Purpose:To compare the functional and patient-reported outcome measures after autologous chondrocyte implantation (ACI) and arthroscopic debridement (AD) in symptomatic, isolated cartilage injuries larger than 2 cm2 in patients aged 18 to 50 years.Methods:Twenty-eight patients were included and randomized to ACI (n = 15) or AD (n = 13) and followed for 2 years. The primary outcome was the change in the Knee injury and Osteoarthritis Outcome Score (KOOS) Quality of Life (QoL) subscale.Results:The mean age at inclusion was 34.1 (standard deviation [SD] 8.5) years. There were 19 (68%) male patients. The mean size of the lesion was 4.2 (SD 1.7) cm2. There was a statistically significant and clinically meaningful improvement in patient-reported outcome measures from baseline to 2 years in both groups. The improvement from baseline to final follow-up for the primary endpoint (the KOOS QoL subscale) was larger for the AD group (39.8, SD 9.4) compared with the ACI group (23.8, SD 6.7), but this difference was not statistically significant (P = .17). However, according to a mixed linear model there were statistically significantly greater scores in the AD group for several KOOS subscales at several time points, including KOOS QoL, KOOS pain, and KOOS sport and recreation at 2 years.Conclusions:This study indicates that AD followed by supervised physiotherapy is equal to or better than ACI followed by supervised physiotherapy in patients with isolated cartilage lesions of the knee larger than 2 cm2. The improvement in KOOS QoL score from baseline to 2 years was clinically meaningful for both groups (23.8 points for ACI and 39.8 points AD), and larger for the AD group by 16 points.Level of Evidence:Level I, prospective randomized controlled trial.

Focal lesions of articular cartilage give rise to pain and reduced joint function and may, if left untreated, lead to osteoarthritis. Implantation of in vitro generated, scaffold-free autologous cartilage discs may represent the best treatment option. Here we compare articular chondrocytes (ACs) and bone marrow-derived mesenchymal stromal cells (MSCs) for their ability to make scaffold-free cartilage discs. Articular chondrocytes produced more extracellular matrix per seeded cell than mesenchymal stromal cells. Quantitative proteomics analysis showed that articular chondrocyte discs contained more articular cartilage proteins, while mesenchymal stromal cell discs had more proteins associated with cartilage hypertrophy and bone formation. Sequencing analysis revealed more microRNAs associated with normal cartilage in articular chondrocyte discs, and large-scale target predictions, performed for the first time for in vitro chondrogenesis, suggested that differential expression of microRNAs in the two disc types were important mechanisms behind differential synthesis of proteins. We conclude that articular chondrocytes should be preferred over mesenchymal stromal cells for tissue engineering of articular cartilage.

Exposure to fine particulate matter (PM2.5 ) from incomplete fossil fuel combustion (coal, oil, gas and diesel) has been linked to increased morbidity and mortality due to metabolic diseases. PM2.5 exaggerate adipose inflammation and insulin resistance in mice with diet-induced obesity. Here, we elucidate the hypothesis that such systemic effects may be triggered by adhered particle components affecting adipose tissue directly. Studying adipocytes differentiated from primary human mesenchymal stem cells, we found that lipophilic organic chemicals (OC) from diesel exhaust particles induced inflammation-associated genes and increased secretion of the chemokine CXLC8/interleukin-8 as well as matrix metalloprotease 1. The oxidative stress response gene haem oxygenase-1 and tumour necrosis factor alpha were seemingly not affected, while aryl hydrocarbon receptor-regulated genes, cytochrome P450 1A1 (CYP1A1) and CYP1B1 and plasminogen activator inhibitor-2, were clearly up-regulated. Finally, expression of β-adrenergic receptor, known to regulate adipocyte homoeostasis, was down-regulated by exposure to these lipophilic OC. Our results indicate that low concentrations of OC from combustion particles have the potential to modify expression of genes in adipocytes that may be linked to metabolic disease. Further studies on mechanisms linking PM exposure and metabolic diseases are warranted.

Objective:MicroRNA-140-3p is the most prevalent form of canonical miR-140 in native chondrocytes. IsomiRs are sequence variants of microRNAs with potentially distinct functionalities. Here we present functional studies of canonical microRNA-140-3p and two of its most prevalent isomiRs, a 5' isomiR and a 3' isomiR, in an inflammation-induced model of osteoarthritis (OA). Method:Canonical miR-140-3p, the 5' isomiR and the 3' isomiR were overexpressed separately in chondrocytes from three donors and subsequently subjected to an inflammatory milieu mediated by interleukin 1 beta and tumor necrosis factor alpha. RNA sequencing was performed on the cells to investigate the altered transcriptomes, RT-qPCR was performed to validate important observations, and western blot analysis was carried out to further study key inflammatory molecules. Results:The three microRNAs downregulated many of the same genes. However, the 5' isomiR showed a much greater target spectrum compared to the other two miRNAs, and downregulated cascades of genes downstream of interferon beta, interferon gamma and interleukin 1 beta as well as genes involved in several other inflammatory and antiviral pathways. In addition the 5' isomiR downregulated practically all HLA class II and class I genes. Conclusion:Introduction of the 5' isomiR led to downregulation of genes essential for some of the most important inflammation cascades and virtual silencing of genes responsible for antigen presentation. These observations may indicate a very promising therapeutic potential for the 5' isomiR for OA and several inflammatory conditions, particularly HLA associated immune conditions including many arthritic diseases.

OBJECTIVE:Despite new strategies in tissue engineering, cartilage repair remains a major challenge. Our aim is to treat patients with focal lesions of articular cartilage with autologous hyaline cartilage implants using a scaffold-free approach. In this article, we describe experiments to optimize production of scaffold-free cartilage discs.DESIGN:Articular chondrocytes were expanded in vitro, seeded in transwell inserts and redifferentiated using established chondrogenic components. Experimental variables included testing 2 different expansion media, adding bone morphogenetic protein 2 (BMP2), insulin-like growth factor 1 (IGF1), growth/differentiation factor 5 (GDF5), or fibroblast growth factor 18 (FGF18) to the differentiation medium and allowing the disc to float freely in large wells. Cartilage discs were analyzed by weight and thickness, real-time RT-qPCR (reverse transcriptase qualitative polymerase chain reaction), fluorescence immunostaining, transmission electron microscopy, second harmonic generation imaging, and measurement of Young's modulus.RESULTS:Addition of BMP2 to the chondrogenic differentiation medium (CDM) was essential for stable disc formation, while IGF1, GDF5, and FGF18 were redundant. Allowing discs to float freely in CDM on a moving platform increased disc thickness compared with discs kept continuously in transwell inserts. Discs cultured for 6 weeks reached a thickness of almost 2 mm and Young's modulus of >200 kPa. There was abundant type II collagen. Collagen fibrils were 25 nm thick, with a tendency to be organized perpendicular to the disc surface.CONCLUSION:Scaffold-free engineering using BMP2 and providing free movement in CDM produced firm, elastic cartilage discs with abundant type II collagen. This approach may potentially be used in clinical trials.

Immunological off-target effects of RNA and RNAi therapy are a considerable challenge in research and the future of RNA-therapy. Here we investigated some of the hurdles we previously encountered when transfecting mircroRNA (miRNA) control sequences into chondrocytes in an inflammatory model simulating osteoarthritis (OA). We investigated different negative control sequences of different technologies; Pre-miR miRNA Precursor and mirVana from Thermo Fisher Scientific. We used RT-qPCR, western blot analysis and mass spectrometry to asses for the effects of the transfected control sequences.The data did not show a global immunological off-target effect, however a specific off-target effect on IL6 and IL8 was observed. IL6 and IL8 were both upregulated by the negative control from the Pre-miR miRNA Precursor technology (Pre-neg #1), and downregulated by the negative control from the mirVana technology (mirVana-neg). Moreover, the results suggested that the effect on IL6 and IL8 was dependent on both sequence and type of chemical modifications in addition to donor variation. We conclude that negative controls should be selected wisely, and suggest that scientists need to test several controls to ensure correct interpretation of data before drawing any conclusions.

Objective To investigate the heterogeneity of in vitro expanded chondrocytes used for autologous chondrocyte implantation. Methods Human articular chondrocytes were expanded in vitro for 14 days, sorted into 86 single cells using fluorescence-activated cell sorting and subjected to single-cell RNA sequencing. Principal component, Cross R2 hierarchical clustering, and differential gene expression analyses were used for data evaluation. Flow cytometry and single-cell RT-qPCR (reverse transcriptase quantitative polymerase chain reaction) was used to validate the results of the RNA sequencing data Polyclonal chondrocyte populations from the same donor were differentiated in vitro toward the osteogenic and adipogenic lineages. Results There was considerable variation in gene expression between individual cells, but we found no evidence for separate cell subpopulations based on principal component, hierarchical clustering, and differential gene expression analysis. Most of the cells expressed all the markers defining mesenchymal stem cells, and as polyclonal chondrocyte populations from the same donor were shown to differentiate into osteocytes and adipocytes in vitro, these cells formally qualify as mesenchymal stem cells. Conclusions In vitro expanded chondrocytes consist of one single population of cells with heterogeneity in gene expression between the cells. Dedifferentiated chondrocytes qualify as mesenchymal stem cells as they fulfill all the criteria suggested by the International Society for Cellular Therapy.

Objective:MicroRNAs (miRNAs) are being launched as biomarkers for various diseases, but a robust biomarker for articular cartilage pathology has yet to be discovered. Here we evaluate plasma extracellular vesicle (EV) miRNAs as possible biomarkers for osteoarthritis (OA).Method:We compared miRNA levels found in plasma EVs from patients with OA with controls without OA using next generation sequencing (NGS) technique. The patient and control pairs were matched for age, gender and body mass index.Results:23 pairs of patients and controls were included. Patients with OA differed significantly from controls in both clinical and radiological assessment of OA. We identified 177 canonical miRNAs in plasma EVs, but found no difference in miRNA levels between the two groups. Interestingly, the concentration of each miRNA in plasma EVs showed minimal difference between the participants, suggesting that the release of miRNAs in EVs from cells within the various organs is a tightly controlled process.Conclusion:This is the first study using NGS in search of a miRNA biomarker in plasma EVs in OA. The levels of each plasma EVs miRNA were surprisingly similar for all participants. No plasma EVs miRNA can be used as a biomarker for OA.

Therapeutic potential of human bone marrow stromal/stem cells (hBMSC) must be developed using well defined xenogenic-free conditions. hBMSC were isolated from healthy donors (n = 3) using different isolation and expansion methods. Donor I was isolated and expanded by either bone marrow directly seeded and cells expanded in 10% AB human serum (AB) + 5 ng/ml fibroblast growth factor-2 (FGF2) [Direct(AB + FGF(low))] or Ammonium-Chloride-Potassium Lysing Buffer was used before the cells were expanded in 10% AB + 5 ng/ml FGF-2 [ACK(AB + FGF(low))] or Lymphoprep density gradient medium was used before the cells were expanded in 10% AB + 5 ng/ml FGF2 [Lympho(AB + FGF(low))] or bone marrow directly seeded and cells expanded in 10% pooled platelet lysate plasma (PL) + heparin (2 I/U/mL) [Direct(PL)]. Groups for donors II and III were: Direct(AB + FGF(low)) or 10% AB + 10 ng/ml FGF2 [Direct(AB + FGF(high))] or Direct(PL). HBMSCs were assessed for viability, multi-potency, osteogenic, inflammatory response and replicative senescence in vitro after 1 and 3 weeks. Pre-selected culture conditions, Direct(AB + FGF(high)) or Direct(PL), were seeded on biphasic calcium phosphate granules and subcutaneously implanted in NOD/SCID mice. After 1 and 11 weeks, explants were analysed for inflammatory and osteogenic response at gene level and histologically. To identify implanted human cells, in situ hybridisation was performed. hBMSC from all conditions showed in vitro multi-lineage potency. hBMSCs expanded in PL expressed stemness markers in vitro at significantly higher levels. Generally, cells expanded in AB + FGF2 conditions expressed higher osteogenic markers after 1 week both in vitro and in vivo. After 11 weeks in vivo, Direct(AB + FGF(high)) formed mature ectopic bone, compared to immature mineralised tissues formed by Direct(PL) implants. Mouse responses showed a significant upregulation of IL-1 alpha and IL-1 beta expression in Direct(PL). After 1 week, human cells were observed in both groups and after 11 weeks in Direct(AB + FGF(high)) only. To conclude, results showed a significant effect of the isolation methods and demonstrated a relatively consistent pattern of efficacy from all donors. A tendency of hBMSC expanded in PL to retain a more stem-like phenotype elucidates their delayed differentiation and different inflammatory expressions.

One promising strategy to reconstruct osteochondral defects relies on 3D bioprinted three-zonal structures comprised of hyaline cartilage, calcified cartilage, and subchondral bone. So far, several studies have pursued the regeneration of either hyaline cartilage or bone in vitro while—despite its key role in the osteochondral region—only few of them have targeted the calcified layer. In this work, we present a 3D biomimetic hydrogel scaffold containing β-tricalcium phosphate (TCP) for engineering calcified cartilage through a co-axial needle system implemented in extrusion-based bioprinting process. After a thorough bioink optimization, we showed that 0.5% w/v TCP is the optimal concentration forming stable scaffolds with high shape fidelity and endowed with biological properties relevant for the development of calcified cartilage. In particular, we investigate the effect induced by ceramic nano-particles over the differentiation capacity of bioprinted bone marrow-derived human mesenchymal stem cells in hydrogel scaffolds cultured up to 21 d in chondrogenic media. To confirm the potential of the presented approach to generate a functional in vitro model of calcified cartilage tissue, we evaluated quantitatively gene expression of relevant chondrogenic (COL1, COL2, COL10A1, ACAN) and osteogenic (ALPL, BGLAP) gene markers by means of RT-qPCR and qualitatively by means of fluorescence immunocytochemistry.

y Osteoarthritis (OA) is the most common degenerative joint disease. One of the main pathogenic factors of OA is thought to be inflammation. Other factors associated with OA are dys-regulation of microRNAs, reduced autophagic activity, oxidative stress, and altered metabolism. microRNAs are small non-coding RNAs that are powerful regulators of gene expression. miR-140-5p is considered a cartilage-specific microRNA, is necessary for in vitro chondrogenesis, has anti-inflammatory properties, and is downregulated in osteoarthritic cartilage. Its passenger strand, miR-140-3p, is the most highly expressed microRNA in healthy cartilage and increases during in vitro chondrogenesis. miR-146a is a well-known anti-inflammatory microRNA. Several studies have illustrated its role in OA and autoimmune diseases. We show that, when human chondrocytes were transfected individually with miR-140-5p, miR-140-3p, or miR-146a prior to stimulation with interleukin-1 beta and tumor factor necrosis-alpha as an inflammatory model of OA, each of these microRNAs exhibited similar protective effects. Mass spectrometry analysis provided an insight to the altered proteome. All three microRNAs downregulated important inflammatory mediators. In addition, they affected different proteins belonging to the same biological processes, suggesting an overall inhibition of inflammation and oxidative stress, enhancement of autophagy, and restoration of other homeostatic cellular mechanisms, including metabolism.

Osteochondral (OC) tissue is a biphasic material comprised of articular cartilage integrated atop subchondral bone. Damage to this tissue is highly problematic, owing to its intrinsic inability to regenerate functional tissue in response to trauma or disease. Further, the function of the tissue is largely conferred by its compartmentalized zonal microstructure and composition. Current clinical treatments fail to regenerate new tissue that recapitulates this zonal structure. Consequently, regenerated tissue often lacks long-term stability. To address this growing problem, we propose the development of tissue engineered biomaterials that mimic the zonal cartilage organization and extracellular matrix composition through the use of a microfluidic printing head bearing a mixing unit and incorporated into an extrusion-based bioprinter. The system is devised so that multiple bioinks can be delivered either individually or at the same time and rapidly mixed to the extrusion head, and finally deposited through a coaxial nozzle. This enables the deposition of either layers or continuous gradients of chemical, mechanical and biological cues and fabrication of scaffolds with very high shape fidelity and cell viability. Using such a system we bioprinted cell-laden hydrogel constructs recapitulating the layered structure of cartilage, namely, hyaline and calcified cartilage. The construct was assembled out of two bioinks specifically formulated to mimic the extracellular matrices present in the targeted tissues and to ensure the desired biological response of human bone marrow-derived mesenchymal stem cells and human articular chondrocytes. Homogeneous and gradient constructs were thoroughly characterized in vitro with respect to long-term cell viability and expression of hyaline and hypertrophic markers by means of real-time quantitative PCR and immunocytochemical staining. After 21 days of in vitro culture, we observed production of zone-specific matrix. The PCR analysis demonstrated upregulated expression of hypertrophic markers in the homogenous equivalent of calcified cartilage but not in the gradient heterogeneous construct. The regenerative potential was assessed in vivo in a rat model. The histological analysis of surgically damaged rat trochlea revealed beneficial effect of the bioprinted scaffolds on regeneration of OC defect when compared to untreated control.

microRNAs (miRNAs) are small double stranded RNA molecules consisting of two complementary strands called the 5p and 3p arms. Following imprecise processing and/or addition of nucleotides at the ends, miRNA biogenesis can give rise to variants called isomiRs. Exosomes are small vesicles released by cells. They have attracted attention due to their potential use in biomarker development because of their content of biomolecules, including miRNAs and isomiRs. Exosomes are found in body fluids such as plasma. In this study we used next generation sequencing to investigate the distribution of 5p and 3p arms of both miRNAs and isomiRs in plasma exosomes from 46 individuals. Among the canonical miRNAs there was similar prevalence between 5p and 3p miRNAs. Most of the miRNAs had isomiRs, and in approximately half of the cases an isomiR was more abundant than the corresponding canonical miRNA. Most of the isomiRs were generated from 5p miRNAs. There were very small differences in the concentration of canonical miRNA and isomiR sequences between donors, suggesting tight control of isomiR generation and sorting into exosomes. IsomiRs are abundant in plasma exosomes and should be included in analysis when plasma exosomal miRNAs are investigated as potential biomarkers for disease development.

Background: Mesenchymal stromal cells (MSCs) can be used in several clinical applications. While MSCs are frequently cultured in fetal bovine serum for in vitro experimentation, human serum supplements are required for cells to be used in patients. Here we show how different human serum supplements and in vitro manipulations used during the cell culture impact on MSC proliferation rate and expression of inflammatory molecules. Methods: MSCs were cultured in medium supplemented with human plasma or serum combined with human platelet lysate (PL) and/or basic fibroblast growth factor (FGF2). Real time RT-PCR and western blot were used to assess expression of inflammatory cytokines. Results: Serum with addition of FGF2 gave the fastest proliferation rate. However, serum with FGF2 also increased expression of genes encoding inflammatory cytokines. The most favorable expansion condition for chondrogenic differentiation and inhibition of cartilage matrix degrading enzymes was plasma supplemented with PL and FGF2. Detachment of cells using trypsin gave considerable upregulation of inflammatory cytokine mRNAs which lasted for up to 24 h, with concomitant increase in protein levels. Even the gentle act of changing medium led to upregulation of cytokine mRNA, caused by addition of fresh serum. Discussion: Different culture conditions and simple cell manipulation influence proliferation rate and expression of inflammatory genes. Supplementing culture medium with allogeneic AB serum and FGF2 during monolayer expansion supported cell expansion better than other supplements, but also induced the highest levels of inflammatory cytokines and gave inferior results for chondrogenic differentiation. The importance of the composition of the culture medium and even gentle in vitro manipulation of the cells should be taken into account in the planning of procedures using in vitro expanded MSCs.

Exposure to traffic-derived particulate matter (PM), such as diesel exhaust particles (DEP), is a leading environmental cause of cardiovascular disease (CVD), and may contribute to endothelial dysfunction and development of atherosclerosis. It is still debated how DEP and other inhaled PM can contribute to CVD. However, organic chemicals (OC) adhered to the particle surface, are considered central to many of the biological effects. In the present study, we have explored the ability of OC from DEP to reach the endothelium and trigger pro-inflammatory reactions, a central step on the path to atherosclerosis.

Vascular disease is a major cause of death worldwide, and the growing need for replacement vessels is not fully met by autologous grafts or completely synthetic alternatives. Tissue engineering has emerged as a compelling strategy for the creation of blood vessels for reconstructive surgeries.One promising method to obtain a suitable vessel scaffold is decellularization of donor vascular tissue followed by recellularization with autologous cells. To prevent thrombosis of vascular grafts, a confluent and functional autologous endothelium is required, and researchers are still looking for the optimal cell source and recellularization procedure.Recellularization of a decellularized scaffold with only a small volume of whole blood was recently put forward as a feasible option. Here we show that, in contrast to the published results, this method fails to re-endothelialize decellularized veins. Only occasional nucleated cells were seen on the luminal surface of the scaffolds. Instead, we saw fibrin threads, platelets and scattered erythrocytes. Molecular remnants of the endothelial cells were still attached to the scaffold, which explains in part why earlier results were misinterpreted.Decellularized vascular tissues may still be the best scaffolds available for vascular tissue engineering. However, for the establishment of an adequate autologous endothelial lining, methods other than exposure to autologous whole blood need to be developed.

Aims Intracoronary infusion of autologous nucleated bone marrow cells (BMCs) enhanced the recovery of left ventricular ejection fraction (LVEF) after ST-segment elevation myocardial infarction (STEMI) in the randomised-controlled, open-label BOOST trial. We reassessed the therapeutic potential of nucleated BMCs in the randomised placebo-controlled, double-blind BOOST-2 trial conducted in 10 centres in Germany and Norway. Methods and results Using a multiple arm design, we investigated the dose-response relationship and explored whether γ-irradiation which eliminates the clonogenic potential of stem and progenitor cells has an impact on BMC efficacy. Between 9 March 2006 and 16 July 2013, 153 patients with large STEMI were randomly assigned to receive a single intracoronary infusion of placebo (control group), high-dose (hi)BMCs, low-dose (lo)BMCs, irradiated hiBMCs, or irradiated loBMCs 8.1 ± 2.6 days after percutaneous coronary intervention (PCI) in addition to guideline-recommended medical treatment. Change in LVEF from baseline (before cell infusion) to 6 months as determined by MRI was the primary endpoint. The trial is registered at Current Controlled Trials (ISRCTN17457407). Baseline LVEF was 45.0 ± 8.5% in the overall population. At 6 months, LVEF had increased by 3.3 percentage points in the control group and 4.3 percentage points in the hiBMC group. The estimated treatment effect was 1.0 percentage points (95% confidence interval, -2.6 to 4.7; P = 0.57). The treatment effect of loBMCs was 0.5 percentage points (-3.0 to 4.1; P = 0.76). Likewise, irradiated BMCs did not have significant treatment effects. BMC transfer was safe and not associated with adverse clinical events. Conclusion The BOOST-2 trial does not support the use of nucleated BMCs in patients with STEMI and moderately reduced LVEF treated according to current standards of early PCI and drug therapy.

The two mesenchymal stem cell (MSC) populations that have gained most attention in relation to bone tissue engineering are adipose tissue (AT) MSCs and bone marrow (BM) MSCs. The purpose of this study was to investigate the ability of human BM-MSCs and AT-MSCs to survive, proliferate and deposit collagen type 1 when cultured on polycaprolactone nanofiber scaffolds and to ascertain the effect of medium composition on collagen type 1 formation and expression of osteogenic genes. The cells were seeded on polycaprolactone nanofiber scaffolds and cultured in three different types of media that differed by the presence of ascorbic acid,beta-glycerophosphate and dexamethasone, that are typical components used for osteogenic differentiation of MSCs in vitro. In summary, AT-MSCs were proliferating significantly faster than BM-MSCs. AT-MSCs also showed better ability to deposit collagen type 1 and had a higher expression of early osteogenic markers, whereas BM-MSCs had higher expression of late osteogenic markers. This suggests that MSCs from diverse sources have different attributes and with respect to osteogenic differentiation, AT-MSCs are more immature compared to BM-MSCs. Collagen formation was depending on medium composition and the organization of collagen type I appeared to be influenced by the presence of dexamethasone. (C) 2016 Elsevier Ltd. All rights reserved.

Purpose: Cell therapy using expanded articular chondrocytes (ACs) may be used for cartilage regeneration in osteoarthritis. The purpose of this study was to use single cell RNA sequencing to characterize expanded ACs. Methods: ACs were isolated and cultured for 14 days before analysis. Individual ACs were sorted using FACS. cDNA libraries was prepared according to the SmartSeq2 protocol. Data were aligned using Tophat and transcripts were quantified using Cufflinks. R and Graphpad were used for data analysis and graphics. Results: Hierarchical clustering of all expressed genes showed that cultured ACs are a heterogeneous group of cells. Many genes were stably expressed, but genes such as SOX9, ACAN, COL1A1, COL1A2, IL1B, TNFA, IL1R1, MMP3, MMP13, ADAMTS5 and many other genes associated with osteoarthritis (OA) and chondrogenesis were differently expressed between most cells. Some cells had high expression of these genes, while other had low or no expression of these genes. However, it was not possible to identify any groups of cells with similar expression pattern of these genes. The OA-related gene, CHI3L1, was found to be one of the most highly expressed genes in many of the cells, but there was also a group of cells that did not express CHI3L1. Comparing these two groups of cells we found 20 genes to be differently expressed. Other results from ongoing analysis will also be presented. Conclusions: Cultured ACs consist of a heterogeneous group of cells. Many genes were stably expressed, but a significant number of genes associated with cartilage biology and osteoarthritis were differently expressed between most cells. For cell therapy purposes it might be possible to use single-cell RNAseq to identify different cell populations with varying potential to inhibit or respond to the inflammatory environment in the joint.