Chenyu Huang1, Rei Ogawa. 1. Department of Plastic, Reconstructive and Aesthetic Surgery, Nippon Medical School, Tokyo, Japan.
Abstract
BACKGROUND: Mechanics is increasingly being recognized as the fourth essential factor in bone tissue engineering next to cell, scaffold, and growth factors. The development of bioprocessors has made it possible to simulate the in vivo mechanics that are needed to generate three-dimensional (3D) bone constructs. However, although hydrostatic pressure (HP) is a dominant and constant mechanical strain on bone cells in vivo, little is known about the effect of HP applied via perfusion bioprocessors on in vitro human bone marrow-derived mesenchymal stem cell (hMSC) behavior. METHODS: hMSCs underwent primary culture for three passages before being seeded into hydroxyapatite (HA) scaffolds. The scaffolds were incubated for 3 weeks in an automated bioprocessor under cyclic HP. Scaffolds exposed to atmospheric pressure (AP) served as the comparator. Osteogenic differentiation medium was employed for both the HP and AP groups. Immediately before and 1, 2, and 3 weeks after incubation, the scaffolds were harvested for histological, immunohistochemical, and gene expression analyses. RESULTS: Cells were only found in the AP scaffold surfaces, whereas in the HP group, they were distributed evenly throughout the scaffolds. Immunohistochemical analysis revealed that the HP group expressed higher levels of osteocalcin (OC), osteopontin (OP), osteonectin (ON), and collagen type 1 (Col1) than the AP group during the 3-week process. Gene expression analysis revealed that the HP group expressed higher levels of ON, Col1, alkaline phosphatase, and integrin β5 than the AP group at the 1-, 2-, and 3-week timepoints. The HP group also expressed higher levels of core-binding factor α-1 (Cbfa1) at the 2- and 3-week timepoints and higher levels of OP and OC at the 1-week timepoint. Their proliferating cell nuclear antigen levels were lower at the 1- and 2-week timepoints. CONCLUSIONS: HP enhances cellular viability and improves osteogenic differentiation and maturation, although somewhat at the expense of proliferation and self-renewal of MSCs. Possible negative effects of the bioprocessor-induced HP on bone regeneration were not observed. Further, the mechanotransductive molecule integrin β5 was expressed at high levels after HP stimulation and may enhance migration, promote differentiation, and inhibit osteoclast maturation during HP-driven osteogenesis in vitro.
BACKGROUND: Mechanics is increasingly being recognized as the fourth essential factor in bone tissue engineering next to cell, scaffold, and growth factors. The development of bioprocessors has made it possible to simulate the in vivo mechanics that are needed to generate three-dimensional (3D) bone constructs. However, although hydrostatic pressure (HP) is a dominant and constant mechanical strain on bone cells in vivo, little is known about the effect of HP applied via perfusion bioprocessors on in vitro human bone marrow-derived mesenchymal stem cell (hMSC) behavior. METHODS: hMSCs underwent primary culture for three passages before being seeded into hydroxyapatite (HA) scaffolds. The scaffolds were incubated for 3 weeks in an automated bioprocessor under cyclic HP. Scaffolds exposed to atmospheric pressure (AP) served as the comparator. Osteogenic differentiation medium was employed for both the HP and AP groups. Immediately before and 1, 2, and 3 weeks after incubation, the scaffolds were harvested for histological, immunohistochemical, and gene expression analyses. RESULTS: Cells were only found in the AP scaffold surfaces, whereas in the HP group, they were distributed evenly throughout the scaffolds. Immunohistochemical analysis revealed that the HP group expressed higher levels of osteocalcin (OC), osteopontin (OP), osteonectin (ON), and collagen type 1 (Col1) than the AP group during the 3-week process. Gene expression analysis revealed that the HP group expressed higher levels of ON, Col1, alkaline phosphatase, and integrin β5 than the AP group at the 1-, 2-, and 3-week timepoints. The HP group also expressed higher levels of core-binding factor α-1 (Cbfa1) at the 2- and 3-week timepoints and higher levels of OP and OC at the 1-week timepoint. Their proliferating cell nuclear antigen levels were lower at the 1- and 2-week timepoints. CONCLUSIONS: HP enhances cellular viability and improves osteogenic differentiation and maturation, although somewhat at the expense of proliferation and self-renewal of MSCs. Possible negative effects of the bioprocessor-induced HP on bone regeneration were not observed. Further, the mechanotransductive molecule integrin β5 was expressed at high levels after HP stimulation and may enhance migration, promote differentiation, and inhibit osteoclast maturation during HP-driven osteogenesis in vitro.