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INTRODUCTION: Platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs) serve as two of the most popular potential therapies to enhance cartilage regeneration. Platelets contain alpha granules that house a plethora of growth factors, cytokines, and other molecules that may potentiate tissue healing. Some studies posit that MSCs exert regenerative effects via paracrine signaling  which may be mediated by exosomes. Defined as extracellular vesicles released from the fusion of a cytosolic multivesicular body with the cell membrane, exosomes have been implicated in intracellular communication and molecular transport . Early evidence suggests that MSC-derived exosomes possess chondroprotective and anti-inflammatory properties . Similarly, PRP has been shown to increase chondrocyte proliferation and gene expression indicative of extracellular matrix (ECM) production . However, no studies have examined the concomitant effects of PRP and MSC-derived exosomes on chondrocyte gene expression in both a neutral and inflammatory environment. It was hypothesized that chondrocytes cultured in a combination of PRP and exosomes would demonstrate enhanced gene expression suggestive of ECM synthesis and decreased inflammation than chondrocytes grown with PRP or exosomes in isolation. METHODS: Articular chondrocytes were harvested from articular surfaces of bovine ankle joints and cultured in DMEM-F12 medium with 10% fetal bovine serum (FBS) and 1% antibiotic-antimycotic (AA) solution. MSCs were isolated from the bone marrow of Sprague-Dawley rats and cultured in low-glucose DMEM medium with 15% FBS and 1% AA. Both cell lines were incubated with 5% CO2 at 37°C. PRP was obtained from one healthy donor using a Regen PRP kit. Following a previously published protocol , exosomes were obtained from MSCs as a 100K pellet. Chondrocytes were seeded at 50,000 cells/well into 24-well plates. They were partitioned into one control (media only) and five experimental conditions: exosomes alone (Exo), 1% unactivated PRP (1u), 5u, combined exosomes and 1% uPRP (Exo1), and Exo5. Exosomes were added to the appropriate treatment groups at a concentration of 10 μg/ml. All conditions were further allocated into one of two environments: with or without IL-1β (10 ng/ml) to simulate an osteoarthritic or neutral environment respectively. Following 1 and 3 days, RNA was harvested, isolated, and reverse transcribed into cDNA. Quantitative RT-PCR was then performed (n = 3 per condition), using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as the endogenous control, for the following target genes: aggrecan (ACAN), sex-determining region Y box 9 (SOX9), alpha-1 chain of type I collagen (Col1), alpha-1 chain of type II collagen (Col2), and matrix metalloproteinase 3 (MMP3), thioredoxin-interacting protein (TXNIP), and proliferating cell nuclear antigen (PCNA). The data were analyzed according to the 2-ΔΔCT method. Because significant deviations from normality were observed, within-day comparisons across conditions and within-condition comparisons across days were analyzed with Kruskal-Wallis tests followed by Conover-Iman post hoc tests. Due to the large number of comparisons performed, the Benjamini-Hochberg procedure was employed to control the false discovery rate. Significance was determined if p < 0.05. RESULTS: After 3 days following IL-1β exposure (Day 3 + IL-1β), expression of ACAN decreased relative to the control in 1u, 5u, Exo1, Exo5 but not in Exo (Figure 1A). In contrast, expression of SOX9 increased in these groups following 1 day with IL-1B (Day 1 + IL-1β) as well as in 5u, Exo1, and Exo5 after 3 days with no IL-1β (Day 3 – IL-1β) (Figure 1B). Additionally, 5u and Exo5 showed reduced expression of Col1 in Day 3 – IL-1β while 5u also demonstrated it in Day 3 + IL-1β (Figure 1C). 5u continued to exhibit significant differences in gene transcription with decreased expression of TXNIP in Day 1 + IL-1β (Figure 1D) but elevated expression of PCNA in Day 3 – IL-1β (Figure 1E). Similarly, Exo5 showed greater expression of PCNA in Day 3 – IL-1β both relative to control and the identical condition after 1 day without IL-1β (Day 1 – IL-1β). Likewise, increased expression of TXNIP was observed in Exo1 in Day 3 – IL-1β compared to control as well as across time points and environments in the same condition. Greater expression was found in Day 1 + IL-1β versus Day 1 – IL-1β while the reverse was seen in Day 3 + IL-1b versus Day 3 – IL-1β. No significant changes in expression of Col2 or MMP3 were observed. DISCUSSION: This study is the first to report effects of combined PRP and MSC-derived exosomes on chondrocyte gene expression. The results suggest that after 3 days in a neutral environment, exosomes with 1% uPRP was able to sustain elevated expression of SOX9, a transcription factor for genes involved in ECM synthesis (e.g., ACAN, Col2), compared to either biologic in isolation. When the volume of uPRP was increased to 5%, however, an additive rather than a synergistic effect was observed. Also in Day 3 – IL-1β, TXNIP expression was higher in Exo1 while no difference was seen in Exo or 1u. This result provides evidence for a synergistic relationship between 1% uPRP and exosomes at enhancing chondrocyte autophagy. Previous investigations support this finding, having concluded that PRP  and exosomes  separately upregulate cell autophagy. TXNIP is used as an indirect marker of cell autophagy due to its inhibitory effect on proteosomal degradation of DNA-damage-inducible transcript 4 (DDIT4) which normally attenuates the mTOR-mediated inhibition of autophagy. Considering that autophagy, a form of recycling dysfunctional cell components, is partially responsible for maintaining proper chondrocyte function and avoiding senescence, downregulation of autophagic mechanisms may contribute to cartilage degeneration . SIGNIFICANCE/CLINICAL RELEVANCE: Combined 1% uPRP and MSC-derived exosomes may exhibit synergistic effects for upregulating (a) the transcription of factors related to ECM production and (b) genes involved in increasing chondrocyte autophagy. Clinical application of these results has the potential to optimize cartilage regeneration and improve the symptomatology of osteoarthritis.
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