Improving the practicality and safety of artificial corneas: Pre-assembly and gamma-rays sterilization of the Boston Keratoprosthesis

Purpose

To make the Boston keratoprosthesis (B-KPro), together with its carrier corneal graft, more easily procured, transported and stored, as well as less expensive, easier for the surgeon to implant and safer for the patient, it is proposed that the B-KPro-graft combination be pre-assembled by an expert technician, followed by sterilization with gamma ray irradiation (GI) allowing long-term storage at room temperature. For this to be possible, it must be shown that the B-KPro itself (not only the graft) remains unharmed by the irradiation.

Changing pattern of utilization of human donor cornea in India

Purpose:To review the changing pattern of donor, corneal utilization in an eye bank at a Tertiary Care Center in Northern India by analyzing the trend in the years 2003, 2008, and 2011.Methods:A retrospective review of eye bank records for 3 years (2003, 2008, and 2011) was performed at the National Eye Bank. Details including a clinical grade of donor cornea, indication of corneal transplantation (therapeutic or optical), type of procedure (penetrating or lamellar keratoplasty [LK]), and clinical diagnosis of the graft recipients were recorded. Primary outcome measure was to observe any preference toward LK, judicious usage of donor corneal tissue, and impact of lamellar corneal transplant in the usage of donor corneas. Secondary outcomes included overall utilization rate and change in trend of indication for keratoplasty.Results:A total of 673, 745, and 864 corneas were retrieved in the years 2003, 2008, and 2011, respectively. The percentage of donor corneal utilization increased significantly over time with the rate being 65.08%, 70.06%, and 68.29%, respectively, in the years 2003, 2008, and 2011 (P = 0.014); however, this change was reflected only in the usage of nonoptical grade corneas and not for the optical grade corneas. There was an overall increase in lamellar corneal procedures for any clinical grade of cornea (P = 0.0019); number of Descemet''s stripping automated endothelial keratoplasty (DSAEK) procedures increased significantly (P < 0.001), particularly for pseudophakic corneal edema (PCE) (P = 0.0085) and failed graft (P = 0.002). Significant increase in the utilization of nonoptical grade corneas was observed over the years (P = 0.005), though the utilization did not increase significantly for optical purposes viz., LK (P = 0.08).Conclusions:Utilization rate of donor corneas increased over the years, primarily due to increase in usage of nonoptical grade corneas for therapeutic purposes. There was a procedural shift toward DSAEK for PCE and failed graft. However, an increase in usage of nonoptical grade corneas for LK, a single donor corneal tissue for two recipients, and retrieval or utilization of optical grade cornea was not observed.     

Evaluation of corneal cell growth on tissue engineering materials as artificial cornea scaffolds

The keratoprosthesis (KPro; artificial cornea) is a special refractive device to replace human cornea by using heterogeneous forming materials for the implantation into the damaged eyes in order to obtain a certain vision. The main problems of artificial cornea are the biocompatibility and stability of the tissue particularly in penetrating keratoplasty. The current studies of tissue-engineered scaffold materials through comprising composites of natural and synthetic biopolymers together have developed a new way to artificial cornea. Although a wide agreement that the long-term stability of these devices would be greatly improved by the presence of cornea cells, modification of keratoprosthesis to support cornea cells remains elusive. Most of the studies on corneal substrate materials and surface modification of composites have tried to improve the growth and biocompatibility of cornea cells which can not only reduce the stimulus of heterogeneous materials, but also more importantly continuous and stable cornea cells can prevent the destruction of collagenase. The necrosis of stroma and spontaneous extrusion of the device, allow for maintenance of a precorneal tear layer, and play the role of ensuring a good optical surface and resisting bacterial infection. As a result, improvement in corneal cells has been the main aim of several recent investigations; some effort has focused on biomaterial for its well biological properties such as promoting the growth of cornea cells. The purpose of this review is to summary the growth status of the corneal cells after the implantation of several artificial corneas.     

Endothelial cell transplantation and growth behavior of the human corneal endothelium

Background: The human corneal endothelium has a limited proliferative capacity in vivo. Until now it has only been possible to replace damaged endothelium by transplantation of a donor cornea. After establishing methods for the isolation and in vitro cultivation of human corneal endothelial cells, transplantation of these cells my be an alternative therapeutic option. Materials and methods: In this review methods for the in vitro cultivation of human corneal endothelial cells and their transplantation on the Descemet membrane of donor corneas are described. Results: In vitro proliferation of human adult corneal endothelial cells was achieved by the development of defined cell culture conditions, including supplementation of culture medium with specified growth factors and substances. Dependent on the culture conditions, as well as independent of them, in vitro cultured endothelial cells showed phenotypic changes and different proliferative behavior. Thus, molecular biological examinations revealed a different expression pattern of growth factor receptors in fibroblast-like endothelial cells (dedifferentiated) compared to typical endothelial cells (differentiated). Moreover, the proliferative capacity of the cells differed, dependent on their corneal location. Cells isolated from the peripheral part of donor corneas have a higher proliferative capacity than cells obtained from the central part. The propagation of corneal endothelial cells in vitro offered the possibility of their transplantation on donor corneas in an in vitro model. After transplantation, these cells formed a monolayer whose morphology and cell density depended on the differentiation of the cells. DNA synthesis was predominantly detectable in cells of the corneal periphery. Conclusions: Our findings are the basis of the following hypothesis: the periphery of the cornea represents a regenerative zone of the corneal endothelium. The fact that early after transplantation corneal endothelial cells form a monolayer on the natural extracellular matrix (ECM), which shows contact inhibition, suggests that inhibitory factors are released by the Descemet membrane that influence the proliferation of the cells. Further studies on the regulation of the proliferation and differentiation of human corneal endothelial cells in vitro and after transplantation might offer the possibility to establish a selective procedure for the treatment of corneal endothelial cell loss in the near future.      

Die Gefrierkonservierung des Homologen Spendermaterials für Die Durchgreifende Keratoplastik

High cell metabolism above 0° C prevents prolonged storage of donor tissue for penetrating keratoplasty. Long-term preservation is only possible at low temperatures. In deep freeze storage, however, the addition of a protective agent such as dimethylsulphoxyd dmso or glycerol to the suspending medium is required so that cells are protected against damage during cooling to banking at and thawing from low temperatures.In experiments on rabbits intact donor eyes were frozen and the cut corneas gave good results after transplantation. The anterior chambers of the donor eyes had been injected with a solution containing dmso and the eyes had been suspended in a solution containing glycerol prior to cooling. The above deep freeze technique was also used for the preservation of human donor eyes, and 7 of the 9 fulll thickness corneal grafts which had been stored up to 29 days gave good results.The preservation of intact eyes was replaced by the preservation of the cornea with a scieral rim and since the rabbit cornea was found to be unsuitable for these experiments dog corneas were used. Human deep frozen corneas were flown to Ethiopia and transplanted, these transplants proved more successful than those for which fresh donor eyes had been used.In final experiments varying concentrations of the protective agent dmso were examined and the rate of thawing was investigated. Animal experiments confirmed the results of in-vitro tests and donor material which had been cooled slowly but thawed out rapidly gave best full thickness grafts. The investigations also showed that endothelial cells of fresh and of frozen corneal donor tissue only partially survived transplantation.Aus der Universitäts-Augenklinik Würzburg, Direktor: prof. Dr. W. Leydhecker.     

Stress-strain measurements of human and porcine corneas after riboflavin-ultraviolet-A-induced cross-linking

PURPOSE: To evaluate the biomechanical effect of combined riboflavin-ultraviolet A (UVA) treatment on porcine and human corneas. SETTING: Department of Ophthalmology, Technical University of Dresden, Dresden, Germany. METHODS: Corneal strips from 5 human enucleated eyes and 20 porcine cadaver corneas were treated with the photosensitizer riboflavin and irradiated with 2 double UVA diodes (370 nm, irradiance = 3 mW/cm2) for 30 minutes. After cross-linking, static stress-strain measurements of the treated and untreated corneas were performed using a microcomputer-controlled biomaterial tester with a prestress of 5 x 10(3) Pa. RESULTS: There was a significant increase in corneal rigidity after cross-linking, indicated by a rise in stress in treated porcine corneas (by 71.9%) and human corneas (by 328.9%) and in Young's modulus by the factor 1.8 in porcine corneas and 4.5 in human corneas. The mean central corneal thickness was 850 microm +/- 70 (SD) in porcine corneas and 550 +/- 40 microm in human corneas. CONCLUSIONS: Riboflavin-UVA-induced collagen cross-linking led to an increase in mechanical rigidity in porcine corneas and an even greater increase in human corneas. As collagen cross-linking is maximal in the anterior 300 microm of the cornea, the greater stiffening effect in human corneas can be explained by the relatively larger portion of the cornea being cross-linked in the overall thinner human cornea.     

In situ corneal excision – Experience of the Lions Cornea Bank of Nordrhein-Westfalen in 1995 and 1996

Summary Background: Donor corneas are normally obtained by whole globe enucleation – a procedure often refused by the bereaved. To increase the acceptance of cornea donation, we have exclusively obtained donor corneas by in situ excision since the end of 1994. There have been reports of increased endothelial damage and higher contamination rates. We report our experience in 1995 and 1996. Methods: The in situ excision was performed by staff trained in microsurgical techniques. Only donor corneas with negative end-storage cultures after at least 10 days and an endothelial cell count of more than 2500 cells/mm² were used for transplantation. Results: In all, 705 corneoscleral buttons were excised from 1/95 to 12/96. The bereaved consented in 34 % in 1996. A total of 30.5 % of the corneas were ineligible for transplantation which corresponds to the discard figures from all cornea banks with culture methods. We did not observe any primary transplant failure nor endophthalmitis after 444 perforating keratoplasties. Conclusion: In situ corneal excision is safe, and helps to reduce the shortage in donor corneas.      

Pars plana vitrectomy with corneal transplantation -- combined procedure

The aim of our study is to present own observations with Eckardt temporary keratoprosthesis, during combined pars plana vitrectomy and corneal transplantation. We operated on two aphakic patients with unclear corneas and retinal detachment--I case, phthisis bulbi after recurrent uveitis--II case. Eckardt temporary keratoprosthesis was sutured to the corneal bed with 4 or 6 Ethilon 10.0 bites, pars plana vitrectomy was performed followed by corneal transplantation and silicone oil tamponade. We obtained good transplant clarity only in the I case, in the II case because of hipotony and persistent contract of silicone oil with cornea, transplant was cloudy and collapsed with small exception in the central area. After 3-rd month we noticed local retinal detachment in the periphery, which was suppressed with laser photocoagulations. We think, that Eckardt temporary keratoprosthesis gives possibility to do vitrectomy in patients with undear cornea, which was in the past impossible. However, combined procedure requires surgical skills in both: anterior and posterior segments of the eye.     

Influence of donor age,post‐mortem time and cold storage on metabolic profile of human cornea

Purpose: Limited knowledge exists about the influence of donor age and death‐to‐preservation interval (DPI) on the metabolic properties of the cornea. The aim of this study is to investigate the relationship between both factors and metabolite content of the cornea. Methods: Corneas from 15 human donors (age: 41–78 years) were obtained within 16 hrs post‐mortem and kept in cold storage for 8 days. The metabolic profiles of the samples were investigated using high‐resolution, magic angle spinning ¹H nuclear magnetic resonance spectroscopy before and after 8 days of preservation. Results: Twenty‐two metabolites were detected and assigned in the corneal spectra. The significant metabolic differences before and after hypothermic storage were revealed between younger and older donors. DPI‐related significant differences revealed before preservation of the corneas were not displayed after 8 days of cold storage. Conclusions: Age of donor as well as post‐mortem time influences the biochemical properties of the cornea. Cold storage decreases the metabolite differences between the tissues collected at different post‐mortem time.     

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 cornea's 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.     

Implantation of a Keratoprosthesis of Novel Design in Rabbits

Purpose To evaluate a keratoprosthesis, implanted by penetrating keratoplasty, in rabbits.Methods We implanted our keratoprosthesis (optics and flange portions of polymethylmethacrylate and a polyurethane skirt with micropores) into 14 eyes of 14 rabbits. In four eyes, we evaluated histologically the junction between the keratoprosthesis and host cornea. Long-term keratoprosthesis survival was evaluated in ten eyes by slit-lamp biomicroscopy.Results The histological study showed good approximation of the keratoprosthesis to the host cornea in the junction area, with overlying superficial corneal stroma on the skirt, keratocyte and collagen fiber ingrowth into the micropores, and partial migration of epithelial cells onto the skirt. However, in the long-term survival study, eight out of ten eyes developed acute suture-related inflammation, considered to be from bacterial infection, requiring enucleation 30 ± 18 weeks after implantation. The remaining two eyes have survived for 70 and 76 weeks.Conclusions Our keratoprosthesis was well tolerated in the short term. However, further modifications are necessary to avoid corneal infection. Jpn J Ophthalmol 2004;48:448–453 © Japanese Ophthalmological Society 2004     

AlphaCor™ keratoprosthesis: postoperative development of six patients

Purpose  AlphaCor™ (Argus Biomedical Pty. Ltd., Perth, Australia) is an artificial, soft, one-piece keratoprosthesis (KPro) indicated for severe corneal conditions not treatable by a donor graft. To evaluate the efficacy and visual restoring of six patients with complete corneal opacification and deep neovascularizations treated with AlphaCor™. Methods  A retrospective review of six patients with a history of corneal opacification treated with keratoprosthesis surgery. AlphaCor™ synthetic cornea was implanted into the corneal stroma. During the follow-up time, slit-lamp and ultrasound examinations, best corrected visual acuity (BCVA), and intraocular pressure measurements were performed. Results  Six eyes with corneal scarring and vascularizations in three to four quadrants of six patients to years of age underwent a keratoprosthesis procedure. The follow-up time was 13–36 months; mean 23 months. The operation procedure was not limited by severe complications. The preoperative BCVA was hand movement to light perception. The postoperative BCVA ranged between 20/200 and 80/100. Intraocular pressure was controlled in all cases. Three patients developed a melting of the anterior corneal lamella. The keratoprosthesis had to be explanted (15–34 months after implantation) and was replaced by a donor cornea. Conclusions  Further evaluation is needed to evaluate the role of AlphaCor™ as a keratoprosthesis.     

不同保存方法对角膜形态结构及朗格罕斯细胞的影响

角膜的正常结构及内皮细胞功能的稳定对于角膜的透明极为重要，术前供体角膜材料的处理和保存方法直接影响穿透角膜移植术后的成功与失败。良好的中长期角膜保存为适时进行穿透角膜移植手术提供了便利的条件。综述了短期、中期和长期保存的供体角膜材料在保存过程中以及使用该材料行穿透角膜移植术后角膜的厚度、内皮细胞密度及角膜缘的HLA—DR阳性朗格罕斯细胞的变化情况。     

Corneal endothelial regeneration and tissue engineering

Human corneal endothelial cells (HCECs) have a limited proliferative capacity. Descemet stripping with automated endothelial keratoplasty (DSAEK) has become the preferred method for the treatment of corneal endothelial deficiency, but it requires a donor cornea. To overcome the shortage of donor corneas, transplantation of cultured HCEC sheets has been attempted in experimental studies. This review summarizes current knowledge about the mechanisms of corneal endothelial wound healing and about tissue engineering for the corneal endothelium. We also discuss recent work on tissue engineering for DSAEK grafts using cultured HCECs and HCEC precursor cell isolation method (the sphere-forming assay). DSAEK grafts (HCEC sheets) were constructed by seeding cultured HCECs on human amniotic membrane, thin human corneal stroma, and collagen sheets. The pump function of the HCEC sheets thus obtained was approximately 75%–95% of that for human donor corneas. HCEC sheets were transplanted onto rabbit corneas after DSAEK. While the untransplanted control group displayed severe stromal edema, the transplanted group had clear corneas throughout the observation period. The sphere-forming assay using donor human corneal endothelium or cultured HCECs can achieved mass production of human corneal endothelial precursors. These findings indicate that cultured HCECs transplanted after DSAEK can perform effective corneal dehydration in vivo and suggest the feasibility of employing the transplantation of cultured HCECs to treat endothelial dysfunction. Additionally, corneal endothelial precursors may be an effective strategy for corneal endothelial regeneration.     

改良钛支架人工角膜生物相容性的实验研究

目的 探讨改良钛支架人工角膜的生物相容性和角膜生物愈合的过程.方法 实验研究.设计与制备改良钛支架人工角膜,钛支架采用喷砂、羟基磷灰石涂层处理,镜柱用聚甲基丙烯酸甲酯为原料按设计好的图纸手工磨制.将18只正常新西兰白兔采用单纯随机抽样方法分为A、B、C组,每组6只.18只新西兰白兔角膜碱烧伤模型采用单纯随机抽样方法分为E、F、G组,每组6只.其中A、E组和B、F组兔右眼角膜基质层内分别植入改良支架和对照支架(无涂层),C、G组仅做角膜板层切口;另取4只(分别为2只正常动物模型、2只碱烧伤模型)即D、H组分别作为空白对照;术后1、3个月取材,取出支架做拉出实验和扫描电镜检查;角膜组织行苏木素-伊红(HE)染色、透射电镜检查.另取8只兔(其中2只碱烧伤模型)植入改良支架,3个月后植入镜柱.两组剪切力比较采用t检验.结果 改良后的人工角膜支架表面呈鳞片状微孔结构,植入兔角膜反应轻,术后1、3个月取材,组织学检查见A、E组支架与角膜界面处成纤维细胞数多于B、F组支架.3个月时拉出试验测定剪切力A与B组比较、E与F组比较差异均有统计学意义(t=3.297,P<0.05;t=4.237,P<0.05),改良支架植入角膜3个月后的剪切力较对照支架强.扫描电镜检查显示A、E组支架表面被细胞外基质包裹,而B、F组支架细胞附着量明显少.透射电镜检查显示支架周围的胶原纤维束紊乱,A、E组支架与角膜的结合方式垂直或成一定角度,结合更牢固.支架植入后碱烧伤模型组角膜基质较正常兔角膜组愈合延迟.结论 改良钛支架人工角膜促进了材料与组织界面愈合,无论正常还是碱烧伤动物体内,改良钛支架人工角膜提高了材料的生物相容性.     

A specular microscopic viewing system for donor corneas

A system for routine specular microscopic assessment of donor corneal endothelium including a chamber that allows both storage and viewing of the donor tissue was evaluated. A specular microscope modified to enable noncontact scanning of donor cornea from the endothelial side was used. Forty-five human corneas were examined upon arrival at the eyebank. Storage in the new chamber was compared with that in standard vials for 13 paired corneas. This study demonstrates the feasibility and simplicity of routine specular microscopy on donor corneal tissue, and discusses its advantages.     

AlphaCor人工角膜移植治疗兔角膜碱烧伤

目的评价AlphaCor人工角膜移植对兔角膜碱烧伤的治疗效果。方法用1mol/LNaOH烧伤兔左眼建立角膜碱烧伤模型,选择角膜白斑、角膜缘新生血管〉2个象限的5只兔眼行人工角膜移植术。AlphaCor人工角膜移植手术分2期进行：Ⅰ期手术,将AlphaCor人工角膜植入角膜板层间;Ⅱ期手术,暴露人工角膜光学区。术后观察人工角膜、受体角膜及眼前节情况。结果碱烧伤后2～6周形成带新生血管的角膜白斑。Ⅰ期术后5例人工角膜均在位,2例人工角膜出现白色沉积物,其中1例并发角膜浅层基质溶解,另3例无并发症发生。Ⅱ期手术中麻醉死亡1例,其余术后人工角膜均在位,1例术后6周发生角膜感染、穿孔,另3例术后4～6周发生人工角膜前膜增生,未见其他并发症发生。结论 AlphaCor人工角膜移植治疗兔角膜碱烧伤具有良好的生物相容性,效果较好。     

Graft failure in human donor corneas due to transmission of herpes simplex virus

AIM: To report the clinical consequences of contamination of human donor corneas by herpes simplex virus (HSV) in organ culture. METHODS: Two patients without previous history of ocular HSV infection underwent penetrating keratoplasty (PK), one for keratoconus and the other for Fuchs' endothelial dystrophy. One patient suffered primary graft failure while the other developed a persistent epithelial defect, ultimately resulting in graft failure. Viral culture of swabs taken from both corneas during the early postoperative period was undertaken. The failed donor corneas were examined histopathologically by immunohistochemistry (IHC) for HSV-1 antigens, transmission electron microscopy (TEM), and by polymerase chain reaction (PCR) for HSV DNA. Both failed corneas were replaced within 6 weeks of the initial surgery. The records of the fellow donor corneas were also examined for evidence of infection. RESULTS: HSV was cultured from both corneas during the early postoperative period. Histology of both donor corneas demonstrated a thickened corneal stroma with widespread necrosis of keratocytes and loss of endothelial cells. IHC showed keratocytes positive with antibodies to HSV-1 antigens. TEM demonstrated HSV-like viral particles within degenerating keratocytes. PCR performed on the failed corneal grafts was positive for HSV-1 DNA, whereas PCR performed on the excised host corneal buttons was negative in both patients. Records of the fellow donor corneas showed that one cornea was successfully transplanted into another recipient after 18 days in organ culture, whilst the other was discarded because of extensive endothelial cell necrosis noted after 15 days in organ culture. CONCLUSION: HSV within a donor cornea may cause endothelial destruction in organ culture and both primary graft failure and ulcerative keratitis after transplantation. Endothelial necrosis of a donor cornea in culture also raises the possibility of HSV infection within the fellow cornea.