In previous in vitro and in vivo studies, decellularized adipose tissue (DAT) has demonstrated unique bioactivity, but little is known about the bioactive components preserved in the decellularized scaffold. With the goal of characterizing the bioactive components in the DAT, protein was extracted from DAT samples from 3 donors using 5 different buffers. The resulting DAT extracts were found to have very low protein content so molecular weight fractioning centrifugation was used to concentrate the samples. Concentrated extracts were screened for the presence of the bioactive components adiponectin, vascular endothelial growth factor A (VEGF-A), bone morphogenetic protein 2 (BMP-2) and Dickkopf related protein 1 (DKK-1) using Western blotting. Positive signal for BMP-2 was found for one donor in Roger’s Sample Buffer and Urea Buffer, but all other proteins investigated with Western blotting went undetected for all extraction buffers. Immunohistochemistry (IHC) was also used to determine the presence and distribution of bioactive components in the DAT samples. Sections of DAT were probed for the same bioactive components; adiponectin, VEGF-A, BMP-2 and DKK-1, with positive signals detected for adiponectin and VEGF-A. In order to develop an injectable, DAT-derived hydrogel for soft tissue regeneration, DATgels were fabricated from an enzymatically treated and homogenized DAT pre-gel suspension, which was neutralized to physiological pH and salt concentration. The highly-hydrated DATgels, containing up to 97% water, were found to degrade substantially over 14 days in simulated physiological fluid, with only a slight reduction in the overall scaffold size. Histology and SEM showed no major structural differences between the two formulations evaluated (40 mg/mL and 50 mg/mL), with both containing intact DAT features and small void spaces scattered heterogeneously throughout the scaffold. Preliminary in vitro cell work with adipose-derived stem cells (ASCs) showed that the 40 mg/mL formulation DATgel supported cell attachment and viability greater than 70% for all scaffolds up to 7 days after seeding. Further, migration into the scaffold was observed over time, indicating that the adhesive properties of the native ECM were retained through the processing steps required to fabricate the DATgels. / Thesis (Master, Chemical Engineering) -- Queen's University, 2013-12-19 16:22:32.717

No description available.

Degenerative disc disease (DDD) is a common medical issue among human adults, leading to back pain and potentially, disability, decreasing an individual’s quality of life. In the United States alone, huge economic impacts are apparent with an estimated $50- 100 billion attributed to lost productivity and medical costs related to DDD. Spinal degeneration occurs in the intervertebral disc (IVD) and once damaged, the IVD is incapable of adequate self-repair. A regenerative therapy incorporating nucleus pulposus (NP) tissue engineering may provide an answer to spinal degeneration. The objective of this in vitro study was to evaluate the potential of a thiol-modified hyaluronan (TMHA) and elastin-like polypeptide (ELP) as a hydrogel scaffold for nucleus pulposus tissue engineering. Two materials, one composed of TMHA only and one a 3:1 TMHA/ELP, crosslinked with polyethylene glycol diacrylate (PEGDA), were seeded with cultured human NP cells and cyclic hydrostatic loading was applied at 1MPa for 3 hours a day for 3 consecutive days. Cell viability and gene expression were analyzed. A decreasing trend in cell viability with time and cyclic hydrostatic pressure loading was observed and statistically significant differences were observed between the TMHA unloaded treatment group at day 0 and the TMHA loaded treatment group at day 4 and between the TMHA unloaded treatment group at day 0 and the 3:1 TMHA/ELP loaded group at day 4. Comparisons between TMHA only and 3:1 TMHA/ELP hydrogels for the same treatment indicate similar trends and no statistically significant differences in biological effects were observed. Gene expression analysis indicated low frequency expression of NP extracellular matrix (ECM) molecules regardless of time point or cyclic hydrostatic pressure application. These results are revealing in that the 3:1 TMHA/ELP hydrogel did not support NP cells significantly better than the TMHA hydrogel, though cell source and hydrostatic pressure generation issues may have impacted this finding. Additional studies with alternative cell type and a refined hydrostatic pressure application method may better illuminate the efficacy of a 3:1 TMHA/ELP hydrogel as for NP tissue engineering. / Thesis (Master, Chemical Engineering) -- Queen's University, 2011-03-17 14:16:28.83

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White adipose tissue (WAT) is now recognised as an endocrine organ through its secretion of hormones and protein factors - ‘adipokines’.  This thesis examined the regulation of two adipose expressed genes, retinol binding agent (RBP) involved in retinol transport, and tissue factor (TF) which initiates the extrinsic coagulation cascade.  RNA was isolated and RBP and mRNA levels determined by chemiluminescence-based Northern blotting.  TF and mRNA levels were determined by real-time PCR.  WAT RBP mRNA levels were second only to liver, and TF mRNA levels were highest in WAT depots.  RBP and TF mRNA were detected predominantly from mature adipocytes.  Obesity was not associated with altered RBP and TF gene expression except of for a significant (<i>p</i><0.05) decrease in RBP mRNA from subcutaneous WAT of obese rodent models.  Primary adipocytes were treated with <span lang=EN-GB style='font-family:Symbol'>b-agonists, dexamethasone or leptin.  Only dexamethasone significantly (<i>p</i><0.05) reduced RBP mRNA levels.  TF mRNA levels were unaltered following <span lang=EN-GB style='font-family:Symbol'>b-agonists, forskolin, or dexamethasone treatment except for a significant (<i>p</i><0.05) increase with a high dose of BRL 37344 (a <span lang=EN-GB style='font-family:Symbol'>b₃ agonist).  Administration of two isoforms of retinoic acid significantly decreased RBP gene expression, with 9-<i>cis</i> showing more potency (<i>p</i><span lang=EN-GB style='font-family:Symbol'>£ 0.001) that all-<i>trans</i> (<i>p</i><0.05.  The thiazolidinediones ciglitazone and rosiglitazone were administered, high doses significantly reducing RBP gene expression (<i>p</i> <span lang=EN-GB style='font-family:Symbol'>£ 0.001 and <i>p </i><span lang=EN-GB style='font-family:Symbol'>£ 0.05 respectively).  Fasting and cold exposure are two physiological stimuli which stimulate substrate flux and the release of fatty acids from WAT.  RBP gene expression in WAT was unaltered with fasting, cold exposure and <span lang=EN-GB style='font-family:Symbol'>b-agonist injection.  These studies suggest WAT may be an important source of RBP and TF.  In contrast to lipolysis and leptin production, the SNS does not significantly regulate RBP and TF gene expression.  The high TF gene expression in rodent WAT suggests an association between TF and the cardiovascular disease seen with obesity.

Effects of homologous extracts prepared from mature avian skeletal muscle on the development of isolated myoblasts from the thigh muscle of 11-12 day old chick embryoswere studied in vitro under previously unpublished culture conditions. Results of these experiments indicate that:1) fusion of myoblasts can occur in a predictable manner under the culture conditions described in this report2) some factor(s) present in extracts of homologous adult organ is able to partially inhibit this fusion3) such inhibition occurs maximally between 12 and 24 hours after myoblasts are placed in an in vitro environment, and is only partially reversible by the re-establishment of optimal culture conditions4) the inhibitory factor(s) is apparently long lived, non-dialyzablQ, heat labile, and subject to inactivation by proteolytic enzymes.

No description available.

Following a review of methods for measuring the optical properties of tissue, the majority of this thesis is concerned with the design, construction, calibration and use of a solid, tissue equivalent phantom. The phantom material is a clear polyester plastic. This is obtained in unpolymerised form, scattering particles and absorbing dyes are added to it, and it is then polymerised to form a stable solid. Purely scattering and absorbing phantoms were made separately, and their optical properties were measured using a specially built system. This has a co-linear collimated light source and detector, and measures the unscattered light transmitted through a sample as a function of its thickness. Other methods of measuring the optical coefficients of tissue were tested with this phantom. One of these uses integrating spheres to measure the transmitted and reflected light from a sample. A model of light transport (in this case a Monte Carlo model) is used to convert these measurements into scattering and absorption coefficients. It was found that the measurement of scattering coefficient was reasonably accurate, but that the absorption coefficient was overestimated at the low values typical of tissue. A measurement of the optical properties of bone was made with this system. The other system investigated uses the diffusion theory to calculate optical properties from measurements made through a thick slab. The material was also employed to create a test phantom for near infrared spectroscopy machines. This provides a diffusing medium with an attenuation that is variable in discrete steps over three orders of magnitude. The relative attenuation between steps is totally wavelength independent. This phantom was adopted by the EC concerted action on near infrared spectroscopy and imaging. Finally, the phantom was used to create test objects with which to investigate the potential of imaging with infrared light.