Rheology measurements were made with an AR-G2 stress controlled rheometer (TA Instruments). Alginate gels were deposited directly onto the surface plate of the rheometer immediately after mixing with the crosslinker. A 20 mm plate was immediately brought down, forming a 20 mm disk of gel with an average thickness of ~1.8 mm. The mechanical properties were then measured over time until the storage modulus reached an equilibrium value. The storage modulus at 0.5% strain and at 1 Hz was recorded periodically for 45 minutes. Then, a strain sweep was performed to confirm this value was within the linear elastic regime, followed by a frequency sweep. No prestress was applied to the gels for these measurements.
The initial elastic moduli and stress relaxation properties of alginate gels were measured from compression tests of the gel disks (15 mm in diameter, 2 mm thick, equilibrated in DMEM for 24 hr) using a previously published method4 (link),35 (link). The gel disks were compressed to 15% strain with a deformation rate of 1 mm/min using an Instron 3342 single column apparatus. Within 15% compression, the stress vs. strain relations of the gels are almost linear, and the slope of the stress-strain curves (first 5–10% of strain) gives the initial elastic modulus. Subsequently, the strain was held constant, while the load was recorded as a function of time. Compression and stress relaxation measurements of polyacrylamide hydrogels and biological tissues were performed using the same procedure. Polyacrylamide hydrogels were formed following previously established protocols28 (link). In brief, 0.2 g of acrylamide and 0.02 g of bis-acrylamide were dissolved in 2 mL of water. Then 60 μL of 137 mg/mL ammonium persulfate and 60 μL of 70 mg/mL tetramethylethylenediamine (TEMED) were added into above mixture. The solution was mixed and allowed to gel for 6 hours. The hydrogel was then equilibrated in PBS for 24 hours before mechanical testing. We note that some stress relaxation of the covalently crosslinked hydrogels is observed at longer timescales, but this was previously found to arise from water leaving the hydrogel under bulk compression35 (link). Sprague Dawley Rats (male, 7 weeks of age, Charles River Lab) were euthanized in compliance with National Institutes of Health and institutional guidelines. Brain, liver, and adipose were collected immediately after euthanization and tested with Instron 3342 single column apparatus. Bone marrow from multiple rat femurs and tibias were collected fresh after euthanization and allowed to coagulate for 1 hr before compression testing. A fracture hematoma from human patient was retrieved from the bone fracture site at the moment of bone stabilization surgery. The surgery took place 7 days after occurrence of the fracture, when the surrounding soft tissue trauma around the fracture gap was sufficiently stabilized. The complete hematoma was collected and processed for mechanical testing within 1h after surgery. The same procedure of compression and relaxation measurements was performed as with the rat samples but on a Bose TestBench LM1 system using a 250g load cell. Care was taken to not test samples that contained bone chips. Fracture hematoma collection was approved by the Institutional Review Board of the Charité University Hospital Berlin, where the collection and testing were performed, and the participant gave written informed consent. Stress relaxation tests that were noisy were smoothed with a Savitzky-Golay filter in Igor Pro (Wavemetrics) with a 4s window.
The initial elastic moduli and stress relaxation properties of alginate gels were measured from compression tests of the gel disks (15 mm in diameter, 2 mm thick, equilibrated in DMEM for 24 hr) using a previously published method4 (link),35 (link). The gel disks were compressed to 15% strain with a deformation rate of 1 mm/min using an Instron 3342 single column apparatus. Within 15% compression, the stress vs. strain relations of the gels are almost linear, and the slope of the stress-strain curves (first 5–10% of strain) gives the initial elastic modulus. Subsequently, the strain was held constant, while the load was recorded as a function of time. Compression and stress relaxation measurements of polyacrylamide hydrogels and biological tissues were performed using the same procedure. Polyacrylamide hydrogels were formed following previously established protocols28 (link). In brief, 0.2 g of acrylamide and 0.02 g of bis-acrylamide were dissolved in 2 mL of water. Then 60 μL of 137 mg/mL ammonium persulfate and 60 μL of 70 mg/mL tetramethylethylenediamine (TEMED) were added into above mixture. The solution was mixed and allowed to gel for 6 hours. The hydrogel was then equilibrated in PBS for 24 hours before mechanical testing. We note that some stress relaxation of the covalently crosslinked hydrogels is observed at longer timescales, but this was previously found to arise from water leaving the hydrogel under bulk compression35 (link). Sprague Dawley Rats (male, 7 weeks of age, Charles River Lab) were euthanized in compliance with National Institutes of Health and institutional guidelines. Brain, liver, and adipose were collected immediately after euthanization and tested with Instron 3342 single column apparatus. Bone marrow from multiple rat femurs and tibias were collected fresh after euthanization and allowed to coagulate for 1 hr before compression testing. A fracture hematoma from human patient was retrieved from the bone fracture site at the moment of bone stabilization surgery. The surgery took place 7 days after occurrence of the fracture, when the surrounding soft tissue trauma around the fracture gap was sufficiently stabilized. The complete hematoma was collected and processed for mechanical testing within 1h after surgery. The same procedure of compression and relaxation measurements was performed as with the rat samples but on a Bose TestBench LM1 system using a 250g load cell. Care was taken to not test samples that contained bone chips. Fracture hematoma collection was approved by the Institutional Review Board of the Charité University Hospital Berlin, where the collection and testing were performed, and the participant gave written informed consent. Stress relaxation tests that were noisy were smoothed with a Savitzky-Golay filter in Igor Pro (Wavemetrics) with a 4s window.
