Per Fagerholm

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.

EXCIMER LASER PHOTOREFRACTIVE KERATECTOMY FOR MYOPIA; CLINICAL RESULTS IN SIGHTED EYES

PURPOSE To evaluate the refractive results of excimer laser photorefractive keratectomy (PRK) performed on normal, sighted myopic eyes; to assess the role of postoperative topical steroid treatment in patients with PRK; and to study the regression of effect. METHODS An argon fluoride 193-nm excimer laser was used. Photorefractive keratectomy was performed on 420 eyes with preoperative refraction ranging from -1.25 to -7.50 diopters (D). Minimum follow-up time was 12 months, and 194 of the eyes were followed for 15 months. Postoperative treatment generally consisted of topical dexamethasone for 3 months, but in a sub-study, some eyes were treated for only 5 weeks. RESULTS Mean refraction (spherical equivalent +/- standard deviation) at 12 months was -0.04 +/- 0.84 D and at 15 months -0.22 +/- 0.78 D. At 12 months postoperatively, 86% of the eyes were within 1.00 D of emmetropia, at 15 months 87%. At 12 months, 91% of the eyes had an uncorrected visual acuity of at least 20/40, at 15 months 87%. Eyes treated with dexamethasone for 3 months regressed significantly less than those treated for only 5 weeks (P < 0.01). Dexamethasone also was effective in reversing regression later in the postoperative course. Eyes with preoperative myopia up to 4.90 D had significantly better refractive results at 12 months than eyes with myopia ranging from 5.00 to 7.50 D (P < 0.01). CONCLUSION These data show that excimer laser PRK can correct myopia with good predictability. Results at 12 and 15 months tend to suggest stability of postoperative refraction. Regression of effect was more common in higher myopes. Topical steroids postoperatively seem to play a crucial role for the refractive result.

Normal human lens—the distribution of protein

The distribution of dry mass and protein was determined in 21 normal human lenses of different age. The lenses were obtained from enucleated tumor eyes and were immediately prepared for quantitative microradiography. The measurements were performed along the lens axis. In the anterior and posterior subcapsular cortex the dry mass content was about 0·2 g/cm 3 From this region there was an even and continuous increase over a distance of about 0·5 mm to a level of about 0·45 g/cm 3 . This level was fairly constant in the center of the lens. These results allowed a subdivision of the lens into a central lens core with a constant high concentration of dry mass and a peripheral zone with an increasing concentration from the periphery. No major changes with age in the dry mass levels could be found.

Apoptosis in UV-exposed rabbit corneas.

Purpose. Apoptosis was studied in rabbit corneas as a possible mechanism of cell death after photokeratitis induced by different UV wavelengths. Method. Fourteen albino rabbit corneas were exposed to 280-and 310-nm UV radiation (UVR) in 10-nm full wavebands at doses that cause biomicroscopically significant keratitis (0.12 J/cm2 for 280 nm and 0.47 J/cm2 for 310 nm). Animals were killed 24 and 76 h after exposure. Corneas were processed for light and transmission electron microscopy and in situ end labeling of fragmented DNA by using a modification of the TUNEL technique. Results. Corneas exposed to 280-nm UVR showed TUNEL-positive staining only in epithelial cells and superficial keratocytes at 24 and 76 h after irradiation. Twenty-four hours after 310-nm UVR exposure, TUNEL-positive staining was present in the epithelial cells, keratocytes throughout the entire thickness of the central stroma, and in endothelial cells. Seventy-six hours after exposure to 310-nm UVR, keratocytes disappeared throughout the whole thickness of the damaged stroma. Only a few epithelial cells were TUNEL positive at that time. Transmission electron microscopy (TEM) verified the occurrence of apoptotic nuclei and cells. Conclusion. Apoptosis appears to be a mechanism of corneal cell death after UVR. The 310-nm UVR caused more extensive damage to the corneal stroma and endothelium than did the 280-nm UVR.

Twenty-four-month Follow-up of Excimer Laser Photorefractive Keratectomy for Myopia: Refractive and Visual Acuity Results

PURPOSE To evaluate the 24-month refractive outcome of excimer laser photorefractive keratectomy (PRK) performed on normal, sighted myopic eyes; and to assess the evolution of postoperative refraction, the accuracy of predicted correction, and the results in terms of uncorrected visual acuity. METHODS Photorefractive keratectomy was performed on 495 eyes, with a preoperative refraction ranging from -1.25 to -7.50 diopters (D). Ablation zone diameters of 4.3 and 4.5 mm were used. All patients were treated with a standard topical steroid regimen postoperatively. Minimum follow-up time was 24 months. RESULTS Mean refraction (spherical equivalent +/- standard deviation) at 24 months was -0.27 +/- 0.74 D, which was significantly (P < 0.01) different from the mean at 12 months (0.01 +/- 0.78 D). There was also a significant (P = 0.01) difference between the 12- and 18-month (-0.15 +/- 0.82 D) mean refractions. But there was no significant difference between the means at 18 and 24 months postoperatively. Subgroup analysis at 24 months showed that patients with low to moderate myopia (up to -3.90 D) had significantly better refractive outcomes than those with higher myopia. Also at 24 months, 91% of the eyes had an uncorrected visual acuity of at least 20/40, and 81.5% had an uncorrected visual acuity of at least 20/30. Correspondingly, 87.5% of the eyes were within 1.00 D of emmetropia, and 71.7% were within 0.50 D. Only 0.4% lost one line of best-corrected visual acuity, no eye lost two lines or more. CONCLUSIONS Refraction after PRK is slow to stabilize, but appears to reach stability by 18 to 24 months after surgery. The refractive results are reasonably predictable and compare well with those achieved with radial keratotomy.

Intraocular PMMA lenses modified with surface-immobilized heparin: evaluation of biocompatibility in vitro and in vivo

Intraocular lenses (IOL) were surface modified with covalently linked heparin. The surface-bound heparin could not be removed by incubation in solutions known to be effective in breaking non-covalent bonds, nor by incubation in a solution of proteinase K and only to a limited extent by incubation with heparinase. In vitro studies demonstrated improved biocompatibility by the heparin surface-modified lens with respect to outgrowth of fibroblasts and macrophages, activation of granulocytes and adhesion of platelets. These results were subsequently verified in vivo in terms of less inflammatory cells on the lens surface and fewer incidences of synechiae after 3 and 6 wk IOL implantation in the rabbit eye.

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.

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.