Abstract
Osteoarthritis (OA) is a degenerative disease that causes chronic pain and disability worldwide. This disease is mainly caused by IL-1β and TNF-α, which lead to cartilage degradation and inhibit the repair capacity of damaged cartilage. Recent studies have shown that amniotic fluid mesenchymal stem cells (AF-MSCs) secrete proteins that can effectively help in the treatment of cartilage damaged by OA. However, the underlying mechanism is still unclear. Therefore, the aim of this study was to investigate the effects and mechanisms behind the healing properties of the AF-MSC secretome (AFS-se) under OA conditions. This study involved growing chondrocyte progenitor cells (CPCs) and traumatized cartilage tissues in the presence of the cytokines IL-1β and TNF-α, which mimic OA conditions. AFS-se was then added to the culture medium to determine its effect on the CPCs and cartilage. Cell migration, endogenous cell outgrowth, the expression of chondrogenic and anabolic genes, and the mechanism of proteins in the NF-κB and MAPK signaling pathways were examined in this study. AFS-se inhibited the inflammatory effects of IL-1β and TNF-α by significantly reducing ERK phosphorylation in the MAPK signaling pathway and decreasing downstream proinflammatory COX2 products. The impaired CPCs recovered their ability to migrate, and endogenous CPCs in injured osteoarthritic cartilage were able to regrow in response to inflammatory stimuli. Additionally, the expression of anabolic genes such as Col I, Col II, and IGF1 was restored in defective CPCs. In conclusion, this study demonstrated that AFS-se has therapeutic effects on OA by inhibiting the inflammatory functions of IL-1β and TNF-α through protein phosphorylation in the MAPK pathway while also promoting the regenerative and self-repair functions of CPCs in traumatized cartilage.
Keywords: Amniotic fluid, Extracellular vesicle, Osteoarthritis, Regenerative medicine, Stem cell
Introduction
Osteoarthritis (OA), a chronic progressive degenerative disease of the articular cartilage, is characterized by knee pain, disrupted daily life, and a vast range of complications in elderly people. The pathophysiological process of OA is inflammation modulated by interleukin-1β (IL-1β) and tumor necrosis factor α (TNF-α)[1–4]. Generally, IL-1β and TNF-α are produced by the synovium and chondrocytes to accumulate in knee synovial fluids and induce inflammation through the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways; subsequently, it produces downstream molecules, including inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2). In OA, iNOS and COX2 act to upregulate a catabolic response and downregulate an anabolic action, resulting in extracellular matrix degradation and cartilage degeneration[1–5]. Joos et al.[6] revealed that the presence of IL-1β and TNF-α inhibited the migration of chondrocytes. This information suggests halting the inflammatory action of the cytokines IL-1β and TNF-α could be a strategy for treating OA. Transplantation of mesenchymal stem cells (MSCs) is a therapeutic option for treating incurable disorders such as degenerative diseases. A primary benefit of MSC transplantation is its paracrine effect[7–9], which is known to trigger growth factors, cytokines, and chemokines in microvesicular exosomes secreted from MSCs[7–9]. The paracrine molecules secreted from MSCs can heal injured tissues, stimulate tissue regeneration, and play a role in anti-inflammation[8,9]. The knowledge of stem cell secretory factors provides a novel therapeutic option for non-cell-based therapy. Generally, a strategy for exogenous stem cell therapy involves the use of suitable types of stem cells for precise treatment. MSCs of different origins can secrete unique proteins of specific tissue origin[9–11]. Angulski et al.[12] demonstrated that MSCs from different routes secrete 60% similar proteins but 40% different proteins.
Amniotic fluid mesenchymal stem cells (AF-MSCs) represent a new class of MSCs with an intermediate phenotype that reside between adult MSCs and embryonic stem cells, resulting in adult MSCs and pluripotent features. AF-MSCs have emerged as an attractive type of MSCs for regenerative medicine because of their distinguishing characteristics, such as a low immunogenic profile, multilineage differentiation potential, genomic stability of HLA-G, and high proliferation ability but not tumorigenicity[13,14]. A strength of AF- MSCs is that they secrete various growth factors, and anti- inflammatory cytokines and uniquely express interleukin-4 (IL-4), which is rarely expressed in other MSC types[15,16]. IL-4 has the potential to downregulate the action of IL-1β and TNF-α, exert remarkable pro-survival and anti-apoptotic effects, and promote tissue repair[2,4]. The secretory proteins derived from AF-MSCs (AFS-se) have healing effects on degenerative diseases and OA[16–21]. Zavatti et al.[17] revealed that AFS-se enhances pain tolerance, improves histological scores, and restores cartilage surface area, which is characteristic of hyaline cartilage, in animal models. Based on this information, identifying the secretory proteins of AF-MSCs can contribute to the development of OA treatment. However, the underlying process and other roles of AFS-se in OA therapy are still undefined and must be understood.
To explore the function and underlying mechanism of AFS-se in arresting inflammation and healing in OA cartilage, we investigated the ability of AFS-se to repair CPCs cultured in IL-1β and TNF-α inflammatory cytokines via the study of cell migration using scratch assay and the outgrowth of endogenous CPCs. The anti-inflammatory effect of AFS-se was determined by studying inflammatory signaling proteins in the nuclear factor-κB (NF-κB) pathway and mitogen- activated protein kinase (MAPK) signaling pathway via western blotting. The effect of AFS-se on homeostatic balance was observed via anabolic gene activity using real-time RT-PCR.
Materials and methods
Human materials
Samples of articular cartilage were obtained from the knees of seven patients with osteoarthritis who were 60-75 years old. These patients had undergone total knee replacement at the Department of Orthopeadic Surgery, Faculty of Medicine Siriraj Hospital, Mahidol University. Before the samples were taken, the participants were fully informed about the study and signed a document allowing their specimens to be used for investigation. The study was approved by the IRB of Siriraj Hospital (Si 075/2014), Mahidol University, Thailand.
Culture medium
The experiment utilized three different culture media conditions: (1) a control with 10% DMEM, (2) IL-1β/TNF-α induced medium (induction medium or induced medium), and (3) IL-1β/TNF-α induced medium supplemented with AF-MSC secretome (AFS-se medium). The 10% DMEM included Dulbecco's modified Eagle’s medium (DMEM; Gibco, Invitrogen, CA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin-streptomycin (Sigma-Aldrich, St. Louis, MO). The IL-1β/TNF-α induced medium was created by adding 1 ng/ml IL-1β and 10 ng/ml TNF-α to the 10% DMEM. The AFS-se medium was developed by supplementing the IL-1β/TNF-α induced medium with 50% v/v AF-MSC secretome.
AF-MSC secretome preparation
To construct the AF-MSC secretome (AFS-se), AF-MSCs were cultured in a T75 tissue culture flask (Nunc) at an initial density of 7,000 cells/cm2 density and incubated in 10% α-MEM. The medium contained alpha minimum essential medium (α-MEM; Gibco, Invitrogen, CA), supplemented with 10% ES-FBS (Gibco) and 1% penicillin-streptomycin (Sigma-Aldrich). The MSCs were incubated until they reached 80% confluence. The medium was then replaced with 5 ml of α-MEM without serum supplementation for 24 hours at 37 °C and 5% CO2. The culture medium was collected, filtered through a 0.22 μm filter membrane (Sartorius Stedim Biotech GmbH, Heidelberg, Germany), and designated AFS-se; it was stored at -80 °C for later use. AFS-se was examined for its protein composition by fragmenting it through gel electrophoresis 15% SDS-PAGE, followed by mass spectrometry analysis and protein classification using the PANTHER classification system version 13.
Isolation, characterization, and culture of chondrocyte progenitor cells (CPCs)
Healthy cartilage tissues were obtained from non-weight-bearing areas with smooth surfaces and no evidence of erosion from seven patients who underwent total knee replacement surgery for experimental uses (Figure 1). These cartilage tissues were cut into small pieces of approximately 3x3 mm from intact and nonfibrillated regions. Then, they were placed in a 60 mm tissue culture dish (Nunc, Denmark) with 10% DMEM at 37 °C and 5% CO2. The medium was changed every four days, and the tissues were removed after the outgrowing cells migrated from the cartilage tissues to the culture dish. These cells were scaled up by repetitive subcultures passaged to passage 4 and were utilized for the study. Flow cytometry was used to characterize the presence of CD29, CD34, CD73, CD90, and CD105 surface proteins on the cells. The population doubling time (PDT) was measured to determine the cell growth rate and was calculated with the formula (log2)T/log(Y)- log(X), where (X) represents the number of cells at seeding and (Y) represents the number of harvested cells.
Figure. 1. Schematic diagram of experimental design.
Migration study using scratch assay
In a 35 mm tissue culture dish, CPCs were initially plated at a density of 1.5 x 104 cells/cm2 in 10% DMEM. The dish was then incubated at 37 °C and 5% CO2 until a 90% confluent monolayer was achieved. The CPC monolayer was scraped to create a 0.5 cm gap for cell migration, and a straight line was made in the migration area using a sterile blade. The culture was incubated at 37°C and 5% CO2 with a daily medium change.
Next, we optimized the appropriate concentrations of IL-1β and TNF-α by performing a migration test using a scratch assay. IL-1β (ImmunoTools, Friesoythe, Germany) at concentrations of 0.02, 0.2, and 1 ng/ml and TNF-α (ImmunoTools) at concentrations of 0.05, 0.5, 5, and 10 ng/ml were added to the culture medium, and their inhibitory effects on CPC migration were determined. The appropriate concentrations of IL-1β and TNF-α were then used to prepare the IL-1β/TNF-α induced medium.
To determine the anti-inflammatory effects of AFS-se, a migration test was used. Three different media were used for comparison, namely10% DMEM (control), IL-1β/TNF-α induced medium (induction medium), and IL-1β/TNF-α induced medium supplemented with the AF-MSC secretome (AFS-se medium). Cell migration was observed for seven days, and the number of cells that migrated across the straight line in the migration area was observed and compared among the three different media on days one, three, five, and seven using phase contrast inverted microscopy. The number of cells that moved into the migration area and migrated across a straight line was observed, and the cells were finally counted using the ImageJ program. The experiment was conducted in five replicates.
Observation of endogenous CPC outgrowth
We investigated how the AF-MSC secretome affects cartilage repair. Human cartilage tissues harvested from the area of the OA lesion in seven patients, were cut into small pieces (3x3 mm) and then incubated in three different media: control medium, induction medium, and AFS-se medium. The incubated cartilage tissues were observed under an inverted microscope to check for CPC outgrowth. The medium was changed twice a week. We recorded the number of cartilage tissues exhibiting CPC outgrowth and counted the number of outgrowing cells from each tissue until Day 21 of culture using Image. The experiment was performed in triplicate.
Protein extraction and western blot analysis
CPCs were grown to 70% confluence in a monolayer. These cells were divided into three groups for three different conditions. The first group, which served as the control, was cultured under unstimulated conditions in control culture medium (10% DMEM). The second group was cultivated in IL-1β/TNF-α-induced medium for 3 hours before being cultured in 10% DMEM for 24 hours. The third group was cultivated in IL-1β/TNF-α induced medium for 3 hours and subsequently transferred to AFS-se medium for 24 hours. After culture, cytoplasmic and nuclear proteins were extracted from the CPCs to investigate signaling proteins in the MAPK and NF-κB pathways.
To extract the proteins, the CPCs were harvested via mechanical scraping from plastic tissue culture dishes and washed with cold PBS before being resuspended in a hypotonic buffer solution by pipetting and incubating for 15 minutes on ice. The cell membrane was lysed by adding 10% NP-40 (Abcam, Cambridge, UK) before vortexing and centrifugation. The supernatant was collected for cytoplasmic protein analysis. The pellet was further lysed by vortexing in ice-cold nuclear extraction buffer. Chromatin was removed by centrifugation, and the supernatant was collected for soluble nuclear protein extraction.
For western blot analysis, proteins (10 micrograms) were separated via 10% SDS-PAGE and transferred onto PVDF membranes (Bio- Rad, Hercules, CA). Nonspecific protein was blocked with 5% skim milk (Himedia, Mumbai, India) in TBST buffer before overnight incubation with primary antibodies against iNOS (ab178945), COX2 (ab62331), NF-κB p65 (ab32536), pNF-κB p65 (ab76302), p38 (ab170099), pp38 (ab195049), ERK1/ERK2 (ab184699), pERK (ab76299), and beta actin (ab8226) (all from Abcam), and histone H3 (3638S, Cell signaling, Denvers, MA). The immunoblots were incubated with anti-rabbit IgG (HAF008, R&D systems, Minneapolis, MN) or anti-mouse IgG (ab97046, Abcam) and detected using the ImageQuant LAS4010 system. The protein band intensities were quantified using the ImageJ program. Protein expression levels were analyzed relative to those of housekeeping proteins as the mean±SEM and computed as the fold change. The experiment was performed in triplicate.
Gene expression analysis
We determined how the expression of genes involved in anabolic activity and chondrogenesis is affected by the combination of IL- 1β/TNF-α and AFS-se on CPCs at the molecular level. To do this, we extracted total RNA from CPCs cultured in 10% DMEM, induction medium, and AFS-se medium for 96 hours using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA). cDNA was synthesized using iScript Reverse Transcription Supermix (Bio-Rad Laboratories, Hercules, CA) and amplified using selective primers (Table 1). qRT- PCR was performed on a LightCycler 480 using SYBR Green I master mix (Roche Diagnostics GmbH, Mannheim, Germany), and the data were analyzed using the 2-△△CT method relative to the quantity of beta-actin. The experiment was repeated in triplicate.
Table 1. Primers of chondrocyte-specific and anabolic activity genes.
| Gene | Primer sequences (5’-3’) | Accession No. | Annealing Temp (°C) | Product size (bp) |
|---|---|---|---|---|
| β-actin | F: 5’-ATGTGGCCGAGGACTTTGATT-3’ R: 5’-AGTGGGGTGGCTTTTAGGATG-3’ |
NM_001101.5 | 60 | 107 |
| ACAN | F: 5’-ACAGCTGGGGACATTAGTGG-3’ R: 5’-GTGGAATGCAGAGGTGGTTT-3’ |
NM_001135.4 | 57 | 189 |
| Col I | F: 5’-AGGACAAGAGGCATGTCTGGTT-3’ R: 5’-GGACATCAGGCGCAGGAA-3’ |
NM_000088.3 | 57 | 122 |
| Col II | F: 5’- GGCAATAGCAGGTTCACGTACA-3’ R: 5’- CGATAACAGTCTTGCCCCACTT-3’ |
NM_033150.3 | 60 | 79 |
| IGF1 | F: 5’- AAGATGCACACCATGTCCTCC-3’ R: 5’- AGCCTCCTTAGATCACAGCTCC-3’ |
NM_001111285.3 | 58 | 248 |
| RUNX2 | F: 5’- ATGCTTCATTCGCCTCAC-3’ R: 5’- ACTGCTTGCAGCCTTAAAT-3’ |
NM_001024630.3 | 57 | 156 |
| SOX9 | F: 5’- CCCAACAGATCGCCTACAG-3’ R: 5’- TTCTGGTGGTCGGTGTAGTC-3’ |
NM_000346.4 | 57 | 97 |
| TGFβ1 | F: 5’- GGGACTATCCACCTGCAAGA-3’ R: 5’- CCTCCTTGGCGTAGTAGTCG-3’ |
NM_000660.7 | 59 | 239 |
Statistics
The data (the mean ± standard error of the mean; SEM) were analyzed using Student’s t-test and one-way ANOVA with Tukey’s post hoc test using PRISM software version 8.0 (GraphPad Software). P values < 0.05 were considered to indicate statistically significant differences.
Results
Chondrocyte progenitor cell (CPC) characteristics
On Day 3 of incubation, a few cells grew from the cartilage tissue pieces. These cells then continued to grow and multiply with a fibroblastic morphology, as shown in Figure 2A. The cells were scaled up to passage 3 and had an average population doubling time of 2.73±0.38 days, with a range of 2.39 to 3.14 days. The CPCs were confirmed to express high levels of CD29 (98.4%), CD73 (99.8%), CD90 (99.9%), and CD105 (99.9%) but low levels of CD34 (1.4%) and CD45 (0.9%) surface markers.
Figure 2. Cell migration study using scratch assay.
(A) Morphological appearance of CPC migrating from healthy cartilage was observed under 10X magnification using inverted microscopy in a fibroblast type.
(B) The concentrations of IL-1β and TNF-α were optimized in triplicate using a scratch assay to inhibit CPC migration. CPC migration was impaired on day seven after exposure to 0.2 or 1 ng/ml IL-1β or 0.5, 5, or 10 ng/ml TNF-α. The number of CPCs in the migration area of the studied medium was compared to that in the control medium. The mean ± SEM of the cell counts were significantly different at *P<0.05 and **P<0.01, as determined by Student's t test. (C) CPCs were observed migrating in a straight line using 10X inverted microscopy in three different media. (D) The mean ± SEM of CPCs (n=5) that migrated across the straight line on Day 7 were analyzed by one-way ANOVA, with * P<0.05 and *** P<0.001 indicating statistical significance.
AF-MSC secretome (AFS-Se) characteristics
AFS-se is a substance derived from the conditioned medium of cultured AF-MSCs. AF-MSCs exhibited typical MSC characteristics, including a spindle fibroblast-like morphology and a low population doubling time (PDT) of approximately 1.2 ± 0.21 days. These cells also expressed specific MSC surface proteins, including CD29 (99.6%), CD44 (99.6%), CD73 (99.9%), CD90 (98.6%), and CD105 (99.5%). Additionally, they demonstrated the ability to differentiate into adipogenic-, chondrogenic-, and osteogenic lineages in vitro. AFS-se contains various proteins, with a total protein concentration of 0.69 ± 0.11 mg/ml according to the Bradford assay. Mass spectrophotometry analysis revealed that AFS-se is composed of proteins related to different biological processes (Table 2), including cell growth and maintenance, protein metabolism, energy pathways, signal transduction, and cell communication. In total, 41 proteins were found to be involved in cell growth and maintenance, 17 in protein metabolism, 17 in energy pathways, and 12 in signal transduction and cell communication.
Table 2. Biological molecules found in AFS-se.
| Molecules in cell growth/maintenance/signal transduction/cell communication/protein metabolism/energy pathways | |||
| Accession | Protein description | Accession | Protein description |
| Related to cell growth/maintenance | |||
| P68032 | Actin, alpha cardiac muscle 1 | P35749 | Myosin-11 |
| P60709 | Actin, cytoplasmic 1 | Q7Z406 | Myosin-14 |
| P61160 | Actin-related protein 2 | P07737 | Profilin-1 |
| P61158 | Actin-related protein 3 | Q01082 | Spectrin beta chain, non-erythrocytic 1 |
| Q9P1U1 | Actin-related protein 3B | Q9Y490 | Talin-1 |
| Q01518 | Adenylyl cyclase-associated protein 1 | A6NHL2 | Tubulin alpha chain-like 3 |
| P12814 | Alpha-actinin-1 | P68363 | Tubulin alpha-1B chain |
| O43707 | Alpha-actinin-4 | Q9BQE3 | Tubulin alpha-1C chain |
| P61163 | Alpha-centractin | Q13748 | Tubulin alpha-3C/D chain |
| Q14019 | Coactosin-like protein | P68366 | Tubulin alpha-4A chain |
| P23528 | Cofilin-1 | Q9NY65 | Tubulin alpha-8 chain |
| P15311 | Ezrin | P07437 | Tubulin beta chain |
| Q16658 | Fascin | Q9BVA1 | Tubulin beta-2B chain |
| P21333 | Filamin-A | Q13509 | Tubulin beta-3 chain |
| O75369 | Filamin-B | P04350 | Tubulin beta-4A chain |
| Q14315 | Filamin-C | P68371 | Tubulin beta-4B chain |
| P26038 | Moesin | Q9BUF5 | Tubulin veta-6 chain |
| P14649 | Myosin light chain 6B | Q3ZCM7 | Tubulin beta-8 chain |
| P60660 | Myosin light polypeptide 6 | Q8IUG5 | Unconventional myosin-XVIIIb |
| P35579 | Myosin-9 | P18206 | Vinculin |
| P35580 | Myosin-10 | ||
| Molecules in signal transduction/cell communication pathways | |||
| P04083 | Annexin A1 | Q8WZ94 | Olfactory receptor 5P3 |
| P08758 | Annexin A5 | Q8IUH5 | Palmitoyltransferase ZDHHC17 |
| P08133 | Annexin A6 | O00750 | Phosphatidylinositol 4-phosphate 3-kinase C2 domain-containing subunit beta |
| Q5KU26 | Collectin-12 | P31150 | Rab GDP dissociation inhibitor alpha |
| P08238 | Heat shock protein HSP 90-beta | P24821 | Tenascin |
| Q8NGS5 | Olfactory receptor 13C4 | Q6ZQQ6 | WD repeat-containing protein 87 |
| Molecules in the metabolic pathway | |||
| P11021 | 78 kDa glucose-regulated protein | P34931 | Heat shock 70 kDa protein 1-like |
| P68104 | Elongation factor 1-alpha 1 | P17066 | Heat shock 70 kDa protein6 |
| P26641 | Elongation factor 1-gamma | P11142 | Heat shock cognate 71 kDa protein |
| P13639 | Elongation factor 2 | P07900 | Heat shock protein HSP 90-alpha |
| P14625 | Endoplasmin | P54652 | Heat shock-related 70 kDa protein 2 |
| P60842 | Eukaryotic initiation factor 4A-I | Q9NZJ4 | Sacsin |
| Q14240 | Eukaryotic initiation factor 4A-II | Q9UHB9 | Signal recognition particle subunit SRP68 |
| P0DMV8 | Heat shock 70 kDa protein 1A | P09936 | Ubiquitin carboxyl-terminal hydrolase isozyme L1 |
| P0DMV9 | Heat shock 70 kDa protein 1B | ||
| Molecules in energy production/glycolysis pathway | |||
| P23526 | Adenosylhomocysteinase | Q6ZMR3 | L-lactate dehydrogenase A-like 6A |
| P06733 | Alpha-enolase | Q9BYZ2 | L-lactate dehydrogenase A-like 6B |
| P13929 | Beta-enolase | P07195 | L-lactate dehydrogenase B chain |
| P04075 | Fructose-bisphosphate aldolase A | Q8IXY8 | Peptidyl-prolyl cis-trans isomerase-like 6 |
| P35557 | Glucokinase | P00558 | Phosphoglycerate kinase 1 |
| P06744 | Glucose-6-phosphate isomerase | P14618 | Pyruvate kinase PKM |
| P09211 | Glutathione S-transferase P | Q16881 | Thioredoxin reductase 1, cytoplasmic |
| P04406 | Glyceraldehyde-3-phosphate dehydrogenase | P60174 | Triosephosphate isomerase |
| P00338 | L-lactate dehydrogenase A chain | ||
AFS-se arrests the effects of IL-1β and TNF-α in migration studies.
We studied CPCs and their response to IL-1β and TNF-α stimuli. We found that when CPCs were cultured with 0.2 or 1 ng/ml IL-1β and 0.5, 5, or 10 ng/ml TNF-α, cell migration significantly decreased compared to that in the control group. This inhibitory effect of IL-1β and TNF-α prevented CPCs from moving to the free space in the migration area (Figure 2B). Therefore, we used 1 ng/ml IL-1β and 10 ng/ml TNF-α as the concentrations used to treat the induced medium.
We then observed the effect of AFS-se on CPCs under IL-1β and TNF-α stimulation and compared the cell migration activity to that of CPCs incubated in a control medium or induced medium (Figure 2C). At 24 hours in the scratch assay, the CPCs under the three conditions started to move into the free space of the migration area. On Day 3, the number of CPCs in the migration area in the AFS-se medium (50.6±4.9 cells) was significantly greater than that in the induced medium (21.2±2.6 cells, P <0.0001) and control medium (34.3±3.9 cells, P<0.05). On Day 5, the CPCs in the AFS-se medium still moved and were scattered in the migration area, while the CPCs in the induced medium stopped migrating and remained as a cluster in a limited area. The number of CPCs that moved through the straight line during migration significantly differed among the three conditions, with the number of migrating CPCs in AFS-se medium (145.9±13.3 cells) being significantly greater than that in induced medium (45.4±4.8 cells, P <0.0001) and control medium (68.5±7.7 cells, P <0.0001). On Day 7, the CPCs in the induced medium still showed no improvement in cell migration. The number of migrating CPCs in the AFS-se medium (217.5±17.8 cells) was approximately four times greater than that in the induced medium (55.2±6.0 cells) and two and one-half times greater than that in the control medium (86.6±11.1 cells). Our study demonstrated that IL-1β and TNF-α inhibited the migratory functions of CPCs, while AFS-se arrested the inhibitory effects of IL-1β and TNF-α on CPCs and restored the migratory properties of CPCs in the presence of IL-1β and TNF-α.
AFS-se promotes self-repair in traumatized OA cartilage
We evaluated the regenerative effects of AFS-se on damaged cartilage tissues. We cultured forty-eight pieces of cartilage from OA lesions under three different conditions: control medium, induced medium, and AFS-se medium (as shown in Figure 3A). We observed that CPCs started to outgrow 1-3 of the 48 pieces of cartilage after ten days of culture, regardless of the conditions. By Day 14, the cells had migrated to areas on the tissue culture dish. The CPCs that grew in the induced medium was found in only seven of the 48 pieces of cartilage (14.8%), while the CPCs that grew in the AFS-se medium was found in 12 of the 48 pieces of cartilage (25%). The control medium yielded 11 outgrowth cartilages (22.9%). The CPCs that grew had a typical fibroblastic appearance. By Day 21 of culture, we observed 15 outgrowth cartilages under AFS-se medium (31.3%), while the outgrowth tissue under induced medium (7 of 48 pieces of cartilage; 14.8%), and control medium (11 of 48 pieces of cartilage; 22.9%) did not improve beyond Day 14 (as shown in Figure 3B). We also found that the endogenous CPCs in the induced medium had limited migration ability, enlarged size, and entered senescence. In contrast, the outgrowing CPCs in the AFS-se medium and control medium showed a typical fibroblastic appearance with migration potential. The number of outgrowing CPCs in the AFS-se medium was significantly greater (559.7±222 cells) than that in the control medium (104.4±47.1 cells; P<0.001) and induced medium (72.7±56.9 cells; P<0.001) (as shown in Figure 3C).
Figure 3. AFS-se promotes cartilage outgrowth.
(A) The regenerative capability of AFS-se in inducing self-repair and tissue regeneration in OA cartilage tissues was demonstrated through the proliferation and outgrowth of endogenous CPCs. (B) The quantity of cartilage that exhibited endogenous CPC outgrowth is presented. (C) The number of CPCs in the tissue culture dish on Day 21 was determined, and presented as the mean ± SEM (n=3). Statistical evaluation was conducted using one-way ANOVA with * P<0.05.
These findings suggest that IL-1β and TNF-α inhibit the regenerative and repair functions of OA cartilage. AFS-se has the ability to re- energize these functions in OA defective cartilage under inflammatory stimuli.
AFS-se arrests inflammation via the NF-κB and MAPK signaling pathways
We investigated the effects of AFS-se on CPCs and the mechanisms underlying these effects. We examined the levels of various signaling proteins in the NF-κB and MAPK pathways, including NF-κB, pNF-κB, p38, pp38, ERK, pERK, COX2, and iNOS, using western blotting (Figure 4).
Figure 4. Genes and protein analysis.
(A) The relative changes in chondrogenic and anabolic gene expression levels in CPCs cultured for 96 hours in IL-1β/TNF-α-induced media and in those cultured in induced media supplemented with AFS-se are presented as the mean ± SEM. Statistical significance was found when compared with the control group, using Student’s t test with * P<0.05. (B) The phosphorylation levels of signaling proteins in the NF-κB and MAPK pathways in CPCs that were pretreated in IL-1β/TNF-α-induced medium for three hours and then cultured for 24 hours in either 10% DMEM or AFS-se medium. One-way ANOVA was used to analyze the mean ± SEM of band intensity in triplicate experiments. Statistical significance was indicated by * P<0.05 and ** P<0.01
Compared with control CPCs, CPCs incubated in induction medium exhibited significantly greater levels of NF-κB (2.76-fold), pNF-κB (1.35-fold), p38 (1.35-fold), pp38 (1.36-fold), ERK (1.92-fold, P<0.05), pERK (1.7-fold), COX2 (1.35-fold), and iNOS (1.52-fold, P<0.05). However, after treatment with AFS-se, the production of these inflammatory signaling proteins decreased and became similar to that of the control CPCs. Specifically, the expression of NF-κB (0.99-fold), pNF-κB (1.1-fold), p38 (1.1-fold), pp38 (1.1-fold), ERK (1-fold), pERK (1.2-fold), COX2 (1.0-fold), and iNOS (1.1- fold) was similar to that in the control CPCs.
The NF-κB and MAPK pathways were elevated in CPCs incubated in the induction medium but decreased after treatment with AFS-se, and we observed statistically significant differences in the levels of nuclear pERK and COX2. Specifically, CPCs incubated in the induction medium had significantly greater levels of pERK than those treated with AFS-se (P<0.01). This finding suggested that the ERK/MAPK pathway plays an essential role in the anti- inflammatory effects of AFS-se on IL-1β and TNF-α and downstream production of COX2 and iNOS.
AFS-se promotes chondrogenic and anabolic gene expression
We analyzed the gene expression of the cartilage-specific genes ACAN, Col I, Col II, RUNX2, and SOX9, and the anabolic genes IGF1 and TGFβ1 in CPCs cultured in different media. Real-time RT-PCR was used to measure gene expression levels, and the results are presented in Figure 4A.
When CPCs were incubated in the induced medium, the expression of Col II was significantly lower (0.5-fold, P<0.05) than that in untreated CPCs in the control medium. However, there was an increase in the expression of IGF1 (3.4-fold), TGFβ1 (1.6-fold), Col I (2.2-fold), and ACAN (2.2-fold).
Figure 5. Proposed mechanisms of AFS-se in inhibiting the inflammatory MAPK pathway and promoting cartilage regeneration and repair.
However, when CPCs were incubated in induction medium supplemented with AFS-se (AFS-se medium), the expression of Col II and SOX9 increased to the same levels as those in the control. Compared with those in control CPCs, there was also significantly greater expression of IGF1 (20.4-fold) and RUNX2 (1.6-fold, P<0.05), but lower expression of TGFβ1 (0.5-fold, P<0.001) and ACAN (0.4- fold, P<0.001).
Furthermore, CPCs cultured in AFS-se medium had higher expression of IGF1 (6-fold, P<0.05), Col II (2.1-fold), and RUNX2 (1.5-fold), but lower expression of TGFβ1 (3-fold), ACAN (5.5-fold), and Col I (1.8- fold) than CPCs cultured in induction medium.
Overall, our results suggest that CPCs cultured in AFS-se medium had elevated anabolic IGF1 gene expression and were able to maintain the expression of the cartilage-specific genes Col I, Col II, RUNX2, and SOX9.
Discussion
The use of exogenous MSCs in cell therapy is supported by two theories: the “differentiation theory” and the “paracrine theory”. The differentiation theory suggests that stem cells can directly differentiate into specific cells and replace damaged tissue, while the paracrine theory proposes that stem cells secrete bioactive trophic factors that trigger the biological behavior of endogenous stem cells[22,23]. However, it has been proven difficult to achieve stem cell differentiation and replacement of lesions because only a few cells survive for a few weeks after transplantation[9,22–25]. Recent studies have suggested that extracellular vesicles containing secretory paracrine factors have advantages for medical therapy, including OA therapy[17]. This approach avoids the injection of cells and instead relies on the paracrine effect of therapeutic agents to benefit stem cell therapy. Furthermore, this approach has more advantages than stem cell transplantation, such as safety, no side effects due to the natural lipid and surface protein composition, simple preservation methods, few ethical issues, and better crossing of some biological barriers than MSCs[21].
AF-MSCs possess unique characteristics, such as superior proliferation ability and healing effects. These cells originate during organ development and secrete special anti-inflammatory cytokines. They also release various growth factors that support tissue generation and organ development. Because of these properties, AF- MSCs may have advantages over MSCs from neonatal and adult sources for regenerating degenerative organs such as OA cartilage.
The tissue healing process involves reducing inflammation, achieving homeostasis, and repairing the damaged tissue. To examine therapeutic potentials of AFS-se in treating cartilage osteoarthritis, we developed an in vitro model that mimics OA. This model uses IL-1β and TNF-α cytokines, which are known to have major inflammatory effects in OA. We observed the effects of AFS- se on ability to reduce inflammation through proteins in the inflammatory pathway, restore homeostasis by promoting anabolic and chondrogenic gene expression, act on CPCs in healthy sites, and CPCs in injury sites to restore the self-repair function.
Our study showed that IL-1β and TNF-α inhibit the self-repair and regenerative functions of cartilage by blocking the growth and migration of endogenous CPCs. This finding is consistent to the report by Joos et al.[6]. This is why patients with OA knees cannot regenerate and repair themselves naturally. However, our work revealed that AFS-se can arrest the inhibitory action of IL-1β and TNF-α and liberate CPCs to migrate and outgrow cartilage tissues even under inflammatory stimuli. This implies that AFS-se has the potential to be used as a tool for cartilage regeneration and repair in OA. According to a report by Joos et al. (2013)[6], cartilage tissue that had spontaneously regrown from the nonfibrillated region of OA knee patients had a success rate of 97%. Based on this information, our study utilized a culture technique to generate CPCs, and our results consistently showed that almost 100% of the cells from the cartilage tissue obtained from the healthy region of OA knee patients were successfully regenerated. However, our study revealed that cartilage tissue obtained from the traumatic OA region was less successful at spontaneously outgrowing. Therefore, we conducted a study to observe the growth of endogenous CPCs from cartilage using OA cartilage tissue in traumatized regions from seven donors. The tissue was cut into small pieces, pooled together, and randomly divided into three groups for each culture medium to reduce variability in human cartilage explants. In a separate study using CPCs obtained from healthy cartilage regions, cell samples were collected from individual donors. Experiments were carried out in 3-5 replications, which is consistent with previous studies[6,26] to observe cell migration, cell outgrowth, and protein signalling.
During our experiment, we used culture medium consisting of a 50% AF-MSC secretome to investigate how it affects CPCs and cartilage. According to a study by Kukumberg et al. (2021)[16], human cardiomyocyte proliferation was found to be dose-dependent when influenced by AFS-se. The study showed that 100% AFS-se was better than the addition of 50% AFS-se to the culture medium. They also found that AFS-se collected after 24 hours of culture under normoxic conditions resulted in good cell proliferation. Based on this literature, we attempted to replace the CPC culture medium with AFS-se obtained after 24 hours of culture under normoxic conditions to substitute 50% and 100% of the medium. However, we discovered that CPCs could not survive under 100% AFS-se replacement (data not shown). Therefore, we used selected AFS-se collected after 24 hours under normoxic conditions to replace 50% of the CPC culture medium throughout our experiment.
The cytokines IL-1β and TNF-α are crucial for the inflammation associated with osteoarthritis (OA). They activate the MAPK inflammatory pathway by increasing the phosphorylation of signaling proteins, including p38 and ERK[27]. Our study showed that AFS-se significantly reduced the phosphorylation of signaling proteins in the ERK/MAPK pathways. This finding is consistent with the reports by Li et al.[27] and Qi et al.[28], who found that secretory proteins from bone marrow-derived MSCs can reduce the production of phosphorylated ERK in an in vitro study. A decrease in the phosphorylation of ERK/MAPK and p38/MAPK leads to a reduction in the production of downstream proinflammatory COX2 and iNOS, which suggests that this decrease is a cause of the reduction in inflammation in OA. Although our results did not show a statistically significant decrease in the p38/MAPK pathway, the evidence suggests that AFS-se blocks inflammation in OA via the MAPK pathway and its downstream proinflammatory proteins.
In cases of OA, the cytokines IL-1β and TNF-α are released from chondrocytes and synovial cells in the affected area, causing quiescent progenitor chondrocytes to produce matrix-degrading enzymes known as MMPs; this leads to extracellular matrix (ECM) breakdown and obstructs chondrogenesis[1,2]. MMPs, along with IL-1β and TNF-α, also disrupt collagen type II synthesis, replacing hyaline cartilage with fibrocartilage and making the knee structurally weak. Our study revealed that the presence of the cytokines IL-1β and TNF-α in culture reduced the amount of collagen type II. However, when AFS- se was introduced to the culture, it counteracted the negative effects of cytokines, and the amount of collagen type II increased. This finding is consistent with that of Vonk et al.[26], who examined the effect of bone marrow MSC-derived EVs on OA chondrocytes. Moreover, AFS-se treatment increased the mRNA levels of anabolic and chondrogenic genes, indicating the restoration of cartilage homeostasis.
Conclusion
This work demonstrated that AFS-se has therapeutic potential in an in vitro model of OA. AFS-se works by inhibiting the inflammation caused by IL-1β and TNF-α through the MAPK pathway, promoting tissue repair, and increasing the expression of anabolic genes to maintain the balance of cartilage homeostasis.
Acknowledgments
The authors thank Grace Alinpisa McGuin for diligently editing the manuscript with resilience.
Conflict of interests
The authors declare no conflicting interests
Funding
The Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand funded this work.
References
- 1.Kuppa SS, Kim HK, Kang JY, Lee SC, Seon JK. Role of mesenchymal stem cells and their paracrine mediators in macrophage polarization: An approach to reduce inflammation in osteoarthritis. Int J Mol Sci. 2022;23((21)):13016. doi: 10.3390/ijms232113016. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Molnar V, Matišić V, Kodvanj I, Bjelica R, Jelec Ž, Hudetz D, Rod E, Cukelj F, Vrdoljak T, Vidovic D, Starešinic M, Sabalic S, Dobricic B, Petrovic T, Anticevic D, Boric I, Košir R, Zmrzljak UP, Primorac D. Cytokines and chemokines involved in osteoarthritis pathogenesis. Int J Mol Sci. 2021;22((17)):9208. doi: 10.3390/ijms22179208. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Pei YA, Chen S, Pei M. The essential anti-angiogenic strategies in cartilage engineering and osteoarthritic cartilage repair. Cell Mol Life Sci. 2022;79((1)):71. doi: 10.1007/s00018-021-04105-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Liu S, Deng Z, Chen K, Jian S, Zhou F, Yang Y, Fu Z, Xie H, Xiong J, Zhu W. Cartilage tissue engineering: From proinflammatory and anti inflammatory cytokines to osteoarthritis treatments (Review). Mol Med Rep. 2022;25((3)):99. doi: 10.3892/mmr.2022.12615. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Ahmad N, Ansari MY, Haqqi TM. Role of iNOS in osteoarthritis: Pathological and therapeutic aspects. J Cell Physiol. 2020;235((10)):6366–76. doi: 10.1002/jcp.29607. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Joos H, Wildner A, Hogrefe C, Reichel H, Brenner RE. Interleukin-1 beta and tumor necrosis factor alpha inhibit migration activity of chondrogenic progenitor cells from non-fibrillated osteoarthritic cartilage. Arthritis Res Ther. 2013;15:R119. doi: 10.1186/ar4299. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Alvites R, Branquinho M, Sousa AC, Lopes B, Sousa P, Maurício AC. Mesenchymal stem/stromal cells and their paracrine activity-immunomodulation mechanisms and how to influence the therapeutic potential. Pharmaceutics. 2022;14((2)):381. doi: 10.3390/pharmaceutics14020381. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Mancuso P, Raman S, Glynn A, Barry F, Murphy JM. Mesenchymal stem cell therapy for osteoarthritis: The critical role of the cell secretome. Front Bioeng Biotechnol. 2019;7:9. doi: 10.3389/fbioe.2019.00009. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Daneshmandi L, Shah S, Jafari T, Bhattacharjee M, Momah D, Saveh-Shemshaki N, Lo KW, Laurencin CT. Emergence of the stem cell secretome in regenerative engineering. Trends Biotechnol. 2020;38((12)):1373–84. doi: 10.1016/j.tibtech.2020.04.013. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Wang ZG, He ZY, Liang S, Yang Q, Cheng P, Chen AM. Comprehensive proteomic analysis of exosomes derived from human bone marrow, adipose tissue, and umbilical cord mesenchymal stem cells. Stem Cell Res Ther. 2020;11((1)):511. doi: 10.1186/s13287-020-02032-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Shin S, Lee J, Kwon Y, Park KS, Jeong JH, Choi SJ, Bang SI, Chang JW, Lee C. Comparative proteomic analysis of the mesenchymal stem cells secretome from adipose, bone marrow, placenta and Wharton's jelly. Int J Mol Sci. 2021;22((2)):845. doi: 10.3390/ijms22020845. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.Angulski AB, Capriglione LG, Batista M, Marcon BH, Senegaglia AC, Stimamiglio MA, Correa A. The protein content of extracellular vesicles derived from expanded human umbilical cord blood-derived CD133+ and human bone marrow-derived mesenchymal stem cells partially explains why both sources are advantageous for regenerative medicine. Stem Cell Rev Rep. 2017;13((2)):244–57. doi: 10.1007/s12015-016-9715-z. [DOI] [PubMed] [Google Scholar]
- 13.Phermthai T, Odglun Y, Julavijitphong S, Titapant V, Chuenwattana P, Vantanasiri C, Pattanapanyasat K. A novel method to derive amniotic fluid stem cells for therapeutic purposes. BMC Cell Biol. 2010;11:79. doi: 10.1186/1471-2121-11-79. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Phermthai T, Pokathikorn P, Wichitwiengrat S, Thongbopit S, Tungprasertpol K, Julavijitphong S. P53 mutation and epigenetic imprinted IGF2/H19 gene analysis in mesenchymal stem cells derived from amniotic fluid, amnion, endometrium, and Wharton's jelly. Stem Cells Dev. 2017;26:1344–54. doi: 10.1089/scd.2016.0356. [DOI] [PubMed] [Google Scholar]
- 15.Mareschi K, Castiglia S, Sanavio F, Rustichelli D, Muraro M, Defedele D, Bergallo M, Fagioli F. Immunoregulatory effects on T lymphocytes by human mesenchymal stromal cells isolated from bone marrow, amniotic fluid, and placenta. Exp Hematol. 2016;44((2)):138–50. doi: 10.1016/j.exphem.2015.10.009. [DOI] [PubMed] [Google Scholar]
- 16.Kukumberg M, Phermthai T, Wichitwiengrat S, Wang X, Arjunan S, Chong SY, Fong CY, Wang JW, Rufaihah AJ, Mattar CNZ. Hypoxia-induced amniotic fluid stem cell secretome augments cardiomyocyte proliferation and enhances cardioprotective effects under hypoxic-ischemic conditions. Sci Rep. 2021;11:163. doi: 10.1038/s41598-020-80326-w. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Zavatti M, Beretti F, Casciaro F, Bertucci E, Maraldi T. Comparison of the therapeutic effect of amniotic fluid stem cells and their exosomes on monoiodoacetate-induced animal model of osteoarthritis. Biofactors. 2020;46:106–17. doi: 10.1002/biof.1576. [DOI] [PubMed] [Google Scholar]
- 18.Zhang Y, Yan J, Liu Y, Chen Z, Li X, Tang L, Li J, Duan M, Zhang G. Human amniotic fluid stem cell-derived exosomes as a novel cell-free therapy for cutaneous regeneration. Front Cell Dev Biol. 2021;9:685873. doi: 10.3389/fcell.2021.685873. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 19.Costa A, Balbi C, Garbati P, Palamà MEF, Reverberi D, De Palma A, Rossi R, Paladini D, Coviello D, De Biasio P, Ceresa D, Malatesta P, Mauri P, Quarto R, Gentili C, Barile L, Bollini S. Investigating the paracrine role of perinatal derivatives: human amniotic fluid stem cell-extracellular vesicles show promising transient potential for cardiomyocyte renewal. Front Bioeng Biotechnol. 2022;10:902038. doi: 10.3389/fbioe.2022.902038. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Katifelis H, Filidou E, Psaraki A, Yakoub F, Roubelakis MG, Tarapatzi G, Vradelis S, Bamias G, Kolios G, Gazouli M. Amniotic fluid-derived mesenchymal stem/stromal cell-derived secretome and exosomes improve inflammation in human intestinal subepithelial myofibroblasts. Biomedicines. 2022;10((10)):2357. doi: 10.3390/biomedicines10102357. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Nguyen TH, Duong CM, Nguyen XH, Than UTT. Mesenchymal stem cell-derived extracellular vesicles for osteoarthritis treatment: extracellular matrix protection, chondrocyte and osteocyte physiology, pain and inflammation management. Cells. 2021;10((11)):2887. doi: 10.3390/cells10112887. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 22.Jiang S, Tian G, Li X, Yang Z, Wang F, Tian Z, Huang B, Wei F, Zha K, Sun Z, Sui X, Liu S, Guo W, Guo Q. Research progress on stem cell therapies for articular cartilage regeneration. Stem Cells Int. 2021;2021:25. doi: 10.1155/2021/8882505. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Stoddart MJ, Bara J, Alini M. Cells and secretome--towards endogenous cell re-activation for cartilage repair. Adv Drug Deliv Rev. 2015;84:135–45. doi: 10.1016/j.addr.2014.08.007. [DOI] [PubMed] [Google Scholar]
- 24.Quintavalla J, Uziel-Fusi S, Yin J, Boehnlein E, Pastor G, Blancuzzi V, Singh HN, Kraus KH, O'Byrne E, Pellas TC. Fluorescently labeled mesenchymal stem cells (MSCs) maintain multilineage potential and can be detected following implantation into articular cartilage defects. Biomaterials. 2002;23((1)):109–19. doi: 10.1016/s0142-9612(01)00086-2. [DOI] [PubMed] [Google Scholar]
- 25.Emans PJ, Pieper J, Hulsbosch MM, Koenders M, Kreijveld E, Surtel DA, van Blitterswijk CA, Bulstra SK, Kuijer R, Riesle J. Differential cell viability of chondrocytes and progenitor cells in tissue-engineered constructs following implantation into osteochondral defects. Tissue Eng. 2006;12((6)):1699–709. doi: 10.1089/ten.2006.12.1699. [DOI] [PubMed] [Google Scholar]
- 26.Vonk LA, van Dooremalen SFJ, Liv N, Klumperman J, Coffer PJ, Saris DBF, Lorenowicz MJ. Mesenchymal stromal/stem cell-derived extracellular vesicles promote human cartilage regeneration in vitro. Theranostics. 2018;8((4)):906–20. doi: 10.7150/thno.20746. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Li S, Stöckl S, Lukas C, Götz J, Herrmann M, Federlin M, Grässel S. hBMSC-derived extracellular vesicles attenuate IL-1β-induced catabolic effects on OA-chondrocytes by regulating pro-inflammatory signaling pathways. Front Bioeng Biotechnol. 2020;8:603598. doi: 10.3389/fbioe.2020.603598. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Qi H, Liu DP, Xiao DW, Tian DC, Su YW, Jin SF. Exosomes derived from mesenchymal stem cells inhibit mitochondrial dysfunction-induced apoptosis of chondrocytes via p38, ERK, and Akt pathways. In Vitro Cell Dev Biol Anim. 2019;55((3)):203–10. doi: 10.1007/s11626-019-00330-x. [DOI] [PubMed] [Google Scholar]