Eva Skiöldebrand

Support of Concept That Migrating Progenitor Cells From Stem Cell Niches Contribute to Normal Regeneration of the Adult Mammal Intervertebral Disc: A Descriptive Study in the New Zealand White Rabbit

Study Design. Descriptive experimental study performed in rabbits of 2 age groups. Objective. To study and investigate presence of prechondrocytic cells and cell migration routes (MR) in the intervertebral disc (IVD) region to gain knowledge about the normal IVD regeneration pattern. Summary of Background Data. Disc degeneration is thought to play a major role in patients with chronic lumbar pain. Regeneration processes and cell migration within the IVD have been sparsely described. Therefore, it is of interest to increase knowledge of these processes in order to understand pathological conditions of the IVD. Methods. At the beginning of the experiment, 5-bromo-2-deoxyuridine (BrdU) in vivo labeling was performed in 2 groups of rabbits, 3 and 9 months old (total 27 rabbits). BrdU is incorporated into DNA during mitosis, and then it is gradually diluted with each cell division until it finally disappears. Incorporation of BrdU was then visualized by immunohistochemistry (IHC) at different time points providing cell division pattern and presence of slow-cycling cells in the IVD region. IVD tissue was investigated by IHC for growth and differentiation factor-5 (GDF5), SOX9 (chondrogenic lineage markers), SNAIL homolog 1 (SNAI1), SNAIL homolog 2 (SLUG) (migration markers), and &bgr;1-INTEGRIN (cellular adhesion marker). In addition, GDF5, SOX9, and BMPRIB expression were investigated on genetic level. Results. BrdU+ cells were observed in early time points in the IVD niche, adjacent to the epiphyseal plate, at later time points mainly in outer region of the annulus fibrosus for both age groups of rabbits, indicating a gradual migration of cells. The presence of SLUG, SNAI1, GDF5, SOX9, and &bgr;1-INTEGRIN was found in same regions. Conclusion. The results suggest a cellular MR from the IVD stem cell niche toward the annulus fibrosus and the inner parts of the IVD. These findings may be of importance for understanding IVD regenerative mechanisms and for future development of biological treatment strategies.

Similar cellular migration patterns from niches in intervertebral disc and in knee-joint regions detected by in situ labeling: an experimental study in the New Zealand white rabbit.

IntroductionPotential stem cell niches (SNs) were recently reported in intervertebral discs (IVDs) and knee joints (KJs) in different mammals (located adjacent to the epiphyseal plate; EP). The aim here was to examine further possible cellular migration and migration directions of cells originating from niches possibly involved in regeneration of cartilaginous tissues in the IVD and in the KJ regions in adult mammals.MethodsIn total, 33 rabbits were used in studies A through C.A.IVD cells were sorted; fluorescence-activated cell sorting (FACS) by size (forward scatter; ≤10 μm or >10 μm or GDF5+ cells (anti-GDF5 antibody). Sorted cells, labeled with cell tracer (carboxyfluorescein-diacetate-succinimidyl ester; CDFA-SE) were applied on IVD explants in vitro. Migrating cells/distance was evaluated by fluorescence- and confocal-microscopy (FC).B.DNA labeling was performed with BrdU (oral administration). Animals were killed (14 to 56 days), KJs collected, and BrdU+ cells visualized with immunohistochemistry (IHC)/anti-BrdU antibody in SN and articular cartilage (AC).C.Cell tracer: (Fe-nanoparticles: Endorem) were injected into SNs of IVDs (LI-LV) and KJs (tibia). Animals were killed after 2 to 6 weeks. Fe-labeled cells were traced by ferric-iron staining (Prussian blue reaction; Mallory method).ResultsA. GDF5+ cells and ≤10-μm cells displayed the best migration capability in IVD explants. GDF5+ cells were detected at a tissue depth of 1,300 μm (16 days). B. BrdU+ cells were observed in early time points in niches of KJs, and at later time points in AC, indicating a gradual migration of cells. C. Fe+ cells were detected in IVDs; in annulus fibrosus (AF) in 11 of 12 animals and in nucleus pulposus (NP) in two of 12 animals. In AC (tibia), Fe+ cells were detected in six of 12 animals. In the potential migration route (PMR), from niches toward the IVD, Fe+ cells (three of 12 animals) and in PMR toward AC (KJs) (six of 12 animals) were detected.ConclusionsResults indicate similar cellular migration patterns in cartilage regions (IVD and KJs) with migration from stem cell niche areas into the mature cartilaginous tissues of both the KJs and the IVD. These findings of a cellular migration pattern in mature cartilage are of interest from tissue-repair and engineering perspectives.

Quantitative proteomics reveals regulatory differences in the chondrocyte secretome from human medial and lateral femoral condyles in osteoarthritic patients

BackgroundOsteoarthritis (OA) is a destructive joint disease and there are no known biomarkers available for an early diagnosis. To identify potential disease biomarkers and gain further insight into the disease mechanisms of OA we applied quantitative proteomics with SILAC technology on the secretomes from chondrocytes of OA knees, designated as high Mankin (HM) scored secretome. A quantitative comparison was made between the secretomes of the medial and lateral femur condyle chondrocytes in the same knee since the medial femur condyle is usually more affected in OA than the lateral condyle, which was confirmed by Mankin scoring. The medial/lateral comparison was also made on the secretomes from chondrocytes taken from one individual with no clinically apparent joint-disease, designated as low Mankin (LM) scored secretome.ResultsWe identified 825 proteins in the HM secretome and 69 of these showed differential expression when comparing the medial and lateral femoral compartment. The LM scored femoral condyle showed early signs of OA in the medial compartment as assessed by Mankin score. We here report the identification and relative quantification of several proteins of interest for the OA disease mechanism e.g. CYTL1, DMD and STAB1 together with putative early disease markers e.g. TIMP1, PPP2CA and B2M.ConclusionsThe present study reveals differences in protein abundance between medial/lateral femur condyles in OA patients. These regulatory differences expand the knowledge regarding OA disease markers and mechanisms.

Effects of high mobility group box protein-1, interleukin-1β, and interleukin-6 on cartilage matrix metabolism in three-dimensional equine chondrocyte cultures.

The effects of high mobility group box protein (HMGB)-1, interleukin (IL)-1β, and IL-6 on equine articular chondrocytes were investigated, with emphasis on detecting differences between anatomical sites exposed to different loading in vivo, using three-dimensional (3D) cell cultures established with chondrocytes from dorsal radial facet (DRF, highly loaded) and palmar condyle (PC, less loaded) of the third carpal bone (C3). Expression of important genes involved in cartilage metabolism, presence of glycosaminoglycans and cartilage oligomeric matrix protein (COMP) in pellets, and concentrations of matrix metalloproteinase (MMP)-13 and aggrecan epitope CS 846 were evaluated. Compared to controls, IL-1β treatment increased gene expression of versican, matrix-degrading enzymes, and tissue inhibitor of metalloproteinase (TIMP)-1, and decreased aggrecan and collagen type I and type II expression. In addition, IL-1β-treated pellets showed decreased safranin O staining and increased COMP immunostaining and MMP-13 concentrations in culture supernatants. Effects of IL-6 and HMGB-1 on gene expression were variable, although upregulation of Sry-related high-mobility group box 9 (Sox9) was often present and statistically increased in HMGB-1-treated pellets. Response to cytokines rarely differed between DRF and PC pellets. Thus, site-associated cartilage deterioration in equine carpal osteoarthritis (OA) is not explained by topographically different responses to inflammatory mediators. Differences in gene expressions of structural matrix proteins in untreated DRF and PC pellets were noted in the youngest horses, which may indicate differences in the chondrocytes potential to produce matrix in vivo. Overall, a strong catabolic response was induced by IL-1β, whereas slight anabolic effects were induced by IL-6 and HMGB-1.

An inflammatory equine model demonstrates dynamic changes of immune response and cartilage matrix molecule degradation in vitro

Abstract The molecular aspects of inflammation were investigated in equine articular cartilage explants using quantitative proteomics. Articular cartilage explants were stimulated with interleukin (IL)-1β in vitro for 25 days, and proteins released into cell culture media were chemically labeled with isobaric mass tags and analyzed by liquid chromatography-tandem mass spectrometry. A total of 127 proteins were identified and quantified in media from explants. IL-1β-stimulation resulted in an abundance of proteins related to inflammation, including matrix metalloproteinases, acute phase proteins, complement components and IL-6. Extracellular matrix (ECM) molecules were released at different time points, and fragmentation of aggrecan and cartilage oligomeric matrix protein was observed at days 3 and 6, similar to early-stage OA in vivo. Degradation products of the collagenous network were observed at days 18 and 22, similar to late-stage OA. This model displays a longitudinal quantification of released molecules from the ECM of articular cartilage. Identification of dynamic changes of extracellular matrix molecules in the secretome of equine explants stimulated with IL-1β over time may be useful for identifying components released at different time points during the spontaneous OA process.

Coupled cell networks are target cells of inflammation, which can spread between different body organs and develop into systemic chronic inflammation

Several organs in the body comprise cells coupled into networks. These cells have in common that they are excitable but do not express action potentials. Furthermore, they are equipped with Ca2+ signaling systems, which can be intercellular and/or extracellular. The transport of small molecules between the cells occurs through gap junctions comprising connexin 43. Examples of cells coupled into networks include astrocytes, keratinocytes, chondrocytes, synovial fibroblasts, osteoblasts, connective tissue cells, cardiac and corneal fibroblasts, myofibroblasts, hepatocytes, and different types of glandular cells. These cells are targets for inflammation, which can be initiated after injury or in disease. If the inflammation reaches the CNS, it develops into neuroinflammation and can be of importance in the development of systemic chronic inflammation, which can manifest as pain and result in changes in the expression and structure of cellular components. Biochemical parameters of importance for cellular functions are described in this review.

Characterisation of lubricin in synovial fluid from horses with osteoarthritis

Reason for performing study: The glycoprotein lubricin contributes to the boundary lubrication of the articular cartilage surface. The early events of osteoarthritis involve the superficial layer where lubricin is synthesised. Objectives: To characterise the glycosylation profile of lubricin in synovial fluid from horses with osteoarthritis and study secretion and degradation of lubricin in an in vitro inflammation cartilage model. Study design: In vitro study. Methods: Synovial fluid samples collected from horses with joints with normal articular cartilage and structural osteoarthritic lesions; with and without osteochondral fragments, were analysed for the lubricin glycosylation profiles. Articular cartilage explants were stimulated with or without interleukin‐1&bgr; for 25 days. Media samples collected at 3‐day intervals were analysed by quantitative proteomics, western blot and enzyme‐linked immunosorbent assay. Results: O‐glycosylation profiles in synovial fluid revealed both Core 1 and 2 O‐glycans, with Core 1 O‐glycans predominating. Synovial fluid from normal joints (49.5 ± 1.9%) contained significantly lower amounts of monosialylated Core 1 O‐glycans compared with joints with osteoarthritis (53.8 ± 7.8%, P = 0.03) or joints with osteochondral fragments (57.3 ± 8.8%, P = 0.001). Additionally, synovial fluid from normal joints (26.7 ± 6.7%) showed higher amounts of disialylated Core 1 O‐glycan than from joints with osteochondral fragments (21.2 ± 4.9%, P = 0.03). A C‐terminal proteolytic cleavage site in lubricin was found in synovial fluid from normal and osteochondral fragment joints and in media from interleukin‐1&bgr; stimulated and unstimulated articular cartilage explants. Conclusions: This is the first demonstration of a change in the glycosylation profile of lubricin in synovial fluid from diseased equine joints compared with that from normal joints. We demonstrate an identical proteolytic cleavage site of lubricin both in vitro and in vivo. The reduced sialation of lubricin in synovial fluid from diseased joints may affect the boundary lubricating ability of the superficial layer of articular cartilage and could be one of the early events in the progression of osteoarthritis.

Effects of interleukin-6 and interleukin-1β on expression of growth differentiation factor-5 and Wnt signaling pathway genes in equine chondrocytes

OBJECTIVE To determine the effects of interleukin (IL)-6 and IL-1β stimulation on expression of growth differentiation factor (GDF)-5 and Wnt signaling pathway genes in equine chondrocytes. SAMPLE Macroscopically normal articular cartilage samples from 6 horses and osteochondral fragments (OCFs) from 3 horses. PROCEDURES Chondrocyte pellets were prepared and cultured without stimulation or following stimulation with IL-6 or IL-1β for 1, 2, 12, and 48 hours; expression of GDF-5 was determined with a quantitative real-time PCR assay. Expression of genes in various signaling pathways was determined with microarrays for pellets stimulated for 1 and 2 hours. Immunohistochemical analysis was used to detect GDF-5, glycogen synthase kinase 3β (GSK-3β), and β-catenin proteins in macroscopically normal cartilage samples and OCFs. RESULTS Chondrocytes stimulated with IL-6 had significantly higher GDF-5 expression within 2 hours versus unstimulated chondrocytes. Microarray analysis of Wnt signaling pathway genes indicated expression of GSK-3β and coiled-coil domain containing 88C increased after 1 hour and expression of β-catenin decreased after 2 hours of IL-6 stimulation. Results of immunohistochemical detection of proteins were similar to microarray analysis results. Chondrocytes in macroscopically normal articular cartilage and OCFs had immunostaining for GDF-5. CONCLUSION AND CLINICAL RELEVANCE Results indicated IL-6 stimulation decreased chondrocyte expression of the canonical Wnt signaling pathway transactivator β-catenin, induced expression of inhibitors of the Wnt pathway, and increased expression of GDF-5. This suggested IL-6 may inhibit the Wnt signaling pathway with subsequent upregulation of GDF-5 expression. Anabolic extracellular matrix metabolism in OCFs may be attributable to GDF-5 expression. This information could be useful for development of cartilage repair methods.

Ultrastructural immunolocalization of cartilage oligomeric matrix protein (COMP) in the articular cartilage on the equine third carpal bone in trained and untrained horses.

The present study was designed to delineate the presence of COMP at the ultrastructural level comparing concentrations between two areas of articular cartilage from the equine third carpal bone, subjected to different loading, from trained and untrained horses. We also analyzed the fibril thickness of collagen type II in the same compartments and zones. Samples were collected from high load-bearing areas of the dorsal radial facet (intermittent high load) and an area of the palmar condyle (low constant load) in five non-trained and three trained young racehorses. The data show that COMP is much less abundant in the matrix in intermittent high loaded areas of articular cartilage from trained horses as compared to the untrained horses (p=0.036). On the other hand, the untrained horses often displayed a higher immunolabeling in loaded areas compared to unloaded areas, indicating that an adequate dynamic load promotes COMP synthesis and/or retention, while an excessive load may have an opposite effect. The collagen fibril diameter showed marked variation between individuals. The present study indicates that dynamic in vivo compression at high load and frequency lowers matrix content of COMP in the articular cartilage of the third carpal bone. It also indicates that the collagen network is influenced by mechanical load following by strenuous exercise.

Cell and matrix modulation in prenatal and postnatal equine growth cartilage, zones of Ranvier and articular cartilage.

Formation of synovial joints includes phenotypic changes of the chondrocytes and the organisation of their extracellular matrix is regulated by different factors and signalling pathways. Increased knowledge of the normal processes involved in joint development may be used to identify similar regulatory mechanisms during pathological conditions in the joint. Samples of the distal radius were collected from prenatal and postnatal equine growth plates, zones of Ranvier and articular cartilage with the aim of identifying Notch signalling components and cells with stem cell‐like characteristics and to follow changes in matrix protein localisation during joint development. The localisation of the Notch signalling components Notch1, Delta4, Hes1, Notch dysregulating protein epidermal growth factor‐like domain 7 (EGFL7), the stem cell‐indicating factor Stro‐1 and the matrix molecules cartilage oligomeric matrix protein (COMP), fibromodulin, matrilin‐1 and chondroadherin were studied using immunohistochemistry. Spatial changes in protein localisations during cartilage maturation were observed for Notch signalling components and matrix molecules, with increased pericellular localisation indicating new synthesis and involvement of these proteins in the formation of the joint. However, it was not possible to characterise the phenotype of the chondrocytes based on their surrounding matrix during normal chondrogenesis. The zone of Ranvier was identified in all horses and characterised as an area expressing Stro‐1, EGFL7 and chondroadherin with an absence of COMP and Notch signalling. Stro‐1 was also present in cells close to the perichondrium, in the articular cartilage and in the fetal resting zone, indicating stem cell‐like characteristics of these cells. The presence of stem cells in the articular cartilage will be of importance for the repair of damaged cartilage. Perivascular chondrocytes and hypertrophic cells of the cartilage bone interface displayed positive staining for EGFL7, which is a novel finding and suggests a role of EGFL7 in the vascular infiltration of growth cartilage.