PuraStat RADA16 Self-Assembling Peptide Reduces Postoperative Abdominal Adhesion Formation in a Rabbit Cecal Sidewall Injury Model

Abstract

Objective: To evaluate the effect of PuraStat (2.5% RADA16) administration on postoperative abdominal adhesion formation in an in vivo model. Methods: Anesthetized New Zealand white rabbits underwent cecal sidewall abrasion surgery in which the cecal serosa and juxtaposed parietal peritoneum were abraded after access through an abdominal midline incision. Eight animals were randomized to receive PuraStat administration at the interface of the injured tissues before incision closure, and five animals served as untreated controls. Treated animals received 3-12 ml PuraStat solution per lesion. Animals were sacrificed 14 days after surgery and examined for adhesion formation at the wound site. Results: At study terminus, adhesions were identified in 90% (9/10) of abraded cecum/peritoneal wound sites in untreated controls versus 25% (4/16) of PuraStat-treated sites (p = 0.004). Mean ± SD Total Adhesion Score (average of the values for extent + strength of the adhesion in both defects per animal; maximum score = 14 points) was significantly 76% lower in PuraStat-treated animals (2.0 ± 3.0 points) compared to untreated controls (8.2 ± 1.9 points) (p = 0.029). Mean adhesion coverage area of wound sites was 79% lower in PuraStat-treated animals than controls (p < 0.001), and mean adhesion durability was 72% lower in PuraStat-treated animals versus controls (p = 0.005). Remnant hydrogel was observed at the wound sites of 75% of treated animals at postoperative Day 14. Conclusion: PuraStat treatment has a positive protective effect in the cecal sidewall injury model, and significantly reduces abdominal adhesion formation at the interface of the injured cecum and overlying peritoneal sidewall defect.

Keywords: PuraStat; RADA16; abdominal adhesions; cecal sidewall injury; hydrogel; nanofiber; self-assembling peptide; wound healing.

Conflict of interest statement

Authors EG, EA, and LS were employed by the company 3-D Matrix, Inc.

Copyright © 2021 Gil, Aleksi and Spirio.

Figures

FIGURE 1

Chemical structure and self-assembly of RADA16 into higher-order hydrogels. (A): The RADA16 peptide has 16 amino acids organized as repeated 4-amino acid sequences containing “R” (positively-charged arginine), “A” (hydrophobic alanine), and “D” (negatively-charged aspartic acid) residues. (B): RADA16 undergoes spontaneous and revisable self-assembly in acidic solutions to generate nanofibers. RADA16 molecules with β-sheet conformation interact through face-to-face hydrophobic interactions and edge-to-edge hydrogen bonding to form layered and extended nanofibers, ∼ 6 nm wide. These extracellular matrix-like nanofibers form a viscous and transparent aqueous solution at a relatively low concentration range (e.g., 0.1–2.5% weight/volume). (C): Illustration of RADA16 structure and properties as it is applied to and gels on a wound site. Acidic aqueous solutions of RADA16 are viscous and exhibit thixotropic disassembly/reassembly, which allows their easy administration to wound sites through catheters and syringes, with viscosity returning immediately after administration. Upon contact with the physiological pH of body fluids including blood, lymph and interstitial fluid, the surface net charges of RADA16 nanofibers become zero resulting in the physical crosslinking by hydrophobic interactions between neighboring RADA16 nanofibers, so that RADA16 solution forms in-situ hydrogels on the wound site and act as a physical adhesive that is hemostatic and supports wound healing. Adopted from Sankar et al., Front. Bioeng. Biotechnol. 2021; doi: 10.3389/fbioe.2021.679525, in accordance with Creative Commons Attribution License CC-BY.

FIGURE 2

Abdominal adhesion formation at the cecal serosa/peritoneal junction in the rabbit cecal sidewall injury model. This model predictably and quickly generates robust abdominal adhesion formation, with grossly visible adhesions typically present by 7–14 days after surgery. Briefly, the abdominal cavity was accessed through a 12-cm ventral midline incision and bleeding injury was induced on the cecal serosa by abrasion with a sterile dry gauze pad and on the juxtaposed peritoneal inner surface by shallow excision and scraping with a scalpel blade. Approximately ≈2 × 4.5 cm wounds were irrigated with saline and then either treated with topical administration of PuraStat solution or left untreated as Controls before midline incision closure and recovery. At postoperative Day 14, adhesions were significantly more numerous and sizeable at the cecum/peritoneum interface in Control [Panel (A)] animals compared to PuraStat-treated animals [Panel (B)] whose abraded tissue surfaces received 3–12 ml/lesion of PuraStat solution (2.5% RADA16) immediately after wound creation and just prior to abdominal closure. The mean ± SD Total Adhesion Score (average of the scores for extent + strength of the adhesions in both defects per animal) was significantly 76% lower in PuraStat-treated animals at 2.0 ± 3.0 points compared to 8.2 ± 1.9 points in untreated control animals (p = 0.029 by unpaired, two-tailed t-test; maximum possible score = 14 points). The self-assembling RADA16-based hydrogel significantly reduced both the adhesion extent (percent of initial wound area covered by adhesions) and adhesion durability. Remnant PuraStat was observed in 75% of treated animals at the 14-days study terminus.