Hydrostatic Pressure Differentially Regulates Outer and Inner Annulus Fibrosus Cell Matrix Production in 3D Scaffolds

Abstract

Mechanical stimulation may be used to enhance the development of engineered constructs for the replacement of load bearing tissues, such as the intervertebral disc. This study examined the effects of dynamic hydrostatic pressure (HP) on outer and inner annulus (OA, IA) fibrosus cells seeded on fibrous poly(glycolic acid)-poly(L-lactic acid) scaffolds. Constructs were pressurized (5 MPa, 0.5 Hz) for four hours/day from day 3 to day 14 of culture and analyzed using ELISAs and immunohistochemistry (IHC) to assess extracellular matrix (ECM) production. Both cell types were viable, with OA cells exhibiting more infiltration into the scaffold, which was enhanced by HP. ELISA analyses revealed that HP had no effect on type I collagen production while a significant increase in type II collagen (COL II) was measured in pressurized OA constructs compared to day 14 unloaded controls. Both OA and IA dynamically loaded scaffolds exhibited more uniform COL II elaboration as shown by IHC analyses, which was most pronounced in OA-seeded scaffolds. Overall, HP resulted in enhanced ECM elaboration and organization by OA-seeded constructs, while IA-seeded scaffolds were less responsive. As such, hydrostatic pressurization may be beneficial in annulus fibrosus tissue engineering when applied in concert with an appropriate cell source and scaffold material. scaffolds. Constructs were pressurized (5 MPa, 0.5 Hz) for four hours/day from day 3 to day 14 of culture and analyzed using ELISAs and immunohistochemistry (IHC) to assess extracellular matrix (ECM) production. Both cell types were viable, with OA cells exhibiting more infiltration into the scaffold, which was enhanced by HP. ELISA analyses revealed that HP had no effect on type I collagen production while a significant increase in type II collagen (COL II) was measured in pressurized OA constructs compared to day 14 unloaded controls. Both OA and IA dynamically loaded scaffolds exhibited more uniform COL II elaboration as shown by IHC analyses, which was most pronounced in OA-seeded scaffolds. Overall, HP resulted in enhanced ECM elaboration and organization by OA-seeded constructs, while IA-seeded scaffolds were less responsive. As such, hydrostatic pressurization may be beneficial in annulus fibrosus tissue engineering when applied in concert with an appropriate cell source and scaffold material.

Fibrocartilage Tissue Engineering: The Role of the Stress Environment on Cell Morphology and Matrix Expression

“…However, experimental results indicate a role for hydrostatic compression in the enhancement of matrix synthesis relevant to fibrocartilage. 38 Our experimental results supported the initial hypothesis with regard to the fibrous components of fibrocartilage but did not support our hypothesis with regard to the cartilaginous components of fibrocartilage. Consistent with reports in the literature, 16,36 production of fibrous collagenous matrix and changes in cell shape and orientation into a tendon fibroblast morphology was enhanced by both the dynamic and the tensile aspects of loading.…”

Transcriptional profiling distinguishes inner and outer annulus fibrosus from nucleus pulposus in the bovine intervertebral disc

“…Few studies have addressed inner and outer AF cell phenotypes [27][28][29]. In our study, bAcan, bCol2a1, bCD24, bCol1a1, bAdamts17, bSfrp2, bCol5a1 and bCol12a1 did not distinguish inner AF from NP tissue.…”

The effects of dynamic loading on the intervertebral disc

“…It has been shown in various in vivo and in vitro models that a low magnitude of dynamic compression [70,122] and static/dynamic hydrostatic pressure [31,36,43,46,50,65,86,98,124] at a physiological frequency could increase anabolic response, but a high magnitude of dynamic loading causes early signs of disc damage and catabolic remodelling (Tables 2, 3). …”