A method for generating zonular tension in the murine eye by embedding and compressing the globe in a hydrogel.

The ocular lens is the primary organ within the eye responsible for accommodation. During accommodation, the lens is subject to biomechanical forces. We previously demonstrated that stretching the porcine lens can increase lens epithelial cell proliferation. Although murine lenses are commonly employed in lens research, murine lens stretching has remained unexplored. Murine lens stretching thus represents a novel source of potential discovery in lens research. In the present study, we describe a method for stretching the murine lens by compressing the murine globe embedded in a hydrogel. We hypothesized that, as the eye is compressed along the optic axis, the lens would stretch through zonular tension due to the equatorial region of the eye bulging outward. Our results showed that this led to a compression-dependent increase in murine lens epithelial cell proliferation, suggesting that compression of the embedded murine globe is a viable technique for studying the mechanobiology of the lens epithelium.

Mathematical Modeling of Drug Delivery from Bi-Layered Core-Shell Polymeric Microspheres.

Chronic diseases usually require repetitive dosing. Depending on factors such as dosing frequency, mode of administration, and associated costs this can result in poor patient compliance. A better alternative involves using drug delivery systems to reduce the frequency of dosing and extend drug release. However, reaching the market stage is a time-consuming process. In this study, we used two numerical approaches for estimating the values of the critical parameters that govern the diffusion-controlled drug release within bilayered core-shell microspheres. Specifically, the estimated parameters include burst release, drug diffusion coefficient in two polymers, and the drug partition coefficient. Estimating these parameters provides insight for optimizing device design, guiding experimental efforts, and improving the device's effectiveness. We obtained good agreement between the models and the experimental data. The methods explored in this work apply not only to bi-layered spherical systems but can also be extended to multi-layered spherical systems.

Computer Modeling of Bevacizumab Drug Distribution After Intravitreal Injection in Rabbit and Human Eyes.

Age-related macular degeneration (AMD) is a condition brought on by macular deterioration caused primarily by inflammation and cell death in the retina. There is no cure for the disease and current treatments for advanced (wet) AMD rely on intravitreal injections of anti-vascular endothelial growth factor (anti-VEGF) therapeutics. One common off-label anti-VEGF drug used in AMD treatment is bevacizumab. There have been experimental efforts to investigate the pharmacokinetic (PK) behavior of bevacizumab in the vitreous and aqueous humor. Still the quantitative effect of elimination routes and drug concentration in the macula are not well understood. In our study, we developed two spatial models representing rabbit and human vitreous humor to better understand the PK behavior of bevacizumab. We explored convective effects on the vitreous while considering the anterior elimination alone or coupled with posterior elimination. We compared our models with available experimental data and calculated an approximate macula concentration. Our results show that both anterior and posterior elimination play a role in bevacizumab clearance from the eye. Furthermore, an effective bevacizumab concentration close to the macula region is maintained for shorter time periods when compared to the whole vitreous region. This model can improve knowledge and understanding of AMD treatment.

Experimental and mathematical approaches for drug delivery for the treatment of wet age-related macular degeneration.

Several chronic eye diseases affect the posterior segment of the eye. Among them age-related macular degeneration can cause vision loss if left untreated and is one of the leading causes of visual impairment in the world. Most treatments are based on intravitreally injected therapeutics that inhibit the action of vascular endothelial growth factor. However, due to the need for monthly injections, this method is associated with poor patient compliance. To address this problem, numerous drug delivery systems (DDSs) have been developed. This review covers a selection of particulate systems, non-stimuli responsive hydrogels, implants, and composite systems that have been developed in the last few decades. Depending on the type of DDS, polymer material, and preparation method, different mechanical properties and drug release profiles can be achieved. Furthermore, DDS development can be optimized by implementing mathematical modeling of both drug release and pharmacokinetic aspects. Several existing mathematical models for diffusion-controlled, swelling-controlled, and erosion-controlled drug delivery from polymeric systems are summarized. Compartmental and physiologically based models for ocular drug transport and pharmacokinetics that have studied drug concentration profiles after intravitreal delivery or release from a DDS are also reviewed. The coupling of drug release models with ocular pharmacokinetic models can lead to obtaining much more efficient DDSs for the treatment of age-related macular degeneration and other diseases of the posterior segment of the eye.

3D Bioprinting of Acellular Corneal Stromal Scaffolds with a Low Cost Modified 3D Printer: A Feasibility Study.

PURPOSE: Loss of corneal transparency is one of the major causes of visual loss, generating a considerable health and economic burden globally. Corneal transplantation is the leading treatment procedure, where the diseased cornea is replaced by donated corneal tissue. Despite the rise of cornea donations in the past decade, there is still a huge gap between cornea supply and demand worldwide. 3D bioprinting is an emerging technology that can be used to fabricate tissue equivalents that resemble the native tissue, which holds great potential for corneal tissue engineering application. This study evaluates the manufacturability of 3D bioprinted acellular corneal grafts using low-cost equipment and software, not necessarily designed for bioprinting applications. This approach allows access to 3D printed structures where commercial 3D bioprinters are cost prohibitive and not readily accessible to researchers and clinicians. METHODS: Two extrusion-based methods were used to 3D print acellular corneal stromal scaffolds with collagen, alginate, and alginate-gelatin composite bioinks from a digital corneal model. Compression testing was used to determine moduli. RESULTS: The printed model was visually transparent with tunable mechanical properties. The model had central radius of curvature of 7.4 mm, diameter of 13.2 mm, and central thickness of 0.4 mm. The compressive secant modulus of the material was 23.7 ± 1.7 kPa at 20% strain. 3D printing into a concave mold had reliability advantages over printing into a convex mold. CONCLUSIONS: The printed corneal models exhibited visible transparency and a dome shape, demonstrating the potential of this process for the preparation of acellular partial thickness corneal replacements. The modified printing process presented a low-cost option for corneal bioprinting.

The Roles of Vitreous Biomechanics in Ocular Disease, Biomolecule Transport, and Pharmacokinetics.

PURPOSE: The biomechanical properties of the vitreous humor and replication of these properties to develop substitutes for the vitreous humor have rapidly become topics of interest over the last two decades. In particular, the behavior of the vitreous humor as a viscoelastic tissue has been investigated to identify its role in a variety of processes related to biotransport, aging, and age-related pathologies of the vitreoretinal interface. METHODS: A thorough search and review of peer-reviewed publications discussing the biomechanical properties of the vitreous humor in both human and animal specimens was conducted. Findings on the effects of biomechanics on vitreoretinal pathologies and vitreous biotransport were analyzed and discussed. RESULTS: The pig and rabbit vitreous have been found to be most mechanically similar to the human vitreous. Age-related liquefaction of the vitreous creates two mechanically unique phases, with an overall effect of softening the vitreous. However, the techniques used to acquire this mechanical data are limited by the in vitro testing methods used, and the vitreous humor has been hypothesized to behave differently in vivo due in part to its swelling properties. The impact of liquefaction and subsequent detachment of the vitreous humor from the posterior retinal surface is implicated in a variety of tractional pathologies of the retina and macula. Liquefaction also causes significant changes in the biotransport properties of the eye, allowing for significantly faster movement of molecules compared to the healthy vitreous. Recent developments in computational and ex vivo models of the vitreous humor have helped with understanding its behavior and developing materials capable of replacing it. CONCLUSIONS: A better understanding of the biomechanical properties of the vitreous humor and how these relate to its structure will potentially aid in improving clinical metrics for vitreous liquefaction, design of biomimetic vitreous substitutes, and predicting pharmacokinetics for intravitreal drug delivery.

Sustained release of heme-albumin as a potential novel therapeutic approach for age-related macular degeneration.

Globally, age-related macular degeneration (AMD) is the third most common visual impairment. Most often attributed to cellular fatigue with aging, over expression of reactive oxygen species (ROS) causes ROS accumulation in the retina, leading to chronic inflammatory immune signaling, cellular and tissue damage, and eventual blindness. If left uncontrolled, the disease will progress from the dry form of AMD to more severe forms such as geographic atrophy or wet AMD, hallmarked by choroidal neovascularization. There is no cure for AMD and treatment options are limited. Treatment options for wet AMD require invasive ocular injections or implants, yet fail to address the disease progressing factors. To provide more complete treatment of AMD, the application of a novel anti-inflammatory heme-bound human serum albumin (heme-albumin) protein complex delivered by antioxidant ROS scavenging polydopamine (PDA) nanoparticles (NPs) for sustained treatment of AMD was investigated. Through the induction of heme oxygenase-1 (HO-1) by heme-albumin in retinal pigment epithelial (RPE) cells, anti-inflammatory protection may be provided through the generation of carbon monoxide (CO) and biliverdin during heme catabolism. Our results show that the novel protein complex has negligible cytotoxicity towards RPE cells (ARPE-19), reduces oxidative stress in both inflammatory and ROS in vitro models, and induces a statistically significant increase in HO-1 protein expression. When incorporated into PDA NPs, heme-albumin was sustainably released for up to 6 months, showing faster release at higher oxidative stress levels. Through its ability to react with ROS, heme-albumin loaded PDA NPs showed further reduction of oxidative stress with minimal cytotoxicity. Altogether, we demonstrate that heme-albumin loaded PDA NPs reduce oxidative stress in vitro and can provide sustained therapeutic delivery for AMD treatment.

Tunable alginate hydrogels as injectable drug delivery vehicles for optic neuropathy.

Many disease pathologies, particularly in the eye, are induced by oxidative stress. In particular, injury to the optic nerve (ON), or optic neuropathy, is one of the most common causes of vision loss. Traumatic optic neuropathy (TON) occurs when the ON is damaged following blunt or penetrating trauma to either the head or eye. Currently, there is no effective treatment for TON, only management options, namely the systematic delivery of corticosteroids and surgical decompression of the optic nerve. Unfortunately, neither option alleviates the generation of reactive oxygen species (ROS) which are responsible for downstream damage to the ON. Additionally, the systemic delivery of corticosteroids can cause fatal off-target effects in cases with brain involvement. In this study, we developed a tunable injectable hydrogel delivery system for local methylene blue (MB) delivery using an internal method of crosslinking. MB was chosen due to its ROS scavenging ability and neuroprotective properties. Our MB-loaded polymeric scaffold demonstrated prolonged release of MB as well as in situ gel formation. Additionally, following rheological characterization, these alginate hydrogels demonstrated minimal cytotoxicity to human retinal pigment epithelial cells in vitro and exhibited injection feasibility through small-gauge needles. Our chosen MB concentrations displayed a high degree of ROS scavenging following release from the alginate hydrogels, suggesting this approach may be successful in reducing ROS levels following ON injury, or could be applied to other ocular injuries.

Considerations for Polymers Used in Ocular Drug Delivery.

PURPOSE: Age-related eye diseases are becoming more prevalent. A notable increase has been seen in the most common causes including glaucoma, age-related macular degeneration (AMD), and cataract. Current clinical treatments vary from tissue replacement with polymers to topical eye drops and intravitreal injections. Research and development efforts have increased using polymers for sustained release to the eye to overcome treatment challenges, showing promise in improving drug release and delivery, patient experience, and treatment compliance. Polymers provide unique properties that allow for specific engineered devices to provide improved treatment options. Recent work has shown the utilization of synthetic and biopolymer derived biomaterials in various forms, with this review containing a focus on polymers Food and Drug Administration (FDA) approved for ocular use. METHODS: This provides an overview of some prevalent synthetic polymers and biopolymers used in ocular delivery and their benefits, brief discussion of the various types and synthesis methods used, and administration techniques. Polymers approved by the FDA for different applications in the eye are listed and compared to new polymers being explored in the literature. This article summarizes research findings using polymers for ocular drug delivery from various stages: laboratory, preclinical studies, clinical trials, and currently approved. This review also focuses on some of the challenges to bringing these new innovations to the clinic, including limited selection of approved polymers. RESULTS: Polymers help improve drug delivery by increasing solubility, controlling pharmacokinetics, and extending release. Several polymer classes including synthetic, biopolymer, and combinations were discussed along with the benefits and challenges of each class. The ways both polymer synthesis and processing techniques can influence drug release in the eye were discussed. CONCLUSION: The use of biomaterials, specifically polymers, is a well-studied field for drug delivery, and polymers have been used as implants in the eye for over 75 years. Promising new ocular drug delivery systems are emerging using polymers an innovative option for treating ocular diseases because of their tunable properties. This review touches on important considerations and challenges of using polymers for sustained ocular drug delivery with the goal translating research to the clinic.

Glutathione Improves the Antioxidant Activity of Vitamin C in Human Lens and Retinal Epithelial Cells: Implications for Vitreous Substitutes.

PURPOSE: Tissues in the eye are particularly susceptible to oxidative damage due to light exposure. While vitamin C (ascorbic acid) has been noted as a vital antioxidant in the vitreous humor, its physiological concentration (1-2 mM) has been shown to be toxic to retinal and lens epithelial cells in in vitro cell culture. We have explored adding vitamin C to hydrogel vitreous substitutes as a potential therapeutic to prevent oxidative damage to intraocular tissues after vitrectomy. However, vitamin C degrades rapidly even when loaded at high concentrations, limiting its long-term effectiveness. Glutathione, another antioxidant found abundantly in the lens at concentrations of 2-10 mM, was proposed to be used in conjunction with vitamin C. METHODS: Cell viability and reactive oxygen species activity of human retinal and lens epithelial cells treated with various combinations of vitamin C, glutathione, hydrogen peroxide, and a hydrogel vitreous substitute were determined using CellTiter-Glo luminescent cell viability assay and dichlorofluorescein assay, respectively. The vitamin C remaining in hydrogel vitreous substitute or glutathione-vitamin C solutions was determined using a microplate reader at 265 nm wavelength, compared against standard solutions with known concentrations. RESULTS: Glutathione protected the lens and retinal cells from the negative effect of vitamin C on cell viability and prolonged the antioxidant effect of vitamin C in vitro. While the detected reading of pure vitamin C solution decreased rapidly from 100% to 10% by 3 days, glutathione provided a significant extension to vitamin C stability, with 70% remaining after 14 days when the glutathione was used at physiological concentrations found in the lens (2-10 mM). CONCLUSIONS: These results indicate glutathione might be an effective addition to vitamin C in intraocular implants, including potential vitreous substitutes, and warrants additional studies on the effectiveness of the vitamin C - glutathione combination in preventing oxidative stress post-vitrectomy.

Macro- and Microscale Properties of the Vitreous Humor to Inform Substitute Design and Intravitreal Biotransport.

Research on the vitreous humor and development of hydrogel vitreous substitutes have gained a rapid increase in interest within the past two decades. However, the properties of the vitreous humor and vitreous substitutes have yet to be consolidated. In this paper, the mechanical properties of the vitreous humor and hydrogel vitreous substitutes were systematically reviewed. The number of publications on the vitreous humor and vitreous substitutes over the years, as well as their respective testing conditions and testing techniques were analyzed. The mechanical properties of the human vitreous were found to be most similar to the vitreous of pigs and rabbits. The storage and loss moduli of the hydrogel vitreous substitutes developed were found to be orders of magnitude higher in comparison to the native human vitreous. However, the reported modulus for human vitreous, which was most commonly tested in vitro, has been hypothesized to be different in vivo. Future studies should focus on testing the mechanical properties of the vitreous in situ or in vivo. In addition to its mechanical properties, the vitreous humor has other biotransport mechanisms and biochemical functions that establish a redox balance and maintain an oxygen gradient inside the vitreous chamber to protect intraocular tissues from oxidative damage. Biomimetic hydrogel vitreous substitutes have the potential to provide ophthalmologists with additional avenues for treating and controlling vitreoretinal diseases while preventing complications after vitrectomy. Due to the proximity and interconnectedness of the vitreous humor to other ocular tissues, particularly the lens and the retina, more interest has been placed on understanding the properties of the vitreous humor in recent years. A better understanding of the properties of the vitreous humor will aid in improving the design of biomimetic vitreous substitutes and enhancing intravitreal biotransport.

Controlled release of anti-VEGF by redox-responsive polydopamine nanoparticles.

Reactive oxidative species (ROS) are the primary mediator of angiogenesis by upregulating the expression of vascular endothelial growth factor (VEGF) in the development of wet age-related macular degeneration (AMD). However, the current treatment of AMD currently relies on monthly intravitreal injection of anti-angiogenic therapeutics to inhibit new choroidal angiogenesis. However, repeated injections have been associated with side-effects, are costly, and may lower patient compliance. Moreover, the intraocular oxidative stress-dependent angiogenesis is not alleviated by current treatments, which limits the overall efficacy of the treatment strategy. Recently, nanoparticle-based devices present potential in sustained delivery of angiogenesis inhibitors and excellent capability of scavenging reactive oxygen species (ROS). Nevertheless, limited efforts have been dedicated to the treatment of oxidative stress-related diseases via a combined anti-angiogenesis and anti-oxidization pathway. For this purpose, we developed anti-angiogenetic protein-loaded polydopamine (PDA) nanoparticles for the enhanced treatment of AMD. Remarkably, the PDA nanoparticles could efficiently scavenge ROS to reduce the expression of angiogenic agents. In parallel, the particles were able to controllably release loaded anti-angiogenic drugs in response to oxidative stress.

Chitosan-Polycaprolactone Core-Shell Microparticles for Sustained Delivery of Bevacizumab.

The current therapy for treating neovascular age-related macular degeneration requires monthly intravitreal injection of angiogenesis inhibitors such as bevacizumab or ranibizumab via a 31-gauge needle to inhibit choroidal neovascularization. However, repeated intravitreal injections are associated with poor patient compliance and potential side effects. Microparticle-based injectable devices have shown great promise to address this issue by sustained delivery of protein therapeutics, but critical barriers remain, including limited loading capacity and steady long-term release without compromising the anti-angiogenic activity of drugs. Addressing these challenges, we developed a unique method for synthesizing biodegradable polymer-based core-shell microparticles with sizes around 10 µm, high physical integrity, and uniform size. Subsequent electrostatic and physical interactions to control protein diffusion were designed for the core-shell microparticles to effectively increase the capacity of drug loading to 25%, reduce burst release by almost 30%, and extend the period of drug release from 3 to 6 months. Remarkably, the microparticles enabled a longer-term drug administration and maintained high drug potency up to 6 months in vitro, representing significant advancement compared to conventional microparticle-based delivery platforms or currently commercialized devices. Additionally, the microparticles presented minimal toxicity to human retinal cells in vitro with over 90% cell viability, and they also exhibited good injection feasibility through 31-gauge needles in an ex vivo porcine eye model. These results warrant further studies to evaluate the clinical potential for treating posterior ophthalmic diseases as well as other conditions or injuries requiring long-term local drug administration.

Injectable biodegradable bi-layered capsule for sustained delivery of bevacizumab in treating wet age-related macular degeneration.

Vascular endothelial growth factor (VEGF) is a key regulator of abnormal blood vessel growth. As such, bevacizumab-based inhibition of VEGF has been the clinically adopted strategy to treat colorectal and breast cancers as well as age-related macular degeneration (AMD). However, as the treatment of vascular diseases often requires a high drug concentration for a long period, the burst release of bevacizumab remains a critical limitation in anti-VEGF-based therapies. Maintaining bevacizumab at high concentrations over extended periods remains challenging due to insufficient drug loading capacity and drug-device interactions. We report the development of a polymeric based bi-layered capsule that could address these challenges by extending the release over one year, thereby providing an effective platform enabling treatment of chronic vascular diseases. Remarkably, the developed capsules have a bi-layered structure which ensures the structural integrity of the injectable capsules and appropriate diffusion of bevacizumab by providing optimal physical trapping and electrostatic interaction. Meanwhile, the central hollow design enables a higher drug loading to meet the need for long-term release of bevacizumab for several months to one year. Using an in vitro drug release assay, we demonstrated that the bi-layered capsule could produce longer-term local drug administration by intravitreal injection compared to previously reported devices. The capsules also present minimal toxicity and maintain anti-VEGF potency, suggesting that our approach may have the potential to treat vascular-related diseases using bevacizumab.

Accommodative tissues influence the shape of the cornea and potentially drive corneal morphogenesis.

This study investigates whether the presence of accommodative tissues biomechanically influences the shape of the cornea and potentially drives corneal morphogenesis during embryonic ocular development. Porcine eyes were subjected to an internal pressure simulating intraocular pressure. Ocular geometry was evaluated using a corneal topographer and digital cameras before and after dissection of the accommodative tissues. A computational model of the porcine eye was constructed and loaded by an internal pressure representing intraocular pressure. Eye shape was evaluated in models with and without the ciliary body. The porcine model was generalized to the human model, simplified model, or embryonic model with different ocular tissue shapes, sizes, and stiffnesses. Experimental data showed that, even in the six-month-old pig eye, the average corneal radius of curvature increased after the removal of accommodative tissues compared to sham controls (p = 0.002). Computational results agreed with the experimental data and further suggested that the change in corneal radius is greater when the tissue stiffness is low and the intraocular pressure is high, regardless of the geometry and size of the eye components. Using a combined in vitro and in silico approach, this study explores the biomechanical influence of the accommodative tissues and related loads on the cornea and offers additional factors that might influence the shape of the cornea.

A Hydrogel Vitreous Substitute that Releases Antioxidant.

Current experimental vitreous substitutes only replace the physical functions of the natural vitreous humor. Removal of the native vitreous disrupts oxygen homeostasis in the eye, causing oxidative damage to the lens that likely results in cataract formation. Neither current clinical treatments nor other experimental vitreous substitutes consider the problem of oxidative stress after vitrectomy. To address this problem, biomimetic hydrogels are prepared by free radical polymerization of poly(ethylene glycol) methacrylate and poly(ethylene glycol) diacrylate. These hydrogels have similar mechanical and optical properties to the vitreous. The hydrogels are injectable through small-gauge needles and demonstrate in vitro biocompatibility with human retinal and lens epithelial cells. The hydrogels and added vitamin C, an antioxidant, show a synergistic effect in protecting ocular cells against reactive oxygen species, which fulfills a chemical function of the natural vitreous. These hydrogels have the potential to prevent post-vitrectomy cataract formation and reduce the cost of additional surgeries.

Rheological Properties and Age-Related Changes of the Human Vitreous Humor.

The vitreous humor is a fragile, transparent hydrogel situated between the lens and the retina, occupying 80% of the eye's volume. Due to its viscoelastic behavior, the vitreous serves as a mechanical damper for the eye, absorbing impacts, and protecting the lens and retina. The vitreous liquefies with age, which compromises its function as a shock absorber and causes complications including retinal detachment, macular holes, and vitreous hemorrhage. Studies on the viscoelastic properties of the vitreous have been limited. Rheological testing of the vitreous has commonly been done on non-primate mammalian species. Human vitreous rheological properties have been previously reported; however, various measurement techniques were used, resulting in data that differed by orders of magnitude. Shear rheometry is commonly used to characterize soft tissues and hydrogels such as the vitreous humor. However, no human vitreous rheological data have been reported using this technique, preventing direct comparison to other published work. Additionally, no age-related changes in the mechanical properties of the human vitreous humor have been reported. Human vitreous samples (n = 39, aged 62 ± 15 years) were tested using a shear rheometer. Small amplitude oscillatory shear and creep experiments were performed. The linear viscoelastic region of the human vitreous was found to be below 1% strain. The solid phase of the old human vitreous was found to be stiffer than the young human vitreous and the porcine vitreous. The stiffness of the human vitreous gel also appeared to be positively correlated with age. Vitreous dehydration due to a decrease in hyaluronic acid concentration with age was proposed to cause the stiffening of the solid phase of the vitreous gel. Vitreous liquefaction, therefore, might be characterized as a simultaneous increase in liquid volume and localized stiffening of the vitreous gel. The phase separation of the vitreous humor with age has been hypothesized as the cause of many vitreous-related complications. This study provides viscoelastic properties and age-related changes of the human vitreous humor, which will aid in the design of biomimetic vitreous substitutes, enhancement in analyzing intravitreal transport of therapeutics, and understanding the pathological conditions of the vitreous humor.

Electrospun poly(caprolactone)-elastin scaffolds for peripheral nerve regeneration.

Peripheral nerve regeneration can be enhanced by chemical and mechanical cues for neurite growth. Aligned and randomly oriented electrospun nanofibers of poly(ε-caprolactone) (PCL) or a blend of PCL and elastin were fabricated to test their potential to provide contact guidance to embryonic chick dorsal root ganglia for peripheral nerve regeneration. Scanning electron microscopy was used to analyze the fiber diameter. Fiber diameter was found to be significantly smaller when elastin was incorporated into the scaffold (934 ± 58 nm for PCL and 519 ± 36 nm for PCL:elastin). After 24 h in culture, there was preferential cell attachment and neurite extension along the fibers of the elastin-containing scaffolds (average neurite extension 173.4 ± 20.7 µm), indicating that the presence of elastin promotes neurite outgrowth on electrospun scaffolds.

The impact of laminin on 3D neurite extension in collagen gels.

The primary goal of this research was to characterize the effect of laminin on three-dimensional (3D) neurite growth. Gels were formed using type I collagen at concentrations of 0.4-2.0 mg mL(-1) supplemented with laminin at concentrations of 0, 1, 10, or 100 µg mL(-1). When imaged with confocal microscopy, laminin was shown to follow the collagen fibers; however, the addition of laminin had minimal effect on the stiffness of the scaffolds at any concentration of collagen. Individual neurons dissociated from E9 chick dorsal root ganglia were cultured in the gels for 24 h, and neurite lengths were measured. For collagen gels without laminin, a typical bimodal response of neurite outgrowth was observed, with increased growth at lower concentrations of collagen gel. However, alteration of the chemical nature of the collagen gel by the laminin additive shifted, or completely mitigated, the bimodal neurite growth response seen in gels without laminin. Expression of integrin subunits, α1, α3, α6 and ß1, were confirmed by PCR and immunolabeling in the 3D scaffolds. These results provide insight into the interplay between mechanical and chemical environment to support neurite outgrowth in 3D. Understanding the relative impact of environmental factors on 3D nerve growth may improve biomaterial design for nerve cell regeneration.

Rabbit study of an in situ forming hydrogel vitreous substitute.

PURPOSE: An in situ forming hydrogel was evaluated as a potential vitreous substitute in rabbits. METHODS: The hydrogel used a disulfide cross-linker that was then reduced to produce an injectable thiol-containing polymer solution. The disulfide cross-links reformed by air oxidation of the thiols and produced a stable hydrogel once inside the eye. The polymer was clear, autoclavable, and could be stored easily in the presence of nitrogen gas. Capillary rheometry was used to measure the viscoelastic properties of the hydrogels and the porcine vitreous. Fourteen black rabbits underwent a pars plana, 25-gauge, three-port vitrectomy by a single surgeon with injection of a vitreous substitute. RESULTS: The refractive indices of the hydrogels were measured by refractometry and were shown to be close to 1.33, and the 2% hydrogel matched the mechanical properties of the natural vitreous humor. The reduced polymeric hydrogel was easily injectable through a small-gauge needle into the vitreous cavity and did not show any fragmentation. The material underwent gelation within the eye, remained optically clear, and appeared well tolerated clinically. Slit lamp examination, dilated fundus examination, and electroretinograms showed no evidence of vitritis, uveitis, or endophthalmitis after 1 week. Histopathologic evaluation did not reveal any overt toxicity or gross morphologic changes in the retina. CONCLUSIONS: The fact that this process of in situ gelation gives rise to hydrogels that are biocompatible and physically and optically similar to the natural vitreous suggests its suitability as a permanent vitreous substitute. Hydrogel candidates will be further studied to evaluate long-term biocompatibility and degradation in vivo.