Development of biodegradable silkworm cocoon derived silk membrane for GTR in the treatment of grade II furcation

Thiswas a preliminary study to develop a biodegradablemembrane from silkworm Bombyx.mori. The silk ibroin membrane prepared was analyzed for tensile strength and used in patients with grade II furcation defects, which was compared with collagen membrane. Though collagen is the gold standard, it has disadvantages, one of them being slow biodegradability. Hence this research was carried out to see if silk ibroin was good as a regenerative material in providing bone ill in the furcal area. As it is commonly accepted that treatment in the furcal area is better with bone graft and GTRmembrane compared with just bone graft and lap surgery, this clinical trial with a common bone graft was carried out in addition to barrier membrane, which were of different sources. The patients ful illing the inclusion criteriawere randomized into test and control sites. The test sites received bone graft(colocast) and silk ibroinmembranewhile the control site received bone graft(colocast) and collagen membrane(cologide). The bone ill was analyzed radiographically using AUTOCAD software 2017. The results of the study showed that both the sites did not show any adverse effects, but improvement were noted clinically and radiographically. Radiographically signi icant improvement was seen in silk ibroin sites indicating that silk ibroin with bone graft would be better as a regenerative material.


INTRODUCTION
A challenge faced in the management of the periodontal disease is furcation involvement, which mainly affects the bifurcation and trifurcation of multirooted teeth. It is indeed a challenge to the clin-ician as furcally involved teeth are dif icult to treat as it is inaccessible for instrumentation as well as personal home care (Figure 1).
Various treatment approaches have been advocated for the treatment of furcation, which includes conservative, resective and regenerative techniques (Sanz et al., 2015). Of the regenerative materials, bone grafts have been used as a gold standard of which xenograft has been quite popular. It has structural components similar to human bone, thereby improving the osteoconductive capability when compared to that of synthetically derived materials.
There is enough evidence which has proved that the combination of bone graft and barrier membrane helps in better regeneration of bone when compared to lap surgery alone or lap surgery plus bone graft (Mcclain and Schallhorn, 1993a;Lekovic et al.,  1990; Wallace et al., 1994). Though collagen is the standard biodegradable membrane which has been most studied and versatile material in dentistry, it has disadvantages. This had led to researchers seeking a material which would be more biocompatible, cost-effective with better mechanical properties and has slow degradability since collagen degrades within 3-4months. For ideal regeneration to take place, the barrier material should stay for a mini-mum of 4 weeks to 6 months.
With the advent of various technologies and with the emergence of tissue engineering, the barrier membranes have undergone various modi ications according to the recent trend. The requisites of an ideal barrier membrane include biocompatibility, better handling properties, ability to adhere to root surface, promote tissue coverage with reduced barrier exposure rates, resist bacterial contamination, promote selective cell proliferation within the defect and should be absorbed at a rate that parallels regenerative tissue formation within 4 weeks to 6 months. There are very few membranes which fulill these requirements. Hence the search is on for an innovative material from a natural source and with minimal side effects.
New insights have shown that silk could be the futuristic regenerative material due to its remarkable mechanical properties, biocompatibility and biodegradability as it degrades after a period of 12 months (Melke et al., 2016). It is a novel biomaterial which has evolved from insects such as silkworm, mites, spiders and beetles and has proved to be a promising material as a scaffold which could help in tissue engineering as it has been used as a scaffold for periodontal ligament cells (Yang et al., 2013).
Silk can be processed into different forms such as hygrogels, ilms, nano ibers and nanoparticles. The degradation rate can also be adjusted by controlling the (beta-sheet content) during processing (Nagal and Singla, 2013). Though silk ibroin has been studied as a scaffold for periodontal regeneration, these studies have been done only in vitro. There are no studies done in humans though this material has been extensively studied in regenerative medicine using animals. Thus this has kindled an interest in seeing it as a possible periodontal regenerative material. Though various technologies have been used to treat furcations, it is still an enigma for the clinician. So a comparative evaluation of silk ibroin over the collagen membrane with xenograft in the treatment of grade II furcation defects was carried out.  India. All the materials used for the study were of pharma grade and were generally regarded as safe(GRAS).

Extraction of Silk ibroin from Bombyx mori
5 grams of Bombyx mori silkworm cocoons were cut into small pieces with titanium scissors.
Pieces of cut cocoons were placed in a beaker of 1liter capacity containing ultrapure water and boiled for half an hour. To the heated silk ibers,2grams of sodium bicarbonate was added to the beaker and occasionally stirring with a glass rod was done to obtain a dispersed phase of ibers. This process is called degumming. After cooling, the silk ibers were washed with water to remove an excess of sodium bicarbonate followed by squeezing of silk ibers to remove water. The degummed silk iber was stored at room temperature( can be stored for a long term period) and wrapped in aluminium foil( ig 2).

Dialysis of degummed silk iber
A dialysis bag(cellulose membrane) was presoaked for 24 hours. The silk ibers were soaked in a medium consisting of 1:2:8 ratio of Calcium Chloride-Ethanol-Water, which was used as a predissolving solvent mixture for the silk ibers. The soaked silk ibers were then placed in the cellulose membrane(dialysis cassette was replaced ) and dialysed against ultrapure water for 4 days ( Figure 3).

Centrifugation
Silk ibers and the supernatant was removed from the dialysis bag and centrifuged in an ultra-low centrifuge at 4000rpm at 4º C for 40 minutes. The resultant viscous solution was then stored in a screwcapped tube and placed in a vacuum driver (Figure 4).

Preparation of the membrane
The extracted viscous solution was mixed with 0.2% hydroxyl proplyl methylcellulose(HPMC), a copolymer to get a membrane. The solution was poured into Petri dishes and gently tapped for even dispersion of contents. The Petri dishes were placed in a vacuum driver(Memmertcompany ) for one day at 39º C ( Figure 5). The ilm/membrane, thus formed was stored in a sterile container and evaluated for

Characterization
Tensile strength of the membrane was determined using a universal testing machine at a speed of 1mm/minute. A rectangular-shaped silk ibroin membrane with a thickness of 1.1mm (10cm in length) was taken and ixed to Universal Testing Machine(UTM). This machine determined the tensile strength at peak load and break load with %elongation and yield strength.

Sterilization
The membrane was sterilized using gamma radiation using cobalt 60 at 15kGy

Clinical trial design
The patients were selected from the outpatient's department of Periodontology, JSS Dental College and Hospital, Mysuru. By purposive sampling, patients of both sexes who ful illed the inclusion criteria and those who had given the informed consent were enrolled for this study. Randomization was done by lottery method and the patients were allocated into two sites, the test site (A) and control site(B).
The inclusion criteria comprised of systemically healthy patients within the age group of 30-50 years both males and females, patients who were willing and had given the informed consent, patients who could maintain their oral hygiene, clinical and radiographic evidence of mandibular buccal furcation defect ≥ 3mm, vital teeth, presence of at least 2mm of keratinized gingiva on selected tooth, vertical probing depth ≤4mm.
The exclusion criteria comprised of patients on any medication, patients on antibiotic therapy within last 3 months, medically compromised or under therapeutic regimen that could have altered the probability of tissue and bone healing, pregnancy and lactation, smokers, acute infections-HIV, metabolic bone diseases-osteoporosis, rheumatoid arthritis, teeth with mobility and hopeless prognosis.

Sample size calculation
This was estimated using hypothesis testing for 2 means(equal variances) using n masters software. The sample size was computed to be 11 per group at 5% α error, 80%power with an effect size of 1.23 to identify a mean difference of 0.8 to be statistically signi icant. The sample size was inally rounded off to 15, anticipating some degree of dropouts.

Study design
This was a randomized controlled interventional study with a split-mouth design. By purposive sampling, 34 defect sites in 17 patients were selected. The selected sites were randomized using the lottery method into test and control sites ( Figure 6). The randomization process was performed by an examiner blinded to the study. The allocation was concealed within opaque envelopes with numbers on it until immediately before surgery to determine which groups would receive test and control procedures.
Test site(A) -Flap surgery and the Silk Fibroin membrane with Xenograft(COLOCAST).

Ethical Clearance and Informed Consent"
Institutional Review Board (IRB) of the JSS Academy of Higher Education and Research approved the study protocol. A prior written informed consent was taken from the participants. All principles outlined in the Declaration of Helsinki(1964, revised in 2008) on experimentation involving humans were observed. This trial was registered at clinical trials.gov as CTRI/2018/12/016509.The duration of the study was for12 months.

Pre Surgical Procedure
Patients ful illing the inclusion criteria and have willingly given informed consent underwent routine hematological investigations and Phase I therapy comprising of scaling along with root planing. Mobility was checked for teeth with furcation involvement along with radiographic investigations.

Radiographic parameters
Conventional intraoral periapical radiographs(IOPAR) with an X-ray grid were taken with standardized parameters, exposure and processing. The ilms used were Kodak E speed of size 2. The grid, dental (from Bluedent India, Chennai) with RINN XCP ilm holder, was used. Exposures were ixed at 8mA, 70kvp with 2mm of aluminium iltration and underwent processing in an automatic processor. Radiographic assessments with grid were further analyzed using AutoCAD software 2017.

Clinical data collection
All clinical parameters were assessed by a caliberated examiner who was masked to the treatment provided. The recordings were done on the day of surgery (baseline) 3months, 6months and 12 months. The following were assessed:

Probing depth(PD)
the distance between the gingival margin and the base of the pocket

Clinical attachment level( CAL)
the distance between the gingival margin and the base of the pocket

Plaque index(PI)
A mouth mirror and an explorer were used after air drying of teeth and gingiva to assess plaque. Four areas of the teeth (distofacial, facial, mesiofacial and lingual surfaces) were examined systematically for each tooth.

Gingival index(GI)
A mouth mirror and William's graduated periodontal probe were used after drying the gingiva. Four areas of the teeth (distofacial papilla, facial margin, mesiofacial papilla and lingual gingival margin) were examined systematically for each tooth.

Vertical probing depth
The UNC-15 probe was inserted into the periodontal pocket along the root surface to locate the initial luting of the furcation. The distance from the gingival margin to the opening of the furcation was noted. The probe was then advanced apically until resistance was felt and the distance from the gingival margin to the vertical depth of probing was noted and the reading was recorded to the nearest millimeter as the difference between the two values.

Horizontal probing depth
A customized stent as a ixed reference point was made using self-cure clear resin extending up to the attached gingiva to go beyond the furcal entrance. A hole at the buccal extension of the stent coinciding with the furcal entrance to guide the probe penetration (UNC-15) in the same direction every time it is inserted for measurements was made. The outer surface of the hole served as the reference point for horizontal probing depth (Figure 7) Surgical Procedure After asepsis, the sites with furcal defects were anesthetized using 2% lidocaine with 1:80,000 adrenaline.
Following intracrevicular incisions with blade 12, a full-thickness lap was raised using blunt dissection. The tooth, including the furcation area and bone surfaces, were carefully debrided with Gracey curettes to clear off all granulation tissue following which the root surfaces were thoroughly planed until it attained a hard and glassy surface. After instrumentation, the surgical area was irrigated thoroughly with saline to remove the remaining detached fragments. The test site received xenograft (colocast) and silk ibroin as a guided tissue regeneration(GTR)membrane ( Figure 8). The contralateral control site received xenograft(COLOCAST) and collagen membrane(COLOGIDE), which was performed in less than 2 weeks (Figure 9). The laps were repositioned and suturing was done using 3-0 silk sutures and periodontal dressing (Coe-Pak ) was placed.

Postoperative care
Manifestations of any allergic reaction like irritation, itching, etc. were observed for 1-hour post-surgery, subsequent to which patients were discharged along with postoperative instructions. The patients were instructed to maintain their oral hygiene with regular home care, except in the operated area for a week. Antibiotics were prescribed for 5 days and analgesics were advised to be taken only if it was necessary on a 'need to treat' basis. Patients were asked to report after 7 days for suture removal and check-up.

Post-surgical evaluation
At one week follow up, periodontal pack and sutures were removed. The clinical parameters were evaluated at "3months, 6months and 12 months". Radiographic parameters IOPAR with a grid which has markings of 1mm 2 was used as it can make linear measurements easy. These parameters were analyzed at "6months and 12 months "using AUTO-CAD 2017 version which is a "commercial computeraided design and drafting software application (Figure 10).

RESULTS AND DISCUSSION
The tensile strength of 1.1mm thick silk ibroin membrane was determined by the universal testing machine and was found to be 0.04Newton/sqmm at peak load, and 0.001Newton/sqmm at break load with a yield strength of 0.04Newton/sqmm which is superior when compared to other polymers and synthetic materials (Figure 11).
The recordings from clinical and radiographic parameters were subjected to statistical analysis using SPSS version 22 software. The results were analyzed using descriptive statistics, repeated measures ANOVA and paired t-test. A total of 15 participants were selected for study, 11 of which were males and 4 were females. The mean age of males were 40.00±5.2. The mean age of females were 36±6.6 (Table 1) The mean plaque scores at baseline was 1.39±0.38, which reduced to 0.55±0.30 at 12 months.
The results show statistically signi icance at p<0.05 indicating that the patients were maintaining good oral hygiene which had an effect on the overall outcome (Table 2) The mean pocket probing depth in the test site at baseline was 4.4±2.6, 2.45±1.03 at 3 months, 2.09±0.83 at 6 months and 1.63±0.67 at 12 months. The mean pocket probing depth in the control sites at baseline was 3.2±1.4, 2.81±0.87 at 3 months, 2.3±0.80 at 6 months and 1.9±0.70 at 12 months. Both sites have shown a reduction in pocket depth. Repeated measures ANOVA has shown statistical signi icance where (p=0.013)( Table 3)( Figure 12) The reduction could be due to the complete debridement of the defect as furcations are inaccessible for instrumentation and thereby, lap surgery would have facilitated incomplete debridement of the area, thereby causing a reduction in pocket probing depth.
The mean CAL at baseline is 2.3±1.2, at 3 months it was 1.8±0.87, at 6 months was 1.2±0.46 and at 12 months is 1.00±0.63. The mean CAL at the test site had reduced from baseline to 12 months. At the control sites, it was 2.5±1.5 at baseline, at 3 months 2.4±1.3, at 6 months 2.00±1.41 and at 12 months 1.4±1.1. This shows a mean reduction in CAL at the control sites as well over a period of 12 months. Repeated measures ANOVA shows statistically insigni icance as p=0.25 (Table 3     A statistical signi icance was also noted at the 3 rd to,6 th and 12 months in the test sites (p=0.006 and p=0.01) and in the control sites only at the 6 th and 12th months (p=0.006, p= 0.00). This could be a result of true periodontal regeneration via new attachment or of healing by repair. This is in accordance to a study where bilateral Class II furcation defects on lower irst molars showed improvement in clinical attachment level (CAL) and change in class of clinically detectable furcation involvement when treated with bone graft and a barrier membrane (Leonardis et al., 1999).
The test site shows a mean horizontal probing depth(HPD) of 3.6±0.92 at baseline, 3.18±1.16 at 3 months, 2.00±0.77 at 6 months and 1.36±0.50 at 12 months. The control site shows a mean HPD of 3.8±1.07 at baseline, 3.5±1.2 at 3 months, 2.9±1.0 at 6 months and 2.4±1.0 at 12 months. Both the sites have shown a reduction in mean HPD from baseline to 12 months with a greater reduction observed in the test sites. Repeated measures ANOVA showed a statistical signi icance of (p=0.01) in the test sites (Table 3) (Figure 14) This has been explained by factors such as partially resorbed particles of the bone graft used which can hinder probe penetration, true regeneration of connective attachment, which may have created and maintained a larger space underneath the barrier, allowing for selective cell repopulation (Urist, 1965) or increased bone formation mainly induced by the xenograft and silk ibroin. Although this is supported by some studies, it requires further investigation.

Radiographic parameters
All the changes in bone ill were detected radiographically using consecutive radiographs. Conven-tional IOPAR with grid were used as reduces scattered radiation and improves image contrast (Deshpande and Bhargava, 2014). The radiographs were subjected to AUTOCAD version 2017 for analysis. The radiographs were enlarged to twice the original dimension and the area of interest was marked digitally so that any differences between preoperative and postoperative changes could be calculated. Although the X-ray grid can evaluate bone changes, AUTOCAD is more precise and can give values upto decimal points (Melke et al., 2016;Gowda et al., 2011). This is in accordance to a study done by (Yajamanya et al., 2017). The result is in accordance to a study by (Mobini et al., 2013;Bhumiratana et al., 2011) who showed the growth of mineralized bone matrix on silk scaffolds was a result of enhanced differentiation and faster recruitment of cells. These results are in accordance to the results of a meta-analysis of 10 studies that showed statistically signi icant greater vertical and horizontal bone ill for GTR + Osseous graft(OG) with OFD in furcation defects (Chen et al., 2013;Mcclain and Schallhorn, 1993b). It is also in accordance to a study where bone volume in the Silk Fibroin group at 4 weeks were greater than in the Bio-Gide group in Sprague-Dawley rats (Lu et al., 2015;Seok et al., 2014) who showed that New Zealand white rabbits when treated with silk membranes of 0.5mm thickness, resulting in effective bone regeneration.
From the results of the study, it has been clearly stated that silk ibroin throws light to expand the family of regenerative barrier membranes with an enhanced performance for bone regeneration in the ield of dentistry. It can thus be considered as a better material for periodontal regeneration with further longitudinal clinical trials.

Future perspectives
Factors in luencing the furcation ill, such as divergence of roots and interproximal bone height in association to the fornix of the furcation, needs to be evaluated. To con irm the regenerative potential of silk ibroin, a histological study needs to be done. Biodegradability studies would throw better light to the function of silk as a barrier membrane.

Limitations of this study
Considering the prevalence of mandibular grade II furcation defects, the sample size was small. The degree of dropouts were high. The study did not compare xenograft with open lap debridement vs.
silk ibroin and open lap debridement to know which among these components had lead to clinical and radiographic improvements. Though evidence from animal studies have shown that silk is highly biocompatible, nontoxic and degrades over a period of 1 year, cytotoxicity and biodegradability studies would have con irmed the results (Wang et al., 2008). Histological assessment of regeneration was not done.

CONCLUSIONS
In this study, silk ibroin membranes were prepared and compared to collagen as it has advantages like good biocompatibility, better mechanical properties and slow biodegradability. Preclinical and clinical studies were carried out in animals to establish its ef icacy in tissue engineering. This was the irst human trial where silk ibroin as a scaffold was used in periodontal regeneration. Within the limitations, the study has proved that silk ibroin with xenograft alone is better when compared to collagen with xenograft. This study has also proved the ef icacy of silk ibroin with xenograft over collagen with xenograft in treating grade II furcation defects. However, further longitudinal studies with larger sample size, longer follow up and detailed characterization is essential to prove its potential in periodontal regeneration.