Adult male Sprague Dawley (SD) rats (180–220 g) were purchased from Shanghai SIPPR-Bk Lab Animal Co., Ltd. and maintained in specific pathogen-free laboratory animal facilities of Shanghai Ninth People’s Hospital, and housed in individual cages at room temperature (23 ± 1°C), relative humidity, and a 12 h light/dark cycle with free access to food and water. All animal experiments complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were reviewed and approved by the Institution of Animal Care and Use Committee (IACUC) of the Ninth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine.

First, 9 ml blood from the artery of each donor animal (10-week male rat) was pumped and then mixed with 1 ml of 3.8% sodium citrate. An extra 1 ml blood sample was set apart to determine the concentration of platelets. Subsequently, the blood samples were centrifuged at 200 g for 10 min at 4°C. The supernatant and the interlayer of the buffy coat were transferred to another centrifuge tube and centrifuged at 1,000 g for 10 min at 4°C. A total of 3/4 of the supernatant was discarded, PRP was achieved through resuspension of the remaining sample, and the concentration of platelets was approximately 2 × 10⁶/µl. PRP was stored at -80°C and was activated by the addition of 10% CaCl₂ before use.

Posterior joint capsules were obtained from the knee joints of healthy SD rats (male, 4 weeks old) after euthanization and subsequently washed with Dulbecco’s minimum essential medium (DMEM). The tissue fragments were cut into 2–3 mm, arranged in a 6-well culture plate containing 1 ml DMEM supplemented with 1% penicillin/streptomycin and 10% fetal bovine serum (FBS), and cultured in an incubator at 5% CO₂ and 37°C. The medium was changed every couple of days. Fibroblasts started to migrate from the subsections after 3–5 days and tissues were removed on approximately the seventh day when the culture reached 90% confluence. Primary joint capsule fibroblasts were cultured to passages 2 to 4 and identified by cells exhibiting a characteristic morphology and positive staining for the fibroblast marker vimentin (Abcam) before used in subsequent experiments.

Fibroblasts were cultured in serum-low medium (basic medium with 0.3% FBS) for 16 h and then induced with 10 ng/ml TGF-β1 for 24 h. The groups of experiments were set as follows: 1) CON: fibroblasts with DMEM; 2) PRP: fibroblasts with DMEM and PRP (1:50); 3) TGF-β1: fibroblasts with DMEM and 10 ng/ml TGF-β1; and 4) TGF-β1 + PRP: fibroblasts with DMEM, 10 ng/ml TGF-β1, and PRP (1:50) (Sassoli et al., 2018).

Fibroblasts were plated in a 96-well plate at a density of 1× 10⁵ cells/mL overnight and subsequently treated with TGF-β1 (0, 0.5, 1, 5, 10 ng/ml) for 24 h. Then, 10 µl of Cell Counting Kit-8 (CCK-8; Dojindo) was added to the cells and incubated for an additional 2 h at 37°C. Cell viability was measured using a spectrophotometer by detecting absorbance of each well at 450 nm. Assays were performed three times using triplicate wells.

As previously described (Zhang et al., 2021a; Zhang et al., 2021b), rats were anaesthetized via intraperitoneal injection of sodium pentobarbital (50 mg/kg) and placed in a supine position for surgery. The knee joint of the right hind leg underwent a midline skin incision and a lateral parapatellar arthrotomy was performed. The patella was moved medially and the femoral condyle was exposed by flexing the knee joint. Two 1.5 × 1.5-mm cortical windows were made from non-articulating cartilaginous regions of the lateral and medial femoral condyles using a 1.5-mm drill bit. The anterior and posterior cruciate ligaments were incised, and the posterior joint capsule was disrupted by hyperextension of the knee at −45°. The right knee was immobilized at 135°- flexion with a 0.5-mm steel wire passing through the holes made by a drill bit in the proximal femur and the distal tibia and buckled at the subcutaneous medial knee joint. Finally, after patellofemoral joint reduction, the muscles and skin were sutured with silk threads. For drug delivery, activated PRP was injected into the joint cavity after sutured. After surgery, rats were allowed unrestricted daily activity in cages. Rats were euthanized at 0 days and 4 weeks, internal fixation was removed, and the knee extension ROM was measured within 15 min of euthanasia. Affected knee joint and posterior joint capsules were collected for subsequent analysis.

After euthanization using an overdose of sodium pentobarbital, rat knee joints and posterior capsule tissues were removed, fixed with 4% paraformaldehyde, decalcified in 10% EDTA, and then embedded in paraffin. Samples were embedded in paraffin and cut into 4-μm thick sections. Hematoxylin-eosin (HE) staining and Masson trichrome staining were used for qualitative observation. The tissues were deparaffinized and hydrated in distilled water and then covered with hematoxylin and eosin, respectively, by using an HE staining kit (Abcam). Masson staining was conducted using tissue covered by Weigert iron hematoxylin, Biebrich scarlet-acid fuchsin and aniline blue (Sigma-Aldrich) according to the manufacturer’s instructions.

Posterior joint capsule tissues were removed and quickly frozen for subsequent immunohistochemical staining of TGF-β1, α-SMA, COL-Ⅰ, and matrix metalloproteinase (MMP)-9 (Servicebio). Briefly, the slides were incubated with primary antibodies overnight at 4°C. Secondary goat anti-rabbit antibodies were applied to the slides after washing at room temperature for 1 h. DAB solution was applied after the washing step and then incubated in the dark for 30 min followed by counterstaining with hematoxylin. All images were captured using a Zeiss LSM710 confocal microscope (Zeiss).

Total RNA of was extracted with TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions and was then reverse transcribed using the Omniscript Reverse Transcription Kit (QIAGEN). qRT-PCR was carried out on a real-time PCR system (Applied Biosystems) using SYBR® Premix Ex Taq™ (Takara Bio). Gapdh was used as a reference gene and calculated the relative levels using 2^−ΔΔCt. The primers were designed and synthesized by Sangon Biotech (Shanghai, China) as follows: Gapdh: forward primer 5ʹ-ACA GCA ACA GGG TGG TGG AC-3ʹ, reverse primer 5ʹ-TTT GAG GGT GCA GCG AAC TT-3ʹ; a-sma: forward primer 5ʹ-ATC GTC CAC CGC AAA TGC-3ʹ, reverse primer 5ʹ-AAG GAA CTG GAG GCG CTG-3ʹ; Tgf-β1: forward primer 5ʹ-AGC AAC AAT TCC TGG CGT TAC-3ʹ, reverse primer 5ʹ-TGT ATT CCG TCT CCT TGG TTC A-3ʹ; Col-Ⅰ: forward primer 5ʹ-TGT ATC ACC AGA CGC AGA AGT-3ʹ, reverse primer 5ʹ-ACC AGG AGG ACC AGG AAG T-3ʹ; Mmp-9: forward primer 5ʹ-CCT ACT GCT GGT CCT TCT GAG-3ʹ, reverse primer 5ʹ-TGG CTT CCT CCG TGA TTC G-3ʹ.

Proteins were extracted from the cells and the posterior capsules. Samples were lysed in RIPA buffer containing protease and phosphatase inhibitors. After centrifugation, protein concentrations in the supernatants were determined using the BCA Protein Assay Kit (Beyotime). Western blots was performed as previously described (Zhang et al., 2019a; Zhang et al., 2021a). Briefly, proteins were separated by electrophoresis on NuPAGE® 4–12% Bis-Tris Gel (Invitrogen) for 40 min at 200 V and transferred onto polyvinylidene difluoride (PVDF) membranes (Invitrogen). The membranes were blocked with 5% skim milk for 30 min on a rotary shaker at room temperature. They were then incubated overnight at 4°C with the following primary antibodies: β-actin (ProteinTech, 1:5000), TGF-β1 (Abcam, 1:1000), α-SMA (Abcam, 1:1000), COL-Ⅰ (Abcam, 1:1000), MMP-9 (Abcam, 1:1000), Smad2/3 (Abcam, 1:1000), and p-Smad 2/3 (Abcam, 1:1000). Secondary antibodies included Goat anti-Rabbit IgG (H + L) (DyLight 800 4X PEG) (Invitrogen, 1:20000) and Goat anti-Mouse IgG (H + L) (DyLight 680) (Invitrogen, 1:20000). The fluorescent signals were determined with an Odyssey imaging system (Li-Cor, Lincoln, NE, United States). Relative protein expression levels were expressed as the ratio of the band intensity of the target protein to that of β-actin.

All results are expressed as the mean ± standard deviation after analysis using SPSS 22.0 statistical software (SPSS Inc., Chicago, IL, United States). Parametric data were analyzed via Student’s t-test or one-way analysis of variance (ANOVA) followed by Tukey’s post hoc analysis for comparisons between two groups. Calculations were performed using GraphPad Prism software 4.0 (GraphPad, CA, United States). A level of p < 0.05 was considered statistically significant.

Fibroblast abnormal activation is a critical process in joint capsule fibrosis. To determine the effect of PRP on fibroblast abnormal activation, we obtained primary joint capsule fibroblasts from the posterior joint capsule of 4-week-old rat knee joints (Figure 1A). Numerous studies have reported that TGF-β1 promotes the differentiation of fibroblasts into myofibroblasts, collagen hyperplasia, and ECM protein accumulation (Sasabe et al., 2017; Vallee and Lecarpentier, 2019; Weiskirchen et al., 2019; Zhou et al., 2020). As shown by CCK-8, TGF-β1 slightly enhanced fibroblast cell viability (Figure 1B), indicating that this concentration range was applicable to subsequent experiments. Therefore, we selected 10 ng/ml TGF-β1 to establish an in vitro fibroblast abnormal activation model and explored whether PRP inhibits activated fibroblast phenotypic transformation. The mRNA expression levels of fibrotic markers (α-sma, Tgf-β1, Col-Ⅰ, and Mmp-9) were determined by qRT-PCR (Figures 1C–F). The results showed that inactive joint capsule fibroblasts expressed very low levels of fibrosis related genes. After incubation for 24 h with 10 ng/ml TGF- β 1, the mRNA expression of fibrotic markers significantly increased, which was strongly inhibit by PRP. Interestingly, PRP alone did not significantly change the expression of fibrotic markers compared to the CON group. These results indicated that PRP suppressesed TGF-β1-induced fibrotic marker expression in joint capsule fibroblasts at the gene level in vitro.

Based on the observation that PRP suppresses the transformation of joint capsule fibroblasts to myofibroblasts induced by TGF-β1 and reduces the mRNA expression of α-sma, Tgf-β1, Col-Ⅰ, and Mmp-9, we hypothesized that PRP may also suppress fibrotic markers at the protein level. Consistent with the qRT-PCR results, western blot showed that the expression of α-SMA, TGF-β1, COL-Ⅰ, and MMP-9 was significantly increased after 10 ng/ml TGF-β1 treatment. Co-incubation with PRP reversed the TGF-β1-induced fibrosis process (Figure 2). These results indicated that PRP suppressed TGF-β1-induced fibrotic marker expression in joint capsule fibroblasts at the protein level in vitro.

Considering that Smad2/3 is the major signal transduction pathway responsible for mediating profibrotic effect of TGF-β1, we therefore evaluated whether PRP exerts its antifibrotic effect through the suppression of the Smad2/3 signaling pathway. As shown in Figure 3A, western blot showed that Smad2/3 in joint capsule fibroblasts was quickly induced 15 min after stimulation with 10 ng/ml TGF-β1 and remained continuously activated for 120 min. The addition of PRP to TGF-β1-conditioned medium led to a significant decrease in Smad2/3 phosphorylation levels (Figure 3C). These results indicate that PRP suppressed TGF-β1-induced transformation of joint capsule fibroblasts to myofibroblasts and fibrotic marker expression possibly by blocking the TGF-β1/Smad2/3 pathway (Figure 4A).

To examine whether PRP can inhibit the process of joint capsule fibrosis after PTJC in vivo, we established the PTJC model as previously described (Figure 4B) and measured knee extension ROM after myotomies of the trans-articular muscles. The results showed that the normal knee extension ROM was 159.9 ± 1.3° and was reduced to 73.8 ± 3.2° at 4 weeks postinduction of PTJC, while the group of rats that received an injection of PRP was increased to 85.5 ± 5.1° (Figure 4C). In addition, we further checked the expression level of fibrosis-related proteins in the rat posterior joint capsule by western blot (Figure 5) and immunohistochemistry staining (Figure 6). Consistent with the observation results of in vitro cell experiments, the expression of α-SMA, TGF-β1, COL-Ⅰ, and MMP-9 was significantly induced in the posterior joint capsule following PTJC, which was effectively reversed by PRP injection at the lesion sites, but was still higher than that of the CON group. These data indicated that PRP can reduce fibrotic marker expression both in vitro and in vivo.

To further evaluate the influence of PRP on PTJC, PRP/saline was injected into the lesion site. Four weeks after the injection, we performed histological analysis of the posterior joint capsule sections. The immunohistochemical staining of the experimental knees is shown in Figure 7. Histological analyses of the posterior joint capsule revealed increased numbers and density of inflammatory cells (HE) (Figure 7A), along with collagen fiber hyperplasia and disordered arrangement (Masson) (Figure 7B). These changes mirrored those found in humans and indicated that inflammation and fibrosis are closely related to PTJC. Injection of PRP at the lesion sites significantly attenuated joint capsule inflammatory cell infiltration and collagen fiber hyperplasia (Figures 7A,B). Interestingly, HE staining showed that PRP injection has the potential to delay degeneration of articular cartilage in PTJC (Figure 7C). These data indicated that PRP has anti-inflammatory, antifibrotic, and cartilage protective effects during PTJC, which may be achieved by inhibiting the production of fibrosis-related proteins and matrix degrading enzymes.