The effects of dynamic compressive loading on human mesenchymal stem cell osteogenesis in the stiff layer of a bilayer hydrogel

Abstract

Bilayer hydrogels with a soft cartilage-like layer and a stiff bone-like layer embedded with human mesenchymal stem cells (hMSCs) are promising for osteochondral tissue engineering. The goals of this work were to evaluate the effects of dynamic compressive loading (2.5% applied strain, 1 Hz) on osteogenesis in the stiff layer and spatially map local mechanical responses (strain, stress, hydrostatic pressure, and fluid velocity). A bilayer hydrogel was fabricated from soft (24 kPa) and stiff (124 kPa) poly (ethylene glycol) hydrogels. With hMSCs embedded in the stiff layer, osteogenesis was delayed under loading evident by lower OSX and OPN expressions, alkaline phosphatase activity, and collagen content. At Day 28, mineral deposits were present throughout the stiff layer without loading but localized centrally and near the interface under loading. Local strains mapped by particle tracking showed substantial equivalent strain (~1.5%) transferring to the stiff layer. When hMSCs were cultured in stiff single-layer hydrogels subjected to similar strains, mineralization was inhibited. Finite element analysis revealed that hydrostatic pressures ≥~600 Pa correlated to regions lacking mineralization in both hydrogels. Fluid velocities were low (~1-10 nm/s) in the hydrogels with no apparent correlation to mineralization. Mineralization was recovered by inhibiting ERK1/2, indicating cell-mediated inhibition. These findings suggest that high strains (~1.5%) combined with higher hydrostatic pressures negatively impact osteogenesis, but in a manner that depends on the magnitude of each mechanical response. This work highlights the importance of local mechanical responses in mediating osteogenesis of hMSCs in bilayer hydrogels being studied for osteochondral tissue engineering.

Keywords: bilayer hydrogel; compressive strain; dynamic loading; hydrostatic pressure; mesenchymal stem cells; osteochondral; osteogenesis.

Figure 1.

Bilayer hydrogel formation and characterization. A) Schematic of fabrication method for forming the bilayer hydrogel through a sequential and repeating process involving deposition of the precursor solution and polymerization using light to form each hydrogel layer. B) Relaxation behavior of soft and stiff hydrogels after being subjected to a 15% compressive strains applied at 2 mm/s. Force was recorded as a function of time. C) The true modulus for poly(ethylene glycol) hydrogels used to form the soft and stiff layers of the bilayer hydrogel. D) Permeability determined from the force relaxation experiments for the soft and stiff hydrogel. Data are reported as mean with standard deviation as error bars or listed parenthetically (n=3).

Figure 2.

Osteogenesis of hMSCs in stiff layer of bilayer hydrogel. A) Schematic of experiment. The hydrogel was either cultured under no loading (unloaded) or subjected to 2.5% amplitude strain at 1 Hz applied 1 hour per day for up to 28 days in osteogenic medium. B-D) Gene expression of hMSCs encapsulated in the stiff layer of the bilayer hydrogel for B) OSX (osterix), C) ALPL (alkaline phosphatase), and D) OPN (osteopontin). Relative gene expression for each gene was normalized to the pre-encapsulated hMSCs, denoted as day 0. E-G) Biochemical assessment of hMSCs encapsulated in the stiff layer of the bilayer hydrogel for E) alkaline phosphatase (ALP) activity, F) total collagen content, and G) total calcium content. Data in B-G are presented as mean with standard deviation as error bars (n=3). P-values are reported from either a two-way ANOVA for each factor of time and loading (ALPL, ALP activity, total calcium) or from simple main effects for time due to a significant two-way interaction (OSX, OPN, total collagen). In B-D, p-values denoted at a time point indicate the significance between unloaded and loaded from post-hoc analysis. In E-G, the * above a column indicates pairwise comparisons with the corresponding p-value from day 1 and the + indicates pairwise comparisons with the corresponding p-value from unloaded at the same time point (* is p<0.05, ** and ++ are p<0.01, and *** is p<0.001) from post-hoc analysis. H) Representative brightfield microscopy of the bilayer hydrogels stained for mineral deposits by von Kossa. Scale bar is 0.5 mm. Inset red boxes are magnified and shown to the right of each image. I) Line plots showing relative mineral intensity (0=white, 1 = black) from the center line of the unloaded (black) and loaded (gray) constructs normalized to distance in the bone layer only.

Figure 3.

Strain heat maps in the bilayer hydrogel under an externally applied compressive strain. A) Schematic of experimental set-up showing silica beads encapsulated throughout the bilayer hydrogel, hemi-sectioned, and then placed into a custom straining rig that sits on the stage of a microscope. B) Brightfield microscopy images of the hemi-sectioned bilayer hydrogel prior to applying an external strain (<0% strain), 15% strain and 25% strain. C) Heat maps of strain prior to applying an external strain (<0% strain), 15% strain and 25% strain in the x-direction (E_xx) parallel to the direction of the applied strain. D) Heat maps of strain at an 25% external applied strain in the normal y-direction (E_yy) perpendicular to the direction of the applied strain, shear xy-direction (E_xy), and the equivalent strain (E_eqv).

Figure 4.

Heat maps of equivalent stress (A,B), hydrostatic pressure (C,D), and fluid velocity (E,F) in the bilayer hydrogel under an externally applied dynamic compressive strain. Finite element analysis was performed on a bilayer hydrogel with soft, stiff and an interfacial layer under unconfined dynamic compression externally applied at an 2.5% amplitude strain at 1 Hz. Heat maps are shown at steady state (during the 18^th cycle) for 1.25% strain during compression and at 2.5% maximum strain. Line plots from the center line of the bone layer for equivalent stress (B), hydrostatic pressure (D) and fluid velocity (F) are shown and overlaid with a representative line plot of mineralization from the hMSC experiment under loading (see Figure 2).

Figure 5.

Biochemical assessment of hMSCs encapsulated in a stiff single layer hydrogel. The hydrogel was subjected to dynamic compressive strains applied at 1 Hz for up to 28 days in osteogenic medium under one of three experimental conditions: 0.15% amplitude strain, 1.5% amplitude strain, and 1.5% amplitude strain with 10 μM PD98059. A) Schematic of experiment. B-C) Biochemical assessments were made for B) alkaline phosphatase (ALP) activity and C) total calcium content. Data are presented as mean with standard deviation as error bars (n=3). P-values are reported from a two-way ANOVA for each factor of time and loading (ALP activity) or from simple main effects for time due to a significant two-way interaction (total calcium). P-values for pairwise comparisons from post-hoc analysis are denoted. The * and ^# symbols above a column indicate the significance level from day 1 and day 7, respectively and the ⁺ symbol above a column indicates the significance level from the 1.5% strain condition. Lines denote pairwise significance between culture condition. The ** and ⁺⁺ denotes p<0.01, ***, ^### and ⁺⁺⁺ denote p<0.001. E) Representative brightfield microscopy of the single layer hydrogels stained for mineral by von Kossa. Scale bar is 100 μm.

Figure 6.

Heat maps of equivalent stress (A,B), hydrostatic pressure (C,D), and fluid velocity (E,F) in single layer hydrogels under an externally applied dynamic compressive strain at a maximum of 0.15% applied strain (A,C,E) or 1.5% applied strain (B,D,F) at 1 Hz. Heat maps are shown at steady state (during the 18^th cycle) for the half-way point during compression and at the maximum strain.