PREPARATION OF FIBRIN ENRICHED NANOHYDROXYAPATITE/CHITOSAN/EUGENOL GEL FOR PERIODONTAL REGENERATION

Abstract

A periodontal regenerative dental biomaterial gel named as F+CH+nHAP+E that was prepared for hampering the biofilm formation on the material and was highly enhanced for improving the antioxidant property of the same along with being homogenous, stable, and hemobiocompatibility in nature with the presence of particles at 421.3 d.nm in size as well as has the ability to neutralize the acidity and can encapsulate 100% eugenol. Since the gel has its properties regarding biofilm inhibition, antioxidant, and anti-inflammatory, the gel is utilized in the dental biomaterial gel process for periodontal regeneration. Because of this property, the gel has an immense potential to be an excellent dental restorative material.

Specification

Description:SUMMARY OF THE INVENTION
Out of various ratios of chitosan (CH), nanohydroxyapatite (nHAP), eugenol (E) and platelet rich fibrin (F) taken equal dry weight ratio of CH, nHAP, E taken 100 times increase in wet weight ratio of fibrin was taken and was found to be effective in forming gels that can be used as periodontal regenerative materials.
F+CH+nHAP+E gel was formed through crosslinking technique using nanohydroxyapatite as crosslinker
Zeta potential measurements for CH, nHAP, CH+nHAP , CH+nHAP+E, F+CH+nHAP+E was found to be 30.9 mV, -10.3mV, 37.9 mV, 33.4mV, 44.9 mV respectively and hydrodynamic diameter of particles for nHAP, CH+nHAP , CH+nHAP+E, F+CH+nHAP+E was found to be 565 d.nm, 347 d.nm, 526.1 d.nm, 421.3 d.nm respectively and was found to be in nanometer range.
CH+nHAP , CH+nHAP+E, F+CH+nHAP+E was characterised UV-Vis spectroscopy by shift in the spectra and CH, nHAP, CH+nHAP , CH+nHAP+E, F+ CH+nHAP+E, E and F was characterised by FTIR by changes in the molecular bonding vibrations
Acid neutralization studies of F+CH+nHAP+E in artificial saliva and lactic acid indicated its ability to neutralize acid.
Swelling of CH, nHAP, CH+nHAP, CH+nHAP+E, F+CH+nHAP+E, was noted and F+CH+nHAP+E and good swelling that enabled sustained release of E
CH+nHAP , CH+nHAP+E, F+CH+nHAP+E, E was found to be hemobiocompatible when compared to CH, nHAP and E alone group
F+CH+nHAP+E had better growth inhibition of bacteria S. aureus and E. coli and prevention of formation of biofilm thereby suggesting its use as dental regenerative materials
STATEMENT OF THE INVENTION
As a result, the current invention is primarily concerned with fibrin-enriched gel of nanohydroxyapatite/chitosan/eugenol is involved with periodontal regeneration, the gel is prepared to facilitate repair and regeneration of periodontal tissues. This is achieved by a) It combines the osteoconductive potential of nanohydroxyapatite, biocompatibility, and antimicrobial properties of chitosan, anti-inflammatory and antibacterial properties of eugenol, and the healing properties of fibrin, b) This composite gel present a new approach to periodontal therapy, which provides for a bioactive scaffold that supports cell adhesion, proliferation, and integration into the tissue while reducing infection and inflammation in the affected regions.
In an embodiment of the present invention, F+CH+nHAP+E crosslinking using nanohydroxyapatite was based on the gel formation at proper ratio.
In yet another embodiment of the present invention, CH+nHAP, CH+nHAP+E, F+CH+nHAP+E was characterized by UV-Vis spectroscopy and FTIR and compared to single counterparts CH, nHAP, E and F.
In yet another embodiment of the present invention, characterized by UV-Vis spectroscopy the entrapment of E in CH+nHAP+E and F+CH+nHAP+E entrapment efficiency was 72.26 and 100% respectively.
In yet another embodiment of the present invention, F+CH+nHAP+E treated denture swelling increased when compared to acrylic denture (D) or denture treated D+CH, D+nHAP, D+CH+nHAP, D+CH+nHAP+E, D+E groups suggesting sustained release of E from F+CH+nHAP+E
In yet another embodiment of the present invention, DPPH scavenging activity of CH, nHAP, E, CH+nHAP, CH+nHAP+E, F+CH+nHAP+E was found to be 32.7, 28.4, 58.6, 60.8, 67.3 and 71.1 % respectively, indicating synergistic efficacy of F+CH+nHAP+E
In yet another embodiment of the present invention, F+CH+nHAP+E, CH+nHAP+E, CH+nHAP was better hemocompatible CH, nHAP or E alone treated groups.
In yet another embodiment of the present invention F+CH+nHAP+E had acid neutralizing capacity in lactid acid and maintained salivary (artificial) pH.
In yet another embodiment of the present invention, F+CH+nHAP+E when compared to CH, E,nHAP, CH+nHAP, CH+nHAP+E inhibited S.aureus bacterial growth at 87.42, 38.32, 53.89, 14.9, 43.11, and 61.67 % respectively; also F+CH+nHAP+E when compared to CH, E,nHAP, CH+nHAP, CH+nHAP+E inhibited E.coli bacterial growth at 78.57, 21.42, 35.71, 14.28, 54.14, 64.28 respectively indicating synergistic efficacy of F+CH+nHAP+E.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
Since the inventions described hereafter are only intended to serve as illustrative examples of the present invention, they are not to be construed as representing the only embodiments of the current invention. The invention is further illustrated with the aid of the following examples.
Example-1 Fibrin (F) preparation and homogenization, 5 mL of blood was derived and centrifuged for 30 min at 3000 rpm. The F was then separated. Chitosan (CH) 10 mg of CH (low molecular weight) was weighed and added to 15 mL of 1% acetic acid and kept for stirring until CH gets dissolved. The solution was stored at 2-8 ℃. Chitosan + nano hydroxyapatite (CH+nHAP) 10 mg of CH (low molecular weight) was weighed and added to 15 mL of 1% acetic acid and kept for stirring until CH gets dissolved. Once CH gets dissolved, 10 mg of nHAP is dissolved in 5 mL milli Q water and was added to CH solution. The solution was kept for 2 h of stirring followed by centrifugation for 20 min at 3000 rpm. The supernatant and pellets were separated and were stored at 2-8 ℃. Chitosan + nano hydroxyapatite + eugenol (CH+nHAP+E) 10 mg of CH (low molecular weight) was weighed and added to 15 mL of 1% acetic acid and kept for stirring until CH gets dissolved. Once CH gets dissolved, 10 mg of eugenol was dissolved in 2 mL of milli Q water and added to the CH solution. After 1 h of stirring, 10 mg of nHAP, dissolved in 3 mL milli Q water and was added to the above solution. The solution was kept for 2 h of stirring followed by centrifugation for 20 min at 3000 rpm. The supernatant and pellets were separated and were stored at 2-8 ℃. Group 5 - F + chitosan + nano hydroxyapatite + eugenol (F+CH+nHAP+E) 10 mg of CH (low molecular weight) was weighed and added to 15 mL of 1% acetic acid and kept for stirring until CH gets dissolved. Once CH gets dissolved, the homogenized F (2mL) was added to it and stirred for one hour. Followed by this, 10 mg of EUG, dissolved in 1 mL of milli Q water was added. After 1 h of stirring, 10 mg of nHAP, dissolved in 2 mL milli Q water and was added. The solution was kept for 2 h of stirring followed by centrifugation for 20 min at 3000 rpm. The supernatant and pellets were separated and were stored at 2-8 ℃. A UV-Vis spectrophotometric study was performed, and the spectra were recorded afterward. Measurements of the zeta potential were carried out in order to gain an understanding of the degree of stability and the charge of the molecules that are bound to the surface. The hydrodynamic diameter procedure was carried out in order to gain an understanding of the size distribution of. Analysis was carried out using FT-IR studies.
Example-2 An experimental hemolysis assay was performed on all of the CH, E, nHAP, CH+nHAP, CH+nHAP+E, and F+CH+nHAP+E samples. To determine whether or not the nanocomposite is stable in a variety of pH levels, an acid neutralization study was carried out, and a lactic acid pH acid neutralization study was also carried out. The compounds are submerged in a solution consisting of lactic acid and artificial saliva at this stage of the process. First, the nanocomposite F+CH+nHAP+E was immersed in artificial saliva for twenty-four hours, and then it was submerged in lactic acid for one hour. This process was repeated for a total of twenty days. On day 1, day 3, day 5, day 10, and day 20, a change in the pH of artificial saliva and lactic acid was noticed and documented. Furthermore, this change was observed and recorded. In addition to that, the pH of the synthetic saliva was evaluated. When each pH test was finished, the artificial saliva solution was instantly replaced. This was done immediately after the fact. For a period of ten days, the dentures D+CH, D+E, D+nHAP, D+CH+nHAP+E, and D+F+CH+nHAP+E were immersed in a solution of PBS. This was done in order to assess the swelling of acrylic dentures as well as dentures that contained other components. At regular intervals for a period of one day, three days, five days, and ten days, the weight of the groups was measured, and the difference in weight was recorded. The measurements were taking place over the course of the study.
Example-3 Biofilm formation inhibition assay A growth inhibition experiment was conducted to identify the biofilm inhibition concentration of the synthesized nanoparticles. To prepare a fresh Nutrient broth, weigh 0.65 grams of Nutrient broth and dissolve in fifty millilitres of milli-Q water. Prepare the broth. Autoclave the prepared broth for an hour. All the experiments were done inside a biosafety cabinet, and before beginning the experiment, all the materials required were exposed directly to ultraviolet radiation. Then, once the fresh broth was added to each group, sterile 24-well plates were appropriately labeled with the necessary information. The cultured microorganisms, such as S. aureus and E. coli, were left overnight. A concentration of 100 µL was added into the broth, and after that, samples CH, E,nHAP, CH+nHAP, CH+nHAP+E, F+ CH+nHAP+E were added. Then into all the wells d.H2O was added to get a volume of 1 mL. After incubation for six hours, OD of the plates was measured at 540 nm. Crystal violet and safranin stains were used to carry out the gram staining of gram-positive (S. aureus) and gram-negative E. coli bacteria. This was done by the staining microscopy of each glass slide where two hundred microliters of the incubated samples are spread out to dry. Immediately after that, the slides were passed directly over a flame, and the heating then set. One minute after adding crystal violet to these slides they were washed with milli-Q water to eliminate any remaining precipitate. After adding Gram's iodide, the mixture was allowed to stand for one minute before it was washed using milli-Q water. This smear was briefly rinsed after the addition of the decolorizing chemical, that was acetone. A final counter stain that contained safranin was applied to each and it was left for one minute. Then all the slides were washed using milli-Q water, dried and then examined using a microscope at a magnification of one hundred times. A quantitative analysis was also performed on the data, in which the outcomes were reported in percentage.
ADVANTAGES OF THE INVENTION
A biocompatible periodontal regenerative dental biomaterial gel containing fibrin enriched nanohydroxyapatite/chitosan/eugenol.
The most significant benefit of these gels is that they have the ability to deliver eugenol in a continuous manner.
Crosslinking with nHAP in the suitable manner can be of benefit in the swelling of gel F+CH+nHAP+E.
The presence of chitosan in gel can help with adhesion, and the presence of fibrin in gel can help with swelling that is considered suitable.
The presence of eugenol in gel F+CH+nHAP+E can prevent inflammation and provide antioxidant activity.
The presence of fibrin in gel F+CH+nHAP+E can aid in rendering hemobiocompatibility to the gel and also aid in acid neutralization ability.
The presence of multicomponents gel F+CH+nHAP+E can aid the gel in contributing to the antibiofilm and antibacterial activity thereby contributing to the synergistic efficacy.
EXPLANATION OF FIGURES
Figure 1 Fig 1 A represents the mechanism of formation of biocompatible periodontal regenerative biomaterial gel made of CH, F, nHAP and E. fig 1 B indicated the UV-vis spectra of nanocomposite after addition of E and F. fig 1 C indicates the hydrodynamic diameter d.nm of CH, nHAP, CH+nHAP, CH+nHAP+E, F+CH+nHAP+E. Fig 1 D indicated the zeta potential of CH, nHAP, CH+nHAP, CH+nHAP+E, F+CH+nHAP+E. fig 1 E indicates the IR spectra of CH, nHAP, E, F, CH+nHAP, CH+nHAP+E, F+CH+nHAP+E which indicates the changes in the vibrational band after addition of E (Example 1).
Figure 2 Fig 2 A indicates the acid neutralization study done for F+CH+nHAP+E gel wherein the nanocomposite was placed in lactic acid and artificial saliva for 20 d. Fig 1 B indicates the hemolysis assay where there is no hemolysis for CH+nHAP, CH+nHAP+E, F+CH+nHAP+E when compared to CH, nHAP, E, negative control (NC) and positive control (PC). Fig 2 C indicates the data of swelling assay of D and D with/ without nanocomposite which was monitored for 10 d. Fig 2 D indicates the DPPH scavenging activity of CH, nHAP, E, CH+nHAP, CH+nHAP+E, F+CH+nHAP+E when compared to control and fig 2 E indicates the DPPH % of inhibition of CH, nHAP, E, CH+nHAP, CH+nHAP+E, F+CH+nHAP+E (Example 2).
Figure 3 Fig 3 A indicates the % of biofilm formation inhibition of CH, E, nHAP, CH+nHAP, CH+nHAP+E, F+CH+nHAP+E when compared to control. Fig 3 B indicates the gram staining of CH, nHAP, E, CH+nHAP, CH+nHAP+E, F+CH+nHAP+E done for S. aureus and E.coli (Example 3).
, Claims:I/We Claim:
1. A periodontal regenerative dental biomaterial gel (F+CH+nHAP+E) with antioxidant capability for preventing biofilm formation comprises following characteristics: -
i) Gel F+CH+nHAP+E was found to be a homogeneous material with good stability as determined by the zetapotential measurement (44.9 mV).
ii) The particles in gel F+CH+nHAP+E were found to be 421.3 d.nm as determined by the DLS.
iii) Gel F+CH+nHAP+E 100% eugenol entrapping efficacy and is hemobiocompatible
iv) Gel F+CH+nHAP+E exhibits acid neutralisation ability and swelling characteristics resulting in sustained release of eugenol
v) Periodontal regenerative dental biomaterial gel F+CH+nHAP+E exhibits biofilm inhibition property (78.57 - 87.42%), antioxidant (DPPH scavenging activity 71.1%) and anti-inflammatory property
2. Periodontal regenerative dental biomaterial gel (F+CH+nHAP+E) as claimed in claim 1, method of preparation comprising of steps:-
i) Chitosan (CH) 10 mg of low molecular weight was weighed and added to 15 mL of 1% acetic acid and kept for stirring until CH gets dissolved.
ii) Fibrin (F) was homogenized and 2 mL containing wet weight 100 times more than CH taken that is 1000 mg was added to dissolved CH solution and stirred for one hour.
iii) Followed by this, 10 mg of eugenol (E), dissolved in 1 mL of milli Q water was added to the solution containing CH and F.
iv) After one hour of stirring, 10 mg of nanohydroxyapatite (nHAP), dissolved in 2 mL milli Q water and was added an.
v) The solution was kept for 2 hours in stirring followed by centrifugation for 20 min at 3000 rpm.
vi) The supernatant and pellets (F+CH+nHAP+E) were separated and were stored at 2-8 ℃.

Documents