241 Fibroblast remodeling of extracellular matrix is directed by the fibrotic nature of the threedimensional microenvironment was used to calculate Young’s modulus (E, stiffness) (Equation (v)) until the peak point for the highest measurement observed). Relaxation values were calculated starting from the time point at which highest stiffness was observed by using the formula (Equation (vi)). Time duration for reaching 100% relaxation was recorded as ‘Relaxation time’. Time duration for reaching 50% of the total relaxation was recorded as ‘Time to Reach 50% Stress Relaxation’. All calculations were performed using Microsoft Excel 2016. (iii) Stiffness values and viscoelastic relaxation properties of the hydrogels at day 7 and 14 were measured using a Low Load Compression Tester as previously described [32, 39]. The LLCT analysis was performed on three different randomly selected locations on each hydrogel using 20% fixed strain rate. The measurement locations had at least 1.5 mm distance between them and 0.5 mm from the edges to ensure robustness and representativeness of the measurements. The stress ((Equation (iii)) and strain (Equation (iv)) values were calculated as described below and the slope of the line was used to calculate Young’s modulus (E, stiffness) (Equation (v)) until the peak point for the highest measurement observed). Relaxation values were calculated starting from the time point at which highest stiffness was observed by using the formula (Equation (vi)). Time duration for reaching 100% relaxation was recorded as ‘Relaxation time’. Time duration for reaching 50% of the total relaxation was recorded as ‘Time to Reach 50% Stress Relaxation’. All calculations were performed using Microsoft Excel 2016. (iii) = (iv) = ℎ (v) ( ) = (vi) ( ) = ( 0)− ( ) ( 0) (iv) Mechanical testing with Low Load Compression Testing (LLCT) Stiffness values and viscoelastic relaxation properties of the hydrogels at day 7 and 14 were measured using a Low Load Compression Tester as previously described [32, 39]. The LLCT analysis was performed on three different randomly selected locations on each hydrogel using 20% fixed strain rate. The measurement locations had at least 1.5 mm distance between them and 0.5 mm from the edges to ensure robustness and representativeness of the measurements. The stress ((Equation (iii)) and strain (Equation (iv)) values were calculated as described below and the slope of the line was used to calculate Young’s modulus (E, stiffness) (Equation (v)) until the peak point for the highest measurement observed). Relaxation values were calculated starting from the time point at which highest stiffness was observed by using the formula (Equation (vi)). Time duration for reaching 100% relaxation was recorded as ‘Relaxation time’. Time duration for reaching 50% of the total relaxation was recorded as ‘Time to Reach 50% Stress Relaxation’. All calculations were performed using Microsoft Excel 2016. (iii) = (iv) = ℎ (v) ( ) = (vi) ( ) = ( 0)− ( ) ( 0) (v) Mechanical testing with Low Load Compression Testing (LLCT) Stiffness values and viscoelastic relaxation properties of the hydrogels at day 7 and 14 were measured using a Low Load Compression Tester as previously described [32, 39]. The LLCT analysis was performed on three different randomly selected locations on each hydrogel using 20% fixed strain rate. The measurement locations had at least 1.5 mm distance between them and 0.5 mm from the edges to ensure robustness and representativeness of the measurements. The stress ((Equation (iii)) and strain (Equation (iv)) values were calculated as described below and the slope of the line was used to calculate Young’s modulus (E, stiffness) (Equation (v)) until the peak point for the highest measurement observed). Relaxation values were calculated starting from the time point at which highest stiffness was observed by using the formula (Equation (vi)). Time duration for reaching 100% relaxation was recorded as ‘Relaxation time’. Time duration for reaching 50% of the total relaxation was recorded as ‘Time to Reach 50% Stress Relaxation’. All calculations were performed using Microsoft Excel 2016. (iii) = (iv) = ℎ (v) ( ) = (vi) ( ) = ( 0)− ( ) ( 0) (vi) alignment of fibers [38]. Mechanical testing with Low Load Compression Testing (LLCT) Stiffness values and viscoelastic relaxation properties of the hydrogels at day 7 and 14 were measured using a Low Load Compression Tester as previously described [32, 39]. The LLCT analysis was performed on three different randomly selected locations on each hydrogel using 20% fixed strain rate. The measurement locations had at least 1.5 mm distance between them and 0.5 mm from the edges to ensure robustness and representativeness of the measurements. The stress ((Equation (iii)) and strain (Equation (iv)) values were calculated as described below and the slope of the line was used to calculate Young’s modulus (E, stiffness) (Equation (v)) until the peak point for the highest measurement observed). Relaxation values were calculated starting from the time point at which highest stiffness was observed by using the formula (Equation (vi)). Time duration for reaching 100% relaxation was recorded as ‘Relaxation time’. Time duration for reaching 50% of the total relaxation was recorded as ‘Time to Reach 50% Stress Relaxation’. All calculations were performed using Microsoft Excel 2016. (iii) = (iv) = ℎ (v) ( ) = (vi) ( ) = ( 0)− ( ) ( 0) Statistical Analysis Statistical analyses of the measured parameters were performed using an interaction analysis in a mixed model analysis in IBM SPSS Statistics 26 (IBM, Armonk, New York, USA). For each parameter analyzed, the interactions between the disease status of the hydrogels and disease status of the fibroblasts, as well as fibroblast seeding status of the hydrogels were used. For all analyses presented, a random effect was used for the intercept per experimental batch (i.e. same combination of control or IPF fibroblasts and control or IPF hydrogel for the 6 experimental conditions (n=6 batches)). TWOMBLI results were analyzed using 6 different images generated per sample to address the sample heterogeneity. Mechanical characterization results were analyzed using triplicated measurements performed on the same sample to tackle the heterogeneity within samples. Presented results show estimate ± 95% confidence interval for all results. ACKNOWLEDGMENTS Authors thank Mr. Albano Tosato for assistance in preparation of visuals. Funding: MN, TK, BNM, IHH and JKB receive unrestricted research funds from Boehringer Ingelheim. JKB also acknowledges support from the Nederlandse Organisatie voor Wetenschappelijk Onderzoek (NWO) (Aspasia 015.013.010). This collaboration project is co-financed by the Ministry of Economic Affairs and Climate Policy, the Netherlands, by means of the PPP-allowance made available by the Top Sector Life 9
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