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NOVEL APPROACHES TO TESTING GASTRO INTESTINAL FUNCTION IN VITRO: CONTROLLING SIGNAL ACQUISITION, TISSUE COMPOSITION, OR THE PLATFORM BY Dylan Thomas Knutson A Thesis Submitted to the Graduate Faculty of WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES In Partial Fulfillment of the Requirements for the Degree of Master of Science Biomedical Engineering May, 2017 Winston-Salem, North Carolina Approved by: Khalil N. Bitar, Ph.D., AGAF, Advisor, Chair Adam R. Hall, Ph.D Aleksander Skardal, Ph.D

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NOVEL APPROACHES TO TESTING GASTRO INTESTINAL

FUNCTION IN VITRO: CONTROLLING SIGNAL ACQUISITION,

TISSUE COMPOSITION, OR THE PLATFORM

BY

Dylan Thomas Knutson

A Thesis Submitted to the Graduate Faculty of

WAKE FOREST UNIVERSITY GRADUATE SCHOOL OF ARTS AND SCIENCES

In Partial Fulfillment of the Requirements

for the Degree of

Master of Science

Biomedical Engineering

May, 2017

Winston-Salem, North Carolina

Approved by:

Khalil N. Bitar, Ph.D., AGAF, Advisor, Chair

Adam R. Hall, Ph.D

Aleksander Skardal, Ph.D

ii

DEDICATION

To my family

iii

ACKNOWLEDGEMENTS

I would like to thank my advisor, Dr. Khalil N Bitar for the opportunity to

work in his lab and full support. This project was his vision for me, and I thank

him for the guidance, expertise, mentorship. I would also like to thank my

committee, Dr. Adam R. Hall and Dr. Aleks Skardal for their willing collaboration,

mentorship, and commitment to join my committee. I am so grateful for your kind

help.

I am also quite thankful to both Dr. Prabhash Dadhich and Elie Zakhem for

their mentorship during my studies. Without their guidance, the success of my

project would not have been possible. Last, the support of family and friends

gave me the strength to continue pushing myself to work harder.

iv

TABLE OF CONTENTS

DEDICATION ............................................................................................................................................. II

ACKNOWLEDGEMENTS ........................................................................................................................... III

TABLE OF CONTENTS ............................................................................................................................... IV

LIST OF FIGURES ....................................................................................................................................... V

LIST OF TABLES ........................................................................................................................................ IX

LIST OF ABBREVIATIONS........................................................................................................................... X

INTRODUCTION ........................................................................................................................................ 1

CHAPTER I: DO INTERSTITIAL CELLS OF CAJAL PLAY A ROLE IN PYLORIC FUNCTION? ................................ 7

CHAPTER II: AN IN VITRO MODEL OF THE GASTRIC NEUROMUSCULAR APPARATUS USING ENGINEERED

PYLORUS TO UNDERSTAND GASTRIC PATHOPHYSIOLOGY ..................................................................... 25

CHAPTER III: ELECTROPHYSIOLOGY OF AN IN VITRO MODEL OF THE GASTRIC NEUROMUSCULAR

APPARATUS ........................................................................................................................................... 51

CHAPTER IV: MICRO-SENSITIVE MOLDED SILICONE TISSUE PILLAR PLATFORM, FABRICATED BY 3-D

PRINTING FOR SIMPLER PHYSIOLOGICAL STUDIES ................................................................................. 76

CHAPTER V: SUMMARY AND CONCLUSIONS ........................................................................................ 100

APPENDIX ............................................................................................................................................ 101

SCHOLASTIC VITA ................................................................................................................................. 102

v

LIST OF FIGURES

Figure 1- An explanation on the methods for analyzing tissues for slow-wave associated

contractions. Ultimately, the signal is filtered and can be analyzed for both amplitude and

frequency. 14

Figure 2- Cryosections of pyloric tissue, stained with DAPI (Blue) and ICC marker Ano1.

Several ICC were visualized in the tissue, including ICC-SM (submucosal), ICC-MY

(Myenteric), and ICC-IM (Intramuscular). Scale Bars are 100 micrometers. 16

Figure 3- An explanation on the methods for physiology. The thick pyloric sphincter is

isolated from the distal stomach (A). It is stretched and equilibrated in the tissue bath

(B), where an isometric force transducer senses strain and reports changes in stress

(newtons) from stimuli (C). The data is acquired real–time and sent to a computer (D). 17

Figure 4- A(A) Maximum Response to Ach and EFS with and without inhibitor T16Ainh-

A01 incubation. Contraction following exogenous ACh was significantly reduced (p<.05,

n=5). EFS-induced relaxation remained similar. (B) Tissue Response to incubation of

Inhibitor. There was a large increase in basal tone upon the incubation of T16. This

response makes the assessment of gross tissue responses unfair. 18

Figure 5- One minute segments from baseline recordings. It was observed that small

oscillations were being reduced when adding the inhibitor. This would be investigated

quantitatively. 19

Figure 6- Frequency and amplitude with and without inhibition. There was a significant

decrease in both frequency and amplitude of phasic contraction when incubated with

T16Ainh-A01 (25% and 20% respectively, n=5). 20

Figure 7- Characterization of isolated GFP-ICC in Culture. (A) ICCs attached and

acquired a star-shaped morphology. (B) ICCs stained positive for calcium-activated

chloride channel Ano1 confirming a population of isolated ICC (red). DAPI stained nuclei

with blue. All scale bars are 100 µm. 36

Figure 8- Microscopic Evaluation of Engineered Constructs. (A) Engineered pylorus

formed dense muscular tissues around the central post in culture vessel. (B) GFP-ICC

was visualized in the engineered tissues. (C) ICC formed networks within the tissues. All

scale bars are 100 µm. 37

Figure 9- Organ Bath Studies: Basal Tone and KCl: (A trace, before dashed line) All

engineered pylorus constructs established spontaneous basal tone. (B) Tone was similar

in maximum, area, and the rate of establishment (p>.05, n=5) among all constructs. (A,

after dashed line) KCl induced tissue contraction. Tissues contracted to a similar extent

(maximum, area, and the rate (p>.05, n=4)). 38

Figure 10- Organ Bath Studies: Electrical Field Stimulation EFS: EFS induced smooth

muscle relaxation in (A) SMC + NS + ICC and (C) SMC + NS constructs only. There was

vi

no relaxation in (B) SMC + ICC or (D) SMC only constructs. (E) EFS-induced relaxation

was significantly larger in SMC + NS + ICC than all others (1.9x, p<.05, n=5). 40

Figure 11- Organ Bath Studies: Inhibitors for EFS response: TTX abolished EFS-

induced relaxation in the engineered constructs (p<.05, n=3). nNOS neuronal blocker L-

NAME yielded similar extent of inhibition in the SMC + NS + ICC and SMC + NS

constructs, confirming there was a large functional population of differentiated NO donor

neurons (p>.05, n=3). 41

Figure 12- Kinetics EFS-Induced Relaxation: All relaxation responses over time were

averaged, computed and graphed. (A) A representative EFS-induced relaxation graph in

SMC + NS + ICC (purple trace) and SMC + NS (blue trace) constructs. EFS induced

relaxation was faster in (B) area and (C) rate in the SMC + NS + ICC constructs

compared to SMC + NS constructs (1.7x n=5, p=.30 and 2.5x, n=5 p=.12 respectively).

42

Figure 13 L-NAME inhibition of EFS-Induced relaxation: L-NAME completely abolished

EFS-induced relaxation in (A) SMC + NS constructs (dashed blue line) and (B) SMC +

NS + ICC constructs (dashed purple line). 44

Figure 14- Leads for detecting a differential current on the engineered tissue within the

organ bath. 60

Figure 15- Raw recordings from force transducer (blue trace) and/or electrodes (voltage,

mV, green) of within tissue bath. It was necessary for the electrodes to remain in the

bath; otherwise a differential current could not be achieved (arrowed, A) through tissue.

Voltage pulses from EFS (5V, 5Hz, 0.5ms) were detected upon stimulation (B). Without

tissue or electrical activites, the liquid reading from electrodes remained approximately 0

volts (C). 61

Figure 16- Recording from electrodes (voltage, mV, green) of SMC + ICC + NS within

tissue bath. Perturbances of either washing (A, after dashed line) or adding reagents (B,

highlighted) could obscure recordings for a short period. Washing increased perceived

voltage during and for several seconds following. Adding reagents sometimes resulted in

a voltage drop. Analyses and tests were performed following. 62

Figure 17- Recording from electrodes (voltage, mV, green) of SMC only tissue within

tissue bath. Perturbances of either washing (A, after dashed line) or adding reagents (B,

highlighted) could obscure recordings for a short period. Only with tissue did voltage

exceed 1 mV in these tests, and nifedipine blocked electrical activity recorded from the

tissues. Therefore the recordings of activity from the bath are accurate. 63

Figure 18- Raw simultaneous recordings of force (blue trace) and voltage of engineered

tissues within tissue bath. Both graphs show establishment of basal tone, with expected

electrical spikes (arrowed, green trace) preceeding contractions (arrowed, blue trace).

Engineered tissue with ICC had a higher frequency and amplitude, were more rhythmic.

64

Figure 19 - Raw simultaneous recordings of engineered neuromuscular tissues from

EFS response within tissue bath. Scales (force and voltage) are NOT the same. EFS

Response Is much larger, and more immediate in ICC tissues Electrical Changes: SMC

+ NS- increased peak activities following EFS, then nothing. SMC + NS + ICC, slowing

vii

of rhythm, maintain rhythm. Upon the release of NO, ICC slow waves may reduce in

order to facilitate relaxation from tonic state. 67

Figure 20- Spectral analyses graphs of electrical Activity of both SMC + NS and SMC +

NS + ICC tissues 69

Figure 21- Final CAD drawings to create Stomach on a chip design. Features: (1) Top

ABS plate provides windowed view, inlet, and outlet, and thermal resistance. (2) PDMS

Center channel protects 3-D printed parts from leaks, and width for low stress, lowered

flow around tissues. Cutouts allow for gating when culturing tissues. (3) 3 sets of PDMS

tissue pillars allow for the combination of 3 different tissues, and report strains visually to

quantify force. (4) Bottom plate allows sufficient room for added elements and thermal

resistance. 85

Figure 22- CAD drawings are printed using a desktop 3-D printer. Molds for pillars (A)

and bath walls (B) fit into stage (C, shown with part of mold component A). After printing,

M5 screws are tapped through the bottom of the stage for raising molded substrate out

of the assembly. Some bonding surfaces are sanded or epoxy-coated for smooth casts.

The complete assembly allows for the molding of either the pillars or walls in the stage

(D). 85

Figure 23- CAD drawings are printed using a desktop 3-D printer. Final products are

rigid, accurate, and fit together tightly. Right: top plate and bottom plate before

assembly. Lower Left: Assembled molds for PDMS products. 86

Figure 24- The stage allows for the fitment of either the pillar (A) or bath wall mold (B). 87

Figure 25- Molding process to make tissue pillars- (1) desired negative mold is placed in

stage and PDMS is poured over mold. (2) After curing, molded piece and negative are

driven out of stage by set screws. (3) The base is loosely peeled from face of product.

(4) For tissue pillars, the negative mold is disassembled into pieces. (5) Molded pillars

are revealed. (6) A PDMS base with a single set of pillars. 87

Figure 26- All components of the assembly. The bath with pillars (above) or the full

assembly for fluidic experiments (below). 88

Figure 27- (A) Gates were produced from 3D printed mold which fit into slotted walls to

isolate/separate hydrogels cultured in vessel. (B) Gates isolated 1 mL of 1.9 mg/mL

Collagen hydrogel. Gates could be removed after 40 minutes of gelation at 37˚C to add

medium. 89

Figure 28- (A) The bath with pillars representing the assembly to be characterized by

formula, B. Three mixtures of hardener to elastomer were characterized (C). Ultimately

the 1:30 mixture was chosen, and a linear curve was plotted to represent the relationship

of force to pillar distension (D). 90

Figure 29- Microscopy of tissues in culture reveal muscle cells are captured in the tissue,

and are aligned between the silicone pillars. Staining with Smooth muscle Actin

confirmed alignment of muscle filaments. 91

Figure 30- Calibration using a set of masses was used to deflect the pillars and

determine Hooke’s constant for each. 92

Figure 31- A Stereomicroscope can measure the deflection of the pillars in response in

order to calculate forces (A). A force transducer measures strain and reports force

generated by tissues over time (B). The basal tone measured by both devices was

viii

similar (C, n=3, paired, p>.05), but the measured response to KCl was less by the pillar

baths (D). 93

Figure 32- Comparison of the KCl response to sphincteric and non-sphincteric (small

intestine) muscle tissues (IAS). Tissues were washed with fresh buffer, and 60mM KCl

was added following equilibration period. Sphincteric tissues exerted significantly more

force within the device compared to non-sphincteric (n=3-4, p<.05). Therefore the

contractility of muscle when depolarized is different between sphincteric and non-

sphincteric muscle. 94

Figure 33- KCl responses from 3 human pylorus sphincteric tissues. Force from the

tissues was measured every 15 seconds while electrical activity was measured

continuously. Large spikes in EMG activity are associated with the onset of contraction.

Larger or more frequent activites resulted in higher measured forces. 95

ix

LIST OF TABLES

Table 1- Summary of the maximum, duration, and rate of basal tone and KCl

contractions. 39

Table 2- Summary of the minimum, duration, and rate of EFS-induced relaxation in the

absence and presence of inhibitors. 44

x

LIST OF ABBREVIATIONS

Ach Acetylcholine

CAD Computer aided design

ChAT Choline acetyltransferase

DMEM Dulbecco’s modified Eagle medium

EFS Electrical Field Stimulation

ENS Enteric nervous system

GI Gastrointestinal

HBSS Hank’s balanced salt solution

KCl Potassium chloride

LES Lower esophageal sphincter

L-NAME Nω-Nitro-L-arginine methyl ester hydrochloride

nNOS neuronal nitric oxide synthase

PBS Phosphate buffer saline

SEM Scanning electron microscopic

TTX Tetrodotoxin

VIP Vasoactive Intestinal Peptide

xi

1

INTRODUCTION

Motility in the gut involves a complex interplay of tissues and cells to break

down and propel matter throughout each segment. Propulsion is orchestrated by

a complex called the neuromuscular apparatus which includes smooth muscle,

enteric neurons, and interstitial cells of Cajal (ICC) (Kenton M. Sanders, Hwang,

& Ward, 2010). Disruption of this apparatus characterizes some gastrointestinal

(GI) diseases, however both the etiology and pathophysiology of dysmotility

patterns are not well understood (Goldstein, Thapar, Karunaratne, & De Giorgio,

2016). Gastroparesis, or delayed gastric emptying, is a disease in which patients

are left with debilitating symptoms such as nausea and vomiting, early satiety

and malnutrition, and general discomfort. Treatment options are limited and

include surgeries, prokinetics, and pacemaking devices (Bielefeldt, 2012). These

interventions have limited efficacy and are only effective on specific subsets of

patients.

Depletions of ICC and/or enteric neurons have been associated with

clinical cases of gastroparesis. Histological analysis shows patients may be

missing one or both cell types (Grover et al., 2011). It is estimated that at least

50% of Type 1 diabetics and 30% of Type 2 develop this disease. It is well known

that diabetics suffer from neuropathies and other complicated symptoms, but a

significant amount of gastroparesis patients have no other diseases present and

are classified as ‘idiopathic’. Newer studies have mapped altered electrical

waveform patterns, recorded associated symptoms, and the extent of ICC

2

depletions in gastric tissues (Angeli et al., 2015). Furthermore, the specific

location of cell depletions is associated with different symptoms and disease

phenotypes (Moraveji et al., 2016). However, at present there are no cause and

effect studies that can attribute the effect of these cell depletions to disease

pathophysiology.

Furthermore, it is established that ICC generate electrical activity, which

regulates and transmits muscle potentials for contraction. Yet, the role that both

ICC and enteric neurons perform in transmitting cholinergic and nitrergic signals

to direct muscle motility is very controversial (Kenton M Sanders, Kito, Hwang, &

Ward, 2016; Wood, 2016). Some investigators argue that ICC have no role in

transducing responses (Chaudhury, 2016), while others have demonstrated that

they enhance it (Lies et al., 2014). Although tremendous strides have been made

in investigating the role of these cells using clinical populations, isolated tissues,

and gene knockouts (Mashimo, Kjellin, & Goyal, 2000; Sivarao, Mashimo, &

Goyal, 2008; Wagner, Sullins, & Dunn, 2014; Zarate et al., 2003), there is still a

lack of in vitro models to quantitatively deplete and reinstate cell populations in

order to relate cell depletions and measure functional changes in tissues.

In the thesis, provided first is a novel tissue engineering approach to

develop an in vitro model of gastric neuromuscular tissues using different

combinations of sphincteric smooth muscle, enteric neurons, and for the first

time- ICC. Previously, autologous intrinsically innervated pyloric sphincters were

bioengineered using human pyloric smooth muscle and neural progenitor cells.

3

The cells in the constructs demonstrated normal phenotypes, muscular

alignment, and function (Rego, Zakhem, Orlando, & Bitar, 2015). The first goal

of this study was to incorporate ICC into the model. The second goal was to

analyze the contribution of each cell type to basic functions. Physiology of the

engineered constructs showed a specific contribution from each cell type. (i)

Tone was myogenic trait and independent of neuronal or ICC contribution, (ii)

relaxation of tone was dependent on a functional population of nitrergic neurons,

(iii) ICC significantly increased the neural-mediated relaxation in both peak and

rate, and increased electrical rhythms. This study provides a promising new

model to understand, generate, and study new hypotheses regarding

gastrointestinal pathophysiology.

Similarly, mechanisms of gastric emptying disorders are characterized by

an inability for tissue relaxation such as pylorospasm or diabetic neuropathies

and are associated with abnormal manometric data and gastroparesis (Camilleri,

2016). Studying persistent relaxation in ICC knockouts tissues led to the

dismissal of the role of ICC in transducing muscle relaxations (Huizinga et al.,

2008). We confirmed that while these functions indeed persist, the addition of

ICC changes the magnitude of the relaxation response significantly. The

magnitude of relaxation may be an important mechanism for accomodation and

expedition of gastric emptying. Therefore, the ‘persistence’ of responses may not

be physiologically relevant, and so the specific character of these responses

should be investigated before ruling out these key cells.

4

The final aim of the thesis was to develop an open physiology platform to

make studying tissues more accessible and to test innovative hypotheses

regarding functional tissue interactions. In biomedical research, the study of

molecular markers is well characterized, but physiological analysis of tissues

remains less investigated. This is because testing physiological responses takes

tremendous timing, resources for equipment, and multidisciplinary skills. Clever

designs using silicone micro pillars which sense tissue forces have emerged, but

they are too small for simple culture, require complex lithography to produce, and

enhanced imaging using fluorescence to detect forces (Boudou et al., 2011). The

present investigation provides a simpler open-source platform for 3-D printing

molds to make micro-sensitive pillars in a novel fluidic bath. The pillar device

allows for controlled engineering of multiple muscle tissues, and records

muscular tone and response phenomena similar to expensive physiology

equipment. Further novelty includes the capacity for the interaction of several

tissues, measurement of electrical or other phenomena simultaneously, and flow

perfusion for studies like high throughput drug testing on functional tissues.

In conclusion, this study provides novel insights on the effects of gastric

cell depletions on physiological function and offers more accessible and

hypothesis generating platforms which greatly impact future research

propositions and the rectification of disease pathophysiology.

REFERENCES

Angeli, T. R., Cheng, L. K., Du, P., Wang, T. H.-H., Bernard, C. E., Vannucchi, M.-G., . . . O’Grady, G. (2015). Loss of Interstitial Cells of Cajal and Patterns of Gastric Dysrhythmia in Patients

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With Chronic Unexplained Nausea and Vomiting. Gastroenterology, 149(1), 56-66.e55. doi:http://dx.doi.org/10.1053/j.gastro.2015.04.003

Bielefeldt, K. (2012). Gastroparesis: Concepts, Controversies, and Challenges. Scientifica, 2012, 19. doi:10.6064/2012/424802

Boudou, T., Legant, W. R., Mu, A., Borochin, M. A., Thavandiran, N., Radisic, M., . . . Chen, C. S. (2011). A microfabricated platform to measure and manipulate the mechanics of engineered cardiac microtissues. Tissue Engineering Part A, 18(9-10), 910-919.

Camilleri, M. (2016). Novel Diet, Drugs, and Gastric Interventions for Gastroparesis. Clinical Gastroenterology and Hepatology, 14(8), 1072-1080. doi:http://dx.doi.org/10.1016/j.cgh.2015.12.033

Chaudhury, A. (2016). Furthering the debate on the role of interstitial cells of Cajal in enteric inhibitory neuromuscular neurotransmission. American Journal of Physiology - Cell Physiology, 311(3), C479-C481. doi:10.1152/ajpcell.00067.2016

Goldstein, A. M., Thapar, N., Karunaratne, T. B., & De Giorgio, R. (2016). Clinical aspects of neurointestinal disease: Pathophysiology, diagnosis, and treatment. Developmental Biology, 417(2), 217-228. doi:http://dx.doi.org/10.1016/j.ydbio.2016.03.032

Grover, M., Farrugia, G., Lurken, M. S., Bernard, C. E., Faussone–Pellegrini, M. S., Smyrk, T. C., . . . Pasricha, P. J. (2011). Cellular Changes in Diabetic and Idiopathic Gastroparesis. Gastroenterology, 140(5), 1575-1585.e1578. doi:http://dx.doi.org/10.1053/j.gastro.2011.01.046

Huizinga, J. D., Liu, L. W., Fitzpatrick, A., White, E., Gill, S., Wang, X.-Y., . . . Starret, T. (2008). Deficiency of intramuscular ICC increases fundic muscle excitability but does not impede nitrergic innervation. American Journal of Physiology-Gastrointestinal and Liver Physiology, 294(2), G589-G594.

Lies, B., Gil, V., Groneberg, D., Seidler, B., Saur, D., Wischmeyer, E., . . . Friebe, A. (2014). Interstitial cells of Cajal mediate nitrergic inhibitory neurotransmission in the murine gastrointestinal tract. American Journal of Physiology - Gastrointestinal and Liver Physiology, 307(1), G98-G106. doi:10.1152/ajpgi.00082.2014

Mashimo, H., Kjellin, A., & Goyal, R. K. (2000). Gastric stasis in neuronal nitric oxide synthase–deficient knockout mice. Gastroenterology, 119(3), 766-773.

Moraveji, S., Bashashati, M., Elhanafi, S., Sunny, J., Sarosiek, I., Davis, B., . . . McCallum, R. (2016). Depleted interstitial cells of Cajal and fibrosis in the pylorus: novel features of gastroparesis. Neurogastroenterology & Motility.

Rego, S. L., Zakhem, E., Orlando, G., & Bitar, K. N. (2015). Bioengineered human pyloric sphincters using autologous smooth muscle and neural progenitor cells. Tissue Engineering Part A, 22(1-2), 151-160.

Sanders, K. M., Hwang, S. J., & Ward, S. M. (2010). Neuroeffector apparatus in gastrointestinal smooth muscle organs. The Journal of Physiology, 588(23), 4621-4639. doi:10.1113/jphysiol.2010.196030

Sanders, K. M., Kito, Y., Hwang, S. J., & Ward, S. M. (2016). Regulation of Gastrointestinal Smooth Muscle Function by Interstitial Cells. Physiology, 31(5), 316-326.

Sivarao, D. V., Mashimo, H., & Goyal, R. K. (2008). Pyloric sphincter dysfunction in nNOS−/− and W/W V mutant mice: animal models of gastroparesis and duodenogastric reflux. Gastroenterology, 135(4), 1258-1266.

Wagner, J. P., Sullins, V. F., & Dunn, J. C. Y. (2014). A novel in vivo model of permanent intestinal aganglionosis. Journal of Surgical Research, 192(1), 27-33. doi:http://dx.doi.org/10.1016/j.jss.2014.06.010

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Wood, J. D. (2016). Enteric Nervous System: Neuropathic Gastrointestinal Motility. Digestive Diseases and Sciences, 61(7), 1803-1816. doi:10.1007/s10620-016-4183-5

Zarate, N., Mearin, F., Wang, X., Hewlett, B., Huizinga, J., & Malagelada, J. (2003). Severe idiopathic gastroparesis due to neuronal and interstitial cells of Cajal degeneration: pathological findings and management. Gut, 52(7), 966-970.

7

CHAPTER I: DO INTERSTITIAL CELLS OF CAJAL PLAY A

ROLE IN PYLORIC FUNCTION?

Dylan T. Knutson1,2, B.S., Elie Zakhem2, Ph.D, Khalil N. Bitar1,2,3, Ph.D., AGAF.

1Virginia Tech-Wake Forest School of Biomedical Engineering and Sciences, Winston Salem, NC

2Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston Salem, NC

3Section on Gastroenterology, Wake Forest School of Medicine, Winston-Salem, NC, USA

This chapter explores the physiological contribution of ICC in ex vivo Gastric tissues

8

ABSTRACT

BACKGROUND

Ano1 (TMEM16A) is a Ca2+-activated Cl− channel on Interstitial Cells of

Cajal (ICC) which regulates gastrointestinal motility. Absences of ICC and

consequently Ano1 are associated with several motility disorders, since they

pace smooth muscle with electrical conduction called the slow-wave. Several

Ano1 inhibitors have been identified such as T16Ainh-A01. Using the inhibitor has

therapeutic potential to help control cancerous proliferation of cells

overexpressing Ano1; however, it also has been shown to reduce slow waves in

isolated ICC cultures.

AIM

To investigate the effect of Ano1 inhibitor, T16Ainh-A01, on pyloric

function, a gastrointestinal sphincter responsible for regulating the passage of

stomach contents into the duodenum.

METHODS

Whole rat pyloric sphincter was excised and placed in an organ bath to

measure force generation. The effect of T16Ainh-A01 (10μM) was added to the

buffer. The changes in frequency and amplitude of phasic contractions were

measured. Muscarinic response to Ach (10μM) and electrical field stimulation

(EFS) to induce relaxation was measured in organ bath.

9

RESULTS

Phasic contractions in normal pylorus indicated a frequency of 4.2 +/- 0.25

contractions per minute. T16Ainh-A01 significantly reduced the frequency of

phasic contractions of the pyloric tissue to 3.2 +/- 0.11 (72% of control, n=5, P <

.05, t-test). The amplitude of phasic contractions was reduced. Muscarinic

response from Acetylcholine also decreased with the presence of T16Ainh-A01

(n=5). Pyloric relaxation remained similar with T16Ainh-A01 (n=4). We think the

effect of the inhibitor was not specific to ICC only. Some physiological responses

were retained, but reduced rhythmic contractions suggest the underlying slow

wave was being diminished with T16Ainh-A01.

CONCLUSION

The Ano1 inhibitor T16Ainh-A01 inhibited Ca2+-activated Cl− currents,

reduced the phasic contractions, increased tone, and altered some

neurotransmitter responses in healthy pyloric tissue. These data support the

notion that T16Ainh-A01 could alter pyloric function, inhibit ICC function in tissue,

and consequently gastric emptying in therapeutic uses. However, better models

must be made to further understand the ICC contribution.

Keywords: Interstitial cells of Cajal, ICC, TMEM16A, T16Ainh-A01, cancer,

pylorus

10

INTRODUCTION:

Ano1 (TMEM16A) is a Ca2+-activated Cl− channel on Interstitial Cells of

Cajal (ICC) which regulates gastrointestinal motility by activating pacemaker

currents (Zhu, Sung, O'Driscoll, Koh, & Sanders, 2015). Absences of ICC are

associated with several motility disorders, since they pace smooth muscle with

electrical conduction, also called the ‘slow wave’. Recently, it has also been

shown that Ano1 is responsible for regulating cellular proliferation and is up-

regulated in several forms of gastrointestinal cancer. Therefore, several Ano1

inhibitors have been identified such as T16Ainh-A01 (Hwang, Basma, Sanders, &

Ward, 2016; Mazzone et al., 2012). Using the inhibitor has therapeutic potential

to help control cancerous proliferation of cells overexpressing Ano1. However, it

also has been shown to reduce slow waves in isolated ICC cultures. Thus, the

inhibitor’s effects on whole gastrointestinal tissue function should be studied as

this may provide insight on the contribution of ICC within gastric tissues. The role

that ICC perform in transmitting cholinergic and nitrergic signals and regulating

muscular tissue function is controversial (Sanders, Kito, Hwang, & Ward, 2016;

Wood, 2016).

In the stomach, motility is dependent on the effective accomodation,

trituration, and emptying of reduced foods into the duodenum- carried out by the

fundus, antrum, and pylorus respectively. It is thought that phasic muscular

patterns are driven by electrical activity that is propagated by ICC, which

originate in the greater curvature of the stomach and diminish after the pylorus

11

(O'Grady et al., 2010). The pylorus contracts and maintains tone to facilitate

mixing, and subsequently relaxes to enhance emptying of broken down particles.

In the present investigation, the purpose was to inhibit ICC and study the

function of pyloric tissues in an organ bath. The first goal of this study was to

ensure the presence of ICC in the isolated pylorus. The second goal was to

analyze the contribution of ICC to basic pyloric physiology. The results show that

the Ano1 inhibitor T16Ainh-A01 inhibited Ca2+-activated Cl− currents, reduced the

phasic contractions associated with ICC, and altered neurotransmitter responses

in healthy pyloric tissue. These data support the notion that T16Ainh-A01 could

alter pyloric function and consequently gastric emptying in therapeutic uses.

12

MATERIALS AND METHODS:

Pylorus Tissues

Pylori from rat were removed by sharp dissection. Pylorus tissues were

manually cleaned by removing fat and mucosa with a surgical blade. The pyloric

sphincter was studied in its entirety (the band of muscle). Tissues were

approximately 6-7mm in diameter and 3mm in width.

Preparation of Cryosections and Ano1 Staining

Cryosections were prepared as previously described (Chen et al., 2011).

Briefly, tissue was placed on filter paper in 4% paraformaldehyde (PFA) for 2

hours at room temperature, and then placed in 30% sucrose in PBS overnight at

4 °C. The following day, cryosections were prepared in cryomolds and O.C.T.

Tissues were stained with 1:200 Anti-TMEM16A (Abcam, Cambridge, UK)

overnight and then incubated with 1:200 secondary antibody (tritC) diluted in

blocking buffer for 2 hours at room temperature.

Physiological Analysis of Ex Vivo Pylorus

All different combinations of engineered pyloric constructs were analyzed

for physiological functionality. Constructs were hooked in an organ bath between

a fixed arm, and the measuring arm of an isometric, magnetoresistive force

transducer in a horizontal tissue bath (F10; Harvard Apparatus, Holliston, MA).

Force data was acquired using LabChart 7 software (ADInstruments, Colorado

Springs, CO). Constructs were maintained in 4-(2-hydroxyethyl)-1-

piperazineethanesulfonic acid (HEPES) buffered solution at 37°C throughout all

13

experiments. All force generation studies were performed after the establishment

of stable basal tone. Then, acetylcholine (ACh; 1 μM) and electrical field

stimulation (EFS; 5 Hz, 0.5 ms, for 30 seconds) were administered. Constructs

were washed between each treatment, incubated in fresh buffer and allowed to

return to baseline. Pretreatment with T16Ainh-A01 was administered 8 minutes

prior to EFS stimulation to inhibit ICC. GraphPad Prism 7.00 software for

Windows (GraphPad Prism 7.00, San Diego, CA) was used to analyze collected

data. Second-order Savitsky–Golay smoothing was applied. Quantification of

physiologic data was performed relative to basal tone for contraction and

relaxation as maximum/minimum peak response (Delta Force of basal tone),

area (magnitude * time), and rate (linear regression from onset to peak

response).

14

Filtering Slow Wave Associated Contractions

Figure 1

As shown in Figure 1, in order to analyze the amount of slow-wave

associated contractions in the tissue, a FFT style analysis and reconstruction

were perfomed in MATLAB (Mathworks, Natick, MA). 3x1-minute segments, at

3x10 minute intervals, were taken to establish a baseline frequency and

amplitude. Each individual segment was de-trended, smoothed, filtered, and

reconstructed. Peak analyses could then count the amount of peaks and their

respective amplitudes. The baseline was compared to 3 1-minute segments after

T16Ainh-A01 incubation.

Statistical analysis:

Data were expressed as mean ± standard error of mean (SEM) unless

noted otherwise. Alpha was set at p<.05. One-way ANOVA followed by Tukey’s

Oscillations/min

Average Amplitude

1. De-trended (removal of floating baseline).

2. Smooth, Fast-Fourier transform and low-pass (.01 cut-off) filtering.

3. Reconstruction of filtered myograph and analysis.

3

15

test was used to compare all groups. Only up to three t-tests were used to

evaluate a priori hypotheses on the groups. Normality was assessed by fisher

skewness, and for ANOVA a Brown-Forsythe test was used to ensure

homogeneity of variance (Prism 7.00, GraphPad Software, Inc., La Jolla, CA,

USA).

16

RESULTS:

Immunofluorescence evaluation of ICC:

Figure 2

First, cryosections of pyloric tissue were prepared to ensure that the

excised tissues contained ICC (Figure 2). Then the sections were stained with

DAPI (Blue) and ICC marker Ano1. Several ICC could be visualized in the tissue,

including ICC-SM (Submucosal), ICC-MY (Myenteric), and ICC-IM

(Intramuscular). Scale Bars are 100 micrometers. After the presence of ICC was

confirmed in excised tissues, the main procedures of the study could be

performed.

17

Figure 3

Thick pyloric sphincter was isolated from the distal stomach for analyses

(Figure 3, A). It was stretched and equilibrated in the tissue bath (B), where an

isometric force transducer sensed strain and reported changes in stress

(newtons) from stimuli (C). The data was acquired real–time and sent to a

computer (D).

A. B.

C. D. Tensile Strain / Compressive Force

18

Figure 4

After the establishment of a stable baseline, the maximum response to

Ach was assessed. The same experiment was conducted with incubation of

T16Ainh-A01. Contraction following exogenous ACh was significantly reduced

(Figure 4, A, p<.05, n=5). EFS-induced relaxation remained similar with and

without the inhibitor.

In regards to the experiments with the incubation of T16Ainh-A01 inhibitor,

there was a large increase in basal tone upon the addition of inhibitor (B). This

would stabilize after five minutes at a new baseline. The increase was 390.16 ±

36.11 µN on average.

The addition of the inhibitor with exogenous Ach contraction leads to a

contraction change in tone higher than the response to only exogenous Ach.

*

Ex Vivo Rat Pylorus

T16Ainh-A01 response A. B. Ex Vivo Rat Pylorus

NT responses

19

Therefore, comparing gross responses with or without the inhibitor may not be a

suitable comparison.

Analysis of Slow-Wave Associated Contractions

Instead, the presence of slow-wave associated contractions was assessed

using fast Fourier transforms and peak analysis for both the amplitude of

contraction and frequency. Samples were taken with and without the presence of

inhibitor at baselines. Examining samples independently revealed that baselines

had more oscillations before the addition of the T16Ainh-A01 inhibitor,

suggesting that pacing, associated with the regulation of muscle contractility, was

being blocked (Figure 5, 3 different samples with excerpts of force over time).

Figure 5

Pylo

rus S

am

ple

1 minute

100 µN

1

t = 10 min 20 min T16Ainh-A01 30 min

2

3

20

Analyzing phasic contractions in normal pylorus indicated a frequency of

4.2 +/- 0.25 contractions per minute. This validates the filter methods since

normal gastric slow waves in the rat are recorded from three to five cycles per

minute. T16Ainh-A01 significantly reduced the frequency of phasic contractions

of the pyloric tissue to 3.2 +/- 0.11 (Figure 6, 72% of control, n=5, P < .05, t-test).

The amplitude of phasic contractions was reduced.

Figure 6

DISCUSSION:

The present investigation provides an advanced method for studying the

contribution of ICC to pyloric tissue function. The pylorus is a sphincteric tissue

which controls trituration periods and facilitates the passage of foods to the

duodenum from the stomach. The advanced analysis of force signals revealed

that the phasic contraction of the pylorus might be reliant on Ano1 and ICC

function. Physiology of the responses to exogenous Ach and EFS was

inconclusive, since the baseline was altered before the addition of other stimuli.

*

Frequency Amplitude

21

The first purpose of this study was to ensure that the target cells could be

found within the tissue being excised. After making cryosections and staining

them with ICC marker Ano1, ICC could be visualized within several layers of

pylorus tissues including the submucosa, myenteric plexus, and muscle layers.

The next purpose was to assess the contribution of ICC to pyloric

functions of contraction and relaxation, by incubating the tissue with an Ano1

inhibitor, T16Ainh-A01. The results were inconclusive because the incubation of

the inhibitor caused a tremendous increase in basal tone. The conclusion that the

blockade of ICC caused an increased in basal tone contradicts the results of

many prior experiments (Cobine et al., 2017). Rather, we doubt the inhibitors

specificity to target calcium activated chloride channels in only ICC. At this point,

this inhibitor has only been used on isolated ICC, and not whole gastrointestinal

tissues.

Furthermore, the use of this inhibitor as a therapeutic agent could have

tremendous GI side effects, as it caused a robust contraction in the tissue.

Perhaps, it could cause gastroparesis in patients by inducing pylorospasm, or

prolonged pyloric contractions. Therefore, we moved to assessing the function of

the tissue at baseline, instead of to responses only.

A method of examining phasic contractions at baseline was developed.

The novelty in this method was that it allowed us to not only derive the specific

frequency of contractions, but also measure the amplitude of each oscillation. For

us, this was a better method than examining only frequency and power, as we

22

would lose the measurement of force using traditional analyses. Analyzing phasic

contractions in normal pylorus indicated a frequency of 4.2 +/- 0.25 contractions

per minute, validating the filter methods since normal gastric slow waves in the

rat are recorded from 3-5 cycles per minute (Albertí et al., 2007). Here, we were

able to significantly decrease the amount of phasic contractions when incubated

with the inhibitor. There was a slight reduction in amplitude, but it was not

significant.

Other studies have shown that the slow wave or pacemaker activity

frequencies decrease when incubating ICC cultures with T16 Ainh-A01. We were

studying phasic contractions, which are understood to be the result of coupling

pacemaker activity of ICC with smooth muscle, in order to depolarize populations

of smooth muscle in coordination (Sun et al., 1995). At the concentration used in

this study (1 µM), the results of phasic contractions match rigorous assays of

pacemaker activity within ICC cultures using patch clamp techniques (Hwang et

al., 2016).

This study provides promising insights on GI pathophysiology, but there

are several limitations that need to be considered in the future. The data from this

study confirms that the phasic contractions were reduced, but a better method of

measuring ICC activity should instead measure electrical activity or frequency in

tissues, and match it with contractions or changes in tone. Also, the results are

inconclusive on the results of gross physiological responses such as contraction

and relaxation. Thus, better methods are needed to control specific cell

populations in tissues. In this study, as the specificity of the inhibitor was

23

uncertain, possible conclusions are limited. However, this study provides some

promising insights using advanced analyses of phasic contractions.

To conclude, the Ano1 inhibitor T16Ainh-A01 inhibited Ca2+-activated Cl−

currents, reduced the phasic contractions associated with ICC, and altered some

neurotransmitter responses in healthy pyloric tissue. Clinically, T16Ainh-A01

could alter pyloric function, and consequently gastric emptying, in therapeutic

uses. Future research should consider new models to study the contribution of

these key cells to gastrointestinal physiology.

ACKNOWLEDGMENTS:

This work was supported by Wake Forest School of Medicine Institutional Funds.

24

REFERENCES:

Albertí, E., Mikkelsen, H. B., Wang, X. Y., Díaz, M., Larsen, J. O., Huizinga, J. D., & Jiménez, M. (2007). Pacemaker activity and inhibitory neurotransmission in the colon of Ws/Ws mutant rats. American Journal of Physiology - Gastrointestinal and Liver Physiology, 292(6), G1499-G1510. doi:10.1152/ajpgi.00136.2006

Chen, Y., Shamu, T., Chen, H., Besmer, P., Sawyers, C. L., & Chi, P. (2011). Visualization of the Interstitial Cells of Cajal (ICC) Network in Mice. Journal of Visualized Experiments : JoVE(53), 2802. doi:10.3791/2802

Cobine, C. A., Hannah, E. E., Zhu, M. H., Lyle, H. E., Rock, J. R., Sanders, K. M., . . . Keef, K. D. (2017). ANO1 in intramuscular interstitial cells of Cajal plays a key role in the generation of slow waves and tone in the internal anal sphincter. The Journal of Physiology, n/a-n/a. doi:10.1113/JP273618

Hwang, S. J., Basma, N., Sanders, K. M., & Ward, S. M. (2016). Effects of new‐generation inhibitors of the calcium‐activated chloride channel anoctamin 1 on slow waves in the gastrointestinal tract. British journal of pharmacology.

Mazzone, A., Eisenman, S. T., Strege, P. R., Yao, Z., Ordog, T., Gibbons, S. J., & Farrugia, G. (2012). Inhibition of Cell Proliferation by a Selective Inhibitor of the Ca(2+)-activated Cl(−) Channel, Ano1. Biochemical and biophysical research communications, 427(2), 248-253. doi:10.1016/j.bbrc.2012.09.022

O'Grady, G., Du, P., Cheng, L. K., Egbuji, J. U., Lammers, W. J. E. P., Windsor, J. A., & Pullan, A. J. (2010). Origin and propagation of human gastric slow-wave activity defined by high-resolution mapping. American Journal of Physiology - Gastrointestinal and Liver Physiology, 299(3), G585-G592. doi:10.1152/ajpgi.00125.2010

Sanders, K. M., Kito, Y., Hwang, S. J., & Ward, S. M. (2016). Regulation of Gastrointestinal Smooth Muscle Function by Interstitial Cells. Physiology, 31(5), 316-326.

Sun, W. M., Smout, A., Malbert, C., Edelbroek, M. A., Jones, K., Dent, J., & Horowitz, M. (1995). Relationship between surface electrogastrography and antropyloric pressures. American Journal of Physiology - Gastrointestinal and Liver Physiology, 268(3), G424-G430.

Wood, J. D. (2016). Enteric Nervous System: Neuropathic Gastrointestinal Motility. Digestive Diseases and Sciences, 61(7), 1803-1816. doi:10.1007/s10620-016-4183-5

Zhu, M. H., Sung, T. S., O'Driscoll, K., Koh, S. D., & Sanders, K. M. (2015). Intracellular Ca2+ release from endoplasmic reticulum regulates slow wave currents and pacemaker activity of interstitial cells of Cajal. American Journal of Physiology - Cell Physiology, 308(8), C608-C620. doi:10.1152/ajpcell.00360.2014

25

CHAPTER II: AN IN VITRO MODEL OF THE GASTRIC

NEUROMUSCULAR APPARATUS USING ENGINEERED

PYLORUS TO UNDERSTAND GASTRIC

PATHOPHYSIOLOGY

Dylan T. Knutson1,2, B.S., Elie Zakhem2, Ph.D, Khalil N. Bitar1,2,3, Ph.D., AGAF.

1Virginia Tech-Wake Forest School of Biomedical Engineering and Sciences, Winston Salem, NC

2Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston Salem, NC

3Section on Gastroenterology, Wake Forest School of Medicine, Winston-Salem, NC, USA

This chapter describes the validation and physiological analysis of engineered neuromuscular tissues

This manuscript was submitted for publication

26

ABSTRACT

INTRODUCTION

Neuro-muscular disorders of the gut can result from the depletion of

neurons and/or interstitial cells of Cajal (ICC) populations. There is a lack of

controlled in vitro models to study the controversial contribution of these cellular

populations to gastrointestinal physiology.

OBJECTIVE

The aims of this study were (1) to bioengineer pyloric sphincter tissues

containing different combinations of cells and (2) to compare their physiological

functions.

METHODOLOGY

Neuro-muscular tissues were engineered using different combinations: (1)

SMC, (2) SMC + ICC, (3) SMC + NPC, and (4) SMC + ICC + NPC. Engineered

tissues were characterized by immunohistochemistry and physiology.

RESULTS

Isolated ICC exhibited normal morphology in culture and stained positive

for Ano1. Live microscopy of engineered tissues revealed GFP-positive ICC

networks in respective constructs. Organ bath studies demonstrated: (i)

Establishment of basal tone in all tissues to a similar extent. (ii) Increase in basal

tone in response to potassium chloride to a similar extent. (iii) Electric field

stimulation (EFS) induced relaxation in tissues that contained NPC (-244.3 ±

33.41 µN. p<.05, n=10). (iv) Relaxation in response to EFS was blocked by

27

inhibitors Tetrodotoxin and Lω-Nitro Arginine, confirming relaxation was mainly

dependant on a functional nNOS neuronal population. (v) Neural-mediated

relaxation was amplified in the presence of ICC in both peak and rate (1.9x,

p<.05, and 2.5x respectively, p=.12, n=5), indicating that there was an interaction

between the two populations to promote relaxation.

CONCLUSION

This study demonstrates the first successful engineering of a neuro-

muscular apparatus that incorporates the three major cells (smooth muscle,

enteric neurons and ICC). Real time force generation showed functional

contribution from all cell types, and an interaction from neurons and ICC which

enhanced relaxation. This model enables a new way to quantitatively deplete and

reinstate cell populations, in order to model, study, and repair gastrointestinal

pathophysiology.

Keywords: Gastroparesis, Pylorus, Diabetes, Bioengineering, Interstitial Cells of

Cajal (ICC), Disease Models

28

INTRODUCTION

Motility in the gut involves a complex interplay of tissues and cells to break

down, aid in effective absorption, and propel matter throughout each segment.

Propulsion is orchestrated by a complex interplay of the components of the

neuromuscular apparatus which include smooth muscle, enteric neurons, and

interstitial cells of Cajal (ICC) (Kenton M. Sanders, Hwang, & Ward, 2010).

Disruption of this apparatus characterizes some gastrointestinal (GI) diseases;

however, both the etiology and pathophysiology of dysmotility patterns are not

well understood (Goldstein, Thapar, Karunaratne, & De Giorgio, 2016).

Gastroparesis, or delayed gastric emptying, is a disease in which patients suffer

from debilitating symptoms consisting of nausea and vomiting, early satiety and

malnutrition, and discomfort. These incapacitating symptoms contribute to a

decreased health-related quality of life among these patients. Treatment options

are limited and include surgeries, prokinetics, and pacemaking devices

(Bielefeldt, 2012). Not only is the efficacy of these interventions limited, but they

are also only effective on certain subsets of patients.

Normal function of the stomach is dependent on the effective

accomodation, trituration, and emptying of reduced foods into the duodenum-

carried out by the fundus, antrum, and pylorus respectively. Phasic muscular

patterns are driven by electrical activity that is propagated by ICC which originate

in the greater curvature of the stomach and diminish after the pylorus (O'Grady et

al., 2010). The pylorus contracts and maintains tone to facilitate mixing, and

subsequently relaxes to enhance emptying of broken down particles.

29

Gastroparesis is thought to occur from a dysfunction occurring in one or more of

these tissues, including the pylorus, but this hypothesis is not entirely clear

(Camilleri, 2016).

Depletions of ICC and/or enteric neurons have been associated with

clinical cases of gastroparesis. Histological analysis shows patients may be

missing one or both cell types (Grover et al., 2011). It is well known that diabetics

suffer from neuropathies and complicated symptoms, including gastroparesis.

However, a significant amount of patients diagnosed with gastroparesis have no

other diseases present, and are thus classified as ‘idiopathic’. Recent studies

have mapped altered waveform patterns and associated them with both clinical

symptoms and with the extent of ICC depletions in stomach tissues (Angeli et al.,

2015). Furthermore, it has also been shown the specific location of cell

depletions is associated with different symptoms and disease phenotypes

(Moraveji et al., 2016). Yet, at present there are no cause and effect studies that

can attribute the effect of these cell depletions to disease pathophysiology.

The role that both ICC and enteric neurons perform in transmitting

cholinergic and nitrergic signals, and regulating muscular tissue function is

controversial (Kenton M Sanders, Kito, Hwang, & Ward, 2016; Wood, 2016).

Some investigators argue that ICC do not have a role in transducing nitrergic

responses (Chaudhury, 2016), while others have demonstrated that they

enhance it (Lies et al., 2014). Even though tremendous strides have been made

in investigating the role of these cells using clinical studies, isolated tissues, and

knockout models (Mashimo, Kjellin, & Goyal, 2000; Sivarao, Mashimo, & Goyal,

30

2008; Wagner, Sullins, & Dunn, 2014; Zarate et al., 2003), there is a lack of in

vitro models that quantitatively deplete and reinstate cell populations in order to

relate cell depletions with real functional changes in tissues.

The present investigation provides a novel tissue engineering approach to

develop an in vitro model of pyloric neuromuscular tissues using different

combinations of sphincteric smooth muscle, enteric neurons, and for the first

time- ICC. Prior to this study, autologous intrinsically innervated pyloric

sphincters were bioengineered, using human pyloric smooth muscle and neural

progenitor cells. The cells in these constructs demonstrated normal phenotypes,

muscular alignment, and function (Rego, Zakhem, Orlando, & Bitar, 2015). The

first goal of the present study was to incorporate ICC into the aforementioned

model. The second goal was to analyze the contribution of each cell type to basic

pyloric physiology. The results showed that physiology of the engineered

constructs consisted of a significant contribution from all cell types. (1) Tone was

myogenic trait and independent of neuronal or ICC contribution, (2) relaxation of

tone was dependent on a functional population of nitrergic neurons, (3) ICC

significantly increased the neural-mediated relaxation in both peak and rate. This

study provides a promising new model to aid in further research regarding the

investigation and understanding of gastrointestinal pathophysiology.

MATERIALS AND METHODS:

Isolation of cells:

Primary Smooth Muscle Cells (SMC)

31

Human tissues were ethically obtained from organ donors through

Carolina Donor Services and Wake Forest Baptist Medical Center (IRB#:

IRB00007586).

Smooth muscle cells were isolated as previously described (Rego et al.,

2015). Briefly, human pylori were removed by sharp dissection. Pylorus tissues

were manually cleaned by removing fat and mucosa with a surgical blade.

Tissues were extensively washed with HBSS solution containing 2X

antibiotics/antimycotic and then minced in sterile conditions. Tissues were

subjected to 2 digestions with HBSS containing 1 mg/mL collagenase type II

(Worthington Biochemicals, Lakewood, NJ) at 37°C for 1 hour each. Tissue

pellets were then resuspended in SMC growth media and plated in tissue culture

dishes at 37°C with 5% CO2.

Neural Progenitor Cells (NS)

Neural progenitors were isolated as previously described (Raghavan,

Gilmont, & Bitar, 2013). Briefly, duodenum from rats was harvested and

extensively washed with HBSS containing 2X antibiotics/antimycotic, followed by

mincing and additional washing. Minced tissue was subjected to 2 digestions in a

mixture containing 0.85 mg/ml type II Collagenase, 0.85 mg/ml Dispase II with 40

µg/ml DNAase I at 37oC for 45-60 minutes. The cells were then passed through a

70 µm nylon cell strainer and then through a 40 µm nylon cell strainer. Cells were

plated in Neurobasal Medium containing 1X N2 supplement, 20 ng/ml

recombinant human Epidermal Growth Factor, 20 ng/ml recombinant basic

Fibroblast Growth Factor, 1.0mM L-glutamine and 1X antibiotics/antimycotic.

32

Primary ICC

GFP-expressing mice were kindly provided by Dr. Frank C. Marini at

Wake Forest School of Medicine. GFP-ICC were isolated as previously described

(Zhu et al., 2009). Briefly, small intestine was harvested and washed extensively

with sterile HBSS containing 2X antibiotics/antimycotic in sterile conditions. After

mincing, tissues were again washed with HBSS three times. Tissues were

digested in HBSS containing 1.3 mg/mL collagenase type II (Worthington

Biochemicals), 2 mg/mL bovine serum albumin, 2 mg/mL trypsin inhibitor, and

0.27 mg/mL Adenosine triphosphate (Sigma-Aldrich, St. Louis, MO) for 23

minutes at 37°C . After digestion, tissue pellets were centrifuged at 600 g for 5

minutes and washed three more times. Pellets were then resuspended in SMC

growth media containing 0.01 µg/mL human stem cell factor (Peprotech, Rocky

Hill, NJ) and plated on 2.5 µg/mL collagen coated dishes at 37°C with 5% CO2.

Microscopic and immunofluorescence evaluation of isolated ICC:

Isolated ICC were grown on collagen-coated plates and evaluated

microscopically. At 80% confluency, cells were fixed with 10% normal buffered

formalin followed by permeabilization and blocking. ICC were stained against

ANO1 (Rabbit anti-Mouse T16mem/ANO1), followed by incubation with

secondary antibody (goat anti-Rabbit TRITC conjugate) (Abcam, Cambridge,

UK). Negative control received secondary antibody only. Cells were mounted

with mounting medium containing DAPI (Vector Labs, Burlingame, CA). Images

were processed using a fluorescence microscope (Nikon Instruments, Melville,

NY).

33

Bioengineered in vitro models of Pylorus

The process of engineering the sphincters was described previously

(Gilmont, Raghavan, Somara, & Bitar, 2014; Raghavan et al., 2011; Raghavan et

al., 2014). For all sphincters, 35 mm dishes were coated with sylgard. Eight

millimeter cylindrical sylgard posts were placed in the center of each plate,

followed by sterilization using ethanol and UV. Four types of sphincters were

engineered using different combinations of cells. Combinations included the

following and are described below: 1) SMC only, 2) SMC + ICC, 3) SMC + NS,

and 4) SMC + NS + ICC.

SMC only

Smooth muscle cells were trypsinized and 500,000 cells were collected

per construct. Cells were resuspended in a collagen gel of 0.4 mg/ml final

concentration. The mixture was then poured in the culture dish around the post.

The muscle gel was left to gel at 37ºC and then supplemented with differentiation

media.

SMC + ICC

Smooth muscle cells (500,000 per construct) and ICC (75,000 per

construct) were obtained and resuspended in collagen gel mixture with a final

concentration of 0.4 mg/ml. The mixture was left to gel 37ºC and then

supplemented with differentiation media.

SMC + NS

Neural progenitor cells (NS) were dissociated with Accutase (Invitrogen,

Carlsbad, CA) and 200,000 cells per construct were obtained. The cells were re-

34

suspended in a solution with a final concentration of 0.4mg/ml type I rat tail

Collagen (BD Biosciences) and 10ug/ml mouse laminin (Invitrogen, Carlsbad,

CA). The mixture was poured on the prepared Sylgard culture dishes. The

mixture was allowed to gel at 37ºC. Smooth muscle cells were then prepared by

collecting 500,000 cells and mixing them in the collagen gel mixture. The smooth

muscle mixture was poured on top of the first NS gel layer and allowed to gel at

37C. Following gelation of both layers, differentiation media was supplemented.

SMC + NS + ICC

Neural progenitor cells (NS, 200,000 cells per construct) were obtained

and re-suspended in a collagen/laminin solution as described above. The mixture

was poured on the prepared culture dishes coated with Sylgard and allowed to

gel. A total of 500,000 smooth muscle cells and 75,000 ICC per construct were

obtained and suspended in a collagen mixture as described above. The mixture

was overlaid on top of the first NS layer and allowed to gel, followed by the

addition of differentiation media.

Microscopic evaluation of the constructs:

Constructs were evaluated microscopically prior to physiological testing on

day 10 of culture. Constructs were imaged under FITC filter to visualize GFP-

expressing ICC. Images were processed using NIS Elements software (Nikon

Instruments, Melville, NY).

Physiological analysis of bioengineered pylorus

All different combinations of engineered pyloric constructs were analyzed

for physiological functionality. Constructs were hooked in an organ bath between

35

a fixed arm, and the measuring arm of an isometric, magnetoresistive force

transducer in a horizontal tissue bath (F10; Harvard Apparatus, Holliston, MA).

Force data was acquired using LabChart 7 software (ADInstruments, Colorado

Springs, CO). Constructs were maintained in 4-(2-hydroxyethyl)-1-

piperazineethanesulfonic acid (HEPES) buffered solution at 37°C throughout all

experiments. All force generation studies were performed after the establishment

of stable basal tone. Force generation was evaluated following the addition of

potassium chloride (KCl; 60 mM), acetylcholine (ACh; 1 μM), and electrical field

stimulation (EFS; 5 Hz, 0.5 ms, for 30 seconds). Constructs were washed

between each treatment, incubated in fresh buffer, and allowed to return to

baseline. Pretreatment with the neuronal blocker tetrodotoxin (TTX; 1 μM) was

done to evaluate the relative contribution of neurons to EFS response. Nitric

oxide synthase (NOS) inhibitor Nω-Nitro-l-arginine methyl ester hydrochloride

(LNAME; 300 μM) was administered for 12 minutes prior to EFS stimulation to

inhibit nitrergic neurons. GraphPad Prism 7.00 software for Windows (GraphPad

Prism 7.00, San Diego, CA) was used to analyze collected data. Second-order

Savitsky–Golay smoothing was applied. Quantification of physiologic data was

performed relative to basal tone for contraction and relaxation as

maximum/minimum peak response (delta force of basal tone), area (magnitude *

time), and rate (linear regression from onset to peak response).

Statistical analysis

Data were expressed as mean ± SEM unless noted otherwise. Alpha was

set at p<.05. One-way ANOVA followed by Tukey’s test was used to compare all

36

groups. Only up to three t-tests were used to evaluate a priori hypotheses on the

groups. Normality was assessed by fisher skewness, and for ANOVA a Brown-

Forsythe test was used to ensure homogeneity of variance (Prism 7.00,

GraphPad Software, Inc., La Jolla, CA, USA).

RESULTS:

Microscopic and immunofluorescence evaluation of isolated ICC

Isolated GFP-ICC were grown on collagen-coated plates. ICC adhered

and proliferated. Microscopic images at day 7 showed normal star-like

morphology with multiple projections, and exhibited bright green fluorescence

under FITC filter (Figure 7, A). Expression of ICC functional channel Ano1 was

confirmed by a positive ANO1 stain on the cells, (Figure 7, B, red) indicating a

pure ICC population in the culture.

Figure 7

DAPI ANO1 Merge

A. B.

37

Incorporation of ICC into bioengineered pylorus

Pyloric constructs were bioengineered. Smooth muscle cells (SMC),

neural progenitor cells (NS), and interstitial cells of Cajal (ICC) were

incorporated, representing structures as described in the methods; i.e. constructs

with 1) SMC, 2) SMC and ICC, 3) SMC and NS, and 4) SMC, NS, and ICC. All

four types of gels containing the combinations contracted and formed dense

circular muscle tissues around the central post (Figure 8, A). At day 10,

microscopic evaluation of SMC + ICC constructs showed GFP expressing ICC

incorporated in the construct with evidence of ICC forming networks (Figure 8, B

and C).

Figure 8

A. B. C.

38

Physiology of bioengineered pylorus

Basal tone:

All four types of constructs established a spontaneous basal tone (Figure

9, A, before dashed line, representative). SMC constructs generated a basal tone

of 384.6 ± 31.58 µN, SMC + ICC generated 328.9 ± 5.26 µN, SMC + NS

generated 384.9 ± 39.9 µN, and SMC + NS + ICC generated 294.4 ± 14.21 µN.

There was no significant difference in the peak of tone established in all

constructs. Both the rates and area of tone establishment were also similar

(Figure 9, B, p>.05). Results for the maximum, duration, and rate of basal tone

are summarized in Table 1. Establishment of maximum basal tone was similar

among all tissues regardless of the composition of the constructs. This indicates

that the establishment of basal tone is purely myogenic, and is dependent only

on the presence of a functional smooth muscle population.

Figure 9

Contractile response to potassium chloride:

All constructs established baseline before any treatment. Addition of 60

mM potassium chloride resulted in a robust increase of the tone which reached a

Response to KCl

A. B.

C.

Spontaneous Basal Tone

39

plateau (Figure 9, A, after dashed line, representative). Maximal average

contraction in each type of construct was 382.3 ± 49.17 µN for SMC only, 320.5 ±

51.68 for SMC + ICC µN, 269.4 ± 61.28 for SMC + NS µN, and 342.1 ± 107.9 for

SMC + NS + ICC µN. This additional increase in tone in response to KCl was

similar between the different groups (Figure 9, C, p>.05). The area and the rate

of contraction were also similar (p>.05). Results for the maximum, duration, and

rate of KCl contractions are summarized in Table 1.

SMC SMC + ICC SMC + NS SMC + NS + ICC

Tone

n= 5 5 5 5

Maximum (µN)

384.6 ± 31.58 328.9 ± 5.26 384.9 ± 39.9 294.4 ± 14.21

Area (µN x s)

106865 ± 34581 86116 ± 16136 106683 ± 37681 82694 ± 16511

Rate (dF/dt)

1.327 ± 0.3487 1.25 ± 0.2737 1.394 ± 0.226 1.141 ± 0.3952

KCl

n= 4 4 4 4

Maximum (µN)

382.3 ± 49.17 320.5 ± 51.68 269.4 ± 61.28 342.1 ± 107.9

Area (µN x s)

110018 ± 19642 102614 ± 14492 69430 ± 21438 98870 ± 34947

Rate (dF/dt)

0.8524 ± 0.233 0.5191 ± 0.1221 0.5885 ± 0.2624 0.7227 ± 0.1523

Table 1

40

EFS-induced Relaxation of Engineered Tissues

After the constructs established baseline, EFS was applied (Figure 10, A-

D, dashed lines). Average smooth muscle relaxation response in the SMC group

was -94.83 ± 10.01 µN, SMC + ICC was -115.3 ± 23.37 µN, SMC + NS was -

170.1 ± 25.97 µN, and SMC + NS + ICC was -303.7 ± 40.37 µN (Figure 10, E) .

Full EFS analyses are summarized in Table 2. While all minimums were

recorded over 10 minutes, and there was no perceivable relaxation (a decrease

in force) in constructs without neural progenitor cells (SMC and SMC + ICC),

EFS did induce relaxation in the constructs that contained neural progenitor cells

(SMC + NS and SMC + NS + ICC groups). This suggests a necessity of the

neural component for relaxation.

Figure 10

To confirm the dependence of EFS-induced relaxation response on

innervation, neural-blocker TTX was incubated with tissues before EFS

stimulation. TTX abolished EFS-induced relaxation in constructs, bringing

minimum innervated responses similar to those without (SMC, SMC + ICC).

*

41

nNOS neuronal blocker L-NAME yielded similar inhibition, confirming there was a

large functional population of differentiated neurons that were NO donors (Figure

11, p>.05). These results suggest that a differentiated population of nNOS

donated neurons, which were the driving force in the EFS response.

Figure 11

The innervated constructs (SMC +NS and SMC + NS + ICC) responded

with relaxation and returned to stable baseline. Further analysis of relaxation

responses revealed SMC + NS + ICC constructs had the most prominent

relaxation versus SMC + NS alone (Figure 4, E, 1.9x SMC + NS group, p<.05),

suggesting a role of ICC by magnifying EFS-induced relaxation.

42

Figure 12

To investigate the role of ICC on relaxation, rate and area of relaxation

were examined on the innervated groups only. An average of all relaxation

responses over time was computed and graphed to investigate the specific

cellular contributions to relaxation (Figure 12, A, traces, n=5). Here, the shape of

the response is similar- containing distinct relaxation with an effect ending at ten

minutes. However, the rate at which innervated tissues relaxed was markedly

increased with ICC (-0.27 ± 0.08 µN /s for SMC + NS and -0.64 ± 0.20 µN/s for

SMC + NS + ICC peak, figure 6, C, p=.12). Area of the responses remained

increased in similar fashion to the peak response (1.7x, SMC + NS: 49918 ±

14570 µNxs versus SMC + NS + ICC: 82639 ± 23594 µNxs, p=.30). The

difference between the onset rate and duration are represented graphically: The

SMC + NS + ICC tissues have faster rates, are time advanced, and have greater

magnitudes at virtually all time points. In summary, the addition of ICC created

A.

B.

C.

43

neuromuscular tissues which relaxed to a greater extent and reached peak

values at a faster rate.

The average response to nNOS blocker L-NAME was computed, graphed,

and compared to the corresponding EFS response (Figure 7, A and B, dashed

lines). L-NAME inhibition completely abolished relaxation in both construct types.

Summarizing, nNOS neurons were responsible to initiate most prominent

relaxation. ICC transduced inputs, including NO to increase relaxation throughout

the entirity of the response.

44

Figure 13

SMC SMC + ICC SMC + NS SMC + NS + ICC

EFS

n= 4 4 5 5

Minimum (µN)

-94.83 ± 10.01 -115.3 ± 23.37 -170.1 ± 25.97 -303.7 ± 40.37

Area (µN x s)

12905 ± 7733 24274 ± 8888 49918 ± 14570 82639 ± 23594

Rate (dF/dt)

0.26 ± 0.23 -0.09 ± 0.06 -0.27 ± 0.08 -0.64 ± 0.20

TTX EFS

n= 3 3 3 3

Minimum (µN)

-77.24 ± 14.22 -65.00 ± 26.00 -111.7 ± 33.17 -93.4 ± 16.5

L-NAME EFS

n= 3 3 3 3

Minimum (µN)

-58.59 ± 29.32 -60.00 ± 4.00 -50.11 ± 18.17 -72.92 ± 36.11

Table 2

A.

B.

45

DISCUSSION:

In this study, we provide a tissue engineering approach to model clinically

relevant cell depletions in engineered neuromuscular pylorus tissues using

smooth muscle, enteric nerves, and interstitial cells of Cajal. As previously

mentioned, the pylorus is a sphincteric tissue which controls trituration periods

and facilitates the passage of foods to the duodenum from the stomach. The

engineering technique used in this study allowed the formation of pyloric

constructs with different cell combinations for physiological studies. All cell types

contributed significantly to functional changes. Physiology of the engineered

sphincters demonstrated that capacity to establish tone was myogenic, since all

groups results were similar. Prominent relaxation was dependent on the

presence of NO-donating neurons, and the addition of ICC enhanced this neural

mediated relaxation in both the onset rate and peak response.

The first objective of this study was to create and validate a bioengineered

pylorus model similar to those in previous studies, in order to build additional

cells onto it. Following electric field stimulation, sphincters that were innervated

relaxed significantly more than those engineered only with smooth muscle. This

response could also be blocked by TTX and L-NAME, indicating that the base

model by Rego et al. in 2016 was both repeatable and functioned similarly.

Responses were also within magnitudes and shape of similarly sized ex vivo

tissues.

At the same time, we isolated and characterized ICC positive for Ano1.

Engineered ICC could be visualized in the constructs forming dense networks on

46

the day of physiology. These results indicate that the engineered constructs are

feasible, and could be used as representative models for pyloric tissues.

Conveniently, relaxations were initiated from similar basal tone, which reduces

error that can be introduced when studying changes in physiological responses.

ICC were involved in increasing rate and peak components of neural relaxation.

This indicates that relaxation is a fully incorporated mechanism and any change

in these populations creates a measurable change in function. Therefore, the

results of this model can be used to understand what may happen to a tissue as

a result of a complete ICC or neuron depletion.

Many pyloric diseases are characterized by an inability for pyloric

relaxation, such as pylorospasm or diabetic neuropathies, and are associated

with abnormal manometric data and gastroparesis (Camilleri, 2016). Studying

persistent relaxation in ICC knockout tissues has led to the dismissal of the role

of ICC in muscle function (Huizinga et al., 2008). The results of the present

investigation confirm that while these functions indeed persist, the addition of ICC

change the magnitude of the relaxation response. The magnitude of relaxation

may be an important mechanism for the pylorus to expedite gastric emptying.

Thus, the ‘persistence’ of responses may not be physiologically relevant, and the

character of these responses should be investigated further.

Previous research and models rely on the use of inhibitors, knockouts, or

toxins to produce quantitative observations on tissue function (Cobine et al.,

2017; Fujimura et al., 2016; Hwang, Basma, Sanders, & Ward, 2016). While

these may be useful tools, they lack specificity and enhanced control over tissue

47

cellular compositions. This contributes to the controversy regarding the role of

cells in tissue function. The in vitro model of the present study allows for

complete or partial depletion of cell populations, and is also capable of matching

and modeling clinically relevant conditions unique to patient populations.

This study provides a promising model to alter and test GI

pathophysiology, but there are several limitations that need to be considered in

the future. The data confirms that the ICC were positive for Ano1 when isolated

and cultured. Yet, the different connectivities between cells in the constructs

were not assessed. Also, there was no change in the establishment of basal

tone, but there may be cellular contributions to the maintenance of this tone. A

key attribute of ICC is the generation and transduction of electrical signals, and

this was not addressed in this study. Further investigations should examine

electrical phenomena in these tissues. Nevertheless, this study provides proof

that the engineered neuromuscular constructs may provide a basis for modeling

and researching pathophysiology resulting from cell depletions.

In conclusion, this tissue engineering approach has the potential to

generate further advanced research surrounding gastrointestinal

pathophysiology, by allowing cell types associated with disease to be

quantitatively depleted and replenished. The present investigation is the first of

its kind to demonstrate physiology of gut-derived neuromuscular tissues

engineered with ICC. Future research characterizing other qualitative and

quantitative changes, by adding, altering, or partially depleting cells should be

performed using this promising model.

48

ACKNOWLEDGMENTS:

This work was supported by Wake Forest School of Medicine Institutional

Funds. The authors would like to thank Dr. Frank Marini at Wake Forest School

of Medicine for providing the GFP mice.

49

REFERENCES:

Angeli, T. R., Cheng, L. K., Du, P., Wang, T. H.-H., Bernard, C. E., Vannucchi, M.-G., . . . O’Grady, G. (2015). Loss of Interstitial Cells of Cajal and Patterns of Gastric Dysrhythmia in Patients With Chronic Unexplained Nausea and Vomiting. Gastroenterology, 149(1), 56-66.e55. doi:http://dx.doi.org/10.1053/j.gastro.2015.04.003

Bielefeldt, K. (2012). Gastroparesis: Concepts, Controversies, and Challenges. Scientifica, 2012, 19. doi:10.6064/2012/424802

Camilleri, M. (2016). Novel Diet, Drugs, and Gastric Interventions for Gastroparesis. Clinical Gastroenterology and Hepatology, 14(8), 1072-1080. doi:http://dx.doi.org/10.1016/j.cgh.2015.12.033

Chaudhury, A. (2016). Furthering the debate on the role of interstitial cells of Cajal in enteric inhibitory neuromuscular neurotransmission. American Journal of Physiology - Cell Physiology, 311(3), C479-C481. doi:10.1152/ajpcell.00067.2016

Cobine, C. A., Hannah, E. E., Zhu, M. H., Lyle, H. E., Rock, J. R., Sanders, K. M., . . . Keef, K. D. (2017). ANO1 in intramuscular interstitial cells of Cajal plays a key role in the generation of slow waves and tone in the internal anal sphincter. The Journal of Physiology, n/a-n/a. doi:10.1113/JP273618

Fujimura, T., Shibata, S., Shimojima, N., Morikawa, Y., Okano, H., & Kuroda, T. (2016). Fluorescence visualization of the enteric nervous network in a chemically induced aganglionosis model. PloS one, 11(3), e0150579.

Gilmont, R. R., Raghavan, S., Somara, S., & Bitar, K. N. (2014). Bioengineering of physiologically functional intrinsically innervated human internal anal sphincter constructs. Tissue Engineering Part A, 20(11-12), 1603-1611.

Goldstein, A. M., Thapar, N., Karunaratne, T. B., & De Giorgio, R. (2016). Clinical aspects of neurointestinal disease: Pathophysiology, diagnosis, and treatment. Developmental Biology, 417(2), 217-228. doi:http://dx.doi.org/10.1016/j.ydbio.2016.03.032

Grover, M., Farrugia, G., Lurken, M. S., Bernard, C. E., Faussone–Pellegrini, M. S., Smyrk, T. C., . . . Pasricha, P. J. (2011). Cellular Changes in Diabetic and Idiopathic Gastroparesis. Gastroenterology, 140(5), 1575-1585.e1578. doi:http://dx.doi.org/10.1053/j.gastro.2011.01.046

Huizinga, J. D., Liu, L. W., Fitzpatrick, A., White, E., Gill, S., Wang, X.-Y., . . . Starret, T. (2008). Deficiency of intramuscular ICC increases fundic muscle excitability but does not impede nitrergic innervation. American Journal of Physiology-Gastrointestinal and Liver Physiology, 294(2), G589-G594.

Hwang, S. J., Basma, N., Sanders, K. M., & Ward, S. M. (2016). Effects of new‐generation inhibitors of the calcium‐activated chloride channel anoctamin 1 on slow waves in the gastrointestinal tract. British journal of pharmacology.

Lies, B., Gil, V., Groneberg, D., Seidler, B., Saur, D., Wischmeyer, E., . . . Friebe, A. (2014). Interstitial cells of Cajal mediate nitrergic inhibitory neurotransmission in the murine gastrointestinal tract. American Journal of Physiology - Gastrointestinal and Liver Physiology, 307(1), G98-G106. doi:10.1152/ajpgi.00082.2014

Mashimo, H., Kjellin, A., & Goyal, R. K. (2000). Gastric stasis in neuronal nitric oxide synthase–deficient knockout mice. Gastroenterology, 119(3), 766-773.

Moraveji, S., Bashashati, M., Elhanafi, S., Sunny, J., Sarosiek, I., Davis, B., . . . McCallum, R. (2016). Depleted interstitial cells of Cajal and fibrosis in the pylorus: novel features of gastroparesis. Neurogastroenterology & Motility.

50

O'Grady, G., Du, P., Cheng, L. K., Egbuji, J. U., Lammers, W. J. E. P., Windsor, J. A., & Pullan, A. J. (2010). Origin and propagation of human gastric slow-wave activity defined by high-resolution mapping. American Journal of Physiology - Gastrointestinal and Liver Physiology, 299(3), G585-G592. doi:10.1152/ajpgi.00125.2010

Raghavan, S., Gilmont, R. R., & Bitar, K. N. (2013). Neuroglial differentiation of adult enteric neuronal progenitor cells as a function of extracellular matrix composition. Biomaterials, 34(28), 6649-6658. doi:http://dx.doi.org/10.1016/j.biomaterials.2013.05.023

Raghavan, S., Gilmont, R. R., Miyasaka, E. A., Somara, S., Srinivasan, S., Teitelbaum, D. H., & Bitar, K. N. (2011). Successful implantation of bioengineered, intrinsically innervated, human internal anal sphincter. Gastroenterology, 141(1), 310-319.

Raghavan, S., Miyasaka, E. A., Gilmont, R. R., Somara, S., Teitelbaum, D. H., & Bitar, K. N. (2014). Perianal implantation of bioengineered human internal anal sphincter constructs intrinsically innervated with human neural progenitor cells. Surgery, 155(4), 668-674.

Rego, S. L., Zakhem, E., Orlando, G., & Bitar, K. N. (2015). Bioengineered human pyloric sphincters using autologous smooth muscle and neural progenitor cells. Tissue Engineering Part A, 22(1-2), 151-160.

Sanders, K. M., Hwang, S. J., & Ward, S. M. (2010). Neuroeffector apparatus in gastrointestinal smooth muscle organs. The Journal of Physiology, 588(23), 4621-4639. doi:10.1113/jphysiol.2010.196030

Sanders, K. M., Kito, Y., Hwang, S. J., & Ward, S. M. (2016). Regulation of Gastrointestinal Smooth Muscle Function by Interstitial Cells. Physiology, 31(5), 316-326.

Sivarao, D. V., Mashimo, H., & Goyal, R. K. (2008). Pyloric sphincter dysfunction in nNOS−/− and W/W V mutant mice: animal models of gastroparesis and duodenogastric reflux. Gastroenterology, 135(4), 1258-1266.

Wagner, J. P., Sullins, V. F., & Dunn, J. C. Y. (2014). A novel in vivo model of permanent intestinal aganglionosis. Journal of Surgical Research, 192(1), 27-33. doi:http://dx.doi.org/10.1016/j.jss.2014.06.010

Wood, J. D. (2016). Enteric Nervous System: Neuropathic Gastrointestinal Motility. Digestive Diseases and Sciences, 61(7), 1803-1816. doi:10.1007/s10620-016-4183-5

Zarate, N., Mearin, F., Wang, X., Hewlett, B., Huizinga, J., & Malagelada, J. (2003). Severe idiopathic gastroparesis due to neuronal and interstitial cells of Cajal degeneration: pathological findings and management. Gut, 52(7), 966-970.

Zhu, M. H., Kim, T. W., Ro, S., Yan, W., Ward, S. M., Koh, S. D., & Sanders, K. M. (2009). A Ca2+-activated Cl− conductance in interstitial cells of Cajal linked to slow wave currents and pacemaker activity. The Journal of Physiology, 587(20), 4905-4918. doi:10.1113/jphysiol.2009.176206

51

CHAPTER III: ELECTROPHYSIOLOGY OF AN IN

VITRO MODEL OF THE GASTRIC NEUROMUSCULAR

APPARATUS

Dylan T. Knutson1,2, B.S., Elie Zakhem2, Kenneth L. Koch, M.D.3, Ph.D, Khalil N. Bitar1,2,3, Ph.D., AGAF.

1Virginia Tech-Wake Forest School of Biomedical Engineering and Sciences, Winston Salem, NC

2Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston

Salem, NC

3Section on Gastroenterology, Wake Forest School of Medicine, Winston-Salem, NC, USA

This chapter describes the validation and electrophysiological analysis of engineered neuromuscular tissues

52

ABSTRACT

INTRODUCTION

Neuro-muscular disorders of the gut can result from the depletion of

neurons and/or interstitial cells of Cajal (ICC) populations. There is a lack of

controlled in vitro models to study the controversial contribution of these cellular

populations to gastrointestinal physiology.

OBJECTIVE

The aims of this study were to develop a measurement method to record

force and physiology simultaneously, and to study responses from an in vitro

model of the pyloric neuromuscular apparatus.

METHODOLOGY

Recordings were made using silver coated wire in an organ bath. Neuro-

muscular tissues were engineered using different combinations: (1) SMC, (2)

SMC + ICC, (3) SMC + NPC, and (4) SMC + ICC + NPC. Engineered tissues

were characterized by force and electrical activity.

RESULTS

The recording methodology was validated using a barrage of tests.

Tissues containing ICC had higher frequency (A, 6.0 ± .34 Cpm) and amplitude

(B, 1029 ± 65 µV) at baseline compared to tissues without ICC (frequency; 2.0 ±

.34 Cpm and amplitude; 425 ± 8.3 µV). The increase was significant (p<.05,

53

n=2). ICC tissues had more precise rhythm, and EFS prompted further decrease

in rhythmic frequency and increase in rhythmic precision.

CONCLUSION

This study was the first to demonstrate simultaneous electrical and force

recordings of a neuro-muscular apparatus that incorporated the three major cells

(smooth muscle, enteric neurons, and ICC). These results show that ICC play a

direct role in generating electrical phenomena and transducing neural relaxation

to muscle.

Keywords: Gastroparesis, Pylorus, Diabetes, Bioengineering, Interstitial Cells of

Cajal (ICC), Disease Models

54

INTRODUCTION

A hallmark symptom of gastroparesis is abnormal pacemaker activity in

the stomach, detected by electrogastrogram (EGG) (Koch & Stern, 2004). Phasic

muscular patterns are driven by electrical activity that is propagated by interstitial

cells of Cajal (ICC), which originate in the greater curvature of the stomach and

diminish after the pylorus (O'Grady et al., 2010). It has been shown that

depletions of ICC and/or enteric neurons are associated with clinical cases of

gastroparesis (Bashashati & McCallum, 2015; Penagini, 1998). Patients may

have bradygastria (slowing of rhythms), tachygastria (increased rhythm), or an

absence of activity altogether (Penagini, 1998). More recent studies have

mapped altered waveform patterns, and shown them to be associated with both

clinical symptoms and the extent of ICC depletions in stomach tissues (Angeli et

al., 2015). Furthermore, the specific location of cell depletions is associated with

different symptoms and disease phenotypes (Moraveji et al., 2016). As

previously explained, ICC may play a role in transducing nitrergic signaling in

order to boost sphincteric relaxation in the pylorus model. However, the

aforementioned study did not consider pacemaker activity.

In this continuation, for the first time, electrical phenomena of engineered

tissue with ICC were examined. The first goal of this study was to validate that

true signals were being received. The second goal was to evaluate the

contribution of each cell type to electrical activity, and then investigate changes

that may occur during responses of interest. The data showed that recorded

electrical activity was indeed true, and ICC were the main contributor to electrical

55

phenomena at baseline. When treated with EFS, ICC rhythm decreased in

frequency in order to facilitate a relaxation. This study provides a promising

foundation for understanding comprehensive physiology of GI tissues.

MATERIALS AND METHODS

Isolation of cells:

Primary Smooth Muscle Cells (SMC)

Human tissues were ethically obtained from organ donors through

Carolina Donor Services and Wake Forest Baptist Medical Center (IRB#:

IRB00007586). Smooth Muscle cells were isolated as previously described

(Rego, Zakhem, Orlando, & Bitar, 2015). Briefly, human pylori were removed by

sharp dissection. Pylorus tissues were manually cleaned by removing fat and

mucosa with a surgical blade. Tissues were extensively washed with HBSS

solution containing 2X antibiotics/antimycotic and then minced in sterile

conditions. Tissues were subjected to two digestions with HBSS containing 1

mg/mL collagenase type II (Worthington Biochemicals, Lakewood, NJ) at 37°C

for 1 hour each. Tissue pellets were then resuspended in SMC growth media and

plated in tissue culture dishes at 37°C with 5% CO2.

Neural Progenitor Cells (NS)

Neural progenitors were isolated as previously described (Raghavan,

Gilmont, & Bitar, 2013). Briefly, duodenum from rats was harvested and

extensively washed with HBSS containing 2X antibiotics/antimycotic, followed by

mincing and additional washing. Minced tissue was subjected to two digestions in

a mixture containing 0.85 mg/ml type II Collagenase, and 0.85 mg/ml Dispase II

56

with 40 µg/ml DNAase I at 37oC for 45-60 minutes. The cells were then passed

through a 70 µm nylon cell strainer and then through a 40 µm nylon cell strainer.

Cells were plated in Neurobasal Medium containing 1X N2 supplement, 20 ng/ml

recombinant human Epidermal Growth Factor, 20 ng/ml recombinant basic

Fibroblast Growth Factor, and 1.0mM L-glutamine and 1X antibiotics/antimycotic.

Primary ICC

GFP-expressing mice were kindly provided by Dr. Frank C. Marini at

Wake Forest School of Medicine. GFP-ICC were isolated as previously described

(Zhu et al., 2009). Briefly, small intestine was harvested and washed extensively

with sterile HBSS containing 2X antibiotics/antimycotic in sterile conditions. After

mincing, tissues were again washed with HBSS three times. Tissues were

digested in HBSS containing 1.3 mg/mL collagenase type II (Worthington

Biochemicals), 2 mg/mL bovine serum albumin, 2 mg/mL trypsin inhibitor, and

0.27 mg/mL Adenosine triphosphate (Sigma-Aldrich, St. Louis, MO) for 23

minutes at 37°C . After digestion, tissue pellets were centrifuged at 600 g for 5

minutes and washed three more times. Pellets were then resuspended in SMC

growth media containing 0.01 µg/mL human stem cell factor (Peprotech, Rocky

Hill, NJ) and plated on 2.5 µg/mL collagen coated dishes at 37°C with 5% CO2.

Bioengineered in vitro models of Pylorus

The process of engineering the sphincters was described previously

(Gilmont, Raghavan, Somara, & Bitar, 2014; Raghavan et al., 2011; Raghavan et

al., 2014). For all sphincters, 35 mm dishes were coated with sylgard. Eight

millimeter cylindrical sylgard posts were placed in the center of each plate

57

followed by sterilization using ethanol and UV. Four types of sphincters were

engineered using different combinations of cells. Combinations included the

following and are described below: 1) SMC only, 2) SMC + ICC, 3) SMC + NS,

and 4) SMC + NS + ICC.

SMC only

Smooth muscle cells were trypsinized and 500,000 cells were collected

per construct. Cells were resuspended in a collagen gel of 0.4 mg/ml final

concentration. The mixture was then poured in the culture dish around the post.

The muscle gel was left to gel at 37ºC and then supplemented with differentiation

media.

SMC + ICC

Smooth muscle cells (500,000 per construct) and ICC (75,000 per

construct) were obtained and resuspended in collagen gel mixture with a final

concentration of 0.4 mg/ml. The mixture was left to gel 37ºC and then

supplemented with differentiation media.

SMC + NS

Neural progenitor cells (NS) were dissociated with Accutase (Invitrogen,

Carlsbad, CA) and 200,000 cells per construct were obtained. The cells were re-

suspended in a solution with a final concentration of 0.4mg/ml type I rat tail

Collagen (BD Biosciences) and 10ug/ml mouse laminin (Invitrogen, Carlsbad,

CA). The mixture was poured on the prepared Sylgard culture dishes. The

mixture was allowed to gel at 37ºC. Smooth muscle cells were then prepared by

58

collecting 500,000 cells and mixing in the collagen gel mixture. The smooth

muscle mixture was poured on top of the first NS gel layer and allowed to gel at

37C. Following gelation of both layers, differentiation media was supplemented.

SMC + NS + ICC

Neural progenitor cells (NS, 200,000 cells per construct) were obtained

and re-suspended in a collagen/laminin solution as described above. The mixture

was poured on the prepared culture dishes coated with Sylgard and allowed to

gel. A total of 500,000 smooth muscle cells and 75,000 ICC per construct were

obtained and suspended in a collagen mixture as described above. The mixture

was overlaid on top of the first NS layer and allowed to gel followed by the

addition of differentiation media.

Microscopic evaluation of the constructs:

Constructs were evaluated microscopically prior to physiological testing on

day 10 of culture. Constructs were imaged under FITC filter to visualize GFP-

expressing ICCs. Images were processed using NIS Elements software (Nikon

Instruments, Melville, NY).

Physiological analysis of bioengineered pylorus

All different combinations of engineered pyloric constructs were analyzed

for physiological functionality. Constructs were hooked in an organ bath between

a fixed arm, and the measuring arm of an isometric, magnetoresistive force

transducer in a horizontal tissue bath (F10; Harvard Apparatus, Holliston, MA).

Force and electrical data was acquired using LabChart 7 software

59

(ADInstruments, Colorado Springs, CO). Electrical activity was recorded

simultaneously in bath using electrode fixture using 3 silver coated wires, with a

differential amplifier. The electrode sat in a fixed position, to prevent changes in

amplitudes between tissues. Band Pass filters were applied: High pass: 0.03 Hz

and Low pass: 3 Hz, sampled at 2kHz.Constructs were maintained in 4-(2-

hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered solution at 37°C

throughout all experiments. All studies were performed after the establishment of

stable basal tone. Force and electrical phenomena were evaluated following the

addition of potassium chloride (KCl; 60 mM), and electrical field stimulation (EFS;

5 Hz, 0.5 ms, for 30 seconds). Constructs were washed between each treatment,

incubated in fresh buffer and allowed to return to baseline. Pretreatment with the

nifedipine (100 μM) was done to evaluate the risk of false signals. GraphPad

Prism 7.00 software for Windows (GraphPad Prism 7.00, San Diego, CA) was

used to analyze collected data. Second-order Savitsky–Golay smoothing was

applied. Quantification of physiologic data was performed relative to basal tone

for contraction and relaxation as maximum/minimum peak response (Delta Force

of basal tone), area (magnitude * time), and rate (linear regression from onset to

peak response).

Statistical analysis:

Data were expressed as mean ± SEM unless noted otherwise. Alpha was

set at p=.05. One-way ANOVA followed by Tukey’s test was used to compare all

groups. Only up to 3 t-tests were used to evaluate a priori hypotheses on the

groups. Normality was assessed by fisher skewness, and for ANOVA a Brown-

60

Forsythe test was used to ensure homogeneity of variance (Prism 7.00,

GraphPad Software, Inc., La Jolla, CA, USA).

RESULTS:

Validation of Electrical Signals

Figure 14

Silver coated wire leads were placed for detecting a differential current on

the engineered tissue within the organ bath (Figure 14). They remained fixed to

the bath for the following experiments.

61

Figure 15

First, a series of phenomena were recorded and documented, which was

unrelated to tissues within the bath. Raw recordings from force transducer

(Figure 15, B, blue trace) and/or electrodes (voltage, mV, green) of within tissue

bath were recorded simultaneously. It was necessary for the electrodes to

remain in the bath, otherwise a differential current could not be achieved

(arrowed, A) through tissue. Voltage pulses from EFS (5V, 5Hz, 0.5ms) were

detected upon stimulation (B). Without tissue or electrical activites, the liquid

reading from electrodes remained approximately 0 volts (C).

A. B. C.

62

Figure 16

Some disturbances within the bath could be perceived via the electrodes.

Recordings from electrodes of SMC + ICC + NS within the tissue bath (Figure 16,

mV, green). Perturbances of either washing (A, after dashed line) or adding

reagents (B, Highlighted) could obscure recordings for a short period. Washing

increased perceived voltage during and for several seconds following. Adding

reagents occasionally resulted in a voltage drop. Tests or analyses and were

performed following or by removing these false signals.

A.

B.

63

Figure 17

To fully validate and isolate any false recordings, a sequence of tests was

performed in a bath with electrodes with and without tissues (Figure 17). Since

the contractility of the tissues was dependent only on muscle, SMC Only tissue

was prepared for the series. First, perturbances of either washing or adding

reagents (1, after dashed line, carrier only) were recorded. This was done using

only buffer. Only small perturbances were recorded. Next, KCl was added, and

there was a tremendous increase in electrical signals (2 and 3, dashed line). This

test was repeated. Finally, the activity was blocked by nifedipine, confirming the

electrical depolarization was blocked (4, dashed lines). Finally, the same tests

were performed without any tissue in the same bath (5 and 6). No activity was

recorded, except for a small, short 2 mV peak upon the addition of KCl. This

64

barrage confirmed that the electrical signals were true, and that the electrical

signals were coming from functioning muscle cells in engineered tissue.

Physiology of bioengineered pylorus

Basal Tone:

Figure 18

We bioengineered pyloric constructs smooth muscle (SMC), neural

progenitor cells (NS), and interstitial cells of Cajal (ICC). The constructs are

previously described in the methods; i.e. constructs with 1) SMC, 2) SMC and

ICC, 3) SMC and NS, and 4) SMC, NS, and ICC. First, raw simultaneous

recordings of force (Figure 18, blue trace) and voltage (green trace) of

engineered tissues within tissue bath were recorded. Both graphs show

establishment of basal tone, with expected electrical spikes (arrowed, green

trace) preceeding contractions (arrowed, blue trace).

All four types of constructs established a spontaneous basal tone (Figure

3, A, before dashed line, representative). In review, SMC constructs generated a

basal tone of 384.6 ± 31.58 µN, SMC + ICC generated 328.9 ± 5.26 µN, SMC +

*

* *

* A.

B.

SMC + NS SMCC

SMC + ICC SMC + NS + ICC 1 min

2 m

V

65

NS generated 384.9 ± 39.9 µN, and SMC + NS + ICC generated 294.4 ± 14.21

µN. There was no significant difference in the peak of tone, area, and rate

established in all constructs. Establishment of maximum basal tone was similar

among all tissues regardless of the composition of the constructs. This indicates

that the establishment of basal tone was purely myogenic, and was dependent

only on the presence of a functional smooth muscle population.

However, there was a difference in the electrical activity of the tissues.

Tissues containing ICC had higher frequency (A, 6.0 ± .34 Cpm) and amplitude

(B, 1029 ± 65 µV) at baseline compared to tissues without ICC (frequency; 2.0 ±

.34 Cpm and amplitude; 425 ± 8.3 µV). The increase was significant (p<.05,

n=2). This change resulted in voltages with continuous rhythmic peaks each

minute. Therefore the ICC were generating electrical (pacemaker) activity within

the gastric tissue.

EFS-induced Relaxation of Engineered Tissues

Next, further investigation was made of the changes resulting from EFS.

Raw simultaneous recordings of engineered neuromuscular tissues from the EFS

response within the tissue bath were made. As explained in the previous chapter,

EFS did induce relaxation in the constructs that contained neural progenitor cells

(SMC + NS and SMC + NS + ICC groups). This suggests a necessity of the

neural component for relaxation. The relaxation of SMC + NS was -170.1 ± 25.97

µN, and of SMC + NS + ICC was -303.7 ± 40.37 µN.

66

After the constructs established baseline, EFS was applied (Figure 4, A-D,

dashed lines). EFS did induce relaxation in the constructs that contained neural

progenitor cells (SMC + NS and SMC + NS + ICC groups). The results in the

previous chapter confirmed a necessity of the NO donating neural component for

relaxation using inhibitors. Further analysis of relaxation responses revealed

SMC + NS + ICC constructs had the most prominent relaxation versus SMC +

NS alone (Figure 10, E, 1.9x SMC + NS group, p<.05). This suggests that ICC

play a role in relaxation by magnifying EFS-induced relaxation. The SMC + NS +

ICC tissues had faster rates, were time advanced, and had greater magnitudes

at virtually all time points. In summary, the addition of ICC created

neuromuscular tissues which relaxed further and reached peak values at a faster

rate.

67

Figure 19

Electrical Activity was recorded following EFS simultaneously with force

(Figure 19, n=2 per group). In the raw graphs, are two tissues tested on the same

day. scales (force and voltage) are the same. The EFS relaxation response is

greater in rate and magnitude in ICC tissues. In regards to Electrical Changes:

SMC + NS increased peak activities following EFS, then there was a drop-off in

activity. In SMC + NS + ICC, a slowing of rhythm occurs, and then rhythm is

SMC + NS + ICC

SMC + NS

68

maintained. Upon the release of NO, reduction of ICC slow waves may occur in

order to facilitate relaxation from a tonic state.

Following EFS stimulation, a plateau of activities occurs in SMC + ICC

+NS tissue. When comparing it to the control SMC +NS tissue, this .5 mV

plateau can be attributed to the resultant of both the 4-5 CPM, 1 mV signal

recorded following EFS, and the 4-6 CPM, 1 mV ICC signal recorded. Therefore,

each signal does involve the initiation of a neural component, but then the ICC

continue with a slowed, cyclic signal that occurs throughout the duration of

relaxation. This data confirms ICC transduced inputs, including NO to increase

relaxation throughout the entirity of the response, and perhaps became coupled

with muscle to facilitate a coordinated reduction in tone.

69

To confirm these qualitative observations, running spectral analysis was

performed. This analysis shows the changes and powers of different frequencies

over time.

Figure 20

Spectral analyses graphs of electrical Activity of both SMC + NS and SMC

+ NS + ICC tissues (figure 7, A). Strong frequencies are highlighted in bright

colors. Dense blue is indicative of a signal which has more rhythm. A 4 minute

segment was analyzed for spectral density(shaded), and is shown in graph A on

top. The dominant frequencies of these 4 minute segments before and after were

graphed (B).

There was a decrease in frequency in only ICC tissues following EFS from

3.7 cpm to 2.22 cpm. This lage decrease was also accompanied by a prominent

SMC + NS

SMC + NS + ICC

A. B.

1. 2.

70

increase in rhythm (2). This change was not present in SMC + NS only tissues,

who had a continuous, and abberrant signal. At any point, the dominant

frequency of these tissues was .72 CPM. There was no change before or after

EFS. This is summarized in figure 7, graph B.

There was also higher frequncy, low rhythm band in both tissues following

EFS, which could be attributed to Neural Activity (1). This activity is present in

both signals. Since we know that EFS caused no response in muscle only

tissues, and was present in both of the tissues incorporating neurons, then it is

suitable to attribute this activity to a neural component

71

DISCUSSION

The present investigation provides insight on a tissue engineering model

of clinically relevant cell depletions in neuromuscular pylorus tissues using

smooth muscle, enteric nerves, and interstitial cells of Cajal. The pylorus is a

sphincteric tissue which controls trituration periods and facilitates the passage of

foods to the duodenum from the stomach. The acquisition of electrical signals

within the organ bath yielded results that were valid, containing minimal false

recordings. In the prior study, physiology of the engineered sphincters

demonstrated that the capacity to establish tone was myogenic, and prominent

relaxation was dependant on the presence of NO-donating neurons. Also, the

addition of ICC enhanced this neural mediated relaxation in both the onset rate

and peak response.

The first aim of this study was to create and validate a method for

measuring electrical phenomena within the organ bath. After identifying

perturbances in signals, isolation of signals from tissues versus the environment

could reliably be recorded, observed, and analyzed.

Next, both electrical and force phenomena of the engineered tissues were

simultaneously measured. These results indicate that the engineered constructs

exhibited increased cyclic peaks and increased amplitudes, characteristic of the

pacemaker activity of both in vivo and cultured ICC. It is known that ICC play a

role in increasing rate and peak components of neural relaxation. This indicates

that relaxation is a fully comprehensive mechanism, and any change in these

populations creates a measurable change in function. However, when examining

72

the force and electrical phenomena simultaneously, a decrease in the cyclic

pacemaker pattern following the depolarization of the neural component by EFS

occurs.

It is well known that ICC and smooth muscle cells are electrically coupled

(Daniel & Wang, 1999). Though we found previously establishment of tone was

myogenic, maintenance of tone or changes in tone may depend on this

interaction. ICC contain guanylate cyclase, which is involved in a muscle

relaxation cascade in response to reception of NO (Groneberg, Aue, Lies, &

Friebe, 2015). The slowing of cycles from ICC may coordinate the relaxation of

muscle, through electrical coupling. A greater amount of muscle is hypopolarized

simultaneously, and the tissue relaxes at a greater magnitude and faster rate

(Furness & Costa, 1987).

Many pyloric diseases are characterized by an inability of pyloric

relaxation, such as pylorospasm or diabetic neuropathies, and are associated

with abnormal manometric data and gastroparesis (Camilleri, 2016). Studying

persistent relaxation in ICC knockouts tissues has led to the dismissal of the role

of ICC in muscle function (Huizinga et al., 2008). The present investigation

confirms that while these functions indeed persist, the addition of ICC change the

magnitude of the relaxation response. The magnitude of relaxation may be an

important mechanism for the pylorus to expedite gastric emptying. Therefore, the

‘persistence’ of responses may not be physiologically relevant, and the character

of these responses should be further investigated.

73

The innovation of the in vitro model used in the present study allows for

complete or partial depletion of cell populations. In addition, it allows for

controlled phenomena, which makes it possible to make further advancements in

the study of physiological responses. This model also makes it possible to

simulate clinically relevant conditions unique to patient populations. This

investigation is the first of its kind to comprehensively examine the contribution of

each key cell type, in both force and electrical phenomena.

Furthermore, even though this study provides a promising new model to

alter and test GI pathophysiology, there are several limitations that need to be

considered in future research. The data from this study confirm that the ICC were

likely responsible for increasing the electrical activity. Yet, there are currently no

suitable inhibitors available for entirely providing additional controls for this

contribution of the ICC without affecting other components. In this study, only

EFS of a NO donating (nNOS) neuron population was examined. There are

several other neural components in GI tissue including cholinergic, VIPergic, and

nicotinic neurons and receptors (Furness & Costa, 1987). Controlling the

differentiation of other neural populations could provide other models to study.

The paucity of data regarding cellular contributions to tissue function could

also arise from the complicated process of recording physiological phenomena.

As analysis becomes more in-depth, the modalities, expense, and skill required

become greater. Newer methods to create tissue engineered controls and record

phenomena must be produced. Nonetheless, this study provides proof that

74

engineered neuromuscular constructs could provide the basis for modeling and

studying pathophysiology resulting from cell depletions.

In conclusion, this tissue engineering approach has the potential to

generate and test unexplored hypotheses surrounding gastrointestinal

pathophysiology, by quantitatively depleting and replenishing cell types

associated with disease. This study is the first to demonstrate physiology of gut-

derived neuromuscular tissues engineered with ICC. Future studies should use

this promising model to characterize other qualitative and quantitative changes,

by adding, altering, or partially depleting cells.

ACKNOWLEDGMENTS:

This work was supported by Wake Forest School of Medicine Institutional

Funds.

REFERENCES:

Angeli, T. R., Cheng, L. K., Du, P., Wang, T. H.-H., Bernard, C. E., Vannucchi, M.-G., . . . O’Grady, G. (2015). Loss of Interstitial Cells of Cajal and Patterns of Gastric Dysrhythmia in Patients With Chronic Unexplained Nausea and Vomiting. Gastroenterology, 149(1), 56-66.e55. doi:http://dx.doi.org/10.1053/j.gastro.2015.04.003

Bashashati, M., & McCallum, R. W. (2015). Is Interstitial Cells of Cajal–opathy Present in Gastroparesis? Journal of Neurogastroenterology and Motility, 21(4), 486-493. doi:10.5056/jnm15075

Camilleri, M. (2016). Novel Diet, Drugs, and Gastric Interventions for Gastroparesis. Clinical Gastroenterology and Hepatology, 14(8), 1072-1080. doi:http://dx.doi.org/10.1016/j.cgh.2015.12.033

Daniel, E. E., & Wang, Y. F. (1999). Gap junctions in intestinal smooth muscle and interstitial cells of Cajal. Microscopy research and technique, 47(5), 309-320.

Furness, J. B., & Costa, M. (1987). The enteric nervous system: Churchill Livingstone Edinburgh etc.

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Gilmont, R. R., Raghavan, S., Somara, S., & Bitar, K. N. (2014). Bioengineering of physiologically functional intrinsically innervated human internal anal sphincter constructs. Tissue Engineering Part A, 20(11-12), 1603-1611.

Groneberg, D., Aue, A., Lies, B., & Friebe, A. (2015). Cell-specific modulation of gastrointestinal NO-induced relaxation by phosphodiesterases. BMC Pharmacology and Toxicology, 16(1), A56. doi:10.1186/2050-6511-16-s1-a56

Huizinga, J. D., Liu, L. W., Fitzpatrick, A., White, E., Gill, S., Wang, X.-Y., . . . Starret, T. (2008). Deficiency of intramuscular ICC increases fundic muscle excitability but does not impede nitrergic innervation. American Journal of Physiology-Gastrointestinal and Liver Physiology, 294(2), G589-G594.

Koch, K. L., & Stern, R. M. (2004). Handbook of electrogastrography: Oxford University Press. Moraveji, S., Bashashati, M., Elhanafi, S., Sunny, J., Sarosiek, I., Davis, B., . . . McCallum, R.

(2016). Depleted interstitial cells of Cajal and fibrosis in the pylorus: novel features of gastroparesis. Neurogastroenterology & Motility.

O'Grady, G., Du, P., Cheng, L. K., Egbuji, J. U., Lammers, W. J. E. P., Windsor, J. A., & Pullan, A. J. (2010). Origin and propagation of human gastric slow-wave activity defined by high-resolution mapping. American Journal of Physiology - Gastrointestinal and Liver Physiology, 299(3), G585-G592. doi:10.1152/ajpgi.00125.2010

Penagini, R. (1998). Practical Guide to Gastrointestinal Function Testing. European Journal of Gastroenterology & Hepatology, 10(7), 623.

Raghavan, S., Gilmont, R. R., & Bitar, K. N. (2013). Neuroglial differentiation of adult enteric neuronal progenitor cells as a function of extracellular matrix composition. Biomaterials, 34(28), 6649-6658. doi:http://dx.doi.org/10.1016/j.biomaterials.2013.05.023

Raghavan, S., Gilmont, R. R., Miyasaka, E. A., Somara, S., Srinivasan, S., Teitelbaum, D. H., & Bitar, K. N. (2011). Successful implantation of bioengineered, intrinsically innervated, human internal anal sphincter. Gastroenterology, 141(1), 310-319.

Raghavan, S., Miyasaka, E. A., Gilmont, R. R., Somara, S., Teitelbaum, D. H., & Bitar, K. N. (2014). Perianal implantation of bioengineered human internal anal sphincter constructs intrinsically innervated with human neural progenitor cells. Surgery, 155(4), 668-674.

Rego, S. L., Zakhem, E., Orlando, G., & Bitar, K. N. (2015). Bioengineered human pyloric sphincters using autologous smooth muscle and neural progenitor cells. Tissue Engineering Part A, 22(1-2), 151-160.

Zhu, M. H., Kim, T. W., Ro, S., Yan, W., Ward, S. M., Koh, S. D., & Sanders, K. M. (2009). A Ca2+-activated Cl− conductance in interstitial cells of Cajal linked to slow wave currents and pacemaker activity. The Journal of Physiology, 587(20), 4905-4918. doi:10.1113/jphysiol.2009.176206

76

CHAPTER IV: MICRO-SENSITIVE MOLDED SILICONE

TISSUE PILLAR PLATFORM, FABRICATED BY 3-D

PRINTING FOR SIMPLER PHYSIOLOGICAL STUDIES

Dylan Knutson1, 2, and Khalil N. Bitar1, 2, 3

1Virginia Tech-Wake Forest School of Biomedical Engineering and Sciences, Winston Salem, NC

2Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston Salem, NC

3Section on Gastroenterology, Wake Forest School of Medicine, Winston-Salem, NC, USA

This chapter describes a new innovation for physiological studies

77

ABSTRACT

INTRODUCTION

Physiological studies are a tremendous resource for understanding cell

or tissue function, repair, or engineering. However, these studies require

expensive equipment to record signals, vast knowledge to acquire the right

signals, and have limited capabilities.

OBJECTIVE

The aim of this study were to fabricate a multifunctional tissue pillar

platform using 3-D printing and silicone molding.

METHODOLOGY

Pillars were molding using a printed device. Muscle tissues were

engineered inside using a collagen gel mixture.

RESULTS

The assembly and pillars could be made with high success. (>90%). The

sensitivity of the pillars fabricated reached 1.91 µN/µm of deflection. The

engineering process demonstrated control over the tissues, and cells expressed

contractile phenotype Actin and aligned uniformly between the pillars. Organ bath

studies validated that the pillars were almost as sensitive traditional techniques.

Electrical activity could be recorded simultaneously, and the pillars could

differentiate between sphincteric and non-sphincteric tissues.

78

CONCLUSION

This method provides a simple, powerful, multifunctional tool to investigate

physiology using engineered tissue.

Keywords: micro pillars, sensitive, physiology, tissue engineering, fluidic,

disease Models

79

INTRODUCTION:

Physiological studies are a tremendous resource for understanding cell or

tissue function, repair, or engineering. However, these studies require expensive

equipment to record signals, vast knowledge to acquire the right signals, and

have limited capabilities. Due to the challenge and inaccessibility of these

technologies, there is a lack of research involving the characterization of the

phenomena that whole tissues or cells generate (Polacheck & Chen, 2016). Even

though new techniques have emerged to record forces, these same hurdles of

cost, complexity, and inadequate equipment still exist.

Moreover, one favorable solution involves micro pillars, which provide a

simpler approach to quantifying cellular forces (Boudou et al., 2011; Stephen L

Rego, Raghavan, Zakhem, & Bitar, 2015; Stephen L Rego, Zakhem, Orlando, &

Bitar, 2016). Small silicone pillars were fabricated using lithography. Their

deflection was characterized, and then small collagen tissues containing

myocytes were engineered around them. The data from this abovementioned

study showed that the alignment of the cells was dependent on the experienced

tensile strain, and that each cell exerted nanonewton scale forces in order to

deflect the pillars. These results demonstrate simpler physiology approaches can

be used to investigate the formation of tissues and cellular function.

Nevertheless, not only is lithography an advanced method of fabrication, but the

tissues were engineered in a complex manner in that centrifugation of the entire

pillar platform in very small isolated wells was performed. Therefore, challenges

80

exist among fabricating the pillars, engineering the tissue, and isolating each

bath containing the pillars.

Recently, the accessibility of 3-D printers has increased. Simple and

accurate printers that can be used out-of-the-box can be acquired for fewer than

300 dollars. This modality provides new opportunities and methods for capturing

physiological data. Thus, an aim of the present investigation was to mold silicone

pillar tissues, using a basic 3-D printer, and subsequently characterize and

validate them. The final aim was to perform small physiological studies using

these silicone pillar tissues. Micro-sensitive pillars in a fluidic-ready bath were

created, which allowed for differentiation between muscle tissue types, and

simultaneous recordings of both electrical and forces phenomena. This type of

technology provides promising new techniques for further research regarding

cellular and tissue function.

METHODS:

Primary Smooth Muscle Cells (SMC)

Human tissues were ethically obtained from organ donors through

Carolina Donor Services and Wake Forest Baptist Medical Center (IRB#:

IRB00007586). Smooth muscle cells were isolated as previously described

(Stephen Lee Rego, Zakhem, Orlando, & Bitar, 2015). Briefly muscle tissues

were removed by sharp dissection. Tissues were manually cleaned by removing

fat and mucosa with a surgical blade. Tissues were extensively washed with

HBSS solution containing 2X antibiotics/antimycotic and then minced in sterile

conditions. Tissues were subjected to two digestions with HBSS containing 1

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mg/mL collagenase type II (Worthington Biochemicals, Lakewood, NJ) at 37°C

for 1 hour each. Tissue pellets were then resuspended in SMC growth media and

plated in tissue culture dishes at 37°C with 5% CO2. Other smooth muscle

tissues were isolated using the same process.

Bioengineered Muscle Tissues

The process of engineering the sphincters was similar to methods

described previously (Gilmont, Raghavan, Somara, & Bitar, 2014; Raghavan et

al., 2011; Raghavan et al., 2014), with changes outlined below. Smooth muscle

cells were trypsinized and 500,000 cells were collected per construct. Cells were

resuspended in 1 mL of collagen gel of 1.9 mg/ml final concentration. The

mixture was then poured into the pillar vessel with ‘gates’. The muscle gel was

left to gel at 37ºC and then supplemented with differentiation media after 1 hour.

Microscopic evaluation of the constructs:

Constructs were evaluated microscopically prior to physiological testing on

day 4 of culture. A Stereomicroscope was used to collect data on the force of

tissues. Tissues were monitored by a time-lapse of 15 second intervals over 10

minutes. Changes in pillar deflection were measured at each time point, force

was then calculated, and then the output was plotted. Images were processed

using NIS Elements software (Nikon Instruments, Melville, NY).

Characterization of Pillar Stiffness

The stiffness of pillars was characterized using the following equation:

Some pillar deflection, d (µm) that corresponds to a force, F, µN; directly

82

determined by constant, k (µN / µm). Defined, 𝐹 = 𝑘𝑑. Pillars were submerged in

water, to account for swelling of silicone elastomer in liquids. Then, a hanging

mass was hooked to a string and pulley which was fastened to the pillar. The

deflection was measured and a k-value was computed for the translation of

deflection to a force. The k-value was characterized for each pillar set.

Physiological Analysis of Bioengineered Tissues

All muscle tissues were analyzed for physiological functionality. Tissues

were monitored by a time-lapse of 15 second intervals over 10 minutes. Changes

were measured at each point and then plotted following the calculation of force.

For the validation using the organ bath, constructs were hooked between a fixed

arm, and the measuring arm of an isometric, magnetoresistive force transducer

in a horizontal tissue bath (F10; Harvard Apparatus, Holliston, MA). Force data in

the organ bath and electrical activity was acquired using LabChart 7 software

(ADInstruments, Colorado Springs, CO). Electrical phenomena were acquired

using band pass filter (High pass: 0.03 Hz and Low Pass: 3 Hz, at 2000 kHz

sampling rate). Constructs were maintained in 4-(2-hydroxyethyl)-1-

piperazineethanesulfonic acid (HEPES) buffered solution at 37°C throughout all

experiments. All force generation studies were performed after the establishment

of stable basal tone. Force generation was evaluated following the addition of

potassium chloride (KCl; 60 mM). Constructs were washed between each

treatment, incubated in fresh buffer and allowed to return to baseline. GraphPad

Prism 7.00 software for Windows (GraphPad Prism 7.00, San Diego, CA) was

used to plot and analyze collected data. Second-order Savitsky–Golay smoothing

83

was applied. Quantification of physiologic data was performed relative to basal

tone for contraction and relaxation as maximum/minimum peak response (Delta

force of basal tone).

Fabrication:

CAD Drawings were made using AutoDesk Fusion 360 (AutoDesk, Mill

Valley, CA). Assembly and molds were made on a Printrbot Simple Metal using

PLA filament (Printrbot, Lincoln, CA). Pillars were molded using Sylgard 184

(Dow Corning, Greensboro, NC).

Statistical analysis:

Data were expressed as mean ± SEM unless noted otherwise. Alpha was

set at p.05. One-way ANOVA followed by Tukey’s test was used to compare all

groups. Only up to three t-tests were used to evaluate a priori hypotheses on the

groups. Normality was assessed by fisher skewness, and for ANOVA a Brown-

Forsythe test was used to ensure homogeneity of variance (Prism 7.00,

GraphPad Software, Inc., La Jolla, CA, USA).

84

RESULTS:

Fabrication:

CAD drawings were made to create the final design, which needed to be

simple, and allow for fluid flow and tissue interaction (Figure 21). The design

featured: 1. a top ABS plate providing thermal resistance and a windowed view,

inlet, and outlet. 2. A PDMS center channel allowed 3-D printed parts to be

protected from leaks, provided width for lower stresses, and lowered flow velocity

around tissues. Notches in the side allowed for ‘gating’ with silicone walls, so

that when tissues were cultured, barriers could be put in between each tissue. 3.

Several pairs of PDMS tissue pillars allow for the construction of different tissues,

and report strains visually to quantify force. (4) The bottom plate provides

sufficient room for added elements and thermal resistance.

37.5 mm

Tissue Pillar Device

1 2 3 4

85

Figure 21

CAD drawings are printed using a desktop 3-D printer. Final products are rigid,

accurate, and fit together tightly. Right: top plate and bottom plate before

assembly. Figure 22 shows assembled molds for PDMS products and parts for

the assembly.

Figure 22

86

Figure 23

CAD drawings are printed using a desktop 3-D printer. Molds for pillars

and bath walls (Figure 23, A and B, respectively) fit into the stage (C, shown with

part of mold component A). Some molds were coated with epoxy to provide a

smoother finish on silicone parts. After printing, M5 screws were tapped through

the bottom of the stage in order to raise molded pillars out of the stage. The

stage allows for the fitment of either the pillar (figure 24, A) or bath wall mold (B).

CAD drawings of mold components to be 3-D printed:

Mold assemblies after printing

A.

B.

C.

D.

87

Figure 24

The following figure outlines the molding process used to make tissue

pillars (Figure 25)- (1) the desired negative mold is placed in stage and PDMS is

poured over mold. (2) After curing, molded piece and negative are driven out of

stage by set screws. (3) The base is loosely peeled from face of product. (4) For

tissue pillars, the negative mold is disassembled into pieces. (5) Molded pillars

are revealed. (6) A PDMS base with a single set of pillars. Molding was complete

after only a day and the rate of success for this process was 90%.

Figure 25

A.

B.

1 2 3

4 5 6

88

All components of the assembly (Figure 26). The two silicone parts were

bonded with a final layer of silicone. The bath with pillars (below, top) or the full

assembly for fluidic experiments (below, bottom) is shown.

Figure 26

89

Tissue Engineering and Characterization:

Gates were produced from 3D printed mold which fit into slotted walls to

isolate/separate hydrogels cultured in vessel (Figure 27, A). These silicone‘gates

isolated 1 mL of 1.9 mg/mL Collagen hydrogel with a 92% percent success rate

(B). Gates could be easily removed with forceps after 40 minutes of gelation at

37˚C to add medium.

Figure 27

A.

B.

90

Figure 28

The bath with pillars, (Figure 28, A), representing the assembly to be

characterized for sensing force. These small pillars would operate like cantilevers

in order to report forces as they are deflected. Hooke’s Law describes the

deflection of an elastic material which corresponds to a linear change in force

(B). This approximation is generally accurate to a threshold, called the elastic

limit, where the deflection in relation to force loses its linearity. PDMS is a tunable

substrate where solids of different elasticity can be molded.

Two common mixtures of hardener to elastomer were characterized as an assay-

1:20 and 1:30 (Figure 29). In this experiment each pillar deflection was tested

against a series of known calibration masses to determine linear constant, k, and

the elastic limit of each.

Hooke’s Law Some pillar deflection, d (µm)

that corresponds to a force, F, µN; directly determined by constant, k (µN / µm).

Defined: 𝐹 = 𝑘𝑑

A. B.

91

0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0

0

2 0 0

4 0 0

6 0 0

8 0 0

1 0 0 0

P D M S M ix tu re S e n s it iv ity

(H a rd e n e r :E la s to m e r )

C a lib ra t io n F o rc e ( N )

Dis

ten

sio

n o

f P

illa

r (

m

)

1 :2 0

1 :3 0

1:2

0

1:3

0

0 .0

0 .5

1 .0

1 .5

2 .0

2 .5

H o o k e s L a w :

1 s t-O rd e r A p p ro x im tio n o f

S t if fn e s s , K

H a rd e n e r : E la s to m e r o f P il la r

N

/

m

0 2 0 0 4 0 0 6 0 0 8 0 0 1 0 0 0

0

2 0 0

4 0 0

6 0 0

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C a lib ra t io n F o rc e ( N )

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E la s tic L im it o f

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Figure 29

Constants from each calibration test were calculated. For example a mass which

exerts 102 µN was hung from a tether to the pillar, and the change deflection was

recorded on the stereomicroscope. At some point, masses no longer deflected

the pillars within a linear confidence, representing their elastic limits. The 1:20

mixture resulted in constant, k= 0.58 ± 0.03 µN/µm, that is- each micrometer of

deflection measured represented 0.58 micronewtons. The 1:30 mixture resulted

in a more sensitive pillar at 1.909 ± 0.22 µN/µm. Linearity fell off at 600 and 400

micronewtons respectively, representing each vessels elastic limit. For the

experiments that followed a 1:30 pillar mixture was used, for the anticipation of

forces that were less than 400 micronewtons.

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Microscopy of tissues in culture revealed muscle cells captured in the

tissue and aligned between the silicone pillars (Figure 30). Alignment was

uniform, and could be seen at high objectives. Staining with smooth muscle actin

(green) confirmed alignment of muscle filaments, and expression of a contractile

phenotype. Filaments in the tissues, with only 500k cells culture for 4 days, were

only beginning to connect.

Figure 30

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Validation and investigation:

Forces were compared between a traditional organ bath and the pillars

(Figure 31). Tissues were tested first on the pillars they were cultured in, and

then they were shifted to an organ bath to perform the same test. A

stereomicroscope could measure the deflection of the pillars in response in order

to calculate forces (A). Deflections were small, but easy to perceive and measure

on the microscope. In the bath a force transducer measures strain and reports

force generated by tissues over time (B). The forces measured on the pillars

were: Basal Tone: 346.7 ± 80.91 and KCl: 67.39 ± 10.71. Forces measured in

the bath from the same tissues were 291 ± 12.31 and 95 ± 20.82 respectively.

The basal tone measured by both devices was similar (C, n=3, paired, p>.05),

but the measured response to KCl was slightly less by the pillar baths and was

approaching significance (D, n=3, paired, p=0.16).

Figure 31

A

.B

C

DD

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With the forces measured by the pillars compared to a standard,

comparison of the KCl response of sphincteric and non-sphincteric (rabbit small

intestine) muscle tissues (rabbit internal anal sphincter) was made. Tissues were

washed with fresh buffer, and 60mM KCl was added following 30 minute

equilibration period. Sphincteric tissues exerted significantly more force within the

device compared to non-sphincteric (Figure 32, n=3-4, p<.05). Therefore, the

contractility of muscle when depolarized is different between sphincteric and non-

sphincteric muscles.

Figure 32

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Next both force and electrical phenomena were measured simultaneously

in the bath. Below are KCl responses from three muscular tissues (Figure 33).

Force from the tissues was measured every 15 seconds while electrical activity

was measured continuously. Peak forces and large spikes in EMG activity are

associated with the onset of contraction. The peak EMG activity was 1.676 ±

0.8522 mV, and peak forces were 45 ± 7.211 µN. Larger or more frequent

activites resulted in higher measured forces.

Figure 33

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DISCUSSION:

This study provides a simple and easy to fabricate platform for

investigating physiology of engineered tissues and cells. It allows for control over

engineering and reported phenomena of the tissues. In the study, the tissues had

muscle cells aligned between the pillars, and the pillars themselves could read

forces, though slightly lower than other physiology device standards. With this in

mind, the pillars still had the capacity to read micro-scale forces, which allows for

open ended investigations such as those of electrical activity.

The first aim of this investigation was to effectively fabricate the device.

This yielded high success, and provided a strong foundation for tissue

engineering. Parts were large enough to print successfully with a desktop printer,

and then molded silicone pillars were easily retrieved from the stage with high

success. These molded pillar vessels were sensitive enough to detect micro

scale forces.

Next, the tissues were engineered, which contracted into neatly aligned

muscular tissues which had a contractile phenotype. In the present studies, only

500K smooth muscle cells were used in the constructs. While these tissues were

functional, future studies might look to improve the density and consequently

force exerted by increasing cell numbers.

Last, validation of the apparatus was made by testing previously known

hypotheses on the tissues. For example, it is well know that the contractility of

sphincteric and non-sphincteric muscles are different (Stephen L Rego et al.,

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2015; Stephen L Rego et al., 2016). The pillars in the apparatus were able to

differentiate between tissues cultured with either muscle type. We confirmed that

sphincteric smooth muscle exerted significantly more force than non-sphincteric

muscle when depolarized. Increased force is important to controlling the passage

of food between segments by increasing luminal pressure and limiting the size by

decreasing the luminal diameter. Ultimately, this study demonstrates the

feasibility and practicality of such technologies for generating and testing new

research regarding contractile tissues.

However, one drawback to these pillars was the discrepancy between

muscular responses when comparing it to a force transducer. Testing between

the standard horizontal bath and the pillar tissues showed that the pillar tissues

read a response on the same tissues that was less. This discrepancy could be

due to the stretch of the tissues. Upon setting tissues in an organ bath it is

recommended that the tissue be stretched, so that the transducer is immediately

feeling strain from the tissue. While the pillar tissue are cultured on the device,

and have some small ‘slack’ which is not accounted for immediately. So any

strain generated at first with the tissue is not experienced by the pillars, but

instead is used to pick up ‘slack’.

Another challenge is the seal of 3-D printed parts to water. Printing with a

large nozzle at lower infills produced parts that had to be patched with epoxy. So

waterproofing is another potential drawback of this fabrication. More involved

investigators performing long-term fluidic studies might produce platens made

from sheets of plastic instead.

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In conclusion, this technology offers a simpler, novel approach to studying

the physiology of both engineered tissues and cells. It also provides an open

source platform for future research on tissues.

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REFERENCES

Boudou, T., Legant, W. R., Mu, A., Borochin, M. A., Thavandiran, N., Radisic, M., . . . Chen, C. S. (2011). A microfabricated platform to measure and manipulate the mechanics of engineered cardiac microtissues. Tissue Engineering Part A, 18(9-10), 910-919.

Gilmont, R. R., Raghavan, S., Somara, S., & Bitar, K. N. (2014). Bioengineering of physiologically functional intrinsically innervated human internal anal sphincter constructs. Tissue Engineering Part A, 20(11-12), 1603-1611.

Polacheck, W. J., & Chen, C. S. (2016). Measuring cell-generated forces: a guide to the available tools. Nat Meth, 13(5), 415-423. doi:10.1038/nmeth.3834

Raghavan, S., Gilmont, R. R., Miyasaka, E. A., Somara, S., Srinivasan, S., Teitelbaum, D. H., & Bitar, K. N. (2011). Successful implantation of bioengineered, intrinsically innervated, human internal anal sphincter. Gastroenterology, 141(1), 310-319.

Raghavan, S., Miyasaka, E. A., Gilmont, R. R., Somara, S., Teitelbaum, D. H., & Bitar, K. N. (2014). Perianal implantation of bioengineered human internal anal sphincter constructs intrinsically innervated with human neural progenitor cells. Surgery, 155(4), 668-674.

Rego, S. L., Raghavan, S., Zakhem, E., & Bitar, K. N. (2015). Enteric neural differentiation in innervated, physiologically functional, smooth muscle constructs is modulated by bone morphogenic protein 2 secreted by sphincteric smooth muscle cells. Journal of tissue engineering and regenerative medicine.

Rego, S. L., Zakhem, E., Orlando, G., & Bitar, K. N. (2015). Bioengineered human pyloric sphincters using autologous smooth muscle and neural progenitor cells. Tissue Engineering Part A, 22(1-2), 151-160.

Rego, S. L., Zakhem, E., Orlando, G., & Bitar, K. N. (2016). Bioengineering functional human sphincteric and non-sphincteric gastrointestinal smooth muscle constructs. Methods, 99, 128-134.

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CHAPTER V: SUMMARY AND CONCLUSIONS

In conclusion, these tissue engineering approaches have the potential to

generate and test new hypotheses surrounding pathophysiology, prompted by

the first study. By quantitatively depleting and replenishing cell types associated

with disease, the second study was the first of its kind to demonstrate physiology

of gut-derived neuromuscular tissues engineered with ICC. The development of

the final platform using micro pillars provides a promising and innovative model

to characterize other qualitative and quantitative changes of tissues.

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APPENDIX

MATLAB CODING FOR SLOW WAVE ASSOCIATED CONTRACTION ANALYSIS:

close all %Detrends Sample Data force = force5; time = time5; [p,s,mu] = polyfit((1:numel(force))',force,6); f_y = polyval(p,(1:numel(force))',[],mu); %plot detrended data x = force - f_y; figure plot(x); hold on %Fast-Fourier Transform and Low-Pass Filter %figure [a, b] = butter(4, .01, 'low'); %filter cutoff at frequency fx = filter(a,b,x); %application of butterworth filter %plot(fx) %hold on % Reconstruction of Plot [Maxfreq, Loc] = max(periodogram(x)); figure periodogram(fx) hold on %Counting Peaks figure findpeaks(fx, time,'MinPeakProminence',20,'MinPeakDistance',5); xlabel('time(s)'); ylabel('microNewtons'); ylim([-200 200]); title('1 Min'); [pks, locs] = findpeaks(fx),

time,'MinPeakProminence',20,'MinPeakDistance',5);

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SCHOLASTIC VITA

Dylan Knutson

Minneapolis, MN

Graduate Student + Biomedical Engineer

Education

Master of Science Candidate (Biomedical Engineering) 2017

Virginia Tech-Wake Forest School of Biomedical Engineering Sciences (SBES, Winston Salem,

NC)

Thesis:

Novel Approaches to Testing Gastro Intestinal Function In Vitro: Controlling Signal

Acquisition, Tissue Composition, or The Platform.

Advisor: Dr. Khalil Bitar.

Bachelor of Science (Health Sciences, Mathematics) 2014

Lees-McRae College (Banner Elk, NC)

Thesis:

Static and Kinematic Slip Properties of Wax Emulsions

Publications

DT Knutson, E Zakhem, KN Bitar, An In Vitro Model of the Gastric

Neuromuscular Apparatus Using Engineered Pylorus to Understand Gastric

Pathophysiology (Submitted).

DT Knutson, KN Bitar, Micro-sensitive Molded Silicone Tissue Pillar Platform,

fabricated by 3D printing, for simple physiological analysis (in preparation).

Awards

Awarded Alvarez Award, for ‘Best Overall Abstract’ and invited to do an extended

oral presentation by International Gastrointestinal Electrophysiology Society

Awarded 1st place poster presentation, people’s choice for “An Engineered In

Vitro Model of Pylorus Neuro-Muscular Apparatus.”, WFIRM Retreat, 01/2017

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Presentations

DT Knutson, E Zakhem, KN Bitar, Interstitial Cells of Cajal Increase Neural-

Mediated Relaxation and Electrical Phenomena within In Vitro Model of Pylorus

Neuromuscular Apparatus.

o (Extended Oral presentation, International Gastrointestinal

Electrophysiology Society, 05/2017)

DT Knutson, E Zakhem, KN Bitar, An Engineered In Vitro Model of Pylorus

Neuro-Muscular Apparatus.

o (Oral, Wake Forest-Virginia Tech Graduate Research Seminars, 02/2017)

o (Poster, WFIRM Retreat, 01/2017)

DT Knutson, E Zakhem, KN Bitar, Incorporation of Interstitial Cells of Cajal in

Engineered Innervated Smooth Muscle Pyloric Sphincters.

o (Poster, International Conference of Biofabrication, 10/2016)

DT Knutson, E Zakhem, KN Bitar, Role of Interstitial Cells of Cajal (ICC) on

Sphincteric Tone in the Pylorus.

o (Poster, Graduate Symposium, 02/2016)

KN Bitar, E Zakhem, J Bohl, R Tamburrini, P Dadhich, C Scott, DT Knutson, J Gilliam Implantation of Autologous Biosphincters in a Non-Human Primate (NHP) Model of Fecal Incontinence.

o (Oral, Digestive Disease Week, 04/2017)

E. Petran, P. Dadhich, DT Knutson, E. Zakhem, K. N. Bitar, Cell Therapy for

Neo-innervation and Restoration of Neural Function in the Pylorus.

o (Poster and Oral presentation, WFIRM, 08/2016)

Departmental/Institutional Citizenship

Mentored summer student to complete a research project in 3 months.

o Taught and oversaw safe lab practices necessary for project.

o Assisted with collection, analysis, and interpretation of data.

o Edited and reviewed abstract and presentations.

Collaborates with other investigators to quickly engineer new components and

assemblies for projects

o Modeled and fabricated components to house innovative device which

will be used for high throughput drug screening.

o Created molding kits which can expedite the processing of bioengineered

tissues.

Restarted, refurbished, and manages entire physiology core.

o Instructs other scientists how to setup, record and analyze tissue

functionality.

o Improve scientific outreach by explaining and displaying physiology

equipment in departmental tours

o Saved institute tens of thousands of dollars on equipment and expensive

installation of new devices.

Volunteered at international and institutional conferences

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o International Conference on Biofabrication- coordinated projection of

presentations.

o Volunteered as representative of Biomedical Engineering Society at 2016

SBES Symposium.

o 2016 Graduate School Symposium- helped to set-up and tear-down.