May Griffith

PEG-stabilized carbodiimide crosslinked collagen–chitosan hydrogels for corneal tissue engineering

Implantable biomaterials that mimic the extracellular matrix (ECM) in key physical and physiological functions require components and microarchitectures that are carefully designed to maintain the correct balance between biofunctional and physical properties. Our goal was to develop hybrid polymer networks (HPN) that combine the bioactive features of natural materials and physical characteristics of synthetic ones to achieve synergy between the desirable mechanical properties of some components with the biological compatibility and physiological relevance of others. In this study, we developed collagen-chitosan composite hydrogels as corneal implants stabilized by either a simple carbodiimide cross-linker or a hybrid cross-linking system comprised of a long-range bi-functional cross-linker (e.g. poly(ethylene glycol) dibutyraldehyde (PEG-DBA)), and short-range amide-type cross-linkers (e.g. 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), and N-hydroxysuccinimide (NHS)). Optimum hybrid hydrogel demonstrated significantly enhanced mechanical strength and elasticity by 100 and 20%, respectively, compared to its non-hybrid counterpart. It demonstrated excellent optical properties, optimum mechanical properties and suturability, and good permeability to glucose and albumin. It had excellent biocompatibility and when implanted into pig corneas for 12 months, allowed seamless host-graft integration with successful regeneration of host corneal epithelium, stroma, and nerves.

A Biosynthetic Alternative to Human Donor Tissue for Inducing Corneal Regeneration: 24-Month Follow-Up of a Phase 1 Clinical Study

A biosynthetic cornea is stably integrated with host tissues for 2 years after implantation and produces nerve regeneration and vision improvement. More Windows on the World We are visual animals, and our ability to see depends on a tiny piece of transparent tissue that covers the surface of our eyes—the cornea. Constructed from parallel strands of the protein collagen, it refracts light to focus images on the retina, assisted by the adjustable lens, which modulates the focal length. The see-through nature of the cornea is easily destroyed by trauma or infection, but replacement human corneas can be inserted and reliably restore vision. The problem is that a shortage of donated corneas leaves millions of people likely to go blind. An alternative source of corneas could make a big difference. In a 2-year follow-up study of 10 patients, Fagerholm and his colleagues show that biosynthetic corneas that closely mimic the natural one are readily incorporated into the eye. They become reinnervated, restoring sensitivity to the cornea and restoring vision to the patients. Recombinant human collagen, synthesized in yeast and chemically cross-linked, was molded into a biosynthetic cornea by the authors. They used these facsimiles to replace the distorted corneas of nine patients with keratoconus and one patient who had had a corneal infection. By monitoring the patients carefully for 2 years, they were able to see how the implants were incorporated into the existing eye. First, a normal-appearing protective layer of epithelial cells, derived from the patient, covered the surface. Then, in 9 of the 10 patients, nerves that had been cut during surgery regrew into the biosynthetic cornea, and the cornea was again sensitive to mechanical stimulation, an essential response that protects the eye from injury. Because the cornea must be transparent, it has no blood supply and oxygen must come from the film of tears that bathes the tissue. This essential element was also restored, with the tears having normal osmolarity. Although without corrective contact lenses, the 10 patients on average did not have as good visual acuity 2 years after receiving their implants as did a group of patients with donated human corneas, with contact lenses (which they could not wear before surgery) the 10 patients’ vision was equivalent. The authors suggest that lessons learned in this initial trial will improve the vision of the next set of patients to receive the biosynthetic implants. The sutures used in this study caused problems with the epithelialization process, blocking cell migration and inducing haziness, as well as causing roughness on the surface. Less disruptive sutures should correct this problem. These biosynthetic—but also biomimetic—corneas may soon allow many patients who need corneal transplants but do not have donors to regain normal sight. 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.

Collagen-phosphorylcholine interpenetrating network hydrogels as corneal substitutes

A biointeractive collagen-phospholipid corneal substitute was fabricated from interpenetrating polymeric networks comprising 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide and N-hydroxysuccinimide crosslinked porcine atelocollagen, and poly(ethylene glycol) diacrylate crosslinked 2-methacryloyloxyethyl phosphorylcholine (MPC). The resulting hydrogels showed an overall increase in mechanical strength beyond that of either original component and enhanced stability against enzymatic digestion (by collagenase) or UV degradation. More strikingly, these hydrogels retained the full biointeractive, cell friendly properties of collagen in promoting corneal cell and nerve in-growth and regeneration (despite MPCs known anti-adhesive properties). Measurements of refractive indices, white light transmission and backscatter showed the optical properties of collagen-MPC are comparable or superior to those of the human cornea. In addition, the glucose and albumin permeability were comparable to those of human corneas. Twelve-month post-implantation results of collagen-MPC hydrogels into mini-pigs showed regeneration of corneal tissue (epithelium, stroma) as well as the tear film and sensory nerves. We also show that porcine collagen can be substituted with recombinant human collagen, resulting in a fully-synthetic implant that is free from the potential risks of disease transmission (e.g. prions) present in animal source materials.

Tissue-Engineered Injectable Collagen-Based Matrices for Improved Cell Delivery and Vascularization of Ischemic Tissue Using CD133+ Progenitors Expanded From the Peripheral Blood

Background— The use of stem and/or progenitor cells to achieve potent vasculogenesis in humans has been hindered by low cell numbers, implant capacity, and survival. This study investigated the expansion of CD133+ cells and the use of an injectable collagen-based tissue engineered matrix to support cell delivery and implantation within target ischemic tissue. Methods and Results— Adult human CD133+ progenitor cells from the peripheral blood were generated and expanded by successive removal and culture of CD133− cell fractions, and delivered within an injectable collagen-based matrix into the ischemic hindlimb of athymic rats. Controls received injections of phosphate-buffered saline, matrix, or CD133+ cells alone. Immunohistochemistry of hindlimb muscle 2 weeks after treatment revealed that the number of CD133+ cells retained within the target site was >2-fold greater when delivered by matrix than when delivered alone (P<0.01). The transplanted CD133+ cells incorporated into vascular structures, and the matrix itself also was vascularized. Rats that received matrix and CD133+ cells demonstrated greater intramuscular arteriole and capillary density than other treatment groups (P<0.05 and P<0.01, respectively). Conclusions— Compared with other experimental approaches, treatment of ischemic muscle tissue with generated CD133+ progenitor cells delivered in an injectable collagen-based matrix significantly improved the restoration of a vascular network. This work demonstrates a novel approach for the expansion and delivery of blood CD133+ cells with resultant improvement of their implantation and vasculogenic capacity.

The biocompatibility and antibacterial properties of collagen-stabilized, photochemically prepared silver nanoparticles.

Spherical 3.5 nm diameter silver nanoparticles (AgNP) stabilized in type I collagen (AgNP@collagen) were prepared in minutes (5-15 min) at room temperature by a photochemical method initiated by UVA irradiation of a water-soluble non-toxic benzoin. This biocomposite was examined to evaluate its biocompatibility and its anti-bacterial properties and showed remarkable properties. Thus, while keratinocytes and fibroblasts were not affected by AgNP@collagen, it was bactericidal against Bacillus megaterium and E. coli but only bacteriostatic against S. epidermidis. In particular, the bactericidal properties displayed by AgNP@collagen were proven to be due to AgNP in AgNP@collagen, rather than to released silver ions, since equimolar concentrations of Ag are about four times less active than AgNP@collagen based on total Ag content. This new biocomposite was stable over a remarkable range of NaCl, phosphate, and 2-(N-morpholino)ethanesulfonic acid concentrations and for over one month at 4 °C. Circular dichroism studies show that the conformation of collagen in AgNP@collagen remains intact. Finally, we have compared the properties of AgNP@collagen with a similar biocomposite prepared using α-poly-L-Lysine and also with citrate stabilized AgNP; neither of these materials showed comparable biocompatibility, stability, or anti-bacterial activity.

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.

Artificial human corneas: Scaffolds for transplantation and host regeneration

Purpose To review the development of artificial corneas (pros-theses and tissue equivalents) for transplantation, and to provide recent updates on our tissue-engineered replacement corneas. Methods Modified natural polymers and synthetic polymers were screened for their potential to replace damaged portions of the human cornea or the entire corneal thickness. These polymers, combined with cells derived from each of the three main corneal layers or stem cells, were used to develop artificial corneas. Functional testing was performed in vitro. Trials of biocompatibility and immune and inflammatory reactions were performed by implanting the most promising polymers into rabbit corneas. Results Collagen-based biopolymers, combined with synthetic crosslinkers or copolymers, formed effective scaffolds for developing prototype artificial corneas that could be used as tissue replacements in the future. We have previously developed an artificial cornea that mimicked key morphologic and functional properties of the human cornea. The addition of synthetic polymers increased its toughness as it retained transparency and low light scattering, making the matrix scaffold more suitable for transplantation. These new composites were implanted into rabbits without causing any acute inflammation or immune response. We have also fabricated full-thickness composites that can be fully sutured. However, the long-term effects of these artificial corneas need to be evaluated. Conclusions Novel tissue-engineered corneas that comprise composites of natural and synthetic biopolymers together with corneal cell lines or stem cells will, in the future, replace portions of the cornea that are damaged. Our results provide a basis for the development of both implantable temporary and permanent corneal replacements.

Bioengineered corneas for transplantation and in vitro toxicology

Bioengineered corneas have been designed to replace partial or the full-thickness of defective corneas, as an alternative to using donor tissues. They range from prosthetic devices that solely address replacement of the corneas function, to tissue engineered hydrogels that permit regeneration of host tissues. In cases where corneal stem cells have been depleted by injury or disease, most frequently involving the superficial epithelium, tissue engineered lamellar implants reconstructed with stem cells have been transplanted. In situ methods using ultraviolet A (UVA) crosslinking have also been developed to strengthen weakened corneas. In addition to the clinical need, bioengineered corneas are also rapidly gaining importance in the area of in vitro toxicology, as alternatives to animal testing. More complex, fully innervated, physiologically active, three-dimensional organotypic models are also being tested.

Bioengineered corneas: how close are we?

Bioengineered corneas are substitutes for human donor tissue that are designed to replace part or the full thickness of damaged or diseased corneas. They range from prosthetic devices that solely address replacement of the corneas function to tissue-engineered hydrogels that allow some regeneration of the host tissue. In addition, there are also bioengineered lenticules that may be implanted into the cornea to improve vision by altering the refractive properties of the eye, an alternative procedure to refractive surgery. In recent years, there have been significant developments in many areas of bioengineered corneas, such as the clinical trials of an artificial cornea designed as a prosthesis, the development of completely natural corneal replacements, and the development of biosynthetic matrices that permit host tissue regeneration. For correction of refractive errors, a synthetic corneal onlay that allows stable overgrowth of epithelium appears to be promising.

Tissue-Engineered Recombinant Human Collagen-Based Corneal Substitutes for Implantation: Performance of Type I versus Type III Collagen

PURPOSE To compare the efficacies of recombinant human collagens types I and III as corneal substitutes for implantation. METHODS Recombinant human collagen (13.7%) type I or III was thoroughly mixed with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide. The final homogenous solution was either molded into sheets for in vitro studies or into implants with the appropriate corneal dimensions for transplantation into minipigs. Animals with implants were observed for up to 12 months after surgery. Clinical examinations of the cornea included detailed slit lamp biomicroscopy, in vivo confocal microscopy, and fundus examination. Histopathologic examinations were also performed on corneas harvested after 12 months. RESULTS Both cross-linked recombinant collagens had refractive indices of 1.35, with optical clarity similar to that in human corneas. Their chemical and mechanical properties were similar, although RHC-III implants showed superior optical clarity. Implants into pig corneas over 12 months show comparably stable integration, with regeneration of corneal cells, tear film, and nerves. Optical clarity was also maintained in both implants, as evidenced by fundus examination. CONCLUSIONS Both RHC-I and -III implants can be safely and stably integrated into host corneas. The simple cross-linking methodology and recombinant source of materials makes them potentially safe and effective future corneal matrix substitutes.