Purpose

The long-term success of visual rehabilitation in patients with severe conjunctival scarring is reliant on the reconstruction of the conjunctiva with a suitable substitute. The purpose of this study is the development and investigation of a re-epithelialized conjunctival substitute based on porcine decellularized conjunctiva (PDC).

Methods

PDC was re-epithelialized either with pre-expanded human conjunctival epithelial cells (PDC + HCEC) or with a human conjunctival explant placed directly on PDC (PDC + HCEx). Histology and immunohistochemistry were performed to evaluate epithelial thickness, proliferation (Ki67), apoptosis (Caspase 3), goblet cells (MUC5AC), and progenitor cells (CK15, ΔNp63, ABCG2). The superior construct (PDC + HCEx) was transplanted into a conjunctival defect of a rabbit (n = 6). Lissamine green staining verified the epithelialization in vivo. Orbital tissue was exenterated on day 10 and processed for histological and immunohistochemical analysis to examine the engrafted PDC + HCEx. A human-specific antibody was used to detect the transplanted cells.

Results

From day-14 in vitro onward, a significantly thicker epithelium and greater number of cells expressing Ki67, CK15, ΔNp63, and ABCG2 were noted for PDC + HCEx versus PDC + HCEC. MUC5AC-positive cells were found only in PDC + HCEx. The PDC + HCEx-grafted rabbit conjunctivas were lissamine-negative during the evaluation period, indicating epithelial integrity. Engrafted PDC + HCEx showed preserved progenitor cell properties and an increased number of goblet cells comparable to those of native conjunctiva.

Conclusion

Placing and culturing a human conjunctival explant directly on PDC (PDC + HCEx) enables the generation of a stable, stratified, goblet cell-rich construct that could provide a promising alternative conjunctival substitute for patients with extensive conjunctival stem and goblet cell loss.

中文翻译：

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脱细胞猪结膜可作为组织工程上皮化结膜的替代底物。

目的

严重结膜瘢痕形成患者视觉康复的长期成功依赖于用合适的替代物重建结膜。本研究的目的是开发和研究基于猪脱细胞结膜（PDC）的上皮化结膜替代物。

方法

PDC用预先扩增的人结膜上皮细胞（PDC + HCEC）或直接放置在PDC上的人结膜外植体（PDC + HCEx）重新上皮。进行组织学和免疫组织化学以评估上皮厚度，增殖（Ki67），凋亡（胱天蛋白酶3），杯状细胞（MUC5AC）和祖细胞（CK15，ΔNp63，ABCG2）。将优良的构建体（PDC + HCEx）移植到兔子的结膜缺损中（n = 6）。Lissamine green染色证实了体内上皮化。在第10天取出眼眶组织，并进行组织学和免疫组织化学分析，以检查植入的PDC + HCEx。使用人类特异性抗体来检测移植的细胞。

结果

从第14天开始，PDC + HCEx与PDC + HCEC相比，上皮明显更厚，表达Ki67，CK15，ΔNp63和ABCG2的细胞数量更多。仅在PDC + HCEx中发现MUC5AC阳性细胞。在评估期间，PDC + HCEx移植的兔结膜为lissamine阴性，表明上皮完整性。植入的PDC + HCEx表现出保留的祖细胞特性，并且杯状细胞的数量与天然结膜相当。

结论

将人结膜外植体直接放置和培养在PDC（PDC + HCEx）上可以生成稳定的，分层的，富含杯状细胞的构建体，可以为结膜干和杯状细胞大量丧失的患者提供有希望的结膜替代品。