Effect of nano-crystalline silver membrane on early wound healing after periodontal surgery: A comparative randomized study.

BACKGROUND: Periodontal dressings are used for wound protection and patient comfort. Nano-silver particles have the ability to promote wound healing through anti-inflammatory properties. Hence, the present study aims to evaluate early wound healing parameters following periodontal surgery using nano-crystalline silver membrane as periodontal dressing.
MATERIALS AND METHODS: Forty-two systemically healthy patients diagnosed with chronic periodontitis indicated for periodontal flap surgery were enrolled for the present study. Post surgery, the patients were randomly allocated to either a nano-crystalline silver dressing (Acticoat™) group (test group) or only the noneugenol dressing group (control group). Plaque index (PI) and wound healing index were recorded at the 7th- and 14th-day postsurgery. The microbiological analysis and vascular endothelial growth factor (VEGF) levels were evaluated at baseline and 7th-day postsurgery.
RESULTS: The healing index was significantly higher in the test group as compared to the control group at days 7 and 14 (P < 0.001; P < 0.001). The colony-forming units/ml count of bacteria were significantly reduced postsurgery in the test group (P = 0.019). VEGF levels increased significantly 7th-day postsurgery in the test group (P = 0.001). There was no statistically significant difference in the PI on the 7th-day postsurgery between the two groups (P = 0.173).
CONCLUSION: The results of the study revealed that silver can be used as a potent periodontal dressing ingredient that can decrease the microbial colonization beneath the pack and promote faster healing postsurgery due to its antimicrobial activity. Copyright:

INTRODUCTION

Periodontal dressing is a commonly used dressing placed following various periodontal surgical procedures including gingivectomies, flap procedures, crown lengthening, mucogingival procedure, and most commonly after periodontal regeneration.[1] These dressings help in holding the flap or graft in place; protecting, shaping, or molding the newly formed tissue; minimizing the experience of postoperative pain, infection, and hemorrhage; protecting the surgical site from trauma from mechanical insults like during eating and drinking, and also, supporting the mobile teeth during the healing process.[23]

However, a number of clinical trials have reported plaque accumulation beneath these dressings, leading to inflammation in the tissues and transient bacteremia.[4] This encouraged the invention of drug-loaded dressings, which act as a barrier to microorganisms when applied on a wound, thus stimulating the wound-healing environment by preventing secondary infections. Various agents have been tested such as chlorhexidine digluconate salts, zinc bacitracin, and tetracycline.[25] Among the different antimicrobial agents, silver has been used widely due to many benefits, including multilevel antibacterial effect on cells with low systemic toxicity. It also reduces the chances of developing resistance and is effective against multidrug-resistant organisms.[6]

The silver nanoparticles (NPs) are being increasingly used nowadays as wound dressings, contraceptive devices, surgical instruments, and bone prostheses.[7] There is increasing literature to support that silver NPs can promote wound healing through anti-inflammatory properties.[89] Furthermore, the levels of pro-inflammatory markers have been found to be significantly reduced in animals treated with nAg.[9] These results suggest that nAg can be involved in altering or suppressing inflammatory events in the early phases of wound healing, and the use of nAg may be clinically applicable to many conditions. A systematic review by Song and Ge also compiled in sufficient details about their applications in prevention of oral diseases including periodontal infections and peri-implant infections inhibiting both Gram-positive and Gram-negative species.[10]

Keeping in mind, the above-mentioned advantages; it might be prudent to utilize the antimicrobial properties of silver NPs as an integral component of periodontal dressings. The present study was carried out to clinically, microbiologically, and biochemically evaluate early wound healing under Nano-crystalline silver membrane placed following periodontal flap surgery.

MATERIALS AND METHODS

Forty-two subjects (18 males, 24 females) indicated for periodontal flap surgeries within the age range from 25 to 60 years were enrolled for the study. The subjects with a history of allergy to silver, presence of any active infectious diseases, immunocompromised condition, and steroid medication were excluded from the study. Following flap surgery, the subjects were randomly assigned to test or control groups using the lottery method. In test group (n = 21) subjects, the postsurgical wound was covered with a combination of nano-crystalline silver membrane and Coe-Pak, whereas control group subjects (n = 21) received only Coe-Pak dressing postsurgery. The study protocol was explained to each subject and written informed consent was taken. The study was approved by Panjab University Institutional Ethics Commitee, Chandigarh, India, and registered in the clinical trial registry, India under ID no. (CTRI/2019/07/020454).

Baseline assessment

On the day of surgery (baseline), before starting with surgical procedure, plaque index (PI) using Löe and Silness Index (1967) was recorded. Gingival Crevicular Fluid (GCF) was collected from the deepest site of the selected area for surgery and plaque sample was obtained using the standard pocket out collection method.[11] Immediately after collection, the swab was put in a sterile transport media (Thioglycollate broth), plugged, and sent to microbiology laboratory.

Surgical procedure and dressing application

Periodontal surgical procedure was performed on an outpatient basis under aseptic conditions. After administration of local anesthesia, full-thickness buccal and palatal/lingual flaps were elevated using a periosteal elevator. The area was debrided of the granulation tissue using curettes to provide access and visibility to the root surfaces. The remaining plaque and calculus were gently removed with ultrasonic scalers and root planing was done using curettes to achieve a clean smooth surface. The flaps were approximated to the original level and secured using sutures. The subjects were randomly allocated to receive either nano-crystalline silver membrane with Coe-Pak postsurgery or Coe-Pak dressing only.

Eugenol free periodontal pack was prepared using 2 cm of each paste (base paste and accelerator paste), measured with a sterile endodontic ruler and mixed with a sterile spatula on a sterile glass plate. In control group, only this dressing was given and for test group, the predetermined size of the silver membrane (25 mm × 5 mm) was cut, activated for the release of antibacterial silver ions using sterile distilled water, and then placed on the inner surface of the mixed eugenol free pack before placing it on the surgical site [Figure 1]. The pack was mechanically interlocked between the teeth for retention up to 7 days. Postoperative instructions were given. Patients were instructed to establish their manual oral hygiene measures 7 days postoperatively.

Figure 1

(a) Eugenol-free dressing (Coe-Pak); (b) Silver dressing (Acticoat™); (c) Predetermined size of silver dressing (25 mm × 5 mm); (d) Silver dressing along with eugenol-free dressing before placement; (e) Periodontal dressing in place

Postoperative assessment

Sutures and packs were removed after 7 days and PI as well as healing (healing index by Landry, et al.) was assessed.[12] PI and wound healing index were also assessed after 14 days postsurgery. All the patients were also subjected to the collection of microbial flora using a sterile cotton swab after the removal of the packs. GCF/periwound fluid was collected for immunological biomarker vascular endothelial growth factor (VEGF) evaluation postsurgically at the day of pack removal.

Vascular endothelial growth factor marker analysis

The concentration of VEGF in the GCF samples was determined by a quantitative sandwich enzyme-linked immunoassay kit (Bioassay Technology Laboratory) as per the instructions of the manufacturer. The concentrations of VEGF in the tested samples were estimated using the standard curve plotted using the optical density values with the standards. The results were expressed as VEGF ng/ml.

Microbiological analysis

Microbial load analysis was done by culturing the plaque samples collected from the operatory sites aseptically by standard procedure. Spread plate technique using a glass rod/metal rod-shaped like a hockey stick was used to grow and isolate colonies on nutrient and MacConkey plates. These plates were then incubated overnight at 37°C. The plates were then observed for colony-forming units (CFU) at 18 h up to 48 h. Colonies were counted under a colony counter and expressed as CFU/ml. Gram staining was performed for the assessment of bacterial species. Various biochemical tests were also carried out to identify and confirm the particular species.[13]

Statistical analysis

Statistical analysis was carried out using Statistical Package for Social Sciences (SPSS Inc., Chicago, Illinois, USA, IBM, version 17.0 for windows). All quantitative variables were estimated using measures of central location (mean) and measures of dispersion (standard deviation). For time-related comparisons, Paired t-test and Mann–Whitney tests were applied. For comparison of the change in clinical parameters, Wilcoxan signed-rank test was applied. All statistical tests were two sided and performed at a significance level of α =0.05 and confidence interval was kept at 95%. The results of this analysis were tabulated and plotted as graphs.

RESULTS

The mean age of patients was 43.57 years with mean CAL of 2.9 mm. PI at baseline (PI-1) showed fair scores (1.12; 1.03) in both the groups. Seventh-day postsurgery showed that the PI scores (PI-2) increased highly significantly (1.41; 1.55) in both the groups (P = 0.001; P < 0.001) with no statistically significant difference among the groups (P = 0.173), then reduced significantly (0.63; 0.86) at 14th-day postsurgery (PI-3) (P < 0.001, P < 0.001).

The wound healing scores showed mean values of 3.52 in test group and 2.90 in controls at 7th-day postsurgery. This difference between the two groups was highly significant with P < 0.001. On the 14th day, the scores were statistically higher in test group than controls (P < 0.001) [Table 1].

Table 1

Comparative evaluation of Healing Index† between Test group (n=21) and Control group (n=21) at different periods of observation

Periods of observation	(Mean+/-Standard Deviation)
	Test Group	Control Group	P
Healing index† at day 7 (HI 1)	3.52+/-0.5	2.90+/-0.301	<0.001**
Healing index† at day 14 (HI 2)	4.90+/-0.301	3.90+/-0.301	<0.001**

*P<0.05 was considered as statistically significant. **P<0.001 was considered as highly significant. † Healing Index by Landry, Turnbull and Howley Healing Index (1988). Very poor (2 or more signs present): ≥ 50% of red gingiva, incision margins not epithelialized, presence of bleeding on palpation, granulation tissue and suppuration. Poor: ≥ 50% of red gingiva, incision margins not epithelialized with exposed connective tissue, presence of bleeding on palpation, granulation tissue and absence of suppuration. Good: 25-50% of red gingiva, incision margins without exposed connective tissue, absence of bleeding on palpation, granulation tissue and suppuration. Very Good: 25% of red gingiva, incision margins without exposed connective tissue, absence of bleeding on palpation, granulation tissue and suppuration. Excellent: All pink tissue, incision margins without exposed connective tissue, absence of bleeding on palpation, granulation tissue and suppuration; P: Probability;

There was a significant reduction (P = 0.019) in CFU/ml postsurgery in test group as compared to presurgery [Table 2]. No evident change was seen between presurgery and postsurgery mean values in the control group (P = 0.68). Gram staining revealed that presurgery plaque demonstrated predominant Gram-positive facultative anaerobes with 85.7% in test group and 80.9% in control group [Table 3 and Figure 2]. Gram-negative facultative anaerobes dominated in postsurgery samples in both the groups (76.1% in test group and 66.6% in control group) [Table 3 and Figure 2]. There were some cases wherein no growth was seen on the agar plates, thereby labeled as no growth. Species identified in all samples are tabulated in [Figure 3].

Table 2

Comparative evaluation of CFU/ml and VEGF between Test Group (n=21) and Control Group (n=21) at different periods of observation

Periods of observation	Mean+/-Standard deviation		P
	Test Group	Control Group
CFU/ml
Pre-surgery (x106)	3.3 +/- 4.7	1.7 +/-3.9	0.06
Post-surgery (x106)	1.6 +/- 2.5	1.9 +/- 3.4	0.211
Pre Surgery - Post surgery VEGF	0.019*	0.687
Pre-surgery (VEGF 1)	0.0292+/-0.007	0.0286+/-0.006	0.92
Post-surgery (VEGF 2)	0.0342+/-0.005	0.0260+/-0.006	<0.001**
VEGF 1 – VEGF 2	0.001**	0.520

CFU expressed as Colony Forming Units/ml; VEGF: Vascular Endothelial Growth Factor. *P<0.05 was considered as statistically significant. **P<0.001 was considered as highly significant. P: Probability; n: total number of patients;

Table 3

Comparison between the type of bacteria in the plaque before and after the application of two periodontal dressings

Type of bacteria	Test Group (n=21); n (%)	Control Group (n=21); n (%)	χ 2	P
Pre-surgery
Gram Positive	18 (85.7%)	17 (80.9%)	0.36	0.83
Gram Negative	2 (9.5%)	2 (9.5%)
No growth	1 (4.7%)	2 (9.5%)
Post-Surgery
Gram positive	3 (14.2%)	7 (33.3%)	3.73	0.15
Gram Negative	16 (76.1%)	14 (66.6%)
No growth	2 (9.5%)	0 (0%)
P	<0.0001**	<0.0005**

χ2 : Value for Chi square; *P<0.05 was considered as statistically significant. **P<0.001 was considered as highly significant; P: Probability; n: Total number of patients.

Figure 2

(a) Presurgery slide showing Gram-positive cocci in test group; (b) Postsurgery slide showing Gram-negative cocci in test group; (c) Presurgery slide showing Gram-positive cocci in control group; (d) Postsurgery slide showing Gram negative bacilli in control group

Figure 3

(a) Pie diagram showing type of bacteria in presurgery plaque within test group; (b) Pie diagram showing type of bacteria in postsurgery plaque within test group; (c) Pie diagram showing type of bacteria in presurgery plaque within control group; (d) Pie diagram showing type of bacteria in postsurgery plaque within control group

The mean levels of VEGF-A were comparable in both the groups at baseline (P = 0.92). The postsurgery levels of VEGF-A showed a highly significant difference (P < 0.001) between test group (0.0342 ng/μl) and control group (0.0260 ng/μl). Intragroup evaluation showed a highly significant increase in the VEGF-A levels at 7th-day postsurgery from baseline in the test group (P = 0.001), while no evident change was seen postsurgery in the control group (P = 0.52) [Table 2].

DISCUSSION

Of the various adverse outcomes that may be encountered following periodontal surgery, the risk of infection is of primary concern to the surgeon, since oral wound is in constant exposure to outer environment and any infection can lead to morbidity and poor healing outcomes. A surgical dressing allows uninterrupted healing to occur.[2]

In our study, we used a periodontal dressing with a nano-crystalline silver membrane to assess the antimicrobial effect of silver particles along with the effect of their nano-size on the postsurgical healing. The healing index by Landry et al., has been used by various investigators to assess healing following the periodontal surgeries.[141516] The mean values of the Healing Index showed a significantly higher healing score in test group as compared to controls at day 7 and at day 14 postsurgery (P < 0.001, P < 0.001). A better healing response can be contributed to the innate potential of silver NPs which play a positive role in fibroblast maturation and proliferation by providing an inflammation-free environment in addition to their antimicrobial effects.[17] The efficacy of silver NPs in promoting wound healing through their antimicrobial effect has been also demonstrated by various researchers.[18192021] The enhanced healing response by silver NPs was also seen by Habiboallah et al. in 2014, wherein authors used the same dressing as a postoperative dressing following gingivectomy surgery in vitro and assessed healing through histologic sections. They found a statistically significant difference (P = 0.034, 0.05) in the inflammatory and repair parameters after the use of the silver membrane.[22]

VEGF levels were significantly increased from baseline to 7th-day postsurgery, beneath silver pack as compared to the controls (P = 0.001). There is sufficient evidence in literature to commemorate the angiogenic potential and rapid connective tissue repair of VEGF in human wound fluid.[23242526] Alzahrani (2017) and Pirebas et al. evaluated healing after a periodontal surgery through VEGF levels and found that the VEGF levels increased till 14th-day postsurgery indicating better healing.[2728]

There was a significant increase in PI score (P = 0.001; P < 0.001) at 7th-day postsurgery in both the groups from the baseline; however, the scores among the groups were comparable (P = 0.173). The plaque retentive property of the hardened noneugenol pack is due to its rough and flint-like surface texture which attracts more plaque on its irregular surface.[14] A high PI score at week 1 after pack application was also seen by Kumar et al. who compared PI scores following collagen dressing, light cure dressing, and eugenol-free dressing.[29] Various other authors have also reported significant retention of plaque beneath the packs, showing the PI scores between 1.7 and 1.9 at 7th-day postsurgery.[1430]

In our study, we found that this increase in PI on the 7th-day postsurgery, however, did not comply with a concomitant increase in the CFU/ml in test group. We found that CFU/ml decreased significantly (P = 0.019) in test group postsurgery. However, there was almost a similar value in the control group, depicting no evident change postsurgery (P = 0.687). This result showed that though the plaque levels increased beneath the packs, the number of viable microorganisms beneath the silver pack decreased tremendously due to the antibacterial effect of the silver particles. Literature suggests that the silver NPs possess significant antibacterial properties due to their large surface area to volume ratio and through various interactions with the bacterial components.[31] Shawky et al.(2015) introduced silver NP solution into the periodontally involved deep pockets and found a significant reduction in the number of microorganisms 1 month post procedure.[32]

Gram staining showed that Gram-positive microorganisms predominated the outer surface of gingival mucosa in both the groups at presurgery. The genus included Streptococcus mutans, Enterococcus faecalis, and Staphylococcus aureus. Postsurgery plaque showed Gram-negative bacteria, namely Klebsiella oxytoca, Acinetobacter, Enterobacter (Citrobacter), Escherichia coli, and Pseudomonas aeruginosa. S. mutans, E. faecalis, and S. aureus were also found.

The above finding can be explained by the presence of huge microbiological diversity among periodontal pathogens. Although the literature shows a shift of Gram-positive bacteria to Gram-negative bacteria in progressing periodontal disease condition, the shift is seen in the strict anaerobes, especially in the areas of increasing disease severity like deeper pockets. Furthermore, the sample analyzed by most of the studies was a sub-gingival plaque.[33] In our study, we have examined supragingival plaque as well as the gingival mucosa because this was the region where the pack is usually placed. Studies clearly mention that supragingival plaque show more of the aerobes than anaerobic bacteria accounting for about 62%–67% of whole flora.[3435] These microaerophiles have also been recently discussed as potential pathogenic microorganisms in periodontal diseases.[32] Hence, our study suggests that the shift of Gram-positive bacteria into Gram-negative is also seen in facultative anaerobes/aerobes on the gingival surface, but it is happening when the periodontal condition is reverting back to health. The presence of Gram-negative bacteria in health has been also supported by Kumar et al., wherein they concluded that Gram-negative bacteria were associated with periodontal health.[36]

The silver has been seen to exhibit its antibacterial action against both Gram-positive and Gram-negative microorganisms, rendering a decrease in the total CFU of viable microflora with the least systemic side effects.[3738] Hence, we observed an overall reduction in the pathogenic microorganisms implicated for periodontitis on using silver dressing and at the same time, a significant increase in the physiological microflora.

The strength of our study is that it is a novel study to test silver in periodontal wound healing. Second, we have analyzed the antibacterial action of silver by quantitative and qualitative estimation of the supragingival plaque microorganisms. Third, we have tried to evaluate healing not only clinically but also with the help of a very potential healing biomarker, i.e., VEGF. A small sample size and absence of split-mouth design could be considered as the limitation of our study.

SUMMARY AND CONCLUSION

Within its limitations, the present study revealed a highly potent effect of nano-crystalline silver particles as a periodontal dressing with a good antibacterial effect on the gingival surface promoting early healing postsurgery. Further large population-based clinical trials can be suggested to evaluate healing with the novel formulation of periodontal dressing with silver incorporation.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.