Immunomodulatory effects of amniotic membrane matrix incorporated into collagen scaffolds

Abstract

Adult tendon wound repair is characterized by the formation of disorganized collagen matrix which leads to decreases in mechanical properties and scar formation. Studies have linked this scar formation to the inflammatory phase of wound healing. Instructive biomaterials designed for tendon regeneration are often designed to provide both structural and cellular support. In order to facilitate regeneration, success may be found by tempering the body's inflammatory response. This work combines collagen-glycosaminoglycan scaffolds, previously developed for tissue regeneration, with matrix materials (hyaluronic acid and amniotic membrane) that have been shown to promote healing and decreased scar formation in skin studies. The results presented show that scaffolds containing amniotic membrane matrix have significantly increased mechanical properties and that tendon cells within these scaffolds have increased metabolic activity even when the media is supplemented with the pro-inflammatory cytokine interleukin-1 beta. Collagen scaffolds containing hyaluronic acid or amniotic membrane also temper the expression of genes associated with the inflammatory response in normal tendon healing (TNF-α, COLI, MMP-3). These results suggest that alterations to scaffold composition, to include matrix known to decrease scar formation in vivo, can modify the inflammatory response in tenocytes. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1332-1342, 2016.

Keywords: 3D scaffold; amniotic membrane; collagen; inflammation; tissue engineering.

FIGURE 1

Amniotic membrane histological analysis, scaffold mechanical and microstructural analysis. A: Placental section stained with H&E. Arrows indicate amniotic membrane. Scale bar: 200 μm. B: Masson’s Trichrome staining of placental section. Arrows indicate amniotic membrane. Scale bar: 200 μm. C: Elastic modulus of scaffold variants under compression. (*) significance (p <0.05) between scaffold groups. D: SEM images of scaffold variants; collagen:chondroitin sulfate (C:CS), collagen:hyaluronic acid (C:HA), collagen: amniotic membrane (C:AM) (left to right). Scale bar: 100 μm. E: Histological staining of vimentin in cell-seeded scaffolds with varying composition at day 7. Left to right: collagen:-chondroitin sulfate (C:CS), collagen:hyaluronic acid (C:HA), collagen: amniotic membrane (C:AM). Scale bar: 200 μm.

FIGURE 2

Tenocyte metabolic activity in scaffolds cultured in (A) control media, (B) inflammatory media containing 0.1 ng/mL IL-1β, and (C) high inflammatory media supplemented with 1 ng/mL IL-1β. (*) significance between groups indicated (p<0.05). The same data has been grouped by scaffold type and plotted in (D) collagen:chondroitin sulfate, (E) collagen:hyaluronic acid, and (F) collagen:amniotic membrane. (**) significance compared with all groups at given time point (p<0.05). (N.S.) no significant differences between groups at a given time point (p >0.05).

FIGURE 3

Gene expression of tumor necrosis factor-alpha (TNF-α) in tenocytes cultured in three scaffold variants in (A) inflammatory (0.1 ng/mL IL-1β) and (B) high inflammatory (1 ng/mL IL-1β) media conditions. Gene expression was normalized to tenocytes cultured in respective scaffold in control (no inflammatory factors) media. (*) significance between groups indicated (p <0.05). (**) significance compared with all groups at given time point (p <0.05).

FIGURE 4

Tenocyte gene expression levels of collagen I (COL1A2; A, B), matrix metalloproteinase-3 (MMP-3; C, D), and scleraxis (SCXB; E, F) as a function of scaffold content at days 1, 4, and 7 of culture in inflammatory (0.1 ng/mL IL-1β) (A, C, E) and high inflammatory (1 ng/mL IL-1β) (B, D, F) media. Gene expression was normalized to tenocytes cultured in respective scaffold in control (no inflammatory factors) media. (*) significance between groups indicated (p <0.05). (∧,∨) significant increase, decrease (respectively) compared with corresponding control (same scaffold type, same time point) (p <0.05).

FIGURE 5

Transforming growth factor-beta 1 (TGF-β1) release from tenocyte-seeded scaffolds of varying compositions. Measurements were made after 24 h of culture in serum-free medias: (A) inflammatory (0.1 ng/mL IL-1β) and (B) high inflammatory (1 ng/mL IL-1β). (*) significance between groups indicated (p <0.05).

FIGURE 6

Pull down of IL-1β by scaffolds with varying compositions. The degree of pull down was normalized to the concentration of IL-1β in a solution without scaffolds.