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

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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 a greater degree (21x compared to 14x with pH control). Most interestingly, both EDC treatments were equally effective in stabilizing ACM arteries against collagen solubilization by collagenase, cathepsin B, trypsin, and acetyltrypsin in vitro. The observed differences between EDC treatments under different pH conditions is attributed to differences in the location and types of the exogenous crosslinks formed. Furthemore, the location or type of crosslinks differentidy affected the mechanical behaviour of ireated materials without affecting the increase in resistance to enzymatic degradation. In summary, chemical modification without crosslinking can alter solubilization. This effect however, is not as broad-based as that produced by EDC crosslinking. The effects of chemical modification depended on the modifying reagent, the amino aQd(s) modified, and architecture of the substrate. The ability to modulate the enzyme degradation of tissue-derived materials as demonstrated in this study may, however, facilitate the design of novel engineering scaffolds for tissue regeneration or collagen based drug delivery systems. This thesis is dedicated to my wife Julie and daughter Katie. 1 would like to express rny sincerest appreciation to my wife for aU of her support, understanding, love, and patience. 1 would like to thank Katie for al1 of her hugs, kisses, laughter, and srniles. 1would like to express my deepest gratitude to my supervisors and mentors, Drs. J. Michael Lee and J. Paul Santerre. Their guidence, enthusiasm, and understanding have been key to the completion of this work and the start of my academic career. 1 would also lïke to thank Dr. Greg Wilson, Dr. George Adams, and Dr. Fred Keeley for their guidence and support. 1 would like to express my gratitude to technicians Chris Pereira and Rey Intenor for their help, and to Dr. Steve Thorpe for the use of hsmicroscope and digital camera. Finally, 1 wish to thank all of rny colleagues in Dr. Santerre's lab and Cathie Bellingham for their stimulamg conversations and much needed diversion. TABLE OF CONTENTS .. ABSTRACT ...... II

ACKNOWLEDGEMENTS ...... v

TABLE OF CONTENTS...... vi

LIST OF FIGURES ...... x ... LIST OF TABLES ...... XIII

1.O INTRODUCTION ...... 1 1.1 Tissue Derived Materials ...... 1 1.2 Structure and Properties of Soft Tissues ...... 6 1.2.1 Collagen ...... 7 1 .2.2 Elastic Fibres...... 12 1.2.3 Proteoglycans. Glycosarninoglycans...... 14 and Glycoproteins 1.2.4 Mechanical Properties of Tissue.Derive d...... 16 Materials 1.3 Degradation of Tissue-Derived Materials ...... 19 1.3.1 Collagen Degradation...... 20 1 .3.2 Elastin Degradation...... 22 1-4 Preimplantation Processing of Tissue-Derived ...... 24 Materials 1. 4.1 Crosslinking Techniques ...... 24 1.4. 1.1 Evaltration of Crossli~zkitig...... 26 1.4.2 Biochemical and Physical Manipulation ...... 28 1-5 Justification of Study and Hypotheses ...... 30

2.0 MATERIALS AND METHODS ...... 32 2.1 Preparation of Samples ...... 32 2.1 -1 Acellular Matrix (ACM) Processing...... 32 2.1 -2 Purified Type I Collagen...... 34 2.1 -3 Crosslinking with EDCINHS ...... 35 2.1 -4 Carboxyl Capping : EDCMHS and ...... 37 Glycine Methyl Ester 2.1.5 Lysine Capping : Monofunctional Epoxide Treatments.... 37 2.1 -6 Arginine Capping: Methylglyoxal Treatment ...... 40 2.2 Characterization of Materials ...... 42 2.2.1 Biochemical Properties...... 42 2.2.1.1 Anzino Acid Analysis ...... 42 2.2.1.2 Lysine Determination by TNBS Assay ...... 43 2.2.1.3 Thermal Dena tu ration Testing ...... 43 2.2.2 Physical Properties ...... 44 2.2.2.1 lemile Mecltnn icd lesting ...... 44 2.2.2.2 Tende Mechanical Data Aruilyses ...... 47 2.2.2.3 Resitizial Stress Mensir rements ...... 48 2.2.2.4 Bending (Sltear) Stiffness Measu rentozts ...... 49 2.2.2.5 Swelling Measureinents ...... 49 2.3 Enzyme Degradation Studies ...... 52 2.3.1 Enzyme Degradation Assays ...... 52 2.3.1.1 Trypsin and AcetyIhypsin ...... 53 2.3.1.2 Microbinl Collagenase ...... 53 2.3.1.3 Cathepsin B ...... -....-..-.-...53 2.3.2 Gel Chromatography Analysis of Enzyme Degradation.. 53 Products 2.3.3 Hydroxyproline Analysis for Collagen Content ...... 55 2.4 Statistical Analyses ...... 56

3.0 RESULTS ...... 57 3.1 Verification of TNBS Assay for Lysine ...... 57 3.2 EDC Crosslinking...... 59 3.2.1 Thermal Denaturation and Biochemical Analyses ...... 59 3.2.2 Mechanical Testing ...... 63 3.2.3 Enzyme Degradation Assays ...... 63 3.3 Carboxyl Capping: EDCMHS and Glycine Methyl Ester ...... 66 3.3.1 Thermal Denaturation and Biochemical Analyses ...... 66 3.3.2 Tençile Mechanical Testing ...... 66 3.3.3 Enzyme Degradation Assays ...... 70

vii 3 -4 Lysine and Arginine Capping: Epoxide and Methylglyoxal ...... 72 Treatment of ACM Arteries 3.4.1 Thermal Denaturation and Biochemical Analyses ...... 72 3.4.2 Tensile Mechanical Testing ...... 76 3.4.3 Enzyme Degradation Assays ...... 83 3.4.3.1 Autolysis of Trypsin ...... 83 3.4.3.2 Trypsiri Degradation ...... 85 3.4.3.3 Acetylhjpsin Degradation ...... 85 3.4.3.4 Cathepsin B Degradation ...... 85 3 .4.3 -5 Collagenase Degradatio n ...... 88 3.4.4 Gel Chromatography of Degradation Products ...... 88 3.4.4.1 Calibrafion of Gel Colii~nnwitlz Globrrlar Proteiiz ..... 88 Standards 3.4.4.2 280 nm UV Absorbance Elution Profiles ...... 92 3.4.4.3 Collagtm Contents of Clirontatosaphy Fractions ..... 94 3.5 Lysine and Arginine Capping : Epoxide and Methylglyoxal ...... 103 Treatment of Purified Type I Collagen 3.5.1 Amino Acid Analyses and Swelling Measurements...... 103 3.5.2 Enzyme Degradation Assays ...... 103 3.5.3 Gel Chromatography of Degradation Products ...... 103 3.5.3.1 280 nrn UV Absorbniice Elution Profiles ...... 103 3.5.3 -2 Collagoz Contents of Chro?natogrnplzyFractions ... 1O8

4.0 DISCUSSION...... 115 4.1 EDC Crosslinking...... 115 4.2 Capping of Amino Acid Side-Chain Functionalities...... 123 4.2.1 Carboxyl Capping: EDC/NHS and ...... 123 Glycine Methyl Ester 4.2.2 Lysine and Arginine Capping: Epoxide and ...... 126 Methylglyoxal Treatments 4.2.2.1 Effects of Lysine and Arginine Capping on ...... -127 Collagen Structure 4.2.2.2 Effects of Lysine and Arginine Cnpping on ...... 129 Enzyme Degradation

AMAR 5.1 Relating Results of the Study Back to the Original Hypotheses .142

viii 6.0 CONCLUSIONS ...... 143 6.1 EDC Crosslinking and pH Control ...... 143 6.2 Chernical Modification of Amino Acid Side-Chains ...... 143

7.0 RECOMMENDATIONS...... 144

8.0 REFERENCES...... 145

9.0 APPENDIX: LIST OF CHEMICALS AND EQUIPMENT ...... 176 Schematic Diagram of a Blood Vessel ...... 8 Structural Hierarchy of Fibrous Collagen ...... 10

Typical Stress-Strain and Stress Relaxation Behaviour of ...... 17 Tissue-Derived Materials Flowchart of Sample Preparation and Testing ...... 33 Crosslinking Reaction of EDCINHS with Collagen ...... 36

EDCINHS Mediated Capping of Carboxyl Groups ...... 38 Epoxide Modification of Amino Groups of Lysine ...... 39

Methylglyoxal Modification of Arginine ...... 41 Example of Output from Thermal Denaturation Testing ...... 45 Schematic Diagrarn of Mechanical Testing Apparatus For Loops ..... 46 Definition of Opening Angle ...... 51 Plot of pH vs . Time for EDC Crosslinking Without pH Control ...... 60 lncrease in Denaturation Temperature vs . Time for ...... 61 EDC Crosslinking of ACM Arteries

Percent lncrease in Denaturation Temperature vs ...... 62 Percent Lysine Residues Modified for EDC Crosslinking Stress-Strain Response for EDC+G-Treated ACM Arteries ...... 68 Stress Relaxation Behaviour for EDC+G-Treated ACM Arteries ...... 69 In-Vitro Degradation of EDC+G-Treated ACM Arteries ...... 71 Denaturation Temperatures for Epoxide and Epoxide Plus ...... 73 Methylgloxal-Treated ACM Arteries

Percent Change in Denaturation Temperature for Epoxide-Treated 74 ACM Arteries vs . Percent Lysines Modified Stress-Strain Response of Glycidol-Treated ACM Arteries ...... TI Stress-Strain Response of n-Butylglycidylether-Treated...... 78 ACM Arteries 22 Stress Relaxation Behaviour of Glycidol-Treated ACM Arteries ...... 79 Stress Relaxation Behaviour of n-Butylglycidylether-Treated...... 80 ACM Arteries Stress-Strain Response of Epoxide and Methylglyoxal-Treated...... 81 ACM Arteries

Stress Relaxation Behaviour of Epoxide and ...... 82 Methylglyoxal-Treated ACM Arteries

280 nm Absorbance Elution Profiles of Trypsin and Acetyltrypsin .... 84 Before and After 48 hour Incubation

Percent Collagen Solubilized From Epoxide and Epoxide Plus ...... 86 Methylglyoxal Treated ACM Arteries by Trypsin Percent Collagen Solubilized From Epoxide and Epoxide Plus ...... 87 Methylglyoxal Treated ACM Arteries by Acetyltrypsin Percent Collagen Solubilized From Epoxide and Epoxide Plus ...... 89 Methylgl yoxal Treated ACM Arteries by Cathepsin B Percent Collagen Solubilized From Epoxide and Epoxide Plus ...... 90 Methylglyoxal Treated ACM Arteries by Collagenase

280 nm Absorbance Elution Profiles of Trypsin, Trypsin After ...... 93 48 hours, and Trypsin Degradation Products From Untreated ACM 280 nm Absorbance Elution Profiles of Collagenase and ...... 95 Collagenase Degradation Products From Untreated ACM

280 nm Absorbance Elution Profile of Collagenase Degradation ...... 96 Products frorn Untreated, GMG-, and BMG-Treated ACM Arteries Site Distribution of Collagen Solubilized by Trypsin From...... 98 Untreated, GMG-, and BMG-Treated ACM Arteries Size Distribution of Collagen Solubilized by Acetyltrypsin From ...... 100 Untreated, GMG-, and BMG-Treated ACM Arteries Site Distribution of Collagen Solubilized by Collagenase From...... 101 Untreated, GMG-, and BMG-Treated ACM Arteries Size Distribution of Collagen Solubilized by Cathepsin B From ...... 102 Untreated, GMG-, and BMG-Treated ACM Arteries Samples of Untreated, GMG-, and BMG-Treated ...... 105 Type I Collagen Before and After Swelling in Acid Percent Collagen Solubilized by Enzymes From Untreated, ...... 106 GMG- and BMG-Treated Type I Collagen 280 nm Absorbance Elution Profiles of Acetyltrypsin, ...... 107 Acetyltrypsin after 48 hours, and Acetyltrypsin Degradation Products From Untreated Type I Collagen

280 nm Absorbance Elution Profiles of Collagenase and ...... 109 Collagenase Degradation Products From Type I Collagen

280 nm Absorbance Elution Profiles of Collagenase Degradation .. 110 Products from Untreated, GMG-, and BMG-Treated Type I Collagen

Sire Distribution of Collagen Solubilized by Acetyltrypsin From ...... 1 11 Untreated, GMG-, and BMG-Treated Type I Collagen

Size Distribution of Collagen Solubilized by Collagenase From...... 1 13 Untreated, GMG-, and BMG-Treated Type I Collagen

Size Distribution of Collagen Solubilized by Cathepsin B From ...... 1 14 Untreated, GMG-, and BMG-Treated Type I Collagen EDCINHS Crosslinking and Side-Reactions ...... 120 Formation of CO, by a Reaction Between EDC and NHS ...... 121 Hypothesized Inhibition of Enzyme Binding Through Blocking of .... 139 Primary and Secondary Binding Sites Hypothesized Rotation of Side-Chahs of Collagen Due to ...... 139 Chemical Modifications Hypothesized Changes in Collagen Conformation Due to ...... 139 Chemical Modifications

xii LIST OF TABLES Comparison of Lysine Contents for Epoxide Modified ...... 58 ACM Arteries as Determined by Amino Acid Analysis and TNBS Analysis

Properties of ACM Arteries Before and After EDC Crosslinking ...... 64 In-Vitro Degradation of ACM Arteries Before and After ...... 65 EDCINHS Crosslinking

Properties of ACM Arteries Before and After Carboxyl Modification 67 With EDC and Glycine Methyl Ester Properties of ACM Arteries Before and After Epoxy Treatrnent ...... 75 With or Without Methylglyoxal Treatment

Molecular Weights and Corresponding Elution Volumes for ...... 91 Gel Chrornatography Globular Protein Standards

Effects of Epoxy and Methylglyoxal Treatrnent on the Swelling of .. 104 Type I Collagen

Sumrnary of the Effects of EDC Crosslinking With and Without ...... 1 16 pH Control on ACM Arteries

xiii 1. INTRODUCTION

1.1 Tissue Derived Materials Tissue-denved materiais may be defined as "... any tissue or tissue component which has undergone pre-implantation processing, such as high level disinfection with antibiotics, protein cross-Lùiking, protein isolation and purification, anticdafication treatment or ayopreservation" [Hilbert et al. (l989)I. Therefore, tissue-derived materials include both viable and non-viable tissues (eg. heart valves and vascular grafts) as well as individual tissue components (e.g. collagen), which have undergone some type of pre- implantation treatment. A variety of tissues-derived materials have been used to hbricate devices to replace parts of the human body. These materials have been derived from autologous (self), allograft (same speaes), or xenograft (cross- species) sources. Although tissues indude soft and hard (bone) materials, this review will focus only on the history and use of soft tissue-derived materials. The use of natural tissues to replace damaged or diseased parts of the human body began with the use of allograft materids. Vascdar allografts were investigated as early as 1906 by Goyanes; however it wasn't until the Iate 1940's to early 1950's and the Korean War that vascular allografts were extensively used to treat h-aumatic wounds [Dennis (1987); Callow (1982)j. Early on, vascular allografts were exased from dying trauma victims and implanted with the hope of the grafts being incorporated into the host tissue. Cells within the grafts, however, disappeared soon after implantation and the grafts remained non- viable. Later, as demand increased and supplies of fresh gr& were Limited, methods for the sterilization and storage of vascular allografts were investigated. Immunological reactions to the fresh gr& and degradation, however, resulted in very poor long-term performance which was only exacerbated by treahents aimed at presening and storing harvested tissues [Callow (1986)].During the sarne time period, the use of allograft materials to repIace aortic heart valves was also investigated. In 1962, Sir Bnan Barratt-Boyes and Donald Ross independently reported on the use of allografts to successhilly replace failing aortic valves [Shim and Lenker (1988); Black et al. (1983)l. As with vascular allog-rafts, problems with availability, steriiization, storage and irnrnunological recognition and degradation prevented the general use of allografts to replace aortic valves. In early 1965, Binet and Carpentier reported on the first dinical use of xenograf t (inter-species)heart valves [Carpentier et al. (1969); Carpentier (l977)I. Interestingly, it was French regulations that forbade the removal of cadaver tissue within 48 hours of death that prompted Carpentier to investigate the use of xenograft tissue. These first valves were obtained from pigs and rendered iess antigenic through the use of organic mercurial salts in the hopes of overcoming the problem of rejection. Unfortunately, initial investigations showed that al1 mercurial xenograft valves failed within 4 years due to irnmunological reaction or degradation [Carpentier (1977)l. Carpentier later determined that the integrity of prosthetic tissue valves was dependent solely on their own structure-as opposed to providing a framework ont0 which the body could regrow a valve. From this, Carpentier proposed the use of a crosslinking agent for electron microscopic analysis (glutaraldehyde) in xenograft preparation. During the treatment process, all of the cells within the tissue were destroyed and any host cellular ingrowth was inhibited. In 1968, early studies demonstrated that glutaraldehyde treatment also decreased antigenicity and increased tissue durability. [Carpentier (1977); Shim and Lenker (1988)]. Carpentier referred to these chemicaily treated, non-viable implants as "bioprostheses". As a result of the introduction of glutaraldehyde by Carpentier, nurnerous other glutaraldehyde-treated tissue-derived devices were investigated. These included bovine pericardial (tissue that foms a sac around the heart) aortic valves and pericardial patches, human dura mater (tissue surrounding the brain) heart valves, human umbilical cord vein grafts, bovine carotid and mammary artery grafts, and bovine ligament grafts to narne a few [Hilbert et al. (1988); Khor (1997); Sawyer et al. (1982); Dardik et al. (1982); Chvapil et al. (1987); Dardik (1987); Sawyer et al. (1987)l. The discovery of problems linked to the use of glutaraldehyde, induding release of cytotoxic glutaraldehyde monomers, calcification, significant changes in tissue mechanical properties after treatment, mechanical Wear, and degradation, prevented the exclusive use of most of these devices [Wiebe et al. (1988); Dahm et al. (1990); Lee and Boughner (1991); Hilbert et al. (1988)l. Currently, only porcine (glutaraldehyde-treated) aortic valves and bovine pericardial valves are generdy used for specific situations such as in the elderly and in women of child-bearing age. Problems associated with glutaraldehyde treahent including durability and calcification, have yet to be resolved. Anti-calafication treatments for glutaraidehyde-treated tissues are currently under investigation and will be discussed later in this review [Vyavahare et al. (1998), (1997); Walther et al. (1998)l. Due to the problems associated with glutaraldehyde, alternative crosslinking treatments for tissue-derived materials have been sought. Alternative crosslinking heahnents that have been investigated indude carbodiimides, diisocy ana tes, epoxides, and oxidative photo-crosslinking [Moore et al. (1998); Naimark et al. (1995); Chamlatha and Rajram (1997); Petite et al. (1990); Lee et al. (1996), (1994)l. A more detailed overview of these crosslinking alternatives will be given later in this review. Despite the use of the above alternative crosslinking treatments, the problems associated with changes in mechanical behaviour following treatment, immunological recognition, and degradation still persist. Thus far, only devices manufactured from whole tissues have been discussed. Tissue-denved materials, however, indude the purified components of tissues such as the structurai proteins collagen and elastin and the glycosaminoglycans heparan sulphate and chondroitin sulphate. By far, collagen has been the most studied and utilized tissue component for medical and surgical applications. Collagen can be extracted and purified from tissues such as tendon and skin, and be formed into a variety of preparations induding solutions, gels, powders, films, and sponges [Gorham (1991); Chvapil et al. (1973); Chvapil (1977); Nimni et al. (1987)l. These preparations, alone or in combination with heparan sulphate and chondroitin sulphate, have been used to produce bum dressings, wound dressings, haemostats, soft tissue augmentation materials, contraceptives, synthetic vascular graft Iinings, tendons, and dnig delivery systems [Gorham (1991); Chvapil et al. (1973)l. Collagen preparations were treated in a similar manner to whole tissue grafts with the goal of preventing immunologic recognition and degradation. Early fxeahnents induded crosslinking with reagents such as glutaraldehyde, cyanimide (a carbodümide), and dehydration [McPherson et al. (1986); Collins et al. (1991); Nimni et al. (1987); Wang et al. (1994)l. Problems observed earlier in whole tissue gr& due to crosslinking treatments (calcification, cytotoxic monomer rdease, immunologic recognition, and degradation) were also evident in collagen prepara tions. Recently, with the advent of tissue engineering, there has been a renewed interest in using tissue-derived materials as scaffolds to encourage the regeneration of tissues [Hubbell and Langer (1995); Langer and Vacanti (1993); Nerem (1992)]. The application of tissue-derived materials as tissue engineering scaffolds has the advantages that material degradation products can be easily metabolized and the structures can provide a natural guide for tissue regeneration. Ongoing research with tissue-derived scaffolds has been broad in scope and has included skin grafts, vascular grafts, neural grafts, periodontai regeneration, ligament prostheses, and abdominal wall patches [Zacdu et ai (1998); Woerly et al. (1996); Bladc et al. (1998); Dunn et al. (1995); Peulve et al. (1997); van Wachem et al. (1994); Berthaume et al. (1994)l. While most work has been conducted using purïfied tissue components alone or in combination (e.g. collagen and chordroitin sulfate), the use of whole tissues as scaffolds for tissue regeneration has also been investigated. Methods of removing cells (the main source of immunological recognition) from tissues, while leaving stmcturai proteins intact, have been developed and utilized to produce 'acellularized' heart valves, tendons, vascular grafts, and bladders [Cartmell and Dunn (1998); Wilson et al. (1995); Dahms et al. (1998)l. The development of novel synthetically- produced natural polymeric materials are also being investigated [Deming (1997); Tire11 (1997); Bellingham et al. (1998)j. These novel materials are derived from precursors of proteins (eg. tropoelastin) which have been produced by genetically manipulated bacteria. As with earlier 'bioprostheses', imrnunological recognition and degradation of materials are important considerations in the production of new tissue engineering scaffolds from tissue-derived materials. Contrary to the principles of preventing cellular invasion and maintaining structural in tegrity, tissue-engineering scaffolds must encourage infiltration and degradation, eventually leading to regeneration by host cells. Careful controt over the degradation process is therefore required to maintain the strength of the implant during the regeneration process whiIe allowing heding and remodeling to take place [Berthiaume et ai. (1994); van Wachem et al. (1994); Osborne et al. (l9W)j. Thus, new techniques are required to stabilize tissue-denved materiais utilized for engineering scaffolds. 1.2 Structure and Properties of Soft Tissues Soft tissues can be described as biological composites comprised of cells and the extracellular matrix (ECM). The ECM consists of insoluble fibres which resist tensile forces and interfibrillar hydrated polymers which are referred to as the 'ground substance". The insoluble fibres (coilagen and elastic fibres) and ground substance (glycosaminoglycans and proteoglycans) of the ECM form a cornplex intercomected system whose architecture determines the mechanical properties of the tissue [Viidik et ai. (1982); Naimark et al. (1992)l. The basic geometncal forms of tissues can be seen through out the body and uidude cables, sheets, and tubes [Silver and Doillon (1989)l. Cable-like geometries are found in tissues such as nerves, tendons, and ligaments where forces or electrical signals are required to be transmitted from one place to another. The ECM of these tissues is mainly comprised of rope-like collagen fibres aligned dong the axis of the cable structures [Harkness (1968); Silver and Doillon (1989); Amiel et al. (1984); Kielty et al (1993)l. A tube-like covering of loose comective tissue is also present on the outside of the cable stnictures forming a protective sheath. Sheet-like geometries are found in tissues such as skin, pericardium, and dura mater. The ECM in this geometry is designed to support sheets of cells (e.g. epi thelial or epidermd cells) while aiso resisting mechanical forces and acting as a semi-permeable barrier. Layers of collagen and elastin fibres are generally arranged in an interconnected and randomly onented 'wicker work' structure [Birk and Linsenmayer (1994); McGarvey et al. (1984)j. This allows a force from any direction to be borne dong the axis of some fibres and the intenveave within and behveen layers halitates the transfer of force ont0 fibres not directly acted upon [Harkness (1968); (1971)].

- Originally a mistranslation of the German "grundsubstantz" or "fundamental substance". Tubular structural forxns are found in tissues such as blood vessels, intestine, spinal chord, and the reproductive system. These tube-like geometries generaily consist of three concentric layers which reinforce the tube wail and separate the lumen from the extemal environment. A biood vessel for example, is comprised of an inner (hinica)intima, midde media, and outer adventitia (Figure 1) [Kucharz (1992); Harkness (1968); Hilbert et al. (1988)l. The intima is a layer of collagen and elasün lined with endotheliai cells which allows for the maintenance of blood flow. Collagen, elastin, and smooth muscle ceils (which actively regulate vessel diameter) are found in altemating concentric rings located in the media which, together with the adventitia, prevents destructive over-dilation of the blood vessel. The intricate interactions between aiternating layers of collagen and elastin in the media are also responsible for the viscoelastic transmission of pressure pulse waves which propel blood throughout the body [Viidik et al. (1982)l. The outer adventitia, is comprised of loose layers of collagen and elastic fibres. Collagen and elastic fibres in blood vessels dso have unique alignrnents. The fibres run in three different directions; dong the ais, helically dong the axis, and arcumferentially [Canham et al. (1997)l.

1 -2.1Collagen Collagen is recognized as the most widespread protein found in mamrnals and one which plays a major role in defining the form and integriv of the body [Pany (1988);Ricard-Blum and Ville (1988)J.At least twenty types of chemically distinct collagens are known at the present time [Labat-Robert et al. (1990)l; Kielty et al. (1993)j. Types 1, II, and III collagens are the fibrillar, penodic collagens found in tendon, skin, bone, vessel walls, cartilage, and the pericardium. Type 1 and III collagens are by far the most abundant in most tissues-the exception being cartilage. For example, by dry weight, arteries contain 20-50% collagen of which 8&9û% is type 1 and III collagens, tendons contain approximately 87% collagen of which 90% is type 1 and 10% is type III, and skin contains 77% collagen of which type III accounts for 15% with the remaining being almost all type 1 [Mayne (1986); Amiel et al. (1984); Odland Intii lntemal Elastic

Smooth Muscle

Figure 1. Schematic diagram of a blood vesse1 showing its concentric ring structure. The advetitia consists of a basement membrane and endothelial cells which line the lumen. An intemal elastic lamella defines the start of the media comprised of alterating wavy laye= of collagen, elastin. and smooth muscle cells. An external elastic larnella marks the transition to the adventitia which consists of loose collagen and elastin fibras. (1983)l. CoUagen fibres are not formed £rom just one collagen type but iwtead are CO-polymersof two or more fibril-formirtg collagens [Kadler et al. (1996); Kielty et al. (1993)l. The basic unit or molecule of collagen (- 300 nm in Iength and 1.5 nm in diameter) consists of three left-handed polypeptide chahs which ïntertwine and form a triple-helical coi1 with an overall right-handed twist (tertiq structure) (Figure 2.) [Parry (1988); Rarnadiandran (1988); Chapman et al. (1990); Nimni and Harkness (1988); Gorham (1991); Kielty et al. (1993); Kadler et al. (1996)l. Types II and III collagen consist of three identical a-&& known as al(II)and al(m),respectively. Type 1 couagen, however, consists of two different a-chahs; two al(1)and one a2(I). Each a-chain (secondary structure) of collagen consists of more than 1000 amino aads (primary structure). The unique triple-helix forming ability of the chahs is derived from a sequence in whch every third amino aad is glycine and the repeat unit is (Gly-X-Y) [Gorham (1991); Pany (1988); Rarnachandran (1988); Nimni and Harkness (l988)]. The components X and Y in the repeat sequence cm be any amino aud; however, they are frequently proline and hydroxyproline, respectively. Proline occurs almost exclusively at the X-position and accounts for 130 residues per 1000, whereas hydroxyproline occurs at the Y-position, accounting for about 90 residues per 1000 [Gorharn (1991)l. Proline and hydroxyproline are rigid due to their five- membered ring and 'stiffen' the a-chains where they occur by limiting C-N bond rotation [Kielty et al. (1993); Gorham (1991)l. This Gly-X-Y repeat unit, and hence the triple-helical shucture, is found only in the helical or cenhd part of the collagen molecule; hvo smaü non-helical regions, or telopeptide regions (- 2% of the molecule) are located at either end [Kadler et aI. (1996)l. The collagen triple helical configuration is stabilized mainly through hydrogen bonds via the hydroxyl groups of hydroxyproline residues [Nemethy (1988); Ramadiandran (1988); Kielty et al. (1993); Gorham (1991)l. Collagen type III, however, also contains covalent intrahelical disulphide (S-S) bonds behveen Collagen Crimp Flgun 2. Structural hierarchy of fibrous collagen. Structures represented include: amino acid sequence (primary structure), a-helical chain (secondary structure), collagen molecule (teitiary structure), and collagen fibrils and fibre bundles (quaternaiy structure). Collagen crimp is associated with collagen fibres. alpha chains via the amino aad cysteine [Nimni and Harkness (1988); Eyre et al. (1984); Ricard-Blum and Ville (1988)l. Polar groups tend to form hydrogen bonds with one another when located withthe interior of the protein, but usually form hydrogen bonds with the surrounding solvent when located on the surface of the molecule. At physiological pH, most ionizable groups, such as -NH3+ and - COO-, are charged and highly hydrated. Such groups tend to be localized on the solvent-accessible exterior of the molecule [Nemethy (I988)l. Non-polar side groups of arnino aads, however, are also present on the surface of the collagen molecule, resulting in altemating polar and non-polar domains. The altemating polar and non-polar regions on the surface of the collagen molecule facilitates the formation of higher forms of collagen structure (pentafibrils, fibrils, fibres, and fibre bundles) (Figure 2.). In a process calIed "fibrillogenesis" collagen molecules aggregate together in groups of five through electrostatic and hydrophobic interactions between polar and non-polar regions dong each molecule to form a pentafibril [Chapman et al. (2990); Reiser et ai. (1992); Nimni and Harkness (2988); Miller (1988); Kader et al. (1996)l. The interaction of the polar and non-polar regions displaces each molecule in the pen tafibril by - 67 nm, resulting in what is known as a "quarter-staggered" array [Kadler et al. (1996); Kielty et al. (1993); Chapman et al. (1990) 1. The pentafibrils then laterally and axially aggregate together to form collagen fibrils. Although collagen fibrils have Iittle strength in either flexion or torsion, they exhibit high tensile strength due to the presence of interhelical covalent crosslinks which link collagen molecules both axially and laterally [Davidson and Brennan (1983); Parry (1988); Nimni and Harkness (1988)l. The covalent crosslinking of collagen molecules occurs through the action of the enzyme lysyl oxidase whose action depends upon the presence of copper. The enzyme oxidatively de-aminates speafic &-carbonsof lysine and hydroxylysine to yield corresponding semi-aldehydes (allysine or hydroxyallysine), respectively [Kucharz(1992); Ricard-Blum and Ville (1988); Kielty et al. (1993)l. Only four locations for native crosslinking have been established in types 1, II, and III collagens: two in the non-helical or telopeptide regions (one at each end) and two within the helical region [Eyre et al. (1984); Ricard-Blum and Ville (1988); Zimmermann et al. (1973); Zimmermann et al. (l%'O)]. Intrahelical crosslinks may form through the aldol condensation reaction of allysine or hydroxydysine residues in the telopeptide region on two adjacent a-chains in the same collagen molecule [Eyre et al. (1984); Nimni and Harkness (1988)l.Crosslinks behveen different collagen molecules are formed through the aldol condensation reaction of adjacent allysine or hydroxyallysine residues or through the reaction of these compounds with unmodified lysine or hydroxylysine residues to form a Schiff Base (aldimine crosslink) [Reiser et al. (1992); Davidson and Breman (1983); Nirnni and Harkness (1988); Gorham (1991); Eyre et al. (1984)j. The quarter stagger array facilitated by the interaction of polar and non-polar regions dong the collagen molecde ensures the proper alignment and overlap of the crosslinking regions [Kielty et al. (1993); Ricard- Blum and Ville (1988)l. Once formed, collagen fibnls band together to form collagen fibres. The fibrils are not confined to a single collagen fibre. They can cross over to different fibres, residing in different fibres for part of their total length [Parry (1988)]. This acts to mechanically link or cement fibres together to form even larger structural units, such as fibre bundles. Both collagen fibres and fibre bundles display a macroscopic penodic aimp or "waviness" of approximately lpm to several 100's of Pm which is visible under the light microscope (Figure 2.) [Gathercole and Keller (1991); Parry (l988)I.

1.2.2 Elastic Fibers Elastin is an amorphous-appearing, relatively insoluble and lightly crosslinked protein rich in hydrophobie amino acids. It is the main component (- 90% dry weight) of elastic fibres which are responsible for the elasticity of the arterial wall, skin, lungs and other comective tissues which must store and absorb energy [Urry (1983); Gosline (1976); Kadar (1989)j. The elastin content of tissues can be as high as 50% by dry weight (aorta) to as little as 24%(skin) [Pasquali-Ronchetti et al. (1995); Pratt and Madri (1987); Rosenbloom et al. (1993)l. Elastin is comprised mainly of non-polar amino aads: glycine accounts for approximately one-third of the total and hydroxyproline and proline account for 1043%of the total [Urry (1983); Gosline (1976); Rosenbloom (1993)l. Fewer than 5% of the amino aads in elastin have charged side groups Glycoprotein rnicrofibrüs are the secondary component of elastic fibres and are found surrounding the central elastin core. The exact structure and composition of microfibrils is still under investigation; however two major components, fibrillin and microfibril-associated glycoprotein (MAGP), have been well characterized [Brown-Augsburgeret al. (1996); Zhang et al. (l995)I. Mimofibrils have a "tubuiar" profile in cross-section approximately 10-12 run in diameter. The fibres appear as 5 or 6 small doughnut-like electron-dense filaments arranged around the outside of an electron-lucent zone approximately 4 mn in diameter [Cleary et al. (1989); Rosenbloom (l993)j. Microfibrils appear to act as a scaffold during the formation of elastic fibres ensuring the proper alignment of crosslinking regions [Gosline (1976); Cleary et al. (1989); Sandberg (1976); Rosenbloom (1993)). MAGP has been shown to faalitate the interaction behveen tropoelastin and microfibrils through its N- and C-terminal regions respectively [Brown-Augsburgeret al. (1996)l. Furthemore, the glycoprotein emilin has also been shown to have an important role in elastin deposition [Bressan et al. (1993)l. Microfibrils were originally thought not to play a role in the mechanical behaviour of the elastic fibres [Ross and Bomstein (1969)l; however a recent study of lobster artenai wall revealed that microfibrils have a measurable elastic modulus which is similar to elastin [McConnell et al. (l996)I. Elastin in mature elastic fibres is an insoluble, aosslinked protein. Before deposition ont0 microfibrils however, it is produced by cells as a soluble precursor: tropoelastin. Each tropoelastin molecule is approximately 800 amino acid residues in length and is almost identical in composition to mature elastin-with the exception of a higher quantity of lysine [Urry (1983); Sandberg (1976); Rosenbloom (1993)l. Lysine, as in collagen, is utilized in the crosslinking of elastin and is responsible for the protein's insolubility. The unique form of the crosslinks in elastin, together with its hydrophobic nature, give it the mechanical behaviour of a lightly uosslinked polyrner: i.e. a rubber [Gosline (1976); Urry (1983)j. The crosslinks in eiastin are comprised of 4 lysines (desrnosine and isodesmosine), 3 lysines (merodesrnosine), or 2 lysines residues (lysinoniorleuane) and occur between hohopoelastin moledes [Eyre et al. (1984); Partridge (1989); Reiser et al. (1992)l. Although the exact mechanism of crosslink formation remains undear, the crosslinks begin as 37 lysines within eadi tropoelastin molecule, with all but about five partiapating in crosslinking [Rosenbloorn et ai. (1993); Francis et al. (1973)j . The lysines are fourid in alpha-helical-alanine rich regions such as -Lys- Ala-Ala-Lys- or -Lys-Na-Ala-Na-Lys- occurring approximately 12 times dong the fropoelastin molecule [Urry (1983); Sandberg (1976); Foster et ai. (1976)l. As in collagen, some of the lysines are oxidatively de-aminated to form corresponding semi-aldehydes (allysine) through the action of the enzyme lysyl oxidase [Eyre et al. (1984); Kagan et al. (1995)l. The allysines cm either read with one another (condensation reaction) to form an allysine aldol, or can read with an unconverted lysine to form a Schïff base (dehydrolysinonorleucine)[Eyre et al. (1984); Reiser et al. (1992); Partridge (1989); Viidik (1982)j.

1 -2.3Proteoglycans, Glycosaminoglycans and Glycoproteins Proteoglycans (PGs), glycosaminoglycans (GAGS), and glycoproteins (GPs) are part of the interfibrillar matrix of tissues. Their roles indude faalitating connections between cells and/or ECM components and acting as a water reservoir. The hydration of the ECM is important for cellular function as well as contribu ting to the mechanical properties of tissues. Glycosaminoglycans are linear polymers of repeating disaccharides that contain one hexosamine and either a carboxylate and/or sulfate ester [Wight et al. (1991); Heinegiird and Oldberg (1993); Labat-Robert et al. (1990)l. They include hyaluronic aad, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, and heparin sulfate. The size of a single GAG molecule cm be as little as 10-20 kDa (keratan sulfate) or as high as 10 mülion Daltons (hyaluronic aad) [Wight et al. (1991); Heinegârd and Oldberg (1993)J.Most GAGs are assotiated with proteoglycans; however, hyaluronic aad is synthesized without being covalently linked to a protein core and is found mostly in cartilage and synovial fluid [Wight et al. (1991); Labat-Robert et al. (1990)l. Proteoglycans have been desmbed as having the structure of a 'bottle brush' and consist of GAG side-diains that are attached to a core polypeptide chain (protein) ranging in weight from a few thousand Daltons to 250 kDa [Siiver and Doillon (1989); Wight et al. (1991)l. Thus, PGs contain reactive side-groups within their carbohydrate chains and within the core protein. GAG side-chains are comected to the core protein via a xylose residue linked by an O-glycosidic bond to a senne residue or by an N-glycosylamine bond to an asparagine residue [Wight et al. (1991); Labat-Robert et al. (1990)l. PGs are found on the surface and orthogonal to coilagen fibrils keeping them apart and holding them in specific orientations [Scott (1991), (1988); SiIver and Doillon (1989)l. PGs interad at speafic binding sites on collagen via electrostatic interactions with their gIycosaminoglycan side-chahs and protein-protein interactions involving the protein core [Scott (1991); (l988)I. The number and type of GAGs Vary depending upon the type of PG, but can be generally divided into two classes: large and small [Nietfeld (1993); Wight et al. (WB)].Although PGs are present in cells, on cell surfaces, and in the ECM, only PGs in the ECM will be discussed in this review. The large PGs of the ECM include aggrecan, versican and perlecan. The srnall PGs of the ECM indude biglycan, decorin, and fibromodulin. These PGs contain smaller protein cores and only one or a few GAG chahs. The structural glycoprotein family of the ECM indudes fibronectin, Iaminin, nidogen (entactin), thrombospondin, and tenasan [Yamada (1991); von der Mark and Goodman (1993)l. This group of molecules is heterogeneous in size, structure, and tissue origin but has common features: distinct functionally active dornains speaalized for binding cells and ECM, and the ability to form oligomers or polymers through either disulphide bonds or by non-covalent self- association [Yamada (1991)l. The details of the domains for each glycoprotein and their self-assoaation is beyond the scope of this review and WUnot be discussed. In general, these glycoproteins help to link components of the ECM together as well as to connect them to ceils via integruis and receptors.

1 -2.4Mechanical Properties of Tissue-Derived Materials Sof t tissues can be described mechanically as biological composites consisting of couagen and elastin fibres encased in a highly hydrated proteoglycan-based maMx. As indicated in the previous sections, co~ective tissue proteins and the ground substance form a complex network of interconnecting components that define mechanical behaviour. Properties are not only defined by the quantities of each component, but also by their structures and architectures [Viidik et ai. (1982)j. Therefore, the response of a bIood vesse1 to a ggiven stress field will not be the same as that of skin. In generai however, soft tissues display comrnon mechanical characteristics. Soft-tissues al1 display non-linear viscoelastic behaviour [Fung (1981)1. That is, they display both viscous and elastic behaviours at the same tirne. Typical stress-strain and stress relaxation behaviours for tissue-derived materials are shown in Figure 3. Stress is defined as the load per unit aoss- sectional area and strain as the percent increase in length of a material. The non- linear stress-stain curve can be divided into three regions: toe, heel and linear (Figure 3a). When a uniaxial tensile load is applied to intact tissue, collagen and elastin fibres slowly extend. Collagen in a relaxed state possess a natural crïmp of approximately 1pm to several 100's of pm which is reduced, but not eliminated by the application of a tensile force [Gathercole and Keller (1991); Parry (1988); Broom (1978)l. Elastic fibres aid in the recovery of collagen aimp when an imposed force is removed. In the toe region of the stress-strain cuve, as elastin is stretched, the natural crimp assoaated with collagen begins to be reduced (Figure 3a(i)) [Oxland and Ancireassen (1980); Fratzl et al. (1997); Parry (1988); Broom (1978)j. Under the smail strains associated with the toe region, collagen and elastin act "elastically": i.e. their original architecture can be recovered if the tensile force is removed. In the heel region, as strain increases, collagen aimp is %- ( ii)

STRESS

(b)

STRESS

Figure 3 Typical (a) stress-strain and (b) stress relaxation behaviour for tissue-derived materials. The stress-strain respanse can be divided into three regions: Toe, Heel, and Linear. As a tensile force is applied, (i) collagen crimp is removed and elastic fibres are stretched. As crimp is removed. (ii) collagen fibres are directly acted upon, reorienting in the direction of the applied force. At high strains, collagen fibres begin to slide past one another. The stress-relaxation behaviour of tissue-derived materials is viscoelastic. When the tissue is stretched and held at a fixed length, stress decays with time as collagen and elastin fibres reorient in the direction of the applied force. A perfectly elastic material (---) would show no decay in stress. further reduced and the fibres begin to become aiigned in the direction of stress (Figure 3a(ii)). At this point, the ability of elastin and colIagen to regain their original architecture may be comprornised and viscoelastic or permanent deformations may occur. FinaIIy, in the hear region as strain continues to increase, collagen and elastin fibres continue to align in the direction of the applied force and eventually begin to slide past one another (yielding) unti1 the material breaks (fracture) (Figure 3a(ii)). Thus, the ultimate strength of the tissue is strongly dependent upon the interconnections within collagen (crosslinking) [Parry (1988)].The non-linear stress-strain behaviour of intact tissues is also displayed by purified collagen and elastin. Purified elastin however, displays a higher degree of extensibility, ability to recover elasticdy, and lower strength cornpared to collagen [Frantzl et. al. (1997); Gosiine (1976); Viidik et al. (l982)I. The stress relaxation behaviour of tissue-derived materials is a feature of viscoelastiaty. As shown in Figure 3(b), the stress measured from tissue held at a fixed extension will decay over tinte. For a perfectly elastic material, no such decay in stress would be observed. The stress relaxation behaviour of tissue- derived materials is related to the ability of collagen and elastin fibres within a proteoglycan matnx to reonent in the direction of the applied force [Lee and Bougnher (1991); Purslow et al. (1998); Naimark et al. (1992)]. As tirne progresses, stress deaeases as more collagen and elastin fibres are recruited to the direction of the applied force (Figure 3b). Fibre re-orientation appears to play a predominant role in the stress relaxation behaviour with small deformations; hotvever fibre interactions with the proteoglycan matrix appears to be more important at Iarger deformations [Purslow et al. (1998); Naimark et al. (1992)l. 1.3 Degradation of Tissue Derived Materials Upon implantation, tissue-denved materials are exposed to a barrage of cellular and non-cellular components of the normal host response to injury. During the inflammatory process, cells such as polymorphonudear leukocytes, neutrophils and macrophages release extracellular enzymes by exocytotic or lysitic mechanisms [Anderson (1988); Bainton (1980); Shoshan (1981); Gorham (1991)l. These enzymes are active in neutral or acidic pH and are directed toward the components of the implant in an attempt to degrade and resorb the foreign materid. In addition to inflammation, latent enzymes can also be present within the tissue-derived material itself, or be of bacteriai origin if infection is present [Simionescu et al. (1993),(1994),(1996);Gristina et al. (l994)j. Also during the inflammatory process, cellular and non-cellular components of the lymphatic systern (T-cells ,B-cells and antibodies) seek out foreign antigen and either eliminate their source or identify it for destruction by non-lymphatic cells. The interaction of lymphocytes and leukocytes is synergistic with each stimulating and responding to the signals of the other [Anderson (1988); Bainton (1980); Raghow (1994)j. It is during this initial idammatory process that the Çate of the implant is decided. The implant is ultimately accepted by the host through direct integration or encapsulation or the implant is rejected-resulting in its resorption or, if it persists, the initiation of a chronic inflammatory response [Anderson (1988)l.Thus, it is important for the implant to resist the initial destructive attack and to minimize interactions that may aggravate the natural heding process. The degradation of tissue-derived materids, and in particular of collagen and elastin, occurs via the action of hydrolytic proteinases. These proteinases can be divided into families based upon their enzymatic hydrolytic mechanism: metalloproteinases, cysteine proteinases, aspartic proteinases and serine proteinases [Sellers and Murphy (1981); Kurcharz (1992); Pol@ (1989)J. Regardless of the type of proteinase, al1 enzymes utilize variations of an acyl- group transfer reaction to induce eledron density changes in the substrate and facilitate bond deavage and formation [Polgb (1989); Page (1987)l. The point of deavage within the target protein, however, is different for each enzyme. In some cases, it changes from substrate to substrate. Chernical composition (amino acid side-chain) and conformation of the binding surface of the enzyme are responsible for the specifiaty or recognition of the substrate [PolgAr (1989); Page (1987); Gross (1981)l. Preferential binding of the enzyme and substrat-both at the catalytic site and outside of the catalytic site-enables reacting groups to be brought into dose proximity and the activation energy for the reaction to be lowered [PolgAr (1989); Page (l987)J.Thus, the conformation and complimentary chernicd composition of the substrate are both equally important in enzyme- mediated hydrolysis of proteins.

1 -3.1Collagen Degradation Collagen, in its fibrous form, is resistant to most proteinases. This resistance is derived from the high stability of its triple helical and suprarnolecular structure [Kucharz (1992); Sellers and Murphy (1981); Gorham (1991)l. Collagen degradation, however, has been demonstrated both in vivo and in vitro through the action of a number of specific and non-specific proteinases. Speafic collagenases which cmdegrade native collagen are members of the rnetalloproteinase farnily. These enzymes contain a metal ion (zinc) and a glutamic acid residue at their cataiytic site and require calcium for activity and stability [Polgzir (1989); Sellers and Murphy (1981); Auld (1987)J.Speafic collagenases have neutral pH optima and cleave coilagen at a speafic site in the helical domain of each of the three polypeptide chains (adjacent to glycyl residues), 3/4 of the distance from the arnino terminus [Shicklin and Hibbs (1988); Sellers and Murphy (1981); Gross (1981)l. Collagenase can be obtained from mamrnalian (interstitial and neutrophil matrix metalloproteinases (MMP-1 and -8)) and miaobial (Clostridiz~mkisto1yticuni -which produces six individual collagenases) sources [PolgAr (1989); Auld (1987); Kurchan (1992); Bond and Van Wart (19&1)].Each type of collagenase has its own preference for the various types of collagen and residues associated with glycine at the site of deavage (e.g. Na, Ile, Pro, Hyp, Lys, Arg) [Auld (1987); Woessner (1991); Sellers and Murphy (198 1); Ma trisian (1992); French et ai. (l992)J.Other non-specific collagen- degrading metalloproteinases indude thennolysin from B. thmoproteolytiars, the stromelysins 1 and II (MMP-3 and-10) which deave collagen withui the globular domains, and gelatinases A and B (MMP-2 and -9) [Matrisian (1992); Woessner (1991); Nagase (1994)]. The active site of the cysteine proteinase family indudes cysteine and histidine residues [Polgb (1989); Broddehurst (l987)J.Collagenolytic enzymes in Hus family indude the lysozomal cathepsins 8,L, N, and S [Kurcharz (1992); Sellers and Murphy (1981); Maaewiu and Ethenngton (1988)]. These enzymes exhibit their collagenolytic activity below pH 5.0 and attadc the non-helical ends of the molecule. Macrophages have been shown to generate aadic microenvironments and to release cathepsins in the attadunent zone between the ce1 1 and substrate (Silver et al. (1988); Sellers and Murphy (1981)]. Studies have reported that cathepsins L and N are more effective at deaving insoluble collagen than are cathepsins B and S. The speafic deavage points on collagen for cathepsins S and N have yet to be defined; however, studies using synthetic substrates have shown cathepsins B and L to preferentially deave bonds adjacent to Arg and Phe [Maciewicz and Ethenngton (1988); PolgAr (1989)]. The remaining collagenolytic enzymes are members of the serine proteinase family which have serine and histidine residues at their active site [Polgar (1989); Fink (1987)l. These enzymes indude trypsin, chymotrypsin, and cathepsin G, dlof which attadc collagen in the non-helical domains [Polg* (1989); Fink (1987); Sellers and Murphy (1981); Gustavson (1956a)j. Chymotrypsin and cathepsin G hydrolyze the peptide bonds adjacent to tyrosine, tryptophan, phenylaianine, leucine and methionine, whereas trypsin hydrolyzes bonds adjacent to arginine and lysine [Polgbr (1989); Gustavson (l956a)I. Within the serine proteinase family is an elastase which has collagenolytic activity: polymorphonudear leukoctye (Pm)elastase. This elastase is able to degrade a number of collagens, induding; types 1, Il, III, IV, V, and X [Bieth (1989a); Kucharz (1992); Sellers and Murphy (1981)]. Interestingly, PMNL elastase has been reported to have different actions on different types of collagen. Collagen types 1 and II are deaved in the non-helical globular domains, whereas types III and IV are deaved in theV helical domains, thus mimicking the action of the "true collagenases"-the metalloproteinases [Bieth (1989a); Kudiarz (1992)].

1.3.2Elastin Degradation Like collagen, elastin in its insoluble form is highly resistant to most proteinases. The protein is rich in hydrophobic amino aads and contains unique types of crosslinks which lends it resistance to enzymatic degradation. Elastin, however, is susceptible to enzymatic degradation in vivo and in vitro in ih insoluble form by hydrolytic enzymes from the serine-, cysteine-, and metalloproteinase families. These enzymes corne from both rnammalian and non- rnarnmalian sources with pH optima ranging from approximately 2 to 9 [Wallach and Hornebedc (1989); Bieth (1989b)j. Only enzymes which solubilize elastin near or at neutral pH, however, are referred to as true "elastases". Elastases are not specific for elastin since they can degrade other proteins-as in the case of collagen mentioned previously. True elastases of mammalian origin indude pancreatic elastases, neutrophil elastases, and macrophage elastases. Neutrophil and pancreatic elastases are members of the senne proteinase family with most of the pancreatic elastases targeting similar peptide bonds to those of neutrophil elastases-adjacent to alanine and valine [Bieth (1989b); Banda and Senior (l99O)I. Human pancreatic elastase is the exception, having a preference for peptide bonds adjacent to leuane, phenylalanine, and tyrosine. Elastases of macrophage origin fall under the category of metalloproteinases. Macrophage elastases preferentially deave peptide bonds adjacent to leuane [Lafuma and Hornebedc (1989); Banda and Senior (1990)j. In comparison, the elastase secreted by neutrophils is more active at degrading elastin than is macrophage elastase. This may be explained by the difference in amino aad sequence recognized by the two elastases. Elastin is rich in alanine, valine, and glycine, but relatively low in Ieuane [Branda and Senior (1990)J.Thus, the number of deavage sites in elastin recognized by macrophage elastase would be lower than those recognized by neutrophil elastase. Other elastolytic enzymes not dassified as true "elastases" include cathepsin L and pepsin which operate at aadic pH [Wallach and Homebeck (1989); Branda and Senior (1990)l. One of the most important non-mamrnalian elastases is produced by the bacteria Psn~domonasaenrginosa and is found particularly in infections of heart valves and vascular grafts [GVstina et al. (1994)j. This enzyme has a pH optimum of 7 to 8 and preferentially deaves peptide bonds close to aromatic or bulky groups such as phenylalanine, leucine and tyrosine [Morihara (1989); Bieth (1989b)I. Pseudovzonas aeruginosa elastase has been demonstrated to deave collagen types 1, III and IV [Monhara (1989)j. 1.4 Preimplantation Processing of Tissue Derived Materials As indicated previously, pre-implantation treatment of tissue-derived materials began with treatments aimed at prese~ngthe viability of allograft tissues in the hopes of integration with the host [Callow (1986); Senning (1982)l. Problems availability and maintainance of cellular viability however, led to the use of xenograft materials and crosslinking treatments to reduce immunogenicity and increase degradation resistance. The mechanisrn by which crosslinking adueves reduced antigeniaty and inaeased degradation resistance is not known. It has been hypothesized that crosslinking may (i) block sites recognized by enzymes and antibodies, (ii) stencdy prevent enzyme and antibody binding, or (iii) prevent solubilization of comective tissue proteins [Lee et al. (1996)l. In general, however, crosslinking treatments leave materials non-viable. Today, lvith the advent of tissue engineering, new treatments for tissue-derived materials are being sought which produce materials supporting integration and regeneration.

1-4.1 Crosslinking Techniques Al1 crosslinking treatments involve the introduction of exogenous linkages into the structural proteins of tissue-derived materials-collagen being the main target for modification. Gosslinks are generated betrveen reactive side-groups of amino aads (e.g. lysine or aspartic acid) by reagents which either (i) faalitate the reaction or (ii) become part of the crosslink. The earliest crosslinking reagent utilized for tissue-denved materials was glutaraldehdye [Carpentier (1977)l. Glutaraldehyde is a 5-carbon, dialdehyde molecule which reacts with the &-aminogroups of lysine [Kohrn et al. (1972); Cheung and Nirnni (1982a), (1982b); Cheung et al. (1982)l. Glutaraldehyde can polymerize to span between distant lysine residues and thus more effectively crosslink materials. Although glutaraldehyde is still in use today in the preparation of bioprosthetic heart valves, the treatment is far from ideal. Problems with calafication, depolymerization and release of cytotoxic glutaraldehyde monomers, mechanical property changes in treated tissues, and tissue degeneration persist Efforts to circumvent the problem of calcification through post-crosslinking treahnents, however, have been investigated. Treatments such as metallic salts, sodium dodecyl sulfate, monosodium glutamate, diphosphonates. and ethano1 have show some promise [Hirsch et al. (1993a), (1993b); Liao et al. (1992); Vyavahare et al. (1997), (1998); Walther et al. (l99S)l. In order to avoid the problems assoaated with glutaraldehyde, other bifunctional and polyfunctional crosslinking reagents have been investigated. Like glutaraldehyde, epoxide, diisocyanate and dimethylsuberimidate reagents react rvith the &-aminogroups of lysine. Epoxide crosslinking occurs via reactive epoxide ring moieties which are attadced on their less hindered carbon by the nudeophilic nitrogen of &-aminogroups of lysine. Epoxide reagents are available in a number of different structures with various numbers of reactive epoxide rings [Lee et al. (1994); Tu et al. (1994); Sung et al. (1996)]. The initial resdts with epoxide treated tissue-denved materials has been promising, however the long- term durability of these devices remains to be determined. The crosslinking of tissue-denved materials with hexarnethylene diisocyanate (HMDC) requires a unique approach due to the insolubility of the reagent in aqueous media [Naimark et al. (1995); Chvapil et al (1983)l. HMDC crosslinking has been possible only through the use of organic solvents (iso-propanol) [Naimark et al. (1995)l and detergent solutions [Olde Damink (1993)l. Dimethylsuberimidate crosslinking via its imidoester groups has been studied in purified collagen only [Hey et al. (1990); Charulatha and Rajarm (1997)l. Like HMDC however, the cytotoxicity of dimethylsuberimidate has prevented its dinical use. In order to prevent the potential release of cytotoxic reagents from tissue- denved materials, a new group of crossiinking reagents has been investigated. These reagents are referred to as "zero-length" crosslinking reagents due to their ability to faalitate the formation of a crosslink between amino aad side-chains without being incorporated into the link [Wong (1991)l. These reagents indude carbodiimides and acyl azide, as weii as dye-mediated photo-oxidation methods (Olde Damink et al. (1996); van Wachem et ai. (1994); Lee et al (1996); Petite et al. (1%O),(l994); Ramshaw et al. (1994); Moore et al. (1998), Weadock et ai. (1984)l. Carbodiimide and acyl azide treatments faalitate the aosslinking of carboxyl side-chahs of aspartic and glutarnic aads with the &-aminogroups of lysine. Although early biocompatibility studies with carbodiimides are promising, problems with altered mechanical properties of tissues after carbodiimide h-eatment must be resolved. Reaction conditions such as pH and concentration have been shown to affect carbodümide aosslinkùig, however the effect of these parameters on the mechanical properties (specificdy shear properties) and enzyme degradation behaviour of treated materials has not been studied. Dye- mediated photo-oxidation involves the formation of a free radical on aromatic residues such as histidine, tryptophan, or tyrosine which can then read with other nearby side-chahs. The exact mechanism(s) of crosslinking by this technique are yet unknown [Moore et al. (1998)j.

1.4.1.1 Evnlzmî-ion of Crosslinking An important factor in assessing treatments for tissue-derived materials is the effectiveness of reagents in forming crosslinks. The direct measurement of aosslinking in tissue-denved materials is not currently possible; however a number of teduuques are available that act as a proxies for crosslinking. These techniques indude measurement of thermal denaturation temperature, enzyme degradation assays, chernical degradation by cyanogen brornide (CNBr), and the analysis of unreacted amino aad side-chahs. These techniques do not al1 measure the same attributes of crosslinking and as such, should be used to complernent one another where possible. The denaturation temperature (T,) is a measure of the thermal energy required to break the intrahelical hydrogen bonds which stabilize the coUagen triple helix [Gustavson (1956b); Lee et al. (1995b)j. Because the triple helix is further stabilized by exogenous crosslinks, T, can also be used as an indirect measure of crosslinking with a higher value indicating a greater degree of intrahelical aosslinking [Naimark et al. (1992); Main et al. (1978)l. Measurement of T, cmbe performed by mechanical meam (See Lee et ai. (1995); Weadodc et al. (1996) and Methods section 2.2.1.3) or by differential scanning calorimetry @SC) [Finch and Ledward (1972); Kopp et al. (1989)l. Degradation studies involve incubating known masses of treated materials with either degradative enzymes or the chemical cyanogen bromide (CNBr) which deaves at methionine residues in proteins [Vasudev and Chandy (1997); Cheung and Nimni (1982b)l. After incubation, the amount of material remaining will refiect the amount of aosslinking rvith a higher amount of crosslinking being refiected in a higher residual mass. The type(s) of aosslinking that is identified by ths measurement in unknown. Furthemore, these type of assays only reflect the amount of material solubiiized and not the extent of degradation. In order to assess the effed of degradation on stmctural integrity, methods involving tensile mechanical testing of materials after exposure to enzymes have been utilized [Weadodc et al. (1996)l. For enzyme assays, the choice of enzyme used in degradation assays has been varied and has included hypsin, chymotrypsin, pepsin, cathepsin B, and microbiai collagenase [Vasudev and Chandy (1997); Weadodc et ai. (1996); Weadodc et al. (1984); Lee et al. (1996)l. Enzyme degradation assays have dso been utilized as a proxy for the in vivo degradation performance of treated materials. The more aggressive microbial collagenase has been used more often for this purpose. The use of chemical and amino acid analyses to determine the amount of umeacted side-groups of amino aads targeted by crosslinking reagents is the most direct measure of crosslinking [Cheung and Nimni (1982b); Olde Damink et al. (1996)j. Nthough the type and location of the uosslinks camot be determined, the amount of side-chains in tissue-derived materials involved in crosslinks can be estimated. For carbodiimide reagents which facilitate iso- peptide bond formation, amino aad analysis cannot be used and therefore, a chemical technique (2,4,6, trinitrobenzenesulfonic aad (TNBS) method-See Olde Damink et al. (1996) or Methods section here) is required. 1.4.2 Biochemical and Physical Manipulation

One of the first hue bioprosthetic grafts developed uivolved the biochemical processing of a bovine carotid artery to remove antigenic smooth musde cells followed by a crosslinking technique. The enzyme fich (derived from the fig tree) removed smooth muscle cells and elastin, but nominally left collagen intact [Rosenberg et al. (1970)l. The graft was then crosslinked ivith dialdehyde starch. Unfomuiatelyy the use of ficin and loss of elastin caused structural weakness in the wall of the graft resulting in marked degradation and aneurysm [Guidoin et al. (1989)l. Other investigators have looked at removing cells from tissue-derived materials in order to deuease their antigenicity. These acellular matrïx (ACM) techniques involve the use of a combination of hypotonie, hypo-osmotic, and enzyme extractions to remove all cellular materials while leaving comective tissue fibres intact with differential extraction of proteoglycans [Wilson et al. (1995); Malone et al. (1984); Courtman et ai. (1994)l. Designed as hue scaffolds for tissue regeneration, initial results with ACM-processed allografts have been promising. ACM xenografts however, require further refinement due to thrombosis and degeneration [Courtman et al. (l992)J. Cryopreservation has also been utilized to treat and preserve tissue- denved materials. In ayopresewation, cells of the tissues are preserved in an attempt to retain tissue viabiiity. Large tissue bankç, such as Cryolife laboratories in Georgia, USA, have been established to bank allograft tissues. Cryopreservation is performed within 24 hours after harvesting using antibiotic solutions containing dirnethyl sulfoxide (DMSO) as a a-yoprotectant [Goumier et al. (1993)l. Although cryopreserved allograft tissue is currently used, the availability of donor tissue and immunologie problems still remain as unresolved problems. As alternatives to diemical crosslinking, other biochemical and physicai processing techniques have been investigated to increase the degradation resistance of tissue-denved materials. Physical techniques such as dehydration and ultraviolet treatment have been studied; however, results have shown these methods to inadequately stabilize and strengthen purified collagen materials [W eadock et al. (1996). (l984)].An interesting biochemical technique has been investigated as an alternative to crosslinking. It involved the chernical modification of side-chains of amino aads as a method of altering the degradation resistance of tissue-derived materials. The side-chains of amino atids wi thin pun fied collagen were aitered via methylation, acetylation, and sucanylation [Diarnond et al. (1991)l.The resuits of this preluninary investigation were incondusive due to the la& of material characterization after each treatment and conflicting data from in vitro enzyme degradation and in vivo implantation assays. With Merinvestigation, ths technique may provide a unique method of rnodulating the degradation of tissue-derived materials and may be directly applied to the development of tissue-engineering scaffolds. 1.S Justification of Study and Hypotheses The literature ated in the preceding sections has demonstrated that the current methods for pre-implantation treatment of tissue-derived materials are inadequate. Furthennore, the emerging field of tissue engineering requires the development of new approaches for pre-implantation treahnents if tissue- derived materials are to be used as scaffolds for tissue regeneration. In this study, the effects of speafic chemical modifications of the amino aad side-chains of lysine, arginine, and aspartic and giutamic aads on the in vitro degradation of 'native' collagen and 'purifie& type 1 collagen were investigated. 'Native' is used to desaibe couagen obtained from natural tissues ivithout alteration of its architecture (fibres, fibre bundles) and structure (tertiq quaternary). In conhast, 'p~~rified'collagen has been extraded from natural tissues and has undergone some architectural and structural changes as a result of the purification process. The use of ACM technology in ths study provided a unique opportunity to study the effeds of modifications on the degradative properties of collagen in a native form (without alteration of quaternary or tertiary sû-ucture) and in the absence of cellular components. Although ACM materials are a more complex system, information obtained may aid the understanding of the host response to tissue-derived materids and may have direct implications for implant design. The reagent 1-ethyl-3-(3- dimethylaminopropy1)-carbodiimide(EDC) was used as a basis for cornparison of the effeds of crosslinking with chemical modification of side-chahs. EDC was selected due to its reactivity with the same targeted amùlo aad side-chains (lysine, aspartic and glutamic aads) and pseudo-catalytic iso-peptide crosslinking mechanism. EDC treatment was also 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 assays, thennomechanical, tende mechanical, and shear stiffness testing were utilized to assess physical property effects and multi-enzyme in vitro enzyme analyses were used to determine the degradative property effects of each chemical modification.

For this study, the following hypotheses are presented:

(1) The extent of enzymatic solubilization of collagenous materials will be reduced by specific chemical modification of the functional side-groups of amino aads which are recognized by degadative enzymes.

(2) Alteration of the chemical moiety (hydrophilic or hydrophobic) or size of amino acid side-chains will modulate the reduced solubilization of collagenous materials to different degrees. 2. METHODS AND MATERIALS

An overview of the treatment and testing methods utilized in this study is shown in Figure 4. Bnefly, acellular maMx (ACM) processed carotid arteries were prepared and subjected to crosslinking, carboxyl, Iysine, and arginine capping treatmentç. Purified type 1 collagen was subjected to lysine and arginine modifications only for comparison with equivalently treated native collagen within ACM arteries. To determine the extent of each modification, samples of untreated and treated acellular arteries and pwified type 1 collagen were analyzed with either arnino acid analysis or 2,4,6-trinitrobenzenesulfonicaad (TNBS)lysine analysis. Thermal denaturation temperature and mechanical (tensile and shear) properties were determined for untreated and treated acellular artenes only. In vitro enzyme degradation experiments were performed with untreated and treated sarnples from both ACM arteries and purified type 1 collagen. The solubilized products of degradation were analyzed for total collagen content and the size distribution of solubilized collagen fragments determined by gel chromatography. The details of sample preparation and testing are given in the foilowing sections.

2.1 Preparation of Samples

2.1 .1 Acellular Matrix (ACM) Processing The plu& of adult sheep-containing the trachea, esophagus, and attached carotid arteries-were obtained from slaughter 0. and J. Meat Padcers: Nobleton, Ontario) and transported to the laboratory in room temperature phosphate-buffered saline solution containing 5 mL/ L of penicillin/ streptomycin (10,000 U/mL/10,000 mg/ mL). In the laboratory, the carotid arteries were carefully dissected from the pluck and deaned of adherent fat and loose connective tissue. The arteries were then carehlly mounted ont0 0.06 inch OD tygon tubing which acted as a support during the processing of the vessels. 'Native' Coliagen ular Carotid Arteries

EDC Crosslinklng Lysine Modification - Glycidol, n-Buty/g/ycidy/ether Carboxyl Modification - Arginhe Modification Methylglyoxal Glyche Methyl Ester -

Amino Acid Analysis or TNBS Lysine Analysis 1

Thermal Denaturation Tests for Collagen Stability

Mechanical Testing - Loop Sarnples

In-vitro Enzymatic Degradation (48 hm.) (Acetyl)Trypsin, Collagenase, Cathepsin 'B'

Figure 4. Flowchart indicating the treatments and testing methods utilized for native (ACM processed aiteries) and purified (type 1) collagen substrates. Native ACM collagen was subjected to al1 chernical modifications and testing methods. Purified collagen was modified by epoxides (glycidol, n-butylglycidylether) and rnethylglyoxal and subjected to amino acid analysis and in vitro degradation assays only. Acehhr Matrix (Am)processing of ovine carotid artenes followed the methods of Wilson et al. [Wilson (1990), (1995)j. In this process, cellular components are extracted, leaving the fibrous proteins collagen and elastin intact and without alteration of native structure [Courtman et al. (1994)l. Bnefly, carotid artenes were hersed in hypotonie 10 mM Tris buffer (pH 8.0) containing a protease inhibitor (5% phenylmethyIsulfonyl fluoride (PMSF) in ethanol, 0.35 mL/L) and 5 mL/ L of penicillin/ streptomycin (10,000 U/mL/ 10,000 mg/mL), for 36 hours at 25 OC with constant stirring. The second stage utilized a 1% solution of Triton X-100 (odyl phenoxy polyethoxyethanol) in Tris-buffered sait solution (1.5 M KCI, pH 8.0) with protease inhibition and antibiotics, for 48 hours at 25 OC with constant stimng. Arteries were thoroughly rinsed in Hanks' physiological solution before digestion with DNAse and RNAse at 37OC for 5 hours under gentle agitation. This was followed by a 48 hour extraction at 25OC with 1%Sodium Dodecyl Sulphate (SDS) in Tris-buffer contaking antibiotics. Finally, al1 samples were washed for 24 hours in phosphate buffered saline (PBS) containing antibiotics and then bottied under sterile conditions in sterile PBS with 20mL/L of peniciIlin/ streptomycin (10,000 U/mL / 10,000 mg/ ml). The acellularized arteries were finally stored at CC until required.

2.1.2 Purified Type I Collagen Purified and lyophilized insoluble type I collagen from bovine Achilles tendon was obtained from Sigma Chernical Co. (product number C-9879) and utilized as received. The collagen was purified following the protocol of Einbinder and Schubert (1951). Fresh tendons were deaned of non-collagenous tissue, cut into smdpieces and extracted for 6 days at O OC with several changes of 3% (w/ w) sodium phosphate (Na,HPO,) to remove soluble proteins. GIycosaminogIycans and proteoglycans were removed by immersion in a 25% (w/ W)potassium chloride solution for 6 days at O OC. Findy, the residue was thoroughly washed with water, dehydrated with absolute alcohol, and air-dried. The insoluble collagen was comprised of short fibre bundles of various lengths and widths.

2.1.3 Crosslinking with EDCAJHS Zero-length crosslinking between carboxyl groups of aspartic and glutamic aads and &-aminogroups of lysine and hydroxylysine was conducted using the reagent 1-eth yl-3-(3-dimeth ylaminoprop y l ) carbodiimide (EDC) [Lundblad (1991); Carraway and Koshland (1972); Olde Damink (1993) and Lee et al. (1996)l. The reaction scherne is shown in Figure 5. EDC activates the carboxyl functionalities of glutamic or aspartic acid residues within collagen to yield O-acylisourea groups. N-hydroxysuccinirnide (NHS) replaces the EDC converting the O-acylisourea groups to more stable succinirnidyl esters which prevents side-reactions from occuhg which do not form crossiinkages. A crosslink is formed through the nudeophilic attack of a nearby amine group of lysine or hydroxylysine at the carbonyl carbon. EDC was seleded because of its ability to react with highly reactive and abundant diemical moieties in collagen (amino and carboxyl groups), its well characterized chemistry, and prornising preliminary in vivo performance [Olde Damink (1993); Wilson (1995)j. Sarnples were treated using the optimized conditions of Lee et al. (1996). Briefly, a 1.15% (w/ w) unbuffered solution of EDC in distilled water kvas prepared and a 2:l molar ratio (EDC:NHS) of N-Hydroxysuccinimide was added. The reaction was conduded either with or without maintenance of pH at 5.5 using 1M Haor NaOH at E°C. After treatment, samples were washed 3 times (10 minutes each) in 20 mL of cold 4% NaHCO, to quench the reaction. Time-series experurients were conducted to determine the rate of reaction and to determine the time period required for maximal aosslinking as assessed by 2,4,6-trinitrobenzenesulfonicaad (TNBS) lysine analysis and thermal denaturation temperature analyses. 7' NH I C-OH + C C-O-c II N I

O II + R2-NH-C-NH-Rl (A Urea) O

Figure 5. The1 -ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) crosslinking reaction of coliagen utilizing N-hydroxysuccinimide (NHS) as a blocking agent. 2.1 -4Carboxyl Capping: EDC/NHS and Glycine Methyl Ester The carboxyl groups of aspartic and glutamic aads were capped with the free arnino aad ester of glycine using EDC as a coupling reagent [Lundblad (1991); Carraway and Koshland (1972)l. The reaction scheme is shown in Figure 6. The reaction is identical to EDC/ NHS crosslinking desaibed above, however, the nudeophilic amino group is the a-amino group of glycine methyl esters supplied in excess during the reaction. This method was selected because of the previous characterizaiion of EDC reactiviv ivith collagen by Lee et al. (1996) and the flexibility in the types of modifications possible by utilizing various methyl esters of amino aads. Samples were treated based upon the methods of Hoare and Koshland (1967), (1966) and Lee et al. (1996). Samples were immersed in a solution containing 1.15% EDC in distilled water with a 1:3 molar ratio of EDC to glycine methyl ester and the pH was maintained at 4.75 with 1 M HCI and NaOH. Based upon previous studies, the use of a lower pH, la& of NHS, and a molar excess of the methyl esters would favour the capping reaction over crosslinking [Lee et al. (1996); Olde Damink (1993); Carraway and Koshland (1972)]. After treatment, samples were washed 3 times (10 minutes each) in 20 mL of cold 4% NaHCO, to quench the reaction. The samples were finally dialyzed against 3 changes of 0.001 M HCI at 4 OC for a total period of 24 hours to remove unreacted methyl ester. The extent of modification of aspartic and glutamic acids with EDC/glycine methyl ester was determined by amino aQd analysis from the increase in glycine denved from capping of carboxyl side-chah hnctionalities with glycine methyl ester.

2.1-5 Lysine Capping: Monofunctional Epoxide Treatments

The &-aminogroups of lysine and hydroxylysine residues were modified using two rnonofunctional epoxides: glyadol (oxiranemethanol)and n- butylglycidylether (1,2 -epoxy-3-methoxy-butane) as shown in Figure 7. The two reagents were selected because of their contrasting size and chernistry. Modification of lysines and hydroxylysines occurs through the nudeophilic

OH I INH -CH2-CH -CH2 -OH b(Srnall, relatively hydrophilic) n - Butyl Glycic

Figure 7. Epoxide modification of amino groups of lysine residues in collagen. Two epoxides of different size and chemistry were utilized: glycidol and n-butylglycidylether. attack of the less hindered electrophilic epoxide carbon by the nitrogen of an E- amino group. Methods were based upon the work of Chadira et al. (1996) and Gratzer (1990) to maximize the arnount of residues modified. The epoxide reaction with arnino groups of collagen has been well charaderized and is very specific [Tu et al (1994); Wong (1991)l. Polymerization of monofundional epoxides after reaction with secondary amines and reactions involving other groups (-OH, -COOH) has been shown to be negligible under the conditions used in this study [Shechter et al. (1956)j. Briefly, samples of ACM carotid arteries or purified type 1 coliagen were immersed in 0.6% (w/ w) solutions of each epoxide buffered with 0.1M sodium phosphate (Na,PO,), pH 11.0, at E°C for 1 week. After treatment, samples were washed three times for 10 minutes each, with 20 ml of cold (T- 4°C) 0.1 M sodium phosphate. The amount of lysine and hydroxylsine modified and the specificity of the reagents to these residues were determined by amino aad analysis.

2.1.6 Arginine Capping: Methylglyoxal Treatment Methylglyoxal was utilized to modify the guanido side-diain of arginine residues based on the method of Cheung and Fonda (1979). The reaction scheme is shown in Figure 8. The reaction initially involves the nuudeophilic attadc of an amino group of arginine on the more hindered carbonyl carbon of one methylglyoxal molecule. A second methylglyoxal molecule is subsequently attacked by arginine and after an intemal remangement, the final stable form of the modification is obtained. The protocoi for arginine modification consisted of immersion of samples in 0.24 M methylglyoxal in a 83 mM bicarbonate buffer (pH 11) for 1 hour. After treahnent, samples were rinsed three times for 10 minutes each, in 20 ml of cold (T- 4°C) 0.1 M sodium phosphate. The amount of arginine modified and the specificity of the methylglyoxal to arginine was determined by amino aad analysis. C Arginine Methylglyoxal

Figure 8. Reaction behnreen the side-chain of the amino acid arginine and methylglyoxal. Two methylglyoxal groups react with one arginine residue. 2.2 Characterization of Materials

2.2.1 Biochemical Properties

2.2.1.1 Amino Acid Annlysis Amino aad analyses were conducted at the Hospital for Sick Children (HSC)/ Pharmaaa Bioteduiology Senice Centre and Amino Aad Analysis Faality. The protocol utilized was as foliows:

The amino acid analyses were perforrned on a Waters PICO-TAG S system. Each protein sample was hydrolyzed by vapour phase/liquid phase reaction, using 6h.I HCl with 1% phenol at 100° C for 24 hours. The drying, evacuation of air, flushing with nitrogen and hydrolysis were done at a Waters Work Station. After hydrolysis, the excess HCl was removed from the reaction via1 under vacuum and the sample was redried using a solution consisting of methanol:rvater:triethylamine (2:2:1). The sample was then derivatized for 20 minutes at room temperature using methano1:water:triethylamine:phenylisothioanate (PITC) (7:l:l:l). The derivitizing solution was removed under vacuum and the sample was redried to remove any traces of PiTC which may interfere with the quantification of some of the amino acid residues. The derivatized sample was dissolved in a given arnount of sampIe diluent (pH 7.40) and placed on the Waters reversed phase

High Performance Liquid Chromatograph (HPLC) system at 35 OCcolumn temperature. The amino auds are detected at 254 nm at 0.01 AUFS range. The Waters PICO-TAG system employed consisted of a data processor and controller, Digital VAX station 2000 with Waters 845 software, System Interface Module, bvo Mode1 510 pumps, Lamba-Max 481 UV Detector, Sample Processor WISP 7106, Column Heater, Temperature Control Module and PICO-TAG column (3.9 mm X 15 cm). (Courtesy of HS C/Plzarmacia) The raw counts obtained for each sarnple were converted into residues per 1000 arnino acids. The amount of each residue before and after chemicai treatments were compared for ACM processed carotid arteries and purified type I collagen. 2.2.1.2 Lysine Determination by TNBS Assay Due to the aad labile nature of EDC-induced aosslinks (iso-peptide bonds), the extent of EDC crosslinking could not be determined from amino aad anal y sis and was therefore determined using the 2,4,6-trini trobenzenesulfonic aad (TNBS) anaiysis for unreacted &-aminogroups of lysine [Habeeb (1966); Kakade and Liener (1969)l. The extent of crosslinking was assessed by comparing the amounts of lysine in treated and untreated ACM arteries. Samples of lyophilized, treated and untreated ACM arteries (8.2 I 0.3 mg) were immersed in a solution consisting of 1 mL each of 0.1% TM35 and a 4%

(W / V) sodium bicarbonate buffer (pH 9). The samples were dlowed to react for 2 hours at 40 OC to produce a TNBTNBSlyshe product which absorbs at 345 m. The samples were then hydrolyzed with 3 mL of 12 M XC1 at llO°C for 12-15 hours. Excess TNBS and trinitrophenyl N-terminal amino aads were removed from cooled samples by washmg twice with 10 mL of diethyl ether. Sarnples were then diluted to 10 mL with distilled water and the absorbance read against a blank treated in a similar manner, but containing no protein. The normalized amount of lysine (mollg) was determined from the molar absorptivity of E- tnnitrophenyl-lysine (1.46 x IO4 l-mol-'an-') and the dry weight of each sample.

2.2.1.3 Thermal Denaturation Testing Denaturation temperature tests were performed on radial strips of ACM ovine carotid arteries approximately 0.5 cm x 13 mm using a 6-sample denaturation temperature tester (Centre for Biomaterials, University of Toronto and EDC samples at Dalhousie University) following the method of Lee et. al. [Lee et. al. (1995)l. The denaturation temperature (T,) is a measure of the thermal energy required to break the intrahelical hydrogen bonds which stabilize the collagen triple helix [Gustavson (1956a)l. Because the triple helix is further stabilized by exogenous crosslinks, "id can also be used as an indirect measure of crosslinking with a higher value indicating a greater degree of intrahelical Bnefly, each sarnple was held between hvo spring loaded damps and hurig verticdy between a rigid and movable fixture. A knurled adjustment nut, located on the movable fixture, was tightened until an average tensile load of - 75 g was recorded by a strain-gauged cantiiever Ioad cell. The sample was then rnaintained at that length (isometric constraint). A distilled water bath surrounding the samples was slowly heated (at - 2 OC/ min) and the temperature monitored by a centrdy Iocated thermister probe. At 1OC intervals, the load remaining on each sample was automaticdy recorded by an Apple Macintosh IIfx cornputer running custom-developed software (Chris Pereira). The test was completed when the temperature of the bath reached 100 O C. A plot of load remaining on the sample versus temperature was then prepared (Figure 9). The point where the load remaining on the sample suddenly increased (due to the disruption of the collagen triple helix) was identified as the dennhuntion terrtpernture T,. The T, value for untreated ACM processed carotid artery in this study was utilized as the baseline for comparisons: i-e. 62.0 * 1.2 OC.

2.2.2 Physicat Properties

2.2.2.2 Tensile Mechanical Testing Mechanical tests were performed on loop sarnples of ACM-processed carotid artenes using an Instron 4301 electro-mechanical unit (Faculv of Dentisfry, University of Toronto). The average width of each loop sample (4.8 r 0.1 mm) was calculated from three measurements made at different locations around the circumference of each loop using a der. Loops of artery were mounted between a pair of brass grips secured with two steel pins (Figure 10). The sarnples were then immersed in a tank containing Hanks' solution maintained at 37 I 1O C during testing. The testing method used was a variant on the technique desaibed by Lee and Boughner (1981) and Gratzer and Lee (1997). Each sample was brought to 75 g load and photographed to obtain a loaded length (See next section). Prior to testing, the loops of artery were preconditioned from 1to 75 g for 15 cycles as

1 1- Immersion Heater Steel

Figure 10. Schematic diagram of the mechanical testing apparatus used for testing loops of ACM arteries. Exploded view indicates the definition of gauge length (Lo). determined by hysteresis measurements on ACM arteries. The maximum load of 75 grams used was equivalent to the maximum normal human systolic arterial blood pressure (120 mm Hg). Stress-strain experiments were performed using rarnp extension at 10 mm/min from 1 to 75 g for one cycle. The tissue was then loaded to 75 g and held at a fixed extension for 1000 seconds while the decay in stress with respect to time was recorded (stress relaxation). Finaiiy, the samples were returned to O g and extended to fracture at 10 mm/min.

2.2 -2.2 T'ensile Mechanical Data Analyses Al1 mechanical test data were analyzed on an Apple De cornputer utilizing software developed in-house (Chris Pereira). Photographs of samples were enlarged so as to fill a 5" x 8" piece of photographie paper. The photographs of loop samples under 75 g load were digitized using a Kurta Series 2 digitizer with a resolution of 1,000 points/ inch. The magnification of the photographs of samples under load were determined using a 15 mm scale attached to the lower @P. The caldation of gauge-length was based upon methods we have published previously [Gratzer and Lee (1997)l. The digitized grip-to-grip distance of the sample at 75g load (defined by the inside edge of the sample in the upper grip to the inside edge of the sample in the lower grip, Figure 10) was determined first. Using the load-deformation curve from the instron chart recorder, the distance traveled by the crosshead was determined from the point just pnor to a load being recorded above zero (defined as O grams) to a 75 g load. To account simply for tensile loading, the gauge length (mean = 3.3 I 0.2 mm) was calculated as the digitized grip-to-grip distance of each sample at 75 g minus the distance traveled by the cross-head from 0-75 g. A mean thickness was obtained by sampling six points at equal intervals dong the length of each loop sample. The cross-sectional area was calculated as twice the product of the mean digitized width (2.9 k 0.2 mm) and thidcness.

Strain (E) in each sample was calculated as the percent change in length of the sample: where AL is the extension and Lo is the gauge Iength as defined above. Stress a was calculated as: a=F/A where F is the force applied (in Newtons) and A is the 75g-loaded cross-sectionai area. Tissue modulus was calculated as the slope of the linear region of the stress-sixain cuve just pnor to fracture. Stress relaxation data were calculated as the ratio of the stress at tirne t, o(t), to the initial stress at time zero (ao):

Stress Remaining (%) = o(t) / 00 x 100 % Time zero was taken at the point of maximum stress which was reached after a loading time of approximately 10-32 seconds (dependent upon the sample tested). A total of 17 data points were calculated from the ratio of a(t) to 00 for each sample and plotted on a semi-log plot from 1 to 1000 seconds on a loglo scale. Data were considered to be reliable after 410 times the loading tirne had elapsed [Turner (1983)l. Shess-strain curves for each sample were aeated using load-deformation data stored on disk and converted to stress and strain as described above. Mean and standard error calculations for stress relaxation and stress-strain data were conducted using software developed in-house on an Apple IIe cornputer (Midiael Lee). Ultirnate tensile strength (UTS) and strain at fracture (SM)were defined as the highest stress and strain achieved before faiIure respectively.

2.2.2.3 Residzrnl Stress Measurements Residual stress experiments were used to monitor the unloaded sûuctural changes in carotid arteries due EDC heatment with or without pH control (Matt Kujath, Dalhousie University). The opening angle of paired (untreated and treated) samples were measured using the method described by Liu and Fung (1989); (1992). Paired sarnples were obtained by using two adjacent rings of ACM artery per test. One sample remained untreated and the other sample was heated in a carbodiimide solution. Samples (with or without treatment) were placed cross-sectional face up in a large petri dish fiiled with enough distilled water to allow the sample to Boat freely. Each set of paired circular segments were cut radially in the same location (position marked with a dye) and photographed after 15 minutes. This delay permitted viscoelastic creep of the opening hoop. Landmarks on each sample were digitized using a Kurta Series 2 digitizer (1000 points/ inch resolution) and an opening angle calculated using software developed in-house (Chris Pereira). The angle between two radii comecting the midpoint of the inner wail to the tips of the inner wall of the sector was defined as the opening angle (Figure 11).

2.2.2.4 Bending (Shear) StzfFiess Measzirements Diametral bending tests were carried out on 5 mm wide hoop samples of ACM artenes before and after EDC crosslinking using the methods and apparatus of Lee et al. (1998) (Matt Kujath. Dalhousie University). The very low load apparatus was partially submerged in Hanks solution at 37 OC and buoyancy and surface tension effects were compensated. Bending stiffness was calculated using proving ring analysis based on Castigliano's [Higdon (1978)] theorem as: P?(n2-8) Bending Stiffness (El) =

ivhere E is the "elastic" modulus taken tangent to the initial loading curve. I is the moment of inertia. P is the applied load, CD is the extemal hoop diameter and r is the mid-wall radius.

In order to assess the possible role of swelling of epoxy and methylglyoxal-treated materials on degradation. samples of untreated, GMG and BMG-h-eated purified type 1 collagen were immersed in 0.1 M acetic aad for 24 hours as per Hu et al. (1996). The length and width of samples of fibre bundles (n=6 per treatment group) of untreated and treated type 1 coIiagen, ktin a dry state and then after exposure to acetic aad, were measured using a miaoscope with CCD camera and associated image analysis software (LEC0 3000). Changes due to swelling were then obtained by comparing the dimensions of samples before and after immersion in acetic acid.

2.3 Enzyme Degradation Studies

2.3.1 Enzyme Degradation Assays The effect of crosslinking and chernical capping on the in vitro degradation of ACM artenes and purified type 1 collagen was investigated using trypsin, acetylated trypsin, collagenase and cathepsin B. The enzymes were selected to represent enzymes which speafically target (i) both modified residues- trypsin, acetyltrypsin (targets: lysine and arginine), (ii) one of the modified residues- cathepsin B (targets: arginine and phenylalanine) and (iii) none of the modified residues-collagenase (targets: isoleucine and hydrophobic amino aads) [Add (1987); Kucharz (1992); Maaewicz and Etherington (1988); Walsh (1970); Gustavson (1956)j. Acetyltrypsin was utilized as weil as trypsin (lysine and arginine-speafic) due to the susceptibility of trypsin to undergo partial degradation (pseudotrypsin form), altering the specificity of the enzyme to include those substrates associated with chymotrypsin (large hydrophobic residues) [Rice et al. (1977); Keil-Dlouha et al (l!Vl)]. The autolytic degradation of trypsin was confirmed in ths study (refer to Results section). Pnor to enzymatic degradation, al1 samples of treated and untreated ACM arteries or purified type 1 collagen were lyophilized and accurately weighed (duplicate measurements). At the end of the degradation experiments, each sample was centrifuged at 30,000 rpm for 20 minutes at 10 OC, and the supernatants were transferred to glass hydrolysis tubes. One milliliter of distilled water was then added to the pellets and the samples were centrifuged again. The supernatants and pellets (ACM arteries only) were then transferred to glass hydrolysis tubes. Supernatants and pellets (ACM arteries only) were finally analyzed for collagen content using hydroxyproline analysis. Initial experiments for dl degradation assays were conducted on untreated ACM artenes and purified type 1 collagen (based on published methods) to determine the optimal conditions necessary to obtain 3060% collagen degradation as determined by hydroxyproline analysis. Furthemore, due to the inability of trypsin to significantly digest undenatured tissue, samples of untreated and EDC treated ACM arteries were first denatured by heating in 0.1 M Tris-buffer (pH 7.5) containing 20 mM CaCI, as per Lee et al. (1996) before exposure to the enzymes acetyltrypsin and trypsin.

2.3.1.1 Tnypsin and Acefyltrypsin Degradation experiments using trypsin and acetyltrypsin were based upon the methods of Diarnond et al. (1991) and Lee et al. (1996). Samples were incubated with bovine trypsin (Sigma Type 1) or acetyltrypsin (Sigma Type V) at a ratio of 20:l w/ i.v (tissue to enzyme) in a 0.1 M Tris-buffer (pH 7.5) containing 20 mM CaCl, for 48 hours at 37 OC.

2.3.1.2 Microbial Collagenase Degradation experiments using collagenase were based on the methods of Naimark et al. (1995) and Lee et al (1996). Samples were incubated with microbial collagenase (C. Histolyficum, Sigma Type 1) at a ratio of 100:l w/ w (tissue to enzyme) in 0.05 M Tris-buffer (pH 7.4) containing 10 mM CaCl, for 48 hours at 37 OC.

Degradation experiments using cathepsin B were based upon the methods of Burleigh et al. (1974) and Etherington (1972). Samples were incubated with cathepsin B from bovine spleen at 0.15 units enzyme per mg of tissue in a 0.1 M sodium formate buffer (pH 4.0) containing 1mM cysteine and 1 mM EDTA for 48 hours at 37 OC.

2.3.2Gel Chromatography Analysis of Enzyme Degradation Products Solubilized enzyme degradation produds from unrnodified and epoxy and rnethylglyoxal-modified ACM and purified type 1 collagen were separated by gel chromatography based upon the methods of Rao and Adams (1975); Davidson (1978); Burleigh et al. (1974). The equipment used induded: a Water's High Performance Liquid Chromatography (WLC) pump a manual HPLC injedor with a sample injection volume of 200 pL

a Bio-Rad 1 an x 120 an Econo-Column with flow adapter Pharmacia Biotech Sephracryl S-200 HR padcing (fractionation range for globular proteins = 5-250 kDa) a Pharmacia Biotech single path UV-1 absorbance detector with 280 nm fil ter a Pharmacia Biotech FRAC-100 automated fraction collecter a Pharmacia Biotech single-channel REC-481 chart recorder Freeze-dried supernatants of enzyme digests were first re-hydrated in 250 PL of distilled water and centrifuged at 10,000 r.p.m. for 15 minutes. Two hundred microlitres of each sample was then injected into the column and eluted at a cons tant 0.3 ml/min. with 0.05 M Tris-buffer containing 0.15 M NaCl, pH 7.5. Fractions were collected from the bottom of the column at 10 minute intervals (3 mL volume per fraction) and were analyzed by (i) a 280 nm in-line W absorbance detector and (ü) hydroxyproline analysis of elutant fractions for collagen content. The range of fractions selected for hydroxyproline analyses were deterrnined from the 280 nm elution profiles of enzyme digest supematants of untreated ACM or purified type 1 collagen. The void volume (- 45 mL) and total volume (-110 mL) of the colurnn were determined by the standards blue dextran (2,000,000 Da) and vitamin B-12 (1,350 Da) respectively [Stellwagen (1990)j. If the 280 nm elution profile began slightly ahead of the void-volume, or ended slightly after the total volume peaks, the fraction range was adjusted accordingly for each enzyme series. For analysis, fractions were pooled into groups of 3 tubes (equivalent to a 9 mL total volume of elutant and a period of 30 minutes) and were freeze dried and then analyzed for collagen content. The collagen content of each fraction range was calculated as a percentage normalized to the collagen contents of all the fraction ranges analyzed. Three samples per treatment group, per enzyme were analyzed (n=3).

2.3.3 Hydroxy Proline Analysis for Collagen Content A spectrophotometric hydroxyproline analysis foliowuig the method of Woessner (1976) was utilized to determine the collagen contents of supernatants and pellets (ACM arteries only) from al1 degradation experirnents using ACM carotid artenes or type I collagen. Freeze dried supernatants or pellets were first hydrolyzed with 2 mL of 6 M Hafor 24 hours at llO°C. Aftenvard, the hydrolyzates were allowed to cool and 2 drops of methyl red indicator were added. The solution was then neutralized with approximately 4.8 mL of 2.5 M NaOH and the find volume adjusted to 15 mL using distilled water. Color development was conduded on 150 PL aliquots adjusted to 2 mL total volume with distilled water. A senes of standard solutions containing 0-5 pg hydroxy-L-proline in distilled water (2 mL total volume) were also prepared. One millilitre of a 0.05 M Chloramine-T solution was first added to each aliquot, which was then vortexed and ailowed to stand at room temperature for 20 minutes. The Chlorarnine-T was then destroyed by addition of 1 mL of 3.15 M perchloric aad. After 5 minutes at room temperature, 1mL of Ehrlich's Reagent (p-dirnethylarninobenzaldehyde)in methyl cellosolve (0.2 g/ mL) was added and the samples were heated at 60 OC for 20 minutes in a water bath. Finally, the sarnples were cooled in a bath containing cold tap water for 5 minutes and the absorbance read at 561 nm against a biank containing no hydroxyproline. The amount of hydroxyproline in each sample was determined from a linear calibration cunre prepared from the standards containing 0-5 pg hydroxyproline. The amount of collagen in each sample was then calculated based upon 14% hydroxyproline content in type 1 collagen (Ethenngton (1972)l. 2.4 Statistical Analyses Analysis of variance (ANOVA) with Fisher's Ieast significant ciifference tests for multiple cornparisons were performed on ail data at the 95% confidence level(pc0.05) or 90% confidence level (p<0.10) where noted. The analyses were conducted using a commerdaily available statistical program (~tatviewa4.01 Abacus Software) running on an a Macintosh cornputer. Ail data are presented as the mean k standard error of the rnean (SEM). 3. RESULTS

3.1 Verification of TNBS Assay for Lysine Lysine contents of untreated and n-butylglyadylether treated ACM arteries were compared using the 2,4,6-trinitrobenzenesulfonic aad (TNBS) method and standard amino aad analysis. The monofunctional epoxide modification was seleded due to the aad stable nature of its lysine derived product. Samples for TNBS and arnino aQd analysis were taken from two adjacent ring-segments of an artery. Six arteries were used for each treatment (total = 12 samples). The results of the comparison are shown in Table 1.

Differences behveen measured lysine content (mol/ g) for the TNBS and arnino acid analysis ranged from 21 + 4.1% (untreated segment) to a high of 65 c 7.7% (treated segment) in terms of absolute numbers (see Table 1).Relatively speaking, however, the difference in the calculated percentage of lysines modified measured using the TNBS and amino aad analysis data was only - 8%. In this study, the extent of EDC crosslinking is reported as a relative value (percent of lysine modified). Furthmore, no other type of analysis (e.g. amino aad anaiysis) for the direct determination of EDC modification of substrates is available due to the acid-labile nature of EDC crosslinkages. The TNBS method therefore, is a valid pragmatic estimate of the relative changes in lysine content of ACM arteries due to EDC treatment as judged by comparison with standard amino adanalysis. I Sample I TNBS Method 1 Amino Acid Analysis . -- --

Untreated 1.1 xlO4 k 3.1 xlO* mou g 1.4 x1 O4 f 9.5~1O6 moVg Treated 7.9 xiO6 k 1.9 xlO* moVg 2.3 x10" f 1.4 xl Od moVg % Lys. Modified 91.8 + 1.9 % 83.6 + 1.2 %

Table 1. Cornparison of lysine contents from untreated and epoxy modified ACM arteries as assessed by 2,4,6-trinitrobenzenesulfonic acid and amino acid analyses. 3.2 EDC Crosslinking

3.2.1 Thermal Denaturation and Biochemical Analyses Without controi, the pH of the carbodümide solution dropped over 3 hours, from a high of 5.78 i 0.04 to a low of 5.38 I0.04 (Figure 12). The maximal decrease in pH however, occurred prior to the achievement of maximai crosslinking. Thermal denaturation measurements of carbodiimide-crosslinked ACM, with and without pH control, reveaied significant differences in the time course of changes in collagen thermal stability. ACM crosslinked with pH control (EDC- pH) showed significantly greater denaturation temperatures at every time point assessed (5 minutes to 48 hours) than was seen in ACM treated without pH control (EDC-No pH, Figure 13). In each case, the denaturation temperatures reached a plateau after 3 hours of treabnent. EDC-pH treatment increased thermal stability by a maximum of 24.3 + 0.6 OC compared to 21.7 i 0.7 OC for EDC-No pH treated ACM. The rate of crosslinking was also significantly higher for EDC-pH treatment with a 29% increase in thermal stability adiieved after only 15 minutes compared to the 1hour required for EDC-No pH treatment to obtain the same increase in thermal stability. By contrast, for the same increase in T,, EDC-No pH treatment modified a greater number of lysine residues (one target of carbodiirnide-mediated crossIinking) than did EDC-pH treatment (Figure 14). in agreement with Olde Damink et al., a linear correlation was found between the amount of lysine residues modified and the increase in thermal denaturation temperature temperature-regardless of pH control [Olde Damink et al. (1996)l. No significant changes in the nurnber of lysine residues modified were found after the maximum increase in thermal denaturation temperature had been achieved (following 3 hours of treatment) for both groups. I Mean ISEM

I O 20 40 60 80 100 120 140 160 180 200 Time (minutes)

Figure 12. Plot of pH versus time for an EDC crosslinking solution without pH control. The pH rapidly decreases and then reaches a plateau after 2 hours. Mean SEM EDC - pH l

50 1O0 150 200 250 300 Time of Treatment (min.)

Figure 13. Plot of increase in denaturation temperature versus time for EDC treatment of ACM arteries with (EDCpH) or without (EDC-No pH) pH control. The EDC crosslinking reaction reaches a plateau regardless of pH control. EDC- pH treatment increases the denaturation temperature of ACM arteries to a greater degree at al1 time points when compared to EDC-No pH treatment. Percent lncrease in Denaturation Temperature 3.2.2Mechanical Testing Differences in the shear properties of EDC-pH and EDC-No pH treated materials rvere revealed upon analysis of opening angle and hoop bending data. Bo th carbodiimide treatments affected the opening angle in a similar manner (i .e. by decreasing the ability of ACM arteries to relieve hoop stresses upon cutting). Al1 the EDC-No pH treated hoops failed to open at al1 upon cuttùig and only one sample in the EDC-pH group opened (Table 2). Differences behveen treatment groups ivas demonstrated by bending (shear stiffness) tests, which assessed the ability of Iayers of comective tissue fibres to slide past one another in the presence of radial compression and resulting circumferential shear stresses. AIthough both carbodiimide treatments increased bending stifhess, EDC-No pH treated ACM was 21x stiffer than untreated ACM, while EDC-pH treated ACM kvas 14x times stiffer (Table 2).

3.2.3Enzyme Degradation Assays Carbodiimide treatment significantly increased the resistance of collagen in ACM arteries to solubilization by al1 proteases used (Table 3). There were no significant differences, however, between EDC-pH and EDC-No pH treatments for any of the enzymes used. Collagen solubilization by trypsin was reduced from 94.3 k 0.4 % to 30.2 a 6.9 % and 34.0 & 8.4 % for EDC-pH and EDC-No pH treatments respectively. Similar values were obtained for coilagen solubilization by acetyltrypsin with 93.8 + 0.8 % soIubiIization for untreated ACM and 27.4 I 2.6 % and 28.2 t 2.0 % for EDC-pH and EDC-No pH treatments respectively. Collagen solubilization by collagenase was reduced most by carbodiimide treatment from 97.0 k 0.4 O/a for untreated ACM to 5.7 + 0.7 % and 6.1 + 0.9% for EDC-pH and EDC-No pH treatments respectively. Interestingly, carbodiimide-treated ACM was totally resistant to cathepsin B under the conditions used in this shidy (Table 3.) * - Samples significantîy different trom untreated with pz 0,0004 # - Al16 samples did not open t - 5 of 6 samples did not open V - Signiticantly different from each other with pr 0.02

Table 2. Properties of ACM aiteries before and alter EDC crosslinking, with (EDC-pH) and without (EDC-No pH) pH control. EDC crosslinking, significantly altered the properties of ACM arteries. EDC-No pH treatrnent altered the shear properties of ACM arteries (as shown by bending stiffness) to a greater degree than did EDCpH treatrnent, Table 3. Percent collagen solubilized by enzymes from ACM arteries before and after EDC crossinking with (EDC-pH) and without (EDC-No pH) pH control. EDC crosslinking, regardless of pH control, significantly increased the lesistance of collagen in ACM aiteries to solubilization by enzymes in-vitro. 3.3 Carboxyl Capping: EDC/NHS and Glycine Methyl Ester

3.3.1 Thermal Denaturation and Biochemical Analyses EDC plus glycine methylester (EDC+G) treahnent of ACM arteries was found to modify carboxyl moieties of aspartic and glutamic acids without EDC crosslink formation or the detectable destabilization of native collagen Amino aad analysis of EDC+G-treated ACM reveaied that an average of 23.4 I 0.9% of carboxyl groups were capped after treatment. TNBS lysine anaiysis was utilized in an attempt to determine if and to what extent, crosslinkùlg rnay have occurred dunng treatment. Since lysine is only detected with the TNBS method and the EDC+G treahnent is intended for carboxyl groups of aspartic and glutamic acids, any loss of lysine after treatment may be assoaated with EDC crosslinking. The analysis was performed on two different occasions using six different samples of unh-eated and treated arteries (n=24 total). The results of the analysis revealed that no signifiant loss of lysine had occurred; lysine contents were 1.09 x104 5 6.30 x 10" mol/g and 1.11 x104 + 3.53 x 10" mol/g for untreated and EDC+G-treated ACM respectively. Thermal denaturation measurements failed to reveal any differences between EDC+G and untreated ACM arteries and thus confirrned that EDC crosslinking had not occurred (Table 4).

3.3.2Tensile Mechanical Testing EDC + glyane methyl ester did not significantiy alter extensibility at fracture, tissue modulus, or ultimate tensile strength as shown in Table 4. At lower physiological loads (120 mm Hg), however, EDC + glycine rnethyl ester treated ACM displayed a slightly decreased extensibility as revealed in the stress-strain response (Figure 15). There was, however, no difference in the stress relaxation behaviour before and after EDC+G treahnent (Figure 16). - --

ûenaturation Tissue UTS Strain at Stress Remalning Treatment Temperature (OC) Modulus (MPa) (MPa) Fracture (Oh) 0 1000 s (%)

Untreated 62.0 I0.7 1.3210.17 0,7910.12 170.0î14.4 68.6 I3.0

EDC + Glycine 63.5 I 0.8 1-17 I 0.22 1 .O1 î 0.24 206.2 I21.5 69.7 I1.6

Table 4. Properties of ACM arteries before and after modification of carboxyl groups with EDC plus glycine methyl ester treatment. Capping of carboxyl groups with glycine methy ester did not alter the properties of ACM arteries. Mean SEM Untreated ACM

EDC + Glycine Methyl

60 80 Strain (%)

Figure 15. Stress versus strain for ACM arteries before and after capping of carboxyl gcoups with EDC plus glycine methyl ester treatment. EDC plus glycine methyl ester treated ACM arteries were less extensible than untreated ACM arteries as shown by the shift of the stress-strain curve to the left. Mean A SEM

EDC+ Glycine Methyl Estei

Untreated ACM

Figure 16. Stress relaxation behaviours of untreated and EDC plus glycine rnethyl ester treated ACM arteries. Capping of carboxyl groups with glycine rnethyl ester did not alter the stress relaxation behaviour of ACM arteries. 3.3.3Enzyme Degradation Assays With the exception of cathepsin B, EDC+G h-eatment of ACM arteries increased the amount of collagen solubilized by enzymes. Collagen solubilization was significantly increased by EDC+G treahnent between 30 % and 48 % for trypsin and acetylû-ypsin respectively (Figure 17). In conhast, degradation of native ACM collagen by cathepsin B was significantly (~~0.05)reduced by 12 % af ter treatment. Mean î SEM *

Trypsin Acetyltrypsin Cathepsln B Collagenase Enzyme

Figure 17. In vitro solubilization of collagen from ACM arteries by enzymes before and after capping of carboxyl groups with EDC plus glycine methyl ester treatment. With the exception of cathepsin B, the amount of collagen solubilized by enzymes was increased in comparison to untreated ACM arteries. 3.4 Lysine and Arginine Capping: Epoxide and Methylglyoxal Treatment of ACM Arteries

3.4.1 Thermal Denaturation and Biochemical Analyses Amino capping of lysine using epoxy compounds glyadol and n- butylglycidylether, resuited in significant decreases in thermal denaturation temperatures. Mer a maximal 7 days of treatment, glyadol (Td = 58.7 i. 1.5 OC), and n-butylglycidylether (T, = 50.3 f 1.2 OC)treated samples displayed Td values 7.1 10.9% and 20.6 5 1.0%lower than untreated ACM (T, = 63.4 + 1.0 OC) respectively (Figures 18 and 19). The thermal stability of ACM arteries was found to decrease with the amount of lysine modified for both epoxy compounds (Figure 19). Although both epoxides modified over 80% of lysine residues, n- butylglyadylether was found to be more effective in destabiliizing collagen within the ACM-as would be predicted from its bulky and relatively hydrophobic structure (Figure 19).

After subsequent arginine modification with methylglyoxal however, glycidol-treated ACM (GMG) recovered collagen stability equivalent to that of untreated ACM arteries (T, = 63.6 + 2.1 OC). A smaller increase in the thermal stability of collagen was also noted for n-butylglyadylether freated ACM (T, = 53.2 2 1.8 OC) after methylglyoxal treatment (Figure 18). Equivalent modification of arginine residues to well over 70 % were obtained regardless of prior epoxide treahnent. The recovery of thermal stability in monofunctional epoxide-treated ACM after modification of arginine residues may be hypothesized to occur as a result of increased hydrogen bonding within the collagen triple helix faalitated by the methylglyoxal treatment (See Discussion section 4.2.2.1). Mean * SEM

*

- Untreated Glycidol GMG BMG

Figure 18. Denaturation temperatures for untreated, epoxide (Glycidol, n-Butylgly.), and epoxide plus methylglyoxal (glycidol+methylglyoxal - GMG, n-butylglycidylether + methylglyoxal - BMG)-treated ACM arteries. Epoxide modification of lysine residues alone decreased denaturation ternperatures. Combined epoxide lysine and methylglyoxal arginine modifications lowered the denaturation temperature of ACM arteries to a lesser degree than epoxide treatments alone. O 20 40 60 80 100 Percent of Lysines Modified

Figure 19. Percent change in denaturation temperature of ACM arteries versus percent of lysine residues modified by epoxide treatments. As the percent of lysine residues modified by epoxides increases, the denaturation temperature (thermal stability) of ACM arteries is lowered with the large, hydrophobic n-butylglycidylether modification having the greater effect. Denaturation Tissue UTS Strain at Stress Rernaining Treatment Temperature (OC) Modulus (MPa) (MPa) Fracture (%) O 1000 s (%) L

Fresh (Glycidol) 63.3 I1.3 2.4 I0.3 1.4 I0.1 248.2 î 15.2 73.8 I2.4

Glycidol 58.8 I1.5* 1.5 î 0.3** 1.3 * 0.2 232.7 * 31.8 76.8 î 1.5 r Fresh (n-Butyl.) 63.4 î 1.1 2.5 î 0.4 1.3 î 0.2 200.5 * 13.8 75.6 I2.1

n-6utylGly.Ether 50.4 I1.2* 1.2 I0.2* 1.O î 0.1 249.4 I 17.5 80.0 A- 2.5

I Fresh (Epoxy + MG) 63.3 * 0.8 1.6 I0.2 1.1 10.1 21 9.4 I10.5 66.7 I1.5

Glycidol + MG 63.7 I2.1 0.7 î 0.1* 0.8 î 0.1 235.5 * 14.5 71.6 * 12

n-ButyîGly. + MG 53.2 1.8" 1.1 10.2" 0.9 I0.1 236.6 * 22.8 74.0 * 0.4*

4' *- signficantly different from corresponding untreated at ps 0.05. ** - significantly different from corresponding untreated at 0.10.

Table 5. Properties of ACM arteries before and after epoxide treatrnent with or without methylglyoxal treatment. All treatments were found to alter tissue moâulus; however, lysine epoxide modification in combination with arginine modification with methylglyoxal was found to alter the stress relaxation behaviour of ACM aiteries. 3.4.2Tensile Mechanical Testing Treatrnent with monofunctional epoxides was found to alter low stress mechanical behaviours of ACM arteries and was found to depend upon the size/ chemistry of the epoxide utüized. The effects on mechanical properties was more pronounced when combined with arginine modification using methylglyoxal. Epoxide capping of amino groups of lysine with glyadol or n- butylglyadylether reduced the tissue modulus of ACM artenes, but did not alter strain at fracture (SAF) or ultimate temile strength (UTS) (Table 5). The tissue modulus of arteries treated with n-butylglyadylether was reduced on average by 52 % compared to a reduction of 38 % for glyadol treatment. Reductions in tissue modulus were also found after subsequent arginine modification with methylglyoxal. A greater reduction after glyadol plus methylglyoxal treatment of 56 % however, was found in cornparison to 31 O/o for n-butylglyadylether plus methylglyoxal treatment (Table 5).

The stress-strain behaviour of glyadol and n-butylglycidylether treated XCM arteries differed, with glyadol slightly decreasing tissue extensibiiity and n-butylglyadylether increasing tissue extensibility (Figures 20 and 21). This is most likely a reflection of the different size and/or polarities of the two reagents. The stress relaxation behaviour of ACM arteries was not significantly altered after glytidol and n-butylglyàdylether treatments (Figures 22 and 23).

In contrast to epoxide treabnent only, the stress-strain and stress relaxation behaviour of epoxide plus methylglyoxal treated ACM arteries were both significantly different from untreated ACM artenes. Decreased extensibility (Figure 24) and more elastic stress relaxation behaviours (Figures 25 and Table 5) tvere found for both glycidol plus methylglyoxai and n-butylglyadylether plus methylglyoxal treated materials. The decrease in extensibility and stress relaxation were greatest for glyadol plus methylglyoxal treatment. The changes in mechanical behaviour after methylglyoxal capping combined with monofunctional epoxide treatments may be due to increased hydrogen bonding Mean * SEM

Untreated ACM

Strain (%)

Flgure 20. Stress-strain response for ACM arteries before and after lysine residues have been modified using the srnail, hydrophilic epoxide glycidol. The glycidol treatment producecl a material with decreased extensibility as indicated by the shift of the stress-strain response to the left after modification. Mean ISEM - Untreated ACM

n-Butylglycidyl ether

O 20 40 60 80 100 120 Strain (%)

Figure 21. Stress-strain response for ACM arteries before and after lysine residues have benmodified using the large, hydrophobie epoxide n-butylglycidylether. The n-butylglycidylether treatment produced a more extensible material as indicated by the shift of the stress-strain response to the right after modification. Untreated ACM

100 Time (seconds)

Figure 22. Stress relaxation behaviour of ACM aiteries before and after modification of lysine residues with the small, hydrophilic epoxide glycidol. The gylcidol-treated ACM arteries displayed more elastic behaviour as indicated by the higher percent stress remaining after 1O00 seconds. Untreated ACM

œ œ 1 œ - 1-11 m 1 III I 1 w m muII 10 100 1 Time (seconds)

Figure 23. Stress relaxation behaviour of ACM arteries before and after modification of lysine residues with the large, hydrophobic epoxide n-butylglycidylether. The n-butylglycldylether-treated ACM arteries displayed a more elastic behaviour as indicated by the higher percent stress remaining after 1O00 seconds. Mean ISEM

Figure 24. Stress-strain response for ACM aiteries before and after methglyoxal and glycidol (GMG) or n-butylglycidylether (BMG) treatments. Epoxide modification of lysine residues in combination with arginine modification with methylglyoxal decreased the extensibility of ACM arteries as indicated by the shift of the stress-strain curve to the left after treatment. GMG treatment had the greater eflect. Figure 25. Stress relaxation behaviours for ACM arteries before and after methglyoxal and glycidol (GMG) or n-butylglycidylether (BMG) treatments. Epoxide modification of lysine residues in combination with arginine modification with methylglyoxal increased the elasticity of ACM arteries as indicated by the higher amount of stress remaining after 1000 seconds. BMG-treatment had the greater effect. within collagen. Furthemore, the collagen in glyadol-treated materials may have been disrupted to a lesser degree (due to its smaller size and/ or hydrophilicity ), and thus rnay have a greater ability to form hydrogen bonds after methylglyoxal treatment than wouid n-butylglycidylether-treatedACM arteries.

3.4.3Enzyme Degradation Assays

3.4.3.1 Autolysis of Trypsin

Trypsin and acetyltrypsin were incubated alone in buffer for 48 hours under similar conditions used for degradation experiments with ACM arteries and punfied type 1 collagen. After centrifugation, the supernatants from these incubations were analyzed by gel chromatography and compared to the elution patterns of trypsin and acetylû-ypsin before incubation. In Figure 26 the 280 nm absorbante cuves from the chromatography of trypsin and acetyltrypsin before and after 48 hours of incubation are shown. Clearly, the autolysis of trypsin is complete as the peak for the intact protein completely disappears and is replaced by hvo lower molecuiar weight peaks appearing at higher elution volumes (Figure 26). In contrast, after acetyltrypsin is incubated for 48 hours, a peak associated with the intact protein is present in conjunction with two similar lower molecular weight peaks attributed to degraded enzyme. Using relative peak area to detennine the quantity of speaes in each peak, the amount of intact acetylbypsin can be estimated at around 2533%after 48 hours. Although not totally resistant to autolysis, the presence of a small amount of intact acetylbypsin after 48 hours appears to have been enough to produce a difference in the degradation behaviour of ACM artenes and purified collagen when compared to trypsin. This is shown in the results below. ------Trypsin

-mm---i Trypsin after 48 hrs. -Acetyltrypsin

------iAcetyltrypsin after 48 hrs.

1111-1- 1111111~111 20 40 60 80 100 1 Elution Volume (ml)

Figure 26. Gel chromatography elution profiles for trypsin and acetyltrypsin before and after incubation for 48 hours. The presence of new peaks in the elution profiles of the two enzymes after 48 hours of incubation indicates that autolytic degradation has occurred. 3.4.3.2 Trypsin Degradation The degradation of ACM arteries by trypsin (targeting lysine and arginine) was found to generdy be greater after monohuictional epoxy (lysine) and methylglyoxal (arginine) treatments. Modification of amino groups of lysine alone with glycidol or n-butylglycidylether signtficantly increased the amount of collagen degradation compared to untreated tissue (Figure 27). Most interestingly, in contrast to glyadol treatment aione, glycidol treatment with subsequent arginine modification by methylglyoxal did not alter the degradation of collagen in ACM arteries. N-butylglytidylether plus methylglyoxal-treated ACM however, increased collagen degradation to weU over 90 %-equivalent to that of n-butylglycidylether treatment alone (Figure 27).

Only the effects of n-butylglycidylether and BMG treatments on the solubilization of native collagen from ACM artenes by acetyltrypsin were similar to those of trypsin (Figure 28 and Figure 27). When compared to untreated material, glycidol only treatnent reduced, although not significantly, the arnount of collagen solubilized by acetyltrypsin. GMG treated native ACM collagen, however, was found to be significantly more resistant to solubilization by acetyl trypsin. in contrast, n-butylglyadylether modification with or without arginine capping with methylglyoxal. increased collagen solubilization by acetyltrypsin to over 90 %. The results of combined epoxy and methylglyoxal heahnents appears to indicate that the chernical nature (size/ chemistry) of the lysine modifying epoxides determined the change (increase or decrease) in the degradation of ACM arteries.

3.4.3.4 Cafhepsin B Degradation Like acetyltrypsin and trypsin, increased solubilization of native coilagen from ACM artenes by cathepsin B to well over 90 % was observed after n-B or Mean ISEM * *

Untreated Glycidol BMG

Figure 27. Percent of native collagen solubilized from ACM arteries exposed to tiypsin before and after epoxide modification of lysine residues without (Glycidol, nBuîylgly.) or with arginlne modification using methylglyoxal (glycidot + methylglyoxal - GMG, n-butylglycidylether + methylglyoxal - BMG). lncreased native collagen solubilization by trypsin was found with n-butylglycidylether and BMG-treated ACM arteries. Glycidol only treated ACM arteries also displayed increased native collagen solubilization by trypsin. Mean i SEM

- Untreated Glycidol Untreated GMG BMG

Figure 28. Percent of native collagen solubilized from ACM arteries exposed to acetyitrypsin before and after epoxide modification of lysine residues without (Glycidol, n-Butylgly.) or with arginine modification using rnethylglyoxal (glycidol t methylglyoxal - GMG, n-butylglycidylether t methylglyoxal - BMG). lncreased native collagen solubilization by acetyltvpsin was found with BMG and ndutylglyAreated ACM aiteries. GMG treatment was found to decrease native collagen solubilization by acetyltrypsin. BMG treatments (Figure 29). In contrast, glyadol treatment alone resulted in a 28 % deuease in collagen solubilized. Interestingly, glyadol plus methylglyoxd treahent did not significdy reduce coiiagen degadation by cathepsin B. The la& of reductions in collagen solubilization after GMG or BMG treatment of ACM arteries is curious due to the fact that methylglyoxal modifies the amino atid arginine which is a primary binding site for cathepsin B.

3.4.3.5 Collagenase Degradation The arnount of coilagen solubilized by coiiagenase from ACM arteries was unaltered after epoxide only or epoxide and methylglyoxal treabnents, with the exception of BMG treatment (Figure 30). BMG treatment of ACM artenes increased the amount of collagen solubilized by collagenase by 21%.

3.4.4 Gel Chromatography of Degradation Products

3.4.4.7 Cnlibration of Gel Coliimn with Globlifar Protein Standards The resolution and separation capacity of the gel duomatography column used in this study was verified with Bio-Rade Gel Filtration standards. The molecular weights and elution volumes obtained for these globular protein standards is shown in Table 6. The elution volumes listed in Table 6 are vdid for estimahg the molecular weights of globular proteins only. Polypeptide chains of collagen under the elution conditions used in this study, can adopt various conformations cvhich alter their Stokes radii in solution [Butkowski et al. (1982); Rao and Adams (1973) 1. Thus, depending upon their conformation in solution, two equal mass collagen fragments rnay elute at different volumes. Proper estimates of molecular weights for collagen peptide hagments from gel chromatography can be obtained only with spetial techniques. These indude the use of denaturing elution solvents such as guanidine hydrochlonde and sodium dodecyl sulfate complexing [Butkowski et al. (1982); Rao and Adams (1975); Miller and Rhodes Mean ISEM

Intreateâ (0) Glycidol Untreatd - n-Butylgly. - Untreated ' GMG BMG (n-8) (GMG,BMG)

Figure 28. Percent of native collagen çolubilized from ACM arteries exposed to cathepsin B before and after epoxide modification of lysine residues without (Glycidol, nButylgly.) or with arginine modification using methylglyoxal (glycidol .trnethylglyoxal - GMG, n-butylglycidylether + methylglyoxal - BMG). lncreased native collagen solubilization by cathepsin B was found with n-butylglycidylether-treated ACM arteries regardless of arginine modification. Glycidol-treated ACM arteries displayed decreased native collagen solubilization by cathepsin 8. Mean * SEM

Untreated Glycidol GMG BMG

Figure 30. Percent of native collagen solubilized from ACM arteries exposed to collagenase before and after epoxide modification of lysine residues without (Glycidol, nButylgly.) or with arginine modification using methylglyoxal (glycidol t methylglyoxal - GMG, n-butylglycidylether + methylglyoxal - BMG). lncreased native collagen solubilization by collagenase was found with BMG-treated ACM aiteries only. Molecular Wt. (Da) Protein Elution Volume (mL) 675,000 Thyroglubulin (bovine) 44 - 1 58,000 Gamma Globulin 52 (bovine) w 44,000 Ovalbumin (chicken) 61

17,000 Myoglobulin (horse) 70 , 1.350 Vitamin B-12 98

Table 6. Mofecuiar weights and corresponding elution volumes for globuiar protein standards run on gel the chrornatography system used in this study. (1982)j. Therefore, in this study, any estimation of the molecular weight obtained by cornparison of elution volumes for coliagen fragments to those of globular standards located in Table 6 is purely qualitative.

3.4.4.2 280 nm UV Absurbance Elution Profies The absorbance at 280 nm wavelength of protein or protein fragments eluted from the bottorn of the column is dependent upon the polypeptides containing either of the aromatic amino aads phenylalanine (Phe) or tyrosine (Tyr). Unfortunately, neither coiIagen (Phe - 1.5%,Tyr - 0.5%) nor elastin (Phe -2.8%, Tyr- 0.8%)contain many of these residues and thus the probability of solubilized fragments from these proteins containing these residues is low. In conhast, the enzymes used in the degradation experiments, trypsh (Phe - 2.5%, Tyr- 4%), cathepsin B (Phe - 4%, Tyr- 5%), and microbial collagenase (Phe - 5%, Tyr- 7%), contain significantly more of these amino acids and generdy remain intact at the completion of the degradation experiments [Bond and Van Wart (1984); DNA SMderTMv. 1.1; L. Polgar (1989)l. As a resdt, the 280 nm elution profiles of products from the enzymatic degradation of untreated and modified ACM arteries and purified coIlagen appears to represent the profiles of the enzymes and not the products of ACM degradation. Alternatively, a wavelength of 230 run (peptide bond absorbance) could have been used to monitor for protein content, however; the in-line W monitor utilized in this study was of fixed wavelength [Scopes (l98î)I.

In Figure 31, the elution profile at 280 nm for trypsin by itself and after autolysis is compared to the elution profile of the degradation products of untreated ACM. The plot illustrates that dl of the peaks assoaated with the elution profile for the enzyme aione are also found in the degradation product elu tion profile of untreated ACM. Furthemore, the elution profiles of degradation products from untreated, GMG-and BMG-treated ACM digests are also similar. Thus, the elution profiles of degradation products monitored by absorbance at 280 nm are really those of the enzymes. This was found to be tme 1 -Untreated ACM I

O 20 40 60 80 100 120 Elution Volume (ml)

Figure 31. Gel chromatography elution profiles for trypsin before and after incubation for 48 hours, and the degradation products from an untreated ACM artery exposed to trypsin. The peaks in the elution profile for the untreated ACM artery is comprised of the intact trypsin and its autolytic degradation products. for the 280 nm elution profiles from trypsin, acetyltrypsin and cathepsin B digests of treated and untreated ACM arteries.

The one exception to this trend was the elution profiles of degradation products from collagenase digests of treated and untreated ACM. In Figure 32, the profile for collagenase by itself is plotted dong with the profile obtained for the degradation products of untreated ACM incubated with collagenase. Clearly, the enzyme alone produces a single strong peak at approximately 85 mL elution volume. Although the profile for digest products of untreated ACM also contains a peak at - 85 mL, there iç another unique peak whidi appears at approxhately 40 mL elution volume (Figure 32). When the collagenase degradation profiles for untreated, GMG, and BMG-treated ACM are compared, the unique peak at 40 mL elution volume is much reduced for GMG and BMG-treated ACM (Figure 33). Ln some other samples, the profiles for GMG and BMG ACM do not contain the 40 mL peak at all. This 40 mL peak in the elution profile for ACM arteries degraded by collagenase corresponds to the void volume of the padung material which has a molecular weight cut-off of 250 kDa for globular proteins. Thus, the peak may be assoaated with large f-ragments from other extracellular matrix components sudi as elastin or proteoglycans since collagen molecules are initially deaved by collagenases into 1/3 and 2/3 fragments. An intact collagen molecule has a molecular weight of - 285 kDa [Kielty et al. (1993); PinneIl and Murad (1983)l.

3.4.4.3 Collngen Contents of Chrornatography Fractions In order to determine the possible influence of solubilized materiais on the collagen contents of fractions determined by hydroxyproline analysis, samples of untreated, GMG and BMG-treated ACM were incubated in buffer for 48 hours at 37°C without enzymes. After incubation, the samples were cenûifuged, the supernatants and pellets collected, and then freeze-dried. Hydroxyproline analysis was conducted on the supematants and pellets to determine collagen content. The infiuence of the enzymes themselves on the outcome of - Untreated ACM .-.-. ., Collagenase

O 20 40 60 80 100 120 Elution Volume (ml)

Figure 32. Gel chromatography elution profiles for collagenase and the degradation products from an untreated ACM arteiy exposed to collagenase. The peaks in the elution profile for the untreated ACM aitery correspond with the exception of a peak at approximately 40 mL. Untreated -ACM

O 20 40 60 80 100 120 Elution Volume (ml)

Figure 33. Gel chromatography elution profiles for the degradation products from untreated, glycidol + methylglyoxal (GMG) and n-butylglycidylether + methylglyoxal @MG)-treated ACM aiteries exposed to collagenase. The peaks in the elution profites correspond with the exception of a peak at approximately 40 mL which diminishes after GMG and BMG treatments. hydroxyproline analyses was also investigated by performing the analysis on the enzymes alone. The percentage collagen solubilized by the incubation of ACM artenes in buffer was approximately 4% for untreated and GMG-treated ACM and 7% for BMG-treated ACM. These values are low compared to the 15-98% and 80-98% collagen contents of supematants from degradation experiments performed with untreated and GMG-treated, and BMGheated ACM artenes, respectively. The enzymes alone also produced a small but measurable response in the hydroxyproline assay. Trypsin accounted for approximate~y5%, cathepsin B 1.2%, and collagenase 1.9% of the hydroxyproline measured in digest fractions of untreated, GMG-treated, and BMG-treated ACM. Overail, the hydroxyproline contents of enzymes and solubilized materials from ACM artenes were negligible and therefore were not deducted from the results of degradation experiments. With the exception of trypsin, GMG and BMG treaûnent did not significantly alter the pattern of collagen solubilized from ACM arteries by degradative enzymes used in this study. The quantity of collagen degraded in identical fractions however, was altered by GMG and BMG treatments in al1 cases, with GMG treahent generdy decreasing and BMG treatment generally increasing collagen solubilization when compared to untreated materials. The distribution of collagen from trypsin digest supematants of untreated, GMG, and BK-treated ACM are shown in Figure 34. Both GMG and BMG treatments produced different patterns of collagen fragments solubilized compared to untreated ACM arteries. For untreated KM, the bulk of collagen soIubiIized is contained within the fraction range from 75-84 mL. According to Table 6, this corresponds to fragments with a molecular weight of approximately 15 kDa or less. GMG-treated ACM, however, produced a collagen fragmentation pattern with equivalent amounts of coilagen contained in al1 of the fraction ranges analyzed. The pattern of collagen solubilized from BMG-treated ACM however, was different from both GMG-treated and untreated ACM with the +Untreated ACM Mean î SEM -- GMG ACM BMGACM >

Elution Volume Range (mL)

Figure 34. The percent of native collagen solubilized from untreated, glycidol + methylglyoxal (GMG) and n- butylglycidylether t methylglyoxal (BMG)-treated ACM arteries exposed to trypsin separated by gel chromatography. Fragments of larger size are located in lower elution volumes. GMG and BMG treatments altered the pattern of collagen cleavage by trypsin as indicated by the changes In the size distribution of collagen fragments after treatment. bulk of collagen solubilized spread between 66 and 84 mL, corresponding to an approximate molecular weight of less than 35 kDa (Table 6). Ln Figure 35, the patterns of collagen fragments solubilized by acetylbypsin from untreated, GMG, and BMG-treated ACM arteries are shown. The pattern for GMG-treated and untreated ACM arteries appeared similar, but were different from those produced by the trypsin degradation of untreated and GMG-treated ACM (compare to Figure 34). In contrast, the pattern of coilagen solubilized from the exposure of BMGtreated ACM arteries to acetyltrypsin was different from untreated and GMG-treated ACM arteries, but similar to trypsin degradation of BMG-treated ACM arteries. The distribution of collagen solubilized by collagenase for untreated, GMG, and BiMG-treated ACM arteries is shown in Figure 36. The patterns of collagen solubilized were similar for dl treahnents. The only difference obsewed kvas a higher amount of coilagen solubilized from untreated ACM arteries in the fraction range 93-102 mL elution volume corresponding to a n approximate molecular weight of less than 1.5 kDa. Like collagenase, the pattern of collagen solubilized from ACM by cathepsin B was not signihcantiy altered by GMG and BMG treatments (see Figure 37). In terms of overall collagen degradation, however, BMG-treated ACM was shown to be more susceptible to cathepsin B and collagenase. This may be reflected in the slightly higher collagen contents in the fraction range 90-99 (~2.5kDa) and 93-102 mL (4.5 kDa) for cathepsin B and collagenase respectively (see Figures 37 and 36). - Mean t SEM I Untreated ACM GMGACM -+-• BMGACM

Elution Volume Range (ml)

Figure 35. The percent of native collagen solubilized from untreated, glycidol + methylglyoxal (GMG) and n- butylglycidylether + methylglyoxal (BMG)-treated ACM arteries exposed to acetyltrypsin separated by gel chromatography. Fragments of larger size are located In lower elution volumes. BMG treatment altered the pattern of collagen cleavage by acetyltrypsin as indicated by the change in the size distribution of collagen fragments in the elution volume range 66 to 75 mL. Untreated ACM Mean A SEM -cw- GMG ACM

-+-a BMGACM

Elution Volume Range (mL)

Figure 36. The percent of native coll gen solubilized from untreated, glycidol + methylglyoxal (GMG) and n- butylglycidylether t methylglyoxal (BMG)-treated ACM aiteries exposed to collagenase separated by gel chromatography. Fragments of larger size are located in lower elution volumes. GMG and BMG treatments altered the amount of collagen solubilized between 93 and 102 mL elution volume. Mean SEM Untreated ACM -+- GMG ACM -*-• BMGACM

Elution Volume Range (mL)

Figure 37. The percent of native collagen solubilized from untreated, glycidol t methylglyoxal (GMG) and n- butylglycidylether t methylglyoxal (6MG)-treated ACM arteries exposed to cathepsin B separated by gel chromatography. Fragments of larger size are located in lower elution volumes. GMG and BMG treatments did not alter the pattern of collagen solubilized by cathepsin B. 3.5 Lysine and Arginine Capping: Epoxide and Methylglyoxal Treatment of Purified Type I Collagen

3.5.1 Amino Acid Analyses and Swelling Measurements Amino aad analysis revealed that equivalent amounts of lysine and arginine were modified by GMG and BMG treatment of purified type 1 collagen modified (Table 7). Changes in the length (taken as the direction dong the long axis of collagen fibres) after swelling of GMG-treated purified type 1 collagen fibre bundles was significantly less than untreated and BMG -treated type 1 collagen (Table 7). Changes in width (taken perpendicular to the long axis of collagen) after swelling however, was significantly less for both GMG and BMG treatments when compared to untreated collagen (Table 7). The gross differences between treated and untreated collagen could be noted by eye and without the aid of measurement equipment (all photographs are taken at the same magnification) as shown in example photos in Figure 38. From the swelling measurements, it is dear that both treatments involving arginine blocking (GMG and BMG) significantly altered the ability of collagen to uptake solvent.

3.5.2 Enzyme Degradation Assays Solubilization of purified type 1 collagen by trypsin, acetyltrypsin, cathepsin B and collagenase was significantly altered by GMG and BMG treatments (Figure 39). GMG treatment deueased purified type 1collagen solubilization when exposed to acetiytrypsin and collagenase. BMG treahnent of purified type 1 collagen was found to inaease collagen solubilization by al1 enzymes utilized in the in vitro assays with the exception of coilagenase.

3.5.3Gel Chromatography of Degradation Products

3.5.3.1 280 iim UV Absorbance Elirfion Profiles In Figure 40, the elution profile at 280 nm for acetyltrypsin by itself and after 48 hours of incubation by itself (autolysis) is compared to the elution profile Lysine Arginine Change In Length Change in Wldth Treat ment Modifieci (O/) Modified (O/o) After Swelling (%) After Swelling (76)

Untreated Collagen O O -7.81 2.4 251 î46

GMG Collagen 92.7 a.4 82.5 + 4.1 -1.8 I0.7' 50.5 I8.8*

1 BMG Collage" 1 95.1 I1.2 1 81.6 I1.5 1 -6.1 I1.9 1 51.5 î 8.6* 1 * - Significantly different from untreated collagen at psO.05

Table 7. Percent of lysine and arginine residues modified in purified type I collagen and their effect on dimensional changes due to swelling in acid. Glycidol plus methylglyoxal (GMG) and n-butylglycidylether plus methylglyoxal (BMG) treatments were found to significantty reduce swelling. Untreated Purified Type I Untreated Purified Type I Collagen (Dry) Collagen Swollen in 0.1 M Acetic Acid

GMG Treated Purified Type I Collagen (Dry) GMG Treated Purified Type I Collagen Swollen in 0.1 M Acetic Acid

BMG Treated Purified Type I Collagen (Dry) BMG Treated Purified Type I Collagen Swollen in 0.1 M Acetic Acid

Figure 38. Samples of purified type I collagen before and after swelling in 0.1 M acetic acid. Length was defined along and width perpendicular to the long axis of the collagen bundles. Measurement resolution = 23 p.The lengths of samples are indicated in mm. 1O5 Mean 4 SEM

- - - Trypsin Acety ltrypsin Cathepsin B Collagenase Enzyme

Figuie 39. Percent of purified type I collagen solubilized by enzymes before and after epoxide modification of lysine residues and arginine modification using methylglyoxal (glycidol + methylglyoxal - GMG, n-butylglycidylether t methylglyoxal - BMG). lncreased collagen solubilization after BMG treatment was found for all enzymes except collagenase. GMG treatrnent decreased the amount of collagen solubilized by acetyltrypsin and collagenase. -Acetyltrypsin

LI-. . -W.SrA<- Acetyltrypsin after 48 hrs. - - - Untreated Collagen

60 80 Elution Volume (mL)

Figure 40. Gel chromatography elution profiles for acetyltrypsin before and after incubation for 48 hours, and the degradation proâucts from untreated purified type I collagen exposed to acetyltrypsin. The peaks in the elution profile for the degradation proâucts from untreated purified type 1 collagen is comprised of the intact enzyme and ils autolytic degradation products. of the degradation products of untreated purified type 1 collagen. The plot illustrates that alI of the peaks associated with the elution profile for the enzyme alone were also found in the degradation product elution profile of untreated purified type 1 collagen. Furthexmore, the elution profiles of degradation products from untreated, GMG and BMG-treated purified type 1 collagen digests were also similar. Thus, the elution profiles of degradation products monitored by absorbance at 280 nm are really those of the enzymes. This was also true for the 280 nm elution profiles from cathepsin B digests of treated and untreated purified type I collagen. The only exception to hswas, as was the case with ACM arteries, the elution profiles of collagenase degradation products. In Figure 41, collagenase is shown as a single peak at an elution volume of - 85 mL whereas the degradation produds of untreated purified collagen contains three peaks at approximately 80,95, and 115 mL. When the elution profiles at 280 nm for untreated, GMG and BMG-treated purified collagen exposed to collagenase are compared however, only the peak at - 80 mL is common to al1 three samples: the 95 mL and 115 mL peaks in the untreated collagen profile are not present in the GMG and BMG-treated samples (Figure 12). Thus, the common peak at - 80 ml may be due to the enzyme coilagenase even though it does not appear in exactly the sarne place as the enzyme alone (= 85 ml). The exact nature of the other peaks in purified collagen collagenase digests is unknown.

3.5.3.2 Collngerz Contents of Chrornntogrnphy Frnctiom With the exception of acetyltrypsin, GMG and BMG treatments did not affect the pattern of solubilized degradation products from purified type 1 collagen. Interestingly, as with trypsin degradation of ACM arteries, solubilization of purified collagen by acetylûypsin after GMG and BMG û-eatments resulted in a shift in the pattern of solubilized products to smaller elution volumes and thus, higher molecular weights (Figure 43). In contrast, although overall collagen solubilization was affected by GMG treahnent, the pattern of degradation of purified type 1 collagen by collagenase was unaltered Elution Volume (ml)

Figura 41. Gel chromatography elution profiles for collagenase and the degradation products from untreated purified type I collagen exposed to collagenase. The peaks in the elution profile for the untreated collagen correspond to collagenase with the exception of two peaks at approximately 95 and 115 mL. Untreated-. .-.--.- - -Collagen

-' -- GMG Collagen I 1 1 - - -m BMG Collagen 1 1 \

0 20 40 60 80 100 120 140 Elution Volume (ml)

Figure 42. Gel chromatography elution profiles for the degradation products from untreated, glycidol + methylglyoxal (GMG) and n-butylglycidylether + rnethylglyoxal (6MG)-treated purified type I collagen exposed to collagenase. The peaks in the elution profiles correspond with the exception of peaks at approximately 95 and 115 rnL which are not present after GMG and BMG treatments. Mean î SEM -m- Untreated Collagen

-9- GMG Collagen - +- BMG Collagen

Elution Volume Range (ml)

Figure 43. The percent of collagen solubilized from untreated, glycidol + methylglyoxal (GMG) and n-butytglycidylether + methylglyoxal @MG)-treated purified type I collagen exposed to acetyltrypsin separated by gel chromatography. Fragments of larger size are located in lower elution volumes. BMG and GMG treatments altered the pattern of collagen cleavage by acetyltrypsin as indicated by the change in the size distribution of collagen fragments in the elution volume range 66 to 75 mL. by chemicai modification (Figure 44). A significantly higher colIagen content of 84-93 mL elution volume, however, was observed for untreated coilagen. The degradation of purified type I collagen by cathepsin B was the least affected by GMG and BMG treatments with identical distributions and quantities of collagen found in all fraction ranges (Figure 45). - -- Mean SEM FA- Untreated Coltagen 1

Elution Volume Range (ml)

Figure 44. The percent of collagen solubilized from untreated, glycklol t methylglyoxal (GMG) and n-butylglycidylether + methylglyoxal (BMG)-treated purified type I collagen exposed to collagenase separated by gel chromatography. Fragments of larger size are tocated in lowet elution volumes. GMG and BMG treahents altered the amount of collagen solubMzed between 84 and 93 rnL elution volume. +Untreated Collagen Mean SEM --b- GMG Collagen - -i - BMG Collagen

Elution Volume Range (ml)

Figure 45. The percent of collagen solubilized frorn untreated, glycidol + methylglyoxal (GMG) and n-butylglycidylether + methylglyoxal (BMG)-treated purified type I collagen exposed to cathepsin B separated by gel chromatography. Fragments of larger size are located in lower elution volumes. GMG and BMG treatments did not alter the pattern or amounts of collagen solubilized by cathepsin 6 in al1 fractionation ranges analyzed. 4.1 EDC Crosslinking Control of pH during EDC/NHS treahent of tissue derived materials is of industrial interest since most buffers render EDC treatment ineffective due to cross-reactions [Lee et ai. (1996)j. The reaction must therefore be carried out in unbuffered solution and, if pH is to be controlled, it must be adjusted during the reaction by small additions of aad and base [Lee et al. (1996); Olde Damink et al. (I996)J.This problem led to the present exploration of differences in properties which might be produced if pH was pragrnatically left uncontrolled after the reaction began, rather than being maintained at the optimal pH of 5.5. Initial resuih were surprising enough that a more complete study of the treatment effects on extracted acellular rnatrix (ACM) arteries was undertaken. A rich picture of the effects of EDC-induced crosslinking under different pH conditions was achieved using a spechum of testing techniques: e.g. biochemical analysis, thennomechanical analysis, and shear bending testing (Table 8). EDC crosslinking with pH control produced a larger increase in thermal denaturation temperature, whereas EDC crosslinking without pH control consurned more lysine residues. EDC crosslinking under either pH condition significantiy altered the shear properties of ACM arteries; however resistance to stress relief was most affected by treatment without pH control. The observed differences in material properties may be attributed to the preferential formation of particular types of uosslinks in collagen or between other molecular species induding proteoglycans, glycosaminoglycans, elastin or microfibrillar proteins of elastin. ACM arteries possess a high content of elastic tissue (4û-60% dry w/ w), however elastin itself contains few reactive amino and carboxyl groups and thus would not have played a significant role in the differences observed with EDC crosslinking. Therefore, the observed differences may be attributed to (i) crosslinking with pH control having favoured intrahelical crosslinking, rapidly inueasing T, and (ii) crosslinking without pH Property EDC-pH EDC-No pH Dena tura tion Faster rise; Higher final Temperature value Lysine Residues More consumed Bendina Stiffness Greater increase

Table 8. Summary of the effects of EDC crosslinking with and without pH king maintained at 5.5 on ACM arteries. The treatment having the greater effect on each propeRy is indicated. control having favoured interheücal crosslinking and/ or mslinking with proteoglycans, glycosaminoglycans or miaofibrillar proteiw of elastin, reducing the ability of collagenous layers to slide by each other in shear deformation. These ideas are supported by the following discussion. Previous EDC crosslinking studies using intact (unextraded) tissue and extracted collagen had both shown EDC crosslinking at constant pH to be essentiaily complete after 3 hours of treatment [Lee et al. (1996); Olde Damink et al. (1996)j. The results obtained here agreed with these studies-regardless of pH conbol. Compared to the uncontrolled pH case, crosslinking under a constant pH of 5.5 produced a lower rate of lysine consumption but the fastestflargest increase in the hydrothermal stability of ACM arteries. This difference was the first due as to the differences in crosslinking produced (Figure 14, Table 2). Thermal denaturation is the uncoiling transition of collagen from the rod-like triple helix to a nearly random coi1 [Flory and Garrett (1958); Von Hippel(1967)]. It has been previously shown that thermal denaturation temperatures do not change with maturation of mammalian collagen despite large increases in interhelical mosslinking [Naimark et al. (1992); Allain et al. (1978)]. Thus, exogenous intrahelicd crosslinks have the greatest impact on the thermal stability of ACM arteries. With pH control, the greater increase in thermal stability-achieved with a smaller number of lysines modified-suggests that the majority of EDC crosslinks formed within the hiple helix of collagen. The most striking differences between pH treahnents were found in the shear mechanicd behaviours of EDC-crosslinked ACM arteries. EDC treatment without pH conbol had the greatest overall effect on the shear properties of ACM artenes. Shear properties of tissue-derived materials are dependent upon the ability of layers of collagen and elastin fibres to slide past one another under load [Lee and Boughner (1991); Tahan and Boughner (1996)l. This feature exclusive1y determines the bending stiffness of soft tissues since they canno t effectively support compression in true bending. Furthermore, exogenous crosslinking has been shown to alter the shear properties of tissues [Talman and Boughner (1995) 1. In the present study, the shear behaviour of ACM arteries was assessed via bending measurements utilizing the diametral hoop compression approach developed by Lee et al. (1998). Without pH control, EDC crosslinking increased bending stiffness in diametral hoop compression drarnaticdy by a factor of 21x (compared to 14x for pH-controlled treahnent (Table 2)). This suggests the preferential formation of crosslinkç which can restnct interlayer shear deformation. These linkages may be interfibrillar coilagen crosslinks, due to crosslinking of artenal ground substance through the carboxyl side-groups of gIycosaminoglycans or proteoglycans, or due to the crossluiking of microfibrilku proteins of elastin. Indeed, the partiapation of glycosaminoglycaw or proteoglycans is IikeIy given their location on the surface of coliagen fibrils and the short distance over which EDC crosslinkages can form. EDC crosslinking (in collagen alone) has been compared to charge or "salt" linkages between acidic (aspartic and glutamic aads) and basic groups (lysine and arginine). These Iinkages can span a distance of no more than two amino aad residues [Lee et al. (1996)l.As well, the alteration of mechanical behaviour of tissues due to the crosslinking of miaofibrillar proteins of elastin has been previously dernonstrated with another

Figure 46. EDClNHS crosslinking reaction. Crosslinking can accur via reaction (iii) or the action of NHS. Side reactions include the formation of a stable N-acylurea (i) or the rapid hydrolysis of the O-acylisourea to reform the protein carboxyl side-chain (ii).

EDC-induced crosslinking with cokiagen, forming a proteoglycan or glycosaminoglycan "glue" between fibre layers. This hypothesis has the appeal of explaining the Iimited interfibriilar reach of shear-resistant crosslinks. With decreased pH, dose apposition of nudeophilic hydroxyl groups from the carbohydrate component of glycosaminoglycans to very reactive adivated carboxyls may also have provided opportunity for enhanced crosslinking. The EDC crosslinking of carbohydrates of glycosaminogIycans has been demonshated previously [Tomihata and Ikada (1997)l. While attractive, the above hypotheses remain spedative and await specific, mechanistic investigation. In surnmary, the pragmatic choice to control or not control pH during EDC/ NHS crosslinking had significant and interesting effects on the properties of the resulting bioprosthetic materials. Shear properties of ACM artenes were differentially affected by the two treatmenh. Viewed together, the results of biochemical, thermomechanicai, and shear property analyses revealed characteristics which may be attributed to differences in the location of the crosslinks formed. Preferential formation of interhelical crosslinks without pH control is hypothesized to occur as a result of an aad-enhanced increase ui: (i) the reactivity of both EDC- and NHS-activated esters and (ii) the nucleophilitity of hydroxyl groups from carbohydrate chah of glycosaminoglycans. 'Ihis may have enhanced interhelical/ interfibrillar crosslinking, producing enhanced resistance to shear and stiffening of the tissue in bending. The involvement of other ECM components (e-g. elastic tissue, proteoglycans, glycosaminoglycans)may also have contributed to this effect. Perhaps most importantly, the type of crosslinking had no effect whatsoever on resistance to enzymatic solubilization. 4.2 Capping of Amino Acid SideChain Functionalities Very Little data exists on amino acid side-chain modifications and their effects on the proteolysis of coliagenous materials. In fact, there has been little work conducted for atzy protein subshate on the effects of diemical modification of amino aad side-chahs and enzymatic degradation. Interestingly, of the Little work available, most studies are in the area of food-stuff stabilization and nutrition enhancement [Murakami and Etlinger (1987); Puigsewer et al. (1979); Lee et al. (1978); Lin et al. (1969)l. The one study most relevant to this work however, is an investigation into the in vivo and in vitro degradation behaviours of insoluble bovine type I coliagen modified by methylation (target group= COOH), sucanylation (target group= NH,),and acetylation (target group= NH2)[Diamond et al. 19911. Direct cornparison of this work to the present study is difficult due to the la& of material characterization by these workers and, in particular, a failure to determine the amount by which each treahnent modified the targeted amino aad side-chain hctionalities. Therefore, the present study can only be compared to the limited body of work in the literature in, at best, a qualitative manner.

4.2.1 Carboxyl Capping: EDCINHS and Glycine Methyl Ester In this study, the modification of carboxyl moieties of aspartic and glutamic aads by EDC plus glyane methyl ester treatment (EDC+G) appeared to have no hinctional effect on the shiicture of the native collagen in ACM processed arteries. The effects of treatment on structure were determined by mechanicd and thermal denaturation temperature measurements which indirectly assessed changes in the macro- and mo1ecula.r structure of collagen respectively. Mechanical tests revealed that the high (fracture) and low (physiological) stress properties of ACM artenes remained unaitered after EDC+G treatment. Similar values of tissue modulus, UTS, SAF, and stress remaining at 1000s (Table 4) were obtained for treated and untreated ACM arteries. The one mechanicai behaviour aitered after EDC+G treatment was the sbess-strain response under simulated physiological loads, showing a slight decrease in ex tensibility after keatment (Figure 15). One possible explanation for this decrease in extensibility is the formation of EDC uosslinks during the carboxyl capping treatment. EDC crosslinking, however, was discouraged by careful selection of reaction conditions previously shown to favour capping over crosslinking [Hoare and Koshland (1966); Lewis and Shafer (1973); Carraway and Koshland (1972); Hoare and Koshland (1967)l. The absence of crosslinking kvas also confirmed indiredly since thermal denaturation temperature measurements did not show an increase (a proxy for crosslinking) after EDC+G treatment (Table 4). The absence of crosslinks in EDC+G treated ACM was also directly confirmed by TNBS analysis which showed that the amount of lysine (the other target of EDC aossluiking) did not decrease after treatment. Another possible explanation for the decrease in extensibility under simulated physiological loads may be related to the aiteration of the charge balance in collagen: i.e. removal of negatively charged carboxyl groups. The registration of collagen molecules in the characteristic D-bandïng pattern within fibrils and fibres is dependent upon the matdùng of polar and nonpolar regions in adjacent molecules [Chapman et al. (1990); Parry (1988)l.The removal of some negative charge from collagen may have increased the level of hydrophobic interactions. The obstacle for increased hydrophobic (van der Waals) interactions has been hypothesized to be the electrostatic repulsion of charged residues in the same region that are opposite each other when triple helices are in register [Kajava (1991)l. The increased hydrophobic interactions may have been enough to cause a slight increase in resistance to extension (unmmping or uncoiling) within the low-stress region of the stress-strain curve. Furthemore, the limited extent of modification of carboxyls (- 23%) together with the weak nature of van der Waals interactions (1 kcal / mol) in cornparison to hydrogen and electrostatic bonds (3-7 kcd/ mol), may also explain the assoaated la& of change in high- stress mechanical properties or thermal stability [Stryer (1988)j. The lack of gross structural changes in colIagen of ACM arteries after EDC+G treatment does reflect the effect of the low extent of carboxyl modification (- 23%). Previous studies of methylated coliagen (using acidified methanol to add methyl groups to carboxyl moieties) have shown that modifications of 70% only slightly destabilized coilagen. This was indicated by a reduction in thermal denaturation temperature of only 1.5 OChom that of untreated collagen [Rauterberg and Kühn (1968); Balleisen et al. (1976)]. Significant loss of helicai stability indicated by a 13-20 OC decrease in thermal stability measurements occurred only after more than 70% of carboxyl groups were modified. Although physical property measurements (mechanical and thermal stability) indicated that the shucture of native collagen was not significantly altered after EDC+G h-eahnent, the results of in vitro enzymatic degradation experiments indicate othenvise. A signifiant increase in the in vitro solubilization of native ACM collagen after EDC+G treatment was observed for trypsin, acetyltrypsin and collagenase (Figure 17). This change may have been due to a slight perturbation of the collagen structure as a result of carboxyl capping with glycine methyl esters which did not alter physical properties but ruas recognized by degradative enzymes. The disruption of the helical conformation of collagen (Le. denaturation) has been shown to increase its susceptibility to both trypsin and microbial collagenase [Burleigh (1977); Kucharz (1992); French et al. (1992); Mal1ya et al. (l99î)I. Indeed, increased susceptibility to trypsin and pepsin was noted by Diamond et al. for type 1 collagen after methylation [Diamond et ai. (1991)l. Most interesting however, was the decreased susceptibility of native ACM collagen to cathepsin B after EDC+G treatment in the present study compared to the imaltered susceptibility of type 1 coIlagen to cathepsin B after methylation found in Diamond's study [Diamond et al. (1991)j. The reason for this difference is undear and may be reiated to a number of factors induding: the methylation of carboxyl moieties versus capping with glycine methyl ester, the use of native ACM collagen versus pwified type 1 collagen, and fïnally the number of carboxyl groups modified. AIthough Diamond did not directly measure the modification of carboxyls, he reported a drop in thermal denaturation temperature of approximately 19 OCfor type 1collagen after methylation. This would suggest that more than 70% modification was achieved when compared with 23% modification achieved using EDC+G treatment. As mentioned previously, a 13- 20" C drop in denaturation temperature observed in other studies for methylated collagen was only found with greater than 70% modification [Rauterberg and Kühn (1968); Balleisen et al. (1976)j. More importantly, in this study, the decreased solubilization of collagen after EDC+G heabnent by cathepsin B contrasts with the incremed solubilization by trypsin, acetyltrypsin and microbial collagenase. Again one possible explanation may be the rotation of amino aad side-diains involved in cathepsin B binding due to a slight perturbation in the structure of collagen caused by the capping of negatively charged carboxyl groups. As a result of this smail rotation, the arnino acid side-chains involved in primas, and/or secondary binding of cathepsin B may have become preferentially (through happenstance) buried and/ or disordered and therefore unrecognizable. For example, covalent attachment of arnino acids to the protein casein has been shown to alter conformation and reduce degradation by enzymes [Puigserver et al (1979)l. The binding of antibodies (which bind to proteins in a similar manner to enzymes) has also been shown to be prevented through alterations in collagen conformation via diemical modifications [Beil et al. (1973); Glattauer et al. (1991)l. The challenge remains to predict how a given treatment will influence binding and activiv of a given enzyme on a given substrate.

4.2.2 Lysine and Arginine Capping: Epoxide and Methylglyoxal Treatments Capping of lysine residues using mono-functional epoxides of different molecular structures had significant and different effects on both the physical (mechanical and thennomechanical) and enzyme degradative properties of both native ACM collagen and purified type 1 collagen. These differences appeared to be more distinct when lysine epoxide modifications were combined with methylglyoxal modification of arginine. The changes in the physical properties of native ACM collagen and enzyme degradative properties of native ACM and purified type 1 collagen were dependent upon the chernistry and/or size differences of the epoxide used to modify lysine residues. The structure of collagen and/ or presence of other ECM components was also found to significantly influence the effed of chernicd modifications on the in vitro enzymatic soiubilization of collagen.

4.2.2.1 Effects of Lysine and Arginine Capping on Collogen Shîcbrre Epoxide modifications of lysine residues significantly altered the structure of native ACM collagen. Treatment with the smd, hydrophilic glyadol (G)or the large, hydrophobic n-butylglycidylether (n-B) epoxide resulted in modification of the same amount (over 80%)of lysine residues in the native collagen of ACM artenes. Both epoxides destabilized native ACM collagen. Thermal denaturation temperatures were lower when compared to untreated ACM; however n-B treahnent had the greater effect (Figure 18, Table 5). Sirnilar observations on the effect of collagen modification with a mono-fundional epoxide were noted by Tu et ai. in their study on the kinetics of epoxide treatment of bovine internai thoraac arteries [Tu et al. (1994)j. Treatment usïng an industrial equivalent to glyadol (Denacol EX-131) for 6 days resulted in 86.5% lysine modification and a reduction in thermal denaturation temperature of - 15 OC compared to - 5 OC in this study. Other observations of various chemicai capping treatments confirm that, in generai, collagen structure is disrupted after lysine modification. One hundred percent succinylation of acid-soluble collagen or acetylation of insoluble calf collagen, was found to reduce thermal denaturation temperatures by 4 OC and 9 OC respectively [Rauterberg and Kühn (1968); Fujimoto (1970)j. These values are similar to G treatment and may refled the general effect of the hydrophiüc modification of lysine residues on collagen thermal stabiliîy. When epoxide modification of lysine was combined with arginine modification by methylglyoxal, glyadol plus methyIglyoxa1 (GMG) treatment did not alter the thermal stability of native ACM coIlagen; however, thermal stability was significantly reduced with n-butylglyadylether plus methylglyoxal (BMG) treatment (Figure 19, Table 5). Clearly the large, hydrophobic nature of n- B and BMG treatments destabilized the structure of collagen to a greater degree than did the smaller, hydrophilic G and GMG treatments. In comparison to each epoxide treatment alone, increases in thermal denaturation temperature were observed after subsequent methylglyoxal modification (Table 5). It may be hypofhesized that methylglyoxal treatment inueases collagen thermal stability through hydrogen bond formation within the triple-helix via introduced oxygens. Furthemiore, methylglyoxaI may have been more effective in forming hydrogen bonds with glyadol-treated ACM artenes given glyadol's Iesser destabilizing effect (compared to n-B). Further evidence of differences in the effects of G and n-B epoxide modifications on collagen sh-ucture are found in the results of mechanical testing. With the exception of tissue modulus, the hgh (fracture) stress properties remained und tered after trea tment. Significant changes in mechanical behaviour of ACM artenes after epoxy treatment with or without methylglyoxal treatment were only observed at low (simulated physiological) stresses. G treahnent of native ACM collagen produced a less extensible material (curve shifted to the left) in comparison to untreated ACM (Figure 20). The decreased extensibility at low stresses may be the result of minor alterations in collagen sbcture combined with increased interaction among collagen chains via introduced polar groups (hydroxyls).The capping of lysine with acetyl (CH,CO-) groups, another oxygen-containing polar group, has been shown to lower thermal stability and thus to destabilize collagen; nonetheless, resistance to the enzyme pronase was retained [Fujimoto (1970)l.It was hypothesized that acetylation introduced oxygen-mediated interactions between collagen chains, possibly through aosslink formation. In contrast to G treatment, n-B treatment increased the extensibility (curve shifted to the right) of native ACM collagen (Figure 21). It is likely that the large, hydrophobic modification grossly altered collagen conformation (as shown by reduced denaturation temperatures), thereby reducing the interaction between collagen chahs, and allowing more freedom of movement and increasing extensibility a t low stresses. Interestingly, the tirne-dependent stress relaxation curves for of G and n-B treated ACM showed equally more elastic behaviour than was seen in untreated ACM (Figures 22,23). This feature may have been more sensitive to disruptions in collagen structure. Even the small perturbation of collagen structure caused by G treahnent was apparently enough to equal the large alterations caused by n-B modification. When subsequently combined with methylglyoxal treatment of arginine residues, both epoxide treatments produced similar changes in the stress-strain and stress relaxation behaviours of native ACM collagen: reduced extensibility and greater elastiaty than was seen with the epoxide treatments alone. These effects may be explained under the sarne hypothesis presented earlier to address the changes in thermal stability. The methylglyoxal modification of arginine likely introduced oxygen atoms into the collagen structure whidi faolitated hydrogen bonding and increased collagen diain interactions. Furthermore, this may have occurred in conjunction with an alteration in coUagen fibre conformation as a result of the loss of nearly al1 positive charges in collagen through amino and guanido group capping. The removal of &-aminogroups of lysine with nitrous aad and/or capping of arginine with cydohexandione in rat type 1 collagen has been shown to interfere with collagen alignment and alter fibre morphology [Hu et al. (1996)J.

4.2.2.2 Effects of Lysine and Arginine Capping on Enzyme Degradatiort The capping of amino functionalities of lysine with mono-iünctional epoxides (with or without arginine modification) altered the enzymatic solubility of both native ACM collagen and purified type 1 collagen. The most striking and profound effects were observed with enzymes which primarily target lysine and / or arginine: i.e. trypsin, acetyltrypsin, and cathepsin B. Collagen structure and / or the presence of other extracellular matrix components (i.e. elastin, proteoglycans (PGs), glycosaminoglycans (GAGS)) were also found to play roles in determining the effectç of diemical modïfïcationç on the enzymatic solubilization of coilagen. These observations were primarily denved £rom measurements of total collagen and gelchromatography molecular weight distribution analyses of collagen bagments solubilized by enzymes during in vitro assays. Chemical modi£ication of Lysine and arginine (except with BMG) did not significantly effect the solubilization of native ACM coilagen by microbial collagenase (Figure 30). This was expected since rnicrobial collagenase deaves at hydroxyproline and glutamine as well as arginine and lysine [French et al. (1992)l. BMG treatment increased solubilization by collagenase without altering the pattern of deavage (Figure 36). It is known kom the physical charaderization data presented earlier that BMG treatment destabilizes collagen structure. Therefore, the increased susceptibility of BMG treated native ACM collagen may be due to the gross perturbation of collagen structure. Unlike mammalian colIagenases, Clostriditun histolyriczirn collagenases have been shown to be more effective on denatured collagen or gelatin [Bond and Van Wart (1984)l. In contrast to native ACM collagen, only GMG treatment altered solubilization of purified type 1 collagen by collagenase. In this case, however, a decrease in solubilization was observed (Figure 39). An assnaated change in the pattern of collagen deavage was not observed, however. This indicates that only the amount of collagen solubilized was affected (Figure 44). Interestingly, although 2 out of 3 deavage sites in type 1 collagen which are susceptible to Clostriditm hystolytinrm collagenase involve arginine residues, only GMG- and not BMG-treatment decreased collagen solubilization. Thus, the features of the epoxide modification, in this case glyadol, had a unique effed on collagen degradation. It may be hypothesized that the glyadol modification slightly perturbed the conformation of type 1 collagen so as to alter or bury residues involved in collagenase binding. Spatial and conformational characteristics of amino aads around collagenase deavage sites play important roles in degradation; however, their exact characteristics remain unknown [French et al. (1992); Mallya et al. (1992)]. Interestingly, the changes in GMGtreated type 1 collagen that affeded collagenase binding was not apparent in native ACM collagen. Therefore, this observation was unique to purifïed type 1 collagen. The extracted and purified collagen may have had more freedom to reach a conformation which prevented collagenase binding whereas native ACM collagen was affected by the presence of other extraceiidar mahix components (i.e.elastin, GAGS, and PGs). Solubilization of collagen by cathepsin B could be either increased or decreased by lysine and arginine modification. BMG or n-B treatments inaeased the solubilization of collagen, regardless of the type of collagen modified (Figures 29 and 39). The increased solubilization must be attributed to the dismption of collagen structure caused by n-B and BMG treatments. Cathepsin B has been shown to require the disruption of collagen structure if it is to be effective on insoluble collagens [Etherington and Evans (1977); Burleigh et al. (1974)j. The disruption of collagen is usually obtained through swelling under aadic conditions, however this did not play a role in the observed results. Although a reduced amount of swelling was observed in purified type 1 collagen after GMG and BMG treatments, BMG treatment increased and GMG treatment decreased solubilization of collagen by cathepsin B (see Figure 38 and Table 7). In contrast to n-B treatment, G treahnent of native ACM collagen decreased the solubilization of collagen by cathepsin B (Figure 29). Interestingly, GMG treatment did not alter collagen degradation by cathepsin B in either native ACM or purified type 1 collagens. This observation was curious since one of the pnmary targets for cathepsin B is arginine whch was modified by methylglyoxal treatment. This result may again be a case (as presented earlier for (i) EDC+G treatment and cathepsin B and (ii)GMG treatment and collagenase) where G treahent apparentiy imparts unique conformational changes in collagen which alter or bury binding sites recognized by cathepsin B. In their study, Diamond et al. obsewed that the modification of lysine in type 1 collagen by acetylation decreased degradation by cathepsin B [Diarnond et al. (2991)l. Modification of lysine by succinylation, however, did not affect degradation. This suggests that acetylation may also have a unique effect on coiiagen conformation-as in this study with G treatment. Any interpretation and cornparison of Diamond's work to the present study, however, is difficult since those workers did not determine the extent of modification of lysine by each treahnent. By far the most dramatic and intriguing effects of lysine and arginine capping on collagen degradation, were obtained with trypsin and acetyltrypsin digests. Both trypsin and acetyltrypsin recognize lysine and arginine as sites for collagen deavage and these residues are also the targets for epoxide and methyiglyoxal modification. Interestingly, the susceptibility of native ACM collagen or purified type 1 collagen to trypsin was either unaltered or increased-regardless of treahnent (Figure 27 and 39). This was espeaally surprising for GMG and BMG treatments considering that over 87 % of lysine and over 72 % of arginine residues had been modified for both treatments. Modification of lysine by methylation and arginine by cydohexanedione in proteins has been shown previously to prevent degradation by trypsin [Lin et al. (1969); Lee et al. (1978); Toi et al. (1965)l. Interestingly, in the present study, GMG and BMG treatments also altered the pattern of collagen solubilization by trypsin (Figure 34). The inability of GMG and BMG treatments to lower the solubilization of native ACM collagen by trypsin may be attributed to the self-degradation of trypsin during in vitro enzyme degradation assays. The self-degradation of trypsin occurred even with the addition of calcium cations (CaCl,) to prevent autolysis during in vitro experiments (Figure 26) [Rice et al. (1977); Walsh (1970)l.Partial degradation of trypsin has been shown to produce a form of trypsin (pseudotrypsin) which has chyrnotryptic properties, thus recognizing tyrosine and hydrophobie amino aads as primary targets for collagen deavage [Keil-Dlouha et al. (1971)l. The self-degradation of trypsin may have also played a role in producing the altered deavage patterns of collagen found for GMG and BMG beated ACM artenes. Consequently, in order to eliminate the influence of trypsin self-degradation from the results of the in vitro degradation assay, a fonn of trypsin resistant to autolysis (acetyltrypsin) was utilized. It is interesthg to note that acetyltrypsin is a form of trypsin in which lysine residues have been chemically modified through acetylation to prevent self-degradation [Labouesse and Gervais (1967)l. When exposed to acetyltrypsin, epoxy-treated native ACM collagen responded in a similar manner as observed for trypsin. Capping of lysines with glycidol did not alter the solubilization of native collagen, whereas n-B treatment significantly increased the amount of collagen solubilization (to well over 90%) (Figure 28). The increased solubilization of collagen after n-B treatment by acetyltrypsin may be atû-ibuted to the destabilization of coLiagen and an associated increase in the access of enzyme to unmodified arginine containing deavage si tes. Most interesting, however, were the effects of combined epoxide and methylglyoxal treatments on the degradation of collagen by acetyltrypsin. For both native ACM and purified type 1 collagens, GMG treatment significantly reciilced collagen solubilization by acetyltrypsin whereas BMG treatment significantly increased collagen solubilization (Figures 28 and 39). Although epoxy and methylglyoxal treatment of both native ACM and purified type 1 collagen produced similar dianges in acetyltrypsin degradation, the pattern of collagen deavage was altered for purified type 1 collagen oniy (Figures 35 and 43). This difference may reflect the presence of other foms of collagen (i-e.types III, IV, V etc.) and/or the presence of other extracellular matrix components in native ACM collagen. The reason for the vast difference in solubilization of collagen by acetlytrypsin after GMG and BMG treatments is undear. Differences in the extent of modification of arginine with methylglyoxal and lysine with each epoxide could not have been responsible since each amino aad was modified to the same extent with both treatments of both collagen types. Therefore, the different structures and/ or chernistries of the two epoxides used to modify lysine residues must be responsible for the contrasting effects on collagen solubilization. Although û-ypsin has been one of the most studied degradative enzymes, a dear and complete understanding of its recognition, binding, and catalytic deavage of pro teins remains elusive. Furthermore, to the best of this author's knowledge, similar observations on the effects of diemical modification of lysine and arginine in collagen akin to this study have never been reported. Thus, any hypothesis presented here is speculative at best and is formulated £rom indirect observations derived from the published literature. That being said, a possible explanation for the differences between the degradation of GMG and BMG treated collagens by acetyltrypsin may be derived from three considerations: (i) the sbuchire of the catalytic podcet of trypsin, (ii) the requirements for hydrolytic deavage of a substrate by trypsin as is currently understood, and (iii) the proposed flexibility of trypsin. First, the skcture of trypsin has been determined through X-ray crystallography and the attributes of its catalytic site are known. The wails of the catalytic podcet contain hydrophobic amino aads which are lacking bulky side-chains to permit the access to large substrate side-chahs such as that of lysine [Perona et al. (1993); Kawaguchi et al. (1997); Fink (1987)l. Second, it is now known that the initial binding of the substrate is less important than the £irst attadc of the catalytic machinery on the scissile bond (acylation step) in the hydrolysis of peptide bonds by trypsin [Hedstrom et al. (1994a),(1994b); Perona et al. (1995); Hedstrom (1996); Fink (1987)l.Thus, the binding of enzyme to substrate does not necessarily have to be a perfect fit as long as the sassile bond is properly positioned to the catalytic groups which are responsible for peptide hydrolysis. In this study, it appears that the long, hydrophobic n-B modification of lysine is readily recognized by acetyltrypsin whereas the small hydrophilic modification is not. Therefore, the hydrophilic nature of the glyadol modification of lysine derived from the presence of two hydroxyl groups in the epoxide must sornehow prevent the correct aiignrnent of the scissile bond to the catalytic machinery of the enzyme. nie exact manner by which glyadol modification prevents a correct alignment in the catalytic podcet remains undear. It is intriguing how the large, hydrophobic n-B modification of lysine is still recognized by acetyltrypsin. A degree of flexibility in the primary binding podcet of trypsin has been demonstrated through the binding of a wide range of side-chains of various configurations uiduding the samino of lysine, guanido of arginine, and the inhibitor benzarnidine [Perona et al. (1993)l. Furthermore, the Iength of the carbon chain before an amino group has also been shown to be important to ûypsin-mediated hydrolysis of proteins [Hatton and Regoeui (1975); Baird et al. (1965); Walsh (1970); Baines et al. (1964)l.These studies however, have only investigated diain lengths up to 5 carbons long (equivalent to lysine) and the exact importance of the hydrophobic nature of the diain is unknown. Further information for a possible hypothesis cm be gained from the investigation of enzymes which can recognize both polar and non-polar side- diains. The mechanism of recognition of acidic and hydrophobic substrates by Eschericllin coli aspartate aminotransferase and E. coli aromatic amino aad aminotransferase has been attributed to requirements of hydrophobiaty of the active site and enzyme flexibility [Kawaguchi et al. (1997)j. These charaderistics are also found in trypsin. It is known that the correct positioning of the sassile bond is more important to hydrolysis than the proper side-diain functionality being present within the catalytic podcet of trypsin [Hedstrom et ai. (1994a),(1994b);Perona et al. (1995); Hedstrom (1996); Fink (1987)l. Furthennore, it is known that extended substrate binding sites, which may have remained unaltered after epoxy modifications in this study, contribute substantially to subsbate specifiaty. Thus, a hydrophobic and flexible catalytic podcet, together with optimal scissile bond placement facilitated by extended substrate binding sites, may have been responsible for the ability of acetlytrypsin to bind and cleave n-B modified lysine residues wi thin collagen. One main point of contention with the hypothesis presented that cannot be addressed, is exactly how the n-B modified lysine remains a target for acetyltrypsin. Questions remain as to the conformation of the 13 carbon long n-B modified lysine side-chain (linear or cydic) and how acetyltrypsin accommodates a diain seven carbons longer than lysine within its catalytic pocket. Regardless, the demonstrated inaease in solubilization of BMG-treated collagens and decreased solubiiization of GMG-treated coilagens by acetyltrypsin raises intriguing new questions regarding enzyme binding and recognition that dearly need to be addressed. In order to answer these questions, studies involving detailed molecular modeling of (i) enzyme binding and (ii) conformational changes due to chernical modifications would be required. However, this is beyond the scope of this thesis. Tissue-derived materials require treatment to increase their stability and to prevent degradation by enzymes after implantation. The standard technique used to prevent the degradation of tissue-derived materials has been chemical crosslinking. Gosslinking has been hypothesized to work by sterically inhibiting the abitity of enzymes to attach to and degrade implanted materials. The purposes of this study were (i) to determine if increased resistance to degradation can be obtained by chemically modifying speafic amino aad side- chains witliolit uosslfizhing and (ii) to determine if increased resistance to enzymatic degradation is not merely due to stenc effects, but indudes direct interference with enzyme-substrate binding. Ln this study, the effects of chemical modification of speafic amino acid side-chahs on the physicd and enzyme degradative properties of native ACM and purified type 1collagens was investigated. First, carboxyl groups of aspartic and glutarnic acids were rnodified by capping with glyane methylester mediated by the crosslinking reagent l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). Next, (i) arginine residues were modified with methylglyoxal and (ii) two stnicturally different epoxides were utilized to modify the amino groups of lysine and hydroxylysine: glyadol (small, hydrophilic) and n-butylglycidylether (large, hydrophobic). Third, in order to compare the effeds of chemical modification to crosslinking, the EDC crosslinking of native ACM collagen with and without pH control was also investigated. EDC crosslinking involved the same arnino aads targeted by the reagents used for chemical modifications: aspartic and glutamic aads, lysine and hydroxylysine. EDC crosslinking without pH control was found to significantly increase the shear stiffness, to increase the amount of lysine residues consumed in the crosslinking reaction, and to produce less increase in the thermal stability of native ACM arteries (compared to EDC crosslinking at a fixed pH of 5.5). In vitro enzyme degradation by trypsin, acetyltrypsin, cathepsin B, and collagenase however, were similar for EDC-treated ACM artenes-regardless of pH control. The differences in shear stiffness, thermal stability, and lysine residues consumed with EDC treatment under the two pH conditions, is attributed to a change in the type and location of exogenous crosslinks. A drop in pH during EDC treatment without pH control-due to protonationl deprotonation of EDC during the crosslinking reaction-facili tated the formation of interfîbrillar crossiinkages. As a result, layers of fibres within the structure of ACM artenes became more intercomected. These interconnections likely involved collagen and other extracellular matrix components such as proteoglycans, elastic tissue, and glycosaminoglycans. Most importantly, differences in the type of crosslinking obtained with and without pH control did not affect the increased resistance of the EDC-treated ACM arteries to enzymatic solubilization. The speufic chemical modification of amino aad side-chahs was found to significantly alter the in vitro enzymatic solubiiization of both native ACM and purified type 1 collagens. Modification of carboxyl groups of aspartic and glutamic acids by - 25% increased solubilization of native collagen by trypsin, acetyltrypsin, and collagenase. Solubilization of native ACM collagen by cathepsin B, however, was significantly reduced. Although no gross changes in the structure of coliagen could be detennined, smd conformational changes in collagen structure caused by the alteration of carboxyl side-chahs are hypothesized to have occurred. These conformational changes could have opened up new sites of collagen deavage to -sin, acetyltrypsin and collagenase (Figure 50). Similady, conformational changes may have altered or buned some of the collagen deavage sites recognized by cathepsin B (Figure 49). Furthemore, the blodcage of secondary binding sites involved in cathepsin B binding may have been responsible for deueased solubilization of collagen after carboxyl capping only with EDC+G treatment (Figure 48b). The most striking effects on the enzyme degradation of collagen was observed with lysine modification using glyadol (G) and n-butylglycidylether (n-B) treatment, with or without arginine modification using methylglyoxal. The alteration of collagen susceptibility to enzymes was dependent upon the properties of the lysine modifying reagent. Overall, the large, hydrophobie (a) Primary Blocking (b) Secondary Blocking

Prirnary Binding Site A~econdar~Binding Site 0 Chemical Modification

Figure 48. Hypothesized inhibition of enzyme binding through blocking of (a) primary sites in the catalytic domain or (b) secondary sites.

Modification

Figure 49. Hypothesized rotation of collagen polypeptide chains within the triple helix due to amino acid side-chain modification exposes or bunes cleavage sites recognized by enzymes.

Chemical

Figure 50. Hypothesized changes in polypeptide conformation due to amino acid sidechain modification opens-up the structure of collagen to enzymes. n-B modification inaeased the susceptibility of both native ACM and purified type 1 collagens to solubilization by enzymes. In contrast, the smd, hydrophilic reagent G, did not alter--or significantly reduced-solubilization of native ACM and purified type 1collagens. The effect of chernical modification of amino aad side-chahs on degradation was most significant when modified residues of lysine and arginine were speafic targets of the degradative enzymes. Both the amount of solubilization and the deavage sites of collagen were found to be altered by modification of lysine and arginine. The architedure of collagen (i.e. fibre, fibre bundles) and presence of other extraceiiular matrix components influenced the effect of chemical modifications on degradative behaviour. This was shown by contrasting results obtained with native ACM and purified type 1 collagen. Changes in the solubilization of collagen may be due to (i) the direct modification of residues recognized by enzymes (trypsin and acetylbypsin) (Figure 48a) or (ü) interference with secondary enzyme binding; either by altering side-chain functionaiities or through coLlagen conformational changes induced by side-chah modifications (cathepsin B and collagenase) (Figures 48b, 49, 50). Ultimately, the results of this study have shown that enzymatic solubilization of collagenous materials cm be increased or decreased by the chemical modification of amino acid side-chains directly or indirectly involved in enzyme binding. In cornparison to EDC crosslinking, chemical modification of arnino acids was not as broadly effective at reducing degradation by enzymes; however significant reductions in collagen solubilization for some enzymes was observed. Therefore, the ability of crosslinked tissue-derived materials to resist enzyme degradation in vivo may be (at least in part) dependent upon the masking or alteration of recognizable sites of deavage. The effediveness of treatments such as glycidol plus methylglyoxal in preventing collagen degradation in vivo remains to be determined. Effective stabilization of a tissue-denved device (for example a vascular graft) may involve a combination of techniques, such as lysine and arginine capping to prevent collagen degradation in conjunction with carboxyl capping showpreviously to reduce elastolysis [Hall and Czerkawski (1961)l. It should also be considered that the demonstrated ability to modulate (increase or decrease) the enzymatic breakdoivn of collagenous materials may faahte the design of new engineering scaffolds for tissue regeneration (skin, nerve, tendon, etc.) and bug and peptide delivery systems. The modulation of degradation rates may dowfor proper remodeling of tissue engineering scaffolds by host cells or facilitate the design of unique dmgdelivery profiles from collagen or other protein based systems. 5.1 Relating Results of the Study Back to the Origlnal Hypotheses (1) 7he extent of enzymatic dubilization of collagenous matends will be reduced by specific chemical modification of the functional side-groups of amho aads which are recognized by degradative enzymes.

Valid for certain cases: Capping of amino aad side-chains recognized by degradative enzymes did not reduce the solubilization of collagen in ali cases. However, chemical modification of side-chains of amino acids which (i) are or (ii) are not specificaily targeted by enzymes produced inaeased and deaeased sotubilization of collagen. Furthemore, both the patterns of deavage of collagen by enzymes and the amounts of collagen solubilized were aitered.

(2) Alteration of the chernical moiety (hydrophilic or hydrophobic) or size of amino acid side-chains will modulate the reduced solubilization of collagenous materials to different degrees.

Validated: Significant and extreme differences were observed for the enzymatic degradation of materials modified by the small, hydrophilic epoxide glycidol and large, hydrophobic epoxide n-butylglycidylether. Direct evidence of this is shown by results obtained with trypsin and acetyltrypsin where despite ~90%modification of lysine and = 80% of arginine in each case, BMG-treated matenals displayed over 90% and GMG-treated materials less than 20% collagen solubilization. 6. CONCLUSIONS

6.1 EDC Crosslinking and pH Control 1. EDC crosslinking significantly increases the shear stifhess of ACM-processed carotid arteries; this effect is greatest without pH control.

2. EDC lreatment of ACM-processed artenes without pH conh-ol preferentially encourages formation of interfibrillar crosslinkages between coLIagen and other extracellular matrix components. Treatment at pH 5.5 encourages greater intraheiicai / intrahelical crosslinking.

3. The type of crosslinks formed (intrahelical or interfibrillar) does not al ter the ability of EDC treated tissue-derived materials to resist enzymatic solubilization in vitro.

6.2 Chemical Modification of Amino Acid Side-Chains 1. Chemical modification of speafic amino aad side-chains can increase or decrease the solubilization of collagen by enzymes in vitro.

2. Chemical modification of amino acid side-chahs can alter the fragmentation pattern in enzymatically solubilized collagen.

3. A small, hydrophilic modification of lysine does not alter (or decreases) collagen solubilization while a large, hydrophobie lysine modification increases collagen solubilization.

4. The structure of collagen (native or purified type 1) and the presence of other extracellular matrix components influences the effect of diemical modifications on enzyme degradation. 1. Glycidol plus methylglyoxal and n-butylglyadylether plus methylglyoxal treatments should be studied to determine if the observed in vitro results are seen in vivo.

2. Epoxide and methylglyoxal treatments should be investigated with other protein substrates (for example albumin) to determine if the effects on degradation observed are collagen-specific.

3. The mechanism by which a smd, hydrophilic modification of lysine is not recognized and a large, hydrophobie modification of lysine is recognized by (acety1)trypsinshould be investigated further through molecular modeling. This may lead to new insights into enzyme recognition and binding.

4. The effects of chernical modification of speafic amino aad side-chah on the antigenicity and immunogenitity of collagenous materials be determined first through in vitro antibody binding assays and then by in vivo shidies.

5. The mechanisms by which collagen may be aosslinked with other tissues components (e.g. elastic tissue, proteoglycans) should be investigated in order to isolate undesired mechanical changes from resistance to enzyrnatic solubilization. This may be investigated by the removal of proteoglycans from tissues using guanidine hydrochloride treatment. 8. REFERENCES Allain, J.C., LeLouis, M., Bazin, S. Bailey, A,J., and Delaunay, A. (1978) "Isometric Tension Developed During Heating of Collagenous Tissues: Relationships

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Prodrict

Ace ty l trypsin Calaum chionde (dihydrate) Cathepsin B Chlorarnine-T Collagenase (type 1- Clostridkirn Histolytincm) Cysteine Diethyl ether DNAse Ethylenediaminetetraacetic acid (EDTA) 1-Ethyl-3-(3-dimethylaminopropyl)carbodümide (EDC) Glyadol Glyane methyl ester Hydroxy-L-proline Me thyl cellosolve Methylglyoxal n-Butylglyad ylether n-Hydrox y sucunimide p-Dimethylaminobenzaldehyde (Ehrlich's Reagent) ~henyImethylsulfonylfluonde(PMSF) Potassium chloride RNAse Sodium bicarbonate Sodium Dodecyl Sulfate (SDS) Sodium formate Sodium phosphate 2,4,6-Trinitrobenzenesdfonicacid (TNBS) Triton X-IO0 Trizma base Trizma hydrochloride Trypsin (type 1- bovine pancreas) Type I collagen (bovine Achilles tendon)

Gi bco-BRL, Life Technologies Inc., New York, USA

Prodrict

Peni cillin-streptomycin (10,000U / ml, 10,000 mg / ml) 15140-22 Sterile phosphate buffered saline (PBS) 14190-29 VWWCanlab, Mississauga, Ontario

Hydrochloric acid (6N) Perchloric aad (70%) Sodium hydroxide (ION)

Biorad Laboratories, Mississauga, Ontario

Prodtlcf Cotalog Number

Econo chromatography column (1 x 120 an) Econo-pac flow adapter Gel filtration standards

Pharmacia Biotech Inc., Baie D'urfé, Quebec

Prodrict Catalog Nurnber

Sephacryl S-200 HR column packing 17-0584-10 FRAC 100 Automated fraction collector 18-1000-17 UV4 In-line ultraviolet absorbante detector 18-1003-66 1 Channel chart recorder (mode1 #481) (discontinued)

Waters-miIli pore, Massachusetts, USA

Waters 501 HPLC solvent delivery pump