ArticleLiterature Review

Regenerative approaches for the cornea

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Abstract

The cornea is the transparent front part of the eye that transmits light to the back of the eye to generate vision. Loss of corneal transparency, if irreversible, leads to severe vision loss or blindness. For decades, corneal transplantation using human donor corneas has been the only option for treating corneal blindness. Despite recent improvement in surgical techniques, donor cornea transplantation remains plagued by risks of suboptimal optical results and visual acuity, immune rejection and eventually graft failure. Furthermore, the demand for suitable donor corneas is increasing faster than the number of donors, leaving thousands of curable patients untreated worldwide. Here, we critically review the state of the art of biomaterials for corneal regeneration. However, the lessons learned from the use of the cornea as a disease model will allow for extension of the biomaterials and techniques for regeneration of more complex organs such as the heart.

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... Millions of people worldwide suffer from corneal blindness [1,2]. Severe corneal blindness resulting from thermal or chemical burns, is characterized by the loss of limbal epithelial stem cells (LESCs), the tissue-resident stem cells of the cornea, which leads to chronic inflammation, overgrowth of blood vessels and conjunctival cells, as well as stromal scarring [3,4]. ...
... However, delivery of two therapeutic stem cell populations compartmentalized in a tissue-specific arrangement is a daunting task, which we have previously sought to solve by using laser-assisted bioprinting [16]. So far, only few applications have emerged combining different cells to regenerate both the scarred stromal tissue as well as the surface epithelium [17,18], but their implantation requires suturing, which can increase inflammation and neovascularization of the cornea [2]. ...
... J K −1 mol −1 ) and T is the temperature (298 K). Average molecular weight between crosslinks, M c , was calculated using Equation (2) (2) ...
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Regeneration of a severely damaged cornea necessitates the delivery of both epithelium-renewing limbal epithelial stem cells (LESCs) and stroma-repairing cells, such as human adipose-derived stem cells (hASCs). Currently, limited strategies exist for the delivery of these therapeutic cells with tissue-like cellular organization. With the added risks related to suturing of corneal implants, there is a pressing need to develop new tissue adhesive biomaterials for corneal regeneration. To address these issues, we grafted dopamine moieties into hydrazone- crosslinked hyaluronic acid (HA-DOPA) hydrogels to impart tissue adhesive properties and facilitate covalent surface modification of the gels with basement membrane proteins or peptides. We achieved tissue-like cellular compartmentalization in the implants by encapsulating hASCs inside the hydrogels, with subsequent conjugation of thiolated collagen IV or laminin peptides and LESC seeding on the hydrogel surface. The encapsulated hASCs in HA-DOPA gels exhibited good proliferation and cell elongation, while the LESCs expressed typical limbal epithelial progenitor markers. Importantly, the compartmentalized HA-DOPA implants displayed excellent tissue adhesion upon implantation in a porcine corneal organ culture model. These results encourage sutureless implantation of functional stem cells as the next generation of corneal regeneration.
... How novel technologies impact on stem cell research and regenerative medicine was the topic of a recent Journal of Internal Medicine (JIM) symposium entitled 'Human Models and Technology for Regenerative Medicine'. Five important and rapidly emerging areas of stem cell-technology interface that were discussed at the symposium are considered in more detail in review articles in the current issue of JIM [1][2][3][4][5]. ...
... Furthermore, although transplantation of donated human corneas generally works well, a perfect match is rarely provided, and there is thus considerable interest in exploring biomaterial-based corneas for transplantation. This topic is addressed by May Griffith and colleagues from Link€ oping University, who provide an update in the current issue of JIM on corneal transplantation and consider the prospect of using biosynthetic corneas [5]. ...
Article
Content List – Read more articles from the symposium: Human models and technology for regenerative medicine.
... In order to address the global shortage of transplantable donor corneas, recent efforts have been expended towards the development of protocols for the expansion of human corneal endothelial cells for injection into the anterior cornea [35] or for seeding onto natural or synthetic scaffolds for transplantation [20,23,32,56]. These approaches require a readily accessible source of a sufficient number of viable and functional corneal endothelial cells. ...
... The % membrane integrities (MI) shown are of fresh cells and of cells that were slowly cooled to various sub-zero temperatures in the presence of 5% dimethyl sulfoxide (Me 2 SO) and 6% hydroxyethyl starch (HES), plunged and stored in liquid nitrogen and then rapidly thawed. development of tissue-engineered endothelial grafts [20,23,32,45,56,62] or cell-based injection therapies [22,35] as alternatives to full donor corneal transplantation for the treatment of corneal endothelial dysfunction. ...
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Impairment of the corneal endothelium causes blindness that afflicts millions worldwide and constitutes the most often cited indication for corneal transplants. The scarcity of donor corneas has prompted the alternative use of tissue-engineered grafts which requires the ex vivo expansion and cryopreservation of corneal endothelial cells. The aims of this study are to culture and identify the conditions that will yield viable and functional corneal endothelial cells after cryopreservation. Previously, using human umbilical vein endothelial cells (HUVECs), we employed a systematic approach to optimize the post-thaw recovery of cells with high membrane integrity and functionality. Here, we investigated whether improved protocols for HUVECs translate to the cryopreservation of corneal endothelial cells, despite the differences in function and embryonic origin of these cell types. First, we isolated endothelial cells from pig corneas and then applied an interrupted slow cooling protocol in the presence of dimethyl sulfoxide (Me2SO), with or without hydroxyethyl starch (HES). Next, we isolated and expanded endothelial cells from human corneas and applied the best protocol verified using porcine cells. We found that slow cooling at 1 °C/min in the presence of 5% Me2SO and 6% HES, followed by rapid thawing after liquid nitrogen storage, yields membrane-intact cells that could form monolayers expressing the tight junction marker ZO-1 and cytoskeleton F-actin, and could form tubes in reconstituted basement membrane matrix. Thus, we show that a cryopreservation protocol optimized for HUVECs can be applied successfully to corneal endothelial cells, and this could provide a means to address the need for off-the-shelf cryopreserved cells for corneal tissue engineering and regenerative medicine.
... Recent advances allow for the selective replacement of affected defective or opacified corneal layers without the removal of unaffected layers. Due to the worldwide limited availability of suitable donor tissues and the high costs involved in donor transplantation there is a high demand for alternative treatment strategies that reduce the demand for donor cornea by supplying ready-to-use materials [4,5]. Despite efforts with a range of artificial and polymer materials, there is no corneal equivalent that effectively hits the 'Goldilocks zone' of combined biological and biomechanical properties of the cornea. ...
... Challenges associated with their use include poor integration into the host cornea, acute immune responses, lack of epithelialization, mechanical property mismatch, device extrusion, infection and pressure build up behind the https://doi.org/10.1016/j.biomaterials.2018. 10 [4,5,16]. ...
Article
Damaged corneas can lead to blindness. Due to the worldwide shortage of donor corneas there is a tremendous unmet demand for a robust corneal replacement that supports growth of the major corneal cell types. Commercial artificial corneas comprise plastic polymers that do not adequately support diverse cell growth. We present a new class of protein elastomer-dominated synthetic corneas with attractive performance that intimately couple biologically active tropoelastin to mechanically robust and durable protein silk. Fabricated films substantially replicate the natural cornea physically and by interacting with both key cells types used in cornea repair. Performance encompasses optical clarity at high transmittance, compatible refractive index, substantial glucose permeability, compliant mechanical properties, and support of both growth and function of corneal epithelial and endothelial cells.
... The increasing importance of monolayers in tissue-engineered constructs and in transplantation has led to a need to develop protocols for successful cryopreservation of cells in monolayers. For example, because of the global shortage of donor corneas, recent approaches involve the use of tissue-engineered corneal implants that employ endothelial monolayers [18,28]. The complexity of monolayer cryopreservation arises from the cell configuration [2,7]. ...
... In the short-term, cryopreserved vascular and corneal endothelial monolayers may find application in drug testing and disease modeling. In the future, corneal endothelial monolayers may be used in tissue-engineered constructs for transplantation [18,28]. ...
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Cryopreservation of endothelium is one of the major challenges in the cryopreservation of complex tissues. Human umbilical vein endothelial cells (HUVECs) in suspension are available commercially and recently their post-thaw cell membrane integrity was significantly improved by cryopreservation in 5% dimethyl sulfoxide (Me2SO) and 6% hydroxyethyl starch (HES). However, cryopreservation of cells in monolayers has been elusive. The exact mechanisms of damage during cell monolayer cryopreservation are still under investigation. Here, we show that a combination of different factors contribute to significant progress in cryopreservation of endothelial monolayers. The addition of 2% chondroitin sulfate to 5% Me2SO and 6% HES and cooling at 0.2 or 1 °C/min led to high membrane integrity (97.3 ± 3.2%) immediately after thaw when HUVECs were cultured on a substrate with a coefficient of thermal expansion similar to that of ice. The optimized cryopreservation protocol was applied to monolayers of primary porcine corneal endothelial cells, and resulted in high post-thaw viability (95.9 ± 3.7% membrane integrity) with metabolic activity 12 h post-thaw comparable to unfrozen control.
... A variety of bioengineered implants designed to promote corneal regeneration have been tested in preclinical and clinical studies (Griffith et al., 2016). These include limbal epithelial cells on amniotic membranes (Sangwan et al., 2011), corneal stromal cells on polyglycolic acid (PGA) fibrous scaffolds (Hu et al., 2006) or silk substrates (Ghezzi et al., 2017), and carrier-free corneal epithelial and endothelial cell sheets generated on a thermoresponsive polymer (Umemoto et al., 2013). ...
Article
Ocular regenerative therapies are on track to revolutionize treatment of numerous blinding disorders, including corneal disease, cataract, glaucoma, retinitis pigmentosa, and age-related macular degeneration. A variety of transplantable products, delivered as cell suspensions or as preformed 3D structures combining cells and natural or artificial substrates, are in the pipeline. Here we review the status of clinical and preclinical studies for stem cell-based repair, covering key eye tissues from front to back, from cornea to retina, and including bioengineering approaches that advance cell product manufacturing. While recognizing the challenges, we look forward to a deep portfolio of sight-restoring, stem cell-based medicine. VIDEO ABSTRACT.
... All three prostheses are associated with potentially serious side effects and are regarded as a last resort treatment for patients with severe pathology or previous conventional graft failures [5]. Given the shortcomings of conventional corneal transplantation and insufficient performance of prostheses, various research groups have focused on the development of a range of corneal replacements [6,7]. ...
Article
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Purpose To determine feasibility of plant-derived recombinant human collagen type I (RHCI) for use in corneal regenerative implants Methods RHCI was crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to form hydrogels. Application of shear force to liquid crystalline RHCI aligned the collagen fibrils. Both aligned and random hydrogels were evaluated for mechanical and optical properties, as well as in vitro biocompatibility. Further evaluation was performed in vivo by subcutaneous implantation in rats and corneal implantation in Göttingen minipigs. Results Spontaneous crosslinking of randomly aligned RHCI (rRHCI) formed robust, transparent hydrogels that were sufficient for implantation. Aligning the RHCI (aRHCI) resulted in thicker collagen fibrils forming an opaque hydrogel with insufficient transverse mechanical strength for surgical manipulation. rRHCI showed minimal inflammation when implanted subcutaneously in rats. The corneal implants in minipigs showed that rRHCI hydrogels promoted regeneration of corneal epithelium, stroma, and nerves; some myofibroblasts were seen in the regenerated neo-corneas. Conclusion Plant-derived RHCI was used to fabricate a hydrogel that is transparent, mechanically stable, and biocompatible when grafted as corneal implants in minipigs. Plant-derived collagen is determined to be a safe alternative to allografts, animal collagens, or yeast-derived recombinant human collagen for tissue engineering applications. The main advantage is that unlike donor corneas or yeast-produced collagen, the RHCI supply is potentially unlimited due to the high yields of this production method. Lay Summary A severe shortage of human-donor corneas for transplantation has led scientists to develop synthetic alternatives. Here, recombinant human collagen type I made of tobacco plants through genetic engineering was tested for use in making corneal implants. We made strong, transparent hydrogels that were tested by implanting subcutaneously in rats and in the corneas of minipigs. We showed that the plant collagen was biocompatible and was able to stably regenerate the corneas of minipigs comparable to yeast-produced recombinant collagen that we previously tested in clinical trials. The advantage of the plant collagen is that the supply is potentially limitless.
... One approach to overcome the shortage of donor corneas is to develop biomaterial scaffolds that mimic the cornea. Scaffolds manufactured from collagen type 1, the most abundant protein in the cornea, have been extensively investigated for replacing and regenerating the corneal stroma [9][10][11]. While these scaffolds have good transparency and can support the growth of keratocytes and epithelial cells, they often exhibit inferior mechanical strength and stiffness compared to the native tissue [12,13]. ...
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Alternatives to donor cornea transplantation based on tissue engineering are desirable to overcome the current severe donor tissue shortage. Many natural polymers have good biological properties but poor mechanical properties and degradation resistance; while synthetic polymers have good mechanical properties but do not contain biochemical molecules normally found in the real tissue. In addition, both fiber orientation and composition play a key role in dictating cell behavior within a scaffold. In this study, the effect on corneal stromal cells of adding decellularized corneal extracellular matrix (ECM) to an electrospun polymer with differing fiber organizations was explored. Electrospun matrices were generated using polycaprolactone (PCL) and PCL combined with ECM and electrospun into random, radial and perpendicularly aligned fiber scaffolds. Human corneal stromal cells were seeded onto these scaffolds and the effect of composition and orientation on the cells phenotype was assessed. Incorporation of ECM into PCL increased hydrophilicity of scaffolds without an adverse effect on Young's modulus. Cells seeded on these matrices adopted different morphologies that followed the orientation of the fibers. Keratocyte markers were increased in all types of scaffolds compared to tissue culture plastic. Scaffolds with radial and perpendicularly aligned fibers promoted enhanced cell migration. Aligned scaffolds with incorporated ECM show promise for their use as cell-free implants that promote endogenous repopulation by neighboring cells.
... Due to the structural similarities of extracellular matrix and the three-dimensional framework supporting cell proliferation and survival, hydrogels have unique desirable properties for cornea regeneration [14]. Cornea regeneration biomaterials, including recombinant human collagen-based, fibrin-based, silk-based, and self-assembled corneal implants, have been previously described in the literature [15]. ...
Article
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Transparent composite hydrogel in the form of a contact lens made from poly(vinyl alcohol) (PVA) and cellulose nanocrystals (CNCs) was subjected to in vitro biocompatibility evaluation with human corneal epithelial cells (HCE-2 cells). The cell response to direct contact with the hydrogels was investigated by placing the samples on top of confluent cell layers and evaluating cell viability, morphology, and cell layer integrity subsequent to 24 h culture and removal of the hydrogels. To further characterize the lens–cell interactions, HCE-2 cells were seeded on the hydrogels, with and without simulated tear fluid (STF) pre-conditioning, and cell viability and morphology were evaluated. Furthermore, protein adsorption on the hydrogel surface was investigated by incubating the materials with STF, followed by protein elution and quantification. The hydrogel material was found to have affinity towards protein adsorption, most probably due to the interactions between the positively charged lysozyme and the negatively charged CNCs embedded in the PVA matrix. The direct contact experiment demonstrated that the physical presence of the lenses did not affect corneal epithelial cell monolayers in terms of integrity nor cell metabolic activity. Moreover, it was found that viable corneal cells adhered to the hydrogel, showing the typical morphology of epithelial cells and that such response was not influenced by the STF pre-conditioning of the hydrogel surface. The results of the study confirm that PVA-CNC hydrogel is a promising ophthalmic biomaterial, motivating future in vitro and in vivo biocompatibility studies.
... Medical grade polymethyl methacrylate (PMMA) with the ability to block UVA/UVB is used for the front and back plates. The idea of using PMMA was conceived due to its ability to induce only minimal inflammatory responses in the eye (Griffith et al., 2016). PMMA is a transparent thermoplastic polymer, also known as acrylic glass, which can be modified to achieve desired mechanical properties, like toughness and stiffness. ...
Article
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The cornea is a unique tissue and the most powerful focusing element of the eye, known as a window to the eye. Infectious or non-infectious diseases might cause severe visual impairments that need medical intervention to restore patients' vision. The most prominent characteristics of the cornea are its mechanical strength and transparency, which are indeed the most important criteria considerations when reconstructing the injured cornea. Corneal strength comes from about 200 collagen lamellae which criss-cross the cornea in different directions and comprise nearly 90% of the thickness of the cornea. Regarding corneal transparency, the specific characteristics of the cornea include its immune and angiogenic privilege besides its limbus zone. On the other hand, angiogenic privilege involves several active cascades in which anti-angiogenic factors are produced to compensate for the enhanced production of proangiogenic factors after wound healing. Limbus of the cornea forms a border between the corneal and conjunctival epithelium, and its limbal stem cells (LSCs) are essential in maintenance and repair of the adult cornea through its support of corneal epithelial tissue repair and regeneration. As a result, the main factors which threaten the corneal clarity are inflammatory reactions, neovascularization, and limbal deficiency. In fact, the influx of inflammatory cells causes scar formation and destruction of the limbus zone. Current studies about wound healing treatment focus on corneal characteristics such as the immune response, angiogenesis, and cell signaling. In this review, studied topics related to wound healing and new approaches in cornea regeneration, which are mostly related to the criteria mentioned above, will be discussed.
... Other than the approach to utilize the artificial polymers, natural polymers, implants and bio-engineered cornea can also be used in a regenerative manner to replace the damaged cornea. Some examples are fibrinbased corneal implants, recombinant human collagen corneal implants, silk-based corneal implants, self-assembled corneal implants, grafting of corneal limbus pieces, 2methacryloxyethyl phosphorylcholine [60,61]. With the advent of new technology, new alternatives to artificial polymers keep on arising. ...
Chapter
Ophthalmology focuses on health issues related to eye, vision, anatomy and concerned ailments. Hitherto, a variety of materials such as ceramic, metal, glass and polymers have been extensively explored that modify optic adherence, residing time, targeted release with minimum downsides. Modern ophthalmic implants including contact lens, ocular endotamponades, viscoelastic replacements etc. are developed by polymers owing to their inherent virtues such as biocompatible with eye tissues, ease of manufacturing, flexible in operation, cost effective etc. The chapter emphasizes on biodegradable (chitosan, poly-lactic acid) and non- biodegradable (PVA, silicones) polymeric ocular preparations such as artificial cornea, orbital ball, glaucoma drainage device etc.
... B, In vivo corneal injury models used to study cornea fibrosis have shown that when mesenchymal stem cells (MSCs) are injected into the stroma, corneal scarring is reduced significantly after injures that typically cause fibrosis (Modified and Reproduced with permission from Mittal et al., Stem Cell Rep, 2016, 7, 583-590). also being investigated ( Griffith et al., 2016;Ong et al., 2016;Ghezzi et al., 2017;Stern et al., 2018); fibrotic tissue would be removed and replaced with scaffolds to maintain the structure of the cornea and promote regeneration of corneal transparency via normal healing mechanisms. ...
Article
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Regenerative repair in response to wounding involves cell proliferation and migration. This is followed by the reestablishment of cell structure and organization and a dynamic process of remodeling and restoration of the injured cells' extracellular matrix microenvironment and the integration of the newly synthesized matrix into the surrounding tissue. Fibrosis in the lungs, liver, and heart can lead to loss of life and in the eye to loss of vision. Learning to control fibrosis and restore normal tissue function after injury repair remains a goal of research in this area. Here we use knowledge gained using the lens and the cornea to provide insight into how fibrosis develops and clues to how it can be controlled. The lens and cornea are less complex than other tissues that develop life‐threatening fibrosis, but they are well characterized and research using them as model systems to study fibrosis is leading towards an improved understanding of fibrosis. Here we summarize the current state of the literature and how it is leading to promising new treatments. This article is protected by copyright. All rights reserved.
... [3][4][5] For this reason alternatives to donor allografts are being developed such as cellbased therapies and tissue engineering strategies. 6,7 To successfully develop new cell-based therapies, a suitable cell source is required. For injuries or diseases affecting the corneal stroma, keratocytes, the main cell type in the stroma that has characteristics associated with mesenchymal stromal cells, are the logical choice of cells for corneal regeneration. ...
Article
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Purpose: To identify biochemical cues that could promote a keratocyte-like phenotype in human corneal stromal cells that had become fibroblastic when expanded in serum-supplemented media while also examining the effect on cell proliferation and migration. Methods: Proliferation was assessed by PrestoBlueTM, morphology was monitored by phase contrast microscopy, phenotype was analysed by real-time polymerase chain reaction (qPCR), immunochemistry and flow cytometry, and migration was studied with a scratch assay. Results: Ascorbic Acid (AA), Retinoic Acid (RA), Insulin-Transferrin-Selenium (ITS), Insulin-like Growth Factor 1 (IGF-1) and 3-isobutyl-1-methylxanthine (IBMX) promoted a dendritic morphology, increased the expression of keratocyte markers, such as keratocan, aldehyde dehydrogenase 3 family member A1 (ALDH3A1) and CD34, and prevented myofibroblast differentiation, while in some cases increasing proliferation. Transforming Growth Factor beta 1 (TGF-β1) and 3 (TGF-β3) promoted the differentiation towards myofibroblasts, with increased expression of α-SMA. Fibroblast Growth Factor 2 (FGF-2) supported a fibroblastic phenotype while Platelet-Derived Growth Factor Homodimer B (PDGF-BB) induced a pro-migratory fibroblastic phenotype. A combination of all the pro-keratocyte factors was also compared to the serum-free only, which significantly increased CD34 and keratocan expression. Conclusions: Partially recovery towards a quiescent keratocyte-like phenotype was achieved by the removal of serum and the addition of AA, IGF-1, RA, ITS and IBMX to a basal medium. These findings can be used to develop cell-based corneal therapies and to study corneal diseases in vitro.
Chapter
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The human cornea is an avascular and transparent tissue which is responsible for the three-fourths of the total refractive power of the eye. It undergoes continuous stress due to the dust, pollution, infection and other environmental insults which may lead to dryness and abrasion injuries. Diseases of the cornea show wide spectrum of manifestations including corneal opacity, conjunctivalization, scarring, limbal stem cell deficiency and immune disorders and may result in blindness. The field of regenerative medicine has shown a great promise in the last two decades. Almost 30 years have been passed since the corneal epithelial stem cells were first reported to be localized in the limbus, a transition zone between the transparent cornea and opaque sclera. During these years, various efforts have been made for the corneal regeneration, including the cell and tissue engineering-based approaches and development of surgical modalities. However, a successful therapy for bilateral corneal diseases remains elusive. We put in here our perspective about the past, the present and the foreseeable future of regeneration and reconstruction of the human cornea.
Article
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For many years, corneal transplantation has been the first-choice treatment for irreversible damage affecting the anterior part of the eye. However, the low number of cornea donors and cases of graft rejection highlighted the need to replace donor corneas with new biomaterials. Tissue engineering plays a fundamental role in achieving this goal through challenging research into a construct that must reflect all the properties of the cornea that are essential to ensure correct vision. In this review, the anatomy and physiology of the cornea are described to point out the main roles of the corneal layers to be compensated and all the requirements expected from the material to be manufactured. Then, a deep investigation of alginate as a suitable alternative to donor tissue was conducted. Thanks to its adaptability, transparency and low immunogenicity, alginate has emerged as a promising candidate for the realization of bioengineered materials for corneal regeneration. Chemical modifications and the blending of alginate with other functional compounds allow the control of its mechanical, degradation and cell-proliferation features, enabling it to go beyond its limits, improving its functionality in the field of corneal tissue engineering and regenerative medicine.
Article
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The role of biomaterials in tissue engineering and regenerative medicine strategies to treat vision loss associated with damage to tissues in the anterior segment of the eye has been studied for several years. This has mostly involved replacement and support for the cornea and conjunctiva. These are complex tissues with specific functional requirements for different parts of the tissue. Amniotic membrane (AM) is used in clinical practice to transplant autologous or allogenic cells to the corneal surface. Fibrin gels have also progressed to clinical use under specific conditions. Alternatives to AM such as collagen gels, other natural materials, for example keratin and silks, and synthetic polymers have received considerable attention in laboratory and animal studies. This experience is building a body of evidence to demonstrate the potential of tissue engineering and regenerative medicine in corneal and conjunctival reconstruction and can also lead to other applications in the anterior segment of the eye, for example, the trabecular meshwork. There is a real clinical need for new procedures to overcome vision loss but there are also opportunities for developments in ocular applications to lead to biomaterials innovations for use in other clinical areas.
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Die Risikobewertung von Chemikalien ist für die öffentliche Gesundheit von entschei-dender Bedeutung, weshalb strenge Testverfahren zu deren toxikologischer Begutach-tung angewandt werden. Die ursprünglich tierbasierten Testverfahren werden aufgrund von neuen wissenschaftlichen Erkenntnissen und wegen ökonomischer Ineffizienz sowie ethischer Fragwürdigkeit immer mehr durch alternative Methoden ohne Tiermodelle ersetzt. Für den toxikologischen Endpunkt der Augenreizung wurden bereits die ersten alternativen Testsysteme auf der Basis von ex vivo- oder in vitro-Modellen entwickelt. Jedoch ist bis dato kein alternatives Testsystem in der Lage, das gesamte Spektrum der verschiedenen Kategorien der Augenreizungen nach dem global harmonisierten System zur Einstufung und Kennzeichnung von Chemikalien (GHS) vorherzusagen und damit den tierbasierten Draize-Augenreizungstest vollends zu ersetzen. Gründe hierfür sind fehlende physiologische Merkmale im Modell sowie eine destruktive Analysemethode. Aufgrund dessen wurden in dieser Studie die Hypothesen getestet, ob ein verbessertes In-vitro-Modell oder eine zerstörungsfreie, hochsensitive Analysemethode die Vorher-sagekraft des Augenreizungstests verbessern können. Dafür wurden zunächst neue Mo-delle aus humanen Hornhaut- und Hautepithelzellen entwickelt. Die Modelle aus pri-mären cornealen Zellen zeigten eine gewebespezifische Expression der Marker Zytokera-tin 3 und 12 sowie Loricrin. In beiden Modellen konnte durch die Verkürzung der Kul-turdauer die Ausbildung einer Hornschicht verhindert werden. Die Modelle wiesen dadurch eine sensiblere Barriere vergleichbar der nativen Cornea auf. Darüber hinaus konnte durch die chemische Quervernetzung mit Polyethylenglykolsuccinimidylglutara-tester ein transparentes, nicht kontrahierendes Stroma-Äquivalent etabliert werden. Der Stroma-Ersatz konnte zur Generierung von Hemi- und Voll-Cornea-Äquivalenten einge-setzt werden und lieferte somit erste Ansatzpunkte für die Rekonstruktion der nativen Hornhaut. Parallel dazu konnte ein zerstörungsfreies Analyseverfahren basierend auf der Impe-danzspektroskopie entwickelt werden, das wiederholte Messungen der Gewebeintegri-tät zulässt. Zur verbesserten Messung der Barriere in dreidimensionalen Modelle wurde hierfür ein neuer Parameter, der transepitheliale elektrische Widerstand (TEER) bei der Frequenz von 1000 Hz, der TEER1000 Hz definiert, der eine genauere Aussage über die Integrität der Modelle zulässt. Durch die Kombination der entwickelten cornealen Epithelzellmodelle mit der TEER1000 Hz-Messung konnte die Prädikitivität des Augenrei-zungstests auf 78 - 100 % erhöht werden. Von besonderer Bedeutung ist dabei, dass die nicht destruktive Messung des TEER1000 Hz zum ersten Mal erlaubte, die Persistenz von Irritationen durch wiederholte Messungen in einem in vitro-Modell zu erkennen und somit die GHS-Kategorie 1 von GHS-Kategorie 2 zu unterscheiden. Der wissenschaftli-che Gewinn dieser Forschungsarbeit ist ein neues Testverfahren, das alle GHS-Kategorien in einem einzigen in vitro-Test nachweisen und den Draize-Augenreizungstest gänzlich ersetzen kann.
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The search for alternatives to allotransplants is driven by the shortage of corneal donors and is demanding because of the limitations of the alternatives. Indeed, current progress in genetically engineered (GE) pigs, the introduction of gene-editing technology by clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9, and advanced immunosuppressants have made xenotransplantation a possible option for a human trial. Porcine corneal xenotransplantation is considered applicable because the eye is regarded as an immune-privileged site. Furthermore, recent non-human primate studies have shown long-term survival of porcine xenotransplants in keratoplasty. Herein, corneal immune privilege is briefly introduced, and xenogeneic reactions are compared with allogeneic reactions in corneal transplantation. This review describes the current knowledge on special issues of xenotransplantation, xenogeneic rejection mechanisms, current immunosuppressive regimens of corneal xenotransplantation, preclinical efficacy and safety data of corneal xenotransplantation, and updates of the regulatory framework to conduct a clinical trial on corneal xenotransplantation. We also discuss barriers that might prevent xenotransplantation from becoming common practice, such as ethical dilemmas, public concerns on xenotransplantation, and the possible risk of xenozoonosis. Given that the legal definition of decellularized porcine cornea (DPC) lies somewhere between a medical device and a xenotransplant, the preclinical efficacy and clinical trial data using DPC are included. The review finally provides perspectives on the current standpoint of corneal xenotransplantation in the fields of regenerative medicine.
Article
Corneal grafting is one of the most common forms of human tissue transplantation. The corneal stroma is responsible for many characteristics of the cornea. For these reasons, an important volume of research has been made to replicate the corneal stroma in the laboratory to find an alternative to classical corneal transplantation techniques.There is an increasing interest today in cell therapy of the corneal stroma using induced pluripotent stem cells or mesenchymal stem cells since these cells have shown to be capable of producing new collagen within the host stroma and even to improve its transparency.The first clinical experiment on corneal stroma regeneration in advanced keratoconus cases has been reported and included. Fourteen patients were randomized and enrolled into 3 experimental groups: (1) patients underwent implantation of autologous adipose-derived adult stem cells alone, (2) patients received decellularized donor corneal stroma laminas, and (3) patients received implantation of recellularized donor laminas with adipose-derived adult stem cells. Clinical improvement was detected with all cases in their visual, pachymetric, and topographic parameters of the operated corneas.Other recent studies have used allogenic SMILE implantation lenticule corneal inlays, showing also an improvement in different visual, topographic, and keratometric parameters.In the present report, we try to summarize the available preclinical and clinical evidence about the emerging topic of corneal stroma regeneration.
Article
Background: To investigate the transparency, biocompatibility, and safety of human-derived acellular dermal matrix for application in corneal stromal transplantation. Methods: Twenty-four patients (24 eyes) with pellucid marginal corneal degeneration were enrolled, and intrastromal keratoplasty was performed with human-derived acellular dermal matrix. The ocular symptoms and signs as well as graft characteristics were evaluated at baseline and at 1 day, 1 week, 1 month, 3 months, and 6 months postoperatively. Photography by a slit lamp, topography by Pentacam, anterior segment-optical coherence tomography (AS-OCT), and corneal confocal microscopy were conducted at baseline and during the follow-up period. Results: Postoperative discomfort was relieved during the follow-up period. No abnormal ocular signs were observed at 6 months, indicating the safety of the procedure. Desirable and improved transparency of the grafts was demonstrated, and all the grafts healed without dissolution or fall at 6 months postoperatively. Reepithelization was completed, and confocal microscopy revealed that keratocytes and nerve repopulated in all the grafts at 6 months postoperatively. The thinning of the marginal corneal stroma was eliminated following the transplantation, and the curvature and corneal regularity remained stable at 6 months compared with baseline. Conclusions: The present study demonstrated the transparency, biocompatibility, and safety of human-derived acellular dermis matrix in intrastromal keratoplasty. With further improvements, human-derived acellular dermis matrix could be applied in central lamellar keratoplasty and ultimately solve the shortage of donor grafts.
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Mechanically challenged tissue-engineered organs, such as blood vessels, traditionally relied on synthetic or modified biological materials for structural support. In this report, we present a novel approach to tissue-engineered blood vessel (TEBV) production that is based exclusively on the use of cultured human cells, i.e., without any synthetic or exogenous biomaterials. Human vascular smooth muscle cells (SMC) cultured with ascorbic acid produced a cohesive cellular sheet. This sheet was placed around a tubular support to produce the media of the vessel. A similar sheet of human fibroblasts was wrapped around the media to provide the adventitia. After maturation, the tubular support was removed and endothelial cells were seeded in the lumen. This TEBV featured a well-defined, three-layered organization and numerous extracellular matrix proteins, including elastin. In this environment, SMC reexpressed desmin, a differentiation marker known to be lost under standard culture conditions. The endothelium expressed von Willebrand factor, incorporated acetylated LDL, produced PGI(2), and strongly inhibited platelet adhesion in vitro. The complete vessel had a burst strength over 2000 mmHg. This is the first completely biological TEBV to display a burst strength comparable to that of human vessels. Short-term grafting experiment in a canine model demonstrated good handling and suturability characteristics. Taken together, these results suggest that this novel technique can produce completely biological vessels fulfilling the fundamental requirements for grafting: high burst strength, positive surgical handling, and a functional endothelium.
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According to the World Health Organization, globally 4.9 million are blind due to corneal pathology. Corneal transplantation is successful and curative of the blindness for a majority of these cases. However, it is less successful in a number of diseases that produce corneal neovascularization, dry ocular surface and recurrent inflammation, or infections. A keratoprosthesis or KPro is the only alternative to restore vision when corneal graft is a doomed failure. Although a number of KPros have been proposed, only two devices, Boston type-1 KPro and osteo-odonto-KPro, have came to the fore. The former is totally synthetic and the latter is semi-biological in constitution. These two KPros have different surgical techniques and indications. Keratoprosthetic surgery is complex and should only be undertaken in specialized centers, where expertise, multidisciplinary teams, and resources are available. In this article, we briefly discuss some of the prominent historical KPros and contemporary devices.
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Cardiac malformations and disease are the leading causes of death in the United States in live-born infants and adults, respectively. In both of these cases, a decrease in the number of functional cardiomyocytes often results in improper growth of heart tissue, wound healing complications, and poor tissue repair. The field of cardiac tissue engineering seeks to address these concerns by developing cardiac patches created from a variety of biomaterial scaffolds to be used in surgical repair of the heart. These scaffolds should be fully degradable biomaterial systems with tunable properties such that the materials can be altered to meet the needs of both in vitro culture (e.g. disease modeling) and in vivo application (e.g. cardiac patch). Current platforms do not utilize both structural anisotropy and proper cell-matrix contacts to promote functional cardiac phenotypes and thus there is still a need for critically sized scaffolds that mimic both the structural and adhesive properties of native tissue. To address this need, we have developed a silk-based scaffold platform containing cardiac tissue-derived extracellular matrix (cECM). These silk-cECM composite scaffolds have tunable architectures, degradation rates, and mechanical properties. Subcutaneous implantation in rats demonstrated that addition of the cECM to aligned silk scaffold led to 99% endogenous cell infiltration and promoted vascularization of a critically sized scaffold (10 × 5 × 2.5 mm) after 4 weeks in vivo. In vitro, silk-cECM scaffolds maintained the HL-1 atrial cardiomyocytes and human embryonic stem cell-derived cardiomyocytes and promoted a more functional phenotype in both cell types. This class of hybrid silk-cECM anisotropic scaffolds offers new opportunities for developing more physiologically relevant tissues for cardiac repair and disease modeling.
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The retina is a highly sophisticated piece of the neural machinery that begins the translation of incoming light signals into meaningful visual information. Several degenerative diseases of the retina are characterized by photoreceptor loss and eventually lead to irreversible blindness. Regenerative medicine, using tissue engineering-based constructs to deliver progenitor cells or photoreceptors along with supporting carrier matrix is a promising approach for restoration of structure and function. Fresh fibrin glue (FG) produced by the CryoSeal(®)FS system in combination with mouse retinal progenitor cells (RPCs) were evaluated in this study. In vitro expanded RPCs isolated from postnatal mouse retina were encapsulated into FG and cultured in the presence of the protease inhibitor, tranexamic acid. Encapsulation of RPCs into FG did not show adverse effects on cell proliferation or cell survival. RPCs exhibited fibroblast-like morphology concomitantly with attachment to the encapsulating FG surface. They expressed α7 and β3 integrin subunits that could mediate attachment to fibrin matrix via an RGD-independent mechanism. The three-dimensional environment and the attachment surface provided by FG was associated with a rapid down-regulation of the progenitor marker SOX2 and enhanced the expression of the differentiation markers cone-rod homeobox and recoverin. However, the in vitro culture conditions did not promote full differentiation into mature photoreceptors. Nevertheless, we have shown that autologous fibrin, when fabricated into a scaffold for RPCs for delivery to the retina, provides the cells with external cues that could potentially improve the differentiation events. Hence, transient encapsulation of RPCs into FG could be a valid and potential treatment strategy to promote retinal regeneration following degenerative diseases. However, further optimization is necessary to maximize the outcomes in terms of mature photoreceptors.
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Tissue engineering by self-assembly uses the cells' secretome as a regeneration template and biological factory of trophic factors. Despite the several advantages that have been witnessed in preclinical and clinical setting, the major obstacle for wide acceptance of this technology remains the tardy extracellular matrix formation. Herein, we assessed the influence of macromolecular crowding / excluding volume effect, a biophysical phenomenon that accelerates thermodynamic activities and biological processes by several orders of magnitude, in human corneal fibroblast culture. Our data indicate that the addition of negatively charged galactose derivative (carrageenan) in human corneal fibroblast culture, even at 0.5% serum, increases by 12-fold tissue-specific matrix deposition, whilst maintaining physiological cell morphology and protein / gene expression. Gene analysis indicates that a glucose derivative (dextran sulphate) may drive corneal fibroblasts towards a myofibroblast lineage. Collectively, these results indicate that macromolecular crowding may be suitable not only for clinical translation and commercialisation of tissue engineering by self-assembly therapies, but also for the development of in vitro pathophysiology models.
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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.
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Defects in corneal stroma caused by trauma or diseases such as macular corneal dystrophy and keratoconus can be detrimental for vision. Development of therapeutic methods to enhance corneal regeneration is essential for treatment of these defects. Here, we report a bioactive peptide nanofiber scaffold system for corneal tissue regeneration. These nanofibers are formed by self-assembling peptide amphiphile molecules containing laminin and fibronectin inspired sequences. Human corneal keratocyte (HTK) cells cultured on laminin-mimetic peptide nanofibers retained their characteristic morphology and their proliferation was enhanced compared to cells cultured on fibronectin-mimetic nanofibers. When these nanofibers were used for damaged rabbit corneas, laminin-mimetic peptide nanofibers increased keratocyte migration and supported stroma regeneration. These results suggest that laminin-mimetic peptide nanofibers provide a promising injectable, synthetic scaffold system for cornea stroma regeneration.
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Fibroin extracted from silkworm cocoon silk provides an intriguing and potentially important biomaterial for corneal reconstruction. In this chapter we outline our methods for producing a composite of two fibroin-based materials that support the cocultivation of human limbal epithelial (HLE) cells and human limbal stromal (HLS) cells. The resulting tissue substitute consists of a stratified epithelium overlying a three-dimensional arrangement of extracellular matrix components (principally "degummed" fibroin fibers) and mesenchymal stromal cells. This tissue substitute is currently being evaluated as a tool for reconstructing the corneal limbus and corneal epithelium.
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Corneal diseases represent the second leading cause of blindness in most developing world countries. Worldwide, major investments in public health infrastructure and primary eye care services have built a strong foundation for preventing future corneal blindness. However, there are an estimated 4.9 million bilaterally corneal blind persons worldwide who could potentially have their sight restored through corneal transplantation. Traditionally, barriers to increased corneal transplantation have been daunting, with limited tissue availability and lack of trained corneal surgeons making widespread keratoplasty services cost prohibitive and logistically unfeasible. The ascendancy of cataract surgical rates and more robust eye care infrastructure of several Asian and African countries now provide a solid base from which to dramatically expand corneal transplantation rates. India emerges as a clear global priority as it has the world's largest corneal blind population and strong infrastructural readiness to rapidly scale its keratoplasty numbers. Technological modernization of the eye bank infrastructure must follow suit. Two key factors are the development of professional eye bank managers and the establishment of Hospital Cornea Recovery Programs. Recent adaptation of these modern eye banking models in India have led to corresponding high growth rates in the procurement of transplantable tissues, improved utilization rates, operating efficiency realization, and increased financial sustainability. The widespread adaptation of lamellar keratoplasty techniques also holds promise to improve corneal transplant success rates. The global ophthalmic community is now poised to scale up widespread access to corneal transplantation to meet the needs of the millions who are currently blind.
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Although many regenerative cell therapies are being developed to replace or regenerate ischaemic muscle, the lack of vasculature and poor persistence of the therapeutic cells represent major limiting factors to successful tissue restoration. In response to ischaemia, stromal cell-derived factor-1 (SDF-1) is up-regulated by the affected tissue to stimulate stem cell-mediated regenerative responses. Therefore, we encapsulated SDF-1 into alginate microspheres and further incorporated these into an injectable collagen-based matrix in order to improve local delivery. Microsphere-matrix impregnation reduced the time for matrix thermogelation, and also increased the viscosity reached. This double-incorporation prolonged the release of SDF-1, which maintained adhesive and migratory bioactivity, attributed to chemotaxis in response to SDF-1. In vivo, treatment of ischaemic hindlimb muscle with microsphere-matrix led to increased mobilisation of bone marrow-derived progenitor cells, and also improved recruitment of angiogenic cells expressing the SDF-1 receptor (CXCR4) from bone marrow and local tissues. Both matrix and SDF-1-releasing matrix were successful at restoring perfusion, but SDF-1 treatment appeared to play an earlier role, as evidenced by arterioles that are phenotypically older and by increased angiogenic cytokine production, stimulating the generation of a qualitative microenvironment for a rapid and therefore more efficient regeneration. These results support the release of implanted SDF-1 as a promising method for enhancing progenitor cell responses and restoring perfusion to ischaemic tissues via neovascularisation.
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Ocular burns can damage the corneal epithelial stem cells located at the limbus. This study evaluated the efficacy of xeno-free autologous cell-based treatment of limbal stem cell deficiency. This retrospective study included 200 patients, above 8 years of age, with clinically diagnosed unilateral total limbal stem cell deficiency due to ocular surface burns treated between 2001 and 2010. A small limbal biopsy was obtained from the unaffected eye. The limbal epithelial cells were expanded ex vivo on human amniotic membrane for 10-14 days using a xeno-free explant culture system. The resulting cultured epithelial monolayer and amniotic membrane substrate were transplanted on to the patient's affected eye. Postoperative corneal surface stability, visual improvement and complications were objectively analysed. A completely epithelised, avascular and clinically stable corneal surface was seen in 142 of 200 (71%) eyes at a mean follow-up of 3 ± 1.6 (range: 1-7.6) years. A two-line improvement in visual acuity, without further surgical intervention, was seen in 60.5% of eyes. All donor eyes remained healthy. Autologous cultivated limbal epithelial transplantation using a xeno-free explant culture technique was effective in long-term restoration of corneal epithelial stability and improvement of vision in eyes with ocular surface burns.
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Cell therapy for the treatment of cardiovascular disease has been hindered by low cell engraftment, poor survival, and inadequate phenotype and function. In this study, we added chitosan to a previously developed injectable collagen matrix, with the aim of improving its properties for cell therapy and neovascularization. Different ratios of collagen and chitosan were mixed and chemically crosslinked to produce hydrogels. Swell and degradation assays showed that chitosan improved the stability of the collagen hydrogel. In culture, endothelial cells formed significantly more vascular-like structures on collagen–chitosan than collagen-only matrix. While the differentiation of circulating progenitor cells to CD31+ cells was equal on all matrices, vascular endothelial-cadherin expression was increased on the collagen–chitosan matrix, suggesting greater maturation of the endothelial cells. In addition, the collagen–chitosan matrix supported a significantly greater number of CD133+ progenitor cells than the collagen-only matrix. In vivo, subcutaneously implanted collagen–chitosan matrices stimulated greater vascular growth and recruited more von Willebrand factor (vWF+) and CXCR4+ endothelial/angiogenic cells than the collagen-only matrix. These results indicate that the addition of chitosan can improve the physical properties of collagen matrices, and enhance their ability to support endothelial cells and angiogenesis for use in cardiovascular tissue engineering applications.
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There is a clinical need for a functional tissue-engineered blood vessel because small-caliber arterial graft (<5 mm) applications are limited by the availability of suitable autologous vessels and suboptimal performances of synthetic grafts. This study presents an analysis of the mechanical properties of tissue-engineered vascular constructs produced using a novel single-step self-assembly approach. Briefly, the tissue-engineered vascular media were produced by culturing smooth muscle cell in the presence of sodium l-ascorbate until the formation of a cohesive tissue sheet. This sheet was then rolled around a tubular support to create a media construct. Alternatively, the tissue-engineered vascular adventitia was produced by rolling a tissue sheet obtained from dermal fibroblasts or saphenous vein fibroblasts. The standard self-assembly approach to obtain the two-layer tissue-engineered vascular constructs comprising both media and adventitia constructs consists of two steps in which tissue-engineered vascular media were first rolled on a tubular support and a tissue-engineered vascular adventitia was then rolled on top of the first layer. This study reports an original alternative method for assembling tissue-engineered vascular constructs comprising both media and an adventitia in a single step by rolling a continuous tissue sheet containing both cell types contiguously. This tissue sheet was produced by growing smooth muscle cells alongside fibroblasts (saphenous vein fibroblasts or dermal fibroblasts) in the same culture dish separated by a spacer, which is removed later in the culture period. The mechanical strength assessed by uniaxial tensile testing, burst pressure measurements, and viscoelastic behavior evaluated by stepwise stress relaxation tests reveals that the new single-step fabrication method significantly improves the mechanical properties of tissue-engineered vascular construct for both ultimate tensile strength and all the viscoelastic moduli.
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To determine whether the implementation of Routine Notification and Request (RNR) has been effective in increasing the amount of donor corneal tissue available and reducing wait times for corneal transplant (CT) surgeries. Survey of the CT surgeons and eye banks in Canada.Participants: CT surgeons and representatives of the 10 eye banks in Canada. Voluntary, anonymous questionnaires were distributed between May 1 and September 30, 2006. Following the implementation of RNR, 3 eye banks had an increase in the amount of corneal tissue available: Manitoba, 81% (from 42 tissues in 2004 to 76 tissues in 2006); Ontario, 25% (from 1304 tissues in 2005 to 1626 tissues in 2006); New Brunswick, 129% (from 86 tissues in 2005 to 197 tissues in 2006). British Columbia, where RNR was implemented in 1999, had a 6% increase (from 766 in 2005 to 812 in 2006). There has been a significant decrease in wait times from the time of diagnosis by CT surgeons to the time of surgery in British Columbia (from 48+/-18 weeks in 2004 to 39+/-20 weeks in 2006), Manitoba (from 82+/-56 weeks in 2004 to 32+/-23 weeks in 2006), Ontario (from 82+/-56 weeks in 2004 to 31+/-34 weeks in 2006), and Nova Scotia (from 44+/-12 weeks in 2004 to 32+/-28 weeks in 2006). RNR has been effective in increasing corneal tissue availability and decreasing wait times in provinces where it has been implemented. We recommend similar legislative changes to be considered in those provinces where corneal tissue shortage is delaying the availability of CT surgery.
Book
Despite enormous advances made in the development of external effector prosthetics over the last quarter century, significant questions remain, especially those concerning signal degradation that occurs with chronically implanted neuroelectrodes. Offering contributions from pioneering researchers in neuroprosthetics and tissue repair, Indwelling Neural Implants: Strategies for Contending with the In Vivo Environment examines many of these challenges, paying particular attention to how the healing of tissues surrounding an implant can impact the intended use of a device. The contributions are divided into four sections • Part one examines wound healing from the initial insertion trauma through the inflammatory and repair process, explaining how the action of healing varies throughout different areas of the body. • Part two considers various performance issues specific to particular implant components, including those that arise from the chemical, mechanical, thermal, and electrical impact on surrounding tissues. It discusses challenges that result from chronic tissue stimulation and heat effects that occur with on-chip and telemetric processing. • Part three presents both in vitro and in vivo approaches to assessing wound healing response to materials. It includes the contribution of the developer of a chronic hollow fiber membrane implant who explains how an in vivo model is used to assess molecular transport in brain tissue surrounding the implant. • The final section evaluates molecular and materials strategies for intervening in CNS wound repair and enhancing the electrical communication between the electrode surface and the surrounding tissue. It also presents novel approaches to nerve regeneration and repair. This seminal work provides researchers with an up-to-date account of the progress in the field that they can build upon to bring us closer to realizing the full value of neural implants in combating otherwise intractable human health problems.
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Corneas with severe pathologies have a high risk of rejection when conventionally grafted with human donor tissues. In this early observational study, we grafted bioengineered corneal implants made from recombinant human collagen and synthetic phosphorylcholine polymer into three patients for whom donor cornea transplantation carried a high risk of transplant failure. These patients suffered from corneal ulcers and recurrent erosions preoperatively. The implants provided relief from pain and discomfort, restored corneal integrity by promoting endogenous regeneration of corneal tissues, and improved vision in two of three patients. Such implants could in the future be alternatives to donor corneas for high-risk patients, and therefore, merits further testing in a clinical trial. Clin Trans Sci 2015; Volume #: 1-5. © 2015 Wiley Periodicals, Inc.
Article
Globally there are ≈4.9 million bilaterally corneal blind and 23 million unilaterally corneal blind. Majority of this blindness exists in the developing countries, where resources for corneal banking and transplant surgery are less than adequate. Survival of corneal grafts gradually declines over the long term. Corneal transplantation has poor prognosis in vascularised corneal beds, ocular surface disease and viral keratitis. Keratoprosthesis (KPro) remains as a final option for end-stage ocular surface disease, multiple corneal transplant failures and high-risk corneal grafts. Boston type-1 KPro and osteo-odonto-keratoprosthesis are the two devices proven useful in recent years. Choice of a keratoprosthetic device is patient specific based on the underlying diagnosis, ocular morbidity and patient suitability. KPro surgery demands a high level of clinical and surgical expertise, lifelong commitment and extensive resources. Improvements in techniques and biomaterials may in the future provide retainable KPros that do not need regular follow-up of patients, have low complications but high retention rates and may be produced at a low cost on a mass scale to be available as 'off the shelf' devices. Because KPros have the potential to effectively address the burden of surgically treatable corneal blindness and may also eliminate the problems of corneal transplantation, more research is required to develop KPros as substitutes for corneal transplantation even in low-risk cases. In those countries where corneal blindness is a major liability, we need a two pronged approach: one to develop eye donation, eye banking and corneal transplantation and the second to establish centres for keratoprostheses, which are affordable and technically not challenging, in a population where default on follow-up visits are high. Until the latter is achieved, KPros should be viewed as a temporary means for visual restoration and be offered in national and supraregional specialised centres only.
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The implant-host interface is a critical element in guiding tissue or organ regeneration. We previously developed hydrogels comprising interpenetrating networks of recombinant human collagen type III and 2-methacryloyloxyethyl phosphorylcholine (RHCIII-MPC) as substitutes of the corneal extracellular matrix that promote endogenous regeneration of corneal tissue. To render them functional for clinical application, we have now optimized their composition and thereby enhanced their mechanical properties. We have demonstrated that such optimized RHCIII-MPC hydrogels are suitable for precision femtosecond laser cutting to produce complementing implants and host surgical beds for subsequent tissue welding. This avoids the tissue damage and inflammation associated with manual surgical techniques, thereby leading to more efficient healing. Although we previously demonstrated in clinical testing that RHCIII-based implants stimulated cornea regeneration in patients, the rate of epithelial cell coverage of the implants needs improvement, e.g. modification of the implant surface. We now show that our 500 μm thick RHCIII-MPC constructs comprising over 85% water, are suitable for microcontact printing with fibronectin. The resulting fibronectin micropatterns promote cell adhesion, as compared to the bare RHCIII-MPC hydrogel. Interestingly, a pattern of 30 μm wide fibronectin stripes enhanced cell attachment and showed highest mitotic rates, an effect that potentially can be utilized for faster integration of the implant. We have therefore shown that laboratory-produced mimics of naturally occurring collagen and phospholipids can be fabricated into robust hydrogels that can be laser profiled and patterned to enhance their potential function as artificial substitutes of donor human corneas.
Article
Purpose: The aim of this study was to evaluate the outcome of penetrating keratoplasties, at the University Eye Hospital, Ludwig-Maximilians-University, Munich, Germany, using organ-cultured donor corneas and to identify preoperative risk factors, which may influence the event of graft failure. Methods: In this study, 377 medical records of patients, who underwent penetrating keratoplasty between 2001 and 2011, were reviewed. Organ-cultured donor corneas were obtained from the eye bank, Ludwig-Maximilians-University, Munich, Germany. Donor-related and preoperative recipient-related risk factors for graft failure were analyzed by univariate and multivariate analyses. Results: Graft failure occurred in 26% of patients. The following preoperative factors were significantly associated with graft failure by multivariate analyses: high donor age, low donor endothelial cell density, high patient age, indications of infectious keratitis, acute perforation of noninfectious keratitis, prior graft failure, chemical burn, trauma, glaucoma-associated corneal decompensation, high-risk graft indications, corneal edema, anterior chamber lens, diabetes mellitus, atopy, and autoimmune diseases. Conclusions: This study demonstrated a success rate of 74%, which is consistent with previous studies. Various preoperative recipient-related factors seem to influence the outcome of penetrating keratoplasties, whereas few donor-related factors have a significant association with graft failure.
Article
Purpose: Corneal tissue shortage has become a major concern worldwide, which has motivated the search for alternative solutions to eye bank human eyes for corneal transplantation. Minimally invasive lamellar transplantation and tissue engineering may offer new opportunities for the rehabilitation of diseased corneas. The aim of this study was to evaluate the biocompatibility and functionality of stromal lamellar grafts tissue-engineered (TE) in vitro and transplanted in vivo in the cornea of a feline model. Methods: The corneal stromas were engineered in culture from corneal stromal cells using the self-assembly approach, without the addition of exogenous material or scaffold. Eight healthy animals underwent two intrastromal grafts in one eye and the contralateral eye was used as a control. Animals were followed with slit-lamp ophthalmic examination, corneal esthesiometry and optical coherent tomography. Confocal microscopy, immunofluorescence, histology, and transmission electron microscopy (TEM) were performed at 4 months. Results: Four months after transplantation, the TE-stromal grafts were transparent, functional, and well tolerated by the eye. All grafts remained avascular, with no signs of immune rejection, despite a short course of low-dose topical steroids. Corneal sensitivity returned to preoperative level and reinnervation of the grafts was confirmed by confocal microscopy and immunofluorescence. Histology and TEM of the TE-grafts showed a lamellar stromal structure with regular collagen fibril arrangement. Conclusions: These results open the way to an entirely new therapeutic modality. Intracorneal filling using a biocompatible, transparent, and malleable TE-stroma could be the basis for multiple types of novel therapeutic options in corneal interventional surgery.
Article
Silk proteins represent a unique choice in the selection of biomaterials that can be used for corneal tissue engineering and regenerative medical applications. We implanted helicoidal multilamellar arginine–glycine–aspartic acid-functionalized silk biomaterials into the corneal stroma of rabbits, and evaluated its biocompatibility. The corneal tissue was examined after routine hematoxylin–eosin staining, immunofluorescence for collagen I and III, and fibronectin, and scanning electron microscopy. The silk films maintained their integrity and transparency over the 180-day experimental period without causing immunogenic and neovascular responses or degradation of the rabbit corneal stroma. Collagen I increased, whereas Collagen III and fibronectin initially increased and then gradually decreased. The extracellular matrix deposited on the surface of the silk films, tightly adhered to the biomaterial. We have shown this kind of silk film graft has suitable biocompatibility with the corneal stroma and is an initial step for clinical trials to evaluate this material as a transplant biomaterial for keratoplasty tissue constructs. © 2014 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2014.
Article
Emulating corneal stromal tissue is believed to be the most challenging step in bioengineering an artificial human cornea because of the difficulty in reproducing its highly ordered microstructure, the key to the robust biomechanical properties and optical transparency of this tissue. We conducted a comparative study to assess the feasibility of human corneal stromal stem cells (hCSSCs) and human corneal fibroblasts (hCFs) in the generation of human corneal stromal tissue on groove-patterned silk substrates. In serum-free keratocyte differentiation medium, hCSSCs successfully differentiated into keratocytes secreting multilayered lamellae with orthogonally-oriented collagen fibrils, in a pattern mimicking human corneal stromal tissue. The constructs were 90-100 μm thick, containing abundant cornea-specific extracellular matrix (ECM) components, including keratan sulfate, lumican, and keratocan. In contrast, hCFs tended to differentiate into myofibroblasts that deposited less organized collagen in a pattern resembling that of corneal scar tissue. RGD surface coupling coupling was an essential factor in enhancing cell attachment, orientation, proliferation, differentiation and ECM deposition on the silk substratum. These results demonstrated that an approach of combining hCSSCs with an RGD surface-coupled patterned silk film offers a powerful tool to develop highly ordered collagen fibril-based constructs for corneal regeneration and corneal stromal tissue repair.
Article
One of the major challenges in the field of regenerative medicine is how to optimize tissue regeneration in the body by therapeutically manipulating its natural ability to form scar at the time of injury or disease. It is often the balance between tissue regeneration, a process that is activated at the onset of disease, and scar formation, which develops as a result of the disease process that determines the ability of the tissue or organ to be functional. Using biomaterials as scaffolds often can provide a "bridge" for normal tissue edges to regenerate over small distances, usually up to 1 cm. Larger tissue defect gaps typically require both scaffolds and cells for normal tissue regeneration to occur without scar formation. Various strategies can help to modulate the scar response and can potentially enhance tissue regeneration. Understanding the mechanistic basis of such multivariate interactions as the scar microenvironment, the immune system, extracellular matrix, and inflammatory cytokines may enable the design of tissue engineering and wound healing strategies that directly modulate the healing response in a manner favorable to regeneration.
Article
Despite present optimal standard treatment of lower-extremity ulceration, a high incidence of recurrence and treatment failure is observed. The objective of this project was to evaluate the effect of a self-assembled skin substitute (SASS) made by tissue engineering as a temporary cutaneous dressing in the treatment of hard-to-heal chronic ulcers. The prospective uncontrolled case study includes patients suffering from venous or mixed ulcers lasting more than 6 months and unresponsive to compression therapy, with an Ankle Brachial Index greater than 0.5. Compression therapy was combined with the weekly application of SASS, produced from the patient's own skin cells, until healing. A weekly follow-up recorded wound size, skin aspect, pain, drainage, and percentage of wound healing. Photographs were also taken to assess ulcer evolution. Fourteen ulcers present on 5 patients were treated. A mean of 6.7 SASS depositions by ulcer was required for healing. Two ulcers developed a minor wound infection, which was treated with oral antibiotics; another 2 ulcers recurred, and 1 healed with a second course of treatment, whereas 1 ulcer had a small recurrence treated with local wound care. The authors' study suggests that the SASS used as a biological dressing is a promising treatment for hard-to-heal chronic venous and mixed ulcers that are unresponsive to compression therapy.
Article
Purpose: To identify risk factors for corneal graft rejection and rejection irreversibility. Design: Retrospective cohort study. Methods: setting: Institutional. patients: A total of 1438 consecutive eyes of 1438 patients who underwent corneal transplantation for optical indication at the Centre Hospitalier National d'Ophtalmologie des XV-XX, Paris, France, between December 1992 and December 2010 were studied. Surgical technique was penetrating keratoplasty (PK) in 1209 cases, anterior lamellar keratoplasty (ALK) in 165 cases, and Descemet stripping with endothelial keratoplasty in 64 cases. main outcome measures: Cumulative incidence of rejection episodes and rejection irreversibility rate. Results: A total of 299 cases of rejection episodes were identified, of which 145 (48.5%) were irreversible after treatment. In multivariate analysis, the cumulative incidence of rejection episodes was influenced by recipient age (P = .00002), recipient rejection risk (P = .0003), lens status (P = .00003), and surgical group (P = .035). A higher incidence of rejection episodes was observed in young patients (<20 years) and patients aged from 41 to 50, high-risk recipients, aphakic eyes and eyes with anterior chamber intraocular lens, and eyes with PK (compared with eyes with ALK). Rejection episodes were more likely to be irreversible for high-risk recipients (P = .02), for eyes with preoperative hypertony (P = .009), and for eyes with poor visual acuity at presentation (P = .002). Conclusions: Recipient rejection risk and surgical group are the main risk factors for rejection as they both influence the incidence of rejection and the reversibility rate. Recipient age and lens status are predictive factors for the occurrence of rejection. Preoperative hypertony is a predictive factor for rejection irreversibility.
Article
The structural stability of skin substitutes is critical to avoid aesthetic and functional problems after grafting, such as contractures and hypertrophic scars. The present study was designed to assess the production steps having an influence on the contractile behaviour of the tissue-engineered skin made by the self-assembly approach, where keratinocytes are cultured on tissue-engineered dermis comprised of fibroblasts and the endogenous extracellular matrix they organized. Thus, different aspects were investigated, such as the assembly method of the engineered dermis (various sizes and anchoring designs) and the impact of epithelial cell differentiation (culture submerged in the medium or at the air-liquid interface). To evaluate the structural stability at the end of the production, the substitutes were detached from their anchorages and deposited on a soft substrate, and contraction was monitored over 1 week. Collected data were analysed using a mathematical model to characterize contraction. We observed that the presence of a differentiated epidermis significantly reduced the amount of contraction experienced by the engineered tissues, independently of the assembly method used for their production. When the epidermis was terminally differentiated, the average contraction was only 24 ± 4% and most of the contraction occurred within the first 12 h following deposition on the substrate. This is 2.2-fold less compared to when the epidermis was cultured under the submerged condition, or when tissue-engineered dermis was not overlaid with epithelial cells. This study highlights that the maturation at the air-liquid interface is a critical step in the reconstruction of a tissue-engineered skin that possesses high structural stability. Copyright © 2012 John Wiley & Sons, Ltd.
Article
Limbal tissues can be cultured on various types of scaffolds to create a sheet of limbal-corneal epithelium for research as well as clinical transplantation. An optically clear, biocompatible, biomimetic scaffold would be an ideal replacement graft for transplanting limbal stem cells. In this study, we evaluated the physical and culture characteristics of the recombinant human cross-linked collagen scaffold (RHC-III scaffold) and compared it with denuded human amniotic membrane (HAM). Optical/mechanical properties and microbial susceptibility were measured for the scaffolds. With the approval of the institutional review board, 2 mm fresh human limbal tissues were cultured on 2.5 x 2.5 cm(2) scaffolds in a medium containing autologous serum in a feeder cell-free submerged system. The cultured cell systems were characterized by morphology and immunohistochemistry for putative stem cells and differentiated cell markers. The refractive index (RI) and tensile strength of the RHC-III scaffold were comparable to human cornea, with delayed in vitro degradation compared to HAM. RHC-III scaffolds were 10-fold less susceptible to microbial growth. Cultures were initiated on day 1, expanded to form a monolayer by day 3 and covered the entire growth surface in 10 days. Stratified epithelium on the scaffolds was visualized by transmission electron microscopy. The cultured cells showed p63 and ABCG2 positivity in the basal layer and were immunoreactive for cytokeratin K3 and K12 in the suprabasal layers. RHC-III scaffold supports and retains the growth and stemness of limbal stem cells, in addition to resembling human cornea; thus, it could be a good replacement scaffold for growing cells for clinical transplantation.
Article
RGD-coupled silk protein-biomaterial lamellar systems were prepared and studied with human cornea fibroblasts (hCFs) to match functional requirements. A strategy for corneal tissue engineering was pursued to replicate the structural hierarchy of human corneal stroma within thin stacks of lamellae-like tissues, in this case constructed from scaffolds constructed with RGD-coupled, patterned, porous, mechanically robust and transparent silk films. The influence of RGD-coupling on the orientation, proliferation, ECM organization, and gene expression of hCFs was assessed. RGD surface modification enhanced cell attachment, proliferation, alignment and expression of both collagens (type I and V) and proteoglycans (decorin and biglycan). Confocal and histological images of the lamellar systems revealed that the bio-functionalized silk human cornea 3D constructs exhibited integrated corneal stroma tissue with helicoidal multi-lamellar alignment of collagen-rich and proteoglycan-rich extracellular matrix, with transparency of the construct. This biomimetic approach to replicate corneal stromal tissue structural hierarchy and architecture demonstrates a useful strategy for engineering human cornea. Further, this approach can be exploited for other tissue systems due to the pervasive nature of such helicoids in most human tissues.
Article
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.
Article
Shortages in transplantable corneas are common, yet little appears in the medical literature about patterns of tissue donation and factors affecting procurement. We have analyzed data on eye donations and taken measures to improve procurement rates based on our findings. Fifty consecutive Cardiovascular Intensive Care Unit (CVICU) deaths were reviewed to compare the number of transplant-eligible donors to the amount of tissue received. An anonymous survey of 250 house staff and nurses was undertaken to identify obstacles to donor eye procurement. Although 12 of 50 potential donors in the CVICU met transplant eligibility criteria, only 1 became a donor. A required request policy notwithstanding, the most common reason for nonprocurement was failure to make a request. According to the survey, the most significant impediments to making the request were (a) not thinking to ask, (b) unfamiliarity with eligibility criteria, (c) unfamiliarity with enucleation procedures, (d) feeling that someone else should make the request, and (e) reluctance to impose on a grieving family. Very few cited religious reasons or being too busy. Education based on the specific concerns listed in the survey was undertaken. During the 12 months after this initiative, the number of transplantable corneas donated from our facility doubled, as compared with the same period in 1992. Despite required request laws and regulations, failure to request tissue donation is common in our facility and may be common elsewhere. Systematic analysis of obstacles to donor eye procurement and their solutions may help to improve our country's performance in this area.
Article
Complete loss of the corneal-limbal epithelium leads to re-epithelialisation by bulbar conjunctival cells. Since conjunctival and corneal-limbal epithelial cells represent two different cell lines, this conjunctival healing of the cornea is followed by stromal scarring, decreased visual acuity, and severe discomfort. Unilateral corneal-limbal epithelial defects can be resolved by the transplantation of limbal grafts taken from the uninjured eye. However, this procedure requires a large limbal graft to be taken from the healthy eye, and is not possible for bilateral lesions. We investigated the possibility of restoring the human corneal surface with autologous corneal epithelial sheets generated by serial cultivation of limbal cells. Cells were cultivated from a 1 mm2 biopsy sample taken from the limbus of the healthy eye of two patients with severe alkali burns, and thus complete loss of the corneal-limbal surface, of one eye. Normal corneal differentiation was tested with a specific biochemical marker. Autologous cultured corneal sheets were then grafted onto the damaged eyes of the two patients. The patients were followed up at more than 2 years after grafting. We have shown that corneal progenitor cells are localised in the limbus, that cultured limbal cells generate cohesive sheets of authentic corneal epithelium, and that autologous cultured corneal epithelium restored the corneal surface of two patients with complete loss of the corneal-limbus epithelium. Long-term follow-up showed the stability of regenerated corneal epithelium and the striking improvement in patients' comfort and visual acuity. The cultivation of corneal epithelium might offer an alternative to patients with unilateral lesions and a therapeutic chance to patients with severe bilateral corneal-limbal epithelial defects. Our findings give a new perspective on the treatment of ocular disorders characterised by stem-cell deficiency.
Article
To compare repeat penetrating keratoplasty (PKP) with primary PKP with respect to patient characteristics, survival rates, and risk factors for graft failure. Retrospective, consecutive, noncomparative case series of 116 patients who underwent repeat PKP and who were identified from a cohort of 696 PKPs performed by one surgeon over a 7.5-year period. Compared with patients who underwent primary PKP, regraft patients were 5 years older, had a higher rate of peripheral anterior synechiae (PAS), were more likely to require intraocular pressure (IOP)-lowering medications prior to surgery, were more likely to develop postoperative corneal neovascularization, were less likely to be phakic, and were more likely to undergo PKP in conjunction with a lens procedure. There was no difference between the two groups with respect to the distribution of original diagnoses leading to PKP and the rate of graft rejection. Two- and 5-year survival rates for repeat PKP were 63.9% and 45.6%, respectively. In a multivariate analysis, the original diagnosis leading to corneal transplantation, the presence of preoperative PAS, intraoperative anterior vitrectomy, and postoperative corneal neovascularization were identified as risk factors for graft failure in patients undergoing a regraft. Patients undergoing PKP for the first and second time share common risk factors for graft failure, namely, the original diagnosis leading to corneal transplantation, the presence of preoperative PAS, and the occurrence of postoperative corneal neovascularization. The difference in graft survival rates between the two groups can be partially explained on the basis of higher rates of the latter two risk factors among regrafts.
Article
To evaluate the French waiting list and the indications of registered patients, to compare the rates of registration, graft, and procurement between French regions. In France, each patient with an indication for penetrating keratoplasty should be registered on the waiting list with his or her clinical characteristics. Those registered during 2000 and 2001 were included in the study. Data on transplantation activity from the waiting list were compared to data collected by a questionnaire on graft and procurement activities completed each year by medical teams. In 2000 and 2001, 6093 and 5505 waiting patients, respectively, were registered. For the same years, 3984 and 3457 keratoplasties were declared for the patients registered, but the questionnaires reported 4514 and 4388 grafts, respectively. The national registration rate was 96 per million population (pmp). The extreme values between regions ranged from 53 to 143 pmp. There was a significant correlation between regional procurement and transplantation rates (r=0.75, p=0.001) but not for registration and procurement rates, and not for registration and transplantation rates. The national registration rate was 27 pmp for pseudophakic and aphakic corneal edema, with extreme values of 12-64 pmp. The national registration rate was 24 pmp for keratoconus (11-37 pmp). A high patient turnover was observed between regions. Among the 11,598 patients registered, the most common indications were pseudophakic and aphakic corneal edema (27.7%), keratoconus (25.3%), and Fuchs'endothelial dystrophy (9.1%). Mean recipient age was 57+/-22 years (0-103 years). Among these patients, 14.1% had already received transplants at least once for the same eye (7.8% for keratoconus, 14.3% for pseudophakic and aphakic corneal edema, and 6.1% for Fuchs'dystrophy). Ophthalmologists will be able to register their patients directly on the waiting list, which will improve data quality for transplantation notification. Regional policies should be developed to decrease the inequalities of graft shortages between regions.
Article
Adipose tissue is an accessible and abundant source of mesenchymal stem cells for soft-tissue reconstruction. In an attempt to create a novel, entirely autologous tissue-engineered adipose substitute, we extracted human stromal cells from either lipoaspirated or resected fat, and assessed their capacity to produce a three-dimensional adipose tissue using an adapted "self-assembly" culture methodology. This strategy involved a concomitant induction of adipogenic differentiation whilst ascorbic acid supplementation stimulated the stromal cells to produce and organize their own "biomaterial" in the form of extracellular matrix, forming manipulatable sheets that are then assembled into thicker reconstructed adipose tissues. When compared to resected fat, lipoaspiration-derived cells featured an increased adipogenic potential and the enhanced ability to recreate an adipose substitute in vitro. When viewed by scanning electron microscopy, the appearance of these reconstructed adipose tissues was strikingly similar to subcutaneous fat. Furthermore, these substitutes secreted adipokines and mediated beta-adrenergic receptor-stimulated lipolysis, hence reproducing known major biological functions of white adipose tissue. Therefore, our cell-based tissue engineering strategy led to the production of a functional and entirely natural reconstructed adipose tissue, which offers the potential to be used for specific in vitro applications as well as for autologous soft-tissue reconstruction.
Biocompatibility and functionality of a tissue-engineered living corneal stroma transplanted in the feline eye
  • Pankert
Allogeneic Tissue Engineering (Nanostructured Artificial Human Cornea) in Patients With Corneal Trophic Ulcers in Advanced Stages
  • Clinicaltrials
  • Gov
ClinicalTrials.gov. Allogeneic Tissue Engineering (Nanostructured Artificial Human Cornea) in Patients With Corneal Trophic Ulcers in Advanced Stages, Refractory to Conventional (Ophthalmic) Treatment. Available from: https://clinicaltrials.gov/ct2/show/NCT01765244.
Corneal Regenerative Medicine: Methods and Protocols
  • B Wright
  • Cannon Cjeds
In: Wright B, Cannon CJEds. Corneal Regenerative Medicine: Methods and Protocols. Methods in Molecular Biology 1014.New York: Humana Press, 2013; 165-78.
Eye Bank Association of America
Eye Bank Association of America, Eye Banking Statistical Report 2010: Washington, DC, USA: Eye Bank Association of America, 2009.
Outcomes of repeat penetrating keratoplasty and risk factors for graft failure
  • Weisbrod