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Background
Curcumin has anti-inflammatory effects and qualifies as a potential candidate for the treatment of osteoarthritis (OA). However, curcumin has limited bioavailability. Extracellular vesicles (EVs) are released by multiple cell types and act as molecule carrier during intercellular communication. We assume that EVs can maintain bioavailability and stability of curcumin after encapsulation. Here, we evaluated modulatory effects of curcumin-primed human (h)BMSC-derived EVs (Cur-EVs) on IL-1β stimulated human osteoarthritic chondrocytes (OA-CH).
Methods
CellTiter-Blue Viability- (CTB), Caspase 3/7-, and live/dead assays were used to determine range of cytotoxic curcumin concentrations for hBMSC and OA-CH. Cur-EVs and control EVs were harvested from cell culture supernatants of hBMSC by ultracentrifugation. Western blotting (WB), transmission electron microscopy, and nanoparticle tracking analysis were performed to characterize the EVs. The intracellular incorporation of EVs derived from PHK26 labeled and curcumin-primed or control hBMSC was tested by adding the labeled EVs to OA-CH cultures. OA-CH were pre-stimulated with IL-1β, followed by Cur-EV and control EV treatment for 24 h and subsequent analysis of viability, apoptosis, and migration (scratch assay). Relative expression of selected anabolic and catabolic genes was assessed with qRT-PCR. Furthermore, WB was performed to evaluate phosphorylation of Erk1/2, PI3K/Akt, and p38MAPK in OA-CH. The effect of hsa-miR-126-3p expression on IL-1β-induced OA-CH was determined using CTB-, Caspase 3/7-, live/dead assays, and WB.
Results
Cur-EVs promoted viability and reduced apoptosis of IL-1β-stimulated OA-CH and attenuated IL-1β-induced inhibition of migration. Furthermore, Cur-EVs increased gene expression of BCL2, ACAN, SOX9, and COL2A1 and decreased gene expression of IL1B, IL6, MMP13, and COL10A1 in IL-1β-stimulated OA-CH. In addition, phosphorylation of Erk1/2, PI3K/Akt, and p38 MAPK, induced by IL-1β, is prevented by Cur-EVs. Cur-EVs increased IL-1β-reduced expression of hsa-miR-126-3p and hsa-miR-126-3p mimic reversed the effects of IL-1β.
Conclusion
Cur-EVs alleviated IL-1β-induced catabolic effects on OA-CH by promoting viability and migration, reducing apoptosis and phosphorylation of Erk1/2, PI3K/Akt, and p38 MAPK thereby modulating pro-inflammatory signaling pathways. Treatment of OA-CH with Cur-EVs is followed by upregulation of expression of hsa-miR-126-3p which is involved in modulation of anabolic response of OA-CH. EVs may be considered as promising drug delivery vehicles of curcumin helping to alleviate OA.
Objective
Cartilage defect treatment strategies are dependent on the lesion size and severity. Osteochondral explant models are a platform to test cartilage repair strategies ex vivo. Current models lack in mimicking the variety of clinically relevant defect scenarios. In this controlled laboratory study, an automated device (artificial tissue cutter, ARTcut®) was implemented to reproducibly create cartilage defects with controlled depth. In a pilot study, the effect of cartilage defect depth and oxygen tension on cartilage repair was investigated.
Design
Osteochondral explants were isolated from porcine condyles. 4 mm chondral and full thickness defects were treated with either porcine chondrocytes (CHON) or co-culture of 20% CHON and 80% MSCs (MIX) embedded in collagen hydrogel. Explants were cultured with tissue specific media (without TGF-β) under normoxia (20% O\(_2\)) and physiological hypoxia (2% O\(_2\)). After 28 days, immune-histological stainings (collagen II and X, aggrecan) were scored (modified Bern score, 3 independent scorer) to quantitatively compare treatment outcome.
Results
ARTcut® represents a software-controlled device for creation of uniform cartilage defects. Comparing the scoring results of the MIX and the CHON treatment, a positive relation between oxygen tension and defect depth was observed. Low oxygen tension stimulated cartilaginous matrix deposition in MIX group in chondral defects and CHON treatment in full thickness defects.
Conclusion
ARTcut® has proved a powerful tool to create cartilage defects and thus opens a wide range of novel applications of the osteochondral model, including the relation between oxygen tension and defect depth on cartilage repair.
Background: Human bone marrow-derived mesenchymal stromal cells (hBMSCs) provide a promising therapeutic approach in the cell-based therapy of osteoarthritis (OA). However, several disadvantages evolved recently, including immune responses of the host and regulatory hurdles, making it necessary to search for alternative treatment options. Extracellular vesicles (EVs) are released by multiple cell types and tissues into the extracellular microenvironment, acting as message carriers during intercellular communication. Here, we investigate putative protective effects of hBMSC-derived EVs as a cell-free approach, on IL-1β-stimulated chondrocytes obtained from OA-patients.
Methods: EVs were harvested from the cell culture supernatant of hBMSCs by a sequential ultracentrifugation process. Western blot, scanning electron microscopy (SEM), and nanoparticle tracking analysis (NTA) were performed to characterize the purified particles as EVs. Intracellular incorporation of EVs, derived from PHK26-labeled hBMSCs, was tested by adding the labeled EVs to human OA chondrocytes (OA-CH), followed by fluorescence microscopy. Chondrocytes were pre-stimulated with IL-1β for 24 h, followed by EVs treatment for 24 h. Subsequently, proliferation, apoptosis, and migration (wound healing) were analyzed via BrdU assay, caspase 3/7 assay, and scratch assay, respectively. With qRT-PCR, the relative expression level of anabolic and catabolic genes was determined. Furthermore, immunofluorescence microscopy and western blot were performed to evaluate the protein expression and phosphorylation levels of Erk1/2, PI3K/Akt, p38, TAK1, and NF-κB as components of pro-inflammatory signaling pathways in OA-CH.
Results: EVs from hBMSCs (hBMSC-EVs) promote proliferation and reduce apoptosis of OA-CH and IL-1β-stimulated OA-CH. Moreover, hBMSC-EVs attenuate IL-1β-induced reduction of chondrocyte migration. Furthermore, hBMSC-EVs increase gene expression of PRG4, BCL2, and ACAN (aggrecan) and decrease gene expression of MMP13, ALPL, and IL1ß in OA-CH. Notably, COL2A1, SOX9, BCL2, ACAN, and COMP gene expression levels were significantly increased in IL-1β+ EV groups compared with those IL-1β groups without EVs, whereas the gene expression levels of COLX, IL1B, MMP13, and ALPL were significantly decreased in IL-1β+ EV groups compared to IL-1β groups without EVs. In addition, the phosphorylation status of Erk1/2, PI3K/Akt, p38, TAK1, and NF-κB signaling molecules, induced by IL-1β, is prevented by hBMSC- EVs.
Conclusion: EVs derived from hBMSCs alleviated IL-1β-induced catabolic effects on OA-CH via promoting proliferation and migration and reducing apoptosis, probably via downregulation of IL-1ß-activated pro-inflammatory Erk1/2, PI3K/Akt, p38, TAK1, and NF-κB signaling pathways. EVs released from BMSCs may be considered as promising cell-free intervention strategy in cartilage regenerative medicine, avoiding several adverse effects of cell-based regenerative approaches.
Objective
As native cartilage consists of different phenotypical zones, this study aims to fabricate different types of neocartilage constructs from collagen hydrogels and human mesenchymal stromal cells (MSCs) genetically modified to express different chondrogenic factors.
Design
Human MSCs derived from bone-marrow of osteoarthritis (OA) hips were genetically modified using adenoviral vectors encoding sex-determining region Y-type high-mobility-group-box (SOX)9,transforming growth factor beta (TGFB) 1or bone morphogenetic protein (BMP) 2cDNA, placed in type I collagen hydrogels and maintained in serum-free chondrogenic media for three weeks. Control constructs contained unmodified MSCs or MSCs expressing GFP. The respective constructs were analyzed histologically, immunohistochemically, biochemically, and by qRT-PCR for chondrogenesis and hypertrophy.
Results
Chondrogenesis in MSCs was consistently and strongly induced in collagen I hydrogels by the transgenesSOX9,TGFB1andBMP2as evidenced by positive staining for proteoglycans, chondroitin-4-sulfate (CS4) and collagen (COL) type II, increased levels of glycosaminoglycan (GAG) synthesis, and expression of mRNAs associated with chondrogenesis. The control groups were entirely non-chondrogenic. The levels of hypertrophy, as judged by expression of alkaline phosphatase (ALP) and COL X on both the protein and mRNA levels revealed different stages of hypertrophy within the chondrogenic groups (BMP2>TGFB1>SOX9).
Conclusions
Different types of neocartilage with varying levels of hypertrophy could be generated from human MSCs in collagen hydrogels by transfer of genes encoding the chondrogenic factorsSOX9,TGFB1andBMP2. This technology may be harnessed for regeneration of specific zones of native cartilage upon damage.
The topical application of tranexamic acid (TXA) helps to prevent post-operative blood loss in total joint replacements. Despite these findings, the effects on articular and periarticular tissues remain unclear. Therefore, this in vitro study examined the effects of varying exposure times and concentrations of TXA on proliferation rates, gene expression and differentiation capacity of chondrocytes and human mesenchymal stromal cells (hMSCs), which underwent osteogenic differentiation. Chondrocytes and hMSCs were isolated and multiplied in monolayer cell cultures. Osteogenic differentiation of hMSCs was induced for 21 days using a differentiation medium containing specific growth factors. Cell proliferation was analyzed using ATP assays. Effects of TXA on cell morphology were examined via light microscopy and histological staining, while expression levels of tissue-specific genes were measured using semiquantitative RT-PCR. After treatment with 50 mg/mL of TXA, a decrease in cell proliferation rates was observed. Furthermore, treatment with concentrations of 20 mg/mL of TXA for at least 48 h led to a visible detachment of chondrocytes. TXA treatment with 50 mg/mL for at least 24 h led to a decrease in the expression of specific marker genes in chondrocytes and osteogenically differentiated hMSCs. No significant effects were observed for concentrations beyond 20 mg/mL of TXA combined with exposure times of less than 24 h. This might therefore represent a safe limit for topical application in vivo. Further research regarding in vivo conditions and effects on hMSC functionality are necessary to fully determine the effects of TXA on articular and periarticular tissues.