Uterine Tissue Engineering

BY
S R I V I D YA H
MIRM-MAHE
Tissue Engineering-General Concepts
“An interdisciplinary field that applies the principles of engineering and life sciences
towards the development of biological substitutes that restore, maintain, or improve
tissue function or a whole organ”
Langer and Vacanti, Science 1993

 Tissue engineering is the use of a combination

Cells
(Stem cells, Progenitor of cells, materials, and suitable factors to
cells, Differentiated Cells) improve or replace biological tissues.

Biomaterials
(Synthetic, Bioactive molecules
natural, (Growth factors,
Extracellular Chemotactic
matrix) factor)

2
A collagen scaffold loaded with human umbilical cord-derived mesenchymal stem
cells facilitates endometrial regeneration and restores fertility
Liaobing Xin et,al. 1742-7061/ 2019 Acta Materialia Inc. Published by Elsevier Ltd

Statement of Significance:
 Intrauterine adhesions due to severe endometrium injuries happen frequently in clinic and become one of
the crucial reasons for women’s infertility or miscarriage.
Therefore, to regenerate the damaged endometrium is a big challenge.
 In this study, a collagen scaffold (CS) loaded with human umbilical cord-derived mesenchymal stem cells
(UC-MSCs) was fabricated and applied for endometrium regeneration.
Herein, UC-MSCs, known for low immunogenicity and high proliferative potential, exhibit promising
potential for endometrium regeneration; and collagen scaffolds provide suitable physical support.
It was proved that transplantation with CS/UC-MSCs promoted endometrial regeneration and fertility
restoration.
 It suggested that topical administration of CS/UC-MSCs in uterus could be a promising strategy for patients
suffering severe intrauterine adhesion and infertility.
 Materials and methods:
1.UC-MSC monoculture Frozen at passage 3 were purchased
2. Human endometrial stromal cell (HESC) were obtained from fresh
endometrial specimens
3.Fabrication of CS/UC-MSCs
Collagen type I was obtained from bovine tendon by tryptic digestion
and acid dissolution and was injected into a polytetrafluoroethylene
mold, frozen
4. . Effect of CS/UC-MSCs on HESC proliferation
UC-MSCs was placed on top of the HESC-seeded plate and checked for
apoptosis and proliferation.
5. Rat model of endometrial damage and transplantation of
CS/UCMSCs
A 3-cm longitudinal incision was made in the uterus to expose the
inner endometrium. The endometrium was scraped using a T10 scalpel
blade until the surface of the uterus was rough
, a CS/UC-MSCs (2.5 cm 0.5 cm) was transplanted onto the damaged
endometrial surface
6. Histological and immunohistological analysis
Samples with a length of 0.5 cm were harvested from uterine wound sites near the cervix at
each time interval. The sections were stained with HE and Masson trichrome using conventional
protocols. The thickness of the regenerated endometrium was measured
7.Fertility test A total of 60 rats (120 uterine horns) were used in the fertility test.
 Results

. Conclusions A CS/UC-MSCs was fabricated and applied for endometrial regeneration. In vitro studies showed that the CS/UC-
MSCs promoted HESC proliferation and inhibited apoptosis through paracrine effects. The transplanted CS/UC-MSCs maintained
luminal structures, and promoted endometrium regeneration and collagen remodeling in a rat model of endometrial damage
 Regeneration of Uterine Horns in Rats Using Collagen Scaffolds Loaded with
Human Embryonic Stem Cell-Derived Endometrium-Like Cells
Tianran Song et,al. TISSUE ENGINEERING: Part A Volume 21, Numbers 1 and 2, 2015 ª Mary Ann Liebert, Inc

This study aimed at efficiently generating endometrium-like cells from the hESCs and at using these cells with
collagen scaffold to repair uterine damage.
The hESCs were induced by co-culturing with endometrial stromal cells, and simultaneously added cytokines:
epidermal growth factor (EGF), platelet-derived growth factor-b (PDGF-b), and E2.
Expression of cell specific markers was analyzed by immunofluorescence and reverse trascription-polymerase chain
reaction to monitor the progression toward an endometrium-like cell fate.
Then established the uterine full-thickness-injury rat models to test cell function in vivo.
 hESC-derived cells were dropped onto collagen scaffolds and transplanted into the animal model.
 Twelve weeks after transplantation, discovered that the hESC-derived cells could survive and recover the structure
and function of uterine horns in a rat model of severe uterine damage.
 The experimental system presented here provides a reliable protocol to produce endometrium-like cells from
hESCs. These results encourage the use of hESCs in cell replacement therapy for severe uterine damage in future.
Transplantation experiment. (A) hESC-derived cells were
isolated, resuspended, and dropped on to the collagen
scaffold.
(B) Collagen scaffolds with or without hESC-derived cells
were grafted to the remaining uterine horns.
(C) Gross view of intrauterine adhesions and distal
hydrometra in natural regeneration group at 4 weeks after
grafting. The arrowheads indicate hydrometra.
(D, E) Gross view of reconstructed uterine horns in
cells/collagen group at 4 weeks (D) and at 12 weeks (E) after
grafting. The arrowheads indicate repair sites. (F)
Immunostaining analyses on uterine slices of grafted rats
killed at 12 weeks after transplantation in the cells/ collagen
group. Anti-human nucleus antigen (HN) was used to
demonstrate the presence of human cells. HN staining is in
red, and nuclei were stained with DAPI (blue).. (G–I)
Pregnancy in uterine horns at 12 weeks after surgery.
Pregnancies in the natural regeneration (G) and collagen
group (H) generally implanted in the normal tissue. In the
cells/collagen group (I), embryos can be implanted in the
grafted tissue. The arrowheads show the margins of the
grafted tissue.
 Allo- and Xeno-Reassembly of Human and Rat Myometrium from Cells and
Scaffolds
Roger C. Young, MD, PhD, and Gabriela Goloman, MD, TISSUE ENGINEERING: Part A Volume 19, Numbers 19 and 20, 2013 ª Mary Ann Liebert, Inc.

Introduction
The dual purpose of the uterus is to accommodate the growing fetus, and then expel the fetus at term by phasically
contracting it.
 The first function requires physical robustness as well as the ability to expand and remodel.
Congenital anomalies or previous surgeries can mechanically compromise the uterus and lead to major complications in
pregnancy.
The second function utilizes multiple interactions of complex physiological mechanisms that have yet to be fully
elucidated, and this knowledge gap contributes to the continuation of serious complications of pregnancy.
To address both problems, reconstructed myometrium from isolated myocytes and scaffold
The ideal scheme to repair defects of the uterus is to create an autologous neo-myometrium that is of a greater volume
than a harvested tissue specimen.
While the number of myocytes can be greatly increased using monolayer culture techniques, scaffold size cannot be
easily increased. Hence, it may be necessary to use a nonautologous donor scaffold., therefore, culture human
myometrial myocytes into rat myometrial scaffold to create xeno-neo-myometrium as proof of principle for repair of
uterine defects.
 Allo- and Xeno-Reassembly of Human and Rat Myometrium
from Cells and Scaffolds
Roger C. Young, MD, PhD, and Gabriela Goloman, MD

Objective: To create neo-myometrium that retains the ability to generate force Histology. hematoxylin and eosin (H + E),
Materials and Methods: desmin, and Masson’s trichrome staining.
Isolation of scaffold (decellularization):
Live/dead assays.
Human- Human myometrium Strips were placed on Petri dish. Cells were destroyed by
exposing the strips to 70% ethanol. Isometric contractility studies
Rat: Full-thickness myometrium was trimmed into a rectangle shape and cells were destroyed
by using ethanol.
Isolation of human and rat myocytes (dispersion): By trypsinization and mechanical
method
Three-dimensional culture of myocytes into myometrial scaffolds
Human myocytes onto human scaffolds
Human myocytes onto rat scaffolds
Rat myocytes onto rat scaffolds
 Results

1.Human cells attached and grew in thin layers on the surface of human myometrial scaffolds.
 the myocytes overgrew each other, but they then detached, retracted, and never achieved a thick layer of
myocytes on the scaffold surface.
Even though individual myocytes penetrated the scaffold to moderate depths, they did not associate with each
other or create structures that resembled native bundles.
 The inability to directly repopulate the human scaffold into bundle-like structures is perhaps because the
physical collapse of the scaffold that occurred after the cells were removed did not allow enough space for the
cells to easily penetrate or proliferate
. These observations are consistent with our failure to observe coordinated contractions in allo-neo-
myometrium.
2. Human myocytes on rat myometrial scaffold,
overgrew themselves and formed multicellular layers on the surface.
the surface multicellular layers were thicker, and crucially, clusters of cells were observed within the depths of
the rat scaffold.
 This xeno-neo-myometrium demonstrated structural integrity, good cellularity, and excellent cellular viability.
The observation of coordinated contractions, although not as forceful as native myometrium, provides proof of
principle that neo-myometrium can potentially be used to systematically study the relationship between
scaffold and myocytes in the generation of phasic contractions.
3.Rat myocytes onto rat scaffolds. Interestingly, using the scaffold preparation described earlier, rat scaffold did
not support culture of rat myocytes as well as human myocytes.
 Isometric contractile
test
Native myometrium contracts in a coordinated, rhythmic
pattern when placed on tension in a muscle bath.
Attempted to observe similar contractions from human
myocytes cultured into human scaffold (allo-neo-
myometrium). These strips were able to withstand 900
mg of applied tension, but coordinated phasic
contractions were not appreciated , although small
irregularly appearing forces were observed
When human myocytes were cultured into rat scaffold,
The peak forces are much less than what is typically
observed in native tissues
 Reconstruction of Engineered Uterine Tissues Containing Smooth
Muscle Layer in Collagen/Matrigel Scaffold In Vitro
Shuang-Hong Lü et,al. 2008, TISSUE ENGINEERING: Part A, Mary Ann Liebert, Inc

Objective: This study attempted to reconstruct engineered uterine tissues (EUTs) containing smooth muscle
layer, akin to the normal uterine wall.
Methods
Methods:

Agar
Agar mold
mold method
method Collagen
Collagen
Matrigel
Matrigel method
method

1. Agar mold method: Sterile agar molds are prepared and poured on the well plates
smooth muscle cells were mixed with type I collagen containing 10% Matrigel The mixture was then pipetted
into the mold and allow hardening.
After that, the stromal cells were mixed with the same collagen=Matrigel mixture and pipetted on top of the
previously formed smooth muscle layer to form a stromal layer, the epithelial cells were seeded on the surface of
the stromal layer.
  2. Collagen/ Matrigel method: the smooth muscle layer was reconstructed exactly in the same
as that in the first method.
 the epithelial cells were not seeded on the surface of the stromal layer, but in the stromal layer;
 the epithelial and stromal cells were mixed in 1:2 before mixing with the collagen=Matrigel
 pipetted on top of the smooth muscle layer to form an epithelial–stromal layer.
 To determine whether the epithelial cells could self-assemble to form an epithelial layer and a
glandular structure when cultured in vitro.

 After 14 days of culture, the structure of the EUTs was analyzed histologically and immunohistochemically, or by scanning
electron microscopy (SEM) and transmission electron microscopy (TEM).
 The expression of integrin b3 subunit, heparin-binding epidermal growth factor (EGF)–like growth factor (HB-EGF), and
HOXA-10 was detected by reverse transcription–polymerase chain reaction (RT-PCR).
 The ability of the EUTs supporting the development of embryos was estimated by coculturing embryos on the EUTs
Results : The results found that the constructed EUTs with the first and
the second method showed three-layered structures.
The epithelial layer, stromal layer, and smooth muscle layer were
stained by cytokeratin 18, vimentin, and a-actin,
The coculture system of EUTs improved the development rate and
quality of murine embryo significantly in comparison with those of
control Chatot Ziomek Bavister culture.
 In the EUTs reconstructed by the second method, the epithelial cells
demonstrated self-assembling ability and formed epithelial cell layer on
top of the stromal layer and glandular tube–like structures in the stromal
layer.
Conclusion:
Engineered EUTs containing smooth muscle layer by two methods. The
reconstructed EUTs could support the development of embryos. The
epithelial cells showed self-assembling ability in the EUT
 A tissue-engineered uterus supports live births in rabbits
Renata S. Magalhaes et,al. Nat Biotechnol (2020)

Bioengineered uterine tissue could provide a treatment option for women with uterine factor infertility.
In large animal models, reconstruction of the uterus has been demonstrated only with xenogeneic tissue grafts.
 Here in this paper biodegradable polymer scaffolds seeded with autologous cells to restore uterine structure and
function in rabbits.
 Rabbits underwent a subtotal uterine excision and were reconstructed with autologous cell-seeded constructs,
with nonseeded scaffolds or by suturing.
 At 6 months postimplantation, only the cell-seeded engineered uteri developed native tissue-like structures,
including organized luminal/ glandular epithelium, stroma, vascularized mucosa and two-layered myometrium.
 rabbits with cell-seeded constructs had normal pregnancies (four in ten) in the reconstructed segment of the
uterus and supported fetal development to term and live birth.
With further development, this approach may provide a regenerative medicine solution to uterine factor
infertility.
 Materials and
methods

full excision of one uterine horn and a

subtotal excision of the remaining
uterine horn,

a tissue-engineered uterine edges

a nonseeded were sutured
scaffold
scaffold together
 Methods
Characterization of cultured primary uterine cell.
Cells used for seeding the scaffolds were collected
from a full-thickness biopsy of the excised uterine
horn.
Fabrication of the uterine constructs. PGA/PLGA
scaffolds were tailor-made in semicircular shapes .
Scanning electron microscopy (SEM) micrographs of
representative samples of the synthetic polymer
scaffolds depicted a three-dimensionally
interconnected porous network
Implantation of the uterine constructs.
In vivo analyses of uterine constructs.
Immuno-histological/molecular findings. Tissue cross-
sections were stained with Masson’s trichrome and
used to calculate the relative collagen and smooth
muscle content in samples retrieved at 1, 3 and
6months.
 Results
Pregnancy outcomes. To investigate the in vivo functionality of the engineered uterine

tissue
Pregnancy studies were done in 42 of the 78 rabbits.
 Pregnancies were identified 29–30d after successful mating using X-rays.
Location of the fetuses inside the uterus was investigated via computed tomography), which was then confirmed macroscopically
during surgical delivery .
 Only the rabbits receiving the tissue-engineered constructs had normal pregnancies inside the reconstructed segment of the uterus
(four out of ten rabbits).
There were no congenital malformations found at necropsy , and the average delivered fetus body weights were comparable to
normal controls , suggesting that the engineered uteri supported normal fetal development.
The nonseeded scaffold constructs and subtotal excision controls had no fetal development or placentation at the reconstructed
segment of the uterine horn.
The results indicate that the tissue-engineered uteri achieved reproductive function, and responded to the expansion and
mechanical strains that occur during pregnancy, allowing appropriate growth and intrautero survival of the fetuses until late stages,
supporting live births.
Thank You