BackgroundNotochordal cell conditioned medium (NCCM) derived from non-chondrodystrophic dogs has pro-anabolic and anti-catabolic effects upon nucleus pulposus (NP) cells. Here, for the first time, we assessed the ability of NCCM to influence the production of extracellular matrix and inflammatory proteins by healthy and osteoarthritic human chondrocytes within engineered cartilage tissues. We hypothesized that, similar to its action on NP cells, NCCM exerts metabolic and anti-catabolic effects on human articular chondrocytes and has the potential to significantly counteract inflammatory mediators.MethodsChondrocytes from nine non-osteoarthritic patients and from six osteoarthritic (OA) donors at the time of total knee arthroplasty were chondro-differentiated in pellets for 2 weeks. Non-OA pellets were exposed for 72 hours to IL-1β/TNF-α and then cultured up to 14 days in 2 % FBS-supplemented NCCM or 2 % FBS-supplemented medium (control (ctr)). OA pellets were cultured in NCCM or ctr medium without pro-inflammatory treatment. Tissues after each culture phase were analyzed biochemically (GAG/DNA), (immuno-) histologically (collagen I, II and GAG) and by Western blotting. Supernatants were analyzed by ELISA.ResultsResponse to NCCM was age and disease dependent with healthy chondrocyte pellets (from donors >55 years of age) recovering their glycosaminoglycan (GAG) contents to baseline levels only with NCCM. OA pellets treated with NCCM significantly increased GAG content (1.8-fold) and levels of hyaluronic acid link protein (HAPLN), fibromodulin and SOX-9. The catabolic proteins (matrix metalloproteinase (MMP)-3 and MMP-13) and pro-inflammatory enzyme levels (cyclooxygenase-2 (COX-2)) were markedly reduced and there was significantly reduced secretion of pro-inflammatory chemokines (IL-6 and IL-8).ConclusionsNCCM restores cartilage matrix production of end-stage human OA chondrocytes towards a healthy phenotype and suppresses the production of inflammatory mediators. Harnessing the necessary and sufficient factors within NCCM that confers chondroprotection and regenerative effects could lead to a minimally invasive agent for treatment of degenerative and inflammatory joint diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13075-016-1026-x) contains supplementary material, which is available to authorized users.

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Objective Cellular and molecular events occurring in cartilage regions close to injury are poorly investigated, but can possibly compromise the outcome of cell-based cartilage repair. In this study, key functional properties were assessed for cartilage biopsies collected from the central part of traumatic joint lesions ( central) and from regions surrounding the defect ( peripheral). These properties were then correlated with the quality of the initial cartilage biopsy and the inflammatory state of the joint. Design Cartilage samples were collected from knee joints of 42 patients with traumatic knee injuries and analyzed for cell phenotype (by reverse transcriptas-polymerase chain reaction), histological quality, cellularity, cell viability, proliferation capacity, and post-expansion chondrogenic capacity of chondrocytes (in pellet culture). Synovium was also harvested and analyzed for the expression of inflammatory cytokines. Results Cartilage quality and post-expansion chondrogenic capacity were higher in peripheral versus central samples. Differences between these 2 parameters were more pronounced in joints with high inflammatory features characterized by >100-fold difference in the mRNA levels of IL6 and IL8 in the corresponding synovium. Peripheral chondrocytes isolated from good- versus bad-quality biopsies expressed higher levels of collagen II/I and aggrecan/versican and lower levels of MMP13 and ADAMTS5. They also exhibited reduced proliferation and enhanced cartilage-forming capacity. Conclusions Chondrocytes at the periphery of traumatic lesions better maintain properties of healthy cartilage compared to those isolated from the center, even when derived from bad-quality tissues harvested from highly inflamed joints. Future studies are necessary to investigate the change of functional properties of peripheral chondrocytes over time.

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Figure 1. Histology (Safranin-O staining) of Day 21 harvest constructs cultured in different glucose concentrations. Scale bar: 100mm. Top right corner shows the overall glucose consumption in mg. Toronto Western Res. Inst., Toronto, ON, CanadaPurpose: It has recently been shown that notochordal cell conditioned medium (NCCM), derived from non-chondrodystrophic canines protect animal and human nucleus pulposus cells from degradation and induced apoptosis. Here we assessed whether the post-inflammatory recovery of human articular chondrocytes within an engineered cartilage tissue is enhanced by NCCM. Methods: Chondrocytes harvested from cartilage samples of 9 nonosteoarthritic (healthy) patients (group I: <55 years [N ¼ 4], group II: >55 years [N ¼ 5]) and from 7 osteoarthritic donors (mean age 75.6y) that underwent total knee replacement were expanded for two passages and then chondro-differentiated in pellets for 3 weeks. Resulting tissues were exposed for 72 hours to IL-1b/TNF-a and then cultured up to 14 days in medium containing 2%NCCM or 2%FBS (ctr). Pellets generated with OA chondrocytes without inflammatory pretreatment were cultured in NCCM-or ctr-medium. Tissues after each culture phase were analyzed biochemically (glycosaminoglycans -GAG -and DNA), (immune/histologically (collagen II and GAG) and by Western Bolts. Results: Inflammatory treatment resulted in a significant loss of GAG from the pellets generated by healthy chondrocytes. Response to NCCM was age and disease-dependent: GAG contents (i) of group I cell-pellets was not significantly influenced by NCCM, (ii) of group II cell-pellets recovered to baseline levels only in NCCM medium, (iii) of OA chondrocytes-pellets drastically increased in response to NCCM to levels 2fold higher than baseline. (Immuno/histological results confirm these trends. NCCM treatment of OA chrondrocytes increased the expression of anabolic proteins (HAPLN, Fibromodulin), the chrondrogenic transcription factor SOX-9, significantly suppressed thelevels of the catabolic proteins (MMP-3 and -13) and the pro-inflammatory mediator Cox2. Conclusions: NCCM enhances cartilage matrix production by IL-1b/ TNFa-treated chondrocytes from healthy patients >55 years and by end stage OA chondrocytes. Research activities are ongoing to investigate the mechanisms whereby NCCM confers such chondroprotection. In vivo studies are necessary to assess the feasibility of using NCCM-based therapeutics for the treatment of inflammatory/degenerative joint diseases. TISSUE ENGINEERING IN AN IN VITRO MODEL OF HUMAN CARTILAGE REPAIRPurpose: The purpose was to study the cartilage repair of in vitro lesion models using human bone marrow mesenchymal stromal cells (BMSCs) and different types of collagen scaffolds. Methods: 3 mm lesions were made in human cartilage biopsies. 2x10 5 BMSCs were seeded on type I collagen (C1), C1 and heparan sulfate (C1HS) and type I and II collagen and HS (C1C2HS) biomaterials. They were introduced inside the lesion, and cultured during 60 days in chondrogenic medium with 1...

Background. It is generally assumed that traumatic cartilage lesions affect the whole joint homeostasis. However, it remains unknown to which extent the properties of chondrocytes within lesions are affected compared to cells from adjacent locations. To unravel cellular and molecular events occurring in cartilage regions close to injury sites, we collected cartilage biopsies from the central part of the lesions ( central ) and from regions closely surrounding the lesion ( peripheral , 2-5mm distance from defect) of traumatic joints and assessed their key functional properties. Additionally, we investigated the correlation of these properties with the inflammatory features of the joint and the quality of the initial cartilage biopsy. Methods. Cartilage samples were collected from the knee joints of 42 patients (male:female = 7:3, age range: 18-60y) with traumatic knee injuries and analysed for cell phenotype (by RT-PCR), histological quality (using a grading score based on glycosaminoglycan staining), cellularity (cell numbers/gram tissue, isolated after enzymatic digestion), cell viability, proliferation capacity (cell doublings/day) and post-expansion chondrogenic capacity of chondrocytes (Bern score of chondrogenically cultured cells in pellets). In addition, synovial tissues were harvested and analysed for the expression of inflammatory cytokine genes. Results. Cartilage quality and post-expansion chondrogenic capacity were higher in peripheral vs central samples. Differences between these two parameters were more pronounced in joints with high (vs low) inflammatory features, as characterised by >100-fold difference in the mRNA levels of IL-6 and IL-8 in the corresponding synovial tissues. Peripheral chondrocytes isolated from initially good compared to bad quality biopsies expressed higher levels of chondrogenic markers (type II/I collagen and aggrecan/versican ratios) and lower levels of cartilage degrading markers MMP13 and ADAMTS5. They also exhibited reduced proliferation and enhanced cartilage-forming capacity. Conclusions. Chondrocytes at the periphery of traumatic lesions better maintain properties typical of healthy cartilage as compared to those isolated from central parts even when derived from bad quality tissues harvested from highly inflamed joints. Future studies will be necessary to investigate the change of functional properties of peripheral chondrocytes over time and, consequently, to identify a “point of no return” (since onset of symptoms) for a possible use of such chondrocytes in cartilage repair strategies.