Enhanced Regeneration of Corneal Tissue via a Bioengineered Collagen Construct Implanted by a Nondisruptive Surgical Technique

Abstract

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 crosslinker ratio, BPCs were fabricated into hydrogel corneal implants with over 90% transparency and fourfold increase in strength and stiffness compared to previous versions. Remarkably, optical transparency was achieved despite the absence of collagen fibril organization at the nanoscale. In vitro testing indicated the 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.3

Composite core-and-skirt collagen hydrogels with differential degradation for corneal therapeutic applications

“…We previously reported the fabrication process for collagen-based corneal implants [14,28]. Nippon Ham Meat Packers (Tokyo, Japan) and SE Eng Co. (Seoul, South Korea).…”

“…To evaluate in vitro degradation rate of biomaterials, collagenase Type I (from Clostridium histolyticum) was used as in a previously reported protocol [14]. Briefly, 80 mg samples (150 µm thick) from each biomaterial group were incubated in collagenase-buffer solution.…”

“…Briefly, 80 mg samples (150 µm thick) from each biomaterial group were incubated in collagenase-buffer solution. The hydrogels were weighed at different time points after the surface water was gently blotted Operations were performed according to the technique of femtosecond laser-assisted intrastromal keratoplasty (FLISK), reported previously [14]. An Intralase iFS 150kHz femtosecond laser (Abbott Medical Optics, Solna, Sweden) was used to cut corneal buttons of purely stromal tissue (not including any part of the epithelium or endothelium).…”

“…Natural collagen-derived corneal scaffolds are the most mature of these [13][14][15][16][17][18][19], and we recently reported results from collagen-based scaffolds four years after implantation into diseased human corneas [13]. These cell-free scaffolds replaced the extracellular matrix, allowing host cells and nerves to eventually grow over and around the scaffold [13].…”

“…The composites consist of a natural collagen-based core-and-skirt design that functions as a corneal stromal substitute (core) and a biodegradable reservoir for therapeutic drugs and cells (skirt). The central core is a transparent and stable cross-linked collagen hydrogel called the bioengineered porcine construct (BPC) which we have recently reported [14], while the skirt is composed of mechanically-compressed collagen embedded within the BPC scaffold by in situ copolymerization. Physical properties of the skirt (thickness, porosity, mechanical properties) can be tuned for optimal degradation and transparency while the core remains stable over a longer period as a regenerative scaffold.…”