Data of a stiffness softening mechanism effect on proliferation and differentiation of a human bone marrow derived mesenchymal stem cell line towards the chondrogenic and osteogenic lineages

This article contains data related to the research article entitled “Stiffness memory of indirectly 3D-printed elastomer nanohybrid regulates chondrogenesis and osteogenesis of human mesenchymal stem cells” [1] (Wu et al., 2018). Cells respond to the local microenvironment in a context dependent fashion and a continuous challenge is to provide a living construct that can adapt to the viscoelasticity changes of surrounding tissues. Several materials are attractive candidates to be used in tissue engineering, but conventional manufactured scaffolds are primarily static models with well-defined and stable stiffness that lack the dynamic biological nature required to undergo changes in substrate elasticity decisive in several cellular processes key during tissue development and wound healing. A family of poly (urea-urethane) (PUU) elastomeric nanohybrid scaffolds (PUU-POSS) with thermoresponsive mechanical properties that soften by reverse self-assembling at body temperature had been developed through a 3D thermal induced phase transition process (3D-TIPS) at various thermal conditions: cryo-coagulation (CC), cryo-coagulation and heating (CC + H) and room temperature coagulation and heating (RTC + H). The stiffness relaxation and stiffness softening of these scaffolds suggest regulatory effects in proliferation and differentiation of human bone-marrow derived mesenchymal stem cells (hBM-MSCs) towards the chondrogenic and osteogenic lineages.

development and wound healing. A family of poly (urea-urethane) (PUU) elastomeric nanohybrid scaffolds (PUU-POSS) with thermoresponsive mechanical properties that soften by reverse selfassembling at body temperature had been developed through a 3D thermal induced phase transition process (3D-TIPS) at various thermal conditions: cryo-coagulation (CC), cryo-coagulation and heating (CC þ H) and room temperature coagulation and heating (RTC þ H). The stiffness relaxation and stiffness softening of these scaffolds suggest regulatory effects in proliferation and differentiation of human bone-marrow derived mesenchymal stem cells (hBM-MSCs) towards the chondrogenic and osteogenic lineages.  Compression mechanical testing along with histological assessment was sensitive to elucidating how stiffness softening affects stem cell differentiation.  Table 1 shows the effect of the infill density (i.e. 3D priting) and the variou 3D-TIPS thermal conditions (i.e. CC, CC þ H and RTC þ H) on the mechanical properties of the scaffolds. Table 2 demonstrates the isothermal stiffness softening behaviour of 50% infill density scaffolds after a 28-day period incubation in vitro at body temperature (37°C), with all scaffold groups reaching their intrinsic elasticity (i.e. 'stiffness memory' concept). Table 3 shows viscoelastic behaviours of 50% infill density scaffolds during dynamic compression testing, all reaching their intrinsic elasticity. Fig. 2 and Table 4 demonstrate the hierarchical micro-/nano-porous structure of the various scaffold groups. Figs. 3 and 5 show his- Table 1 Physical, tensile and compression mechanical properties of 3D-TIPS PUU-POSS scaffolds with various infill densities.

Scaffold
Infill density, %    tological sectioning demonstrating chondrogenic and osteogenic differentiation, respectively, on the various scaffolds. Fig. 4 and Tables 5 and 6 show elemental mapping analysis after chondrogenic and osteogenic differentiation on the various scaffolds.     Table 4 Pore size and pore size distribution of 50% infill density scaffolds [2].

3D-TIPS PUU-POSS scaffold manufacturing
3D-TIPS PUU-POSS scaffolds at different thermal conditions (Cyo-coagulation, CC; cryo-coagulation and heating, CC + H; and room temperature coagulation and heating, RTC + H) were manufactured by a 3D confined thermal induced phase separation process (3D-TIPS) based on selfassembly, phase transition and phase separation of the polymeric solution at controlled temperatures as described in [1,2].

Cell expansion and differentiation
A human bone marrow derived mesenchymal stem cell line was expanded, seeded and differentiated (Table S1-S2) on 3D-TIPS PUU-POSS scaffolds with stiffness softening as described in [1].

Physico-mechanical characterization of the scaffolds prior to cell seeding
Static mechanical testing of the scaffolds under tensile and compression mode, for different infill densities, before and after incubation over 28 days at body temperature in vitro (37°C), was performed with an Instron 5655 tester as described previously [1].
A mercury intrusion porosimeter (PoreMaster 60GT, Quantachrome, UK) was used to characterise the pore structure including the pore size, pore volume, size distribution and surface area of freeze-dried scaffolds (50% infill density).

Chondrogenic and osteogenic assessment
Element detection on cell-laden 50% infill density scaffolds after differentiation was quantified via Energy-dispersive X-ray (EDX) analysis as described in [1].
Histological section and staining of the scaffolds (50% infill density) was performed after chondrogenic and osteogenic differentiation as previously described [1]. HFH-C, human femoral head cartilage.