In this study we fabricated free standing multilayer films of polyelectrolyte complexes for potential use in tissue engineering of corneal stroma by using the layer-by-layer (LbL) approach. In the formation of these LbL films negatively charged, photocrosslinkable (methacrylated) hyaluronic acid (MA-HA) was used along with polycations polyethyleneimine (PEI) and poly(L-lysine) (PLL). Type I collagen (Col) was blended in with PLL for improving the water absorption and cell attachment properties of the films. It was shown that the LbL films could be easily peeled off from glass substrates due to the photocrosslinking of one of the LbL components, the hyaluronic acid. Film growth and composition were monitored with FTIR-ATR. Heights of peaks at 3383 cm-1, and 2958 cm-1increased along with the bilayer number confirming the polymer build-up. Film integrity and thickness were investigated by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). Films thicker than 5 bilayers (BLs) were found to be uniform in appearance and 10 BL (PEI/MeHA) films were calculated to be ca. 6 &mu / m thick. Atomic force microscopy (AFM) revealed that as the number of BLs increased, surface roughness decreased. Activity of methacrylated hyaluronic acid was shown by the increased resistance of photocrosslinked multilayer films against hydrolysis by hyaluronidase. Patterns could be created on the films by photocrosslinking further proving that the crosslinking step is successful. Since the ultimate goal was to construct a corneal stroma PEI/MA-HA films were tested with corneal stroma cells, keratocytes. Cell proliferation on PEI/MA-HA films was quite poor in comparison to TCPS. In order to improve the cell adhesion the tests were repeated with PLL/MA-HA. Collagen was added to decrease the hydrophilicity and introduce cell adhesion sequences (Arg-Gly-Asp, RGD) to improve cell proliferation on the films and thus PLL+Col/MA-HA films were also tested. Introduction of collagen to the PLL/MA-HA films was found to decrease water retention of the multilayer films and improve cell viability and proliferation. Col+PLL/MA-HA LbL thus appear to be a promising platform for tissue engineering, especially of corneal stroma.

The work presented in this thesis focuses on the development of strategies and smart bioactive materials for the treatment of osteoporosis. High and low molecular weight soluble hyaluronic acid-bisphosphonate (HA-BP) derivatives were investigated for their ability to inhibit osteoclasts. Low molecular weight HA-BP (L-HA-BP) was most effective in inhibiting active resorption of both murine and human osteoclasts (without affecting osteoblasts) compared to free bisphosphonate (BP). Precursor monocytes were unaffected, suggesting the specificity of HA-BP towards osteoclasts. This new class of functionalized hyaluronic acid could lead to rapid development of tailor-made pro-drugs for targeted treatment of osteoporosis. Polyphosphoesters (PEP) have been widely studied for their pro-osteoblast effects, primarily due to their involvement in cellular energy production pathway leading to the formation of inorganic phosphates that contribute to mineralized bone. Given that the effect of PEP on human osteoclasts is little studied, this work on poly(ethylene sodium phosphate) (PEP.Na) explores the potential to use PEP.Na as an inhibitor of osteoclast activity for the first time. PEP.Na exposure led to a dose-dependent toxicity of osteoclasts with reduction in their capacity to form resorption pits over 24h. Currently, there is a dearth of in vitro cell-culture systems that can study osteoclast-related resorption and osteoblast-related mineralization in a single co-culture system, and to simultaneously quantify the effects of soluble factors on these processes. Described here, is the development of a novel and simple two-sided co-culture system that can overcome these limitations with reliable and quantifiable readouts. In comparison with traditional one-sided co-culture systems, the two-sided co-culture was able to generate similar readouts for alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) markers. There is also the advantage of distinctly separate and quantifiable readouts for mineralization and resorption, which has been demonstrated using Pamidronate. Finally, HA-BP was synthesized with pre-determined amounts of BP groups. The BP groups attached to HA allowed the tunable incorporation of BMP-2 in hydrogels. The charge-based affinity of BMP-2 and BP allowed stable incorporation of specific amounts of BMP-2, which could be tuned by the ratio of BP groups. 125I-labelled BMP-2 was loaded into hydrogels and their release was studied. Radioactive measurements revealed the tunable sequestration and controlled release of protein over time. This result was corroborated by ALP measurements of cells exposed to released BMP-2. ALP production was found to be almost 5-fold higher in HA-BP hydrogels loaded with BMP-2 which suggested that the sequestered BMP-2 is not only available to cells but also remains highly potent, even in entrapped form, The release of BMP-2 is dependent upon the rate of diffusion, swelling in hydrogels and degradation pattern of the gels and may assist in the long-term and rapid regeneration of osteoblasts in vitro.

Hyaluronic acid (HA) is a naturally occurring polymer that holds considerable promise for tissue engineering applications. Current cross-linking chemistries often require a coupling agent, catalyst, or photoinitiator, which may be cytotoxic, or involve a multistep synthesis of functionalized-HA, increasing the complexity of the system. With the goal of designing a simpler one-step , aqueous-based cross-linking system, we synthesized HA hydrogels via Diels-Alder “click” chemistry. Furan-modified HA derivates were synthesized and cross-linked via dimaleimide poly(ethylene glycol). By controlling the furan to maleimide molar ratio, both the mechanical and degradation properties of the resulting Diels-Alder cross-linked hydrogels can be tuned. Rheological and degradation studies demonstrate that the Diels-Alder click reaction is a suitable cross-linking method for HA. These HA cross-linked hydrogels were shown to be cytocompatible and may represent a promising material for soft tissue engineering.

Hyaluronic acid (HA) is a naturally occurring polymer that holds considerable promise for tissue engineering applications. Current cross-linking chemistries often require a coupling agent, catalyst, or photoinitiator, which may be cytotoxic, or involve a multistep synthesis of functionalized-HA, increasing the complexity of the system. With the goal of designing a simpler one-step , aqueous-based cross-linking system, we synthesized HA hydrogels via Diels-Alder “click” chemistry. Furan-modified HA derivates were synthesized and cross-linked via dimaleimide poly(ethylene glycol). By controlling the furan to maleimide molar ratio, both the mechanical and degradation properties of the resulting Diels-Alder cross-linked hydrogels can be tuned. Rheological and degradation studies demonstrate that the Diels-Alder click reaction is a suitable cross-linking method for HA. These HA cross-linked hydrogels were shown to be cytocompatible and may represent a promising material for soft tissue engineering.

Synthetic glycopeptides have attracted much interest in the biomedical field due to their structural similarities to the natural glycopeptides or glycoproteins. It is still difficult to synthesize glycopeptides with greater efficiency and ring opening polymerization remains an effective way to do so. Proteoglycans are a special class of glycoproteins with glycosaminoglycan chains. In this study, I tried to do controlled ring opening polymerization of Hyaluronic acid derivatives with smaller to higher molecular weight while avoiding side reactions. It is challenging to work with higher molecular weight molecules and do a click reaction in water effectively. Making nanopolymers with a desired size, studies of the characteristics, and how to build nanocarriers for drug delivery application was the focus of this work. Polymeric characteristics, e.g., modification and polymer formation were studied by nuclear magnetic resonance technique; Particle size was studied by dynamic light scattering and the loading of rhodamine B encapsulated into the polymer was measured by confocal imaging technique.

The effect of absorbable packing on the healing of nasal respiratory epthelium after endoscopic sinus surgery (ESS) was examined in a diseased sheep model. Full thickness injuries were created on the lateral nasal wall of sheep infested with Oestrus ovi. Sites of injury were packed on one side with hyaluronic acid (HA) packing or hyaluronic acid packing impregnated with insulin-like growth factor- 1 (HA+IGF1) in a randomized fashion. The opposite side was left unpacked as a control. Biopsies were obtained for light microscopy, scanning electron microscopy, and ciliary beat frequency (CBF) analysis over a period of 16 weeks. Statistical analysis of results was performed in order to determine if any intervention had any impact on healing and to determine if there was any correlation between extent of regeneration as assessed by electron microscopy and CBF. Furthermore assessment of the effect of isotonic and hypertonic saline on ciliary beat frequency was performed in healthy human volunteers. Reepithelialization was increased in the HA+IGF1 group compared to the HA group and controls at eight weeks after injury but not at later time points. Cilial regeneration was improved in the HA+IGF1 group compared to the HA group and controls at 16 weeks. CBF was noted to be worse at the eight week time point with the HA+IGF1 group compared to the HA group and controls, but no other statistically significant effects on CBF were noted. This most likely represents a spurious finding. Wide distributions of CBF results were noted, reflecting numerous missing data points due to methodological difficulties. There was a trend noted toward increased CBF with improved grades of reciliation, although this correlation was not statistically significant. However this trend was supported by the finding of statistically significant differences between individual and combined grades of reciliation. Hypertonic saline was found to have a ciliostimulatory effect when compared to normal saline at 5 minutes after administration in healthy human subjects. This effect had disappeared by 60 minutes after administration. It is suggested that the presence of insulin-like growth factor- 1 at the time of mucosal injury improves epithelial regeneration in the short term, but is not sufficient for this effect to be sustained. This improved early epithelial regeneration forms a foundation for cilial regeneration, as is reflected in an improved grade of reciliation at 16 weeks. Our interventions had no effect on CBF, and various experimental problems made it difficult to provide further comment on CBF results. There is evidence that CBF improves as the grade of cilial regeneration improves following ESS. Furthermore, hypertonic saline appears to also have a positive impact on CBF, which is likely to reflect changes in the rheological properties of mucous. A number of possible avenues of enquiry are delineated and recommendations for future research are outlined. / Thesis (M.S.)--Department of Surgery, 2005.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniversitet, 2006. / Härtill 5 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed June 5, 2008). Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (p. 117-132).