IntroductionThe repair capability of traumatized articular cartilage is highly limited so that joint injuries often lead to osteoarthritis. Migratory chondrogenic progenitor cells (CPC) might represent a target cell population for in situ regeneration. This study aims to clarify, whether 1) CPC are present in regions of macroscopically intact cartilage from human osteoarthritic joints, 2) CPC migration is stimulated by single growth factors and the cocktail of factors released from traumatized cartilage and 3) CPC migration is influenced by cytokines present in traumatized joints.MethodsWe characterized the cells growing out from macroscopically intact human osteoarthritic cartilage using a panel of positive and negative surface markers and analyzed their differentiation capacity. The migratory response to platelet-derived growth factor (PDGF)-BB, insulin-like growth factor 1 (IGF-1), supernatants obtained from in vitro traumatized cartilage and interleukin-1 beta (IL-1β) as well as tumor necrosis factor alpha (TNF-α) were tested with a modified Boyden chamber assay. The influence of IL-1β and TNF-α was additionally examined by scratch assays and outgrowth experiments.ResultsA comparison of 25 quadruplicate marker combinations in CPC and bone-marrow derived mesenchymal stromal cells showed a similar expression profile. CPC cultures had the potential for adipogenic, osteogenic and chondrogenic differentiation. PDGF-BB and IGF-1, such as the supernatant from traumatized cartilage, induced a significant site-directed migratory response. IL-1β and TNF-α significantly reduced basal cell migration and abrogated the stimulative effect of the growth factors and the trauma supernatant. Both cytokines also inhibited cell migration in the scratch assay and primary outgrowth of CPC from cartilage tissue. In contrast, the cytokine IL-6, which is present in trauma supernatant, did not affect growth factor induced migration of CPC.ConclusionThese results indicate that traumatized cartilage releases chemoattractive factors for CPC but IL-1β and TNF-α inhibit their migratory activity which might contribute to the low regenerative potential of cartilage in vivo.

In the context of this ex vivo study, we showed not only remarkable cell- and chondroprotective features, but also revealed new encouraging findings concerning the therapeutically effective concentration and treatment-time regimen of NAC. Its defense against chondrocyte apoptosis and catabolic enzyme secretion recommends NAC as a multifunctional add-on reagent for pharmaceutical intervention after cartilage injury. Taken together, our data increase the knowledge on the therapeutic potential of NAC after cartilage trauma and presents a basis for future in vivo studies.

BackgroundProgenitor cells display interesting features for tissue repair and reconstruction. In the last years, such cells have been identified in different cartilage types. In this study, we isolated a migrative subpopulation of adult human nasoseptal chondrocytes with progenitor cell features by outgrowth from human nasal septum cartilage. These putative progenitor cells were comparatively characterized with mesenchymal stem cells (MSC) and human nasal septum chondrocytes with respect to their cellular characteristics as well as surface marker profile using flow cytometric analyses. Differentiation capacity was evaluated on protein and gene expression levels.ResultsThe migrative subpopulation differentiated into osteogenic and chondrogenic lineages with distinct differences to chondrocytes and MSC. Cells of the migrative subpopulation showed an intermediate surface marker profile positioned between MSC and chondrocytes. Significant differences were found for CD9, CD29, CD44, CD90, CD105 and CD106. The cells possessed a high migratory ability in a Boyden chamber assay and responded to chemotactic stimulation. To evaluate their potential use in tissue engineering applications, a decellularized septal cartilage matrix was either seeded with cells from the migrative subpopulation or chondrocytes. Matrix production was demonstrated immunohistochemically and verified on gene expression level. Along with secretion of matrix metalloproteinases, cells of the migrative subpopulation migrated faster into the collagen matrix than chondrocytes, while synthesis of cartilage specific matrix was comparable.ConclusionsCells of the migrative subpopulation, due to their migratory characteristics, are a potential cell source for in vivo regeneration of nasal cartilage. The in vivo mobilization of nasal cartilage progenitor cells is envisioned to be the basis for in situ tissue engineering procedures, aiming at the use of unseeded biomaterials which are able to recruit local progenitor cells for cartilage regeneration.

In osteoarthritis (OA), cartilage destruction is associated not only with an imbalance of anabolic and catabolic processes but also with alterations of the cytoskeletal organization in chondrocytes, although their pathogenetic origin is largely unknown so far. Therefore, we have studied possible effects of the proinflammatory cytokine IL-1β on components of the cytoskeleton in OA chondrocytes on gene expression level. Using a whole genome array, we found that IL-1β is involved in the regulation of many cytoskeleton-related genes. Apart from well-known cytoskeletal components, the expression and regulation of four genes coding for LIM proteins were shown. These four genes were previously undescribed in the chondrocyte context. Quantitative PCR analysis confirmed significant downregulation of Fhl1, Fhl2, Lasp1, and Pdlim1 as well as Tubb and Vim by IL-1β. Inhibition of p38 mitogen-activated protein kinase (MAPK) by SB203580 counteracted the influence of IL-1β on Fhl2 and Tubb expression, indicating partial involvement of this signaling pathway. Downregulation of the LIM-only protein FHL2 was confirmed additionally on the protein level. In agreement with these results, IL-1β induced changes in the morphology of chondrocytes, the organization of the cytoskeleton, and the cellular distribution of FHL2. We conclude that L-1β is involved in the regulation of various cytoskeletal components in human chondrocytes including the multifunctional protein FHL2. This might be relevant for the pathogenesis of OA.

Cartilage injury can trigger crucial pathomechanisms, including excessive cell death and expression of matrix‐destructive enzymes, which contribute to the progression of a post‐traumatic osteoarthritis (PTOA). With the intent to create a novel treatment strategy for alleviating trauma‐induced cartilage damage, we complemented a promising antioxidative approach based on cell and chondroprotective N‐acetyl cysteine (NAC) by chondroanabolic stimulation. Overall, three potential pro‐anabolic growth factors – IGF‐1, BMP7 and FGF18 – were tested comparatively with and without NAC in an ex vivo human cartilage trauma‐model. For that purpose, full‐thickness cartilage explants were subjected to a defined impact (0.59 J) and subsequently treated with the substances. Efficacy of the therapeutic approaches was evaluated by cell viability, as well as various catabolic and anabolic biomarkers, representing the present matrix turnover. Although monotherapy with NAC, FGF18 or BMP7 significantly prevented trauma‐induced cell dead and breakdown of type II collagen, combination of NAC and one of the growth factors did not yield significant benefit as compared to NAC alone. IGF‐1, which possessed only moderate cell protective and no chondroprotective qualities after cartilage trauma, even reduced NAC‐mediated cell and chondroprotection. Despite significant promotion of type II collagen expression by IGF‐1 and BMP7, addition of NAC completely suppressed this chondroanabolic effect. All in all, NAC and BMP7 emerged as best combination. As our findings indicate limited benefits of the simultaneous multidirectional therapy, a sequential application might circumvent adverse interferences, such as suppression of type II collagen biosynthesis, which was found to be reversed 7 days after NAC withdrawal.