[Show abstract][Hide abstract] ABSTRACT: Disease modifying anti-rheumatic drugs (DMARDs) may interfere with bone healing. Previous studies give conflicting advice regarding discontinuation of these drugs in the peri-operative setting. No consensus exists in current practice especially with the newer DMARDs such as Leflunomide, Etanercept, and Infliximab. The aim of this study was to assess the in-vitro effect of these drugs alone and in relevant clinical combinations on Osteoblast activity.
Osteoblasts were cultured from femoral heads obtained from five young otherwise healthy patients undergoing total hip replacement. The cells were cultured using techniques that have been previously described. A full factorial design was used to set up the experiment on samples obtained from the five donors. Normal therapeutic concentrations of the various DMARDs were added alone and in combination to the media. The cell proliferation was estimated after two weeks using spectrophotometric technique using Roche Cell proliferation Kit. Multilevel regression analysis was used to estimate which drugs or combination of drugs significantly affected cell proliferation.
Infliximab and Leflunomide had an overall significant inhibitory effect (p < 0.05). Dexamethasone had a small stimulatory effect that was however strongly donor-dependent. The cox-2 inhibitor Etoricoxib was found to negate or increase the action of two other drugs (Leflunomide and Dexamethasone). Methotrexate and Etanercept had no discernable donor-dependant or donor-independent effect on osteoblast proliferation.
Our study indicates that in-vitro osteoblast proliferation can be inhibited by the presence of certain DMARDs. Combinations of drugs had an influence and could negate the action of a drug on osteoblast proliferation. The response to drugs may be donor-dependent.
Journal of Orthopaedic Surgery and Research 01/2009; 4:17. · 1.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Membrane-bound heparan sulphate proteoglycans (HSPGs) act as co-receptors and presenters of cytokines and are involved in cell-matrix and cell-cell adhesion.
To investigate which HSPGs are expressed in knee joint synovia from patients with different forms of arthritis and normal individuals.
Synovial samples were obtained from patients with early rheumatoid arthritis (n = 8), longstanding rheumatoid arthritis (n = 13), psoriatic arthritis (n = 7), osteoarthritis (n = 6) and normal joints (n = 12). Expression of syndecan-1, -2, -3 and -4 and glypican-1, -3 and -4 was analysed by immunohistochemistry and dual label immunofluorescence.
The expression of HSPGs in chronically inflamed synovium exhibited a differential distribution. Syndecan-1 was present in the mononuclear infiltrates of synovia from patients with rheumatoid and psoriatic arthritis where it was expressed by plasma cells. Syndecan-2 was present mainly in blood vessels where it occurred on endothelial cells, pericytes and smooth muscle cells. Syndecan-3 stained intensely in endothelial cells but also occurred in sublining macrophages and the lining layer. Glypican-4 occurred in the lining layer and blood vessels. Increased expression of these HSPGs was apparent in rheumatoid and psoriatic compared to osteoarthritic and normal synovia. Little or no staining for syndecan-4, glypican-1 and glypican-3 was seen in all samples.
Selected HSPGs, such as syndecan-1, -2 and -3 and glypican-4, could play a part in the pathophysiology of arthritis, such as the migration and retention of leukocytes and angiogenesis in the chronically inflamed synovium.
Annals of the rheumatic diseases 06/2008; 67(5):592-601. · 8.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Previous studies have shown that transplantation of bone marrow stromal cells (MSCs) in animal models of spinal cord injury (SCI) encourages functional recovery. Here, we have examined the growth in cell culture of MSCs isolated from individuals with SCI, compared with non-SCI donors.
Centre for Spinal Studies, Midland Centre for Spinal Injuries, RJAH Orthopaedic Hospital, Oswestry, UK.
Bone marrow was harvested from the iliac crest of donors with long-term SCI (>3 months, n=9) or from non-SCI donors (n=7). Mononuclear cells were plated out into tissue culture flasks and the adherent MSC population subsequently expanded in monolayer culture. MSC were passaged by trypsinization at 70% confluence and routinely seeded into new flasks at a density of 5 x 10(3) cells per cm(2). Expanded cell cultures were phenotypically characterized by CD-immunoprofiling and by their differentiation potential along chondrocyte, osteoblast and adipocyte lineages. The influence of cell-seeding density on the rate of cell culture expansion and degree of cell senescence was examined in separate experiments.
In SCI, but not in non-SCI donors the number of adherent cells harvested at passage I was age-related. The proliferation rate (culture doubling times) between passages I and II was significantly greater in cultures from SCI donors with cervical lesions than in those with thoracic lesions. There was no significant difference, however, in either the overall cell harvests at passages I or II or in the culture doubling times between SCI and non-SCI donors. At passage II, more than 95% of cells were CD34-ve, CD45-ve and CD105+ve, which is characteristic of human MSC cultures. Furthermore, passage II cells differentiated along all three mesenchymal lineages tested. Seeding passage I-III cells at cell densities lower than 5 x 10(3) cells per cm(2) significantly reduced culture doubling times and significantly increased overall cell harvests while having no effect on cell senescence.
MSCs from individuals with SCI can be successfully isolated and expanded in culture; this is encouraging for the future development of MSC transplantation therapies to treat SCI. Age, level of spinal injury and cell-seeding density were all found to relate to the growth kinetics of MSC cultures in vitro, albeit in a small sample group. Therefore, these factors should be considered if either the overall number or the timing of MSC transplantations post-injury is found to relate to functional recovery.
[Show abstract][Hide abstract] ABSTRACT: Mesenchymal stem cells (MSCs) are non-haematopoeitic, stromal cells that are capable of differentiating into mesenchymal tissues such as bone and cartilage. They are rare in bone marrow, but have the ability to expand many-fold in culture, and retain their growth and multi-lineage potential. The properties of MSCs make them ideal candidates for tissue engineering. It has been shown that MSCs, when transplanted systemically, can home to sites of injury, suggesting that MSCs possess migratory capacity; however, mechanisms underlying migration of these cells remain unclear. Chemokine receptors and their ligands play an important role in tissue-specific homing of leukocytes. Here we define the cell surface chemokine receptor repertoire of murine MSCs from bone marrow, with a view to determining their migratory activity. We also define the chemokine receptor repertoire of human MSCs from bone marrow as a comparison. We isolated murine MSCs from the long bones of Balb/c mice by density gradient centrifugation and adherent cell culture. Human MSCs were isolated from the bone marrow of patients undergoing hip replacement by density gradient centrifugation and adherent cell culture. The expression of chemokine receptors on the surface of MSCs was studied using flow cytometry. Primary murine MSCs expressed CCR6, CCR9, CXCR3 and CXCR6 on a large proportion of cells (73+/-11%, 44+/-25%, 55+/-18% and 96+/-2% respectively). Chemotaxis assays were used to verify functionality of these chemokine receptors. We have also demonstrated expression of these receptors on human MSCs, revealing some similarity in chemokine receptor expression between the two species. Consequently, these murine MSCs would be a useful model to further study the role of chemokine receptors in in vivo models of disease and injury, for example in recruitment of MSCs to inflamed tissues for repair or immunosuppression.
PLoS ONE 02/2008; 3(8):e2934. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Successful healing of a nine-year tibial nonunion resistant to six previous surgical procedures was achieved by tissue engineering. We used autologous bone marrow stromal cells (BMSCs) expanded to 5 x 10(6) cells after three weeks' tissue culture. Calcium sulphate (CaSO4) in pellet form was combined with these cells at operation. The nonunion was clinically and radiologically healed two months after implantation. This is the description of on healing of a long-standing tibial nonunion by tissue engineering. The successful combination of BMSCs and CaSO4 has not to our knowledge been reported in a clinical setting.
Journal of Bone and Joint Surgery - British Volume 11/2007; 89(10):1382-6. · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Meniscus deficient knees develop early osteoarthritis in the knee. Autologous Chondrocyte Implantation has provided a new dimension to the treatment of chondral defects in the knee, with 85% good to excellent results and a long-term durable outcome of up-to 11 years. However, it is contraindicated in meniscus deficient knees. Allogenic Meniscus Transplantation gives good symptomatic relief in meniscus deficient knees, with a success rate of 89%. However, it is contraindicated in advanced cartilage degeneration. We hypothesized that combination of these two might be a solution for bone-on-bone arthritis in young individuals. We studied a consecutive series of eight patients, with mean age of 43 years, presenting with large kissing chondral defects, secondary to the previous meniscectomy. All the patients were treated with a combination of Autologous Chondrocyte Implantation and Allogenic Meniscus Transplantation. Mean pre-operative Lysholm score was 49, which rose to mean of 66 at 1 year, an average increase by 16.4 points. Six patients showed significant improvement at one year. MRI scans showed good integration of the menisci with the capsule, without any rejection. Histology confirmed the integration. All the patients could lead an active life-style. Five patients maintained the improvement at a mean follow-up of 3.2 years. We could not find any deleterious effects of the combination of these two techniques. So we conclude that the combination of these two techniques together may act a one step towards a true biological knee replacement.
The Knee 11/2007; 14(5):361-8. · 2.01 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MSCs are nonhematopoietic stromal cells that are capable of differentiating into, and contribute to the regeneration of, mesenchymal tissues such as bone, cartilage, muscle, ligament, tendon, and adipose. MSCs are rare in bone marrow, representing ∼1 in 10,000 nucleated cells. Although not immortal, they have the ability to expand manyfold in culture while retaining their growth and multilineage potential. MSCs are identified by the expression of many molecules including CD105 (SH2) and CD73 (SH3/4) and are negative for the hematopoietic markers CD34, CD45, and CD14. The properties of MSCs make these cells potentially ideal candidates for tissue engineering. It has been shown that MSCs, when transplanted systemically, are able to migrate to sites of injury in animals, suggesting that MSCs possess migratory capacity. However, the mechanisms underlying the migration of these cells remain unclear. Chemokine receptors and their ligands and adhesion molecules play an important role in tissue-specific homing of leukocytes and have also been implicated in trafficking of hematopoietic precursors into and through tissue. Several studies have reported the functional expression of various chemokine receptors and adhesion molecules on human MSCs. Harnessing the migratory potential of MSCs by modulating their chemokine-chemokine receptor interactions may be a powerful way to increase their ability to correct inherited disorders of mesenchymal tissues or facilitate tissue repair in vivo. The current review describes what is known about MSCs and their capacity to home to tissues together with the associated molecular mechanisms involving chemokine receptors and adhesion molecules.Disclosure of potential conflicts of interest is found at the end of this article.
[Show abstract][Hide abstract] ABSTRACT: The use of adult stem cells to regenerate damaged tissue circumvents the moral and technical issues associated with the use of those from an embryonic source. Mesenchymal stem cells (MSC) can be isolated from a variety of tissues, most commonly from the bone marrow, and, although they represent a very small percentage of these cells, are easily expandable. Recently, the use of MSC has provided clinical benefit to patients with osteogenesis imperfecta, graft-versus-host disease and myocardial infarction. The cellular cues that enabled the MSC to be directed to the sites of tissue damage and the mechanisms by which MSC then exert their therapeutic effect are becoming clearer. This review discusses the relative therapeutic importance of the ability of MSC to differentiate into multiple cell lineages or stimulate resident or attracted cells via a paracrine mode of action. It also reviews recent findings that MSC home to damaged tissues in a similar, but somewhat distinct, manner to that of leucocytes via the utilisation of adhesion molecules, such as selectins and integrins, and chemokines and their receptors in a manner reminiscent of leucocytes trafficking from the blood stream to inflammatory sites.
British Journal of Haematology 07/2007; 137(6):491-502. · 4.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In animal models, transplantation of bone marrow stromal cells (MSC) into the spinal cord following injury enhances axonal regeneration and promotes functional recovery. How these improvements come about is currently unclear. We have examined the interaction of MSC with neurons, using an established in vitro model of nerve growth, in the presence of substrate-bound extracellular molecules that are thought to inhibit axonal regeneration, i.e., neural proteoglycans (CSPG), myelin associated glycoprotein (MAG) and Nogo-A. Each of these molecules repelled neurite outgrowth from dorsal root ganglia (DRG) in a concentration-dependent manner. However, these nerve-inhibitory effects were much reduced in MSC/DRG co-cultures. Video microscopy demonstrated that MSC acted as "cellular bridges" and also "towed" neurites over the nerve-inhibitory substrates. Whereas conditioned medium from MSC cultures stimulated DRG neurite outgrowth over type I collagen, it did not promote outgrowth over CSPG, MAG or Nogo-A. These findings suggest that MSC transplantation may promote axonal regeneration both by stimulating nerve growth via secreted factors and also by reducing the nerve-inhibitory effects of the extracellular molecules present.
Biochemical and Biophysical Research Communications 04/2007; 354(2):559-66. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Fresh frozen bone allograft is available for human recipients after at least 6 months of quarantine at -80 degrees C. It is assumed that cryopreservation without cryoprotectant removes all viable donor cells.
We studied the in vitro cell growth from samples of fresh frozen human femoral head allografts after they had been released for patient use, and compared it with cell growth from a control group of fresh cancellous bone specimens from excised femoral heads (8 samples in each group).
Cell outgrowths were seen in all of the fresh cancellous bone specimens (100% of replicates, 48 replicates per specimen) but only in a small minority of replicates from 4 of the allograft samples (mean 3.1%). Reverse transcription quantitative polymerase chain reaction (RT-qPCR) investigations revealed that cell outgrowths from both groups contained mRNA for transcription factors Runx2 and Osterix, and also for matrix proteins collagen type I, osteocalcin and bone sialoprotein. This is consistent with the cells being osteoblast-related.
This study confirms that fresh frozen human bone allograft cells have the potential to grow in vitro, but the significance of this in recipients is currently unknown.
[Show abstract][Hide abstract] ABSTRACT: To identify and characterize which endothelial heparan sulfate proteoglycans (HSPGs) bind the chemokine CXCL8 (interleukin-8) in human rheumatoid arthritis (RA) and nonrheumatoid synovia.
CXCL8 binding to endothelial HSPGs in RA and nonrheumatoid synovia was determined by heparinase treatment followed by an in situ binding assay and autoradiography. Endothelial HSPGs were characterized by immunohistochemical analysis and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Phosphatidyinositol-specific phospholipase C (PI-PLC) and antibodies to HSPGs were used in in situ binding experiments to identify which HSPGs bound CXCL8.
The expression of heparan sulfate on microvascular endothelial cells was demonstrated in RA and nonrheumatoid synovia. Using antibodies to syndecan-1-4 and glypican-1, -3, and -4, the selective expression of syndecan-3 by endothelial cells was detected in RA and nonrheumatoid synovia. In addition, RT-PCR showed the presence of syndecan-3 messenger RNA in endothelial cells extracted from RA and nonrheumatoid synovia. (125)I-CXCL8 bound to venular endothelial cells; treatment with heparinases I and III significantly reduced this binding in RA but not nonrheumatoid synovia. (125)I-CXCL8 binding was not reduced after treatment with PI-PLC, which cleaves glycosyl phosphatidylinositol linkages, suggesting that CXCL8 did not bind to glypicans. Treatment of synovia with a syndecan-3 antibody reduced CXCL8 binding to RA but not nonrheumatoid endothelial cells; however, no reduction in binding was observed with syndecan-2 or glypican-4 antibodies.
Our results show the selective induction of a CXCL8 binding site on endothelial syndecan-3 in RA synovium. This site may be involved in leukocyte trafficking into RA synovial tissue.
[Show abstract][Hide abstract] ABSTRACT: To evaluate magnetic resonance (MR) imaging features of autologous chondrocyte implantation (ACI) grafts and compare these with graft histologic features 1 year after ACI for treatment of femoral condylar defects.
This study was approved by the regional ethics committee, and all patients gave informed consent. Forty-one patients (mean age, 35 years; 30 men, 11 women) underwent ACI for treatment of femoral condylar defects. One year later, knee joint MR imaging and graft biopsy were performed. Graft biopsy results were categorized into those showing hyaline, mixed fibrohyaline cartilage, fibrocartilage, and fibrous tissue. Standard T1-, T2-, T2*-, and intermediate-weighted sequences were performed, as well as three-dimensional (3D) fast low-angle shot (FLASH) and double-echo steady-state sequences for cartilage assessment. ACI grafts were assessed for signal intensity (with FLASH sequence), thickness, overgrowth, surface smoothness, integration to adjacent cartilage and underlying bone, bone marrow edema underneath graft, and contour of bone underneath graft. MR images were assessed by two observers, first independently and then in consensus. MR imaging findings were correlated with histologic findings.
All 41 grafts were present at 1-year follow-up. The graft consisted of hyaline cartilage in four, mixed fibrohyaline cartilage in 10, fibrocartilage in 25, and fibrous tissue in two cases. Graft signal intensity was virtually always lower than adjacent normal cartilage signal intensity, and there was no relationship between graft signal intensity and histologic appearance (P = .34). Graft thickness (P = .83), overgrowth (P = .69), surface smoothness (P = .28), and integration with adjacent cartilage and underlying bone (P = .90); edema in bone marrow underneath graft (P = .63); and bone contour underneath graft (P = .94) at MR imaging had no correlation with graft histologic appearance. Graft overgrowth (n = 16; 39%) and edema-like signal in bone marrow underneath graft (n = 23; 56%) were common. The origin of graft overgrowth remains unclear.
With the methods presented here, MR imaging findings cannot predict ACI graft histologic features, and graft histologic appearance determined at biopsy was not related to graft signal intensity, graft thickness, overgrowth, surface smoothness, integration with adjacent cartilage or underlying bone, signal intensity change in underlying bone marrow, or underlying bone contour. Overgrowth and bone marrow changes underneath the graft were common.
[Show abstract][Hide abstract] ABSTRACT: In patients with rheumatoid arthritis (RA), chemokine and chemokine receptor interactions play a central role in the recruitment of leukocytes into inflamed joints. This study was undertaken to characterize the expression of chemokine receptors in the synovial tissue of RA and non-RA patients. RA synovia (n = 8) were obtained from knee joint replacement operations and control non-RA synovia (n = 9) were obtained from arthroscopic knee biopsies sampled from patients with recent meniscal or articular cartilage damage or degeneration. The mRNA expression of chemokine receptors and their ligands was determined using gene microarrays and PCR. The protein expression of these genes was demonstrated by single-label and double-label immunohistochemistry. Microarray analysis showed the mRNA for CXCR5 to be more abundant in RA than non-RA synovial tissue, and of the chemokine receptors studied CXCR5 showed the greatest upregulation. PCR experiments confirmed the differential expression of CXCR5. By immunohistochemistry we were able to detect CXCR5 in all RA and non-RA samples. In the RA samples the presence of CXCR5 was observed on B cells and T cells in the infiltrates but also on macrophages and endothelial cells. In the non-RA samples the presence of CXCR5 was limited to macrophages and endothelial cells. CXCR5 expression in synovial fluid macrophages and peripheral blood monocytes from RA patients was confirmed by PCR. The present study shows that CXCR5 is upregulated in RA synovial tissue and is expressed in a variety of cell types. This receptor may be involved in the recruitment and positioning of B cells, T cells and monocytes/macrophages in the RA synovium. More importantly, the increased level of CXCR5, a homeostatic chemokine receptor, in the RA synovium suggests that non-inflammatory receptor-ligand pairs might play an important role in the pathogenesis of RA.
Arthritis research & therapy 02/2005; 7(2):R217-29. · 4.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The aim of the study was to compare the ability of the human Duffy antigen to bind homeostatic and inflammatory chemokines. Homeostatic chemokines did not bind to the Duffy antigen on erythrocytes with high affinity. In contrast, 60% of inflammatory chemokines bound strongly to Duffy, with no obvious preference for CXC or CC classes. It was investigated if this binding profile was reflected in the binding pattern of endothelial cells. Two examples of homeostatic (125I-CXCL12 and 125I-CCL21) and inflammatory (125I-CXCL8 and 125I-CCL5) chemokines were incubated with human synovia. In agreement with the erythrocyte binding data, intense specific signals for CXCL8 and CCL5 binding were found on endothelial cells, whereas CXCL12 and CCL21 showed only weak binding to these cells. Our study provides evidence that the human Duffy antigen binds selected inflammatory, but not homeostatic, chemokines and that this binding pattern is reflected by endothelial cells within inflamed and non-inflamed tissue.
Biochemical and Biophysical Research Communications 09/2004; 321(2):306-12. · 2.28 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Freshly isolated bovine articular chondrocytes were seeded into a resorbable gelatin sponge and cultured in the absence or presence of extrinsic high molecular weight hyaluronan (HA) for up to 1 month. The gelatin sponge could be uniformly and reproducibly loaded with chondrocytes. Immunostaining demonstrated that accumulation of pericellular HA increased in the presence of extrinsic HA. However, this approach could not differentiate between extrinsic and endogenous HA. More chondrocytes were retained within the loaded sponges in the presence of HA. Both cell number and matrix synthesis were increased in the presence of high molecular weight HA throughout the time course. Proteoglycan synthesis per cell increased by 22-fold in the presence of HA at 500 microg/mL. Our model demonstrates that HA can be used as a tool not only to expand freshly isolated chondrocyte numbers but also to increase matrix synthesis and deposition within a resorbable gelatin sponge. Autologous chondrocytes for tissue engineering are always in short supply, so this could be a useful tool with which to increase the retention of cells seeded into other types of scaffold matrices before implanting them into a cartilage defect.
[Show abstract][Hide abstract] ABSTRACT: Risk of viral and/or prion disease transmission associated with the use of fetal bovine serum in clinical cell culture has led to the increasing use of autologous human serum in tissue engineering. A relatively large volume of blood is needed and so, to decrease patient discomfort, we have investigated the feasibility of taking blood when the patient is anesthetized. Two serum samples were prepared from each of 22 patients: (1). from the awake patient (PRE) and (2). from the patient 5 min after induction of general anesthesia (PER). The sera were compared for their ability to support the in vitro proliferation of primary human chondrocytes, determined by cell counting. The effects of anesthetic agents on the PER/PRE cell number ratio were established by analysis of variance and stepwise multilinear regression analysis. The PER sample supported higher growth in 2 of 22 patients, equivalent growth in another 11, and significantly lower growth in the remaining 8. Only the opiate analgesics (fentanyl [Sublimaze], alfentanyl [Rapifen], and diamorphine) had a significant and inhibitory effect on chondrocyte proliferation. It is suggested that opiate analgesics be avoided when blood is taken to support the in vitro growth of human cells.
[Show abstract][Hide abstract] ABSTRACT: At sites of inflammation and in normal immune surveillance, chemokines direct leukocyte migration across the endothelium. Many cell types that are extravascular can produce chemokines, and for these mediators to directly elicit leukocyte migration from the blood, they would need to reach the luminal surface of the endothelium. This article reviews the evidence that endothelial cells are active in transcytosing chemokines to their luminal surfaces, where they are presented to leukocytes. The endothelial binding sites that transport and present chemokines include glycosaminoglycans (GAGs) and possibly the Duffy antigen/receptor for chemokines (DARC). The binding residues on chemokines that interact with GAGs are discussed, as are the carbohydrate structures on GAGs that bind these cytokines. The expression of particular GAG structures by endothelial cells may lend selectivity to the type of chemokine presented in a given tissue, thereby contributing to selective leukocyte recruitment. At the luminal surface of the endothelium, chemokines are preferentially presented to blood leukocytes on the tips of microvillous processes. Similarly, certain adhesion molecules and chemokine receptors are also preferentially distributed on leukocyte and endothelial microvilli, and evidence suggests an important role for these structures in creating the necessary surface topography for leukocyte migration. Finally, the mechanisms of chemokine transcytosis and presentation by endothelial cells are incorporated into the current model of chemokine-driven leukocyte extravasation.