Stable corneal regeneration four years after implantation of a cell-free recombinant human collagen scaffold.

We developed cell-free implants, comprising carbodiimide crosslinked recombinant human collagen (RHC), to enable corneal regeneration by endogenous cell recruitment, to address the worldwide shortage of donor corneas. Patients were grafted with RHC implants. Over four years, the regenerated neo-corneas were stably integrated without rejection, without the long immunosuppression regime needed by donor cornea patients. There was no recruitment of inflammatory dendritic cells into the implant area, whereas, even with immunosuppression, donor cornea recipients showed dendritic cell migration into the central cornea and a rejection episode was observed. Regeneration as evidenced by continued nerve and stromal cell repopulation occurred over the four years to approximate the micro-architecture of healthy corneas. Histopathology of a regenerated, clear cornea from a regrafted patient showed normal corneal architecture. Donor human cornea grafted eyes had abnormally tortuous nerves and stromal cell death was found. Implanted patients had a 4-year average corrected visual acuity of 20/54 and gained more than 5 Snellen lines of vision on an eye chart. The visual acuity can be improved with more robust materials for better shape retention. Nevertheless, these RHC implants can achieve stable regeneration and therefore, represent a potentially safe alternative to donor organ transplantation.

A biosynthetic alternative to human donor tissue for inducing corneal regeneration: 24-month follow-up of a phase 1 clinical study.

Corneas from human donors are used to replace damaged tissue and treat corneal blindness, but there is a severe worldwide shortage of donor corneas. We conducted a phase 1 clinical study in which biosynthetic mimics of corneal extracellular matrix were implanted to replace the pathologic anterior cornea of 10 patients who had significant vision loss, with the aim of facilitating endogenous tissue regeneration without the use of human donor tissue. The biosynthetic implants remained stably integrated and avascular for 24 months after surgery, without the need for long-term use of the steroid immunosuppression that is required for traditional allotransplantation. Corneal reepithelialization occurred in all patients, although a delay in epithelial closure as a result of the overlying retaining sutures led to early, localized implant thinning and fibrosis in some patients. The tear film was restored, and stromal cells were recruited into the implant in all patients. Nerve regeneration was also observed and touch sensitivity was restored, both to an equal or to a greater degree than is seen with human donor tissue. Vision at 24 months improved from preoperative values in six patients. With further optimization, biosynthetic corneal implants could offer a safe and effective alternative to the implantation of human tissue to help address the current donor cornea shortage.

Recombinant microbial systems for the production of human collagen and gelatin.

The use of genetically engineered microorganisms is a cost-effective, scalable technology for the production of recombinant human collagen (rhC) and recombinant gelatin (rG). This review will discuss the use of yeast (Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha) and of bacteria (Escherichia coli, Bacillus brevis) genetically engineered for the production of rhC and rG. P. pastoris is the preferred production system for rhC and rG. Recombinant strains of P. pastoris accumulate properly hydroxylated triple helical rhC intracellularly at levels up to 1.5 g/l. Coexpression of recombinant collagen with recombinant prolyl hydroxylase results in the synthesis of hydroxylated collagen with thermal stability similar to native collagens. The purified hydroxylated rhC forms fibrils that are structurally similar to fibrils assembled from native collagen. These qualities make rhC attractive for use in many medical applications. P. pastoris can also be engineered to secrete high levels (3 to 14 g/l ) of collagen fragments with defined length, composition, and physiochemical properties that serve as substitutes for animal-derived gelatins. The replacement of animal-derived collagen and gelatin with rhC and rG will result in products with improved safety, traceability, reproducibility, and quality. In addition, the rhC and rG can be engineered to improve the performance of products containing these biomaterials.

Expression and characterization of a low molecular weight recombinant human gelatin: development of a substitute for animal-derived gelatin with superior features.

Gelatin is used as a stabilizer in several vaccines. Allergic reactions to gelatins have been reported, including anaphylaxis. These gelatins are derived from animal tissues and thus represent a potential source of contaminants that cause transmissible spongiform encephalopathies. We have developed a low molecular weight human sequence gelatin that can substitute for the animal sourced materials. A cDNA fragment encoding 101 amino acids of the human proalpha1 (I) chain was amplified, cloned into plasmid pPICZalpha, integrated into Pichia pastoris strain X-33, and isolates expressing high levels of recombinant gelatin FG-5001 were identified. Purified FG-5001 was able to stabilize a live attenuated viral vaccine as effectively as porcine gelatin. This prototype recombinant gelatin was homogeneous with respect to molecular weight but consisted of several charge isoforms. These isoforms were separated by cation exchange chromatography and found to result from a combination of truncation of the C-terminal arginine and post-translational phosphorylation. Site-directed mutagenesis was used to identify the primary site of phosphorylation as serine residue 546; serine 543 was phosphorylated at a low level. A new construct was designed encoding an engineered gelatin, FG-5009, with point mutations that eliminated the charge heterogeneity. FG-5009 was not recognized by antigelatin IgE antibodies from children with confirmed gelatin allergies, establishing the low allergenic potential of this gelatin. The homogeneity of FG-5009, the ability to produce large quantities in a reproducible manner, and its low allergenic potential make this a superior substitute for the animal gelatin hydrolysates currently used to stabilize many pharmaceuticals.

The application of recombinant human collagen in tissue engineering.

Collagen is the main structural protein in vertebrates. It plays an essential role in providing a scaffold for cellular support and thereby affecting cell attachment, migration, proliferation, differentiation, and survival. As such, it also plays an important role in numerous approaches to the engineering of human tissues for medical applications related to tissue, bone, and skin repair and reconstruction. Currently, the collagen used in tissue engineering applications is derived from animal tissues, creating concerns related to the quality, purity, and predictability of its performance. It also carries the risk of transmission of infectious agents and precipitating immunological reactions. The recent development of recombinant sources of human collagen provides a reliable, predictable and chemically defined source of purified human collagens that is free of animal components. The triple-helical collagens made by recombinant technology have the same amino acid sequence as human tissue-derived collagen. Furthermore, by achieving the equivalent extent of proline hydroxylation via coexpression of genes encoding prolyl hydroxylase with the collagen genes, one can produce collagens with a similar degree of stability as naturally occurring material. The recombinant production process of collagen involves the generation of single triple-helical molecules that are then used to construct more complex three-dimensional structures. If one loosely defines tissue engineering as the use of a biocompatible scaffold combined with a biologically active agent (be it a gene or gene construct, growth factor or other biologically active agent) to induce tissue regeneration, then the production of recombinant human collagen enables the engineering of human tissue based on a human matrix or scaffold. Recombinant human collagens are an efficient scaffold for bone repair when combined with a recombinant bone morphogenetic protein in a porous, sponge-like format, and when presented as a membrane, sponge or gel can serve as a basis for the engineering of skin, cartilage and periodontal ligament, depending on the specific requirements of the chosen application.