Joint disease is one of the most prevalent disorders in both companion and athletic horses, which occurs by continuous injuries in joint tissues including the cartilage, synovium, and subchondral bone [1
]. Articular cartilage is a hyaline tissue that enables bones to contact and glide over, ultimately providing resilience, support, and movement with almost no friction [2
]. Lameness caused by joint injuries can lead to reduced training days, decreased performance, and ultimately an early retirement. It was reported that a substantial number of thoroughbred horses that had been euthanized were diagnosed with arthritis in metacarpophalangeal joints [3
]. Similarly, one prospective observational study revealed that the occurrence of injuries in metacarpo- and metatarso-phalangeal joints were an important cause of morbidity, based on the results of daily exercise performance and diagnosis [4
Currently, several medications are available for joint diseases in equine, e.g., hyaluronic acid, chondroitin sulfate, nonsteroidal anti-inflammatory drugs (NSAIDs), and corticosteroids [5
]. However, these methods mostly are effective for reducing the inflammatory symptom. Thus, other alternatives, i.e., cellular therapies using autologous or allogenic origins are now being developed, mostly due to their biological characteristics and therapeutic potential for tissue regeneration [6
]. Recently, Luis et al. reported that repeated intra-articular injection of allogenic adipose-derived MSCs was able to reduce the lameness for 90 days, while reducing the administration of anti-inflammatory drugs [7
]. Other study demonstrated that a combination of chondrogenic-induced MSCs and equine allogenic plasma led to an improvement of lameness score, lower glycosaminoglycan concentration and higher viscosity in synovial fluid in experimental groups [8
]. The successes of MSC-based therapies in equine osteoarthritis (OA) was reported, however, several limitations such as uncontrolled differentiation potential, early cellular senescence in vivo, immunogenicity, and tumorigenicity, remains to be solved to make MSCs become safer and therapeutically feasible [9
Extracellular vesicles (EVs) are cell-derived nanoparticles enclosed with lipid-bilayer membrane, and it is being recognized that they play multiple role in intercellular communication [11
]. They contain various bioactive cargos such as various species of RNA (messenger RNA and small RNA), proteins, enzymes, lipids, and short DNA sequences [12
]. Given that EVs contain bioactive molecules that can represent the physiological or molecular characteristics of the original cells, the potential of their use as an alternative way as cell-free therapeutics has been recently increased [14
]. Not surprisingly, the therapeutic function of MSC-derived EVs on joint or cartilage diseases have been reported in various studies conducted in vitro and in vivo. Mechanistically, their therapeutic potentials have been reported to be mainly due to the ability to enhance the proliferation and attenuate the apoptosis of chondrocytes, and to modulate the immune reactivity [15
]. For example, treatment of MSC-derived exosomes led to an activation of ERK and AKT signaling in cultured chondrocytes in vitro [17
]. Also, infiltration of M2 over M1 macrophages was increased in the osteochondral tissue in a surgical defect created on the model [18
]. In addition, EVs from mouse BM-MSCs was able to reduce the progression of osteoarthritis in a collagenase-induced arthritis model, as indicated by the reduction in osteoarthritis damage and apoptotic cells, together with a significant improvement of cartilage tissue regeneration. Additionally, it was shown that exosomal miR-92a-3p inhibited the expression of WNT5A expression, which led to a reduction in cartilage degradation [19
]. Another study reported that EVs from mouse BM-MSCs inhibited the proliferation of T lymphocytes in a dose-dependent manner, and that contributed to formation of a fewer plasmablasts and more Breg-like cell in lymph nodes [20
Despite the potential usefulness of cell-derived nanoparticles or EVs for cartilage regeneration, no study has conducted on their reparative role in equine chondrocytes. Herein, we isolated and investigated the basic characteristics of equine bone marrow-derived cells (BMCs) from fetal bone marrow. Subsequently, the stimulatory effect of BMC-derived nanoparticles (BMC-NPs) on the growth as well as the survival of apoptotic equine chondrocytes under inflammatory damage were demonstrated.
2. Materials and Methods
2.1. Isolation and Culture of Bone Marrow Cells (BMCs)
This study was approved by Institutional Animal Care and Use Committees of Seoul National University (SNU-171103-2), and was conducted in accordance with approved guidelines. The pregnancy of a 3-year-old Jeju mare was terminated on day 190 upon detecting several complications, including lack of fetal mobility, nearly absent fetal heartbeat, and severe uterine torsion. Briefly, the mare was administered xylazine (0.8 mg/kg, intravenously [iv]), followed by diazepam (0.04 mg/kg, iv), and ketamine (2.2 mg/kg, iv). After sedation, anesthesia was maintained with isoflurane (2.0–2.5%) in oxygen. The uterus was exteriorized using the ventral midline approach and the fetus was pulled out, followed by closure of the uterus and abdomen. The gross morphology of the fetus was normal, without any lesions of microbial infection or severe tissue necrosis. After both proximal and distal ends of the femurs were cut open, the BMCs were harvested through flushing with a 50 mL DMEM (Dulbecco’s modified eagle medium, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (Fetal bovine serum, Altas Biologicals, Fort Collins, CO, USA) and 1% Antibiotics-Antimycotics (Genedirex, Taoyuan, Taiwan) using a syringe attached with a 16-gauge needle. Harvested BMCs were cultured in MesenPRO™ RS (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 0.1% Mycozap (Lonza, Basel, Switzerland). Cells were replenished with fresh culture medium every four days. When the cell growth reached 90% confluence, BMCs were split into 1:4 by being treated with 0.05% Trypsin-EDTA (Genedirex, Taoyuan, Taiwan). The cells passaged 8–10 times were used in the experiments.
2.2. Culture of Equine Chondrocytes
Equine primary chondrocytes (purchased from Cellider biotech, Zaragoza, Spain) were cultured in DMEM (Thermo Fisher Scientific, Waltham, MA, USA) with 10% FBS with 1% Antibiotics-Antimycotics (Genedirex, Taoyuan, Taiwan) under 5% CO2 condition at 37 °C. When the cells reached 90% confluence, chondrocytes were split into 1:2 or 1:3 by being treated with 0.05% Trypsin-EDTA (Genedirex, Taoyuan, Taiwan).
2.3. Co-Culture of Equine Chondrocytes with BMCs
Chondrocytes and BMCs (3.0 × 105 cells/well for both cells) were separately cultured overnight on the bottom and the upper insert, respectively, in a 6-well transwell system (SPLInsert™ Hanging, SPL Life Sciences, Pocheon-si, Korea). After overnight culture, upper insert (BMCs) were subsequently hanged over the well where chondrocytes are being cultured. Afterwards, chondrocytes were co-cultured with BMCs in serum-free DMEM (Thermo Fisher Scientific, Waltham, MA, USA) for designated periods. Chondrocytes cultured in DMEM supplemented with (2.0%) or without FBS were used as positive and negative controls, respectively. To measure the growth of chondrocytes, the cell viability was measured by using the CellTiter-Glo 3D cell viability assay kit (Promega, Madison, WI, USA). The intensity of luminescence by cellular ATP was measured by Cytation 5 (BioTek, Winooski, VT, USA).
2.4. Flow Cytometry
BMCs were trypsinized and washed twice before resuspension in PBS containing 4% FBS. All stainings were performed using 1 × 106 in 100 μL. Cell suspensions were incubated at 4 °C for 30 min with antibodies (1:100) against mouse anti-human CD29 (clone TS2/16, BioLegend, San Diego, CA, USA), mouse anti-CD34 (clone 4H11, Invitrogen, Carlsbad, CA, USA), mouse anti-CD90 (clone MRC OX-7, Abcam, Cambridge, UK), mouse anti-human CD105 (clone SN6, Bio-rad, Hercules, CA, USA), and rat anti-CD44 (clone IM7, Invitrogen, Carlsbad, CA, USA). Goat anti-mouse IgG H&L Dylight 488 (Abcam, Cambridge, UK) and anti-rat IgG Alexa Fluor 488 (BioLegend, San Diego, CA, USA) were used for secondary antibodies. The reactivity was analyzed using BD FACS Canto™ II Cytometer and FACS DIVA software (Ver 6.1.3, BD Bioscience, Franklin Lakes, NJ, USA).
2.5. Cytokine Treatment in BMCs and qRT-PCR
BMCs (4 × 105
cells in 6-well plate) were treated with TNF-α (10 ng/mL, Peprotech US, Cranbury, NJ, USA) or IL-1β (10 ng/mL, Peprotech US, Cranbury, NJ, USA) for 24 or 48 h, and then the total RNA was extracted by Trizol®
(Invitrogen, Carlsbad, CA, USA). The concentration of total RNA was measured using DeNovix DS-11 (DeNovix, Wilmington, DE, USA), and then RNA was reverse transcribed with cDNA Synthesis Kit (PhileKorea, Daejeon-si, Korea). qPCR was performed using the AccuPower®
2X GreenStar qPCR Master Mix (Bioneer, Daejeon-si, Korea) in StepOne™ Real-Time PCR System (Applied Biosystems, Waltham, MA, USA). After the expression of each gene was normalized against Gapdh, the relative expression of each gene was calculate by the 2−ΔΔCt
]. The sequences of primers are listed in Table S1
2.6. In Vitro Differentiation of BMCs
BMCs were plated in triplicate in 4-well plate (SPL Life Sciences, Pocheon-si, Korea) for chondrogenic a pellet cultured incubated at 37 °C and 5% CO2. After one day, the culture medium was removed, and then replaced with StemPro chondrogenesis (Thermo Fisher Scientific, Waltham, MA, USA) medium. Osteogenic and Adipogenic differentiation were incubated at 37 °C and 5% CO2. After reaching 80% confluence, the culture medium was removed, and StemPro osteogenesis and StemPro adipogenesis (Thermo Fisher Scientific, Waltham, MA, USA) were added to the cultures. All differentiation medium was renewed every three days, and their differentiation potential was examined after 15 days of differentiation. Chondrogenic differentiation was examined by staining with Alcian Blue staining kit (Lifeline Cell Technology, Frederick, MD, USA) to identify sulfated proteoglycans deposits. Osteogenic differentiation was examined by staining with 2% Alizarin Red staining kit (Lifeline Cell Technology, Frederick, MD, USA) to identify calcium deposits. Adipogenic differentiation was examined by staining with Oil Red O staining (Sigma-Aldrich, St. Louis, MO, USA) to identify lipid droplets.
2.7. Establishment of In Vitro Model of Chondrocyte Injury
Equine chondrocytes were cultured in DMEM supplemented with 0.1% FBS for 24 h. Cells were then harvested and re-seeded with various concentration of TNF-α or IL-1β (0.1, 1, 10, 100, and 200 ng/mL, Peprotech US, Cranbury, NJ, USA) in FBS-free DMEM for 24, 48, or 72 h. The viability of cells were measured by using CCK-8 assay.
2.8. CCK-8 Assay
Other than the co-culture experiments between chondrocytes and BMCs (Section 2.3
), all analysis of cell viability/proliferation was measured by Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan). Cells were seeded into 96-well plate (SPL Life Sciences, Pocheon-si, Korea) at a density of 5.0 × 103
cells per well with 100 μL medium and incubated at 37 °C. At the designated time point, 10 μL CCK-8 solutions were added. After further incubation for 3 h at 37 °C, the amount of formazan generated by cellular dehydrogenase activity was measured (450 nm) by a microplate reader (TECAN, Mannedorf, Switzerland).
2.9. Collection of NPs
EV-depleted FBS was prepared by ultracentrifuge at 40,000× g for 8 h at 4 °C. Upon reaching 80% confluency, the culture media from BMCs were replaced with fresh medium supplemented with EV-depleted FBS (2%) and the cells were subsequently cultured for an additional 48 h. After incubation, the BMC culture medium was harvested, centrifuged 2000× g for 20 min at 4 °C, and the supernatants were filtered through 0.2 μm pore filters to remove the particle larger than 200 nm. And then, the supernatants were concentrated using Vivaspin 20 (100,000 MWCO) (Sartorius, Gottingen, Germany). Next, NPs were isolated by using Exo2 D™ Kit (Exosome plus, Suwon-si, Korea). The mixture was mixed by rocking for 30 min at 4 °C, followed by centrifugation at 3000× g for 30 min at 4 °C. Pellet was then resuspended in EV-free PBS that had been filtered through a 20 micrometer-pose sized syringe filter. The protein concentration was measured by the Pierce™ BCA Protein Assay kit (Thermo Fisher, Waltham, MA, USA). The aliquots were then stored at −80 °C.
An aliquot (4 μL) of BMC-NPs were applied to glow-discharged (Glow discharge system, PELCO EasiGlow™, TED PELLA, Redding, CA) carbon-coated grids (Quantifoil, R2/2, 200 mesh, EMS, Hatfield, PA, USA). After the grids were then blotted for 90 s at 4 with 100% humidity, the samples were plunge-frozen for vitrification (Vitrobot mark IV, FEI, Hillsboro, Oregon). Images were collected on TEM microscope (Talos L120C, FEI, Hillsboro, Oregon) at 120 kV.
2.11. NTA Assessment
NPs were suspended EV-free distilled water, and then their particle size and concentration were measured using NanoSight300 (Malvern Panalytical, Malvern, UK).
2.12. Apoptosis Assay
Chondrocytes (2 × 105 cells in 6-well plate) were treated with TNF-α (200 ng/mL) or IL-1β (200 ng/mL) for 24 or 48 h in the presence or absence of BMC-NPs (100 μg/mL). The apoptosis was measured with the Annexin V-FITC Apoptosis Detection Kit I (BD Bioscience, Franklin Lakes, NJ, USA). Cells were trypsinized and washed twice with cold PBS and then resuspended 1 × 106 cells in 500 μL of binding buffer. Five microliters of Annexin V-FITC and 5 μL of PI (Propidium Iodide, 1 mg/mL) were added into the cell resuspended for 30 min at room temperature in the dark. The cells were analyzed BD FACS Canto™ II Cytometer and FACS DIVA software (Ver 6.1.3, BD Bioscience, Franklin Lakes, NJ, USA).
NPs and cellular protein total protein concentration evaluated by PierceTM BCA Protein Assay Kit (Thermo Fisher). NP and cell lysates were loaded into each well and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, 10%). The protein bands were transferred to nitrocellulose membranes after which were blocked in 5% skim milk or 5% BSA. Primary antibodies (dilution 1:1000) against CD 63 (Abcam, Cambridge, UK), HSP 70, phospho-AKT, AKT, phospho-ERK, ERK (Santa Cruz, Dallas, TX, USA), or β-Actin (Abcam, Cambridge, UK) were incubated with the membrane at 4 °C overnight. After being washed three times with TBST (TBS with 0.1% Tween20®), membranes were incubated with horseradish peroxidase-linked anti-mouse or anti-rabbit secondary antibody for 1 h at room temperature. After being washed three times with TBST for 15 min each, the reactivity was examined by an enhanced chemiluminescence kit (Thermo Fisher Scientific, Waltham, MA, USA). The image of the membrane was taken using UV or white light on a Davinci-K Gel Imaging System (Davinch-K, Seoul, Korea). The density of bands was quantified by Image J (Version 1.50, National Institutes of Health, Bethesda, MD, USA).
2.14. BMC-NPs Uptake Study
Equine chondrocytes were cultured for 2 days at a density of 1.0 × 104 cells per well in 8-well coated cover slides (SPL Life Sciences, Pocheon-si, Korea). BMC-NPs were labeled with PKH26® red fluorescent dye according to the manufacturer’s instruction (Sigma-Aldrich, St. Louis, MO, USA). Labeled NPs (10, 50, 100 μg/mL) were co-cultured with equine chondrocytes for 24 h. For counterstaining, the nuclei and F-actin were stained with 4′,6-diandino-2-phenylindole (DAPI) (Sigma-Aldrich, St. Louis, MO, USA) and phalloidin (Abcam, Cambridge, UK), respectively. Images were obtained using the Cytation 5 (Biotek, Winooski, VT, USA).
2.15. Statistical Analysis
All data were presented as mean ± S.D. of at least three replications. For pairwise comparison, Student’s t-test was used. For more than three groups, one-way analysis of variation (ANOVA) followed by Tukey’s multiple comparison tests were conducted. All analyses were conducted by using GraphPad Prism software (Ver. 5.0 GraphPad Software, San Diego, CA, USA). P values less than 0.05 were considered as significantly different.
During last several decades, the reparative role of MSCs in joint diseases has been evidenced by various preclinical studies [25
]. However, the possibility of immune rejection from allogenic origin, as well as the risk of tumor generation, can become a significant concern, which necessitates the development of other strategy equipped with less safety issue [27
]. Also, difficulties in long-term storage and maintenance, while sustaining its consistency and viability, should be overcome to make these cells feasible for use in veterinary practice [28
]. Initially, studies reported that MSCs take part in cartilage repair by the unique ability of MSCs to differentiate into several mesenchymal lineages, i.e., chondrocytes, which may replace the dead or injured chondrocytes [29
]. However, it is now becoming accepted that the secretome of MSCs also plays a vital role in the repair of injured chondrocytes [30
]. Since then, EVs including exosomes or microvesicles, which occupies a certain part of secretome, were found to be efficacious against cartilage regeneration. In particular, MSC-EVs have been effective in cartilage regeneration in a rodent model of osteoarthritis [18
]. Despite such success and potential uses of MSC-EVs, no attempt was made on the use of MSC-derived nanoparticles or in cartilage repair in equine species. In this study, we investigated whether BMC-NPs have potential to stimulate the growth of equine chondrocytes, and found that BMC-NPs were readily taken up by equine chondrocytes, and that they promoted the proliferation of chondrocytes. Further, we found that BMC-NPs were able to rescue the apoptotic death of chondrocytes. Finally, we demonstrated that BMC-NPs led to an increased level of phosphorylated AKT as well as a reduction of ERK1/2 activation in equine chondrocytes that are under inflammatory stimuli.
Studies found that cellular characteristics of MSCs from fetal and adult tissues differ, and several advantages can be obtained from MSCS from fetal origin. It was reported that fetal MSCs have an increased proliferative activity [39
], and an better therapeutic effects such as enhanced anti-inflammatory capacity, fitness, and homing ability compared with those obtained from adults [39
]. It was also reported that in human, the ratio of the number of MSCs in newborns and aged adults are one out of a thousand and two million, respectively [43
]. Thus, MSCs of fetal origin are found at a higher frequency in tissues than in adult MSCs, and are readily available in fetal and extra fetal tissues such as fetal liver, umbilical cord, umbilical cord blood, placenta, and amniotic fluid [39
]. In addition, studies showed that fetal MSCs are more effective in avoiding immune recognition than adult MSCs [46
]. Specifically, fetal MSCs are known to be less immunogenic than adult MSCs, because fetal MSCs have a lower surface of HLA Class I [41
], and they do not express HLA class II on their surface. Lastly, fetal MSCs can be more suitable for obtaining specific mesenchymal lineages, e.g., due to an increased osteogenic potential compared with adult MSCs [49
]. Indeed, a comparative study showed that many osteogenic genes were more abundantly expressed fetal than adult MSCs [50
]. The differences in the potential of growth, differentiation, and immunoregulation between adult bone marrow-derived MSCs and fetal MSCs or BMCs remain unexplored in horses, thus further side-by-side comparison between two cell types is needed.
Signals downstream of stem cell-derived exosomes are often mediated via AKT or ERK pathways in various cells including chondrocytes [17
]. For example, recent study demonstrated that exosomes from human embryonic stem cell-derived MSCs were able to facilitate the repair of osteochondral defect by 1) stimulating the proliferation by activating AKT signaling, 2) decreasing the apoptotic death of chondrocytes in the chondral tissue as well as IL-1β and TNF-α in synovial fluid in rats [18
]. We found that BMC-NPs induced an increase of AKT signaling, while reduced the activity of ERK in the presence of IL-1B and TNF-α, respectively. These results suggest that BMC-NPs can promote the survival of chondrocytes via AKT, and that they also can inhibit the TNF-α/ERK1/2 inflammatory pathway. Further studies are warranted to clearly delineate the mechanisms how BMC-NPs can reduce the cell death induced by inflammatory cytokines in equine chondrocytes.
Studies have shown a varying degree of cell surface marker expression in horse MSCs. It was reported that the reactivity of CD90 was 67.7% in umbilical cord-derived-MSCs [54
], while more heterogeneous findings are available regarding the population of CD105-postivie cells among study groups; Barberini et al. showed that adult MSCs from bone marrow, adipose tissue, and umbilical cord tissue was positive for CD105 [54
], while other group reported that CD105 was not detectable in MSCs from bone marrow and adipose tissue [55
]. Other study showed that adipose tissue-derived CD105+
MSCs were within the range of 21 to 74.4%, depending on the tissue sources (subcutaneous and intraperitoneal tissues) and passages [7
]. Meanwhile, our results on the expression of CD29 and CD44 was comparable against other studies [55
]. An extensive, comparative study also showed that the expression of CD29, CD90, and CD105 was 48.4, 11.4, and 35%, respectively, in adipose tissue derived MSCs, while 52.9, 0.3, 33.7%, respectively, in tendon-tissue derived MSCs [57
]. Not surprisingly, these results demonstrate that immunophenotyping results differ significantly among investigations, indicating that various factors including cell sources and protocols for isolation and culture may account for these differences. Also, it should be considered that a more detailed studies for determining the specificity of common epitope and responding antibody is needed because the antibodies that work efficiently on equine species are not always commercially available.
Although the guidelines and general consensus are being renewed by scientific communities, the protocol for isolating, characterizing, and functional testing of EVs still remains heterogeneous among study groups. In equine species, few reports are available on the identification of MSC-derived EVs [58
]. Mostly, EVs were collected by ultracentrifugation of the culture supernatant obtained during adipose tissue-derived MSCs (Ad-MSCs) culture. An early study showed that membrane vesicles from equine Ad-MSCs, as shown by scanning electron microscopy, had ability to stimulate the growth of endothelial cells [59
]. Regarding the EV markers, it was recently shown that equine Ad-MSC-derived EVs were positive with CD63, CD9, and CD90 [60
], while another study demonstrated the expression of CD90 and Flotillin-1, although the degree of reactivity was not clearly shown [58
]. BMC-NPs from our study were positive with CD63 and HSP70, which are also markers used for exosome characterization [61
]. As for the purification protocol, Klymiuk et al. reported that ultrafiltration method is superior to other methods (ultracentrifugation or precipitation) for isolating equine ADSC-exosomes in terms of the total yield as well as the aggregation issue during resuspension, although the biological functions of EVs from each preparation were not tested. The particle diameter was 178.7 ± 62.3 and 116.2 ± 38.3 nm in equine ADSC-exosomes isolated by ultracentrifugation and ultrafiltration, respectively [60
], creating a significant difference on their diameter depending on the isolation methods. The method used in our present study is based on the aqueous two-phase system (ATPS), which is based on the incompatibility-based separation between the phases of two polymeric molecules, polyethylene glycol (PEG) and dextran [65
]. Our data on the diameter of BMC-NPs was slightly larger than the previous study [60
], indicating different physical characteristics among investigations. Since the isolation protocol can often create biological differences, standardization and further optimization is needed to improve the yield and to minimize the changes among batches.
Also, determining the optimal regime of nanoparticle treatment, e.g., concentration, frequency, and total dosage, is a critical not only for maximizing the therapeutic effect but also for reducing the toxicity. We noticed that higher concentration (100 μg/mL) of BMC-NPs led to a reduced cell survival compared with those treated with 50 μg/mL at 72 h. Given that the effects of BMC-NPs on injured chondrocytes were examined in the absence of other survival factors (e.g., FBS), the reduced survivability at high BMC-NP concentration may be related with toxic effect of lipid components (e.g., ceramide) that is derived from biogenesis processes [67
]. Thus, it is suggested that further optimization is needed to minimize the toxic effect that can be possibly caused by a higher dosage of BMC-NPs.
Together, our results demonstrate that NPs from equine fetal bone marrow-derived cells can simulate the growth as well as the survival of apoptotic chondrocytes under inflammatory stimuli. The therapeutic effect of equine fetal BMC-NPs may be utilized to develop a novel strategy for treating joint disease in horses.