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The Effect of Chemical Modification on the Enzymatic Degradation of Acellular Matrix (ACM) Processed Biomaterials Paul F. Gratzer A thesis submitted in canformity with the requirements for the degree of Dodor of Philosophy Graduate Departmentof Metallurgyand Materials Scienœ Engineering and the Institute of Biomaterialsand Biomedical Engineering University of Toronto O Copyright by Paul F. Gratzer 1999 National Library Bibliothèque nationale l*! of Canada du Canada Acquisitions and Acquisitions et Bibiiographic Services services bibliographiques 395 Wellington Street 395. rue Wellington OtlawaOfU K1AW OttawaON KlAûN4 Canada CaMda The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microform, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/nlm, de reproduction sur papier ou sur fcmat électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts fiom it Ni la thèse ni des extraits substantiels may be prhted or otherwise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. The Effect of Chemical Modification on the Enzymatic Degradation of Acellular Matrix (ACM) Processed Biomaterials Paul F. Gratzer Doctor of Philosophy, 1999 Department of Metallurgy and Materials Science and lnstitute of Biomaterials and Biomedical Engineering, University of Toronto In this study, the effects of specific chemical modifications of amino add side- chains on the in vitro degradation of 'native' collagen (obtained from acellular maûix (ACM) processed arteries) and 'purified' type 1 collagen (extracted from bovine Achilles tendon) was studied. Two monofunctional epoxides of different size and chemistry were used to modify lysine with or without methylglyoxai modification of argiriine. Carboxyl groups of aspartic and glutamic acids were modified with glycine methyl ester. The reagent 1-ethyl-3-(3-dimethylaminopropyl)- carbodiimide (EDC) was used as a basis for cornparison of the effects of crosslinking ivith chemical modification. EDC treatment was studied under two different pH conditions: (i) pH controlled at an optimal value of 5.5 and (ii) a simpler-but industrially significant-uncontrolled pH system. Biochemical, thennomechanical, tensile mechanical, shear stiffness and multi-enzyme (collagenase, cathepsin B, (acety1)bypsin)in viîro enzyme analyses were used to determine the effects of each modification. Carboxyl capping had no effect on the structure of native ACM collagen as determined by thermo- or tensile medianical testing. Increased collagen solubilization by enzymes, with the exception of cathepsin B, was observed after carboxyl capping. In contrast, lysine modification destabilized native ACM collagen kvith the larger, hydrophobic epoxide having the greater effect. In general, enzymatic solubilization of collagen was either unaltered or decreased after modification with the smaller, hydrophilic epoxide, whereas the larger, hydrophobic epoxide increased solubilization. Analysis of collagen fragments solubilized by trypsin and acetyltrypsin revealed that sites of deavage were altered after lysine and arginine modification. Differences were dso observed in the solubilization of (i) purified type 1 collagen and (ii) native ACM collagen by collagenase. In cornparison, both EDC treahnents were equaily effective in stabilizing native ACM collagen against solubilization by enzymes in vitro. EDC crosslinking of ACM artenes significantly increased thermal denaturation temperatures, treahnent with pH control having the greatest effect. Crosslinking without pH control, however, consumed more lysine residues and increased shear stiffness to a greater degree (21x compared to 14x with pH control). The observed differences are athibuted to differences in the location or type of EDC crosslinks formed which differentially affected mechanical behaviour without affecting the increase in resistance to enzymatic degradation. In summary, diemical modification without crosslinking can prevent degradation. This effect however, is not as broad-based as that produced by EDC crosslinking. The effects of chernical modification depended on the modifying reagent, the amino aad(s) modified, and architecture of the substrate. The ability to modulate the enzyme degradation of tissue-derived materiais as demonstrated in this study may, however, facilitate the design of novel engineering scaffolds for tissue regeneration or collagen based drug delivery systems. ABSTRACT A new approadi in the preparation of xenograft vasdar prostheses is the acellular matrix (ACM) process which removes cellular components (the main source of immunological recognition) while leaving the extracellular stnictural components of the tissue intact (collagen and elastin). While ACM processed nilografs have been shown to remain patent for more than 4 years, ACM xetiopnfis have proven to be more challenging with respect to biodegradation. Simple crosslinking with industry-standard bifunctional or polyhinctional reagents (e.g. gIutaraldehyde, polyepoxy compounds) has yielded undesired cellular and thrombogenic responses. Since proteolytic enzymes respond to specific sites on proteins, an alternative treatment could involve the 'masking' of collagen from degradative enzymes through the terminal capping of recognizable amino aad side-diain functionalities without formation of a crosslink. In this study, the effects of specific chernical modifications of amino acid side-chahs on the in vitro degradation of 'native' ACM artenal and 'purified' collagens were investigated. Two mono~ctionalepoxides (glyadol and n- butylglycidylether) of different size and chemistry were used to modify the &-aminogroup of lysine, with or without methylglyoxal modification of arginine. Carboxyl groups of aspartic and glutamic auds were modified with glycine me thyl es ter. The reagent 1-ethy1-3-(3-dimethylaminoprop yl)-carbodide (EDC) was also examined as a basis for cornpanson of the effects of crosslinking xvith chernical modification of side-chahs. EDC was selected due to its reactivity with the same targeted amino aad side-diains (lysine, aspartic and glutamic acids) and its zero-length crosslinking mechanism. EDC treatment was studied under hvo different pH conditions: (i) pH controlled at an optimal value of 5.5 and (ii) a simpler-but industrially significant-uncontroued pH system. Biochemical assays, thermomechanical, tensile mechanical, and shear stiffness testing were utilized to assess physical property effects, and mdti-enzyme in vitro enzyme analyses were used to determine the degradative property effects of each chernical modification. Carboxyl capping had no significant effect on the thermal denaturation temperature or tensile mechanical behaviour of ACM arteries. However, increased collagen solubilization by collagenase, hypsin, and acetyltrypsin was observed after carboxyl capping, whereas collagen solubilization by cathepsin B was reduced. In contrast, lysine modification reduced thermal denaturation temperatures of ACM artenes, indicating destabilization of the structure of native ACM collagen. The larger, more hydrophobic n-butylgiycidylether having the greater effect. After subsequent arginine modification, oniy n-butylglycidylether treatment reduced collagen thermal stability. Tensile mechanical behaviour (induding stress-strain, stress-relaxation, and fracture properties) was al tered after lysine modification of ACM arteries, again wi th the larger, hydrophobic epoxide having the greater effect. Subsequent arginine modification increased the differences obse~edbehveen the mechanical behaviours of untreated and epoxide treated ACM arteries. Differences in collagen solubilization by enzymes were found to depend upon the size and chemistry of epoxides used to modify lysine residues. In general, the solubilization of native ACM collagen by collagenase, cathepsin B, trypsin, and acetyltrypsin was either unaltered or decreased after modification with glyadol. In contrast, n-butylglycidylether treatment increased solubilization by al1 enzymes. Subsequent arginine modification significantly reduced collagen solubilization by acetyltrypsin for glyadol-treated ACM arteries, whereas increased collagen solubilization was observed for n-butylglyadylether treated ACM arteries with al1 enzymes. Gel chromatographie analyses of collagen fragments solubilized by trypsin and acetyltrypsin from native ACM collagen and purified type 1 collagen revealed that both the amount and sites of deavage were altered after lysine and arginine modification. Furthemore, differences in the solubilization of (i) purified type 1 collagen and (ii) native ACM collagen by collagenase obtained after lysine and arginine modification indicated that iii collagen structure and/ or the presence of other extraceilular matrix proteins infIuence the effect of chemical modifications. In cornparison to chemical modifications, EDC crosslinking of ACM arteries significantly increased thermal denaturation temperatures, treatxnent with pH control having the greatest effect. Crosslinking without pH control, however, consumed more lysine residues and increased shear stiffness to

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