Evaluation of Omega-3 piscine acellular dermal matrix membrane as a soft tissue alternative for the treatment of mucogingival defects

Abstract

BACKGROUND:

The natural Omega-3 lipids in the OADM serve to reduce inflammation. Preliminary results in a human model reported no adverse events and favorable healing and esthetic outcomes.

OBJECTIVE:

The primary objective of this animal model study was to histologically evaluate the use of Omega-3 piscine acellular dermal matrix (OADM) as a soft tissue alternative in surgically created mucogingival defects.

METHODS:

Bilateral maxillary canines in 6 adult beagle dogs were randomly assigned to the test (OADM) and control sub-epithelial connective tissue graft (SCTG) groups. Dehiscence defects 4 $\times$ 6 mm were created surgically on the buccal surfaces. The OADM/SCTG were placed to completely cover the root surface to the level of the cemento-enamel junction and sutured with resorbable sling sutures. The gingival flap was repositioned to cover the grafts. At two months follow-up, the dogs were sacrificed, and block samples were retrieved, including the whole canine and periodontium. The histological outcomes were evaluated using qualitative analysis.

RESULTS:

The qualitative histological analysis revealed the oral, sulcular and junctional epithelium had healed with normal appearance on both test and control sites. None of the test (OADM) samples presented with any foreign body reaction.

CONCLUSION:

The use of this new piscine xenograft resulted in minimal complications and the attachment apparatus healed normally.

Keywords

Acellular dermal matrix mucogingival defects connective tissue graft xenograft dentistry

1. Background

Gingival soft tissue augmentation is a procedure used to increase the width of keratinized tissue (KT) and/or achieve root coverage around teeth [1, 2, 3, 4], The gold standard for this procedure is the use of autogenous tissue harvested from the palate [5, 6]. The limitations include the inconsistent quantity and quality of the harvested autogenous tissue, the increased healing time for the donor site and patient discomfort [1, 2].

Various soft tissue alternatives to autogenous sub-epithelial connective tissue grafts (SCTG) and free gingival grafts (FGG) have been developed [7, 8, 9, 10]. Acellular dermal matrix allografts and xenografts have been used with some success. The wound healing response of soft tissue alternatives has been evaluated histologically. The histological outcomes used to determine the successful integration of a soft tissue alternative graft are characterized by the absence of elastic fibers, presence of rete pegs at the epithelial-connective tissue interface and a layer of keratin in the epithelium [11, 12, 13, 14, 15]. The majority of the xenografts are processed from bovine or porcine species [10, 11, 12, 13]. However, religious considerations or the risk of viral transmission may limit the use of these materials. This piscine xenograft can be an alternative for those clinical scenarios.

The Omega-3 piscine acellular dermal matrix (OADM) – Kerecis, Iceland is derived from acellular fish skin from North Atlantic cod fish (Gadus morhua), harvested immediately from the caught fish [16]. The graft contains lipids and proteins, that in a concerted manner may help the body regenerate damaged tissue. This piscine acellular dermal matrix acts as a scaffold for revascularization and repopulation of cells, during epithelial wound healing. The natural Omega-3 lipids in the OADM also serve to reduce inflammation allowing wound healing to occur in a more favorable cellular environment [15]. Preliminary results in a human model reported no adverse events and favorable healing and esthetic outcomes [17].

The primary objective of this study in an animal model (beagle dog) was to evaluate and compare the histological wound healing response of OADM compared with the SCTG in surgically created mucogingival defects. The histological outcomes were evaluated using qualitative analysis.

2. Material and methods

After receiving institutional ethical approval (Dental Implant Research Chair, College of Dentistry, King Saud University, Riyadh – Saudi Arabia; #DIORC 00123), six healthy beagle dogs and free of periodontal disease were enrolled in this split-mouth design animal study. The study was carried at King Saud University, Riyadh – Saudi Arabia and the ethics committee that granted approval for the study has the jurisdiction to authorize studies using animals. The mean age of the dogs was 12 months at the beginning of the study and they belonged to the academic institution, King Saud University Bilateral maxillary canines in each animal were randomly assigned to the test and control groups. The groups assigned were either a SCTG (control) or OADM (test). The randomization was performed using a computer generated sequence (www.random.org).

General anesthesia was achieved using Ketalar (10 mg/kg body weight; Pfizer Inc, New York, NY). Prior to the surgery, the dogs received supra-gingival scaling using an ultrasonic scaler (Hu-Friedy, Chicago, IL). On the day of the surgery, a preoperative injection of local anesthetic 2% Xylocaine (Astra, Westborough, MA, USA) with epinephrine 5 mg/mL was administered. At the beginning of the surgical procedure clinical photographs were taken for both groups. Following standard of care procedures, the procedures were performed in a similar manner with the only difference being the material used to correct the surgically created mucogingival defect.

A sulcular and two vertical releasing incisions with full-thickness flaps were reflected (Fig. 1A–C). The denuded root surfaces were scaled and root planed to ensure the removal of the cementum and fibers. Dehiscence defects 4 $\times$ 6 mm were created surgically on the buccal maxillary canines in all the beagle dogs.

Figure 1.

Overview of the surgical procedure for the OADM group.

2.1 Test group

The piscine xenograft was measured and adjusted to fit the surgical defect. The material was hydrated in saline for one minute, according to the manufacturer’s recommendations. The xenograft was placed to completely cover the root surface to the level of the cemento-enamel junction and sutured with resorbable sling sutures (Fig. 1D). The material was covered by the gingival flap with minimum coronal flap advancement. Simple interrupted non-absorbable sutures were used to approximate the flaps in the initial position (Fig. 1E). Primary wound closure was achieved.

2.2 Control group

The autogenous SCTG was grafted from the palatal vault. Using a U-shaped incision in the lateral part of the palatal vault and a mucoperiosteal flap, the SCTG was retrieved following the dimensions of the created defect to be restored. The graft was inspected, removing glandular and adipose tissues. The 2.5 mm thickness SCTG was placed in the defect created and the same suturing technique as for the test group was used. The elevated buccal flap was re-positioned and simple interrupted sutures were used to immobilize the graft and reposition the buccal flap, with minimum coronal flap advancement. Primary wound closure was achieved in both the recipient and the donor sites.

Post-operative care was carried out for all the six dogs involved in this study. Daily plaque control was reinforced using 2.0% chlorhexidine by topical application. The first seven days the dogs were fed a soft diet. After a period of two weeks, anesthesia was administered and the sutures were removed and the areas debrided. Three weeks after the surgery, one dog passed away due to an infection not related to the project. At two months follow-up, the remaining five dogs were sacrificed and block samples were retrieved, including the whole canine and periodontium. The grafted soft tissue areas were retrieved with the adjacent healthy gingival tissue and the supporting bony structures. The maxillary canines and surrounding tissues were sectioned and prepared for histological assessment in 10% neutral formalin solution. The specimens were coded and decalcified in Morse solution, followed by routine processing for paraffin-embedded samples. Histomorphometry of the sections with vestibular-palatal orientation were obtained in order to visualize the buccal surfaces (Radboud University Medical Center, Nijmegen, The Netherlands). Hematoxylin and eosin (H&E) staining was used to highlight the histological characteristics of the retrieved sections.

2.3 Histological outcomes

Two investigators (TW, ID) screened the histological samples and only those histological samples free of any technical artifacts were selected for further analysis. Therefore, five samples were analyzed histologically. The analysis was performed on digital images determined on a light microscope with a 2X/0.05 objective utilizing SPOT imaging software 5.0. All the samples were confirmed by a third examiner (NK).

For the qualitative analysis, two calibrated blinded examiners (ID, NK) performed the descriptive assessment. The assessment was performed on digital images determined on a light microscope with a 2X and 40X/0.05 objective. The categories evaluated were: quantification of the inflammatory reaction in the graft side (absent, minimal, abundant); the presence or absence of a foreign body reaction; the degree of connective tissue organization of the graft (adequate, inadequate); changes to the appearance of the epithelium (normal, abnormal).

Figure 2.

8 week healing period for both the “control” and the “test” groups.

3. Results

At the eight-week time period (at the time of sacrifice) all the remaining sites were evaluated (Fig. 2). All five test sites healed normal. Only two of the control sites had healed normally, three of the control sites had failed to heal (there was severe recession). Therefore, only two samples from the control and five samples from the test group were available for the histological study.

3.1 Clinical analysis

All five test sites healed within normal limits. Only two of the control sites had healed normally, three of the control sites had failed to heal. The pre and post-operative photographs (time 0 and time 8 weeks) for the five test and two control specimens were compared. All included samples presented soft tissue integration and re-epithelialization of the grafts.

3.2 Histological analysis

The qualitative histological analysis revealed the oral, sulcular and junctional epithelium with normal appearance in both test and control sites. The blinded examiners were not able to differentiate the test from the control samples.

3.3 Test OADM samples

Figure 3.

Test sample. A. Magnification 2X, hematoxylin and eosin – The graft is adhering to tooth structure. Graft material comprised of stratified squamous epithelium and fibrous connective tissue. Star marks the alveolar bony crest. B. Magnification 40X, hematoxylin and eosin – The graft contains stratified squamous epithelium with underlying loose fibrous connective tissue containing numerous chronic inflammatory cells (marked by arrow). Star denotes stratified squamous epithelium.

At low power magnification (2X) – (Fig. 3A), the gingival tissues had reattached to the tooth structure. None of the samples had any foreign body reaction. The attachment apparatus appeared to have healed normally. The gingival and sulcular epithelium appeared normal and there was new connective tissue attachment to the root surface. The alveolar crest of the bone and the periodontal ligament appeared normal.

At high power magnification (40X), the connective tissue organization presented with a loose appearance. Overall, the inflammatory reaction present was minimal and was commonly located in the cervical area of most of the samples. In some of the samples evaluated there was stratified squamous epithelium with underlying loose fibrous connective tissue containing numerous chronic inflammatory cells (Fig. 3B). Less commonly observed was the presence of tight organization with connective tissue fibers that were parallel with less space/blood vessels from sulcular epithelium and attachment to the gingival epithelium. New bone formation and cementum was very limited and difficult to differentiate.

3.4 Control SCTG samples

Figure 4.

Control sample. A. Magnification 2X, hematoxylin and eosin – The sub-epithelial connective tissue graft is comprised of stratified squamous epithelium and fibrous connective tissue adhering to tooth structure. Star marks the alveolar crest. B. Magnification 40X, hematoxylin and eosin – The sub-epithelial connective tissue graft is comprised of stratified squamous epithelium underlying loose fibrous connective tissue. Chronic inflammatory cells (arrows) and a blood vessel filled with red blood cells (erythrocytes) (denoted by star) are noted.

At low power magnification (2X), (Fig. 4A), the attachment apparatus appears to have healed normally – similar with the test site. At higher power (40X), (Fig. 4B) the connective tissue organization was deemed normal, and was comprised of stratified squamous epithelium and underlying loose fibrous connective tissue. Chronic inflammatory cells and a blood vessel filled with red blood cells (erythrocytes) were noted.

4. Discussion

For the test group, the use of this new piscine xenograft resulted in no adverse effects and the attachment apparatus appeared to have healed normally. The piscine graft allowed for tissue integration and revasculation of the graft. The current study presented favorable results, similar results to other alternative materials when compared with SCTG [11, 13, 15].

Similar with Werfully et al., the surgical techniques followed standard of care procedures for root coverage [15]. Since wound healing was one of the outcomes evaluated in this study, epithelialization of the wound and connective tissue closure were critical to the procedures. Compared with the study by Werfully et al., which evaluated the periodontal wound healing in a beagle dog model for the first 28 days (4 weeks), our study evaluated outcomes at 8 weeks. In our study, 3 of the “control” samples failed to heal and presented with severe recession. This could have been due to the surgical challenges of harvesting and suturing SCTG in the animal model.

Compared with other xenograft materials, the piscine alternative handled relatively well was not friable during suturing and it did not tear. However, the second surgical site in this animal model healed very slowly, which has been reported previously. ${}^{18}$ We found it extremely challenging to harvest consistent SCTG from the palate in this model. Bleeding during the procedure was an issue that added to the challenge. In this study, the poor outcomes of the control sites were due to technical issues faced. The two samples that did heal allowed for some comparison.

When conducting a histomorphometric analysis one can expect that depending on the orientation of the cuts, there might be a slight discrepancy in the angulation of the cut and made quantitative analysis difficult. The qualitative histological analysis revealed the oral, sulcular and junctional epithelium with normal appearance in both test and control sites. The blinded examiners were not able to differentiate the test from the control samples. Histological analysis revealed an inflammatory reaction, but considering that the dogs did not have a regular plaque control protocol, that is expected. The histological outcomes of this study are encouraging, as Omega-3 Wound™piscine xenograft might be an alternative graft material that showed no adverse effects, allowed for normal healing. Therefore this material can be used as a substitute for SCTG. The clinical outcomes showed relatively normal healing post-operatively and that the width of KT was relatively stable.

One of the limitations of the study was the animal model study design – three weeks after the surgery, one dog passed away due to an infection not related to the project. Another limitation was the small sample size, but the report certainly provided valuable information regarding the safety and efficacy of the material investigated. Although there was minimal inflammation noted, future studies might include a plaque control protocol.

This soft tissue alternative is not recommended for any patients with known hypersensitivity to fish or any piscine products. There were no adverse effects seen clinically or histologically. The safety and efficacy of OADM for oral application will allow for pilot studies in other models (human).

5. Conclusion

The use of this new piscine xenograft resulted in minimal complications and attachment apparatus healed normally. There were no adverse events reported in any of the cases. The clinical and histological analysis revealed the oral, sulcular and junctional epithelium with normal appearance on both test and control sites. Further studies are recommended to investigate the safety and efficacy of OADM for oral application in human model.

Consent for publication

N/A. All data were de-identified and our manuscript does not contain data from any individual person.

Availability of data and material

All datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Funding

The study was supported exclusively by the academic institutions. TUSDM has covered the publication costs.

Author contributions

ID helped with the analysis and interpretation of the data and major compilation of the manuscript preparation. TW helped with data interpretation and compilation of the manuscript. FA, LH, UM helped with the data collection/blinded measurements and the descriptive statistics. AN helped with the study design and study implementation. NK helped with study design, implementation and overview of the manuscript compilation. MA helped with the study design and implementation. All authors read and approved the final manuscript.

Footnotes

Acknowledgments

The authors would like to acknowledge Professor John Jansen from Radboud University Medical Center (The Netherlands) for sharing his expertise with the preparation of the histological samples.

Conflict of interest

Dr. Dragan and Dr. Karimbux have consulted with Kerecis on developing a protocol for human studies.

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