Title: Assessment of Crosslink Density in Collagen Models and Ultrafast Laser Crosslinking of Corneal and Cartilage Tissues as Novel Treatment Modalities
Description: Osteoarthritis (OA) is a progressive and complex joint disease that results from breakdown of articular cartilage and remodeling of underlying bone, which affects millions of Americans. While the expected lifetime of the load-bearing cartilage tissue should coincide with the lifespan of an individual, it has a limited ability to self-repair and the damage to the tissue can accumulate severely. One of the major challenges in OA treatment is its long asymptomatic period. Symptoms usually become noticeable when the disease is reaching advanced stages, and currently there is no effective intervention for early stages of OA. This may be due to lack of a reliable diagnostic method for detecting early OA. While OA is a degenerative joint disorder that may lead to gross cartilage loss and morphological damage to other joint tissues, a lot of subclinical, subtle biochemical changes occur in the early stages of OA progression. The degradation of collagen type II matrix in articular cartilage extracellular matrix (ECM) network may corelate with the progression of cartilage OA.

During onset of OA, with the loss of collagen crosslinks, the collagen matrix in cartilage ECM becomes more disorganized and the cartilage can become susceptible to disorder and thus aggravate the degeneration. Raman spectroscopy has been utilized in studies of components of connective tissues, such as OA, and cartilage degradation. Although studies have demonstrated the potential of applying Raman for diagnosing cartilage degeneration, the analysis of Raman spectrum obtained from articular cartilage is rather complicated and so far there is no generally accepted quantitative analysis for diagnosing early stages of OA. The first stage of this doctoral study aims to extend the capability of Raman spectroscopy to quantitatively characterize collagen network in articular cartilage, to investigate the possible correlation with the degeneration of OA. The first part of this doctoral dissertation is focused on developing a novel, non-destructive, quantitative diagnostic modality, based on Raman spectroscopy that has potential to detect changes in biochemical composition of articular cartilage.

The study is focused on the basic research associated with quantification of crosslink density and kinetics of the crosslinking process. A theoretical and computational framework for characterization of collagen crosslinks has been established and applied onto two models, 2-dimensional collagen type I thin films, and immature bovine, proteoglycan depleted, articular cartilage. Glutaraldehyde solution has been applied onto the model as a convenient method to introduce various levels of collagen crosslinks. Refractive error is a problem with focusing light accurately onto the retina due to the shape or other misfunctioning of the eye, rather than on the retina for the normal vision. The most common types of refractive errors are near-sightedness, far-sightedness, astigmatism, and presbyopia. Refractive errors have become a growing public health problem worldwide. Its incidence has doubled over the last 50 years in the United States and Europe. It is even more significant issue in some East Asian countries, where its prevalence reaches 70 to 90%.

Most affected individuals use spectacles or contact lenses, which generally provides adequate refractive error correction. However, both are subject to limitations. Glasses do not work well in the rain and mist may form on them following changes in temperature or humidity. Contact lenses improve the field of vision and acuity, but many people find their presence on ocular surfaces intolerable. Over the last two to three decades, refractive surgery for the permanent correction of vision has thus emerged as an attractive choice for many patients. However, such a surgery is an invasive procedure that may compromise corneal structure, and postsurgical complications have been reported. In the second stage of this doctoral work, a novel, non-invasive femtosecond laser collagen crosslinks manipulation method is studied. This laser collagen crosslinking treatment is applied on corneal tissue for vision correction. Two examples of the laser treatment on an ex vivo porcine eyes model are proposed in the study: corneal flattening, which is used to correct refractive errors due to myopia, and corneal steepening, which is used to treat hyperopia.

The effective refractive power is used to evaluate the effectiveness of the two different treatments. The depth of the crosslinked region in the cornea is assessed by two-photon autofluorescence (TPF) imaging. TPF imaging can be used to visualize changes induced in the cornea, because collagen is a primary extracellular source of nonlinear emissions. The safety of the proposed treatment methods is examined by haematoxylin and eosin (H&E) stained histological sections of corneas. The ex vivo porcine corneas are also cultured for one week after treatment, to determine whether crosslink density remains stable, and to check for degradation in the crosslinked layers of the stromal matrix, and further prove the safety of the proposed laser treatment method through the evaluation of cell viability after one week of treatment. An in vivo rabbit animal model, widely used as a model for the correction of refractive errors, is further utilized to demonstrate the stability and safety of the induced changes.

The effective refractive power of live rabbits is assessed 24 h, seven days, and then weekly up to three months after the laser crosslinking treatment. The safety of the laser treatment is first evaluated by histology staining, and further confirmed by in vivo confocal laser scanning microscopy. This laser treatment approach could expand the pool of patients eligible for permanent vision correction, while simultaneously eliminating the adverse effects associated with current forms of surgery. Furthermore, the approach described is also suitable for the treatment of other disease for collagenous tissues. The last chapter of in this doctoral dissertation have discussed the results of applying this laser treatment techniques for the treatment of progressive OA. Finally, in a preliminary study, the proposed femtosecond laser treatment modality developed for corneal tissue has been applied onto articular cartilage towards slowing down or retarding progression of early osteoarthritis. We hypothesize that degradation of the articular cartilage extracellular matrix can be slowed down or reversed in the collagen network crosslinked with a femtosecond laser.

We further theorize that the crosslinking mechanism introduced in the corneal tissue, which relies on laser ionization and dissociation of the tissue interstitial water to produce of refractive oxygen species, can increase crosslink density of collagen network in an articular cartilage. In the study, the treatment has been applied onto devitalized and live immature bovine cartilage explants, as well as cartilage plugs obtained from OA afflicted human cadaver joints. The preliminary results have shown that the proposed treatment has potential to enhance tissue mechanical properties, and increase wear resistance, an important factor in slowing down the progression of OA. Furthermore, preliminary imaging of live/dead stained tissue has shown that the laser treatment has minimal adverse effects up to two weeks after the laser irradiation.