Evaluation of novel decellularizing corneal stroma for cornea tissue engineering applications

Abstract

AIM

To develop a new decellularization method depended upon the natural corneal structure and to harvest an ideal scaffold with good biocompatibilities for corneal reconstruction.

METHODS

The acellular cornea matrix (ACM) were prepared from de-epithelium fresh porcine corneas (DFPCs) by incubation with 100% fresh human sera and additional electrophoresis at 4°C. Human corneal epithelial cells (HCEs) were used for the cytotoxicity tests of ACM. ACM were implanted into the Enhanced Green Fluorecence Protein (eGFP) transgenic mouse anterior chamber for evaluation of histocompatibility.

RESULTS

HE and GSIB4 results showed fresh porcine cornea matrix with 100% human sera and electrophoresis could entirely decellularize stromal cell without reducing its transparency. ACM had no cytotoxic effect ex vivo. Animal test showed there was no rejection for one month after surgery.

CONCLUSION

These results provide a decellularizing approach for the study of corneal tissue engineering and had the broader implications for the field of biological tissue engineering in other engineered organ or tissue matrix.

INTRODUCTION

Cornea disease is a major cause of vision loss. Although corneal transplantation using human cornea stroma is the most appropriate therapeutic method for keratoconus^[1], its supply is in a grave shortage in China. These facts have prompted various efforts to develop several corneal substitutes, including keratoprosthesis (KPro), natural and synthetic corneal replacement^[2]-^[4]. The physial properties and safety of these substitutes were improved to some degree but were still remain many problems, which have restricted their clinical applications^[5]-^[8]. The method of decellularization in porcine cornea has been widely used, because of its advantages in preserving the natural extracellular matrix, which is difficult to imitate using synthetic methods. Therefore, we examined the efficacy of a novel method of decellularizing porcine corneal stroma and the feasibility of using them in vivo and ex vivo. To our best knowledge, a study of this novel decellularizing method has not been previously reported.

MATERIALS AND METHODS

Materials

Preparation of fresh human sera and decellularized porcine cornea

All studies using human sera were in accordance with the tenets of the Declaration of Helsinki and the policies of the institutional review board for human subjects from Xiamen Eye Center (Xiamen, China). Under aseptic conditions, 90mL of human blood was collected from healthy volunteers and centrifuged at 2000r/min for 10 minutes, the supernatant sera which preserved complement activity was then harvested and stored in -20°C before use. Sera from blood hemotype A was applied in this study.

Porcine cornea was immediately transported to the laboratory in moist chambers at 4°C. Cornea was washed in the phosphate buffer saline (PBS, pH 7.4) containing antibiotics (penicillin 60mg/L, streptomycin 100mg/L, Invitrogen, USA) three times. Corneal epithelium and endothelium was then removed by cell scraper, and the epithelium side was marked using 10-0 nylon (Alcon, USA). Cornea lamellar stroma (about 300µm in thickness) was incubated with 100% sterile human sera for duration up to 1 day. After washed 3 times with PBS for 15 minutes, lamellar stroma (including DNA fragmentation with electric charge) were subjected to electrophoresis (150V/cm,) in sterilized buffer (pH 7.4, 320 mosmol/kg) containing 40mmol/L Tris-base, 18mmol/L glacial acetic acid and antibiotics for 1 hour at 4°C, followed by 3 washes with PBS for 20 minutes at 4°C with continuous shaking in thermostat-controlled water bath. Cornea stroma went through such procedure were defined as acellular corneal matrix (ACM).

Methods

α-Gal epitopes binging assay

GSIB4 staining was performed to detect α-Gal epitopes as previously described^[9]. In brief, ACM section were reacted with Alexa fluor 568 conjugated GSIB4 (5µg/mL in PBS, Invitrogen, USA) for 10 minutes at room temperature. After 3 times washes with PBS for 15 minutes, the nuclei were counterstained with DAPI (Vector Laboratories, USA). The samples were investigated and photographed with laser confocal microscopy (Fluoview 1000, Olympus, Japan).

Extract cytotoxicity assay

The ACM were tested for their potential cytotoxicity (cytotoxicity test: ISO 1099317) to the proliferation of human corneal epithelial cells (HCE, ADL Company, USA; culture medium: DMEM supplemented with 10% Fetal Bovine Serum). The scaffolds were extracted at 37°C for 24 hours in 10mL medium consisting of HCE culture medium under constant shaking in thermostat-controlled water bath. HCE (1.0×10⁴/cm²) were seeded onto 96-well plates and incubated for 24 hours at 37°C in 5% CO₂ and air, after that the culture medium was aspirated, and 100µL of culture medium accordingly plus 100µL of the aseptic scaffolds extracts (pH 7.2) were added to five replicate wells, respectively. Controls included 40% (v/v) di-methyl sulfoxide (positive control) in media and media alone (negative control). The plates were incubated for further 12, 24, 36 and 48 hours. The effect of the extracts was determined using the Cell Counting Kit-8 (CCK-8, Dojindo, Japan) following the manufacturer's instructions. Within 2 hours, the optical density (OD) at 450nm was determined with a microplate reader (ELX800, BIO-TEK Corporation, USA).

Mouse anterior chamber implant assay

Animal experiments were performed in accordance with the ARVO Statement on the Use of Animals in Ophthalmic and Vision Research and the Guidelines of the Animal Experimental Committee of Xiamen University (Fujian, China). Enhanced Green Fluorecence Protein (eGFP) transgenic mouse (20g-30g, 6 weeks old, female) was anesthetized with intraperitoneal pentobarbital sodium (30mg/kg) and topical 0.5% proparacaine hydrochloride. After sterilization, The square ACM (1mm×1mm, n=5) were implanted into the anterior chamber in the left eye of mice. One month after the transplantation, the left eye was enucleated and the cornea was stained with HE for histological study.

Histology analysis

Tissue samples (6µm) were prepared and fixed, HE staining and DAPI staining were performed as described in previous report^[5]. Sections were observed under light microscope and Eclipse epi-fluorescence microscope (TE-2000U, Nikon, Japan) and photographed.

Statistical Analysis

Data were expressed as mean±SD and analyzed statistically by Student's t-test. P<0.05 was regarded as statistically significant. The statistical analysis was conducted with commercial software (GraphPad Prism for Windows, ver. 5.00; GraphPad Software. Inc., USA).

RESULTS

Morphology of ACM

Compared the fresh porce cornea (Figure 1B, D), HE staining revealed the maintenance the excellent transparence and uniform structure of collagen fibrils in the ACM (Figure 1A, C). The GSIB4 binding assay and DAPI staining showed the absence of cells and α-Gal in ACM (Figure 1E) but fresh porce cornea (Figure 1F).

Figure 1. Representative photographs, histology-stained sections of the fresh porcine cornea and ACM (GSIB4, red; DAPI, blue;(C-F), magnification, ×200).

The left column shows the transparency (A), HE staining(C), GSIB4 and DAPI staining (E) of fresh porcine cornea. The right column shows the transparency (B), HE staining (D), GSIB4 and DAPI staining (F) of ACM.

Ex vivo evaluation of cytotoxicity

Evaluation of the safety of ACM was performed in vitro by cells seeding on leaching liquor of ACM. The OD values after 12, 24, 48 and 72 hours of culture were showed in Figure 2A. There were no significant differences of the proliferation of HCE between ACM extract and negative control at predetermined time points (P>0.05).

Figure 2. CCK-8 assay for proliferation of HCE in ACM extract/control medium after in vitro culture for different hours (A). There are no significant differences in the proliferation of HCE between ACM extract and negative control (P>0.05). One month postoperatively, ACM did not invoke an immune response in anterior chamber in eGFP mouse anterior chamber (B). HE staining showed that there were no eGFP marker cells (bright dot) infiltrated the transplanted ACM (C). (n=5 for A. dash line:ACM).

In vivo evaluation of histocompatibility

One month postoperatively, ACM did not invoke an adverse immune response by the host in anterior chamber (Figure 2B). HE staining showed that the transplanted ACM could be recognized in the mouse anterior chamber and that there were no eGFP marker cells infiltrated the transplanted ACM (Figure 2C).

DISCUSSION

This study has identified that our method of decellularization using with 100% sterile human sera and electrophoresis entirely decellularize the porcine cornea stromal cell without reducing its transparency and inducing rejection in an anterior chamber implant models. But its mechanism was not detected since the study was focused on developing a new decellularization method.

The α-Gal epitope is one of the most abundant carbohydrate epitopes on cells of non-primate mammals and New World monkeys, where it is synthesized by expressing a functional α1, 3 galactosyltransferase enzyme. On the contrary, in humans, apes and Old World monkeys, this epitope is absent. However, the gastrointestinal bacterium that express α-Gal can induce anti-Gal antibodies in humans ^[10]. It is known that the major immunologic barrier in xenotransplantation is the interaction between anti-Gal IgM and IgG antibodies and α-Gal epitope. Previous studies have shown that the hyperacute rejection of vascularized xenografts in the in vitro porcine-to-human combination is triggered by the binding of human preformed natural antibodies (PNAbs) to the α-Gal epitope in pig endothelium and the subsequent activation of complement^[11] and it is induced by the combining of the complement and antibody dependent cell mediated cytotoxicity process in vivo^[12],^[13]. However, serum support the colloid osmotic pressure, the antiprotease component (trypsin inhibitor), the growth factors (fibroblast growth factor, epidermal growth factor and platelet growth factor), and the nutrient substances (amino acid, vitamin, lipoid and inorganic substance). Serum also protects the fiber tissue and cell to away from the damage of mechanic and enzymatic treatment. Since α-Gal epitope is expressed in several keratocytes in the most anterior corneal stroma^[14],^[15], our study was to use human serum, which contains anti-Gal, to decellularize the porcine corneal stromal cells.

As we know, decellularized biological scaffolds have been successfully used in clinical applications including heart valves, blood vessels, skin, nerves, skeletal muscle, tendons, ligaments, small intestinal submucosa, urinary bladder and liver for tissue engineering and regenerative medicine applications^[16],^[17]. However, transparency is an essential property of the cornea. Most of decellularized procedure use detergents or snap freezing to remove cells from tissue^[18],^[19]. Detergents are generally toxic and may lead to the tissue denaturation and cornea opacification^[20]. Freezing alone did not decellularize the corneal stroma^[21]. Our decellularization method resulted in no cell residual in the corneal stroma and the transparency was well preserved. The extract cytotoxicity was demonstrated that HCE cells had obvious proliferation tendency. These results suggest that the ACM do not retard the cell proliferation and have no cytotoxic effects ex vivo. Histologic evaluation revealed that few cells entered the ACM 1 month after they were transplanted into the mouse anterior chamber. Since keratocytes may play some role in metabolism of the corneal matrix, the degradation of ACM must be observed for a longer period.

In conclusion, we found the acellular method without destroying stroma structure, and the nontoxic ACM that was transplanted into the mouse anterior chamber for one month. This decellularization method may be valuable for preparation of xenogenic corneal matrix equivalent for clinical application.