Gerjo J.V.M. van Osch

Effect of Arthritic Synovial Fluids on the Expression of Immunomodulatory Factors by Mesenchymal Stem Cells: An Explorative in vitro Study

Background: In diseased joints, the catabolic environment results in progressive joint damage. Mesenchymal stem cells (MSCs) can have immunomodulatory effects by secreting anti-inflammatory factors. To exert these effects, MSCs need to be triggered by pro-inflammatory cytokines. To explore the potential of MSCs as a treatment for diseased joints, we studied the effect of synovial fluid (SF) from donors with different joint diseases and donors without joint pathology on the immunomodulatory capacities of human MSCs in vitro. We hypothesized that SF of diseased joints influences the immunomodulatory effects of MSCs. Materials and Methods: MSCs were cultured in medium with SF of six osteoarthritis (OA) or six rheumatoid arthritis (RA) donors and three donors without joint pathology were used as control. Gene expressions of IL-6, HGF, TNFa, TGFb1, and indoleamine 2,3-dioxygenase (IDO) were analyzed. l-kynurenine concentration in conditioned medium (CM) by MSCs with SF was determined as a measure of IDO activity by MSCs. Furthermore, the effect of CM with SF on proliferation of activated lymphocytes was analyzed. Results: Addition of SF significantly up-regulated the mRNA expression of IL-6 and IDO in MSCs. SF(OA) induced significantly higher expression of IDO than SF(control), although no difference in IDO activity of the MSCs could be shown with a l-kynurenine assay. Medium conditioned by MSCs with SF(OA or RA) suppressed activated lymphocyte proliferation in vitro more than medium conditioned by MSCs without SF or with SF(control). Discussion: SF can influence the expression of genes involved in immunomodulation by MSCs and the effect on lymphocyte proliferation. We found indications for disease-specific differences between SFs but the variation between donors, even within one disease group was high. These data warrant further research to examine the potential application of MSC therapy in arthritic joints.

Can Platelet-Rich Plasma Enhance Tendon Repair? A Cell Culture Study

Background Autologous platelet-rich plasma (PRP) application appears to improve tendon healing in traumatic tendon injuries, but basic knowledge of how PRP promotes tendon repair is needed. Hypothesis Platelet-rich plasma has a positive effect on cell proliferation and collagen production and induces the production of matrix-degrading enzymes and endogenous growth factors by human tenocytes. Study Design Controlled laboratory study. Methods Human tenocytes were cultured 14 days in 2% fetal calf serum medium complemented with 0%, 10%, or 20% vol/vol platelet-rich clot releasate ([PRCR] the active releasate of PRP) or platelet-poor clot releasate (PPCR). At day 4,7, and 14, cell amount, total collagen, and gene expression of collagen Iα1 (COL1) and Mod (COL3), matrix metalloproteinases ([MMPs] MMP1, MMP3, and MMP13), vascular endothelial-derived growth factor (VEGF)-A, and transforming growth factor (TGF)-β1 were analyzed. Results Platelet numbers in PRP increased to 2.55 times baseline. Growth-factor concentrations of VEGF and platelet-derived growth factor (PDGF)-BB were higher in PRCR than PPCR. Both PRCR and PPCR increased cell number and total collagen, whereas they decreased gene expression of COL1 and COL3 without affecting the COL3/COL1 ratio. PRCR, but not PPCR, showed upregulation of MMP1 and MMP3 expression. Matrix metalloproteinase 13 expression was not altered by either treatment. PRCR increased VEGF-A expression at all time points and TGF-β1 expression at day 4. Conclusion In human tenocyte cultures, PRCR, but also PPCR, stimulates cell proliferation and total collagen production. PRCR, but not PPCR, slightly increases the expression of matrix-degrading enzymes and endogenous growth factors. Clinical Relevance In vivo use of PRP, but also of PPP to a certain extent, in tendon injuries might accelerate the catabolic demarcation of traumatically injured tendon matrices and promote angiogenesis and formation of a fibrovascular callus. Whether this will also be beneficial for degenerative tendinopathies remains to be elucidated.

Association between weight or body mass index and hand osteoarthritis: a systematic review

Objective To investigate the association between weight or body mass index (BMI) and the development of hand osteoarthritis. Methods Systematic review of observational studies. Medical databases were searched up to April 2008. Articles that presented data on the association between weight and hand osteoarthritis were selected. The qualities of these studies were then assessed by two independent reviewers using a 19 criteria scoring system. Using the mean scores of all studies as a cut-off value, the studies were deemed as high or low quality. Study quality and study designs were combined to determine the level of evidence using best-evidence synthesis, which consisted of five levels of evidence. Results From the 25 studies included, two had cohort, three case–control and 20 cross-sectional study designs. Fifteen studies were considered high-quality studies. Of these high-quality studies, one cohort, two case–control and seven cross-sectional studies showed a positive association between weight or BMI and hand osteoarthritis. Based on three high-quality studies with preferred study designs (one cohort and two case–control) with a positive association, the level of evidence of the association between overweight and developing hand osteoarthritis is moderate. The approximate risk ratio of this association is 1.9. Conclusion Weight or BMI is associated with the development of hand osteoarthritis. The level of evidence of published studies is moderate according to best-evidence synthesis. Further high-quality cohort or case–control studies are needed to elucidate the role of weight in hand osteoarthritis.

Platelet-Rich Plasma Releasate Inhibits Inflammatory Processes in Osteoarthritic Chondrocytes

Background: Platelet-rich plasma (PRP) has recently been postulated as a treatment for osteoarthritis (OA). Although anabolic effects of PRP on chondrocytes are well documented, no reports are known addressing effects on cartilage degeneration. Since OA is characterized by a catabolic and inflammatory joint environment, the authors investigated whether PRP was able to counteract the effects of such an environment on human osteoarthritic chondrocytes. Hypothesis: Platelet-rich plasma inhibits inflammatory effects of interleukin-1 (IL-1) beta on human osteoarthritic chondrocytes. Study Design: Controlled laboratory study. Methods: Human osteoarthritic chondrocytes were cultured in the presence of IL-1 beta to mimic an osteoarthritic environment. Medium was supplemented with 0%, 1%, or 10% PRP releasate (PRPr, the active releasate of PRP). After 48 hours, gene expression of collagen type II alpha 1 (COL2A1), aggrecan (ACAN), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)4, ADAMTS5, matrix metalloproteinase (MMP)13, and prostaglandin-endoperoxide synthase (PTGS)2 was analyzed. Additionally, glycosaminoglycan (GAG) content, nitric oxide (NO) production, and nuclear factor kappa B (NFκB) activation were studied. Results: Platelet-rich plasma releasate diminished IL-1 beta–induced inhibition of COL2A1 and ACAN gene expression. The PRPr also reduced IL-1 beta–induced increase of ADAMTS4 and PTGS2 gene expression. ADAMTS5 gene expression and GAG content were not influenced by IL-1 beta or additional PRPr. Matrix metalloproteinase 13 gene expression and NO production were upregulated by IL-1 beta but not affected by added PRPr. Finally, PRPr reduced IL-1 beta–induced NFκB activation to control levels containing no IL-1 beta. Conclusion: Platelet-rich plasma releasate diminished multiple inflammatory IL-1 beta–mediated effects on human osteoarthritic chondrocytes, including inhibition of NFκB activation. Clinical Relevance: Platelet-rich plasma releasate counteracts effects of an inflammatory environment on genes regulating matrix degradation and formation in human chondrocytes. Platelet-rich plasma releasate decreases NFκB activation, a major pathway involved in the pathogenesis of OA. These results encourage further study of PRP as a treatment for OA.

Achilles Tendinosis: Changes in Biochemical Composition and Collagen Turnover Rate

Background Understanding biochemical and structural changes of the extracellular matrix in Achilles tendinosis might be important for developing mechanism-based therapies. Hypothesis In Achilles tendinosis, changes occur in biochemical composition and collagen turnover rate. Study Design Descriptive laboratory study. Methods From 10 patients undergoing surgery for Achilles tendinopathy, 1 tendinosis biopsy specimen and 1 biopsy specimen of macroscopically healthy tendon tissue adjacent to the lesion were collected. Furthermore, biopsy samples were collected from 3 donors with asymptomatic Achilles tendons. Water content, collagen content, percentage of denatured collagen, amount of lysine hydroxylation, number of enzymatic and nonenzymatic crosslinks, matrix metalloproteinase activity, and matrix metalloproteinase and collagen gene-expression levels were analyzed. Results In tendinotic lesions, the water content was highest, and collagen content was subnormal with higher amounts of denatured/damaged collagen. Low pentosidine levels in tendinotic tissue indicated the presence of relatively young collagenous matrix. More hydroxylated lysine residues were present in tendinotic samples, but enzymatic crosslinks revealed no differences between tendinotic, adjacent, and healthy samples. In tendinotic specimens, matrix metalloproteinase activity was higher, matrix metalloproteinase gene-expression profile was altered, and collagen type I and III gene expression were upregulated. Conclusion In Achilles tendinosis, the collagen turnover rate is increased, and the natural biochemical composition of the collagenous matrix is compromised. Clinical Relevance Although tendon tissue directly adjacent to an Achilles tendinosis lesion looks macroscopically healthy, histological and biochemical degenerative changes in adjacent tissue are evident, which may have implications for surgical interventions.

Cartilage repair: past and future--lessons for regenerative medicine.

•  Introduction: the impaired repair capacity of cartilage and the beginning of cell therapy ‐  Autologous chondrocyte implantation ‐  Combination products ‐  Randomized controlled studies •  Choice of cell types •  Regulating cellular activities ‐  Influence of growth factors on chondrogenic differentiation ‐  Influence of growth factors on matrix deposition ‐  Supportive effect of biomaterials •  Interference of inflammation with cartilage repair •  Tracking of transplanted cells in vivo •  Conclusion and future directions

Effects of iron oxide incorporation for long term cell tracking on MSC differentiation in vitro and in vivo

Successful cell therapy will depend on the ability to monitor transplanted cells. With cell labeling, it is important to demonstrate efficient long term labeling without deleterious effects on cell phenotype and differentiation capacity. We demonstrate long term (7 weeks) retention of superparamagnetic iron oxide particles (SPIO) by mesenchymal stem cells (MSCs) in vivo, detectable by MRI. In vitro, multilineage differentiation (osteogenic, chondrogenic and adipogenic) was demonstrated by histological evaluation and molecular analysis in SPIO labeled and unlabeled cells. Gene expression levels were comaparable to unlabeled controls in adipogenic and chondrogenic conditions however not in the osteogenic condition. MSCs seeded into a scaffold for 21 days and implanted subcutaneously into nude mice for 4 weeks, showed profoundly altered phenotypes in SPIO labeled samples compared to implanted unlabeled control scaffolds, indicating chondrogenic differentiation. This study demonstrates long term MSC traceability using SPIO and MRI, uninhibited multilineage MSC differentiation following SPIO labeling, though with subtle but significant phenotypical alterations.

Tissue-engineered cartilage using serially passaged articular chondrocytes. Chondrocytes in alginate, combined in vivo with a synthetic (E210) or biologic biodegradable carrier (DBM).

In vitro multiplication of isolated autologous chondrocytes is required to obtain an adequate number of cells to generate neo-cartilage, but is known to induce cell-dedifferentiation. The aim of this study was to investigate whether multiplied chondrocytes can be used to generate neo-cartilage in vivo. Adult bovine articular chondrocytes, of various differentiation stages, were suspended in alginate at densities of 10 or 50 million/ml, either directly after isolation (P0) or after multiplication in monolayer for one (P1) or three passages (P3). Alginate with cells was seeded in demineralized bovine bone matrix (DBM) or a fleece of polylactic/polyglycolic acid (E210) and implanted in nude mice for 8 weeks. The newly formed tissue was evaluated by Alcian Blue and immunohistochemical staining for collagen type-II and type-I. Structural homogeneity of the tissue, composed of freshly isolated as well as serially passaged cells, was found to be enhanced by high-density seeding (50 million/ml) and the use of E210 as a carrier. The percentage of collagen type-II positive staining P3-cells was generally higher when E210 was used as a carrier. Furthermore, seeding P3-chondrocytes at the highest density (50 million/ml) enhanced collagen type-II expression. This study shows promising possibilities to generate structurally regular neo-cartilage using multiplied chondrocytes in alginate in combination with a fleece of polylactic/polyglycolic acid.

In vitro redifferentiation of culture-expanded rabbit and human auricular chondrocytes for cartilage reconstruction

To construct an autologous cartilage graft using tissue engineering, cells must be multiplied in vitro; they then lose their cartilage-specific phenotype. The objective of this study was to assess the capacity of multiplied ear chondrocytes to re-express their cartilage phenotype using various culture conditions. Cells were isolated from the cartilage of the ears of three young and three adult rabbits and, after multiplication in monolayer culture, they were seeded in alginate and cultured for 3 weeks in serum-free medium with insulin-like growth factor 1 (IGF-1) and transforming growth factor-beta2 (TGF-beta2) in three different dose combinations. As a control, cells were cultured in 10% fetal calf serum, which was demonstrated in previous experiments to be unable to induce redifferentiation. Chondrocytes from the ears of young, but not adult, rabbits, synthesized significantly more glycosaminoglycan when serum was replaced by insulin-like growth factor-1 and transforming growth factor-beta2. The number of collagen type II-positive cells was increased from 10 percent to 97 percent in young cells and to 33 percent in adult cells. Using human ear cells from 12 patients (aged 7 to 60 years), glycosaminoglycan synthesis could also be stimulated by replacing serum with insulin-like growth factor and transforming growth factor-beta. Although the number of collagen type II-positive cells could be increased under these conditions, it never reached above 10 percent. Data from five patients showed that further optimization of the culture conditions by adding ITS+ and cortisol significantly increased (doubled or tripled) both glycosaminoglycan synthesis and collagen type II expression. In conclusion, this study demonstrates a method to regain cartilage phenotype in multiplied ear cartilage cells. This improves the chances of generating human cartilage grafts for the reconstruction of external ears or the repair of defects of the nasal septum.

Effect of Glucosamine Sulfate on Hip Osteoarthritis: A Randomized Trial

Context Although many patients use glucosamine to treat osteoarthritis, available studies have reported inconsistent effects of glucosamine on symptoms and joint changes. In addition, previous studies have more often included patients with knee than with hip osteoarthritis. Contribution The investigators randomly assigned 222 patients with hip osteoarthritis to glucosamine, 1500 mg/d, or placebo. After 2 years of treatment, no clinically significant effect on pain, function, or joint space narrowing was found. Caution Twenty of the patients in the trial had joint replacement during the study. The Editors The effectiveness of glucosamine sulfate for treating osteoarthritis is controversial. A 2005 systematic review of 20 trials found evidence to be inconclusive (1). In the 15 trials comparing glucosamine with placebo, the overall effect on pain favored glucosamine, but 8 of the trials found no effect on pain. More recent trials (24) have also yielded inconclusive results. In the Netherlands and other countries, glucosamine is sold as an over-the-counter dietary supplement and is used by many patients, often on the advice of their physicians. Given the prevalent use of glucosamine, definitive evidence about its effectiveness is needed. Some studies suggest that glucosamine may provide greater benefit to patients with less severe radiographic osteoarthritis than to patients with more severe disease (5, 6). Most previous trials have studied only patients with knee osteoarthritis, with the exception of 3 early trials that included patients with other affected joints (79). Trials specifically testing glucosamine in patients with hip osteoarthritis have not been available. Although osteoarthritis of the knee is more common than hip osteoarthritis, hip osteoarthritis is common enough to warrant assessment of glucosamine for this condition. To date, only 2 trials have published data on the effects of glucosamine sulfate on joint structure (10, 11). Some expressed concern about the radiography protocol used in these trials (1214), and further study is needed to clarify these findings. To explore some of the uncertainties regarding the effectiveness of glucosamine sulfate, we conducted a 2-year, blinded, randomized, placebo-controlled trial to evaluate the effect of glucosamine sulfate on the symptomatic and radiographic progression of hip osteoarthritis in patients recruited from primary care settings. Methods Study Design In this trial, all outcome assessors, patients, data analysts, and researchers were blinded to group assignment. The Medical Ethics Committee of the Erasmus Medical Center, Rotterdam, the Netherlands, approved the study design, and patients provided written informed consent. We reported the detailed study protocol in 2005 (15) and summarize it here. Setting and Participants General practitioners in the Rotterdam area recruited study patients. Patients were eligible for inclusion if they met the American College of Rheumatology clinical criteria for hip osteoarthritis (16) during a screening examination at the research center. Patients who had undergone or were awaiting hip replacement surgery were not eligible. We excluded patients who had a Kellgren and Lawrence score of 4 (17), renal disease, liver disease, diabetes mellitus, or a disabling comorbid condition that would make visits to the research center impossible, as well as patients already receiving glucosamine and those unable to fill out Dutch questionnaires. We encouraged patients who violated study protocol and those who had total hip arthroplasty during the study to complete data collection to limit the loss to follow-up. Randomization and Intervention Eligible patients were randomly assigned to receive either 1500 mg of oral glucosamine sulfate (administered once daily as two 750-mg tablets) or placebo for 2 years. The glucosamine used in this study was provided by Numico Research BV (Wageningen, the Netherlands) but was manufactured by Nutricia Manufacturing USA (Greenville, South Carolina). It contained 2000 mg of D-glucosamine sulfate 2-potassium chloride, which results in a net content of 1500 mg of glucosamine sulfate per 2 pills. The placebo pills were identical in appearance, smell, and taste. We used a computer-generated, blinded randomization list provided by an independent researcher to randomly assign patients to glucosamine sulfate or placebo. This list, which was randomized per block of 6 numbers, stratified patients by radiologic findings (Kellgren and Lawrence score <2 vs. 2) and by local versus generalized osteoarthritis; patients received a number in chronological order (15). Assignment of patients to the right stratum of the random assignment list was done by the main researcher, who was blinded to therapy. To evaluate blinding, patients had to indicate in the last questionnaire to which treatment they thought they were randomly assigned. Outcomes and Follow-up Primary outcome measures were WOMAC 3.1 (5-point Likert format) pain and function over 24 months and joint space narrowing after 24 months (18, 19). Secondary outcome measures were WOMAC pain, function, and stiffness after 3, 12, and 24 months; overall WOMAC stiffness; a visual analogue scale (VAS) to measure pain in the past week; and pain medication use. The WOMAC subscales are presented as normalized scores (0 to 100, where 0 equals no symptoms). We recorded the use of pain medication; classified patients as never, occasional, or daily users; and then determined whether people increased, decreased, or did not change their use of pain medication from baseline. In the case of patients with bilateral hip symptoms, we asked patients to indicate their most affected hip for our analyses of joint space narrowing. For patients who were undecided, we used the hip with the highest Kellgren and Lawrence score or the smallest internal rotation during a physical examination. We used QBone Planner 5.4 (Medis, Leiden, the Netherlands) to measure joint space width on calibrated digital radiographs of the hip joints. We read radiographs from both time points (baseline and 24 months) side by side. One researcher measured joint space width manually on predefined lateral, superior, axial, and medial sites (20). In addition to these 4 points, we visually identified and measured the minimal joint space width on both the baseline and 24-month radiograph. We used the smallest of these 6 measurements as the actual minimum joint space width for analyses. A second observer also measured the joint space width in a random subset of 28 study patients, and we found high interobserver agreement (intraclass correlation coefficient of minimal joint space width, 0.98). We collected data for the primary and secondary outcome measures at different time points throughout the study. At baseline and after 24 months, patients came to the Erasmus Medical Center for radiography and to complete study questionnaires. Weight-bearing, anteroposterior digital radiography of the pelvis was performed according to a highly standardized protocol to allow reliable measurement of joint space narrowing (15). At baseline and then every 3 months through month 24, we asked patients to complete the WOMAC instrument, a VAS for pain in the past week (score range, 0 to 100; 0 equals no pain), and a checklist for specific adverse events and to answer questions regarding pain medication and adherence. We mailed the intermediate questionnaires to the patients for completion at home. A researcher visited patients every 6 months to deliver a new supply of study medication and evaluate adherence by using the Brief Medication Questionnaire (BMQ) (21) and a pill count. The BMQ monitors the amount of days per week that patients have taken their study medication. For overall effect, we considered patients to be adherent if they ingested more than 80% of the total study medication. Statistical Analysis We used the data from all nine 3-month questionnaires (at baseline and 3, 6, 9, 12, 15, 18, 21, and 24 months). We also report outcomes for measurements at 3, 12, and 24 months and a mean effect of the therapy over 24 months incorporating all scores. We performed the analyses by using SPSS 11.0.1 (SPSS, Chicago, Illinois) and SAS 8.2 (SAS Institute, Cary, North Carolina). We used linear mixed models to analyze the data, assuming that data were missing at random. We chose an unstructured covariance structure to model the covariance of repeated measures by patients, because this yielded the lowest Akaike information criterion. Fixed effects were time, time by therapy, and the covariates we adjusted for. For patients who had total hip arthroplasty during the trial, we included observed data before surgery in the analysis and assumed data after surgery to be missing. For patients who were lost to follow-up, we included all observed data in the analysis. We adjusted the WOMAC and VAS pain analyses for body mass index, sex, and agefactors that may have influenced symptoms (22, 23). We also adjusted analyses for pain medication use and Kellgren and Lawrence score. The analyses for joint space narrowing were adjusted for Kellgren and Lawrence score (24), age, and sex (25). We used ordinal regression analysis to assess the effect of glucosamine sulfate on pain medication use by using data from all patients who completed the study and did not have total hip arthroplasty. We performed additional analyses to assess the effect of adherence on the outcome. To explore the validity of the missing-at-random data assumption for patients who underwent total hip arthroplasty during the study, we did sensitivity analyses on the WOMAC pain data. In 5 scenarios, the missing data for patients who underwent total hip arthroplasty were imputed with extreme scores: mean of the 5 best scores for the glucosamine sulfate recipients and that of the 5 worst scores for the placebo recipients (traditional best case); mean of the best scores for placebo recipients and