Reconstruction of engineered uterine tissues containing smooth muscle layer in collagen/matrigel scaffold in vitro.
OBJECTIVE
This study attempted to reconstruct engineered uterine tissues (EUTs) containing smooth muscle layer, akin to the normal uterine wall.


METHODS
EUTs were reconstructed by seeding epithelial cells on top of the constructed stromal layer over smooth muscle layer. A self-made mold was used to keep the EUTs from contraction. At the same time, it provided static stretch to the EUTs. 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 beta3 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. We also tried a new method to reconstruct EUTs by mixing epithelial cell and stromal cells (1:2) in collagen/Matrigel to form an endometrial layer and putting it on top of the smooth muscle layer. The self-assembling ability of the endometrial epithelial cells and stromal cells in the reconstructed EUTs was analyzed histologically and immunohistochemically.


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 alpha-actin, respectively. TEM showed that the cells in the EUTs reconstructed by the first method were attached to each other by apical tight junctions and rivet-like desmosomes. SEM showed protruded pinopodes, microvilli, and cilium of epithelial cells. The RT-PCR analysis showed that integrin beta3 subunit, HB-EGF, and HOXA-10 were expressed in EUTs. 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. Columnar epithelial cells existed in some parts of the epithelial layer.


CONCLUSIONS
We 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 EUTs.
Lü
Shuang-Hong
SH
Department of Tissue Engineering, Beijing Institute of Basic Medical Sciences and Tissue Engineering Research Center, Academy of Military Medical Sciences, Beijing, PR China.
Wang
Hai-Bin
HB
Liu
Hui
H
Wang
He-Ping
HP
Lin
Qiu-Xia
QX
Li
De-Xue
DX
Song
Yu-Xuan
YX
Duan
Cui-Mi
CM
Feng
Li-Xin
LX
Wang
Chang-Yong
CY
D000818:Animals; D003094:Collagen / Q000378:metabolism*; D004338:Drug Combinations; D004622:Embryo, Mammalian / Q000166:cytology; D005260:Female; D018507:Gene Expression Regulation, Developmental; D007150:Immunohistochemistry; D007797:Laminin / Q000378:metabolism*; D051379:Mice; D009130:Muscle, Smooth / Q000502:physiology* / Q000648:ultrastructure; D011509:Proteoglycans / Q000378:metabolism*; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013314:Stress, Mechanical; D023822:Tissue Engineering; D054457:Tissue Scaffolds; D014599:Uterus / Q000166:cytology / Q000502:physiology* / Q000648:ultrastructure
D004338:Drug Combinations; D007797:Laminin; D011509:Proteoglycans; C059485:matrigel; D003094:Collagen