TY - JOUR
T1 - Establishment of a bi-layered tissue engineered conjunctiva using a 3D-printed melt electrowritten poly-(ε-caprolactone) scaffold
AU - Xie, Jiajun
AU - Gao, Qi
AU - del Prado, Zelmira Nuñez
AU - Venkateswaran, Nandini
AU - Mousa, Hazem M.
AU - Salero, Enrique
AU - Ye, Juan
AU - De Juan-Pardo, Elena M.
AU - Sabater, Alfonso L.
AU - Perez, Victor L.
PY - 2023/1
Y1 - 2023/1
N2 - Purpose: To utilize melt electrowriting (MEW) technology using poly-(ε-caprolactone) (PCL) coupled with a 2-step co-culturing strategy for the development of a conjunctival bi-layer synthetic construct. Methods: Melt electrowritten scaffolds using PCL were fabricated using an in-house-built MEW printer. Human conjunctival stromal cells (CjSCs) and epithelial cells (CjECs) were isolated from donor tissue. A 2-step co-culture method was done by first seeding the CjSCs and culturing for 4 weeks to establish a stromal layer, followed by CjECs and co-culturing for 2 more weeks. Cultured cells were each characterized by morphology and marker expression on immunofluorescence and qPCR. The produced construct was assessed for cellular proliferation using viability assays. The bi-layer morphology was assessed using scanning electron microscopy (SEM), confocal microscopy, and immunofluorescence imaging. The expression of extracellular matrix components and TGF-b was evaluated using qPCR. Results: CjSCs were spindle-shaped and vimentin + while CjECs were polygonal and CK13 +. CjSCs showed consistent proliferation and optimal adherence with the scaffold at the 4-week culture mark. A 2-layered construct consisting of a CjSC-composed stromal layer and a CjEC-composed epithelial layer was appreciated on confocal microscopy, SEM, and immunofluorescence. CjSCs secreted collagens (types I, V, VI) but at differing amounts from natural tissue while TGF-b production was comparable. Conclusion: The 3D-printed melt electrowritten PCL scaffold paired with the 2-step co-culturing conditions of the scaffold allowed for the first approximation of a bi-layered stromal and epithelial reconstruction of the conjunctiva that can potentially improve the therapeutic arsenal in ocular surface reconstruction.
AB - Purpose: To utilize melt electrowriting (MEW) technology using poly-(ε-caprolactone) (PCL) coupled with a 2-step co-culturing strategy for the development of a conjunctival bi-layer synthetic construct. Methods: Melt electrowritten scaffolds using PCL were fabricated using an in-house-built MEW printer. Human conjunctival stromal cells (CjSCs) and epithelial cells (CjECs) were isolated from donor tissue. A 2-step co-culture method was done by first seeding the CjSCs and culturing for 4 weeks to establish a stromal layer, followed by CjECs and co-culturing for 2 more weeks. Cultured cells were each characterized by morphology and marker expression on immunofluorescence and qPCR. The produced construct was assessed for cellular proliferation using viability assays. The bi-layer morphology was assessed using scanning electron microscopy (SEM), confocal microscopy, and immunofluorescence imaging. The expression of extracellular matrix components and TGF-b was evaluated using qPCR. Results: CjSCs were spindle-shaped and vimentin + while CjECs were polygonal and CK13 +. CjSCs showed consistent proliferation and optimal adherence with the scaffold at the 4-week culture mark. A 2-layered construct consisting of a CjSC-composed stromal layer and a CjEC-composed epithelial layer was appreciated on confocal microscopy, SEM, and immunofluorescence. CjSCs secreted collagens (types I, V, VI) but at differing amounts from natural tissue while TGF-b production was comparable. Conclusion: The 3D-printed melt electrowritten PCL scaffold paired with the 2-step co-culturing conditions of the scaffold allowed for the first approximation of a bi-layered stromal and epithelial reconstruction of the conjunctiva that can potentially improve the therapeutic arsenal in ocular surface reconstruction.
KW - Melt electrowriting
KW - Ocular surface
KW - Ophthalmology
KW - Tissue engineering
UR - http://www.scopus.com/inward/record.url?scp=85135488882&partnerID=8YFLogxK
U2 - 10.1007/s10792-022-02418-y
DO - 10.1007/s10792-022-02418-y
M3 - Article
C2 - 35932420
AN - SCOPUS:85135488882
SN - 0165-5701
VL - 43
SP - 215
EP - 232
JO - International Ophthalmology
JF - International Ophthalmology
IS - 1
ER -