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1.  Soluble Mediators in Posttraumatic Wrist and Primary Knee Osteoarthritis 
New discoveries about the pathophysiology changed the concept that all forms of osteoarthritis are alike; this lead to the delineation of different phenotypes such as age, trauma or obese related forms. We aim to compare soluble mediator profiles in primary knee and posttraumatic wrist osteoarthritis. Based on the general faster progression rate of wrist osteoarthritis, we hypothesize a more inflammatory profile.
We collected synovial fluid from 20 primary osteoarthritic knee and 20 posttraumatic osteoarthritic wrist joints. 17 mediators were measured by multiplex enzyme-linked immunosorbent assay: chemokine ligand 5, interferon-γ, leukemia inhibitory factor, oncostatin-M, osteoprotegerin, tumor necrosis factor-α, vascular endothelial growth factor, interleukin (IL)-1α, IL-1β, IL-1 receptor antagonist, IL-4, IL-6, IL-7, IL-8, IL-10, IL-13 and IL-17.
Ten mediators were higher in posttraumatic osteoarthritic synovial fluid: tumor necrosis factor-α (TNFα), IL-1α, IL-1RA, IL-6, IL-10, IL-17, oncostatin-M, interferon-γ, chemokine ligand 5 and leukemia inhibitory factor (P<0.001). IL-1ß, IL-4, IL-7 were not detected, TNFα was not detected in knee osteoarthritic synovial fluid. IL-8, IL-13, osteoprotegerin and vascular endothelial growth factor levels did not differ between the synovial fluid types.
In general wrist osteoarthritis seems characterized by a stronger inflammatory response than primary knee osteoarthritis. More pronounced inflammatory mediators might offer a paradigm for the faster progression of posttraumatic osteoarthritis. Increase of specific mediators could form a possible target for future mediator modulating therapy in wrist osteoarthritis.
PMCID: PMC4225017  PMID: 25386573
Cytokines; Knee; Osteoarthritis; Posttraumatic; Wrist
2.  Concise Review: Unraveling Stem Cell Cocultures in Regenerative Medicine: Which Cell Interactions Steer Cartilage Regeneration and How? 
This review provides a systematic overview of the literature on stem cell cocultures with cartilage cells and tries to elucidate the mechanisms that lead to chondrogenesis. It could serve as a basis for research groups and clinicians aiming to design and implement combined cellular technologies for single-stage cartilage repair and treatment or prevention of osteoarthritis.
Cartilage damage and osteoarthritis (OA) impose an important burden on society, leaving both young, active patients and older patients disabled and affecting quality of life. In particular, cartilage injury not only imparts acute loss of function but also predisposes to OA. The increase in knowledge of the consequences of these diseases and the exponential growth in research of regenerative medicine have given rise to different treatment types. Of these, cell-based treatments are increasingly applied because they have the potential to regenerate cartilage, treat symptoms, and ultimately prevent or delay OA. Although these approaches give promising results, they require a costly in vitro cell culture procedure. The answer may lie in single-stage procedures that, by using cell combinations, render in vitro expansion redundant. In the last two decades, cocultures of cartilage cells and a variety of (mesenchymal) stem cells have shown promising results as different studies report cartilage regeneration in vitro and in vivo. However, there is considerable debate regarding the mechanisms and cellular interactions that lead to chondrogenesis in these models. This review, which included 52 papers, provides a systematic overview of the data presented in the literature and tries to elucidate the mechanisms that lead to chondrogenesis in stem cell cocultures with cartilage cells. It could serve as a basis for research groups and clinicians aiming at designing and implementing combined cellular technologies for single-stage cartilage repair and treatment or prevention of OA.
PMCID: PMC4039458  PMID: 24763684
Bone marrow stromal cells; Cellular therapy; Chondrogenesis; Clinical translation; Marrow stromal stem cells; Mesenchymal stem cells; Skeleton
3.  Intradiscal application of rhBMP-7 does not induce regeneration in a canine model of spontaneous intervertebral disc degeneration 
Strategies for biological repair and regeneration of the intervertebral disc (IVD) by cell and tissue engineering are promising, but few have made it into a clinical setting. Recombinant human bone morphogenetic protein 7 (rhBMP-7) has been shown to stimulate matrix production by IVD cells in vitro and in vivo in animal models of induced IVD degeneration. The aim of this study was to determine the most effective dose of an intradiscal injection of rhBMP-7 in a spontaneous canine IVD degeneration model for translation into clinical application for patients with low back pain.
Canine nucleus pulposus cells (NPCs) were cultured with rhBMP-7 to assess the anabolic effect of rhBMP-7 in vitro, and samples were evaluated for glycosaminoglycan (GAG) and DNA content, histology, and matrix-related gene expression. Three different dosages of rhBMP-7 (2.5 μg, 25 μg, and 250 μg) were injected in vivo into early degenerated IVDs of canines, which were followed up for six months by magnetic resonance imaging (T2-weighted images, T1rho and T2 maps). Post-mortem, the effects of rhBMP-7 were determined by radiography, computed tomography, and macroscopy, and by histological, biochemical (GAG, DNA, and collagen), and biomolecular analyses of IVD tissue.
In vitro, rhBMP-7 stimulated matrix production of canine NPCs as GAG deposition was enhanced, DNA content was maintained, and gene expression levels of ACAN and COL2A1 were significantly upregulated. Despite the wide dose range of rhBMP-7 (2.5 to 250 μg) administered in vivo, no regenerative effects were observed at the IVD level. Instead, extensive extradiscal bone formation was noticed after intradiscal injection of 25 μg and 250 μg of rhBMP-7.
An intradiscal bolus injection of 2.5 μg, 25 μg, and 250 μg rhBMP-7 showed no regenerative effects in a spontaneous canine IVD degeneration model. In contrast, intradiscal injection of 250 μg rhBMP-7, and to a lesser extent 25 μg rhBMP-7, resulted in extensive extradiscal bone formation, indicating that a bolus injection of rhBMP-7 alone cannot be used for treatment of IVD degeneration in human or canine patients.
Electronic supplementary material
The online version of this article (doi:10.1186/s13075-015-0625-2) contains supplementary material, which is available to authorized users.
PMCID: PMC4443547  PMID: 26013758
4.  Non-invasive monitoring of BMP-2 retention and bone formation in composites for bone tissue engineering using SPECT/CT and scintillation probes 
Non-invasive imaging can provide essential information for the optimization of new drug delivery-based bone regeneration strategies to repair damaged or impaired bone tissue. This study investigates the applicability of nuclear medicine and radiological techniques to monitor growth factor retention profiles and subsequent effects on bone formation. Recombinant human bone morphogenetic protein-2 (BMP-2, 6.5 μg/scaffold) was incorporated into a sustained release vehicle consisting of poly(lactic-co-glycolic acid) microspheres embedded in a poly(propylene fumarate) scaffold surrounded by a gelatin hydrogel and implanted subcutaneously and in 5-mm segmental femoral defects in 9 rats for a period of 56 days. To determine the pharmacokinetic profile, BMP-2 was radiolabeled with 125I and the local retention of 125I-BMP-2 was measured by single photon emission computed tomography (SPECT), scintillation probes and ex vivo scintillation analysis. Bone formation was monitored by micro-computed tomography (μCT). The scaffolds released BMP-2 in a sustained fashion over the 56-day implantation period. A good correlation between the SPECT and scintillation probe measurements was found and there were no significant differences between the non-invasive and ex-vivo counting method after 8 weeks of follow up. SPECT analysis of the total body and thyroid counts showed a limited accumulation of 125I within the body. Ectopic bone formation was induced in the scaffolds and the femur defects healed completely. In vivo μCT imaging detected the first signs of bone formation at days 14 and 28 for the orthotopic and ectopic implants, respectively, and provided a detailed profile of the bone formation rate. Overall, this study clearly demonstrates the benefit of applying non-invasive techniques in drug delivery-based bone regeneration strategies by providing detailed and reliable profiles of the growth factor retention and bone formation at different implantation sites in a limited number of animals.
PMCID: PMC3974410  PMID: 19105972
Drug delivery; Controlled release; Bone morphogenetic protein-2; Single photon emission computed; tomography; Scintillation probes; Micro-computed tomography
5.  Osteoarthritis treatment using autologous conditioned serum after placebo 
Acta Orthopaedica  2015;86(1):114-118.
Background and purpose
Autologous conditioned serum (ACS) is a disease-modifying drug for treatment of knee osteoarthritis, and modest superiority over placebo was reported in an earlier randomized controlled trial (RCT). We hypothesized that when given the opportunity, placebo-treated patients from that RCT would now opt for ACS treatment, which would result in a greater clinical improvement than placebo.
Of 74 patients treated with placebo in the previous trial, 20 opted for ACS treatment. Patients who did not choose further treatment were interviewed about their reasons. Clinical improvement of the 20 ACS-treated patients was measured using knee-specific clinical scores, as was “response shift” at 3 and 12 months.
In the 20 patients who did opt for ACS, the visual analog scale (VAS) score for pain improved; but after 12 months, clinical results were similar to those after placebo treatment. Response shift measurement demonstrated that the 20 patients had adapted to their disabilities during treatment.
Placebo-treated patients from an earlier trial were reluctant to undergo ACS treatment, in part due to the laborious nature of the therapy. In a subset of patients who opted for treatment, ACS treatment after placebo did not result in greater clinical improvement than placebo treatment only. However, due to the limited power of the current study and possible selection bias, definite advice on using or refraining from ACS cannot be given.
PMCID: PMC4366668  PMID: 25140983
6.  Effect of Autologous Bone Marrow Stromal Cell Seeding and Bone Morphogenetic Protein-2 Delivery on Ectopic Bone Formation in a Microsphere/Poly(Propylene Fumarate) Composite 
Tissue Engineering. Part A  2008;15(3):587-594.
A biodegradable microsphere/scaffold composite based on the synthetic polymer poly(propylene fumarate) (PPF) holds promise as a scaffold for cell growth and sustained delivery vehicle for growth factors for bone regeneration. The objective of the current work was to investigate the in vitro release and in vivo bone forming capacity of this microsphere/scaffold composite containing bone morphogenetic protein-2 (BMP-2) in combination with autologous bone marrow stromal cells (BMSCs) in a goat ectopic implantation model. Three composites consisting of 0, 0.08, or 8 μg BMP-2 per mg of poly(lactic-co-glycolic acid) microspheres, embedded in a porous PPF scaffold, were combined with either plasma (no cells) or culture-expanded BMSCs. PPF scaffolds impregnated with a BMP-2 solution and combined with BMSCs as well as empty PPF scaffolds were also tested. The eight different composites were implanted subcutaneously in the dorsal thoracolumbar area of goats. Incorporation of BMP-2–loaded microspheres in the PPF scaffold resulted in a more sustained in vitro release with a lower burst phase, as compared to BMP-2–impregnated scaffolds. Histological analysis after 9 weeks of implantation showed bone formation in the pores of 11/16 composites containing 8 μg/mg BMP-2–loaded microspheres with no significant difference between composites with or without BMSCs (6/8 and 5/8, respectively). Bone formation was also observed in 1/8 of the BMP-2–impregnated scaffolds. No bone formation was observed in the other conditions. Overall, this study shows the feasibility of bone induction by BMP-2 release from microspheres/scaffold composites.
PMCID: PMC2810278  PMID: 18925831
7.  Cytokine profiles in the joint depend on pathology, but are different between synovial fluid, cartilage tissue and cultured chondrocytes 
This study aimed to evaluate whether profiles of several soluble mediators in synovial fluid and cartilage tissue are pathology-dependent and how their production is related to in vitro tissue formation by chondrocytes from diseased and healthy tissue.
Samples were obtained from donors without joint pathology (n = 39), with focal defects (n = 65) and osteoarthritis (n = 61). A multiplex bead assay (Luminex) was performed measuring up to 21 cytokines: Interleukin (IL)-1α, IL-1β, IL-1RA, IL-4, IL-6, IL-6Rα, IL-7, IL-8, IL-10, IL-13, tumor necrosis factor (TNF)α, Interferon (IFN)γ, oncostatin M (OSM), leukemia inhibitory factor (LIF), adiponectin, leptin, monocyte chemotactic factor (MCP)1, RANTES, basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), vascular growth factor (VEGF).
In synovial fluid of patients with cartilage pathology, IL-6, IL-13, IFNγ and OSM levels were higher than in donors without joint pathology (P ≤0.001). IL-13, IFNγ and OSM were also different between donors with cartilage defects and OA (P <0.05). In cartilage tissue from debrided defects, VEGF was higher than in non-pathological or osteoarthritic joints (P ≤0.001). IL-1α, IL-6, TNFα and OSM concentrations (in ng/ml) were markedly higher in cartilage tissue than in synovial fluid (P <0.01). Culture of chondrocytes generally led to a massive induction of most cytokines (P <0.001). Although the release of inflammatory cytokines was also here dependent on the pathological condition (P <0.001) the actual profiles were different from tissue or synovial fluid and between non-expanded and expanded chondrocytes. Cartilage formation was lower by healthy unexpanded chondrocytes than by osteoarthritic or defect chondrocytes.
Several pro-inflammatory, pro-angiogenic and pro-repair cytokines were elevated in joints with symptomatic cartilage defects and/or osteoarthritis, although different cytokines were elevated in synovial fluid compared to tissue or cells. Hence a clear molecular profile was evident dependent on disease status of the joint, which however changed in composition depending on the biological sample analysed. These alterations did not affect in vitro tissue formation with these chondrocytes, as this was at least as effective or even better compared to healthy chondrocytes.
PMCID: PMC4201683  PMID: 25256035
8.  Of Mice, Men and Elephants: The Relation between Articular Cartilage Thickness and Body Mass 
PLoS ONE  2013;8(2):e57683.
Mammalian articular cartilage serves diverse functions, including shock absorption, force transmission and enabling low-friction joint motion. These challenging requirements are met by the tissue’s thickness combined with its highly specific extracellular matrix, consisting of a glycosaminoglycan-interspersed collagen fiber network that provides a unique combination of resilience and high compressive and shear resistance. It is unknown how this critical tissue deals with the challenges posed by increases in body mass. For this study, osteochondral cores were harvested post-mortem from the central sites of both medial and lateral femoral condyles of 58 different mammalian species ranging from 25 g (mouse) to 4000 kg (African elephant). Joint size and cartilage thickness were measured and biochemical composition (glycosaminoclycan, collagen and DNA content) and collagen cross-links densities were analyzed. Here, we show that cartilage thickness at the femoral condyle in the mammalian species investigated varies between 90 µm and 3000 µm and bears a negative allometric relationship to body mass, unlike the isometric scaling of the skeleton. Cellular density (as determined by DNA content) decreases with increasing body mass, but gross biochemical composition is remarkably constant. This however need not affect life-long performance of the tissue in heavier mammals, due to relatively constant static compressive stresses, the zonal organization of the tissue and additional compensation by joint congruence, posture and activity pattern of larger mammals. These findings provide insight in the scaling of articular cartilage thickness with body weight, as well as in cartilage biochemical composition and cellularity across mammalian species. They underscore the need for the use of appropriate in vivo models in translational research aiming at human applications.
PMCID: PMC3578797  PMID: 23437402
9.  Gene expression profiling of early intervertebral disc degeneration reveals a down-regulation of canonical Wnt signaling and caveolin-1 expression: implications for development of regenerative strategies 
Early degeneration of the intervertebral disc (IVD) involves a change in cellular differentiation from notochordal cells (NCs) in the nucleus pulposus (NP) to chondrocyte-like cells (CLCs). The purpose of this study was to investigate the gene expression profiles involved in this process using NP tissue from non-chondrodystrophic and chondrodystrophic dogs, a species with naturally occurring IVD degeneration.
Dual channel DNA microarrays were used to compare 1) healthy NP tissue containing only NCs (NC-rich), 2) NP tissue with a mixed population of NCs and CLCs (Mixed), and 3) NP tissue containing solely CLCs (CLC-rich) in both non-chondrodystrophic and chondrodystrophic dogs. Based on previous reports and the findings of the microarray analyses, canonical Wnt signaling was further evaluated using qPCR of relevant Wnt target genes. We hypothesized that caveolin-1, a regulator of Wnt signaling that showed significant changes in gene expression in the microarray analyses, played a significant role in early IVD degeneration. Caveolin-1 expression was investigated in IVD tissue sections and in cultured NCs. To investigate the significance of Caveolin-1 in IVD health and degeneration, the NP of 3-month-old Caveolin-1 knock-out mice was histopathologically evaluated and compared with the NP of wild-type mice of the same age.
Early IVD degeneration involved significant changes in numerous pathways, including Wnt/β-catenin signaling. With regard to Wnt/β-catenin signaling, axin2 gene expression was significantly higher in chondrodystrophic dogs compared with non-chondrodystrophic dogs. IVD degeneration involved significant down-regulation of axin2 gene expression. IVD degeneration involved significant down-regulation in Caveolin-1 gene and protein expression. NCs showed abundant caveolin-1 expression in vivo and in vitro, whereas CLCs did not. The NP of wild-type mice was rich in viable NCs, whereas the NP of Caveolin-1 knock-out mice contained chondroid-like matrix with mainly apoptotic, small, rounded cells.
Early IVD degeneration involves down-regulation of canonical Wnt signaling and Caveolin-1 expression, which appears to be essential to the physiology and preservation of NCs. Therefore, Caveolin-1 may be regarded an exciting target for developing strategies for IVD regeneration.
PMCID: PMC3672710  PMID: 23360510
10.  Interleukin-6 is elevated in synovial fluid of patients with focal cartilage defects and stimulates cartilage matrix production in an in vitro regeneration model 
Arthritis Research & Therapy  2012;14(6):R262.
This study aimed to determine whether, as in osteoarthritis, increased levels of interleukin-6 (IL-6) are present in the synovial fluid of patients with symptomatic cartilage defects and whether this IL-6 affects cartilage regeneration as well as the cartilage in the degenerated knee.
IL-6 concentrations were determined by ELISA in synovial fluid and in conditioned media of chondrocytes regenerating cartilage. Chondrocytes were obtained from donors with symptomatic cartilage defects, healthy and osteoarthritic donors. The effect of IL-6 on cartilage regeneration and on metabolism of the resident cartilage in the knee was studied by both inhibition of endogenous IL-6 and addition of IL-6, in a regeneration model and in osteoarthritic explants in the presence of synovial fluid, respectively. Readout parameters were DNA and glycosaminoglycan (GAG) content and release. Differences between controls and IL-6 blocked or supplemented samples were determined by univariate analysis of variance using a randomized block design.
Synovial fluid of patients with symptomatic cartilage defects contained more IL-6 than synovial fluid of healthy donors (P = 0.001) and did not differ from osteoarthritic donors. IL-6 production of osteoarthritic chondrocytes during cartilage regeneration was higher than that of healthy and defect chondrocytes (P < 0.001). Adding IL-6 increased GAG production by healthy chondrocytes and decreased GAG release by osteoarthritic chondrocytes (P < 0.05). Inhibition of IL-6 present in osteoarthritic synovial fluid showed a trend towards decreased GAG content of the explants (P = 0.06).
Our results support a modest anabolic role for IL-6 in cartilage matrix production. Targeting multiple cytokines, including IL-6, may be effective in improving cartilage repair in symptomatic cartilage defects and osteoarthritis.
PMCID: PMC3674617  PMID: 23206933
11.  Enhanced Bone Morphogenetic Protein-2-Induced Ectopic and Orthotopic Bone Formation by Intermittent Parathyroid Hormone (1–34) Administration 
Tissue Engineering. Part A  2010;16(12):3769-3777.
Bone morphogenetic proteins (BMPs) play a central role in local bone regeneration strategies, whereas the anabolic features of parathyroid hormone (PTH) are particularly appealing for the systemic treatment of generalized bone loss. The aim of the current study was to investigate whether local BMP-2-induced bone regeneration could be enhanced by systemic administration of PTH (1–34). Empty or BMP-2-loaded poly(lactic-co glycolic acid)/poly(propylene fumarate)/gelatin composites were implanted subcutaneously and in femoral defects in rats (n = 9). For the orthotopic site, empty defects were also tested. Each of the conditions was investigated in combination with daily administered subcutaneous PTH (1–34) injections in the neck. After 8 weeks of implantation, bone mineral density (BMD) and bone volume were analyzed using microcomputed tomography and histology. Ectopic bone formation and almost complete healing of the femoral defect were only seen in rats that received BMP-2-loaded composites. Additional treatment of the rats with PTH (1–34) resulted in significantly (p < 0.05) enhanced BMD and bone volume in the BMP-2 composites at both implantation sites. Despite its effect on BMD in the humerus and vertebra, PTH (1–34) treatment had no significant effect on BMD and bone volume in the empty femoral defects and the ectopically or orthotopically implanted empty composites. Histological analysis showed that the newly formed bone had a normal woven and trabecular appearance. Overall, this study suggests that intermittent administration of a low PTH dose alone has limited potential to enhance local bone regeneration in a critical-sized defect in rats. However, when combined with local BMP-2-releasing scaffolds, PTH administration significantly enhanced osteogenesis in both ectopic and orthotopic sites.
PMCID: PMC2991197  PMID: 20666615
13.  Cytokine profile of autologous conditioned serum for treatment of osteoarthritis, in vitro effects on cartilage metabolism and intra-articular levels after injection 
Arthritis Research & Therapy  2010;12(3):R114.
Intraarticular administration of autologous conditioned serum (ACS) recently demonstrated some clinical effectiveness in treatment of osteoarthritis (OA). The current study aims to evaluate the in vitro effects of ACS on cartilage proteoglycan (PG) metabolism, its composition and the effects on synovial fluid (SF) cytokine levels following intraarticular ACS administration.
The effect of conditioned serum on PG metabolism of cultured OA cartilage explants was compared to unconditioned serum. The effect of serum conditioning on levels of interleukin-1beta (IL-1β), IL-4, IL-6, IL-10, IL-13, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), osteoprotegerin (OPG), oncostatin M (OSM), interleukin-1 receptor (IL-1ra) and transforming growth factor beta (TGF-β) were measured by multiplex ELISA. As TNF-α levels were found to be increased in conditioned serum, the effect of TNF-α inhibition by etanercept on PG metabolism was studied in cartilage explants cultured in the presence of conditioned serum. Furthermore, cytokine levels in SF were measured three days after intraarticular ACS injection in OA patients to verify their retention time in the joint space.
PG metabolism was not different in the presence of conditioned serum compared to unconditioned serum. Levels of the anti-inflammatory cytokines IL-1ra, TGF-β, IL-10 as well as of pro-inflammatory cytokines IL-1β, IL-6, TNF-α and OSM were increased. IL-4, IL-13 and IFN-γ levels remained similar, while OPG levels decreased. TNF-α inhibition did not influence PG metabolism in cartilage explant culture in the presence of condtioned serum. Although OPG levels were higher and TGF-β levels were clearly lower in ACS than in SF, intraarticular ACS injection in OA patients did not result in significant changes in these cytokine levels.
ACS for treatment of osteoarthritis contains increased levels of anti-inflammatory as well as pro-inflammatory cytokines, in particular TNF-α, but conditioned serum does not seem to have a net direct effect on cartilage metabolism, even upon inhibition of TNF-α. The fast intraarticular clearance of cytokines in the injected ACS may explain the limited effects found previously in vivo.
PMCID: PMC2911907  PMID: 20537160
14.  Non-invasive screening method for simultaneous evaluation of in vivo growth factor release profiles from multiple ectopic bone tissue engineering implants 
The purpose of this study was to develop and validate a screening method based on scintillation probes for the simultaneous evaluation of in vivo growth factor release profiles of multiple implants in the same animal. First, we characterized the scintillation probes in a series of in vitro experiments to optimize the accuracy of the measurement setup. The scintillation probes were found to have a strong geometric dependence and experience saturation effects at high activities. In vitro simulation of 4 subcutaneous limb implants in a rat showed minimal interference of surrounding implants on local measurements at close to parallel positioning of the probes. These characteristics were taken into consideration for the design of the probe setup and in vivo experiment. The measurement setup was then validated in a rat subcutaneous implantation model using 4 different sustained release carriers loaded with 125I-BMP-2 per animal. The implants were removed after 42 or 84 days of implantation, for comparison of the non-invasive method to ex-vivo radioisotope counting. The non-invasive method demonstrated a good correlation with the ex-vivo counting method at both time-points of all 4 carriers. Overall, this study showed that scintillation probes could be successfully used for paired measurement of 4 release profiles with minimal interference of the surrounding implants, and may find use as non-invasive screening tools for various drug delivery applications.
PMCID: PMC2601638  PMID: 18554743
Controlled drug delivery; non-invasive screening; scintillation detector; radiolabelled growth factor; method validation

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