An Ultra-thin Amniotic Membrane as Carrier in Corneal Epithelium Tissue-Engineering

Abstract

Amniotic membranes (AMs) are widely used as a corneal epithelial tissue carrier in reconstruction surgery. However, the engineered tissue transparency is low due to the translucent thick underlying AM stroma. To overcome this drawback, we developed an ultra-thin AM (UAM) by using collagenase IV to strip away from the epithelial denuded AM (DAM) some of the stroma. By thinning the stroma to about 30 μm, its moist and dry forms were rendered acellular, optically clear and its collagen framework became compacted and inerratic. Engineered rabbit corneal epithelial cell (RCEC) sheets generated through expansion of limbal epithelial cells on UAM were more transparent and thicker than those expanded on DAM. Moreover, ΔNp63 and ABCG2 gene expression was greater in tissue engineered cell sheets expanded on UAM than on DAM. Furthermore, 2 weeks after surgery, the cornea grafted with UAM based cell sheets showed higher transparency and more stratified epithelium than the cornea grafted with DAM based cell sheets. Taken together, tissue engineered corneal epithelium generated on UAM has a preferable outcome because the transplanted tissue is more transparent and better resembles the phenotype of the native tissue than that obtained by using DAM for this procedure. UAM preserves compact layer of the amniotic membrane and maybe an ideal substrate for corneal epithelial tissue engineering.

Figure 1. The characteristics of ultra-thin amniotic membrane.

(A) The thickness change of amniotic membrane after digestion with collagenase type IV for different time durations. (B) H&E and Masson trichrome staining of IAM, DAM and UAM tissues (Bar: 100 μm). (C) Macroscopic views of IAM, DAM and UAM were evaluated by photography scanning in moist form and light microscope in freeze dry form. (D) Hoechst whole mount staining of IAM, DAM and UAM (Bar: 100 μm).

Figure 2. Representative scanning electron microscopic images of IAM, DAM and UAM tissues.

The IAM epithelial side showed hexagonal cells and dense microvilli on the apical surface of the epithelial cells. Both DAM and UAM showed a continuous flattened matrix layer with dense and crossed fibroid structure. On the stromal side, irregular and loosen collagen fibrils were appeared in the IAM and UAM, while the stromal side of UAM exhibited smooth surface and the collagen fibrils were compact (Bar: 100 μm).

Figure 3. Representative transmission electron microscopic images of IAM, DAM and UAM tissues.

The magnified image of superficial layer (red inserts) and bottom layer (blue inserts) of IAM, DAM and UAM tissues were observed respectively. The collagen fibrils of superficial layer in all groups (a,c,e) showed densely packed in a regular orthogonal lamellar array. Loose collagen fibrils packing was showed in bottom layer in both IAM (b) and DAM (d) tissue, while same dense collagen fibrils packed pattern was showed in whole layer of UAM tissue (f).

Figure 4. Immunohistochemical staining of the stromal collagen components collagen I and collagen III, and immunofluorescent staining of the basement membrane components collagen IV, collagen VII, laminin 5, and perlecan in cryosections of the IAM, DAM, and UAM tissues.

Nuclei of the cells were stained with DAPI (Bar: 100 μm).

Figure 5. Morphologic features of tissue engineered corneal epithelium.

(A) Phase-contrast observation of rabbit limbal epithelial cells grown on DAM or UAM carrier for 2 weeks (Bar: 100 μm). (B) Optical property of tissue engineered corneal epithelium grown on DAM and UAM inserts respectively. (C) H&E stained sections of tissue engineered corneal epithelial cell sheets generated on DAM and UAM (Bar: 50 μm). (D) Representative transmission electron microscopic images of tissue engineered corneal epithelial cell sheets generated on DAM and UAM. The superficial cell layer (red insert) and basal cell layer (blue insert) were observed under different magnifications, respectively. Arrow heads in superficial layer demonstrated cell-cell junction space, and arrow heads in basal layer demonstrated basal cell-AM junction space. (E) Connexin 43, β-catenin, and Claudin-1 immunostaining showed weaker expression in DAM based epithelium, compared with that of UAM based epithelium (Bar: 50 μm).

Figure 6. Phenotype evaluation of tissue engineered corneal epithelium grown on DAM and UAM.

(A) Immunofluorescent staining of K12, K3, and K14 of tissue engineered corneal epithelium (Bar: 50 μm). (B) Western blotting demonstrated K12, K3, K14, and ΔNp63 expression in tissue engineered corneal epithelium. β-actin was used as the loading control. (C) Relative quantitative real-time PCR showed that DAM and UAM based epithelium expressed similar level of PCNA, while p63 and ABCG2 genes showed higher expression in UAM based epithelium.

Figure 7. Rabbit ocular surface reconstructed with tissue engineered corneal epithelium grown on DAM and UAM.

(A) Slit lamp microscopy images showed quiescent ocular surface and the surface protected amniotic membrane was not dissolved one week after surgery. Post operatively 2 weeks, the surface protected amniotic membrane was removed, diffused light images (left column), slit light images (middle column) and fluorescein staining images (right column) showed more transparent cornea and intact epithelium after UAM based tissue engineered corneal epithelium transplantation. There was mild neovascularization in the peripheral cornea after DAM based epithelium transplantation (arrow head). (B) CFDA SE successfully labeled epithelial cells (green) before transplantation. Two weeks after surgery, fluorescein dye remained in the central cornea and limbal region, and was confirmed by cryosection observation (Bar: 100 μm). (C) H&E staining of corneal and limbal epithelial cells at 2 weeks after surgery (Bar: 100 μm).

Figure 8. Corneal epithelial phenotype after tissue engineered corneal epithelium transplantation.

Immunofluorescent staining showed that K3 (red) was present in the full thickness of both central and limbal corneal epithelium in both DAM and UAM groups. K14 (red) was highly expressed in the central corneal epithelium, while ΔNp63 (red) was mainly expressed in the basal cells of limbal and central epithelia in both groups. The white arrows indicated undissolved AM tissues in central cornea at postoperatively 2 weeks (Bar: 100 μm). Nuclei of the cells were stained with DAPI.