Papain-gel degrades intact nonmineralized type I collagen fibrils

Abstract

Papain-gel has been utilized as a chemo-mechanical material for caries removal due to its ability to preserve underlying sound dentin. However, little is known about the effect of the papain enzyme on intact type I collagen fibrils that compose the dentin matrix. Here we sought to define structural changes that occur in intact type I collagen fibrils after an enzymatic treatment with a papain-gel. Intact and nonmineralized type I collagen fibrils from rat tail were obtained and treated with a papain-gel (Papacarie) for 30 s, rinsed with water and imaged using an atomic force microscope (AFM). Additionally, polished healthy dentin specimens were also treated using the same protocol described above and had their elastic modulus (E) and hardness (H) measured by means of AFM-based nanoindentation. AFM images showed that the papain-gel induced partial degradation of the fibrils surface, yet no rupture of fibrils was noticed. The distinction between gap and overlap zones of fibrils vanished in most regions after treatment, and overlap zones appeared to be generally more affected. Mechanical data suggested a gradual decrease in E and H after treatments. A significant two-fold drop from the values of normal dentin (E=20+/-1.9, H=0.8+/-0.08 GPa) was found after four applications (E=9.7+/-3.2, H=0.24+/-0.1 GPa) (P<0.001), which may be attributed to the degradation of proteoglycans of the matrix. In summary, this study provided novel evidence that intact nonmineralized type I collagen fibrils are partially degraded by a papain-gel.

Fig 1

The intact nonmineralized collagen fibrils from rat tail before enzymatic degradation from the top (A) and a 3-D perspective (B). Fibrils present the typical staggered pattern with a periodicity of 67 nm of gap (black line) and overlap zones (white doted outline). At this stage the fibrils present a smooth surface and clear distinction between zones.

Fig 2

Collagen fibrils after papain-gel digestion from a top view (A) and a 3-D perspective (B). Images show degradation of the fibrils surface, which appeared roughened, yet no rupture of fibrils was noticed. The distinction between gap and overlap zones vanished in most regions with random preservation of gap zones (arrowheads).

Fig 3

Averaged values of reduced elastic modulus, E, (A) and hardness, H, (B). For each application the papain-gel was allowed to react with the dentin sample for 30 s and rinsed with de-ionized water for 20 s. Treated specimens were significantly different from normal dentin (Po0.001).