Synthesis and characterization of degradable polymer hydrogels for tissue scaffolds

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Synthesis and characterization of degradable polymer hydrogels for tissue scaffolds
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xxx, 315 leaves : ill. ; 29 cm.
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Campbell, Angela L., 1963-
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Thesis:
Thesis (Ph. D.)--University of Florida, 1992.
Bibliography:
Includes bibliographical references (leaves 309-313).
Statement of Responsibility:
by Angela L. Campbell.
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Typescript.
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Vita.

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University of Florida
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SYNTHESIS AND CHARACTERIZATION OF
DEGRADABLE POLYMER HYDROGELS FOR TISSUE SCAFFOLDS


















By

ANGELA L. CAMPBELL


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1992






























The author dedicates this dissertation to her Lord and Savior

Jesus Christ without whom the completion of this project would

have been impossible.














ACKNOWLEDGEMENTS


The author would like to express her appreciation for the

patience, guidance, encouragement, instruction and support she

received from her primary advisor Dr. Christopher D. Batich.

She is also thankful for the support, advice, and

encouragement received from the remaining members of her

supervisory committee, Dr. Eugene P. Goldberg, Dr. Kenneth B.

Wagener, Dr. Fereshteh Ebrahimi, and Dr. Kevin S. Jones.

The author extends her gratitude to other faculty and

staff members who provided invaluable advice during the course

of her studies. These include former Dean Roderick McDavis,

Dr. Saeedur R. Khan, Dr. David F. Senior, Dr. Anthony Brennen,

Dr. Marc S. Cohen, Dr. William Toreki, Dr. Roy King, Dr. Nai

Zheng Zhang, Dr. Lynn Peck, Dr. Jeanne Quigg, Wayner Williams,

Gail D. Anderson, Susan J. Studstill, Barbara E. Folino,

Steven Bourdon, and Paula Shevock.

Special thanks go to those individuals who helped the

author with several analytical techniques. The author thanks

Guy Latorre for his assistance, instruction and guidance with

the FT-IR analysis. The work of John West and Dr. Wallace S.

Brey with the NMR analysis was greatly appreciated, as well as

the assistance of Chris Marmo with the GPC analysis. The

assistance of Fabio Zuguala with the GPC analysis was helpful

iii








as well as valuable. The assistance of Jesse Mitrani with the

UV analysis was greatly appreciated.

The author is thankful for the patience and assistance of

Don DePalma with the computer programs, which made it possible

for her to generate all of the graphs in this dissertation.

The valuable assistance, advice, encouragement and

support received from Dr. Ali Yahiaoui was greatly appre-

ciated. The advice assistance, encouragement and support from

Khalid Mentak and Drew Amery was appreciated as well.

Thanks are extended to the students and staff in the

Batich Research Group for their patience, support and

assistance; Lili Mateo, Kirk Foster, Guang Choi, Ed Morales,

Dr. Yan Jun, Lei Wei, Dr. Maher Elasabee and Dr. I Rezaeian,

Madana Balaji, Ernie Serrano, Ian McFetridge, Shannon Eggers,

Special thanks go to Lili and Kirk for their help in trans-

porting supplies from the Health Center. This was always

timely and very helpful.

A very special heartfelt thanks is extended to Lori

Clayton whose priceless and selfless assistance made the

production of this publication possible. The author is

indebted to her for the time and sacrifices that she offered,

and prays for a one hundred-fold return on her giving. The

author is grateful for the patience of her family in allowing

her to spend the time to complete this dissertation. The

author also thanks Shannanda Caffe for her assistance as

well.







A special thanks goes to Damon Austin for his help

throughout the duration of this project.

The author expresses gratitude to Dr. Israel Tribble, and

the Florida Endowment Fund for the McKnight Doctoral

Fellowship that was awarded to her and for the foundational

financial support, academic support and encouragement that

they provided. Thanks is extended to the Materials Science

and Engineering Department and to the National Institutes of

Health for their financial assistance as well. The author is

also appreciative of the employment opportunity given to her

by the Air Force through the Senior Knight Program which made

the completion of this project possible.

The author expresses her deepest appreciation for the

support, prayers, encouragement and patience of her mother,

Evelyn C. Campbell, and her family, Viola M. Cooper, William

R. Campbell, M.D., Shana M. Campbell, and Kimberley C.

Campbell. The prayers and encouragement of the members of

Agape' Faith Center Ministries were appreciated as well.

Finally, and most importantly, the author gives thanks to

her Lord and Savior Jesus Christ who provided all of the

strength, stamina, understanding, knowledge, wisdom and

assistance necessary to complete this project.
















TABLE OF CONTENTS


ACKNOWLEDGEMENTS . .

LIST OF TABLES . .

LIST OF FIGURES . .

KEY TO ABBREVIATIONS AND SYMBOLS .

ABSTRACT ...............

1 GENERAL INTRODUCTION AND BACKGROUND

1.1 General Introduction .
1.2 Background .
1.2.1 Description of Hydroge
1.2.2 Classifications of Deg


. iii


S. X

. xxvii

S. xxix

. 1


s .
radable


Polymers . .
1.2.3 Description of Tissue Scaffolds
1.2.4 Applications for Biodegradable
Hydrogels . .
1.2.5 Polymers and the Inflammatory
Response . .
1.2.6 Mechanism of In Vivo Degradation f,
Polymers . .

2 MODIFICATION AND CHARACTERIZATION OF ALBUMIN AND
DEXTRAN . .

2.1 Introduction . .
2.2 Materials and Methods . .
2.2.1 Materials . .
2.2.2 Methods . .
2.2.2.1 Modification of albumin .
2.2.2.2 Modification of dextran .
2.2.2.3 FT-IR of modified albumin
2.2.2.4 FT-IR of modified dextran
2.2.2.5 NMR of modified albumin and
modified dextran .
2.3 Results and Discussions .. .
2.3.1 FT-IR of Modified Albumin .
2.3.2 FT-IR of Modified Dextran .


. 4
S. 5

S. 7

11
Dr
.. 14


16

16
29
29
30
30
S. 31
31
. 32


. .


. .




rr



rr
r








2.3.3 IH-NMR of Modified Albumin 42
2.4 Conclusions . . 65

3 POLYMERIZATIONS TO FORM DEGRADABLE POLYMER
HYDROGELS . ...... 66

3.1 Introduction . 66
3.1.1 Hydrogels of Biomedical Applications 66
3.1.2 Polymerization of Hydrogels 68
3.1.3 Factors which Affect Hydrogel
Properties . 69
3.2 Materials and Methods . 73
3.2.1 Materials . 73
3.2.2 Polymerization of Degradable
Hydrogels .. . 74
3.2.2.1 Thermal polymerization .. 74
3.3 Results and Discussion . 77
3.3.1 Thermal Polymerization 77
3.3.1.1 Acrylic acid, acrylic acid/
modified albumin and acrylic
acid/modified dextran hydrogels 77
3.3.1.2 N-vinyl pyrrolidone, N-vinyl
pyrrolidone/modified albumin, and
acrylic acid/N-vinyl pyrrolidone/
modified albumin hydrogels 77
3.3.2 Gamma Polymerization . 83
3.3.2.1 Acrylic acid/modified albumin
and acrylic acid/modified
dextran hydrogels 83
3.3.2.2 N-vinyl pyrrolidone/modified
albumin and N-vinyl pyrrolidone
/modified dextran hydrogels 83
3.4 Conclusions . . 84

4 SWELLING AND DEGRADATION BEHAVIOR OF DEGRADABLE POLYMER
HYDROGELS .. . . 85

4.1 Introduction . . 85
4.2 Materials and Methods . .88
4.2.1 Materials . 88
4.2.2 Procedure for Swelling Studies 89
4.2.3 Degradation Studies with Ultraviolet
Spectroscopy . 94
4.2.4 Gel Permeation Chromatography of the
Degradation Products in the Swelling
Media . 95
4.2.5 Degradation of Hydrogels with 1 M HC1
and Gel Permeation Chromatography 97

4.3 Results and Discussion. . 98
4.3.1 Swelling Studies . 98


vii








4.3.1.1 Preliminary swelling studies
(SS#0-SS#6) 99
4.3.1.2 Control swelling studies 110
4.3.1.3 Acrylic acid/N-vinyl pyrrolidone
copolymer swelling studies (SS#9-
SS#11) . 112
4.3.1.4 Swelling studies of hydrogels with
modified dextran 119
4.3.1.5 Swelling studies with purified
crosslinking agents .. 121
4.3.1.6 Gamma polymerized hydrogel swelling
swelling studies SS#15-SS#18. 123
4.3.1.7 Enzyme swelling studies (ESS) in
in unactivated papain
(ESS#1-ESS#4) .. 127
4.3.1.8 Degradation of polymer hydrogels
with 1 M HC .. 137
4.3.1.9 Mass swelling study #1 140
4.3.1.10 Mass swelling study #2 164
4.3.1.11 Mass swelling study #3 195
4.3.2 Ultraviolet Spectroscopy of the Swelling
Media . 256
4.3.3 Gel Permeation Chromatography of the
Swelling Media and the Hydrogels
Autoclaved with 1 M HC1 267
4.4 Summary and Conclusions . 279

5 SUMMARY, CONCLUSIONS AND FUTURE WORK 286

5.1 Summary . . 286
5.2 Conclusions .. 287
5.3 Future Work . .. 301

APPENDIX . . 303

REFERENCE LIST . . 309

BIOGRAPHICAL SKETCH . . 314


viii










Table 1-1


LIST OF TABLES

Summary of Applications for Hydrogels


Table

Table


Table

Table


1-2

1-3


3-1

3-2


Table 3-3


Table 3-4


Table 3-5


Table 3-6


Table 3-7


Table

Table

Table


The Stages of Inflammatory Response 13

The Four Stages of In Vitro Polymer
Degradation (14) . 15

Components of Hydrogels .. 70

Summary of Monomer Solution
Compositions . 75

Monomer Solution Compositions for
Polymerized Hydrogels 76

Vital Statistics of Hydrogels
Polymerized . .. 78

Vital Statistics for Hydrogels in Mass
Polymerization 1 . .. 80

Vital Statistics for Hydrogels Polymerized in
Mass Polymerization #2 .. 81

Vital Statistics for Hydrogels Polymerized in
Mass Polymerization #3 . 82

Endopeptidases and Exopeptidases 85

Summary of Swelling Studies 91

Results of Autoclaving of the Hydrogels
in 1 M HC1 at 1000C for 24 hours 139

Predictions of Hydrogels Suitable as
Degradable Hydrogels .. 141

Hydrogels Suitable for Use as Degradable
Hydrogels . 282

Comparison of the 3% MA and 10% MA
Hydrogels . 284


4-1

4-2

4-3


Table 4-4


Table 4-5


Table 4-6


S 4








LIST OF FIGURES


Figure 2-1.


Figure 2-2.




Figure 2-3.




Figure 2-4.



Figure 2-5.


Figure 2-6.


Figure 2-7.


Figure 2-8.


Figure 2-9.


Figure 2-10.


Figure 2-11.


Figure 2-12.


Figure 2-13.



Figure 2-14.

Figure 2-15.


Schematic of the linkages between C-1 and
C-6. Source: Reference 24. .. 18

Loop structure of bovine serum albumin
indicating disulphide bonds forming loops,
subdomains and domains. Source:
Reference 30 .. .. 19

Diagram of Albumin primary structure showing
its eight and a half double disulfide loops
and some suggested binding sites. Source:
Reference 28 . 19

Primary structure of the carboxy-terminal
double loop of bovine serum albumin, the
"Phe-fragment". Source: Reference 30 19

Repeat unit of dextran. Source:
Reference 24 . .. 21

Reaction for Modification of Albumin.
Source: Reference 5 . 22

Reaction for the Modification of Dextran.
Source: Reference 6 . 23

FT-IR spectra of 5% pure unmodified albumin
(circle cell). . .. 34

FT-IR spectra of modified albumin 3/1/91
(circle cell). . ... 34

FT-IR spectra of modified albumin 4/25/91
(circle cell). .. 35

FT-IR spectra of UMA-PBS vs. MA-Al-PBS
(circle cell). . .. 36

FT-IR spectra of UMA-PBS vs. MA-B-PBS
(circle cell). . 36

Structures of Free Amino Acid, Amino Acid
Hydrochloride, and Amino Acid Sodium Salt.
Source: Reference 30 .. 37

FT-IR spectra of pure albumin BM (KBr) 39

FT-IR spectra of modified albumin reacted
for 5 hours (KBr). .. . 39








Figure 2-16.


Figure 2-17.


Figure 2-18.


Figure 2-19.

Figure 2-20.

Figure 2-21.

Figure 2-22.


Figure 2-23.


Figure 2-24.


Figure 2-25.



Figure 2-26.



Figure 2-27.


Figure 2-28.



Figure 2-29.



Figure 2-30.


Figure 2-31.


FT-IR spectra of modified albumin reacted for
24 hours (KBr) .. ... .. 40

FT-IR spectra of UMA BM vs. MA 5 hours
(KBr) .. .. . 41

FT-IR spectra of UMA BM vs. MA 24 hours
(KBr) .... .... 41

FT-IR spectra of pure dextran (KBr) 43

FT-IR spectra of modified dextran2 (KBr) 43

FT-IR spectra of modified dextran (KBr) 44

FT-IR spectra of UMD vs. modified
dextran2 (KBr) .. 44

NMR spectra of pure albumin in D20
(H9135, 8.80 ppm 1.3 ppm) 45

NMR spectra of pure albumin in D20 (H9135,
11.38 ppm 3.71 ppm) . 46

NMR spectra of modified albumin in D20
reacted for 5 hours (H9173, 7.99 ppm -
0.07 ppm) . 47

NMR spectra of modified albumin in D20
reacted for 10 hours (H9175, 8.47 ppm -
0.26 ppm) . 48

NMR spectra of pure albumin in D20
(H9135, 3.61 ppm 0.29 ppm) 50

NMR spectra of modified albumin in D20
reacted for 5 hours (H9173, 3.61 ppm -
0.29 ppm) . 51

NMR spectra of modified albumin in D20
reacted for 10 hours (H9175, 3.60 ppm -
0.29 ppm) . 52

NMR spectra of pure albumin in D20
(H9135, 8.24 ppm 6.04 ppm) 53

NMR spectra of modified albumin in D20
reacted for 5 hours (H9137, 7.89 ppm -
6.40 ppm) . 54








Figure 2-32.



Figure 2-33.

Figure 2-34.


Figure 2-35.



Figure 2-36.


Figure 2-37.



Figure 2-38.


Figure 2-39.



Figure 2-40.

Figure 3-1.

Figure 4-1.




Figure 4-2.




Figure 4-3.




Figure 4-4.


NMR spectra of modified albumin in D20
reacted for 10 hours (H9175, 7.89 ppm -
6.4 ppm) . . 55

The ABX system . .. 56

NMR spectra of pure dextran in D20 (H9174,
5.07 ppm 3.38 ppm) . 58

NMR spectra of modified dextran in D20
reacted for 7 days (H9134, 6.57 ppm -
2.88 ppm) . 59

N"MR spectra of pure dextran in D20 (H9174,
4.06 ppm 3.38 ppm) . 60

NMR spectra of modified dextran in D20
reacted for 7 days (H9134, 4.06 ppm -
3.38 ppm) . 61

NMR spectra of pure dextran in D20 reacted
for 7 days (H9174, 6.69 ppm 4.69 ppm) 62

NMR spectra of modified dextran in D20
reacted for 7 days (H9134, 6.69 ppm -
4.69 ppm) . 63

NMR spectra of modified dextran in D20
(H9134, 4.69 ppm 6.69 ppm) 64
Schematic of the Structure of Hydrogels 67

Average Swelling Ratio vs. Time for SS#0 in
PBS. 30% AA/ALB/AIR, tp = 1 hour, 3% MA.
The high and low values represent the
first standard deviation 101

Average Swelling Ratio vs. Time for SS#1 in
PBS. 30% AA/ALB/AIR, tp = 1 hour, 3% MA.
The high and low values represent the
first standard deviation 102

Average Swelling Ratio vs. Time for SS#2 in
PBS. 30% AA/ALB/AIR, tp = 1 hour, 3% MA.
The high and low values represent the
first standard deviation 103

Average Swelling Ratio vs. Time for SS#3 in
PBS. 30% AA/ALB/AIR, tp = 1 hour, 3% MA.
The high and low values represent the first
standard deviation . .. 105


xii








Figure 4-5.




Figure 4-6.




Figure 4-7.




Figure 4-8.


Figure 4-9.




Figure 4-10.




Figure 4-11.




Figure 4-12.




Figure 4-13.




Figure 4-14.


Average Swelling Ratio vs. Time for SS#4 in
PBS. 30% AA/ALB/N2, tp = 1 hour, 3% MA.
The high and low values represent the
first standard deviation .. ... 106

Average Swelling Ratio vs. Time for SSf5 in
PBS. 30% AA/ALB/AIR, tp = 1 hour, 3% MA. The
high and low values represent the first
standard deviation .. 108

Average Swelling Ratio vs. Time for SS#6 in
PBS. 30% AA/ALB/N2, tp = 1 hour, 3% MA. The
high and low values represent the first
standard deviation .. 109

Average Swelling Ratio vs. Time for Comparison
of SS#5 and SS#6 . .. 111

Average Swelling Ratio vs. Time for SSf7, PAA
controls in PBS. 30% AA/ALB/AIR, tp = 1 hour,
3% MA. The high and low values represent the
first standard deviation 113

Average Swelling Ratio vs. Time for SS#8 PAA
Controls in PBS. 30% AA/APS/N2. tp = 1 hour,
3% MA. The high and low values represent the
first standard deviation .. 114

Average Swelling Ratio vs. Time for SS#9 in
PBS. 30% AA/NVP/ALB/N2, tp = 5.75 hours, 3%
MA. The high and low values represent the
first standard deviation . 116

Average Swelling Ratio vs. Time for SS#10 in
PBS. 30% AA/NVP/ALB/N2, tp = 5 hours, 3% MA.
The high and low values represent the first
standard deviation . 117

Average Swelling Ratio vs. Time for SS#11 in
PBS. 40% AA/NVP/ALB/N2, tp = 24 hours, 3% MA.
The high and low values represent the first
standard deviation . .. 118

Average Swelling Ratio vs. Time for SS#12 in
PBS. 30% AA/NVP/DEX/AIR, tp = 18 hours, 3%
MD. The high and low values represent the
first standard deviation . 120


xiii








Figure 4-15.




Figure 4-16.




Figure 4-17.




Figure 4-18.




Figure 4-19.




Figure 4-20.




Figure 4-21.




Figure 4-22.




Figure 4-23.




Figure 4-24.


Average Swelling Ratio vs. Time for SS#13 in
PBS. 40% AA/ALB/N2, tp = 22 hours, 3% MA.
The high and low values represent the first
standard deviation . 122

Average Swelling Ratio vs. Time for SS#14 in
PBS. 40% AA/DEX/N2, tp = 22 hours, 3% MA.
The high and low values represent the first
standard deviation . 124

Average Swelling Ratio vs. Time for SS#15 in
PBS. 40% NVP/ALB/y/N2, tp = 6.33 hours, 3% MA.
The high and low values represent the first
standard deviation . .. 125

Average Swelling Ratio vs. Time for SS#16 in
PBS. 40% NVP/DEX/y/N2, tp = 6.33 hours, 3% MA.
The high and low values represent the first
standard deviation .. 126

Average Swelling Ratio vs. Time for SS#17 in
PBS. 40% AA/ALB/7/N2, tp = 6.33 hours, 3% MA.
The high and low values represent the first
standard deviation . .. 128

Average Swelling Ratio vs. Time for SS#18 in
PBS. 40% AA/DEX/7/N2, tp = 6.33 hours, 3% MA.
The high and low values represent the first
standard deviation . 129

Average Swelling Ratio vs. Time for ESS#1 in
PBS. 30% AA/ALB/AIR, tp = 1 hour, 3% MA. The
high and low values represent the first
standard deviation . 130

Average Swelling Ratio vs. Time for ESS#1 in
SS#5 in unactivated papain. 30% AA/ALB/AIR,
tp = 1 hour, 3% MA. The high and low values
represent the first standard deviation 131

Average Swelling Ratio vs. Time for ESS#2 in
unactivated papain. 30% AA/ALB/N2, tp = 1
hour, 3% MA. The high and low values
represent the first standard deviation 133

Average Swelling Ratio vs. Time for ESS#2 and
SS#6 in unactivated papain. 30% AA/ALB/N2, tp
= 1 hour, 3% MA. The high and low values
represent the first standard deviation 134


xiv








Figure 4-25.


Figure 4-26.




Figure 4-27.


Figure 4-28.


Figure 4-29.


Figure 4-30.



Figure 4-31.


Figure 4-32.


Figure 4-33.



Figure 4-34.



Figure 4-35.


Figure 4-36.


Figure 4-37.



Figure 4-38.


Average Swelling Ratio vs. Time for ESS#3 in
PBS. 40% AA/ALB/N2, tp = 22 hours, 3% MA. The
high and low values represent the first
standard deviation .. 135

Average Swelling Ratio vs. Time for ESS#4 in
unactivated papain. 40% AA/DEX/N2, tp = 22
hours, 3% MA. The high and low values
represent the first standard deviation 136

Average Swelling Ratio vs. Time for MSS#1-1PBS
in PBS. tp = 1 hour, 3% MA 143

Swelling Ratio vs. Time for MSS#1-1H in 1 M
HC1. tp = 1 hour, 3% MA. .. 145

Average Swelling Ratio vs. Time for MSS#1-1PBS
in PBS then trypsin. tp = hour, 3% MA. 147

Average Swelling Ratio vs. Time for MSS#1-1T
in trypsin after 835.17 hours of swelling in
PBS. tp = 1 hour, 3% MA . 148

Average Swelling Ratio vs. Time for MSS#1-4PBS
in PBS. tp = 4 hours, 3% MA 149

Swelling Ratio vs. Time for MSS#1-4H in 1 M
HC1. tp=4 hours, 3% MA .. 150

Average Swelling Ratio vs. Time for MSS#1-1
PBST in PBS then trypsin. tp = 4 hours, 3%
MA . . .151

Average Swelling Ratio vs. Time for MSS#1-4T
in trypsin after 835.17 hours of swelling in
PBS. tp = 4 hours, 3% MA .. 153

Average Swelling Ratio vs. Time for MSS#1-
12PBS in PBS. tp = 12 hours, 3% MA 154

Average Swelling Ratio vs. Time for MSS#1-12
in 1 M HC1. tp = 12 hours, 3% MA 155

Average Swelling Ratio vs. Time for MSS#1-12
PBST in PBS then trypsin. tp = 12 hours, 3%
MA . . 156

Average Swelling Ratio vs. Time for MSS#1-12T
in trypsin after 837 hours of swelling in PBS.
tp = 12 hours, 3% MA .. .. 158








Figure 4-39.


Figure 4-40.


Figure 4-41.



Figure 4-42.



Figure 4-43.



Figure 4-44.


Figure 4-45.





Figure 4-46.





Figure 4-47.





Figure 4-48.





Figure 4-49.


Average Swelling Ratio vs. Time for MSS#1-
24PBS in PBS. tp = 24 hours, 3% MA 159

Swelling Ratio vs. Time for MSS#1-24H in 1 M
HC1. tp = 24 hours, 3% MA 160

Average Swelling Ratio vs. Time for MSS#1-
24PBST in PBS then trypsin. tp = 24 hours,
3% MA . . 162

Average Swelling Ratio vs. Time for MSS#1-24T
in trypsin after 835 hours of swelling in PBS.
tp = 24 hours, 3% MA .. 163

Average Swelling Ratio vs. Time for MSS#2-1T
in activated trypsin. tp = 1 hour,
3% MA . . 165

Average Swelling Ratio vs. Time for MSS#2-1TH
in Tris-HC1. tp = 1 hour, 3% MA 166

Average Swelling Ratio vs. Time for MSS#2-1-
30T in activated trypsin vs. Tris-HCl. 30%
AA/ALB, tp = 1 hour, 3% MA. The high and
low values represent the first standard
deviation. . .. .. 168

Average Swelling Ratio vs. Time for MSS#2-1-
20TA in activated trypsin vs. Tris-HCl. 20%
AA/ALB, tp = 1 hour, 3% MA. The high and
low values represent the first standard
deviation . 169

Average Swelling Ratio vs. Time for
MSS#2-1-20TD in activated trypsin vs. Tris-
HC1. 20% AA/DEX, tp = 1 hour, 3% MA. The
high and low values represent the first
standard deviation. . 171

Average Swelling Ratio vs. Time for MSS#2-1-
30T in activated trypsin vs. Tris-HCl. 30%
AA/ALB, tp = 1 hour, 3% MA. The high and low
values represent the first standard deviation.
. . 172

Average Swelling Ratio vs. Time for MSS#2-1-
30TC in activated trypsin vs. Tris-HCl. 30%
AA/APS, no crosslinking agent, tp = 1 hour, 3%
MA. The high and low values represent the
first standard deviation. .. 174


xvi








Figure 4-50.





Figure 4-51.



Figure 4-52.


Figure 4-53.


Average Swelling Ratio vs. Time for MSS#2-1-
40T in activated trypsin vs. Tris-HC1.
2240%AA/ALB,tp=lhour, 3% MA. The high and low
values represent the first standard
deviation. . 175

Average Swelling Ratio vs. Time for MSS#2-4T
in activated trypsin. tp = 4 hours, 3% MA
. . . 176

Average Swelling Ratio vs. Time for MSS#2-4T
in Tris-HCl. tp = 4 hours, 3% MA 177

Average Swelling Ratio vs. Time for MSS#2-4-
10T in activated trypsin vs. Tris-HCl. 10%
AA/ALB, tp = 4 hours, 3% MA. The high and low
values represent the first standard deviation.


178


Figure 4-54.


Average Swelling Ratio vs. Time for MSS#2-4-
20TA in activated trypsin vs. tris-HCl. 20%
AA/ALB, tp = 4 hours, 3% MA. The high and low
values represent the first standard deviation.


. . . 0


180


Figure 4-55.





Figure 4-56.





Figure 4-57.





Figure 4-58.


Average Swelling Ratio vs. Time for MSS#2-6-
20TD in activated trypsin vs. tris-HCl. 20%
AA/DEX, tp = 6 hours, 3% MA. The high and low
values represent the first standard deviation.
. . . 181

Average Swelling Ratio vs. Time for MSS#2-4-
30T in activated trypsin vs. tris-HCl.
30%AA/ALB, tp = 4 hours, 3% MA. The high and
low values represent the first standard
deviation. . .182

Average Swelling Ratio vs. Time for
MSS#2-4-40T in activated trypsin vs. tris-HCl.
40% AA/ALB, tp = 4 hours, 3% MA. The high and
low values represent the first standard
deviation. . 183

Average Swelling Ratio vs. Time for MSS#2-6-
40TNA in activated trypsin vs. tris-HCl. 40%
NVP/ALB, tp = 6 hours, 3% MA. The high and
low values represent the first standard
deviation . .. 184


xvii


,~








Figure 4-59.





Figure 4-60.


Figure 4-61.


Figure 4-62.





Figure 4-63.





Figure 4-64.





Figure 4-65.


Figure 4-66.


Figure 4-67.





Figure 4-68.


Average Swelling Ratio vs. Time for MSS#2-6-
40TND in activated trypsin vs. tris-HC1. 40%
AA/DEX, tp = 6 hours, 3% MA. The high and low
values represent the first standard deviation.
. . . 185

Average Swelling Ratio vs. Time for MSS#2-12T
in activated trypsin. tp = 12 hours, 3% MA


187


Average Swelling Ratio vs. Time for MSS#2-12TH
in Tris-HCl. tp = 12 hours, 3% MA 188

Average Swelling Ratio vs. Time for MSS12-12-
10T in activated trypsin vs. tris-HCl. 10%
AA/ALB, tp = 12 hours, 3% MA. The high and
low values represent the first standard
deviation . 189

Average Swelling Ratio vs. Time for
MSS#2-12-20TA in activated trypsin vs. Tris-
HC1. tp = 12 hours, 3% MA. The high and low
values represent the first standard
deviation. .. 190

Average Swelling ratio vs. Time for
MSS#2-12-20TD in activated trypsin vs. Tris-
HC1. tp = 12 hours, 3% MA. The high and low
values represent the first standard
deviation. . 191

Average Swelling Ratio vs. Time for MSS#2-24T
in activated trypsin. tp = 24 hours, 3% MA.


192


Average Swelling Ratio vs. Time for MSS#2-24TH
in tris-HC1. tp = 24 hours, 3% MA 193

Average Swelling Ratio vs. Time for MSS12-24-
10T in activated trypsin vs. tris-HCl. 10%
AA/ALB, tp = 24 hours, 3% MA. The high and
low values represent the first standard
deviation . .. 194

Average Swelling Ratio vs. Time for MSS#2-24-
20TA in activated trypsin vs. tris-HCl. 20%
AA/ALB, tp = 24 hours, 3% MA. The high and
low values represent the first standard
deviation . 196


xviii


~


. . 0 .








Figure 4-69.





Figure 4-70.





Figure 4-71.



Figure 4-72.



Figure 4-73.



Figure 4-74.




Figure 4-75.





Figure 4-76.





Figure 4-77.



Figure 4-78.


Average Swelling Ratio vs. Time for MSS#2-24-
20TD in activated trypsin vs. tris-HCl. 20%
AA/DEX, tp = 24 hours, 3% MA. The high and
low values represent the first standard
deviation . .. 197

Average Swelling Ratio vs. Time for MSS#2-24-
40TC in activated trypsin vs. tris-HCl. 40%
AA/APS, no crosslinking agent, tp = 24 hours,
3% MA. The high and low values represent the
first standard deviation . 198

Average Swelling Ratio vs. Time for MSS#3P-1
in activated papain. tp = 1 hour, 10% MA.


200


Average Swelling Ratio vs. Time for MSS#3PB-1
in the activating solution for papain. tp = 1
hour, 10% MA . 201

Average Swelling Ratio vs. Time for MSS#3T-1
in unactivated trypsin. tp = 1 hour, 10% MA.
. 202

Average Swelling Ratio vs. Time for MSS#3TB-1
in PBS-NaN3. tp = 1 hour, 10% MA. The high
and low values represent the first standard
deviation . 203

Average Swelling ratio vs. Time for MSS#3P-1-
10 in activated papain vs. buffer. 10%
AA/ALB, tp = 1 hour, 10% MA. The high and low
values represent the first standard deviation.
. . 204

Average Swelling Ratio vs. Time for MSS#3T-1-
10 in unactivated trypsin vs. buffer. 10%
AA/ALB, tp = 1 hour, 10% MA. The high and low
values represent the first standard
deviation . 205

Average Swelling Ratio vs. Time for MSS#3P-1-
20 in activated papain vs. buffer. 20%
AA/ALB, tp = 1 hour, 10% MA 206

Average Swelling Ratio vs. Time for MSS#3T-1-
20 in unactivated trypsin vs. buffer. 20%
AA/ALB, tp = 1 hour, 10% MA. The high and low
values represent the first standard
deviation .. .. 208


xix


. 0 . .








Figure 4-79.





Figure 4-80.





Figure 4-81.




Figure 4-82.





Figure 4-83.



Figure 4-84.



Figure 4-85.



Figure 4-86.


Figure 4-87.





Figure 4-88.


Average Swelling Ratio vs. Time for MSS#3P-1-
30 in activated papain vs. buffer. 30%
AA/ALB, tp = 1 hour, 10% MA. The high and low
values represent the first standard
deviation . 209

Average Swelling Ratio vs. Time for MSS#3P-1-
30 in unactivated trypsin vs. buffer. 30%
AA/ALB, tp = 1 hour, 10% MA. The high and low
values represent the first standard
deviation . 210

Average Swelling Ratio vs. Time for MSS#3P-40
in activated papain vs. buffer. 40% AA/ALB,
tp = 1 hour, 10% MA. The high and low values
represent the first standard deviation 211

Average Swelling Ratio vs. Time for MSS#3T-1-
40 in unactivated trypsin vs. buffer. 40%
AA/ALB, tp = 1 hour, 10% MA. The high and low
values represent the first standard
deviation . .. 213

Average Swelling Ratio vs. Time for MSS#3P-4
in activated papain, tp = 4 hours, 10%
MA . . 214

Average Swelling Ratio vs. Time for MSS#3PB-4
in the activating solution for papain, tp = 4
hours, 10% MA . ... 215

Average Swelling Ratio vs. Time for MSS#3T-4
in unactivated trypsin. tp = 4 hours, 10% MA.
. . 216

Swelling Ratio vs. Time for MSS#3PB-4 in PBS-
NaN3. tp = 4 hours, 10% MA 217

Average Swelling Ratio vs. Time for MSS#3P-4-
10 in activated papain vs. buffer. 10%
AA/ALB, tp = 4 hours, 10% MA. The high and
low values represent the first standard
deviation . 218

Average Swelling Ratio vs. Time for MSS#3T-4-
10 in unactivated trypsin vs. buffer. 10%
AA/ALB, tp = 4 hours, 10% MA. The high and
low values represent the first standard
deviation . 219








Figure 4-89.





Figure 4-90.





Figure 4-91.





Figure 4-92.





Figure 4-93.





Figure 4-94.





Figure 4-95.



Figure 4-96.



Figure 4-97.



Figure 4-98.


Average Swelling Ratio vs. Time for MSS#3P-4-
20 in activated papain vs. buffer. 20%
AA/ALB, tp = 4 hours, 10% MA. The high and
low values represent the first standard
deviation . ... .. 220

Average Swelling Ratio vs. Time for MSS#3T-4-
20 in unactivated trypsin vs. buffer. 20%
AA/ALB, tp = 4 hours, 10% MA. The high and
low values represent the first standard
deviation . 222

Average Swelling Ratio vs. Time for MSSi3P-4-
30 in activated papain vs. buffer. 30%
AA/ALB, tp = 4 hours, 10% MA. The high and
low values represent the first standard
deviation . 223

Average Swelling Ratio vs. Time for MSS#3T-4-
30 in unactivated trypsin vs. buffer. 30%
AA/ALB, tp = 4 hours, 10% MA. The high and
low values represent the first standard
deviation . .. 224

Average Swelling Ratio vs. Time for MSS#3P-4-
40 in activated papain vs. buffer. 40%
AA/ALB, tp = 4 hours, 10% MA. The high and
low values represent the first standard
deviation . 226

Average Swelling Ratio vs. Time for MSS#3T-4-
40 in unactivated trypsin vs. buffer. 40%
AA/ALB, tp = 4 hours, 10% MA. The high and
low values represent the first standard
deviation. . .. 227

Average Swelling Ratio vs. Time for MSS#3P-14
in activated papain. tp = 14 hours, 10% MA.
S . .. 228

Average Swelling Ratio vs. Time for MSS/3PB-14
in the activating solution for papain.
tp = 14 hours, 10% MA .. 229

Average Swelling Ratio vs. Time for MSS#3T-14
in unactivated trypsin. tp = 14 hours, 10%
MA . . 230

Swelling Ratio vs. Time for MSS#3TB-14 in PBS-
NaN3. tp = 14 hours, 10% MA 231


xxi








Figure 4-99.





Figure 4-100.





Figure 4-101.





Figure 4-102.





Figure 4-103.





Figure 4-104.





Figure 4-105.





Figure 4-106.





Figure 4-107.


Average Swelling Ratio vs. Time for MSS#3P-14-
10 in activated papain vs. buffer. 10%
AA/ALB, tp = 14 hours, 10% MA. The high and
low values represent the first standard
deviation. . 232

Average Swelling Ratio vs. Time for MSS#3T-14-
10 in unactivated trypsin vs. buffer. 10%
AA/ALB, tp = 14 hours, 10% MA. The high
and low values represent the first standard
deviation . .. 234

Average Swelling Ratio vs. Time for MSS#3P-14-
20 in activated papain vs. buffer. 20%
AA/ALB, tp = 14 hours, 10% MA. The high and
low values represent the first standard
deviation. .. 235

Average Swelling Ratio vs. Time for MSS#3T-14-
20 in unactivated trypsin vs. buffer. 20%
AA/ALB, tp = 14 hours, 10% MA. The high and
low values represent the first standard
deviation . 236

Average Swelling Ratio vs. Time for MSS#3P-14-
30 in activated papain vs. buffer. 30%
AA/ALB, tp = 14 hours, 10% MA. The high and
low values represent the first standard
deviation. .. 237

Average Swelling Ratio vs. Time for MSS#3T-14-
30 in unactivated trypsin vs. buffer. 30%
AA/ALB, tp = 14 hours, 10% MA. The high and
low values represent the first standard
deviation . 238

Average Swelling Ratio vs. Time for MSS#3P-14-
40 in activated papain vs. buffer. 40%
AA/ALB, tp = 14 hours, 10% MA. The high and
low values represent the first standard
deviation. . 240

Average Swelling Ratio vs. Time for MSS#3T-14-
40 in unactivated trypsin vs. buffer. 40%
AA/ALB, tp = 14 hours, 10% MA. The high and
low values represent the first standard
deviation . 241

Average Swelling Ratio vs. Time for MSS#3P-24
in activated papain. tp = 24 hours, 10% MA.
. . 242


xxii








Figure 4-108.



Figure 4-109.



Figure 4-110.


Figure 4-111.





Figure 4-112.





Figure 4-113.




Figure 4-114.





Figure 4-115.





Figure 4-116.





Figure 4-117.


Average Swelling Ratio vs. Time for MSS#3PB-24
in the activating solution for papain. tp =
24 hours, 10% MA .. ... 243

Average Swelling Ratio vs. Time for MSS#3T-24
in unactivated trypsin. tp = 24 hours, 10%
MA . . 244

Swelling Ratio vs. Time for MSS#3TB-24 in PBS-
NaN3. tp = 24 hours, 10% MA 245

Average Swelling Ratio vs. Time for MSS#3P-24-
10 in activated papain vs. buffer. 10%
AA/ALB, tp = 24 hours, 10% MA. The high and
low values represent the first standard
deviation . .. 246

Average Swelling Ratio vs. Time for MSS#3T-24-
10 in unactivated trypsin vs. buffer. 10%
AA/ALB, tp = 24 hours, 10% MA. The high and
low values represent the first standard
deviation . 248

Average Swelling Ratio vs. Time for MSS#3P-24-
20 in activated papain. 20% AA/ALB, tp = 24
hours, 10% MA. The high and low values
represent the first standard deviation. 249

Average Swelling Ratio vs. Time for MSS#3T-24-
20 in unactivated trypsin vs. buffer. 20%
AA/ALB, tp = 24 hours, 10% MA. The high and
low values represent the first standard
deviation . 250

Average Swelling Ratio vs. Time for MSS#3P-24-
30 in activated papain vs. buffer. 30%
AA/ALB, tp = 24 hours, 10% MA. The high and
low values represent the first standard
deviation. . 251

Average Swelling Ratio vs. Time for MSS#3T-24-
30 in unactivated trypsin vs. buffer. 30%
AA/ALB, tp = 24 hours, 10% MA. The high and
low values represent the first standard
deviation. . .... 253

Average Swelling Ratio vs. Time for MSS#3P-24-
40 in activated papain vs. buffer. 40%
AA/ALB, tp = 24 hours, 10% MA. The high and
low values represent the first standard
deviation. . 254


xxiii








Figure 4-118.





Figure 4-119.



Figure 4-120.



Figure 4-121.



Figure 4-122.



Figure 4-123.



Figure 4-124.



Figure 4-125.



Figure 4-126.



Figure 4-127.



Figure 4-128.



Figure 4-129.


Average Swelling Ratio vs. Time for MSS#3T-24-
40 in unactivated trypsin vs. buffer. 40%
AA/ALB, tp = 24 hours, 10% MA. The high and
low values represent the first standard
deviation .. .. 255

Maximum Absorbance vs. Time for UV
Spectroscopy of the swelling media from SS#5.
. . 257

Maximum Absorbance vs. Time for UV
Spectroscopy of the swelling media from SS#6.
. . 258

Maximum Absorbance vs. Time for UV
Spectroscopy of the swelling media from SS#5
and SS#6 (250C) . 259

Maximum Absorbance vs. Time for UV
Spectroscopy of the swelling media from SS#5
and SSf6 (370C) 260

Maximum Absorbance vs. Time for UV
Spectroscopy of the swelling media from SS#12.
. 262

Maximum Absorbance vs. Time and Wavelength vs.
Time for UV Spectroscopy of the swelling
medium from sample 165 in SSf12 (250C) 263

Maximum Absorbance vs. Time and Wavelength vs.
Time for the swelling medium from sample 168
in SS#12 (250C) . 264

Maximum absorbance vs. Time and Wavelength vs.
Time for the swelling medium from sample 171
in SS#12 (370C) . 265

Maximum absorbance vs. Time and wavelength vs.
time for the swelling medium from sample 174
in SS#12 (370C) . 266

Maximum Absorbance vs. Time for UV
Spectroscopy of the swelling media from SS#13.
. . . 268

Maximum Absorbance vs. Time and Wavelength vs.
Time for the swelling medium from sample 177
in SS#13 (250C) .. 269


xxiv








Figure 4-130.



Figure 4-131.



Figure 4-132.



Figure 4-133.



Figure 4-134.



Figure 4-135.



Figure 4-136.




Figure 4-137.



Figure 5-1.


Figure 5-2.


Figure 5-3.

Figure 5-4.


Figure 5-5.


Figure 5-6.


Maximum Absorbance vs. Time and Wavelength vs.
Time for the swelling medium from sample 180
in SS#13 (250C) . 270

Maximum Absorbance vs. Time and Wavelength vs.
Time for the swelling medium from sample 183
in SS#13 (370C) . 271

Maximum Absorbance vs. Time and Wavelength vs.
Time for the swelling medium from sample 186
in SS#13 (370C) . .. 272

Calibration curve for gel permeation
chromatography, which was generated using
poly(ethylene glycol) standards 273

Chromatograph of the swelling medium from
sample 87 in SS#5. This sample was taken
after 3 hours of swelling 275

Chromatograph of the swelling medium from
sample 99 in SS#6. This sample was taken
after 3 hours of swelling 276

Chromatograph of the solution of the from the
40% monomer AA/ALB disc. (tp = 24 hours)
which was autoclaved in 1M HC1 at 1000C for 24
hours. . 277

Chromatograph of the solution from the 10%
monomer AA/DEX disc which was autoclaved in 1M
HC1 at 100oC for 24 hours .. 278

Typical Average Swelling Ratio vs. Time curve
for degradable hydrogels . 290

Maximum Average Swelling Ratio (Qm) vs. Volume
Fraction (Vf) of Monomer. . 291

Time to Qm vs. Volume Fraction of Monomer.292

Degradation Time (td) vs. Volume Fraction of
Monomer. tp = 1 hour. .. .294

Degradation Time vs. Volume Fraction of
Monomer. tp = 12 hours. . .295

Maximum Average Swelling Ratio vs. Volume
Fraction of Monomer. . .296


xxV








Figure 5-7.


Figure 5-8.


Figure 5-9.


Time to Qm vs. Volume Fraction of Monomer in
PBS-NaN3. . . 298

Degradation Time vs. Volume Fraction of
Monomer. tp = 1 hour .. 299

Degradation Time vs. Volume Fraction of
Monomer. tp = 14 hours .. .300


xxvi










AA
AA/ALB
AA/DEX
APS
AM
BM
BSA
CaC12
EDTA
FT-IR
GA
HPLC
KBr
MA
MA-PBS
MWCO
NMR
NaN3
NNMBA
NVP/ALB
NVP/DEX
NVP
PAA
PBS
PEG
PNVP
Q
Qavg
RT
Tg
TRIS-HCI


KEY TO ABBREVIATIONS AND SYMBOLS

Acrylic Acid
Acrylic acid/modified albumin
Acrylic acid/modified dextran
Ammonium Persulfate
Albumin Acrylamide
Boehringer Mannheim
Bovine Serum
Calcium Chloride
Ethylenediamine tetraacetic acid
Fourier Transform Infrared Spectroscopy
Glycidyl Acrylate
High Performance Liquid Chromatograph
Potassium Bromide
Modified Albumin
Subtraction spectra of MA minus PBS
Molecular Weight Cut Off
Nuclear Magnetic Resonance Spectroscopy
Sodium Azide
N,N'-methylene Bisacrylamide
N-vinyl pyrrolidone/modified albumin
N-vinyl pyrrolidone/modified DEXTRAN
N-vinyl Pyrrolidone
Poly(acrylic acid)
Phosphate Buffered Saline
Poly(ethylene glycol)
Poly(N-vinyl pyrrolidone)
Swelling Ratio
Average Swelling Ratio
Room Temperature
Glass transition temperature
Tris(hydroxymethyl) aminomethane
hydrochloride buffer


xxvii








KEY TO ABBREVIATIONS AND SYMBOLS (CONTINUED)


Time of Polymerization
Trimethylsilylpropionate
Subtraction spectra of UMA minus PBS
Unmodified Dextran
Weight of the hydrogel dry
Weight of the hydrogel wet


xxviii


tp
TSP
UMA-PBS
UMD
Wd (DRY)
Wd (WET)













Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

SYNTHESIS AND CHARACTERIZATION OF
DEGRADABLE POLYMER HYDROGELS FOR TISSUE SCAFFOLDS

By

Angela L. Campbell

December, 1992

Chairperson: Dr Christopher D. Batich
Major Department: Materials Science and Engineering

Degradable polymer hydrogels of several types were

polymerized by thermal solution polymerization or gamma

solution polymerization of the vinyl monomers acrylic acid

(AA) and N-vinyl pyrrolidone (NVP) to be used as coatings for

tissue scaffolds. It was attempted to synthesize hydrogels

which would last for 2-4 weeks. The solvent for the

polymerizations was phosphate buffered saline (PBS). The

crosslinking agents used were bovine serum albumin (BSA) or

dextran which were alkylated by stirring them in PBS with

glycidyl acrylate (GA). This introduced a double bond into

their structures and allowed them to participate in the free

radical polymerization of the AA and NVP, and resulted in

their being covalently bound to the three dimensional polymer

hydrogel networks. These modified natural polymers introduced

enzymatic and hydrolytic degradability into the hydrogels.


xxix








The initiator used in the thermal solution polymerizations was

ammonium persulfate (APS).

The modification of the BSA and dextran was verified by

FT-IR and NMR. Evidence of the conjugated ester stretch was

found in the modified albumin (MA) spectrum in FT-IR as well

as the C-H out-of-plane bending peak for the terminal vinyl

group. The olefinic protons introduced into the BSA and MD

were seen in the NMR spectra between 7.5 ppm and 6.6 ppm, and

between 6.3 ppm and 5.9 ppm, respectively.

Thermal solution polymerizations were successful with AA.

Acrylic acid was simultaneously polymerized in PBS in the

presence of MA or MD, and APS at 600C. Attempts to polymerize

NVP and mixtures of AA and NVP by thermal solution polymeriza-

tion failed and it was concluded that APS is not a suitable

initiator for the polymerization of NVP. Gamma solution

polymerization of AA and NVP in PBS in the presence of MA or

MD yielded degradable hydrogels.

The swelling and degradation behavior of the hydrogels

was investigated by swelling them in PBS, activated papain,

activated trypsin, trypsin in PBS, 1 M HC1, and TRIS-HC1

buffer. They were also degraded in 1 M HC1 at 1000C for

-24 hours. Acrylic acid/MA, AA/MD, NVP/MA, and NVP/MD

hydrogels were found to degrade in the presence of activated

papain, activated trypsin, trypsin in PBS, and in 1 M HC1 at

1000C. Degradable hydrogels were synthesized with a range of

lifetimes from 1 day to 4 weeks.


xxx














CHAPTER 1
GENERAL INTRODUCTION AND BACKGROUND


1.1 General Introduction


Damage to natural tissues and organs due to disease or

injury may require repair or total replacement. Tissue

scaffolds can be used to repair or replace tissues and organs

by utilizing the natural healing processes of the host.

Natural tissue-derived scaffolds may require degradable

hydrogel coatings to prevent premature degradation by

proteolytic enzymes released in the normal inflammatory

response, to prevent encrustation by mineral deposits, to fill

spaces and to retard cell ingrowth (1, 2). The degradable

hydrogels should remain as a coating on the scaffold for the

duration of the inflammatory response. The molecular weight

of the degradation products should be less than 68,000 (3).

This is the molecular weight cut off point for the perme-

ability of proteins through the glomerular capillaries of the

kidney (3). This time period of degradation can be tailor

made by variations in monomer, initiator, and crosslinking

agent concentrations, in the monomer solution, and the time of

polymerization (4).

The purpose of this study was to synthesize degradable

hydrogels, and characterize their swelling and degradation

1








2

behavior. Variations in the components included in the

monomer solution were studied. The hydrogels were designed to

be used as coatings for scaffolds in a variety of

applications.

Degradable hydrogels of several types were synthesized.

The biodegradable hydrogel components were made from poly-

merization of acrylic acid (AA) or N-vinyl pyrrolidone (NVP)

and were crosslinked with bovine serum albumin or dextran

functionalized with glycidyl acrylate (GA). The acrylic acid

was polymerized by thermal polymerization with ammonium

persulfate as initiator or by gamma (7) polymerization. The

NVP was polymerized by 7 polymerization only (5, 6).

The swelling and degradation behavior of the gels were

investigated by swelling them in phosphate buffered saline

(PBS), activated papain solution, activated trypsin solutions,

IM HC1, and Tris-HCl buffer. They were also degraded in 1 M

HC1 at 1000C -24 hours.


1.2 Background


1.2.1 Description of Hvdrogels


Hydrogels are three dimensional crosslinked polymeric

networks which swell in the presence of water. They are

insoluble in water and may have water contents between 30% and

95% (7, 8, 9, 10). They are synthesized by polymerization of

hydrophilic natural or synthetic vinyl-type monomers or by








3

chemical modification of pre-existing polymers (7, 8, 10).

Hydrophobic monomers can also be included in the polymeri-

zation to improve their mechanical properties (8).

Hydrogels are soft, highly permeable, hydrophilic, and

are easily cast into desired shapes. These properties as well

as their water content make them good materials for biomedi-

cal, agricultural, and food industry applications (7, 8, 9,

10). Hydrogels have good biocompatibility due to their

hydrophilicity and water content; however, these properties

also cause them to have poor mechanical stability. They are

often utilized as coatings for polymer substrates with good

mechanical properties to improve their surface characteris-

tics. A specific example of this would be a hydrogel coating

for a preformed device or implant that will come in contact

with biological fluids (8, 10). Table 1-1 provides a summary

of the various applications reported for hydrogels (7, 8, 9).














COATINGS

Sutures
Catheters
IUD's
Blood Det

Sensors (
Tissue De
Vascular
Artificia
Degradabl


BULK MATE

Contact L
Artificia
Estrus-In
Breast an
Soft T
Burn Dres


TALE1-


TABLE 1-1
Summary of Applications for Hydrogels

DRUG RELEASE DEPOTS

Antibiotics
Anticoagulants
Anticancer Drugs
:oxicant Antibodies

electrodes) Drug Antagonists
srived Prostheses Enzymes
Prostheses Contraceptives
il Skin Estrus-Inducer
.e Coatings Antibacterial Agents
Dead or Living Cells

TRIALS OTHER APPLICATIONS

senses Soil Modifiers
1l Corneas (Agriculture)
ducer Moisture-Releasing Agents
id Other (Agriculture)
issue Substitutes Thickening Agents
sings (Food industry)


Bone Ingrowth Sponge
Dentures
Ear Drum Plug
Absorbent Material
(hygienic aids, wound
dressings)


Source: Reference 7, 8 and 9


1.2.2 Classifications of Degradable Polymers


Monomers and/or crosslinking agents which are susceptible

to hydrolytic or enzymatic attack can be included in the

polymerization process to introduce biodegradability into

hydrogels which would otherwise not be degradable (9). There

are several terms used to describe polymers that purposely

degrade in a short period of time upon exposure to biological








5

agents (organisms, organelles, or enzymes). These terms are

biodegradable, bioerodible or bioassimilable, bioresorbable,

and bioabsorbable (11, 12). Biodegradable polymers undergo

enzymatic degradation in a given physiological or microbial

environment. Enzymes capable of degrading the polymers are

able to cause a chemically recognizable cleavage of the

molecular structure of the polymer. As a result of the

cleavage, the integrity of the polymer is lost, leaving

fragments or other degradation products (11, 12).

According to Kumar, bioerodible or bioassimilable

polymers are degraded by specific hydrolytic processes with no

participation of enzymes (12). Bioresorbable polymers are

those which degrade into low molecular weight materials

normally involved in metabolic pathways. Examples of

bioresorbable polymers are poly(a-hydroxy acids) such as

poly(lactic acid) and poly(8-hydroxy acids) such as poly(f-

malic acid) (12). Bioabsorbable polymers are degraded by

solubilization and elimination with no degradation of the

macromolecules. An example would be slow dissolution of water

soluble implants in body fluids (12).


1.2.3 DescriDtion of Tissue Scaffolds


Non-degradable or biodegradable hydrogels can be used as

coatings for tissue scaffolds. They can be used to improve

biocompatibility, to protect the scaffold from the normal in-

flammatory response, and to prevent encrustation with








6

minerals. Tissue scaffolds are biomedical devices used to

promote regeneration and repair of biological tissues within

living organisms. They can be made of degradable natural or

synthetic polymeric materials. They provide temporary

replacement for tissues which have been damaged due to disease

or injury. Initially, the scaffold provides support for

ingrowth of surrounding tissue and mechanical strength. With

time, the scaffold degrades, ultimately disappears, and is

replaced by normal tissue from the host (2, 6, 13).

Tissue scaffolds can be used in various applications such

as regeneration of nerve, periodontal, urinary, and vascular

tissues. They can also be used as artificial skin, ligaments,

and tendons. The rate of degradation of polymers used partly

determines the success of the scaffold. In some applications,

a rapidly degrading scaffold may be needed while in others

slow degradation is needed. This creates a need to control

the rate of degradation of the polymer.

The porosity of the scaffold is also important. The

percent of porosity as well as pore size determines the extent

of tissue ingrowth. In addition, porosity controls the

transport of materials (fluids, nutrients, oxygen) to and from

the tissue or organ (14, 15).

Surface properties of the scaffold material influence

cell adhesion and tissue ingrowth as well and are especially

critical for controlling nucleation and growth of mineral

deposits, and absorption of proteins.








7

Tissue scaffolds must also meet the basic requirements

for biomaterials. They must have the physical properties,

strength, elasticity, and permeability necessary for a

particular application. They must be easily purified,

fabricated, and sterilized, and maintain physical properties

and function in vivo over a desired period of time. They

should not induce undesirable host reactions such as clotting,

tissue necrosis, carcinogenesis, allergenic responses or im-

munogenic responses (16).


1.2.4 ADDlications for Biodearadable Hvdroaels


Degradable hydrogels have been used in many applications.

Bioerodible hydrogels have been used as microspheres to entrap

and immobilize water soluble molecules such as proteins,

enzymes, antigens, insulin, albumin, and various drugs (11,

17, 18, 19). Dextran hydrogels have been made by reacting the

dextran hydroxyl groups with GA to form a 3-acryloyl-2-hydroxy

propyl ether, and crosslinking with N,N'-methylene bisacryl-

amide (NNMBA) (17). Starch hydrogels have also been

synthesized by the same method. Starch xanthide gels have

been used to encapsulate pesticides (17).

Another bioerodible hydrogel system, described by Heller,

(17, 18) is composed of a polyester prepared from a water

soluble linear polymer containing backbone or pendant

unsaturation, which is crosslinked by copolymerization with a

water soluble monomer such as NVP or AA. The system is eroded








8

by hydrolysis of ester linkages which leads to the production

of poly(ethylene glycol) (PEG), and a poly(N-vinyl

pyrrolidone) (PNVP) modified by vicinal carboxylic acid

groups. These hydrogels were used as microspheres to entrap

and immobilize bovine serum albumin (BSA), for sustained

release by erosion of the hydrogel matrix. The rate of

release was controlled by varying the crosslink density, and

by incorporating activating diacids with an electron

withdrawing group, with fumaric acid and polyesters which had

unsaturation in their backbone. The diacids enhanced the

hydrolytic instability of the polyester and increased the rate

of BSA release by matrix erosion (17, 18).

Bioerodible hydrogels based on NVP or acrylamide (AM)

crosslinked with NNMBA were found to be inadequate as

bioerodible microspheres for immobilization of macromolecules

in a study by Heller et al. (17, 18). The rate of cleavage of

the hydrogel matrix by way of hydrolysis was too slow if the

crosslinking agent was present at a concentration of more than

1%. The macromolecules were released by diffusion from the

matrix when the concentration of the crosslinking agent was

less than 1%. This was unacceptable. Microspheres of this

system with crosslink densities which would prevent diffusion

of macromolecules did not erode at rates which are suitable

for the application (17, 18).

Biodegradable hydrogels susceptible to enzymatic

degradation have been made with composites of natural and








9

synthetic materials. Hydrogels for oral drug delivery were

developed in a study by Park, involving hydrophilic synthetic

monomers (AA, AM) polymerized in the presence of BSA modified

with GA (as crosslinking agent) and ammonium persulfate (APS)

as an initiator. These hydrogels degraded in the presence of

trypsin or pepsin by cleavage of the albumin crosslinks. The

rate of degradation was affected by the concentration of the

monomer and the modified BSA (5).

Hydrogels synthesized by Subr et al. (19) from

N-(2-hydroxypropyl) methacrylamide crosslinked with

oligope tMde sequences degraded in the presence of lysosomal

enzymes or chymotrypsin. Their degradation was dependent on

the equilibrium degree of swelling, the length of the

oligopeptide chain, and the structure of the amino acid

residues. These were developed for use in controlled delivery

of anticancer agents (19).

In studies by Dickinson et al., a poly(a-amino acid) was

found to degrade in the presence of the enzymes papain and

pronase in vitro, and also during the first two weeks of

implantation in rats in vivo. In the in vivo study, peptide

bonds were cleaved by proteolytic enzymes released during the

acute and chronic stages of the inflammatory response.

Poly(2-hydroxylethyl-L-glutamine) was prepared from

poly(7-benzyl-L-glutamate) in the presence of benzene,

ethanolamine and crosslinking agent diaminododecane (20, 21).








10

Gelatin hydrogels have been made by three methods which

include heat treatment, aldehyde treatment, and chromic acid

treatment as pointed out by Heller et al. They have been used

as nanospheres and microspheres for delivery of mitromycin C

to the liver, spleen, and lungs. The presence of residual

aldehyde in treated gelatins causes a toxicity problem

resulting in tissue death (17).

Crosslinked albumin hydrogel microspheres are produced by

either heat treatment (1000C) or gluteraldehyde crosslinking

(17). The microspheres can be readily removed from the

vascular system by phagocytes and provide a useful means of

drug delivery to endocytic cells (17).

Gelatin hydrogels have also been prepared by grafting

vinyl monomers to the polypeptide backbone (17). The gels

were degraded by an inoculum of Pseudomonas aeruginosa,

Bacscillus subtilis and Serratia marcescens. The amount of

biodegradation decreases with an increase in the amount of

grafting sites on the gelatin backbone (17).

Hydrogels made from copolymerzation of hydrophilic and

hydrophobic a-amino acids were found to have biodegradation

properties based on the concentration of the hydrophilic

component (17). They were made from mixtures of the

hydrophilic monomer L-aspartic acid and the hydrophobic

monomers L-leucine, 8-methyl-L-aspartate, and

P-benzyl-L-aspartate. The biodegradability of the resulting

hydrogels increased with an increase in hydrophilicity of the








11

copolymers. A similar polymer hydrogel system based on a

copolymer of glutamic acid and ethyl glutamate was found to

increase biodegradation as the concentration of the

hydrophilic glutamic acid increased (17).

Chitin, a hydrophobic polysaccharide, can be made

hydrophilic by partial deacetylation of the N-acetyl

glucosamine units in strong alkali (17). Hydrogels of chitin

are made by reaction of the free amino groups of the partially

deacetylated chitin with gluteraldehyde. These gels are

lysozyme degradable (17).


1.2.5 Polymers and the Inflammatory Response


Natural or synthetic polymeric materials which are intro-

duced into a living organism are treated as foreign bodies and

will induce an inflammatory response (16, 20, 21). This

response is the major reaction to foreign bodies in the

extravascular system (16). Inflammation occurs in the

vicinity of the foreign body where proteins and cells try to

digest, enzymolyse, and convert it into metabolites which can

be eliminated by the organism (16). If the organism is unable

to metabolize the foreign body, it will attempt to wall the

foreign body off by encapsulating it with fibrous collagen

scar tissue (16). Fibrous ingrowth will occur in porous

foreign body materials (16, 20, 21).

Inflammatory cells release enzymes that may cause

degradation of polymeric materials. This could cause loss of








12

mass, loss of mechanical properties, and loss of the specific

function that the polymers are intended to provide. The acute

inflammatory response occurs during the first three or four

days of implantation (20, 21). This response is designed to

destroy biological materials. Neutrophils are the major group

of phagocytic cells which proliferate during this period and

are found immediately adjacent to the foreign body. These

cells can initiate rapid degradation of the major components

of the extracellular matrix in tissue-derived scaffolds by

releasing low levels of proteolytic enzymes. The enzymes

released encompass proteases which include collagenase,

elastase, and cathepsins. These enzymes attack polypeptide

bonds in both natural and synthetic polymers (20, 21, 22).

According to Dinkinson et al., after the first four days

of implantation, the chronic inflammatory response begins (20,

21). The neutrophils are replaced by granulation tissue which

consists of histiocytes which release higher levels of proteo-

lytic enzymes than the neutrophils. The histiocytes present

are mast cells, macrophages, and proliferating fibroblasts.

Capillaries are also found in the granulation tissue. Mast

cells release chymotrypsin-like proteases. Macrophages

release high levels of cathepsins, carboxypeptidase,

collagenase, and leucine amino peptidase. The chronic

inflammatory response lasts two weeks unless the implant or

foreign body irritates the host tissue (20, 21).








13

The enzymes released during the acute and chronic

inflammatory responses are a threat to the stability of

tissue-derived scaffolds which are primarily made of collagen.

Protection for such scaffolds can be obtained by coating them

with enzyme degradable hydrogels that must hold their

integrity at least 2 to 4 weeks. In doing so, they would

protect the scaffolds from the acute and chronic

inflammatory responses. Table 1-2 summarizes the stages of

the inflammatory response (16, 20, 20, 23).


TABLE 1-2
The Stages of the Inflammatory Response

ACUTE INFLAMMATORY RESPONSE

Duration: 3-4 days after implantation
Cells Involved: Neutrophils
Enzymes Released: Collagenase
Elastase
Cathepsins

CHRONIC INFLAMMATORY RESPONSE

Duration: 4-12 days and longer after
implantation
Cells Involved: Graunulation tissue
Mast Cells
Macrophages
Proliferating
Fibroblasts
Capillaries
Enzymes Released: Chymotrypsin-like
Proteases
Cathepsins
Carboxypeptidase
Leucine Amino-
peptidase

Source: References 20 and 21








14
1.2.6 Mechanism of In Vivo Degradation for Polymers


Polymers which are introduced into a living organism have

been observed to undergo physiological degradation in a

systematic manner as Kumar pointed out (11). There are four

stages of degradation, which include hydration, biodegra-

dation, bioerosion, and solubilization. The hydration stage

involves swelling of the polymer. In this stage, there is an

immediate reduction in the mechanical strength. There are

very few broken bonds, however, disruption of the secondary

and tertiary structures occurs by the breaking of van der

Waals' forces and hydrogen bonds (11). The extent of

hydration which occurs in a given polymer depends on the

hydrophilic nature of the polymer (11).

The biodegradation stage involves cleavage of covalent

bonds in the polymer backbone. The extent of biodegradation

depends on the molecular structure of the polymer and the

types of enzymes involved (11).

Bioerosion is characterized by loss of polymer mass, and

absorption of the polymer (11). Here the polymer is further

degraded into oligomers. This stage is also marked by loss of

the physical and mechanical integrity of the polymer. The

polymer may become a friable or gelatinized mass. The extent

of bioerosion that occurs is a function of the Tg of the

polymer, its conformation, and its crystallinity.

The final stage of in vivo degradation is that of

solubilization (11). This stage can also be called the








15

bioassimilation stage. Oligomers of the bioeroded polymer are

either solubilized into intracellular fluids or engulfed by

phagocytic cells and eventually transported to the lymphatic

system. Eventually, the total mass of the polymer is lost as

a result of this solubilization process. Table 1-3 summarizes

the stages of in vivo polymer degradation (11).



TABLE 1-3
The Four Stages of In Vivo Polymer Degradation (14)


1. HYDRATION



2. STRENGTH LOSS



3. LOSS OF MASS




4. SOLUBILIZATION


Disruption of van der Waals'
forces and hydrogen bonds


Initial cleavage of backbone
covalent bonds
-Biodegradation

Further cleavage of covalent
bonds to low molecular weight
levels
Bioerosion

Dissolution of low molecular
weight species and phagocytosis
of small fragments
-Bioassimilation

Source: Reference 11













CHAPTER 2
MODIFICATION AND CHARACTERIZATION OF ALBUMIN AND DEXTRAN


2.1 Introduction


The hydrogels in this study were made degradable by

introducing enzymatically and hydrolytically degradable

crosslinks. Albumin and dextran were functionalized with GA

to introduce double bonds into their structure by methods

similar to those introduced in studies by Park (5) and Edman

et al. (6). This enabled these natural polymers to partici-

pate in the solution free radical chain polymerization of AA

and NVP. The results of the polymerizations were hydrogels

with albumin or dextran crosslinks which were covalently

bonded to the poly(acrylic acid) (PAA) or PNVP matrices. The

hydrogels were designed to degrade when introduced into living

organisms. The hydrogels with albumin crosslinks were

expected to degrade due to the proteolytic enzymes released

during the inflammatory response (16, 20, 21, 22).

The hydrogels with dextran crosslinks were expected to

degrade due to hydrolytic mechanisms by enzymes that catalyze

the degradation of polysaccharides (11). Hydrolases which

catalyze the hydrolytic cleavage of ester, amide or ether

linkages, should degrade dextran at the ether linkages located

at carbons 1 and 6 or at the third carbon of the chain linking








17

unit (Figure 2-1) (11, 24). Lysosomal enzymes from the

reticuloendothelial system will degrade dextran (acid

hydrolases) (17, 25, 26).

Serum albumin (MW 67,000 69,000) is a low molecular

weight protein which is the major component (60%) found in

mammalian blood plasma besides water (27, 28, 29). It

provides important transport functions for fatty acids, drugs,

antigens, K+, Na+, Ca+, hormones, and other hydrophobic

substances (27, 28, 29). The colloid osmotic pressure of

blood is also regulated by this protein which is a single

polypeptide chain consisting of 585 amino acids (27, 28, 29,

30).

Serum albumin has adjacent cysteine residues which are

the basis for its structure. It has a repeating loop

structure formed by disulfide bridges, with a total of nine

loops which form three domains. These three domains each

contain two large loops and one small loop. The large loops

are forty-six amino acid residues long and the small loops are

sixteen amino acids long (28, 29, 30).

Serum albumin is soluble in water and diluted salt

solutions in the pH range between 4 and 8.5, and assumes

several conformational structures over this pH range (28, 29).

It has an acidic character due to the large percentage (20-

25%) of amino acids, glutamic acid and aspartic acid (28, 29).

Figures 2-2, 2-3, 2-4 show portions of the structure of

albumin (28, 30).

















iD
I
w


I
u
61



0




0'
I


.94



ft<

C)
g


^i





r4
S
'4









Albumin as a SDecific Binding Protein for Drugs and Endogenous Compounds


DOMAINS

SUBDOMAINS

LOOPS


Fig.2.2.








Cu2'
NI2+ /

Fig.2.3.


1

1AB 1C

1 2 3
a 1


2 I

2AB 2C
45 6
- I


3AB
7 8


Loop structure of bovine serum albumin indica-
ting disulphide bonds forming loops, subdomains
and domains. Source: Reference 30


(Aspirin)'
Diagram of albumin primary structure showing its
eight and a half double disulfide loops and some
suggested binding sites. Source: Reference 28


I2L L HN
IL L
3 4

igE .4 7 1 1 3
Y L L Y L AL LR T V 8 A
A


Fig.2.4.


Primary structure of the carboxy-terminal double
loop of bovine serum albumin, the "Phe fragment"
Note the proposed overlapping configuration of
the loops at the Cys-Cys pair.
Source: Reference 30








20

Dextran is a high molecular weight glucose polymer

produced by the action of Leuconostoc meseteroides on sucrose

(31). This poly(a-D-glucopyranoside) has been used as plasma

substitutes or extenders (24, 31). Figure 2-5 shows the

structure of the repeat unit of dextran (24).

Glycidyl acrylate modifies albumin and dextran by

attaching pendant groups containing double bonds onto their

structure. Figures 2-6 and 2-7 show the reactions involved in

the modification. Glycidyl acrylate probably reacts with free

amine groups which might be provided by amino acids trytophan,

asparagine, glutamine, lysine, arginine, and histidine (32).

All of these a-amino acids have one or more amine groups

present in their R group (32). The reaction results in a 3-

acryloyl-2-hydroxypropyl amine. Glycidyl acrylate reacts with

the hydroxyl groups of dextran to form a 3-acryloyl-2-

hydroypropyl ether (6).

Verification of the modification of albumin and dextran

with GA, can be obtained by analyzing them with FT-IR and NMR.

Infrared spectroscopy and NMR are two spectroscopic methods

which can be used to determine the molecular structure of a

compound (33). Infrared spectroscopy is a vibrational

spectroscopy which detects the normal minute vibrations that

occur naturally within molecules with bonds that possess a

dipole moment (33, 34). It provides a fingerprint for

identification by producing information about the molecular

structure, symmetry and functional groups present in a given































OH


Figure 2.5. Repeat unit of dextran. Source: Reference 24















































E




g I
. C


On
I


"I
(Y



0-E
o--U

yM
=-?


III O
0
0
*rl

*M






*64
U





rl
5
[24









































































U)

0
CC








24
molecule (33). The sample is irradiated with infrared (IR)

radiation which causes a quantum of mechanical transition to

occur between two vibrational energy levels. The energy

difference AE between the two vibrational energy levels is

directly related to the frequency of IR electromagnetic

radiation incident on the sample and is given by:

AE = E2 El = hy = photon

A plot of absorbance or transmittance vs. wavelength is

recorded and the positions and intensities of the absorbance

peaks found provides information about the structure of the

molecule (33).

Infrared spectroscopy can be used to distinguish between

the types of bonds that are present in a molecule (33). Each

bond can be modelled as a system involving two balls with

masses ml and m2, held together by a spring with a force

constant k. The normal mode of vibration for this system is

simple harmonic oscillation about its equilibrium position

with all atoms moving in phase at the same frequency. The

center of gravity of the system does not move. Each bond has

a characteristic frequency at which it vibrates given by:

v = 11w V(k/l)

where A is the reduced mass, k is the spring force constant

(Nm-1). The bond will absorb IR radiation which has the same

frequency as its characteristic frequency of vibration. The

amount of energy absorbed by the molecule is given by:

A = logl01o/I








25
The spectral transitions of the molecule are detected by

scanning the frequency of IR radiation and continuous

monitoring of the transmitted or absorbed light intensity.

The range of frequencies of interest is from 4000 400 cm-1

or 2.5 25 um (34).

Nuclear magnetic resonance spectroscopy can provide

information about the types and numbers of nuclei that are

present in a molecule (33). Information is obtained from the

magnetic properties of the nuclei present. Magnetic nuclei

have spins which move about in a circular motion. This

circulation causes a magnetic moment (Iz) to form due to the

positive charge of the protons in the nucleus. The

distribution of the charge about the spinning magnetic nuclei

determines the value of Iz. Magnetic nuclei with a spherical

charge distribution have an Iz of 1/2 (1H, 13C, 15N, 19F, 29Si

and 31P). Magnetic nuclei with nonspherical (quadrapolar)

charge distributions have magnetic moments of 1, 3/2, 2, etc.

(2D, 7Li,11B, 14N, 170, and 2Na) (33).

Samples in NMR spectroscopy are placed between the poles

of a strong laboratory magnet with magnetic field Bo (telsa).

In the absence of this field all of the spin orientations in

the magnetic nuclei are the same. When the field is applied

in 1H-NMR the magnetic moments of the nuclei with spin Iz =

1/2 can align parallel (Iz = +1/2) or antiparallel (Iz = -1/2)

with the direction of the applied field. These two states








26

have well defined energies E2; Iz = -1/2 and E1; Iz = +1/2.

The Iz = -1/2 state is the less favorable state and has a

higher energy. The difference between these two energy states

(AE) in a magnetic nucleus is used to determine the structure

of a molecule. This energy is given by:

AE = E2 El (h/2w)7yGBo

where h is Planck's constant and 7G is the gyromagnetic ratio.

Each distinct nucleus has a characteristic AE which depends on

the nuclear properties which are found in the gyromagnetic

ratio and the value of the applied field B0. Every nucleus

has a distinct gyromagnetic ratio (33).

Once the energy separation between the spin states is

achieved, a second magnetic field B1 is applied to the sample

to detect the presence of the two different spin states

present in each distinct nucleus. The field B1 has a

characteristic frequency P. This frequency can be adjusted

until it reaches a frequency vo which has the same energy hvo

as the energy difference between the two spin states of the

nucleus. When this energy is absorbed by the nucleus and a

conversion of the spin state from +1/2 to -1/2 occurs. This

resonance energy is detected electronically and provides

information about the nucleus. The resonance frequency is:

0o = YG(Bo)/21

The B1 frequency is varied so that each type of nucleus comes

into resonance and gives a spike like signal (33).








27
Each nucleus in a molecule can be characterized according

to a parameter known as the chemical shift (6) (33). This is

the variation of the resonance frequency vP with the

electronic structure of the nucleus. The resonance frequency

of a given nucleus is a function of its electronic

environment, the applied field Bo and 7G. The electronic

structure of a nucleus can affect its resonance frequency by

interaction of its electron cloud with Bo. The electron cloud

can either increase or decrease the strength of Bo. The field

around the nucleus is actually given by:

Bocl = Bo(l-a)

where a is the shielding or the measure of the ability of the

electrons to alter Bo. The shielding of a nucleus varies from

compound to compound and within compounds due to changes in

the environment which surrounds it. With this in mind the

resonance frequency of a nucleus is given by:

vo = 7GBo(1-a)/2v

The chemical shift is measured in terms of the parts per

million of the magnetic field (33). In 1H a reference,

usually tetramethylsilane (TMS) is used. The distance delta

in parts per million of the resonance of the nuclei in the

compound from the resonance of TMS is the standard expression

for chemical shift. The equation for 6 is given by:

S(ppm) = distance from TMS in Hz
value of B1 in MHz








28

Interaction between nuclei can be determined by the

splitting of the resonance peaks at a given chemical shift

(33). The nucleus of interest has one magnetic environment

when a neighboring magnetic nucleus is at +1/2 and another

when it is at -1/2. This results in the resonance of the

nucleus containing two peaks. This influence of neighboring

spins on the multiplicity of the peaks is called spin-spin

coupling. The quantity J is the distance between the two

peaks for the resonance of one nucleus split by another and is

called the coupling constant (33).

The quantity of a particular type of nucleus present in

a compound can be determined by electronic or mechanical

integration of the resonance peaks (33). The absorbance of

energy when the nuclear spins of the protons flip from the

+1/2 state to the -1/2 state is directly proportional to the

number of spins present in the molecule (33).

FT-IR should be able to detect the ester carbonyl which

is in conjugation with the double bond. The c=o stretch will

be between 1715-1730 cm-1 (35). There should also be evidence

of the terminal vinyl with the c=c stretch between 1648 cm"1

and 1638 cm'l and C-H out of plane bending bands at 995-985 cm-

1 and 915-905 cm- (35). A c-c(=o)-o stretch should be present

between 1300-1000 cm'1 (35). The MD should have a c-o-c

stretch between 1150-1085 cml1 since an ether is formed in the

reaction (6, 35).








29

NMR is expected to show the presence of the terminal

vinyl with chemical shifts 5 ppm for CH2= protons and 6 ppm

for the =c -H proton for both modified albumin (MA) and MD (35).
The modification of albumin and dextran with GA introduces an

ABX system to the macromolecules.


2.2 Materials and Methods

2.2.1 Materials


Phosphate buffered saline (PBS) was prepared from A.C.S.

reagent grade, sodium phosphate dibasic heptahydrate, sodium

phosphate monobasic monohydrate, and sodium chloride obtained

from Aldrich. The PBS was adjusted to pH 7.2 if necessary by

sodium hydroxide and hydrochloric acid (Fisher Scientific).

The modified albumin (MA) was prepared from bovine serum

albumin (BSA) [Fisher Biotech or Boehringer Mannheim

Biochemicals BSA fraction V, heat shock, MW 68,000] modified

with GA (Aldrich). Glycine (Aldrich) was used to quench the

reaction. Dextran (MW 225,000, Sigma) was also modified with

GA. The MA and MD were dialyzed using Spectra/-Por Dialysis

Membrane from Spectrum (reorder # 132128) with a molecular

weight cut off (MWCO) of 50,000. All water used for solutions

was purified by the Barnstead Nanopure Ultrapure Water System.

The MA and MD were prepared for analysis with Nuclear Magnetic

Resonance Spectroscopy (NMR) by lyopholyzing them with the

Labconco Lyph-Lock 4.5 liter Freeze Dryer. They were








30
dissolved in D20 (Aldrich) and analyzed with a General

Electric QE-300 or NT-300 1H-NMR Spectrometer at 7.05 Telsa in

narrow bore 5 mm sample tubes. Trimethylsilylpropionate (TSP)

was used as an external reference. Fourier Transform Infrared

Spectroscopy (FT-IR) of the MA and MD were carried out with a

Nicolet 20SXB FT-IR Spectrometer. The spectra were run with

2 wavenumber resolution and triglyceryl sulfate as a

reference. The circle cell used for solution MA samples was

from Spectratech with a ZnSe crystal. The spectrophotometer

was purged with liquid nitrogen before the spectra were run.

Anhydrous potassium bromide (KBr) from Fisher was used to make

KBr pellets for analysis.


2.2.2 Methods


2.2.2.1 Modification of albumin

Bovine serum albumin (BSA) was modified using a procedure

by Park (5). A 5% solution of BSA was prepared with PBS as

the solvent. In the study by Park 5 ml of the 5% BSA solution

was modified by adding 200 sl GA and stirring for 5 hours (5).

In our study the GA was stirred with the 5% BSA solution for

5-24 hours. After 5 to 24 hours 1 ml of glycine was added to

the MA to quench the reaction between the BSA solution and the

GA, and the solution was stirred for an additional 30 minutes.

The solution was then purified by dialysis for at least 24 -

72 hours or until the smell of the GA was eliminated.

Phosphate buffered saline was used as the dialyzing medium.








31

Unreacted GA and glycine was removed from the MA solution by

dialysis. The solution was dialyzed in 1500 2000 ml of PBS.

The PBS was changed three times or more at least every 6

hours. After dialysis the MA was placed in vials and frozen.

2.2.2.2 Modification of dextran

Dextran was modified using a combination of the

procedures by Edman et al. and Park (5, 6). A 14.7% solution

of dextran was prepared (MW 225,000) with PBS as the solvent

(5 g dextran in 29 g PBS). Two ml of GA were added into the

solution and allowed to stir at room temperature (RT) for

seven days. The MD was dialyzed with 3 4 changes of the PBS

dialyzing medium (1500 2000 ml for each change. Dialysis was

continued until the smell of the GA was eliminated). After

dialysis the MD was placed in vials and stored in the

refrigerator at 40C.

2.2.2.3 FT-IR of modified albumin

FT-IR was used to verify the modification of the albumin.

The presence of the ester carbonyl or the c=c bond from the GA

was investigated, as well as the ester c-(c=o)-o stretch.

Modified albumin was analyzed in solution form using a

circle cell sample holder and in solid form by KBr pellets.

The circle cell was used to obtain spectra of the solutions of

MA, PBS, un-modified albumin (UMA), GA in PBS, and glycine in

PBS. Subtraction spectra were obtained. The PBS spectrum was








32

subtracted from the MA and UMA spectra making the assumption

that all of the free GA and glycine were removed from the MA

during dialysis.

MA was also analyzed by FT-IR spectroscopy in KBr pellets

(0.0015 g MA in 0.150 g KBr). The MA in liquid form was

lyopholized to yield solid MA. Spectra were run on pure

albumin from both Fisher and Boehringer Mannheim (BM), MA

modified with GA for 5 hours, and MA modified with GA for 24

hours. No subtraction spectra were generated.

2.2.2.4 FT-IR of modified dextran

Modified dextran was analyzed by FT-IR in KBr pellets

(0.0015 g MD in 0.150 g KBr). Spectra of pure unmodified

dextran (UMD) and MD were obtained and compared. No

subtraction spectra were generated. These spectra were run to

verify the c=c double bond, the c-o-c stretch and the ester

c=o stretch which should have been introduced by the GA.

2.2.2.5 NMR of modified albumin and modified dextran

Crosslinking agents (MA and MD) modified with GA were

analyzed with NMR to verify that the modification was

successful. The MA and MD solutions were lyopholized and then

dissolved in D20. A concentrated solution -1 g in 5 ml was

used for the MA while a very dilute solution was used for the

MD. The presence of peaks indicating terminal vinyl groups

was investigated since GA introduces a double bond to the

albumin and dextran.








33

2.3 Results and Discussions

2.3.1 FT-IR of Modified Albumin


The FT-IR spectra of liquid samples of UMA and MA

appeared to be very similar (figures 2-8, 2-9, 2-10). (The MA

samples were reacted for 5 hours.) The subtraction spectra of

UMA minus PBS (UMA-PBS) and MA minus PBS (MA-PBS) were also

compared (figures 2-11, 2-12). In both the MA-PBS and UMA-PBS

spectra there is a peak at 1550 cm-1. This peak is stronger

in the UMA spectra. The MA-PBS spectra has a strong peak at

about 1650 cml1 which is not present in the UMA spectra. There

is a peak in the UMA spectra at about 1610 cm'l which seems to

appear as a shoulder on the 1650 cm"1 peak in the MA-PBS

spectra. Information about the FT-IR spectra of amino acids

should give some insight on the bands found in the FT-IR

spectra obtained. Amino acid sodium salts have a strong

carboxylate asymmetrical stretch between 1600 cm1 and 1590 cm

1 and a weak symmetrical stretch at 1400 cml1. Amino acid

hydrochloride or other salts have a weak asymmetrical bending

band near 1610-1590 cm'1 and a relatively strong symmetrical

NH3+ bending at 1500-1481 cm-1. There is also a strong band

at 1220-1190 cm-1 due to the stretching. Strong carbonyl

absorption occurs between 1755 cm-1 and 1700 cm-1 for amino

acid hydrochlorides (Figure 2-13) (35).










5% ALBUMIN


FROM 6/7/91

















1525 700


NAVENUMBER


Figure 2-8. FT-IR spectra of 5% pure unmodified albumin (circle cell).


MODIFIED ALBUMIN 3/1/91

















175 2350 1525 700
NAVENUMBER


FT-IR spectra of modified albumin 3/1/91 (circle cell).


L)
U

H-


Zr-
[-


a:


4
H-


Li


Figure 2-9.























M MODIFIED ALBUMIN (ACCI
0o



Zu





Z cc




C I
rr-
F-





o

q000 3175 2350 1525 700
WAVENUMBER


Figure 2-10. FT-IR spectra of modified albumin 4/25/91 (circle cell).









UMA VS.


DO 100
NAVENUMBER


Figure 2-11. FT-IR spectra of UMA-PBS vs. MA-Al-PBS (circle cell).


0
(O UMA
1


UMA VS. MA-B


DO 1400
NAVENUMBER


FT-IR spectra of UMA-PBS vs MA-B-PBS (circle cell).


;00


800


MA-A1


Figure 2-12.




















z

C14








CI
0










C'J
Cm) C.)










0 +0










0+










CV)

z


0



r-I


01
14


054
40












rfl



540






*0

C4:
r4
(se



0 '
54








38
Albumin is made of 20-25% glutamic acid and aspartic

acids, which have carboxylate anions. This could be the cause

of the peak at 1550 cam1. The shift of the peak out of 1590-

1600 cm-1 for the amino acid carboxylate anion could be due to

the fact that albumin is a polymer of amino acids which have

been condensed into amide bonds. There is also a peak in both

the MA and UMA spectra at 1400 cm"- which may correspond to the

weak symmetrical stretching (35). The peak at 1650 cm'l in the

MA spectra was probably due to the conjugated ester carbonyl

introduced by the GA.

The MA and UMA samples analyzed in KBr pellets are shown

in figures 2-14, 2-15, 2-16, 2-17 and 2-18. All of the

spectra have the bands characteristic for carboxylate anion

(1650 cm-1-1550 and 1400 cm-1). The MA spectra, have two peaks

which were not present in the UMA spectra. These are peaks at

1000 cm-1 and 1080 cm"1. The peak at 1000 cm'l is probably due

to the out-of-plane C-H bending vibrations of the vinyl (-

CH=CH2) double bond added to the albumin due to the GA. The

peak at 1080 cm"l was probably due to the alcoholic C-O stretch

which for secondary alcohols occurs between 1124 cm"- and 1087

cm-1. This peak could also be due to the C-C(=O)-O ester

stretching which occurs between 1300 cm-1 and 1000 cm-1 (35).

The ester carbonyl added to the BSA by GA was probably over-

shadowed by the peak at 1650 cm-1 from the carboxylate anion.

The MA spectra have a slight shoulder at this peak which is








ALBUMIN B


2350
VENUMBER


FT-IR spectra of pure albumin BM (KBr).


MODIFIED


e'J

It


ALBUMIN 5


WAVENUMBER


Figure 2-15.


FT-IR spectra
(KBr).


of modified albumin reacted for 5 hours


OL

U-
Z


: CD
CC -
H-
^


4000


3175
WA


Figure 2-14.


1525


HRS















700


PUR


6/3/92


cI?




j

























MODIFIED


WAVENUMBER


Figure 2-16.


FT-IR spectra of modified albumin reacted for 24 hours
(KBr).


OJ

LI
U


C)
F-<

cU
S1
to-
MI0


ALBUMIN


24 HRS








41

UMA BM VS. MA 5HRS


U
-
OD
Z


cn
2-


z
(r -
t-


' 1000


UMA BM


j
~.


2350
VENUMBER


FT-IR spectra UMA BM vs MA 5 hours (KBr).


UMA BM


VS.


MA 2;4HRS


in

LU
U
z

H-


z
C Ln
cr-
H-


00


WAVENUMBER


FT-IR spectra of UMA BM vs MA 24 hours (KBr).


3175
WA


Figure 2-17.


1525


Figure 2-18.








42

probably due to the conjugated ester carbonyl. The shoulder

in the 24 hour MA is more prominent than that in the 5 hour

MA.


2.3.2 FT-IR of Modified Dextran


Pure dextran and modified dextran (MD) were analyzed in

KBr pellets only. Their spectra are shown in figures 2-19, 2-

20, 2-21, 2-22. The MD spectra have a peak which was not

present in the unmodified dextran (UMD) spectra at 1700 cm"1.

This was probably due to the conjugated ester carbonyl

introduced by the GA. This peak is the only difference

between the MD and UMD spectra.


2.3.3 1H-NMR of Modified Albumin


Pure unmodified albumin and MA were analyzed in D20 by

1H-NMR with TSP as an external reference. Figure 2-23 is a

spectra of pure UMA in the range between 8.80 ppm and -1.33

ppm. Figure 2-24 is an expanded spectra of pure UMA. The

large peak at -4.7 ppm is the water peak. Figure 2-25 is a

spectra of MA in D20 which was modified by stirring for 5

hours with GA as described in section 2.2.2.1. The peaks in

the range between 6.7 ppm and 7.4 ppm are evidence of the CH2=

and C-CH= protons, introduced by the double bond in GA.

These peaks were not present in pure albumin spectra (Figures

2-23 and 2-24). Figure 2-26 is a spectra of MA modified by








43
PURE DEXTRAN


/
/


./


3175 2350
WAVENUMBER


Figure 2-19.


FT-IR spectra of pure dextran (KBr).


MODIFIED


DEXTRAN2


1 I
3175 2350
WAVENUMBER


Figure 2-20. FT-IR spectra of modified dextran2


UL)


000


1525


I00
700


.4


4000


1525
1525


00
700


1


(KBr) .








44

MODIFIED


DEXTRAN


6/3/92


1\/i^


'I


I I
3175 2350
WAVENUMBER


q00


Figure 2-21.




N(J
0 ]


FT-IR spectra of modified dextran (KBr).



UMD VS. MD2
MD


3175 2350
NAVENUMBER


FT-IR spectra of UMD vs modified dextran2 (KBr).


0


15
1525


700


00


Figure 2-22.











45
















00
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0



C
a









S r-






4-







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49
stirring with GA for 10 hours. The peaks in this spectra were

very broad compared to those in Figure 2-25. This was

probably due to the fact that more free -NH2 groups were

modified at this longer reaction time and the various

locations in the albumin had different chemical shifts. There

also may have been some crosslinking of the albumin structure.

The broad peak between 6.6 ppm and 7.3 ppm was due to the

presence of the CH2= and C-CH= protons. Figures 2-27, 2-28

and 2-29 are expansions of the peaks between 3.61 ppm and

0.29 ppm for pure UMA, MA (5 hours), and MA (10 hours),

respectively. Similarities in these three spectra can be seen

if they examined and compared. Figures 2-30, 2-31 and 2-32,

are spectra expansions of the peaks between 7.89 ppm and 6.04

ppm, for pure UMA, MA (5 hours), and MA (10 hours), respec-

tively. No significant peaks show up in the pure UMA spectra.

The MA (5 hours) and MA (10 hours) spectra show indications of

the presence of olefinic protons, in the region between 6.85

ppm and 7.45 ppm. The MA (10 hours) spectra has a large broad

band in the region between 6.75 ppm and 7.45 ppm. In figure

2-31 the peaks at 6.85 ppm and 7.15 ppm are probably those for

the A and B protons of the ABX system introduced by the GA.

The peaks between 7.25 ppm and 7.5 ppm are probably due to the

X proton in the ABX system, in Figure 2-31. Figure 2-33 shows

a schematic of the ABX structure formed in modification of the

albumin.












50













a
a




(r
























0 0
N N









04
,4


N












I-
44
0




o,
0a
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= /HA
Dextran >C = C
HB




Figure 2.33. The ABX System.
Source: Reference 35








57
2.3.4 IH-NMR of Modified Dextran


Pure UMD and MD were analyzed in D20 by 1H-NMR with TSP

as an external reference. The MD spectra showed chemical

shifts characteristic of an ABX system involving terminal

vinyl protons. Figure 2-34 is a spectra of pure UMD in the

range between 5.07 ppm and 3.38 ppm. There were no signifi-

cant peaks present above 5.4 ppm when the pure UMA was

analyzed. Figure 2-35 is a spectra of MD in the range between

6.57 ppm and 2.88 ppm. The peak at 4.75 ppm occurred due to

water. Peaks above 5.5 ppm showed evidence of the ABX system

introduced into the dextran during modification. The peaks at

5.61 ppm were probably due to the A and B protons and the

peaks around 6.3 ppm to 5.9 ppm are probably due to the X

protons. Figure 2-36 is an expansion of region between 4.06

ppm and 3.38 ppm in pure UMD, and figure 2-37 is an expansion

of that region for MD. The peaks in the UMD spectra are

sharper than those in the MD spectra but the chemical shifts

and shapes of the peaks are the same in both spectra. Figure

2-38 and 2-39 are spectra of UMD and MD, respectively in the

region from 6.69 ppm to 4.69 ppm. Once again it can be seen

that there was nothing significant above 5.4 ppm for UMD and

that the presence of olefinic protons is indicated above 5.3

ppm in MD. Figure 2-40 is an expansion of the MD spectra in

the region between 6.69 ppm and 4.69 ppm showing more detail

in the olefinic proton peaks.





















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65

2.4 Conclusions

Albumin and dextran were modified with GA to enable them

to participate in the radical polymerization of AA and form

covalently bonded degradable crosslinks. The modifications

were verified by comparing FT-IR and NMR spectra of these pure

and modified natural polymers. The results showed evidence

that the GA was covalently bonded to the albumin and dextran,

and that the amount increased with reaction time.













CHAPTER 3
POLYMERIZATIONS TO FORM DEGRADABLE POLYMER HYDROGELS


3.1 Introduction

3.1.1 Hvdroaels of Biomedical Applications


Hydrogels are three dimensional crosslinked polymeric

networks which have a high affinity for water (36). (Figure

3-1 gives a schematic structure for hydrogels). They are held

together by crosslinks which may be due to cohesive forces,

hydrogen bonds, ionic bonds or covalent bonds (36).

When placed in water and other aqueous solutions, dry

hydrogels can swell up to as much as 200 times their original

weight without dissolving. Some gels may also be swellable in

organic solvents and are termed organogels (36). The amount

of swelling and the properties of the hydrogels depend on

hydrogel composition, crosslink density and the swelling

medium (8, 36, 37).

Hydrogels are ideal for use in biomedical applications

because they have physical properties which are similar to

natural tissues (38). They have high permeabilities for water

and small ions or molecules (8). Their high water content (30

- 95%) gives them similar diffusive, mechanical, interfacial,

and adsorption properties as living tissues. These









67




















0
0
0
43



0




43
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A
43




C,


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68
properties allow biomolecules to bind to hydrogel surfaces and

retain their biological activity (39).

Hydrogels have a soft rubbery consistency, low

interfacial tension, are pliable when they are wet, and are

easily machinable when they are dry. They can also be

fabricated into various geometrical shapes during the

polymerization process, and are easily purified due to the

ability to extract unwanted reaction by-products and unreacted

monomer before use, due to the open structure and rapid

diffusive transport (8, 37, 39).


3.1.2 Polymerization of Hvdroaels


Hydrogels can be fabricated by several polymerization

processes. They can be formed by the bulk or solution

polymerization of vinyl monomers or mixtures of vinyl monomers

in the presence of crosslinking agents and appropriate

initiators (free radical, redox, anionic, cationic) (8, 38,

40). Pre-existing polymers can be fabricated into hydrogels

by modifying them chemically or physically to introduce

crosslinks (8, 40). Polymers can be crosslinked chemically in

bulk or in solution by mixing them with initiators and cross-

linking agents (8, 40). Gamma-irradiation can also be used to

crosslink linear homopolymers and or copolymers to form

hydrogels (40). Mixtures of polymers with opposite charges

can form physically crosslinked polyelectrolyte complex

hydrogels (8, 38, 40).








69

Four classes of vinyl monomers have been used in the

polymerization of hydrogels which are neutral, anionic or

acidic, cationic or basic, and crosslinkers (8). Table 3-1

summarizes the various types of monomers that have been cited

in the literature, some of the initiators that have been used

as well as other solvents (8, 37, 40, 41).

In this study AA and NVP were solution polymerized with

ammonium persulfate as the initiator, MA or MD as the cross-

linking agent and PBS as the solvent at 600C.


3.1.3 Factors Which Affect Hvdroael Properties


The monomers used in polymerization of hydrogels

determine their physical and chemical properties. The

hydrophilicity of the monomers will determine their mechanical

properties as well as the amount of swelling. More hydro-

philic monomers produce hydrogels with a high water content,

good biocompatibility and poor mechanical properties (8, 40).

More hydrophobic monomers produce hydrogels with good mech-

anical properties, low water content and poor biocompatibility

(8, 40). Mixtures of hydrophilic and hydrophobic monomers can

be used to optimize biocompatibility and mechanical properties

for a given biomedical application (8, 40).

Peppas et al. found that the concentration of the solvent

(H20) during solution polymerization of hydroxyethyl methacry-

late determines whether the structure of the resulting hydro-

gel is optically homogeneous or heterogeneous (38). There is













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