Clinical and radiographical evaluation of a bioresorbable collagen membrane of fish origin in the treatment of periodontal intrabony defects: A preliminary study.

BACKGROUND: Recently, there has been interest in non-mammalian collagen sources such as fish collagen in periodontal regeneration. In the present study, collagen barrier membrane of fish origin was assessed in the treatment of periodontal intrabony defects.
MATERIALS AND METHODS: Ten systemically healthy chronic periodontitis patients having a paired osseous defect in the mandibular posterior teeth were selected and randomly assigned to receive a collagen membrane (test) or open flap debridement (control) in a split mouth design. Clinical parameters such as Plaque index, Gingival bleeding index, Probing pocket depth, Relative attachment level, and Recession were recorded at baseline, 3, 6, and at 9 months, while radiographic evaluation was done to assess alveolar crestal bone level and percentage of defect fill at 6 and 9 months using autoCAD 2007 software. Student's t test (two-tailed, dependent) was used to find the significance of study parameters on continuous scale. Significance was set at 5% level of significance. Wilcoxon signed rank test was used to find the significance of percentage change of defect fill.
RESULTS: The comparison between the two groups did not show any statistically significant differences in the parameters assessed (P > 0.05) but, within each group, clinical parameters showed statistically significant differences from baseline to 9 months (P < 0.05).
CONCLUSION: Within the limits of the study, it can be inferred that no significant differences were found either by using collagen membrane of fish origin or open flap debridement in the treatment of periodontal intrabony defects.

RCT Entities:   Population Interventions Outcomes

INTRODUCTION

Periodontal therapy involves controlling of periodontal infection and aims at regeneration of the lost periodontium.[1] The two techniques with the most successful documentation of periodontal regeneration are osseous grafting and guided-tissue regeneration (GTR).[2] The regeneration of the periodontium with new connective fiber insertion, new cementum, and new bone formation constitutes ideal healing. The regeneration process can only be initiated by periodontal ligament cells derived from the remaining periodontium as these are the cells capable of differentiating into new fibroblasts, cementoblasts, and osteoblasts.[34] Nyman et al.[3] suggested the placement of a physical barrier between the flap and the root surface to exclude gingival connective tissue and epithelium from the healing process, giving the periodontal ligament cells the opportunity to repopulate the coagulum on the root surface. This technique was named GTR.

The barriers recommended for the use in GTR, regardless of the material used, must be safe, biocompatible, and non-toxic, not induce any inflammatory response, and be designed for clinical applicability based on the morphology of the osseous defects.[5] The barrier used are either nonresorbable or bioresorbable membranes. Resorbable membranes are preferred as nonresorbable membranes require second surgical procedures to remove it. Furthermore, gingival recession, device exposure, infection, and inflammation are frequently experienced with nonresorbable membranes.[6]

Among resorbable barriers, investigators have examined type-I collagen for the use in GTR procedures as it is a major extracellular macromolecule of the periodontal connective tissue. Also collagen is known to be a weak immunogen, has a chemotactic property for fibroblasts, and acts as a barrier for migrating epithelial cells in vitro.[7] Collagen from mammalian sources, primarily bovine skin, has been utilized in foods, cosmetics, and biomaterials and has the advantage of biodegradability and low toxicity. Therefore, there have been various attempts to use mammalian collagen in the medical field as a scaffold for developing artificial organs. However, the use of bovine collagen has been reconsidered, as some reports have shown the risk of transmission of bovine spongiform encephalopathy (BSE) to human beings. Recently, there has been interest in non-mammalian collagen sources, primarily in fish collagen such as that of shark and salmon, as it is known to have low risk for transmission of infectious disease to humans than bovine collagen.[8]

Hence, an attempt was made to assess, a bioresorbable collagen membrane of fish origin (Periocol™, Eucare pharmaceuticals Private limited, Chennai, India) in the treatment of periodontal intrabony defects. The study was planned with the following objectives.

To assess the clinical parameters like probing pocket depth (PPD), relative attachment level (RAL) and recession (R) with and without a bioresorbable collagen barrier membrane in the treatment of intrabony defects

To assess the alveolar crestal bone level (ABL) and percentage of defect fill (DF) radiographically in intrabony defects with and without a bioresorbable collagen barrier membrane.

MATERIALS AND METHODS

The study employed a total of 10 patients (6 males and 4 females) aged between 21 and 50 years in a split mouth manner to assess the collagen membrane both clinically and radiographically. Subjects having bilateral isolated intra osseous defects in the mandibular posterior teeth were screened based on the following inclusion and exclusion criteria.

Inclusion criteria: (a) patients of either sex having chronic periodontitis, (b) patients who were systemically healthy with no contraindication to periodontal surgery, (c) non smokers, (d) presence of an intrabony defect with residual PPD of five millimeters (mm) and with radiographic evidence of the affected site, (e) patients having 2 to 3 mm band of keratinized tissue to allow surgical manipulation and suturing, and (f) patients who were co-operative and able to come for regular follow up. Exclusion criteria: (a) patients allergic or sensitive to any medication or any ingredient of the test material, (b) patients showing unacceptable oral hygiene compliance during or after phase I therapy, and (c) pregnant and lactating mothers.

Following selection of subjects based on inclusion criteria, patients were given an explanation of the study, and an informed consent was obtained. The study was analyzed and approved by the Institutional Review Board and the Ethical Clearance Committee of the institution. All patients underwent initial therapy consisting of oral hygiene instructions and scaling and root planing. Randomization was done by a coin toss to select the sites to be treated by GTR using a bioresorbable membrane (i.e., test group) and control sites treated by conventional open flap debridement (OFD). All the surgeries were performed by the same operator, and the measurements were recorded by a blinded examiner.

Clinical parameters assessment

Plaque index,[9] gingival bleeding index,[10] RAL, PPD, and R were recorded from baseline, 3, 6, and at 9 months using University of North Carolina-15 (UNC) probe. A customized acrylic occlusal stent with a vertical groove was made for proper guidance and orientation of the periodontal probe in the same plane [Figure 1]. The stents were preserved in the study casts to minimize distortion for follow-up measurements[11]. RAL was measured from the reference point (RP) on the stent to the base of the pocket (BOP). Recession was measured from RP to the free gingival margin (FGM). PPD was recorded by noting the difference between measurements from RP to FGM and RP to the BOP.

Figure 1

Baseline clinical parameters were assessed by using UNC-15 probe on a custom-made acrylic stent in relation to 46

Radiographic parameters assessment

Radiographs were taken at baseline, 6, and 9 months postoperatively. Intra-oral periapical radiographs were taken using a long cone/extension cone paralleling technique with the positioning device (Rinn® XCP, Dentsply, IL, USA.) and a size 2E speed intra-oral periapical radiographic film (Kodak X-ray film, USA) was used in a roentgen machine operating at 70 Kilo Voltage Power (Kvp), 0.6 milli amperes (mA). Bite registration was done using rubber base impression material and stored for follow-up assessment. Radiographs were scanned using a scanner (HP 3010G) at an input of 400 dpi and 100% scale. The scanned images were then analyzed with the help of auto CAD 2007 software. Radiographic parameters were recorded as follows: (1) distance from cementoenamel junction to base of the defect (CEJ to BD) =A, (2) distance from cementoenamel junction to alveolar crest (CEJ to AC) =B, (3) defect depth = A-B, (4) DF was measured as the difference in defect depth from baseline to 6 months and 9 months.[12]

Surgical procedure

Patients were anaesthetized with 2% lignocaine solution at the surgical site. Intracrevicular incisions were given both buccally and lingually and full thickness mucoperiosteal flap was elevated. After the reflection of the flap and exposure of the osseous defect, a thorough surgical debridement was carried out to remove sub-gingival plaque, calculus, diseased granulation tissue, and pocket epithelium [Figure 2]. The surgical sites were irrigated with sterile saline, and care was taken to keep the area free of saliva. The GTR membrane was removed from the sterile package and hydrated in normal saline for few seconds before placement on to the defect to improve adhesion properties and malleability. The membrane design was custom prepared chair side to receive for an intrabony defect and then carefully tweezed through the interproximal contact area [Figure 3] and extended 2 to 3 mm beyond the bony margin, to provide a broad base during placement. Prior to closure of mucoperiosteal flaps, decortication of the osseous defect was done to induce bleeding. Surgical flaps were repositioned to the pre-surgical level and sutured with 3-0 silk suture (Mersilk -Ethicon, Division of Johnson and Johnson Ltd, Aurangabad, India) utilizing an interrupted direct loop suturing technique thereby achieving primary closure [Figure 4]. Care was taken not to displace the GTR membrane during suturing. A non-eugenol periodontal dressing (Coe-pack - GC America INC. ALSIP, IL, USA) was placed on the surgical area following which the post-operative instructions were given. Control site was treated with only OFD.

Figure 2

Following incision, a full thickness mucoperiosteal flap was reflected and the defect debridement was done

Figure 3

The collagen barrier membrane was trimmed to the required size and shape and placed over the interproximal vertical defect

Figure 4

Suturing was done using 3-0 silk with a simple interrupted direct loop technique

Post-operative medication included Amoxicillin (Novomox-Cipla Ltd, India) 500 mg three times daily for 5 days, non-steroidal anti-inflammatory agent; three times daily for 5 days and 0.2% chlorhexidine gluconate (Hexidine®-ICPA Health Products Ltd, Mumbai, India) mouth rinse for 3 to 4 weeks. Periodontal dressing and sutures were removed 1 week following surgery. All patients were recalled at a 1-month interval initially to monitor wound healing and to reinforce oral hygiene instructions. Later, they were followed up at 3, 6, and at 9 months to assess clinical and radiographic parameters postoperatively [Figure 5].

Figure 5

Postoperative follow up at 9 months in relation to the test site

Statistical analysis

The Statistical software SPSS 15.0, Stata 8.0, MedCalc 9.0.1 and Systat 11.0 were used for the analysis of the data. Descriptive statistical analysis was carried out in the present study. Student t test (two-tailed, dependent) was used to find the significance of study parameters on a continuous scale within each group. Significance was set at 5% level of significance. Wilcoxon signed-rank test was used to find the significance of percentage change of DF.

RESULTS

Ten patients participated in the study. Of the 10 patients, 1 patient failed to return for the 9 months recall, and 9 patients completed the study. Over the course of the study, there was no membrane exposure or dehiscence or infectious episodes, or any other adverse complications in sites treated with GTR.

The mean plaque index scores in test sites at baseline were 0.79 ± 0.37, which reduced to 0.49 ± 0.31 at 3 months, and further reduced to 0.35 ± 0.24 and 0.32 ± 0.11 at 6 and 9 months respectively. This difference was found to be highly statistically significant with P = 0.008, P < 0.001, P = 0.002 at 3, 6, and 9 months, respectively. The mean plaque index scores in control sites at baseline were 0.91 ± 0.35, which reduced to 0.54 ± 0.36 at 3 months and further reduced to 0.29 ± 0.23 and 0.32 ± 0.11 at 6 and 9 months, respectively. This difference was found to be highly statistically significant with P = 0.011, P = 0.001, P < 0.001 at 3, 6, and 9 months, respectively.

The mean gingival bleeding index scores in test sites at baseline were 0.72 ± 0.22, reduced to 0.28 ± 0.17 at 3 months and further to 0.18 ± 0.11 and 0.22 ± 0.44 at 6 and 9 months, respectively. This difference was found to be highly statistically significant with P = 0.003, P = 0.001, P = 0.001 at 3, 6, and 9 months, respectively. Similarly, in control sites the mean gingival bleeding index scores at baseline were 0.69 ± 0.22, reduced to 0.30 ± 0.26 at 3 months and further reduced to 0.18 ± 0.14 at 6 months. There was a slight increase to 0.23 ± 0.14 at 9 months. This difference was found to be highly statistically significant with P = 0.016, P = 0.001, P = 0.002 at 3, 6, and 9 months, respectively.

The mean RAL scores in test sites at baseline were 12.33 ± 2.83 mm, reduced to 9.67 ± 3.00 mm at 3 months and further reduced to 9.00 ± 2.00 mm at 6 months. There was a slight increase to 9.78 ± 2.28 mm at 9 months. This difference was found to be highly statistically significant with P = 0.011, P = 0.004, P = 0.006 at 3, 6, and 9 months, respectively. The mean RAL scores in control sites at baseline were 12.44 ± 3.00 mm, reduced to 11.00 ± 3.08 mm at 3 months and further reduced to 10.22 ± 2.91 mm at 6 months. There was a slight increase to 10.89 ± 2.47 mm at 9 months. This difference was found to be statistically significant with P = 0.089, P = 0.017, P = 0.071 at 3, 6, and 9 months, respectively.

The mean PPD scores in test sites at baseline were 7.22 ± 2.28 mm, reduced to 4.33 ± 3.16 mm at 3 months. At 6 months, PPD further reduced to 3.00 ± 1.80 mm. There was a slight increase to 4.00 ± 1.94 mm at 9 months. This difference was found to be highly statistically significant with P = 0.007, P = 0.001, P < 0.001 at 3, 6, and 9 months, respectively. The mean PPD in the control site at baseline was 7.00 ± 2.55 mm, reduced to 4.33 ± 2.12 mm and 4.11 ± 2.15 mm at 3 and 6 months, respectively. There was a slight increase in PPD to 4.33 ± 1.73 mm at 9 months. This difference was found to be highly statistically significant with P = 0.018, P = 0.020, P = 0.008 at 3, 6, and 9 months, respectively.

The mean distance from the lower border of the stent to the gingival margin was 7.22 ± 4.24 mm at baseline in test sites. At the end of 3 months, it increased to 7.33 ± 4.44 mm and then at 6 and 9 months it reduced to 7.11 ± 3.44 mm and 6.89 ± 3.29 mm, respectively. This difference was found not to be statistically significant. The mean distance from the lower border of the stent to the gingival margin was 5.44 ± 1.94 mm at baseline in control sites. At the end of 3 months, it increased to 6.89 ± 2.52 mm and then at 6 and 9 months; it reduced to 6.55 ± 2.35 mm, and 6.56 ± 2.55 mm, respectively. This difference was found not to be statistically significant.

The comparison from baseline to 3 months, 6 months, and 9 months with respect to clinical parameters showed significant statistical difference within the test and control groups (P < 0.05), but the inter group comparison did not show significant statistical differences [Table 1].

Table 1

Clinical parameters assessed (mean±standard deviation) at baseline, 3 months, 6 months, and 9 months for test and control sites

The percent of DF in test sites at 6 months was 51.85 median % and 46.66 median % at 9 months. At control sites, the percent of DF at 6 months was 46.03 median % and 78.46 median % at 9 months. Radiographical evaluation with respect to ABL and percentage of DF from baseline to 6 months and at 9 months did not show significant statistical differences both within and between the groups (P > 0.05) [Table 2].

Table 2

Radiographic parameters assessed (mean±standard deviation) at baseline, 6 months, and 9 months for test and control sites

DISCUSSION

The regeneration of the periodontium is the result of elective cellular events that are facilitated by tissue exclusion using bioabsorbable or non-resorbable barriers.[1] The results of our study demonstrated that no significant differences were found either by using a bioabsorbable membrane or OFD in the treatment of intrabony defects both clinically and radiographically. This study was designed as a spilt-mouth investigation to facilitate the comparison of both GTR and OFD under similar healing conditions by eliminating patient-specific characteristics, which might impact on the results of the conventional and regenerative surgeries.[13]

The clinical methods used to evaluate therapeutic end points include various assessments of gingival inflammation, periodontal probing, radiographs, and re-entry procedures. The true endpoint is determined by histology. However, due to ethical reasons and patient concerns, re-entry procedures and histological analysis are not feasible in clinical trials. Hence, clinical criteria that provide surrogate evidence of periodontal regeneration were considered.[14]

The plaque and gingival bleeding indices were assessed to monitor patient's oral hygiene and its effect on soft tissues. There was a reduction in mean plaque index and gingival bleeding index for the test and control group from baseline to 9 months with a high statistical significance (P < 0.05). There was no statistically significant difference between test and control groups with respect to plaque and gingival scores thereby showing that there was a good maintenance of oral hygiene throughout the study. Cortellini and Pini-prato[15] et al. have reported the clinical effect of plaque control and the influence of increased bacterial contamination on the outcomes to GTR.

The clinical parameters on the sites treated with the barrier were similar to those of open flap surgery. PPD in the test group at baseline was 7.22 ± 2.28 mm which reduced to 4.00 ± 1.94 mm at the end of 9 months. In the control group, the mean PPD reduced from 7.00 ± 2.55 to 4.33 ± 1.73 mm at 9 months. The clinical attachment level or RAL has become widely accepted as the primary clinical endpoint of regenerative attempts around natural teeth. Significant loss in clinical attachment levels is reflected in histological loss of the tooth's attachment apparatus.[16] The mean RAL in the test group reduced from 12.33 ± 2.83 to 9.78 ± 2.28 mm at 9 months. At the control sites, the mean RAL reduced from 12.44 ± 3.00 to 10.89 ± 2.47 mm at the end of 9 months. Although these clinical changes were statistically significant between baseline and final measurements within the groups, no statistically significant differences were found between the test and control groups [Table 1]. Cortellini[17] and Falk[18] et al. have reported greater gain in the clinical attachment level after GTR; however, these authors were dealing with deeper initial intrabony defects. The defects in this study were smaller as compared to other studies. This may suggest that clinical attachment level after GTR is dependent up on the initial defect depth.

During the surgical procedures, efforts were made to preserve the interproximal soft tissues. In relation to gingival recession, there was no significant statistical difference in test and control sites. There was a decrease of 0.3 mm of recession from baseline to 9 months in GTR-treated sites. The possible explanation could be due to non-exposure of the barrier membrane following placement in any of the test sites. This would have prevented undue exposure of the barrier to oral environment, thereby preventing infection or soft-tissue inflammation, which would lead to faster resorption of membrane and cause recession. In contrast, studies by Cortellini[19] and Becker[20] et al. in 1996 have shown that sites treated with GTR have increased amount of recession between 1.8 and 2 mm. Gottlow[21] et al. in 1986 has postulated that the greater the degree of gingival recession, shorter the root surface area that is provided for the repopulation of the periodontal ligament cells thereby negatively influences new attachment formation.

New bone formation is frequently used as a primary outcome variable in controlled clinical trials of regenerative therapy. Radiographic monitoring of alveolar bone changes following regenerative procedures is a non-invasive painless alternative to direct bone measurement. The radiographic variables assessed were the levels of crestal bone and extent of DF. Crestal bone resorption is a characteristic feature after the flap procedures. Most of the alveolar bone changes following regenerative therapy of intrabony defects occur in the intrabony component while crestal resorption may be minimal or may not occur at all.[14] In our study, no significant change in the level of the alveolar crest was seen in both test and control groups after 6 and 9 months. This was in accordance with a similar study done by Becker[22] et al. where in 0.33 mm of crestal resorption was noted in GTR-treated sites.

There was a significant difference in defect depth from baseline to 9 months in test and control groups (P < 0.05) while an inter group comparison did not show any significant differences in defect depth. Similarly, no significant difference was found in percentage of DF both within and between the groups [Table 2 and Figures 6–11]. This observation is in accordance to the findings by Gottlow[21] et al. in 1986, Becker[22] et al. in 1993, and Micheal[23] et al. in 2000. In our study, conventional radiography with image analysis software (Auto CAD 2007) was used to assess radiographic parameters as applicability and reliability of the image analysis system in alveolar bone measurement have been studied previously by Micheal,[24] Hausmann[25] et al., and Verdonschot[26] et al. However, advanced radiographic techniques like subtraction digital radiography remain the method of choice if subtle changes in mineralization of alveolar bone need to be detected.[26]

Figure 6

Radiograph of test site at baseline in relation to 46 and 47

Figure 11

Radiograph of control site at 9 months in relation to 36 and 37

Radiograph of control site at baseline in relation to 36 and 37

Radiograph of test site at 6 months in relation to 46 and 47

Radiograph of control site at 6 months in relation to 36 and 37

Radiograph of test site at 9 months in relation to 46 and 47

The GTR material utilized in this study is an orange-brown color type-I fish collagen. It is available in dimensions of 1 × 1 cm and 1 × 2 cm and can be easily manipulated and adapted to the root surface. It has a resorption time of 6 weeks. In fish, the largest concentration of collagen is found in the skeleton, fins, skin, and air bladder. Use of this barrier in our study did not show any case reporting with any form of allergy or hypersensitivity reaction.

The results of the study is difficult to compare due to a number of possible differences like in small sample size, study design, patient/defect selection, percentage of one/two/three wall defect components, baseline depth of defects, probing forces, and evaluation method. Hence, the regenerative potential and beneficial effects of this GTR membrane should be further evaluated with larger sample size, longer follow ups, use of advanced radiographic aids (like cone-beam-computed tomography, etc.) and with combination therapy involving bone grafts.

CONCLUSION

Within the limits of the study, it can be inferred that no significant differences were found either by using a collagen membrane of fish origin or open flap debridement in the treatment of periodontal intrabony defects both clinically and radiographically.