Enteric Nerve Regeneration for Hirshsprung Disease using Four Emerging Rules for Organ Regeneration

1. ENTERIC NERVE
REGENERATION FOR
HIRCHSPRUNG DISEASE
MUNIRA SHAHBUDDIN
MASSACHUSETTS INSTITUTE OF TECHNOLOGY
NORTHEASTERN UNIVERSITY
27TH MARCH 2018

2. OUTLINE
ENTERIC NERVOUS
REGENERATION
• WHAT IS ENS?
• HIRSHSPRUNG DISEASE
• CURRENT INTERVENTION FOR
HIRSHSPRUNG DISEASE
• BIOMATERIALS AND CELLS
INTERVENTION
• WORKS BY JAPANESE AND GERMAN
GROUPS ON ANIMAL MODEL
4 EMERGING RULES FOR
ORGAN REGENERATION
• In an injured organ epithelial tissues and
the basement membrane regenerate
spontaneously; the stroma does not
• The required reactants for inducing
regeneration are an appropriate scaffold
and, optionally, epithelial cells
• Scaffolds are regeneratively active if they
inhibit contraction and scar formation
• Structural features in scaffolds that are
required for regenerative activity: pore
structure, degradation rate, surface
chemistry

3. NERVOUS
SYSTEM
The nervous system is a complex
network of nerves and cells that
carry messages to and from the
brain and spinal cord to various
parts of the body.
The nervous system includes both
the Central nervous system and
Peripheral nervous system.

4. The enteric nervous
system (ENS) or
intrinsic nervous
system is one of the main
divisions of the
autonomic nervous
system and consists of a
mesh-like system of
neurons that governs the
function of the
gastrointestinal tract.
ENTERIC
NERVOUS
SYSTEM

5. GASTROINTESTINAL
SYSTEM
The GI tract is a series of
hollow organs joined in a
long, twisting tube from
the mouth to the anus.
The hollow organs that
make up the GI tract are
the mouth, esophagus,
stomach, small intestine,
large intestine, and anus.

6. HIRCHSPRUNG
DISEASE
Hirchsprung's disease is a
congenital disorder of the
colon in which certain
nerve cells, known as
ganglion cells, are absent,
causing chronic
constipation.

8. CURRENT
INTERVENTIONS
FOR
HIRCHSPRUNG
DISEASE
Removal of
agangliosection of the
intestine
Reconstruction of
intestine

9. CAN TISSUE
ENGINEERING BE
APPLIED FOR
HIRSHSPRUNG’S
PATIENTS?
1. Biomaterials
2. Cell and Tissue
Engineering approach

10. CELL AND TISSUE
ENGINEERING
APPROACH
From lab to organ
approach

11. COMBINATION OF SCAFFOLD AND
CELLS

12. Tissue Engineering of
Small Intestinal Tissue
Using Collagen Sponge
Scaffolds Seeded with
Smooth Muscle Cells
FIG. 1. Surgical technique and collagen scaffolds
for SMC (-) and SMC (+) groups. (A) SMC (-) or
SMC (+) scaffolds were implanted as patch grafts
into defects created in two isolated ileal loops. (B)
The patch graft was covered with omentum, and
the two ileal loops were used to construct a double
ileostomy on the anterior abdominal wall
bilaterally. (C) At 8 weeks after implantation, the
ileal loops were reanastomosed to avoid disuse
atrophy. (D and E) Hematoxylin and eosin staining
of SMC (-) (D) and SMC (+) scaffolds (E). SMC
were seeded in the lattice spaces of the collagen
sponge in the SMC (-) scaffolds (arrows). (F) Most
cells of the SMC (+) scaffolds are labeled with Di
I (arrows). (D–F) Original magnification 100X.

13. Macroscopic findings on the luminal side of the
graft area. The SMC (-) group (A) and SMC (+
group (B) at 4 weeks after implantation, and the
SMC (-) group (C) and SMC (+) group (D) at 12
weeks after implantation. (A and B) In both groups,
the luminal surface of the graft area was not covered
with mucosa at 4 weeks, but had an ulcerative
appearance. (C) At 12 weeks, the graft surfaces in
the SMC (-) group were covered by regenerated
mucosa that was depressed relative to the adjacent
mucosa. (D) At 12 weeks in the SMC (+) group, it
was difficult to macroscopically distinguish the
appearance and contour of the regenerated mucosa
from that of the normal mucosa.

14. SMC (-). The luminal surface of the graft site was
covered with a monolayer of mucosal columnar
epithelial cells. Collagen sponge scaffolds were
absorbed, and myofibroblasts had disappeared. A
thin smooth muscle layer, the muscularis mucosae,
was stained immunohistochemically with anti--
SMA and anti-basic calponin (Fig. 6 A, C, and E).
SMC (+). The luminal surface of the graft site was
covered with a regenerated epithelial cell layer
that included goblet cells and had a villus-like
configuration, although these villi were shorter
than those in adjacent normal mucosa.
Immunohistochemical staining with anti-SMA and
basic calponin and detection of the labeled cells
using a fluorescence microscope showed the
presence of implanted SMCs in the lamina propria
and formed a deeper smooth muscle layer (Fig.
6B, D, and F). The collagen sponge scaffolds were
absorbed, and myofibroblasts had disappeared.
Implanted SMCs were multilayered, and the
surface area of the graft site shrank.

15. Histologic, immunohistochemical, and
immunofluorescence features of the graft
area at 12 weeks after SMC (+) scaffold
implantation. (A) Hematoxylin and eosin
staining. (B and C) High-power views of A:
H&E staining (B); immunohistochemical
staining for basic calponin (C). The graft
site is shown by the bar. (D–I) High-power
views of B: a portion of the lamina propria
of the graft site(D–F); a portion of the
smooth muscle layer of the graft site(G–I).
(D and G) H&E staining; (E and H)
immunofluorescence staining for basic
calponin. (F and I) The location of SMC
labeled by DiI. The luminal surface of the
graft site was completely covered by a
relatively well-developed epithelial layer
with numerous villi and an orderly
smooth muscle layer. It was observed that
implanted SMCs were organized into a
circumferential smooth muscle layer and
were present in a part of the lamina propria
(arrows, E and F).

16. SUMMARY OF
THIS PAPER • Collagen Scaffold : Pelnac (Kyoto, Japan) 3 mm thick, with a pore
size of 70–110 μm and pore volume fraction of 80–95%
Problems:
• Mechanical stability and integrity
• Cells – the authors only examined the presence of SMC, not other
cells or formation of enteric nerves.
• Scarring
Improvements:
- 2D vs 3D – arguments are in the following slides.
- Increase population/concentration of cells or different type of cells
– like Glial cells transplantation, or look into the study of ileal
and bowel conduit for urinary tract regeneration – and compare
that to PNS work.
- Use different measurement and observations for motility – study
Cajal method for staining -
The scaffolds had shrunk about 10% from their
original size at 12 h after seeding.
SMCs were seeded on lattice spaces of collagen
sponge scaffolds.
Without collagen solution, far fewer cells
remained in the sponge scaffolds because they
passed through the pores of the sponge.
Immersion in collagen solution prior
transplantation caused the formation of fibrosis
and scarring.
At 4 weeks after implantation, Vicryl sutures had
nearly disintegrated, and silicone sheets had
almost come off the luminal surface of the graft
site.

17. Two differentially
structured collagen
scaffolds for potential
urinary bladder
augmentation: proof
of concept study in a
Göttingen minipig model
Both collagen scaffold (2D and 3D)
prototypes in vivo had good ingrowth
capacity into the bladder wall including a
quick lining with urothelial cells. The
ingrowth of detrusor muscle tissue, along
with the degradation of the scaffolds were
observed throughout the study period.

18. Implantation
procedure of a
seeded OptiMaix 2D
into the minipig
bladder. a Seeded
OptiMaix 2D in the
custom made
seeding ring.
b Creation of the
serosal flap.
c Setting of marks
with non-degradable
sutures.
d Implanted
OptiMaix 2D.
e Sealed
implantation site.
Implantation of
OptiMaix 3D was
performed similarily
(not shown)

21. SUMMARY OF
THE PAPER
OptiMaix 3D scaffolds had a greater
potential for leakages than the 2D
scaffolds which occurred in two of the
six pigs.
OptiMaix 3D, was more delicate
because of its highly porous structure.
Preliminary attempts to suture the 3D
scaffold in the same precut oval shape as
the 2D failed due to rupturing of the
scaffold at the suture points.
Pre-seeding with SMCs did not improve
the ingrowth process – compared to the
native control and cause the formation
of scarring as was seen before in the
Japanese experiment with canine.
At 22 weeks, OptiMaix 3D scaffolds
were covered by fibrous tissues but not
on 2D scaffold.
The pore structure of the OptiMaix 3D
was a disadvantage concerning the
urothelium.The urothelial cells, like
in vitro, lined the inside of these pores
and were not able to build a closed layer
on top of the scaffolds.
OptiMaix 2D was relatively easier to
handle and additionally one week after
operation, the implantation sites of the
2D scaffolds were closed.
3D scaffolds needed longer recovery

22. FOUR
EMERGING
RULES FOR
ORGAN
REGENERATION
Rule 1 distinguishes between
tissues that regenerate
spontaneously and those that
do not
PNS - Transverse section through myelinated fibers within a
nerve fascicle (Triple stain). Blue endoneurium surrounds
myelin sheaths.
Epidermis
Dermis
Spontaneous regeneration
Non-regenerative
http://vanat.cvm.umn.edu/neurHistAtls/cataPages/cataPNS.html

23. THE CONCEPT OF
TISSUE TRIAD
Nature of injured tissue determines
the reversibility of injury. Epithelial
tissues and basement membrane
regenerate spontaneously. In adults,
the stroma never regenerates
spontaneously
Epidermis
Dermis

24. The gut provides another contrast in healing
mode between a superficial vs. a deep injury
in a hollow organ. Gastric epithelium
responds to superficial injury (erosion) by
rapid reepithelialization. A much deeper
wound that has penetrated through the thin
basement membrane into the underlying
layers (submucosa and muscularis propria)
leads to scar formation (ulcers; Graham et
al. 1992).

25. Rule 2 selects the
two reactants that are
required for
regeneration
Rule 3 recognizes the
essential modification of the
wound healing process that
must be realized prior to
regeneration
M. Nakao et. al 2015
Modification of
Bianchi method
for intestinal
regeneration

26. BIANCHI
METHOD
- Procedure to lengthen
the intestine by cutting
it into halves and then
connect the other half at
the other end to make
the length longer.
- The semicircular
intestine will be sutured
to mesentry.

27. SURGERY –
SPONTANEOUS
WOUND
HEALING -
SCAR

28. Rule 4 identifies
three structural
features of scaffolds
that are required for
regenerative activity
1. Pore size
2. Degradation rate
3. Surface chemistry
I.V. Yannas. 2013

29. Cell-cell cluster inside the MFB
capsule in 400 um average pore size
Cell-cell cluster inside the MFB
capsule in 40 um average pore size
I.V. Yannas. 2013

30. I.V. Yannas. 2013

31. DECELLULARIZED
MATRICES
Imperfections in function of
regenerated organs were noted by
several investigators who used
decellularized matrices. These
have included abnormally high
stiffness of a regenerated bladder
[67] and lack of restoration of
physiologic voiding of the bladder
[66].
66. Horst M et al. Engineering functional bladder tissues. 2012.
67. Brown AL et al. 22 week assessment of bladder acellular
matrix in a porcine model. 2002.
Decellularized skin
Right. M. Shahbuddin et al. Inhibition of TE skin model
contraction in preparation

32. Right. M. Shahbuddin et al. Inhibition of contraction in TE skin
model in preparation
0 2 4 6 8 10 12 14 16
40
50
60
70
80
90
100
110
***
***
%reduction
ofTEskin
Time (Day)
Control
1 mg.mL
-1
KGM
5 mg.mL
-1
KGM
0.5% crosslinked KGM
1% crosslinked KGM
Graph conetwork (24% KGM and 1% Ce(IV)
***

33. IN VIVO or IN VITRO SYNTHESIS?
SKIN NERVE
I.V. Yannas. 2013

34. CONCLUSION
At this time, organ transplantation is
on the decline, autografting is very
active though limited largely to skin
and heart bypass surgery, permanent
prostheses are used more and more,
in vitro organ synthesis has greatly
frustrated investigators while in vivo
studies have led to the emerging
field of regenerative medicine.
I.V. Yannas. 2013

35. RISKS AND FUTURE
OF TISSUE
ENGINEERING FOR
ENTERIC NERVOUS
-Risks associated with the
performance of the final product
such as malabsorption and motility
is a major concern.
- Risks that the regeneration
process may not yield tissue with
adequate mechanical or physical
properties, which could result in
life-threatening situations.