Introduction: Healing following traumatic tissue injury, with or without surgical intervention, results in scar formation characterized by disorganized collagen fibers and decreased ultimate tendon strength. However, scar formation is reduced in the absence of extensive inflammatory response[1]. Our lab is developing collagen scaffolds for musculoskeletal regeneration. Here we examine how incorporating fetal-wound inspired matrix, amniotic membrane (AM) derived matrix or hyaluronic acid (HA), can modulate cell response to inflammatory challenge. AM is the innermost layer of the placenta with known anti-inflammatory, anti-microbial, and anti-scarring properties while HA plays a central role in the fetal (scarless) wound healing cascade[2]. We examined how incorporating AM matrix or HA within the scaffold alters the bioactivity of equine tenocytes in response to the pro-inflammatory factor interleukin-1 beta (IL-1β). We subsequently examined short-term healing response in a porcine craniofacial defect.
Materials and Methods: Amniotic membranes were isolated from human placentas following uncomplicated vaginal births using an IRB approved protocol. Membranes were washed, decellularized, and dried. Scaffolds were fabricated via lyophilization from a suspension of type I collagen with dried AM powder, HA, or chondroitin sulfate (CS; control). Equine tenocytes (P4; 250,000 cells/scaffold) were cultured in the scaffolds in complete growth media for 24h. The media was then exchanged with media supplemented with 0 (control), 0.1 (inflammatory) or 1 ng/mL (high inflammatory) IL-1β. alamarBlue and PCR assays were used to monitor metabolic activity and gene expression over the 7 day study. Scaffolds were implanted into 8mm dia. porcine mandibular defects; inflammatory response was examined after 3, 7, 14 days via histology and gene expression.
Results and Discussion: Tenocyte metabolic activity increased over time regardless of scaffold or media condition. However, the metabolic activity of cells in HA and AM scaffolds was significantly higher than the control, especially in media supplemented with high levels of IL-1β (Fig. 1). Further, cells in AM and HA scaffolds showed significant down-regulation of TNFα at day 7 (Fig 2). Additional findings show that cells in AM scaffolds under the inflammatory challenge exhibit gene expression profiles (COLI, MMPs) most similar to cells grown in normal growth media.
Conclusions: Collagen scaffolds containing HA or AM support and promote cellular metabolic activity as well as temper the effect of a pro-inflammatory signal on gene expression. Current work studying these scaffolds in vitro is challenging cells (macrophages, tenocytes) with additional pro-inflammatory factors (TNFα), singly or in combination. Ongoing analyses of the in vivo porcine mandibular defect implants is comparing the cellular and inflammatory response in AM and CS scaffolds.
References:
[1] Beredjiklian PK, Favata M, Cartmell JS, Flanagan CL, Crombleholme TM, Soslowsky LJ. Regenerative versus reparative healing in tendon: a study of biomechanical and histological properties in fetal sheep. Ann Biomed Eng. 2003;31:1143-52.
[2] Larson BJ, Longaker MT, Lorenz HP. Scarless fetal wound healing: a basic science review. Plast Reconstr Surg. 2010;126:1172-80.