PRP-Exos extraction

Exosomes were extracted carefully from PRP using the exoEasy Maxi Kit (cat. no. 76064). It was recommended to use only pre-centrifuged PRP. Briefly, PRP obtained as described above was centrifuged in conical tubes for 15 min at 3000×g at 4 °C to remove additional cellular fragments and cell debris. The cleared supernatant was carefully transferred to a new tube without disturbing the pellet, which formed a smear along the outer side/bottom of the centrifugation tube. Then, 1 volume of buffer XBP was added to 1 volume of PRP. The sample was mixed well by gently inverting the tube five times to allow the mixture to warm to room temperature. After that, we obtained a total of 16 mL of a mixture of PRP/XBP, which was added onto the exoEasy spin column and centrifuged at 500×g for 1 min. After discarding the flow-through, we placed the column back into the same collection tube. The above steps were repeated until the entire volume was no more than 8 mL, and the entire volume was centrifuged at 5000×g for 1 min to remove residual liquid from the membrane. Then, 10 mL of buffer XWP was added to the volume, and the volume was centrifuged at 5000×g for 5 min to remove residual buffer. Then, the spin column was transferred to a fresh collection tube, and the flow-through and collection tube were discarded. Next, 400 μL Buffer XE was added to the membrane and incubated for 1 min. The eluate was collected by centrifuging at 500×g for 5 min and once again was added to the exoEasy spin column membrane and incubated for 1 min. Finally, the eluate was gathered after centrifuging at 5000×g for 5 min. The eluate was used for isolation of exosomes by the exoEasy Maxi Kit [47–49]. All steps were performed at 4 °C. The exosomes were carefully resuspended in sterile PBS and stored at −80 °C for subsequent experiments.

In this experiment, we designed activated PRP and PRP-Exos to observe the effects in treating OA, and each group contained the same total protein content, which was determined by the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific Inc.) and standardized [50]. The activated PRP containing the main active constituents was named PRP-As. To observe the effect on cells in vitro, we used protein concentrations of 5 μg/mL and 50 μg/mL, and the in vivo concentrations were 10 μg/mL and 100 μg/mL [8].

Identification of PRP-Exos

First, Particle Metrix ZetaView® nanoparticle tracking analysis (NTA) technology (Particle Metrix GmbH, Germany) was used to carefully estimate the size and concentration distribution of PRP-Exos. Second, after the exosomes were coated onto a 2 nm copper grid and stained using 2% uranyl acetate, a Hitachi H-7650 transmission electron microscope (TEM) was employed to observe the morphology of exosomes derived from PRP. Finally, Western blotting was used to examine the specific exosome biomarkers CD9, CD63, CD81, and HPS101 [13]. Activated PRP (PRP-As) was prepared to act as a control, according to the methods described by Torreggiani et al. [51].

Chondrocyte isolation and culture

Under aseptic conditions, cartilage slices derived from the terminal of tibia and femur of 4-week-old New Zealand white rabbits, were cut into small pieces, washed with PBS three times, added to 0.25% trypsin (Gibco 25200-056) to digest for 1–2 h, and then digested with 0.2% collagenase II (Sigma V900892) in a 37 °C incubator with 5% CO₂. After overnight digestion, the entire mixture was filtered through a 200 mesh strainer, and the filtrate was centrifuged at 190×g for 5 min. The supernatant was discarded, and the residue was resuspended in the remaining culture medium and plated onto T25 flasks (Corning 430639) with DMEM/F12 medium (Gibco 11320033) containing 10% FBS (Gibco C20012500BT) and 1% penicillin/streptomycin (Gibco 15140-122). The P1 chondrocytes were used for identification, and the P2 chondrocytes were used for the following experiments and preserved at −80 °C [52].

Alcian blue staining and immunocytochemistry of chondrocytes

The articular chondrocytes were identified by alcian blue staining. In brief, P1 chondrocytes were digested with 0.25% trypsin-EDTA and transferred onto glass cover slides. After fixing with 4% paraformaldehyde for 30 min, the chondrocytes were soaked in alcian acidizing fluid for 3 min, stained with alcian dyeing liquid for 30 min, rinsed with PBS, re-stained with nuclear solid red dye for 5 min, and washed with PBS for an additional 1 min. Then, the slides were dehydrated in absolute ethanol with different gradients and rinsed in xylene for 10 min. Finally, the samples were observed under a microscope.

The P2 chondrocytes were harvested and fixed in 4% (w/v) buffered paraformaldehyde solution for 15 min, followed by permeabilization with 0.2% (w/v) Triton X-100 for 10 min. Then, mouse anti-Col II (1:100; EMD Millipore, Cat. #MAB8887) and rabbit anti-ADAMTS5 (1:100; Abcam, Cat. #ab41037) primary antibodies were added to the fixed chondrocytes and incubated overnight at 4 °C. Then, the cells were completely washed with PBS. The corresponding goat anti-mouse IgG (H + L) cross-adsorbed secondary antibody conjugated to Alexa Fluor 488 (1:250; Invitrogen, Carlsbad, CA, USA, Cat. #A11001) and F(ab′)2-goat anti-rabbit IgG(H + L) cross-adsorbed secondary antibody conjugated to Alexa Fluor 555 (1:250; Invitrogen; Cat. #21430) were added to these cells for incubation. Finally, the cell nuclei were counterstained with DAPI (1:5000; Beyotime; Cat. #C1002) and observed with confocal microscopy (Olympus, Tokyo, Japan, BX61W1-FV1000) [53].

Enzyme-linked immunosorbent assay

In vitro, chondrocytes were stimulated with IL-1β (10 ng/mL) in the presence or absence of different concentrations of PRP-As or PRP-Exos for 24 h. The supernatant of the cells was centrifuged and stored at −80 °C until analysis. Tumor necrosis factor-α (TNF-α) was measured by enzyme-linked immunosorbent assay (ELISA) kits from eBioscience (San Diego, CA, USA) according to the manufacturer’s protocol [54]. The plates were read at 450 nm. The experiment was independently repeated three times.

Proliferation of chondrocytes

The proliferation of chondrocytes was observed using a Cell Counting Kit-8 (CCK-8, Dojindo, ck04) [55]. Briefly, P2 chondrocytes at an initial density of 3000 cells/well were cultured with 100 μL of various media containing PBS (as the blank), interleukin 1 (IL-1, as a control), IL-1, and 5 μg/mL or 50 μg/mL PRP-Exos, or IL-1 and 5 μg/mL or 50 μg/mL PRP-As and were plated onto 96-well plates and cultured at 37 °C. At 24, 48, 72, and 120 h, 10 μL CCK-8 solution and 100 μL fresh culture medium was added to each well at each time point, avoiding air bubbles, and the plate was incubated for 1–4 h at 37 °C. Finally, the optical density (OD) values were measured by a spectrophotometric microplate reader (Bio-Rad 680, Bio-Rad, Hercules, CA, USA) at a wavelength of 450 nm. The survival/proliferation of chondrocytes is expressed as the value of the optical density (OD) of the test wells minus the absorbance of the blank wells.

Apoptosis of chondrocytes

We used flow cytometry to detect the apoptosis of chondrocytes in vitro. After different treatments as described previously, the chondrocytes were harvested by centrifugation at 300×g for 5 min, washed twice with PBS at 4 °C, and then centrifuged at 300×g once for an additional 5 min. The chondrocytes were resuspended in 250 μL 1× Binding Buffer and adjusted to 1 × 10⁶ cells/mL. Then, 100 μL of cell suspension was added into 5 mL of fluid and mixed with 5 μL of annexin V/PE and 10 μL of 7-AAD. Afterwards, the sample was gently mixed at 4 °C under darkness for 15 min and analyzed by flow cytometr y[53] within 1 h of being added to 400 μL of 1× Binding Buffer.

Migration of chondrocytes

Chondrocyte migration was assessed with Transwell cell culture chambers (Corning, 3422, diameter 8 μm). Briefly, 2 × 10⁴ cells were added into the upper chamber, with 700 μL of low serum culture medium (DMEM-F12, Gibco 11320033) containing 10% FBS (Gibco, 15240-112). Then, the same volume of “Drugs” containing PBS (as the blank), interleukin 1 (IL-1, as a control), IL-1, and 5 μg/mL or 50 μg/mL PRP-Exos, or IL-1 and 5 μg/mL or 50 μg/mL PRP were added into the lower chamber. After 24-h incubation at 37 °C, the chondrocytes were fixed with 1 mL 4% formaldehyde for 15 min at room temperature. After discarding the fixed liquid, the chondrocytes were washed twice with PBS, and 1 mL of 0.5% crystal violet was added to each well to stain the chondrocytes for 60 min. Then, the chondrocytes were washed with PBS three times. Finally, the upper surface of the Transwell filters was swabbed to remove cells. The chondrocytes were observed and counted at ×100 magnification in five randomly selected fields. The cells in five randomly selected fields at ×100 magnification were counted. The percentage of chondrocyte migration was calculated by normalizing the number of cells on the underside of the filter to the initial number of cells seeded and compared to that of the control, which was set at 100% [56].

Scratch wound assay

The P2 chondrocytes were harvested to analyze the effect of PRP-Exos and PRP on the migration of chondrocytes by the scratch wound assay [57]. Briefly, 5 × 10⁵ cells were seeded onto 6-well plates and incubated at 37 °C overnight to maintain a confluent monolayer of cells. Each well was scratched using the spear along the ruler and washed twice with PBS to remove the free cells. The medium was then replaced with serum-free medium and “drugs,” containing PBS (as the blank), interleukin 1 (IL-1, as a control), IL-1 and 5 μg/mL or 50 μg/mL PRP-Exos, or IL-1 and 5 μg/mL or 50 μg/mL PRP, as described previously. We used an inverted microscope to monitor the wound closure of the chondrocytes at 0, 6, 12, and 24 h after the scratch. The images were obtained at the same position before and after incubation. Scratched areas were measured using ImageJ software.

Western blotting analysis

Western blotting analysis was performed as described previously [58]. Protein from chondrocytes treated with PBS, IL-1β, IL-1β + PRP-As, and IL-1β + PRP-Exos was extracted with RIPA lysis buffer (Solarbio, Beijing, China), and the total protein concentration was determined with a BCA protein assay kit (Pierce, Rockford, IL, USA). Then, 30 μg extracted cellular protein was mixed with loading buffer, denatured in boiling water for 5 min, and separated by 10% (w/v) sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). After electrophoresis, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane and blocked in 5% (w/v) bovine serum albumin (BSA, Sangon Biotech, Shanghai, China) for 1 h at room temperature. The membrane was probed with Wnt5a (1:1000 dilution, Cell Signaling Technology), RUNX2 (1:1000 dilution, Cell Signaling Technology), β-catenin (1:1000 dilution, Cell Signaling Technology), and β-actin (1:1000 dilution, Santa Cruz Biotechnology Inc.) overnight at 4 °C. After washing in Tris-buffered saline with Tween-20 (TBST), the membranes were treated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3000 dilution, Cell Signaling Technology) at room temperature for 1 h. The excess secondary antibody was washed off by TBST, and the chemiluminescent signal was generated by the ECL imaging kit (Thermo Fisher Scientific, Waltham, MA, USA). β-actin was used as an internal control.

Animal model and groupings

Twenty-four male New Zealand white rabbits (4-week-old, 2–3 kg) purchased from Jinan Xinjian biological technology company were utilized to establish the OA model and undergo experiments. All rabbits were randomly divided into four groups: (1) control group (without surgery; received normal saline by intra-articular(IA) injection once a week, n = 6); (2) OA model group (with surgery; received normal saline by intra-articular injection once a week, n = 6); (3) OA + PRP-Exos group (with surgery; received 100 μg/mL PRP-Exos by intra-articular injection once a week, n = 6); and (4) OA + PRP-As group (with surgery; received 100 μg/mL PRP by intra-articular injection once a week, n = 6). The OA model rabbits were anaesthetized by injection of 10% chloral hydrate (4 mL/kg) and underwent surgery of left knee, in which the medial collateral ligament and the anterior cruciate ligament (ACL) were cut off and the medial meniscus was excised [1, 53, 59]. After surgery, penicillin potassium (8 million U/kg, Shandong Lukang Biotechnology, China) was used to prevent infection by intramuscular injection for 7 days. After the operations, all of the rabbits were housed in individual cages at 21 ± 0.5 °C and allowed to move freely for 6 weeks. Soon afterwards, all of the rabbits were treated by intra-articular injection of drugs according to the previously described groups. Six weeks later, all the New Zealand white rabbits were sacrificed by injecting an overdose of air, and the left knee samples were harvested to assess cartilage degeneration and undergo follow-up experiments.

Tissue staining, immunohistochemistry, and OARSI score

The rabbit joints were fixed in 4% buffered paraformaldehyde for 24 h after the samples were harvested, decalcified in 10% (w/v) EDTA (pH 7.4) for 30 days (d) at 4 °C, and embedded in paraffin. The samples were processed at 5 μm thickness by sagittal joint sections and stained with hematoxylin-eosin (HE). The expression of Collagen II and RUNX2 was analyzed by immunohistochemistry. The Osteoarthritis Research Society International (OARSI) scoring syste m[60] was used to evaluate the degree of cartilage destruction according to the percentage of the vertical clefts/erosion in the calcified cartilage and the recommended OA grading table supplied by Glasson et al. Chondrocyte counts at randomly high magnification and OARSI scores were used to evaluate cartilage conditions, and the higher the score, the more serious the destruction of articular cartilage with the total possible score was 24 [61].

Isolation and identification of chondrocytes

The second-generation rabbit chondrocytes presented as triangles, polygons, and spindles after 24 h of culture, and the cells were gradually integrated (Fig. ​(Fig.2a,2a, b). Alcian blue staining was performed to assess proteoglycan deposition in chondrocytes, which presented as blue (Fig. ​(Fig.2c).2c). Immunohistochemistry staining for Col II was performed to identify that the cells we extracted were chondrocytes (Fig. ​(Fig.2d).2d). The nuclei were blue, and the cytoplasm was tan. This staining showed the morphology and properties of chondrocytes.

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Fig. 2

Isolation and identification of chondrocytes: The chondrocytes were obtained from New Zealand white rabbits (4 weeks). Morphology of chondrocytes observed by inverted microscope (a, ×40 and b ×200, bar 100 μm). Identification of chondrocytes performed by Alcian blue staining (c) and immunohistochemistry staining for Col II (d), bar 50 μm

PRP-Exos inhibit the release of TNF-α

To establish the osteoarthritis chondrocyte model in vitro, chondrocytes were incubated with IL-1β (10 ng/mL) in medium without serum for 24 h. We detected the release of the pro-inflammatory cytokines (TNF-α) into the culture medium compared with that of non-stimulated control cells by ELISA (Fig. ​(Fig.3).3). The release of TNF-α was obviously increased. Treatment with PRP-As (5 μg/mL), PRP-Exos (5 μg/mL), PRP-As (50 μg/mL), and PRP-Exos (50 μg/mL) significantly reduced the levels of TNF-α. The strongest effect was exhibited by PRP-Exos (50 μg/mL), followed by PRP-As (50 μg/mL), which was not significantly different. The inhibitory effect of PRP-Exos (5 μg/mL) was better compared with that of PRP-As (5 μg/mL).

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Fig. 3

Cytokine release (TNF-α) of chondrocytes: In vitro, the chondrocytes were co-culture with PBS, IL-1β (10 ng/mL), IL-1β (10 ng/mL) added different concentration of PRP-Exos and PRP-As for 24 h. Osteoarthritis chondrocytes were established with TNF-α release increasing significantly(&<0.05). Both PRP-Exos and PRP-As with different concentration could inhibit TNF-α release significantly (*P < 0.05). Results were detected by ELISA with presenting as mean ± SEM from three independent experiments

PRP-Exos promoted chondrocyte proliferation

In vitro, we explored the cell viability of chondrocytes treated with IL-1β (10 ng/mL) with different concentrations of PRP-Exos and PRP-As by CCK-8 assay. The proliferation of chondrocytes cultured for 24 h, 48 h, 72 h, and 120 h is shown in Fig. ​Fig.4.4. Moreover, PRP-Exos and PRP-As both promoted osteoarthritis chondrocyte proliferation, and the cell viability was inhibited by IL-1β. However, chondrocytes cultured in PRP-Exos (50 μg/mL) proliferated significantly better than those cultured in PRP-As (50 μg/mL), PRP-As (5 μg/mL), and PRP-Exos (5 μg/mL) at all timepoints assessed, but the proliferation of chondrocytes cultured with PRP-Exos (5 μg/mL) showed no significant difference compared with that of the PRP-As (5 μg/mL) groups, which had similar proliferation rates.

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Fig. 4

Chondrocytes proliferation. In vitro, the cell viability of chondrocytes treated with IL-1β (10 ng/mL) togehter with different concentration of PRP-Exos and PRP-As were detected at different times(24 h, 48 h, 72 h, 120 h) by CCK-8 analysis. Quantitative data were presented as means ± standard deviations of three independent experiments, P < 0.05. Both PRP-Exos and PRP-As significantly promoted the proliferation of osteoarthritis chondrocytes induced by IL-1β, *P < 0.05. Beyond that, the effect of proliferation stimulated by PRP-Exos compared with PRP-As in the same protein concentration was significantly better (#P < 0.05)

PRP-Exos inhibit the chondrocyte apoptosis induced by IL-1β

In vitro, apoptosis of the chondrocytes induced by IL-1β (10 ng/mL) with or without PRP-Exos and PRP-As was assessed with annexin V-PE-H staining with flow cytometric analysis (Fig. ​(Fig.5a).5a). The results suggest that the chondrocytes induced by IL-1β had a dramatically higher apoptotic rate than that of the control group. In contrast, the chondrocytes co-cultured with high concentrations of PRP-Exos and PRP-As inhibited the apoptotic effect of IL-1β. Furthermore, the chondrocytes treated with PRP-Exos exhibited a lower apoptotic rate compared to that of the PRP-As group (Fig. ​(Fig.55b).

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Fig. 5

Apoptosis of chondrocytes: In vitro, the apoptosis of the chondrocytes induced by IL-1β (10 ng/mL) with or without PRP-Exos, PRP-As were assessed through Annexin V-PE-H staining with flow cytometric analysis (a). In vitro, IL-1β significantly increased apoptotic rate of chondrocytes (&P < 0.05, compared with control group). PRP-Exos and PRP-As both inhibited apoptosis of chondrocytes (*P < 0.05, compared with IL-1Β group), but the effect of PRP-Exos significantly was better than PRP-As, #P < 0.05.And the low concentration of PRP-Exos and PRP-Ase either obviously inhibited apoptosis of chondrocytes (b)

PRP-Exos promote the migration of chondrocytes

As shown in Fig. ​Fig.6a,6a, IL-1β (10 ng/mL) significantly inhibited the migration of chondrocytes. However, the effect could be reversed by co-culturing the chondrocytes with PRP-Exos and PRP-As. Moreover, higher concentrations of PRP-Exos or PRP-As had a more obvious effect compared with that of the low concentration groups. Furthermore, the chondrocytes treated with PRP-Exos exhibited more migration cells compared to that of the PRP-As group (Fig. ​(Fig.66b).

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Fig. 6

Migration of osteoarthritis chondrocytes: In vitro, the migration of osteoarthritis chondrocytes induced by IL-1β (10 ng/mL) stimulated by different concentration of PRP-Exos and PRP-As were detected by transwell assay. Scar bar: 100 μm (a). Both PRP-Exos and PRP-As significantly promoted the migration of osteoarthritis chondrocytes induced by IL-1β (*P < 0.05, compared with IL-1β group), the effect of migration dramatically responded to the concentration (&P < 0.05). Beyond that, the effect of migration stimulated by PRP-Exos compared with PRP-As in the same protein concentration was significantly better ($P < 0.05, b). Quantitative data were presented as meams ± standard deviations from three independent experiments, P < 0.05

Scratch wound assays (Fig. ​(Fig.7a)7a) were used to detect the motility of chondrocytes and indicated that only PRP-Exos (50 μg/mL) significantly enhanced the motility of chondrocytes induced by IL-1β (10 ng/mL) at both 6 h and 12 h (Fig. ​(Fig.7b),7b), and other groups did not show significant differences compared with each other.

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Fig. 7

Migration rate of chondrocytes. Scratch wound assays presented the migration rate of osteoarthritis chondrocytes induced by IL-1β (10 ng/mL) stimulated by different concentration of PRP-Exos and PRP-As. Scar bar:1 00 μm (a). The PRP-Exos (50 μg/mL) significantly promoted the migration of osteoarthritis chondrocytes induced by IL-1β, compared with other groups (*P < 0.05), not only at 6 h but also at 12 h (b). Quantitative data were presented as meams ± standard deviations from three independent experiments, P < 0.05

PRP-Exos promoted chondrocyte proliferation and migration by the Wnt/β-catenin signal pathway

To verify that the Wnt/β-catenin signaling pathway and the associated effector proteins respond to PRP-Exo stimulation, Western blotting was performed to evaluate the protein levels of β-catenin, RUNX2, and Wnt5a (Fig. ​(Fig.8a).8a). In this experiment, the protein expression levels of β-catenin and Wnt5a were upregulated in chondrocytes exposed to IL-1β; furthermore, RUNX2, a downstream effector protein, was also upregulated. This result indicated that the Wnt/β-catenin signaling pathway is activated and is involved in the development of osteoarthritis. Furthermore, PRP-As and PRP-Exos reduced the protein levels to alleviate osteoarthritis. The PRP-Exo-induced inhibition was more noticeable compared with that of PRP-As for RUNX2 and Wnt5a. In contrast, PRP-As had a stronger effect on β-catenin than that of PRP-Exos (Fig. ​(Fig.88b–d).

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Fig. 8

Responses of chondrocytes to stimulation by PRP-As and PRP-Exos via Wnt/β-catenin signal pathway. The chondrocytes were induced by IL-1β (10 ng/mL) stimulated and protein expression levels of β-catenin, RUNX2 and Wnt5a were estimated by Western blotting (a). Quantitative data are presented as means ± standard deviations of three independent experiments. β-actin was used as an internal control. Protein expression levels of β-catenin (b), RUNX2 (c), and Wnt5a (d) was upregulated in osteoarthritis chondrocytes, compared with control group (#P < 0.05), depressed by PRP-As and PRP-Exos(*P < 0.05). The depressed effect of PRP-Exos was more obvious than PRP-As; however, protein expression levels of β-catenin was opposite ($P < 0.05)

Not applicable.