Bio‐Orthogonally Crosslinked, In Situ Forming Corneal Stromal Tissue Substitute

Abstract

In this study, an in situ forming corneal stromal substitute based on collagen type I crosslinked by bio-orthogonal strain-promoted azide-alkyne cycloaddition (SPAAC) is presented. The crosslinked collagen gel has greater transparency compared to non-crosslinked collagen gels. The mechanical properties of the gels are controlled by changing functional group ratios and conjugated collagen concentrations. Higher concentrations of conjugated collagen yield enhances mechanical properties, where the storage modulus increases from 42.39 ± 8.95 to 112.03 ± 3.94 Pa after SPAAC crosslinking. Encapsulated corneal keratocytes grow within the SPAAC-crosslinked gels and corneal keratinocytes are supported on top of the gel surfaces. SPAAC-crosslinked gels support more favorable and stable keratinocyte morphology on their surface compared to non-crosslinked gels likely as a result of more optimal substrate stiffness, gel integrity, and resistance to degradation. SPAAC-crosslinked collagen gels with and without encapsulated keratocytes applied to rabbit corneas in an organ culture model after keratectomy exhibit surface epithelialization with multilayered morphology. The novel in situ forming gel is a promising candidate for lamellar and defect reconstruction of corneal stromal tissue.

In situ-forming collagen hydrogel crosslinked via multi-functional PEG as a matrix therapy for corneal defects

“…In prior work, we developed in situ-forming collagen hydrogels crosslinked by strain-promoted azide-alkyne cycloaddition (SPAAC) using bovine collagen type 1. These hydrogels were able to sustain the growth of both human keratinocytes and keratocytes 12 . Here we have developed in situ forming collagen hydrogels crosslinked by multi-functional PEG-N-hydroxysuccinimide that exhibited suitable mechanical and biological properties to fill and repair corneal stromal defects.…”

“…The non-crosslinked collagen is a physical crosslinked hydrogel formed by the neutralization of collagen at 37 °C. Although it is not a covalently crosslinked system, it does form a physically crosslinked gel that is relatively strong and allow cell studies in these systems over several days 12,33 . To visualize the hydrogel for the in vivo experiments, Alexa Fluor 647 NHS Ester (Thermo Fisher Scientific, A20006) was conjugated to the neutralized collagen following the manufacturer's instructions.…”

“…Recently, matrix therapies of various kinds have been developed 2 – 4 , 8 , 12 , 13 where a flowable material is applied and cured over corneal wounds. The goal of a matrix therapy is to sustain epithelial and stromal cell ingrowth 4 , and specific priorities are transparency and biocompatibility to improve host-graft interaction 6 , 14 .…”

Characterizing the impact of 2D and 3D culture conditions on the therapeutic effects of human mesenchymal stem cell secretome on corneal wound healing in vitro and ex vivo

“…For the air/liquid organ culture system, the same method as a previous study was used [18] which has also been used extensively by other institutions [19][20][21]. Following wounding, the injured corneas were excised from the globes with a 1 mm scleral rim, grasping only scleral rims and not the clear cornea.…”

Bioengineering Approaches for Corneal Regenerative Medicine

“…A stratified epithelium was regenerated on the top of the scaffold and delivered keratocyte cells elongated within the hydrogel after 6 days. Further in vivo investigation is required to evaluate the role of delivered cells in the regeneration process and study the impact of host cells migration on the corneal repair [124].…”