Bone is constantly exposed to a range of macro-scale loading which creates a complex mechanical microenvironment for its resident cells. One such mechanical stress generated is hydrostatic pressure (HP) which plays an important role in cell function and fate determination. Although HP is a constant mechanical cue for bone resident cells, little is known about the effect of this external stimuli in a 3D microenvironment. Inspired by native bone mechanical microenvironment, this research studied for the first time the effect of different ranges of cyclic HP on human adipose-derived mesenchymal stem cells (hASCs) encapsulated in a 3D liquefied microcapsule. In the proposed system, while encapsulated hASCs were free in a liquid environment, surface functionalized microparticles were provided as cell attachment sites. In the first step of the study, different ranges of HP (10-250 MPa) were applied to the hASCs for 10 minutes, to find the maximum magnitude that cells could survive. According to the results, 50 MPa was the highest applicable pressure without jeopardizing cellular viability. Then, cyclic HP (6 cycles of 10 minutes) was applied to the hASCs encapsulated in microcapsules in a low (5 MPa) or a high (50 MPa) magnitude.
The electrospraying technique was employed to produce alginate microgels encapsulating hASCs and microparticles in a calcium chloride bath . Using alginate microgels as templates, a multilayered membrane made of poly(L-lysine), chitosan, and alginate polyelectrolytes were produced via layer-by-layer assembly technology (n=12-layers). After a mild core liquefaction process, liquefied microcapsules were cultured in basal (BAS) or osteogenic (OST) media up to 21 days. Taking advantage of the liquefied core environment of microcapsules, hASCs were exposed to cyclic HP at 5 or 50 MPa magnitudes 3 times/week.
Biological tests including MTS and live-dead assays indicated that hASCs remained viable up to 21 days of culture in all tested conditions. The fluorescence staining of F-actin filaments demonstrated a noticeable increase in cell-cell interactions and network formation of hASCs in the pressurized groups, compared to the non-pressurized group. Being this phenomenon more pronounced in OST condition, the observation confirmed by fluorescent staining of vinculin. Results showed that vinculin distribution increased in response to pressurization, specifically in OST group. More interestingly, a significantly higher alkaline phosphatase activity was detected in 50 MPa group. Furthermore, a greater staining of osteopontin, and hydroxyapatite markers was observed in 50 MPa/OST group.
Overall, this study demonstrated that the proposed liquefied encapsulation system holds great potential as an effective platform for studying the impact of various magnitudes of HP for numerous differentiation purposes. Moreover, results revealed that the beneficial effect of HP for osteogenic differentiation is magnitude dependent. Finally, the highest differentiation effect was observed when both biochemical and mechanical cues were combined (50 MPa, OST).
1. Correia C.R. and Ghasemzadeh-Hasankolaei M. et al., Plos One (2019).
The authors acknowledge support of the doctoral grant (SFRH/BD/147418/2019), the FCT projects CIRCUS (PTDC/BTM-MAT/31064/2017), TETRISSUE (PTDC/BTM-MAT/3201/2020), the European Research Council REBORN (ERC-2021-AdG883370), and the project CICECO-Aveiro Institute of Materials, POCI-01-0145-FEDER-007679 (UID/CTM /50011/2013) and LAQV Research Unit (FCT UID/QUI/50006/2019).