there is a long history of reconstructing ocular function with biomaterials , particularly in the outer segment of the eye where various lens materials have been employed as contact and intraocular lenses .
the discovery of the possible use of biomaterials in the eye started after the discovery of windshield plexiglass materials lodged in the cornea of a world war ii fighter pilot without major side effects .
since then , a number of synthetic materials have been explored as biomaterials for the eye . while millions of intraocular lenses , contact lenses or various devices have been utilized on patients around the world , development of a synthetic corneal substitute has had little success . in the meantime , the field of tissue engineering and regenerative medicine developed as a major force in creating tissue substitutes .
the general approach of tissue engineering is to employ biomaterials as temporary scaffolds to support and direct cells to form new tissue .
biological cues , in the form of growth factors , small molecules or even gene therapy can also play important roles in directing cell behavior and tissue development and repair .
while tissue engineering has reached clinical testing and even commercialization for applications , including skin , bone and cartilage , it has been slow to reach ophthalmology and corneal reconstruction . here
, we will present a number of strategies by which a regenerative medicine approach can be introduced to provide biological repair and reconstruction of the cornea .
there are a number of degrees and levels of complexity in which smart biomaterials and tissue engineering can be introduced for treating tissue repair and loss in the cornea [ figure 1 ] .
regenerative technologies can be simple and stimulate local tissue repair , such as the case in small injuries or incisions . for example
, a biological adhesive can be designed with properties that allow it to  glue  tissue together while simultaneously stimulating tissue growth . at the next level
, regenerative strategies can be employed to replace corneal layers , such as the epithelium or endothelium .
finally , a number of strategies are being employed to create biological substitutes for the complete cornea .
while corneal transplantation has an excellent record of success , there remain challenges in cases of rejection or graft failure , along with inadequate donor supply in the developing world .
ultimately , using biomaterials to direct cell function and new tissue development in the cornea and other ocular tissues represents a paradigm shift in clinical treatment that will allow patients to rebuild physiologically normal tissues instead of relying on synthetic materials that can not mirror native structure or function . finally , by taking a materials engineering perspective in characterizing the complex structure of the cornea , we can better understand the matrix properties of the tissue during disease and , ultimately , design more efficient therapeutic interventions .
application of biomaterials for biological reconstruction of the cornea cells , in particular , stem cells , are critical components required to reconstruct and rebuild tissues .
there are significant , ongoing research efforts by a number of groups to elucidate the identity and function of stem cells in practically all tissues of the body , including the many tissue compartments of the eye .
these cells can be isolated from small biopsies , expanded in culture and delivered to a site of injury , oftentimes using a biomaterial carrier . in another , more subtle use of stem cells , biomaterials can be designed to attract and concentrate stem cells directly in tissues , frequently in conjunction with biological signals . in this way ,
this strategy of modulating endogenous stem cell behavior is simple and more practical for widespread clinical application ; however , aged patients or those with severe disease may need the addition of exogenous stem cells for adequate repair .
the recognition that stem cells possess the ability to both self renew and differentiate , is leading to the generation of adequate numbers of cells for delivery to patients . in the eye ,
limbal stem cells ( lscs ) were identified as the source of cells that continually generate the corneal epithelial layer .
more recently , corneal stromal stem cells , which can differentiate to keratocytes , were discovered .
finally , progenitor cells of the endothelium and trabeculum ( pcet ) , which seem to differentiate into endothelial cells , were also identified . as in organ transplantation ,
although use of lifelong or long - term immunosuppressive medication is possible , the best way to prevent rejection is to use the patient s own cells , which may be possible in unilateral eye disease or trauma .
the classic corneal epithelial cell transplantation procedure employs grafting of a healthy limbal biopsy , either from a healthy contra lateral eye or from a donor , onto the damaged cornea .
the technique was developed in the late 1980s and early 1990s and proved useful in severe surface diseases ( now narrowed to limbal stem cell deficiencies , ) .
the need for a greater number of cells for transplantation was soon evident , and efforts began to culture epithelial and limbal cells in vitro .
the goal of in vitro culture procedures was to obtain a layer of cells that could be used as a replacement for the native corneal epithelium .
denuded amniotic membrane is currently employed to improve culture conditions and facilitate culture and transplantation . in a recent review of the literature , baylis et al .
they also found that results differ according to disease etiology , with inflammatory conditions having the best outcomes ( 86% ) and congenital ones producing the worst ( 60% ) .
some of the most common complications reported for cell transplantation include infections , conjunctivalization and corneal perforations .
clear biomaterials capable of fighting bacterial or cellular invasions through drug release or surface modifications , could be manufactured to improve on current results .
furthermore , as discussed later , we are developing new biological membranes with complex fibrillar architecture to enhance cell proliferation while remaining transparent . the quest for alternative ,
autologous cell sources for regeneration has led to the use of different cell types for corneal epithelial reconstruction .
dental pulp - derived stem cells share some markers with lscs , and were effective for cornea reconstruction in a rabbit model of moderate and severe chemical burns .
another recently proposed source of stem cells for cornea epithelium is the hair follicle , cells from which were induced to express cytokeratin 12 in a mouse model of cornea regeneration .
mesenchymal stem cells ( mscs ) from bone marrow and umbilical cord were used to regenerate damaged corneas in animal models , with overall promising results .
mscs also were used in animal models of acute cornea surface damage to reduce inflammation and promote wound healing .
direct incorporation of these autologous stem cell sources on surface biomaterial implants may eventually become commonplace , especially for elderly or bilaterally affected patients , but further research is necessary and clinical practicality must be considered .
the most widespread alternative cell type that was tried clinically for corneal regeneration , however , is not a stem cell .
cultivated oral mucosal epithelial transplantation ( comet ) was successfully used to treat lsc deficiency in japan . in recent long - term results of comet , at 36 months , 42% of patients had at least a two - line improvement in best corrected visual acuity ( bcva ) and 53% had at least a one - line improvement .
another report shows a stable cornea surface in 53% of patients at three - year follow up , with failure - free survival of the surface in approximately 78% of patients .
these results are remarkable because , unlike stem cells , these cells are not expected to differentiate into corneal epithelial cells .
however , these cells provided adequate functionality to be clinically relevant , but are not regenerative per se . utilizing cells from tissues other than the cornea , however , is not free from complications and concerns .
there is an increased risk of peripheral cornea neo - vascularization and there are doubts as to the long - term survival of the cells .
a recent report has proven survival of the oral mucosal cells for at least one year after the procedure , but with only four patients reported and a very short follow up time . while comet will continue to be a viable clinical alternative for now
, there will be development of new sources of more efficient and effective cells or stem cells and means of delivery .
in contrast to corneal epithelial cells , endothelial cells can not be renewed or replaced during a lifetime .
this means that , as far as we know , there are no adult stem or progenitor cells routinely repopulating the endothelial layer . if damaged , it was generally believed that endothelial cells migrate , grow in size and cover the space left open , but do not proliferate .
there is the possibility of a dormant stem cell population on the schwalbe s ring region ( the transition between endothelium and the anterior portion of the trabecular meshwork ) capable of producing both endothelial and trabecular cells .
the existence of these putative stem cells does not change that the corneal endothelial cell layer does not regenerate in vivo , but it provides hope for the existence of a cell population that can be leveraged to generate a native endothelial cell layer for transplantation in the near future .
the traditional treatment for endothelial disorders is a full - thickness penetrating keratoplasty ( full cornea transplant ) .
more recently , deep lamellar endothelial keratoplasty ( transplant of the endothelium with descemet s membrane and a thin layer of stroma ) and even descemet membrane endothelial keratoplasty have been adopted .
there have been many different protocols and media compositions used to expand endothelial cells in vitro , with equally varying results .
so far , expansion seems dependent on the presence of serum in the media , which limits the clinical potential of any cells cultured .
different cell carriers have been tested in animal models , but problems remain regarding biomaterial transparency , structural strength and integration with native tissues . taken together , all these factors have prevented the development of clinically relevant cell therapies for reconstructing the corneal endothelial layer , but researchers continually search for solutions . corneal disease affects more than 10 million people worldwide and , after cataracts , is the second leading cause of blindness .
corneal transplantation is currently the standard treatment for restoring vision in the most severe cases .
the success rate is high . with the notable exception of north america , however , in most of the world the demand for high - quality donors exceeds the supply .
the future for maintaining the current high quantity and high quality of donors is uncertain because of aging populations , the increase in corneal refractive surgeries and infectious diseases .
such devices must fulfill the natural functions of the cornea : maintain transparency ; have an adequate refractive index ; protect the inner ocular structures from external hazards , including pathogens and ultraviolet ( uv ) light ; have proper mechanical properties to tolerate the intraocular pressure and allow diffusion of oxygen and nutrients .
in addition , as biomaterials , they must be biocompatible , nontoxic , neither immunogenic nor mutagenic and integrate well with the recipients surrounding tissues and cells .
there are two main substitute categories : synthetic keratoprostheses and , our main interest , tissue engineered corneal substitutes .
they consist of two main parts : the transparent optical center and the surrounding skirt designed to support the implant . depending on the type of kpro , additional supporting components may be found .
the optical region is commonly made of plastic polymers , including poly ( methyl methacrylate ) ( pmma ) , poly(2-hydroxyethyl methacrylate ) ( phema ) and polydimethylsiloxane ( pdms ) , which do not integrate well with the native tissue .
the major complications derive from integration problems and include device extrusion , stromal melting , epithelial thinning and persistent epithelial defects .
the boston kpro ( dohlman - doane keratoprosthesis ) is fda approved and is considered the gold standard in synthetic corneal substitutes .
type 1 consists of a central pmma optical region , a porous back plate and a titanium locking ring that locks on native corneal tissue .
the type 2 kpro was designed for patients suffering from severe diseases of the ocular surface , such as stevens 
johnson syndrome ( sjs ) and ocular cicatricial pemphigoid ( ocp ) , who are unable to maintain eyelid function . except for the additional anterior protruding rod in the central area ,
recent reports indicate high retention rates ( 84% at 17 months ) , good visual prognosis ( 75% better than 20/200 at one year ) and no endophthalmitis , excluding sjs and ocp patients . however , there are still relatively high complication rates in the sjs groups , presenting as retroprosthetic membrane formation and persistent epithelial defects . the osteo - odonto keratoprosthesis ( ookp ) , like the boston type 2 kpro , is primarily used for patients with severe dry eye .
the concept of the ookp derives from research showing that integration is maximized while immune reactions are minimized through the use of the patient s own tissue , including cartilage , and tibial bone .
the ookp uses a canine tooth , its alveolar bone and ligament as a skirt to support the central optic component . a retrospective study of the ookp shows high retention rates ( 85% ) over 19 years , including in sjs and ocp patients , with low complication rates .
even with the additional complex and invasive surgical procedures and the risk of resorption of the tissue , the ookp is the most successful and well known kpro in use today .
the alphacor kpro is manufactured by altering the water contents in phema during the polymerization of the optical center and the skirt .
unlike other kpros , the design of the alphacor utilizes the interpenetrating polymer network to permanently connect the two regions .
the porous phema allows for the vigorous invasion of keratocytes , resulting in a better integration with host tissue .
because of this stromal skirt repopulation , the retention rate is relatively high ( 87% , 58% and 42% at 1 , 2 and 3 years post - implantation , respectively ) .
unfortunately , even though there are promising short - term results , the fixation failures caused by stromal melting and intraoptic deposits still limit the usefulness of the alphacore kpro in the long term .
tissue engineering corneal substitutes promise to overcome the many challenges and deficiencies of synthetic materials .
tissue - engineered corneal substitutes are constructed by naturally generating an extracellular matrix ( ecm ) component with or without corneal cells .
it is well established that the ecm directs the fate of cells , therefore , the fabrication of the correct ecm components could produce an ideal corneal substitute , able to mimic the native corneal function . since the components of the ecm are natural ( biological ) polymers , there is higher biocompatibility and integration with host tissues when compared to synthetic polymeric materials , such as those in the different kpros .
in addition , these substitutes can be used to generate a targeted corneal layer , such as the endothelium for descemet s stripping endothelial keratoplasty ( dsek ) .
cosmetically , the tissue - engineered cornea is more similar to native cornea than the kpros and has the potential to provide a long - term or even permanent tissue replacement , indistinguishable from the original tissue .
they are highly versatile materials that allow for the diffusion of oxygen and nutrients , have adjustable mechanical properties , can be designed to contain large volumes of water and are relatively biocompatible . the corneal stroma comprises roughly 85% of the corneal thickness and consists of keratocytes and ecm . since collagen type i is the predominant component of the corneal stromal ecm ( approximately 70% of dry weight ) , it has been considered as the natural component to reconstruct it . as such
, a corneal substitute was developed using highly cross- linked collagen hydrogels using n-(3-dimethylaminopropyl)-n-ethylcarbodiimide ( edc ) and n - hydroxysuccinimide ( nhs ) technology .
it was successfully transplanted into several animal models , including guinea pig , dog and pig , either as a deep lamellar or full thickness keratoplasty .
recently , these collagen - based corneal substitutes were transplanted into 10 patients in a phase i clinical trial .
after 24 months , there were no severe adverse effects and the implants allowed for epithelial and keratocyte migration in all patients , as well as slow nerve invasion in the younger patients .
eight of the patients , however , developed focal hazing , which may critically reduce vision quality .
a long - term clinical study , with a large number of patients and further development of the material , will be needed if the material is to be used in clinical practice .
non - cross - linked collagen hydrogels are generally composed of loosely packed collagen fibers , strongly contrasting with the highly condensed and well organized collagen fibers found in the stromal layer of the cornea . recently , takezawa et al . , developed a type i collagen hydrogel membrane using a novel processing method , called vitrification , that we have been optimizing for ocular applications .
the collagen hydrogel membrane was named vitrigel , and is made from a type i collagen solution prepared through a three - stage sequence : gelation , vitrification and rehydration .
the vitrification step is unique in that it allows the collagen hydrogel to dry slowly so the collagen fibers can be reorganized and develop new hydrogen bonding interactions with each other . more importantly , unlike the opaque and comparably weak non - cross - linked collagen hydrogels , vitrigels are clear and possess greatly enhanced mechanical properties that are critical for ocular applications .
most important , with this method we begin to approach the more complex structure of the native cornea matrix with collagen fibers that are critical for both physical and biological function of the tissue [ figure 2c ] .
keratocytes cultured on vitrigels maintain a native dendritic morphology in serum - free [ a ] and 10% serum media [ b ] tem images of collagen vitrigel highlighting the fibrillar nanostructure of the matrix [ c ] bar = 500 nm using vitrigel , we evaluated the reconstruction of the three different corneal layers : epithelium , stroma and endothelium .
human lscs were cultured successfully on the vitrigel , demonstrating maintenance and upregulated expression of the putative stem cell markers abcg2 and p63 , respectively , thus indicating that vitrigels can preserve the lsc phenotype . in vivo , keratocytes exhibit a characteristic dendritic morphology with extensive intercellular contacts . however , when the corneal stroma is wounded , the cells transform either to wound - healing fibroblasts or to scar - forming myofibroblasts , which form a disorganized and opaque ecm .
therefore , it is desirable to promote the fibroblast phenotype and proliferative activity of keratocyte wound healing .
we found that keratocytes cultured on vitrigels exhibit enhanced dendritic branch density and length , compared to tissue culture plate control [ figure 2a - b ] .
an enhanced expression of aldehyde dehydrogenase ( aldh ) and keratocan ( important markers of keratocyte phenotype and function ) was also observed .
these results suggest that the use of vitrigel materials may benefit corneal stromal regeneration , as well as promote better integration and cellular function in vivo .
finally , promising results were obtained for endothelial culture on the vitrigels . in short ,
vitrigels demonstrate unique advantages over other materials ; these include optical transparency , mechanical strength and handling convenience .
these properties make vitrigels novel and very promising options for corneal tissue engineering and regeneration .
furthermore , as we analyze the collagen fibrillar structure of the vitrigels , we are gaining important insight into how fibers form .
we can apply this to understanding stroma structure - function properties in normal and diseased states . engineering a new cornea or subset of the cornea
however , we can also look to other strategies , including animal sources , to provide surgeons with a corneal substitute that has a structure similar to native tissue .
in particular , we can apply materials science and engineering strategies to process and create an implant from animal sources .
xenotransplantation can be employed to obtain corneal substitutes , particularly in developing countries that suffer from low donor rates because of regional , cultural and other institutional issues .
although rejection is the greatest obstacle to widespread use of xenotransplantation , fully decellularized components of the animal tissue may be clinically applied without immune rejection .
decellularization can be applied , not only to xenogeneic , but also to allogeneic cornea tissues for transplantation .
this could , in theory , improve the clinical outcome and reduce the complications associated with allogeneic cornea transplants .
a decellularized tissue provides a three - dimensional ecm structure for corneal reconstruction that fully mimics the native tissue and can be applied with or without the addition of a cellular component .
corneal decellularization has been performed on porcine corneas using various methods : sodium dodecyl sulfate ( sds ) , triton - x , hypertonic nacl solution , and high hydrostatic pressure .
these methods have been used by several groups in asia,[4651 ] and on bovine corneas by our group .
there are mixed results in the literature , however , with some reports failing to provide proper quantitative data on the presence of cell debris , which may cause immune reactions and affect transparency .
the relevance of cell debris can be seen in a recent article reporting that both untreated corneas as well as corneas in which cell debris was not removed experienced immune rejection in a primate model .
recently , our group successfully engineered decellularized corneas from both porcine and bovine tissue with a novel method , combining chemical and enzymatic treatments to remove the cellular components , but also incorporating vitrification and cross - linking steps to improve the optical and structural properties of the device . with this novel procedure , we were able to create acellular porcine and bovine corneas containing almost no cellular debris , while maintaining impressive light transmission rates and biocompatibility .
moreover , the curvature can be manipulated in this novel , engineered decellularized cornea , allowing for control of the refractive index , which is essential to corneal function .
 a recent study reported the results of 150 patients who received anterior lamellar keratoplasty with this product .
they showed high levels of epithelialization within a few days and no postoperative infection or rejection in all but four cases , all of which occurred in patients with preexisting corneal melting .
finally , instead of utilizing a biomaterials approach , researchers have also harnessed the capacity of cells to self - assemble to form biological corneal substitutes .
keratocyte - derived fibroblasts produce stromal ecm , and epithelial cells produce basement membrane . using this concept ,
a translucent ecm from corneal fibroblasts has been produced and coated with epithelial cells . even though the thickness of the ecm is
far from that of the native cornea , the model was able to mimic the microstructure and wound healing processes of the corneal surface . with further development
traumatic defects or even cataract incisions would benefit greatly from a repair strategy that includes both physical apposition ( i.e. tissue bonding ) and biological stimulation to promote new tissue development .
such technologies for local corneal repair includes tissue adhesive ( ta ) biomaterials that can form chemical , biological or physical bonds with the molecular residues from local tissue . before the development of tas for the eye ,
nylon sutures were the standard method to close corneal wounds caused by cataract incision , corneal ulcer , trauma or transplant .
these new materials offer to improve on the past regenerative results of sutures for surgical and non - surgical wounds by reducing operation time , minimizing scar formation . and providing a vehicle for delivery of biological signals that can stimulate tissue development .
numerous additional corneal adhesives have been developed over the past 10 years to address the unique requirements for sealing different types of wounds .
the material is prepared by processing plasma from autologous blood or from plasma pools of volunteer donations . in corneal surgery ,
fibrin glue has been employed to seal corneal perforations and it melts along with adhering amniotic membrane to the ocular surface ( alone and with lscs).the adhesive can also be applied in different kinds of keratoplasty and even to temporarily stabilize keratoprosthetics .
however , as a blood product , it carries the risk of disease transmission and its low tensile strength and fast degradation are significant drawbacks .
thus , there is a significant need for an ocular adhesive that has the biological benefits and compatibility of a biological adhesive but has the increased mechanical strength and durability specifically needed for ocular applications .
synthetic adhesives provide faster polymerization ( i.e. cure rate ) and higher tensile strength , making them good candidates for corneal wound closure devices . among all of the synthetic adhesives for corneal wound healing , polymeric hydrogels
for example , a dendritic linear copolymer based on poly ( ethylene glycol ) ( peg ) , glycerol and succinic acid was shown to be effective in closing linear , full - thickness corneal incisions .
after photocross linking using uv light , the adhesive produced a firm seal , matching the strength of sutures .
another version of the material that does not require light has been applied clinically in europe .
while these materials have the strength and durability required clinically , they have little biological activity to stimulate new tissue growth . to create an ocular adhesive that combines the benefits of both synthetic and biological materials
the first generation adhesive we developed was composed of a modified chondroitin sulfate ( cs ) , cs - aldehyde that , when combined with polyvinyl alcohol co - vinylamine ( pva - a ) , cross - linked together and with the surrounding tissue .
the aldehyde - amine reaction provided the basis for this adhesive ; however , the biocompatibility was not good .
therefore , we created a second generation material that combines cs and peg . in this case
, the cs is modified with succinimide groups and is chemically cross - linked with peg - amine molecules through a michael addition .
in addition to being more biocompatible , the cs - peg adhesive formed tight seals on corneal incisions to maintain a normal intraocular pressure . ultimately , the combination of the biological benefits of the cs , which includes reducing scar formation and inflammation , with the physical benefits of the synthetic peg , which provides durability , allows the creation of an optimal ocular tissue adhesive .
drugs , such as antibiotics or anti - inflammatory molecules , can also be incorporated into the adhesives based on clinical need . in hydrogel adhesives , biological factors
could either be encapsulated in the material or covalently bonded to the macromolecule , and dual or multiple delivery systems could also be developed .
our approach to developing new strategies for ocular reconstruction and regeneration is based on understanding the physical structure of a tissue or biomaterial and the resulting impact on biological function .
this structure - function principle is key , not only to developing new therapies , but also for understanding disease processes and designing more efficient therapies .
even the most advanced biological products today do not take into account the microscopic and molecular structure of the cornea , providing natural corneal cells with unnatural , cross - linked substrates .
if successful corneal regeneration will ever be accomplished , we must pay close attention to the natural structure and function of the native tissue .
there is a delicate balance in the relationship of cornea cells and their local micro- and nano - environments that allow the corneal tissue to maintain function throughout a lifetime . at the same time
, the loss of this homeostatic balance gives rise to different disease states , some of which can be addressed by taking a new , engineering design approach .
for example , given the fibrillar nature of the cornea stroma , the transparency of the tissue is a remarkable engineering feat .
understanding the matrix design , we are engineering biomimetic materials for the cornea with geometrically oriented biological nanofibers .
such reinforced materials could be used as stromal fillers or to treat conditions of stromal weakness , such as keratoconus .
future collagen materials for cornea reconstruction will likely be composed of recombinant human molecules containing the telopeptides to maximize the proteoglycan effect and fibril control .
if truly mimetic materials can be made , they will offer an option for patients who currently are unable to undergo refractive surgery because of a thin stroma .
furthermore , the importance of proteoglycans and their relationship to collagen fibril formation is evident in diseases such as keratoconus , where an increase in their stromal contents is accompanied by thinning and disarray of the fibers . ultimately , harnessing the biological cues that direct cell function during tissue repair will lead to improvements in repair and regeneration therapies targeted for the cornea .
the future of regeneration in the eye is , indeed , very promising . in the future
, we aim to control cell migration and remodeling of biomimetic materials to promote the restoration of a natural corneal structure , fully functional and useful for a lifetime .
furthermore , these strategies will be extended to engineering and replacing conjunctiva , lacrimal glands and other ocular and periocular tissue .
the recognition that stem cells possess the ability to both self renew and differentiate , is leading to the generation of adequate numbers of cells for delivery to patients . in the eye , limbal stem cells ( lscs ) were identified as the source of cells that continually generate the corneal epithelial layer .
more recently , corneal stromal stem cells , which can differentiate to keratocytes , were discovered .
finally , progenitor cells of the endothelium and trabeculum ( pcet ) , which seem to differentiate into endothelial cells , were also identified . as in organ transplantation ,
although use of lifelong or long - term immunosuppressive medication is possible , the best way to prevent rejection is to use the patient s own cells , which may be possible in unilateral eye disease or trauma .
the classic corneal epithelial cell transplantation procedure employs grafting of a healthy limbal biopsy , either from a healthy contra lateral eye or from a donor , onto the damaged cornea .
the technique was developed in the late 1980s and early 1990s and proved useful in severe surface diseases ( now narrowed to limbal stem cell deficiencies , ) .
the need for a greater number of cells for transplantation was soon evident , and efforts began to culture epithelial and limbal cells in vitro .
the goal of in vitro culture procedures was to obtain a layer of cells that could be used as a replacement for the native corneal epithelium .
denuded amniotic membrane is currently employed to improve culture conditions and facilitate culture and transplantation . in a recent review of the literature , baylis et al .
they also found that results differ according to disease etiology , with inflammatory conditions having the best outcomes ( 86% ) and congenital ones producing the worst ( 60% ) .
some of the most common complications reported for cell transplantation include infections , conjunctivalization and corneal perforations .
clear biomaterials capable of fighting bacterial or cellular invasions through drug release or surface modifications , could be manufactured to improve on current results .
furthermore , as discussed later , we are developing new biological membranes with complex fibrillar architecture to enhance cell proliferation while remaining transparent . the quest for alternative ,
autologous cell sources for regeneration has led to the use of different cell types for corneal epithelial reconstruction .
dental pulp - derived stem cells share some markers with lscs , and were effective for cornea reconstruction in a rabbit model of moderate and severe chemical burns .
another recently proposed source of stem cells for cornea epithelium is the hair follicle , cells from which were induced to express cytokeratin 12 in a mouse model of cornea regeneration .
mesenchymal stem cells ( mscs ) from bone marrow and umbilical cord were used to regenerate damaged corneas in animal models , with overall promising results .
mscs also were used in animal models of acute cornea surface damage to reduce inflammation and promote wound healing .
direct incorporation of these autologous stem cell sources on surface biomaterial implants may eventually become commonplace , especially for elderly or bilaterally affected patients , but further research is necessary and clinical practicality must be considered .
the most widespread alternative cell type that was tried clinically for corneal regeneration , however , is not a stem cell .
cultivated oral mucosal epithelial transplantation ( comet ) was successfully used to treat lsc deficiency in japan . in recent long - term results of comet , at 36 months , 42% of patients had at least a two - line improvement in best corrected visual acuity ( bcva ) and 53% had at least a one - line improvement .
another report shows a stable cornea surface in 53% of patients at three - year follow up , with failure - free survival of the surface in approximately 78% of patients .
these results are remarkable because , unlike stem cells , these cells are not expected to differentiate into corneal epithelial cells .
however , these cells provided adequate functionality to be clinically relevant , but are not regenerative per se . utilizing cells from tissues other than the cornea , however , is not free from complications and concerns .
there is an increased risk of peripheral cornea neo - vascularization and there are doubts as to the long - term survival of the cells .
a recent report has proven survival of the oral mucosal cells for at least one year after the procedure , but with only four patients reported and a very short follow up time . while comet will continue to be a viable clinical alternative for now
, there will be development of new sources of more efficient and effective cells or stem cells and means of delivery .
in contrast to corneal epithelial cells , endothelial cells can not be renewed or replaced during a lifetime .
this means that , as far as we know , there are no adult stem or progenitor cells routinely repopulating the endothelial layer .
if damaged , it was generally believed that endothelial cells migrate , grow in size and cover the space left open , but do not proliferate .
there is the possibility of a dormant stem cell population on the schwalbe s ring region ( the transition between endothelium and the anterior portion of the trabecular meshwork ) capable of producing both endothelial and trabecular cells .
the existence of these putative stem cells does not change that the corneal endothelial cell layer does not regenerate in vivo , but it provides hope for the existence of a cell population that can be leveraged to generate a native endothelial cell layer for transplantation in the near future .
the traditional treatment for endothelial disorders is a full - thickness penetrating keratoplasty ( full cornea transplant ) .
more recently , deep lamellar endothelial keratoplasty ( transplant of the endothelium with descemet s membrane and a thin layer of stroma ) and even descemet membrane endothelial keratoplasty have been adopted .
there have been many different protocols and media compositions used to expand endothelial cells in vitro , with equally varying results .
so far , expansion seems dependent on the presence of serum in the media , which limits the clinical potential of any cells cultured .
different cell carriers have been tested in animal models , but problems remain regarding biomaterial transparency , structural strength and integration with native tissues . taken together , all these factors have prevented the development of clinically relevant cell therapies for reconstructing the corneal endothelial layer , but researchers continually search for solutions .
corneal disease affects more than 10 million people worldwide and , after cataracts , is the second leading cause of blindness .
corneal transplantation is currently the standard treatment for restoring vision in the most severe cases .
the success rate is high . with the notable exception of north america , however , in most of the world the demand for high - quality donors exceeds the supply .
the future for maintaining the current high quantity and high quality of donors is uncertain because of aging populations , the increase in corneal refractive surgeries and infectious diseases .
such devices must fulfill the natural functions of the cornea : maintain transparency ; have an adequate refractive index ; protect the inner ocular structures from external hazards , including pathogens and ultraviolet ( uv ) light ; have proper mechanical properties to tolerate the intraocular pressure and allow diffusion of oxygen and nutrients .
in addition , as biomaterials , they must be biocompatible , nontoxic , neither immunogenic nor mutagenic and integrate well with the recipients surrounding tissues and cells .
there are two main substitute categories : synthetic keratoprostheses and , our main interest , tissue engineered corneal substitutes .
they consist of two main parts : the transparent optical center and the surrounding skirt designed to support the implant . depending on the type of kpro
the optical region is commonly made of plastic polymers , including poly ( methyl methacrylate ) ( pmma ) , poly(2-hydroxyethyl methacrylate ) ( phema ) and polydimethylsiloxane ( pdms ) , which do not integrate well with the native tissue .
the major complications derive from integration problems and include device extrusion , stromal melting , epithelial thinning and persistent epithelial defects .
the boston kpro ( dohlman - doane keratoprosthesis ) is fda approved and is considered the gold standard in synthetic corneal substitutes .
type 1 consists of a central pmma optical region , a porous back plate and a titanium locking ring that locks on native corneal tissue .
the type 2 kpro was designed for patients suffering from severe diseases of the ocular surface , such as stevens 
johnson syndrome ( sjs ) and ocular cicatricial pemphigoid ( ocp ) , who are unable to maintain eyelid function . except for the additional anterior protruding rod in the central area ,
recent reports indicate high retention rates ( 84% at 17 months ) , good visual prognosis ( 75% better than 20/200 at one year ) and no endophthalmitis , excluding sjs and ocp patients . however , there are still relatively high complication rates in the sjs groups , presenting as retroprosthetic membrane formation and persistent epithelial defects . the osteo - odonto keratoprosthesis ( ookp ) , like the boston type 2 kpro , is primarily used for patients with severe dry eye .
the concept of the ookp derives from research showing that integration is maximized while immune reactions are minimized through the use of the patient s own tissue , including cartilage , and tibial bone .
the ookp uses a canine tooth , its alveolar bone and ligament as a skirt to support the central optic component .
a retrospective study of the ookp shows high retention rates ( 85% ) over 19 years , including in sjs and ocp patients , with low complication rates .
even with the additional complex and invasive surgical procedures and the risk of resorption of the tissue , the ookp is the most successful and well known kpro in use today .
the alphacor kpro is manufactured by altering the water contents in phema during the polymerization of the optical center and the skirt .
unlike other kpros , the design of the alphacor utilizes the interpenetrating polymer network to permanently connect the two regions .
the porous phema allows for the vigorous invasion of keratocytes , resulting in a better integration with host tissue .
because of this stromal skirt repopulation , the retention rate is relatively high ( 87% , 58% and 42% at 1 , 2 and 3 years post - implantation , respectively ) .
unfortunately , even though there are promising short - term results , the fixation failures caused by stromal melting and intraoptic deposits still limit the usefulness of the alphacore kpro in the long term . tissue engineering corneal substitutes promise to overcome the many challenges and deficiencies of synthetic materials .
tissue - engineered corneal substitutes are constructed by naturally generating an extracellular matrix ( ecm ) component with or without corneal cells .
it is well established that the ecm directs the fate of cells , therefore , the fabrication of the correct ecm components could produce an ideal corneal substitute , able to mimic the native corneal function . since the components of the ecm are natural ( biological ) polymers , there is higher biocompatibility and integration with host tissues when compared to synthetic polymeric materials , such as those in the different kpros .
in addition , these substitutes can be used to generate a targeted corneal layer , such as the endothelium for descemet s stripping endothelial keratoplasty ( dsek ) .
cosmetically , the tissue - engineered cornea is more similar to native cornea than the kpros and has the potential to provide a long - term or even permanent tissue replacement , indistinguishable from the original tissue .
they are highly versatile materials that allow for the diffusion of oxygen and nutrients , have adjustable mechanical properties , can be designed to contain large volumes of water and are relatively biocompatible .
the corneal stroma comprises roughly 85% of the corneal thickness and consists of keratocytes and ecm .
since collagen type i is the predominant component of the corneal stromal ecm ( approximately 70% of dry weight ) , it has been considered as the natural component to reconstruct it . as such
, a corneal substitute was developed using highly cross- linked collagen hydrogels using n-(3-dimethylaminopropyl)-n-ethylcarbodiimide ( edc ) and n - hydroxysuccinimide ( nhs ) technology .
it was successfully transplanted into several animal models , including guinea pig , dog and pig , either as a deep lamellar or full thickness keratoplasty .
recently , these collagen - based corneal substitutes were transplanted into 10 patients in a phase i clinical trial .
after 24 months , there were no severe adverse effects and the implants allowed for epithelial and keratocyte migration in all patients , as well as slow nerve invasion in the younger patients .
eight of the patients , however , developed focal hazing , which may critically reduce vision quality .
a long - term clinical study , with a large number of patients and further development of the material , will be needed if the material is to be used in clinical practice .
non - cross - linked collagen hydrogels are generally composed of loosely packed collagen fibers , strongly contrasting with the highly condensed and well organized collagen fibers found in the stromal layer of the cornea . recently , takezawa et al . , developed a type i collagen hydrogel membrane using a novel processing method , called vitrification , that we have been optimizing for ocular applications .
the collagen hydrogel membrane was named vitrigel , and is made from a type i collagen solution prepared through a three - stage sequence : gelation , vitrification and rehydration .
the vitrification step is unique in that it allows the collagen hydrogel to dry slowly so the collagen fibers can be reorganized and develop new hydrogen bonding interactions with each other . more importantly , unlike the opaque and comparably weak non - cross - linked collagen hydrogels
, vitrigels are clear and possess greatly enhanced mechanical properties that are critical for ocular applications .
most important , with this method we begin to approach the more complex structure of the native cornea matrix with collagen fibers that are critical for both physical and biological function of the tissue [ figure 2c ] .
keratocytes cultured on vitrigels maintain a native dendritic morphology in serum - free [ a ] and 10% serum media [ b ] tem images of collagen vitrigel highlighting the fibrillar nanostructure of the matrix [ c ] bar = 500 nm using vitrigel , we evaluated the reconstruction of the three different corneal layers : epithelium , stroma and endothelium .
human lscs were cultured successfully on the vitrigel , demonstrating maintenance and upregulated expression of the putative stem cell markers abcg2 and p63 , respectively , thus indicating that vitrigels can preserve the lsc phenotype . in vivo , keratocytes exhibit a characteristic dendritic morphology with extensive intercellular contacts . however , when the corneal stroma is wounded , the cells transform either to wound - healing fibroblasts or to scar - forming myofibroblasts , which form a disorganized and opaque ecm .
therefore , it is desirable to promote the fibroblast phenotype and proliferative activity of keratocyte wound healing .
we found that keratocytes cultured on vitrigels exhibit enhanced dendritic branch density and length , compared to tissue culture plate control [ figure 2a - b ] .
an enhanced expression of aldehyde dehydrogenase ( aldh ) and keratocan ( important markers of keratocyte phenotype and function ) was also observed .
these results suggest that the use of vitrigel materials may benefit corneal stromal regeneration , as well as promote better integration and cellular function in vivo .
finally , promising results were obtained for endothelial culture on the vitrigels . in short ,
vitrigels demonstrate unique advantages over other materials ; these include optical transparency , mechanical strength and handling convenience .
these properties make vitrigels novel and very promising options for corneal tissue engineering and regeneration .
furthermore , as we analyze the collagen fibrillar structure of the vitrigels , we are gaining important insight into how fibers form .
we can apply this to understanding stroma structure - function properties in normal and diseased states . engineering a new cornea or subset of the cornea
however , we can also look to other strategies , including animal sources , to provide surgeons with a corneal substitute that has a structure similar to native tissue .
in particular , we can apply materials science and engineering strategies to process and create an implant from animal sources .
xenotransplantation can be employed to obtain corneal substitutes , particularly in developing countries that suffer from low donor rates because of regional , cultural and other institutional issues .
although rejection is the greatest obstacle to widespread use of xenotransplantation , fully decellularized components of the animal tissue may be clinically applied without immune rejection .
decellularization can be applied , not only to xenogeneic , but also to allogeneic cornea tissues for transplantation .
this could , in theory , improve the clinical outcome and reduce the complications associated with allogeneic cornea transplants .
a decellularized tissue provides a three - dimensional ecm structure for corneal reconstruction that fully mimics the native tissue and can be applied with or without the addition of a cellular component .
corneal decellularization has been performed on porcine corneas using various methods : sodium dodecyl sulfate ( sds ) , triton - x , hypertonic nacl solution , and high hydrostatic pressure .
these methods have been used by several groups in asia,[4651 ] and on bovine corneas by our group .
there are mixed results in the literature , however , with some reports failing to provide proper quantitative data on the presence of cell debris , which may cause immune reactions and affect transparency .
the relevance of cell debris can be seen in a recent article reporting that both untreated corneas as well as corneas in which cell debris was not removed experienced immune rejection in a primate model .
recently , our group successfully engineered decellularized corneas from both porcine and bovine tissue with a novel method , combining chemical and enzymatic treatments to remove the cellular components , but also incorporating vitrification and cross - linking steps to improve the optical and structural properties of the device . with this novel procedure , we were able to create acellular porcine and bovine corneas containing almost no cellular debris , while maintaining impressive light transmission rates and biocompatibility .
moreover , the curvature can be manipulated in this novel , engineered decellularized cornea , allowing for control of the refractive index , which is essential to corneal function .
 a recent study reported the results of 150 patients who received anterior lamellar keratoplasty with this product .
they showed high levels of epithelialization within a few days and no postoperative infection or rejection in all but four cases , all of which occurred in patients with preexisting corneal melting .
finally , instead of utilizing a biomaterials approach , researchers have also harnessed the capacity of cells to self - assemble to form biological corneal substitutes .
keratocyte - derived fibroblasts produce stromal ecm , and epithelial cells produce basement membrane . using this concept ,
a translucent ecm from corneal fibroblasts has been produced and coated with epithelial cells . even though the thickness of the ecm is
far from that of the native cornea , the model was able to mimic the microstructure and wound healing processes of the corneal surface . with further development , this approach may be used to engineer a full - thickness corneal substitute .
traumatic defects or even cataract incisions would benefit greatly from a repair strategy that includes both physical apposition ( i.e. tissue bonding ) and biological stimulation to promote new tissue development .
such technologies for local corneal repair includes tissue adhesive ( ta ) biomaterials that can form chemical , biological or physical bonds with the molecular residues from local tissue . before the development of tas for the eye ,
nylon sutures were the standard method to close corneal wounds caused by cataract incision , corneal ulcer , trauma or transplant .
these new materials offer to improve on the past regenerative results of sutures for surgical and non - surgical wounds by reducing operation time , minimizing scar formation . and providing a vehicle for delivery of biological signals that can stimulate tissue development .
numerous additional corneal adhesives have been developed over the past 10 years to address the unique requirements for sealing different types of wounds .
the material is prepared by processing plasma from autologous blood or from plasma pools of volunteer donations . in corneal surgery ,
fibrin glue has been employed to seal corneal perforations and it melts along with adhering amniotic membrane to the ocular surface ( alone and with lscs).the adhesive can also be applied in different kinds of keratoplasty and even to temporarily stabilize keratoprosthetics .
however , as a blood product , it carries the risk of disease transmission and its low tensile strength and fast degradation are significant drawbacks .
thus , there is a significant need for an ocular adhesive that has the biological benefits and compatibility of a biological adhesive but has the increased mechanical strength and durability specifically needed for ocular applications .
synthetic adhesives provide faster polymerization ( i.e. cure rate ) and higher tensile strength , making them good candidates for corneal wound closure devices . among all of the synthetic adhesives for corneal wound healing , polymeric hydrogels
for example , a dendritic linear copolymer based on poly ( ethylene glycol ) ( peg ) , glycerol and succinic acid was shown to be effective in closing linear , full - thickness corneal incisions .
after photocross linking using uv light , the adhesive produced a firm seal , matching the strength of sutures .
another version of the material that does not require light has been applied clinically in europe .
while these materials have the strength and durability required clinically , they have little biological activity to stimulate new tissue growth .
to create an ocular adhesive that combines the benefits of both synthetic and biological materials , we engineered a biosynthetic ocular adhesive .
the first generation adhesive we developed was composed of a modified chondroitin sulfate ( cs ) , cs - aldehyde that , when combined with polyvinyl alcohol co - vinylamine ( pva - a ) , cross - linked together and with the surrounding tissue .
the aldehyde - amine reaction provided the basis for this adhesive ; however , the biocompatibility was not good .
therefore , we created a second generation material that combines cs and peg . in this case
, the cs is modified with succinimide groups and is chemically cross - linked with peg - amine molecules through a michael addition .
in addition to being more biocompatible , the cs - peg adhesive formed tight seals on corneal incisions to maintain a normal intraocular pressure .
ultimately , the combination of the biological benefits of the cs , which includes reducing scar formation and inflammation , with the physical benefits of the synthetic peg , which provides durability , allows the creation of an optimal ocular tissue adhesive .
drugs , such as antibiotics or anti - inflammatory molecules , can also be incorporated into the adhesives based on clinical need . in hydrogel adhesives , biological factors
could either be encapsulated in the material or covalently bonded to the macromolecule , and dual or multiple delivery systems could also be developed .
our approach to developing new strategies for ocular reconstruction and regeneration is based on understanding the physical structure of a tissue or biomaterial and the resulting impact on biological function .
this structure - function principle is key , not only to developing new therapies , but also for understanding disease processes and designing more efficient therapies .
even the most advanced biological products today do not take into account the microscopic and molecular structure of the cornea , providing natural corneal cells with unnatural , cross - linked substrates .
if successful corneal regeneration will ever be accomplished , we must pay close attention to the natural structure and function of the native tissue .
there is a delicate balance in the relationship of cornea cells and their local micro- and nano - environments that allow the corneal tissue to maintain function throughout a lifetime . at the same time
, the loss of this homeostatic balance gives rise to different disease states , some of which can be addressed by taking a new , engineering design approach .
for example , given the fibrillar nature of the cornea stroma , the transparency of the tissue is a remarkable engineering feat .
understanding the matrix design , we are engineering biomimetic materials for the cornea with geometrically oriented biological nanofibers .
such reinforced materials could be used as stromal fillers or to treat conditions of stromal weakness , such as keratoconus .
future collagen materials for cornea reconstruction will likely be composed of recombinant human molecules containing the telopeptides to maximize the proteoglycan effect and fibril control .
if truly mimetic materials can be made , they will offer an option for patients who currently are unable to undergo refractive surgery because of a thin stroma .
furthermore , the importance of proteoglycans and their relationship to collagen fibril formation is evident in diseases such as keratoconus , where an increase in their stromal contents is accompanied by thinning and disarray of the fibers . ultimately , harnessing the biological cues that direct cell function during tissue repair will lead to improvements in repair and regeneration therapies targeted for the cornea .
the future of regeneration in the eye is , indeed , very promising . in the future
, we aim to control cell migration and remodeling of biomimetic materials to promote the restoration of a natural corneal structure , fully functional and useful for a lifetime .
furthermore , these strategies will be extended to engineering and replacing conjunctiva , lacrimal glands and other ocular and periocular tissue .
 repair and reconstruction of the cornea has historically relied on synthetic materials or tissue transplantation . 
 however , the future holds promise for treatments using smart biomaterials and stem cells that direct tissue repair and regeneration to ultimately create new ocular structures that are indistinguishable from the original native tissue . the cornea is a remarkable engineering structure . by understanding the physical structure of the tissue and the resulting impact of the structure on biological function , we can design novel materials for a number of ophthalmic clinical applications . 
 furthermore , by extending this structure - function approach to characterizing corneal disease processes , new therapies can be engineered . 
INTRODUCTION
Cellular therapies
Biomaterials in the cornea
Reconstructive biomaterials
Applying engineering design to corneal disease