Mast Cells Tryptase Promotes Intestinal Fibrosis in Natural Decellularized Intestinal Scaffolds

Abstract

Background:

Standard two-dimensional (2D) culture has confirmed the mechanism of mast cells (MCs) in the pathogenesis of inflammatory bowel disease (IBD), but the regulation of signaling responses of MCs may well differ in three-dimensional (3D) microenvironments. The aim of the study was to develop a 3D culture model based on decellularized intestinal scaffolds (DIS) and verify how MCs influenced fibroblasts phenotype in the 3D model.

Methods:

DIS were achieved using the detergent technique and extracellular matrix (ECM) components were verified by histologic analysis, quantification and scanning electron microscope. After human colon fibroblasts recellularized into the scaffolds and activated by MCs tryptase and TGFβ1, the changes in genes and signaling pathways during fibroblasts activation in 3D were studied and compared with the changes in 2D cell culture on plastic plates.

Results:

Decellularization process effectively removed native cell debris while retaining natural ECM components and structure. The engrafted fibroblasts could penetrate into the scaffolds and maintain its phenotype. No matter whether fibroblasts were cultured in 2D or 3D, MCs tryptase and transforming growth factor β1 (TGF-β1) could promote the differentiation of fibroblasts into fibrotic-phenotype myofibroblasts through Akt and Smad2/3 signaling pathways. Furthermore, the pro-collagen1α1 and fibronectin synthesis of myofibroblasts in 3D was higher than in 2D culture.

Conclusion:

Our results demonstrated that the DIS can be used as a bioactive microenvironment for the study of intestinal fibrosis, providing an innovative platform for future intestinal disease modeling and screening of genes and signaling pathways.

Introduction

In the past decade, the incidence of inflammatory bowel disease (IBD) has been increasing and it has become a global disease [1]. Although lots of drugs including biological agents have been used to treat IBD, intestinal fibrosis is still inevitable in many patients, which severely affects the physical and psychological status of the patients [2]. To develop effective anti-fibrosis agents, researchers have conducted a lot of researches on the molecular and cellular mechanisms of intestinal fibrosis [3]. Unfortunately, most of the virulence gene identification and drug screening were based on two-dimensional (2D) cell culture, it was revealed that more than 90% of predictions from experiments have failed when translated into human treatments for a variety of diseases [4]. In traditional 2D cell culture, these "tool cells" were difficult to retain the inherent heterogeneity of the original tissues from which they originated because they were adapted to the conditions of plastic plate culture in vitro [5]. Therefore, it is necessary to establish a model of IBD in vitro and to develop a drug screening platform that can more accurately reproduce the complex physiology of human fibrotic intestine.

The rapid development of tissue engineering provides innovative 3D models for the study of pathophysiological mechanisms of gastrointestinal diseases in vitro. Currently, a variety of 3D models have been used to study the pathophysiology of IBD [6–8]. Among them, decellularized scaffolds, as a kind of natural 3D model, have aroused great interest of researchers and led to unanticipated results [9]. For example, decellularized scaffolds have been successfully used to mimic immunologic responses seen in IBD. Currently, decellularized scaffolds have been successfully obtained from decellularized human and mouse tissues [10, 11]. The greatest advantage of decellularized scaffolds is that the preserved complex extracellular matrix (ECM) networks are helpful for cells to simulate their complex physiological conditions in vitro. Now, with the help of proteomic analysis techniques, we can more accurately analyze the protein content of decellularized samples. It was demonstrated that the ECM including collagens I, IV, VI, XII, XIV, fibrinogen, dermatopontin, and fibronectin were well retained after decellularization [12]. The native ECM will provide physiological mechanical stimuli for the cells. Thus, how cells grown in vivo could be simulated in vitro and the actual intracellular signal transduction pathway will be explored.

The characteristic of intestinal fibrosis is that the activated myofibroblasts lead to the accumulation of ECM. In recent years, various studies have found that mast cells (MCs) played a very important role in the pathogenesis of IBD [13]. It is believed that MCs are involved in inflammation and fibrosis of various organs by secreting mediators such as tryptase and histamine. Our previous studies confirmed that the underlying mechanism was that tryptase promoted the differentiation of fibroblasts into fibrotic-phenotype myofibroblasts by activating the PAR-2/Akt/mTOR pathway [14]. However, some researchers have shown that the interaction between MCs and fibroblasts was modulated when cultured in 3D scaffolds compared to conventional 2D cell culture on plastic plates, highlighting the important role of ECM [15]. Therefore, the role of ECM in regulating the interaction between MCs and fibroblasts cannot be ignored, and it is necessary to verify the results of conventional 2D culture by a complex 3D cell culture model.

In our previous study, we demonstrated the feasibility of decellularization and recellularization of decellularized scaffolds [16], which provided a valuable research platform for tissue engineering by engrafting cells on ECM scaffolds. This study aimed to verify the 3D model based on decellularized intestinal scaffolds (DIS), mainly to explore the relationship between MCs and fibroblasts activation in the process of intestinal fibrosis in IBD. Meanwhile, considering that transforming growth factor β1 (TGF-β1) has been proven to be a key factor leading to fibrosis [17], it was included in the study as a control.

Materials and methods

Preparation of DIS

The decellularization procedure for the DIS was performed based on a previously reported method [18]. The study was approved by the Ethics Committee of the Shanghai Tenth People’s Hospital affiliated with the Tongji University School of Medicine (IRB no. 21K190) and conducted in accordance with the tenets of the Declaration of Helsinki. Human terminal ileum was obtained from the right hemicolectomy or partial intestinal resection specimen. Before decellularization, the mesenterium was removed and the tissue sections were cut into pieces (1 × 1 cm²). Then the tissues were decellularized with 4% sodium deoxycholate (Sigma-Aldrich, St. Louis, MO, USA) for at least 24 h using the magnetic stirrer, with solution changed every 6 h. This was followed by a next step: incubation with 200 ug/ml DNase-I (Sigma-Aldrich) for 12 h. The tissues were then washed with PBS for 48 h, with multiple PBS changes throughout. Finally, the DIS were sterilized by 0.1% peracetic acid/4% EtOH for 2 h, followed by a washing step in sterile PBS for at least 10 times.

Characterization of DIS

To verify the decellularization, the native tissues and DIS were fixed with 4% paraformaldehyde overnight. Hematoxylin eosin (HE) staining was performed to confirm the result of decellularization, and masson trichrome, sirius red (SR) staining and scanning electron microscopy (SEM) were adopted to confirm the remaining ECM after the decellularization. Collagen I, collagen IV and fibronectin in native and decellularized samples were further confirmed by immunofluorescence (IF). For the quantification of the DNA and collagen, the samples were analyzed by the DNA Extraction Kit (Solarbio, Beijing, China) and Hydroxyproline assay kit (Nanjing Jiancheng bioengineering institute, Nanjing, China) following the manufacturer’s instructions.

Cell culture

The CCD-18Co fibroblasts were purchased from ATCC and cultured with a complete low-glucose dulbecco’s modified eagle medium (L-DMEM; Gibco, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS; Gibco), 1% penicillin/streptomycin (P/S; Gibco). MCs LAD2 were obtained from the cell bank of the Chinese Academy of Sciences and cultured with a complete 1640 medium (Gibco) containing 10% FBS and 1% P/S.

Collection of tryptase conditioned medium

To collect the tryptase conditioned medium (CM), 2 × 10⁵ LAD2 cells were cultured in T25 flasks in complete 1640 medium for 24 h and then supplemented with FBS free 1640 medium for another 24 h cultivation. The supernatant was collected and centrifuged at 5000 rpm to remove the dead cells and cell debris. The concentration of tryptase in the CM was determined by the Human Mast Cell Tryptase (MCT) ELISA Kit (CUSABIO, Wuhan, China) according to the manufacturer’s instructions.

Fibroblasts treatment with tryptase and TGF-β1

The CCD-18Co fibroblasts were evenly plated in 24-well culture plates (Corning, NY, USA). When the cell density approached about 80%, the medium was changed to FBS free L-DMEM and cultured for another 24 h. For the treatment with tryptase, an appropriate amount of 50% tryptase-CM and 50% L-DMEM supplemented with 20% FBS were added to the culture plates. After cultivation for 48 h, the fibroblasts were collected for a Western blot (WB) assay and the supernatants were collected for an ELISA assay. For the treatment with TGF-β1 (Abcam, Cambridge, UK), TGF-β1 were added to the culture dishes at the concentration of 10 ng/ml and incubated for 48 h. The fibroblasts and supernatants were collected as described previously.

Fibroblasts cultivated in DIS

For cell culture in DIS, 2 × 10⁵ fibroblasts in 200 ul L-DMEM were engrafted into the DIS, fixed with the tailored metal rings (Fig. 1). All 3D cell culture, as with 2D culture, were performed under standard conditions (37 °C, 5% CO₂). To evaluate the cells engraft rate on the DIS, the supernatant was collected and unseeded cells were counted after 4 h of incubation. After an adhesion time of 4 h, another 800 ul medium was added into the plates and cultured for 7 days. The treatment with tryptase and TGF-β1 in the DIS was the same as in plate culture. The cellular viability of fibroblasts in 2D and 3D were assessed using CCK8 (Meilunbio, Dalian, China) following the manufacturer’s instructions. The samples and supernatants were collected for WB, histological and ELISA assay.

Fig. 1.

Conceptual diagram of MCs tryptase promoting fibroblasts differentiation to myofibroblasts on 3D DIS

Immunofluorescence

The sample sections were retrieved in sodium citrate buffer (pH 6.0) (Solarbio, China) using microwave antigen retrieval method. Then, the sections were permeabilized with 0.5% Triton X-100 for 15 min, and blocked with 5% bovine serum albumin (BSA; Sigma) for 30 min. Subsequently, the sections were incubated with primary antibodies at 4 °C overnight. The next day, primary antibodies were removed and the sections were incubated with secondary antibodies for 1 h at room temperature. Finally, the sections were stained with dapi (Meilunbio) for 10 min and visualized using an Olympus fluorescence microscope. The primary antibodies used in this study were as follows: rabbit anti-Collagen I (1:100, Abcam), rabbit anti-Collagen IV (1:100, Abcam), and rabbit anti-Fibronectin (1:100, Abcam), rabbit anti-Ki 67 (1:100, Abcam), rabbit anti-alpha smooth muscle actin (α-SMA; 1:100, Abcam),mouse anti-Vimentin (1:100, Abcam). Secondary antibodies included Alexa Flour 488 (1:200, Abcam) and Cy3 (1:200, Abcam, USA).

Western blot

Total protein was extracted from cells or tissues on ice for 30 min using RIPA lysis buffer, phenylmethanesulfonyl fluoride (PMSF), and phosphatase inhibitor cocktail (Beyotime, Shanghai, China). Protein concentrations were determined using BCA Protein Assay Kit (Beyotime) according to the manufacturer’s instructions. Subsequently, protein samples were separated by polyacrylamide gel electrophoresis (SDS-PAGE; Beyotime) and blotted on polyvinylidene fluoride (PVDF) membranes (Beyotime). After blocked in 5% skim milk, the samples were incubated with primary antibodies at 4 °C overnight. The next day, the membranes were incubated with secondary antibodies and visualized using an Odyssey Scanning system (LI-COR Biosciences). All the primary and secondary antibodies used in this study were purchased from Abcam and were as follows: rabbit anti-β actin, rabbit anti-Collagen I, rabbit anti-Fibronectin, rabbit anti-phospho-Smad 2/3, rabbit anti-Smad 2/3, rabbit anti-phospho-Akt, rabbit anti-Akt, rabbit anti-α-SMA.

Statistics

All data analyses were conducted using GraphPad prime 8.0. Quantitative data were expressed as the mean ± standard deviation. Student’s t-test was used for statistical analysis. Differences were considered significant at p < 0.05.

Results

Preparation and characterization of DIS

Macroscopically, the color of the small intestine changed gradually during the decellularization process. After infusion of 4% sodium deoxycholate for 24 h, the intestine became semitransparent (Fig. 2). HE staining showed a significant reduction in cell bodies in the DIS compared to the natural intestine. At the same time, according to the results of masson trichrome and SR staining, the collagen and microstructure in the DIS were well preserved during the decellularization process (Fig. 2B). It was further confirmed by SEM that the integrity of 3D microstructure of the DIS was comparable to that of native tissue (Fig. 2C). Meanwhile, there were no residual cells in the DIS. In order to better reflect the expression and distribution of different components of ECM in the DIS, IF was used to highlight the key structural ECM proteins. A total of three proteins including collagen I, collagen IV and fibronectin were examined and the distribution of each ECM protein in the DIS were completely preserved when compared to native tissues (Fig. 2D). Quantitative DNA analysis demonstrated that the DNA content of DIS was 58.47 ± 6.37 ng/mg dry weight in contrast to 681.87 ± 39.60 ng/mg for the native intestine (Fig. 3B) (p < 0.05). The collagen content in DIS was 279.07 ± 21.30 ng/mg dry weight compared to 352.57 ± 40.29 ng/mg dry weight in native intestine (Fig. 3A) (p > 0.05). Together, these results showed that after complete decellularization, the DNA in the DIS was effectively removed and the ECM was effectively preserved.

Fig. 2.

Human intestine harvest and decellularization. A The intestine became semitransparent after decellularization. B HE, masson trichrome and SR staining showed the absence of cells in the DIS compared to the native intestine, while the collagen was well preserved. C SEM showed that the integrity of 3D microstructure of the DIS was comparable to that of native intestine. D The key structural ECM proteins including collagen I, collagen IV and fibronectin were examined by IF

Fig. 3.

Collagen and DNA analysis in native intestine and DIS. A Quantitative collagen analysis. B Quantitative DNA analysis

Repopulation of fibroblasts in the DIS

After 7 days of culture, the repopulated scaffolds were harvested for histological analysis. HE staining demonstrated that the fibroblasts could not only be engrafted into the DIS, but also migrated through the scaffolds (Fig. 4). Meanwhile, CCK-8 assay showed that the proliferation ability of fibroblasts in the scaffolds was significantly slower than that in the plate culture (Fig. 4C). Moreover, IF demonstrated that engrafted fibroblasts exhibited the ability to proliferate in DIS, which was confirmed by Ki67 and Vimentin (Fig. 4D). Moreover, fibroblasts in the DIS exhibited a higher positive rate for Ki67 and Vimentin not only in the group of normal groups, but in the group of tryptase and TGF-β1.

Fig. 4.

Fibroblasts cultivated in DIS. A Fibroblasts were engrafted into the fixed DIS. B HE staining demonstrated that fibroblasts were engrafted into the DIS after 7 days of culture. C CCK-8 assay showed that the proliferation ability of fibroblasts in the DIS. D IF demonstrated that engrafted fibroblasts exhibited the ability to proliferate in the group of NC, Tryptase and TGF-β1, which was confirmed by Ki67 and Vimentin

Differentiation of fibroblasts to myofibroblasts on plastic plates vs. 3D DIS

The concentration of tryptase in the tryptase CM was 14.52 ± 2.45 ng/mL. Our previous results showed that MCs tryptase and TGF-β1 promoted the expression of α-SMA protein in fibroblasts, thereby activating the differentiation of fibroblasts into fibrotic-phenotype myofibroblasts (Figs. 5, 6). Similarly, fibroblasts in the DIS treated with MCs tryptase and TGF-β1 increased the expression of α-SMA protein and differentiated into fibrotic-phenotype myofibroblasts (Figs. 5B, 6A). In further verification of intracellular signaling pathways, WB showed that fibroblasts in both the tryptase and TGF-β1 groups were activated into myofibroblasts via the Akt and Smad2/3 pathways in both 2D and 3D models, compared with the NC group (Fig. 6A). In 2D and 3D culture models, the concentrations of collagen 1α1 in NC group, MCs tryptase group and TGF-β1 group were respectively: 8.98 ± 0.69 ng/mL versus 11.26 ± 1.25 ng/mL, p > 0.05; 14.96 ± 1.57 ng/mL versus 19.07 ± 1.01 ng/mL, p < 0.05 and 16.77 ± 1.22 ng/mL versus 21.58 ± 1.49 ng/ml, p < 0.05. Noteworthy, the concentration of pro-collagen 1α1 in MC tryptase and TGF-β1 group was significantly different (p < 0.05) (Fig. 6B). The concentrations of fibronectin in NC group, MCs tryptase group and TGF-β1 group were 367.65 ± 35.83 ng/mL versus 405.72 ± 23.06 ng/mL, p > 0.05; 539.70 ± 31.98 ng/mL versus 634.35 ± 33.33 ng/mL, p < 0.05 and 743.72 ± 26.09 ng/mL versus 863.84 ± 25.93 ng/ml, p < 0.05 for 2D versus 3D, respectively. Notably, the concentration of fibronectin in MCs tryptase and TGF-β1 group was significantly different between 2D and 3D (p < 0.05) (Fig. 6C).

Fig. 5.

Fibroblasts differentiate to myofibroblasts. A MCs tryptase and TGF-β1 promoted the expression of α-SMA protein in fibroblasts in plastic plates. B MCs tryptase and TGF-β1 promoted the expression of α-SMA protein in fibroblasts in 3D DIS

Fig. 6.

Fibroblasts activation in 2D vs. 3D. A WB showed that fibroblasts in both the MCs tryptase and TGF-β1 groups were activated into myofibroblasts via the Akt and Smad2/3 pathways in both 2D and 3D models, compared with the NC group. B The concentrations of pro-collagen1α1 and fibronectin in MCs tryptase and TGF-β1 group was significantly different in 2D and 3D models (p < 0.05)

Discussion

There are increasing evidences that in the study of intestinal fibrosis, 3D cell culture in vitro shows similar characteristics to the occurrence of intestinal fibrosis in vivo. By simulating the complex microenvironment in which intestinal fibrosis develops in vivo, 3D culture has the potential to provide an ideal platform for future studies of the cellular and molecular mechanisms of intestinal diseases in vitro. Here, we established a 3D culture model that can simulate the internal environment of the human gut and explore mechanism of intestinal fibrosis. We recellularized human colon fibroblasts in the DIS and simulated the process of fibroblasts activation by MCs tryptase and TGF-β1 leading to intestinal fibrosis. Our results showed that MCs tryptase and TGF-β1 promoted the differentiation of fibroblasts into fibrotic-phenotype myofibroblasts through Akt and Smad2/3 signaling pathways in the 3D environment, which was consistent with the results of conventional 2D culture. However, the pro-collagen1α1 and fibronectin synthesis of myofibroblasts in 3D culture was higher than in 2D culture.

For the past few years, various materials including natural and synthetic have been used as 3D scaffolds to replicate a more physiologically relevant microenvironment for cell culture in vitro [19–22]. Compared with other tissue engineering scaffolds, decellularized scaffolds possess natural ECM with micro-architecture that resembles native organs, and allow the introduction of mechanical stimuli, suggesting that decellularized scaffolds are innovative platforms for in vitro modeling of healthy and diseased tissues. A number of major studies have demonstrated that decellularized scaffolds could be generated into transplantable and functional grafts through the recellularization [23–25], which laid the foundation for their applicability as 3D scaffolds for cell culture. Currently, many methods have been used as decellularization strategies [26, 27]. Appropriate decellularization process will maximize the retention of native ECM structure and content, which play an important role in intercellular signaling [9, 18]. Our study additionally showed that after standard decellularization of human small intestine, DNA in the original tissue was basically removed. At the same time, major ECM proteins such as collagen and fibronectin were well preserved. SEM showed that the native spatial structure of the small intestine was preserved, which created a suitable environment for cell engraftment and provided an ideal platform for further experiments. Subsequently, fibroblasts were engrafted in the DIS and cultured for 1 week, confirming that fibroblasts could proliferate in the scaffolds. In contrast, compared with 2D culture, the cell growth was lower in 3D, but cell function was not affected (Fig. 4), which was consistent with most existing research. Due to the differential exposure to oxygen and nutrients of cells in 2D and 3D culture systems, the proliferation of cells in 2D usually displayed higher rate [28]. On the other hand, considering the more complex internal environment of 3D scaffolds, cell migration was usually accelerated [29].

At present, a large amount of evidences shows that MCs play a prominent role in the process of fibrosis, such as skin [30], heart [31], lung [32], liver [33] and kidney [34]. MCs are characterized by an abundance of electron-dense vesicles/granula filled with various preformed compounds including proteases, chymase, tryptase, histamine and growth factors. The diversity of mediators leads to their involvement in a variety of biological activities in vivo, such as immunoregulation, anti-infections and maintaining normal homeostasis. As for the mechanism of MCs promoting fibrosis, it may vary in different organs [35]. In the study of intestinal fibrosis by Hamilton et al., MCs were believed to be involved in the regulation of epithelium permeability, inflammatory responses, and remodeling of inflammatory tissue [36]. Our previous study has confirmed the role of MCs tryptase in IBD-induced intestinal fibrosis. The underlying mechanism was that tryptase promoted the differentiation of fibroblasts into fibrotic-phenotype myofibroblasts by activating the PAR-2/Akt/mTOR pathway [14]. But whether the 2D model based on the plastic plates can really simulate the actual situation in vivo. In fact, a number of studies have begun to use decellularized scaffolds for 3D culture. One of the advantages is that the complex spatial structure formed by native ECM proteins in the tissue provides a suitable microenvironment for cell proliferation and growth. It is believed that specific environment determines consequently the way cells grow and thus affects intracellular signal transduction pathways. Secondly, ECM stores a large number of growth factors involved in the regulation of cell differentiation and gene expression. Therefore, decellularized scaffolds are promising natural biomaterials that can simulate the microenvironment in vivo to the greatest possible extent in vitro compared to other materials.

Currently, decellularized intestine tissue derived trial models have mainly focused on the research of colorectal cancer (CRC). DIS not only provided similar tumor microenvironment (TME) for CRC, but also promoted epithelial mesenchymal transition (EMT) in CRC [22, 37]. Furthermore, CRC showed different drug resistance in DIS when compared to 2D culture [38, 39]. For intestinal fibrosis studies, Giuffrida et al. first engrafted human intestinal myofibroblasts in DIS, and compared myofibroblasts cultured in 3D scaffolds with those cultured on plastic dishes. The results showed that the effect of TGF-β1 on fibroblasts in 3D was more similar to that in vivo [40]. Another study showed that recellularized DIS could be used as a model mimicking epithelial immunologic responses in IBD [8]. In the studies related to pulmonary fibrosis, fibrotic matrices have been shown to significantly promote myofibroblast differentiation compared with normal matrices, highlighting the important role of ECM in driving cellular phenotypic responses in fibrotic disorders [41]. Therefore, the mechanical and biochemical cues in native ECM are indispensable for inducing specific cellular behavior [42]. In this study, IF and WB showed that, compared with the NC group, fibroblasts engrafted in the DIS were positive for α-SMA when stimulated by MCs tryptase and TGFβ1. At the same time, two fibrosis related signaling pathways, Akt and Smad2/3, were activated. These results were consistent with 2D, which indicated that the experimental data from 2D culture were authentic and worthy of further application. However, pro-collagen1α1and fibronectin secretion by fibroblasts showed differences in 2D and 3D. Although the synthesis of procollagen 1α1 and fibronectin in 2D and 3D did not reach statistical significance in the NC group, we showed that fibroblasts stimulated by MCs tryptase and TGF-β1 could secrete more pro-collagen1α1 and fibronectin in 3D.

In summary, the study successfully established a 3D cell culture system based on DIS, and verified the similarities and differences in cell biology between 3D ECM scaffolds and standard 2D culture. DIS based 3D culture models have potential significance in studying the role played by fibroblasts, intestinal epithelial cells and immune cells in intestinal diseases. In the future, decellularized scaffolds will be correlated to various human disease, making it a useful tool to explore the mechanism of disease and cellular function.

Declarations

Conflict of interest

The authors declare that there is no conflict of interests regarding the publication of this paper.

Ethical statements

The study was approved by the Ethics Committee of the Shanghai Tenth People’s Hospital affiliated with the Tongji University School of Medicine (IRB no. 21K190) and conducted in accordance with the tenets of the Declaration of Helsinki.