Individual Collagen Fibers Research Articles

Mitral annular reduction with subablative therapeutic ultrasound: pre-clinical evaluation of the ReCor device

To evaluate the potential for mitral annular (MA) size reduction using a novel device utilising therapeutic ultrasound (TU).The ReCor device (ReCor Medical, Inc., Ronkonkoma, NY, USA, Investigational device, not for use in human application) was studied in a closed chest canine animal model (35 dogs). Under fluoroscopy, a 12 Fr TU balloon catheter was advanced into the left atrium (transseptal approach). The TU balloon was inflated with contrast-saline, positioned at the MA and energy delivered circumferentially, to heat the tissue locally. Five TU applications were delivered (at least 60W for at least 40 sec). Relative to baseline, mitral valve annular diameter reduction (measured by transthoracic echocardiography) was 8.4% immediately post procedure(p<0.001), 8.6% at one week (p<0.001), 8.8% at two weeks (p<0.001), 9.3% at three weeks (p<0.001), 10.8% at four weeks (p<0.001), 8.6% at three months (p<0.001) and 5.7% at six months (p<0.001). Histology showed an increase in elastin associated with tissue thickening at the annular level. Transmission electron microscopy demonstrated a decrease in diameter of individual collagen fibres in treated regions compared to controls.Therapeutic ultrasound (TU) energy application to the mitral annulus is feasible percutaneously. A reduction in annular dimensions occurs immediately and appears to be durable without peri-annular damage.

Probing cellular mechanobiology in three-dimensional culture with collagen–agarose matrices

The study of how cell behavior is controlled by the biophysical properties of the extracellular matrix (ECM) is limited in part by the lack of three-dimensional (3D) scaffolds that combine the biofunctionality of native ECM proteins with the tunability of synthetic materials. Here, we introduce a biomaterial platform in which the biophysical properties of collagen I are progressively altered by adding agarose. We find that agarose increases the elasticity of 3D collagen ECMs over two orders of magnitude with modest effect on collagen fiber organization. Surprisingly, increasing the agarose content slows and eventually stops invasion of glioma cells in a 3D spheroid model. Electron microscopy reveals that agarose forms a dense meshwork between the collagen fibers, which we postulate slows invasion by structurally coupling and reinforcing the collagen fibers and introducing steric barriers to motility. This is supported by time lapse imaging of individual glioma cells and multicellular spheroids, which shows that addition of agarose promotes amoeboid motility and restricts cell-mediated remodeling of individual collagen fibers. Our results are consistent with a model in which agarose shifts ECM dissipation of cell-induced stresses from non-affine deformation of individual collagen fibers to bulk-affine deformation of a continuum network.

Morphological parameters for assessment of burn severity in an acute burn injury rat model

Determination of burn severity (i.e. burn depth) is important for effective medical management and treatment. Using a recently described acute burn model, we studied various morphological parameters to detect burn severity. Anaesthetized Sprague-Dawley rats received burns of various severity (0- to 14-s contact time) followed by standard resuscitation using intravenous fluids. Biopsies were taken from each site after 5 h, tissues fixed in 10% neutral-buffered formalin, processed and stained with haematoxylin and eosin. Superficial burn changes in the epidermis included early keratinocyte swelling progressing to epidermal thinning and nuclear elongation in deeper burns. Subepidermal vesicle formation generally decreased with deeper burns and typically contained grey foamy fluid. Dermal burns were typified by hyalinized collagen and a lack of detectable individual collagen fibres on a background of grey to pale eosinophilic seroproteinaceous fluid. Intact vascular structures were identified principally deep to the burn area in the collagen. Follicle cell injury was identified by cytoplasmic clearing/swelling and nuclear pyknosis, and these follicular changes were often the deepest evidence of burn injury seen for each time point. Histological scores (epidermal changes) or dermal parameter depths (dermal changes) were regressed on burn contact time. Collagen alteration (r(2) = 0.91) correlated best to burn severity followed by vascular patency (r(2) = 0.82), epidermal changes (r(2) = 0.76), subepidermal vesicle formation (r(2) = 0.74) and follicular cell injury was useful in all but deep burns. This study confirms key morphological parameters can be an important tool for the detection of burn severity in this acute burn model.

Late Posthemorrhagic Structural and Functional Changes in Pulmonary Circulation Arteries

Objective: to reveal the major regularities and mechanisms of morphological changes in the rat pulmonary circulation arteries in the late posthemorrhagic period and to compare them with age-related features of the vessels. Materials and methods: experiments to generate graduated hemorrhagic hypotension with the blood pressure being maintained at 40 mm Hg were carried out on young (5—6-month) albino male Wistar rats. Throughout hypotension and 60 days after blood loss, the blood was tested to determine low and average molecular-weight substances by spectrophotometry and the pro- and antioxidative systems by chemiluminescence. Pulmonary circulation arteries were morphologically studied in young animals, rats in the late posthemorrhagic period and old (24—25-month) rats. Results. Sixty-minute hemorrhagic hypotension leads to the development of endotoxemia and imbalance of the pro- and antioxidative systems, the signs of which are observed in the late periods (2 months) after hypotension. At the same time, the posthemorrhagic period is marked by the significant pulmonary circulation arterial morphological changes comparable with their age-related alterations in old rat. This shows up mainly in the reorganization of a connective tissue component in the vascular wall: the elevated levels of individual collagen fibers, their structural changes, elastic medial membrane destruction and deformity. At the same time, there is a change in the morphometric parameters of vessels at all study stages while their lowered flow capacity is only characteristic for intraorgan arteries. Conclusion: The increased activity of free radical oxidation and endotoxemia may be believed to be one of the causes of morphological changes in pulmonary circulation arteries in the late posthemorrhagic period, which is similar to age-related vascular alterations. Key words: hemorrhagic hypotension, pulmonary circulation arteries, free radical oxidation, endotoxemia, remodeling, late posthemorrhagic period, age-related vascular changes.

A constitutive model for fibrous tissues considering collagen fiber crimp

A micromechanically based constitutive model for fibrous tissues is presented. The model considers the randomly crimped morphology of individual collagen fibers, a morphology typically seen in photomicrographs of tissue samples. It describes the relationship between the fiber endpoints and its arc-length in terms of a measurable quantity, which can be estimated from image data. The collective mechanical behavior of collagen fibers is presented in terms of an explicit expression for the strain-energy function, where a fiber-specific random variable is approximated by a Beta distribution. The model-related stress and elasticity tensors are provided. Two representative numerical examples are analyzed with the aim of demonstrating the peculiar mechanism of the constitutive model and quantifying the effect of parameter changes on the mechanical response. In particular, a fibrous tissue, assumed to be (nearly) incompressible, is subject to a uniaxial extension along the fiber direction, and, separately, to pure shear. It is shown that the fiber crimp model can reproduce several of the expected characteristics of fibrous tissues.

Stromal Fibrosis Reaction in Rat Prostates Induced by Alpha 1 Adrenergic Stimulation

Most of the publications dealing with the experimental induction of prostatic neoplasia have focused on the description of epithelial lesions, but little attention has been paid to the involvement of their stromal alterations. The present study is a first attempt to assess the stromal changes in both collagen and elastic fibrils as well as in its cellular constituents, which accompany prostatic intraepithelial neoplastic (PIN)-like lesions induced by phenylephrine (PE) in rats. Adolescent rats received subcutaneous injections of PE daily (10 mg/kg/d) for 1 month. At the end of the experimental period the rats were sacrificed; the dissected ventral prostates were fixed in Stieve solution and paraffin-embedded; and sections were cut and stained accordingly. Most of the stromal cells were identified by immunohistochemistry techniques using primary antibodies to ED2 (resident macrophages), actin (fibrocytes and vascular smooth muscle cells), vimentin (mesenchymal cells), and 5'-bromo-2'-deoxyuridine (S-phase proliferating cells). Collagen stromal mass was visualized by Gomori trichrome and individual collagen fibers by picrosirius red staining under polarized light, whereas the fine fibrils were stained according to the Pinkus method. The untreated rat prostates are characterized by a delicate interacinar stroma with scanty cells and fibrils. The PE-treated prostates showed a significant increase in both cellular and fibrillar elements as well as an increase in arteriolar density, in addition to the typical PIN lesions. The presence of such an interstitial fibrosis, which also includes inflammatory cells, neoangiogenesis, and synthesis de novo of collagen and fibers, might be regarded as a desmoplastic reaction. It is suggested that these changes could be related to a tissue repair process occurring subsequent to the inflammatory exudate that takes place during the incipient phases of the PE treatment.

Lysyl Oxidase Is Essential for Normal Development and Function of the Respiratory System and for the Integrity of Elastic and Collagen Fibers in Various Tissues

Lysyl oxidases, a family comprising LOX and four LOX-like enzymes, catalyze crosslinking of elastin and collagens. Mouse Lox was recently shown to be crucial for development of the cardiovascular system because null mice died perinatally of aortic aneurysms and cardiovascular dysfunction. We show here that Lox is also essential for development of the respiratory system and the integrity of elastic and collagen fibers in the lungs and skin. The lungs of E18.5 Lox(-/-) embryos showed impaired development of the distal and proximal airways. Elastic fibers in E18.5 Lox(-/-) lungs were markedly less intensely stained and more disperse than in the wild type, especially in the mesenchyme surrounding the distal airways, bronchioles, bronchi, and trachea, and were fragmented in pulmonary arterial walls. The organization of individual collagen fibers into tight bundles was likewise abnormal. Similar elastic and collagen fiber abnormalities were seen in the skin. Lysyl oxidase activity in cultured Lox(-/-) skin fibroblasts and aortic smooth muscle cells was reduced by approximately 80%, indicating that Lox is the main isoenzyme in these cells. LOX abnormalities may thus be critical for the pathogenesis of several common diseases, including pulmonary, skin, and cardiovascular disorders.

The effect of uniform heating on the biomechanical properties of the intervertebral disc in a porcine model

Background context The use of minimally invasive lumbar intradiscal heating techniques, including intradiscal electro-thermal therapy (IDET), endoscopic radio-frequency annuloplasty, nucleoplasty and laser discectomy, for chronic lumbar discogenic pain and contained disc herniation has recently gained popularity. The purported therapeutic mechanisms of these interventions include subtotal nuclectomy, annular nociceptor ablation, and stabilization of the annular fibers. Basic science data elucidating the biomechanical and histomorphologic alterations of heat treatments on disc remain sparse. Purpose The purpose of this study is to examine the effects of uniform heating on biomechanical properties and histomorphology of intervertebral disc tissues using a porcine model. Study design/setting In a laboratory setting, porcine functional spinal units consisting of vertebra-nucleus pulposus-vertebra core and porcine hamstring tendons were harvested. Studies were performed on these tissue samples by uniformly heating the specimens in a constant temperature water bath. Ten porcine lumbar disc core and twenty-five porcine hamstring tendons were utilized as the subjects for this study. The effects of uniform heat treatments on disc core and hamstring tendon were measured for shrinkage, stiffness, and load to failure strength. Histomorphological study was also carried on the same specimen. Methods The porcine vertebra-nucleus pulposus-vertebra segments were cored to a uniform 1-cm diameter. The hamstring tendons were cut to uniform 1.2-inch lengths. The tendon specimens were divided into groups of five each and heated in constant temperature water baths of 60°C, 65°C, 70°C, or 75°C for 10 min. Unheated specimens served as controls. The disc core specimens were divided in two group of five each, and tested at room temperature or after immersion in a 70°C bath. The shrinkage was monitored during immersion in the water bath. Biomechanical testing to failure was carried out using mechanical loading on an MTS servohydraulic testing machine operating under stroke control. Strength and stiffness of the tissue was determined. Histomorphology was studied by staining the specimen with hematoxylin and eosin (H&E), and examined under 200 times magnification. Non-heated controls were used for comparisons. Results The porcine hamstring tendons had no measurable shrinkage in specimens heated up to 65°C. At temperatures above 65°C, the shrinkage was concluded within 2 min of immersion and 70°C appeared to be the optimal temperature, as temperatures higher than this did not demonstrate incremental effects. The disc core samples were heated to 70°C (optimum temperature), and there appeared to be gross contraction of the disc core circumference to visual inspection, but no measurable lengthwise shrinkage could be appreciated. Histologically, the specimens demonstrated progressive loss of individual collagen fiber outline as the temperature increased. In the tendons, at 75°C all of the fibers appear to be fused together, and the voids between individual collagen fibers were no longer present. Biomechanical testing revealed that the tendons undergo a substantial reduction in stiffness after heating. The mean tendon stiffness for the unheated specimens was 19,356 psi, while the corresponding value for the heated tendons was 1023 psi. These were significantly different using the paired t-test at p=0.0043. For the disc core samples, there was no significant difference in either stiffness (p=0.182) or failure strength (p=0.998) after heating. All failures occurred in mid-substance of the specimen. Conclusions The application of uniform heating to nucleus pulposus disc core caused visible contraction of its circumference but not lengthwise shrinkage. The same heating shrinks the hamstring tendon and reduces its stiffness. Ultimate failure strength of the disc core specimen remains unchanged. The failure data was not obtainable for the tendon due to premature slippage from the fixation apparatus before failure. The results of this study fail to support a biomechanical justification for the application of uniform heat treatment to the whole intervertebral disc. Heating annulus fibrosus and nucleus pulposus separately to specific temperatures may have potential clinical benefits.

Effects of polar solvents on the fracture resistance of dentin: role of water hydration

Although healthy dentin is invariably hydrated in vivo, from a perspective of examining the mechanisms of fracture in dentin, it is interesting to consider the role of water hydration. Furthermore, it is feasible that exposure to certain polar solvents, e.g., those found in clinical adhesives, can induce dehydration. In the present study, in vitro deformation and fracture experiments, the latter involving a resistance-curve (R-curve) approach (i.e., toughness evolution with crack extension), were conducted in order to assess changes in the constitutive and fracture behavior induced by three common solvents—acetone, ethanol and methanol. In addition, nanoindentation-based experiments were performed to evaluate the deformation behavior at the level of individual collagen fibers and ultraviolet Raman spectroscopy to evaluate changes in bonding. The results indicate a reversible effect of chemical dehydration, with increased fracture resistance, strength, and stiffness associated with lower hydrogen bonding ability of the solvent. These results are analyzed both in terms of intrinsic and extrinsic toughening phenomena to further understand the micromechanisms of fracture in dentin and the specific role of water hydration.

Exercise Attenuates Diabetes-Induced Ultrastructural Changes in Rat Cardiac Tissue

Exercise is an effective nonpharmacological treatment in the prevention of mortality and morbidity due to cardiovascular disease in Type I diabetes. This study sought to explore the effects of endurance exercise on the ultrastructural changes seen in diabetic cardiomyopathy. Seven-week-old rats were divided into three groups consisting of sedentary nondiabetic control, sedentary diabetic, and exercised diabetic animals. Diabetes was induced using streptozotocin injection, and the exercised animals were run daily on a treadmill for 9 wk. Changes in heart ultrastructure were analyzed using transmission electron microscopy. Ultrastructural changes in the left ventricle produced by diabetes included changes in myofibrillar arrangements, disrupted mitochondria, and increased cytoplasmic area with an increase in lipid amounts and an increase in individual collagen fiber cross-sectional surface area. Also, an increase in heterochromatin lining the nuclear envelope and an increase in invaginations of the nuclear membrane were observed in cardiomyocytes from diabetic rats when compared with the nuclei from nondiabetic cells. Exercise was found to significantly attenuate the diabetes-induced changes in collagen fibrils, cytoplasmic area, and level of mitochondrial disruption. In contrast, exercise did not appear to significantly influence myofibril volume density, lipid accumulation, or nuclear deformities. These findings indicate that exercise restores specific ultrastructural characteristics of diabetic cardiomyopathy returning them toward nondiabetic phenotypes, particularly in the mitochondria and extracellular matrix proteins.

Dynamic Elastic Modulus of Porcine Articular Cartilage Determined at Two Different Levels of Tissue Organization by Indentation-Type Atomic Force Microscopy

Cartilage stiffness was measured ex vivo at the micrometer and nanometer scales to explore structure-mechanical property relationships at smaller scales than has been done previously. A method was developed to measure the dynamic elastic modulus, | E *|, in compression by indentation-type atomic force microscopy (IT AFM). Spherical indenter tips (radius = ∼2.5 μm) and sharp pyramidal tips (radius = ∼20 nm) were employed to probe micrometer-scale and nanometer-scale response, respectively. | E *| values were obtained at 3 Hz from 1024 unloading response curves recorded at a given location on subsurface cartilage from porcine femoral condyles. With the microsphere tips, the average modulus was ∼2.6 MPa, in agreement with available millimeter-scale data, whereas with the sharp pyramidal tips, it was typically 100-fold lower. In contrast to cartilage, measurements made on agarose gels, a much more molecularly amorphous biomaterial, resulted in the same average modulus for both indentation tips. From results of AFM imaging of cartilage, the micrometer-scale spherical tips resolved no fine structure except some chondrocytes, whereas the nanometer-scale pyramidal tips resolved individual collagen fibers and their 67-nm axial repeat distance. These results suggest that the spherical AFM tip is large enough to measure the aggregate dynamic elastic modulus of cartilage, whereas the sharp AFM tip depicts the elastic properties of its fine structure. Additional measurements of cartilage stiffness following enzyme action revealed that elastase digestion of the collagen moiety lowered the modulus at the micrometer scale. In contrast, digestion of the proteoglycans moiety by cathepsin D had little effect on | E *| at the micrometer scale, but yielded a clear stiffening at the nanometer scale. Thus, cartilage compressive stiffness is different at the nanometer scale compared to the overall structural stiffness measured at the micrometer and larger scales because of the fine nanometer-scale structure, and enzyme-induced structural changes can affect this scale-dependent stiffness differently.

Effects of Aging on the Toughness of Human Cortical Bone: A Study from Nano to Macro Size-Scales

ABSTRACTAge-related deterioration of both the fracture properties and the architecture of bone, coupled with increased life expectancy, are factors leading to the increasing incidence of bone fracture in the elderly. In order to facilitate the development of treatments which counter this increased fracture risk, a thorough understanding of how fracture properties degrade with age is required. The present study describes ex vivo fracture experiments to quantitatively assess the effects of aging on the fracture toughness of human cortical bone in the longitudinal direction. Because cortical bone exhibits rising crack-growth resistance with crack extension, we depart from most previous studies by evaluating the toughness in terms of resistance-curve (R-curve) behavior, measured for bone taken from donors 34 to 99 years old. Using this approach, both the crack-initiation and crack-growth toughness are determined and are found to deteriorate with age; the initiation toughness decreases ∼40% from 40 to 100 years, while the growth toughness is effectively eliminated over the same age range. Evidence from x-ray synchrotron tomography is provided to support the hypothesis that the reduction in crack-growth toughness is associated primarily with a degradation in the degree of extrinsic toughening, in particular involving crack bridging at the microstructural level in the wake of the crack. Atomic force microscope-based nanoidentation of individual collagen fibers revealed changes at the collagen fibrillar level and deep-ultraviolet Raman spectroscopy showed that the cross-linking at the nanostructural level also changes with age. These results should provide for a better mechanistic understanding of the increased propensity for bone fracture with age.

BLUE, BUT NOT SKY BLUE

Attracting a desirable mate is all about beauty in the eye of the beholder!Animals have developed spectacular manes, lustrous scales or brilliant plumage to catch the attention of a passing suitor. Some birds have added vividly coloured skin patches to their repertoire of attractive decorations. However,only a few of these bright skin shades are produced by pigments; many of the colours towards the blue end of the spectrum are generated by the optical properties of the skin itself. Richard Prum explains that it was accepted that blue colours in bird skins were generated by Rayleigh scattering, in the same way as the sky's colour; randomly arranged particles in the atmosphere only scatter short wavelength light to give the sky its familiar tone. But when Prum began looking at the microscopic collagen structures in coloured bird skins, he realised that the skin's colour wasn't produced by randomly scattered light, it was generated by a few wavelengths of light scattered from arrays of collagen fibbers in the skin to produce the blue-green effect(p. 2409); the same as oil-film colours.Prum explains that he first stumbled across this strange class of structural colours because he was intrigued by the ornate decorative structures that birds use to attract a mate. Knowing that these skin colours weren't iridescent, because the colour didn't appear to shimmer, everyone had assumed that the effect was generated by Rayleigh scattering from randomly arranged structures in the skin. But when Prum used transmission electron microscopy to look at the blue skin from a Madagascan velvet aity, he realised that this couldn't be true. The collagen structures were arranged regularly,almost like a crystal, so that light reflected off each layer to produce the blue and green tones. Was the velvet aity unique, or had other birds come up with the same solution to the structural colour problem?Prum sent bottles of `wattle [preserving] juice' out far and wide, asking friends and colleagues to send back samples of skin, gathering vivid samples from more then 30 bird species across the planet. Time and again, the skin's collagen structures were far too ordered to produce the colours by Rayleigh scattering. But were they ordered enough to generate colours in the same way that thin layers of oil only reflect a few wavelengths of light to make rich colours? Prum teamed up with Rodolfo Torres, a mathematician, to see if Torres could derive information from the layered collagen structures, and use it to predict the vivid spectrum of colours that the skin produced.Amazingly, Torres' mathematical analysis of the skins' structures produced predictions that were remarkably similar to the measured spectra. The skins'hues weren't produced by Rayleigh scattering from individual collagen fibers,but constructive interference among blue waves scattered by multiple collagen fibres. And Prum thinks that this effective way of generating skin colours could have evolved more than 50 times over the course of evolution.Prum adds `No one had tested to see whether Rayleigh applied'. He explains that every one assumed that the colours were produced by Rayleigh scattering because they weren't iridescent. `The take home is that biologists were lazy physicists, they had accepted a lousy criterion of accepting [that the colours were generated] by Rayleigh scatterers' concludes Prum.

Automated quantification and reconstruction of collagen matrix from 3D confocal datasets.

The geometrical structure of fibrous extracellular matrix (ECM) impacts on its biological function. In this report, we demonstrate a new algorithm designed to extract quantitative structural information about individual collagen fibres (orientation, length and diameter) from 3D backscattered-light confocal images of collagen gels. The computed quantitative data allowed us to create surface-rendered 3D images of the investigated sample.

A quantitative method to determine the orientation of collagen fibers in the dermis.

We have developed a quantitative microscopic method to determine changes in the orientation of collagen fibers in the dermis resulting from mechanical stress. The method is based on the use of picrosirius red-stained cryostat sections of piglet skin in which collagen fibers reflect light strongly when epipolarization microscopy is used. Digital images of sections were converted into binary images that were analyzed quantitatively on the basis of the length of the collagen fibers in the plane of the section as a measure for the orientation of the fibers. The length of the fibers was expressed in pixels and the mean length of the 10 longest fibers in the image was taken as the parameter for the orientation of the fibers. To test the procedure in an experimental setting, we used skin after 0 and 30 min of skin stretching. The orientation of the fibers in sections of control skin differed significantly from the orientation of fibers in sections of skin that was stretched mechanically for 30 min [76 +/- 15 (n=5) vs 132 +/- 36 (n=5)]. The method described here is a relatively simple way to determine (changes in) the orientation of individual collagen fibers in connective tissue and can also be applied for analysis of the orientation of any other structural element in tissues so long as a representative binary image can be created.

Biaxial mechanical behavior of excised porcine mitral valve leaflets.

Anterior and posterior leaflets from excised porcine mitral valves were mechanically tested under cyclic equibiaxial and strip biaxial stretch protocols at a strain rate of 4-12%/s after preconditioning. Cauchy stress and Lagrangian strain were calculated for both membrane and three-dimensional cases. The leaflets exhibited nonlinearly elastic, anisotropic behavior. Both anterior and posterior leaflets were less extensible in the circumferential than in the radial direction under equibiaxial stretch, with stress ratios of 5.7 and 4.3, respectively. The posterior leaflets exhibited greater extensibility in both directions and lower circumferential posttransitional moduli (ranges 690-820 vs. 2,500-3,200 N/m for anterior). This larger posterior extensibility may be due to the greater number of chordal attachments, which provide additional mechanical stability to this structure. Coupling of radial and circumferential mechanical behavior was evidenced by the response to different stretch protocols, indicating a complex microstructural coupling between individual collagen fibers or bundles. These are the first biaxial data for mitral valves and are a foundation for the development of a more detailed quantitative material description.

Ultrastrucure of the superior oblique tendon.

Individual collagen fibers in the superior oblique tendon of patients with superior oblique palsy studied at 60,000x with transmission electron microscopy are fewer in number and larger in diameter when compared to normal fibers. The relationship of these changes to function and to gross morphologic changes are not clear.

Deposition of cement at reversal lines in rat femoral bone.

Femora from young adult Wistar rats were prepared for both light and electron microscopy. Routinely processed wax sections showed the appearance of cement lines immediately proximal to the resorption surface formed by active osteoclasts and distal to the onset of lamella formation in femoral bone tissue. This early stage of extracellular matrix elaboration at reversal lines was then studied by scanning electron microscopy (SEM) of actively remodeling sites, mainly on trabecular and endosteal surfaces. The resorption surface was shown to comprise a decalcified collagenous mat with individual fibers running either parallel or perpendicular to the surface plane. By examining different, neighboring, areas of resorption lacunae, a temporal sequence of new extracellular matrix production could be established. Before the deposition of new collagen, globular accretions were deposited onto the resorption surface. In areas where individual collagen fibers were oriented perpendicular to the surface plane, this globular matrix was initially deposited on the exposed fiber tips. The globules increased in size and fused laterally to form a continuous cement layer, which not only interdigitated with the collagen mesh of the resorption surface but also provided anchorage for new collagen fibers, which themselves became mineralized. These morphologic results provide a mechanistic explanation of coupling at reversal lines.

Structural abnormalities in the mitis form (type-II) of Ehlers-Danlos syndrome.

ABSTRACTThe ultrastructure of collagen fibers in the dermis of Ehlers‐Danlos syndrome (EDS) has been described in several previous reports. However, discussions of the ultrastructural changes in EDS‐Type II have been infrequent and brief. The behavior of alterative degeneration remains unclear, although there has been one previous report each on EDS‐Type I, Type VI, and Type VII (1, 3, 6). Our ultrastructural studies of EDS‐Type II revealed that bundles of collagen fibers were slightly thinner than in normal human skin, as were the individual collagen fibers also. Two or three serrated collagen fibers (up to 188 nm) were seen within the collagen bundle. The width of these serrated fibers was clearly less than in EDS‐Type I, and these fibers were less frequent than in the severe clinical form of EDS. It could be useful to be able to classify the subtypes of the EDS by structural pattern as well as by clinical appearance.

A scanning electron microscope study of the effects of various agents on instrumented periodontally involved root surfaces.

This study evaluated the effects of chemical and biologic agents on periodontally diseased root surfaces which had been scaled or root planed. The proximal surfaces of 25 teeth were scaled to remove all visible calculus, and the proximal surfaces of another 25 teeth were vigorously root planed to remove all cementum and to achieve a hard, smooth, glass-like surface. Five scaled and five root planed specimens were randomly selected for light microscopic examination to determine the amount of cementum removed. Cementum remained on all scaled surfaces but root planing had removed most of the more coronal cementum. Each of the 40 remaining teeth (20 scaled and 20 root planed) was longitudinally sectioned to obtain an experimental and control specimen. Four scaled and four root planed sections were randomly selected as experimental specimens for a test of each of the following five agents: (1) saturated citric acid for three minutes; (2) 15% EDTA for 5 minutes; (3) sodium hypochlorite for 5 minutes, followed by a 30-second application of 5% citric acid; (4) sodium hypochlorite alone for 5 minutes; and (5) 2% sodium deoxycholate (NAD) for 1 minute, followed by a 1-minute rinse in distilled water, and then a 1-minute application of 5% Cohn's fraction IV1. The control for each experimental specimen was treated with saline. All samples were prepared for SEM and examined at 3,000 X. Areas of particular interest were also examined at 12,000 X. The chemical treatments exposed only individual collagen fibers or irregular fiber bundles on the scaled surfaces. Saturated citric acid, EDTA, and sodium hypochlorite with citric acid neutralization removed debris and exposed openings in the root surfaces. Sodium hypochlorite alone and NaD/Cohn's fraction IV1 were less effective in removing surface debris and had an effect similar to that seen in the saline controls. Application to root planed specimens of saturated citric acid, EDTA, and sodium hypochlorite followed by 30 seconds of citric acid neutralization resulted in surfaces virtually free of debris and with numerous collagen fibers exposed on the surface. EDTA appeared to cause a morphologic change in the collagen fibers. Sodium hypochlorite alone, sodium deoxycholate followed by Cohn's fraction IV1, and physiologic saline were relatively ineffective in surface debridement.