Nicole Kops

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.

Chondrogenic Priming of Human Bone Marrow Stromal Cells: A Better Route to Bone Repair?

The use of bioengineered cell constructs for the treatment of bone defects has received much attention of late. Often, bone marrow stromal cells (BMSCs) are used that are in vitro-stimulated toward the osteogenic lineage, aiming at intramembranous bone formation. The success of this approach has been disappointing. A major concern with these constructs is core degradation and necrosis caused by lack of vascularization. We hypothesized that stimulation of cells toward the endochondral ossification process would be more successful. In this study, we tested how in vitro priming of human BMSCs (hBMSCs) along osteogenic and chondrogenic lineages influences survival and osteogenesis in vivo. Scaffolds that were pre-cultured on chondrogenic culture medium showed collagen type II and collagen type X production. Moreover, vessel ingrowth was observed. Priming along the osteogenic lineage led to a mineralized matrix of poor quality, with few surviving cells and no vascularization. We further characterized this process in vitro using pellet cultures. In vitro, pellets cultured in chondrogenic medium showed progressive production of collagen type II and collagen type X. In the culture medium of these chondrogenic cultured pellets, vascular endothelial growth factor (VEGF) release was observed at days 14, 21, and 35. When pellets were switched to culture medium containing beta-glycerophosphate, independent of the presence or absence of transforming growth factor beta (TGF-beta), mineralization was observed with a concomitant reduction in VEGF and matrix metalloproteinase (MMP) release. By showing that VEGF and MMPs are produced in chondrogenically differentiated hBMSCs in vitro, we demonstrated that these cells produce factors that are known to be important for the induction of vascularization of the matrix. Inducing mineralization in this endochondral process does, however, severely diminish these capacities. Taken together, these data suggest that optimizing chondrogenic priming of hBMSCs may further improve vessel invasion in bioengineered constructs, thus leading to an alternative and superior approach to bone repair.

In vivo imaging of cartilage degeneration using μCT-arthrography

OBJECTIVE In vivo imaging of cartilage degeneration in small animal models is nowadays practically impossible. In the present study, we investigated the use of micro-computed tomography (microCT) in combination with a negatively charged ionic iodine dimer (ioxaglate) for in vivo assessment of cartilage degeneration in a small animal model. METHODS Cartilage degeneration was induced in the right knee of rats by injection of mono-iodoacetate (MIA). We imaged the rat knees with ioxaglate enhanced microCT-arthrography at 4, 16 and 44 days after MIA injection. Subsequently, microCT-arthrographic findings were evaluated and compared with quantitative histology of the patellar cartilage. RESULTS In vivo microCT-arthrography clearly detected cartilage degeneration in the rat knee-joints, in which the ioxaglate diffused into the degenerated cartilage layer. Higher microCT-attenuation values and smaller total volumes of the cartilage layer were detected at longer time periods after MIA injection, which is quantitatively confirmed by histology. CONCLUSION In vivo microCT-arthrography is a valuable tool for detection of minor cartilage alterations and distinguishes different stages of cartilage degeneration in a small animal model. Since microCT, at the same time, also visualizes osteophyte formation and changes in the underlying subchondral bone structures, the technique will be very useful for longitudinal overall assessment of the development of (osteo)arthritis and to study interventions in small animal models.

Intrinsic differentiation potential of adolescent human tendon tissue: an in-vitro cell differentiation study

BackgroundTendinosis lesions show an increase of glycosaminoglycan amount, calcifications, and lipid accumulation. Therefore, altered cellular differentiation might play a role in the etiology of tendinosis. This study investigates whether adolescent human tendon tissue contains a population of cells with intrinsic differentiation potential.MethodsCells derived from adolescent non-degenerative hamstring tendons were characterized by immunohistochemistry and FACS-analysis. Cells were cultured for 21 days in osteogenic, adipogenic, and chondrogenic medium and phenotypical evaluation was carried out by immunohistochemical and qPCR analysis. The results were compared with the results of similar experiments on adult bone marrow-derived stromal cells (BMSCs).ResultsTendon-derived cells stained D7-FIB (fibroblast-marker) positive, but α-SMA (marker for smooth muscle cells and pericytes) negative. Tendon-derived cells were 99% negative for CD34 (endothelial cell marker), and 73% positive for CD105 (mesenchymal progenitor-cell marker). In adipogenic medium, intracellular lipid vacuoles were visible and tendon-derived fibroblasts showed upregulation of adipogenic markers FABP4 (fatty-acid binding protein 4) and PPARG (peroxisome proliferative activated receptor γ). In chondrogenic medium, some cells stained positive for collagen 2 and tendon-derived fibroblasts showed upregulation of collagen 2 and collagen 10. In osteogenic medium Von Kossa staining showed calcium deposition although osteogenic markers remained unaltered. Tendon-derived cells and BMCSs behaved largely comparable, although some distinct differences were present between the two cell populations.ConclusionThis study suggests that our population of explanted human tendon cells has an intrinsic differentiation potential. These results support the hypothesis that there might be a role for altered tendon-cell differentiation in the pathophysiology of tendinosis.

In Vitro Model to Study Chondrogenic Differentiation in Tendinopathy

Background Treatment of midportion Achilles tendinopathy is hampered by limited knowledge of the pathophysiology. Hypothesis Chondrogenic differentiation of tendon cells might take place in midportion Achilles tendinopathy and could be used as a target for drug treatment. An in vitro model for chondrogenic differentiation would be useful to evaluate existing and future treatment opportunities. Study Design Descriptive and controlled laboratory study. Methods Perioperatively harvested tissue from human midportion Achilles tendinotic lesions and healthy Achilles tendons was analyzed by microscopy and real-time reverse transcription polymerase chain reaction. In vitro chondrogenic differentiation of tendon explants was induced using transforming-growth-factor beta. This model was modulated by removing the chondrogenic stimulus or adding triamcinolone or platelet-rich plasma. Results Midportion Achilles tendinotic lesions had increased glycosaminoglycan staining and more rounded cell nuclei. Chondrogenic markers (sex-determining region Y)–box9, aggrecan, collagen 2, and RUNT-related transcription factor 2 were upregulated, but collagen 10 was not. Nondegenerative tendon explants cultured on chondrogenic medium had higher expression of aggrecan, collagen 2, and collagen 10 but not (sex-determining region Y)–box9 and RUNT-related transcription factor 2. Removing the chondrogenic stimulus decreased expression of aggrecan, collagen 2, and collagen 10. Both triamcinolone and platelet-rich plasma influenced the chondrogenic gene expression pattern in the in vitro model. Conclusion Chondrogenic differentiation is present in midportion Achilles tendinopathy. An in vitro model to study this chondrogenic differentiation was developed. Clinical Relevance This model can be used to investigate chondrogenic differentiation as a possible target for drug treatment, contributing to the development of more successful mechanism-based treatment opportunities.

Cell labelling with superparamagnetic iron oxide has no effect on chondrocyte behaviour

BACKGROUND Tissue engineering and regenerative medicine are two rapidly advancing fields of research offering potential for effective treatment of cartilage lesions. Today, chondrocytes are the cell type of choice for use in cartilage repair approaches such as autologous chondrocyte implantation. To verify the safety and efficacy of such approaches it is necessary to determine the fate of these transplanted cells. One way of doing this is prelabelling cells before implantation and tracking them using imaging techniques. The use of superparamagnetic iron oxide (SPIO) for tracking of cells with magnetic resonance imaging (MRI) is ideal for this purpose. It is non-radioactive, does not require viral transfection and is already approved for clinical use as a contrast agent. OBJECTIVE The purpose of this study was to assess the effect of SPIO labelling on adult human chondrocyte behaviour. METHODS Cells were culture expanded and dedifferentiated for two passages and then labelled with SPIO. Effect on cell proliferation was tested. Furthermore, cells were cultured for 21 days in alginate beads in redifferentiation medium. Following this period, cells were analysed for expression of cartilage-related genes, proteoglycan production and collagen protein expression. RESULTS SPIO labelling did not significantly affect any of these parameters relative to unlabelled controls. We also demonstrated SPIO retention within the cells for the full duration of the experiment. CONCLUSIONS This paper demonstrates for the first time the effects of SPIO labelling on chondrocyte behaviour, illustrating its potential for in vivo tracking of implanted chondrocytes.

Quantifying osteoarthritic cartilage changes accurately using in vivo microCT arthrography in three etiologically distinct rat models

In vivo microCT arthrography (µCTa) can be used to measure both quantity (volumetric) and quality (glycosaminoglycan content) of cartilage. This study investigated the accuracy of four segmentation techniques to isolate cartilage from µCTa datasets and then used the most accurate one to investigate if the µCTa method could show osteoarthritic changes in rat models during longitudinal follow‐up. Volumetric measurements and glycosaminoglycan contents of patellar cartilage from in vivo µCTa‐scans were compared with an ex vivo gold standard µCT‐scan. Cartilage was segmented with three global thresholds and one local threshold algorithm. Comparisons were made for healthy and osteoarthritic cartilage. Next, three rat models were investigated for 24 weeks using µCTa. Osteoarthritis was induced by injection with a chemical (mono‐iodoacetate), a surgical intervention (grooves applied in articular cartilage), and via exercise (strenuous running). After euthanasia, all knee joints were isolated for histology. Local thresholds accurately segmented cartilage from in vivo µCTa scans and best measured cartilage quantity and glycosaminoglycan content. Each of the three osteoarthritic rat models showed a specific pattern of osteoarthritis progression. All µCTa results were comparable to histology. In vivo µCTa is a sensitive technique for imaging cartilage degradation. Local thresholds enhanced the sensitivity of this method and will probably more accurately detect disease‐modulating effects from interventional strategies. The data from rat models may serve as a reference for the time sequence of cartilage degeneration during in vivo testing of new strategies in osteoarthritis treatment. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29:1788–1794, 2011

Physiological tonicity improves human chondrogenic marker expression through nuclear factor of activated T-cells 5 in vitro

IntroductionChondrocytes experience a hypertonic environment compared with plasma (280 mOsm) due to the high fixed negative charge density of cartilage. Standard isolation of chondrocytes removes their hypertonic matrix, exposing them to nonphysiological conditions. During in vitro expansion, chondrocytes quickly lose their specialized phenotype, making them inappropriate for cell-based regenerative strategies. We aimed to elucidate the effects of tonicity during isolation and in vitro expansion on chondrocyte phenotype.MethodsHuman articular chondrocytes were isolated and subsequently expanded at control tonicity (280 mOsm) or at moderately elevated, physiological tonicity (380 mOsm). The effects of physiological tonicity on chondrocyte proliferation and chondrogenic marker expression were evaluated. The role of Tonicity-responsive Enhancer Binding Protein in response to physiological tonicity was investigated using nuclear factor of activated T-cells 5 (NFAT5) RNA interference.ResultsModerately elevated, physiological tonicity (380 mOsm) did not affect chondrocyte proliferation, while higher tonicities inhibited proliferation and diminished cell viability. Physiological tonicity improved expression of chondrogenic markers and NFAT5 and its target genes, while suppressing dedifferentiation marker collagen type I and improving type II/type I expression ratios >100-fold. Effects of physiological tonicity were similar in osteoarthritic and normal (nonosteoarthritic) chondrocytes, indicating a disease-independent mechanism. NFAT5 RNA interference abolished tonicity-mediated effects and revealed that NFAT5 positively regulates collagen type II expression, while suppressing type I.ConclusionsPhysiological tonicity provides a simple, yet effective, means to improve phenotypical characteristics during cytokine-free isolation and in vitro expansion of human articular chondrocytes. Our findings will lead to the development of improved cell-based repair strategies for chondral lesions and provides important insights into mechanisms underlying osteoarthritic progression.

Ferumoxides-protamine sulfate is more effective than ferucarbotran for cell labeling: implications for clinically applicable cell tracking using MRI.

The use of superparamagnetic iron oxide (SPIO) for labeling cells holds great promise for clinically applicable cell tracking using magnetic resonance imaging. For clinical application, an effectively and specifically labeled cell preparation is highly desired (i.e. a large amount of intracellular iron and a negligible amount of extracellular iron). In this study we performed a direct comparison of two SPIO labeling strategies that have both been reported as efficient and clinically translatable approaches. These approaches are cell labeling using ferumoxides-protamine complexes or ferucarabotran particles. Cell labeling was performed on primary human bone marrow stromal cells (hBMSCs) and chondrocytes. For both cell types ferumoxides-protamine resulted in a higher percentage of labeled cells, a higher total iron load, a larger amount of intracellular iron and a lower amount of extracellular iron aggregates, compared with ferucarbotran. Consequently, hBMSC and chondrocyte labeling with ferumoxides-protamine is more effective and results in more specific cell labeling than ferucarbotran.

Silencing of Antichondrogenic MicroRNA-221 in Human Mesenchymal Stem Cells Promotes Cartilage Repair In Vivo

There is a growing demand for the development of experimental strategies for efficient articular cartilage repair. Current tissue engineering‐based regenerative strategies make use of human mesenchymal stromal cells (hMSCs). However, when implanted in a cartilage defect, control of hMSCs differentiation toward the chondrogenic lineage remains a significant challenge. We have recently demonstrated that silencing the antichondrogenic regulator microRNA‐221 (miR‐221) was highly effective in promoting in vitro chondrogenesis of monolayered hMSCs in the absence of the chondrogenic induction factor TGF‐β. Here we investigated the feasibility of this approach first in conventional 3D pellet culture and then in an in vivo model. In pellet cultures, we observed that miR‐221 silencing was sufficient to drive hMSCs toward chondrogenic differentiation in the absence of TGF‐β. In vivo, the potential of miR‐221 silenced hMSCs was investigated by first encapsulating the cells in alginate and then by filling a cartilage defect in an osteochondral biopsy. After implanting the biopsy subcutaneously in nude mice, we found that silencing of miR‐221 strongly enhanced in vivo cartilage repair compared to the control conditions (untreated hMSCs or alginate‐only). Notably, miR‐221 silenced hMSCs generated in vivo a cartilaginous tissue with no sign of collagen type X deposition, a marker of undesired hypertrophic maturation. Altogether our data indicate that silencing miR‐221 has a prochondrogenic role in vivo, opening new possibilities for the use of hMSCs in cartilage tissue engineering. Stem Cells 2016;34:1801–1811