Collagen has shown promise as a bioink for extrusion-based bioprinting, but further development of new collagen bioink formulations is necessary to improve their printability. Screening these formulations by measuring print accuracy is a costly and time consuming process. We hypothesized that rheological properties of the bioink before, during, and/or after gelation can be used to predict printability. In this study, we investigated the effects of riboflavin photocrosslinking and pH on type I collagen bioink rheology before, during, and after gelation and directly correlated these findings to the printability of each bioink formulation. From the riboflavin crosslinking study, results showed that riboflavin crosslinking increased the storage moduli of collagen bioinks, but the degree of improvement was less pronounced at higher collagen concentrations. Dots printed with collagen bioinks with riboflavin crosslinking exhibited smaller dot footprint areas than those printed with collagen bioinks without riboflavin crosslinking. From the pH study, results showed that gelation kinetics and final gel moduli were highly pH dependent and both exhibited maxima around pH 8. The shape fidelity of printed lines was highest at pH 8-9.5. The effect of riboflavin crosslinking and pH on cell viability was assessed using bovine chondrocytes. Cell viability in collagen gels was found to decrease after blue light activated riboflavin crosslinking but was not affected by pH. Correlations between rheological parameters and printability showed that the modulus associated with the bioink immediately after extrusion and before deposition was the best predictor of bioink printability. These findings will allow for the more rapid screening of collagen bioink formulations.

An advantage of bioprinting is the ability to incorporate cells into the hydrogel bioink allowing for precise control over cell placement within a construct. Previous work found that the printability of collagen bioinks is highly dependent on their rheological properties. The effect of cell density on collagen rheological properties and, therefore, printability has not been assessed. Therefore, the objective of this study was to determine the effects of incorporating cells on the rheology and printability of collagen bioinks. Primary chondrocytes, at densities relevant to cartilage tissue engineering (up to 100 × 106 cells ml−1), were incorporated into 8 mg ml−1 collagen bioinks. Bioink rheological properties before, during, and after gelation as well as printability were assessed. Cell-laden printed constructs were also cultured for up to 14 d to assess longer-term cell behavior. The addition of cells resulted in an increase in the storage modulus and viscosity of the collagen before gelation. However, the storage modulus after gelation and the rate of gelation decreased with increasing cell density. Theoretical models were compared to the rheological data to suggest frameworks that could be used to predict the rheological properties of cell-laden bioinks. Printability testing showed that improved printability could be achieved with higher cell densities. Fourteen-day culture studies showed that the printing process had no adverse effects on the viability or function of printed cells. Overall, this study shows that collagen bioinks are conducive to bioprinting with a wide range of cell densities while maintaining high printability and chondrocyte viability and function.

Mechanical stimulation is recognized as a potent modulator of cellular behaviors such as proliferation, differentiation, and extracellular matrix assembly. However, the study of how cell-generated traction force changes in response to stretch is generally limited to short-term stimulation. The goal of this work is to determine how cells actively alter their traction force in response to long-term physiological cyclic stretch as a function of cell pre-stress. We have developed, to our knowledge, a novel method to assess traction force after long-term (24 h) uniaxial or biaxial cyclic stretch under conditions of high cell pre-stress with culture on stiff (7.5 kPa) polyacrylamide gels (with or without transforming growth factor β1 (TGF-β1)) and low pre-stress by treating with blebbistatin or culture on soft gels (0.6 kPa). In response to equibiaxial stretch, valvular interstitial cells on stiff substrates decreased their traction force (from 300 nN to 100 nN) and spread area (from 3000 to 2100 μm(2)). With uniaxial stretch, the cells had similar decreases in traction force and area and reoriented perpendicular to the stretch. TGF-β1-treated valvular interstitial cells had higher pre-stress (1100 nN) and exhibited a larger drop in traction force with uniaxial stretch, but the percentage changes in force and area with stretch were similar to the non-TGF-β1-treated group. Cells with inhibited myosin II motors increased traction force (from 41 nN to 63 nN) and slightly reoriented toward the stretch direction. In contrast, cells cultured on soft gels increased their traction force significantly, from 15 nN to 45 nN, doubled their spread area, elongated from an initially rounded morphology, and reoriented perpendicular to the uniaxial stretch. Contractile-moment measurements provided results consistent with total traction force measurements. The combined results indicate that the change in traction force in response to external cyclic stretch is dependent upon the initial cell pre-stress. This finding is consistent with depolymerization of initially high-tension actin stress fibers, and reinforcement of an initially low-tension actin cytoskeleton.

This article describes the compositional, mechanical, and structural differences between collagen gels fabricated from different sources and processing methods. Despite extensive use of collagen in the manufacturing of biomaterials and implants, there is little information as to the variation in properties based on collagen source or processing methods. As such, differences in purity and composition may affect gel structure and mechanical performance. Using mass spectrometry, we assessed protein composition of collagen from seven different sources. The mechanics and gelation kinetics of each gel were assessed through oscillatory shear rheology. Scanning electron microscopy enabled visualization of distinct differences in fiber morphology. Mechanics and gelation kinetics differed with source and processing method and were found to correlate with differences in composition. Gels fabricated from telopeptide‐containing collagens had higher storage modulus (144 vs. 54 Pa) and faster gelation (251 vs. 734 s) compared to atelocollagens, despite having lower purity (93.4 vs. 99.8%). For telopeptide‐containing collagens, as collagen purity increased, storage modulus increased and fiber diameter decreased. As α1/α2 chain ratio increased, fiber diameter increased and gelation slowed. As such, this study provides an examination of the effects of collagen processing on key quality attributes for use of collagen gels in biomedical contexts.

I.C. discussed the results and implications and commented on the manuscript. L.B. supervised all studies of This is the author manuscript accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as