Structural response of human corneal and scleral tissues to collagen cross-linking treatment with riboflavin and ultraviolet A light.

High success rates in clinical trials on keratoconic corneas suggest the possibility of efficient treatment against myopic progression. This study quantitatively investigated the in vitro ultrastructural effects of a photooxidative collagen cross-linking treatment with photosensitizer riboflavin and UVA light in human corneo-scleral collagen fibrils. A total of 30.8 × 2 mm corneo-scleral strips from donor tissue were sagittally dissected using a scalpel. The five analytic parameters namely fibril density, fibril area, corneo-scleral thickness, fibril diameter, and fibril arrangement were investigated before and after riboflavin-UVA-catalyzed collagen cross-linking treatment. Collagen cross-linking effects were measured at the corneo-scleral stroma and were based on clinical corneal cross-linking procedures. The structural response levels were assessed by histology, digital mechanical caliper measurement, scanning electron microscopy, and atomic force microscopy. Riboflavin-UVA-catalyzed collagen cross-linking treatment led to an increase in the area, density, and diameters of both corneal (110, 112, and 103 %) and scleral (133, 133, and 127 %) stromal collagens. It also led to increases in corneal (107 %) and scleral (105 %) thickness. Collagen cross-linking treatment through riboflavin-sensitized photoreaction may cause structural property changes in the collagen fibril network of the cornea and sclera due to stromal edema and interfibrillar spacing narrowing. These changes were particularly prominent in the sclera. This technique can be used to treat progressive keratoconus in the cornea as well as progressive myopia in the sclera. Long-term collagen cross-linking treatment of keratoconic and myopic progression dramatically improves weakened corneo-scleral tissues.

AFM study for morphological characteristics and biomechanical properties of human cataract anterior lens capsules.

The aim of this study was to quantitatively investigate the morphologies (surface roughness) and biomechanical properties (Young's modulus) of human anterior lens capsules (ALCs) for noncataract and cataract groups using atomic force microscopy. Eight human ALCs obtained during phacoemulsification from patients with senile cataracts (72 ± 13 years) were investigated in both the hydrated and dehydrated conditions. The cataract group showed clearly the proliferated lens epithelial cells (LECs) with a monomorphic cell structure, a diameter of 12.54 ± 4.31 µm, and a height of 0.23 ± 0.04 µm, whereas the control group showed no LECs. A substantial amount of false-positive calcification was observed caused by the deposition of remnants of dried salt solution. Cataract group showed significantly higher surface roughness (382.06 nm, p ≤ 0.001) than control group in the anterior side of ALCs, whereas cataract group showed significantly lower surface roughness (353.79 nm, p ≤ 0.001) than control group in their posterior side. Cataract group showed significantly higher Young's modulus (69.52 kPa, p ≤ 0.001) compared to the control group, regardless of the ALC side. Therefore, it is significant that this study provides a new method to examine the nanostructural characteristic and biomechanical property of human ALCs through a nanometer-scale resolution microscopy technique.

Short-term nanostructural effects of high radiofrequency treatment on the skin tissues of rabbits.

The aim of this study is to quantitatively investigate the short-term effects of RF tissue-tightening treatment in in vivo rabbit dermal collagen fibrils. These effects were measured at different energy levels and at varying pass procedures on the nanostructural response level using histology and AFM analysis. Each rabbit was divided into one of seven experimental groups, which included the following: control group, and six RF group according to RF energy (20 W and 40 W) and three RF pass procedures. The progressive changes in the diameter and D-periodicity of rabbit dermal collagen fibrils were investigated in detail over a 7-day post-treatment period. The dermal tissues treated with the RF tissue-tightening device showed more prominent inflammatory responses with inflammatory cell ingrowth compared to the control. This effect showed more prominent with the passage of day after treatment. Although an increase in the diameter and D-periodicity of dermal collagen fibrils was identified immediately after the RF treatment, a decrease in the morphology of dermal collagen fibrils continued until post-operative day 7. Furthermore, RF treatment led to the loss of distinct borders. Increases in RF energy with the same pass procedure, as well as an increase in the number of RF passes, increased the occurrence of irreversible collagen fibril injury. A multiple-pass treatment at low energy rather than a single-pass treatment at high energy showed a large amount of collagen fibrils contraction at the nanostructural level.

Effect of cross-linking with riboflavin and ultraviolet A on the chemical bonds and ultrastructure of human sclera.

This study examined the effect of the cross-linking with riboflavin-ultraviolet A (UVA) irradiation on the chemical bonds and ultrastructural changes of human sclera tissues using Raman spectroscopy and atomic force microscopy (AFM). Raman spectroscopy of the normal and cross-linked human sclera tissue revealed different types of the riboflavin-UVA and collagen interactions, which could be identified from their unique peaks, intensity, and shape. Raman spectroscopy can prove to be a powerful tool for examining the chemical bond of collagenous tissues at the molecular level. After riboflavin-UVA treatment, unlike a regular parallel arrangement of normal collagen fibrils, the AFM image revealed interlocking arrangements of collagen fibrils. The observed changes in the surface topography of the collagen fibrils, as well as in their chemical bonds in the sclera tissue, support the formation of interfibrilar cross-links in sclera tissues.

AFM study for morphological and mechanical properties of human scleral surface.

This study examined the structures and the elastic and viscous properties of human scleral collagen fibrils by atomic force microscopy (AFM). Sample preparation was performed to minimize the sources of artifacts for further imaging. To observe the morphological and property characteristics of human scleral surfaces, AFM was used as a microscopic tool. The AFM topography, phase shift and deflection images of the dehydrated scleral collagen fibrils were obtained. The visco-elasticity of collagen fibrils was determined from the force-distance curves of the AFM. Inspection of the fibril surface in high resolution showed that the D-period spacing along the collagen fibrils was clearly evident. The fibril diameter over a scan size of 5 x 5 microm2 was 145.22 +/- 17.78 nm (n = 178) ranging from 98 to 220 nm, and the D-periodicity was 69.14 +/- 14.15 nm (n = 189), which is similar to the normal 67 nm D-periodicity. Force-distance analysis indicated that human scleral collagen had comparatively high adhesion force and elasticity, to protect the eye from external trauma and to withstand the expansive force made by the intraocular pressure.

Nanostructural investigation of frontalis sling biomaterial surfaces.

This study examined the nanostructural surface of three frontalis sling biomaterials: autogenous fascia lata, preserved fascia lata and silicone rod. The morphological characteristics of the sling biomaterial surfaces were examined qualitatively and quantitatively by scanning electron microscopy and atomic force microscopy, respectively. The autogenous fascia lata showed well-arranged nanostructures of parallel fascia collagen fibrils with clear 67 nm axial periodicity, whereas the preserved fascia lata showed tangled nanostructures of damaged collagen fibril bundles. The silicone rod showed a substantial amount of debris with some scratches and the smoothest roughness compared with the other sling biomaterials, followed by preserved fascia lata. Autogenous fascia lata showed the highest surface roughness. The association between the roughness and cell adhesion suggests that the nanostructure of autogenous fascia lata biomaterials is the best for frontalis sling and that of the silicone rod biomaterials is the worst.

Ultrastructural investigation of intact orbital implant surfaces using atomic force microscopy.

This study examined the surface nanostructures of three orbital implants: nonporous poly(methyl methacrylate) (PMMA), porous aluminum oxide and porous polyethylene. The morphological characteristics of the orbital implants surfaces were observed by atomic force microscopy (AFM). The AFM topography, phase shift and deflection images of the intact implant samples were obtained. The surface of the nonporous PMMA implant showed severe scratches and debris. The surface of the aluminum oxide implant showed a porous structure with varying densities and sizes. The PMMA implant showed nodule nanostructures, 215.56 ± 52.34 nm in size, and the aluminum oxide implant showed crystal structures, 730.22 ± 341.02 nm in size. The nonporous PMMA implant showed the lowest roughness compared with other implant biomaterials, followed by the porous aluminum oxide implant. The porous polyethylene implant showed the highest roughness and severe surface irregularities. Overall, the surface roughness of orbital implants might be associated with the rate of complications and cell adhesion.

The effects of subluxation of the femoral head with avascular necrosis in growing rabbits.

To validate the adverse effects of subluxation of the femoral head in Legg-Calve-Perthes disease, the authors made an experimental model of Perthes disease with subluxation in growing rabbits by interrupting the epiphyseal artery (devascularization) and immobilizing the knee in extension (immobilization). Seventy-two rabbits, 4 to 5 weeks old, were divided into three groups: group A with both devascularization and immobilization (25 rabbits), group B with devascularization only (25 rabbits), and group C with immobilization only (22 rabbits). In each experimental group, four to six rabbits each were killed at 1, 2, 4, 8, and 12 weeks. After reviewing the serial radiographs and gross specimens, the authors found six radiologic and six macroscopic abnormalities. The incidence and the severity of deformity in group A rabbits were higher than that of groups B or C in terms of the overall incidence of deformities (P <0.001), head deformity scores (P <0.001), and the incidence of a total collapse of the capital femoral epiphysis. In conclusion, subluxation of the immature femoral head with avascular necrosis in rabbits increased femoral head deformities.