Endoscopic submucosal dissection (ESD) is an accepted treatment for early esophageal cancer and precancerous lesions, but resection of a large mucosal area often leads to postoperative esophageal stricture. Biomaterials provide a new option for the treatment of post-ESD ulcers. In this study, we developed a well-defined ammonolysis-based tetra-armed poly (ethylene glycol) (Tetra-PEG) hydrogel and investigated its efficacy and related mechanisms for preventing esophageal ESD-induced stricture in a porcine model. In terms of material properties, Tetra-PEG hydrogel present great biocompatibility, great capability to retain moisture, strong tissue adhesion and high mechanical strength. Then, six domestic female pigs were randomly divided into PEG (n = 3) and control groups (n = 3). A 3/4 of the esophageal circumference ESD was performed in all pigs. In PEG group, Tetra-PEG hydrogel was easily delivered via endoscopy and adhered to the ulcer bed tightly. Compared to control group, Tetra-PEG hydrogel accelerated esophageal ulcer healing at an early stage with enhanced epithelium regeneration, milder inflammation and lesser fibrosis by regulating TGF-β/Smad2 signaling. Taken together, our findings reveal Tetra-PEG hydrogel is a promising and attractive candidate for preventing the formation of fibrotic stricture in the process of esophageal ESD-induced ulcer repair.
Research Article
A novel Tetra-PEG Based Hydrogel for prevention of esophageal stricture after ESD in a porcine model
https://doi.org/10.21203/rs.3.rs-2473041/v1
This work is licensed under a CC BY 4.0 License
Journal Publication
published 01 Jun, 2023
Read the published version in Colloids and Surfaces B: Biointerfaces →
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With recent developments in endoscopic techniques, most early esophageal cancer and Barrett’s esophagus with dysplasia can now be diagnosed and resected en-bloc by ESD even if the lesions are large or invade the submucosal layer.[1] ESD is widely accepted, particularly in Asian countries, as a minimally invasive endoscopic approach to curing early-stage cancer without organ resection.[2] However, ESD sizes larger than 3/4 of the esophageal circumference will develop esophageal stenosis frequently.[3] As yet, no specific preventive measurements or treatments had been established for this serious complication.
More recently, endoscopic tissue shielding biomaterial materials received widespread attention for ESD-induced ulcer repair, including polylactic acid,[4] carboxymethyl cellulose (CMC),[5] and autologous oral mucosal epithelial cell (AOMEC),[6]have emerged to protect the submucosa and muscularis propria from endoluminal factors and infection after ESD. However, conventional wound shields used after ESD such as polyglycolic acid meshes may detach from ulcers even with the use of suture or fibrin glue and may lead to potential complications such as chronic inflammation during degradation, and there are difficulties in the delivery processes inside the body simultaneously. [7] Thus, a material that can adhere more firmly to the wound and does not cause foreign-body type reactions during degradation while facilitating intraoperative use is essential to prevent esophageal stricture after ESD.
Normally, an ideal high-performance shield should fulfill these requirements: maintaining moist environment, enhancing epidermal migration, promoting angiogenesis, protection against bacterial infection and easily to be injected.[8] Composed of hydrophilic polymers with polar functional groups, hydrogel possesses high water content, which could swell and absorb liquid.[9] Therefore, the hydrogel produces repeated hydration to absorb water in the tissue, while the excess water on the surface of the hydrogel can act as a lubricant and reduce tissue adhesion. In addition, the hydrogel can be tightly attached to the uneven ESD postoperative wound, reducing food and bacterial irritation, building physical isolation, and effectively preventing inflammation and fibrosis.
In this study, we apply an optimized one (Cys) via Tetra-PEG-NH2 and tetra-armed poly (ethylene glycol) succinimidyl succinate (Tetra-PEG-NH-Cys) with great capabilities to retain moisture and produce high mechanical strength by its strong tissue adhesion property. The efficacy and potential biological mechanism of this new PEG hydrogel for preventing post-ESD esophageal stricture were also verified in our porcine model. (Fig. 1)
2.1 Preparation and source of PEG materials
The product consists of two synthetic new PEG powders (Component A: Tetra-PEG-SS and Component B: Tetra-PEG-NH-Cys) (Hainan Biomedical Device Co,. LTD, Hainan,China)(Fig. 2a). Cysteine on component B forms a thioester bond with component A under mild conditions, accelerating the rate of gel solidification to form a barrier on the wound bed. The thioester bond is then rearranged intramolecularly to form a new amide bond that forms a covalent crosslinked hydrogel through a crosslinking reaction, which makes the hydrogel have better bioadhesion. At the same time, disulfide bonds can continue to be formed between the sulfhydryl groups, forming a network structure to further strengthen the gel (Fig. 2b).
2.2 Hydrogel properties
2.2.1 Rheological tests
The sample was measured at 25℃ in a frequency range of 100–0.1 rad·s− 1 using a Discovery II Rheometer equipped with coneparallel plate geometry (25mm of diameter) .
2.2.2 Morphological analysis
The surface morphology of PEG scaffolds was characterized through cryo-scanning electron microscope (cryo-SEM) (FEI Quanta 450; SU3500). The sample was fixed on the sample stage by electrocarbon glue, inserted into liquid nitrogen slush for 30s, sublimated at -90℃for 10min and then plated gold at a current of 10mA for 60s, finally sent to the scanning electron microscope sample room for observation.
2.2.3 Cytocompatibility
The cytotoxicity of PEG was assessed according to the standard CCK8 assay. Briefly, L929 cells in good condition are collected and cell suspension prepared with cell culture medium. The L929 cells were seeded in each well of a 96-well plate, respectively. Then the cells culture was replaced by PEG extract solution for 24–48 h and cytotoxicity was assessed using Cell Counting KIT-8 according to manufacturers’ instructions (CCK-8, Dojindo). For the Live/Dead staining assay, a mixture of calcein AM and PI in PBS was used to stain the live and dead cells for 30 min. The appearance of Live/Dead stained cells was captured by the Leica DM IL LED inverted phase-contrast microscope to ascertain the cytotoxicity of GelMA/OD/Borax hydrogel.
2.2.4 Water vapor transmission rate
Water vapor transmission rate (WVTR) of the PEG was caculated using a gravimetric method, according to the ASTM E96–00 standard. For this, a plate containing approximately 1kg of anhydrous calcium chloride was placed in a temperature incubator at 36℃ to 38℃. 5 mL of deionized water and 2g of water absorption cotton was added to a closed box made of corrosion-resistant material, and the circular opening of the box was subsequently covered with the PEG. The control group was not coated with PEG. The initial mass (W0) was accurately weighed. The box was placed in a thermostatic incubator at 37◦C and then removed after 12, 24, 36 and 48 h for weighing (Wt). WVTR(g/m2/day) was calculated as follows:
$$WVTR=\frac{{W}_{0}－{W}_{t}}{A\times T}$$
where, A is the cross-sectional area of the box (m2) and T is the duration for which the PEG are placed in the temperature incubator (day). An average of three measurements was taken for each sample.
2.2.5 Adhesive ability test
To test the bio-adhesive properties of the PEG, lap shear joints between hydrogel and porcine esophageal tissues were prepared. A 4
ml PEG was tied to the gap of two pieces tissue which form a junction contact area of 12.5cm2 (5cm×2.5cm), as shown in Fig. 1f. The end of esophageal tissue was clamped to the tensile machine (Zwick Roell Z2.5 TH with a 2.5 kN sensor) for shear adhesive test and the shear velocity 5 mm/min[10]. The shear strength was calculated according to the following equation:
$$shear strenth=\frac{maximum force}{junction contact area}$$
2.3 Animals and preoperative preparation
Six domestic female pigs weighing about 45 kg were randomly divided into PEG(n = 3) and control groups(n = 3). Animals were prepared with a 48-hour half liquid diet and a 24-hour fast and premedicated by intramuscular administration of atropine sulfate at 0.05 mg/kg. They were then anesthetized with 15 mg/kg ketamine hydrochloride and 3 mg/kg xylazine hydrochloride and intubated endotracheally. Halothane and nitrous oxide gas were used for maintenance of anesthesia during the procedure, under mechanical ventilation. All procedures were performed with the animals under continuous cardio-respiratory monitoring. The protocol was approved by the local ethics committee authorized for animal experimentation at the Gateway Medical Innovation Center. (Registration number SH2021 08011).
2.4 Endoscopic submucosal dissection and PEG spraying technique
In animal esophageal ESD experiments, the properties and effects of the gel was evaluated by locally applied onto the post-ESD ulcers. Animals were placed in the left lateral decubitus position. Incisions were made with a hook knife (Olympus Corporation, Tokyo, Japan), and dissection was performed with an insulated-tip (IT) knife (Olympus Corporation, Tokyo, Japan). A two-step procedure was performed in PEG group, consisting of the creation of ESD ulcers in step 1 and spraying of PEG in step 2, while only step 1 was performed in the control group. Six mucosal defects (3/4 of the esophageal circumference ranging from 40 cm to 45 cm from the dental arch) were created by endoscopic submucosal dissection. Beginning at the distal side of the artificial ulcer, PEG was applied to the ulcer floor while pulling the scope out spirally to the proximal side of the ulcer using a spraying tube to apply gel precisely.
2.5 Postoperative care
The porcines were fasted on the day of ESD and on the day after, and then liquid food was given after postoperative day 2. After postoperative day 5, the animals were fed a solid diet. If the porcines showed difficulty in taking the solid diet during the observation period, they were fed a semi-solid or liquid diet, depending on their conditions. If the animals were unable to take liquid, nutritional support at 60 kcal/kg/d was infused through a central venous line or gastric tube could not be passed through it. Weight change, daily food intake, and instances of regurgitation and vomiting were recorded daily by an experienced veterinarian.
2.6 Evaluation of esophageal stricture
2.6.1 Clinical assessment
The rates of weight loss were calculated from body weight measured before ESD and
immediately before sacrifice and were compared between the control and PEG groups. Dysphagia was scored by using the Mellow-Pinkas dysphagia score in a completely blinded manner: 0 = normal swallowing (solid diet can be swallowed), 1 = a proportion of the solids unable to be swallowed, 2 = a semi-solid diet able to be swallowed, 3 = only liquids able to be swallowed, and 4 = complete dysphagia including saliva. [11]Endoscopic analysis was performed at 3 time points (3, 7, 14 days after the ESD procedure) to evaluate the ulcer surface, the effectiveness of PEG, and the ability to pass the gastroscope. Stricture formation was defined as the presence of a stenosis of the esophageal lumen that had progressed to the point where a 9.8-mm diameter upper gastrointestinal endoscope (GIF Q240 or GIF H260; Olympus Co.)
2.6.2Macroscopic assessment
The animals were euthanized on day 14 post-ESD. Then the esophagus was excised, dissected longitudinally on the remaining 1/4 of the normal tissue of the esophagus and examined macroscopically. The degree of stricture was calculated using the previously reported equation to express the mucosal contraction rate (MCR).[11]
2.6.3 Histologic, immunohistochemical and immunofluorescence analysis
After macroscopic assessment, the specimens were fixed in formalin and routinely processed into 4um-thick paraffin-wax-embedded sections. Hematoxylin-eosin (HE) and Masson’s trichrome staining was carried out by conventional methods. For immunostaining, the primary anti-α-SMA antibody (14395-1-AP, proteintech, 1:200) and anti-vimentin antibody (10366-1-AP, proteintech,1:200) were used. After primary antibody incubation, the sections were washed with PBS and incubated with the appropriate fluorescent secondary antibody (#8878, CST,1:200) secondary antibodies (Molecular Probes, Life Technologies, USA, 1:200) for 1h at room temperature. Nuclei were counterstained with 4,6-Diamidino-2-phenyindole dilactate (DAPI). Finally, coverslips were sealed with nailpolish, and the sections were analyzed under a fluorescence microscopy.
Extent of epithelial coverage were evaluated by HE. Inflammatory cell infiltration score: a score of 0 represented no leukocytes in photomicrograph, a score of 1 represented a small amount of inflammatory cell infiltration, a score of 2 represented more inflammatory cell infiltration, a score of 3 represented moderate amount of inflammatory cell infiltration, a score of 4 represented massive inflammatory cell infiltration.[12] The maximal thickness of the submucosal fibrotic tissue in vertical direction was calculated by Masson. Damage to the muscularis propria was assessed by using the following 4-step scoring system: 0 = no atrophic or fibrotic change in the muscularis propria evident in any of the examined sections, 1 = atrophy or fibrosis present but confined to the inner circular muscle layer, 2 = atrophy or fibrosis present but confined to the outer longitudinal muscle layer, and 3 = transmural fibrosis of the muscularis propria.[13]
2.6.4 Western blot
The frozen esophageal tissue was homogenized in the RIPA buffer supplemented with 1% protease inhibitors. After the determination of protein concentration were quantified by a BCA protein assay kit (Beyotime), according to the manufacturer’s protocol. Then the separated bands were transferred to PVDF membrane. The blots were probed with the following primary antibody: Fibronectin(1:500), vimentin(1:1000), collagen I(1:500), collagen III(1:1000), E-cadherin (1:1000), β-actin(1:1000), VEGF(1:1000), TGF-β1 (1:1000),Smad2(1:1000), Smad3(1:1000), pSmad2(1:1000), pSmad3(1:1000),β-catenin(1:1000), wnt3a(1:1000), β-catenin. The membranes were then reacted with Immobilon western kit (Millipore, USA). β-actin was served as internal control.
2.7 Statistical analysis
Normally distributed variables were presented as medians and compared by Independent-samples Student t tests. Categorical variables were compared by Fisher’s exact test. All P values were two sided and P value < 0.05 was considered significantly. Statistical analysis was performed with SPSS 19.0.
3.1 Preparation and source of PEG materials
The PEG gel was made and applied as follows: Component A (0.4g) was dissolved in solvent A (HCl solution,2mL) and component B (0.4g) was mixed with solvent B (Na2HPO4-NaOH solution,2mL). These two components were then sprayed onto the ESD wound through a dual-syringe. (Fig. 2c). And it can be easily delivered by minimally invasive surgical techniques.
3.2 PEG properties
3.2.1Rheological tests
The larger elastic modulus (G′) than viscous modulus (G′′) proved the formation of hydrogel after mixing the two components. (Fig. 2d) The rheology test result further proved that the hydrogel could stay stable in a wide sweeping frequency. Moreover, the storage modulus and loss modulus of the hydrogel remain constant, independent of frequency changes. This illustrates that it is a viscoelastic solid with stable performance and great mechanical properties.
3.2.2 Morphological analysis
It shows that the PEG has a relatively complete and stable three-dimensional cross-linking network structure. The loose and porous structure of PEG may allow the transportation of cellular nutrients and promote the wound healing. (Fig. 2e, f)
3.2.3 Cytocompatibility
After 1, 3, and 5 days of incubation with different concentrations of leaching solutions of hydrogel (100, 50 and 25mg/mL), the survival rate of L929 cells are higher than 95% (Fig. 3a, b), indicating that the great majority of cells survived from this treatment, like cells treated with saline. Further, Dead/Live staining suggested that almost all L929 cells treated with different concentrations of hydrogel leaching solutions survived and were stained green, indicating that PEG has low cytotoxicity.
3.2.4 Water vapor transmission rate
Ideal dressings should have attribute to prevent dehydration and facilitate epithelization and cell migration into the wound.[14] Water vapor transmission rate(WVTR) is an important fact that represents its capability to retain moisture.[15] In this study, the PEG WVTR values was observed to be 1697 g·m− 2·24h− 1, which was close to the requirements needed to maintain a suitable fluid balance for the wound ulcer.(Fig. 3c)
3.2.5 Adhesive ability test
Debonding force-displacement curves for hydrogels and esophageal tissues were shown in Fig. 2d. PEG showed excellent adhesion on esophageal mucosa in vitro. In vivo, it also has been proved that PEG adhered well to ESD-induced ulcers even though in day 3. (Fig. 3d, e)
3.3 PEG prevents stricture after esophageal circumferential ESD
3/4 of the esophageal ESD was performed safely in all pigs, (Fig. 4a-d) and there was no procedure-related mortality or morbidity. (Table 1) In the control and PEG group, the mean operation time was 29.67 ± 6.51 and 31.67 ± 3.21 minutes(P = 0.658), respectively. The mean(± SD) size of the resected specimen was 10.59 ± 1.33 in the control group and 10.57 ± 0.4 in the PEG group(P = 0.981). According to Table 2, there was no difference in weight loss from preoperative to postoperative day 14 between the control and PEG groups (0.50 ± 0.50 vs 0.67 ± 0.58, P = 0.725). Vomiting was showed in two control group pigs on postoperative days 11 and 13, respectively. On the 13th postoperative day, one pig in the PEG group conversely developed brief vomiting. Endoscopy was performed at day 3,7,14 after ESD. (Fig. 5a-l) The PEG group showed that most of the hydrogel remained on the wound at day 3. Although all of the esophagus showed scar formation and slight lumen narrowing, the stricture in the PEG group was significant milder. In contrast, the control group developed more severe stenosis at day 7 and even pin-hole stricture at day 14. For humane considerations, we decided to sacrifice the animals at day 14 after ESD. The esophageal stricture rate was calculated by measuring the removed esophageal specimen. The PEG group showed significantly lower MCR than the control group (50.82 ± 6.01 vs 83.58 ± 7.02, P = 0.049 < 0.05). This result was in accordance with the dysphagia scores (Table 2) and endoscopic assessments (Fig. 5).
3.4 PEG promotes esophageal epithelization after ESD
According to the results of HE staining, the PEG group showed larger epithelized diameters than the control group and exhibited a significantly better level of mucosal re-epithelization compared with the control group(4.10 ± 0.76vs 1.14 ± 0.40, P = 0.037 < 0.05). (Fig. 6a-d) The border between the nascent mucosa and the original mucosal epithelium was determined by the absence of any muscular mucosal tissue (Fig. 6e). No foreign-body type reactions were observed in the regenerative mucosa in the PEG-treated group.
3.5 PEG inhibits inflammation and fibrosis after ESD
We observed a significant difference in the score of inflammatory cells between the control group and the PEG group (3.00 ± 1.00 vs 2.33 ± 0.58, P = 0.374 < 0.05). (Fig. 6a-d) The maximal thickness of fibrosis in the submucosal layer was significantly thinner in the control group as compared to PEG group (1.29 ± 0.19 mm vs 1.03 ± 0.31mm, P = 0.370 < 0.05).(Fig. 7a-d) The median MP damage score in PEG group was also significantly lower than control group (0.67 ± 0.58 vs 2.33 ± 0.58, P = 0.037 < 0.05).(Table 2) The widespread consensus among academics is that a crucial step in tissue healing is the differentiation of fibroblasts into myofibroblasts.[16] Although the high contractile force of myofibroblasts facilitates the reepithelialization of the mucosal epithelium, the excessive function of these cells leads to pathological narrowing of the digestive tube.[17] According with it, our immunofluorescence analysis showed that there are more myofibroblasts were activated by fibroblasts in the control group.(Fig. 8a-d) Meanwhile, immunohistochemical staining of α-SMA in the wound bed on day 14, the submucosa showed more spindle-shaped myofibroblasts in the control group, whereas more neovascularization appeared in the experimental group.(Fig. 9a-d) Among the fibrosis-related indices, western blots analysis uncovered that the protein levels of Fibronectin, Vimentin, Collagen I, Collagen III, as well as inhibited the protein levels of E-cadherin in esophageal tissues, whereas these changes could be suppressed by PEG treatment (Fig. 10a).
3.6 Mechanism of PEG-Induced reduction of esophageal fibrosis
Transforming growth factor-β(TGF-β)is thought to be a myofibroblast transdifferentiator in normal tissue injury repair, and various cells secrete large amounts of TGF-β1, which activates the Smad signaling pathway by regulating the proliferation and differentiation of related cells, so TGF-β1 is also known as the "initiator" of tissue and organ fibrosis.[18] Accordingly, we examined whether PEG treatment could modulate the activation of TGF-β1/ Smad pathway in post esophageal ESD. Western blot analyses revealed that there was no significant difference in the relative levels of Smad2 and Smad3 in esophageal tissues from two groups of pigs while the relative levels of TGF-β1, pSmad2, pSmad3 and in esophageal tissues of the PEG group were remarkably lower than that in the control group. (Fig. 10b) Aside from the TGF-β/Smad signalling pathway, the Wnt/β-catenin pathway has been shown to play a role in fibroblasts activation and fibrosis in a number of organs, including the liver, kidney, lung, and pancreas.[19] However, our western blot showed that there were no significant differences in Wnt3a and β-catenin between two groups. (Fig. 10b)
In recent years, the expansion of the indications for ESD may increase the incidence of post-ESD esophageal stenosis.[20] Endoscopic balloon dilation (EBD) is the traditional common clinical treatment for post-ESD strictures, but serious adverse events such as perforation may still occur.[21] Therefore, the prevention of stricture after esophageal ESD should be paid more attention. To date, steroid therapy is the main method for preventing stricture after esophageal ESD[22]. However, there were severe complications reported by several studies[23, 24]: systemic infection with oral administration and postoperative perforation after focal injections.
Biomaterials are favored in the fields of tissue engineering and regenerative medicine for their excellent biocompatibility and unique physiological properties.[5, 11, 25] Yoshiki [26] et al reported that PGA sheets and fibrin glue covering postoperative wounds after esophageal ESD was a novel approach that significantly decreases the incidence of esophageal stricture and the number of EBD sessions subsequently required. However, there is a lot of work that needs to be prepared in the early stage, and the endoscopic procedure was inconvenient that application time of PGA sheet was 12.8 ± 5.5 minutes compared to only seconds in our study. Meanwhile, fibrin glue has the risk that bio-derived products may transmit viruses and cause adverse immune reactions, while in a humid physiological environment, the adhesive strength of colloids is poor and the mechanical strength is insufficient. Whereas our commercialized synthetic PEG hydrogel are free of these possible risks, and notable feature of tetra-PEG hydrogels is their mechanical toughness. Apart from that, there are some newer emerging methods include that autologous cell sheet transplantation,[27] high-density collagen patch,[28] CMC sheets[5] had better outcomes in preventing stricture and promoting re-epithelialization of the esophagus following extensive ESDs. In addition to requiring the time-consuming and laborious fabrication of shielding materials, these technologies are also connected with an unstable and complicated technology.
At present, a variety of PEG derivative medical gel have been commercialized, including dural seal,[29] sciatic nerve anastomosis,[30] retinal reduction surgery, [31] prevention of surgical adhesions,[32] ophthalmic surgery,[32] etc. As far as we know, few studies have reported the application of PEG in esophagus, particularly in the endoscopic treatment process. For the ammonolysis reaction, amino reacts fast with succinimidyl-active ester without the need of catalysis and special conditions.[22] As described in the material approach, the unique gel-forming mechanism of our gel is based on the chemical reaction between component A and the functional group on component B, especially the sulfhydryl group on component B, and the formation of thioester bonds, amide bonds, disulfide bonds and other chemical bonds increases the degree of cross-linking of the ge. When our hydrogels touched the tissues immediately, the residual succinimidyl-active ester in the hydrogel network reacted with amino groups on tissue proteins that resulted in the formation of new chemical bonds between hydrogels and tissues. [18, 21] So that this makes our items are simple to apply and do not require any additional metal clips or other fixings to remain in place on the wound. Additionally, because of its excellent adhesion in a humid physiological environment, it preserves the wound for a longer time and minimizes oesophageal fibrosis. The endoscopic findings were also consistent with this feature, the majority of the hydrogel was still on the wound on the third postoperative day.
In the present study, we used this synthetic hydrogel compound for reducing the stricture after extensive esophageal ESD. At first, considering the risk of esophageal stricture formation after ESD[33], a > 3/4 esophageal circumference ESD with lesion longitudinal diameter > 5 cm was performed in a porcine model. Two weeks after ESD, the stricture rates were 83.58% vs 50.82% between the control and PEG group. The study conducted by Honda et al.[11] demonstrated that injected adipose tissue-derived stromal cells was successful in a canine model. 8 weeks after ESD, the MCR were 45.3% vs. 75.7% in the experimental group and control group respectively. Zhang et al.[34] investigated fully covered self-expanding metal stent (FCSEMS) placement with an acellular dermal matrix (ADM) for preventing post-ESD esophageal stricture, and demonstrated lesser esophageal stricture on macroscopic evaluation (21.02% vs. 57.41%). Despite discrepancies in the selection of animals and the length of mucosal resection, the MCR was comparable to our findings.
Previous studies have shown that fibrosis and esophageal scarring are the main causes of esophageal stenosis, and fibroblasts are the most important repair cells in this process, so preventive treatment implemented as soon as possible after ESD to promote epithelial formation and reduce inflammatory response is particularly important in inhibiting stricture formation in hollow organs.[35] The PEG-treated animals showed a significantly lower mucosal contraction rate, better mucosal epithelial repair speed, milder submucosal fibrosis, less severe injury to the muscularis propria, and mild clinical dysphagia. It suggests that PEG can effectively prevent stenosis after esophageal ESD and relieve clinical symptoms.
Moreover, because of the similar structure between hydrogels and extracellular matrixs (ECMs), the survival of cells and biologically active molecules could be supported in hydrogels.[36] Vascular endothelial growth factor (VEGF) is essential for vascular development, which increases the endothelial cell proliferation, survival, migration, and promotes angiogenesis.[37] Blood vessel has been considered as the critical factor for tissue regeneration due to its function of providing nutrition and oxygen for cells around wounds[38]. Based on our findings, in the experimental group, VEGF was substantially expressed, and α-SMA immunohistochemistry staining revealed increased neovascularization in the submucosa of the PEG-treated esophagus. We suggested that the PEG may be more efficient for improving wound repairs, increase the quality of regenerated tissues, and decrease excessive fibrosis.
The mechanism by which ulcer healing leads to stenosis have not been elucidated in detail, and most scholars believe that it begins with local recruitment of inflammatory cells and fibroblasts, followed by persistent pathological microenvironment leads to transition or differentiation of myocytes to myofibroblasts, PEG may inhibit the phenomenon. In this cellular environment, a large number of cytokines and growth factors are released, including members of the TGF-β family, which is the main effectors of the fibrotic process. TGF-βto their stem cell surface receptors and initiate downstream signal transduction, ultimately resulting in nuclear translocations of Smad2/3 transcriptional regulators, respectively. Further enhances myofibroblast differentiation and the production and secretion of ECM proteins, including collagen and fibronectin. In contrast, our results indicate that essential wnt pathway proteins were not significantly altered between the two groups. In a comparative cell experiment, Gago LA, et al.[39] concluded that the ability of CMC sheets to suppress fibrosis was likely due to their mechanical barrier function. the quantitative measurements of serum pro-inflammatory and pro-fibrotic cytokines suggested that the application of CMC sheets on the mucosal defect had anti-fibrotic action. So we also think it is more likely that fibrosis is mitigated through the TGF-βpathway. It also has to be confirmed whether or not additional mechanisms are explicitly implicated in the future.
There are still a number of limitations to this study that need to be acknowledged. Foremost, the sample size of this animal study was small, though. Secondly, we did not assess long-term data of esophageal stenosis. Therefore, we were unable to analyse the association of stenosis and histological effects with clinical dysphagia and swallowing function. Thirdly, the specific effect of PEG in preventing esophageal stricture through physical isolation or other means has not been confirmed. We recommend larger sample sizes and longer observation periods to further demonstrate the effectiveness of PEG before applying it to clinical practice. In addition, cell experiments can be performed to further investigate the mechanism of PEG as a mechanism for the prevention of esophageal strictures.
In summary, as a new tissue shielding strategy, the application of PEG in mucosal defects is expected to improve the speed and quality of epithelial healing, reduce fibrosis, and prevent esophageal stricture during extensive postoperative ESD healing.
Ethics approval and consent to participate:
The experimental protocol was established, the protocol was approved by the local ethics committee authorized for animal experimentation at the Gateway Medical Innovation Center (Registration number SH2021 08011). Written informed consent was obtained from individual or guardian participants.
Consent for publication:
This manuscript has not been published previously, is not under consideration for publication elsewhere and this publication is approved by all authors and the responsible authorities where the work has been carried out. This manuscript will not be published elsewhere in the same form.
Availability of data and materials:
The data that support the findings of this study are available on request from the corresponding author upon reasonable request.
Competing interests:
The authors report no conflict of interest.
Funding:
This study was funded by Endoscopic spraying of fibrin binder for the prevention of delayed bleeding after endoscopic mucosal dissection of gastric superficial tumors Research Fund (No.201940082) and grants from the National Nature Science Foundation of China (No. 82000622).
Authors’ contributions section:
Statement of authorship: Feng Liu and Yunlei Wei designed experiments; Yunlei Wei and Jian Tang prepared figures 1-3; Feng Liu, Xiaojia Hou, Jun Li, Duo Chai, Min Zhao and Di Zhang prepared figures 4-5; Yunlei Wei, Jian Tang, Lei Li, Di Zhang prepared figures 6-10; Yunlei Wei and Jian Tang drafted the article; All authors reviewed the manuscript.
Acknowledgment:
We would like to thank all the authors.
Authors’ information
Yunlei Wei2, Jian Tang1, Xiaojia Hou1, Jun Li1, Lei Li1, Di Zhang2, Duo Chai1, Min Zhao3, Feng Liu1
1.Digestive Endoscopy Center, Shanghai Tenth People’s Hospital, Tongji University School of Medicine, Shanghai, China
2.Anhui University of Science and Technology, Huainan, China
3.Bengbu First People's Hospital, Bengbu, China
DISCLOSURE
All authors disclosed no financial relationships.
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Table 1. Data of ESD between PEG group and control group.
|
PEG group |
Control group |
P value |
|
|
ESD procedure time, min, x ± s |
31.67±3.21 |
29.67±6.51 |
P=0.658 |
|
En-bloc resection (%) |
3 |
3 |
- |
|
Size of the resected specimen,(cm2), x±s |
10.57±0.4 |
10.59±1.33 |
P=0.981 |
|
Bleeding (%) |
0 |
0 |
- |
|
Perforation rate (%) |
0 |
0 |
- |
Table 2. Clinical outcomes, gastrointestinal radiography results, and histological findings
|
PEG group |
Control group |
P value |
|
|
Dysphagia score |
2±0 |
2.67±0.58 |
P=0.116 |
|
Weight change (kg) |
0.5±0.5 |
0.67±0.58 |
P=0.725 |
|
Degree of stenosis (%) |
50.82±6.01 |
83.58±7.02 |
P=0.049 |
|
Epithelized diameters |
1.14±0.40 |
4.10±0.76 |
P=0.037 |
|
Thickness of the submucosal layer,μm |
1034.49±313.42 |
1292.487±190.52 |
P=0.290 |
|
MP damage score |
0.67±0.58 |
2.33±0.58 |
P=0.037 |
|
Inflammatory cell infiltration score |
3.00±1.00 |
2.33±0.58 |
P=0.374 |
No competing interests reported.