Abstract
PurposeTo determine feasibility of plant-derived recombinant human collagen type I (RHCI) for use in corneal regenerative implantsMethodsRHCI was crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to form hydrogels. Application of shear force to liquid crystalline RHCI aligned the collagen fibrils. Both aligned and random hydrogels were evaluated for mechanical and optical properties, as well as in vitro biocompatibility. Further evaluation was performed in vivo by subcutaneous implantation in rats and corneal implantation in Göttingen minipigs.ResultsSpontaneous crosslinking of randomly aligned RHCI (rRHCI) formed robust, transparent hydrogels that were sufficient for implantation. Aligning the RHCI (aRHCI) resulted in thicker collagen fibrils forming an opaque hydrogel with insufficient transverse mechanical strength for surgical manipulation. rRHCI showed minimal inflammation when implanted subcutaneously in rats. The corneal implants in minipigs showed that rRHCI hydrogels promoted regeneration of corneal epithelium, stroma, and nerves; some myofibroblasts were seen in the regenerated neo-corneas.ConclusionPlant-derived RHCI was used to fabricate a hydrogel that is transparent, mechanically stable, and biocompatible when grafted as corneal implants in minipigs. Plant-derived collagen is determined to be a safe alternative to allografts, animal collagens, or yeast-derived recombinant human collagen for tissue engineering applications. The main advantage is that unlike donor corneas or yeast-produced collagen, the RHCI supply is potentially unlimited due to the high yields of this production method.Lay SummaryA severe shortage of human-donor corneas for transplantation has led scientists to develop synthetic alternatives. Here, recombinant human collagen type I made of tobacco plants through genetic engineering was tested for use in making corneal implants. We made strong, transparent hydrogels that were tested by implanting subcutaneously in rats and in the corneas of minipigs. We showed that the plant collagen was biocompatible and was able to stably regenerate the corneas of minipigs comparable to yeast-produced recombinant collagen that we previously tested in clinical trials. The advantage of the plant collagen is that the supply is potentially limitless.
Highlights
The human cornea is the transparent front of the eye that focuses incoming light onto the retina for vision
Samples of biomaterials were fixed and prepared for scanning electron microscopy (SEM) and transmission electron microscopy (TEM) as we previously described in Haagdorens et al [14]
The randomly aligned RHCI (rRHCI) hydrogels allowed for light transmittance within the visual spectrum of over 91% (Fig. S1) while Aligning the RHCI (aRHCI) hydrogels showed only 37% light transmission, appearing opaque (Fig. S1, 1a)
Summary
The human cornea is the transparent front of the eye that focuses incoming light onto the retina for vision. Its cellular layers comprise an outermost epithelium, a middle stroma composed of collagenous extracellular matrix (ECM) with interconnected keratocytes, and an innermost endothelium. The only widely used treatment for restoring vision is human donor cornea (HDC) transplantation. The outcome of conventional corneal transplantation is limited by immune rejection, ocular infection, and premature graft failure. All three prostheses are associated with potentially serious side effects and are regarded as a last resort treatment for patients with severe pathology or previous conventional graft failures [5]. Given the shortcomings of conventional corneal transplantation and insufficient performance of prostheses, various research groups have focused on the development of a range of corneal replacements [6, 7]
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