Mesenchymal Stem Cell-Seeded Regenerated Silk Fibroin Complex

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Mesenchymal stem cells seeded regenerated silk fibroin complex matrices for liver regeneration in an animal model of acute liver failure Lijuan Xu, Shufang Wang, Xiang Sui, Yu Wang, Yinan Su, Li Huang, Yunwei Zhang, Zheng Chen, Qianqian Chen, Haitao Du, Yaopeng Zhang, and Li Yan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b02805 • Publication Date (Web): 14 Apr 2017 Downloaded from http://pubs.acs.org on April 15, 2017

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ACS Applied Materials & Interfaces

Mesenchymal Stem Cells Seeded Regenerated Silk Fibroin Complex Matrices for Liver Regeneration in an Animal Model of Acute Liver Failure Lijuan Xu1,†, Shufang Wang2,†, Xiang Sui3,YuWang3,Yinan Su4, Li Huang5, Yunwei Zhang6, Zheng Chen1, Qianqian Chen1, Haitao Du1, Yaopeng Zhang5, *, Li Yan1, * 1

Department of Gastroenterology, Institute of Geriatrics, Chinese PLA General Hospital, Beijing

100853, China; 2

Department of Blood Transfusion, Chinese PLA General Hospital, Beijing 100853, China;

3

Department of Orthopaedics, Chinese PLA General Hospital, Beijing Key Lab of Regenerative

Medicine in Orthopaedics, Beijing 100853, China; 4

Department of Hepatobiliary Surgery, Chinese PLA General Hospital, Beijing 100853, China;

5

State Key Laboratory for Modification of Chemical Fibers and Polymer Materials,

Collegeof Materials Science and Engineering, Donghua University, Shanghai 201620, China. 6

Emergency Department, Chinese Eighteenth PLA General Hospital, Yecheng 844900, China

KEYWORDS: regenerated silk fibroin matrices; biocompatibility; mesenchymal stem cells (MSCs); transplantation; acute liver failure;

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ABSTRACT: The shortage of organ donation is the main limitation to liver transplantation as a treatment for the end-stage liver disease or acute liver failure. In order to develop an alternative therapy for acute liver failure, mesenchymal stem cells (MSCs) seeded regenerated silk fibroin (RSF) matrices were evaluated in vitro and in vivo. Adipose-derived mesenchymal stem cells (ADSCs) and bone marrow-derived mesenchymal stem cells (BMSCs) were planted and grown on RSF scaffolds to form scaffolds complex, respectively. The RSF-MSCs scaffolds complex (the experimental group) and neat RSF scaffolds (the control group) were then placed onto the liver surface of mice induced by CCl4and detected after 5, 7, 14, 28 and 60 days. The growth and distribution of MSCs were tracked by fluorescence microscopy and live small animal fluorescence. Liver functions were tested by automatic biochemistry analyzer. The histological kinetics of RSF complex and liver tissues were observed by hematoxylin eosin. We found that MSCs exhibited good biocompatibility with RSF and differentiated to hepatocyte-like cells in vitro. Liver functions of the mice in the experimental group were significantly improved than the control group. Moreover, angiogenesis and hepatocyte-like cells were discovered in the RSF scaffolds in an animal model of acute liver failure on the fifth day and in the second month, respectively. The MSCs-RSF matrices show an obvious therapeutic ability for injured liver function of mice, which is more efficient than the neat RSF scaffolds.

1. Introduction Liver transplantation is the only effective therapy for acute or chronic liver failure. However, its application is severely limited by the scarcity of liver donor organs, immune rejection, and huge costs 1 . Alternative therapies, such as a stem cell-based regenerative medicine, offer possibilities to overcome problems above2. Previous studies have successfully induced human


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mesenchymal stem cells (MSCs) to differentiate into hepatocyte-like cells with a two-step protocol in vitro3, 4 . However, it is hard to find a suitable environment that allows the multipotent cells to develop toward hepatic differentiation and allows the formation of an implant construct 5. Scaffolds play an important role in providing the environment that is similar to the natural extracellular matrix, which can be used to regenerate, repair or even replace malfunctioning tissues 6. Previous studies showed that both synthetic and natural three-dimensional (3D) scaffolds could provide an environment that supports the maintenance and growth of hepatocytes 3, 7, 8

. However, the selections of an ideal scaffold for hepatic tissue engineering remains a

problem9. Regenerated silk fibroin (RSF) is a well-known natural biomaterial obtained from Bombyx mori cocoons and contains up to 90% of the amino acids glycine, alanine and serine 10-11. Our previous studies have shown that RSF has excellent biocompatibility, low inflammatory potential, and can be degraded completely in vivo within 2-6 months12. Researchers have also reported that RSF contributed to multiple organ reconstructions, including urethra reconstruction 12

, bladder reconstruction13, blood vessels14, cartilages15, and nerve regeneration 10, 16. In this study, we aim to investigate the compatibility between MSCs and RSF matrices and use

the RSF scaffold for MSCs differentiation of hepatocyte-like cells. Moreover, in vivo functionality of MSC-laden RSF was tested in carbon tetrachloride (CCl4) induced fulminant hepatic failure mice models17. 2. Materials and Methods 2.1.Experimental Animals


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Three-day-old and six to eight-week-old male BALB/c mice (Charles River, China) were purchased from the Laboratory Animal Center of the Academy of Military Medical Sciences of China (Beijing, China). All studies were performed after approval of the Ethics Committee of the Animal Facility of Chinese PLA General Hospital and were in agreement with the Guidelines for the care of laboratory animals. The three-day-old mice were used for the preparation of MSCs and the six to eight-week-old mice were used for animal experiments. 2.2. Preparation of electrospun RSF matrices RSF matrices were prepared from all aqueous solution according to the literature10, 18. In brief, B. mori cocoons were degummed and then dissolved in 9.0 M LiBr aqueous solution. After being diluted, centrifugated and filtered, the solution was dialyzed in deionized water to remove the salt. Finally, a 33 wt% RSF aqueous solution was obtained through condensation by forced airflow. A conventional electrospinning process with an aluminum collection plate was applied to prepare RSF mats at a voltage of 20 kV, a flow rate of 1.2 mL/h, and a sample-to-spinneret distance of 10 cm. The obtained mats with a thickness of cica 130 µm were then immersed in 90 vol% ethanol aqueous solution for 30 minutes to make RSF insoluble in water. 2.3. MSCs culture and characterization Preparation of ADSCs: As described in previous studies19, adipose tissues from three-day-old mice inguinal fat pad were minced into pieces smaller than 1 mm3 and digested in 1 mg/mL collagenase (Sigma) for 1h at 37℃. The digestion was terminated with an equal volume of expansion medium (α-MEM, Minimum Essential Medium α, containing 10% fetal bovine serum (FBS, Hyclone) and 1% penicillin/streptomycin). The mixture was further centrifuged at 500 g for 10 minutes and suspended in expansion medium. The cell suspension was filtered through a


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100 µm cell strainer, and then centrifuged at 300 g for 5 minutes. ADSCs were plated at a density of 5 × 105/cm2 in a 6 cm dish with expansion medium. Cells were harvested when aconfluence of cells reached close to 90% with 0.25% trypsin-EDTA (Gibco). ADSCs at passage 2 to passage 4 were used for subsequent experiments. Preparation of BMSCs: Tibia and fibula of the mice were isolated under sterile conditions and washed twice with PBS containing 5% penicillin/streptomycin prior to excluding muscle and fibrous tissues. The tibias and fibulas were minced into pieces smaller than 1 mm3, then cultured in 6 cm culture dishes, and incubated in a humidiﬁed incubator at 37oC and 5% CO2. After 72 hours, half amount of the medium was changed and the bone chips were kept. When the confluence of cells reached 50%, cells were harvested with 0.25% trypsin-EDTA, and then seeded in a 6 cm dish as the first passage. BMSCs at passage 2 to passage 4 were used for subsequent experiments. Characterization of MSCs: Both BMSCs and ADSCs were identified by flow cytometry, and were differentiated into adipogenic lineage and osteogenic lineage, which are consistent with the previous studies 20. 2.4. Biocompatibility between MSCs and RSF matrices in vitro Cell seeding: The RSF matrices were cut into small pieces (1×1 cm2) and put into 24-well culture dishes. Before using, the matrices were sterilized with 75% ethanol for 2 hours, washed three times in sterilized PBS, and filled in 1 mL α-MEM (Gibco) overnight at 37 oC. The ADSCs and BMSCs were seeded on the scaffolds at 3×105 cells/cm2.


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Hematoxylin and eosin (H&E) staining: Cell morphology and proliferation were assessed by H&E staining. The tissue samples were fixed in 4% paraformaldehyde overnight, washed in PBS, dehydrated through graded alcohols and paraffin embedded. Histological sections of 4 µm thick were cut on a microtome and followed by H&E staining. SEM assay: In brief, the scaffolds with MSCs and the blank scaffolds were washed three times with PBS and fixed in 3% glutaraldehyde at 4℃ overnight and in 0.1% osmium acid at 4℃ for 2 hours. The scaffolds were then washed three times with PBS, dehydrated in a gradient series of alcohols, critical point dried, sputter-coated with gold and examined on a Hitachi Model S-3000N scanning electron microscope (SEM, Tokyo, Japan). Live/Dead staining: The Live/Dead staining was stained with fluorescein diacetate (FDA) and propidium iodide (PI). Briefly, on the second day and on the fifth day, the scaffolds with MSCs were washed with PBS for three times, incubated in FDA (5µg/ml) for 5 minutes at room temperature (RT) and then washed with PBS for three times. The scaffolds were then incubated in PI (5 µg/ml) for 5 min and then washed again with PBS for three times. The distribution and viability of cells were observed by LSCM (Laser scanning confocal microscope, Olympus FV1000, Japan). The viable cells were stained under blue light excitation to emit green fluorescence, while the dead cells were stained under green light excitation to emit red fluorescence. 2.5. Characterization of hepatocyte-like cells on RSF scaffolds Cell induction: The MSCs were seeded on the RSF scaffolds at 2×105 cells/cm2. When the cells adhered to the RSF scaffolds, the expansion medium was replaced with hepatocyte culture medium (HCM) (DMEM with 10% FBS), supplemented with 50 ng/ml HGF, 25 ng/ml FGF4, 30


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ng/ml oncostatin M (OSM), 20 ng/ml epidermal growth factor (EGF), 10-6M dexamethasone, 25 ng/ml acidic fibroblast growth factor (aFGF), 10 ng/ml basic fibroblast growth factor (bFGF), 1× insulin-transferrin-selenium (ITS), 2 mmol/L ascorbic acid and 50 µmol/L nicotinamide (Vpp). One microliter of differentiation medium was added to each 24-well culture dish, and changed every three days. Afterward, the cells were cultured for 10 days in a HCM. Immunofluorescenceassay: Cultured cells were fixed with 4% paraformaldehyde (Sigma) for 30 min at RT, washed with PBS twice, and then permeabilized with 1% Triton X-100 (Sigma) for 20 min at RT. Cells were then incubated with blocking solution consisting of PBS and 10% bovine serumal bumin (BSA) at RT for 2 hours. For immunofluorescence staining, primary antibodies (1:200, Santa Cruz) against albumin (ALB), cytokeratin 18 (CK18), cytokeratin 19 (CK19), alpha-fetoprotein (AFP), and cytochrome P450 (CYP) 1A1were used. Followed incubation with the primary antibodies overnight at 4℃, the cells were incubated with secondary antibodies for 2 h at 37℃. Subsequently, the cells were stained with diamidinophenylindole (DAPI) for 5 min at RT, and photographed with a structured illumination fluorescence microscope. 2.6. Transplantation Chloromethyl - benzamidodialkylcarbocyanine (CM-Dil) staining:The passage 3 and passage 4 ADSCs and BMSCs were trypsinized, centrifuged and washed with PBS for once.The cells were adjusted to a concentration of 1×106 ml. After this, we added CM-Dil (Invitrogen) which is a membrane dye and can label all the treated cells into the cell suspension, the concentration CM-Dil is 5 µg/ml, and the labeling rate can be higher than 95%. Next, we incubated the cells in 4 oC fridge for 15 min, and incubated the cells in 37 oC humidiﬁed


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incubator for 10 min. Then we washed the cells twice with PBS. CM-DiL is one common dye used for cell tracing, and the labeling rate was higher than 95%. Finally, the cells were seeded onto the RSF matrices. Animal model: On day 0, an animal model of acute liver failure was established by undergoing i.p. injection of 100 µL/20 g body weight of olive oil containing 10 µL CCl4. On day 1, mice underwent transplantation of ADSCs-RSF, BMSCs-RSF and RSF matrices (n = 12). As control groups, non-transplanted CCl4-treated mice (n = 12) and non-transplanted CCl4 -nontreated (olive oil) (n = 12) mice were used. Transplantation: The surgeries were performed by the same surgeons. The surgical procedures were as the following: briefly, the mice were under general anesthesia by the intraperitoneal injection of pentobarbital. The volume of the left lateral lobe was exposed and the material was stuck onto the liver. The RSF matrices were then sutured and fixed. The wound was closed in layers with a routine method. At different time, the MSCs-RSF on the liver were tested by small animal imaging techniques (Berthold Technologies LB 983 NC 100). Then the liver tissue samples were harvested at different time points after transplantation, fixed in 4% paraformaldehyde, embedded in paraffin, and stained with H&E. Frozen sections (8 µm thick) were used for fluorescence observation of the CM-Dil labeled MSCs. 2.7. In vivo experiments


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Liver function test: Serum samples were tested on the first, second, third and the seventh day after transplantation for alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels with anautomatedanalyzer (Beckman DXC 600, Beckman Counter, Inc., Brea, CA). H&E staining: Liver tissue samples were harvested at different time points after cell transplantation, fixed in 4% paraformaldehyde, embedded in paraffin, and stained with H&E staining. Frozen sections (8 µm thick) were used for fluorescence staining. 2.8. Follow-up of animal experiments In addition, the neat RSF scaffolds were transplanted to the back of the mice to observe the compatibility of the scaffolds. The longest time of observation was 4 months. Degradation at different time points was observed. All animals were alive. 2.9. Statistical analysis Data were presented as mean± SD. Statistical analyses of ALT and AST levels were conducted by means of Student t-test with the use of SPSS software version 19.0. The significance for all statistical analyses was defined as P< 0.05. 3. Results 3.1.Phenotypic characterization and multiple differentiation of MSCs ADSCs and BMSCs were characterized in passage 3. The results of cell surface markers of MSCs showed that both the ADSCs and BMSCs expressed stem cell-associated surface markers CD90 and CD29, while they did not express CD11b and CD45 (Figure S1 a. The adipocytic and osteogenic differentiation of ADSCs and BMSCs was evaluated at P2 and P3. The adipocytic


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differentiation was identified by Oil O Red and the lipid vacuoles were stained as bright red (Figure S1 b). 3.2. Biocompatibility of MSCs in RSF matrices in vitro MSCs were seeded on RSF scaffolds and transferred into a 24-well dish for culture. After being cultured for 7 days, H&E staining images (Figure 1a) show that the cellular layers were easily distinguished. The ADSCs and BMSCs grew well and formed a monolayer epithelium on the surface of the RSF matrices. Some cells grew in the middle of the scaffolds, and the transect of blank scaffolds show porous and lax structures. The SEM images (Figure 1b) show that the MSCs were in fibroblast-like shape and adhered tightly to the surface of the materials. Compared with ADSCs, BMSCs are much smaller in size. The LSCM images (Figure 1c) show that both ADSCs and BMSCs grew in a manner of colony, and the number of cells increased over time. ADSCs show a stronger colony forming ability. The two MSCs show a strong proliferation ability and adhesion ability on the surface of the RSF scaffolds. Therefore, we concluded that MSCs have good biocompatibility with RSF matrices. The RSF scaffolds might be used as a carrier for MSCs in liver tissue engineering.


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Figure 1. Morphology of MSCs seeded on ADSCs-RSF, BMSCs-RSF, and bare RSF scaffolds (control). (a) H&E staining image of MSCs displaying monolayer growth on RSF scaffolds. (b) SEM images of MSCs. (c) 2D and 3D LSCM images of MSCs on the second day and the fifth day. The viable cells were stained by green fluorescence. The scale bars are 200 µm. 3.3. Hepatic differentiation of ADSCs on RSF scaffolds


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Figure 2. Fluorescent images of hepatic markers of ALB, AFP, CK18, CYP1A1 of induced ADSCs on RSF scaffolds. Blue DAPI stained nucleus show that all markers were positive. The scale bars are 200 µm. In order to test whether MSCs could be induced into hepatocyte-like cells on RSF scaffolds, we seeded the ADSCs onto the RSF scaffolds and added hepatic differentiation medium. On the 10th day, the cells were attached to the surface of the material and showed round and oval morphology (Figure 2). Immunofluorescence staining showed that ALB, AFP, CK18, and


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CYP1A1 were positive, which indicates that the ADSCs were successfully differentiated to hepatocyte-like cells on the RSF scaffolds. 3.4. Biocompatibility of RSF matrices in vivo Figure S2a and Figure S2b showed the operation of transplantation of RSF scaffolds (1 × 1 cm2) to the liver and the back of mouse separately. Figure S3a showed the degradation appearance of the scaffolds in the liver at different times. After 3 months, the RSF materials in the liver almost degraded completely. Figure S3b showed that the scaffolds in the back became smaller till disappeared. H&E staining demonstrated the the exsistance of RSF scaffolds in the back of the mouse after 3 and 4 months. As shown in Figure S3 C-1 and C-2, a variety of inflammatory cells infiltrated on the edge of the scaffolds after 3 months. However, the number of inflammatory cells decreased after 4 months (Figure S3 C-3, C-4). 3.5. Engraftment of RSF-MSCs scaffold on liver surface


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Figure 3. (a) In vivo small animal imaging technique results. The fluorescence expression of the ADSCs-RSF group and the BMSCs-RSF group was observed on day 2, 5, 7, 14 and 30, and the fluorescence intensity gradually decreased. No fluorescence was observed for the bare RSF scaffolds. (b) Fluorescence observation of frozen section of mouse liver tissue at different times. Red fluorescence expression of the ADSCs-RSF group and the BMSCs-RSF group was observed


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on day 2, 5, 7, 14 and 30, while no fluorescence was observed for the bare RSF scaffolds. The scale bars are 200 µm. To investigate the long-term functional engraftment of MSCs-RSF scaffolds, the RSF scaffolds seeded with or without MSCs were attached to the surface of mouse liver with acute liver failure. On day 2, 5, 7, 14 and 30, fluorescence coming from MSCs was observed by small animal imaging techniques (Figure 3a). The fluorescence region gradually decreased over time, a small region of fluorescence could still be observed on day 30. On the contrary, there was no fluorescence expression in the blank control group (Figure 3b). The RSF scaffolds with CM-Dil labeled MSCs expressed red fluorescence at different time points, and the control group did not express red fluorescence (Figure 3a). The strong fluorescence intensity on day 2 gradually decreased for the ADSCs-RSF group and the BMSCsRSF group over time, which is consistent with small animal imaging technique results. The RSF scaffolds seeded with CM-Dil labeled MSCs were transplanted onto the surface of the liver of acute liver failure model. This demonstrates that MSCs can grow on the mouse liver for more than one month, which is consistent with the previous studies. 3.6. Improvement of liver function and liver tissues To address whether MSCs-RSF matrices reveal therapeutic abilities to regenerate the injured mouse liver, MSCs-RSF matrices were transplanted to the surface of liver of mice with acute liver failure. Twenty-four hours after CCl4 injection, mice revealed a serious liver injury that the liver of the mice became larger, the color was soil-yellow and the liver capsule became tense. Forty-eight hours after CCl4 injection, the surface of the liver was in granular shape (Figure S4). Biochemical parameters including ALT and AST were detected. The therapeutic abilities of four


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experiment groups were compared at different time points. After transplantation, ALT and AST of the MSCs-RSF groups significantly decreased compared with the CCl4 group (Figure 4). In addition, the RSF group showed a certain therapeutic ability, but much weaker than the MSCsRSF matrices groups. On day 7, the functions of the injured mice almost recovered (Figure 4).

Figure 4. Liver function analysis in CCl4 treated mice after MSCs and MSCs-RSF matrices transplantation. The ALT and AST levels of different experimental groups compared with the CCl4 group. The hepatic function almost recovered on day 7 (*P < 0.05, **P < 0.01 compared with control group).


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Figure 5. Liver tissue pathology H&E staining (100× magnification) at different days after MSCs and MSCs-RSF matrices transplantation. The MSCs and MSCs-RSF matrices groups show smaller congestion areas than CCl4 group. The scale bars are 200 µm. H&E staining reveals that the level of injury is much weaker in the injured transplanted mice than in the injured, non-transplanted mice (Figure 5). The congestion areas of MSCs-RSF scaffolds groups were smaller than the control group and RSF group. On day 7, all the experimental groups almost histologically recovered. However, the obvious damage areas were still observed in the CCl4 group. These observations indicated that ADSCs-RSF and BMSCsRSF provide similar protection against CCl4 -induced liver injury. 3.7. Biocompatibility and degradation of RSF-MSCs on liver surface After transplanting MSCs-RSF scaffolds and RSF scaffolds to the liver of an acute liver failure model, the compatibility of RSF materials with liver and the differentiation of the transplanted MSCs were observed at different times in vivo (Figure 6). An acute liver failure model was made


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to provide a damaged environment of the hepatocytes for the reason that MSCs can be differentiated to hepatocytes in vivo in an injured liver. On day 2, H&E staining showed that the MSCs-RSF scaffolds were attached to the liver surface and the MSCs were located on the surface of the RSF scaffolds. The neat RSF scaffolds also attached tightly to the liver surface. A few inflammatory cells began to infiltrate in the blank material transplantation group. On day 5, angiogenesis was observed in the experimental groups, and the inflammatory cells increased compared with day 2. On day 7, the number of new blood vessels decreased and the boundaries of the liver capsule were still clear. The inflammatory cells infiltrated markedly in the blank RSF scaffolds group. On day 14, the neovascularization disappeared with a small amount of inflammatory cell infiltrates in the experimental groups. However, the control group still had a great number of inflammatory cell infiltrates.


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Figure 6. H&E staining of MSCs-RSF and RSF scaffolds on liver in vivo. Small inserts represent the general view of the RSF scaffolds and liver tissues. Bigger figures represent the border of RSF scaffolds and the liver parenchyma. The RSF matrices layers (the square) and the liver parenchyma (the triangle) were easily distinguished in the sections. Day 5: angiogenesis was observed in the experimental groups (arrowhead). Day 7: the number of new blood vessels decreased and the inflammatory cells infiltrated markedly in control group (arrowhead). Day 14: the neovascular disappeared with a small amount of inflammatory cell infiltrates in the experimental groups and a large number of inflammatory cell infiltrates in the control group. Month 1: a bile canaliculi-like structure was observed at the scaffolds side of the border area of the ADSCs-RSF group (arrowhead). Month 2: in the experiment groups, some hepatocyte-like cells with large nucleus, abundant cytoplasm and light-color were observed in the scaffolds side of the border area (arrowhead). Month 3: the RSF scaffolds degraded obviously in the two experimental group. However, the inflammatory cell infiltration was still obvious in the control group. The scale bars are 200 µm. In the first month, the cell proliferated obviously at the junction area in the experimental group. At the scaffold side of the border area, a bile canaliculi-like structure was observed in the ADSCs-RSF group. In the control group, the junction of the materials and liver was clear, but the inflammatory cell infiltration was obvious. In the second month, some hepatocyte-like cells with large nucleus, abundant cytoplasm and light-color were observed in the scaffolds side of the border area in the experiment groups. In the third month, the RSF scaffolds of the ADSCs-RSF group degraded obviously, and degraded completely in the BMSCs-RSF group. The inflammatory cell infiltration was still obvious in the control group.


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4. Discussion Regenerative medicine offers an alternative therapy for acute liver failure or the end-stage liver disease. Three strategies including cell transplant, bioartificial liver devices, or bioengineered whole organ have shown promising results in the past decade. However, before they could be incorporated into widespread clinical practice, the ideal cell type, suitable biological scaffolds, and the biocompatibility between them should be evaluated. Moreover, safety concerns including tumorigenicity and xenozoonosis should also be addressed. In this study, we chose ADSCs and BMSCs as seed cells, evaluated the biocompatibility between RSF and the cells, and then investigated their effect in animal model of acute liver failure and evaluated side effects. Adult stem cells have been recently undertaken for liver regeneration in clinical trials8, 9. ADSCs and BMSCs have proven to be attractive cell sources for their multiple differentiation potential and high proliferation ability21,

22

. Our previous study proved that adipose-derived

stromal cells resemble bone marrow stromal cells in their hepatocyte differentiation potentials both in vitro and in vivo. Furthermore, current in vivo experiments indicated that the undifferentiated MSCs showed similar potential to suppress the CCl4-induced liver injury with the hepatocyte-like cells from MSCs in rat liver failure model23. Actually, ADSCs can be obtained more easily, less invasive than BMSCs. Therefore, ADSCs might be more suitable seed cells for cell transplant or liver tissue engineering. Thus only ADSCs rather than BMSCs were induced to hepatocyte-like cells in this paper. RSF is an excellent natural biomaterial thathas been widely used in many clinical applications11. But whether it could be one ideal bioartificial liver materialis unclear. In this study, we used RSF matrices as the scaffold material and observed that both ADSCs and BMSCs attached tightly to the scaffolds and proliferated quickly


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in vitro. In vivo, the results of liver function and histopathology showed that RSF-ADSCs resembled RSF-BMSCs in their contribution to the liver regeneration. We attributed the results to the excellent biological effect of RSF scaffolds and the good biocompatibility. We predict that the RSF scaffolds might provide a microenvironment that could promote the proliferation of MSCs, as well as some certain cytokines released from the compounds of RSF-MSCs contributing to the liver regeneration. Moreover, the degradation products of RSF are amino acids that could be conducive to cell growth, which is consistent with the previous results11. Our study demonstrated that RSF scaffold could be an effective biomaterial for liver regeneration. The main side effects of stem cells transplantation are the fewer located cells, vascular embolization, and the immunological rejection. In order to solve these problems, we transplanted the RSF-MSCs onto the surface of the liver of the animal model of acute liver failure. The follow-up results showed that a large amount of MSCs proliferated in liver surface carried by the RSF scaffolds. There were less inflammatory cells around the scaffolds and no immunological rejection. In addtion, there was no vascular embolization observed in the main organs. Therefore, RSF scaffolds would be one of the safe candidate biomaterials for liver tissue engineering. In this study, some interesting findings were observed during the follow-up of the animal experiments. After transplantation of RSF-MSCs, the neovascularization was developed in RSF scaffold on day 5, then a bile canaliculi-like structure was observed in the first month and some hepatocyte-like cells were developed at the border of scaffolds in the second month. These results indicated that RSF-MSCs scaffolds might contribute to chronic liver injuries, such as liver fibrosis and cirrhosis in addition to acute liver failure. Therefore, we will investigate the role of RSF-MSCs scaffold in chronic liver injury in the future study. 5. Conclusions


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In conclusion, we have validated the biocompatibility between MSCs and RSF materials, and two kinds of MSCs of BMSCs and ADSCs were identified as similar biocompatibility with RSF scaffolds through a series of in vitro and in vivo experiments. Moreover, MSCs could be induced into hepatocyte-like cells in the cell seeded RSF complex matrices in vitro. Moreover, the RSF scaffolds helped the MSCs locate on the surface of the injured liver of mice over 3 months, which was proved by the tracing agents of CM-Dil. The long time survival of the MSCs in the scaffolds might contribute to the liver repair by secreting some cytokines. Neovascularization was firstly developed in RSF, and a bile canaliculi-like structure and some hepatocyte-like cells were then developed in the scaffolds. The RSF materials almost degraded completely after implantation for 3 months. This study has shown that MSCs seeded RSF complex matrices has great potential for the liver regeneration of acute liver failure or chronic liver injury. ASSOCIATED CONTENT Supporting Information. The flow cytometry for identification of phenotype of MSCs, and mutiple differentiations of MSCs; transplantation of RSF scaffolds to the liver of an acute failure model of mouse; in vivo degradation results of RSF matrices; appearance of a mouse liver of an acute liver failure model. AUTHOR INFORMATION Corresponding Author * Li Yan and Yaopeng Zhang are co-corresponding authors. E-mail: [email protected]; [email protected], Fax: +86-21-67792855 Author Contributions


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The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. †These authors contributed equally.

ACKNOWLEDGMENT This work is sponsored by the National Natural Science Foundation of China (30900669), Technology Nova Plan of Beijing City (2011117), the Postdoctoral Science Foundation (2016T90994) of Li Yan, the National Natural Science Foundation of China (21674018), the National Key Research and Development Program of China (2016YFA0201702), and the “Shuguang Program” supported by Shanghai Education Development Foundation and Shanghai Municipal Education Commission (15SG30). REFERENCES 1.

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Graphical abstract 35x15mm (300 x 300 DPI)


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Mesenchymal Stem Cell-Seeded Regenerated Silk Fibroin Complex

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