Neil Lagali

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.

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.

Corneal regeneration following implantation of a biomimetic tissue-engineered substitute.

Per Fagerholm, Neil S Lagali, David J Carlsson, Kimberley Merrett and May Griffith, Corneal Regeneration Following Implantation of a Biomimetic Tissue-Engineered Substitute, 2009, CTS-CLINICAL AND TRANSLATIONAL SCIENCE, (2), 2, 162-164. which has been published in final form at: http://dx.doi.org/10.1111/j.1752-8062.2008.00083.x Copyright: Blackwell Publishing http://eu.wiley.com/WileyCDA/Brand/id-35.html

Analysis of generalized Mach-Zehnder interferometers for variable-ratio power splitting and optimized switching

The nonideal integrated optical N/spl times/N generalized Mach-Zehnder interferometer (GMZI) employing multimode interference (MMI) couplers is analyzed using transfer matrix techniques. Deviations in the phase relations and the power splitting ratio of the MMI couplers are included in the theory, along with the effects of phase errors in the interferometer arms. The predictions of the theory are compared to the response of a 4/spl times/4 GMZI which has been fabricated. The device is operated as both a variable-ratio power splitter and a switch by compensating for the phase errors in the interferometer arms, but the performance is ultimately limited by the nonideal imaging in the MMI couplers. The practicality of these applications is investigated by performing a tolerance analysis for the operation of 1/spl times/N power splitters and switches for N up to 10.

Artificial corneas: a regenerative medicine approach

Corneal substitutes are being developed to address the shortage of human donor tissues as well as the current disadvantages in some clinical indications, which include immune rejection. In the past few years, there have been significant developments in bioengineered corneas that are designed to replace part or the full thickness of damaged or diseased corneas that range from keratoprostheses that solely address the replacement of the corneas function, through tissue-engineered hydrogels that permit regeneration of host tissues. We describe examples of corneal substitutes that encourage regeneration of the host tissue. We also contend that it is unlikely that there will be a single “one-size-fits-all” corneal substitute for all indications. Instead, there will most likely be a small range of corneal substitutes ranging from prostheses to tissue-engineered matrix substitutes that are tailored to different clusters of clinical indications. The tissue-engineered matrices can either be produced as sterile acellular matrices, or complete with functional cells, ready for implantation.

In Vivo Morphology of the Limbal Palisades of Vogt Correlates With Progressive Stem Cell Deficiency in Aniridia-Related Keratopathy

PURPOSE To investigate morphologic alterations in the limbal palisades of Vogt in a progressive form of limbal stem cell deficiency. METHODS Twenty Norwegian subjects (40 eyes) with congenital aniridia and 9 healthy family members (18 eyes) without aniridia were examined. Clinical grade of aniridia-related keratopathy (ARK) was assessed by slit-lamp biomicroscopy, and tear production and quality, corneal thickness, and sensitivity were additionally measured. The superior and inferior limbal palisades of Vogt and central cornea were examined by laser scanning in vivo confocal microscopy (IVCM). RESULTS In an aniridia patient with grade 0 ARK, a transparent cornea and normal limbal palisade morphology were found. In grade 1 ARK, 5 of 12 eyes had degraded palisade structures. In the remaining grade 1 eyes and in all 20 eyes with stage 2, 3, and 4 ARK, palisade structures were absent by IVCM. Increasing ARK grade significantly correlated with reduced visual acuity and corneal sensitivity, increased corneal thickness, degree of degradation of superior and inferior palisade structures, reduced peripheral nerves, increased inflammatory cell invasion, and reduced density of basal epithelial cells and central subbasal nerves. Moreover, limbal basal epithelial cell density and central corneal subbasal nerve density were both significantly reduced in aniridia compared to healthy corneas (P = 0.002 and 0.003, respectively). CONCLUSIONS Progression of limbal stem cell deficiency in aniridia correlates with degradation of palisade structures, gradual transformation of epithelial phenotype, onset of inflammation, and a corneal nerve deficit. IVCM can be useful in monitoring early- to late-stage degenerative changes in stem cell-deficient patients.

Biosynthetic corneal implants for replacement of pathologic corneal tissue: performance in a controlled rabbit alkali burn model.

PURPOSE To evaluate the performance of structurally reinforced, stabilized recombinant human collagen-phosphorylcholine (RHCIII-MPC) hydrogels as corneal substitutes in a rabbit model of severe corneal damage. METHODS One eye each of 12 rabbits received a deep corneal alkali wound. Four corneas were implanted with RHCIII-MPC hydrogels. The other eight control corneas were implanted with either allografts or a simple cross-linked RHCIII hydrogel. In all cases, 6.25 mm diameter, 350 μm thick buttons were implanted by anterior lamellar keratoplasty to replace damaged corneal tissue. Implants were followed for nine months by clinical examination and in vivo confocal microscopy, after which implanted corneas were removed and processed for histopathological and ultrastructural examination. RESULTS Alkali exposure induced extensive central corneal scarring, ocular surface irregularity, and neovascularization in one case. All implants showed complete epithelial coverage by four weeks postoperative, but with accompanying suture-induced vascularization in 6 out of 12 cases. A stable, stratified epithelium with hemidesmosomal adhesion complexes regenerated over all implants, and subbasal nerve regeneration was observed in allograft and RHCIII-MPC implants. Initially acellular biosynthetic implants were populated with host-derived keratocytes as stromal haze subsided and stromal collagen was remodeled. Notably, RHCIII-MPC implants exhibited resistance to vascular ingrowth while supporting endogenous cell and nerve repopulation. CONCLUSIONS Biosynthetic implants based on RHC promoted cell and nerve repopulation in alkali burned rabbit eyes. In RHCIII-MPC implants, evidence of an enhanced resistance to neovascularization was additionally noted.