Department of Ophthalmology, University Hospital Kralovske Vinohrady and 3rd Faculty of Medicine, Srobarova 1150/50, Prague 10, 100 34, Czech Republic.
Third Faculty of Medicine, Charles University in Prague, Ruska 87, 10000, Prague 10, Czech Republic.
BMC Ophthalmol. 2023 Jun 29;23(1):294. doi: 10.1186/s12886-023-03048-3.
The goal of our study is to find an optimal approach to the preparation and preservation of corneal stromal tissue. We want to compare different methods of corneal stromal tissue creation and storage to optimize the efficacy of this process under the conditions of an eye bank. After we find the most suitable method to create a safe high quality product, we want to prove the possibility of using a single donor cornea for more than one patient. We would also like to verify the feasibility of making more corneal lenticules after the removal of a corneal endothelium for DMEK transplantation.
We provided morphological (histology, scanning electron microscope) and microbiological analysis in order to compare different methods of corneal lenticule and corneal stromal lamellae preparation and preservation. We also tested the surgical handling of the tissue to secure a safe manipulation of the tissue for clinical use. We compared two methods of corneal lenticule preparation: microkeratome dissection and femtosecond laser. As methods of preservation, we tested hypothermia, cryopreservation at -80 degrees Celsius in DMSO (dimethyl sulfoxide) and storage at room temperature with glycerol. Some intrastromal lenticules and lamellae in each group were previously irradiated with gamma radiation of 25 kGy (KiloGray).
Corneal stromal lamellae prepared with a microkeratome have a smoother cut - side surface compared to lamellae prepared with a femtosecond laser. Femtosecond laser preparation caused more irregularities on the surface and we detected more conglomerates of the fibrils, while lamellae made with microkeratome had more sparse network. Using femtosecond laser, we were able to make more than five lenticules from a single donor cornea. Gamma irradiation led to damage of collagen fibrils in corneal stroma and a loss of their regular arrangement. Corneal tissue stored in glycerol showed collagen fibril aggregates and empty spaces between fibrils caused by dehydration. Cryopreserved tissue without previous gamma irradiation showed the most regular structure of the fibrils comparable to storage in hypothermia.
Our results suggest that formation of a corneal lenticule lamellae by microkeratome results in smoother corneal lenticules, while being much cheaper than formation by femtosecond laser. Gamma irradiation of 25 kGy caused damage of the collagen fibres as well as their network arrangement, which correlated with loss of transparency and stiffer structure. These changes impair possible surgical utilisation of gamma irradiated corneas. Storage in glycerol at room temperature and cryopreservation had similar outcomes and we believe that both methods are appropriate and safe for further clinical use .
我们研究的目的是找到一种制备和保存角膜基质组织的最佳方法。我们希望比较角膜基质组织制备和储存的不同方法，以优化眼库条件下这一过程的效果。在找到最适合的方法来制备安全高质量的产品后，我们希望证明使用单个供体角膜为不止一位患者提供角膜的可能性。我们还想验证在去除角膜内皮细胞后为 DMEK 移植制备更多角膜透镜的可行性。
为了比较角膜透镜和角膜基质瓣制备和保存的不同方法，我们提供了形态学（组织学、扫描电子显微镜）和微生物分析。我们还测试了组织的手术处理，以确保安全操作组织用于临床应用。我们比较了两种角膜透镜制备方法：微角膜刀解剖和飞秒激光。作为保存方法，我们测试了低温保存、在-80°C 下用二甲基亚砜（DMSO）冷冻保存和在室温下用甘油保存。每组中的一些基质内透镜和瓣在之前用 25kGy（千戈瑞）的γ射线进行了辐照。
与飞秒激光制备的角膜基质瓣相比，用微角膜刀制备的角膜基质瓣具有更光滑的切割侧面。飞秒激光制备导致表面更不规则，我们检测到更多的纤维团块，而用微角膜刀制备的瓣具有更稀疏的网络。使用飞秒激光，我们能够从单个供体角膜制备超过 5 个透镜。γ 射线辐照导致角膜基质中胶原纤维的损伤，并导致其排列规则性丧失。未经γ射线辐照冷冻保存的组织显示出胶原纤维最规则的结构，与低温保存相比可比拟。在甘油中储存的组织显示出胶原纤维聚集体和纤维之间的空穴，这是由脱水引起的。
我们的结果表明，用微角膜刀形成角膜透镜瓣可产生更光滑的角膜透镜，而且比用飞秒激光便宜得多。25kGy 的γ 射线照射会导致胶原纤维及其网络排列受损，这与透明度丧失和结构变硬有关。这些变化会损害γ 射线照射角膜的可能的手术应用。室温下甘油储存和冷冻保存的结果相似，我们认为这两种方法都适合且安全用于进一步的临床应用。
