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Xenogeneic acellular conjunctiva matrix as a scaffold of tissue-engineered corneal epithelium.

Zhao H, Qu M, Wang Y, Wang Z, Shi W - PLoS ONE (2014)

Bottom Line: In the present study, we investigated the preparation of xenogeneic acellular conjunctiva matrix (aCM) and evaluated its efficacy and safety as a scaffold of tissue-engineered corneal epithelium.The corneal epithelial cells seeded on aCM formed a multilayered epithelial structure and endured longer than did those on dAM.These findings support the application of xenogeneic acellular conjunctiva matrix as a scaffold for reconstructing the ocular surface.

View Article: PubMed Central - PubMed

Affiliation: Ophthalmology Department, Yijishan Hospital of Wannan Medical College, Wuhu, Anhui, China; State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong Academy of Medical Sciences, Qingdao, Shandong, China.

ABSTRACT
Amniotic membrane-based tissue-engineered corneal epithelium has been widely used in the reconstruction of the ocular surface. However, it often degrades too early to ensure the success of the transplanted corneal epithelium when treating patients with severe ocular surface disorders. In the present study, we investigated the preparation of xenogeneic acellular conjunctiva matrix (aCM) and evaluated its efficacy and safety as a scaffold of tissue-engineered corneal epithelium. Native porcine conjunctiva was decellularized with 0.1% sodium dodecyl sulfate (SDS) for 12 h at 37°C and sterilized via γ-irradiation. Compared with native conjunctiva, more than 92% of the DNA was removed, and more than 90% of the extracellular matrix components (glycosaminoglycan and collagen) remained after the decellularization treatment. Compared with denuded amniotic membrane (dAM), the aCM possessed favorable optical transmittance, tensile strength, stability and biocompatibility as well as stronger resistance to degradation both in vitro and in vivo. The corneal epithelial cells seeded on aCM formed a multilayered epithelial structure and endured longer than did those on dAM. The aCM-based tissue-engineered corneal epithelium was more effective in the reconstruction of the ocular surface in rabbits with limbal stem cell deficiency. These findings support the application of xenogeneic acellular conjunctiva matrix as a scaffold for reconstructing the ocular surface.

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Construction of rabbit corneal epithelium tissue-engineered with acellular conjunctiva matrix.Rabbit primary limbal epithelial cells proliferated well at day 7 (A). Trypan blue-alizarin red staining showed that the cells maintained live activity and grew to confluence (B). The cells could be labeled with CM-Dil (red fluorescence) (C) and stained positive with anti-K3/12 (D). Negative control lacks primary antibody (E). The rabbit corneal epithelial cells formed a 2–3 epithelial layer structure after culture for 14 days, as confirmed by H.E. staining (F) and TEM observation (G). There were tight junctions between cells and the aCM scaffold (H). Scale bar: 100 µm (D, E), 50 µm (B, C, F), 5 µm (G) and 1 µm (H).
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pone-0111846-g006: Construction of rabbit corneal epithelium tissue-engineered with acellular conjunctiva matrix.Rabbit primary limbal epithelial cells proliferated well at day 7 (A). Trypan blue-alizarin red staining showed that the cells maintained live activity and grew to confluence (B). The cells could be labeled with CM-Dil (red fluorescence) (C) and stained positive with anti-K3/12 (D). Negative control lacks primary antibody (E). The rabbit corneal epithelial cells formed a 2–3 epithelial layer structure after culture for 14 days, as confirmed by H.E. staining (F) and TEM observation (G). There were tight junctions between cells and the aCM scaffold (H). Scale bar: 100 µm (D, E), 50 µm (B, C, F), 5 µm (G) and 1 µm (H).

Mentions: Rabbit primary corneal epithelial cells were labeled with CM-Dil and inoculated on the scaffolds of aCM and dAM. At day 7, the rabbit corneal epithelial cells proliferated well on both scaffolds (Fig. 6). Trypan blue-alizarin red staining showed that the cells were alive and grew to confluence on the scaffold (Fig. 6). K3/12 (markers of corneal epithelium) staining was positive (Fig. 6). At day 14, H.E. staining showed that the cells had formed a 2–3 epithelial layer structure (Fig. 6). TEM elucidated tight junctions between these cells and the aCM scaffold (Fig. 6).


Xenogeneic acellular conjunctiva matrix as a scaffold of tissue-engineered corneal epithelium.

Zhao H, Qu M, Wang Y, Wang Z, Shi W - PLoS ONE (2014)

Construction of rabbit corneal epithelium tissue-engineered with acellular conjunctiva matrix.Rabbit primary limbal epithelial cells proliferated well at day 7 (A). Trypan blue-alizarin red staining showed that the cells maintained live activity and grew to confluence (B). The cells could be labeled with CM-Dil (red fluorescence) (C) and stained positive with anti-K3/12 (D). Negative control lacks primary antibody (E). The rabbit corneal epithelial cells formed a 2–3 epithelial layer structure after culture for 14 days, as confirmed by H.E. staining (F) and TEM observation (G). There were tight junctions between cells and the aCM scaffold (H). Scale bar: 100 µm (D, E), 50 µm (B, C, F), 5 µm (G) and 1 µm (H).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4222936&req=5

pone-0111846-g006: Construction of rabbit corneal epithelium tissue-engineered with acellular conjunctiva matrix.Rabbit primary limbal epithelial cells proliferated well at day 7 (A). Trypan blue-alizarin red staining showed that the cells maintained live activity and grew to confluence (B). The cells could be labeled with CM-Dil (red fluorescence) (C) and stained positive with anti-K3/12 (D). Negative control lacks primary antibody (E). The rabbit corneal epithelial cells formed a 2–3 epithelial layer structure after culture for 14 days, as confirmed by H.E. staining (F) and TEM observation (G). There were tight junctions between cells and the aCM scaffold (H). Scale bar: 100 µm (D, E), 50 µm (B, C, F), 5 µm (G) and 1 µm (H).
Mentions: Rabbit primary corneal epithelial cells were labeled with CM-Dil and inoculated on the scaffolds of aCM and dAM. At day 7, the rabbit corneal epithelial cells proliferated well on both scaffolds (Fig. 6). Trypan blue-alizarin red staining showed that the cells were alive and grew to confluence on the scaffold (Fig. 6). K3/12 (markers of corneal epithelium) staining was positive (Fig. 6). At day 14, H.E. staining showed that the cells had formed a 2–3 epithelial layer structure (Fig. 6). TEM elucidated tight junctions between these cells and the aCM scaffold (Fig. 6).

Bottom Line: In the present study, we investigated the preparation of xenogeneic acellular conjunctiva matrix (aCM) and evaluated its efficacy and safety as a scaffold of tissue-engineered corneal epithelium.The corneal epithelial cells seeded on aCM formed a multilayered epithelial structure and endured longer than did those on dAM.These findings support the application of xenogeneic acellular conjunctiva matrix as a scaffold for reconstructing the ocular surface.

View Article: PubMed Central - PubMed

Affiliation: Ophthalmology Department, Yijishan Hospital of Wannan Medical College, Wuhu, Anhui, China; State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong Academy of Medical Sciences, Qingdao, Shandong, China.

ABSTRACT
Amniotic membrane-based tissue-engineered corneal epithelium has been widely used in the reconstruction of the ocular surface. However, it often degrades too early to ensure the success of the transplanted corneal epithelium when treating patients with severe ocular surface disorders. In the present study, we investigated the preparation of xenogeneic acellular conjunctiva matrix (aCM) and evaluated its efficacy and safety as a scaffold of tissue-engineered corneal epithelium. Native porcine conjunctiva was decellularized with 0.1% sodium dodecyl sulfate (SDS) for 12 h at 37°C and sterilized via γ-irradiation. Compared with native conjunctiva, more than 92% of the DNA was removed, and more than 90% of the extracellular matrix components (glycosaminoglycan and collagen) remained after the decellularization treatment. Compared with denuded amniotic membrane (dAM), the aCM possessed favorable optical transmittance, tensile strength, stability and biocompatibility as well as stronger resistance to degradation both in vitro and in vivo. The corneal epithelial cells seeded on aCM formed a multilayered epithelial structure and endured longer than did those on dAM. The aCM-based tissue-engineered corneal epithelium was more effective in the reconstruction of the ocular surface in rabbits with limbal stem cell deficiency. These findings support the application of xenogeneic acellular conjunctiva matrix as a scaffold for reconstructing the ocular surface.

Show MeSH
Related in: MedlinePlus