Recombinant human decorin (143-DE) and TGF-β1 (240-B) were procured from R&D Systems (Minneapolis, MN, USA). The inhibitor of Smad3 (SIS3) was supplied by MCE (New Jersey, USA). SIS3 is a pyrrolopyridine compound and can selectively block TGF-β1-dependent Smad3 phosphorylation and Smad3-mediated cellular pathway. It was stored as 5 mM solution in DMSO, and this solution was used after diluting with medium. Spongostan was purchased from XIANG’EN (XIANG’EN Medical Technology Development Co., Ltd., Nanchang, China). It was a sterile, water-insoluble, and absorbable hemostatic gelatin sponge. The spongostan was trimmed into 3 × 3 × 5 mm cubes for use as the biological scaffold. High glucose Dulbecco’s modified Eagle’s medium (DMEM) and fetal bovine serum (FBS) were acquired from Gibco (Grand Island, NY, USA). The antibodies specific for α-SMA (ab124964) and fibronectin (ab268020) were purchased from Abcam (Cambridge, UK). Antibodies against Collagen I (#72026), Smad2 (#5339), phosphorylated-Smad2 (p-Smad2) (#3108), Smad3 (#9253), p-Smad3 (#9520), Smad2/3 (#8685), and secondary antibodies (Anti-rabbit IgG, #7074 and Anti-mouse IgG, HRP-linked Antibody #7076) were obtained from Cell Signaling Technology (Danvers, MA, USA). Antibody against Collagen III (22734-1-AP) was procured from Proteintech Group (Wuhan, Hubei, China). Antibody specific for GAPDH (BM1623) was obtained from Boster (Wuhan, Hubei, China).

Human fibroblasts were acquired from the Zhong Qiao Xin Zhou Biotechnology (Shanghai, China). According to the protocol provided by the company, the cells were cultured in high glucose DMEM supplemented with 15% FBS and 1% penicillin/streptomycin solution in a 37°C, 5% CO₂ incubator. The culture medium was refreshed every 3 days. After reaching 80–90% confluence, the cells were separated and passaged. Cells of passages 3–4 were used for subsequent experiments.

Cell Counting Kit-8 (CCK-8) assay was used to measure fibroblast viability and proliferation according to the manufacturer’s protocol (Beyotime, Nanjing, China). In brief, fibroblasts were inoculated into a 96-well plate at a density of 1 × 10⁴ cells/well. After adherence, cells were incubated with different concentrations of TGF-β1 (1, 2.5, 5, and 10 ng/ml) or treated with TGF-β1 (5 ng/ml) in combination with different concentrations of decorin (2.5, 5, and 7.5 µg/ml) for 48 h. Subsequently, 100 µl culture medium containing 10 μl CCK-8 solution was added into each well. Then, the plate was incubated for 1 h at 37°C in the dark. The absorbance of the cells at 450 nm in each well was detected by the spectrophotometric microplate reader (Bio-Rad, CA, USA).

In accordance with the manufacturer’s instructions, total ribonucleic acid (RNA) of the fibroblasts was extracted with an RNA isolation kit purchased from Omega (Guangzhou, Guangdong, China). Then, the concentration of RNA samples was detected by spectrophotometry at 260 nm (Bio-Rad, CA, USA). The ratio of A260/A280 was calculated to verify the purity of total RNA. The equal amounts of RNA samples (1 µg) were transcribed to synthesize cDNA using a cDNA synthesis kit (TOYOBO, Osaka, Japan). Afterward, the cDNA was amplified using SYBR Green Real-time PCR Master Mix (TOYOBO). The qPCR reaction was conducted at 95°C for 1 min followed by 39 cycles at 95°C for 15 s and 60°C for 15 s. The expression levels of the target genes were normalized to the corresponding GAPDH and were analyzed using the 2−ΔΔCq method.

The fibroblasts were initially seeded into 24-well plates at a density of 2 × 10⁴ cells/well. Following stimulation with 5 ng/ml TGF-β1 or in combination with 7.5 µg/ml decorin, the cells were fixed in 4% paraformaldehyde for 30 min and permeabilized with 0.1% Triton X-100 for 5 min. Then, the cells were blocked with 5% normal goat serum for 1 h at room temperature. Subsequently, the cells were incubated with antibodies against collagen I, fibronectin, α-SMA, and Smad2/3 at 4°C overnight and probed with FITC-conjugated (green) anti-rabbit IgG antibody for 1 h. Then, the cells were stained with DAPI in PBS for 5 min. Finally, the fluorescence images of the cells were photographed using a fluorescence microscope (Evos Flauto; Life Technologies, USA).

After incubated with TGF-β1 (5 ng/ml) or in combination with decorin (5 and 7.5 µg/ml) for 48 h, the fibroblasts were lysed with RIPA Lysis Buffer (Boster, Wuhan, China) to extract whole-cell proteins. The BCA assay kit obtained from Boster (Wuhan, Hubei, China) was used to determine the protein concentration. Subsequently, equal amounts of each protein samples (25 µg) were separated by gel electrophoresis and transferred to PVDF membranes (Millipore, Billerica, USA). After blocking with 5% skimmed milk for 1 h, the membranes were then incubated with the specific antibodies against collagen I, collagen III, α-SMA, fibronectin, GAPDH, Smad2, p-Smad2, Smad3, and p-Smad3 (all 1:1,000) at 4°C overnight. The membranes were washed three times with TBST and incubated with correspondent secondary antibodies (1:10,000) for 1 h. The proteins were detected using a western electrochemiluminescence substrate kit (Thermo Pierce, MA, USA) and photographed using a Bio-Rad scanner system (CA, USA). The band intensity was quantified by Image Lab 5.1 software (Bio-Rad, CA, USA).

This study was approved by the Institutional Animal Care and Use Committee of Tongji Hospital of Huazhong University of Science and Technology. One hundred and twenty-eight male Sprague–Dawley rats (250–300 g) were purchased from the Laboratory Animal Center of our hospital. The animals were randomly split into four groups. Sham group (n = 32): accepted sham operation without laminectomy; model group (n = 32): only a laminectomy was performed; spongostan group (n = 32): a spongostan impregnated with 0.4 ml of saline solution was retained on the dura mater; decorin treatment group (n = 32): a spongostan impregnated with decorin (100 µg/kg) in 0.4 ml of saline solution was left on the dura mater. This dosage of decorin was selected based on earlier studies (Alan et al., 2011; Turkoglu et al., 2014). The rats were weighed and anesthetized by intraperitoneal injection of pentobarbital (4.0 mg/100 g weight). After the lower backs of the rats were shaved and sterilized, a midline incision of 2–3 cm was made with the first lumbar vertebra (L1) as the center to expose the bony posterior elements. Then, the L1 laminectomy was performed carefully to keep the dura mater intact and fully exposed. After satisfactory hemostasis, the topical agents were applied and the wounds were closed layer by layer. The rats were observed every day for 2 weeks after the operation to monitor their spinal nerve function and wound healing. At the 4 and 8 weeks after operation, half of the rats in each group were randomly selected for further study.

Macroscopic assessments of epidural adhesions were performed at 4 and 8 weeks after operation, respectively. Eight rats from each group were randomly selected and sacrificed using an overdose of anesthetics. The laminectomy site was explored again through the previous operative incision. To prevent deviation, the researchers were blinded to the animal information before exploring. The epidural adhesions were evaluated according to Rydell standard (Rydell, 1970) as follows: grade 0, epidural scar tissues were not adherent to the dura mater; grade 1, epidural scar tissues adhered to the dura mater, but easily dissected; grade 2, epidural scar tissues adhered to the dura mater dissected with difficulty without disrupting the dura mater; grade 3, epidural scar tissues adhered to the dura mater and could not be dissected.

MRI evaluations were conducted at 4 and 8 weeks postoperatively. The MRI examinations were performed with the Bruker 7.0T MR imaging system (Berlin, Germany). The axial and sagittal T2-weighted imaging sequences were carried out on the segment where the laminectomy was performed. The sets of MRIs were accomplished under the following conditions: repetition time, 3,000 ms; echo time, 33 ms; thickness, 0.8 mm; layers, 15; matrix size, 256 × 256. The epidural adhesions and epidural fibrosis in MRIs were evaluated by the radiologists independently. The classification of epidural fibrosis was analyzed based on the methods reported in previous literature (Ross et al., 1999; Wang et al., 2020). In short, the spinal canal was subdivided into four quadrants by drawing two vertical lines from the center of the dura sac. The two quadrants on the dorsal side of the dura sac were our observation areas. The degree of epidural fibrosis was graded according to grade 0–4: grade 0, no scars; grade 1, >0 to ≤25% of the quadrants were full of scars; grade 2, >25 to ≤50% of the quadrants were full of scars; grade 3, >50 to ≤75% of the quadrants were filled with scars; grade 4, 75–100% of the quadrants were full of scars.

Histological analyses were performed following the MRI scans at 4 and 8 weeks. The spine columns, including surrounding muscle tissue and epidural fibrotic tissue, were resected en bloc between the cephalad and caudal vertebral bodies with L1 as the center. The specimens were fixed with 4% paraformaldehyde for 48 h. After being decalcified with 10% EDTA, the samples were dehydrated in graded ethanol. Then, the spine columns were embedded in paraffin and axially cut into 5-µm-thick sections. H&E staining and Masson staining were both performed in the sections. Antibodies specific for collagen I and fibronectin were used for further immunohistochemical staining. The qualitative analyses of fibroblast infiltration were evaluated in H&E-stained sections. The following methods were used to score fibroblast infiltration: grade 1, <100 fibroblasts in each 400× field; grade 2, 100–150 fibroblasts in each 400× field; grade 3, >150 fibroblasts in each 400× field (Coskun et al., 2000). The dural thickness and epidural fibrosis grades were analyzed in Masson-stained sections. The thickness of the dura mater was evaluated by selecting three measurement points at the laminectomy site. The first measurement point was taken from the midpoint of the laminectomy site, and the second and third measurement points were selected at 2 mm from both sides of the first point (Gurer et al., 2015). The average values of the three measurement points were used for statistical analysis. Epidural fibrosis and epidural adhesions in Masson-stained sections were graded as follows: grade 0, there was no scar tissue around the dura mater; grade 1, only thin fibrous bands were observed between the scar tissue and the dura mater; grade 2, continuous adhesions were observed in less than two-thirds of the laminectomy defect; grade 3, scar tissue adhesions were large, affecting more than two-thirds of the laminectomy defect, or the adhesions extending to the nerve roots (He et al., 1995).

Values of the results were shown as mean ± SD. One-way ANOVA was applied for multiple comparisons, and the unpaired t-test was used to compare the statistical significance between the two groups. A p value <0.05 was considered statistically significant. Prism 8 software (GraphPad Software Inc., La Jolla, CA, USA) was used for statistical analysis in this work.

As shown in Figure 1A, decorin exhibited no cytotoxicity on fibroblasts at the concentrations of 1, 2.5, 5, and 7.5 µg/ml. To determine the proliferation of fibroblasts induced by TGF-β1, dose-response experiments with different concentrations of TGF-β1 (1, 2.5, 5, and 10 ng/ml) were conducted. As shown in Figure 1B, TGF-β1 promoted fibroblast proliferation in a dose-dependent manner. This promotion effect peaked when the concentration was 5 ng/ml. Following incubation with different concentrations of decorin (2.5, 5, and 7.5 µg/ml), the proliferation of fibroblasts induced by TGF-β1 (5 ng/ml) was notably suppressed by decorin with the concentrations of 5 and 7.5 µg/ml (Figure 1C). Decorin was used at doses of 5 and 7.5 µg/ml in subsequent studies.

To investigate whether decorin could suppress transdifferentiation of fibroblasts into myofibroblasts induced by TGF-β1, the expression of α-SMA, a widely investigated myofibroblast marker, was detected by qPCR and western blotting. As exhibited in Figures 2A–C, the expression of α-SMA in fibroblasts induced by TGF-β1 was significantly increased, while decorin could significantly inhibit the increase of α-SMA in a dose-dependent manner. Furthermore, the immunofluorescence staining of α-SMA showed the same results (Figure 2D).

Collagen fibers and fibronectin are the major ECM components that lead to the formation of fibrosis. As exhibited in Figures 3A–C, TGF-β1 significantly induced the upregulation of fibronectin, collagen I, and collagen III. However, decorin could reverse these changes. The changes of fibronectin and collagen I were further confirmed by the fluorescent results (Figures 3D, E).

The Smad2/3 signaling pathway is highly involved in the progression of fibrosis. Several time points (0, 1, 3, 6, and 9 h) were selected to verify the activation process after treatment with TGF-β1. The results in Figures 4A, B showed that activation of the smad2/3 signaling pathway induced by TGF-β1 was time dependent. The activation was the strongest after 1 h, and then gradually weakened. Therefore, we selected the activation peak after 1 h to explore the regulation of decorin on the smad2/3 signaling pathway. As exhibited in Figures 4C, D, TGF-β1 could markedly promote the activation of smad2 and smad3. However, when the fibroblasts were treated with decorin (5, 7.5 µg/ml), the phosphorylation levels of Smad2 and Smad3 were significantly reduced compared with TGF-β1 treated group. In addition, immunofluorescence staining was conducted to evaluate smad2/3 nuclear translocation. As exhibited in Figure 4E, smad2/3 was located in the cytoplasm area of unstimulated fibroblasts. Compared with the control group, we observed accumulation of smad2/3 in the nucleus of fibroblasts after stimulation with TGF-β1 for 1 h. However, this process could be partly repressed by decorin (7.5 µg/ml).

To further investigate the role of the Smad2/3 signaling pathway in TGF-β1-induced fibroblast transdifferentiation and ECM synthesis, SIS3 (a novel specific inhibitor of Smad3) was used in this study. As expected, SIS3 could significantly inhibit the phosphorylation of Smad3 (Figures 5A, B). In addition, the induction of α-SMA and Collagen I by TGF-β1 was significantly inhibited by SIS3 (Figures 5C, D). These results were consistent with decorin (7.5 µg/ml) treatment, which indirectly suggested that decorin could at least partially inhibit TGF-β1-induced fibroblast transdifferentiation and ECM production via the Smad2/3 signaling pathway.

To manifest the effects of decorin on the pathogenesis of epidural fibrosis and epidural adhesions in vivo, we established a rat laminectomy model. The process of building the model is shown in Figure 6A. Gross examinations of the wound sites did not reveal infection or poor wound healing. Gross observations of the epidural adhesions were carried out at the position of the original surgical incision. Representative macroscopic observation results of each group at 4 and 8 weeks are shown in Figure 6B. The dura mater in the sham operation group was smooth without adhesion. However, a large number of scar tissues were formed and adhered to the dura mater in model group, which were difficult to dissect and separate. In the spongostan group, the epidural scar tissues sticking with the dura mater were easy to dissect at 4 weeks. By the 8th week, the epidural scar tissues were difficult to dissect. By contrast, the decorin treatment group showed mild epidural adhesions, which can be bluntly separated by minimal manual traction without damaging the dura. Moreover, the surface of the dura mater was smooth after the removal of the epidural scar tissues. The epidural adhesion score was the lowest among all groups at 4 and 8 weeks, respectively (Figures 6C, D).

The axial and sagittal T2-weighted MRIs of the spinal column were obtained after 4 weeks (Figure 7A) and 8 weeks (Figure 7B) of treatment, respectively. In the sham group, the normal spinal structure of the rats could be observed. There was an intact lamina behind the dural sac and no compression on the dural sac. In contrast, the rats in the model group exhibited noticeable epidural scar adhesions and dense scar tissues compression on the dural sac. At the 4th week, a narrow gap between dense scar tissues and dura mater could be observed in the spongostan group. The dura mater was not obviously compressed. However, in the 8th week, the dense scar tissues increased significantly, causing compression on the dural sac. In the decorin treatment group, there was only a small amount of dense scar tissues around the dural sac at 4 and 8 weeks, and there was no compression on the dural sac. Consistently, similar results were found based on the grading scores of MRI (Figures 7C–E).

Histological evaluations were also performed at 4 weeks (Figures 8A, B) and 8 weeks (Figures 9A, B). Notably, no epidural scar formation and epidural scar adhesions around the dura mater were observed in the sham group due to the barrier function of the intact lamina. In contrast, the outside dural canal was filled with abundant scar tissues, and the epidural adhesions were extensive and serious in the model group. In the spongostan group, although there was a fuzzy gap between dura mater and epidural scar tissues, it was filled by many fibrous connections at 8 weeks. By contrast, the gap in histological sections corresponding to the decorin treatment group was more evident at 4 and 8 weeks. In addition, the model and spongostan groups exhibited more abundant fibroblasts than the decorin treatment group. There were significant differences between the decorin and model group, and decorin and spongostan group (Figure 8C and Figure 9C). This indicated that decorin could inhibit the proliferation of fibroblasts in vivo. Furthermore, significantly higher mean thickness values of dura mater were observed in the model and spongostan groups than in the sham group at 4 and 8 weeks (Figure 8D and Figure 9D). There was no significant difference between the model and the spongostan group. However, there were significant differences in the decorin treatment group compared with the model and the spongostan group at 4 and 8 weeks. Accordingly, the lowest epidural fibrosis score was observed in the decorin treatment group, indicating significantly less epidural fibrosis and epidural adhesions in this group (Figure 8E and Figure 9E). The immunohistochemical staining results of collagen I (Figure 10A) and fibronectin (Figure 10B) showed a trend consistent with in vitro studies.