title: Dynamic culturing of cartilage tissue: the significance of hydrostatic pressure.
abstract: Human articular cartilage functions under a wide range of mechanical loads in synovial joints, where hydrostatic pressure (HP) is the prevalent actuating force. We hypothesized that the formation of engineered cartilage can be augmented by applying such physiologic stimuli to chondrogenic cells or stem cells, cultured in hydrogels, using custom-designed HP bioreactors. To test this hypothesis, we investigated the effects of distinct HP regimens on cartilage formation in vitro by either human nasal chondrocytes (HNCs) or human adipose stem cells (hASCs) encapsulated in gellan gum (GG) hydrogels. To this end, we varied the frequency of low HP, by applying pulsatile hydrostatic pressure or a steady hydrostatic pressure load to HNC-GG constructs over a period of 3 weeks, and evaluated their effects on cartilage tissue-engineering outcomes. HNCs (10×10(6) cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 3 weeks: (1) 0.4 MPa Pulsatile HP; (2) 0.4 MPa Steady HP; and (3) Static. Subsequently, we applied the pulsatile regimen to hASC-GG constructs and varied the amplitude of loading, by generating both low (0.4 MPa) and physiologic (5 MPa) HP levels. hASCs (10×10(6) cells/mL) were encapsulated in GG hydrogels (1.5%) and cultured in a chondrogenic medium under three regimens for 4 weeks: (1) 0.4 MPa Pulsatile HP; (2) 5 MPa Pulsatile HP; and (3) Static. In the HNC study, the best tissue development was achieved by the pulsatile HP regimen, whereas in the hASC study, greater chondrogenic differentiation and matrix deposition were obtained for physiologic loading, as evidenced by gene expression of aggrecan, collagen type II, and sox-9; metachromatic staining of cartilage extracellular matrix; and immunolocalization of collagens. We thus propose that both HNCs and hASCs detect and respond to physical forces, thus resembling joint loading, by enhancing cartilage tissue development in a frequency- and amplitude-dependant manner.
authors.affiliation: University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Taipas, Guimarães, Portugal. ccorreia@dep.uminho.pt
authors.forename: Cristina
authors.initials: C
authors.lastname: Correia
authors.forename: Ana L
authors.initials: AL
authors.lastname: Pereira
authors.forename: Ana R C
authors.initials: AR
authors.lastname: Duarte
authors.forename: Ana M
authors.initials: AM
authors.lastname: Frias
authors.forename: Adriano J
authors.initials: AJ
authors.lastname: Pedro
authors.forename: João T
authors.initials: JT
authors.lastname: Oliveira
authors.forename: Rui A
authors.initials: RA
authors.lastname: Sousa
authors.forename: Rui L
authors.initials: RL
authors.lastname: Reis
mesh_terms: D000328:Adult; D002356:Cartilage / Q000166:cytology*; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology*; D020219:Chondrogenesis / Q000502:physiology; D006801:Humans; D006874:Hydrostatic Pressure; D007150:Immunohistochemistry; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D023822:Tissue Engineering / Q000379:methods*