Measurement of adhesion between calcium oxalate monohydrate and model surfaces using a dynamic wet cell

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Title:
Measurement of adhesion between calcium oxalate monohydrate and model surfaces using a dynamic wet cell
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xvii, 192 leaves : ill. ; 29 cm.
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English
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Habeger, Craig F., 1969-
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Materials Science and Engineering thesis, Ph.D   ( lcsh )
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Thesis:
Thesis (Ph.D.)--University of Florida, 1997.
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Includes bibliographical references (leaves 182-191).
Statement of Responsibility:
by Craig F. Habeger.
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Typescript.
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Vita.

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University of Florida
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MEASUREMENT OF ADHESION
BETWEEN CALCIUM OXALATE MONOHYDRATE AND MODEL SURFACES
USING A DYNAMIC WET CELL












By


CRAIG F. HABEGER


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


1997































This work is dedicated to my loving and supportive wife,


Nicole Habeger,



my parents,

James and Sandra Habeger,



and



my brother and sister, niece and nephew,

Larrin Habeger and Bonnie, Miranda, and Ralph Isaac Testa.

You are all the greatest!















ACKNOWLEDGMENTS


I would like to thank all of the people who made this research possible. Sincere

thanks are extended to Dr. James H. Adair, my committee chair and advisor, for his

guidance and patience. I would like to recognize the other members of my committee,

Drs. Christopher D. Batich, Anthony B. Brennan, and Brij M. Moudgil, from the

Department of Materials Science and Engineering, and Dr. Raymond L. Hackett, from the

Department of Pathology. All of these gentlemen contributed to the work reported herein

and I am grateful for their advice. I would also like to thank Dr. Saeed R. Khan, from the

Department of Pathology, who was not lucky enough to be on my committee but

graciously gave me advice on my research anyway. I appreciate the advice given to me by

Dr. John J. Mecholsky, Jr., from the Department of Materials Science and Engineering,

and his willingness to substitute for Dr. Brennan during my defense. I would like to

acknowledge the National Institutes of Health grant number POG 5P01 DK20586-17

which supported the work reported herein.

I would like to acknowledge the staff of the Major Analytical Instrumentation

Center (MAIC) for valuable advice, discussions, and training. I would also like to thank

Paula Scott, Pat Glenton, and Karen Byer for all of their assistance throughout my

research.

I am grateful to all of my friends, those in Dr. Adair's research group and those

not in the group. When I broke my leg, you showed me what friends really are. Special

thanks are extended to Drs. Robert E. Chodelka, Malanie L. Carasso, Tuo Li and soon to

be Drs. Jeffrey A. Kerchner, Nelson S. Bell, and Paul A. Demkowicz for their fruitful

intellectual conversations but mostly for their lousy card playing ability.










I am indebted to my wife, Nicole, who nursed me through a broken leg, twice, and

many other aches and pains too numerous to develop upon. She pushed me to finish,

however, at times, not hard enough. I would like to thank my entire family for being so

supportive. Finally, but most importantly, I would like to thank my personal Lord and

Savior, Jesus Christ, without whom none of this would be possible.















TABLE OF CONTENTS


ACKNOWLEDGMENT..................................... ........................................................ii

LIST O F TA B LES...................................................................................................... viii

LIST OF FIGURES ....................................................................................................... x

ABSTRACT .................................................................................................................. ...xvi

CHAPTERS

1 INTRODUCTION .................................................................................................... 1
1.1 Introduction....................................................................................................... 1
1.2 Literature Review.............................................................................................. 1
1.3 Development and Characterization of Model COM Particles................................2
1.4 Evaluation of the Calcium Oxalate Monohydrate Hamaker Constant..................3
1.5 Development of the Dynamic Wet Cell and Streaming Potential
M easurem ents .................................................................................................. 3
1.6 Measurement of COM Adhesion to Macromolecular Substrates .........................4
1.7 Conclusions and Future Research ..................................................................4

2 LITERATURE REVIEW........................................................................................... 5
2.1 Introduction....................................................................................................... 5
2.2 Particle Interactions........................................................................................... 7
2.2.1 Electrostatic Repulsion ....................................... ...................................9
2.2.2 A ttraction............................................................................................... 14
2.2.4 Interaction Energy.................................................................................. 15
2.3 Aggregation Mechanisms Among Particles and Particles at Surfaces ...................17
2.3.1 Particle Aggregation in Simple Electrolytes............................................... 19
2.3.2 Secondary Minimum Coagulation........................ ....................................19
2.3.3 Heterocoagulation.................................................................................20
2.3.4 Polymer Bridging Flocculation..................................................................23
2.3.5 Flocculation of COM Particles by Phospholipids and other
Intercellular Substances ...........................................................................26
2.3.6 Adhesion of Particles at Surfaces............................................................26
2.4 The Human Kidney.........................................................................................31
2.5 Characterization Techniques.............................................................................34









2.5.1 Electrokinetic Measurements.............................................................34
2.5.1.1 Electrophoresis............................................ ................................36
2.5.1.2 Streaming Potential.............................. ............... ......................... 36
2.5.2 Scanning Probe M icroscopy .................................. ....................................41
2.5.3 Scanning Electron Microscopy ................................................................43
2.5.4 Fourier Transform Infrared Spectroscopy ...........................................43
2.5.5 Particle Size Determination.................................................................. 45
2.5.6 X-Ray Diffraction................................................................................48

3 DEVELOPMENT AND CHARACTERIZATION OF MODEL COM
PA R TIC L E S .......................................................................................................... 49
3.1 Introduction.....................................................................................................49
3.2 Preparation and Characterization of Calcium Oxalate Monohydrate
Particles under Different Conditions................................................................54
3.2.1 COM particles ("33" particles) without seeds............................................ 54
3.2.2 COM particles with seeds .................................................................................56
3.2.3 COM particles precipitated from homogeneous solution at 900C .............56
3.2.4 Characterization ..................................................................................... 58
3.2.5 Crystal and Atomic Structure Modeling Using the Computer
Programs SHAPE and ATOMS .........................................................60
3.3 Results and Discussion .................................................... ............................. 62
3.3.1 COM particles ("33" particles) without seeds............................................ 66
3.3.2 COM particles ("33" particles) with seeds .............................................. 69
3.3.3 COM particles precipitated from homogeneous solution at 900C ..............71
3.3.4 Atomic structures of COM particles as a function of habit plane for
the high tem perature form ........................................................................71
3.3.5 Characterization of COM crystals...................................................................79
3.4 C onclusions..................................................................................................... 82

4 EVALUATION OF THE CALCIUM OXALATE MOMOHYDRATE
HAMAKER CONSTANT BASED ON STATIC DIELECTRIC
CONSTANT DETERMINATION AND ELECTRONIC POLARIZATION.........85
4.1 Introduction..................................................................................................... 85
4.2 M materials and M ethods............................................................. .................... 89
4.3 Results and Discussion ............................................................. .................... 95
4.4 C onclusions................................................................................................... 103

5 DEVELOPMENT OF THE DYNAMIC WET CELL AND STREAMING
POTENTIAL MEASUREMENTS...................................................................... 106
5.1 Introduction.............................. ...................................................................... 106
5.2 M materials and M ethods......................................................... ...................... 109
5.2.1 Adhesion Measurements using the Dynamic Wet Cell............................109
5.2.2 Streaming Potential Measurement.............................................................. 116









5.3 Results and D discussion ....................................................................................... 122
5.3.1 Dynam ic W et Cell...................................... ............. ...........................122
5.3.2 M odel CO M Particles..............................................................................125
5.3.3 Streaming Potential.............................................................................127
5.3.4 A dhesion ....................................................................................................13 1
5.3.5 Theoretical Modeling of Interactions..........................................................133
5.4 C conclusions .........................................................................................................138

6 MEASUREMENT OF COM ADHESION TO MACROMOLECULAR
SU B STRA TES...................................................................................................... 140
6.1 Introduction................................................................................................... 140
6.2 Materials and Methods...............................................................................143
6.2.1 Particle Synthesis and Characterization...................................................143
6.2.2 Substrate Coating ....................................................................................... 143
6.2.3 Adhesion Measurements....................................................................145
6.2.4 Streaming Potential Measurements......................................................... 146
6.3 Results and D discussion ....................................................................................... 146
6.3.1 Substrate Coverage................................................................................... 146
6.3.2 Zeta Potential Determinations ...............................................................149
6.3.3 Adhesion Measurements....................................................................153
6.3.4 Hydrodynamic Model of the Human Kidney..........................................167
6.4 C conclusions .........................................................................................................170

7 SUMMARY AND FUTURE WORK........................................................................173
7.1 Sum m ary ....................................................................................................... 173
7.2 Future W ork..................................................................................................175

APPENDICES

A DETERMINATION OF THE AREA OF COM CRYSTALLOGRAPHIC
FACES USING EQUIVALENT SPHERICAL DIAMETER................................177

B DETERMINATION OF THE STRESS IN THE KIDNEY AS A FUNCTION
OF FLOW AND DISTANCE FROM THE TUBULE WALL................................ 179

R E F E R E N C E S ................................................................................................................. 182

BIOGRAPHICAL SKETCH........................................................................................ 192















LIST OF TABLES


Table page

3.1. Composition of artificial urine ion solution............................................................ 61

3.2. Values of the lattice parameters of calcium oxalate monohydrate...........................63

3.3. Atomic coordinates of W hewellite..........................................................................65

3.4. Crystal forms and corresponding central distance values used to generate the
theoretical shape of COM crystals.............................................................................68

3.5. Values of the crystallographic data for the high temperature form of
W hew ellite........................................................................................................ 73

3.6. Atomic coordinates for high temperature form of Whewellite................................75

4.1. Index of refraction for COM as a function of optical direction and wavelength.......88

4.2. The composite mixing rules which were evaluated ........................................ ...90

4.3. Molecular structures of the silane coupling agents used to disperse COM in
Eccosil 5019 silicone. .............................................................. .....................92

4.4. Dielectric constant values of COM determined by fitting the mixing rules to
the experim mental data........................................................................................ 98

4.5. Calculated values of the UV characteristic frequency and corresponding
dielectric constants and refractive indices as a function of crystallographic
direction determined from Cauchy plots..........................................................102

4.6. Comparison of A131 calculated for COM using Gregory's approximation vs.
the Tabor-W interton relationship. ................................................................... 105

5.1. A list of the physical parameters necessary to calculate zeta potential from
stream ing potential ................................. ............................................................ 121

6.1. The values of zeta potential of the macromolecular substrates determined
using streaming potential measurements in saturated COM solution.................. 151









6.2. Values of adhesion strength for COM particles adhering to macromolecular
substrates as a function of crystallographic habit plane in COM saturated
so lu tio n ................................................................................................................. 16 5

6.3. Values of adhesion strength for COM particles adhering to macromolecular
substrates as a function of crystallographic habit plane in AUIS......................166

6.4. Human kidney tubule dimensions and volumetric flow rates reported by Kok
and K han ......................................................................................................169















LIST OF FIGURES


Figure page
2.1. Pathways to kidney stone formation................................................. ................. 6

2.2. COM particle aggregate attached to the wall of the proximal tubule in a rat
nephron with one end of the constituent crystals joined together (near the
arrow) and the other end free. ................................... .................................... 8

2.3. The electrical double layer structure illustrating the distribution of ions
surrounding an electrostatically charged particle............................................10

2.4. The effect of electrolyte concentration on the ionic cloud and particle
separation distance in a (A) low ionic strength solution and a (B) high ionic
strength solution.....................................................................................................

2.5. The effect of ioic strength on zeta potential or the electric potential at the
shear plane........................................................................................................ 13

2.6. A schematic diagram illustrating the attractive, repulsive, and total energy
curves for two interacting materials as function of separation distance ...............16

2.7. A schematic representation of six aggregation mechanisms which may
contribute to stone form ation.................................................... .....................18

2.8. Heterocoagulation of COM with HU is predicted based on: (A) zeta potential
determinations, (B) mixing of HU and COM at pCa < 5 where COM and
HU are both negatively charged, and (C) mixing at pCa = 4 where COM and
HU are both oppositely charged........................................................................22

2.9. A schematic representation of patch charge flocculation whereby incomplete
macromolecular coverage may create electrostatic shielding or opposite
charge in the case of a charged polymer. ............................. ...........................24

2.10. Scenario depicting the balance of hydrodynamic forces (Fd) and adhesive
forces (Fa) acting on a COM particle bound to the brush border in the human
nephron .......................................................................................................... 29









2.11. A scanning electron photomicrograph of a tubule cross section in the rat
animal model demonstrating association of a COM crystal with the
basem ent m em brane............................................................................................ 32

2.12. A schematic illustration of a human nephron.........................................................33

2.13. A schematic representation of a tubule wall found in a kidney ..............................35

2.14. A schematic representing charged particle movement in an applied electric field
to determine electrophoretic mobility...............................................................37

2.15. A schematic representation of the flow of ions under an applied hydrodynamic
pressure which generate the potential across the capillary or streaming
potential. ............................................... ............. ..................................................39

2.16. A schematic diagram of the SPM .................................... ...... ...........................42

2.17. Multiple internal reflection within the internal reflection element which is
coupled to the sam ple......................................................................................... 44

2.18. Sampling depth as a function of wavenumber for the KRS-5 internal reflection
element having a 450 incident angle......................................................................46

2.19. A schamatic diagram of a Coulter counter, electrical pulse counting instrument......47

3.1. Possible aggregation mechanisms for particles in the urinary environment ............51

3.2. Schematic representation of processing steps for the preparation of COM
crystals ("33" particles) without seeds...........................................................55

3.3. Schematic representation of processing steps for the preparation of COM
crystals ("33" particles) with seeds.......................................................... ...........57

3.4. Schematic representation of processing steps for the preparation of COM
crystals ("32" particles) with heat treatment at 900C.........................................59

3.5. (A) SEM photomicrograph and (B) and (C) theoretical equilibrium shapes of
the COM crystals precipitated without seeds.(B) The equilibrium shape of
COM crystals based on the crystallographic data of Deganello and Piro and
(C) the equilibrium shape of COM crystals based on the crystallographic
data of Tazzoli and Domeneghetti. .......................................................................64









3.6. Theoretical atomic structures of COM crystals as a function of habit plane:
(A) and (B) are the (010) and (100) planes, respectively, drawn using Cocco
and Sabelli atomic coordinates; (C) and (D) are the (010) and (100) planes,
respectively, drawn using Tazzoli and Domeneghetti atomic coordinates; (E)
and (F) are the (010) and (100) planes, respectively, drawn using Deganello
and Piro atomic coordinates. .............................. .............................................67

3.7. SEM photomicrographs and theoretical equilibrium shapes of seed crystals
and the COM crystals precipitated using seeds:(A) seed crystals, (B)
equilibrium shapes of seed crystals generated with the (001) and (010) faces,
respectively, (C) COM crystals grown using seeds, and (D) equilibrium
shape of COM crystals................................. ................ ............................... 70

3.8. SEM photomicrograph and theoretical equilibrium shapes of the COM
crystals precipitated from homogeneous solution at 900C:(A) COM
crystals, (B) equilibrium shapes of an individual COM crystal and a crystal
with a twin generated on the (001) face, and (C) equilibrium shapes of COM
crystals generated using the (010) face as the dominant face...............................72

3.9. Theoretical atomic high temperature structure (stability range: 318-415 K) of
COM crystals as a function of habit plane, (A) (010) and (B) (101)....................76

3.10 A (top) typical experimental COM x-ray diffraction pattern and (bottom) the
JCPDS file for the mineral W hewellite...............................................................77

3.11. The differential frequency vs. equivalent spherical diameter particle size
distribution of the experimentally produced COM crystals grown without
seeds fit to a log-normal probability distribution.........................................79

3.12. The relative linear dimensions of experimentally produced COM crystals..............80

3.13 Zeta potential as a function of pH for COM in saturated COM solution and
zeta potential at pH 6 for COM in 10% AUIS........................................ ...81

3.14 Contact mode SPM scan of the COM (010) crystallographic face showing the
surface roughness. ............................................................................................83

3.15 Contact mode SPM scan of the COM (101) crystallographic face showing the
surface roughness. ............................................................................................84

4.1. Optical photomicrographs of COM particles dispersed in Eccosil 5019 using
(A) no coupling agent, (B) SMAEPS coupling agent, and (C) GPTMS
coupling agent.................................................................................................. 96

4.2. The composite mixing rules fit to experimental dielectric data. ...............................97









4.3. Cauchy plots for water and COM as a function of optical direction....................101

5.1. (A) A picture and (B) a schematic diagram of the dynamic wet cell developed
to measure adhesion of particulate to surfaces.In (B), structural thru-holes
have been omitted from the drawing to improve clarity....................................108

5.2. (A) A picture and (B) a schematic diagram of the dynamic wet cell supporting
equipm ent................................................................................................... 112

5.3. A time lapse sequence of events during an adhesion experiment.The flow rates
are (A) 12 ml/min, (B) 53 ml/min, and (C) 108ml/min.......................................113

5.4. COM particles of controlled morphology shown in a (A) scanning electron
micrograph, (B) modeled using SHAPEc software showing the two
dominant crystallographic faces, (010) and (101), and (C) demonstrating the
relative crystallographic size ratios.................................................................... 114

5.5. Theoretical (010) and (101) crystallographic planes of calcium oxalate
monohydrate.The Ca2+/C2042" ratio is given above each theoretical atomic
structure. .............................................. ....................................................... 15

5.6. A (A) picture and a (B) schematic diagram of the streaming potential cell.............17

5.7. A diagram of the R-C circuit used to eliminate asymmetry and electrode
polarizations......................... ......................................................................... 118

5.8. (A) A picture and (B) a schematic diagram of the streaming potential
instrument including all of the supporting equipment........................................ 119

5.9. A plot of dial setting versus volumetric flow rate used to calibrate the
pump.The error bars represent the standard deviation of five individual
experim ents....................................................................................................... 123

5.10. A plot of flow rate versus Reynolds number indicating the stable flow inside
the dynamic wet cell at all experimental flow rates................................. ...124

5.11. Experimentally determined zeta potential of COM particles in saturated
calcium oxalate monohydrate solution.Each data point is the mean +/- 95%
confidence interval.The ionic strength of the saturated COM solution was
8x10 6 M ... ............................................................................................... ......... 126

5.12. A plot of pressure drop or driving pressure across the streaming capillary
indicating that the flow is laminar under all experimental flow conditions..........128









5.13. A plot of streaming potential vs. driving pressure for fused quartz in COM
saturated solution showing a linear regression with an r2=0.98 and 95%
confidence lim its............................................................................................129

5.14. The probability of an initial particle on the (010) and on the (101) adhering
versus the applied stress acting on the particle at failure as determined using
the dynamic wet cell.The total number of particles counted that were lying
on the (010) was n=255 and on the (101) was n=380.......................................132

5.15. A scanning probe microscopy image of the fused quartz surface under a COM
saturated solution liquid environment.Also given is the image roughness
statistics which include the ra or mean surface roughness of 0.871 nm ...............135

6.1. A plot of wavenumber vs. absorbance for fused quartz, collagen type I,
fibronectin, MATRIGEL, and PEI produced using ATR-FTIR.All of the
macromolecular substrates were coated on fused quartz...................................147

6.2. A plot of wavenumber vs. absorbance for fused quartz, collagen type I,
fibronectin, MATRIGEL, and PEI produced using ATR-FTIR for the
wavenumbers 2100-3600 cm'1.All of the macromolecular substrates were
coated on fused quartz. ................................... ................................................148

6.3. Zeta potential as a function of pH for COM in saturated COM solution and
zeta potential at a pH of 6 for COM in 10% AUIS..........................................150

6.4. A plot of driving pressure vs. streaming potential for the substrates collagen
type I, fibronectin, MATRIGEL and PEI .....................................................152

6.5. The probability of an initial COM particle on the (010) and the (101) adhering
to fused quartz in AUIS versus the applied stress acting on a particle at
failure as determined using the dynamic wet cell.The total number of
particles counted lying on the (010) was n= 13 and on the (101) was n=84......154

6.6. The probability of an initial COM particle on the (010) and the (101) adhering
to collagen type I in COM saturated solution versus the applied stress
acting on a particle at failure as determined using the dynamic wet cell.The
total number of particles counted lying on the (010) was n=82 and on the
(101) w as n= 133. ........................................................................................... 155

6.7. The probability of an initial COM particle on the (010) and the (101) adhering
to collagen type I in AUIS versus the applied stress acting on a particle at
failure as determined using the dynamic wet cell.The total number of
particles counted lying on the (010) was n=84 and on the (101) was n=72........156









6.8. The probability of an initial COM particle on the (010) and the (10i) adhering
to fibronectin in COM saturated solution versus the applied stress acting on
a particle at failure as determined using the dynamic wet cell.The total
number of particles counted lying on the (010) was n=87 and on the (10i)
w as n= 104............................................ ......................................................... 157

6.9. The probability of an initial COM particle on the (010) and the (101) adhering
to fibronectin in AUIS versus the applied stress acting on a particle at failure
as determined using the dynamic wet cell.The total number of particles
counted lying on the (010) was n=95 and on the (101) was n=143...................158

6.10. The probability of an initial COM particle on the (010) and the (101) adhering
to MATRIGEL in COM saturated solution versus the applied stress acting
on a particle at failure as determined using the dynamic wet cell.The total
number of particles counted lying on the (010) was n=57 and on the (101)
w as n=54 ............................................. ........................................................ 159

6.11. The probability of an initial COM particle on the (010) and the (101) adhering
to PEI in COM saturated solution versus the applied stress acting on a
particle at failure as determined using the dynamic wet cell.The total number
of particles counted lying on the (010) was n=61 and on the (101) was n=69....160

6.12. A bar chart summarizing the adhesion data experimentally determined in COM
saturated solution (i.e., low ionic strength).The numbers above each set of
bars is the value of the zeta potential for each substrate...................................161

6.13. A bar chart summarizing the adhesion data experimentally determined in COM
saturated solution (i.e., low ionic strength).The numbers above each set of
bars is the value of the zeta potential for each substrate...................................163

6.14. COM particles (A) coated during an adhesion experiment using fibronectin and
(B) uncoated COM particles used in an adhesion experiment.Both
photomicrographs were taken after flow ...................................................168

6.15. A plot of the stress on a 5 uim to 8 gm radius model COM particle under the
hydrodynamic conditions found in the different areas in the human
kidney.The shaded region represents the range of experimentally measured
values of adhesion of COM to biological materials................................... 171















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


MEASUREMENT OF ADHESION
BETWEEN CALCIUM OXALATE MONOHYDRATE AND MODEL SURFACES
USING A DYNAMIC WET CELL

By

Craig F. Habeger

December, 1997




Chairman: Dr. James H. Adair
Major Department: Materials Science and Engineering


Calcium oxalate monohydrate (COM) is the primary constituent in kidney stones.

COM crystals were synthesized in the laboratory and characterized. Computer

calculations of particle shape have been reconciled to observed shapes of COM crystals

experimentally synthesized under various conditions. Comparison between the

theoretical atomic structures generated by computer calculations are consistent with

previously reported atomic layering sequences.

Composite mixing rules were used to deconvolute the dielectric constant of COM

from a COM/silicone composite. Utilizing the Lichtenecker dielectric mixing model, the

value of the static dielectric constant of COM was determined to be 28.9. Optical and

dielectric data were then used in the Tabor-Winterton relationship to calculate the

Hamaker constant, Ai31, of COM particles interacting in water. The A131 for COM as a









function of crystallographic habit also was examined. The mean value of A131 for COM

was calculated to be 13.7x10'21 J at 370C in an aqueous environment.

A hydrodynamic method for measuring the adhesion of particles to a surface has

been designed for use in the study of kidney stone disease and other pathological

biomineralization phenomena. The hydrodynamic force required to displace a particle

adhering to a fused quartz substrate was calculated via the Poiseuille equation. The

strength necessary to remove 50% of the COM particles adhering to the substrate on the

(010) and (10 ) crystallographic surfaces are 81 and 170 Pa, respectively. The

previously determined Hamaker constant and measured values of zeta potential were used

to calculate the energy of interaction between a COM particle and the fused quartz

substrate which was found to be comparable to experimentally measured values, provided

the separation distance was on the order of 20 nm.

Using the instrument and technique developed, the adhesion of COM to

biologically and non-biologically relevant materials was measured in COM saturated

solution and in an artificial urine ion solution. The biologically significant materials were

the proteins collagen type I, fibronectin, and MATRIGEL, a mixture of basement

membrane proteins. The non-biologically relevant material was polyethyleneimine a

positively charged macromolecule used as a control. MATRIGEL and the positive

control, polyethyleneimine, exhibited the highest adhesion to COM crystals.















CHAPTER 1
INTRODUCTION


1.1 Introduction

Kidney stones effect hundreds of thousands of people each year. In 1991,

895,467 visits were made to doctors for both kidney and ureter stones [NIH97] and in

1993, kidney and ureter stones resulted in approximately 302,00 hospitalizations [Cla95].

The direct and indirect cost of kidney and ureter stones in 1993 was approximately $1.83

billion [Nat95]. Prevention of kidney stones is the focus of much research; however,

before prevention can occur the mechanisms of stone formation must be understood.




1.2 Literature Review

Chapter 2 reviews the pertinent kidney stone literature and gives background on

techniques utilized in the work reported herein. Kidney stone disease has been described

as an opportunistic disease which can rely on many causative mechanisms acting in

concert [Fin78a, Fin84]. One of those possible mechanisms is the growth of an adherent

particle, called a fixed particle, in the lumen of a nephron. Such an adherent particle can

subsequently grow by secondary nucleation and growth and/or aggregation of crystallites

to a size large enough to occlude a renal tubule, which is the basis of the fixed stone

mechanism of kidney stone development. Finlayson and Reid [Fin78b] calculated the

time for a COM crystal to grow to a large enough size to cause an occlusion and

determined that the crystal would not have time enough to grow to such a size.









Therefore, crystal attachment is a necessary process in kidney stone formation [Man91,

Man94].

Renal injury has also been implicated in fixed stone formation [Gil79, Kha84,

Man91]. Khan [Kha82, Kha95a] has discussed that after attachment of a crystal to the

epithelial cell surface, injury to the cell may or may not occur.

A few different results may ensue due to crystal attachment to the epithelial cell.

The cell may die and be washed away in the urine flow taking with it the crystal. The cell

may envelope the crystal and continue to function normally [Lie92, Lie93, Lie94] with no

further growth of the crystal. However, if epithelial cell injury has occurred, basement

membrane proteins may be accessible to adhere to COM crystals by sloughing of the cells

leaving behind the exposed underlying architecture, the basement membrane. The

basement membrane is a layer of proteins that underlies the epithelial sheet and will be

discussed subsequently in more detail. Depending on the extent of injury, other proteins

common to the extracellular matrix found below the basement membrane may also be

accessible for attachment to COM crystals.

Researchers have shown the attachment of COM crystals to epithelial cells and

fibrous proteins characteristic of those found in the basement membrane and extracellular

matrix, yet the importance of these individual materials in kidney stone formation with

respect to COM crystal adhesion is not known. The necessity to determine the

important materials most responsible for COM crystal adhesion will allow researchers to

focus their interests to those more important materials in the search for a possible cause

and therefore relief from kidney stone formation.


1.3 Development and Characterization of Model COM Particles

Chapter 3 demonstrates the laboratory synthesis of COM particles having a

desired size and crystallographic shape to be used in adhesion experiments. The optimal

particle shape is spherical; however, COM has a monoclinic crystal structure and









spherical, crystalline particles cannot be produced. Therefore well-defined shaped,

rectangular particles having a narrow size distribution were produced to limit errors

associated with calculations. The experimentally produced particles were modeled using

computer software to determine the crystallographic face indices and the theoretical

atomic structure of two dominant crystallographic faces. The particles were characterized

using electrophoresis and atomic force microscopy. The zeta potential values for the

particles were determined using electrophoresis in a low ionic strength solution, COM

saturated solution, and a high ionic strength solution, artificial urine ion solution. Finally,

the surface roughness of each of the two dominant crystal faces were measured in

saturated COM solution using contact mode Atomic Force Microscopy.


1.4 Evaluation of the Calcium Oxalate Monohydrate Hamaker Constant


Chapter 4 involves the determination of the Hamaker constant, a measure of the

van der Waals interaction forces, for COM. Attractive van der Waals forces are very

important in the adhesion process and a reliable value of the Hamaker constant is

necessary to determine these forces. The COM particles developed in Chapter 3 were

mixed with a silicone material to produce a composite. The dielectric properties of the

composite were measured and composite mixing rules were used to deconvolute the

dielectric constant of COM from the COM/silicone composite. Optical and dielectric

data were then used to calculate the Hamaker constant, A131, of COM particles interacting

in water.


1.5 Development of the Dynamic Wet Cell and Streaming Potential Measurements

Chapter 5 describes the development of a hydrodynamic method for measuring

the adhesion of particles to a surface for use in the study of kidney stone disease and

other pathological biomineralization phenomena. By using hydrodynamic flow to remove

particles from a model surface, the strength with which particles adhere to a surface can









be measured. The hydrodynamic force required to displace a particle is calculated via the

Poiseuille equation using the dynamic wet cell dimensions and the fluid flow rate. A

model surface consisting of fused, spectroscopic grade quartz was used in the

development of the apparatus. Also developed was an instrument to measure the

electrostatic potential, called zeta potential, at the surface of a flat substrate in solution.

The previously determined Hamaker constant and measured values of zeta potential were

used to calculate the energy of interaction between a COM particle and the fused quartz

substrate. The theoretical values of adhesive pressure were compared to the measured

values of adhesion.


1.6 Measurement of COM Adhesion to Macromolecular Substrates


Chapter 6 investigates the adhesion of COM to both biologically relevant and

non-biologically relevant materials. The adhesion measurements were measured in COM

saturated solution and in an artificial urine ion solution, using the instrument and

technique developed in this research. The use of two different ionic strength solutions

allows interpretation of the types of material-material interactions taking place (i.e., van

der Waals, electrostatic, chemical) to be made. The biologically significant materials were

the proteins collagen type I, fibronectin, and MATRIGEL, a mixture of basement

membrane proteins. The non-biologically relevant material was polyethyleneimine, PEI, a

positively charged macromolecule used as a control.


1.7 Conclusions and Future Research

Chapter 7 summarizes the conclusions of the current research and presents

suggestions for future work.















CHAPTER 2
LITERATURE REVIEW


2.1 Introduction

Kidney stone disease has been called an opportunistic disease because it is

thought that kidney stone disease takes an unfortunate, simultaneous convergence of

factors that may be achieved by several pathways for a stone to form [Con90, Fin78a,

Fin84, Rob86]. Thus, injury, supersaturation (particularly hyperoxaluria), nucleation and

growth mechanisms all play a role in contributing to stone formation as demonstrated in

Figure 2.1. Even so, urolithiasis is idiopathic with no one risk factor providing an obvious

source of stones in chronic stone former. However, both in vivo and in vitro evidence

indicates that the presence of particles is a necessary but insufficient condition for stone

formation. Most non-stone forming individuals, at certain times, have crystalluria yet do

not have a stone incident. For a stone to be created from freely flowing particles, either a

particle must grow to a large enough size to occlude the tubule or aggregation of multiple

particles must occur. Furthermore, if aggregation is responsible for stone formation, the

attractive energy or energies holding the primary particles together in an aggregate must be

great enough to withstand the hydrodynamic shear forces due to the flow of fluid through

the nephron.

The possibility that the growth of a large crystal leads to a stone has been shown

to be unlikely by several teams of investigators [Fin78b, Kok94]. Finlayson and Reid

[Fin78b] predicted that there was not enough time for a freely flowing particle to grow to

a size sufficient to occlude the tubule based upon the hydrodynamics in the nephron and

measured rates of crystal growth for calcium oxalate monohydrate. It was also suggested
















SCatalysis


Cel Aggregation
-cell/ 2
Damage
a F1


C Aggregation


Figure 2.1. Pathways to kidney stone formation.









by Finlayson and Reid that aggregation of freely flowing particles was not likely due to

the relatively low concentration of particles within the nephron at any given point in

time. However, a recent analysis of the hydrodynamic aspects of transit time through the

nephron by Kok and Khan [Kok94], based on better estimates of the diameters of

different regions in the nephron, indicated that aggregation of freely flowing particles is

possible. Regardless, attachment of calcium oxalate particles to the epithelial nephron

wall has been demonstrated in a rat animal model [Fin84, Fin78b], as shown in Figure 2.2.

Thus, the concept of fixed particles at the epithelial wall has become a fundamental

principle in the development of kidney stones. In more recent work [Kha84, Man87,

Man91, Rie88], it has been demonstrated that particle attachment can be induced by

epithelial wall damage in which intercellular species, such as phospholipids, may play a

role.

The objective of this chapter is to review the dynamics of aggregation for particles

and interactions of the particles and/or aggregates with biological surfaces such as the

basement membrane or interluminal wall of the nephron. The nature of the forces among

particles and surfaces will be discussed with respect to the hydrodynamic flow scheme

found in the nephron; however, particle-particle interactions must first be discussed.


2.2 Particle Interactions

Particles in solution are constantly moving. The movement of the smaller

particles (<1 Igm) is due to Brownian motion, also called perikinetic motion. The

movement of larger particles (>5 jgm), defined as orthokinetic motion, is due to gravity

and convection currents. The constant motion in solution causes the particles to interact

with one another ultimately resulting in either stabilization or aggregation. When

repulsive forces dominate the particle-particle interaction, stabilization is the result. The

opposite is the case if attractive forces dominate the particle-particle interaction, called

aggregation. These forces arise from an electrostatic potential on the surface of the



















































Figure 2.2. COM particle aggregate attached to the wall of the proximal tubule in a rat
nephron with one end of the constituent crystals joined together (near the arrow) and the
other end free (bar = 5 gm). Photomicrograph by S.R. Khan [Kha91].









particles and from an inherent dipole interaction of atoms and molecules within one

particle interacting with another particle.


2.2.1 Electrostatic Repulsion

The surface potential, 'Fo, is the potential difference between the solid surface and

the bulk solution. Ions which alter the surface potential are called potential determining

ions. Curreri et al. [Cur79] have hypothesized that Ca2+ and C2042- specifically adsorb to

the COM surface and, thus, are termed the potential determining ions for COM.

The Gouy-Chapman electrical double layer model describes the excess ions

present on the surface of the solid phase and the distribution of ionic charge of opposite

sign in the solution phase surrounding the electrically charged surface which assures

electroneutrality. A schematic diagram of the Gouy-Chapman model is shown in Figure

2.3. The double layer model consists of an inner layer, often called the Ster layer, which

describes ions adsorbed into the compact or inner region of the double layer, and the outer

or diffuse layer [Ove52]. Theoretical analysis of the double layer shows that the charge

density in an aqueous solution decreases rapidly with increasing distance from the solid

surface, and the electrostatic potential for low potentials is given by the Debye-Htickel

approximation [Hun81],


V = ,o exp(-xt), (2.1)


where Vy is the potential as a function of distance into the solution, K is called the Debye-

Htickel parameter (1/length), and x is the distance from the solid surface.

Based on the electrostatic model for aggregation, in the simplest case, the repulsive

contributions between particles in solution arise from the interaction of ionic clouds

surrounding the particles, as demonstrated by Figure 2.4. The extent of the ionic cloud is

governed by the solution ionic strength described by the simplified equation





















p < tStern Layer

S1\ Gouy-Chapman (Diffuse) Layer
o +0
0 ) 0
E)
W) E) Eo oD
+ 0000 0

Distance from Surface










Figure 2.3. The electrical double layer structure illustrating the distribution of ions
surrounding an electrostatically charged particle.


























+
d

(A)






+ ++


.: ""++.y
++




d

(B)






Figure 2.4. The effect of electrolyte concentration on the ionic cloud and particle
separation distance in a (A) low ionic strength solution and a (B) high ionic strength
solution.
solution.












C = kT (2.2)



where e is the elemental charge, nf, is the number of ions of type i per unit volume far

from the surface, z, is the valence of ions of type i, e is the dielectric permittivity of the

solvent, k is the Boltzmann constant, and T is the temperature in Kelvin. The Debye-

Hiickel simplification assumes that the surface potential or stern potential is low (e.g.,

less than 25 mV). The quantity 1/K is referred to as the Debye length and indicates the

thickness of the double layer. The Debye length is dependent only on temperature,

valence of the electrolyte ions, and electrolyte concentration. Solution ionic strength, I, is

given by


I= (cz2)z (2.3)



where c, is the solution concentration (M). As the ionic strength of the solution increases,

the surface charge decay into the solution occurs more rapidly due to the inability of the

solution to support charge. That is, the diffuse double layer becomes compressed due to

the increased concentration of ions in solution as demonstrated in Figure 2.5. The

increased concentration of ions near the charged particle surface causes the electrical

charge to be neutralized by the ions of opposite charge, counterions, in solution. Figure

2.4 schematically describes the ionic cloud surrounding a charged particle in low and high

ionic strength environments. The variable d in Figure 2.4 is the distance of separation for

the two charged particles. At low ionic strength, the ionic cloud is large and electrostatic

repulsion prevent particle-particle contact. At higher ionic strength the particles can
















I SHEAR PLANE


I


IIi


<12< 13


INCREASING IONIC
STRENGTH


SURFACE DISTANCE INTO THE SOLUTION
















Figure 2.5. The effect of ioic strength on zeta potential or the electric potential at the
shear plane.









come into much closer proximity due to collapse of the surrounding ionic cloud. If the

collapse is significant, aggregation may occur.


2.2.2 Attraction


The tendency towards aggregation can result from oppositely charged electrostatic

potentials or from van der Waals forces which are present at all times, irrespective of

solution conditions. The attractive van der Waals forces are composed of multiple

intermolecular interactions between the ions, molecules, and electrons that make up the

particles interacting across a dielectric medium such as water [Hou80, Hun93, Ber90].

The mutual attraction between interacting particles arise from harmonic oscillations at the

molecular, atomic, or subatomic level. Three primary sources of such intermolecular

interactions exist depending upon the nature of the interacting species. Some of the more

important specific dispersion interactions are known as Keesom, Debye, and London

interactions [Isr92]. Keesom forces are due to molecular dipole-dipole interactions in the

particles. Debye interactions occur when molecular dipoles in one particle induce

electronic polarization in the other interacting particle. As such, Keesom and Debye

interactions occur only if a material has one or more dipoles present within its structure.

In contrast, London interactions are more ubiquitous because London interactions are due

to mutual electronic polarization with all atoms, or electron cloud density shift, of

adjacent atoms.

The range of the force due to dipole interactions between two atoms or molecules

is a nanometer or less and varies as 1/r6. However, the interactions are to some degree

additive in that they effect all neighboring atoms or molecules in a particle. Particle-

particle van der Waals forces interact over a much longer range, 1/r2.









2.2.4 Interaction Energy


The Derjaguin and Landau [Der41] and Verway and Overbeek [Ver48] (DLVO)

theory considered colloid stability in terms of the electrical double layer and van der

Waals forces. Figure 2.6 is a plot of interaction energy as a function of separation

distance, which is a potential energy diagram for two interacting materials across a

medium. Positive interaction energy refers to repulsive energy and negative interaction

energy refers to attractive energy. In the diagram both the attractive and repulsive

portions are plotted with respect to separation distance of the two interacting materials.

The third interaction line is the sum of the attractive and repulsive curves, the total

interaction energy curve. Labeled on the total interaction energy curve in Figure 2.6 are

the primary and secondary minimum, and the energy barrier to aggregation.

The primary minimum is the potential energy well that occurs when the two

interacting materials are in contact. The main attractive forces are due to van der Waals

forces. When two interacting particles are at a separation distance such that their

associated interaction energy is in the primary minimum, the attractive energies dominate

the total interaction energy and the two materials cannot be separated. The two particles

are said to be thermodynamically unstable with respect to dispersion.

The secondary energy minimum occurs at larger separation distances, beyond the

energy barrier. When two interacting materials are in the secondary energy minimum,

they form a weak aggregate and may be dispersed with the addition of energy. The

secondary minimum is thermodynamically metastable with respect to aggregation.

The potential energy barrier to aggregation occurs between 1 to 4 nm [Isr92] and is

due to strong electronic repulsion between the ionic clouds of the interacting particles and

is dependent on the surface potential. If the surface potential is high, then the potential

energy barrier will be high; and, if the surface potential is low, the potential energy barrier

will be low. As the electrolyte concentration of the medium increases, the energy barrier
























>1 -roientai energy 5arner

C
w




SSecondary Minimum
Total Energy

? Attactive Energy




Separation Distance











Figure 2.6. A schematic diagram illustrating the attractive, repulsive, and total energy
curves for two interacting materials as a function of separation distance.









decreases and aggregation becomes more and more likely to occur. Finally, at the critical

coagulation concentration, the concentration of electrolyte at which the repulsive energy

barrier falls below zero, aggregation occurs.


2.3 Aggregation Mechanisms Among Particles and Particles at Surfaces

Numerous studies [Fin78a, Fin84, Fin78b, Rie88, Ada81, Coe91, Deg91, Edy86,

Edy87a, Har86a, Har86b, Kok90, Rob85, Rya81a, Rya81b, Rya81c, Rya84, Rya86,

Scu86a, Scu86b, Wie87] have shown that the aggregation and/or adhesion of COM

particles potentially can lead to the ultrastructures composing kidney stones. However,

the actual mechanisms) of aggregation within the biophysical environment of the human

kidney have not been examined in detail. There are at least six aggregation mechanisms, as

shown in Figure 2.7, acting alone or in concert which may contribute to stone formation

[Ada95].

To establish dispersion techniques which may prevent stone formation, first it is

necessary to determine the most important aggregation mechanisms with respect to

interparticle strength. A number of possible mechanisms exist for COM aggregation

within the human kidney. In addition to minimum double layer interactions, other

interactions include secondary minimum coagulation, heterocoagulation, polymer bridging

flocculation, aggregation by secondary nucleation and growth, and immiscible amphiphilic

molecule flocculation. Hydrodynamic factors also need to be considered, including the

size of the tubule and the shear rate associated with fluid flow within the nephron, as

discussed by Finlayson and Reid [Fin78b], and Kok and Khan [Kok94]. Furthermore,

the concentration of the COM particles performs a role whether one is considering fixed

stone disease or aggregation of the particles in a freely flowing state. Finally, the flow

patterns associated with peristaltic compared to continuous flow within the nephron have

not been addressed in past studies but should be to realistically assess aggregate formation






























E-
Q- .-


t 4.


-I


0

E
E0
o u


U.



8


4-
0
8
o
6-5



.RO


o

00
It
c.


+


+ 0


1*









in the kidney. Each of the mechanisms will be discussed with respect to their likelihood

in the human nephron.


2.3.1 Particle Aggregation in Simple Electrolytes


The aggregation of COM particles has traditionally been attributed to the absence of

charge on COM particle surfaces and/or the high ionic strength of urine. However, neither

of these aggregation mechanisms accommodates the observation that COM particles

observed in vivo invariably have organic matter on their surfaces, as shown in Figure 2.2.

Generally, particles in aqueous solution have a surface charge created by a combination of

several different charging mechanisms. In the case of COM, Curreri et al. [Cur79a,

Cur79b, Cur87] showed that surface charge is due to incongruent dissolution of the

constituent Ca2+ and C2042-. These and other species from solution then can adsorb (in

their hydrated form) into an adsorbed ion layer known as the Stern layer. The

electroneutrality of the system, composed of the surface and Ster charges and

surrounding solution, is achieved by the charge in the diffuse cloud of ions that only are

attracted electrostatically toward the surface.


2.3.2 Secondary Minimum Coagulation


As shown in Figure 2.7, a minimum exists at relatively large distances of

separation in the interaction energy for COM particles. As the ionic strength increases,

the magnitude of this secondary minimum increases. The secondary minimum in the

interaction energy curve is a consequence of the longer range of the van der Waals

attractive forces than the electrostatic repulsive interactions [Hou80, Son72]. Thus, even

when the charge at the surfaces of the interacting particles is great enough to produce an

energy barrier at intermediate separation distances, secondary minimum coagulation may

take place because there is no energy barrier for this mechanism of aggregation. The

strength of the interparticle bonds for secondary minimum interactions has been evaluated









theoretically and experimentally by Chan and Halle [Cha84]. It was demonstrated that

the mean lifetime for secondary minimum aggregates increased with increasing ionic

strength of the suspension containing the model spherical polystyrene particles.

Adair [Ada81] showed that COM suspensions composed of relatively coarse

primary particles (-5 gtm equivalent spherical diameter) aggregate over a wide range of

solution and surface charge conditions. Secondary minimum aggregation was implicated in

conditions where primary minimum aggregation was minimized because of low ionic

strength and relatively high zeta potential. However, the results were ambiguous because

only the thermodynamic aspects of the coagulation process were addressed in Adair's

study. The strength of the proposed secondary minimum interaction was not determined

by analyzing the hydrodynamic shear forces required to promote breakup of the

aggregates. Aggregate bond strength measurements as a function of particle size were

suggested since the magnitude of the secondary minimum increases as a function of the

radii of the interacting particles. The increased likelihood of secondary minimum

coagulation with increasing particle size may have important implications to stone disease

since it has been reported by Robertson [Rob69] that stone forming individuals have

particles significantly larger in size than non-stone former.


2.3.3 Heterocoagulation

Heterocoagulation is the aggregation among particles of different materials. We are

not aware of any investigators that have addressed this potential mechanism for stone

formation. However, there have been a number of investigators [Bar78, Der54, Kuo80,

Mat81] within the colloid chemistry community that have developed the theoretical and

practical framework for a study of heterocoagulation. The basis for heterocoagulation is

the difference in surface charge polarity of particles comprised of different materials.

Thus, a positively charged COM particle may be electrostatically (as well as through the

van der Waals interactions) attracted to a negatively charged hydroxyapatite (HAP)









particle. However, even if the surface charge on particles of dissimilar materials are the

same, the van der Waals attractive forces may be strong enough to promote

heterocoagulation (as well as homocoagulation).

Within the human nephron a variety of potential combinations exist that may lead

to heterocoagulation. These interactions include, COM-HAP, COM-HU (uric acid),

COM-NH4U (ammonium urate), COM-COD (calcium oxalate dihydrate), COM-E.coli

and other bacteria, and COM with various macromolecules. Heterocoagulation will be

reversible only when the sign of the surface charge is made the same for all particles and is

sufficient to overcome van der Waals interactions. The sign of the surface charge and

corresponding solution conditions that will promote or inhibit heterocoagulation need to

be evaluated for each system. It has been predicted that epitaxy of the high temperature

form of COM and HAP is unlikely because of incoherent crystal structures [Man81].

However, heterocoagulation can explain the presence of HAP with COM in a stone.

Heterocoagulation has not been addressed for material systems relevant to

urolithiasis. Preliminary experiments have been conducted in our laboratory to determine

the conditions in which particles composed of various materials will be likely to

heterocoagulate. An obvious starting point in this initial evaluation is to examine the

effect of the polarity of the zeta potential for the various materials as a function of

relevant solution conditions. The zeta potential data for COM and HU are summarized

in Figure 2.8(A) as a function of Ca2+ and C2042- concentration. The COM data are from

Curreri et al. [Cur79a] and incorporate the solubility product for COM in the Ca2+

concentrations. The uric acid data from Adair et al. [Ada88] are given as a function of

Ca2+ or C2042-. COM has a point of zero charge (pzc) at pCa = 5.2 (Ca2+ = 6.3x10-6 M)

with COM having negatively charged surfaces above this pCa and positively charged

surfaces at higher Ca2+ concentrations. Adair et al. [Ada88] showed that uric acid is

negatively charged over a wide range of pH and Ca2+ and C2042- concentrations.












40

20

0

-20

-40


i-C

-7 -6 4 4 2 -1
10 10 10 10 10 10 10
CONCENTRATION OF Ca (M)


(B)













(C)


Figure 2.8. Heterocoagulation of COM with HU is predicted based on: (A) zeta potential
determinations, (B) mixing of HU and COM at pCa < 5 where COM and HU are both
negatively charged, and (C) mixing at pCa = 4 where COM and HU are both oppositely
charged (bar = 20 gm) [Ada95].


M COM
* Uric Acid Ca


I


-

-









Thus, mixing COM and HU particles where pCa is less than 5.2 should promote

heterocoagulation between the negatively charged HU and the positively charged COM.

As shown in Figure 2.8(B) and 2.8(C), fine COM particles produced by the dimethyl

oxalate decomposition adhere to the larger, prismatic HU particles when pCa=4. When

COM and HU particles in saturated COM solutions are mixed with pCa=5, the fine

COM particles have a greater affinity toward one another than the HU particles and

heterocoagulation does not occur, as shown in Figure 2.8(C).


2.3.4 Polymer Bridging Flocculation


This mechanism takes place when there is insufficient polymer (or

macromolecule) for full surface coverage on particles. This is the only aggregation

mechanism that explains some ultrastructural observations of Boyce, Khan et al., and

others [Boy68, Kha83a, Kha83b, Kha87, Mey82, Mey71, Pri86] on the role of matrix

macromolecules in the microstructure and ultrastructure of the mature stone. Maximum

flocculation takes place when one-half of the surface of a particle is covered by polymer

[Son72, Hun86], as shown schematically in Figure 2.9. However, flocculation takes place

anywhere between about one-tenth surface coverage to greater than 75 percent coverage.

We are not aware of any studies relevant to urolithiasis that have addressed the role of

flocculation in detail. Kok et al. [Kok90], Finlayson [Fin78a], and Robertson and

Peacock [Rob85] have discussed this mechanism with other possible mechanisms of

aggregation; but, the research emphasis has been on the prevention of aggregates by

employing large concentrations of macromolecules or polymers [Coe91, Deg91, Rya81a,

Rya84, Scu86a, Scu86b, Lan88, Lea77]. This mechanism can also explain the conflicting

reports of inhibition versus promotion [Edy86, Cam89, Gro90]. Thus, macromolecular

species such as uropontin, Tamm-Horsfall mucoprotein, and nephrocalcin may play a

dual role: at sufficiently low concentration, aggregation is promoted through flocculation




















































Figure 2.9. A schematic representation of patch charge flocculation whereby incomplete
macromolecular coverage may create electrostatic shielding or opposite charge in the case
of a charged polymer.









while at higher surface coverage, dispersion of particles is achieved through the protective

colloidal effect of the macromolecular coating [Hou80, Son72].

Although there have been limited studies on flocculation with respect to

urolithiasis, there have been a number of investigations on floc formation and

hydrodynamic breakup because of its importance in wastewater treatment and other

technologies [Dit82, Eis85, Gre81, Gre85, Ray87]. These studies provide a basis for

evaluating the role of flocculation as an aggregation mechanism for COM and other

relevant particles in urine. For example, an excellent starting point is to determine the

macromolecular or polymer dosage for relevant urinary species required to achieve the

maximum flocculation. The critical flocculation concentrations (CFC) for a particular

macromolecule will indicate whether this mechanism is likely by comparison with its

concentration range in urine.

In preliminary studies, we have examined a flocculant commonly used in mineral

recovery. Polyethyleneimine (PEI) is a positively charged highly branched polymer

molecule used by Pelton and Allen [Pel84] to produce positive charge on glass surfaces in

their particle adhesion studies. It has been shown in preliminary studies, using the

apparatus developed by Eisenlauer and Horn [Eis85], to evaluate the aggregation of freely

flowing particles in suspension. This device has the advantage that flow rate and mode of

flow (i.e., continuous versus peristaltic) can be varied. The commercial analogue to

Eisenlauer and Horn's device, known as a photometric dispersion analyzer (Rank

Brothers, Cambridge, UK), has been used extensively to monitor the flocculation of model

systems. Initial experiments have demonstrated that fine COM particles flocculate at

intermediate dosages of PEI. The relative degree of flocculation varies as a function of

charge (as dictated by the concentrations of Ca2+ and C2042).









2.3.5 Flocculation of COM Particles by Phospholipids and other Intercellular Substances


Work by Khan, and Mandel and co-workers [Kha88, Man94] indicates that

interaction of COM particles with phospholipids at either the epithelial surface of cells or

with phospholipids liberated into the bulk solution by cell injury are important features

in the formation of aggregates in the interluminal channel of the nephron. Flocculation by

sparingly soluble, amphiphilic molecules such as the phospholipids forming a major

component of the epithelial cell membrane have not been studied with respect to

aggregation, but Mandel et al. [Man94] clearly show that adhesion of COM particles is

important. The ability of phospholipids to adsorb to the surfaces of sparingly soluble

inorganic particles and surfaces is well established as are the forces arising from the

interaction of phospholipid monolayers on mica and similar surfaces based on force

balance work by Israelachvili and others [Isr92]. Thus, one would expect phospholipids

to demonstrate an effect similar to macromolecules or polymers capable of promoting

flocculation. However, the interparticle strength of particles flocculated in either freely

flowing suspensions or at surfaces containing phospholipids (i.e., cell membranes) would

be expected to depend on the concentration of phospholipids and the efficiency of

adsorption of such species to COM surfaces.


2.3.6 Adhesion of Particles at Surfaces


Adhesion is an important mechanism in the formation of fixed kidney stones and

is dependent on the adhering materials and the suspending medium. One of the first

theories of adhesion between solid particles and surfaces was given by Krupp [Kru67],

where he defined three classes of interactions:


1. Class I interactions include long range attractive interactions resulting from
van der Waals forces and electrostatic forces.









2. Class II interactions are given by short range attractive interactions such as
chemical bonds and hydrogen bonds.

3. Class III interactions involve interfacial reactions occurring at elevated
temperatures including sintering effects, diffusive mixing, and mutual
dissolution and alloying.


Class III interactions may also be important at lower temperatures for polymer or

macromolecular diffusive mixing. Other important forces also exist that were not directly

addressed by Krupp. These forces are solvation forces, structural forces, or in a water

medium called hydration forces. These short range forces involve ordering of the solvent

medium to cause either attraction or repulsion between particles depending on the

hydrophobic/hydrophilic nature of the solids in suspension [Hor90, Isr92].

All of the aforementioned interactions may apply during adhesion; however,

Krupp [Kru67] believed that van der Waals forces and electrostatic forces are the

dominating long range attractive forces between adhering materials. Krupp thought that

only under ultra-high vacuum or extremely pure systems would primary chemical bond

formation take place due to the saturation of bonding sites by contaminants under

ambient conditions. Kallay et al. [Kal87] was also of the opinion that van der Waals

interactions and electrostatic interactions were dominating, but they furthermore

suspected that short range repulsion played a role in the total interaction energy of

adhering bodies, citing solvation forces, as did Israelachvili [Isr92] and also electron cloud

repulsion. These short range forces were said to act at separation distances on the order

of the distance of closest approach of the two surfaces. Theoretical models for both long

range interactions [Der41, Ver48, Hog66, Wil93] and short range [Isr92, Kal87]

interactions are available for varying surface geometry interactions and will be discussed

later.

The study of adhering materials is fundamental and very broad in application.

The adhesion of many materials by several researchers has been documented [Vis76]. For









example, the adhesion of Fe203 to a glass substrate and a glass substrate covered by a

layer of gelatin [Ryd95], red blood cells to glass [Moh74], human fibroblasts to glass

[van92], submicrometer particles to silicon [Bus93], and bacteria to glass [Bus92] have all

been measured. As observed, applications of the adhesion measurement range widely.

Just as a number of applications are in need of the adhesion measurement, a

number of techniques can be used to measure the adhesion of solid particulate material to

solid surfaces. In past studies researchers have used many techniques to evaluate

adhesion [Cor66, Zim82]. These techniques include a rotating disc [Kri94], a packed

column [Kal87], a centrifuge method [Kor60], a vibrating method [Der61], a surface force

apparatus [Isr78], and a hydrodynamic method that utilizes parallel plates [Pel84, Pel82]

to name only a few. The most recent technique for measuring the force with which a

particle adheres to a surface utilizes the scanning probe microscope (SPM) [Duc91].

Other adhesion measuring techniques exist and are used but are best suited for specific

material systems, much like the techniques mentioned above. Further discussion will be

limited to only the hydrodynamic parallel-plate and adhesion measuring technique.

Another approach in evaluating the bond strength of particles adhering to a surface

is to determine the hydrodynamic shear force required to remove particles. Pelton

[Pel84], Busscher [Bus84], Matijevic [Mat80, Mat81], Owens [Owe87], and others

[Ols78] have used this approach to evaluate thermodynamic models for the attachment of

particles to surfaces. The balance of forces proposed for a COM particle attached to the
epithelium is shown schematically in Figure 2.10. The shear stress at the wall (Tw) is

given by [Owe87, Esk68]


dP
rw = b (2.4)
dl


where Tw is the shear stress at the wall, P is the hydrostatic pressure drop across the

conduit of length, 1, and wall separation, b.
































Epithelial
Brush Border
in the Human
Nephron


COM Particle
FLOW

Fd F
{ gM


Figure 2.10. Scenario depicting the balance of hydrodynamic forces (Fd) and adhesive
forces (Fa) acting on a COM particle bound to the brush border in the human nephron.









Determination of the adhesion strength for particles adhering at surfaces is of

fundamental importance in deducing whether the fixed particle mechanism for stone

formation proposed by Finlayson and Reid [Fin78b] is reasonable within the

hydrodynamic system of the nephron. Kok and Khan [Kok92] recently re-evaluated the

hydrodynamics in the kidney and determined that it is not possible to have a stone

occurrence with out the aid of adhesion.

Riese, Mandel, and coworkers and others [Man94, Rie92, Rie88, Yam96] have

shown in vitro attachment of COM crystals to inner medullary collecting duct (IMCD)

epithelial cells of the rat animal model in the static case (i.e., without the presence of

flow). They also proposed that perturbations in the cell membrane structure with a loss

in membrane polarity can enhance crystal attachment [Rie92]. Lieske et al. [Lie95,

Lie96a] has also measured adhesion of COM to MDCK cells and to monkey renal

epithelial cells in a similar manner to Reise et al [Rie88]. They determined that cell

anionic sites can be blocked by specific cations [Lie96a] and the positive sites on a COM

crystal may be blocked by specific anions [Lie95] thereby minimizing adhesion. Bigalow

et al. [Big97] recently demonstrated that COM crystal attachment to IMCD cells was

effected by the cell membrane fluidity. Changes in temperature, cholesterol content, and

cell culture time which increase cell membrane fluidity also increase the ability of COM

crystals to bind to the membranes leading the researchers to conclude that a long range

arrangement in the membrane is created to match the COM crystal structure [Big97]. In

vivo crystal attachment to epithelial cells in the rat animal has been demonstrated by

Khan et al. [Kha82]. Renal injury has also been implicated in fixed stone formation

[Man91, Gil79, Kha84]. Khan [Kha82, Kha95a] has discussed that after attachment of a

crystal to the epithelial cell surface that injury to the cell may occur but may not always

occur.

A few different results may ensue crystal attachment to the epithelial cell. If the

cell dies, the cell and attached crystal may be washed away in the urine flow. The cell









may envelope the crystal and continue to function normally [Lie92, Lie93, Lie94, Koh96]

with no further growth of the crystal. However, if epithelial cell injury has occurred,

basement membrane proteins may be accessible to adhere to COM crystals by sloughing

of the cells leaving behind the exposed underlying architecture, the basement membrane.

The basement membrane is a layer of proteins that underlies the epithelial sheet and will

be discussed subsequently in more detail.

Depending on the extent of injury, other proteins common to the extracellular

matrix found below the basement membrane may also be accessible for attachment to

COM crystals. Khan et al. [Kha84] demonstrated the association of crystals to fibrillar

macromolecular structures by scanning electron microscopy (SEM). Khan [Kha95a] has

also shown crystals passing through the basement membrane into the extracellular space

near the papillary tip as shown in Figure 2.11.

Researchers have shown the attachment of COM crystals to epithelial cells and

fibrous proteins characteristic of those found in the basement membrane and extracellular

matrix, yet the importance of these individual materials in kidney stone formation with

respect to COM crystal adhesion is not known. The necessity to determine the

important materials most responsible for COM crystal adhesion will allow researchers to

focus their interests to those more important materials in the search for a possible cause

and therefore relief from kidney stone formation.


2.4 The Human Kidney

The human kidney contains approximately 1 million nephrons which absorb

nutrients and water back into the body after being filtered out of the blood by the renal

glomeruli [Ham74]. The nephrons consist of tubules through which the waste is

collected. As the waste products travel further down the length of the nephron, the waste

becomes concentrated and supersaturated conditions may exist allowing precipitation of

crystallites, such as COM, to occur. Figure 2.12 shows a nephron.









32









u


0

o

Cd

0
0
c:
-4-3





o
0









0
CO
-o













C!
a
0



ci

S0
o

*M

0


I-
4-,






o v
o











4-





I U
||0
1NI4.
4.5 -i
iu -c
^3













proximal
convoluted
tubule


collecting
tubule /


Figure 2.12. A schematic illustration of a human nephron [Ham74].









The renal tubules are lined with epithelium. Underlying the epithelial cells is a

structure called the basement membrane which is a continuous thin mat of specialized

extracellular matrix that functions as a support, a molecular sieve, and a cell regulator

[Yur90].

The basement membrane consists of the basal lamina and the lamina reticularis.

The basal lamina is also divided into two different sections, the lamina rara and the lamina

densa. The basal lamina is essentially a mat of collagen type-IV with specific molecules

on each side of the mat that help it bind to adjacent cells or matrix materials [Alb89].

Although the composition of the basal laminae varies from tissue to tissue, one of the

molecules always found in the basal lamina is laminin. Another molecule often found in

the basement membrane, in particular the lamina densa [Ino94], is fibronectin. The

structure below the basement membrane is the extracellular matrix, which is primarily

made up of fibrous proteins in a hydrated polysaccharide gel [Alb89]. Collagen type I is

a fibril forming collagen found in skin, tendon, bone, intestine, uterus, and surrounding

organs [Kuc92]. Figure 2.13 is a schematic representation of the renal epithelial cells,

basement membrane, and extracellular matrix showing there relative positions in the tubule

wall.


2.5 Characterization Techniques



2.5.1 Electrokinetic Measurements


Charge characterization at the solid-solution interface is very important when

attempting to discern mechanisms of adhesion. As described in section 2.2.1 of this

chapter, charged materials when immersed in water are surrounded by strongly adsorbed

ions, called the Stem layer, and by a gradient ionic cloud, called the diffuse part of the

double layer, as shown in Figure 2.3. Electrokinetic measurements are useful in

determining the electrostatic potential, often called the zeta potential, i, at the boundary





















































Figure 2.13. A schematic representation of a tubule wall found in a kidney.









between the strongly adsorbed layer and the beginning of the diffuse double layer as

schematically depicted in Figure 2.5. Electrokinetics is the measurement of the movement

of one phase with respect to another phase in which a charged boundary between the two

exists. Two types of electrokinetic measurements, electrophoresis and streaming

potential, are described below.

2.5.1.1 Electrophoresis

Colloidal particles immersed in water can move under an applied electric field as a

result of the charge on the particle surface as shown in Figure 2.14. The electrophoretic

mobility, uE, is defined as the particle velocity per unit static electric field. The mobility

can be determined by measuring the velocity of a particle under an applied electric field.

The mobility is related to the electrical potential at the shear plane surrounding the

particle. The shear plane is defined as the boundary between the bulk solution, where the

ions are free to move, and the inner layer of strongly adsorbed ions which move with the

particle under an applied electric field. The corresponding zeta potential, C, is calculated

from the experimentally determined electrophoretic mobility, UE, according to the

Smoluchowski equation [Hun81]


= tuE (2.5)
DE,



where 77 is the viscosity of the solution, D is the dielectric constant of the

solution, Eo is the permittivity of free space. A number of techniques exist to determine

the electrophoretic mobility under an applied electric field such as a simple optical

method, electrophoretic light scattering method, and electroacoustics to name a few.

2.5.1.2 Streaming Potential

The charge at the surface or the more commonly accepted charge at the Stern

layer, the zeta potential, can be determined by measuring the streaming potential














-'Illl --



+ -
<4 -


4-0


Figure 2.14. A schematic representing charged particle movement in an applied electric
field to determine electrophoretic mobility.


Zb:;









associated with a surface. Streaming potential is the potential generated when a fluid is

forced through a capillary. The hydrodynamic pressure forces the mobile charges in the

double layer in the direction of flow. The accumulation of charge at one end of the

capillary creates an electrical potential across the capillary as demonstrated in Figure 2.15.

In the case of glass or fused quarts, the charge on the surface is negative; therefore, the

mobile charges will be primarily positive causing a positive current in the direction of

flow. The streaming potential can be measured with a high impedance voltmeter as a

function of pressure. If the potential is measured as a function of pressure, the

Smoluchowski equation can be used to calculate the zeta potential [Hun81]


4r=,E' (2.6)
eP


If the usual mixed units are used, the equation is


( 0.1 O (poise) x (~ cm) x 100 E,(mV)
8.854 x10-'2(C )X p(dyn/ 2)



where and E, are the zeta and streaming potentials, respectively, both measured in mV,

P is the pressure drop across the capillary in cm Hg, the conductivity of the elution, ,, is
in units of l-'cm-', and e is the dielectric constant of the elution.

Equation (2.6) is only valid for solutions in which all or almost all of the current

generated due to streaming is carried through the bulk liquid. For solutions having ionic

concentrations less than 10-3 M, the need to account for surface conduction is important

[Rut47]. At low electrolyte concentrations, a large part of the current may be carried

through the double layers near the capillary walls because of the higher charge density in

that region. Equation (2.6) becomes [Hun88]















vDoube Lar (lon
,..\Double Layer Ions (",


0


Neutral ElectrolyteO 0 () ,Accumulation of
0 G 0 0,Olons S C
0OQI OC
0 G Q)" (0 Flow Direction-m 0














Figure 2.15. A schematic representation of the flow of ions under an applied
hydrodynamic pressure which generate the potential across the capillary or streaming
potential.


)









S4,c(A +2A,/r)E,
P = (2.7)



where the term 22s/r is the specific surface conductivity and r is the capillary radius or

the distance between plates for a flat plate system. The variable A, is the conductance of

a square section of material of unit area and constant thickness measured in Q-1. Briggs

[Bri28] suggested a simpler procedure for correcting for surface conduction. The

procedure involves measuring the resistance of the liquid in the capillary at low ionic

concentration, Rexp, and compare with the value of resistance expected from

measurements at high ionic concentration, RcaIc, where surface conduction can be expected

to be negligible. Equation (2.7) becomes


= 4rE R (2.8)
EP Rexp


The general effect of accounting for surface conduction will give an increase in magnitude

of the calculated value of zeta potential at low ionic concentration conditions.

Ball and Fuerstenau [Bal73] performed an extensive review of the streaming

potential literature and found that the measurement should be performed over a

sufficiently wide range of pressures to obtain an accurate estimate of the slope, EJP, the

obtained slope should be linear, and the intercept of the slope should be zero. Ball and

Fuerstenau [Bal73] and Hunter [Hun88, Hun93] have all stressed the importance of the

linear dependence of the streaming potential, Es, on the applied pressure, P. The linearity

of the plot of streaming potential vs. applied pressure is a necessary first step to

determine experimental reliability; however, unless surface conduction is accounted for,

linearity does not ensure accuracy of the resulting zeta potential [Hun88]. The finite

intercept of the slope is an indication of an asymmetry potential or rest potential which is

said to be a function of the electrodes, including their preparation, treatment, and cleaning,









and of the electrolyte and its concentration [Bal73]. No electrode system, Pt, Au,

Ag/AgCI, Ag/AgI, or calomel, has been found to eliminate the asymmetry potential

completely.

Many authors have developed methods for eliminating asymmetry or rest

potentials [Hun62, Mar30, Bul35, Hor77]. Hunter and Alexander [Hun62] found that the

rest potential could be nullified when the liquid was stationary. Martin and Gortner

[Mar30] and Bull [Bul35] avoided an asymmetry potential by working at high applied

pressures where the asymmetry potential is negligible relative to the streaming potential.

Horn and Onoda [Hor77] devised a resistance-capacitance (R-C) circuit to store the

asymmetry potential in a large capacitor and subtract the potential from the streaming

potential when flow begins.


2.5.2 Scanning Probe Microscopy


Scanning probe microscopy (SPM) offers a versatile range of techniques which

can be employed to acquire information about a material surface. Topographical

information can be acquired using Contact Mode Atomic Force Microscopy (AFM),

TappingModeTM AFM, and Non-contact AFM. Other techniques can also acquire

topographical information.

Contact Mode AFM measures topography by sliding the probe tip across the

sample surface in air or in fluid. A laser is focused on the back side of the probe tip also

known as the cantilever as shown in Figure 2.16. The laser reflects from the cantilever to

a mirror onto a photodetector. The sample is mounted on the piezoelectric scanner which

rasters the sample under the tip and surface interactions between the sample and tip cause

the cantilever to deflect. Any motion of the cantilever is registered by the photodetector.

The position of the laser spot is determined by the electronic circuitry which generates a

voltage difference between the photodiode segments and a topographical image is the

result.















Mirrors


Photodiode


Figure 2.16. A schematic diagram of the SPM.


Laser









2.5.3 Scanning Electron Microscopy


Scanning electron microscopy (SEM) is a useful tool to analyze surface

topography of materials. A focused electron beam is rastered across the sample exciting

electrons in the atoms of which the sample is composed. As a result, both secondary and

backscattered electrons are produced from the sample. A detector measures the intensity

of the electrons versus position and displays this information on a cathode ray tube.

Secondary electron contrast is due to the dependence on electron yield on the topography

and the depth of secondary electron emission is about 100 A. Because the sample must

be electrical conductive to be analyzed using SEM, the samples need to be sputter coated

with Au/Pd.


2.5.4 Fourier Transform Infrared Spectroscopy


Infrared spectroscopy (IR) is a useful tool to study polymers and organic

materials as well as inorganic materials. Fourier transform infrared spectroscopy (FT-IR)

uses the principles of interferometry to study bond vibration in materials.

Electromagnetic radiation in the infrared region (7.8x10-5 to 0.1 cm) is passed through or

reflected off the sample. The radiation excites molecular bonds to higher vibrational

levels, absorbing energy. The absorbed energy corresponds to particular vibrational

frequencies characteristic of a molecule or molecular group. Therefore, the IR technique is

useful in determining unknown materials, determining bond orientation, and quantitative

and qualitative analysis of bond types [Mar86].

Attenuated total reflectance (ATR) spectroscopy also called internal reflection

spectroscopy (IRS) may be the most widely utilized adaptation of IR to study inorganic

material surfaces. ATR is performed by coupling the incident electromagnetic radiation

into an IR transparent crystal of high refractive index, as shown in Figure 2.17. When the

beam reaches the interface between the crystal and the materials to be analyzed, it is



























SAMPLE


























Figure 2.17. Multiple internal reflection within the internal reflection element which is
coupled to the sample.









internally reflected. However, 100% internal reflection does not occur and some part of

the wave travels into the sample material to a depth governed by the wavelength of

radiation, the IR crystal, and the refractive index properties of the sample [Knu85].

Figure 2.18 is a plot of wavenumber vs. sampling depth for a thallium bromide-thallium

iodide (KRS-5) IR crystal having a 450 incident angle coupled to a sample assumed to

have an index of refraction of 1.5. As can be seen from Figure 2.18, the sampling depth

increases as function of increasing wavelength (decreasing wavenumber) over the mid-

infrared region.


2.5.5 Particle Size Determination


Many techniques exist to measure particle size. These techniques include

microscopy, sedimentation methods, electrical pulse counting, light scattering methods,

hydrodynamic methods, and electroacoustics. Each of these techniques has its advantages

and limitations, which may be dependent on size, density, optical properties, or other

parameters.

Electrical pulse counting is able to count the number of particles in a known

amount of solution by drawing the suspension through a very small orifice that has an

electrode on either side of it as shown schematically in Figure 2.19 [Hun93]. When a

particle passes through the orifice, interference with the current flowing between the two

electrodes occurs and the resistance changes. The number of changes in current and the

magnitude of change are recorded. Because the change in current is proportional to the

volume of the particle passing through the orifice, the result is a value of particle size

when calibrated against a dispersion of known particle size. The number of current

changes acts as a particle counter generating a particle size distribution.











































I.O


~1.20
a.

O.W
z08


0.40


0
4000 3200 2400 1600 800
WAVENUMBERS (cm'1)


















Figure 2.18. Sampling depth as a function of wavenumber for the KRS-5 internal
reflection element having a 450 incident angle [Knu85].























































Figure 2.19. A schamatic diagram of a Coulter counter, electrical pulse counting
instrument [Hun93].









2.5.6 X-Ray Diffraction


X-Ray diffraction (XRD) is a tool used to investigate the arrangement of atoms in

a crystal. XRD is useful for identifying compounds in crystalline materials, determining

crystallographic information of metals, ceramics, and polymers, and performing

quantitative phase analysis. A monochromatic or near monochromatic beam of x-rays is

focused on a specimen. The incident x-rays are diffracted by the lattice atoms in the

crystal according to Bragg's Law [Cul78]



n = 2dsin9, (2.9)


where n is the order number, A is the wavelength of the incident x-rays, d is the

interplanar spacing, and 0 is half of the angle of diffraction. A scintillation detector is

moved through some angle at a given rate and the number of counts or intensity is

recorded The resulting pattern is a plot of x-ray intensity vs. angle (20) measured with

respect to the incident beam. Because the crystal structure is periodic, the in-phase

scattered electrons create an increase in intensity.















CHAPTER 3
DEVELOPMENT AND CHARACTERIZATION OF MODEL COM PARTICLES


3.1 Introduction

Calcium oxalate monohydrate (COM) is the most thermodynamically stable form

of calcium oxalate and is a major component of human calcium oxalate calculi [Pri47,

Deg81a]. Stone formation in the urinary tract takes place opportunistically with an

unfortunate, simultaneous convergence of factors that may be achieved by several

pathways for a stone to form [Bro92, Ada95]. Many theories have been proposed to

explain the critical factors) responsible for the formation of urinary stones [Bro92,

Ada95, Fin78a, Fin84, Rob86]. Kidney stone disease may take place as a consequence of

injury, supersaturation (particularly hyperoxaluria), nucleation, and growth mechanisms,

which may include adhesion on epithelial cell surfaces and aggregation. All of the

previously mentioned factors may play a role in contributing to stone formation.

Nonetheless, stone formation remains an idiopathic disease with no one risk factor

providing an obvious cause for stones in most cases. However, both the in vivo and

in vitro evidence indicates that the presence of particles is a necessary but insufficient

condition for stone formation. From this perspective, the formation and presence of

crystallites may be a typical renal function directed at concentrating oxalate for more

effective, irreversible elimination from the body.

The abnormal stone-forming condition results from the aggregation and further

growth of such pre-existing crystallites [Fle78, Rya81a, Ada81, Deg91, Edy86, Edy87a,

Har86a, Har86b, Kok90, Rob85, Scu86a, Scu86b]. Indeed, it has been found that while

the solid phase in the urine of normal individuals is made up mainly of individual crystals,









large aggregates are often found in stone former [Fle78]. Therefore, both crystal growth

and aggregation are generally regarded as important steps in the formation of calcium

oxalate renal stones [Fle78, Rya81a]. Numerous studies have shown that the aggregation

and/or adhesion of COM particles may potentially lead to the ultra-structures typical of

kidney stones [Ada81, Deg91, Edy86, Edy87a, Har86a, Har86b, Kok90, Rob85, Scu86a,

Scu86b]. Kok et al. [Kok90] reported that precipitation alone of COM particles cannot

account for the large size particles necessary to cause blockage of the urinary tract.

Aggregation is therefore the only possible mechanism whereby a stone may reach a size

as large as 0.64 cm, the maximum size that can pass through an average adult male urethra

[Fin78b].

Aggregation is the process whereby crystals bind one to another to produce large

clusters. The actual mechanisms) of aggregation within the biophysical environment of

the human kidney is uncertain; however, Adair et al. [Ada95] have classified six

aggregation mechanisms acting alone or in concert which may contribute to stone

formation: (a) coagulation in simple electrolyte solutions; (b) secondary minimum

coagulation; (c) heterocoagulation of particles with different surface charge polarity; (d)

polymer bridging flocculation; (e) secondary nucleation and growth; and (f) flocculation

via immiscible amphiphilic molecules. The previously mentioned mechanisms, which are

summarized in Figure 3.1, stress the importance of knowing the environmental conditions

in the urinary system for formation of calcium oxalate calculi. For example, citrate can

retard the crystallization of stone-forming calcium salts by two broad means [Cha91].

First, citrate complexes calcium and reduces the ionic calcium concentration available for

precipitation in urine, thereby lowering the supersaturation. Second, citrate can directly

inhibit crystallization of both calcium oxalate and calcium phosphate at the growing

crystal-solution interface. Thus, citrate has been shown to inhibit spontaneous

precipitation of calcium oxalate and to retard agglomeration of pre-formed calcium oxalate

crystals. Citrate is believed to also have a modest inhibitory effect against crystal growth



















0

0 0<
c o E,


O )C
8 0 E 3 0

E o





a. 0 o.
8 >



cu g. c

II






C C 4.
U) c




800
+ I 2 I




a e 0


S&E
E : c|
c o



o .o o


So5 0 )- 3
-n c '5
0


Co m
0 0 .
r d =r+o .


1.:.









of calcium oxalate. Cody and Cody [Cod94] reported habit modification of COM

crystals grown with citrate. Interpenetrating crystals grown in distilled water without

citrate have sharply-angled tips and are flattened parallel to the (010) face whereas

crystals grown with citrate have rounded tips and are flattened on the (101) face.

Few papers have dealt with the theoretical equilibrium shapes of COM crystals

synthesized under different conditions or the atomic structure at specific faces [Deg91,

Cod94]. Furthermore, little, if anything, is known at the atomic-molecular level about the
interaction between COM particles and important species such Ca2+, C2042, citrate,

dicitrate, and urea, which are common in the urinary system and known to play a role in

stone disease. Deganello's work [Deg91] is an exception to the lack of data on the

interaction between some species and COM crystals. Adsorption of nephrocalcin, a

protein found by Nakagawa and co-workers [Nak83, Nak87] in human urine, even at the

lowest concentration tested, affects the habit while also inhibiting the growth of COM.

The nephrocalcin protein promotes the preferential development of the (101) faces to

such an extent that the length to width ratio of the crystals decreases by approximately a

factor of three. In the course of this process, the apical planes eventually disappear and

the size of the crystals decrease. Diminution of crystal size eventually becomes extreme

when nephrocalcin reaches a concentration between 1 to 2x 10"5 M.

Mandel [Man94] reported that the molecular surface structures on the prominent

crystal growth faces of COM are very important in determining potential long-range

molecular bonding interactions between the crystals and the lipid-rich regions in the inner

medullary collecting duct (IMCD) cell plasma membrane. Mandel [Man94] also reported

on epitaxial matching calculations for the high temperature form of COM, calcium oxalate

dihydrate (COD), hydroxyapatite (HA), and uric acid (UA) crystal lattices against the

dimensional repeating lattice of phospholipid headgroup structures. The Mandel study

suggests that COD-COM epitaxy may be important relative to COD-lipid interactions in

crystal attachment and stone development. Therefore, the molecular modeling between









specific surfaces of the COM crystal and these additives provide important insight for

specific adsorption sites on COM surfaces, and the growth and aggregation mechanisms

of COM crystals in the urinary system. Other research suggesting the crystallographic

importance of COM was performed by Habeger et al. [Hab97]. A crystallographic

dependence of COM crystals on adhesion to fused quartz glass substrates was observed.

A variation in electrostatic charge as a function of crystallographic habit on the theoretical

COM surface has been used to justify the differences in adhesive strength observed

[Hab97].

In the current work, morphological forms of COM crystals synthesized in

different conditions have been studied by using the commercial computer programs,

SHAPE and ATOMS0. The computer program SHAPED has been used to determine

the face indices of the synthesized COM crystals and generate the equilibrium shape as a

function of central distance. The computer program ATOMS0 has been used to generate

the surface atomic structure of COM crystals as a function of habit plane.

Certain characteristics in a particle system are desirable to rigorously study

theoretical growth and aggregation, and adhesion mechanisms. Ideally, the particles

should be spherical. If spheres cannot be produced, a crystallographically definable

morphology should be produced so that the habit planes can be identified. The

theoretical study of growth and aggregation depends on well-defined particle

morphologies. Unfortunately, COM has a monoclinic crystal structure whose inherent

asymmetry does not lend itself to the formation of spherical particles. Therefore, the

individual COM particles should be at least uniform with narrow particle size and shape

distributions. Several different particle size ranges is also desirable. Thus, the objective

in the current work is to prepare COM particles by several different methods followed

by careful characterization to deduce the habit planes and atomic surface structure.

Lastly, characterization of the particles having the most consistent COM morphology

will be conducted.









3.2 Preparation and Characterization of Calcium Oxalate Monohydrate Particles under
Different Conditions

Reagent grade chemicals were used in all precipitation studies without further
purification. All solutions were prepared with deionized water (specific conductivity less

than 10 gmho.cm'') which was provided by a Milli-Q high purity system (Millipore

Corporation, Bedford, MA) and were passed through a 0.2 [jn filter. For identification

purposes, precipitated COM particles were designated by the negative logarithms of the

initial Ca+2 and C204-2 molar concentrations, respectively (e.g., particles prepared from

10-3 M Ca+2 and 10- M C204-2 were designated "33" particles).


3.2.1 COM particles ("33" particles) without seeds

Reagent grade potassium oxalate monohydrate (K2C204.H20) (Fisher Scientific
Inc., Fair Lawn, NJ) was used to prepare stock 1 molar (M) K2C204 solution at 250C.

After equilibration, stock solutions were passed through a 0.22 gim filter (MAGNA,

nylon, supported, plain, MSI, Westboro, MA). Reagent grade calcium chloride dihydrate

(CaC12.2H20) (Fisher Scientific Inc., Fair Lawn, NJ) was used to prepare 0.01 M CaC12

solution at 250C. The CaC12 solution was prepared the day of use to prevent CaCO3

formation through reaction with CO2 from the atmosphere. Diluted solutions of K2C204

and CaCl2 solutions were volumetrically prepared from the stock solutions.

A schematic representation of the processing steps for the preparation of COM
crystals ("33" particles) without seeds is given in Figure 3.2. Precipitation studies were

performed by mixing equal volumes (10 liter (I)) of 10-3 M CaC12 and 10-3 M K2C204

solutions at 250C. Crystals of COM were aged for at least 24 hours without stirring.
Solutions containing precipitated crystals were 0.22 gm filtered. The filtrate was washed

with a solution saturated with COM to prevent dissolution of the precipitate during
washing. After washing, the recovered powders were freeze dried (Freeze Drier 4.5,

Labconco Corp., Kansas City, MO) and stored in a desiccator.

















10-3 M CaCI2 Solution (10)


10-3 M CaCI2 Solution (10)


Aging
(for at least 24 hours without stirring)




Recovery of COM particles
(concentrate the particles in the centrifuge)


Washing & Filtering
(washing by saturated COM solution with
filtering through 0.22 gm)


Freeze-Drying
(store the particles in a desiccator)




Characterization
(XRD, SEM, and particle size analysis)


Figure 3.2. Schematic representation of processing steps for the preparation of COM
crystals ("33" particles) without seeds.











3.2.2 COM particles with seeds

Seed crystals ("00" particles) of COM were prepared by mixing equal volumes

(50 ml) of 1 M CaCl2 and 1 M K2C204 solutions at 250C. These seed crystals were aged

for at least 24 hours without stirring. Crystals were concentrated by centrifugation and

then washed with saturated COM solution. After washing, the recovered powders were

freeze dried and stored in a desiccator. The seed suspension was prepared by dispersing

0.1 g of seed crystals in 5 ml isopropanol for a crystal growth experiment. The

dispersion was treated by ultrasonication for 3 minutes to break up agglomerates.

A processing schematic for the preparation of COM crystals ("33" particles) with

seeds is given in Figure 3.3. Precipitation studies were performed by mixing equal

volumes (500 ml) of 10-3 M CaCl2 and 10-3 M K2C204 solutions at 250C with various

amounts of seed crystals. Crystals of COM were aged for at least 24 hours without

stirring. Crystals were filtered 0.22 rm and then washed with a solution saturated with

COM. The recovered powders were freeze dried and stored in a desiccator.


3.2.3 COM particles precipitated from homogeneous solution at 900C

Gordon, Salutsky, and Willard have reported a recipe for the production of COM

by the thermal decomposition of dimethyl oxalate [Gor59]. The goal of this section was

to duplicate this procedure and analyze the nature of the particles. Ammonium acetate

(CH3COONH4) (Fisher Scientific Inc., Fair Lawn, NJ) and acetic acid (CH3COOH)

(Fisher Scientific Inc., Fair Lawn, NJ) were used to prepare stock 2.5 M CH3COONH4

(aq) and 2.5 M CH3COOH (aq) solution at 250C. A buffer solution at pH 2.7 was
prepared by mixing equal volumes (500 ml) of the 2.5 M CH3COONH4 (aq) and

CH3COOH (aq) stock solutions at 250C. After equilibration, stock solutions were

passed through a 0.22 gim filter.






































Recovery of COM particles
(concentrate the particles in the centrifuge)


Washing & Filtering
(washing by saturated COM solution with
filtering through 0.22 rnm)


Characterization
(XRD, SEM, and particle size analysis)


Figure 3.3. Schematic representation of processing steps for the preparation of COM
crystals ("33" particles) with seeds.









A schematic representation of processing steps for the preparation of COM

crystals aged at 900C is given in Figure 3.4. Reagent grade CaC12*2H20 was used to

prepare 0.01 M CaCI2 solution at 25C. The CaC12 solution was prepared the day of use

to prevent CaCO3 formation through CO2 from the atmosphere. The dilute CaCl2

solution were volumetrically prepared to obtain 2.5xl0-3 M CaC12 solution. The pH of

the dilute CaC12 solution was adjusted to 4.7 by adding 0.01 M HCI solution. After

equilibration, the dilute CaC12 solution was filtered (0.22 Wm). The 2.5x10"3 M CaCl2

solution (150 ml) was rapidly mixed with buffer solution (100 ml) in a flask and 10 g of

dimethyl oxalate (CH30COCOOCH3) (Fisher Scientific Inc., Fair Lawn, NJ) was added.

The flask was tightly closed and heated at 900C for 1 hour in an oven to equilibrate. The

holding time at 900C was 2.5 hours. After thermal treatment, the solution was rapidly

cooled to room temperature in an ice bath. Crystals were collected via centrifugation

followed by washing with saturated COM solution previously passed through a 0.22 gtm

filter. After washing, the recovered particles were freeze dried and stored in a desiccator.


3.2.4 Characterization


The dried, recovered powders were analyzed for phase composition using x-ray

diffraction (XRD) (APD3720, CuKa, fine tube, 40kV-20mA, Philips Electronics,
Mahwah, NJ) over a 20 range from 10-70 at rate of 2.4'/min.

The morphology of the synthesized crystals were observed using scanning

electron microscopy (SEM) (JSM 6400, JEOL, Boston, MA).

Particle size analysis of typical COM particles without seeds was performed

using an electrical sensing zone technique (ELZONE 80XY, 95 gtm aperture, Particle Data

Incorporated, Elhurst, IL).

Zeta potential determination was performed using Rank Particle

Microelectrophoresis (Apparatus Mark II, Rank Brothers, Cambridge, ENGLAND) as a











0.01M CaCI2 Solution (100ml)

---- Dilutions


2.5 10-3 M CaCI2 Solution (150m)


Aj ustmeat of pH with HCI
(pH = 4.7)


Filtering (0.22gpm)


2.5M CH3COONH4 Solution (50ml)

2.5M CH3COOH
Solution (50ml)

Buffer Solution (100ml)


Filtering (0.22pm)

ZZ


Precipitation
(heat treatment in an oven at 90C
heating time: Ihour
holding time: 2.5 hours)


Cooling
(rapidly cooled to room temperature
by using an ice bath)


Recovery of COM particles
(concentrate the particles in the centrifuge)


Washing & Filtering
(washing by saturated COM solution with
filtering through 0.22pm)


Freeze-Drying
(store the particles in a dessicator)


Characterizat ion
(XRD, SEM, and particle size analysis)


Figure 3.4. Schematic representation of processing steps for the preparation of COM
crystals ("32" particles) with heat treatment at 900C.


I









function of pH in COM saturated solution and in an artificial urine ion solution (AUIS)

buffered to pH 6.0 at 370C. The composition of the AUIS is given in Table 3.1.

Scanning probe microscopy (SPM) (MultiModeTM, Digital Instruments, Santa

Barbara, CA) was performed on the two major crystallographic surfaces of COM to

determine surface roughness using a Nanoscope III controller. The J-type scanner was

operated in contact mode using a Si3N4 cantilever having a spring constant of 0.12 N/m in

saturated COM solution at 250C.


3.2.5 Crystal and Atomic Structure Modeling Using the Computer Programs SHAPE
and ATOMSc


Based on crystallographic data including space groups and lattice parameters, the

computer program SHAPED (Macintosh version 4.0, SHAPE Software, Kingsport TN)

was used to determine the face indices of the COM particles synthesized under various

conditions and to generate the equilibrium shape of the crystals as a function of the

central distance [Coc61]. The central distance is the perpendicular distance from the

center of the crystal to the faces of the corresponding form. The greater the distance, the

less prominent the form (the smaller the area of the faces of that form in the final shape).

Form factors in the program are the least number of face indices needed to generate the

desired equilibrium shape. Twins of COM particles were drawn using the "twin option"

and "epitaxy option" in SHAPE.

The theoretical atomic structure of COM was displayed using the computer

program ATOMS (Macintosh version 2.0, SHAPE Software, Kingsport TN). The slice

option of the computer program ATOMS was used in the process of analyzing and

predicting surface structure as a function of habit plane. To draw an individual surface

atomic structure using this option, it is necessary to enter the crystal class, the space

group, the corresponding unit cell parameters, and the face indices and atomic coordinates.

From the crystal class and space group, the program determines what symmetry





















Table 3.1. Composition of artificial urine ion
solution

Compound Solution
Concentration (M)
NaCI 0.10554
NaHPO4*2H20 0.03654
Na3C6H507-2H20 0.00321
MgSO4 0.0038476
Na2SO4 0.016952
KC1 0.06374
CaC12-2H20 0.001
Na2C204 0.00131
NH4Cl 0.03632
NH40H 0.00062
NaOH 0.005









operators to use in the calculations. In addition to the indices of the face and input atoms,

several other parameters were specified. The thickness of the slice is in fractions of the d-

spacing. Thus, atoms present to a certain depth of unit cell were drawn with the "slice

option" as a function of habit plane based on the atomic coordinates of the high and low

temperature structures.


3.3 Results and Discussion

First, differing reports regarding lattice parameters for COM need to be reconciled.

Table 3.2 summarizes crystallographic data proposed by various authors [Deg81a,

Taz80, Coc61, Coc62, Deg80]. From Table 3.2, the unit cell parameters reported by

Cocco [Coc61] and Tozzoli and Domeneghetti [Taz80] can be seen to have similar values

in contrast to the unit cell parameters of the a and c axes reported by Deganello and Piro

[Deg8la]. The equilibrium structures of COM were generated the crystallographic data

proposed by Deganello and Piro [Deg81a] and Tozzoli and Domeneghetti [Taz80], as

shown in Figure 3.5. Based on comparisons made between the theoretical equilibrium

shape and the morphological form of the experimentally derived crystals, the equilibrium

shape of COM crystals modeled using the lattice parameters of Deganello and Piro

[Deg81a] can be more easily reconciled to the morphology of the experimentally

synthesized COM crystals.

It is also necessary to mention conflicting papers regarding COM atomic

coordinates. Table 3.3 shows atomic coordinates proposed by various authors [Deg81a,

Taz80, Coc62]. From this table, the atomic coordinates reported by Cocco and Sabelli

[Coc62] and Tozzoli and Domeneghetti [Taz80] have similar positions but only the x

coordinates for carbon(l), carbon(2), oxygen(l), oxygen(3), and calcium(l) are different.

The atomic coordinates reported by Deganello and Piro [Deg8 a] are quite different from

those of the previously mentioned authors. The equilibrium atomic structures of COM

were generated using the three different atomic coordinates. Based on the comparison































Cl e!

,It- C
m 00






0 0 0






-








N -N 0
0








00

00 00 00
0 m











u uo
Cl Cl ON


o o
oo








02
S 00 0
U H

o








o c
SH a
0 8 <
U



































(B) (C)
A D

V A: (Oil)
B: (0 11)
C: (010) c
D D: (Oi)
E: (101)
E F F: (0T) E










Figure 3.5. (A) SEM photomicrograph and (B) and (C) theoretical equilibrium shapes of
the COM crystals precipitated without seeds. (B) The equilibrium shape of COM
crystals based on the crystallographic data of Deganello and Piro [Deg81a] and (C) the
equilibrium shape of COM crystals based on the crystallographic data of Tazzoli and
Domeneghetti [Taz80].














0- Uin o t-(ON 0 ON 00
m m In e r- V SC\cM Inoo 0 o 0 c \


o) N m- -: MN Cq q 0t
06609 9669669050 0
ad d Nddddddd d




C> r o [I e-: c14 M -o r-
6 6do dd0 dd0d0d od dV



O ON en N W) W) o^ r- >n o "t W) C1 4
X^ 0c, rC0 M W) m c (^ ,o oO < ^ W cmn 0
( mM-c en o II Co0vso It o
o666666666 66 66 000



S q C4 C n 0 0 Nt r-- t \C r1- N 00
0 oo0 m cn W) o O a\ CM Wm O o C -
9 M M cn m 0 CM o 0t m It n
dddd dddddddd dd d6 oo0


0 o CN000(100 -C.00N m o oN O 1






N r- t- tn -co tcn m- 0nc W) -
0 >,0000 00000000 00 C CC o't00 \ 0



8 8
rf--. mo> .e ..



0 C5 O\ 5 5C CO\5 -ir 0 6 C5* )
oo^ in 2 5-> r- 0 SO C c,4 4 O \ c 3-, m :700- l


o3 cc o-m r-- A -o .(mCm-m-n (- 0



o N rdd d cddddd dd dd dd



o t
CMO0or~- Cr~-tso sr~-r- \0






Soco 00000000 C 0





a uuou c ocococo ou >> x
m cu c ~ ----------------------------- ------









between the theoretical atomic structures generated using ATOMSc and the atomic

structures from the papers by Deganello and Piro [Deg81a] and Tazzoli and

Domeneghetti [Taz80], the (010) and (100) planes generated with the atomic coordinates

published by Cocco and Sabelli [Coc62] are exactly reconciled to the layering sequence

reported by Deganello and Piro [Deg81a] and Tozzoli and Domeneghetti [Taz80] as

shown in Figure 3.6(A) and (B). However, the (010) and (100) planes generated with the

atomic coordinates by Tozzoli and Domeneghetti [Taz80] are not reconciled to the

layering sequence reported by Deganello and Piro [Deg81a] and Tozzoli and

Domeneghetti [Taz80]. The atomic structures of some oxalate ions are incomplete as

shown in Figure 3.6(C) and (D). Furthermore, the (010) and (100) planes generated with

the atomic coordinates of Deganello and Piro [Deg81a] are completely different from the

layering sequence reported by Deganello and Piro [Deg81a] and Tozzoli and

Domeneghetti [Taz80] as shown in Figure 3.6(E) and (F). Thus, the atomic coordinates

of Cocco and Sabelli [Coc62] were used in computer simulations of atomic structure as a

function of habit plane.


3.3.1 COM particles ("33" particles) without seeds

The COM crystals grown in distilled water without seeds are shown in 3. 5(A).

The interpenetrating crystals have sharply-angled tips and a distinctive shape. Figure

3.5(B) shows the equilibrium shapes of these interpenetrating twins with labeled face

indices. For the twin operation, the individual crystal is reproduced according to the

reflection on (101). The crystallographic form factors and central distances for the

interpenetrating twins used in SHAPED calculations are listed in Table 3.4.

Determinations made from observations of the computer calculation and the

morphological form of the experimentally derived particles, the {010} and { 101} faces

appear to be dominant.


















(A) (B)


O: Hydrogen : Water Oxygen : Carbon : Calium
Figure 3.6. Theoretical atomic structures of COM crystals as a function of habit plane:
(A) and (B) are the (010) and (100) planes, respectively, drawn using Cocco and Sabelli
[Coc62] atomic coordinates; (C) and (D) are the (010) and (100) planes, respectively,
drawn using Tazzoli and Domeneghetti [Taz80] atomic coordinates; (E) and (F) are the
(010) and (100) planes, respectively, drawn using Deganello and Piro [Deg81a] atomic
coordinates.


























0 0 kn)


C,

a.
4-


0

to
,-c
o



4a





7-
0







4-
o
M

U










40

Il


4-
ri
o
0










4--






uO0
(6
E^


o o 0o i


O 2
0-


cS

c ,r >.





-. U


o c)o -o o





to
Uo 4






R C
"2 o

8 0 0






CIS -
el e .)

8 c -







0 o+-e 0 -C
4- <-






So


.5u c
4 .
UU U 0
R &^-Si
Uc < -3 2
o b) o-o'G
rr i u
Css. ^


0 -~ I-
--
00-r









3.3.2 COM particles ("33" particles) with seeds


The seed crystals ("00" particles) precipitated by mixing equal volumes (50 ml) of 1 M

CaC12 and 1 M K2C204 solutions are shown in Figure 3.7(A). The particle size varies

between 100-500 nm. Although the shape and size are not uniform, the crystals are

composed of submicron size particles suitable for seed materials. Figure 3.7(B) shows

the equilibrium shapes of the seed crystals with face indices as calculated by SHAPE

with the crystallographic form factors and central distances for COM seed crystals listed

in Table 3.4. The {10O } faces appear to be dominant based on the computer calculation

and the morphological form of the experimentally derived particles. Also, only the

equilibrium shape generated with the (001) face was reconciled with the morphology of

experimentally prepared seed crystals as shown in Figure 3.7(B).

Figure 3.7(C) shows that the influence of seed particles on the size of COM

crystals grown in distilled water. These interpenetrating crystals produced with 0.1 ml of

seeding suspension have similar shape to the COM crystals grown without seeds, but are

smaller in size. The maximum size of the particles is about 1 nm. Thus, it is

demonstrated that the presence of seed particles may provide a low energy epitaxial

surface in solution to lower the overall surface energy contribution to the nucleation

barrier, increasing nucleation frequency, and reducing the particle size of COM crystals

without changing the particle morphology. Also, the seeding studies suggest the

possibility of controlling the size of the COM crystals ("33" particles) by controlling the

amount of seed materials. From the morphological difference between seed crystals and

COM crystals synthesized with seeds, the effect of seed crystals can be gauged from two

aspects: (1) the presence of seed crystals in the solution system probably lowers the

surface energy barrier of the system to the nucleation and growth of particles, and (2) the

transformation in this case is not only influenced by the number of added seed crystals,

















s~C's
-I-- o
0000e-0




o~~ 4l 1)r




rJ u


00













cn


-ecn
o~t

















to 0
c 1_. I I


(/2





9 E! 4-e c




o u
lii
2~b









but also by forming larger numbers of nuclei due to the seeds and possibly secondary

nucleation [Nyv85, Ran88].


3.3.3 COM particles precipitated from homogeneous solution at 90C

Habit modification of COM crystals grown with structurally specific additives

gives further demonstration of specific preferential binding. The COM crystals which

were made using dimethyl oxalate decomposition from 2.5xl0-3 M CaC12 solution at 900C

are shown in Figure 3.8(A). Habit modification is obvious for the COM crystals grown

by dimethyl oxalate decomposition.

Figure 3.8(B) shows the equilibrium shapes of an individual COM crystal and

contact twin with face indices. The crystallographic form factors and central distances for

COM crystals are listed in Table 3.4. For the contact twins, the individual crystal is

reproduced according to the epitaxial operation in SHAPEc. The host crystal is specified

by the (001) face and the [010] vector and the guest crystal is specified by the (001) face

and the [010 ] vector. The central distance for the epitactic face (001) is 2.0. The {101 }

faces appear to be dominant when comparing the computer calculation to the

morphological form of the experimentally derived particles. Also, the equilibrium shape

generated with the (001) face more closely resembles the experimentally prepared COM

crystals in contrast to the equilibrium shape generated with the (010) face, as shown in

Figure 3.8(B) and (C).


3.3.4 Atomic structures of COM particles as a function of habit plane for the high
temperature form

Equilibrium atomic structures of COM particles were generated as a function of habit

plane to predict the atomic structures of specific surfaces. In whewellite, there exist two

different crystal structures: a high temperature structure (stability range 318-415 K) and a

low temperature structure (stability range 293-318 K) [Deg8lb, Deg80]. Table 3.5 and













I- ci1- -'
o I-
-oo@00
- '-





u


-0008


Kf


a 0

0
I00
7 3


8cl











c)
nat




U o


0 ^
c,

a 0








U0 -*'
" -- E5










0U




0.

0 ca-
0 CO *-
^3"7 s
CoT


V






















Table 3.5. Values of the crystallographic data for the high temperature
form [Deg81b, Deg80] of Whewellite (CaC204 H20)

High Temperature Form
a (A) 9.978(1)
b (A) 7.295(1)
c (A) 6.292(1)
Space Group 12/m
Stability Range (K) 318-425
Ca/Ox Ratio
(010) 0.958
(101) 1.658









Table 3.6 summarize the crystallographic data and atomic coordinates of the high

temperature form. Based on the crystallographic data and atomic coordinates, Figure 3.9

shows the equilibrium atomic structures of specific habit planes for the high temperature

form. Habeger et al. [Hab97] has shown that the ratio of calcium atoms to oxalate

molecules may have an important implication in the adhesion properties of the different

crystal faces in a model environment. The ratio of calcium ions to oxalate ions is 0.958 in

the (010) plane whereas the ratio of calcium ions to oxalate ions is 1.658 in the (101)

plane for the high temperature form. Although the temperature in the human body cannot

produce the high temperature phase of COM, the adsorbtion of ions or molecules may

stabilize the high temperature structure at lower temperatures. The stabilization of the

high temperature structure of COM in the urinary system may have a large implication in

that the (101) has a Ca+2/C204-2>1, making the crystal face positively charged and able to

electrostatically interact with predominately negatively charged biosurfaces found in the

human body.


3.3.5 Characterization of COM crystals


The morphologies reported herein were determined to be phase pure COM by

XRD. A typical XRD pattern is shown in Figure 3.10.

As shown by SEM of the various COM morphologies, the "33" particle

synthesizes without seeds were the most consistent morphology produced. Visually,

they had very few differently shaped particles and a relatively narrow size distribution

relative to the other particle morphologies synthesized. Therefore further

characterization will only involve the "33" COM particles synthesized without seeds and

further mention of COM particles refers only to this particular morphology.

Peakfit software was used to fit a log normal curve to the experimentally produced

particle size distribution. The particle size distribution was determined using an electrical

zone sensing technique which results in an equivalent spherical diameter vs. frequency



















Table 3.6. Atomic coordinates for high temperature
form [Deg81 b] of Whewellite (CaC204 H20)

High Temperature Form
Atom x y z

C(1) -0.0652 0.0 0.3984
C(2) 0.0 0.3937 0.0

0(1) 0.1105 -0.3157 0.1096
0(2) -0.0493 0.0 0.2119
0(3) -0.1783 0.0 0.4454

Ca(l) 0.1903 0.0 0.1751
Ca(2)

W(1) -0.3552 0.0 0.0438

H(1) 0.45 0.0 0.04
H(2) -0.33 0.05 0.2




























S: Water : Oxygen : Carbon


O : Calcium


Figure 3.9. Theoretical atomic high temperature structure [Deg81b] (stability range: 318-
415 K) of COM crystals as a function of habit plane, (A) (010) plane and (B) (10 ).


PDO "3DO* S.3G er

0%
0% 0%1 4 re
y y y8"ae4~
X.o


*^ ii *


0 :Hydrogen




















0.81

0.64

0.49

0.36

0.25

0.16

0.09

0.04

0.01


10.0


100.0
80.0:
60.0:
40.0:
20.0
10.0


C2Ca04.H20


50.0


60.0 70.0


WHEWELLITE, SYN
20- 231


60.0


70.0


Figure 3.10 A (top) typical experimental COM x-ray diffraction pattern and (bottom)
the JCPDS file for the mineral whewellite, CaC204*2H20.


50.0


20.0


30.0


40.0


20.0


.L I


, 1 A I -


30.0


40.0


S I,,









distribution as shown in Figure 3.11. The mean and standard deviation in particle size is

13.04 gmn and 1.27 pm, respectively. The least square regression coefficient of the curve

fit is 0.98.

For purposes of calculation, the relative dimensions of the COM particles were

measured by analyzing multiple particles from SEM photomicrographs. The relative

sizes illustrated in Figure 3.12 were used calculating surface areas of the two major

crystallographic faces given the equivalent spherical diameter, refer to Appendix A.

Zeta potential of the COM crystals was determined as a function of pH in

saturated COM solution as shown in Figure 3.13. Also on Figure 3.13, one data point

representing the zeta potential of COM crystals in 10% AUIS at the buffered pH of 6.0.

The zeta potential values for COM in saturated COM solution and in 10% AUIS

at pH 6 are -32 mV and -21 mV, respectively. As can be seen from Figure 3.13, the lower

ionic strength, 5x10-6 M, COM saturated solution has a higher zeta potential than the

higher ionic strength, 3.71x10-2 M, 10% artificial urine at pH 6. This effect is expected

and is due to compression of the electrical double layer.

As the ionic strength of the electrolyte the COM crystals are suspended in

increases (i.e., 10% AUIS to 50% AUIS to 100% AUIS), the value of zeta potential will

decrease. When the ionic strength becomes high, the measurement of zeta potential using

Rank Particle Microelectrophoresis becomes impossible. Compression of the electrical

double layer is extreme and the potential distribution near the particle surface drops

rapidly as a function of distance into the solvent, Figure 2.5. Therefore the particles do

not move under the applied electric field and measurements of electrophoretic mobility

cannot be made.

Contact mode scanning probe microscopy (SPM) was performed on both the

(010) and (101) crystallographic faces of a COM particle in saturated COM solution at

250C to determine the surface roughness. The r, values or the mean roughness values for



























250


200


150



100



50


Equivalent Spherical Diameter (rnm)













Figure 3.11. The differential frequency vs. equivalent spherical diameter particle size
distribution of the experimentally produced COM crystals grown without seeds fit to a
log-normal probability distribution.


......_.._______ _








































A:B:C=1.0:0.43:0.35


Figure 3.12. The relative linear dimensions of experimentally produced COM crystals.


























-10

-20

-30

-40

-50


-60
3 4 5 6 7 8 9 10
pH














Figure 3.13 Zeta potential as a function of pH for COM in saturated COM solution and
zeta potential at pH 6 for COM in 10% AUIS.


S COM Saturated Solution
& 10% Artificial Urine






A\

s^}i









the COM (010), Figure 3.14, and (101), Figure 3.15, are 91nm and 81 nm, respectively,

over a scan area of 5 nm by 5 glm. The high surface roughness of the COM crystal may

be due to the growth mechanism of the twinned COM crystal. The crystals have a

growth ledge at the twin boundary where the crystal may grow more rapidly on one half

of the twin boundary but not on the other causing the roughness to be large across this

boundary.


3.4 Conclusions

The equilibrium shapes with face indices of calcium oxalate monohydrate (COM)

particles synthesized in different conditions have been successfully generated using the

computer program SHAPEC. Computer calculations of theoretical crystallographic

shapes have been reconciled to the observed shapes of experimentally synthesized COM

particles under various conditions. Also, the seeding studies suggest that it is possible to

control the size of COM crystals ("33" particles) by controlling the amount of seed

materials. The computer program SHAPED was demonstrated to be a useful tool to

determine the face indices of COM crystals. The equilibrium shape of COM crystals

based on the lattice parameters of Deganello and Piro [Deg8 la] is reconciled to the

morphology of the experimentally synthesized COM crystals from the comparison

between the calculated shape and the morphological form of the experimentally derived

particles.

Based on the face indices of the equilibrium shapes and atomic coordinates, the

atomic structures of COM particles as a function of habit plane have also been generated

by using the computer program ATOMSc. From the comparison between the theoretical

atomic structures generated by ATOMS, the (010) and (100) planes generated with the

atomic coordinates published by Cocco and Sabelli [Coc62] are reconciled to the layering

sequence reported by Deganello and Piro [Deg81a] and Tozzoli and Domeneghetti

[Taz80].



























-15.0


10.0


Box Statistics


2 range 507.09 nN
Mean 5.475 nm
Rns CRq) 91.527 nM
Mean roughness (Ra) 31.592 nM
Surface area
Box x dimension 4.638 pU
5.0 Box y dimension 4.374 uM


5-.O 0
15.0 imn


Figure 3.14 Contact mode SPM scan of the COM (010) crystallographic face showing
the surface roughness.


0 5.0 10.0