Typical rheograms for gelatin samples G75-100, G175, and G300 in BSS at 37 °C are shown in Figure 1. The flow curves of various gelatin solutions exhibit a linear relationship over the entire rate range. The viscosity of the fluid is defined as the ratio of the shear stress to the shear rate. In this study, all three types of gelatins showed a Newtonian fluid behavior because the viscosities are independent of the shear rate. Furthermore, the viscosity was increased with increasing the Bloom index of gelatin.

It is known that the triple-helical regions of collagen are stabilized by hydrogen bonding and van der Waals attractions between imino residues on different chains [20]. Conversion of collagen to gelatin involves disruption of these noncovalent interactions and leads to a decrease in molecular order. Bigi et al. [18] have shown that the triple-helix content of gelatin increased with its Bloom number. In addition to the molecular weight, the chemical structure can affect the resultant viscosity of gelatin. A recent study from Peng et al. [21] suggested that the increase in viscosity was related to the chain organization of gelatin in solution, e.g., the formation of a helix structure. Our present results were compatible with their findings and indicated that high Bloom strength gelatins have higher viscosity than the samples with low Bloom index. The material degradation occurred rapidly when the gelatin was implanted in the anterior chamber [16] or the subretinal space [13,14]. Since gelatin is viscous, its degradation products residing in an ocular immune-privileged site may contribute to the increase in viscosity of tissue fluids. It should be investigated whether this effect is detrimental to living cells and tissues. Here, we performed an in vitro study to examine the role of Bloom index on the biocompatibility of gelatin.

Figure 2 shows the results of quantitative analysis for ARPE-19 cell growth. After two days of culture, similar levels of mitochondrial dehydrogenase activity (MTS activity) were observed in the control, G75-100, and G175 groups and not statistically different (p>0.05). By contrast, in the G300 groups, the MTS activity was significantly reduced by about 13% (p<0.05) as compared to that of the control groups. In addition, the cells exposed to gelatins with higher Bloom strengths showed lower density. These results indicate that the Bloom index of gelatin may play a crucial role in the regulation of ARPE-19 cell proliferation.

The interaction between cells and biopolymers is the evaluation indicator of cell compatibility of materials [22]. RPE is composed of a monolayer of cuboidal cells separated from the neural retina by the subretinal space. The RPE cells are known to mediate the transport of metabolic intermediates, waste products, ions, and fluid components between the choriocapillaries and the retina [23]. As a protein-based biomaterial, gelatin is substantially nontoxic. We have previously shown that the high Bloom strength gelatins sterilized with gamma irradiation seem to be cytostatic toward rat iris pigment epithelial cells [14]. In this study, similar results were obtained from gelatin samples sterilized by ethanol treatment. Our findings suggest that the higher Bloom index of gelatins is related to the inhibitory effects of ARPE-19 cell growth. One possible explanation for these observations is that the presence of high Bloom strength gelatins in the culture system leads to an increase in the medium viscosity and to problem of mass transfer. The insufficient nutrient availability to the ARPE-19 cells may limit their proliferation and metabolic responses. Therefore, the gelatins with lower Bloom index are more suitable for use as carrier materials for ocular tissue engineering and drug delivery.

Figure 3 is a representative photograph of ARPE-19 cells labeled with Live/Dead stain, where the live cells fluoresce green and the dead cells fluoresce red. In the control groups, the majority of confluent cell cultures are viable (Figure 3A and B). After exposure to gelatin samples G75-100 for two days at 37 °C, the ARPE-19 cultures maintain good viability with only a few dead cells (Figure 3C and D). By contrast, in the G175 and G300 groups, an increased number of red-stained nuclei were noted, indicating that these gelatin samples induced mild cytotoxicity (Figure 3E-H). Figure 4 shows the mean percentage of live cells as determined by the Live/Dead assay. The cell viability was significantly lower in the G300 groups (89.4 ± 1.9%), compared with those of the control (99.2 ± 0.6%), G75-100 (98.5 ± 1.2%), and G175 groups (96.7 ± 1.7%; p<0.05). These findings suggest that the gelatins with low Bloom index are more cytocompatible than their high Bloom strength counterparts.

Because of its in vivo compatibility and safety, gamma-ray-sterilized gelatin has been used as a matrix for harvested RPE [24] and retinal sheets [13,25]. Using a Live/Dead assay kit, the cell viability of RPE sheets exposed to gelatin (Bloom index = 300) was assessed and was found to be more than 85% 24 h from harvesting [24]. Similar to their findings, our results indicate that the viability of RPE cell line cultures is approximately 90% after incubation with high Bloom strength gelatin samples for two days. However, in this study, we have noted the effects of Bloom index of gelatin on cell compatibility of materials in vitro. The negligible cytotoxicity of the gelatins with low Bloom index makes them promising candidates for ophthalmic applications.

Figure 5 shows the results of glutamate uptake of ARPE-19 cells. After 2 days in culture, the glutamate uptake activity did not show a significant difference between the control, G75-100, and G175 groups (p>0.05). However, there was a significant reduction of activity (>20% reduction) in the G300 groups as compared to that of the control groups (p<0.05). These results clearly demonstrate that the Bloom index of gelatin may be a potential factor involved in the regulation of glutamate uptake in RPE cells exposed to foreign materials.

In the present study, the ARPE-19 cells were cultured with gelatins in the medium for in vitro glutamate uptake assays. We have previously shown that gelatin has a high degree of water binding capability and can absorb a tremendous amount of water at up to 10 times its weight [10]. The mechanism of hydration of gelatin is a capillary phenomenon of water molecules penetrating the tiny interstices of a collagen-like four-dimensional structure in the gelatin [26]. During this process, the medium nutrients may be simultaneously entrapped in the gelatin materials. Glutamate is the main excitatory neurotransmitter in the retina but is neurotoxic when present in excessive amounts [27]. Therefore, the glutamate concentration in the subretinal space should be maintained at a fairly constant level. The RPE is responsible for the turnover and renewal of the photoreceptor and participates in regulating the glutamate uptake [28]. The results of this study showed that the exposure to high Bloom strength gelatins markedly reduced the glutamate uptake capacity of ARPE-19 cells. This is probably due to that the presence of gelatin with high viscosity in the culture medium may affect the nutrient availability to RPE cells.

It has been reported that elevated IL-6 level is associated with severe foreign body reaction to the implanted materials [29]. A study from Abe et al. [30] suggested that the IL-6 was involved in the local reaction of retinal tissues after the early days of transplantation of human RPE. In this study, we therefore analyzed the expression of IL-6 in RPE cell cultures for indication of in vitro biocompatibility. Figure 6 shows the pro-inflammatory gene expression of ARPE-19 cells exposed to various gelatin membranes for two days. Similar IL-6 gene expression levels were observed in the control, G75-100, and G175 groups and not statistically different (p>0.05). By contrast, the expressions of these genes in the G300 groups were significantly higher than those of the other groups (p<0.05), indicating that the IL-6 genes are up-regulated after culture with high Bloom strength gelatins.

The ARPE-19 cell secretion of IL-6 in response to various gelatin membranes is also shown in Figure 7. The expression of IL-6 in the control, G75-100, and G175 groups was 187.4 ± 32.5, 206.1 ± 28.2, and 241.6 ± 24.3 pg/mL, respectively. The values did not show a significant difference between these three groups (p>0.05), which indicates that the exposure to gelatins with low Bloom index does not promote inflammation. However, the IL-6 level was significantly higher in the G300 groups (315.8 ± 26.1 pg/mL), compared with those of the low Bloom strength gelatin groups (p<0.05). These findings suggest that the Bloom index of gelatin has an important influence on the stimulation of pro-inflammatory cytokine IL-6 production in RPE cells.

Recently, we reported that the exposure of human corneal endothelial cells to the gelatins of higher molecular weight induces higher levels of IL-6 [10]. In addition to material chemistry, the material morphology has been suspected to be responsible for pro-inflammatory cytokine production. Reichert et al. [31] have investigated the temporal cytokine expression profile from human THP-1 monocytes exposed to phagocytosable Ti particles and to Ti discs of comparable surface roughness and concluded that cells treated by disc samples may produce in many instances a higher cytokine expression than did particles. In this study, cytokine expression analyses for the cell cultures were performed in order to further compare the effects of Bloom index of gelatin. The expression at both mRNA and protein levels were characterized using the techniques of quantitative real-time RT-PCR and ELISA. In comparison with high Bloom strength samples, the gelatins with low Bloom index may cause less cellular inflammatory reaction and have more acceptable biocompatibility.

Gelatins, prepared through an acid process treatment of pig skin, were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Bloom index indicates the gel strength of the gelatin, defined as the weight in grams necessary to apply to the surface of gelatin gel, to produce a 4 mm depth depression [14]. In the present study, the gelatins had different Bloom numbers (75–100, 175, and 300), with the higher value producing stronger gels. A gelatin sample with a Bloom number of 75–100 was designated as G75-100. Balanced salt solution (BSS, pH 7.4) was obtained from Alcon Laboratories (Fort Worth, TX, USA). Phosphate-buffered saline (PBS, pH 7.4) was purchased from Biochrom AG (Berlin, Germany). Dulbecco’s modified Eagle’s medium/Ham’s F12 nutrient mixture (DMEM/F12) and TRIzol reagent were purchased from Gibco-BRL (Grand Island, NY, USA). Fetal bovine serum (FBS) and the antibiotic/antimycotic (A/A) solution (10,000 U/mL penicillin, 10 mg/mL streptomycin and 25 μg/mL amphotericin B) were obtained from Biological Industries (Kibbutz Beit Haemek, Israel). Radioactive [³H]glutamate was purchased from Amersham (Little Chalfont, UK). All the other chemicals were of reagent grade and used as received without further purification.

The gelatin membranes were prepared by solution casting methods as described elsewhere [13,14]. Briefly, an aqueous solution of 10 wt% gelatin was cast into a polystyrene planar mold (5 × 5 cm², 1.5 cm depth), and air-dried for 24 h at 25 °C to obtain membranes (about 30 μm thick). Then, the membrane samples were sterilized in a 70% ethanol solution overnight.

To measure the rheological behavior of gelatin, the membrane samples (1 mg) were dissolved in 1 mL of physiological medium (BSS) at 37 °C. The shear stress versus shear rate curves and flow properties of resulting gelatin solutions were determined at 37 °C in a rheometer (Carri-Med CSL2100; TA Instruments, Newcastle, UK). A double concentric cylinder system and a cone plate sensor with a 2° cone angle and 6 cm in diameter were used for evaluating flow properties. The shear rate was altered in the range from 0.1 to 1000 s⁻¹ during rheological measurements.

ARPE-19 cells, a spontaneously immortalized human cell line (BCRC No. 60383) with morphological and functional characteristics similar to adult human RPE [32], were purchased from the Bioresource Collection and Research Center (Hsinchu, Taiwan, ROC). The cells were maintained in regular growth medium containing DMEM/F12, 10% FBS, and 1% A/A solution. Cultures were incubated in a humidified atmosphere of 5% CO₂ at 37 °C. The cells from passage 32 were used for experiments.

The sterilized gelatin membranes were thoroughly rinsed in PBS and completely dissolved in fresh culture medium at 37 °C. After 48 h of incubation with medium containing gelatin materials, the cultures were examined by various in vitro tests. The gelatin was presented as a soluble protein on the cell layers during biocompatibility testing. ARPE-19 cells in regular growth medium without gelatins served as control groups.

Results were expressed as mean ± standard deviation. Comparative studies of means were performed using one-way analysis of variance (ANOVA). Significance was accepted with p<0.05.