Porous three-dimensional silk fibroin scaffolds for tracheal epithelial

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Tissue Engineering and Regenerative Medicine

Porous three-dimensional silk fibroin scaffolds for tracheal epithelial regeneration in vitro and in vivo Zhongchun Chen, Nongping Zhong, Jianchuan Wen, Minghui Jia, Yongwei Guo, Zhengzhong Shao, and Xia Zhao ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00419 • Publication Date (Web): 21 Jun 2018 Downloaded from http://pubs.acs.org on June 22, 2018

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ACS Biomaterials Science & Engineering

Porous three-dimensional silk fibroin scaffolds for tracheal epithelial regeneration in vitro and in vivo Zhongchun Chen †,#, Nongping Zhong †,#, Jianchuan Wen ‡, Minghui Jia †, Yongwei Guo †, Zhengzhong Shao ‡ and Xia Zhao†,* †

Department of Otorhinolaryngology-Head and Neck Surgery, Huashan Hospital, Fudan

University, 12 Middle Wu Lu Mu Qi Road, Shanghai 200040, China ‡

Department of Macromolecular Science and the Laboratory of Advanced Materials, Fudan

University, Shanghai 200433, China KEYWORDS: tracheal reconstruction, silk fibroin scaffold, airway epithelial cell, cell culture, tissue engineering

ACS Paragon Plus Environment

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ABSTRACT: The regeneration of functional epithelial lining is critical for artificial grafts to repair tracheal defects. Although silk fibroin (SF) scaffolds has been widely studied for biomedical application (e.g. artificial skin), its potential for tracheal substitute and epithelial regeneration is still unknown. In this study, we fabricated porous three-dimensional (3D) silk fibroin scaffolds and co-cultured them with primary human tracheobronchial epithelial cells (HBECs) for 21 days in vitro. Examined by scanning electronic microscopy (SEM) and calceinAM staining with inverted phase contrast microscopy, the SF scaffolds showed excellent properties of promoting cell growth and proliferation for at least 21 days with good viability. In vivo, the porous 3D SF scaffolds (n=18) were applied to repair a rabbit anterior tracheal defect. In the control group (n=18), rabbit autologous pedicled trachea wall without epithelium, an ideal tracheal substitute, was implanted in situ. Observing by endoscopy and computed tomography (CT) scan, the repaired airway segment showed no wall collapse, granuloma formation or stenosis during an 8-week interval in both groups. SEM and histological examination confirmed the airway epithelial growth on the surface of porous SF scaffolds. Both the epithelium repair speed and the epithelial cell differentiation degree in the SF scaffold group were comparable to those in the control group. Neither severe inflammation nor excessive fibrosis occurred in both groups. In summary, the porous 3D SF scaffold is a promising biomaterial for tracheal repair by successfully supporting tracheal wall contour and promoting tracheal epithelial regeneration.


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Introduction Excessive tracheal resection with tracheal reconstruction may be required in cases of cancer or 1

some benign diseases of the trachea.

When extensive tracheal defects exceed 50% of the

trachea length in adults or 30% in infants, the repair/reconstruction is indispensable but very difficult for otolaryngologists. So far, the ideal tracheal substitute has not been found. Autografts and allografts used for tracheal reconstruction bear shortcomings, such as the collapse of the transplanted graft, the lack of donors and the need of lifelong immunosuppressant therapy, which may result in failure. 2 Variable artificial tracheas have also been applied in tracheal repair, but none of them has reached expectation due to airway collapse, overgrowth of granulation tissue or mucus impaction because of lacking epithelium.

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Therefore, it is important to find suitable

replacement to repair tracheal defect. Both natural and synthetic non-biologically derived materials have been used as scaffold materials. A good trachea scaffold should possess good mechanical properties and capability in promoting functional trachea ciliated epithelial regeneration to help mucus clearance and prevent cicatricial constriction and infection.

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Non-biologically derived scaffolds without bioactivity

give excellent mechanical strength but may elicit inflammatory reactions, immunogenic response, and discourage cell growth. Tissue-engineered airway scaffolds using purely natural materials (e.g. type I collagen, hyaluronic acid and gelatin) have not been frequently utilized because of their limited mechanical properties. 6-9 Silk fibroin (SF), a natural macromolecular protein polymer, has good biocompatibility and controllable degradation rates.

10,11

Furthermore, porous 3D SF scaffolds are also found to have

excellent mechanical properties and provide an environment with appropriate nutrients and


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oxygen for cell growth and attachment.

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Our previous work on the porous 3D SF scaffolds

fabricated by a freezing-defrosting process found that the 3D SF scaffolds consistently showed a good

inherent

biocompatibility,

satisfactory

biodegradability as subcutaneous implants in rat.

mechanical 14

properties

and

adjustable

Although SF has been widely studied for

biomedical application, 15,16 its potential for tracheal substitute and epithelial regeneration is still unknown. Thus, the objective of this study is to investigate the potential of the porous 3D SF scaffold in repairing tracheal defect, especially in the aspect of enhancing the regeneration of functional tracheal epithelium. From in vitro and in vivo rabbit model studies, we found that the porous 3D SF scaffold exhibited great performance in tracheal repair by inducing and sustaining the growth of host tracheal epithelial cells with no obvious wall collapse, fibrosis or inflammatory response. Materials and Methods Fabrication of porous 3D SF scaffolds The porous 3D SF scaffolds were prepared using previously established methods. 17,18 Briefly, Bombyx mori cocoon silk was boiled in Na2CO3 (0.5 wt%) aqueous solution for 30 min to remove the outer sericin. After being dried, the degummed silk fibroin was dissolved in 9.5 mol/L LiBr aqueous solutions at 60 °C. After being filtered, the resulting silk solution was dialyzed for 3 days and concentrated to 7% w/w by dialyzing against aqueous polyethyleneglycol solution. Then, n-butanol was added to SF solution (7% w/w) (the volume ratio 1: 1) with gentle stirring at 100 rpm to form a stable oil/water emulsion. Afterwards, the mixture of SF and n-


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butanol was poured into a cylindrical mold and frozen in a freezer for 24 hours at -20 °C. After thawing at room temperature, the porous 3D scaffold was washed thoroughly with deionized water to remove n-butanol. Finally, the scaffold was kept in sterilized water at 4 °C until use. Scaffold characterization Morphology of the porous 3D SF scaffolds Cross sections of the freeze-dried SF scaffolds were obtained by being fractured in liquid nitrogen. The fracture surfaces were sputter-coated with Au and subsequently examined by SEM (TS 5136MM microscope, Tescan, Czech). Pore size of silk scaffolds was analyzed with Atlas software (accompanied with TS 5136MM microscope, Tescan) and more than 50 pores were randomly selected for the statistical analysis. 19 The porous 3D SF scaffolds co-cultured with HBECs in vitro Primary HBECs isolation and culture Primary HBECs were isolated and cultured as previously described.

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Briefly, the normal

bronchial tissues were obtained from surgical specimen 2 cm away from the tumor part of lung cancer patients who underwent lobectomy. After washed three times with phosphate-buffered saline (PBS), the mucosal surface was consecutively brushed seven to eight times with a bronchial brushing (AF-1810XB, Elton, China) to harvest bronchial epithelial cells. The obtained HBECs were incubated with serum-free BEGM medium (Lonza, Walkersville, Md., USA) in culture bottles at 37 °C in a humidified atmosphere of 5% CO2. When confluent, the cells were passaged using trypsin and cultured for in vitro study. The protocol was approved by the Ethics


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Committee of Huashan Hospital, Fudan University with informed consent from each patient in the study. Cell growth on porous 3D SF scaffolds The porous 3D SF scaffolds were punched into cylindrical shape (15 mm diameter × 1 mm height) and were sterilized with 75% ethanol for half an hour. The cell suspension solution was seeded at a density of 2×105 cells per scaffold. HBECs cultured on scaffolds were incubated continuously for 21 days. At different time points of co-culture (3, 7, 14, and 21 days), the morphology and viability of HBECs on scaffolds were monitored with SEM and inverted phase contrast microscope after calcein-AM staining. The porous 3D SF scaffolds in the reconstruction of rabbit anterior tracheal defect in vivo Implantation into tracheal defect in a rabbit model Thirty-six male New Zealand white rabbits (weight, 2.5–3.0 kg, Laboratory Animal Center of Fudan University) were randomly assigned to two groups as follows: (1) autologous pedicled tracheal wall with the mucosa removed for tracheal repair as the control group (n=18), and (2) the porous 3D SF scaffolds group (n=18). Each animal was given an intramuscular injection of ketamine (50 mg/kg) + diazepam (5 mg/kg) as anesthesia and placed in supine position. After shaving and disinfection, a vertical incision was made at midline of the neck. The sternohyoid and sternothyroid muscles were separated to expose the trachea. Thereafter, 4 rings and 1/3 circumferential tracheal wall were resected. A piece of porous 3D silk fibroin scaffold (length of ~12 mm, width of ~10 mm, and thickness of ~ 1 mm) (Fig. 1A) was engrafted and sutured to tracheal wall with 4-0 absorbable sutures (Safil, Germany) (Fig. 1B, C) to repair the defect. In


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the control group, 4 rings and 1/3 circumferential trachea was resected, and the tracheal epithelial lining was peeled, but the lower edge of the trachea was reserved. The residual cartilage was sutured in situ using 4-0 absorbable sutures (Safil, Germany) (Fig. 1D-F). Finally, strap muscles and the skin were sutured in layers using 3-0 sutures (Mersilk, USA). Postoperatively, each animal was observed until awake and no symptom of severe respiratory distress occurred. Two groups of animals were harvested for endpoint evaluations at 1 week (n=6), 4 weeks (n=6), and 8 weeks (n=6). All animal studies were approved by the Animal Care and Use Committee of Fudan University prior to experimentation.

Figure 1. Tracheal repair in rabbit model. (A) The SF scaffold used for tracheal reconstruction. (B-C) The defect of trachea was repaired with a SF scaffold in the SF scaffold group. The arrow denotes the SF scaffold. (D-F) Tracheal repair with autologous anterior trachea wall with the epithelium removed in the control group. The arrow denotes the peeled tracheal mucosa. Endoscopy and CT scan


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At 1, 4, and 8 weeks after implantation, two animals of each group were given the same procedure of anesthesia for endoscopic examination. A rigid ear endoscope (Storz, Germany) was inserted transorally to observe the anterior wall of the reconstructed trachea and to evaluate the formation of any granulation tissue, scar, airway narrowing, collapse, accumulation of sputum, and exposure of scaffolds. At each time point, animals (n=2 per group) in prone position were also examined by highresolution CT scan (Brilliance iCT, PHILIPS, Netherlands) to check the tracheal morphology, scaffold position and occurrence of neck abscess. The anteroposterior and transverse diameters of the narrowest part of the tracheal luminal at graft sites were measured in CT images. The tracheal cross-section dimension = anteroposterior diameter × transverse diameter. The measurement method was referred to Yusu Ni’s study. 21 Histologic analyses and histomorphometric analyses After the endoscopic and CT examinations, animals (n=6, per group) were sacrificed for histologic analyses. The tracheal specimens were dissected into two equal parts horizontally. One was for hematoxylin and eosin (H&E) and Masson's trichrome (MT) stain; the other was for SEM (n=3) and cytokine analysis (n=3). For histologic preparations, tracheal segments were fixed in 4% paraformaldehyde solution, and dehydrated in graded alcohols, and then embedded in paraffin. Afterwards, sections (5 um) were cut and then stained with H&E and MT according to routine histological protocols. Histomorphometric analyses were performed as previously described

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to assess the ratio of

epithelial coverage, the number of inflammatory cells and the degree of fibrosis in both control and SF groups using automatic image analysis software (LEICA QWins Standard). The length of


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regenerated epithelium (L1) and total tracheal defect (L2) were measured respectively in three different slices of each specimen of 1 week. The ratio of epithelial coverage (%) = (L1/ L2) ×100%. Automatic image analysis software was carried out on 5 independent microscopic fields (magnification 400×) equally to calculate the number of submucosal inflammatory cells. In addition, the degree of fibrosis of each MT slice was also quantified using automatic image analysis software to record the degree of blue staining of collagen fiber in 5 independent microscopic fields (magnification 400×). Scanning electron microscopy (SEM) The epithelial regeneration of the tracheal defect at 1, 4, 8 weeks in vivo were also imaged by SEM. Briefly, the samples were fixed in 2.5% glutaraldehyde solution for 24 hours, and then dehydrated in ethanol. Afterward, the specimens were critical-point dried in carbon dioxide and coated with gold platinum and then examined with SEM (SU8010, Hitachi, Japan) at a magnification of 500-3000 times at an accelerating voltage of 10 kV. Analysis of cytokines by Western blot analysis The expression of TGF-β1 and VEGF protein from tracheal tissues in both groups were measured by western blot to evaluate the ability of scaffolds to promote tracheal tissue repair and capillary regeneration. Briefly, the tissues were cut into pieces and lysed by RIPA lysate. Then the tissue lysate was measured by a BCA protein assay kit and boiled for 5 min before loading. The proteins were separated on 12% SDS-PAGE, transferred electronically to PVDF membranes. The membranes were blocked in a 5% BSA solution for 2 hours at room temperature and incubated with primary antibody against TGF-β1 (Boster, 1:200) and VEGF (Boster, 1:200) diluted in PBS overnight at 4 °C, and then with horseradish peroxidase-


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conjugated secondary antibody (Jackson, 1:2000) for 2 hours at room temperature. The signal bands were detected by ECL reagents (32132, Pierce). Statistical analysis All data were expressed as means ± standard deviation. SPSS Statistics software v20.0 was used to do statistical analyses. The ratio of epithelial coverage was analyzed with Independent Samples T Test. Other data were compared using the One-way ANOVA test. Significance levels were defined as p0.05) (Fig. 6b).


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a

b

A

B

C

D

E

F

40 Cross-section dimensions(mm2)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Control 35

SF

30 25 20 15 10 5 0 1W

4W Time

8W

Figure 6. Computed tomography (CT) imaging of rabbit tracheas after implantation. (a) [A-C] The control group at 1, 4, and 8 weeks, respectively. [D-F] The SF scaffold group at 1, 4, and 8


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weeks, respectively. White arrows denote the site of repair. (b) Tracheal cross-section dimensions at graft sites between two groups at 1, 4, and 8 weeks. Histological analysis At 1 week, SF scaffolds were stained red and the pore structure was in good condition without collapse (Fig. 7A). Both groups showed newly regenerated epithelial tissue with single layer pavement epithelium without well-developed cilia partially covering the scaffold surface. The ratio of epithelial coverage in the SF scaffold group (64.42±14.34) was similar to that in the control group (66.92±8.88) (Fig. 7B). Newly regenerated blood vessels were found in submucosa in the SF scaffold group. Besides, inflammatory cells, mainly including neutrophils, lymphocytes, macrophages, and a small amount of fibroblast cells infiltrated into the scaffold pores in the SF scaffold group and the submucosa in the control group. At 4 weeks, the scaffolds were completely covered with epithelium in both groups. The epithelium was stratified and ciliated on the surface of graft-host tissue interface. It gradually evolved from short cilia cubic epithelium to non-ciliated cubic epithelium from the edge to the center of the defect. At 8 weeks, most of the SF scaffolds were degraded with abundant tissue formation and angiogenesis. The mature pseudostratified ciliated columnar epithelium covered the luminal surface. Inflammatory cells decreased significantly in both groups. The inflammatory cells infiltrating at the submucosa of the reconstructed trachea were quantitatively analyzed (Fig. 7C). The data indicated that the numbers of inflammatory cells were gradually decreased from 1 week to 8 weeks in the control group, but no significant differences were found. In the SF scaffold group, the number of inflammatory cells reached the highest at 4 weeks and decreased at 8 weeks with statistical significance. Besides, the number of inflammatory cell infiltration was higher in the SF scaffold


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group than that in the control group respectively at 1, 4, and 8 weeks, but significant differences were found only at 4 weeks between two groups. The fibrosis of the reconstructed trachea was assessed by Masson's trichrome staining (Fig. 7A). At 1 week, a little of blue stained collagen fibers were observed at the submucosa in both groups and significantly increased at 4 weeks and 8 weeks. The collagen fibers arranged parallel to the mucosa without disorder during the implantation in both groups. The quantitative analysis of the degree of fibrosis showed that the amount of collagen fibers increased over time in both groups, and statistical differences were found at 4 and 8 weeks compared with 1 week in the SF scaffold group, but not in the control group. Moreover, at 1 week, the amount of collagen fibers in the SF scaffold group was less than that in the control group, but it exceeded the control group at 4 weeks and 8 weeks. Only at 4 weeks, there were significant differences between the control group and the SF scaffold group (Fig. 7D).


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Figure 7. Histological evaluations of tissue regeneration and histomorphometric analyses in the SF scaffold group and the control group at 1, 4, and 8 weeks after surgery. (A) [1-4 row]


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Photomicrographs of H&E stained sections in both groups. Second row was the magnified boxed area in 1st row. Forth row was the magnified boxed area in 3rd row. [5-6 row] Photomicrographs of Masson's trichrome stained sections in both groups. Black arrows denote the epithelium. White arrows denote regenerated capillaries. White arrowheads denote inflammatory cells. S= SF scaffold; C=collagen fiber; (B-D) Histomorphometric analyses of the epithelial coverage rate at 1 week (B) and the amount of inflammatory cells (C) as well as collagen fibers (D) in both groups at 1, 4 and 8 weeks, respectively. (*) = P

Porous three-dimensional silk fibroin scaffolds for tracheal epithelial

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