Being the most abundant protein in the body, by mass, type I collagen provides the building blocks for tissues such as bone, extra-cellular matrix, tendons, cornea, etc[1-3]. The ability of a single protein to create structures with such various mechanical properties is not fully understood. Before one can engineer and assemble a complex tissue, such as cornea, the mechanisms underlying the formation and assembly, mechanical properties, and structure must be investigated and quantified. The work presented herein contains an extensive study of Type I collagen from the molecular to the tissue level.
The engineering of collagenous tissues that mimic the mechanical and optical properties of native human cornea have been performed by a number of groups[4-7]. In all of these studies, the corneal-mimicking tissues have been created using a number of methods including repeated flow casting. To date, the ability to create self-assembled corneal tissue has not been achieved. Understanding the mechanisms of formation of native cornea will not only bring us closer to achieving self-assembled transplantable corneal tissue but will also aid in the engineering of all collagenous tissues and other structures comprised of filamentous units.
Recently, the study of type I collagen has primarily focused on the tissue, fiber, and fibril scale[2, 8-21]. Grant, et al.[20] measured the elastic modulus of collagen fibrils in various solutions and found that by increasing ion concentration, in the solution around the fibril, the elastic modulus increased. The solution dependent behavior of the elastic modulus of collagen fibrils was measured but the cause of the dependence was unknown. Grant et al. state that due to the complex nature of the interactions between collagen fibrils and aqueous solutions, the exact cause of this effect is difficult to determine. Through work presented herein, not only do we show that this behavior is seen at the molecular level but also quantify the relationship between ionic concentration and molecular stiffness for a variety of ionic species.
Studies of collagen mechanics, on the molecular level, are brief[22-26]. The most prominent of these studies in recent years was performed by Sun, et al.[27] wherein a persistence length of 14.5nm, for human type I procollagen, was measured. The persistence length of the molecule, which is a measure of flexibility, is a highly debated topic with quoted values of 14.5nm[27], 57nm[28], 130nm[29], 175nm[30], 308nm[31], and 544nm[32]. The broad range of values indicates that the flexibility of the collagen molecule is a complex question.
It became apparent that the disagreement of the persistence length of molecular collagen in the literature may be due to the use of different ionic solutions. To address this, an initial atomic force microscope, AFM, study of the persistence length of molecular collagen diluted in DI water and two ionic solutions was conducted. This study showed that there is a strong solution dependence to the flexibility of the molecule. The ionic solutions presented molecules with a large persistence length, a straightened configuration, while the DI water dilution resulted in a persistence length that was a factor of 10 smaller.
Because two different complex ionic solutions in the initial study showed different persistence lengths, an evaluation of the effect of each individual salt was performed. To elucidate the effects of individual ionic species on the conformations and persistence length of Type I collagen varying concentration of monovalent and divalent salts with different cations and anions were tested. It was found that increasing ionic concentration for all species types resulted in a higher persistence length but the rate of change in persistence length as a function of concentration is unique to each species.
In 2002 Leikina, et at.[33] suggested that Type I molecular collagen is unstable at body temperature using differential scanning calorimetry. To examine these results, an AFM study was performed that imaged the collagen molecules after being held at body temperature for varying times. The density of molecules deposited onto mica, above a 200nm length cutoff, was calculated and it shows that the number of molecules above 200nm in length decreases with increasing incubation time.
These environmental studies were performed with an aim to understanding the role of environment in creating a corneal mimicking tissue. Currently, the most promising method of collagen membrane fabrication for corneal replacement was developed by Tanaka, et al.[4]. This unique repeated flow casting method allows for the manufacturing of transparent collagen membranes with controllable thickness and fibrillar alignment. Using the repeated flow casting technique, orthogonally oriented collagen membranes were created and their optical properties were measured using the Generalized High Accuracy Universal Polarimeter, G-HAUP. When engineering a tissue for the eye, the optical properties of the tissue are of the utmost importance. Appropriately for corneal tissues, the measurements for linear birefringence and linear dichroism were negligible.
It was clear, from the literature, that a fundamental understanding of molecular type I collagen was not available. In this work, the mechanical properties and environmentally sensitive behavior of bovine dermal type I molecular collagen is studied. The exploration into the unique behavior of these systems begins with documenting the rich ionic species and concentration dependent flexibility of molecular type I collagen and the temperature dependence on the stability of the molecule is tested. The study concludes with the construction of corneal mimicking tissues using the repeated flow casting method and measuring the complex optical properties of these tissues.
| Identifer | oai:union.ndltd.org:USF/oai:scholarcommons.usf.edu:etd-6231 |
| Date | 03 April 2014 |
| Creators | Harper, Heather |
| Publisher | Scholar Commons |
| Source Sets | University of South Flordia |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | Graduate Theses and Dissertations |
| Rights | default |
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