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Cell sheet-mediated tissue regeneration is a promising approach for corneal reconstruction. However, the fragility of bioengineered corneal endothelial cell (CEC) monolayers allows us to take advantage of cross-linked porous gelatin hydrogels as cell sheet carriers for intraocular delivery. The aim of this study was to further investigate the effects of biopolymer concentrations (5-15 wt%) on the characteristic and safety of hydrogel discs fabricated by a simple stirring process combined with freeze-drying method. Results of scanning electron microscopy, porosity measurements, and ninhydrin assays showed that, with increasing solid content, the pore size, porosity, and cross-linking index of carbodiimide treated samples significantly decreased from 508±30 to 292±42 µm, 59.8±1.1 to 33.2±1.9%, and 56.2±1.6 to 34.3±1.8%, respectively. The variation in biopolymer concentrations and degrees of cross-linking greatly affects the Young's modulus and swelling ratio of the gelatin carriers. Differential scanning calorimetry measurements and glucose permeation studies indicated that for the samples with a highest solid content, the highest pore wall thickness and the lowest fraction of mobile water may inhibit solute transport. When the biopolymer concentration is in the range of 5-10 wt%, the hydrogels have high freezable water content (0.89-0.93) and concentration of permeated glucose (591.3-615.5 µg/ml). These features are beneficial to the in vitro cultivation of CECs without limiting proliferation and changing expression of ion channel and pump genes such as ATP1A1, VDAC2, and AQP1. In vivo studies by analyzing the rabbit CEC morphology and count also demonstrate that the implanted gelatin discs with the highest solid content may cause unfavorable tissue-material interactions. It is concluded that the characteristics of cross-linked porous gelatin hydrogel carriers and their triggered biological responses are in relation to biopolymer concentration effects.

Recent studies reflect the importance of using naturally occurring biopolymers as three-dimensional corneal keratocyte scaffolds and suggest that the porous structure of gelatin materials may play an important role in controlling nutrient uptake. In the current study, the authors further consider the application of carbodiimide cross-linked porous gelatin as an alternative to collagen for corneal stromal tissue engineering. The authors developed corneal keratocyte scaffolds by nanoscale modification of porous gelatin materials with chondroitin sulfate (CS) using carbodiimide chemistry. Scanning electron microscopy/energy dispersive X-ray spectroscopy and Fourier transform infrared spectroscopy showed that the amount of covalently incorporated polysaccharide was significantly increased when the CS concentration was increased from 0% to 1.25% (w/v). In addition, as demonstrated by dimethylmethylene blue assays, the CS content in these samples was in the range of 0.078-0.149 nmol per 10 mg scaffold. When compared with their counterparts without CS treatment, various CS-modified porous gelatin membranes exhibited higher levels of water content, light transmittance, and amount of permeated nutrients but possessed lower Young's modulus and resistance against protease digestion. The hydrophilic and mechanical properties of scaffolds modified with 0.25% CS were comparable with those of native corneas. The samples from this group were biocompatible with the rabbit corneal keratocytes and showed enhanced proliferative and biosynthetic capacity of cultured cells. In summary, the authors found that the nanoscale-level modification has influence on the characteristics and cell-material interactions of CS-containing gelatin hydrogels. Porous membranes with a CS content of 0.112 ± 0.003 nmol per 10 mg scaffold may hold potential for use in corneal stromal tissue engineering.

Carbodiimide cross-linking can easily regulate the functionality of gelatin carriers used for retinal sheet delivery. This paper investigates the effect of cross-linker concentrations (0-0.4 mmol EDC/mg gelatin membrane (GM)) on the properties of the chemically-modified GMs. ATR-FT-IR and ninhydrin analyses results consistently indicated that the EDC cross-linking reaction approaches saturation at concentrations around 0.02 mmol EDC/mg GM. The thermal stability and resistance to water dissolution and collagenase digestion were significantly enhanced with increasing cross-linker concentration from 0.001 to 0.02 mmol EDC/mg GM. In addition, the chemical cross-linking did not affect the ability to form a tissue-encapsulating structure at 37°C. Irrespective of their cross-linking degree, the GMs had an appropriate degradation rate sufficient to allow tissue integration. It was noted that, although high cross-linker concentrations can be used to improve the delivery efficiency of gelatin samples, the treatment with 0.1-0.4 mmol EDC/mg GM may lead to poor biocompatibility. Results of Live/Dead and pro-inflammatory cytokine expression analyses showed that the exposure of ARPE-19 cultures to the test materials cross-linked with a concentration ≥0.1 mmol EDC/mg GM induces significant cytotoxicity and high levels of interleukin-1β and interleukin-6. However, the presence of EDC cross-linked gelatin membranes in the culture medium had no effect on the glutamate uptake capacity. It is concluded that among the cross-linked gelatin samples studied, 0.02 mmol EDC/mg GM is the best cross-linker concentration for preparation of retinal sheet delivery carriers.

An efficient carrier for corneal endothelial cell therapy should deliver and retain the cell sheet transplants at the site of injury without causing adverse effects. Here we introduced a simple stirring process combined with freeze-drying (SFD1) method for the development of gelatin hydrogels with enlarged pore structure that can improve the aqueous humor circulation. Samples fabricated by air-drying (AD) or freeze-drying method were used for comparison. After cross-linking with 1 mM 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC), the discs were investigated to assess their functionality. The simultaneous presence of ice crystals and gas bubbles resulted in large pore size (461 +/- 85 mum) and high porosity (48.0 +/- 1.9%) of SFD1 carriers. Among all of the samples studied, the SFD1 hydrogels showed the most appropriate swelling characteristics without squeezing effect on the anterior segment tissues of the eye. The enlarged pore structure also allowed carriers to contain the highest fraction of mobile water and exhibit the lowest resistance to the glucose permeation. In comparison with AD samples, the SFD1 materials had better cytocompatibility and biocompatibility and more effectively prevented a drastic change of intraocular pressure. Rheological measurements showed that the SFD1 hydrogels behaved like an elastic solid and had a tough (rigid and deformable) texture. As a temporary supporter, the biodegradable gelatin hydrogel could facilitate cell sheet transfer and avoid long-term residence of foreign carriers in the body. Our findings suggest that the gelatin discs with enlarged pore structure have potential as cell sheet carriers for intraocular delivery and corneal tissue engineering.

Retinal transplantation aims to restore vision for patients suffering from retinitis pigmentosa and age-related macular degeneration. Because the retinal sheets are fragile in nature, it is difficult to maintain graft integrity during surgical manipulation and after transplantation. In the present work, we report the feasibility of applying sandwich-like gelatin membranes as encapsulating carriers for retinal sheet transplantation applications. The relationship between the Bloom index of gelatin and the functionality of carrier membranes was studied by determinations of mechanical property, dissolution degree, melting point, cytocompatibility, biocompatibility, and transplant transfer and encapsulation efficiency. Irrespective of their Bloom strength, the gelatin membranes had a thickness sufficient to provide mechanical support for retinal sheets and would be beneficial to overcome the fragility of transplants during intraocular delivery. It was found that the lower the Bloom value of gelatin, the lower melting point of membranes. This allowed for easy fabrication of a stable sandwich-like encapsulating structure at 37 degrees C. The gelatins with lower Bloom strengths could possibly be dissolved to an extent required for the establishment of close contact between the retinal grafts and defective tissues. In addition, the carrier membranes made from the gelatins with low Bloom values showed a relatively higher cytocompatibility and biocompatibility as well as a higher transfer and encapsulation efficiency as compared to those with high Bloom values. It is concluded that the effect of Bloom index of gelatin plays a significant role in the membrane functionality and the gelatins with low Bloom values have substantial potential to be further developed as effective encapsulating carriers for the intraocular delivery of retinal sheets.

Carbodiimide cross-linking can easily regulate the functionality of gelatin carriers used for retinal sheet delivery. This paper investigates the effect of cross-linker concentrations (0-0.4 mmol EDC/mg gelatin membrane (GM)) on the properties of the chemically-modified GMs. ATR-FT-IR and ninhydrin analyses results consistently indicated that the EDC cross-linking reaction approaches saturation at concentrations around 0.02 mmol EDC/mg GM. The thermal stability and resistance to water dissolution and collagenase digestion were significantly enhanced with increasing cross-linker concentration from 0.001 to 0.02 mmol EDC/mg GM. In addition, the chemical cross-linking did not affect the ability to form a tissue-encapsulating structure at 37°C. Irrespective of their cross-linking degree, the GMs had an appropriate degradation rate sufficient to allow tissue integration. It was noted that, although high cross-linker concentrations can be used to improve the delivery efficiency of gelatin samples, the treatment with 0.1-0.4 mmol EDC/mg GM may lead to poor biocompatibility. Results of Live/Dead and pro-inflammatory cytokine expression analyses showed that the exposure of ARPE-19 cultures to the test materials cross-linked with a concentration ≥0.1 mmol EDC/mg GM induces significant cytotoxicity and high levels of interleukin-1β and interleukin-6. However, the presence of EDC cross-linked gelatin membranes in the culture medium had no effect on the glutamate uptake capacity. It is concluded that among the cross-linked gelatin samples studied, 0.02 mmol EDC/mg GM is the best cross-linker concentration for preparation of retinal sheet delivery carriers. [ABSTRACT FROM AUTHOR]

Abstract: The delivery of intact sheet transplants to the subretinal space can prevent cell loss that is generally associated with the injection of cell suspensions or cell aggregates. The aim of this study was to develop chemically modified gelatin matrices that enhance the delivery efficiency and analyze whether the gelatin membranes cross-linked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) can be considered as potential carriers for retinal sheets. The characteristics of EDC cross-linked gelatin membranes were determined by mechanical and in vitro degradation tests, melting point measurements, cell proliferation assays, cytokine expression analyses, and tissue delivery studies. Gelatin membranes without cross-linking and glutaraldehyde cross-linked gelatin samples were used for comparison. Results of this study indicated that introduction of cross-links is capable of rendering the gelatin network more stable against mechanical stresses and deformations as well as rapid hydrolysis during intraocular delivery of delicate tissue sheets. In comparison with the glutaraldehyde treated samples, the EDC cross-linked gelatin membranes showed a better degradation profile and a relatively higher cytocompatibility. In addition, after EDC cross-linking, the gelatin matrices having an acceptable melting point could be used for the fabrication of a sandwich-like carrier with a high transfer and encapsulation efficiency. These findings suggest that the cross-linking agent type gives an influence on delivery functionality of gelatin membranes. In summary, the EDC cross-linked gelatin is an ideal candidate for use as a carrier material in retinal sheet delivery applications. [Copyright &y& Elsevier]