Cytokine profiles in the joint depend on pathology, but are different between synovial fluid, cartilage tissue and cultured chondrocytes.

INTRODUCTION: This study aimed to evaluate whether profiles of several soluble mediators in synovial fluid and cartilage tissue are pathology-dependent and how their production is related to in vitro tissue formation by chondrocytes from diseased and healthy tissue. METHODS: Samples were obtained from donors without joint pathology (n = 39), with focal defects (n = 65) and osteoarthritis (n = 61). A multiplex bead assay (Luminex) was performed measuring up to 21 cytokines: Interleukin (IL)-1α, IL-1ß, IL-1RA, IL-4, IL-6, IL-6Rα, IL-7, IL-8, IL-10, IL-13, tumor necrosis factor (TNF)α, Interferon (IFN)γ, oncostatin M (OSM), leukemia inhibitory factor (LIF), adiponectin, leptin, monocyte chemotactic factor (MCP)1, RANTES, basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), vascular growth factor (VEGF). RESULTS: In synovial fluid of patients with cartilage pathology, IL-6, IL-13, IFNγ and OSM levels were higher than in donors without joint pathology (P ≤ 0.001). IL-13, IFNγ and OSM were also different between donors with cartilage defects and OA (P < 0.05). In cartilage tissue from debrided defects, VEGF was higher than in non-pathological or osteoarthritic joints (P ≤ 0.001). IL-1α, IL-6, TNFα and OSM concentrations (in ng/ml) were markedly higher in cartilage tissue than in synovial fluid (P <0.01). Culture of chondrocytes generally led to a massive induction of most cytokines (P < 0.001). Although the release of inflammatory cytokines was also here dependent on the pathological condition (P < 0.001) the actual profiles were different from tissue or synovial fluid and between non-expanded and expanded chondrocytes. Cartilage formation was lower by healthy unexpanded chondrocytes than by osteoarthritic or defect chondrocytes. CONCLUSIONS: Several pro-inflammatory, pro-angiogenic and pro-repair cytokines were elevated in joints with symptomatic cartilage defects and/or osteoarthritis, although different cytokines were elevated in synovial fluid compared to tissue or cells. Hence a clear molecular profile was evident dependent on disease status of the joint, which however changed in composition depending on the biological sample analysed. These alterations did not affect in vitro tissue formation with these chondrocytes, as this was at least as effective or even better compared to healthy chondrocytes.

Pronounced biomaterial dependency in cartilage regeneration using nonexpanded compared with expanded chondrocytes.

AIM: We aimed to investigate freshly isolated compared with culture-expanded chondrocytes with respect to early regenerative response, cytokine production and cartilage formation in response to four commonly used biomaterials. MATERIALS & METHODS: Chondrocytes were both directly and after expansion to passage 2, incorporated into four biomaterials: Polyactive™, Beriplast®, HyStem® and a type II collagen gel. Early cartilage matrix gene expression, cytokine production and glycosaminoglycan (GAG) and DNA content in response to these biomaterials were evaluated. RESULTS: HyStem induced more GAG production, compared with all other biomaterials (p ≤ 0.001). Nonexpanded cells did not always produce more GAGs than expanded chondrocytes, as this was biomaterial-dependent. Cytokine production and early gene expression were not predictive for final regeneration. CONCLUSION: For chondrocyte-based cartilage treatments, the biomaterial best supporting cartilage matrix production will depend on the chondrocyte differentiation state and cannot be predicted from early gene expression or cytokine profile.

Single-stage cell-based cartilage regeneration using a combination of chondrons and mesenchymal stromal cells: comparison with microfracture.

BACKGROUND: Autologous chondrocyte implantation (ACI) is traditionally a 2-step procedure used to repair focal articular cartilage lesions. With use of a combination of chondrons (chondrocytes in their own territorial matrix) and mesenchymal stromal cells (MSCs), ACI could be innovated and performed in a single step, as sufficient cells would be available to fill the defect within a 1-step surgical procedure. Chondrons have been shown to have higher regenerative capacities than chondrocytes without such a pericellular matrix. PURPOSE: To evaluate cartilage formation by a combination of chondrons and MSCs in vitro and in both small and large animal models. STUDY DESIGN: Controlled laboratory study. METHODS: Chondrons and MSCs were cultured at different ratios in vitro containing 0%, 5%, 10%, 20%, 50%, or 100% chondrons (n = 3); embedded in injectable fibrin glue (Beriplast); and implanted subcutaneously in nude mice (n = 10; ratios of 0%, 5%, 10%, and 20% chondrons). Also, in a 1-step procedure, a combination of chondrons and MSCs was implanted in a freshly created focal articular cartilage lesion (10% chondrons) in goats (n = 8) and compared with microfracture. The effect of both treatments, after 6-month follow-up, was evaluated using biochemical glycosaminoglycan (GAG) and GAG/DNA analysis and scored using validated scoring systems for macroscopic and microscopic defect repairs. RESULTS: The addition of MSCs to chondron cultures enhanced cartilage-specific matrix production as reflected by a higher GAG production (P < .03), both in absolute levels and normalized to DNA content, compared with chondrocyte and 100% chondron cultures. Similar results were observed after 4 weeks of subcutaneous implantation in nude mice. Treatment of freshly created cartilage defects in goats using a combination of chondrons and MSCs in Beriplast resulted in better microscopic, macroscopic, and biochemical cartilage regeneration (P ≤ .02) compared with microfracture treatment. CONCLUSION: The combination of chondrons and MSCs increased cartilage matrix formation, and this combination of cells was safely applied in a goat model for focal cartilage lesions, outperforming microfracture. CLINICAL RELEVANCE: This study describes the bench-to-preclinical development of a new cell-based regenerative treatment for focal articular cartilage defects that outperforms microfracture in goats. In addition, it is a single-step procedure, thereby making the expensive cell expansion and reimplantation of dedifferentiated cells, as in ACI, redundant.

Interleukin-6 is elevated in synovial fluid of patients with focal cartilage defects and stimulates cartilage matrix production in an in vitro regeneration model.

INTRODUCTION: This study aimed to determine whether, as in osteoarthritis, increased levels of interleukin-6 (IL-6) are present in the synovial fluid of patients with symptomatic cartilage defects and whether this IL-6 affects cartilage regeneration as well as the cartilage in the degenerated knee. METHODS: IL-6 concentrations were determined by ELISA in synovial fluid and in conditioned media of chondrocytes regenerating cartilage. Chondrocytes were obtained from donors with symptomatic cartilage defects, healthy and osteoarthritic donors. The effect of IL-6 on cartilage regeneration and on metabolism of the resident cartilage in the knee was studied by both inhibition of endogenous IL-6 and addition of IL-6, in a regeneration model and in osteoarthritic explants in the presence of synovial fluid, respectively. Readout parameters were DNA and glycosaminoglycan (GAG) content and release. Differences between controls and IL-6 blocked or supplemented samples were determined by univariate analysis of variance using a randomized block design. RESULTS: Synovial fluid of patients with symptomatic cartilage defects contained more IL-6 than synovial fluid of healthy donors (P = 0.001) and did not differ from osteoarthritic donors. IL-6 production of osteoarthritic chondrocytes during cartilage regeneration was higher than that of healthy and defect chondrocytes (P < 0.001). Adding IL-6 increased GAG production by healthy chondrocytes and decreased GAG release by osteoarthritic chondrocytes (P < 0.05). Inhibition of IL-6 present in osteoarthritic synovial fluid showed a trend towards decreased GAG content of the explants (P = 0.06). CONCLUSIONS: Our results support a modest anabolic role for IL-6 in cartilage matrix production. Targeting multiple cytokines, including IL-6, may be effective in improving cartilage repair in symptomatic cartilage defects and osteoarthritis.