Rejean Munger

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.

Positive correlation between Tono-pen intraocular pressure and central corneal thickness

OBJECTIVE To examine the relationship between intraocular pressure (IOP) readings taken by the Tono-Pen tonometer (Mentor O&O, Norwell, MA) and central corneal thickness (CCT). DESIGN Prospective cross-sectional population study. PARTICIPANTS There were 651 eyes of 332 healthy subjects. MAIN OUTCOME MEASURES A questionnaire was given to each subject requesting information on gender, age, race, and other factors that can influence IOP. The IOP then was measured using the Tono-Pen followed by measurements of CCT using an ultrasonic pachymeter. RESULTS The IOP was found to increase by 2.9 mmHg/100 microns CCT in males and 1.2 mmHg/100 microns in females. For males, CCT was found to be statistically significant in predicting IOP (P < 0.001 in the right and left eyes) and diabetes was of borderline significance (P = 0.012 in the right eye, P = 0.089 in the left eye). For females, CCT was of borderline significance (P = 0.064 in the right eye, P = 0.019 in the left eye). In females, a family history of glaucoma (P = 0.021 in the right eye, P = 0.022 in the left eye) and hypertension (P = 0.010 in the right eye, P = < 0.001 in the left eye) were also significant in the prediction of IOP. Race was found to be a significant predictor of CCT (P < 0.001 in both right and left eyes) for both males and females. CONCLUSION Clinicians should be aware that, as with the Goldmann applanation tonometer, the Tono-Pen has a systematic error in IOP readings caused by its dependence on CCT. Tono-Pen IOP readings are positively correlated to CCT in males and, to a lesser extent, in females as well. The CCT measurements should be considered to ensure proper interpretation of IOP measurements in the diagnosis and management of disorders in which the CCT or IOP readings are outside normal limits.

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.

Influence of corneal variables on accuracy of intraocular pressure measurement.

The accurate measurement of intraocular pressure (IOP) is a cornerstone of the diagnosis and management of glaucoma. This article reviews various methods of IOP measurement, summarizes the basic principles involved and the advantages and disadvantages of each technique, and discusses factors, focusi

Innervated human corneal equivalents as in vitro models for nerve-target cell interactions

A sensory nerve supply is crucial for optimal tissue function. However, the mechanisms for successful innervation and the signaling pathways between nerves and their target tissue are not fully understood. Engineered tissue substitutes can provide controllable environments in which to study tissue innervation. We have therefore engineered human corneal substitutes that promote nerve in‐growth in a pattern similar to in vivo re‐innervation. We demonstrate that these nerves (a) are morphologically equivalent to natural corneal nerves; (b) make appropriate contact with target cells; (c) can generate action potentials; (d) respond to chemical and physical stimuli; and (e) play an important role in the overall functioning of the bioengineered tissue. This model can be used for studying the more general topics of nerve ingrowth or regeneration and the interaction between nerves and their target cells and, more specifically, the role of nerves in corneal function. This model could also be used as an in vitro alternative to animals for safety and efficacy testing of chemicals and drugs.

Changes in measured intraocular pressure after hyperopic photorefractive keratectomy.

Purpose: To investigate the effect of hyperopic photorefractive keratectomy (PRK) on intraocular pressure (IOP) measurements. Setting: University of Ottawa Eye Institute, Ottawa Hospital, Ottawa, Canada. Methods: In this retrospective cohort study, IOP and central corneal thickness (CCT) were measured preoperatively and at 1, 2, 3, 6, 12, 18, and 24 months in 191 eyes that had hyperopic PRK with the VISX Star excimer laser. All corrections applied were between +1.00 and +6.50 diopters (D) of sphere and less than 3.75 D of cylinder. Results: At all postoperative examinations, the mean IOP in the hyperopic PRK group was 1.0 to 1.8 mm Hg lower than the preoperative IOP (P < .001). A large range of IOP changes was found across the population; eg, at 6 months, 49% of the eyes had a change in IOP from baseline of at least ±3 mm Hg. A mean reduction of 19 &mgr;m of CCT was found with pachymetry after surgery (P < .001). The change in IOP readings postoperatively was not correlated with age, sex, keratometric readings, or applied correction. Changes in IOP were strongly correlated with preoperative IOP at all time points and with preoperative CCT at 18 and 24 months (P < .001). After hyperopic PRK, the measured IOP was more likely to increase in patients with preoperative IOPs less than 14.5 mm Hg and more likely to decrease in patients with preoperative IOPs above 14.5 mm Hg. Conclusion: Changes in IOP after hyperopic PRK were similar to changes after myopic PRK, despite only minimal changes in the CCT. This suggests that hyperopic PRK results in biomechanical effects that modify the elastic properties of the cornea beyond the changes in rigidity expected from central corneal thinning. There was a strong negative correlation between the measured preoperative IOP and the change in IOP postoperatively that was likely the result of regression of the mean effect.

Regeneration of corneal cells and nerves in an implanted collagen corneal substitute.

Purpose: Our objective was to evaluate promotion of tissue regeneration by extracellular matrix (ECM) mimics, by using corneal implantation as a model system. Methods: Carbodiimide cross-linked porcine type I collagen was molded into appropriate corneal dimensions to serve as substitutes for natural corneal ECM. These were implanted into corneas of mini-pigs after removal of the host tissue, and tracked over 12 months, by clinical examination, slit-lamp biomicroscopy, in vivo confocal microscopy, topography, and esthesiometry. Histopathology and tensile strength testing were performed at the end of 12 months. Other samples were biotin labeled and implanted into mice to evaluate matrix remodeling. Results: The implants promoted regeneration of corneal cells, nerves, and the tear film while retaining optical clarity. Mechanical testing data were consistent with stable, seamless host-graft integration in regenerated corneas, which were as robust as the untreated fellow corneas. Biotin conjugation is an effective method for tracking the implant within the host tissue. Conclusions: We show that a simple ECM mimetic can promote regeneration of corneal cells and nerves. Gradual turnover of matrix material as part of the natural remodeling process allowed for stable integration with host tissue and restoration of mechanical properties of the organ. The simplicity in fabrication and shown functionality shows potential for ECM substitutes in future clinical applications.

Increased disk size in glaucomatous eyes vs normal eyes in the Reykjavik eye study

PURPOSE To evaluate the use of disk diameter as an indicator in the identification of glaucomatous optic neuropathy. METHODS We evaluated all available stereofundus photographs for 1,040 right eyes obtained in the Reykjavik Eye Study. Horizontal and vertical disk diameters were determined in a masked manner by a glaucoma specialist (K.F.D.). All disk diameters were corrected for refractive error. RESULTS There were significant differences (P <.05) between the corrected vertical disk diameters of normal subjects (0.189 +/- 0.018 inches) and those suspected of having glaucoma (0.202 +/- 0.020 inches) as well as between the normal and the glaucoma groups (0.206 +/- 0.029 inches). The corrected horizontal measurement showed the same pattern. CONCLUSIONS In the Reykjavik Eye Study, optic disks meeting structural criteria for glaucoma are significantly larger than normal nerves.