Osteoarthrosis is a debilitating disease affecting millions, yet engineering materials for cartilage regeneration has proven difficult because of the complex microstructure of this tissue. Articular cartilage, like many biological tissues, produces a time-dependent response to mechanical load that is critical to cell’s physiological function in part due to solid and fluid phase interactions and property variations across multiple length scales. Recreating the time-dependent strain and fluid flow may be critical for successfully engineering replacement tissues but thus far has largely been neglected. Here, microindentation is used to accomplish three objectives: (1) quantify a materials time-dependent mechanical response, (2) map material properties at a cellular relevant length scale throughout zonal articular cartilage, (3) and elucidate the underlying viscoelastic, poroelastic, and nonlinear poroelastic causes of deformation in articular cartilage. Untreated and trypsin-treated cartilage were sectioned perpendicular to the articular surface and indentation was used to evaluate properties throughout zonal cartilage on the cut surface. The experimental results demonstrated that within all cartilage zones, the mechanical response was well represented by a model assuming nonlinear biphasic behavior and did not follow conventional viscoelastic or linear poroelastic models. Additionally, 10% (w/w) agarose was tested and, as anticipated, behaved as a linear poroelastic material. The approach outlined here provides a method, applicable to many tissues and biomaterials, which reveals and quantifies the underlying causes of time-dependent deformation, elucidates key aspects of material structure and function, and that can be used to provide important inputs for computational models and targets for tissue engineering.

Multi-layer hydrogels are promising for tissue engineering due to the ability to control the local properties within each layer. However, the interface that forms between each layer has the potential to affect the performance of the hydrogel. The goals of this study were to characterize how the interface forms via its thickness and mechanical properties, identify its impact on the overall hydrogel properties, and provide new insights into how to control the interface. A photo-clickable poly(ethylene glycol) hydrogel was used to form bilayer hydrogels that were sequentially polymerized in a step-and-repeat process. Different processing conditions were studied: the time (0-20min) before initiating polymerization of the second layer (soak time, t) and the hydrogel crosslink density (the same, less crosslinked, or more crosslinked) of the first layer as compared to the second layer. Interface thickness was characterized by confocal microscopy, monomer transport by Fickian diffusion, single and bilayer hydrogel mechanics by bulk moduli measurements, and interface moduli measurements using AFM, nanoindentation, and strain mapping. The interface thickness ranged from ~70 to 600μm (1-10% of total height) depending on processing conditions, but did not affect the bulk hydrogel modulus. Analysis of monomer transport revealed that convection, due to changes in hydrogel swelling, and diffusion contribute to interface thickness. Nanomechanical analysis of bilayer hydrogels formed from soft (75kPa) and stiff (250kPa) layers showed a gradient in elastic modulus across the interface, which corresponded to strain maps. In summary, this work identifies that diffusive and convective transport of monomers across the interface controls its thickness and that a mechanically robust interface forms, which does not affect the hydrogel modulus. By controlling the processing conditions, the thickness of the interface can be tuned without affecting the mechanical properties of the bulk hydrogel.

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.

Biomaterial-based tissue engineering strategies hold great promise for osteochondral tissue repair. Yet significant challenges remain in joining highly dissimilar materials to achieve a biomimetic, mechanically robust design for repairing interfaces between soft tissue and bone. This study sought to improve interfacial properties and function in a bilayer, multi-phase hydrogel interpenetrated with a fibrous collagen scaffold. ‘Soft’ 10% (w/w) and ‘stiff’ 30% (w/w) PEGDM was formed into mono- or bilayer hydrogels possessing a sharp diffusional interface. Hydrogels were evaluated as single- (hydrogel only) or multi-phase (hydrogel+fibrous scaffold penetrating throughout the stiff layer and extending >500μm into the soft layer). Including a fibrous scaffold into both soft and stiff single-phase hydrogels significantly increased tangent modulus and toughness and decreased lateral expansion under compressive loading. In multi-phase hydrogels, finite element simulations predict substantially reduced stress and strain gradients across the soft—stiff hydrogel interface. When combining two low moduli constituent material, composites theory poorly predicts the observed, large modulus increases. These results suggest material structure associated with the fibrous scaffold penetrating within the PEG hydrogel as the major contributor to improved properties and function – the hydrogel bore compressive loads and the 3D fibrous scaffold was loaded in tension thus resisting lateral expansion.

One major challenge observed for the expression of therapeutic bispecific antibodies (BisAbs) is high product aggregates. Aggregates increase the risk of immune responses in patients and therefore must be removed at the expense of purification yields. BisAbs contain engineered disulfide bonds, which have been demonstrated to form product aggregates, if mispaired. However, the underlying intracellular mechanisms leading to product aggregate formation remain unknown. We demonstrate that impaired glutathione regulation underlies BisAb aggregation formation in a CHO cell process. Aggregate formation was evaluated for the same clonal CHO cell line producing a BisAb using fed-batch and perfusion processes. The perfusion process produced significantly lower BisAb aggregates compared to the fed-batch process. Perfusion bioreactors attenuated mitochondrial dysfunction and ER stress resulting in a favorable intracellular redox environment as indicated by improved reduced to oxidized glutathione ratio. Conversely, mitochondrial dysfunction-induced glutathione oxidation and ER stress disrupted the intracellular redox homeostasis, leading to product aggregation in the fed-batch process. Combined, our results demonstrate that mitochondrial dysfunction and ER stress impaired glutathione regulation leading to higher product aggregates in the fed-batch process. This is the first study to utilize perfusion bioreactors as a tool to demonstrate the intracellular mechanisms underlying product aggregation formation.