Yes / Biosynthetic materials have emerged as one of the most exciting and productive fields in polymer chemistry due to their widespread adoption and potential applications in tissue engineering (TE) research. In this work, we report the synthesis of a poly(ε-caprolactone)-graft-collagen (PCL-g-Coll) copolymer. We combine its good mechanical and biodegradable PCL properties with the great biological properties of type I collagen as a functional material for TE. PCL, previously dissolved in dimethylformamide/dichloromethane mixture, and reacted with collagen using carbodiimide coupling chemistry. The synthesised material was characterised physically, chemically and biologically, using pure PCL and PCL/Coll blend samples as control. Infrared spectroscopy evidenced the presence of amide I and II peaks for the conjugated material. Similarly, XPS evidenced the presence of C–N and N–C=O bonds (8.96 ± 2.02% and 8.52 ± 0.63%; respectively) for PCL-g-Coll. Static contact angles showed a slight decrease in the conjugated sample. However, good biocompatibility and metabolic activity was obtained on PCL-g-Coll films compared to PCL and blend controls. After 3 days of culture, fibroblasts exhibited a spindle-like morphology, spreading homogeneously along the PCL-g-Coll film surface. We have engineered a functional biosynthetic polymer that can be processed by electrospinning. / The EPSRC Centre in Innovative Manufacturing in Medical Devices (MeDe Innovation; EP/K029592/1).

In the present study, muscle growth and flesh quality have been investigated from both commercially farmed Atlantic halibut (Hippoglossus hippoglossus) (Aga marine AS, Norway)and halibut obtained from small-scale trials at Mørkvedbukta Research Station (Bodø University College, Norway). Morphometric techniques have been utilized to investigate fast muscle growth in halibut ranging from circa 2 g to 100 kg, and it was established that fast muscle fibre recruitment ceases when the fish attain approximately 81 and 177 cm, in the case of males and females, respectively. Different muscle fibre types were distinguished using histochemical (myosin ATPase and succinic dehydrogenase) and immunohistochemical (S-58, an antibody against slow muscle myosin) staining techniques. Females recruit twice as many fast muscle fibres compared to males, which allows them to reach a larger final size. Furthermore, the seasonal growth patterns during a one year production cycle in commercial farmed halibut revealed a winter depression in growth leading to loss of biomass, which was attributed to the maturation of males. Commercial farmed fish of equal size (~1.5 kg) showed sexual dimorphism of fast muscle fibre number, caused by a significantly higher rate of fast muscle fibre recruitment in females. During the winter season fast muscle fibres shrunk significantly, especially in male fish, as a consequence of loss of appetite, low water temperatures and sexual maturation. None of the female fish matured during the trial. Flesh quality of halibut deteriorated during winter and spring, since it had a softer appearance and significantly lower myotomal protein content, particularly in males. Cathepsin activity was measured using spectroscopy and showed a strong negative correlation to protein content, displaying a seasonal variation. The proteolytic depletion of fast muscle proteins affected the water holding capacity of the muscle (determined by centrifugation), which showed concomitant changes with the increase in cathepsin activity and drop in protein content. Despite the soft appearance, the firmness (shear force) of the flesh increased during the winter. The hydroxylysyl pyridinoline cross-link content of the collagen matrix, determined by HPLC, showed a strong correlation to the fillet texture. The increased firmness during the winter, a period of little (female) or negative growth (males), was probably due to an increased cross-linking of the collagen compartment. Partial sequences of IGF-I and IGF-II were cloned from fast muscle of Atlantic halibut, and their relative gene expression levels were determined along with those of cathepsin B, cathepsin D and IGF-IRa in male halibut using qPCR during a fasting and refeeding trial. Transcript levels of cathepsin B and to some extent cathepsin D were significantly higher during fasting than refeeding, suggesting an increased enzyme production during periods of food deprivation. A temporary increase in IGF-I transcripts was observed after 7 days refeeding suggesting that this growth factor is involved in muscle growth control. Both IGF- IRa and IGF-II were down-regulated during refeeding.

Este estudo avaliou a efetividade de uma nova membrana bioabsorvível com propriedades mecânicas e químicas mais adequadas para o uso em tendões. A avaliação foi realizada em coelhos submetidos a reconstrução da porção gastrocnêmia do tendão calcâneo. Foi feita uma avaliação prévia das propriedades mecânicas da membrana com uso de dinamômetro digital que demonstrou capacidade de deformação elástica mínima de 100%. Todos os coelhos foram submetidos a tenotomia e reparo da porção gastrocnêmia do tendão direito, após isso foram randomicamente separados em grupos envelopado com membrana e controle. A extração foi realizada nos seguintes períodos: sete, 14 e 28 dias. A avaliação foi realizada através da macroscopia, histologia, mensuração objetiva do colágeno à luz polarizada pelo image-J®, mensuração de glicosaminoglicanos sulfatados e expressão gênica de proteoglicanos. Na avaliação macroscópica o grupo com membrana apresentou menos aderência e melhor direcionamento das fibras e tecido mais homogêneo em 14 e 28 dias, p=0,02 e 0,03 respectivamente. Na histologia a Classificação de Watkins modificada apresentou as seguintes médias: 14,67±0,42 membrana e 12,67±0,56 sem membrana, p=0,03 em 14 dias, e 19,88±0,83 membrana e 17,25±0,62 sem membrana, p=0,02 em 28 dias. Na mensuração do colágeno as médias dos valores de cinza(mvc) o colágeno tipo III foram de 17,97±1,83 membrana e 12,63±1,07 sem membrana p=0,03 em 14 dias. Para o colágeno tipo I as médias foram de 2,41±0,33mvc membrana e 1,31±0,18mvc sem membrana p=0,01 em 14 dias e 7,30±0,63mvc membrana e 2,92±0,32mvc sem membrana p < 0,0001 em 28 dias. A média dos GAGs foi avaliada em três porções do tendão, proximal, central e distal, em ug/mg de tecido seco. Em sete dias apresentou diferença significativa apenas na porção distal 0,80±0,04 com e 0,38±0,04 sem membrana para condroitin-sulfato em 14 dias não apresentou diferenças entre os grupos. O dermatan-sulfato apresentou diferença estatisticamente significante em 7 dias apenas na porção central 0,42±0,09 com membrana e 1,29±0,67 sem membrana p=0,02. Em 14 dias não foram observadas diferenças entre os grupos. Houve grande variabilidade na expressão gênica no teste das amostras com beta-Actina e GAPDH levando a resultados inconclusivos ou não variação entre os grupos que pode sugerir não variabilidade na expressão gênica dos GAGs no período de 28 dias. Os dados fornecidos por esse trabalho mostram que a envelopagem com a membrana bioabsorvível promoveu aceleração dos processos cicatriciais da porção gastrocnêmia do tendão calcâneo de coelhos / This study assessed the effectiveness of a new absorbable membrane, that presents mechanical and chemical features more suitable to tendons, in rabbit tendons. Before the animal model assessments a mechanical study of the membrane was carried out demonstrating that the minimal capability for elastic deformation of the membrane was more than 100%. All rabbits underwent to tenotomy and reconstruction of the right gastrocnius tendons, thereafter they were randomly divided in tendon wrapped by the membrane and control groups. Extraction was performed in the following periods of time: seven, 14 and 28 days. Assessments used macroscopy, histology, objective collagen assessment by using polarized light and Image-J® program in mean of gray values(mgv), sulphated glycosaminoglycans, genetic expression of proteoglycans. In the macroscopic 14 and 21-day assessments the membrane group presented less adherences p=0.02 and p=0.03 respectively. The modified Watkins classification: 14,67±0,42 membrane and 12,67±0,56 without membrane p=0,03 for 14 days; 19,88±0,83 membrane and 17,25±0,62 without membrane p=0,02 for 28 days. The type III collagen were 17,97±1,83 membrane and 12,63±1,07 without membrane p=0,029 for 14 days. For type I collagen were 2,41±0,33 membrane and 1,31±0,18 without membrane p=0,01 for 14 days and 7,30±0,63 memebrane and 2,92±0,32 without membrane p < 0,0001 for 28 days. The glycosaminoglycans were measured in 3 tendon portions, distal, central and proximal, by using ug/mg of dry tissue. In seven days just the distal part presented statistical differences 0,80±0,04 membrane and 0,38±0,04 without membrane, for 14 days no differences were found for Chondroitin-Sulphate. For Dermatan-Sulphate the central part of the tendon 0,42±0,09 and 1,29±0,67 p=0,02, for 14 days no differences were found. There was high variability for beta-actin and GAPDH for the samples in 28 days with inconclusive results that may mean no variability in gene expression of GAGs at this time period. Results as mentioned above demonstrated that the wrapped tendons by the new membrane presented acceleration in the healing processes for gastrocnemius tendons of New Zealand Rabbits

Background: Among the common malignancies, pancreatic cancer has the shortest long-term survival. The aggressive, rapid, and infiltrative growth pattern of pancreatic cancer, together with the lack of specific symptoms, often leads to late diagnosis. Metastases are frequently found at the time of diagnosis, which prevents curative surgical treatment. Good tumor markers would enable early detection, thus improving the prognosis. Unfortunately, no such markers are available in the clinic. The tumor stroma is defined as the non-malignant cells and the extracellular matrix (ECM) of a cancer. Pancreatic cancer is characterized by an abundant tumor stroma, rich in ECM proteins such as collagens, which have been shown to play important roles in tumor progression. Furthermore, pancreatic cancer cells produce large quantities of ECM proteins, especially the basement membrane (BM) protein type IV collagen. All epithelial cells are anchored to a BM, which must be degraded in order for an in situ cancer to become invasive. Matrix metalloproteinases (MMPs) are enzymes involved in BM degradation. In this thesis, the tumor stroma of pancreatic cancer is studied, focusing on the BM proteins type IV and type XVIII collagen, with the aim to clarify if the stroma could be a source of novel tumor markers for this form of cancer. Additionally, the role of type IV collagen produced by the cancer cells is studied. Methods: Expression patterns of type IV and type XVIII collagen, MMPs involved in collagen degradation, and collagen receptors (integrins) were studied by immunoflourescence in both normal and pancreatic cancer tissue, and in pancreatic cancer cell lines. Circulating plasma levels of type IV and type XVIII collagen and conventional tumor markers (TPS, Ca 19-9, CEA and Ca 125) were measured in controls and pancreatic cancer patients at the time of diagnosis and after treatment. The role of cancer cell produced type IV collagen was studied in human pancreatic cancer cell lines by functional blocking of integrin receptors (integrin a1, a2 and b1) and integrin-binding sites on type IV collagen, and by siRNA-induced down-regulation of type IV collagen synthesis. Proliferation was analyzed by a luminescence based cell viability assay, migration by time-lapse microscopy, and apoptosis by M30-neoepitope detection. Results: MMPs involved in BM degradation were upregulated in pancreatic cancer tissue. The expression of type XVIII collagen shifted from a general BM expression pattern in normal tissue, to mainly being found in the tumor vasculature in pancreatic cancer. Type IV collagen, on the other hand, remained highly expressed in the vicinity of the cancer cells. The a1, a2, and b1 integrin receptors were highly expressed at the cancer cell surface. Both down-regulation of type IV collagen synthesis and blocking the integrin/type IV collagen interaction decreased cell proliferation and migration. The proliferative capacity was rescued by the addition of exogenous type IV collagen. Furthermore, the circulating levels of both type IV and type XVIII collagen were increased in pancreatic cancer patients at the time of diagnosis compared to controls. After treatment, the levels were normalized for type XVIII collagen, whereas the levels of type IV collagen remained high after surgery. High postoperative levels of type IV collagen were associated with short overall survival. A similar association to short survival was found for preoperative type XVIII collagen levels. No such associations to survival could be detected for the conventional markers.   Conclusion: The results of this thesis show that type IV and type XVIII collagens can serve as tumor markers for pancreatic cancer with advantages compared to conventionally used markers. Additionally, evidence is provided of an autocrine loop, involving type IV collagen and its integrin receptors, with importance for retaining a proliferative and migratory phenotype in pancreatic cancer cells.

Thesis (Ph. D.)--University of Missouri-Columbia, 2006. / Title from title screen of research.pdf file (viewed on December 22, 2006). The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "May 2006" Includes bibliographical references.

Este estudo avaliou a efetividade de uma nova membrana bioabsorvível com propriedades mecânicas e químicas mais adequadas para o uso em tendões. A avaliação foi realizada em coelhos submetidos a reconstrução da porção gastrocnêmia do tendão calcâneo. Foi feita uma avaliação prévia das propriedades mecânicas da membrana com uso de dinamômetro digital que demonstrou capacidade de deformação elástica mínima de 100%. Todos os coelhos foram submetidos a tenotomia e reparo da porção gastrocnêmia do tendão direito, após isso foram randomicamente separados em grupos envelopado com membrana e controle. A extração foi realizada nos seguintes períodos: sete, 14 e 28 dias. A avaliação foi realizada através da macroscopia, histologia, mensuração objetiva do colágeno à luz polarizada pelo image-J®, mensuração de glicosaminoglicanos sulfatados e expressão gênica de proteoglicanos. Na avaliação macroscópica o grupo com membrana apresentou menos aderência e melhor direcionamento das fibras e tecido mais homogêneo em 14 e 28 dias, p=0,02 e 0,03 respectivamente. Na histologia a Classificação de Watkins modificada apresentou as seguintes médias: 14,67±0,42 membrana e 12,67±0,56 sem membrana, p=0,03 em 14 dias, e 19,88±0,83 membrana e 17,25±0,62 sem membrana, p=0,02 em 28 dias. Na mensuração do colágeno as médias dos valores de cinza(mvc) o colágeno tipo III foram de 17,97±1,83 membrana e 12,63±1,07 sem membrana p=0,03 em 14 dias. Para o colágeno tipo I as médias foram de 2,41±0,33mvc membrana e 1,31±0,18mvc sem membrana p=0,01 em 14 dias e 7,30±0,63mvc membrana e 2,92±0,32mvc sem membrana p < 0,0001 em 28 dias. A média dos GAGs foi avaliada em três porções do tendão, proximal, central e distal, em ug/mg de tecido seco. Em sete dias apresentou diferença significativa apenas na porção distal 0,80±0,04 com e 0,38±0,04 sem membrana para condroitin-sulfato em 14 dias não apresentou diferenças entre os grupos. O dermatan-sulfato apresentou diferença estatisticamente significante em 7 dias apenas na porção central 0,42±0,09 com membrana e 1,29±0,67 sem membrana p=0,02. Em 14 dias não foram observadas diferenças entre os grupos. Houve grande variabilidade na expressão gênica no teste das amostras com beta-Actina e GAPDH levando a resultados inconclusivos ou não variação entre os grupos que pode sugerir não variabilidade na expressão gênica dos GAGs no período de 28 dias. Os dados fornecidos por esse trabalho mostram que a envelopagem com a membrana bioabsorvível promoveu aceleração dos processos cicatriciais da porção gastrocnêmia do tendão calcâneo de coelhos / This study assessed the effectiveness of a new absorbable membrane, that presents mechanical and chemical features more suitable to tendons, in rabbit tendons. Before the animal model assessments a mechanical study of the membrane was carried out demonstrating that the minimal capability for elastic deformation of the membrane was more than 100%. All rabbits underwent to tenotomy and reconstruction of the right gastrocnius tendons, thereafter they were randomly divided in tendon wrapped by the membrane and control groups. Extraction was performed in the following periods of time: seven, 14 and 28 days. Assessments used macroscopy, histology, objective collagen assessment by using polarized light and Image-J® program in mean of gray values(mgv), sulphated glycosaminoglycans, genetic expression of proteoglycans. In the macroscopic 14 and 21-day assessments the membrane group presented less adherences p=0.02 and p=0.03 respectively. The modified Watkins classification: 14,67±0,42 membrane and 12,67±0,56 without membrane p=0,03 for 14 days; 19,88±0,83 membrane and 17,25±0,62 without membrane p=0,02 for 28 days. The type III collagen were 17,97±1,83 membrane and 12,63±1,07 without membrane p=0,029 for 14 days. For type I collagen were 2,41±0,33 membrane and 1,31±0,18 without membrane p=0,01 for 14 days and 7,30±0,63 memebrane and 2,92±0,32 without membrane p < 0,0001 for 28 days. The glycosaminoglycans were measured in 3 tendon portions, distal, central and proximal, by using ug/mg of dry tissue. In seven days just the distal part presented statistical differences 0,80±0,04 membrane and 0,38±0,04 without membrane, for 14 days no differences were found for Chondroitin-Sulphate. For Dermatan-Sulphate the central part of the tendon 0,42±0,09 and 1,29±0,67 p=0,02, for 14 days no differences were found. There was high variability for beta-actin and GAPDH for the samples in 28 days with inconclusive results that may mean no variability in gene expression of GAGs at this time period. Results as mentioned above demonstrated that the wrapped tendons by the new membrane presented acceleration in the healing processes for gastrocnemius tendons of New Zealand Rabbits

Background: Tissue engineering and its contribution to regenerative medicine has advanced through the years. It has proven its efficacy especially in the treatment of advanced full thickness burn wounds. Tissue engineering is the synergy between biology and engineering. This fairly young science has one common goal and that is to regenerate new tissue. Various commercially available products have appeared on the market and this due to the ground-breaking work of many. One such well known product is Integra® which is the brain child of Yannas and Burke. This is a collagen-glycosaminoglycan copolymer which serves as a bioactive regeneration template or extracellular matrix analogue. Advanced wound healing is promoted along with the prevention of scar tissue formation and consequent contractures. Aims:</p This study provides an extensive review on the development of this dermal regeneration matrix and also aims to develop an equivalent product. Attention will be paid to: the biological building blocks and the motivation for their use; the essential production steps; and the final processing required in order to deliver a sterile product. Materials and Methods: A collagen and chondroitin 6-sulphate coprecipitate was prepared and subjected to either controlled or uncontrolled freezing. The frozen slurry was dried under vacuum for 17 hours after which each sample was coated with a thin silicone film. Glutaraldehyde crosslinking followed after which the product was thoroughly rinsed. The packaged products were then subjected to terminal sterilisation via gamma irradiation under various conditions. Various tests were conducted to evaluate the newly formed regeneration matrices and included scanning electron microscopy, enzymatic degradation by collagenase, and a cytotoxicity assay. Scanning electron microscopic analysis was done in order to reveal the adequacy of the scaffold architecture. Collagenase degradation of the scaffolds was used to project the rate of degradation of each template. Integra® served as the gold standard for each test. Quantifiable data was statistically analysed and any comparison made included the calculation of means, standard deviations and p-values (confidence interval of 95%). Results: Results indicated that highly porous bioactive tissue engineering matrices were obtained by either controlled freezing or uncontrolled freezing. The average pore diameter of the most homogenous scaffolds ranged between 52.47 and 136.44 µm with a mean of 87.34 µm. These templates were formed by using a 0.5% collagen concentration and a controlled freeze rate of 0.92 °C/min. Uncontrolled freezing (1.3 °C/min) of a 0.5% collagen concentration resulted in the formation of an irregular scaffold with an average pore diameter of 174.08 µm. It was found that the architecture of the most equivalent scaffold compared well with that of Integra® with p = 0.424. Scaffolds prepared using higher collagen concentrations (1.0%) and controlled freezing resulted in dense sponges with average pore diameters of 56.51 µm. Statistical analysis upon comparison indicated a significant difference p = 0.000 in the micro architecture. The rate of degradation of the most equivalent scaffold was 1.9 times that of Integra®. This implicates that the crosslinking was insufficient and due to one of the following: poor collagen quality; method of crosslinking; and degradation due to terminal sterilization. The rate of scaffold degradation can be extended, either by additional crosslinking or the prevention of degradation induced by irradiation. Temperature vacuum dehydration crosslinking through esterification or amide formation can be used as an initial crosslinking method in further studies. This form of crosslinking will complete the conventional glutaraldehyde crosslinking that reacts with the free amine groups of lysine or hydroxylysine of the protein backbone of collagen. It should be stressed that the determination of an in vivo degradation rate, in the form of an animal study, will aid to confirm the efficacy of the biologically active regeneration matrix. / Dissertation (MSc)--University of Pretoria, 2008. / Anatomy / unrestricted

The main concern of this thesis is with desmoplasia - a process in which excessive connective tissue is deposited in a neoplasm. This is a common phenomenon in neoplasia but one whose mechanisms are poorly understood. To study the process, I used a human malignant melanoma cell line (UCT-Mel 7) that was established in this laboratory and that, when injected into athymic mice, gave rise to tumours that showed a number of interesting features. Firstly, the tumour induced a marked desmoplastic response as evidenced by a high content of hydroxyproline in tumour lysates, intense staining for reticulin in sections of the tumour and infiltration of the tumour by host mesenchymal cells. Secondly, the desmoplasia was associated in UCT-Mel 7-derived tumours with an unusual phasic pattern of growth that was related to the in vitro passage number of the melanoma cells. On occasions, murine tumours developed at the site of inoculation of human tumour cells. I have identified 2 possible mechanisms by which UCT-Mel 7 cells could have induced the desmoplastic response: either the tumour cells could have exerted their effect indirectly, i.e. via macrophages, or they could have stimulated the host's stromal cells directly. UCT-Mel 7 cells were shown to be chemotactic for mouse macrophages and human foreskin fibroblasts were stimulated, in a dose-dependent manner, to synthesize increased amounts of collagen when co-cultured with mouse peritoneal exudate cells. Stimulation could only be effected by direct cell:cell contact; medium conditioned by macrophages was not effective. The amount of stimulation was not dependent on the state of activation of the peritoneal cells nor on the strain of mouse used. Tumour cells were also found to act directly. Co-culture of UCT-Mel 7 cells and fibroblasts resulted in increased collagen synthesis by the fibroblasts. Increased synthesis of the protein was reflected in an increase in the amount of collagen mRNA. UCT-Mel 7 cell stimulated in a dose-dependent manner with an absolute requirement for intimate cell:cell contact with the fibroblasts. DNA synthesis was not required. Dexamethasone, retinoic acid and the tumour promoter, phorbol myristate acetate, had significant primary effects on fibroblast collagen synthesis but did not modify the response to melanoma cells. Indomethacin, however, had a minimal primary effect upon the fibroblasts but significantly augmented the melanoma cell effect. The nature of the stimulatory cell:cell contact is still uncertain. The gap junction inhibitor, α-glycyrrhetinic acid, did not diminish the melanoma cell effect. Preliminary findings suggested that cell-surface proteoglycans may be important. Removal of the proteoglycans with the inhibitor of proteoglycan assembly, 4-methylumbelliferyl-β-D-xyloside, abrogated the melanoma cell:fibroblast interaction. Recombinant basic fibroblast growth factor did. not seem to be involved in the desmoplastic response. It was of incidental interest to note that this compound inhibited fibroblast collagen synthesis in a manner that was augmented by the concomitant addition of heparin. A surprising finding was the production of a potent inhibitor of collagen synthesis by superinduced cells of the mouse macrophage cell line, P388D₁. This inhibitor has not been fully characterised.

Type II diabetes mellitus is associated with many detrimental health situations including heart complications. The purpose of this study was to identify a role for PKA-dependent Rap1a signaling in the AGE-RAGE cascade. My hypothesis was Rap1a GTPase increased the downstream effects of AGE-RAGE signaling in diabetes via a PKA-dependent pathway leading to elevated ECM remodeling in the heart. Cardiac fibroblasts were isolated from heterozygous (Het) and diabetic (db/db) mice. To test the hypothesis, gain-ofunction and loss-ofunction treatments were used. PKC-Zeta is known as a major signaling hub that potentially links PKA-dependent and AGE-RAGE signaling cascades so PKC-Zeta inhibition to downregulate PKA-dependent cascade at PKC-Zeta was also used. Results showed a downregulation of signaling markers in the AGE-RAGE cascade when disrupting Rap1a crosstalk at PKC-Zeta. By understanding where the PKA-dependent and AGE-RAGE signaling cascades crosstalk, a new molecular mechanism is understood possibly leading to decreasing remodeling in a diabetic heart.

No description available.