Bacterial Cellulose-Based Biomimetic Nanofibrous Scaffold with

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A bacterial cellulose-based biomimetic nanofibrous scaffold with muscle cells for hollow organ tissue engineering XiangGuo Lv, JingXuan Yang, Chao Feng, Zhe Li, ShiYan Chen, MinKai Xie, JianWen Huang, HongBin Li, Huaping Wang, and YueMin Xu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.5b00259 • Publication Date (Web): 16 Nov 2015 Downloaded from http://pubs.acs.org on November 27, 2015

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ACS Biomaterials Science & Engineering

A bacterial cellulose-based biomimetic nanofibrous scaffold with muscle cells for hollow organ tissue engineering XiangGuo Lv1‡, JingXuan Yang2‡, Chao Feng1‡, Zhe Li2, ShiYan Chen 2, MinKai Xie1 ,JianWen Huang1, HongBin Li1, HuaPing Wang 2 *, YueMin Xu1 3 * 1: Department of Urology, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital Shanghai, China 2: State Key Laboratory for Modification of Chemical Fibers and Polymer Materials, College of Materials Science and Engineering, Donghua University, Shanghai, China. 3: Shanghai eastern urological reconstruction and repair institute, Shanghai, China. ‡Co-first author: These authors contributed equally to the work * Corresponding author

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ABSTRACT In this study, we built a bilayer nanofibrous material by utilizing the gelatinization properties of potato starch (PS) to interrupt bacterial cellulose (BC) assembly during static culture to create more free spaces within the fibrous network. Then, muscle cells were cultured on the loose surface of the BC/PS scaffolds to build biomaterials for hollow organ reconstruction. Our results showed that the BC/PS scaffolds exhibited the similar mechanical characters to those in the traditional BC scaffolds. And the pore sizes and porosities of BC/PS scaffolds could be controlled by adjusting the starch content. The average nanofiber diameters of unmodified BC and BC/PS composites is approximately to that of the urethral acellular matrix. Those scaffolds permit the muscle cells infiltration into the loose layer and the BC/PS membranes with muscle cells could enhance wound healing in vivo and vitro. Our study suggested that the use of bilayer BC/PS nanofibrous scaffolds may lead to improved vessel formation. BC/PS nanofibrous scaffolds with muscle cells enhanced the repair in dog urethral defect models, resulting in patent urethra. Improved organized muscle bundles and epithelial layer were observed in animals treated with BC/PS scaffold seeded by muscle cells compared with those treated with pure BC/PS

scaffold. This study suggests that this biomaterial could be suitable for tissue engineered urinary tract reconstruction and this type of composite scaffold could be used for numerous other types of

hollow organ tissue engineering grafts, including vascular, bladder, ureter, esophagus, and intestine. Keywords: bacterial cellulose; nanofibrous scaffold, hollow organ, urethral reconstruction

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Introduction Hollow organs, such as urinary bladders, urethras, ureters, esophaguses, intestines, vaginas, and blood vessels, are similarly organized and consist of epithelium or endothelium on a lumen surrounded by a collagen-rich connective tissue and muscle layer1. Many patients suffer from hollow organ pathologies requiring reconstructive procedures, and the reconstructive method often requires autologous grafts, such as skin2, buccal mucosa3, and bowel4. However, the use of these tissues has numerous problems5-6. Tissue engineering (TE) approaches provide potential strategies for developing biological substitutes to restore and maintain normal functional anatomy7. In TE, biomaterials should mimic the native architecture of extracellular matrix (ECM) in the replaced hollow organ tissue. From a structural perspective, ECM consists of various interwoven protein fibers with diameters ranging from tens to hundreds of nanometers8. Developing scaffolds that imitate the architecture of tissues at the nanoscale is one of the major challenges in the field of tissue engineering9-11. Beyond that, the ECM of hollow organs has its own special characters that it has the asymmetric structure. When designing biomimetic scaffolds for hollow organ tissue engineering, the following aspects should be considered: They must (1) mimic the nanofibrous collagen ECM; (2) be able to accommodate a large number of cells on one side and serve as a barrier on the opposite side, which is the asymmetric structure; and (3) be able to withstand mechanical stresses during tissue neogenesis. Several studies have used stabilizing additives, threaded stitches or other methods to build this architecture1, 12. These composites are simply combinations of one porous material and a relatively denser material. Unlike the native hollow organ architecture, there are no nanofibrous structures. Thus, there remains a critical need to design and fabricate an ideal biomaterial mimicking the native architecture of the hollow organ tissue.

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Bacterial cellulose (BC) , produced by Gluconacetobacter xylinus, is known for its natural origin, moderate biosynthetic process and environmentalally friendly products13. BC combines the important and well-known qualities of cellulose with the outstanding features of nanoscale materials in an excellent manner14. Because of its high mechanical strength, high crystallinity, high water holding capacity, and unique nanostructure, much interest has been given on the development of medical applications based on BC for wound care and implantable tissue engineered biomaterials, such as modern wound dressings, artificial skins15, blood vessels16, and bone17. However, BC is less than ideal for specific biomedical application when serving as an implantable scaffold because of its compact fibrous network surface, which is hard for cell penetration18. A simple and effective way to circumvent the compact structure is to build a 3D porous BC by adding interfering substances into the BC culture media19-20. Herein, we build a nanofibrous material by utilizing the gelatinization properties of potato starch (PS) to interrupt cellulose assembly during static culture to create more free spaces within the fibrous network. Another critical factor in generating tissue-engineered hollow organs for patients is the cell source. Typically, epithelial or endothelial cells and muscle cells are used to construct TE hollow organs. The epithelial or endothelial cells were used to prevent luminal content leakage into the surrounding tissue 1. However, confluent sheets of epithelial or endothelial cells are resource intensive and challenging to successfully culture. Relatively speaking, the isolation and expansion of muscle cells is relatively easy. A few studies found that muscle cells can release a variety of growth factors21-22 which may stimulate the migration and proliferation of epithelial cells23. We hypothesize that by providing a bilayer BC/PS scaffolds with a relatively impermeable barrier, luminal content leakage would be minimized. Then, muscle cells can be co-cultured on the porous surface of the BC/PS scaffolds to build biomaterials for hollow organ reconstruction. If successful, BC/PS scaffolds seeded with muscle cells could also be potentially economically advantageous compare to the BC/PS scaffolds seeded with

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epithelial/endothelial cells and muscle cells. We examined the validity of composite scaffolds in engineering hollow organs using a urethral tissue model. Materials and Methods. Fabrication of the BC/PS nanofibrous scaffolds Gluconacetobacter xylinus (1.1812) was purchased from the Institute of Microbiology, Chinese Academy of Science, and cultured with basal medium (glucose, 5 wt%; yeast extract, 0.5 wt%; bacto-peptone, 0.5 wt%; disodium phosphate, 0.2 wt%; monopotassium phosphate, 0.1 wt%; and citric acid, 0.1 wt%); the pH was adjusted to 5.0 with NaOH. Biochemical grade PS was purchased from Sinopharm Chemical Reagent Co., Ltd., to prepare BC/PS composites. The fabrication process of BC/PS nanofibrous scaffolds is described as follows. Potato starch (PS) was added to the culture medium at concentrations of 0.5, 1.0, 1.5, 2.0, and 4.0 (wt. %). The starch-enriched medium was heated above 90℃ under stirring to form a homogeneous, gelatinized structure, which was subsequently autoclaved at 121 ℃ for 30 minutes. Gluconacetobacter xylinus at a density of 3.8×105/ml were added into the starch-enriched medium. After 7 days of static cultivation, the BC/PS pellicles were collected and boiled in 1 wt. % NaOH for 30 min, and then repeatedly rinsed with distilled water to remove the residual culture medium and microorganisms. Unmodified BC was prepared for comparison. All samples were cut into fixed sizes (i.e., 5 cm × 2 cm) , freeze dried at -50℃ for 24 h and sterilized using ethylene oxide for further characterization. The interaction between gelatinized starch and cellulose network To investigate the interaction between the gelatinized starch and the cellulose network on a supramolecular level, attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) spectra of samples were recorded on a the spectrometer (Nexus 670 Thermo 5 / 34



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Nicolet, Vernon Hills, USA) in the absorption mode with a wave number range of 4,000 to 600 cm-1. The unmodified BC, pure starch, and BC/PS composites at 0.5 wt.%, 2 wt.%, and 4 wt.% were chosen to represent lower, moderate, and higher starch concentrations, respectively. Macro and micro-morphology observation and porosity measurement for BC/PS composites and urethral ECM. In order to understand the structural of urethral ECM, The urethra between the bladder and the pubic symphysis was harvested from anesthetized beagle dogs weighing 11.5 kg. By agitating the urethral tissue into the 1% Triton X-100 (Sigma) and 0.1% (v/v) ammonium hydroxide solution at 37°C , they were completely decellularized after 14 days. Hematoxylin and eosin (H&E) staining was used to identify whether all cells were removed from scaffolds. Then they were stored in 0.25% chloramphenicol solution before use. The surface morphology of all prepared BC/PS scaffolds with different starch contents and the urethral acellular matrices were characterized using an S-4800 FE SEM operated at 10 kV. Prior to analyses, samples were cut into small pieces from the freeze-dried samples and coated with a thin layer of sputtered gold. we used the cross-section of scaffold to measure its pore sizes. Furthermore, the more details of the SEM revealed that the pores existed in the space between the dense layers. Therefore, those closed loops in the SEM were defined as pore. The pore size were obtained by Image J software for at least 100 individual pore. The average fiber diameter were obtained by Image J software for at least individual fiber. The porosity of scaffold sections was measured using a liquid displacement method. Briefly, a scaffold was placed in a cylinder with a known volume of ethanol. A series of evacuation–repressurization cycles were performed to force the liquid into the pores. The

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porosity of scaffold was determined as follows: Porosity =

,

where V1 is the known volume of ethanol that is used to submerge the scaffold, V2 is the volume of the ethanol and ethanol-impregnated scaffold, and V3 is the remaining ethanol volume when the ethanol –impregnated scaffold was removed. Measurement of mechanical properties The tensile strengths of the samples were measured using a WDW 3020 Universal Testing Machine at room temperature and a crosshead speed of 2 mm/min. Wet state samples 50 mm in length, 20 mm in width were used in the measurements. Breaking stress and elongation at break of these samples were measured. All measurements were performed in distilled water at 37℃. Cell isolation and expansion The seeding cells were isolated from healthy dog lingual tissue. A 0.3 cm×0.3 cm section of lingual tissue was harvested using a biopsy punch (Kai Industries, Gifu, Japan); subsequently, the wound was sutured. The tissue was then divided into mucosal and muscle sections using a sterile scalpel. Muscle tissues were minced and digested with 0.5 wt.% type I collagenase (Worthington, USA). After 30 minutes, the suspended cells were filtered through a 74-µm cell strainer and then cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum(FBS). The velocity adherent technique was used to purify the cells within five passages24: the cell suspension was placed into a 10 cm plate for static culture within 15 mins. Then, the suspension was collected and repeated the procedure mentioned above to remove the adherent fibroblasts. After this velocity adherent technique, the final cellular suspension was placed in a new plate for the normal culture. 7 / 34



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The mucosal sections were incubated in dispase Π (Roche, Mannheim, Germany) overnight at 4 ℃. Then, a thin epithelial layer was peeled from the submucosa and treated with 0.05% trypsin (Gibco, USA). The suspended cells were cultured in keratinocyte serum-free medium (KSFM; USA)24. In vitro incubation of muscle derived cell on BC/PS composite scaffolds Before cell seeding, the unmodified BC and BC/PS composites at different starch contents were sterilized with 75% ethanol for 2 h, washed three times in sterilized PBS and then filled with 5 mL DMEM with 10% FB for 24 h at 37℃. 1 ml muscle cells suspension at a density of 106 cells/ml were seeded onto the porous surface of the scaffold (1 cm×2 cm) and the cell-seeded scaffolds were cultured in DMEM with 10% FBS at 37 ℃ in a humidified atmosphere with 5% CO2, and the culture medium was refreshed every day. All compound grafts were washed in PBS, fixed with 4.0% formaldehyde, dehydrated using gradient alcohol solution, and embedded in paraffin. The tissue blocks were sectioned at a thickness of 5 µm, deparaffinized, rehydrated, and stained with hematoxylin and eosin (HE). Muscle cells adhesion on scaffolds for 4 days was imaged using SEM. Unmodified BC and BC/PS (4 wt. %) composites containing muscle cells were fixed with 2.5% glutaraldehyde for >3 h. The substrates were dehydrated in 50–100% ethanol and dried in hexamethyldisilazane. Samples were mounted on SEM stubs, sputter-coated with gold and imaged using SEM (S-4800 FE). The culture medium for immerging the cell-seeded BC/PS were collected at 14 day. Levels of VEGF, KGF, and b-FGF in the medium were analyzed by enzyme-linked immunosorbent assays according to the manufacturer’s protocol (Biosource, USA). The medium for the cell seeded BC was also used for ELISA measurement as control values. In vitro wound healing study by using the BC/PS nanofibrous scaffold

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The effects of the BC/PS scaffold (4 wt. %) with or without muscle cells on the growth and migration of lingual keratinocytes in an in vitro system were analyzed. To mimic the in vitro crawling process of lingual keratinocytes, we cultured lingual keratinocytes on tissue culture polystyrene (TCPS) plates and observed the growth of lingual keratinocytes to confluence. Then, a “wound” was induced on the confluent monolayer cells by scraping a space with a plastic pipette tip (150 µl); images were immediately taken and the widths of the “wound” were measured (W 1). Three methods were tested for the treatment of such wounds :1) a wound was not directly covered by a patch and cocultured with muscle cell-seeded BC/PS (4.0 wt.%) scaffold. The muscle cell-seeded BC/PS (4.0 wt.%) scaffold was physically separated from the epithelial cell layer in a Transwell. So it is impossible that the muscle cells in the porous surface of BC/PS (4.0 wt.%) scaffold could migrate into the "wound" and confound results; 2) a wound was covered by a BC/PS (4.0 wt.%) scaffold with the compact surface directly facing the wound; 3)and a wound was not treated with a patch. Wound closures were observed by phase contrast microscopy for 24 h after initial scratches and the widths of the “wound” were measured (W 2). The distances cell monolayers migrated to close wounded areas during this time period were caculated (W 2 − W 1). In vivo model animal study of urethral regeneration and follow-up For the animal experiment, a total of 18 female beagle dogs (averaging 1.1 y, 11.5 kg) were divided into three groups of six dogs each. Group A was given unmodified BC scaffolds, group B was given BC/PS scaffolds (4.0 wt.%) , and group C was given BC/PS (4.0 wt.%) scaffolds seeded with muscle-derived cells. All dogs were anesthetized with pentobarbital. The urethra between the bladder and the pubic symphysis was exposed, and a 2 cm-long segment was transected and removed. Then, each scaffold was tubularized by suturing an 8F urethral catheter in preparation for urethroplasty. The urethral repair was performed with 5-0 vicryl sutures applied in a continuous fashion. Multiple non-absorbable marking sutures were placed 9 / 34



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at anastomotic margins. The catheter remained for 14 days after operation. After the urethral caliber was assessed with retrograde urethrograms, two animals from each group were killed one, two, and six months post-implantation. The entire urethra was removed, washed in PBS, fixed with 4.0% formaldehyde, dehydrated using gradient alcohol solution, and embedded in paraffin. The tissue blocks were sectioned at a thickness of 5 µm, deparaffinized, rehydrated, and stained with hematoxylin and eosin (HE), and captured (Leica Microsystems, Wetzlar, Germany) at 200 magnifications. Urethra sections were deparaffinised and processed for specific markers of epidermal cell layers, muscle layers and blood vessels. Non-specific bindings were blocked using 5% BSA, 0.3% Triton X100 in PBS for 1 h. Sections were incubated overnight with polyclonal anti-pancytokeratin AE1/AE3 antibody (1:50, Abcam Inc., USA) , anti- desmin antibody (1:100, Abcam Inc., USA) and Factor Ⅷ(1:100, Abcam Inc., USA). Following washing with PBS buffer, the secondary antibody, biotinylated goat anti-mouse tetramethylrhodamine isothiocyanate (TRITC)(Life technology, USA), was applied at

room tempreture for 1

h.

Nuclei

were

counterstained

with

300 nM

4-6-

diamidino-2-phenylindole (DAPI) (Abcam) for 10 min. After rinsing with PBS, the slides were mounted with DABCO-mounting medium (SigmaeAldrich, St. Louis, MO, USA). Analysis was carried out using a laser confocal scanning microscopy (LSM710, Leica Microsystems, Wetzlar, Germany). The positive staining area was quantified using Image-Pro Plus 5.1 software (Media Cybernetics, Inc., MD, USA) in 10 different fields at the same time under same parameters for each tissue sample. The research was conducted in accordance with the Declaration of Helsinki and with the Guide for Care and Use of Laboratory Animals as adopted and promulgated by the United National Institutes of Health. Statistical analysis

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The data are presented as the means ± the standard deviations (SD). Comparisons of mechanical property between BC and BC/PS composites were performed with two-tailed t-tests, comparisons of ELISA results between cell-seeded BC/PS and cell-seeded unmodified BC were performed with two-tailed t-tests and ANOVA was used to determine the differences among three groups using SPSS 17.0 statistics software. Differences were considered statistically significant when the p value was 0.05). Comparison in the tensile strength and the tenacity between the BC/PS composites (2.0 wt.% and 4.0 wt.%; p>0.05) and the urethral acellular matrix revealed no significantly difference (p

Bacterial Cellulose-Based Biomimetic Nanofibrous Scaffold with

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