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. 2023 Jan 4:10:1078527.
doi: 10.3389/fbioe.2022.1078527. eCollection 2022.

Platelet-rich plasma attenuates the severity of joint capsule fibrosis following post-traumatic joint contracture in rats

Yuxin Zhang  1   2   3 , Zengguang Wang  4 , Chenyu Zong  5 , Xiaoding Gu  3 , Shuai Fan  1 , Lili Xu  1 , Bin Cai  1 , Shenji Lu  1   3
Affiliations

Platelet-rich plasma attenuates the severity of joint capsule fibrosis following post-traumatic joint contracture in rats

Yuxin Zhang et al. Front Bioeng Biotechnol. .

Abstract

Background: Post-traumatic joint contracture (PTJC) mainly manifests as excessive inflammation leading to joint capsule fibrosis. Transforming growth factor (TGF)-β1, a key regulator of inflammation and fibrosis, can promote fibroblast activation, proliferation, migration, and differentiation into myofibroblasts. Platelet-rich plasma (PRP) is considered to have strong potential for improving tissue healing and regeneration, the ability to treat joint capsule fibrosis remains largely unknown. Methods: In this study, we aimed to determine the antifibrotic potential of PRP in vivo or in vitro and its possible molecular mechanisms. The TGF-β1-induced primary joint capsule fibroblast model and rat PTJC model were used to observe several fibrotic markers (TGF-β1, α-SMA, COL-Ⅰ, MMP-9) and signaling transduction pathway (Smad2/3) using histological staining, qRT-PCR and western blot. Results: Fibroblasts transformed to myofibroblasts after TGF-β1 stimulation with an increase of TGF-β1, α-SMA, COL-Ⅰ, MMP-9 and the activation of Smad2/3 in vitro. However, TGF-β1-induced upregulation or activation of these fibrotic markers or signaling could be effectively suppressed by the introduction of PRP. Fibrotic markers' similar changes were observed in the rat PTJC model and PRP effectively reduced inflammatory cell infiltration and collagen fiber deposition in the posterior joint capsule. Interestingly, HE staining showed that articular cartilage was degraded after rat PTJC, and PRP injection also have the potential to protect articular cartilage. Conclusion: PRP can attenuate pathological changes of joint capsule fibrosis during PTJC, which may be implemented by inhibiting TGF-β1/Smad2/3 signaling and downstream fibrotic marker expression in joint capsule fibroblasts.

Keywords: fibroblasts; joint capsule fibrosis; platelet-rich plasma; post-traumatic joint contracture; transforming growth factor-β1.

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Figures

FIGURE 1
FIGURE 1
Effects of PRP on the mRNA expression of TGF-β1-induced fibrotic marker. (A) Primary fibroblasts were cultured and verified by immunofluorescence and immunohistochemical staining with vimentin as a marker. (B) Cell viability measured by CCK-8 following different concentrations of TGF-β1 treatment (0, 0.5, 1, 5, 10 ng/ml). (C–F) The mRNA expression of α-SMA (C), TGF-β1 (D), COL-Ⅰ (E), MMP-9 (F) in fibroblasts in response to 10 ng/ml TGF-β1 combined with PRP treatment was determined by qRT-PCR. Error bars represent standard deviation. *p < 0.05 compared with the CON group. #p < 0.05 compared with TGF-β1 group.
FIGURE 2
FIGURE 2
Effects of PRP on the protein expression of TGF-β1-induced fibrotic marker. (A) Western blot analysis of fibrosis-associated proteins α-SMA, TGF-β1, COL-Ⅰ, MMP-9 after fibroblasts treated with 10 ng/ml TGF-β1 combined with PRP. (B) The bar graphs represent the relative expression of these proteins after normalization to β-actin. Error bars represent standard deviation. *p < 0.05 compared with the CON group. #p < 0.05 compared with TGF-β1 group.
FIGURE 3
FIGURE 3
Effects of PRP on the activation of Smad2/3 signaling. (A) Western blot analysis the activation of fibrosis-associated signaling Smad2/3 after fibroblasts treated with 10 ng/ml TGF-β1 for 0, 15, 30, 60, and 120 min, respectively. (B) The bar graphs represent the relative expression of pSmad2/3/Smad2/3. *p < 0.05 compared with the 0 group. (C) Western blot analysis the activation of fibrosis-associated signaling Smad2/3 after fibroblasts treated with 10 ng/ml TGF-β1 combined with PRP. (D) The bar graphs represent the relative expression of pSmad2/3/Smad2/3. Error bars represent standard deviation. *p < 0.05 compared with the CON group. #p < 0.05 compared with TGF-β1 group.
FIGURE 4
FIGURE 4
PRP inhibited the process of fibrosis. (A) Pattern diagram of PRP inhibited TGF-β1-induced fibrosis. TGF-β1 caused phosphorylation of Smad2/3, up-regulating expression of TGF-β1, COL-Ⅰ, α-SMA, MMP-9 and promoted fibroblasts transforming into myofibroblasts. PRP inhibited TGF-β1-induced fibrosis. (B) Schematic of rat knee joint post-traumatic immobilization. (C) Measurement of extension ROM of the affected knee joint. Error bars represent standard deviation. *p < 0.05 compared with the CON group. #p < 0.05 compared with PTJC group.
FIGURE 5
FIGURE 5
Expression of fibrosis-associated proteins in the posterior joint capsule were assessed via western blot. (A) The western blot analysis of COL-Ⅰ, TGF-β1, α-SMA, MMP-9 in the posterior joint capsule. (B) The bar graphs represent the relative expression of these proteins after normalization to β-actin. Error bars represent standard deviation. *p < 0.05 compared with the CON group. #p < 0.05 compared with PTJC group.
FIGURE 6
FIGURE 6
Expression of fibrosis-associated proteins in the posterior joint capsule were assessed via immunohistochemical. (A–D) The immunohistochemical staining of COL-Ⅰ (A), TGF-β1 (B), α-SMA (C), MMP-9 (D) in the posterior joint capsule. Scale bars, 50 μm.
FIGURE 7
FIGURE 7
HE and Masson staining of the affected knee. (A,B) HE (A) and Masson (B) staining of the posterior joint capsule of the affected knee. (C) HE staining of articular cartilage surface. Scale bars, 50 μm.
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