Jürgen Mollenhauer

Human knee and ankle cartilage explants: catabolic differences

The prevalence of osteoarthritis (OA) is lower in some joints, i.e., the ankle, than in the knee. We have compared the cartilages from these two joints of the same limb in adult donors (matched pairs). Our data to date suggest that there are metabolic, biochemical and biomechanical differences between the cartilages of the two joints. The current study has focused on extending the metabolic studies comparing the response of chondrocytes to Interleukin‐1β (IL‐1β) and osteogenic protein 1 (OP‐1) by analyzing changes in sulfate incorporation into glycosaminoglycans (GAGs) as a measure of proteoglycan (PG) synthesis. Human adult chondrocytes from normal knees (tibiofemoral) and ankles (talocrural) joints cultured as explants both responded to IL‐1β after 72 h by decreasing PG synthesis; however, the IC50 for the knee chondrocytes was 6.2 pg/ml, while that for the ankle was 35 pg/ml. When the explants were incubated for 72 h with IL‐1β and allowed to rebound without IL‐1β, synthesis of PG was significantly elevated by ankle chondrocytes within five days; knee chondrocytes were unable to significantly increase synthesis even after eight days. However, in both knee and ankle, application of OP‐1 enhanced PG synthesis in the rebound phase. In response to IL‐1, an upregulation of proteinase activity was detectable by an increase in the neoepitopes proteolytically‐generated by both aggrecanase and matrix metalloproteinases (MMPs), in the deep zone of the knee cartilage. Stromelysin and collagenase were upregulated as well. The data emerging from these studies confirm that the ankle is less responsive to catabolic stimulation and more responsive to anabolic stimulation following IL‐1 removal. These differences in metabolic activity between the cartilages of the two joints could in part help to explain their differences in susceptibility to OA.

Expression of anchorin CII (cartilage annexin V) in human young, normal adult, and osteoarthritic cartilage.

In its tissue-specific function as a collagen receptor of chondrocytes, cartilage annexin V (anchorin CII) occupies a key position in the organization of the cell-extracellular matrix (ECM) junction for the tissue. The general role of annexin V (Anx V) in other tissues suggests involvement in cellular secretory processes and in regulation of apoptosis. Immunohistochemical analysis of Anx V in growth plate cartilage, confirmed by in situ hybridization, suggests that Anx V is prominently expressed and forms a major constituent of growth plate chondrocytes. Anx V epitopes are also located in the pericellular matrix of hypertrophic cartilage. In adult articular cartilage the expression is downregulated, with the highest levels of immunostaining found in the upper third of the articular cartilage layers and almost no antigen found in the deep layers. Osteoarthritic (OA) cartilage is characterized by a significant upregulation of message and protein throughout the entire depth of the tissue, an accumulation of cytoplasmic annexin V epitopes, and a release of epitopes into the pericellular and interterritorial matrix, in part co-localized with granular structures. Therefore, Anx V expression and tissue distribution may serve as a histological marker for metabolic alterations and for changes in the cellular phenotype associated with OA.

Histological and cell biological characterization of dissected cartilage fragments in human osteochondritis dissecans of the femoral condyle

Introduction: Osteochondritis dissecans (OCD) within the weight-bearing femoral condyle carries a high risk of osteoarthritis. The definitive pathogenetic cause is unclear. Therefore biochemical and cellular features of OCD were analyzed and compared to macroscopically normal cartilage of the same joint surface. Materials and methods: Dissected fragments from 14 patients and biopsies of normal cartilage from the intercondylar notch as controls were harvested at arthroscopy. Staining with safranin O to monitor proteoglycan content, alkaline phosphatase activity, and immunohistochemistry with mouse monoclonal antibodies to collagen types I, II, and X. Chondrocytes were isolated for RT-PCR to detect GAPDH, collagen types I, II, X, aggrecan, TGF-β, BMP-7, bFGF, VEGF and IL-1. Results: The dissected cartilage displayed significant variability. Apart from normal cartilage matrix components also atypical molecules such as collagen type X and alkaline phosphatase were detected at the tidemark but also across the entire dissecate, suggesting chondrocyte hypertrophy. Extended fibrous degeneration associated with collagen type I deposition was observed at the surface and may indicate chondrocyte dedifferentiation. Viable cells could be extracted from OCD and notch. Both expressed similar mRNA levels for matrix molecules, growth factors, and interleukin-1 (IL-1), however significantly more Col X mRNA was detected in dissecates. Conclusion: Histology suggests focal alteration of cartilage matrix originating from the basis of the joint cartilage, potentially the mineralized layer or subchondral bone. The molecular analysis indicates a disorganization of cartilage homeostasis across the joint accompanied by embryogenetic processes. The surprisingly high viability and quality of the extracted cells suggests a still preserved intrinsic repair capacity of those vital dissecates.

Overwhelming septic infection with a multi-resistant Staphylococcus aureus (MRSA) after total knee replacement

IntroductionThe incidence of early deep infection after arthroplasty of the knee is very low but could represent a serious future problem.Material and methodsThe authors report on a 71-year-old woman with gonarthritis who was supplied with a total knee endoprosthesis and developed a local infection followed by septic shock.ResultsThe infection was evidently caused by a multiresistant Staphylococcus aureus (Staph. aureus). The primary clinical signs closely resembled a necrotizing fasciitis. Systemic and local application of vancomycin led to an improvement of the symptoms at secondary sites, but only the amputation of the primarily infected leg was lifesaving.ConclusionIn order to prevent such events, the authors recommend a number of additional presurgical measures. Firstly, a swab from the nose and throat should be taken prior to an elective surgery in patients with elevated risk of immunodeficiency, for example in patients with diabetes. The diabetes should be adequately treated before an elective operation is undertaken. Secondly, an early punction of the knee joint should be carried out if there is any doubt regarding inflammation. Isolated infectious agents should be grounds for early revision, which should always be completed with a rinsing procedure and with adequate antibiotics. Immunotherapy should be taken into consideration. Antiepidemic measures are recommended in cases with known Staph. aureus.

[Ankle chondrocytes are more resistant to Interleukin-1 than chondrocytes derived from the knee].

ZusammenfassungHintergrundDie Inzidenz degenerativer Veränderungen und Osteoarthritis ist im Sprunggelenk geringer als im Kniegelenk. Dies kann nicht ausschließlich mit Unterschieden in der Anatomie und den biomechanischen Eigenschaften dieser beiden Gelenke erklärt werden. Frühere Untersuchungen zeigten Unterschiede in der biochemischen Zusammensetzung der extrazellulären Matrix des Gelenkknorpels von Knie- und Sprunggelenk. Das Ziel der vorliegenden Studie war die Identifikation möglicher metabolischer Unterschiede von Knie- und Sprunggelenkchondrozyten an isolierten Zellen, um die sekundären Effekte der vorhandenen extrazellulären Matrix von der primären matrixunabhängigen zellulären Differenzierung zu unterscheiden.MethodenIsolierte Chondrozyten des Knie- und Sprunggelenks humaner Spender wurden in Alginatkügelchen kultiviert und mit dem katabolen Zytokin Interleukin-1 (IL-1) als Modell einer entzündlichen Episode inkubiert. Der Proteoglykan- (PG-)Stoffwechsel wurde über die 35S-Inkorporation in Glykosaminoglykane (GAG) analysiert.ErgebnisseDie Gegenwart von IL-1 induzierte eine Inhibition der PG-Synthese des Knie- und Sprunggelenkknorpels. Dabei war die 50%-Hemmkonzentration (IC50) von IL-1 für das Kniegelenk ca. 5fach geringer als für das Sprunggelenk.SchlussfolgerungSprunggelenkchondrozyten besitzen eine höhere IL-1-Resistenz als Kniegelenkchondrozyten.AbstractBackgroundThe incidence of degenerative changes and osteoarthritis is lower in the ankle than in the knee joints. This cannot be explained exclusively with differences in anatomy and biomechanical properties of these two synovial joints. Previous studies have indicated distinct differences in the biochemical composition of the extracellular matrix of articular cartilage from knee and ankle joints. The aim of this study was to identify potential metabolic differences between knee and ankle joint chondrocytes using isolated cells to distinguish the secondary effects of the resident extracellular matrix from the primary matrix-independent effects of cellular differentiation.MethodIsolated knee and ankle chondrocytes from the same human donor were cultured in alginate beads and subsequently exposed to a three-day pulse of the catabolic cytokine interleukin-1 (IL-1) as a model of an inflammatory episode. The metabolism of proteoglycans (PG’s) was analyzed as expressed changes in 35S-sulfate incorporation into glycosaminoglycans (GAG’s).ResultsThe presence of IL-1 induced an inhibition of PG synthesis in knee and ankle articular chondrocytes. The 50% inhibitory concentration (IC50) of IL-1 was about 5 times lower for knee than for ankle chondrocytes.ConclusionAnkle chondrocytes are more resistant to IL-1 induced inhibition of PG synthesis than chondrocytes from the knee.

Sprunggelenkchondrozyten besitzen eine höhere Interleukin-1-Resistenz als Kniegelenkchondrozyten

ZusammenfassungHintergrundDie Inzidenz degenerativer Veränderungen und Osteoarthritis ist im Sprunggelenk geringer als im Kniegelenk. Dies kann nicht ausschließlich mit Unterschieden in der Anatomie und den biomechanischen Eigenschaften dieser beiden Gelenke erklärt werden. Frühere Untersuchungen zeigten Unterschiede in der biochemischen Zusammensetzung der extrazellulären Matrix des Gelenkknorpels von Knie- und Sprunggelenk. Das Ziel der vorliegenden Studie war die Identifikation möglicher metabolischer Unterschiede von Knie- und Sprunggelenkchondrozyten an isolierten Zellen, um die sekundären Effekte der vorhandenen extrazellulären Matrix von der primären matrixunabhängigen zellulären Differenzierung zu unterscheiden.MethodenIsolierte Chondrozyten des Knie- und Sprunggelenks humaner Spender wurden in Alginatkügelchen kultiviert und mit dem katabolen Zytokin Interleukin-1 (IL-1) als Modell einer entzündlichen Episode inkubiert. Der Proteoglykan- (PG-)Stoffwechsel wurde über die 35S-Inkorporation in Glykosaminoglykane (GAG) analysiert.ErgebnisseDie Gegenwart von IL-1 induzierte eine Inhibition der PG-Synthese des Knie- und Sprunggelenkknorpels. Dabei war die 50%-Hemmkonzentration (IC50) von IL-1 für das Kniegelenk ca. 5fach geringer als für das Sprunggelenk.SchlussfolgerungSprunggelenkchondrozyten besitzen eine höhere IL-1-Resistenz als Kniegelenkchondrozyten.AbstractBackgroundThe incidence of degenerative changes and osteoarthritis is lower in the ankle than in the knee joints. This cannot be explained exclusively with differences in anatomy and biomechanical properties of these two synovial joints. Previous studies have indicated distinct differences in the biochemical composition of the extracellular matrix of articular cartilage from knee and ankle joints. The aim of this study was to identify potential metabolic differences between knee and ankle joint chondrocytes using isolated cells to distinguish the secondary effects of the resident extracellular matrix from the primary matrix-independent effects of cellular differentiation.MethodIsolated knee and ankle chondrocytes from the same human donor were cultured in alginate beads and subsequently exposed to a three-day pulse of the catabolic cytokine interleukin-1 (IL-1) as a model of an inflammatory episode. The metabolism of proteoglycans (PG’s) was analyzed as expressed changes in 35S-sulfate incorporation into glycosaminoglycans (GAG’s).ResultsThe presence of IL-1 induced an inhibition of PG synthesis in knee and ankle articular chondrocytes. The 50% inhibitory concentration (IC50) of IL-1 was about 5 times lower for knee than for ankle chondrocytes.ConclusionAnkle chondrocytes are more resistant to IL-1 induced inhibition of PG synthesis than chondrocytes from the knee.

Chance and limit of imaging of articular cartilage in vitro in healthy and arthritic joints: DEI (diffraction enhanced imaging) in comparison with MRI, CT, and ultrasound

Description of purpose: Treatment of osteoarthritis in stages of reversible disease requires high resolution visualization of early cartilage damage and of subchondral bone. Here, DEI (Diffraction Enhanced Imaging) is compared to MRI, computed X-ray tomography (CT) and ultrasound (UI) in its ability to detect early degeneration of articular cartilage. In contrast to conventional absorptive X-ray examination where cartilage is poorly visible DEI captures cartilage by detection of selected refraction. Methods: Human femoral heads were investigated by macroscopic inspection, conventional X-ray examination, DEI, MRI, CT, UI and histology. DEI is an imaging technique applying a monochromatic parallel synchrotron X-ray beam. Image features were verified by histology. Results: DEI, MRI and ultrasound lead to interpretable images of cartilage. Of all techniques, DEI provided highest image resolution revealing the structural tissue architecture. MRI needs a very long exposure time (more than 5 hours) to achieve comparable quality. Application of ultrasound is limited because of joint geometry and, at high sound frequency, the necessity of close contact between cartilage and transducer. DEI is an experimental technique which needs synchrotron radiation. Conclusion: DEI is a very promising imaging technique for visualization of cartilage and bone. It may serve as an excellent analytical tool for experimental studies. Our pictures show a part of future of optimised techniques for imaging. Synchrotron based DEI may lead the way towards optimisation of improved techniques for imaging. Upon development of adequate small scale X-ray sources, DEI will also be an important supplementation for medical imaging.