A 3D, Dynamically Loaded Hydrogel Model of the Osteochondral Unit to Study Osteocyte Mechanobiology

Abstract

Osteocytes are mechanosensitive cells that orchestrate signaling in bone and cartilage across the osteochondral unit. The mechanisms by which osteocytes regulate osteochondral homeostasis and degeneration in response to mechanical cues remain unclear. This study introduces a novel 3D hydrogel bilayer composite designed to support osteocyte differentiation and bone matrix deposition in a bone-like layer and to recapitulate key aspects of the osteochondral unit's complex loading environment. The bilayer hydrogel is fabricated with a soft cartilage-like layer overlaying a stiff bone-like layer. The bone-like layer contains a stiff 3D-printed hydrogel structure infilled with a soft, degradable, cellular hydrogel. The IDG-SW3 cells embedded within the soft hydrogel mature into osteocytes and produce a mineralized collagen matrix. Under dynamic compressive strains, near-physiological levels of strain are achieved in the bone layer (≤ 0.08%), while the cartilage layer bears the majority of the strains (>99%). Under loading, the model induces an osteocyte response, measured by prostaglandin E2, that is frequency, but not strain, dependent: a finding attributed to altered fluid flow within the composite. Overall, this new hydrogel platform provides a novel approach to study osteocyte mechanobiology in vitro in an osteochondral tissue-mimetic environment.

Keywords: hydrogels; mechanical loading; mechanobiology; osteochondral; osteocytes.

Figure 1.. A bilayer composite hydrogel was designed to mimic the osteochondral unit and achieve near-physiological levels of strain in the bone layer.

(A) Representative healthy human osteochondral tissue stained with Safranin-O Fast Green. (B) Schematic of the bilayer composite hydrogel. The cartilage layer is comprised of a soft, acellular, non-degradable hydrogel. An enlarged section shows the different components of the bone layer: IDG-SW3 cells (purple) encapsulated in a soft, MMP-degradable hydrogel, which is a continuous matrix infill of the 3D-printed pillar scaffold structure. (C) Representative images of live cells stained green by Calcein-AM, scale bar is 50 μm. Arrows denote dendrite-like cellular protrusions. (D) Schematic of the strain in each layer with an applied strain. (E) Cartilage and (F) bone layer strain as a function of applied strain (n=4).

Figure 2.. IDG-SW3 cells differentiated towards mature osteocytes and deposited bone matrix within the bilayer composite hydrogel.

(A) Study design and corresponding assays at each time point. (B-C) Representative confocal microscopy images of Connexin 43 (red, denoted with arrows) counterstained with DAPI for nuclei (blue) on (B) day 35 within hydrogel by immunohistochemistry and (C) day 3 on collagen type I coated glass dish by immunocytochemistry, scale bar = 20 μm. (D) Normalized gene expression (to Day 0) of osteocyte-marker genes Dmp1 and Sost on Days 0 (pre-encapsulated cells), 1, and 35 (n=3). (E) Photograph of bilayer composite at Day 35 depicting a translucent cartilage layer and opaque bone layer. (F) Representative 3D X-Ray Microscope (XRM) images show mineralization (black) on days 14 and 35. At day 1, there was no detectable mineral content and hence an image was not included. (G) Volume fraction of mineral content from the XRM images (n=3). (H-K) Representative images of day 35 Glycol methacrylate-embedded sections stained with von Kossa for mineralization (black) (H-I) and for collagen type I (red) and counterstained with DAPI for nuclei (blue) (J-K). Scale bar = 100 μm (H, J), 20 μm (I), 10 μm (K). In (H) and (J) 3D-printed pillar regions are outlined with dotted circles. Symbols denote significance from post hoc Tukey’s test (D) or two-sided t-test (G): * p<0.05; **p<0.01; ***p<0.001.

Figure 3.. Osteocytes could sense and respond to loading within the bilayer composite hydrogel.

(A) Experimental loading regimes. (B) PGE₂ concentration in the media directly after loading as a function of applied strain, frequency, and applied strain rate. Outliers were removed and experimental sample size of all groups was 6, except for the 20% applied strain/1 Hz frequency condition which represents a sample size of 12. Symbols denote significance of post hoc Tukey’s test: # compared to unloaded condition; * between strain-matched or frequency-matched conditions. (C) PGE₂ in the media was analyzed directly after loading (n=5) or in unloaded controls (n=3) with or without NS-398, a COX2 inhibitor. Symbols denote significance of post hoc Tukey’s test: # compared to unloaded -NS-398, * compared to unloaded +NS-398; % between NS-398 treatment within the same loading condition. Significance levels: one symbol p<0.05; two symbols p<0.01; three symbols p<0.001.