The Network of Excellence in Corneal Regeneration

2015 Research Articles

Szabó DJ, Noer A, Nagymihály R, Josifovska N, Andjelic S, Veréb Z, Facskó A, Moe MC, Petrovski G. Long-Term Cultures of Human Cornea Limbal Explants Form 3D Structures Ex Vivo - Implications for Tissue Engineering and Clinical Applications. PLoS One. 2015 Nov 18;10(11):e0143053.





Notara M, Refaian N, Braun G, Steven P, Bock F, Cursiefen C. Short-term uvb-irradiation leads to putative limbal stem cell damage and niche cell-mediated upregulation of macrophage recruiting cytokines. Stem Cell Res. 2015 Nov;15(3):643-54.






Sidney LE, McIntosh OD, Hopkinson A. Phenotypic Change and Induction of Cytokeratin Expression During In Vitro Culture of Corneal Stromal Cells. Invest Ophthalmol Vis Sci. 2015 Nov;56(12):7225-35.






Use of Fish Scale-Derived BioCornea to Seal Full-Thickness Corneal Perforations in Pig Models.

Chen SC1, Telinius N2, Lin HT3, Huang MC3, Lin CC3, Chou CH3, Hjortdal J2.

PLoS One. 

2015 Nov 24;10(11):e0143511. doi: 10.1371/journal.pone.0143511. eCollection 2015.



The aim of this study was to test the use of BioCornea, a fish scale-derived collagen matrix for sealing full-thickness corneal perforations in mini-pigs. Two series of experiments were carried out in 8 Lan-Yu and 3 Göttingen mini-pigs, respectively. A 2mm central full thickness corneal perforation was made with surgical scissors and 2mm trephines. The perforations were sealed immediately by suturing BioCornea to the wounded cornea. The conditions of each patched cornea were followed-up daily for 3 or 4 days. Status of operated eyes was assessed with slit lamp examination or optical coherence tomography (OCT). Animals were sacrificed after the study period and the corneas operated were fixated for histological examination. Both OCT imaging and handheld slit lamp observations indicated that a stable ocular integrity of the perforated corneas was maintained, showing no leakage of aqueous humor, normal depth of anterior chamber and only mild swelling of the wounded cornea. Hematoxylin and eosin staining of the patched cornea showed no epithelial ingrowths to the perforated wounds and no severe leucocyte infiltration of the stroma. The fish scale-derived BioCornea is capable to seal full-thickness corneal perforation and stabilize the integrity of ocular anterior chamber in pre-clinic mini-pig models. BioCornea seems to be a safe and effective alternative for emergency treatment of corneal perforations.






Biocompatibility of a fish scale-derived artificial cornea: cytotoxicity, cellular adhesion and phenotype, and in vivo immunogenicity

T.H. van Essen, L. van Zijl, T. Possemiers, A.A. Mulder, S.J. Zwart, C.H. Chou, C.C. Lin, H.J. Lai, G.P.M. Luyten, M.J. Tassignon, N. Zakaria, A.El Ghalbzouri, M.J. Jager

Biomaterials, Available online 1 December 2015, ISSN 0142-9612,






To determine whether a fish scale-derived collagen matrix (FSCM) meets the basic criteria to serve as an artificial cornea, as determined with in vitro and in vivo tests.


Primary corneal epithelial and stromal cells were obtained from human donor corneas and used to examine the (in)direct cytotoxicity effects of the scaffold. Cytotoxicity was assessed by an MTT assay, while cellular proliferation, corneal cell phenotype and adhesion markers were assessed using an EdU-assay and immunofluorescence. For in vivo-testing, FSCMs were implanted subcutaneously in rats. Ologen® Collagen Matrices were used as controls. A second implant was implanted as an immunological challenge. The FSCM was implanted in a corneal pocket of seven New Zealand White rabbits, and compared to sham surgery.


The FSCM was used as a scaffold to grow corneal epithelial and stromal cells, and displayed no cytotoxicity to these cells. Corneal epithelial cells displayed their normal phenotypical markers (CK3/12 and E-cadherin), as well as cell-matrix adhesion molecules: integrin-α6 and β4, laminin 332, and hemi-desmosomes. Corneal stromal cells similarly expressed adhesion molecules (integrin-α6 and β1). A subcutaneous implant of the FSCM in rats did not induce inflammation or sensitization; the response was comparable to the response against the Ologen® Collagen Matrix. Implantation of the FSCM in a corneal stromal pocket in rabbits led to a transparent cornea, healthy epithelium, and, on histology, hardly any infiltrating immune cells.


The FSCM allows excellent cell growth, is not immunogenic and is well-tolerated in the cornea, and thus meets the basic criteria to serve as a scaffold to reconstitute the cornea.

Keywords: artificial cornea; keratoprosthesis; collagen; in vitro; in vivo; biocompatiblity






Novel lamellar, flap-based tattooing techniques for corneal opacities in scarred and vascularized blind eyes.

Hos D(1), Heindl LM, Bucher F, Cursiefen C.

Cornea. 2015 Jan;34(1):82-6. doi: 10.1097/ICO.0000000000000259.


PURPOSE: The aim of this study was to describe novel, flap-based tattooing techniques for the treatment of disfiguring corneal scars in blind eyes.

METHODS: Several new modifications of intrastromal corneal tattooing techniques were performed in 6 patients. In corneas with a low risk of perforation, a large limbus-to-limbus lamellar flap was prepared, and the tattooing dyes were spread over the entire stromal bed. After additional puncturing of the dyes into the stroma, the flap was closed and sutured ("large flap technique"). In fragile corneas where convenient preparation of a large flap was not possible, a central small flap was prepared, and the "pupil" was tattooed in analogy ("small flap technique"). Afterward, the corneal periphery corresponding to the "iris" was tattooed, either by puncturing or injecting the dye into peripheral intrastromal tunnels ("tunnel technique").

RESULTS: Two eyes were tattooed using a large flap, and 4 eyes were tattooed using a small flap. Here, the corneal periphery of 3 eyes was tattooed by puncturing, whereas 1 eye was tattooed using the tunnel technique. All tattooing procedures were performed without complications and with good cosmetic results.

CONCLUSIONS: These novel, flap-based tattooing techniques are alternatives to previously reported procedures and can be adapted to the individual corneal constitution. Further, the tunnel technique is an easy-to-perform method that provides good tattooing results.






Antilymphangiogenic therapy to promote transplant survival and to reduce cancer

metastasis: What can we learn from the eye?

Hos D(1), Schlereth SL(1), Bock F(2), Heindl LM(1), Cursiefen C(3).

Semin Cell Dev Biol. 2015 Feb;38:117-130. doi: 10.1016/j.semcdb.2014.11.003.


The lymphatic vasculature is - amongst other tasks - essentially involved in inflammation, (auto)immunity, graft rejection and cancer metastasis. The eye is mainly devoid of lymphatic vessels except for its adnexa, the conjunctiva and the limbus. However, several pathologic conditions can result in the secondary ingrowth of lymphatic vessels into physiologically alymphatic parts of the eye such as the cornea or the inner eye. Therefore, the cornea has served as an excellent in vivo model system to study lymphangiogenesis, and findings from such studies have substantially contributed to the understanding of central principles of lymphangiogenesis also with relevance outside the eye. Grafting experiments at the cornea have been extensively used to analyze the role of lymphangiogenesis in transplant immunology. In this regard, we recently demonstrated the crucial role  of lymphatic vessels in mediating corneal allograft rejection and could show that antilymphangiogenic therapy increases graft survival. In the field of cancer research, we recently detected tumor-associated lymphangiogenesis in the most common malignant tumors of the eye, such as conjunctival carcinoma and melanoma,  and ciliochoroidal melanoma with extraocular extension. These neolymphatics correlate with an increased risk of local recurrence, metastasis and tumor related death, and may offer potential therapeutic targets for the treatment of these tumors. This review will focus on corneal and tumor-associated ocular lymphangiogenesis. First, we will describe common experimentally used corneal lymphangiogenesis models and will recapitulate recent findings regarding the involvement of lymphatic vessels in corneal diseases and transplant immunology. The second part of this article will summarize findings about the participation of tumor-associated lymphangiogenesis in ocular malignancies and their implications for the development of future therapeutic strategies.






Enhanced regeneration of corneal tissue via a bioengineered collagen construct

implanted by a nondisruptive surgical technique.

Koulikovska M(1), Rafat M, Petrovski G, Veréb Z, Akhtar S, Fagerholm P, Lagali N.

Tissue Eng Part A. 2015 Mar;21(5-6):1116-30. doi: 10.1089/ten.TEA.2014.0562.


Severe shortage of donor corneas for transplantation, particularly in developing  countries, has prompted the advancement of bioengineered tissue alternatives. Bioengineered corneas that can withstand transplantation while maintaining transparency and compatibility with host cells, and that are additionally amenable to standardized low-cost mass production are sought. In this study, a bioengineered porcine construct (BPC) was developed to function as a biodegradable scaffold to promote corneal stromal regeneration by host cells. Using high-purity medical-grade type I collagen, high 18% collagen content and optimized EDC-NHS cross-linker ratio, BPCs were fabricated into hydrogel corneal implants with over 90% transparency and four-fold increase in strength and stiffness compared with previous versions. Remarkably, optical transparency was achieved despite the absence of collagen fibril organization at the nanoscale. In vitro testing indicated that BPC supported confluent human epithelial and stromal-derived mesenchymal stem cell populations. With a novel femtosecond laser-assisted corneal surgical model in rabbits, cell-free BPCs were implanted in vivo in the corneal stroma of 10 rabbits over an 8-week period. In vivo, transparency of implanted corneas was maintained throughout the postoperative period, while healing occurred rapidly without inflammation and without the use of postoperative steroids. BPC implants had a 100% retention rate at 8 weeks, when host stromal cells began to migrate into implants. Direct histochemical evidence of stromal tissue regeneration was observed by means of migrated host cells producing new collagen from within the implants. This study indicates that  a cost-effective BPC extracellular matrix equivalent can incorporate cells passively to initiate regenerative healing of the corneal stroma, and is compatible with human stem or organ-specific cells for future therapeutic applications as a stromal replacement for treating blinding disorders of the cornea.



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Action BM1302 "Joining Forces in Corneal Regeneration Research" 2014-2017.