Osteocytes reside within a heavily mineralized matrix, making them difficult to study in vivo and to extract for studies in vitro. IDG-SW3 cells are capable of producing a mineralized collagen matrix and transitioning from osteoblasts to mature osteocytes, thus offering an alternative to study osteoblast to late osteocyte differentiation in vitro. The goal for this work was to develop a 3D degradable hydrogel to support IDG-SW3 differentiation and deposition of bone extracellular matrix. In 2D, the genes Mmp2 and Mmp13 increased during IDG-SW3 differentiation and were used as targets to create a matrix metalloproteinase (MMP)sensitive poly(ethylene glycol) hydrogel containing the peptide cross-link GCGPLG-LWARCG and RGD to promote cell attachment. IDG-SW3 differentiation in the MMP-sensitive hydrogels improved over nondegradable hydrogels and standard 2D culture. Alkaline phosphatase activity at day 14 was higher, Dmp1 and Phex were 8.1-fold and 3.8-fold higher, respectively, and DMP1 protein expression was more pronounced in the MMP-sensitive hydrogels compared to nondegradable hydrogels. Cellencapsulation density (cells/mL of precursor) influenced the formation of dendrite-like cellular processes and mineral and collagen deposition, with 80 × 10 6 cells/mL of precursor performing better than 2 × 10 6 or 20 × 10 6 cells/mL of precursor, while connexin 43 was not affected by cell density. The cell density effects were more pronounced in the MMP-sensitive hydrogels over nondegradable hydrogels. This study identified that high cell encapsulation density and hydrogels susceptible to cell-mediated degradation enhanced the mineralized collagen matrix and osteocyte differentiation. Overall, a promising hydrogel is presented that supports IDG-SW3 cell maturation from osteoblasts to osteocytes in 3D.

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. Osteocytes are mechanosensitive cells that help to orchestrate signaling in bone and cartilage across the osteochondral unit. Yet the mechanisms by which osteocytes regulate osteochondral

Objective: Quantitative, micrometer length scale assessment of human articular cartilage is essential to enable progress toward new functional tissue engineering approaches, including utilization of emerging 3D bioprinting technologies, and for improved computational modeling of the osteochondral unit. Thus the objective of this study was to characterize the structural organization, material properties, and chemical composition of human skeletally mature articular cartilage with respect to depth and defined morphological features: normal to the articulating surface, parallel to the split-line, and transverse to the split-line. Method: Three samples from the lateral femoral condyles of 4 healthy adult donors (55e61 years old) were evaluated via histology, second harmonic generation, microindentation, and Raman spectroscopy. All metrics were evaluated as a function of depth and direction relative to the split-line. Results: All donors presented with intact and healthy tissue. Collagen fiber orientation varied significantly between testing directions and with increasing depth from the articular surface. Both compressive and tensile modulus increased significantly with depth and differed across the middle and deep zones and depended on orthogonal direction relative to the split-line. Similarly, matrix components varied with both depth and direction, where chondroitin sulfate steadily increased with depth while collagen prevalence was highest in the surface layer. Conclusions: Microscale measurements of human articular cartilage demonstrate that properties are both depth-dependent and orthotropic and depend on the underlying tissue structure and composition. These findings improve upon existing knowledge establishing more accurate measurements, with greater degree of depth and spatial specificity, as inputs for tissue engineering and computational modeling.

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