Effect of scaffold elasticity on the gene expression of annulus fibrosus-derived stem cells

This article provides more experimental details and findings of the study as to how the elasticity of scaffold material modulates the gene expression of annulus fibrosus-derived stem cells (AFSCs) (Zhu et al., 2015 [1]). The detailed synthetic route and characterizations of four kinds of biodegradable poly(ether carbonate urethane)ureas (PECUUs) are described. After AFSCs were cultured on electrospun PECUU fibrous scaffolds, the cell proliferation and gene expression analyses were performed to explore the effect of substrate elasticity on the growth and differentiation characteristics of AFSCs.


a b s t r a c t
This article provides more experimental details and findings of the study as to how the elasticity of scaffold material modulates the gene expression of annulus fibrosus-derived stem cells (AFSCs) (Zhu et al., 2015 [1]). The detailed synthetic route and characterizations of four kinds of biodegradable poly(ether carbonate urethane)ureas (PECUUs) are described. After AFSCs were cultured on electrospun PECUU fibrous scaffolds, the cell proliferation and gene expression analyses were performed to explore the effect of substrate elasticity on the growth and differentiation characteristics of AFSCs.  Table   Subject  1. Data

Mechanical properties of PECUUs after in vitro degradation
After the cast PECUU membranes were incubated in PBS for 1, 2, 3, and 4 weeks, their mechanical properties were measured using uniaxial tensile tests ( Table 2).

Immunofluorescence of AFSCs cultured on PECUUs
The morphology of AFSCs cultured on PECUUs for 2 weeks is shown in Fig. 3. The immunofluorescence pictures of F-actin and nuclei indicate that AFSCs spread and grew well on all the electrospun PECUU fibrous scaffolds.

Gene expression of AFSCs cultured on PECUUs for 2 weeks
The gene expression of major matrix components of AF tissue, including collagen-I, collagen-II and aggrecan, after 2 weeks of culture was examined in AFSCs cultured on electrospun PECUU scaffolds using qRT-PCR analysis. The expression of collagen-I gene in AFSCs appeared to increasing with the increase of PECUU elasticity after being cultured for 2 weeks (Fig. 4). For example, the expression of collagen-I in AFSCs cultured on PECUU-1 (E ¼13.4 MPa) was nearly 2 times higher than that of cells on PECUU-4 (E ¼2.5 MPa). In contrast, the gene expression of collagen-II and aggrecan decreased with the increase of substrate elasticity (Fig. 4). The expression levels of collagen-II and aggrecan in AFSCs on PECUU-4 were 2.4 and 1.7 times, respectively, higher than those of cells on PECUU-1.

The cellular biochemistry and mechanics of AFSCs cultured on PECUUs
The biochemical measurements of major AF matrix components in AFSCs cultured on various electrospun PECUU scaffolds were performed after 2 weeks (Table 3). The cell traction forces were measured as well. For convenience of comparison, the corresponding characteristics of different regions of native AF tissue are also provided side by side.

Synthesis of PECUUs with tunable elasticity
The PECUUs were synthesized by a one pot, three-step polymerization process as shown in Scheme 1. In brief, the ring-opening polymerization of trimethylcarbonate (TMC) was carried out at 110°C for 24 h with PEO-PPO-PEO as the initiator and stannous octoate as the catalyst. The resulting polydiols were used as soft segment and reacted with 1,6-diisocyanatohexane (HDI) at 75°C for 4 h under continuous stirring in 15 wt% toluene. Following that, 1 wt% putrescine in DMF was added dropwise as a chain extender to obtain PECUU.

Measurement of the elastic moduli of PECUUs using nanoindentation
The membranes of all four kinds of PECUUs were prepared using solution cast method. Then the modulus of membranes was tested with a nanoindentation system (Nano Indenter G200, Agilent). As previously described [3], dynamic indentations with five running frequencies (10, 5.62, 3.16, 1.78 and 1 Hz) and 50 nm oscillation amplitude were performed using the "G-Series XP CSM flat punch complex modulus'' module of the NanoSuite method at the axial direction using a flat indenter (diameter, 215 mm) at approximately 22°C. At least ten indentations were measured for each sample.

DSC analysis of PECUUs
DSC analysis was performed on a DSC instrument (Perkin-Elmer) within the range of À 80-150°C at a heating rate of 20°C min À 1 .

AF tissue harvest for isolating AFSCs
After the muscles and ligaments were removed, IVDs from T10 to L5 were isolated (Fig. 5). The spinal column was then transversally sectioned around the middle of each disc. After a completed disc was harvested, pure AF tissue was obtained by carefully removing the NP, inner and outer AF tissues.

Gene expression of AFSCs on electrospun PECUU scaffolds
The electrospun scaffolds of PECUUs were fabricated as previously described [4]. The AFSCs were isolated as previously described and used at passage 1 [5]. PECUU substrates of different elasticity were cut to fit into a 96-well plate for cell morphology observation and into a 24-well plate for gene expression tests, respectively. For cell morphology test, AFSCs were seeded at the density of 2000 cells/well in 96-well plate and then cultured in low glucose DMEM supplemented with 10% FBS. After 2 weeks, the morphology of cells was evaluated by sequentially staining with DAPI and phalloidin-FITC for visualizing F-actin and nuclei, respectively. For gene expression analysis, AFSCs were seeded at the density of 5 Â 10 4 cells/well and then cultured in low glucose DMEM supplemented with 10% FBS. After 2 weeks, total RNA were extracted using TRIZOL isolation system (Invitrogen) and analyzed by real-time quantitative PCR (RT-qPCR) (CFX96, Bio-Rad) using the SsoFast EvaGreen supermix kit (Bio-Rad). The primers for GAPDH, collagen-I, collagen-II and aggrecan were designed using the mRNA sequences deposited in Gene Bank as mentioned in our previously published papers [3,4,6].