<p>Knee pain is a common complaint among older Americans, nearly half of whom have developed or will develop painful osteoarthritis. Osteoarthritis is primarily a disease of articular cartilage, the low-friction, shock-absorbing connective tissue that lines long bones at their articulating surfaces. Within these joint tissues and within arthritis, the minor protein collagen VI plays an uncertain role, although it has been implicated in several muscle and ligament disorders. Determination of the collagen VI role in bone and cartilage of the knee is the focus of this dissertation.</p><p>Within articular cartilage, collagen VI exclusively localizes to and delimits the pericellular matrix (PCM), which differs from the extracellular matrix (ECM) in composition and structure. To interact with the cell, a molecule must first pass through the PCM. Fluorescent dextran diffusivities were quantified in the cartilage PCM using a newly developed model of scanning microphotolysis (SCAMP), a line photobleaching technique. Diffusion was slower in the PCM than in the ECM, although not in early-stage arthritic tissue. These results support the hypothesis that diffusivity is lower in the PCM than in the ECM of healthy articular cartilage, presumably due to differences in proteoglycan content. </p><p>Arthritic degradation is partly mediated by interleukin-1 (IL-1), a catabolic cytokine that affects the mechanical properties of articular cartilage and preferentially binds to cell-surface receptors in the surface zone. Since cells are the cartilage metabolic units, matrix degradation is hypothesized to influence molecular transport in the PCM before the ECM. Cartilage was cultured with or without IL-1, soaked in FITC-ovalbumin, and photobleached using SCAMP to measure diffusivity. Over 7 days of culture, IL-1 doubled the diffusivity in both zones (surface, middle) and matrices (PCM, ECM) of the cartilage. Diffusivity within the PCM was slightly lower than within the ECM. No increase in PCM diffusivity relative to ECM diffusivity was detected within either zone, suggesting that PCM-localized degradation either cannot be distinguished at these time points or cannot be detected by measures of ovalbumin diffusion.</p><p>To determine the effects of collagen VI absence on the morphometry and physical properties of the joint, knees of 2-, 9-, and 15-month-old Col6a1+/+ and Col6a1-/- mice were studied. Bone morphometry was evaluated using micro-computed tomography (microCT). Subchondral bone thickness, joint-capsule thickness, and cartilage degradation were assessed by histology. Cartilage elastic modulus, roughness, and coefficient of friction were measured by atomic force microscopy (AFM). Diffusion through the cartilage ECM was determined by SCAMP. Overall, collagen VI absence had profound effects on the morphometry of the proximal tibia and the overall histological structures of the mouse knee, yet minimal effects on the friction, roughness, elastic modulus, and diffusional properties of the articular cartilage. Musculoskeletal abnormalities at the knee do result from collagen VI absence.</p> / Dissertation

This study investigates the changes due to crosslinking treatment in stiffness, permeability, and glycosaminoglycan (GAG) content of bovine intervertebral discs. The objective of this study was to determine the mechanical and biochemical effects of crosslinking treatment on lumbar bovine tissue. Previous studies have found that crosslinking can increase stiffness and permeability in the intervertebral disc. These changes have not yet been investigated by confined compression, stress-relaxation tests of young bovine tissue. Eleven lumbar motion segments were harvested from calf spines and soaked in a saline solution or one of four crosslinking treatments (genipin, methylglyoxal, proanthrocyanidin, and EDC). Five mm diameter samples were removed from the midannulus region at anterior / anterior-lateral locations, confined in a saline bath, swelled to equilibrium, and tested in confined compression stress-relaxation to 15% strain in 5% increments. Radial samples were also harvested, treated with saline solution and EDC, and tested in the same manner. The aggregate modulus and hydraulic permeability were calculated using the nonlinear biphasic theory. Swelling pressure was calculated as the load at swelling equilibrium. GAG content was measured using the dimethylmethylene blue assay. Differences with P value < 0.05 were considered significant. In the axial orientation, all crosslinking treatments except methyglyoxal at least doubled the aggregate modulus relative to soaked controls (P less than 0.05). Genipin treatment resulted in 78% lower axial permeability, proanthrocyanidin (PA) 50% lower, and EDC treatment 84% lower relative to soaked controls (P &lt; 0.05). GAG content measured in the methyglyoxal treatment group was 25% lower than in soaked control group. Genipin (G), proanthrocyanidin (PA), and EDC treatment increased the swelling pressure by at least 65% (P less than 0.05). In the radial orientation, EDC treatment increased the stiffness by 75%, and did not significantly affect the permeability or swelling pressure. Some crosslinking treatments proved effective in increasing the stiffness and swelling pressure of the disc. The increased swelling pressure in G, PA, and EDC treatment groups relative to soaked controls suggests reduced GAG leaching during soaking treatment, further confirmed by the reduction in permeability in these groups.

Arteries possess the ability to grow and remodel in response to sustained alterations in biomechanical loading, likely via mechanisms that are similarly involved in diverse arterial pathologies and responses to treatment. In particular, myriad experminental observations suggest that cell and matrix turnover within vasoaltered states enable arteries to adapt to sustained changes in mechanical stimuli. The goal herein is to show explicitly how altered smooth muscle contractility and matrix growth and remodeling work together to adapt the geometry, structure, stiffness, and function of a representative basilar artery. This work seeks to illustrate the importance of complementary vasoactivity and matrix remodeling for basilar arteries in response to sustained alterations in mechanical stimuli. Toward this end, an extended constrained mixture model of the arterial wall is employed whereby the mass fractions, material properties, and natural configurations of individual constituents can evolve separately and thereby dictate overall growth and remodeling. This approach accounts for fundamentally important behaviors. Simulations provide important intuition and insight regarding constitutive functional forms and model parameters.

Collagen is an important structural protein in living organisms and plays an indispensable role in connecting cells and tissues, such as in musculature, bone, and ligament. The stability and conformation of collagen are, however, strongly influenced by ambient temperature and constitutes an interesting subject of study. Thermally induced conformation change of collagen has been investigated by techniques such as differential scanning calorimetry (DSC) and second harmonic generation. DSC is a powerful method in uncovered important thermal dynamics properties including phase change, enthalpy, and thermal stability of the collagen. However, due to its collective nature, no localized information can be found. For comparison, second harmonic generation, which reflects structural symmetry, can be combined with laser scanning microscopy to investigate localized variation. It has been shown in previous studies that the thermal stability of collagen is strongly influenced by the water content within collagen. For comparison, we are investigating the conformational change of collagen under a vacuum stat with second harmonic microscopy so as to isolate environmental effects, particularly those from water and oxygen. In this way, we have found the conformational change of collagen takes place at a much higher temperature and activation energy. Additionally, the high spatial resolution achieved also allows many further possibilities.

In this study, it was aimed to produce sericine/collagen composite membranes and to investigate their properties as a wound dressing. Different membrane compositions were prepared by casting and solvent evaporation method. After initial studies for optimization of ratios, membrane groups at two different thicknesses were prepared for each selected ratio and cross-linked with 3 % (w/v) glutaraldehyde (GTA). Considering the wound dressing requirements, equilibrium degree of swelling (EDS), water vapor transmission rate (WVTR), oxygen permeability, mechanical properties, in situ degradation, microbial penetration and cytotoxicity of membranes were examined. The EDS of membranes had a range of 14.91 to 4.37 (g/g) and increased significantly with the presence of sericin. There was no obvious relationship between the sericin ratio of membranes and WVTR, but the increase in membrane thickness decreased WVTR significantly. Thin and sericin containing membranes had statistically better oxygen permeabilities. Sericin deteriorated the tensile strength and elongation of membranes statistically. Cross-linked groups were resistant to hydrolytic degradation through 4 weeks of incubations. None of the membranes were penetrable to bacteria owing to their dense structure. For cytotoxicity studies, 3T3 fibroblasts and keratinocytes were seeded on membranes separately, and analyzed with MTT assays, and light microscopy and scanning electron microscopy (SEM). As regards to MTT assay, keratinocytes proliferated significantly on membranes and reached to high confluence within 7 days. Similarly, fibroblasts also showed high proliferation on membranes. Light microscopy and SEM analysis showed that both cells could attach, grow and spread on membranes. Also, cells gained their characteristic morphology after 1 day and formed flattened structure within 7 days.

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成6年3月25日 服部宇氏の博士論文として提出された

Thesis (M.S.)--Worcester Polytechnic Institute. / Keywords: cartilage, tendon, light scattering. Includes bibliographical references (p. 60-61).

Platelet transfusions can be necessary during treatment of patients with thrombocytopenia or impaired platelet function. Platelet function in platelet concentrates (PC) deteriorate with storage time. Studying swirling is often used to control the quality of PC’s before transfusion but the method has some disadvantages. Therefore other methods can be useful, for example impedance aggregometry (IA, Multiplate® Analyzer) to measure platelet function.      In this study the change in platelet function over time was examined in buffy coat and apheresis platelets with IA where aggregation had been induced with adenosine diphosphate (ADP) and collagen. PC’s were tested on day 1, 4 and 7 after donation. One of the main aims of this study was to evaluate if dilution to a standardized platelet concentration (800x109 platelets/L) for IA of PC’s could be used, since platelet concentration has been shown to influence aggregation. The effect of pathogen inactivation (INTERCEPT) on platelet function and the importance of fibrinogen for aggregation were also studied.      The dilution of platelet samples reduced the range of measured values and was suitable to use with collagen but not ADP. The platelet function decreased significantly over time with both agonists. There was a significant difference between pathogen inactivated and gamma irradiated PC’s with collagen activation on day 1. Fibrinogen was shown to be of importance for platelet aggregation, but other factors in plasma seem to be necessary too.      In conclusion, IA is a suitable method for following change in aggregability over time in PC’s and sample dilution reduced variation in results.

published_or_final_version / Dentistry / Master / Master of Orthodontics

published_or_final_version / Dentistry / Master / Master of Orthodontics