Professor University of Pennsylvania, Pennsylvania, United States
Introduction: In cell and tissue engineering, the mechanical properties of biomaterials play critical roles in directing cell behaviors and achieving tissue functions. Yet, biopolymer network-based hydrogels, despite being widely used as mimics of extracellular matrices, fails to capture the mechanical properties of real tissues in many aspects. For example, while most biological tissues exhibit compression stiffening, wherein their stiffness increases under compression, hydrogels based on fibrin and collagen are compression softening. To address this problem, our group and others have identified a mechanism involving the inclusion of cell-mimicking, volume-conserving spherical particles, which induce local fiber stretching during compression near the particles, leading to compression stiffening. While this model is successful, it has primarily focused on monodisperse, uniformly distributed spherical inclusions, overlooking the effects of irregularly shaped and aggregated inclusions, which more closely resemble the cellular components in biological tissues. This study investigates the impact of aggregates of cell-mimicking particles on the mechanical properties of biopolymer networks. We demonstrate that the inclusion of aggregates of carbonyl iron particles (CIPs) induces compression stiffening in fibrin gels, requiring only a small volume fraction. Importantly, this mechanism differs from previously reported mechanisms. Further investigations reveal that CIP aggregation is crucial for this phenomenon, highlighting the significance of the diverse shape of the aggregates, rather than polydisperse size, in achieving tissue-like mechanical properties in hydrogel composites. Our findings shed light on the formation and regulation of tissue stiffness in physiological and pathological contexts, and offer insights into designing biomaterials with more physiologically relevant mechanical properties.
Materials and
Methods: Fibrin gels were prepared using 5 mg/ml fibrinogen, 1 U/ml thrombin, and 2 mM Ca2+ in T7 buffer (50 mM Tris, 150 mM NaCl, pH 7.4), unless otherwise stated. To prevent particle precipitation and ensure rapid gelation, fibrin gels containing polystyrene beads or coffee grounds employed 3.3 U/ml thrombin. CIPs had an average diameter of 4.5 μm. The volume fraction of the CIP solution was determined by the difference in solution volume before and after CIPs dissolution, assuming no water absorption by the CIPs upon dissolution. Polystyrene beads upon purchase had a volume fraction of 10% and were concentrated via centrifugation before mixing with the fibrin pre-gel solution. Polyacrylamide hydrogels were composed of 3.6% acrylamide, 0.25% bis-acrylamide, 0.3% APS, and 0.25% TEMED. All hydrogels were fabricated to a thickness of 1 mm. The mechanical properties of the hydrogels were determined using a stress-controlled Kinexus rheometer equipped with an 8 mm diameter geometry plate. Hydrogel samples were either glued or gelled between parallel plates. Shear modulus was measured at 1 Hz frequency and 1% shear strain. Axial strains were applied by adjusting the gap size between the geometry plate and the bottom plate during shear oscillation (Fig.1A). Each compression step involved applying 3% axial strain stepwise, up to a total compression of 30%. Following each compression step, the shear modulus exhibited an instantaneous increase followed by relaxation. The next compression was applied only after complete relaxation. All syntheses and measurements were conducted at room temperature.
Results, Conclusions, and Discussions: We first synthesize fibrin embedded with CIPs, which aggregate spontaneously within the gels (Fig.1B), transforming their mechanical behavior from compression softening to compression stiffening (Fig.1C-E). Furthermore, the hydrogel composite demonstrates shear strain softening (Fig.1F). Together, the inclusion of CIPs in the fibrin network results in tissue-like mechanical responses. We next examine the possible mechanisms for the compression stiffening. Previously established fiber-stretching-around-inclusion mechanism for compression stiffening typically requires a volume fraction >30%, but our study utilizes only 10% volume fraction. This prompts us to investigate the impact of particle size and quantity, yet introducing monodispersed polystyrene beads does not produce compression stiffening (Fig.2A). The distinct decay kinetics of G’ and normal stress during compressions (Fig.2B-C) and the absence of reliance on non-linear elasticity (Fig.2D) also rule out the mechanism of fiber stretching around inclusions. Furthermore, we eliminate the jamming mechanism, as the CIPs volume fraction falls well below the jamming threshold (64%), and the rheological responses are distinct from a jammed system (Fig.2E-F). Therefore, our study reveals a previously unexplored mode of compression stiffening. To elucidate its mechanism, we investigate the impact of CIP aggregation. A dispersant comprising 2% Tween-20 and 2% polyvinyl pyrrolidone effectively reduces CIP aggregates in both fibrin and polyacrylamide hydrogels (Fig.3A-C), mitigating compression stiffening (Fig.3D-E). These suggest a crucial role of CIP aggregation in the compression stiffening. Aggregation affects the size and shape of inclusions. To isolate these factors, we observe that fibrin with polydisperse-sized but spherical polystyrene inclusions softens during compression (Fig.4A). Conversely, substituting CIP aggregates with coffee grounds, which exhibit diverse shapes, replicates compression stiffening in fibrin (Fig.4B-C). Furthermore, changes in fibrinogen concentration modulate the magnitude of compression stiffening (Fig.4D). Overall, our findings highlight the significant role of shape diversity over size polydispersity. In conclusion, this study demonstrates that embedding cell-mimicking aggregation-forming inclusions induces tissue-like mechanical responses. This stiffening is distinct from established mechanisms and is attributed to the diverse morphologies of the inclusions that interact with the biopolymer network. These findings offer insights into the underlying mechanisms of tissue mechanics and inform strategies for guiding cell behaviors in tissue engineering applications.
