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Collagen cross-linking using oxidized cellulose
Filka, Pavel ; Márová, Ivana (oponent) ; Vojtová, Lucy (vedoucí práce)
Theoretical part of submitted diploma thesis deals with the basic finding of collagen, one of the most representing proteins in a human body, and oxidized cellulose used in medicine for several decades. Main part of the literature search is concerned on the collagen cross-linking, which is necessary for the collagen stability improving, thereby increasing the resistance against its degradation. As a cross-linking agent, the oxidized cellulose having the haemostatic properties as well as the carboxyl functional groups suitable for cross-linking proteins can be used. Experimental work was focused on the investigation of mutual behaviors of mixed water solutions based on oxidized cellulose and collagen. Films or lyophilized sponges prepared from these polymer mixtures could be used in medicine as a haemostatic or antibacterial wound healing coverings. The series of weight ratios between collagen and oxidized cellulose (9:1, 3:1, 5:3, 1:1, 1:2, 1:3, 1:9) involving constant content of collagen and increasing amounts of cellulose were prepared. Their ability to chemical linking, generating the formation of amide bond between carboxyl group of oxidized cellulose and free amino group of collagen, was investigated via two UV-VIS spectroscopic methods using a colored reaction among the chemical reagent (ninhydrin or 2,4,6-trinitrobenzenesulfonic acid) and free amino groups of collagen. Moreover, the carboxyl groups of oxidized cellulose were activated either in the polymer solution (in situ) or in the form of a film using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N hydroxysuccinimide (EDC/NHS). The changes at the secondary structure level were investigated by Fourier-transformed infrared spectroscopy (FT-IR). The stability of thin prepared films from each polymer mixture was determined using hydrolytic degradation at 37 °C. Morphological changes in the two types of samples, one frozen quickly at – 196 °C and second slowly at -30 °C, were observed using scanning electron microscopy (SEM). During the polymer mixture preparation, the precipitation up to the ratio of 1:1 between collagen and cellulose has occurred. UV-VIS analyses confirmed free amino groups reduction resulting in cross-linking between collagen and cellulose as well as increasing in hydrolytical stability obtained from degradation measurement of prepared films. At the ratio higher than 1:2 up to 1:9 the polymer mixture was homogeneous without precipitation, however, from the increasing free amino groups it is presupposed that cellulose acted as physical cross-linker at the small amount and beyond the equilibrium state works more like solvent. It probably caused the changes in collagen at the secondary structure level registered from FT IR analysis. Activation of cellulose carboxyl groups by EDC/NHS was not confirmed. Collagen to cellulose ratio affected as well as the porosity and the pore size of prepared scaffolds. Based on SEM, the porosity of scaffolds froze by liquid nitrogen was between 46 – 60 % up to the ratio of 1:1 and significantly increased with cellulose addition up to 81 % (at ratio of 1:9). Average pore size of neat collagen was very small (14 ± 5 m) in comparison with pure oxidized cellulose (79 ± 24 m). That is why the cellulose addition increased the pore size approximately up to 55 m except for the 1:9 ratio having very large pore size (186 ± 76 m) and very regular structure resembling honeycomb seen at pure cellulose as well.
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Collagen cross-linking using oxidized cellulose
Filka, Pavel ; Márová, Ivana (oponent) ; Vojtová, Lucy (vedoucí práce)
Theoretical part of submitted diploma thesis deals with the basic finding of collagen, one of the most representing proteins in a human body, and oxidized cellulose used in medicine for several decades. Main part of the literature search is concerned on the collagen cross-linking, which is necessary for the collagen stability improving, thereby increasing the resistance against its degradation. As a cross-linking agent, the oxidized cellulose having the haemostatic properties as well as the carboxyl functional groups suitable for cross-linking proteins can be used. Experimental work was focused on the investigation of mutual behaviors of mixed water solutions based on oxidized cellulose and collagen. Films or lyophilized sponges prepared from these polymer mixtures could be used in medicine as a haemostatic or antibacterial wound healing coverings. The series of weight ratios between collagen and oxidized cellulose (9:1, 3:1, 5:3, 1:1, 1:2, 1:3, 1:9) involving constant content of collagen and increasing amounts of cellulose were prepared. Their ability to chemical linking, generating the formation of amide bond between carboxyl group of oxidized cellulose and free amino group of collagen, was investigated via two UV-VIS spectroscopic methods using a colored reaction among the chemical reagent (ninhydrin or 2,4,6-trinitrobenzenesulfonic acid) and free amino groups of collagen. Moreover, the carboxyl groups of oxidized cellulose were activated either in the polymer solution (in situ) or in the form of a film using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and N hydroxysuccinimide (EDC/NHS). The changes at the secondary structure level were investigated by Fourier-transformed infrared spectroscopy (FT-IR). The stability of thin prepared films from each polymer mixture was determined using hydrolytic degradation at 37 °C. Morphological changes in the two types of samples, one frozen quickly at – 196 °C and second slowly at -30 °C, were observed using scanning electron microscopy (SEM). During the polymer mixture preparation, the precipitation up to the ratio of 1:1 between collagen and cellulose has occurred. UV-VIS analyses confirmed free amino groups reduction resulting in cross-linking between collagen and cellulose as well as increasing in hydrolytical stability obtained from degradation measurement of prepared films. At the ratio higher than 1:2 up to 1:9 the polymer mixture was homogeneous without precipitation, however, from the increasing free amino groups it is presupposed that cellulose acted as physical cross-linker at the small amount and beyond the equilibrium state works more like solvent. It probably caused the changes in collagen at the secondary structure level registered from FT IR analysis. Activation of cellulose carboxyl groups by EDC/NHS was not confirmed. Collagen to cellulose ratio affected as well as the porosity and the pore size of prepared scaffolds. Based on SEM, the porosity of scaffolds froze by liquid nitrogen was between 46 – 60 % up to the ratio of 1:1 and significantly increased with cellulose addition up to 81 % (at ratio of 1:9). Average pore size of neat collagen was very small (14 ± 5 m) in comparison with pure oxidized cellulose (79 ± 24 m). That is why the cellulose addition increased the pore size approximately up to 55 m except for the 1:9 ratio having very large pore size (186 ± 76 m) and very regular structure resembling honeycomb seen at pure cellulose as well.
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