Ординатура / Офтальмология / Английские материалы / Biomaterials and regenerative medicine in ophthalmology_Chirila_2010

synthetic hydrogels in an in vitro set-up using human cadaver eyes (Kang et al., 2005). These two dendron-based hydrogels differ in the polymerization technique that has been used; one used an argon laser and the other used self-gelling chemistry. The laser-activated hydrogel sealant was made from a first-generation PGLSA (poly(glycerol-succinic acid)) dendrimer that has methacrylate groups on its outer surface. After applying 60 μL of a 5 wt% adhesive solution by using the bottom side of a keratome blade on the flap edge, low intensity pulses of argon laser (Coherent SE 2000 argon

ion laser, λmax= 488 and 514 nm, 200 mW, 100 1-second exposures) were used to induce the polymerization of the solution into a clear, soft, flexible

hydrogel, which secured the flap even when excessive force was applied with the Merocel sponge. To reduce the laser exposure to the patient and to obtain faster adhesive curing time (5 min in total with the laser-activated hydrogel), a self-gelling adhesive was tested. This two-component selfgelling adhesive cures via a chemical reaction, and it was prepared by mixing aqueous solutions of a cysteine-based dendron (33 wt% in phosphate buffer,

7.4 pH) and PEG di-aldehyde (55 wt% in phosphate buffer, 7.4 pH). After mixing these two solutions for 5 seconds, the bottom side of a keratome blade was used to apply the hydrogel on the flap edge. The hydrogel cured within 30 seconds, resulting again in a clear, soft, flexible hydrogel that was able to secure the flap even when excessive force was applied with the Merocel sponge. Fluorescein was also injected under the flap to verify if this adhesive could, in addition to securing the flap, potentially prevent ocular surface fluid from entering the sealed flap. None of the six eyes used in this study showed fluorescein leakage after application of the hydrogel, demonstrating that this sealant could be an effective protective barrier for the wound site.

Corneal transplants (penetrating keratoplasty and posterior lamellar keratoplasty)

Even though corneal transplants are the most successful human tissue transplants, they still represent a significant surgical challenge for ophthalmic surgeons. The conventional method (use of sutures) requires high surgical skill in order to prevent the occurrence of astigmatisms or infections. Currently, the graft is secured to the recipient’s eye using either multiple sutures (typically 16) or uninterrupted running sutures. Sutures induce additional trauma and lead to corneal distortion, which may, in turn, cause astigmatism. In order to reduce distortion, surgeons usually use one or two uninterrupted running sutures, but these techniques require more technical skills and may still lead to infection through the corneal gap. Hydrogel sealants, therefore, have been envisioned to improve clinical outcomes by preventing astigmatism, reducing or eliminating altogether additional suture trauma, securing

form the hydrogel adhesive. Using a 20% (w/v) of [G1] PGLSA-MA-PEG, containing either 3400, 10 000, or 20 000 PEG cores and 16 sutures, the leaking pressures were measured to be 103 ± 13, 114 ± 7, and 98 ± 14 mmHg, respectively, significantly greater than an autograft sealed with 16 sutures alone that held to 45 ± 10 mmHg. Next, they determined the leaking pressures of corneal autografts, initially secured with only 8 sutures, followed by the application and photocrosslinking of these three hydrogel adhesives at 20% (w/v). These studies were initiated to determine if, by using a sealant, a reduced number of sutures could be used to seal a PKP in order potentially to reduce the likelihood of astigmatism, to decrease overall surgery time, and to reduce tissue damage during the surgery. Using hydrogel sealants of 20%

(w/v) of ([G1]-PGLSA-MA)2-PEG10 000, and ([G1]-PGLSA-MA)2-PEG20 000 gave leaking pressures of 85 ± 22, and 81 ± 30 mmHg, respectively. For

reference, the autografts sealed with 8 sutures had a leaking pressure of less than 5 mmHg. The capability of these adhesives to prevent bacterial infection was also assessed using an India ink method described elsewhere (Mcdonnell et al., 2003). Even after several cycles of increasing and lowering the IOP, no India ink was detected, neither in the wound tissue interface nor inside the ocular cavity. These positive results show the potential benefits (namely, reductions in bacterial infection, astigmatism, and surgical time) of using a hydrogel sealant as a new alternative to secure corneal transplants with the standard number or a reduced number of sutures.

Next, Wathier and coworkers evaluated the ability of a self-crosslinking dendrimer-based hydrogel to seal PKP on human enucleated eyes (Wathier et al., 2006a). For this study, a self-gelling hydrogel made from two components was used. The first component was a solution of modified PEG (MW 3400) bearing ester–aldehyde moieties at each end. The second component was a solution of lysine-based dendrimer bearing cysteine moieties on its periphery. Using this new ester–aldehyde system, the authors were able to use a new hydrogel crosslinking reaction (an intramolecular O,N–acyl rearrangement) that resulted in a hydrogel adhesive more stable (month vs days) than their previously reported self-crosslinking hydrogel (Wathier et al., 2004), and therefore more effective for this type of surgery which involves a longer healing time. In this study, either 16 or 8 sutures were used in addition to the hydrogel sealant to secure the PKP. As a case in point, using 60 μL of a 50 wt% hydrogel mixture and 8 sutures, the PKP held to a leaking pressure of 77 mmHg compared with 5 mmHg for the 8 sutures-treated group). The India ink method mentioned above (Mcdonnell et al., 2003) was again used to assess the ability of these hydrogels to act as a barrier to the flow of surface ocular fluid as a model for prevention of microbial infections. The hydrogel sealant secures the wound site preventing fluid from entering or exiting the wound. These new materials were also evaluated for their ability to decrease the duration

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of the surgical procedure and to reduce the number of sutures to prevent astigmatism. They concluded that the new hydrogels were easy to use and to apply, also they were transparent, soft enough to avoid eye irritation, elastic enough to prevent astigmatisms stable enough to keep the wound sealed during the healing process, strong enough to hold the IOP, and sealed enough to prevent microbial infections. Even though these parameters are all requirements for an ideal ocular hydrogel sealant, no in vivo data have yet been reported on these materials.

Clear corneal laceration cataract

Since Fine introduced the sutureless self-sealing cataract incision (Fine 1994), several papers have reported a correlated increase in endophthalmitis following surgery. The cause of the infections is the lack of a sealed wound and the resulting flow of ocular surface fluid (carrying the bacteria) into the chamber. This ocular surface fluid movement is the result of dynamic changes of the gap geometry, especially at low pressure and IOP fluctuations

(Mcdonnell et al., 2003; Taban et al., 2005; Thoms et al., 2007) or the use of topical anesthesia, which does not prevent long-term extraocular muscle akinesia (Ellis, 2003; Faulkner, 2007). In all cases, endophthalmitis might be prevented if the wound could be secured with a hydrogel adhesive. Thus, to evaluate a hydrogel adhesive for sealing a cataract incision, Grinstaff, in collaboration with Kim at Duke University (North Carolina, USA), used dendritic hydrogels to secure a cataract incision (Wathier et al., 2004; Johnson et al., 2009). A two-component adhesive was used in these studies, one part being a lysine-based dendrimer bearing cysteine moieties on its periphery, and the other part being a PEG di-aldehyde (MW 3400). Even though the early study showed promising results, the system was further improved by using a slightly different PEG and, additionally, using the India ink method (Mcdonnell et al., 2003) it was confirmed that these hydrogel sealants could act as a barrier for microbial infections. In this study the wound was sealed up to 140 mmHg (± 22 mmHg), whereas an untreated wound leaked at 77 mmHg (± 14 mmHg). It was also noteworthy that even at low or high IOP the wound did not leak, confirming the effectiveness of hydrogel sealants in securing and the tissue preventing surface ocular fluid from penetrating the eye with IOP fluctuations.

Given the high number of cataract procedures performed each year and the significant clinical interest in preventing endophthalmitis, Reyes and coworkers also explored the use of a chondroitin sulfate (CS)-based hydrogel adhesive to secure a 3 mm clear corneal laceration (Reyes et al., 2005). Their self-gelling hydrogel was made from CS-aldehyde with poly(vinyl alcohol- co-vinyl amine) (PVA-A) as a bridging reagent. This gel set in less than

30 seconds after mixing the two components as a consequence of a Schiff

base reaction. The authors tested the leaking pressure of their hydrogel by increasing the IOP of the eye using a saline solution on enucleated rabbit eyes and compared their results with two control groups: one using only one 10-0 nylon suture (group 1) and the other using three 10-0 nylon sutures (group 2). Using this hydrogel, they were unable to detect a leak up to 100 mmHg (upper limit of their laboratory set-up), whereas suture groups 1 and 2 leaked at 26 and 44 mmHg, respectively. Once again these results support the use of a hydrogel adhesive to secure corneal wounds. As before, sealants that can effectively seal the tissue and prevent surface ocular fluid from entering the eye will likely reduce the risk of endophthalmitis.

16.3.4 Retinal applications

Retinal detachment may lead to vision loss and blindness if not treated rapidly.

The peeling of the retina is mainly a result of either injury or inflammation; this inflammation is initially more localized before spreading to the whole ocular globe if not treated. The use of a hydrogel sealant for repairing a retinal detachment is more challenging than in the cases of corneal or scleral wounds, since the sealant has to be applied under wet conditions. Margalit and coworkers used a variety of different adhesives to treat retinal detachment, among them four hydrogel adhesives, one fibrin and three PEG- based hydrogels (Margalit et al., 2000). After preliminary in vitro studies, they found the fibrin glue to be inadequate to treat this kind of wound since the hydrogel made from it, in wet conditions, is too weak. Thus, they focused their study on the three PEG-based hydrogels. The three different hydrogel mixtures were: SS-PEG (succinic succinimidyl ester) plus PEG-NH2, SPAPEG (propionic succinimidyl ester) plus PEG-NH2, and ST-PEG (thiol) plus PEG-NHS (succinimidyl ester). First, they checked the strength of the hydrogel adhesive on retinal tissue using a laboratory strain gauge set-up. They found the three formulations to be strong enough to hold the retina in place. The next step was to check the in vivo cytotoxicity of these materials in rabbits by injecting 0.1 mL of the hydrogel into the ocular globe. The

SPA-PEG/PEG-NH2 formulations showed severe inflammation responses, which hampers their potential clinical use. The other two formulations caused only a mild inflammatory response that disappeared after the first week of treatment. Unfortunately, the histology of the ST-PEG/PEG-NHS formulation showed moderate damage to the photoreceptor layer of the retina, which also removed it from the list of potential retinal adhesives. Despite a short lasting time (72 hours), the SS-PEG/PEG-NH2 formulation could be used as new hydrogel sealant in the treatment of those wounds that only require a short healing time.

In 2007, Sueda and coworkers re-examined the use of PEG-NHS in conjunction with an amine (a tripeptide of lysine) as a hydrogel sealant

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to fix retinal breaks (Sueda et al., 2007). In their study, the authors used a modified formulation of a commercially available product, DuraSeal (Confluent Surgical Inc., Massachusetts, USA), which was designed to seal the dura. Unfortunately, even though this new hydrogel did not show signs of inflammation or abnormality by histology after 90 days in rabbit, its use in the in vivo study led to a 67% failure rate (re-detachment of the retina). This high failure rate was explained by poor mixing of the components and by the difficulty of application of this hydrogel under wet conditions. The authors noted that additional studies are under way to fix these drawbacks and to allow this hydrogel to be used for retinal surgery.

Another study evaluated a new gelatin-based adhesive that formed a hydrogel in the presence of an enzyme, microbial transglutaminase (mTG), to repair a retinal detachment (Chen et al., 2006). Using a lap-shear test and scleral tissue flaps, the authors showed that this hydrogel can glue both flaps together up to 15–45 kPa, even under wet conditions. Furthermore, to evaluate the cytotoxicity of this hydrogel, they injected it into the vitreous cavity of rats. After 2 weeks, the animals were killed and histological sections of the eyes were obtained. No signs of necrosis, severe tissue damage, or inflammation were noted in the treated group.

16.4Short commentary on future trends

The future is extremely positive and bright for the use of hydrogel sealants in ophthalmology. Given the successes with both natural and synthetic hydrogel sealants when used to repair corneal wounds, fix retinal detachments, secure amniotic membrane transplants, and seal scleral lacerations, a clear definition of the design requirements has emerged. These requirements, in addition to the obvious one of biocompatibility, include: restoration of the tissue function; adhesion to moist corneal, scleral, or retinal tissue; set-up time to facilitate application to the wound and subsequent sealing; degradation time of sealant to match the healing process; mechanical properties to favor tissue function while preventing further complications (e.g. astigmatism); transparency for clinical observation after treatment; and easy delivery to the tissue site by the clinician.

The next steps for hydrogel sealants are threefold. First, there must be continued research and development of sealants for applications in the back of the eye. Much of the work completed has focused solely on the front of the eye. Second, the use of these hydrogels for drug delivery applications should continue to be explored. Drug delivery in the eye is still a major challenge and the lessons learned in these studies should be applicable to advancing this area. Third, the work in the laboratory must lead to the transition of hydrogel sealant technology for repairing wounds to the clinic and must result in development of a product. There has been significant progress

on this front. The team at HyperBranch Medical Technology (HBMT) has developed and commercialized the first ophthalmic bandage (OcuSealTM). After extensive in vitro and in vivo studies, as well as successful completion of the required pre-clinical safety studies, HBMT conducted a clinical trial for use of OcuSealTM to seal cataract incisions. In the fall of 2007, they obtained a CE Mark to sell the product in Europe. Today, thousands of cataract incisions have been sealed with OcuSealTM. In addition to providing a closed wound, the hydrogel bandage is delivered in a unique device that enables the clinician to ‘paint’ the sealant on the wound.

Continued research, development, and commercial activities with hydrogel sealants will advance our scientific and engineering knowledge of these materials and, perhaps more importantly, help patients through improved ways to treat ocular wounds. What else may be possible in the future? The possibilities are many and, by working together as a multidisciplinary team consisting of chemists, engineers, and clinicians, we will continue to solve current challenges as well as propose innovative solutions for our unmet clinical needs. In the ophthalmic sealant area, we see: (a) task-specific hydrogel sealants designed for a particular wound type, and (b) hydrogel sealants designed for an individual based on his/her own genetics and wound- healing capabilities, i.e. personalized medical sealants.

16.5Sources of further information and advice

Articles have been found using either (a) key words (e.g. hydrogel, ocular sealant or glue, gel, adhesive, etc.) on the following search engines: SciFinder, Google scholar, and Web of Science or (b) publication citations. The articles have been downloaded online directly from the publisher or Science Direct when available, or they have been ordered through the library exchange system. More information about the OcuSeal™ ophthalmic bandage can be found at http://www.hyperbranch.com/. Other resources may be found at the American Academy of Ophthalmology (http://www.aao.org/) or at the Association for Research in Vision and Ophthalmology (ARVO) http:// www.arvo.org.

16.6Acknowledgements

Our research has been supported in part by the National Institutes of Health

(NIH), the Pew Foundation, and Boston University. We would like to thank our collaborator, Dr Terry Kim (Duke University Eye Center), and his fellows and students who worked on these projects. We would also like to thank the following past and current graduate students and postdoctoral fellows from the Grinstaff Laboratory for their hard work and dedication to ophthalmic adhesive research: Jason Berlin, Michael Carnahan, Lovorka

430 Biomaterials and regenerative medicine in ophthalmology

Degoricija, Nathanael Luman, Meredith Morgan, Abigail Oelker, Kimberly Smeds, and Serge Söntjens.

Conflict of interest: M.W.G. is a co-founder of HBMT.

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