Efficacy of self-assembled hydrogels composed of positively or negatively charged peptides as scaffolds for cell culture.

KASEA16(+) and KASEA16(-) peptides, the net charges of which are positive and negative, respectively, under a neutral condition could undergo self-assembly into nanofibers and form transparent hydrogels without peptide aggregation upon rapid pH neutralization. The numbers of NIH3T3 cells attached to the KASEA16(+) hydrogel and KASEA16(-) hydrogel were similar, and cells proliferated with time on both hydrogels. Cells on the KASEA16(+) hydrogel had spindle-like morphology, while cells on the KASEA16(-) hydrogel formed clusters without extending cytoplasmic processes. Comparison of differently charged peptides under a neutral condition suggested that the charges of the scaffolds should be taken into consideration for the best design and selection of scaffolds for cell culture. Since the KASEA16(+) peptide could form a stable hydrogel under a neutral condition and the hydrogel served as a scaffold for cell proliferation, the KASEA16(+) hydrogel will be a useful scaffold for cell culture.

Morphological and biochemical analysis of intact and opaque cornea in dogs.

The arrangement of collagen fibrils and glycosaminoglycans (GAGs) in substantia propria are important for maintaining transparency of the cornea. Interferences in collagen fibrils and GAG production could be adversative to corneal integrity. In this study, six dogs consisting of four Beagles with normal cornea (normal), one Beagles with opaque cornea (sample No. 1) and one Shih Tzu with neovascularization opaque cornea (sample No.2) were used. All samples were observed morphologically by light and electron microscopes to obtain diameter and distribution of collagen fibrils in substantia propria and were performed biochemically to investigate into GAGs and collagen types. The average diameter of collagen fibrils in the intact cornea of normal, sample No.1 and No.2 was 33.2, 35.0 and 25.0 nm, respectively. The percentage of matrix per unit area was 67% in normal, 87% in sample No.1 and 28.3% in sample No.2. The type III collagen ratio was 25.3% in normal, 21.3% in sample No.1 and 35.8% in sample No.2. The relative amount of heparan sulfate, chondroitin sulfate, dermatan sulfate and keratin sulfate was 1.5, 9.7, 51.1 and 37.7% in normal, 3.3, 26.0, 45.7 and 23.7% in sample No.1 and 1.2, 18.0, 16.6 and 54.1% in sample No.2. Hyaluronic acid was found only in sample No.1 with a relative amount of 1.3%. Since there was some relationship between collagen formation and GAGs composition, it might be speculated that disturbance in arrangement of collagen fibrils and GAG metabolism especially in substantia propria would bring up opacity of the cornea.

Site-dependent differences in collagen lamellae in the corneal substantia propria of beagle dogs.

The fine structure in the center and periphery of the cornea of 16 beagle dogs were characterized and compared. The central cornea (about 540 microm) was apparently thinner than the peripheral cornea (about 720 microm). Thickness ratios of the corneal substantia propria to the entire cornea were approximately 86% in both portions. In addition, number of collagen lamellae, collagen fibril diameter, and collagen fibril index of the central substantia propria are different from those of the periphery (253 vs 236 lamellae, 29.1 vs 32.0 nm, and 39.0 vs 41.6%, respectively). These differences are thought to be due to site-dependent accumulation of proteoglycans (decorin and lumican) which are responsible for production of thin fibrils. The central portion with higher proteoglycans would have abundant thin fibrils with less slippage but better elasticity to buffer against the direct impact of intraocular pressure on the cornea. In contrast, thick fibrils in the peripheral substantia propria would contribute to the maintenance of tensile strength acting on the transition zone between the cornea and sclera.

Morphological analysis of corneal opacity in Shiba dog with GM1 gangliosidosis.

GM1 gangliosidosis is one of the inherited metabolic lysosomal storage disorders characterized by neurological symptoms caused by beta-galactosidase deficiency and consequent accumulation of GM1 ganglioside in neuronal cells. Shiba dogs affected with GM1 gangliosidosis have been found to suffer from corneal opacity. In our morphological analysis, keratocyte enlargement was induced by abnormal intracellular accumulation of neutral carbohydrates, resulting in the loss of normal arrangement of collagen fibrils in the opaque cornea was found to be associated with the disorder. We therefore conclude that corneal opacity in this Shiba dog with GM1 gangliosidosis may be caused by neutral carbohydrate accumulation in lysosomes, swelling and dysfunction of keratocytes, and subsequent irregular arrangement of collagen fibrils in the corneal proper substance.

A preliminary study of direct application of atelocollagen into a wound lesion in the dog cornea.

PURPOSE: To evaluate the effectiveness of atelocollagen for canine corneal wound healing. MATERIALS AND METHODS: Atelocollagen was used to fill a transplant bed in the central cornea, which was then covered with a contact lens. The wound healing process was analyzed clinically, morphologically, and biochemically. RESULTS: At the early healing stage, both the pupillary zone and details of the iris were observed. The stromal collagen fibrils normalized in a time-dependent manner. Type III collagen in the wound area was detected faintly throughout the experimental period. CONCLUSIONS: This novel method is advantageous for accelerating wound healing without causing inflammation.

The modulation of collagen fibril assembly and its structure by decorin: an electron microscopic study.

The present study was carried out to determine the effect of decorin in the process of collagen assembly. Collagen fibrils were obtained in vitro by aggregation from commercialized acid-soluble type I collagen with the addition of different concentrations of decorin (0-25 microg/ml). All specimens were observed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The distribution of collagen fibril diameters was also analyzed by TEM. In samples without or with low concentrations of decorin, highly porous collagen fiber networks were formed. On the other hand, dense networks were observed in samples treated with high concentrations of decorin. The influence of decorin secreted by cells on collagen fibrils was observed by SEM, and the fiber network elasticity was measured using a rheometer. SEM images showed that collagen fiber networks without fibroblasts were much looser than those cultured with normal fibroblasts. The networks cultured with the fibroblasts were composed of straight fibers with large diameters. On the other hand, collagen fiber networks cultured with siRNA-decorin-transfected (siDT) fibroblasts had loose, meandering fibers with small diameters. The elasticity of collagen fiber networks cultured with untransfected fibroblasts showed no significant difference over the 7-day incubation period. However, significantly lower elastic values were obtained for collagen fiber networks treated with siDT cells on days 3 and 7. In addition, after treatment with 5.0 or 25 microg/ml decorin, the l collagen fiber networks cultured with siDT cells exhibited an altered structure that showed a dense structure similar to that of the fiber networks cultured with untransfected fibroblasts. In conclusion, this in vitro study showed that decorin is a regulatory and architecturally small leucine-rich repeat proteoglycan in the process of collagen fibril assembly.

Effect of collagen oligopeptide injection on rabbit tenositis.

Effects of the collagen oligopeptide (COP) were examined by its repeat injection into the inflammatory rabbit Achilles tendon (Tendo calcaneus communis), in which tenositis was induced by injection of bacterial collagenase. COP was evaluated 5 times over a period of 3 weeks to 1 month after injection of collagenase. At 1 month after treatment, the therapeutic effect of COP was evaluated by examining the structure of collagen fibrils, amount and components of glycosaminoglycans (GAGs) and matrix metalloproteinases (MMPs), and compared with the saline injection, control, and normal groups. The Achilles tendon of rabbit in the control group (no COP injection) and saline injection group showed a notable increase in the number of fine collagen fibrils, a change in GAG composition and increase in the amount of pro-MMP-2, indicating the weakening of the tendon. In contrast, the size distribution of collagen fibrils, GAG composition and the amount of pro-MMP-2 was similar to those in the normal group. These results suggest that COP injection promotes healing processes of the tendon injury.