Author Affiliations: Department of Hepatobiliary Surgery, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, China (Xiang JX, Zheng XL, Gao R, Wu WQ and Lv Y); Department of Surgical Oncology, Shaanxi Provincial People's Hospital, Xi'an 710061, China (Zhu XL and Li JH)

Corresponding Author: Yi Lv, MD, PhD, Department of Hepatobiliary Surgery, First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, China (Tel/Fax: +86-29-85323904; Email: luyi169@126.com)

 

© 2015, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(15)60423-4

Published online September 17, 2015.

 

 

Contributors: LY proposed the study. XJX, ZXL, GR and WWQ performed research. XJX wrote the first draft. ZXL and LJH collected and analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. LY is the guarantor.

Funding: This study was supported by a grant from the Specialized Research Fund for the Doctoral Program of Higher Education of China (20110201130009).

Ethical approval: This study was approved by the Ethics Committee of the First Affiliated Hospital of Xi'an Jiaotong University.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

BACKGROUND: The potential application of decellularized liver scaffold for liver regeneration is limited by severe shortage of donor organs. Attempt of using heterograft scaffold is accompanied with high risks of zoonosis and immunological rejection. We proposed that the spleen, which procured more extensively than the liver, could be an ideal source of decellularized scaffold for liver regeneration.

 

METHODS: After harvested from donor rat, the spleen was processed by 12-hour freezing/thawing ×2 cycles, then circulation perfusion of 0.02% trypsin and 3% Triton X-100 sequentially through the splenic artery for 32 hours in total to prepare decellularized scaffold. The structure and component characteristics of the scaffold were determined by hematoxylin and eosin and immumohistochemical staining, scanning electron microscope, DNA detection, porosity measurement, biocompatibility and cytocompatibility test. Recellularization of scaffold by 5×10⁶ bone marrow mesenchymal stem cells (BMSCs) was carried out to preliminarily evaluate the feasibility of liver regeneration by BMSCs reseeding and differentiation in decellularized splenic scaffold.

 

RESULTS: After decellularization, a translucent scaffold, which retained the gross shape of the spleen, was generated. Histological evaluation and residual DNA quantitation revealed the remaining of extracellular matrix without nucleus and cytoplasm residue. Immunohistochemical study proved the existence of collagens I, IV, fibronectin, laminin and elastin in decellularized splenic scaffold, which showed a similarity with decellularized liver. A scanning electron microscope presented the remaining three-dimensional porous structure of extracellular matrix and small blood vessels. The porosity of scaffold, aperture of 45.36±4.87 µm and pore rate of 80.14%±2.99% was suitable for cell engraftment. Subcutaneous implantation of decellularized scaffold presented good histocompatibility, and recellularization of the splenic scaffold demonstrated that BMSCs could locate and survive in the decellularized matrix.

 

CONCLUSION: Considering the more extensive organ source and satisfying biocompatibility, the present study indicated that the three-dimensional decellularized splenic scaffold might have considerable potential for liver regeneration when combined with BMSCs reseeding and differentiation.

 

(Hepatobiliary Pancreat Dis Int 2015;14:502-508)

 

KEY WORDS: tissue engineering; liver regeneration; decellularized scaffold; spleen; bone marrow mesenchymal stem cells

End-stage liver diseases such as acute and chronic liver failure, cirrhosis, and liver cancer are serious health threats with high morbidity and mortality. Liver transplantation is currently the only effective treatment but limited by a severe shortage of donor organs, high cost of treatment, and lifetime immunosuppression.^[1] Inquiry into materials for liver transplantation research has been necessary. Researchers intend to reconstruct the liver by tissue engineering, specifically, reseeding hepatocytes or stem cells in scaffolds to reconstruct liver like tissue to compensate liver function.^[2] However, artificial synthesis scaffolds cannot simulate the natural organ structure for many defects such as lack of native vascular network, extracellular matrix, and poor biocompatibility.

 

Decellularized biological scaffold, which is procured by using decellularization technique to remove parenchymal cells from tissue or organ, may be considered an ideal option for liver tissue engineering. The decellularized scaffold retaining site-specific natural extracellular matrices and basic organ skeletal structures, which are difficult to reproduce artificially, has been shown to offer a desirable scaffold for cell engraftment, proliferation, transplantation, and stem cell differentiation.^[3-5] The applications of decellularized scaffold have been demonstrated initially for some organs, such as the bladder,^[6] skin,^[7] blood vessels,^[8] trachea,^[9] and cardiac valves.^[10] Many other parenchymal organs such as the heart,^[11] lung,^[12] liver,^[13] kidney^[14] have also been explored extensively. Using decellularized liver scaffold, Uygun et al^[15] successfully developed a transplantable recellularized liver graft in 2010, this technique has been applied in reconstructing functional liver like tissue, drug testing, and inducing stem cell differentiation since then.

 

However, potential clinical application of this approach is limited by using liver as only source of scaffold due to severe shortage of donor organs, the same as liver transplantation. Heterograft derived decellularized scaffold, which proposed by some researchers,^[16] is hindered by high risks of zoonosis and immunological rejection.^{[17, 18]} Despite the numerous endeavors to produce decellularized matrices from several different approaches, there still has been a lack of objective bioscaffold with reliable immunologic tolerance, biocompatibility, and biosecurity.

 

Microenvironment of the spleen, especially the extracellular matrix and blood sinus, is similar with the liver and suitable for cell adhering and proliferation.^[19-21] Tsuruga et al^[19] transplanted immortalized hepatocyte into immunodeficiency mice spleen for treatment of acute liver failure induced by acetaminophen. The results showed a significantly higher seven-day survival rate (100% vs 30%), higher blood glucose level and lower level of blood ammonia than control group. There are many hurdles for the hepatocyte-based therapies. First is the difficulties obtaining autologous primary hepatocytes; second is the maintenance of phenotype in culture; third is that intrasplenic cell transplantation may lead to cytoclasis and thrombosis in the portal vein and hepatic sinusoid.

 

Considering the strong proliferation and differentiation potential, along with adequate source, easy accessibility, and successful immunological tolerance in the host tissue, bone marrow mesenchymal stem cells (BMSCs) may be more suitable as seed cells. In addition, it has been shown that the transplantation of BMSCs or MSC-derived hepatocyte-like cells improves the liver function in recipients suffering from liver damage.^{[22, 23]}

 

Based on the above mentioned reasons and previous researches, we hypothesized that allogeneic or autologous spleen may be an ideal source of decellularized scaffold for liver regeneration when combined with BMSCs reseeding and differentiation. The spleen can be harvested from patients with portal hypertension, traumatic rupture, idiopathic thrombocytopenic purpura and donation after cardiac death and therefore, is more widely available than the liver. The present study was to explore the feasibility of decellularized splenic scaffold in liver regeneration.

 

 

Thirty-two healthy male Sprague-Dawley rats (weighing 200-300 g) were used for spleen harvest. After anesthetization and heparin peritoneal injection (1000 U), the communication branches between the spleen and stomach, and hepatic artery were isolated and ligated and a 24G intravenous catheter was inserted into the splenic artery. Heparinized PBS (100 U/mL) was infused via the splenic artery by peristaltic pump at a speed of 4 mL/min for 30 minutes, then immersed the isolated spleen into PBS solution. At last, the spleen was frozen at -80?�� to be further decellularized or to serve as a native spleen control group.

 

The frozen spleen was thawed at room temperature and then frozen at -80?�� for another 12 hours to lyze the cells. The spleen was perfused via the splenic artery at 4 mL/min: Deionized water for 1 hour followed by 0.02% trypsin (Amresco)/0.05% EGTA (MP Biomedicals) solution at 37?�� for 2 hours; the perfusion was changed back to deionized water for 15 minutes followed by 15 minutes of 2×PBS. The 3% Triton X-100 (MP Biomedicals)/0.05% EGTA solution was perfused for 30 hours; the solution tank was changed at 1, 4, 16 and 24 hours. After deionized water wash for 30 minutes, the perfusion was changed to PBS wash for twice, 30 minutes each. The decellularized splenic scaffold was perfused with 0.1% (v/v) peracetic acid/4% ethanol for 2 hours, and then neutralized by PBS and deionized water washes for 30 minutes each.

 

For histological examinations, twelve samples (six in decellularized spleens and six in controls) were fixed in 10% neutral buffered formalin, embedded in paraffin, and cut into 4 µm sections for hematoxylin and eosin (HE) staining. For immunohistological analysis, the samples were fixed with 4% paraformaldehyde and were permeabilized with 100% cold acetone. The samples were blocked using 5% BSA and then incubated overnight at 4?�� with anti-collagen I or anti-collagen IV, anti-fibronectin, anti-laminin and anti-elastin (Abcam) antibodies diluted in antibody dilution solution and DAPI (Sigma) were used to stain nuclei. After wash, the samples were incubated with secondary antibody at room temperature for 4 hours. The dilution ratio of the antibodies used was based on the manufacturer's recommendations.

 

The ultrastructure of decellularized splenic scaffold was observed by scanning electron microscopy (SEM) as previously described.^[13] Briefly, six samples were sectioned into small blocks (8 mm³), and fixed in 1% osmium tetroxide for 60 minutes, following dehydrating by graded series of ethanol for 15 minutes each (30%, 50%, 70%, 90%, 100%). The samples were then dried at critical point for 2 hours in absolute ethanol and mounted on an aluminum stub and sputter-coated with gold before viewing under SEM. To observe preserved native vessels after decellularization, the perfusion filler containing red dye was prepared in methyl methacrylate prepolymer and was slowly infused into the splenic artery. The porosity measurement of decellularized spleen scaffold was conducted by ImageJ software (National Institutes of Health) on the basis of SEM images.

 

DNA extraction was performed using Genomic DNA Kit (TIANGEN Biotech) according to the manufacturer's instructions. The total amount of DNA was quantified using the ultramicro ultraviolet spectrophotometer (Quawell Technology) according to the manufacturer's instructions. A total of eight samples were tested for DNA quantification. Remnants base pair analysis of the decellularized splenic scaffold was performed by gel electrophoresis analysis.

 

A total of fifteen healthy male Sprague-Dawley rats (weighing 200-250 g, provided by the Experimental Animal Center of Xi'an Jiaotong University) were used for biocompatibility evaluation. Decellularized splenic scaffold (treatment group, n=12) or native spleen samples (control group, n=3) were cut into slices about 1 mm thick and implanted subcutaneously at the back of the rats. The procedure was carried out under aseptic conditions. Activity, diet, incision condition, and the survival situation of two groups of rats were recorded every day after operation. The implants were histologically observed at day 5, 14, 21, 28 after operation.

 

Our long-term objective was to test the capability of BMSCs differentiation to hepatocytes in decellularized splenic scaffold and finally, liver regeneration. The present study was to test the cytocompatibility. We reseeded BMSCs of third passage from Sprague-Dawley rats in decellularized spleen matrix (DSM) to decellularized splenic scaffold. DMEM/F12 medium with 10% fetal bovine serum (Gibco) containing a total of 5×10⁶ cells were introduced into the scaffold via the splenic artery by peristaltic pump at a speed of 1 mL/min. We repeated three times with 10 minutes intervals, and the perfusate was collected to determine cell retention rate. The sample was then cut into discs measuring 6 mm in diameter and 3 mm thick, and cultured for three days before histological and SEM evaluation. Culture medium was changed daily.

 

Statistical analysis was carried out using SPSS version 21.0 (SPSS Inc., USA). Results were expressed as mean±standard deviation. Variables between the control and treatment groups were compared with Student's t test. A P<0.05 was considered statistically significant.

 

 

We prepared 32 rats splenic decellularized scaffolds among which 30 (94%) were successful. By decellularization, the spleen was changed gradually from deep red to reddish, granophyric, white translucent, and gross morphology was well-preserved with visible vascular structure (Fig. 1). HE and DAPI nuclear staining of the decellularized spleen showed the absence of cellular components and nuclear material compared to native spleen (Fig. 2A-D). DNA content of the decellularized splenic scaffold was 44.1±3.0 ng/mg dry weight, which represents 1.11% of the total content of native spleen (3996.4±175.0 ng/mg dry weight) (P<0.05). Electrophoretic analysis confirmed that the remaining DNA material consisted of fragments <200 bp in length and that the degradation of cell contents was extensive (Fig. 2E, F).

 

SEM images revealed the honey-comb structure of the decellularized rat spleen represents the footprint of splenocyte removal. The porosity of scaffold, aperture of 45.36±4.87 µm and pore rate of 80.14%±2.99%, was similar to decellularized liver scaffold. The pore size was enough for cell implantation, and the high pore rate and collagen fiber provided suitable location and space for cell engraftment. These data demonstrated that all of the residual blood, cells, and other soluble components were removed from the spleen by the decellularization process. There were sufficient spaces in the matrix, considering the legacy for the removal of cells. In addition, the structural and functional characteristics of the native microvascular were still visible in the matrix by red dye perfusion (Fig. 2G-I).

 

Immunolabeling characterization for extracellular matrix proteins, collagen I, collagen IV, fibronectin, laminin and elastin, indicated that both structural and basement membrane components of the extracellular matrix were retained similarly to native spleen (Fig. 3).

 

Considering histocompatibility, the rats implanted with decellularized spleen had milder inflammatory reaction, better wound healing, and higher survival rate compared with the control group in which poor wound healing and necrosis were observed (Fig. 4). In the control group, a large amount of inflammatory cells, mainly neutrophils and structureless necrosis were presented in the embedded sample. By contrast, the number of inflammatory cells, which peaked at day 14 post-operation, was significantly fewer in the treatment group and these inflammatory cells were mainly lymphocytes and monocytes. In addition, angiogenesis was very active and a large number of new capillaries were seen at the contact surface. Gradually, inflammatory cells were reduced but collagen fibers proliferated significantly and well-organized from 21 to 28 days after operation.

 

When reseeding BMSCs in decellularized splenic scaffold, the sample turned into pink from translucent, and the cell retention rate was 91.8%±6.5% (Fig. 5A, B). After cultivation in vitro for three days, the engrafted BMSCs located in the space of porous scaffold, attached on the surface of fibers and exhibited different morphology according to HE and SEM observation (Fig. 5C, D).

 

 

One of the main limiting factors hindering liver tissue engineering application is the lack of an ideal scaffold, which not only has all the necessary microstructural and extracellular matrix such as collagen, mucopolysaccharide and growth factor for cell adhesion, functioning, proliferation, migration, differentiation, but also provides microvascular networks for oxygen and nutrient transport, as well as metabolite excretion.^{[24, 25]} Decellularized organ scaffold is one of the most promising biomaterials in tissue engineering because of its advantages of favorable biocompatibility, native extracellular matrix and vascular system which provide incomparable convenience for tissue culture and in vivo implantation. Researchers from different groups^{[13, 15, 22, 26, 27]} demonstrated the feasibility of recellularization of hepatocytes or stem cells on decellularized liver scaffold and compensatory liver function of organoids in rodent, goat and swine, that creates an decellularization upsurge all over the world. However, promotion and clinical application of this technology is hindered by severe shortage of donor organs.

 

The present study attempted to reconstruct tissue engineered liver by using decellularized splenic scaffold, which is mainly based on the fact that clinical source of the spleen is more extensive than the liver. The spleen harvested from patients with portal hypertension, traumatic rupture, idiopathic thrombocytopenic purpura and donation after cardiac death could be used for establishing decellularized splenic scaffold, realizing the idea "turning the waste into treasure", with less risks of immunological rejection and zoonosis infection. Concerning the possible pathological changes in spleens obtained from diseased or older sources, further researches about the feasibility of cell seeding are needed, and our group has recently begun an experiment to clarify this issue.

 

Microstructure and component of splenic extracellular matrix are similar to those of liver according to our data and previous studies.^{[13, 15]} SEM confirmed that decellularized splenic scaffold has abundant blood sinus to form a sufficient blood supply to satisfy the large amount of cell metabolism and oxygen consumption. Meanwhile, decellularized splenic scaffold has good porosity and applicable pore diameter and therefore, is suitable for cell engraftment and proliferation. Immunohistochemistry staining revealed that the retention of extracellular matrix components, such as collagen I, collagen IV, fibronectin, laminin and elastin, are beneficial to cell engraftment and proliferation. In fact, previous studies^{[19-21,28,29]} demonstrated the feasibility and effectiveness of cell implantation into the spleen. The spleen, used as the site of cell transplantation, has such advantages as simple operation, fewer complications, easy monitoring, and less affected by the liver fibrosis environment. Transplantation of BMSCs or MSC-derived hepatocyte-like cells into the spleen could significantly promote the recovery of damaged liver and improve the survival rate. For above mentioned reasons, it could be inferred that spleen microenvironment might be suitable for cell seeding and reconstructing functional liver tissue in vitro.

 

Previous studies^{[18, 30]} showed that extracellular matrix is highly conservative within species, which means that the allogeneic extracellular matrix could be applied directly to other individuals without long-term use of immunosuppressant. At the same time, seed cells could be derived from mature stem cells of recipients, by which greatly reduced the risk of immunological rejection and treatment costs. The present study demonstrated that decellularized biological splenic scaffold from allogeneic rats showed mild inflammatory reaction and satisfying histocompatibility in vivo. Recellularization of the splenic scaffold also demonstrated that BMSCs could locate and survive in the decellularized matrix. These results provided a possibility for future differentiation of stem cells and liver reconstruction in decellularized splenic scaffold.

 

In summary, the data presented herein supported the fact that the decellularized splenic scaffold preserved the extracellular matrix environment and components, the structural and functional characteristics of the native microvascular network, and presented favorable biocompatibility. Considering the more extensive organ source, the present study indicated that the three-dimentional decellularized splenic biomatrix might have considerable potential in cell-based therapy and liver tissue engineering. 

 

 

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