Data Set for Thermogel Actuation for Driving Directional Stromal Cell Communication and Migration into Columnar Arrays

This dataset presents information on the freeform printing and temperature actuation of a hydrogel. The hydrogel, comprising a blend of poly(N-isopropylacrylamide) grafted with chondroitin sulfate, gelatin, and Carbopol 940, facilitates cell spatial communication through mild temperature changes, a process referred to as actuation. The dataset includes NMR, rheological, SAXS characterization, as well as cell images stained with DAPI/Phalloidin. Poly(N-isopropylacrylamide)-g-chondroitin sulfate Synthesis: Free radical polymerization of pNIPAAm-CS was performed as described previously according to the following publication with a molar ratio of NIPAAm:CS of 2500:1. Christiani, T., Mys, K., Dyer, K., Kadlowec, J., Iftode, C. and Vernengo, A.J., 2021. Using embedded alginate microparticles to tune the properties of in situ forming poly (N‐isopropylacrylamide)‐graft‐chondroitin sulfate bioadhesive hydrogels for replacement and repair of the nucleus pulposus of the intervertebral disc. JOR spine, 4(3), p.e1161. The functionalization degree of CS was assessed by 1H-NMR analysis according to Wang et al. [86] and found to be 35.4%. Wang, L.F., Shen, S.S. and Lu, S.C., 2003. Synthesis and characterization of chondroitin sulfate–methacrylate hydrogels. Carbohydrate Polymers, 52(4), pp.389-396. Preparation of EXPECT Hydrogels Aqueous solutions of 3% (w/v) pNIPAAm-CS + 0.8% (w/v) Carbopol® 940 (CP, Acros Organics Geel, Belgium) + 1% (w/v) gelatin (from porcine skin, 175 g Bloom, type A, Sigma, St. Louis, USA) were prepared. To produce the hydrogel for cell culture, the required reagents were UV-sterilized for 12 hours, and an aqueous solution of 3% (w/v) pNIPAAm-CS + 0.8% (w/v) CP + 1% (w/v) gelatin was prepared. For all formulations, the pH was adjusted to 7.4 with 50% sodium hydroxide (NaOH) and the hydrogel was stored at 4°C until testing. Rheological Characterization Rheological testing was conducted with an Anton Paar MCR-302 rheometer (Anton Paar GmbH, Austria). All measurements were taken with a gap size of 0.5 mm and a parallel plate with a diameter of 25 mm. Unless stated otherwise, the rheological properties were assessed at a temperature of 25°C, with a frequency of 1 Hz and with n = 3 samples per group. To prevent sample drying during the measurements, silicone oil was applied around the plate to seal the hydrogel. A 0.8% (w/v) CP hydrogel served as a control group for all tests. Amplitude Sweep Test An amplitude sweep test with oscillatory strain was used to determine storage modulus (G') and loss modulus (G'') within the range of 0.01% and 1000% strain. The flow stress was calculated at the point of the curves cross-over (G' = G'') using the integrated analysis tool of the RheoCompassTM software (version 1.26.22, Anton Paar GmbH, Austria). Temperature Ramp Test An oscillatory temperature ramp test with 1% constant strain (within linear viscoelastic region (LVR) as established by primary amplitude test) was used to evaluate viscoelastic properties as a function of temperature. The test was divided into three intervals, the first of which was a 60 s measurement at 25°C. After this, the temperature was increased to 37°C in the second interval for 600 s. In the third interval, the temperature was reduced to 25°C and the measurements were taken for 300 s. To calculate the absolute increase of G' caused by the increase of temperature from 25°C to 37°C, the following equation was used: ∆G^'= G_(t=y s)^'- G_(t=x s)^' (1) where G_(t=y s)^' defines the 4th data point of the first interval and G_(t=x s)^' the 30th data point of the second interval. Step Strain Test (Recovery) In an oscillatory step strain test, G' and G'' were measured while the strain was alternated between a low strain, set to 1%, and a high strain, set to 250%, over the course of seven intervals. The step strain test was started with 1% strain in the 1st interval, followed by 250% strain in the 2nd interval, and so on. Each interval was 120 s, except the 7th, which lasted 160 s at 1% strain. Small angle X-ray scattering (SAXS) Analysis SAXS was performed at B21 beamline, Diamond Light Source, UK with sample loading into MPS sticks enclosed with Kapton tape. Briefly, samples were collected and measured at 25 or 37°C, for 21 frames with 1 s exposure each. Relevant methods are here: Cowieson, N.P., Edwards-Gayle, C.J., Inoue, K., Khunti, N.S., Doutch, J., Williams, E., Daniels, S., Preece, G., Krumpa, N.A., Sutter, J.P. and Tully, M.D., 2020. Beamline B21: high-throughput small-angle X-ray scattering at Diamond Light Source. Journal of Synchrotron Radiation, 27(5), pp.1438-1446. Sceglovs, A., Wychowaniec, J.K., Skadins, I., Reinis, A., Edwards-Gayle, C.J., D'Este, M. and Salma-Ancane, K., 2023. Effect of steam sterilisation on physico-chemical properties of antibacterial covalently cross-linked ε-polylysine/hyaluronic acid hydrogels. Carbohydrate Polymer Technologies and Applications, 6, p.100363. Expansion of Human Mesenchymal Stromal Cells This work was done using cells isolated from the same donor (male, born in 1958), at University Hospital of Freiburg in Germany with the full approval of the patient. Furthermore, for supplementary Figure 5, an additional donor was evaluated (female, born in 1961), from the same university hospital with the patient’s consent. For the human MSC growth and expansion prior printing, Minimum Essential Medium Eagle - alpha modification (10.08g α-MEM powder, Gibco, Thermo Fischer, Switzerland and 2.2 g sodium bicarbonate (56014-1KG, Sigma-Aldrich, Switzerland) for 1L of basal medium), supplemented with 10% corning serum (FBS, Corning Incorporated, New York, USA), 1% (v/v) penicillin-streptomycin (Pen/Strep, Gibco, Carlsbad, USA), and 5 ng/ml basic Fibroblast Growth Factor (bFGF, Fitzgerald Industries International, Acton, MA, USA) was used. Cells were cultured at 37°C with 5% CO2 and 90% relative humidity. The expansion medium was changed three times per week, cells were passaged at 80% confluency and used at passage 4 for bioprinting. For MSC aggregate generation, AggreWell™ 400 Microwell Culture Plates (STEMCELLS technologies, Canada) were utilized. The wells were pretreated with AggreWell™ Rinsing solution for the prevention of cell adhesion to the well’s surface and the promotion of efficient spheroid formation. The cells were detached from the flasks using Trypsin-EDTA (Gibco, Thermo Fisher). The viable cells were determined by a hematocytometer and Trypan Blue exclusion, while the seeding of the cells took place at a concentration of 2 million cells per well following the manufacturer’s protocol for the aggregate generation. The cells were kept in the incubator overnight (24 hours) at 37°C with 5% CO2 to form the spheroids prior to 3D printing. 3D Printing For freeform 3D printing, MSCs (single cells or spheroidal aggregate format) were suspended in 6% (w/v) gelatin in the MSC growth medium described above at a total density of 4.0 x 106 cells/mL. The cell-laden bioinks were extruded through a PTFE-lined needle with 0.3 mm inner diameter and 12.7 mm length, inside 1 mL of the patternable hydrogel composed of 3% (w/v) pNIPAAm-CS + 0.8% (w/v) CP + 1% (w/v) gelatin. The printing pattern consisted of three circles with a radius of 5 mm, stacked in Z-direction with 1 mm spacing, and was deposited at a starting height of 2 mm inside the hydrogel. Printing parameters were as follows: 25°C ambient temperature, 16% relative humidity, 0.2 – 0.8 bar printing pressure, 25°C bioink temperature, and 0.5 mm/s deposition velocity. The cells were cultured in expansion medium for 14, 21 or 36 days at 37°C with 5% CO2 and 90% relative humidity. The culture medium was changed three times per week. For the purpose of the chondrogenic induction, chondrogenic medium was used for the MSCs post printing. The chondrogenic medium was composed of High glucose Dulbecco’s Modified Eagle Medium (Hg DMEM, Lot: 2551077,13.38 g H.g DMEM with 3.7 g of sodium bicarbonate (56014-1KG, Sigma-Aldrich, Switzerland) and 0.11 g of sodium pyruvate (P5280-25G, Sigma-Aldrich, Switzerland) for 1L basal medium), supplemented with 1% (v/v) Non-essential amino acid cell culture supplement (NEAA, Lot: 2554642, Gibco, Thermo Fischer, UK), 1% (v/v) insulin, human transferrin and selenous acid (ITS-plus,Lot: 3046004, Corning, Discovery Labware, Inc., Bedford, USA), 1% (v/v) L-Ascorbic Acid 2-phosphate sesquimagnesium salt hydrate (AA2P, A8960-5G, Sigma-Aldrich, Switzerland), 0.1% (v/v) dexamethasone (D2915-100MG, Lot: 029K1187, Sigma-Aldrich, Switzerland) and 0.1% (v/v) Transforming growth factor beta (TGFβ, Fitzgerald Industries International, USA). After the printing, the hydrogels were placed in the incubator (at 37°C with 5% CO2 and 90% relative humidity) for about an hour for solidification and then the chondrogenic medium was added. The culture of the MSCs and spheroids within the hydrogels took place under the same conditions. The medium was changed 3 times per week. Temperature Actuation of Printed EXPECT Samples Immediately post-printing, hydrogel from all groups was removed for a baseline imaging (referred to as day 0). Subsequently, on days 1, 3, and 5, and every 5 days thereafter, temperature actuation was implemented. The final temperature actuation cycle was applied until day 35, after which the hydrogels were equilibrated for 24 hours at 37°C before being harvested for imaging on day 36. The temperature actuation involved exposing culture plates to 25°C (ambient conditions outside the incubator) for 15 minutes. In contrast, static (control) samples were kept outside the incubator under the same conditions, except on a 37°C surface, to prevent the phase transition of the hydrogels.