Gamma-cross-linked nonfibrillar collagen gel as a scaffold for osteogenic differentiation of mesenchymal stem cells.

We fabricated a transparent nonfibrillar collagen gel using gamma irradiation (5 kGy) and cultured rat mesenchymal stem cells (MSCs) on both the gamma-irradiated collagen gel and on unirradiated fibrillar collagen gel. Cells attached well and proliferated with high viability on the surface of both gels. The cells cultured on the gamma-irradiated nonfibrillar gel had a unique elongated shape and adhered to each other in culture. After 21 days of culture in dexamethasone-containing culture medium, the contents of bone-specific osteocalcin and calcium on the gamma-irradiated nonfibrillar gel were 1.4 and 1.9 times higher than those on fibrillar collagen gel, respectively. These data show that osteogenic differentiation of MSCs was promoted more efficiently on the gamma-cross-linked nonfibrillar gel than on the fibrillar gel and demonstrate the potential of the gamma-irradiated collagen gel for use in bone tissue engineering.

Nanoscale helium ion microscopic analysis of collagen fibrillar changes following femtosecond laser dissection of human cornea.

Over the last decade, femtosecond lasers have emerged as an important tool to perform accurate and fine dissections with minimal collateral damage in biological tissue. The most common surgical procedure in medicine utilizing femtosecond laser is LASIK. During the femtosecond laser dissection process, the corneal collagen fibers inevitably undergo biomechanical and thermal changes on a sub-micro- or even a nanoscale level, which can potentially lead to post-surgical complications. In this study, we utilized helium ion microscopy, complemented with transmission electron microscopy to examine the femtosecond laser-induced collagen fibrillar damage in ex vivo human corneas. We found that the biomechanical damage induced by laser etching, generation of tissue bridges, and expansion of cavitation bubble and its subsequent collapse, created distortion to the surrounding collagen lamellae. Femtosecond laser-induced thermal damage was characterized by collapsed collagen lamellae, loss of collagen banding, collagen coiling, and presence of spherical debris. Our findings have shown the ability of helium ion microscopy to provide high resolution images with unprecedented detail of nanoscale fibrillar morphological changes in order to assess a tissue damage, which could not be resolved by conventional scanning electron microscopy previously. This imaging technology has also given us a better understanding of the tissue-laser interactions in a nano-structural manner and their possible effects on post-operative wound recovery.

Histopathological features of tissue alterations induced by low frequency ultrasound with cavitational effects on human adipose tissue.

Low frequency, high energy level ultrasound (US) induces physical effects on tissues called ultrasonic cavitation (UC). Endermic US therapy has recently been proposed as a method to reduce non-invasively the amount of adipose tissue. Very few published studies have dealt with the biological effects of such therapies on human adipose tissue. The aim of this study is to evaluate, through histopathological examination, the effects of various levels of ultrasonic radiation on supravital human adipose tissue. Four ex vivo human tissue samples were treated as follows: sample 1: no US radiation; sample 2: US radiation 2,5 W/cm2 for 15 minutes; sample 3: US radiation 5 W/cm2 for 15 minutes; sample 4: infiltration of 30 ml physiological saline and US radiation, 5 W/cm2 for 15 minutes. For lower levels of ultrasonic energy, interruption of the adipocytic membranes was evident both in the surface and in the deeper cutaneous and subcutaneous layers, with focal dissolution and homogenization of the surface dermal fascia. For higher levels of ultrasonic energy, alterations of the adipocytes and of the collagen fibers were greater, resulting in the dissolution of the cells and of the interlobular fibrous septa. Both effects were amplified by tumescent saline infiltration. The histological lesions demonstrated in adipocytes confirm the theoretical premises of a possible usefulness in the treatment of localized adiposis. The alterations observed in the connective stroma could have positive effects on the structural re-organization and consequently on the in vivo external appearance of the treated areas.

High-resolution multiphoton tomography and fluorescence lifetime imaging of UVB-induced cellular damage on cultured fibroblasts producing fibres.

BACKGROUND: Multiphoton tomography (MPT) is suitable to perform both ex vivo and in vivo investigations of living skin and cell cultures with submicron resolution. Fluorescence lifetime imaging (FLIM) generates image contrast between different states of tissue characterized by various fluorescence decay rates. Our purpose was to combine MPT and FLIM to evaluate fibroblasts and collagen fibres produced in vitro. METHODS: Fibroblast cultures, 2-4 days old, at a subconfluent stage, were evaluated before and after irradiation with a single UVB dose. One month old cultures stimulated with ascorbic acid were also assessed. RESULTS: After UVB radiation, fibroblasts appear irregular in size, lose their alignment and show a decrease in fluorescence lifetime. One month-old fibroblasts, producing collagen fibres after stimulation with ascorbic acid, appear as small roundish structures intermingled by filaments showing a granular arrangement. CONCLUSION: The combination of MPT and FLIM may be useful for the in vitro study of cell modifications induced by injurious or protective agents and drugs.

YAG laser treatment causes rapid degeneration and regeneration of collagen fibres in pig skin and facilitates fibroblast growth.

The non-ablative laser therapies have been speculated to cause microinjury in the dermal collagen fibres and increase collagen synthesis in the fibroblasts, leading to remodelling of the extracellular matrix. This study investigated the effects of neodymium YAG laser treatment on pig skin, especially focusing on its extracellular matrix molecules. The dorsal areas of a minipig were subjected to laser treatment, and samples were obtained by punch biopsies, and histological, immunohistochemical, and biochemical analyses were performed. The laser treatment caused degeneration of collagen fibres and fibrils, which were reconstituted within 24 hours, whereas there was no inflammation and no apparent damage on elastic fibres. Small blood vessels disappeared by the laser treatment, which re-appeared in 3 days. Biochemically, the amounts of collagen decreased up to day 3 after the treatment and then increased at day 7. When fibroblasts in dermal tissue at day 28 were counted, more fibroblasts in the treated tissue were observed than non-treated control. These results suggest that, although the laser treatment transiently degenerates collagen fibres and fibrils, it restores and increases them, mainly by an increase in dermal fibroblasts, assuring its minimal complication of skin.

Morphological analysis of second-intention wound healing in rats submitted to 16 J/cm 2 lambda 660-nm laser irradiation.

BACKGROUND AND OBJECTIVES: Low-level laser therapy (LLLT) has been extensively applied to improve wound healing due to some biostimulatory properties presented by laser arrays apparently able to accelerate the cicatricial repair of soft tissue injuries. However, many controversial results have been reported in the literature, probably as a result of the wide sort of different protocols of photobiomodulation employed in those experiments. The goal of this study was to investigate the effect of a low-dose protocol of LLT on the intensity of the inflammatory response and the pattern of collagen fibers' deposition during second-intention wound healing in rodents. MATERIALS AND METHODS: Standard-sized wounds were carried out in the back of 24 male rats. Half of them underwent LLLT treatment (16 J/cm 2 ) at 660 nm delivered for 7 days. Eight and 14 days after the wounds were performed, the repairing area was removed and stained in HE and Masson's trichrome, and the inflammatory response, epithelization, and collagen fiber depositions were evaluated. RESULTS: We found that LLLT was able to slightly reduce the intensity of the inflammatory reaction as well as to enhance substantially the epithelization process at both 8 th and 14 th days. In addition, it also appeared to stimulate the deposition of collagen fibers at the final stages of wound healing. CONCLUSIONS: The LLLT protocol tested in this study resulted in some improvements in second-intention wound healing in rodents.

Atomic force microscopy and transmission electron microscopy analyses of low-temperature laser welding of the cornea.

Low-temperature laser welding of the cornea is a technique used to facilitate the closure of corneal cuts. The procedure consists of staining the wound with a chromophore (indocyanine green), followed by continuous wave irradiation with an 810 nm diode laser operated at low power densities (12-16 W/cm(2)), which induces local heating in the 55-65 degrees C range. In this study, we aimed to investigate the ultrastructural modifications in the extracellular matrix following laser welding of corneal wounds by means of atomic force microscopy and transmission electron microscopy. The results evidenced marked disorganization of the normal fibrillar assembly, although collagen appeared not to be denatured under the operating conditions we employed. The mechanism of low-temperature laser welding may be related to some structural modifications of the nonfibrillar extracellular components of the corneal stroma.

Effects of laser irradiation on collagen organization in chemically induced degenerative annulus fibrosus of lumbar intervertebral disc.

BACKGROUND AND OBJECTIVE: The number of in vitro experimental studies was carried out with the use of intact tissues to establish a mechanism of laser-tissue interaction. However, in the process of degeneration, both biochemical composition and behavior of the disc were altered drastically. The objective of this study was to evaluate the role of the main matrix components in laser modification of annulus fibrosus (AF) under IR laser irradiation. STUDY DESIGNS/MATERIALS AND METHODS: The samples of AF in a motion segment after hyaluronidase treatment, trypsin digestion and glycation by glyceraldehyde were heated in hydrothermal bath (95 degrees C, 2 min) or irradiated by laser at 1.56 microm. Specimens were imaged by cross-polarization optical coherence tomography (CP-OCT), and then analyzed by differential scanning calorimery (DSC). RESULTS AND DISCUSSION: According to CP-OCT and DSC data non-significant alteration was revealed in AF after hyaluronidase treatment, glycation led to stabilization of annulus collagen and trypsin digestion resulted in a noticeable impairment of collagen fibrils. Laser treatment induced subsequent damages of AF matrix but these damages cannot be explained by laser heating only. The specificity of chemical modification of AF matrix has an influence on a character of collagen network alteration due to IR laser effect. Minimal and maximal alterations are observed for hyaluronidase and trypsin treated samples respectively. Glyceraldehyde fixed samples showed failure of the collagen structure after moderate laser treatment; at the same time thermal denaturation of collagen macromolecules was negligible. We assume that a mechanical effect of laser irradiation plays an important role in laser-induced annulus collagen modification and propose the scheme of physico-chemical process occurring under non-uniform IR laser treatment in AF tissue. CONCLUSION: CP-OCT and DSC techniques allow us to record the alteration of collagen network organization as a result of chemical modification. There were detected significant and specific effects of the biochemical composition and material properties on the response of AF collagen network on laser irradiation. The results go in accordance with our hypothesis that the primary effect of laser influence on collagen network under tension is the mechanical damage of collagen fiber.

Dynamics of femtosecond laser photo-modification of collagen fibers.

In this work, we investigate the non-ablative, non-thermal photo-modification of collagen fibers by femtosecond Ti:Sa laser. The effect was induced and simultaneously registered during the repetitive laser scanning of type I collagen (rat tail and bovine Achilles' tendon), and bovine cornea. An irreversible increase in two-photon autofluorescence and a decrease in second harmonic generation intensities were associated with the collagen femtosecond laser photo-modification. Confocal spectral imaging revealed the formation of new fluorescent species. Controllable nonlinear photo-modification of collagen fibers and bovine cornea with approximately 2 microm spatial resolution was demonstrated.

Increase in dermal collagen fibril diameter and elastogenesis with UVB exposure: an optical and ultrastructural study in albino Balb/c mice.

Cutaneous aging is a complex biological phenomenon, dependent not only on the innate or intrinsic process ("biological clock"), but also on extrinsic elements, primarily chronic sun exposure (photoaging). In order to verify dermal morphological changes in the elastic fiber system and collagen associated with aged skin, we performed a light and electron microscopic study on exposed-shaved albino mice, which were exposed to UVB radiation. The experimental group consisted of 48 exposed animals, randomly distributed in three groups and submitted to different radiation doses (A, 28800 J/m2; B, 57600 J/m2; and C, 86400 J/m2) and studied 0, 30, 60 and 90 days of exposure discontinuation. Nonexposed-shaved and nonexposed-nonshaved animals were included as controls. From the day of exposure discontinuation and subsequently, the elastic system and collagen network were progressively modified. The increase in collagen fibril diameter was prominent in the 60 and 90 day groups (p<0.05), as noticed on electron microscopy. Elastic fiber density also increased after irradiation (p<0.05). On electron microscopy, elastogenesis was seen in the deep dermis. The comparative study among the groups disclosed clear relationship between doses and "elastotic changes". It also showed that chronological aging of mice skin was apparently intensified after UVB exposure. Skin elastogenesis seems to be a major consequence of UVB exposure, apart from elastolysis, and occurs not only in humans but also in hairless mice submitted to continuous, long-term UVB exposure.

The effect of irradiation and hyperbaric oxygenation (HBO) on extracellular matrix of the condylar cartilage after mandibular distraction osteogenesis in the rabbit.

The effects of irradiation and hyperbaric oxygenation (HBO) on the extracellular matrix of condylar cartilage after mandibular distraction were evaluated. Unilateral distraction was performed on 19 rabbits. Five study groups were included: control, low- and high-dose irradiation, and low- and high-dose irradiation groups with HBO. Additionally, four temporomandibular joints (TMJ) were used as control material. The high-dose irradiated animals were given in the TMJ 22.4 Gy/4 fractions irradiation (equivalent to 50 Gy/25 fractions). Low-dose irradiation group received a 2.2 Gy dosage. Two groups were also given preoperatively HBO 18 x 2.5ATA x 90 min. After a two-week distraction period (14 mm lengthening) and four-week consolidation period the TMJs were removed. Proteoglycan (PG) distribution of the extracellular matrix was evaluated using safranin O staining and collagen I and II using immunohistochemistry. The organization of fibrillar network was studied by polarized light microscopy. On the operated side of the control group and on the unoperated side in all, except for high-dose irradiated group, PG distribution and fibrillar network were normal appearing. In the irradiated groups, with or without HBO, the cartilaginous layer was partially or totally devoid of PG and the network structure was severely damaged. In conclusion, irradiation in conjunction with the pressure applied by distraction causes severe damage to extracellular matrix of condylar cartilage.

Simulation of small-angle x-ray scattering from collagen fibrils and comparison with experimental patterns.

Simulation of small-angle x-ray scattering from collagen in healthy and cancerous breast tissue may reveal detailed information on the structural changes in collagen. Collagen fibril is modelled as a cylinder with axially periodic step-function electron density, and packing is approximated by placing the cylinders in small hexagonal bundles. The intensity from a bundle is calculated by summing analytical scattering amplitudes from the cylinders, and intensities from several bundles with varying lattice constants are averaged. Comparisons with more complex models are made to estimate the robustness of the model. The oscillations in the equatorial direction are not significantly affected by added complexity. The relative intensities of the Bragg peaks in the meridional direction can be tuned by modifying the axial electron density distribution. Tests with different fibril radius distributions show that the average radius can be determined with an accuracy of +/-0.5 nm but that the shape of the radius distribution cannot be accurately determined from the scattering patterns. The effect of multiple scattering and the detector point-spread function (PSF) is considered, and the PSF may make a significant contribution to the final slope of the scattering pattern. Comparisons with observed scattering indicate that the model is basically correct at the supra-molecular level.

In vitro study of a neodynium:yttrium aluminum perovskite laser on human nonexposed pulp after cavity preparation.

PURPOSE: The aim of this study was to investigate dental pulp reactions after a neodynium:yttrium aluminum perovskite laser pulse on the dentinal floor of occlusal cavities in an in vitro model. METHODS: A Lokki dt laser was used at 30 Hz, 5 W, and 160 mJ for 0.5 s. The pulp reactions were analyzed in a previously described human tooth slice cultured model. The following markers were identified by immunohistochemistry: collagens I, III, and IV and HLA-DR-positive cells. RESULTS: After 4 days of culture, under laser pulse, a concentration of type III collagen beneath the odontoblast layer, a higher level of vessels and an accumulation of HLA-DR-positive cells were routinely observed subjacent to the cavity. CONCLUSION: This laser treatment leads to the first step of rapid pulp repair under culture conditions.

Collagen as a model for the study of radiation induced side effects: use of image processing.

Collagen can be used as a model system for studying the effects of radiation on a protein. Quantitative studies of collagen fibrils from electron-optical images from radiated and non-radiated material can produce information not only about the structural effects produced by radiation, but also about the actual part of the fibril being affected.