Osteoarthritis Cartilage. 2016 Jan;24(1):124-8.
Limited evidence of chondrocyte outgrowth from adult human articular cartilage
Christian Zingler1, Hans-Dieter Carl2, Bernd Swoboda2, Sebastian Krinner1, Friedrich Hennig1, Kolja Gelse1
1 Dept. of Orthopaedic and Trauma Surgery, University Hospital Erlangen, Germany
2 Dept. of Orthopaedic Rheumatology, University of Erlangen-Nuremberg, Germany
Dr. Kolja Gelse
Dept. of Orthopaedic and Trauma Surgery, University Hospital Erlangen; Krankenhausstr. 12, 91054 Erlangen; Germany
Objective: Cellular outgrowth from articular cartilage tissue has been described in a number of recent experimental studies. The aim of this study was to investigate the occurrence of cellular outgrowth from articular cartilage explants isolated from adult human donors.
Method: Macroscopically intact articular cartilage specimens were isolated from adult human donors and cultured either in their native status, or in a cleansed status achieved by forced washing to minimize attaching cells. Additionally, the effect of chemotactic stimuli including cell lysate, High-Mobility-Group-Protein B1 (HMGB-1), Trefoil-factor 3 (TFF3), bone morphogenetic protein-2 (BMP-2), transforming growth factor-ß1 (TGF-ß1), or three-dimensional fibrin or collagen matrices were investigated. Co-cultures with synovial membrane served as a positive control for a source of cartilage-adjoining cells. The occurrence of cellular outgrowth was analysed by histological examination after a culture period of 4 weeks.
Results: Spontaneous cellular outgrowth from cleansed cartilage specimens was not observed at a relevant level and could not significantly be induced by chemotactic stimuli or three-dimensional matrices either. A forming cartilage-adjoining cell layer was only apparent in the case of native cartilage explants with cellular remnants from surgical isolation or in co-culture experiments with synovial membrane.
Conclusion: The relevance of cellular outgrowth from cartilage tissue is largely absent in the case of adult human articular cartilage samples. A cartilage-adjoining cell layer forming around the explants may instead originate from still attaching cells that remained from surgical isolation.
KEYWORDS: Cartilage regeneration; Chemotaxis; Explant model; Migration
Articular cartilage provides a low-friction surface that is mandatory for joint movement and withstands high biomechanical load for many decades of life. However, once destroyed it has no capabilities for regeneration, which is at least true for the human adult. During ontogenesis, articular cartilage derives from a multipotent mesenchyme and later on gives rise to the adult joint surface by appositional growth. This developmental pattern suggests the persistence of chondrocyte progenitor cells within the tissue. This cell population is supposed to be still present in the adult. Indeed, a couple of recent studies have provided evidence of such progenitor cells (1-4). The outgrowth of repair cells from cultured cartilage tissue has been shown in studies focusing on younger donors from different species. These repair cells were shown to migrate into cartilage lesions or to spontaneously form cartilage-like tissue (3, 4). Thus, this cell population would be the ideal source for cartilage regeneration or repair. In our recent work, we investigated if relevant cellular outgrowth could be observed in adult human articular cartilage samples. Indeed, we could detect cartilage-adjoining cells flanking native cartilage samples, which were able to form an extracellular matrix following a culture period of 3 weeks (5). However, such cartilage-adjoining cells were not detected in cleansed samples treated by forced washing.
In additional experiments, we investigated to which extent the cartilage-adjoining cells derive from the cartilage tissue itself or from other sources, such as cell-contamination during surgery or sample preparation. We, therefore, performed an experiment with cleansed cartilage specimens that were co-cultured with chondrocytes. For this purpose, isolated chondrocytes were labeled in monolayer culture by exposure to iron-oxide nanoparticles with a ﬁnal concentration of 50 µg/ml iron oxide for 24 hrs prior to co-culture. After 3 weeks of co-culture, the cartilage samples were analyzed histologically by Prussian blue-/nuclear fast red-staining. Histological analysis revealed that 100% of the adjacent cells were labelled by iron-oxide nanoparticles, which allows to conclude that no cellular outgrowth of unlabelled matrix-embedded chondrocytes had occurred (Suppl. Figure 1 a+b). These data underline the absence of relevant cellular outgrowth in human adult articular cartilage.
Suppl. Figure 1: Histological section of a cleansed cartilage sample co-cultured for 3 weeks with a monolayer of chondrocytes that were labelled by iron-oxide nanoparticles. Toluidine blue staining (a). Prussian blue-/ nuclear fast red-staining (b). Bar = 50µm.
In our publication, we further demonstrated that cellular outgrowth cannot be induced by chemotactic stimuli, such as TGFß, BMP-2, HMGB-1, TFF3 or cell lysate, either. The dense extracellular matrix may be one major factor that hinders cell migration, and even very superfically located chondrocytes adjacent to a fissure remain entrapped in their pericellular matrix during a cultivation period of 3 weeks (Suppl. Figure 2a).
One hypothetical concept to enable endogenous repair by cellular migration may be based on a short-term enzymatic degradation of the extracellular and/or pericellular matrix. For example, the short-term exposure (1 hour) to 0.2% collagenase allows the release of superfically located chondrocytes (Suppl. Figure 2b). However, the visible destruction of the superficial extracellular matrix impedes with reasonable clinical use. For future concepts, it remains challenging to define an appropriate spatio-temporal pattern for degradative events.
Nevertheless, inflammation and tissue degradation represents one key mechanism for early steps in the wound healing cascade of many other tissues. Therefore, it may be reasonable to focus on this general biological principle also for cartilage repair.
Suppl. Figure 2. Chondrocytes remain entrapped within the dense pericellular matrix even very close to fissures (arrow) during a 3-weeks-culture period. Collagenase treatment allows to release superficially located chondrocytes (arrowheads). Toluidine blue staining. Bar = 50µm.
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- Zingler C, Carl HD, Swoboda B, Krinner S, Hennig F, Gelse K. Limited evidence of chondrocyte outgrowth from adult human articular cartilage. Osteoarthritis Cartilage. 2016;24(1):124-8.