvement in bioactivity of the PEEK implants. However the existing methods for deposition of HA have some disadvantages and damage the PEEK substrate. In our previous study a new method for deposition of HA on PEEK was presented. In this study cell proliferation of mesenchymal stem cell and apatite formation in simulated body fluid (SBF) tests were conducted to probe the effect of this new method in improvement of the bioactivity of PEEK. The mesenchymal stem cell proliferation result showed better cells proliferation on the treated layer in comparison with untreated PEEK. The apatite formation results showed the growth of the HA on the treated PEEK but there was not any sight of the growth of HA on the untreated PEEK even after 2 weeks. The results showed the new method of the HA deposition improved the bioactivity of the treated PEEK in comparison with the bare PEEK

Potential Use of 3D CORAGRAF-Loaded PDGF-BB in PLGA Microsphere Seeded Mesenchymal Stromal Cells in Enhancing the Repair of Calvaria Critical-Size Bone Defect in Rat Model

Our previous study evidenced that the 3D CORAGRAF loaded with PLGA microsphere constitutes PDGF-BB can support cell attachment and proliferation and can induce an osteogenic commitment of mesenchymal stromal cells in the in vitro condition. However, how this construct can perform in pathophysiological conditions in terms of repairing critical bone defects is yet to be understood. A study was therefore conducted to investigate the regeneration potential of calvaria critical-size defects using CORAGRAF + PLGA with PDGF-BB + mesenchymal stromal cells (MSCs) in a rat model. A 5 mm critical bone defect was created on calvaria of 40 male Sprague-Dawley rats. CORAGRAF incorporated either with or without PDGF-BB and seeded with rat bone-marrow-derived MSCs was implanted at the defect region. The bone regeneration potential of implanted constructs was assessed using micro-CT imaging and histological staining in weeks 4 and 8. The micro-CT images indicated a significant closure of defects in the cranial bone of the rats treated with 3D CORAGRAF + PLGA with PDGF-BB + MSCs on week 4 and 8 post-implantation. This finding, further supported with the histology outcome where the rat cranial defect treated with CORAGRAF + PLGA with PDGF-BB + MSCs indicated neo-bony ingrowth with organized and mature bone-like morphology as compared with other groups. The previous in vitro results substantiated with our pre-clinical findings demonstrate that the combination of CORAGRAF + PLGA with PDGF-BB + MSCs could be an ideal construct to support bone regeneration in critical bone defects. Hence, this construct can be further investigated for its safety and efficacy in large animal models, or it can be skipped to human trial prior for commercialization

Calcium-silicate-incorporated gellan-chitosan induced osteogenic differentiation in mesenchymal stromal cells

Gellan-chitosan (GC) incorporated with CS: 0% (GC-0 CS), 10% (GC-10 CS), 20% (GC-20 CS) or 40% (GC-40 CS) w/w was prepared using freeze-drying method to investigate its physicochemical, biocompatible, and osteoinductive properties in human bone-marrow mesenchymal stromal cells (hBMSCs). The composition of different groups was reflected in physicochemical analyses performed using BET, FTIR, and XRD. The SEM micrographs revealed excellent hBMSCs attachment in GC-40 CS. The Alamar Blue assay indicated an increased proliferation and viability of seeded hBMSCs in all groups on day 21 as compared with day 0. The hBMSCs seeded in GC-40 CS indicated osteogenic differentiation based on an amplified alkaline-phosphatase release on day 7 and 14 as compared with day 0. These cells supported bone mineralization on GC-40 CS based on Alizarin-Red assay on day 21 as compared with day 7 and increased their osteogenic gene expression (RUNX2, ALP, BGLAP, BMP, and Osteonectin) on day 21. The GC-40 CS–seeded hBMSCs initiated their osteogenic differentiation on day 7 as compared with counterparts based on an increased expression of type-1 collagen and BMP2 in immunocytochemistry analysis. In conclusion, the incorporation of 40% (w/w) calcium silicate in gellan-chitosan showed osteoinduction potential in hBMSCs, making it a potential biomaterial to treat critical bone defects

Typical response of CD14⁺⁺CD16^– monocyte to knee synovial derived mediators as a key target to overcome the onset and progression of osteoarthritis

OBJECTIVE: Synovitis with increased infiltration of immune cells is observed in osteoarthritis (OA). Given the inflammatory condition of synovitis, we explored the protein profile of OA synovium (OAS) and its effect on circulating monocytes activation, migration, and functional commitments. METHODS: Knee-synovium was acquired from end-stage OA (N = 8) and trauma patients (Trauma baseline control: TBC; N = 8) for characterization using H&E histology, IHC (iNOS), LCMS-QTOF, and MALDI-imaging. Response of peripheral blood monocytes to OAS conditioned-media (OACM) was observed using transwell (n = 6). The migrated cells were captured in SEM, quantified using phase-contrast microphotographs, and their activation receptors (CCR2, CXCR2, CX3CR1, and CD11b), pro-inflammatory genes, and phagocytic potential were studied using flow cytometry, gene expression array/qPCR, and latex beads (LB) phagocytosis assay, respectively. RESULTS: The Venn diagram displayed 119 typical proteins in OAS, while 55 proteins in TBCS. The STRING protein network analysis indicated distinctive links between proteins and gene ontology (GO) and revealed proteins associated with leukocyte-mediated immunity in OAS as compared to TBC. The MALDI-imaging showed typical localized proteins at 2234.97, 2522.61, 2627.21, 3329.50, and 3539.69 m/z and IHC confirmed pro-inflammatory iNOS expression in OA synovium. CD14(++)CD16(–) classical monocytes significantly migrated in OACM and expressed CCR2, CXCR2, and CD11b receptors, TNFRSF11A, MAPK1, S100A8, HSPB1, ITGAL, NFATC1, IL13RA1, CD93, IL-1β, TNF-α, and MYD88 genes and increased LB uptake as compared to SFM. CONCLUSION: Our findings suggest that the differential protein profile of OA synovium and the classical monocytes migrated, activated, and functionally committed in response to these mediators could be of therapeutic advantage