Assessment of Anti-Protease Property of Nutmeg in Causing Delayed Disintegration of Platelet Rich Fibrin – an in vitro Study

Arunachalam, Varghese, and Thangavelu: Assessment of Anti-Protease Property of Nutmeg in Causing Delayed Disintegration of Platelet Rich Fibrin – an in vitro Study

Authors

INTRODUCTION

Periodontal disease is an inflammatory disease characterized by the destruction of periodontal tissues. The aim of periodontal therapy is to regulate inflammatory process, prevent the progression of periodontal disease and to regenerate the lost periodontal tissues. Periodontal regeneration is a multifactorial process involving cell adhesion, migration, proliferation, and differentiation in an orchestrated sequence.1 There are various regenerative therapies available including soft tissue grafts, hard tissue grafts, guided tissue regeneration, root bio modification or a combination of these.2 However, regenerative periodontal therapies have do not attain complete periodontal restoration.2,3 Various biomaterials have been used in periodontal tissue regeneration in addition to bone grafts.

Periodontal wound healing includes a sequence of interactions between epithelial cells, gingival fibroblasts, periodontal ligament cells and osteoblasts. The disruption of vasculature during wound healing leads to fibrin formation, platelet aggregation, and release of several growth factors4 into tissues from platelets through molecular signals which are primarily mediated by cytokines and growth factors. The presence of growth factors and cytokines in platelets are important in inflammation and wound healing.5 Platelets also secrete fibrin, fibronectin, and vitronectin, which act as a matrix for the connective tissue and as adhesion molecules for more efficient cell migration. Thus, it is wise to use platelets as therapeutic tools to improve tissue repair.

Platelet-rich fibrin (PRF) is a platelet concentrate enhances tissue healing. It was first introduced by Choukroun et al.6 with simplified processing and without biochemical blood handling. The use of platelet gel to improve bone regeneration is a recent technique in implantology. However, the biologic properties and real effects of such products remain controversial. It is advantageous over platelet-rich plasma due to its ease of preparation, ease of application and minimal expense.

Another added advantage of PRF is the presence of natural fibrin network in PRF which protects the growth factors from proteolysis. PRF also favours the development of micro vascularization leading to a more efficient cell migration.

J Mironet al.7 in his review article has concluded that the use of PRF has gained tremendous momentum as a low cost biological scaffold capable of improving tissue healing. Patil et al.8 has presented a case report, reporting the successful treatment of radicular cysts using autologous periosteum and platelet-rich fibrin with demineralized freeze-dried bone allograft. Panda et al.9 in his randomised controlled trial has concluded that the adjunctive use of PRF in combination with barrier membrane is more effective in the treatment of intrabony defects in chronic periodontitis as compared with barrier membrane alone.

Hafez et al.10 in his case series study concluded that Platelet rich fibrin as a membrane provided good soft tissue coverage over the immediate implants and it enhanced bone stability. The technique was easy to perform with good esthetic results. Fernando et al. concluded in his case report that the PRF membranes were excellent alternatives to all alloplastic membranes, providing the necessary protection for bone formation. Ahmed et al. in his clinical trial has concluded that addition of PRF to deprotenized bovine bone might be a useful adjunct for alveolar ridge augmentation around immediate implant placement.

Hartshorne et al.11 in his review had concluded that the benefits derived from using PRF in various clinical applications for promoting wound healing and tissue regeneration, its antibacterial and anti-haemorrhagic effects, the low risks with its use, and the availability of easy and low cost preparation methods, should encourage more clinicians to adopt this technology in their practice for the benefit their patients. Aroca et al.12 in her clinical trial where PRF was added to modified coronally advanced flap for treatment of adjacent multiple recession coverage procedures, concluded that the addition of a PRF membrane positioned under the modified coronally advanced flap (MCAF) provided inferior root coverage but an additional gain in gingival/mucosal thickness (GTH) at 6 months compared to conventional therapy.

An in vitro study by Sam et al.13 have proven that PRF has a higher degradability and less rigidity when compared to commercially available barrier membranes. This causes PRF to get resorbed faster and get collapsed thus causing failure in space maintenance in guided tissue regeneration.

Isobe et al.14 in an in vitro study compared various mechanical and chemical properties of PRF and concentrated growth factor (CGF), including degradability, which was tested by a digestive test using Phosphate buffered saline (PBS) solution containing trypsin and Ethelenediaminetetraacetic acid (EDTA) and concluded that both had similar mechanical and chemical properties.

Yamashita et al.15 in their in vivo histological study in rats studied the stability of PRF, and concluded that the histologically and immunohistochemically, the PRF became small and sparse at 21 days, almost disappeared at 28 days. Hence, although PRF can potentially stimulate tissue regeneration, its degradation time should be considered in its clinical applications. It is also obvious that PRF alone cannot work as a scaffold or a barrier membrane for maintaining the space of regeneration.

Myristica fragrans also known as ‘nutmeg’ is popular in most parts of West Africa as a spice. However, the essential oil of nutmeg is used externally for rheumatism and internally as a carminative. It is also used in soups as a postpartum medication. Compounds isolated from the seeds of this plant have been reported to possess strong platelet anti-aggregatory activity.16

In a study by Lee KK et al.17 the inhibitory effects of 150 medicinal plants on elastase activity were investigated. Among the 150 plants, myristica fragrans (nutmeg) and 5 other plant extracts exhibited more than 65% of inhibition of elastase activity. In the present study, we hypothesized that myristica fragrans (nutmeg) may prolong the degradation of PRF.

Thus this in vitro study aimed to assess whether nutmeg has any effect in inhibiting degradabilityof PRF membrane and to compare the degradability of PRF at different concentrations (200 mg, 100 mg, 50 mg) of ethanolic and crude extracts of nutmeg.

MATERIALS AND METHODS

The study was conducted in the dental research lab in saveetha institute of medical and technological sciences. In this study, the degradability of PRF membrane was compared between crude and ethanolic extracts of nutmeg at various concentrations such as 50mg, 100mg, and 200mg against negative control of Phosphate buffer solution(PBS).

The different groups in this study are: Group 1- PBS (negative control) N=5, Group 2- PBS containing crude extract of nutmeg 200 mg N=5, Group 3- PBS containing crude extract of nutmeg 100 mg N=5, Group 4- PBS containing crude extract of nutmeg 50 mg N=5, Group 5- PBS containing ethanolic extract of nutmeg 200 mg N=5, Group 6- PBS containing ethanolic extract of nutmeg 100 mg N=5 andGroup 7- PBS containing ethanolic extract of nutmeg 50 mg N=5.

Commercially available finely grounded nutmeg powder was used for preparation of crude and ethanolic extracts of nutmrg. For the preparation of ethanolic extract of nutmeg, 5 mg nutmeg was dropped in 20 ml ethanol. It could evaporate over a period of 5 days. The resulting extract was collected. 50, 100 and 200mg of these extracts were measured in a micro weighing scale and mixed in 1ml PBS in separate eppendorfs. For the preparation of crude extract of nutmeg, 50, 100 and 200mg was measured in a micro weighing scale and mixed in 1 ml PBS in separate eppendorfs.

The blood donors for PRF procurement selected were systemically healthy student volunteers. 30 ml venous blood from 5 different donors were collected. The preparation of PRF was done based on the procedure described by Choukran et al.6 with simplified processing and without biochemical blood handling. The blood collected was dispensed in 30 test tubes. It was run on a centrifuge at 2500 rates per minute for 15 min in small batches after which it settles into the following layers: red lower fraction containing red blood cells, upper straw coloured cellular plasma and the middle fraction containing the fibrin clot:The upper straw coloured layer was then removed and middle fraction was collected, 2 mm below lower dividing line, which is the PRF. PRF procured from each donor was pressed between sterile gauze pieces. They were cut into equal sizes of 7×3 mm. They were thoroughly dried in a blotting paper and excess water was removed to eliminate errors in weight measurement. The pieces were weighed in a micro weighing machine (REMI 1MLH). They were later dropped in each of the 7 groups of eppendorfs (5 each in group). After 1 week of storing in room temperature, the PRF pieces were retrieved, dried in a blotting paper and weighed again. Percentage of remaining PRF [(weight at 1 week / weight at baseline) × 100] was calculated in each Eppendorf. The mean value of all the percentage was calculated in each group. The data was analysed using SPSS software (Statistical Presentation System Software, 1999, SPSS Inc., New York, version 10.0).ANOVA and post hoc tests were used to statistically compare this data between the groups.

RESULTS

The mean weight of PRF at baseline in the groups 1(PBS) Group 2(PBS + 200 mg crude extract of nutmeg), group 3 (PBS + 100 mg crude extract of nutmeg), Group 4 (PBS + 50 mg crude extract of nutmeg), Group 5 (PBS + 200 mg ethanolic extract of nutmeg), Group 6 (PBS + 100 mg ethanolic extract of nutmeg), Group 7 (PBS + 50 mg ethanolic extract of nutmeg)were 14.6±2.83, 19.74±5.12, 18.36±2.23, 18±4.15, 18.88±2.83, 18.62±4.51, 19.37±3.71 respectively. The mean weight of PRF at 1 week of groups 1-7 were 5.18±0.85, 2.38±2.6, 4.54±3.3, 5.76±1.81, 16.04±2.2, 15.16±4.82, 17.26±3.04 respectively.There was a reduction in the weights of the PRF in all the groups at 1 week when compared to baseline. The mean percentage of remaining weight of PRFin groups 1-7 were 35.59±14.23, 10.7±11.92, 23.71±17.85, 85.09±2.89, 34.86±17.65, 85.09±2.89, 79.81±9.02, 86.01±4.42 respectively. (Table 1)

The percentage of remaining weight of PRF at 1 week was maximum in Group 7 (PBS + 50 mg ethanolic extract of nutmeg), followed by Group 5 (PBS + 200 mg ethanolic extract of nutmeg), Group 6 (PBS + 100 mg ethanolic extract of nutmeg), Group 1 (PBS - Control), Group 4 (PBS + 50 mg crude extract of nutmeg), Group3 (PBS + 100 mg crude extract of nutmeg) andthe least in Group2 (PBS + 200 mg crude extract of nutmeg).(Table 1)

When the remaining weight of PRF in 7 groups were compared statistically using ANOVA, the difference was statistically significant with a P value of 0.00.(Table 2)

On Post hoc comparison, when each of the test groups (Group 2 – 7) were compared against the control group (Group 1) individually, the difference was statistically significant only in the group 2, group 5, group 6 and group 7.On analysing the result, it was found that, all the three concentrations of ethanolic extract were significantly better than control group, whereas group 2 (PBS + 200mg of crude extract of nutmeg) was found to be significantly worser than the control group. However, Group 3 (PBS + 100mg crude extract of nutmeg) and 4 (PBS + 50mg crude extract of nutmeg), did not have any significant difference with the control group.(Table 3)

DISCUSSION

The present study was aimed to assess whether nutmeg has any effect in inhibiting degradability of PRF membrane and to compare the degradability of PRF at different concentrations (200 mg, 100 mg, and 50 mg) of ethanolic and crude extracts of nutmeg.

The rationale behind the use of PRF membrane is that the platelet α granules have many Growth factors (GF)s including Platelet derived GFs, Transforming GF-β, Vascular endothelial GF, and Epidermal GF.18 PRF has to be considered as a fibrin biomaterial that are important in hard and soft tissue repair mechanism. Its molecular structure is an optimal matrix for migration of endothelial cells and fibroblasts. It permits angiogenesis and remodelling of fibrin in a more resistant connective tissue.6 With simplified processing and without biochemical blood handling.

Choukroun et al.6 introduced the usage of PRF and came up with a protocol for its preparation for use in oral and maxillofacial surgery to improve bone healing in implant dentistry. Many studies19-23 have established the clinical and radiographic efficiency of PRF in periodontal defects. The addition of PRF as a membrane to coronally advanced flap showed an increase in width of keratinized gingiva.12,24 Thus, it is wise to extend the use of PRF membrane to guided tissue regeneration (GTR) applications as well. The assessment of the mechanical properties of PRF membrane for GTR procedures is required before its clinical use.

But, the structural integrity of the implanted bio absorbable barrier membrane should be preserved for a sufficient time to ensure desired results. In the study by Sam et al.13 the degradation rate of PRF in vitro was compared with that of two other commercially available collagen membranes to obtain a gross assessment of its degradation profile. The membrane degradation test results showed that PRF membrane was comparable to other membranes in terms of maintaining its physical property up to 6 days. At the end of 1-week, PRF membrane was found to have degraded to about 36% of initial weight, whereas fish collagen to about 8%, and bovine collagen to about 3%. Hence, in the present study, we were looking to find a way to prolong the degradation rate of the PRF.

In an in vitro study17 comparing 150 medicinal plants, nutmeg and 5 other plant extracts was found to have inhibitory activity on porcine pancreatic elastase. Thus, in the present study, we hypothesized that addition of nutmeg may have an inhibitory activity on PRF degradation.

There was a reduction in the weights of the PRF in all the groups at 1 week when compared to baseline. There was least reduction in Group 7, closely followed by Group 5, Group 6, Group 1, Group 4, Group 3 and Group 2 showing the highest reduction. The reduction is due to the physical property of autogenous PRF that leads to resorption. Yamashita et al.15 has shown that PRF completely disappeared in 28 days in Wistar rat models in vivo. The study by Sam et al.13 reports degradation in in vitro condition too. Hence, it is proved that PRF degrades in both in vitro and in vivo conditions.

There was a greater percentage of remaining PRF in the ethanolic extracts when compared to control. The ethanolic extract of nutmeg in all concentrations (200 mg, 100 mg, and 50 mg) showed a greater remaining PRF than the control with the highest being in 50mg ethanolic extract of nutmeg followed by 200 mg and 100 mg. This maybe attributed to the antiprotease effects enumerated in Lee et al.17 and Jangid et al.16

There was a lesser percentage of remaining PRF in the crude extract when compared to control. The crude extract of nutmeg in all concentrations (200 mg, 100 mg, 50 mg) showed a lesser remaining PRF than the control with the highest being in 50 mg crude extract of nutmeg followed by 100mg and 200 mg. A study by Bae et al.25 showed equal antioxidase property in both the water and ethanolic extracts Rumex Acitosa. Whereas a study by Singh et al.26 showed greater antioxidase effect in crude extracts than ethanolic extracts. This may be attributed to the different solubility properties of different plants. In the present study, since nutmeg is insoluble in water, that may be a reason for its inadequate inhibitory effect on PRF degradation when compared to ethanolic extract.

The uniqueness of this study lies in the fact that a plant extract is being used to inhibit the degradation of PRF thus enhancing its property as a GTR membrane. This study is the first of its kind. However, this is an in vitro study. Thus, the property of the PRF and the effect of nutmeg on it may change in an in vivo condition. Also, in this in vitro study, it was not tested if nutmeg has any influence in the growth factor releasing property of PRF. Further cell culture studies, animal and human histological studies are needed to check the same before establishing it as a drug of therapeutic use in inhibiting PRF degradation, thereby enhancing the clinical efficiency of PRF in periodontal regeneration.

CONCLUSION

The ethonolic extract of nutmeg is effective in inhibiting the degradation of PRF membrane thus prolonging its function as a barrier membrane and providing space maintenance for a longer time.

CONFLICT OF INTERESTS

The authors declare no conflict of interest.

ACKNOWLEDGEMENT

We would like to thank Dr. N.M.Veerayan, Chancellor, Saveetha institute of medical and technical sciences, Dr. Deepak Nallaswamy, Director of Academics, Saveetha institute of medical and technical sciences, Dr. N.D.Jayakumar, Dean, Saveetha Dental College, Dr. Gopinath, ex-Head of the Dental research cell, Saveetha institute of medical and technical sciences, for giving us the opportunity to complete the study.

ABBREVIATIONS

PRF: Platelet-rich fibrin; MCAF: Modified coronally advanced flap; GTH: Gingival/mucosal thickness; CGF: Concentrated growth factor; PBS: Phosphate buffered saline; EDTA: Ethelenediaminetetraacetic acid; GF: Growth factor; GTR: Guided tissue regeneration;

GRAPHICAL ABSTRACT

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SUMMARY

  • The aim of this study was to assess whether nutmeg has any effect in inhibiting degradability of PRF membrane and to compare the degradability of PRF at different concentrations (200 mg, 100 mg, and 50 mg) of ethanolic and crude extracts of nutmeg. PRF was procured from 30 ml blood from 5 different donors were cut to equal sizes into 35 pieces. They were measured at baseline and dropped in 7 sets of ependorphs containing PBS, PBS containing 200 mg, 100mg and 50 mg crude extract of nutmeg, PBS containing 200 mg, 100mg and 50 mg ethanolic extract of nutmeg. After 1 week the PRF were retrieved and measured. The percentage of remaining PRF was calculated and data analysed. It was found that there was a difference in percentages of remaining PRF between all the groups when compared to the control group, out of which, crude extract of nutmeg 200 mg group alone had a significantly lesser % of remaining PRF than the control. All ethanolic extract groups had a significantly greater % of remaining PRF when compared to that of the control. Hence, it was found that nutmeg is effective in inhibiting the degradation of PRF membrane.

Table 1

Mean weight at baseline and 1 week in all the groups.

Control Group 1Crude extractEthanolic extract200 mg Group 2100 mg Group 350 mg Group 4200 mg Group 2100 mg Group 350 mg Group 7
Mean weight at baseline 14.6±2.8319.74±5.1218.36±2.2318±4.1518.88±2.8318.62±4.5119.37±3.71
Mean weight at 1 week 5.18±0.852.38±2.64.54±3.35.76±1.8116.04±2.215.16±4.8217.26±3.04
% of remaining PRF(M± SD) 35.59±14.2310.7±11.9223.71±17.8534.86±17.6585.09±2.8979.81±9.0286.01±4.42
Table 2

Comparison of percentage of remaining PRF between the groups by ANOVA.

Control Group 1Crude extractEthanolic extractP200 mg Group 2100 mg Group 350 mg Group 4200 mg Group 5100 mg Group 650 mg Group 7
% of remaining PRF(M± SD)35.59±14.2310.7±11.9223.71±17.8534.86±17.6585.09±2.8979.81±9.0286.01±4.420.00*

*significant at P < 0.05

Table 3

Comparison of percentage of remaining PRF between the groups by post hoc.

Variable IVariable JMean Difference (I-J)Std. ErrorSig.
Group 1Group 226.004007.50078.025*
Group 312.986007.50078.602
Group 41.840007.500781.000
Group 5-48.390007.50078.000*
Group 6-43.108007.50078.000*
Group 7-49.312007.50078.000*

*significant at P < 0.05

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