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Self-assembling RADA16 peptide hydrogel supports hemostasis, synechiae reduction, and wound healing in a sheep model of endoscopic nasal surgery

  • Michael Fook-Ho Lee
    Correspondence
    Corresponding author at: Institute of Academic Surgery, Royal Prince Alfred Hospital, University of Sydney Medical Center, PO Box M40, Missenden Rd NSW 2050 Australia.
    Affiliations
    Institute of Academic Surgery, Royal Prince Alfred Hospital, University of Sydney Medical Center, Sydney, Australia
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  • Arjuna Ananda
    Affiliations
    Institute of Academic Surgery, Royal Prince Alfred Hospital, University of Sydney Medical Center, Sydney, Australia
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Open AccessPublished:October 22, 2022DOI:https://doi.org/10.1016/j.anl.2022.09.012

      Abstract

      Objectives

      Complications of endoscopic sinus/nasal turbinate surgery include postoperative hemorrhage, synechiae formation, and poor wound healing. Our primary objectives were to evaluate whether a topical hydrogel based on self-assembling RADA16 peptides: i) reduces bleeding and synechiae formation, and ii) supports wound healing, using a sheep nasal surgery model.

      Methods

      Thirty sheep received endoscopic surgery-created bilateral nasal mucosal injuries on the middle turbinate/opposing septum. Injuries were randomly assigned RADA16, Gelatin-thrombin, or no treatment. Outcomes included intra-operative hemostasis, scar tissue/synechiae formation and wound healing at 2 weeks and the 6-week study terminus, and histopathology.

      Results

      Intra-operative hemostasis time improved with RADA16 and Gelatin-thrombin versus Control wounds (139.7±56.2 s, 145.4±58.1 s, and 224.0±69.9 s, respectively; p < 0.0001 for both comparisons). Two-week synechiae scores (maximum 4 points) were similar in Controls (2.9±1.8 points) and Gelatin-thrombin (3.1±1.6 points) wounds (p > 0.05), but were reduced in RADA16 sites by 91% versus Controls and 92% versus Gelatin-thrombin treatment (0.3±0.6 points; p < 0.0001 for both comparisons). Six-week synechiae scores were similar in Control (1.1±1.7 points) and Gelatin-thrombin (1.7±2.0 points) wounds (p > 0.05), but reduced 100% in RADA16-treated wounds. Synechiae occurred in fewer RADA16-treated sites at 2 weeks (20%) versus Gelatin-thrombin (80%) and Controls (75%; p < 0.01) and at 6 weeks (0%, 50% and 35%, respectively; p < 0.01). RADA16 was associated with significantly lower 6-week histopathology scores, driven by reduced submucosal fibrosis and angiogenesis.

      Conclusion

      Although RADA16 and Gelatin-thrombin similarly accelerated hemostasis in this sheep endoscopic sinus surgery model, only RADA16 reduced postoperative synechiae formation at 2 weeks with an absence of synechiae at 6 weeks. Histology suggested RADA16 enhanced mucosal regeneration.

      Keywords

      1. Introduction

      Endoscopic surgery of the sinuses and nasal turbinates is commonly used to treat chronic sinusitis that is refractory to management with maximal medical therapy. Following endoscopic nasal surgery, three of the most common complications include postoperative hemorrhage, scar tissue/synechiae formation, and poor wound healing that often leads to inadequate symptom improvement and the need for revision operations [
      • Alaryani RA
      • Alhedaithy RA.
      Preventive measures of middle turbinate lateralization after endoscopic sinus surgery: An updated review.
      ,
      • Bergmark RW
      • Gray ST.
      Surgical management of turbinate hypertrophy.
      . Traditionally, non-absorbable nasal packing has been used to achieve postoperative hemostasis, though tamponade frequently causes patient discomfort and excessive pressure on the operative site may lead to synechiae formation and foreign body reactions in the peri-operative mucosal membranes [
      • Weber R.
      Nasal packing and stenting.
      ]. Contemporary methods to address this issue include the use of bio-absorbable packs and application of topical agents such as gelatin-thrombin combinations; though synechia formation remains problematic [
      • Weber R.
      Nasal packing and stenting.
      ,
      • Iqbal IZ
      • Jones GH
      • Dawe N
      • Mamais C
      • Smith ME
      • Williams RJ
      • et al.
      Intranasal packs and haemostatic agents for the management of adult epistaxis: systematic review.
      ,
      • Baumann A
      • Caversaccio M.
      Hemostasis in endoscopic sinus surgery using a specific gelatin-thrombin based agent (FloSeal).
      ,
      • Wang TC
      • Tai CJ
      • Tsou YA
      • Tsai LT
      • Li YF
      • Tsai MH.
      Absorbable and nonabsorbable packing after functional endoscopic sinus surgery: systematic review and meta-analysis of outcomes.
      ].6
      RADA16 is a synthetic amphiphilic polypeptide composed of 16 amino acids organized as sequentially repeated 4-amino acid sequences containing R (positively-charged arginine), A (hydrophobic alanine), and D (negatively-charged aspartic acid) residues [
      • Sankar S
      • O'Neill K
      • Bagot D'Arc M
      • Rebeca F
      • Buffier M
      • Aleksi E
      • et al.
      Clinical use of the self-assembling peptide RADA16: a review of current and future trends in biomedicine.
      ]. Upon a change in pH or on contact with a physiological ion rich solution, the polypeptide undergoes controlled self-assembly into a clear hydrogel scaffold structure. Recently, RADA16 has shown promise in cell culturing, tissue repair, rapid hemostasis and drug delivery due to the scaffold's inducible rapid assembly, biocompatibility and low immunogenicity, resulting in the formation of a 3-dimensional (3D) mesh hydrogel structure that resembles the human extracellular matrix (ECM) [
      • Sankar S
      • O'Neill K
      • Bagot D'Arc M
      • Rebeca F
      • Buffier M
      • Aleksi E
      • et al.
      Clinical use of the self-assembling peptide RADA16: a review of current and future trends in biomedicine.
      ,
      • Zhang S.
      Discovery and design of self-assembling peptides.
      ,
      • Ellis-Behnke RG
      • Liang YX
      • Tay DKC
      • Kau PWF
      • Schneider GE
      • Zhang S
      • et al.
      Nano hemostat solution: immediate hemostasis at the nanoscale.
      ,
      • Wang R
      • Wang Z
      • Guo Y
      • Li H
      • Chen Z.
      Design of a RADA16-based self-assembling peptide nanofiber scaffold for biomedical applications.
      ].
      This study evaluated PuraStat® (3-D Matrix Medical Technology Pty Ltd, Melbourne, VIC, Australia), a viscous transparent hemostatic material containing 2.5% RADA16 peptides in a sterile aqueous solution that is preloaded into sterile syringes for use [
      3-D Matrix Europe SAS; Caluire et Cuire, France
      PuraStat® Absorbable haemostatic material instructions for use.
      ]. In acidic formulations, RADA16 remains solubilized as discrete β-sheet nanofibers that spontaneously cross-link into complex three-dimensional (3D) hydrogel matrices within seconds after exposure to the physiological pH of blood, interstitial fluid, and lymph. PuraStat® is CE marked in Europe and licensed in Australia for hemostasis during cardiovascular, GI, general and nasopharyngeal surgeries, cleared in the US for sinus and gastrointestinal (GI) use, and approved in Japan for GI use [
      • Sankar S
      • O'Neill K
      • Bagot D'Arc M
      • Rebeca F
      • Buffier M
      • Aleksi E
      • et al.
      Clinical use of the self-assembling peptide RADA16: a review of current and future trends in biomedicine.
      ,
      3-D Matrix Europe SAS; Caluire et Cuire, France
      PuraStat® Absorbable haemostatic material instructions for use.
      ,
      US Food and Drug Administration
      510(k) Premarket Notification K210098, summary.
      . In the EU, PuraStat® is also approved for treating delayed postoperative bleeding after colon endoscopic submucosal dissection surgery. A related RADA16 product, PuraSinus® (also a 2.5% solution) was cleared by the US FDA in 2019 for use in nasal surgery and trauma repair for intraoperative hemostasis and as a wound covering that prevents adhesion formation and supports healing [
      US Food and Drug Administration
      510(k) Premarket Notification K183015, summary.
      ]. In otorhinolaryngology, RADA16 applied topically to raw mucosal surfaces following endoscopic sinus or nasal turbinate surgery can achieve hemostasis without the need for nasal packing.
      We previously demonstrated the hemostatic potential of intra-operative RADA16 in a 2017 case series of 60 patients who underwent endoscopy-powered turbinoplasties [
      • Lee MF
      • Ma Z
      • Ananda A.
      A novel haemostatic agent based on self-assembling peptides in the setting of nasal endoscopic surgery, a case series.
      ]. In that study, intra-operative RADA16 application to surgical sites prevented rebleeding and synechia/adhesion formation in all subjects, and appeared to support good wound healing. Others have reported good hemostasis with no postoperative adhesion formation by administering RADA16 solution to the wound site after endoscopic resection of a nasopharyngeal stenosis [
      • Wong E
      • Ho J
      • Smith M
      • Sritharan N
      • Riffat F
      • Smith MC.
      Use of PuraStat, a novel haemostatic matrix based on self-assembling peptides in the prevention of nasopharyngeal adhesion formation.
      ]. To expand our understanding of how RADA16 hydrogels can promote wound healing at the tissue and cellular level, we used a sheep model of endoscopic surgery that allowed histopathogical analysis that is not typically performed in humans.
      This study tested the hypothesis that RADA16 use during endoscopic nasal surgery is effective for achieving operative hemostasis, reduces the rate of scar tissue/synechiae formation, and enhances wound healing in a sheep model of chronic sinusitis. Our model was based on established sheep synechiae models that use reproducible scoring and photography systems that have been previously reported [
      • Medina JG
      • Das S.
      Sprayable chitosan/starch-based sealant reduces adhesion formation in a sheep model for chronic sinusitis.
      ,
      • Athanasiadis T
      • Beule AG
      • Robinson BH
      • Robinson SR
      • Shi Z
      • Wormald P-J.
      Effects of a novel chitosan gel on mucosal wound healing following endoscopic sinus surgery in a sheep model of chronic rhinosinusitis.
      . Additionally, we compared the hemostatic and wound-healing effects of RADA16 to a gelatin-thrombin topical sealant that is also widely used and approved for post-operative hemostasis in nasal surgery [
      • Baumann A
      • Caversaccio M.
      Hemostasis in endoscopic sinus surgery using a specific gelatin-thrombin based agent (FloSeal).
      ,
      • Wang TC
      • Tai CJ
      • Tsou YA
      • Tsai LT
      • Li YF
      • Tsai MH.
      Absorbable and nonabsorbable packing after functional endoscopic sinus surgery: systematic review and meta-analysis of outcomes.
      .

      2. Materials and methods

      2.1 Study design and ethical oversight

      This was a prospective, randomized, assessor-blinded, controlled study developed in compliance with International Standard ISO 10993-1 “Biological evaluation of medical devices” [

      United States Food and Drug Administration. Center for Devices and Radiological Health. Docket #FDA-2013-D-0350. “Use of International Standard ISO 10993-1, Biological evaluation of medical devices - Part 1: Evaluation and testing within a risk management process”. 2020. Available at: https://www.fda.gov/regulatory-information/search-fda-guidance-documents/use-international-standard-iso-10993-1-biological-evaluation-medical-devices-part-1-evaluation-and (Accessed August 1, 2022).

      ], and all staff and facilities conducted the study in accordance with World Health Organization's “Good Laboratory Practices Handbook” [

      World Health Organization “Good Laboratory Practices (GLP) Handbook, 2nd Ed). 2009. Available at: https://www.who.int/tdr/publications/documents/glp-handbook.pdf (Accessed August 1, 2022).

      ]. Animal ethics approval was gained from the University of Sydney Animal Ethics Committee in accordance with the New South Wales, Australia Animal Research Act (1985) before starting the study.

      2.2 Animal model

      Sheep were used as an established model for studying the healing process of nasal mucosa following endoscopic nasal surgery, due in part to their large size and similar configuration of the paranasal sinuses to that of humans [
      • Shaw CL
      • Cowin A
      • Wormald PJ.
      Standardization of the sheep as a suitable animal model for studying endoscopic sinus surgery.
      ]. Thirty male, adult Merino sheep (Ovis aries) were purchased and housed at the University Veterinary Centre Camden, an accredited veterinary teaching hospital of the University of Sydney. Sheep were acclimated for a minimum of two weeks prior to operating, and tested for the absence of ova, cysts, or parasites likely to affect their well-being and possibly confound study interpretation.

      2.3 Anesthesia

      Twenty-four hours prior to surgery, sheep were individually weighed; food and water were withheld for at least 12 hours prior to surgery to minimize the risk of intra-operative aspiration. Topical fentanyl patches (150 µg/hour) were applied to all sheep 12 hours prior to surgery. On the day of the operation, sheep were manually restrained and sedated with 0.2 mg/kg intravenous (IV) diazepam injected into the internal jugular vein prior to transfer to an animal holding bay. The skin overlying the internal jugular vein was shaved and disinfected with topical iodine and alcohol, local anesthesia was established by intradermal injection of 2 mL 1% lignocaine (lidocaine) solution, and a No. 15 scalpel blade was used to facilitate placement of a 16-gauge IV catheter that was retained with a 2-0 polydioxanone suture and flushed with heparinized saline. The sheep were further sedated with another 0.2 mg/kg IV diazepam and pre-oxygenated by facemask for 5 minutes. General anesthesia was induced by 4 mg/kg IV ketamine and the sheep were placed in sternal recumbency with the head elevated. Intubation was performed using an 8.0-mm diameter endotracheal tube following desensitization of the larynx by topical application of 2 mL 1% lignocaine solution. Once intubated, the sheep were attached to a breathing system at 1‒2 L/min oxygen and anesthesia was maintained using 1.5‒2.0% isoflurane. Simultaneously, the sheep were given 1 mg/kg IV flunixin, an NSAID, to maximize pain relief and decrease post-surgical inflammation and 5 mg/kg intramuscular ceftiofur to reduce the risk of surgical site infection. Vital signs (heart rate, blood pressure, oxygen saturation, respiratory rate, temperature) were continuously monitored throughout the procedure and hydration was maintained with a 10 mL/kg/hr continuous infusion of IV sodium lactate solution.

      2.4 Operative procedure

      Anesthetized sheep were repositioned into the lateral position. This was accounted for with computer randomization to ensure a similar number of surgical sites in all three treatment groups were assigned to the dependent and non-dependent lateral positions. A zero-degree rigid nasal endoscope was inserted to examine the nasal septum for any deviations to ensure uniform space between the septum and middle turbinate. A StraightShot™ M4 Microdebrider (Medtronic Plc, Minneapolis, MN) was introduced through the nostril under endoscopic guidance and used to create a uniform 2 × 2-cm full thickness mucosal and periosteal injury to the middle turbinate nasal mucosa in a position opposite and directly anterior to the nasal vomer. Immediately thereafter, a corresponding 2 × 2-cm mucosal lesion was created on the contralateral nasal septum opposite the initial lesion thereby creating two opposing wounds in close proximity with a high propensity for synechiae formation. Within 10 s after wound creation, a total of 4 mL of either RADA16 or gelatin-thrombin was applied directly and uniformly over the two wound surfaces, using a modified long applicator tip to allow easy access into the nasal cavity. In one-third of nostrils, these paired wounds were left untreated to function as controls. The surgeon was blinded as to which of these three interventions the wound was randomized to receive until immediately after wound creation. As the study aimed to assess the performance of each product in isolation, no other hemostatic adjuncts such as electrocautery or nasal packing were used. Intra-operative hemostasis was measured as the number of seconds from completion of hemostatic agent application, or ten seconds following wound creation in the control group, and the absence of any appreciable active oozing as measured by a treatment-blinded independent observer.

      2.5 Postoperative care

      Following hemostasis, isoflurane was turned off and the sheep were maintained on 2 L/min oxygen for 5 minutes prior to transfer to recovery, with extubation performed on return of the swallowing reflex. After recovery from anesthesia, food and water was re-offered to the sheep. The sheep were then transferred to monitored, closed pens with straw bedding for seven days. 1 mg/kg flunixin and 5 mg/kg ceftiofur were administered daily for three days postoperatively. The sheep were examined twice daily for any significant bleeding from the nose, and nostrils were irrigated once daily with 20 mL of isotonic saline solution. After one week of recovery, sheep were returned to an open paddock with adequate feeding pastures, shade, and water.

      2.6 Initial postoperative follow-up

      Two weeks following the initial surgery, all sheep were sedated with 0.2 mg/kg IV diazepam and 1 mg/kg IV ketamine for re-examination. A zero-degree rigid nasal endoscope was used to examine the surgical sites for any evidence of persistent bleeding and for the presence of scar-tissue/synechiae. Care was taken to avoid disruption of surgical sites or any scar tissue/synechiae to avoid confounding results. Endoscopic photographs were taken of every surgical site to be scored by a blinded independent observer trained in the identification of nasal synechiae using a graded scale that quantifies the degree of scar tissue/synechiae formation between the middle turbinate and the contralateral septum (Table 1, Fig. 1).
      Table 1Synechiae grading criteria.
      GradeScar tissue/Synechiae Extent
      0No scar tissue/synechiae, 0%
      1Scar tissue extends <25% to septum
      2Scar tissue extends 25‒50% to septum
      3Scar tissue extends >50% to septum
      4Completely formed synechiae extending to septum
      Fig 1
      Fig. 1Visual grading scale for nasal scar tissue/synechiae. Full-thickness 2 × 2-cm wounds were created on the mucosae of the middle turbinate and opposing nasal septum anterior to the nasal vomer, using and endoscope-guided Microdebrider (Medtronic Plc, Minneapolis, MN). Wounds were created bilaterally and nostrils then received either no treatment (Controls) or hemostasis by topical application of either 2.5% RADA16 hydrogel solution (PuraStat®; 3-D Matrix Medical Technology Pty Ltd, Melbourne, VIC, Australia) or gelatin-thrombin (FLOSEAL®; Baxter International Inc., Deerfield, IL), with treatment of each nostril having been randomly allocated. After recovering for 2 weeks, all wound sites were photographed by endoscopy and assessed for degree of synechiae formation between wound sites by a blinded independent observer, using a graded scale (range 0‒4). Shown are representative images of untreated Control wounds as examples of how synechiae extent was scored.

      2.7 Termination and post-mortem

      Six weeks following the initial surgery, all sheep were sedated with 0.2 mg/kg IV diazepam followed by euthanasia with an IV injection of >100mg/kg sodium pentobarbital. This timeframe was chosen because four weeks was the shortest timeframe by which point synechiae grade had stabilized in previous studies using a similar model [
      • Medina JG
      • Das S.
      Sprayable chitosan/starch-based sealant reduces adhesion formation in a sheep model for chronic sinusitis.
      ]. At post-mortem, a zero-degree rigid nasal endoscope was used to examine the surgical sites for the presence of scar tissue/synechiae with endoscopic photographs taken for grading by the same blinded independent observer. Following nasal endoscopy, a No. 22 scalpel blade was used to vertically transect the upper lateral cartilage of both nostrils adjacent to the septum to expose the middle turbinate and nasal vomer. The nasal vomer was again used as a landmark to identify the operative region and curved Mayo scissors were used to dissect out the segment of operated nasal mucosa with underlying bone from the middle turbinate. Following excision, the nasal mucosa was fixed in 10% neutral buffered formalin, sectioned, and stained with hematoxylin and eosin. One representative section from each of the 60 total wound sites (n = 20 per treatment) was evaluated, using an Olympus BX51 microscope and an Olympus EP50 camera (both from Olympus, Tokyo, Japan). Prepared slides were then graded by a blinded independent veterinary histopathologist under light microscopy qualitatively by four different histopathological metrics of mucosal inflammation and regeneration, with scores for each parameter graded on a 0‒3 point scale (Table 2). A histopathology composite score (maximum 12 points) was generated by adding together scores for the four individual parameters. The presence and abundance of seromucous cells and goblet cells were also noted, and assigned a qualitative assessment of either normal or abnormal/deficient, with “normal” indicating that secretory cell numbers and morphologies were not discernable from those in adjacent healthy unoperated mucosae.
      Table 2Histopathology grading parameters.
      ParameterGradeHistopathological Observation
      Epithelial erosion0None; 100% re-epithelialization; epithelium appears normal
      1Mild epithelial cell loss; >90%‒99% epithelial cell layer regeneration
      2Moderate epithelial loss; 50%‒90% epithelial cell layer regeneration
      3Severe; <50% re-epithelialisation
      Inflammatory infiltrate0None; no inflammatory cells observed per high-power field (hpf)
      1Mild; ≤15 inflammatory cells/hpf, scattered
      2Moderate; multi-focal, notable infiltrate, >15 inflammatory cells/hpf
      3Severe; overwhelming, marked inflammatory infiltrate
      Submucosal fibrosis0None; normal submucosal structure, typical cell/fibrillar complement
      1Mild; uncommon focal or multifocal fibrosis, non-disruptive
      2Moderate; prominent, multifocal-to-confluent, non-disruptive
      3Severe; extensive fibrosis, disrupts normal submucosal structure
      Angiogenesis0None; normal complement of submucosal microvasculature
      1Mildly increased microvascular density vs surrounding uninjured tissue
      2Moderately increased microvascular density
      3Severely increased microvascular density; grossly atypical.
      0 ‒ 12Total Composite Histopathology Score

      2.8 Statistical analysis

      Data are presented as mean ± SD, mean ± 95% confidence intervals (CIs), or percentages, as indicated. Continuous variables were compared across the three groups using one-way ANOVA followed by Bonferroni's post-test to adjust for multiple corrections. Categorical data were evaluated using Fisher exact test. A two-tailed p-value <0.05 was considered statistically significant. Prism v.5.03 statistical and graphing software (GraphPad Software Inc., San Diego, CA) was used.

      3. Results

      Surgical procedures and post-operative follow-up nasal endoscopy were successfully completed on all thirty sheep for a total of 60 sites. In total, 20 sites each were randomized to RADA16, Gelatin-thrombin, and untreated Control groups. No sheep died or experienced any serious adverse event or significant post-operative bleeding prior to scheduled euthanasia at postoperative six weeks. No surgical site rebleeding events were noted in any animal during the twice-daily inspections through postoperative Week 1, daily observations thereafter until study terminus, or during the Week 2 interim endoscopic evaluation.

      3.1 Intra-operative hemostasis time

      All the RADA16 sites reached complete hemostasis by 290 s. The average time to hemostasis was significantly improved in the RADA16 group (mean 139.7 ± 56.2 s) and Gelatin-thrombin group (mean 145.4 ± 58.1 s) compared to the Control group (mean 224.0 ± 69.9s; p < 0.01 in comparison to either hemostat-treated group). There was no significant difference in time-to-hemostasis between the RADA16 and Gelatin-thrombin groups (Fig. 2).
      Fig 2
      Fig. 2Intra-operative hemostasis time. Full-thickness 2 × 2-cm wounds created on the opposing mucosae of the middle turbinate and nasal septum anterior to the nasal vomer were randomized to receive either no treatment (Controls) or topical application of either gelatin-thrombin (FLOSEAL®; Baxter International Inc., Deerfield, IL) or 2.5% RADA16 hydrogel solution (PuraStat®; 3-D Matrix Medical Technology Pty Ltd, Melbourne, VIC, Australia). Both RADA16 and Gelatin-thrombin application similarly and significantly reduced wound bleeding time by nearly 40% compared to untreated Control wounds. Shown are mean values ± 95% confidence intervals. p-values were determined by 1-way ANOVA followed by Bonferroni's post-test to adjust for multiple comparisons.

      3.2 Synechiae formation at 2 and 6 weeks

      Scar tissue/synechiae formation was significantly reduced at both the postoperative 2- and 6-week timepoints in RADA16-treated nasal passages compared to the Control and Gelatin-thrombin groups, as evaluated by endoscopy (Fig. 3). At 2 weeks, mean (±SD) synechiae scores were similar in Controls at 2.9±1.8 points (maximum 4 points possible) and Gelatin-thrombin wound sites at 3.1±1.6 points (p > 0.05) (Fig. 3A). In contrast, the mean synechiae score in RADA16-treated wounds was only 0.3±0.6 points, representing a 91% reduction versus Controls and a 92% reduction versus Gelatin-thrombin treatment (p < 0.0001 for both comparisons to RADA16). At 2 weeks, a lower percentage of RADA16-treated nasal passages displayed any measure of scar tissue/synechiae formation (20%) compared to both the Gelatin-thrombin (80%) and Control group (75%) frequency (p = 0.0004 and p = 0.0012 by Fisher exact test, respectively, versus RADA16). Among wound sites with demonstrated synechiae formation, at 2 weeks the RADA16 group alone was also associated with significantly lower median grades of scar tissue/synechiae formation (4 sites with synechiae, median grade 1) compared to the Gelatin-thrombin (16 sites, median grade 4) and Control groups (15 sites, median grade 4) (p < 0.01 for both comparisons to RADA16).
      Fig 3
      Fig. 3Nasal synechiae grade at postoperative 2 and 6 weeks. (A) At 2 weeks, mean synechiae scores were similar in Controls (2.9 points) and Gelatin-thrombin wound sites (3.1 points).The mean synechiae score in RADA16-treated wounds was only 0.3 points, representing a 91% reduction versus Controls and a 92% reduction versus Gelatin-thrombin treatment. At 2 weeks, a lower percentage of RADA16-treated nasal passages displayed any measure of scar tissue/synechiae formation (20%) compared to both the Gelatin-thrombin (80%) and Control group (75%) frequency (p = 0.0004 and p = 0.0012 by Fisher exact test, respectively, versus RADA16; not shown). (B) At 6-weeks, synechiae formation was noted only in the Gelatin-thrombin and Control sites, and not the RADA16-treated sites. Mean synechiae scores were statistically similar in Controls (1.1) points and Gelatin-thrombin wound sites (1.7 points). No observed synechiae at 6 weeks in RADA16-treated wounds equated to a significant 100% reduction in mean score versus both Controls and Gelatin-thrombin treatment. At 6 weeks, a significantly lower rate of scar tissue/synechiae formation was noted in the RADA16 group alone (0%) compared to the Gelatin-thrombin (50%) and Control groups (35%) (p = 0.0004 and p = 0.0083, respectively, compared to RADA16 frequency). Shown in graphs are mean values ± 95% confidence intervals. Two-tailed p-values of mean score comparisons were determined by 1-way ANOVA followed by Bonferroni's post-test to adjust for multiple comparisons.
      At the postoperative 6-week study terminus, synechiae formation was noted only in the Gelatin-thrombin and Control sites, and not the RADA16-treated sites (Fig. 3B). Mean synechiae scores were statistically similar in Controls at 1.1±1.7 points and Gelatin-thrombin wound sites at 1.7±2.0 points (p > 0.05) (Fig. 3B). In contrast, no observed synechiae at 6 weeks in RADA16-treated wounds equated to a 100% reduction of mean score versus both Control and Gelatin-thrombin sites (p = 0.0091 and p = 0.0028, respectively, versus RADA16). At 6 weeks, a significantly lower rate of synechiae formation was noted in the RADA16 group alone (0% of sites) compared to the Gelatin-thrombin (50%) and Control groups (35%) (p = 0.0004 and p = 0.0083, respectively, compared to RADA16 frequency). There was no significant difference in the frequency or median grade of scar tissue/synechiae between the Gelatin-thrombin (10 sites, median grade 4) and Control groups (7 sites, median grade 4).

      3.3 Wound healing

      Wound treatment with RADA16 resulted in more favorable 6-week wound healing scores than observed in Controls or the Gelatin-thrombin group (Table 3 and Fig. 4). Average composite histopathology scores were significantly lower in sites treated with RADA16 (4.1±2.5 points; maximum 12), showing a 39% reduction versus the Control score (6.7±2.6 points; set as 100%; p = 0.0032) and a 45% reduction versus the Gelatin-thrombin score (7.4±2.0 points; p < 0.0001). This difference in Total Histopathology Score primarily reflected the significantly decreased submucosal fibrosis and angiogenesis at RADA16-treated wound sites compared to Control and Gelatin-thrombin-treated wounds. While epithelial cell erosion/damage and submucosal inflammatory infiltrate scores were also numerically lower in the RADA16 group versus both the Control and Gelatin-thrombin groups, these differences did not reach mathematical significance (Table 3).
      Table 3Histopathological parameter scoring.
      ParameterControlGelatin-ThrombinRADA16
      ScoreScorep-value vs ControlScorep-value vs Controlp-value vs Gel-Thromb.
      Epithelial erosion1.4±1.21.7±1.10.42681.2±0.90.46370.0931
      Inflammatory infiltrate1.4±0.71.3±0.60.80101.1±0.60.14570.1869
      Submucosal fibrosis2.3±0.82.6±0.80.31751.1±0.8<0.0001<0.0001
      Angiogenesis1.6±1.01.9±1.10.45320.9±0.90.01860.0035
      Total pathology score (Max 12)6.7±2.67.4±2.00.31644.1±2.50.0032<0.0001
      Each of the four histopathological parameters was scored on a 0‒3 scale, with 0 being normal morphology and 3 indicating severe pathology. Shown are mean values ± SD. Two-tailed p-values were determined by 1-way ANOVA with Bonferroni's post-test for multiple comparisons. One representative slide section was evaluated for each of the 60 cumulative wound sites (i.e., n = 20 per treatment).
      Fig 4
      Fig. 4Histomorphology of turbinate mucosal wound sites at the postoperative 6-week study terminus. (A) Normal unoperated middle turbinate mucosae had a confluent pseudostratified columnar epithelial surface interspersed with goblet cells (top margin of images), abundant serosal glands, and no evidence of submucosal inflammation or fibrosis. (B) Control wound sites demonstrated marked postoperative submucosal fibrosis and reduced epithelial and glandular regeneration. (C) Wounds sites in the Gelatin-thrombin group developed thick distinctive bands of submucosal fibrotic tissue and showed incomplete epithelial regeneration. (D) RADA16 treatment of wound sites resulted in significantly reduced submucosal fibrosis compared to wounds in the Control and Gelatin-thrombin groups. RADA16 treatment was associated with good epithelial layer and underlying serosal gland regeneration, with mucosae that resembled unwounded tissue. One representative slide section was evaluated for each of the 60 cumulative wound sites (i.e., n = 20 determinations per treatment).

      3.4 Secretory cell regeneration

      All three groups demonstrated a variable decrease in secretory cell regeneration at the 6-week study terminus compared to normal unoperated nasal mucosae (Fig. 4). Seromucous cells and associated glands returned to normal number and structure in more RADA16 sites (14/20 sites, 70%) than Control sites (8/20, 40%) or Gelatin-thrombin-treated sites (7/20, 35%), though this difference in proportions did not reach statistical significance (p = 0.1110 and p = 0.0562, respectively, versus RADA16). Goblet cells were present in normal numbers and morphology in 55% of RADA16 wounds, 70% of Controls, and 65% of Gelatin-thrombin sites; these values were mathematically indistinguishable from one another.

      4. Discussion

      More than 250,000 endoscopic nasal/sinus surgeries are performed annually in the United States to treat chronic sinusitis [
      • Bhattacharyya N.
      Ambulatory sinus and nasal surgery in the United States: demographics and perioperative outcomes.
      ]. Although these procedures are often very successful and improve patient quality-of-life [
      • Yim MT
      • Smith KA
      • Alt JA
      • Orlandi RR.
      The value of endoscopic sinus surgery in chronic rhinosinusitis.
      ], approximately 10‒15% of patients will need a repeat operation [
      • Alaryani RA
      • Alhedaithy RA.
      Preventive measures of middle turbinate lateralization after endoscopic sinus surgery: An updated review.
      ,
      • Bergmark RW
      • Gray ST.
      Surgical management of turbinate hypertrophy.
      ,
      • Chandra RK
      • Conley DB
      • Kern RC.
      The effect of FloSeal on mucosal healing after endoscopic sinus surgery: a comparison with thrombin-soaked gelatin foam.
      . Postoperative adhesion formation is a frequent cause of surgical failure [
      • Alaryani RA
      • Alhedaithy RA.
      Preventive measures of middle turbinate lateralization after endoscopic sinus surgery: An updated review.
      ,
      • Bergmark RW
      • Gray ST.
      Surgical management of turbinate hypertrophy.
      , and preventative strategies available to otolaryngologists remain very limited. Postoperative bleeding and suboptimal wound healing are also common complications of nasal/sinus surgery. The current study explored the impact of an intra-operatively administered topical RADA16 peptide formulation on postoperative hemostasis, wound healing and synechia formation in a sheep endoscopic sinus surgery model. We report that RADA16 administration to wound sites reduced postoperative bleeding time similar to a hemostatic gelatin-thrombin comparator formulation, but was associated with significantly better reduction of postoperative adhesion formation incidence and extent, and improved wound healing composite scores.
      Both RADA16 and gelatin-thrombin formulations have known topical hemostatic activity [
      • Baumann A
      • Caversaccio M.
      Hemostasis in endoscopic sinus surgery using a specific gelatin-thrombin based agent (FloSeal).
      ,
      • Sankar S
      • O'Neill K
      • Bagot D'Arc M
      • Rebeca F
      • Buffier M
      • Aleksi E
      • et al.
      Clinical use of the self-assembling peptide RADA16: a review of current and future trends in biomedicine.
      ,
      3-D Matrix Europe SAS; Caluire et Cuire, France
      PuraStat® Absorbable haemostatic material instructions for use.
      ,
      US Food and Drug Administration
      510(k) Premarket Notification K183015, summary.
      , and their use after endoscopic sinus surgery may preclude using nasal packing that risks patient discomfort and foreign body reactions at the surgical wound site that negatively impact healing [
      • Iqbal IZ
      • Jones GH
      • Dawe N
      • Mamais C
      • Smith ME
      • Williams RJ
      • et al.
      Intranasal packs and haemostatic agents for the management of adult epistaxis: systematic review.
      ]. Whereas intra-operative RADA16 application was previously shown to prevent adhesion formation in a series of 60 patients who underwent endoscopic turbinectomy [
      • Lee MF
      • Ma Z
      • Ananda A.
      A novel haemostatic agent based on self-assembling peptides in the setting of nasal endoscopic surgery, a case series.
      ], and after surgery for chemoradiotherapy-induced nasopharyngeal stenosis [
      • Wong E
      • Ho J
      • Smith M
      • Sritharan N
      • Riffat F
      • Smith MC.
      Use of PuraStat, a novel haemostatic matrix based on self-assembling peptides in the prevention of nasopharyngeal adhesion formation.
      ], some gelatin-thrombin formulations have been associated with an increased incidence of adhesion formation after endoscopic sinus surgery [
      • Chandra RK
      • Conley DB
      • Kern RC.
      The effect of FloSeal on mucosal healing after endoscopic sinus surgery: a comparison with thrombin-soaked gelatin foam.
      ]. In the current study, RADA16 treatment was associated with marked >90% reductions in 2-week postoperative adhesion scores relative to both the Control and Gelatin-thrombin treatment groups. At the 6-week study terminus, no adhesions were present at any RADA16-treated wound site; by contrast, adhesions were present in 35% and 50% of Control and Gelatin-thrombin sites, respectively. The notable reduction in adhesion formation at wound sites treated with biocompatible RADA16 hydrogel during endoscopic sinus surgery is encouraging.
      Histopathology scores reflecting several individual wound healing parameters were significantly lower in RADA16-treated wound sites than scores in both the Control and Gelatin-thrombin groups at the 6-week postoperative timepoint. During endoscopic sinus surgery, the mucosae of the nasal and sinus compartments will inevitably be damaged. Mucosal wound healing and regeneration can be complicated by chronic inflammation and metaplasia/fibrosis caused by various factors including foreign body reactions and secondary injury from compression/shearing with nasal packing [
      • Selvarajah J
      • Saim AB
      • Idrus RBH
      • Lokanathan Y.
      Current and alternative therapies for nasal mucosa injury: a review.
      ,
      • Rodrigues M
      • Kosaric N
      • Bonham CA
      • Gurtner GC.
      Wound healing: a cellular perspective.
      . While absorbable nasal packing products are effective for hemostasis and improve patient comfort compared to classical non-absorbable packing [
      • Massey CJ
      • Singh A.
      Advances in absorbable biomaterials and nasal packing.
      ], collagen-based devices have been linked to increased reactionary fibrosis in healing mucosa [
      • Massey CJ
      • Singh A.
      Advances in absorbable biomaterials and nasal packing.
      ,
      • Maccabee MS
      • Trune DR
      • Hwang PH.
      Effects of topically applied biomaterials on paranasal sinus mucosal healing.
      . Following initial hemostasis, normal wound healing proceeds into tightly-regulated inflammatory and proliferative stages, followed by tissue reorganization accompanied by appropriate fibrinolysis. Ideally, the original mucosal histology is regained, consisting of a confluent epithelial layer interspersed with secretory cells that has regenerated and is anchored atop a well-organized substratum containing balanced vascularization, innervation, and ECM composition [
      • Selvarajah J
      • Saim AB
      • Idrus RBH
      • Lokanathan Y.
      Current and alternative therapies for nasal mucosa injury: a review.
      ,
      • Rodrigues M
      • Kosaric N
      • Bonham CA
      • Gurtner GC.
      Wound healing: a cellular perspective.
      .
      Prior studies suggest that RADA16-based hydrogels support good wound healing and tissue regeneration [
      • Sankar S
      • O'Neill K
      • Bagot D'Arc M
      • Rebeca F
      • Buffier M
      • Aleksi E
      • et al.
      Clinical use of the self-assembling peptide RADA16: a review of current and future trends in biomedicine.
      ,
      • Wang R
      • Wang Z
      • Guo Y
      • Li H
      • Chen Z.
      Design of a RADA16-based self-assembling peptide nanofiber scaffold for biomedical applications.
      , perhaps by functioning as a temporary biocompatible surrogate ECM that permits recruitment, proliferation, and functional development of replacement cells that are critical to tissue regeneration [
      • Yi S
      • Ding F
      • Gong L
      • Gu X.
      Extracellular matrix scaffolds for tissue engineering and regenerative medicine.
      ,
      • Han C
      • Zhang Z
      • Sun J
      • Li K
      • Li Y
      • Ren C
      • et al.
      Self-assembling peptide-based hydrogels in angiogenesis.
      ,
      • Gelain F
      • Luo Z
      • Zhang S.
      Self-assembling peptide EAK16 and RADA16 nanofiber scaffold hydrogel.
      ,
      • Tsiamoulos ZP
      • Rajaratnam R
      • Sibbons PD
      • Saunders BP.
      Use of a novel synthetic, topical gel to enhance healing post endoscopic mucosal resection: a randomised, blinded preclinical study.
      ]. Gelatin-based hydrogels are low-immunogenicity and non-toxic and have been explored as wound-healing substrates [
      • Shea BS
      • Probst CK
      • Brazee PL
      • Rotile NJ
      • Blasi F
      • Weinreb PH
      • et al.
      Uncoupling of the profibrotic and hemostatic effects of thrombin in lung fibrosis.
      ]; however, the inclusion of thrombin in gelatin-thrombin hemostatic products might inhibit mucosal wound healing due to thrombin's profibrotic nature, promotion of fibroblast proliferation in various pathologies, and activation of multiple inflammatory processes [
      • Shea BS
      • Probst CK
      • Brazee PL
      • Rotile NJ
      • Blasi F
      • Weinreb PH
      • et al.
      Uncoupling of the profibrotic and hemostatic effects of thrombin in lung fibrosis.
      ,
      • Danckwardt S
      • Hentze MW
      • Kulozik AE.
      Pathologies at the nexus of blood coagulation and inflammation: thrombin in hemostasis, cancer, and beyond.
      ,
      • Levi M
      • van der Poll T
      • Buller HR.
      Bidirectional relation between inflammation and coagulation.
      ]. Other absorbable natural and synthetic biopolymer gels have been previously explored in this area including chitosan and oxidized cellulose. While these products have consistently demonstrated reduced bleeding, there is mixed evidence regarding their wound healing properties [
      • Maccabee MS
      • Trune DR
      • Hwang PH.
      Effects of topically applied biomaterials on paranasal sinus mucosal healing.
      ,
      • Antisdel JL
      • Meyer A
      • Comer B
      • Jang D
      • Gurrola J
      • Khabbaz E
      • et al.
      Product comparison model in otolaryngology: equivalency analysis of absorbable hemostatic agents after endoscopic sinus surgery.
      . Chitosan gel in particular has been shown to be effective in reducing the rate of synechiae formation both in a similar sheep model and humans [
      • Medina JG
      • Das S.
      Sprayable chitosan/starch-based sealant reduces adhesion formation in a sheep model for chronic sinusitis.
      ,
      • Valentine R
      • Athanasiadis T
      • Moratti S
      • Hanton L
      • Robinson S
      • Wormald P-J.
      The efficacy of a novel chitosan gel on haemostasis and wound healing after endoscopic sinus surgery.
      ; however, chitosan may impede the re-epithelialization of the nose during the wound healing process [
      • Huang TW
      • Wei CK
      • Su HW
      • Fang KM.
      Chitosan promotes aquaporin formation and inhibits mucociliary differentiation of nasal epithelial cells through increased TGF-β1 production.
      ]. The utility of RADA16 hydrogels in supporting wound healing has also been demonstrated in non-sinonasal mucosae including animal models of intestinal and middle ear mucosal injury [
      • Tsiamoulos ZP
      • Rajaratnam R
      • Sibbons PD
      • Saunders BP.
      Use of a novel synthetic, topical gel to enhance healing post endoscopic mucosal resection: a randomised, blinded preclinical study.
      ,
      • Oumrani S
      • Barret M
      • Bordaçahar B
      • Beuvon F
      • Hochart G
      • Pagnon-Minot A.
      Application of a self-assembling peptide matrix prevents esophageal stricture after circumferential endoscopic submucosal dissection in a pig model.
      ,
      • Akiyama N
      • Yamamoto-Fukuda T
      • Takahashi H
      • Koji T.
      In situ tissue engineering with synthetic self-assembling peptide nanofiber scaffolds, PuraMatrix, for mucosal regeneration in the rat middle-ear.
      , and in clinical trials involving endoscopic resection of gastric ulcers [
      • Uraoka T
      • Ochiai Y
      • Fujimoto A
      • Goto O
      • Kawahara Y
      • Kobayashi N
      • et al.
      A novel fully synthetic and self-assembled peptide solution for endoscopic submucosal dissection-induced ulcer in the stomach.
      ], and esophageal and colorectal mucosae [
      • Subramaniam S
      • Kandiah K
      • Chedgy F
      • Fogg C
      • Thayalasekaran S
      • Alkandari A
      • et al.
      A novel self-assembling peptide for hemostasis during endoscopic submucosal dissection: a randomized controlled trial.
      ]. Thus, multiple experimental and clinical experiences support the usefulness of RADA16-based hydrogels in facilitating postoperative mucosal wound healing.
      Wound sites treated with RADA16 demonstrated significantly lower rates of inappropriate submucosal fibrosis and angiogenesis compared to Control and Gelatin-thrombin sites. These parameters were the primary drivers in the lower Histopathology Composite Score with RADA16, because although mean epithelial erosion and inflammatory infiltrate scores were both numerically lower in the RADA16 group than Control or Gelatin-thrombin groups, those differences were not significant. By histology, fibrosis at 6 weeks was observed in 50% of Gelatin-thrombin wound sites and 0% of RADA16-treated sites; this disparity was also present in the number and extent of grossly observable synechiae with the different biological treatments. Compared to unoperated adjacent mucosae, the 6-week healed epithelial lining of RADA16-treated wound sites had regenerated normal seromucous and goblet cell numbers and morphology in 70% and 55% of wounds, respectively; similar to secretory cell regeneration in the other groups. Thus RADA16 was consistently associated with reduced fibrosis and scarring inherent to adhesion formation, and appropriate submucosal revascularization.
      RADA16 achieves hemostasis by spontaneously forming a clear hydrogel barrier when applied at the point of bleeding, allowing it to operate independently of the coagulation system [
      • Sankar S
      • O'Neill K
      • Bagot D'Arc M
      • Rebeca F
      • Buffier M
      • Aleksi E
      • et al.
      Clinical use of the self-assembling peptide RADA16: a review of current and future trends in biomedicine.
      ,
      • Zhang S.
      Discovery and design of self-assembling peptides.
      ,
      • Ellis-Behnke RG
      • Liang YX
      • Tay DKC
      • Kau PWF
      • Schneider GE
      • Zhang S
      • et al.
      Nano hemostat solution: immediate hemostasis at the nanoscale.
      ,
      • Wang R
      • Wang Z
      • Guo Y
      • Li H
      • Chen Z.
      Design of a RADA16-based self-assembling peptide nanofiber scaffold for biomedical applications.
      ,
      3-D Matrix Europe SAS; Caluire et Cuire, France
      PuraStat® Absorbable haemostatic material instructions for use.
      ]. This property would suggest its efficacy as a hemostatic agent even in patients with known coagulation system disorders. Additionally, the syringe-delivered viscous RADA16 preparation facilitates administration in the setting of endoscopic nasal surgery where vision is often limited to a narrow and easily obscured field [
      US Food and Drug Administration
      510(k) Premarket Notification K183015, summary.
      ,
      • Lee MF
      • Ma Z
      • Ananda A.
      A novel haemostatic agent based on self-assembling peptides in the setting of nasal endoscopic surgery, a case series.
      . Excess unreacted RADA16 gel not in direct contact with the bleeding point can easily be aspirated and cleared from surgical sites intra-operatively. The hydrogel's transparent nature allows good visualization of the wound surface to evaluate if further surgical revision is required. These properties of RADA16 have also led to its use as a hemostatic agent in cardiothoracic and gastrointestinal surgery [
      • Giritharan S
      • Salhiyyah K
      • Tsang GM
      • Ohri SK.
      Feasibility of a novel, synthetic, self-assembling peptide for suture-line haemostasis in cardiac surgery.
      ,
      • Ortenzi M
      • Haji A.
      Safety and feasibility of PuraStat® in laparoscopic colorectal surgery (Feasibility study).
      .
      RADA16 is the first self-assembling peptide-based agent to see clinical use in endoscopic nasal surgery. The current study demonstrated reduced postoperative adhesion formation and good wound healing after RADA16 application to surgical wounds during experimental endoscopic sinonasal procedures, and provided new insight into underlying cellular mechanisms. Primary study limitations were the lack of an inactive hydrogel comparator group, the relatively low n-values that precluded more advanced statistical analysis, and the lack of generalizability of findings from animal studies to the human scenario. This study only evaluated hemostasis and delayed rebleeding at wounds with oozing mild-to-moderate bleeding, the indication for which RADA16 products are approved [
      • Sankar S
      • O'Neill K
      • Bagot D'Arc M
      • Rebeca F
      • Buffier M
      • Aleksi E
      • et al.
      Clinical use of the self-assembling peptide RADA16: a review of current and future trends in biomedicine.
      ,
      3-D Matrix Europe SAS; Caluire et Cuire, France
      PuraStat® Absorbable haemostatic material instructions for use.
      ,
      US Food and Drug Administration
      510(k) Premarket Notification K210098, summary.
      ,
      US Food and Drug Administration
      510(k) Premarket Notification K183015, summary.
      , and not in wounds with heavy or pulsatile bleeding.

      5. Conclusion

      While RADA16 and Gelatin-thrombin preparations were both effective hemostatic agents, only RADA16 treatment was associated with no synechiae presence at the postoperative Week-6 study terminus. Histopathologically, RADA16 treatment was associated with better wound healing scores that were driven primarily by reduced submucosal fibrous tissue deposition and appropriate revascularization.

      Disclosure

      Funding for the sheep, RADA16 and gelatin-thrombin materials, and veterinary surgery facilities was provided by 3-D Matrix Medical Technology Pty Ltd, Melbourne, VIC, Australia. The funder had no role in the study design, data collection, decision to publish, or preparation of the manuscript. The authors declare that they have no other potential conflicts of interest, financial or otherwise, related to the research described in this paper.

      Declaration of Competing Interest

      The authors declare that they have no other potential conflicts of interest, financial or otherwise, related to the research described in this paper.

      Acknowledgements

      The authors thank Maurice Bagot d'Arc, MD (Blue Pharma SAS, Paris, France) with helping create the project's conceptual design, Andrew Dart, DVM, PhD and the staff the the University of Sydney Veterinary Centre (Camden, NSW, Australia) who assisted with the sheep model, Mark Krockenberger, PhD (University of Sydney School of Veterinary Science) for histology analysis, and Matthew Silverman, MSci, PhD (Biomedical Writing Solutions, Wakulla Springs, FL) for scientific, statistical, and writing assistance. Doctor Bagot d'Arc is a medical consultant for 3-D Matrix Medical Technology Pty Ltd, Melbourne, VIC, Australia.

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