TY - JOUR
T1 - Hypoxic Three-Dimensional Cellular Network Construction Replicates Ex Vivo the Phenotype of Primary Human Osteocytes
AU - Choudhary, Saba
AU - Sun, Qiaoling
AU - Mannion, Ciaran
AU - Kissin, Yair
AU - Zilberberg, Jenny
AU - Lee, Woo Y.
N1 - Publisher Copyright:
© 2018, Mary Ann Liebert, Inc.
PY - 2018/3
Y1 - 2018/3
N2 - Osteocytes are deeply embedded in the mineralized matrix of bone and are nonproliferative, making them a challenge to isolate and maintain using traditional in vitro culture methods without sacrificing their inimitable phenotype. We studied the synergistic effects of two microenvironmental factors that are vital in retaining, ex vivo, the phenotype of primary human osteocytes: hypoxia and three-dimensional (3D) cellular network. To recapitulate the lacunocanalicular structure of bone tissue, we assembled and cultured primary human osteocytic cells with biphasic calcium phosphate microbeads in a microfluidic perfusion culture device. The 3D cellular network was constructed by the following: (1) the inhibited proliferation of cells entrapped by microbeads, biomimetically resembling lacunae, and (2) the connection of neighboring cells by dendrites through the mineralized, canaliculi-like interstitial spaces between the microbeads. We found that hypoxia synergistically and remarkably upregulated the mature osteocytic gene expressions of the 3D-networked cells, SOST (encoding sclerostin) and FGF23 (encoding fibroblast growth factor 23), by several orders of magnitude in comparison to those observed from two-dimensional and normoxic culture controls. Intriguingly, hypoxia facilitated the self-assembly of a nonproliferating, osteoblastic monolayer on the surface of the 3D-networked cells, replicating the osteoblastic endosteal cell layer found at the interface between native bone and bone marrow tissues. Our ability to replicate, with hypoxia, the strong expressions of these mature osteocytic markers, SOST and FGF23, is important since these (1) could not be significantly produced in vitro and (2) are new important targets for treating bone diseases. Our findings are therefore expected to facilitate ex vivo studies of human bone diseases using primary human bone cells and enable high-throughput evaluation of potential bone-targeting therapies with clinical relevance.
AB - Osteocytes are deeply embedded in the mineralized matrix of bone and are nonproliferative, making them a challenge to isolate and maintain using traditional in vitro culture methods without sacrificing their inimitable phenotype. We studied the synergistic effects of two microenvironmental factors that are vital in retaining, ex vivo, the phenotype of primary human osteocytes: hypoxia and three-dimensional (3D) cellular network. To recapitulate the lacunocanalicular structure of bone tissue, we assembled and cultured primary human osteocytic cells with biphasic calcium phosphate microbeads in a microfluidic perfusion culture device. The 3D cellular network was constructed by the following: (1) the inhibited proliferation of cells entrapped by microbeads, biomimetically resembling lacunae, and (2) the connection of neighboring cells by dendrites through the mineralized, canaliculi-like interstitial spaces between the microbeads. We found that hypoxia synergistically and remarkably upregulated the mature osteocytic gene expressions of the 3D-networked cells, SOST (encoding sclerostin) and FGF23 (encoding fibroblast growth factor 23), by several orders of magnitude in comparison to those observed from two-dimensional and normoxic culture controls. Intriguingly, hypoxia facilitated the self-assembly of a nonproliferating, osteoblastic monolayer on the surface of the 3D-networked cells, replicating the osteoblastic endosteal cell layer found at the interface between native bone and bone marrow tissues. Our ability to replicate, with hypoxia, the strong expressions of these mature osteocytic markers, SOST and FGF23, is important since these (1) could not be significantly produced in vitro and (2) are new important targets for treating bone diseases. Our findings are therefore expected to facilitate ex vivo studies of human bone diseases using primary human bone cells and enable high-throughput evaluation of potential bone-targeting therapies with clinical relevance.
KW - 3D culture
KW - Primary human osteocytes
KW - bone tissue engineering
KW - hypoxia
KW - microfluidics
KW - sclerostin
UR - http://www.scopus.com/inward/record.url?scp=85032189617&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85032189617&partnerID=8YFLogxK
U2 - 10.1089/ten.tea.2017.0103
DO - 10.1089/ten.tea.2017.0103
M3 - Article
C2 - 28594289
AN - SCOPUS:85032189617
SN - 1937-3341
VL - 24
SP - 458
EP - 468
JO - Tissue Engineering - Part A
JF - Tissue Engineering - Part A
IS - 5-6
ER -