16.1.1 - Continuous Low-Intensity Ultrasound Preserves Chondrogenesis of MSCs and Mitochondrial Potential While Inhibiting NFκB Activation
Abstract
Purpose
Dysregulation of the anabolic processes in a proinflammatory joint environment coupled with impeded chondrogenic differentiation of mesenchymal stromal cells (MSCs) led to inferior cartilage repair outcomes. The preponderance of proinflammatory cytokines activated nuclear factor kappa B (NFκB) impedes the chondrogenesis of MSCs. Strategies that minimize the deleterious effects of activated NFκB while promoting MSC chondrogenesis are of interest. The present study establishes the ability of continuous low-intensity ultrasound (cLIUS) to preserve MSC chondrogenesis impacted by a proinflammatory environment.
Methods and Materials
MSCs were seeded in alginate: collagen hydrogels and cultured for 21-days in ultrasound (US)-assisted bioreactor 14 kPa (4-applications/day) in the presence of IL1β and evaluated by qRT-PCR and immunofluorescence (IF). The differential expression of markers associated with the NFκB pathway under cLIUS was evaluated upon a single exposure of cLIUS and assayed by qRT-PCR, IF, WB, and tetramethylrhodamine methyl ester (TMRM) assay was used to assess the mitochondrial potential under IL1β and cLIUS treatment. Study groups included appropriate non-cytokine treated and non-cLIUS treated controls (Fig.1B).
Results
Chondroinductive potential of cLIUS was preserved as noted by the increased expression of SOX9 and deposition of collagen II (Fig. 2A-I & III). cLIUS extended its chondroprotective effects by stabilizing the NFκB complex in the cytoplasm via engaging the IκBα feedback mechanism, thus preventing its nuclear translocation (Fig. 2A-II). cLIUS acted as a mitochondrial protective agent by restoring the mitochondrial potential and the mitochondrial mRNA expression in a proinflammatory environment (Fig. 2C-VI). Altogether, our results demonstrated the potential of cLIUS for cartilage repair and regeneration under proinflammatory conditions (Fig.1A).
Conclusion
This study establishes the potential of cLIUS to improve and enhance outcomes of in vivo cartilage repair therapies. Translation of promising in vitro findings with cLIUS requires an understanding of the cLIUS propagation in the joint space along with optimal transducer settings.
16.1.3 - Enhanced Cartilage Tissue Yield from Induced Pluripotent Stem Cells by Initial WNT/beta-Catenin Activation
Abstract
Purpose
Induced pluripotent stem cells (iPSCs) are promising for cartilage tissue engineering as they are unlimited in supply. We have established chondrocyte differentiation of iPSCs, but high cell loss currently compromises tissue yield. During embryo development, cell survival and proliferation are regulated by several pathways including WNT/β-catenin. WNT/β-catenin activation initiates mesoderm commitment and hence might be important for chondrocyte differentiation. We here asked, whether short stimulation of WNT/β-catenin signalling at initiation of differentiation would improve cell survival and tissue yield during subsequent iPSC chondrogenesis.
Methods and Materials
Two human iPSC-lines (IMR90,CB) were differentiated towards mesoderm (14days) using bFGF/serum on matrigel/gelatin followed by 42days of chondrogenic 3D-pellet culture with 10ng/ml TGF-β1. One group was stimulated with a WNT/β-catenin pulse (5µM CHIR99021) for 24h at the start of mesoderm differentiation (d0). Effects were analyzed by Western blot, qPCR, cDNA-microarray, histology, aggregation assay and DNA quantification.
Results
Initial CHIR treatment significantly increased the number of PDGFRα-positive cells at d7 compared to controls. Accordingly, mRNA-levels of mesoderm markers were significantly elevated at d14 and common ectodermal markers reduced, demonstrating an enhanced mesoderm commitment of CHIR-treated iPSCs. While controls quickly formed multiple free-floating small aggregates and only few cells attached to the plastic surface, CHIR-treated cells aggregated into a plastic-adherent cell sheet, which subsequently condensed into one large pellet over 2-14days. In line, CHIR-treated cells expressed ECM and adhesion-related genes at higher levels than controls at d14. As a consequence of improved pellet formation, DNA amounts in the CHIR-group remained significantly higher than in controls during chondrogenic culture, resulting in significantly larger pellets.
Conclusion
Initial WNT activation improved mesoderm commitment; and we demonstrate for the first time that, acting via stimulated cell proliferation, ECM-expression and cell-aggregation, WNT-pulsing is key to rescue low tissue yield during chondrogenesis. This advancement can be highly beneficial for clinical cartilage regeneration, disease modelling and drug screening.
16.1.4 - Differential PI3K/AKT Activity in Endochondral vs. Chondral Development In Vitro
Abstract
Purpose
A main limitation of cartilage engineering with multipotent stromal cells (MSC) is their inherent endochondral development. Current treatments like WNT inhibition can only reduce chondrocyte hypertrophy, but not induce an articular chondrocyte (AC)-like phenotype in MSC. PI3K/AKT signaling is essential for cartilage neogenesis and chondrocyte hypertrophy in the growth plate. Yet, its role for hypertrophic differentiation of MSC in vitro remains unclear. Aim was to uncover if different PI3K/AKT activity in MSC-derived hypertrophic chondrocytes vs. non-hypertrophic AC might indicate PI3K/AKT inhibition as promising to reduce hypertrophy during MSC chondrogenesis.
Methods and Materials
Human expanded MSC and AC were subjected to chondrogenic 3D culture for 42 days and AKT activity was detected via phospho-AKT Western blot. PI3K/AKT signaling was inhibited by LY294002 (0.25µM - 25µM) from day 21 on. Differentiation was assessed via histology, qPCR, proteoglycan quantification and alkaline phosphatase activity. To evaluate AKT activity under anti-hypertrophic stimulation, MSC pellets were co-treated with the WNT inhibitor IWP-2 (5µM, d14-d42).
Results
Unlike AC, MSC upregulated AKT activity during chondrogenesis in parallel to hypertrophy (figure 1), reaching significantly higher levels than AC from d21 on. LY dose-dependently reduced hypertrophic (IHH, PTHR1, COL10A1 mRNA) along with chondrogenic markers (COL2A1, ACAN) and proteoglycan deposition. In line, LY reduced TGFβ-induced pSMAD2/3 and SOX9 protein. Importantly, no LY dose was capable to selectively target hypertrophic but not chondrocyte markers. This indicated that PI3K/AKT activity played primarily a pro-chondrogenic role and may not regulate hypertrophy. In line with this observation, pAKT protein levels became also upregulated in MSC under anti-hypertrophic treatment with IWP-2.
Conclusion
Although the increasing AKT activity in MSC but not in AC suggested its relevance for endochondral differentiation, we here demonstrated that MSC chondrogenesis strongly depends on PI3K/AKT activation. This indicates that induction and maintenance of PI3K/AKT activity will be crucial for future therapeutic success of MSC-based cartilage regeneration.
16.1.5 - Mitochondrial Transport Between Chondrocytes and Mesenchymal Stromal Cells
Abstract
Purpose
IMPACT is a one-stage cartilage transplantation for large cartilage defects, in which 10% autologous chondrons are combined with 90% donor mesenchymal stromal cells (MSCs). One year after transplantation, the regenerated cartilage does not contain donor autosomal DNA. This indicates that the MSCs do not differentiate, but act as signalling cells. The aim of this study is to investigate whether transport of mitochondria exists between chondrocytes and MSC and to investigate if the transfer of mitochondria to chondrocytes contributes to the mechanism of action of MSCs.
Methods and Materials
Chondrocytes were isolated from cartilage defect rims during autologous chondrocyte implantation. MSCs were isolated from surplus bone marrow donated for transplantation. Mitochondria were stained with mitotracker and cell trace was used to distinguish between cell types. After 4 to 24 hours of co-culture, the uptake of fluorescent mitochondria was measured using flow cytometry. Transport was visualized using fluorescence microscopy. Mitochondria were isolated from MSCs and transferred to chondrocytes using MitoCeption. Pellets of 100.000 chondrocytes, chondrocytes with transferred MSC mitochondria, and co-cultures (chondrocyte:MSC; 10:90) were cultured for 28 days. DNA content was measured using qubit fluorometric quantification and proteoglycan content using a Dimethylmethylene Blue Assay.
Results
Mitochondrial transport takes place bidirectional in co-cultures of chondrocytes and MSCs and chondrocytes monocultures and reaches a maximum at 16 hours (Figure 1). Mitochondria are transported via tunnelling nanotubes and direct cell-contact. DNA content and proteoglycan deposition are higher in chondrocyte pellets with transferred MSC mitochondria compared to chondrocyte pellets (Figure 2).
Conclusion
We demonstrate and quantify the transport of mitochondria between MSCs and chondrocytes for the first time. We show the positive effect of uptake of mitochondria by chondrocytes on DNA content and proteoglycan deposition. Further insight into the causes and results of mitochondrial transfer between MSCs and chondrocytes could improve the selection of MSCs and increase the effectivity of regenerative therapies.
16.1.6 - Cartilage Cells, Adipose- & Bone Marrow-Derived Stem Cells Release miRNA That Are Involved in Cartilage Damage-related Pathways
Abstract
Purpose
The treatment of early degenerative processes affecting cartilage could prevent further aggravation and the onset of osteoarthritis (OA). The aim of the present study is to analyze the predicted potential of the micro RNAs (miRNAs) produced by cartilage cells (CCs), adipose- (ASCs) and bone marrow-derived (BMSCs) stem cells as a therapy to treat joint degeneration.
Methods and Materials
Articular cartilage, bone marrow and subcutaneous adipose tissue were collected from 3 patients who underwent total hip replacement. Cells were isolated, expanded and the supernatants were differentially centrifuged to obtain extracellular vesicles (EVs). Expression of EV-embedded miRNA were evaluated with the QuantStudio™ 12 K Flex OpenArray® platform. Mientournet and Ingenuity Pathway Analysis (IPA) were used for validated target prediction analysis and to identify miRNAs involved in OA and inflammation.
Results
Among the 428 analyzed miRNAs, 325 were expressed by all the cell populations, 26 shared by CCs and ASCs, 21 shared by CCs and BMSCs and 17 shared by ASCs and BMSCs. The miRNA selectively expressed only in one cell population were 16 by CCs, 12 by ASCs and 11 by BMSCs. Mienturnet revealed no results for miRNA selectively expressed by ASCs, whereas for CCs miR-17-3p, miR-25-5p, miR-200c-3p and miR-449a showed the highest number of interactions and putatively modulate cell cycle, senescence, apoptosis, Wnt, TGFβ, VEGF, Notch, Hippo, TNFα and IL-1β signaling. For BMSCs the highest number of interaction was showed by miR-141-3p, miR-143-5p, miR-363-3p, miR-205-5p and miR-483-3p involved in apoptosis, TGFβ, IGF-1, RUNX2 and endochondral ossification pathways. IPA analysis showed that many differently expressed miRNAs regulate macrophages and T cells activity, the expression of inflammatory mediators, cartilage homeostasis and cell proliferation.
Conclusion
Despite the need to be validated by functional tests, the predicted pathways identified in this study confirm and support the rationale behind the use of cell-based therapy for the treatment of degenerative joint conditions.
16.1.8 - Preclinical Safety of TG-C, a Cell-Based Gene Therapy Over-Expressing TGF-β1
Abstract
Purpose
TG‑C is a cell-mediated gene therapy for the treatment of knee osteoarthritis (OA). In this preclinical study we employed rats and goats to demonstrate the pre-clinical safety of TG-C.
Methods and Materials
All animal studies were conducted with ethical committee permission and approval and in line with the World Medical Association (WMA) Declaration of Helsinki and NC3Rs ARRIVE guidelines to ensure the welfare of animals used for research. 2x106 / ml (20 μl) TG-C cells (48Gy-irradiated) were injected into the knee joints of 70 (35 male and 35 female) Sprague-Dawley (SD) rats. Quantitative PCR (qPCR) was used to detect human cells and RvTGFβ1 vector-transduced cells. Safety studies in goats examined a total of 71 animals. Goats received high and low doses of TG-C; 10 animals received a high single-dose of 5x107 cells/ml (500 μl); 10 animals received a low dose single-dose of 1x107 cells/ml (500 μl); 10 animals received a high multiple-dose 5x107 cells/ml (500 μl) and were followed up for 6 and 12 months.
Results
Real-time qPCR was performed to determine if human TG-C cells can be detected the knee joints and other organs. Treatment with TG-C had no effect on mortality, body weight, or gross or microscopic pathology of the injection site in rats. Safety studies in goats revealed no adverse findings, either systemically or locally at the 6 or 12-month timepoints. TG-C was well tolerated and did not elicit any immune responses in the goats either in the low, high, single or multiple doses.
Conclusion
The radiation inactivation of cell proliferation in the GP2-293 component of TG C underlines the suitability for intra-articular injection into the knee joint. The introduction of a gamma or x-ray radiation inactivation step in the preclinical development pipeline for TG-C highlights the utility of this approach for developing other cell-based approaches for the intra-articular treatment of OA.
16.1.9 - Novel Small Molecule Modulator of the gp130 Receptor shows Dose-Dependent Therapeutic Efficacy in a Canine Model of Osteoarthritis
Abstract
Purpose
The pathogenesis of osteoarthritis (OA) often begins from an injury to articular cartilage, which establishes chronic, low-grade inflammation principally mediated by IL-6 family cytokines that signal through the obligate receptor gp130. However, we and others have shown that gp130 can also regulate anabolic responses in chondrocytes and immune system. Here, we unveil an advanced late pre-clinical regeneration-promoting candidate CX-011 that modulates, rather than completely inhibits, gp130 signaling.
Methods and Materials
Twenty-four purpose bred Foxhound dogs (12 females, 12 males), 10 months of age, (Marshall BioResources) were chosen for this study. All dogs underwent CT imaging of both knee joints then general anesthesia and arthroscopic surgery to induce OA by medial meniscal release (MMR). Three groups received CX-011 by intra-articular injection into the operated limb and the fourth control group (n=6) received an equal volume of vehicle. Injections were done four weeks post-operatively and again at 6 weeks after initial injection. The three experimental groups given the test article received doses of 10 μg, 1 μg or 0.1 μg in a 0.5 mL volume.
Results
Dose dependent effects of CX-11 were documented. In the highest dose of CX-011 (10 mg) group, there was highly significant chondroprotection (p = 0.012). Synovial inflammation was significantly limited by the two highest dose rates (p = 0.016) of the test article. CT imaging showed a dramatic, statistically significant reduction (p < 0.001) in shape alteration in dogs treated with the two highest dose levels of CX-011 compared to controls. Finally, the two highest doses had significantly reduced lameness, indicating lower pain levels with increasing test article concentration (p < 0.001).
Conclusion
The current study has nominated the gp130 modulator CX-011 as an excellent candidate for clinical progression as a disease modifying agent for OA with concurrent reduction in pain.