2.1 Harvesting of injured ACL tissue and cell culture
In this study, we aimed to investigate the effect of hUSC-EX treatment on chronically injured ACL cells. The injured ACL tissues were harvested from skeletally mature New Zealand male rabbits (n = 6) weighing 2.5–3.0 kg 8 weeks after ACL resection. The whole process was approved by the IACUC(KMU No.110232).
In brief, the rabbits were anesthetized via intramuscular injections of 40 mg/kg ketamine and 10 mg/kg xylocaine and maintained under gaseous anaesthesia of isoflurane (2%)/O2.[27, 28] After the knee joint was exposed via a medial parapatellar approach, the ACL was detached from the femoral insertion.[11] The injured ACL tissues were harvested 8 weeks after the initial surgery. Subsequently, the ACL tissues were digested overnight using 5 mg/ml type I collagenase (Sigma-Aldrich, St. Louis, MO, USA) in low glucose Dulbecco’s modified Eagle’s medium (DMEM) (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) in a 5% CO2 incubator at 37℃\ and sub-cultured to obtain injured ACL cells.[29, 30] Passage three injured ACL cells were used for subsequent experiments.
2.2 Preparation and lyophilization exosomes derived from Umbilical Stem Cells
In the present study, the applied exosomes were derived from the culture medium of human umbilical stem cells. Human umbilical cord harvest, stem cell culture, exosome isolation, and lyophilization were conducted by “Precision Biotech Taiwan Corp” (product no. 1110817001).
2.2.1 Harvest of Human Umbilical Cords Stem Cells (hUSC)
Human umbilical cords were obtained from healthy mothers with full-term fetuses at Tri-Service General Hospital, Taiwan, with all donors providing informed consent before delivery. This study was approved by the Ethics Committee of Tri-Service General Hospital (approval no. A202205014). The method used for hUSC isolation and qualification were described in previous papers.[31, 32]
2.2.2 Prepare the lyophilized hUSC Exosomes (hUSC-EX)
The hUSC-EXs were obtained as described previously, with modificationsr.[33] Briefly, we cultured hUSCs in conditioned medium (Dulbecco’s modified Eagle medium (DMEM) containing nutrient mixture F-12 (F-12; Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific), 1% P/S, and 10 ng/mL basic fibroblast growth factor (bFGF; PeproTech, Cranbury, NJ, USA))[33], seeded them in a Nunc™ Cell Factory™ System (Thermo Fisher) at a density of 3000 cells/cm2, and incubated at 37℃ with humidified 5% CO2. When the cells reached 90% confluence, the culture suspension was collected for exosome isolation by serial centrifugation. The culture suspension was transferred to conical tubes for centrifugation at 300×g for 10 minutes at 4℃ to obtain the pellet. The supernatant was centrifuged at 2,000 ×g for 10 minutes at 4°C for the second time to remove cell debris, followed by centrifugation at 10,000 ×g for 30 minutes at 4°C for the third time to remove apoptotic bodies and other organelles. Finally, the supernatant was centrifuged at 120,000 ×g for 90 minutes at 4°C to obtain the exosome pellet.
The pellet was then resuspended in PBS and centrifuged again at 120,000 ×g for 90 minutes at 4°C for lyophilization preparation.[26] The lyophilization process was carried out as follows: the exosomes were prepared in distilled water and then frozen at -50°C to allow conversion to ice. Subsequently, over a period of 3 days, the pressure was reduced, and heat was applied to prevent sublimation of the frozen water in the material, resulting in a dry, structurally intact powder product. The final products were aseptically aliquoted into bottles and stored at 2°C ~ 30°C (Fig. 1).
2.2.3 Qualification of the lyophilized hUSC-EXs
The hUSC-EX powder was resuspended in PBS and subsequently quantified for size distribution and exosome markers.
2.2.3.1 Size distributions
Particle size measurements and data analysis were performed with a particle analyzer (qNano platform, iZON® Science) and Control Suite software v2.2 (iZON® Science), respectively, according to the manufacturer's protocol.
2.2.3.2 Exosome markers
We investigated the presence of exosomes positive for the markers Alix, TSG101, CD9, CD63, and CD81 and negative for the marker α-Tubulin.[34–37] The primary antibodies used in this study included CD9 (cat#60232-1-1AP), CD63(cat# 25682-1-AP), CD81 (cat# 66866-1-AP), Alix (cat# 12422-1-AP), TSG101 (cat #14497-1-AP), and α-Tubulin (cat# 66031-1-Ig) and were obtained from Proteintech (Chicago, IL, USA). The labeled proteins were visualized using a ChemiDoc™ XRS imaging system (Bio-Rad, Hercules, Cal, USA.). Western blotting was performed using a standard protocol.
2.3 Investigation of the change in cell activities of 8 weeks injured ACL cells after hUSC-EX uptake
2.3.1 Confirming the hUSC-EX uptake and internalization by injured ACL cells
hUSC-EXs were added to the 8 weeks injured ACL cells (1010 EVs particles/104 cells) culture medium for 6 h. The treated cells were washed with PBS and fixed in 4% paraformaldehyde for 10 min. After washing with PBS twice, 100 µL of 1X Phalloidin conjugate working solution (cat# ab176753; Abcam Cambridge, MA, USA) was added to stain the cytoskeleton of the treated cells. The cell slides were mounted, counterstained with Hoechst 33342 (Sigma-Aldrich, St. Louis, MO) and observed via confocal microscopy (Olympus IX-81-FV100, Olympus, Tokyo, Japan).
2.3.2 Changes in cell activity (viability, proliferation, migration, and gene expression) after 8 weeks of injury in ACL cells after hUSC-EX uptake.
2.3.2.1 Cell viability
The cell viability of 8 weeks injured ACL cells was assessed in two groups: those treated with hUSC-EXs (n = 6) and those not treated with exosomes (control group; n = 6). The evaluation was performed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Cat. No. M2003; Millipore Sigma).[29] We measured the 570 nm absorbance using a Bio-Rad Microplate Manager Benchmark Plus Reader (Bio-Rad Laboratories, USA).
2.3.2.2 Cell proliferation: EdU, Ki67 gene expression
The cell proliferation rate was measured with a Click-iT EdU (cell proliferation) assay kit (Thermo Fisher Scientific, Waltham, MA, USA) and the expression of the Ki67 gene, was assessed following the manufacturer’s instructions. The cell slides were mounted and counterstained with Hoechst 33342 (Sigma-Aldrich, St. Louis, MO, USA). The cell proliferation rate was calculated for five randomized areas per sample using ImageJ (64-bit Java v. 1.6.0_24; National Institutes of Health (NIH), Bethesda, Maryland, USA). The expression of the Ki67 gene was calculated and is described in the RT-PCR section.
2.3.2.3 Transwell migration
The transwell migration assay was performed in a 6.5-mm Transwell™ chamber with an 8-µm pore diameter (Millipore EMD, Billerica, MA, USA). These cells were seeded at a density of 3 × 104 cells/per well in the upper chamber compartment with serum-free medium. DMEM supplemented with 10% (v/v) FBS was added to the lower chamber as a chemoattractant. After incubating at 37°C for 20 h, the cells were migrated to the lower membrane. The migrated cells were fixed and counted in each chamber under microscope observation. The relative migration rate was calculated as the ratio of the migration of each treatment group to that of the control group (% of the control).
2.3.2.4 Scratch assay
The hUSC-EX treated ACLs and cells not treated with exosomes were seeded at a density of 3 × 105 cells/per plate and incubated at 37°C with 5% CO2. When the cells reached > 90% confluence, the plate was scratched with a sterile 200-µL pipette tip. Cell migration status was observed under a microscope by closing the scratch gap at regular intervals (6 h, 12 h, 18 h, and 24 h). The relative changes in the cell migration rate were calculated with ImageJ (64-bit Java v. 1.6.0_24; National Institutes of Health (NIH), Bethesda, Maryland, USA).
2.3.3 Changes in immunofluorescence and gene expression:were evaluated for the following: Collagen I and III, VEGF, TGFΒ, tenogenic markers in 8 weeks injured ACL cells after hUSC-EX uptake.
We assessed the metabolic activity of injured ACL cells after hUSC-EX uptake by quantitatively measuring immunofluorescence and RT-PCR for Collagen Type I and III, TGFβ, VEGF and tenogenic markers (Tenascin C (TNC), and Tenomodulin (TNMD)).
2.3.3.1 Immunofluorescence
The hUSC-EX treated ACL cells and non-exosome treated cells were seeded at a density of 2 × 104 cells/well. After 24h of culture, the cells were fixed in 4% paraformaldehyde in PBS for 20 min, permeabilized with 0.5% Triton X-100 in PBS for 10 min and blocked with 5% bovine serum albumin (BSA) in PBS. These cells were assessed for Collagen types I and III, TGFβ, and VEGF expression. These cells were stained with primary antibodies (all from Arigo Biolaboratories, Hsinchu, Taiwan; 1:200) overnight at 4°C, and then stained with the following fluorescent secondary antibodies: donkey anti-goat IgG (H + L)-FAM (Leadgene Biomedical, Tainan, Taiwan; 1:250) for Collagen types I and III and goat anti-mouse IgG (H + L)-TAMRA (Leadgene Biomedical; 1:250) for TGFβ and VEGF for 1h. The cells were subsequently rinsed thrice with PBS.[29] The slides were counterstained with DAPI (Thermo Fisher Scientific), mounted, and observed under a confocal microscope (Olympus FV-100, Olympus America, Inc)
2.3.3.2 RT-PCR
Total RNA from the ACL cells treated with hUSC-EXs (n = 6) and without exosomes (n = 6) was extracted using RNAzol reagent (Cat. No. RN-190; Molecular Research Center, USA), and 2 µg of total RNA was reverse-transcribed using the Maxima First Strand cDNA Synthesis Kit (Cat. No. K1642, Thermo Fisher Scientific) according to the manufacturer’s instructions and described by Lu.[38] Real-time polymerase chain reaction (PCR) was carried out using SYBR Green PCR Master Mix (Cat. A25780, Thermo Fisher Scientific). The complementary DNA samples were amplified using primers according to Lu’s study on ACL cells.[29] The cycling procedure was as follows: 95°C for 15 minutes, followed by 40 cycles of 95°C for 15 seconds, 60°C for 20 seconds, and 72°C for 30 seconds. Threshold cycles (Ct) for each gene tested were normalized to the housekeeping gene GAPDH value (ΔCt), and every experimental sample was referred as its control (ΔΔCt). The experimental data of the hUSC-EX treated ACL cells are expressed as fold-changes (2 − ΔΔCt) compared to those of the nonexosome treated ACL cells, which were set as 1.
Statistical analysis
The differences between hUSC-EX treated ACL cells and non-exosome treated cells were analyzed using the t-test. All datas were presented as mean ± standard deviation (SD) with triple measurements. Statistical significance was set at p < 0.05. All statistical analyses were performed using SPSS software version 20 (IBM, USA).