Figure 1
DAPS In Vitro Growth Trajectory. (A) Study design: Engineered intervertebral disc analogs (disc-like angle ply structures, DAPS) composed of a nanofibrous AF and a hydrogel NP, were seeded with either AF and NP cells or MSCs and cultured in chemically defined media with TGF-β3 separately for 2 weeks, and then combined. The in vitro maturation of DAPS was evaluated mechanically, compositionally, and histologically at regular intervals over the course of 15 weeks. (B) Alcian blue, picrosirius red, type I and type II collagen staining: DAPS matured compositionally in terms of proteoglycan (Alcian blue) and collagen (picrosirius red) deposition over this time period, and stained strongly for type II collagen after 15 weeks. (C) Composition: Histological findings were confirmed by measurements of GAG and collagen content, which exceeded or were within range of native tissue values. GAG content consistently increased over 15 weeks, while collagen content increased early and then reached a plateau. Types I and II collagen-staining indicated a chondrogenic phenotype at 15 weeks. Native values are from34,54. (*p < 0.05 vs week. 2.5; + p < 0.05 vs. AF/NP at corresponding time point) (D) Mechanical Function : DAPS demonstrated a nonlinear stress/strain response in compression, and elastic mechanical properties (moduli) that were stable over 15 weeks. Viscoelastic properties (creep strain) became more dominant over the 15-week period, stabilizing by 10 weeks. In both cases, there were no differences between AF/NP and MSC/MSC DAPS, and mechanical properties were within range of native tissue. Native values are from34,54. (*p < 0.05 vs. week 2.5; + p < 0.05 vs. AF/NP at corresponding time point). AF/NP DAPS were also tested to determine their fatigue behavior in compression after 10 weeks of culture. The peak strain asymptotically plateaued with increasing cycle number (plotted on a log scale), indicating stiffening of the construct. This was confirmed by measurements of the linear region compressive modulus, which increased from cycle 20 to cycle 1000 (#p < 0.1 vs. cycle 20).
