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I. Introduction
Periodontal disease is a multifactorial, inflammatory condition that cause attachment loss and bone loss1. Plaque, calculus and faulty tooth brushing techniques are some of the concerns related to the occurrence of this disease2. Treatment of periodontitis has been established as non-surgical therapy (NST) which includes scaling and root planing (SRP) and surgical therapy (ST) which involves flap surgery or open flap debridement (OFD) with or without the utilization of biomaterials such as bone grafts, guided tissue regeneration materials with resorbable/non-resorbable membranes and platelet concentrates (PCs)3. Researchers have achieved good results regarding decreased probing pocket depth (PPD), gain in clinical attachment level (CAL) and improved defect fill post-surgically on regular follow-ups4. Post-operatively in both NST and ST, systemic antibiotics were used based on the oral inflammation state and the patient’s systemic outcome. However, despite the various advantages of controlling the infection or disease, there may be several side effects affecting the functions of the body and its organs due to their higher concentrations in the blood stream5. Additionally, systemic drug administration may not maintain its levels in the gingival crevicular fluid to control the periodontal infection at the crevice6.
Hence site-specific treatment strategies have been tried and one such modality is local drug delivery systems (LDDs). LDD prevents systemic toxicity and maintains higher levels of drug concentration7. Various drugs like doxycycline, metronidazole (MTZ), azithromycin, metformin, atorvastatin, rosuvastatin etc. have been tried in periodontal pockets with and without SRP and achieved greater PPD reduction and CAL gain8. Studies done by Pradeep et al.9 used metformin in gel forms (1% and 1.5%), rosuvastatin (1.2%) versus atorvastatin (1.5%)10 in OFD and achieved greater defect fill and CAL gain in treating intra bony defects (IBDs) of periodontal disease. Herbal products like neem (NE) and aloe-vera gel extracts were also tried for treating the periodontitis due to their inherent antimicrobial efficacy11. Though the results obtained were beneficial, long-term results were lacking so, specific drug delivery systems were started and used Carbopol polymers, collagen sponges, fibres and PC for constant release of drug with longer duration and sustainability12.
Generations of PC have been established in both dental and medical fields. In the present scenario, PCs are being considered a boon to the dental field. Initially first-generation PC’s such as fibrin glue (FG) and platelet-rich plasma (PRP) have been tried. This FG which has been tried triggered the antigenicity because of bovine antithrombin addition and when human derived FG was tried there might be higher chances of cross reactions such as viral infections. Even, PRP has certain drawbacks such as robust release of growth factors within half an hour and the addition of bovine anti-thrombin for the activation of platelets led to the search of better autologous material. This led to the introduction of leukocyte platelet rich fibrin (L-PRF). This L-PRF has a three-dimensional (3D) fibrin structure with sustained hold and release of GF’s. But because of their drawbacks such as a short life span of 7-11 days, loose fibrin structure and meshwork (for L-PRF), the search for better biomaterial search was not stopped13. During this research, researchers were attracted to titanium metal which is highly biocompatible, non-corrosive, non-breakable, regularly used in implant dentistry and surgical medical fields. Hence Tunalı et al.14 introduced titanium platelet-rich fibrin (T-PRF) using the same centrifugation protocol as that of L-PRF which was autologous, and activation of platelets was done by the titanium dioxide layer formed within the tube itself.
Studies conducted by various researchers14-18 concluded that T-PRF had a better fibrin mesh work, greater cellular entrapment, thicker fibrin membrane and border area which helps in holding GF and had a longer resorption time of 21 days than that of L-PRF. With the evidence of histological research, some clinical studies16,19-22 for treating IBDs and achieved good results regarding the increased bone fill, gain in CAL and decreased PPD. Even gingival recession defects with T-PRF as a biomaterial below the soft tissue flap23-26 and achieved greater reduction in recession depth, recession width and gain in width of keratinized tissue equivalent to that of subepithelial connective tissue graft. Recent systematic review done by Oza et al.27 concluded T-PRF is a better alternative biomaterial with superior properties for various periodontal regenerative surgical outcomes. Because of this positivity of T-PRF, Ercan et al.28, tried this T-PRF as a sustained drug delivery system (SDDS) by injecting doxycycline liquid and assessed for drug kinetics, scanning electron microscopy and anti-microbial efficacy with collagen sponge as control. They have concluded that T-PRF released the doxycycline drug at constant pace for longer duration making T-PRF as a better candidate for SDDS. Polak et al.29 in their study used PRF as a delivery system for antibiotics like MTZ, clindamycin and penicillin and checked for antimicrobial efficacy against Fusobacterium nucleatum (Fn) and Staphylococcus aureus (Sa) where they concluded that PRF incorporated with antibiotics helped in long term antimicrobial effect of 4 days along with healing properties. Recent histological analysis study done by Gummaluri et al.30 reported a comparison of T-PRF+NE gel with T-PRF alone and concluded that NE incorporated T-PRF had a thin fibrin network with scattered or layered pattern, but it was not significantly different from plain T-PRF. Further Gummaluri et al.31 in their histological study regarding comparisons of T-PRF alone T-PRF injected with amoxicillin+clavulanic acid, MTZ and NEs individually concluded that gels incorporated T-PRF clots maintained intact fibrin meshwork, network without hampering the cells distribution. Scanning electron microscopy reported the incorporation of these antibiotic gels and NE which was visualized on surface morphology as coating. Hence it opens a gateway to dental treatments to utilize T-PRF as a SDDS.
In vitro analysis always play an important role in directing a researcher whether their protocol that was prepared going on right path or not and it also acts as first step in authenticating the protocol so that further clinical trials can be established or formulated32. In the present study antibiotic gels were formulated using MTZ, Augmentin and NE drugs. These gels were incorporated in T-PRF clots (test group) and collagen sponge (control group) individually for assessing the drug release using spectrophotometric analysis. Antimicrobial efficacy was also conducted for T-PRF injected with these gels using Porphyromonas gingivalis (Pg), Aggregatibacter actinomycetemcomitans (Aa), Fn, and Sa. Null hypothesis of our study states that T-PRF incorporated drugs such as amoxicillin+clavulanic acid gel, NE extract gel and MTZ gel and cannot be used as SDDS and additionally these extracts will hamper the growth factors release with minimal antimicrobial efficacy. Hence present study aimed to evaluate the drug kinetics, antimicrobial efficacy and growth factor release of T-PRF injected with antibiotics and herbal extract separately.
II. Materials and Methods
1. Study design, sample size estimation and study population
The present study was an in vitro analysis. Sample size calculated for antimicrobial efficacy and drug kinetics based on effect size of 2.50 (This was calculated from formula mean difference/ one of the standard deviations by considering values from Ercan et al.28, study). Further, calculated effect size was placed in G*Power software ver. 3.1.9.4 with alpha value error of 5% and power of 80% a sample of 12 was sufficient (4 per each group). Further for growth factors release additional four volunteers were recruited. A total of 12 healthy volunteers (6 males and 6 females) (4 for drug kinetics, 4 for antimicrobial efficacy and 4 for growth factor release) with mean age of 29.1±5.6 years in males and 30.6 ±4.08 years in females were recruited for the study was based on previous trial conducted by Ercan et al.28. The study consisted of three parts: spectrophotometric analysis along with Fourier transformed infrared spectroscopy (FT-IR) for drug release, antimicrobial efficacy and growth factor release. Ethical concern played a crucial role in the recruitment of volunteers for each part of the in vitro analysis hence, a proper ethical clearance was obtained from the institutional review board. Volunteers participated in the study underwent a thorough explanation of pros and cons of the study that will be carried out. This study has followed the Checklist for Reporting In-vitro Studies (CRIS) to as much extent as possible.
For assessing drug kinetics, 20 mL of venous blood was drawn from each volunteer from the antecubital vein and transferred to four sterile medical grade titanium test tubes (MGTTs) (Grade II titanium test tubes; Supra Alloys company) with 5 mL of blood in each tube. This standardization ensured that the obtained clots were of equal size. T-PRF was briefly prepared as follows; blood was drawn from the patient’s median cubital vein using a sterile technique to maintain purity and prevent contamination. Approximately 5 mL of blood each was collected in 4 sterile, MGTTs without any anticoagulants. The blood-filled titanium tubes were immediately placed into the centrifuge to prevent premature clotting. The centrifuge machine (Remi R 8C; Remi Electrotechnik) was set to 3,500 rpm17-20 at 400×g and blood samples were spun for 15 minutes at room temperature. Using, sterile forceps, the T-PRF clot was gently extracted from the middle layer17-20. Antibiotic gels and herbal extract were then injected separately and checked for drug release at 0 hours 2, 24, 48 and 72 hours. Collagen sponge (Fix- Plug; Synerheal Pharmaceuticals) were used as the control group for drug kinetics. Samples of T-PRF alone, T-PRF+ with injected antibiotic gels and T-PRF+ with herbal extract (4 samples in total) were subjected to FT-IR. For anti-microbial efficacy, 4 separate healthy volunteers were recruited and 5 mL of venous blood was drawn to prepare clots. The clots were cut into 3 pieces to obtain triplicate for checking the zone of inhibition on agar plates impregnated with ATCC strains of Pg, Aa, Fn and Sa at 48 hours. Furthermore, for checking the growth factor release 4 healthy volunteers were recruited and 5 mL of blood was and drawn injected with antibiotic gels and herbal extract to check for growth factors at 3rd, 7th and 10th day.
The entire study protocol was thoroughly reviewed and ethical clearance was obtained from the Institutional Ethics Committee of GITAM Dental College and Hospital with a protocol number 50086060923. The study was registered with the Clinical Trials Registry of India (CTRI) with the number CTRI/2024/01/061891. The study was conducted during the period from June 2023 to June 2024. Antibiotic gels (amoxiclav gel [amoxicillin+clavulanic acid], MTZ and herbal extract [NEs]) were prepared at a private lab (PerioBiologics). Study was performed according to CRIS protocol to as much extent possible.
2. Inclusion and exclusion criteria
Subjects who were systemically healthy, over 20 years of age, and had not used systemic antibiotics within the last 6 months were included in the study. Individuals who were unwilling to donate blood or participate in the study were excluded.
3. Antibiotic gels and herbal extract preparations
The amoxiclav gel was prepared by mixing amoxicillin and clavulanic acid powders in a 7:1 weight ratio. Sodium carboxy methyl cellulose and magnesium stearate were added, and all ingredients were blended into a mixture. Polycarbophil powder and glycerine were added to form an 18% w/v final concentrated gel. The MTZ gel, was prepared with poloxamer 123, propylene glycol, methylisothiazolinone, MTZ and distilled water to make a 30% w/v concentrated MTZ gel. NE extract was prepared by boiling NE leaves in water to obtain 100 gm NE extract. Carbopol 934p was added to water to obtain a base gel. Further calcium chloride (CaCl2) and sodium chloride (NaCl) were added to prepare the 12%w/v concentrated NE gel30,33. We have injected 1mL of each drug in to the respective T-PRF clots and collagen plug samples which had a drug concentration of about 2.2 mg/mL for amoxiclav gel, 2.8 mg/mL for MTZ and 1.8 mg/mL for NE gel All raw materials were procured from Sigma-Aldrich company, Mumbai, India.
4. Procedure for spectrophotometric analysis and FT-IR
Blood was drawn from the antecubital vein, and transferred to MGTTs, then centrifuged according to previous studies17,18,20 at 3,500 rpm for 15 minutes (Remi R8C). Drug kinetics of T-PRF injected clots was performed as per established protocols28,33,34. Drug release was estimated at various time intervals using a UV-VIS-NIR spectrophotometer (Shimadzu 2600i; Shimadzu). The same procedure was followed for the control group collagen sponge (Fix-Plug; Synerheal Pharmaceuticals). The drug kinetics release was monitored for 72 hours to determine the release of the entire drug within that time frame. Aligning with typical antibiotic prescription durations in India.
Falcon tubes (containing 10 mL of phosphate buffer saline [PBS]) with test or control samples were placed on a shaker at a rate of 250 rpm in a water bath at 37°C. Drug release amounts were monitored over time at specific wavelengths: 240-280 nm for amoxicillin, 218 nm for clavulanate, 276-332 nm for MTZ and 310-330 nm for NE. These values were then compared with standardized calibration curves previously prepared for antibiotic gels or herbal extract in PBS solution. At each time interval 2 mL of PBS solution was extracted from the falcon tubes containing the test samples and absorbance values were recorded. Fresh PBS was then added to replenish the lost quantity filled into the falcon tubes to replenish the lost quantity. This process was repeated four times for both test and control groups to accurately calculate the optical density values and subsequently the drug release percentages.(Fig. 1, 2) The drug kinetic release of amoxiclav was checked separately because they (amoxicillin and clavulanic acid [amoxiclav]) released separately and absorbance frequency range was different.
For FT-IR analysis, 4 samples of T-PRF injected with gels were separated from the drug kinetic samples and exposed to different gradients of alcohol (25%, 50%, 75% and 100%) each for 20 seconds each. These samples were then placed in a desiccation jar with CaCl2 for 24 hours. Subsequently, they were analyzed under an FT-IR instrument (ATR FTIR Spectrophotometer; Bruker Company) to confirm drug impregnation. The data obtained was graphically represented and the peaks were compared against the available standards.(Fig. 3)
5. Antimicrobial efficacy procedure
To assess the antimicrobial efficacy against Pg, Aa, Fn, and Sa, individual drug and herbal extract gels were injected into clots and checked for zone of inhibition diameters at 24 and 48 hours in millimetres. Anaerobic culture media mixed with agarose gel were prepared and poured into culture plates. The strains of Pg (ATCC No. 33277), Aa (ATCC No. 43718), Fn (ATCC No. 23726) and Sa (ATCC No. 25923) were inoculated into the agar culture plates (tryptic soy-serum bacitracin vancomycin agar medium was used for Pg and Aa, whereas Sabourauds agar medium was used for Sa and tryptic soy broth supplemented with 1% bactopeptone and 0.25% autoclaved cysteine was used for culture of Fn). Well preparations were done in the culture plates where test samples containing T-PRF incorporated with amoxiclav, MTZ, NE gel were placed at various locations around the petri dishes and incubated for 48 hours at 37°C in an anaerobic and aerobic environment as needed35. After incubation, the diameter of the clear zone around each disk was measured in mm using a ruler.(Fig. 4) All materials were procured from HiMedia Company, Mumbai, India.
6. Growth factors release procedure
Growth factors (GFs) such as vascular endothelial growth factor (VEGF), platelet derived growth factor-BB (PDGF-BB) and insulin like growth factor (IGF) were assessed on the 3rd, 7th and 10th day to evaluate the efficacy of T-PRF injected with antibiotic gels (amoxiclav, MTZ gels) or NE. The levels of VEGF, PDGF and IGF were measured using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s instructions (ELISA Kits; Biocompare and Miltenyi Biotec, Inc.). The sensitivity and evaluation range for each parameter were as follows- human IGF-1: (0.94 ng/mL and 1.56-100 ng/mL), human PDGF-BB (18.75 pg/mL and 31.2-2,000 pg/mL) and human VEGF A (18.75 pg/mL and 31.2-2,000 pg/mL).
7. Statistical analysis
Entire data was collected and transferred to Microsoft Excel 2019 spread sheet. Drug release using spectrophotometric analysis were expressed in mean and standard deviation frequency percentages. Normality of data was assessed by Shapiro–Wilk test and present study showed a non-normal distribution. Hence non-parametric tests were performed. Intra group comparisons for test and control groups were done using Kruskal–Wallis test. Multiple time frame comparisons of drug release for test and control group were performed by Dunn’s post-hoc test. For adjusting these multiple comparisons and reduce the risk of false positives Bonferroni’s correction was applied For Inter group comparison among test and control groups Mann–Whitney U test was performed. Antimicrobial efficacy (zone of inhibitions) of bacteria were expressed in mean and standard deviation diameters at 48 hours. GF’s release was expressed in mean and standard deviations in ng/mL, pg/mL based on the amounts released at 3, 7 and 10 days respectively. P-value <0.05* and <0.001*** were considered statistically significant and highly significant.
III. Results
In the present study, intra group comparisons in T-PRF group injected with antibiotic gels or herbal extract there was a statistical significance recorded at all the time frames with sustained release of clavulanic acid at 0 hour (P=0.003*), 2 hours (P=0.003*). While sustained release was gradually shifted to amoxicillin from 24 hours to 72 hours (P=0.005*, P=0.003* and P=0.003*).(Table 1) In case of collagen sponge group for intra group comparisons, there was statistical significance at all the time frames with greater release of clavulanic acid at 0 hour (0.003*), amoxicillin at 2 hours (0.042*), 24 hours (0.039*) and 48 hours (0.014*) while at 72 hours MTZ showed sustained release (P=0.004*).(Table 2) The Kruskal–Wallis test results overall for both groups there was a statistic value of 14.14 and statistical significance was recorded indicating a significant difference between the groups. When comparisons were performed at different time frames of T-PRF and collagen sponge injected with amoxiclav/MTZ/NE gel showed a statistical significance for all comparisons except at 24 hours of amoxicillin and clavulanic acid comparison (P=0.8857) and at 72 hours of clavulanic acid and MTZ comparison (P=0.0571). Whereas, there was no significant difference between the groups after adjusting for multiple comparisons using bonferroni’s correction.(Table 3)
For inter group comparisons, regarding drug kinetics of T-PRF/collagen sponge injected with antibiotic gels or herbal extract, under spectrophotometric analysis, there was a statistical significance regarding the percentage release of drug for amoxicillin in both T-PRF and collagen sponge groups (0 hours: P=0.020*, 2 hours: P=0.021*, 24 hours: P=0.021*, 48 hours: P=0.021*, 72 hours: P=0.017*) whereas clavulanic acid showed significant percentage release at 0 (P=0.020**), 24 (P=0.042*) and 72 hours (P=0.003***) and non-significance was recorded at 2 (P=0.248) and 48 hours (P=0.248). For MTZ gel there was a significant release (0 hours: P=0.021*, 24 hours: P=0.020*, 48 hours: P=0.021* and 72 hours: P=0.021*) of drug reported at all the time intervals except at 2 hours (P=0.146). While coming to NE release there was a statistical significance at all the time periods with greater release of collagen sponge group than T-PRF group (0 hour: P=0.020*, 2 hours: P=0.020*, 24 hours: P=0.021*, 48 hours: P=0.018* and 72 hours: P=0.019*).(Fig. 2-4) Regarding antimicrobial efficacy, T-PRF+amoxiclav gel showed higher inhibition zone diameters (IZDs) for Aa then an order of Sa, Fn and Pg at 48 hours was recorded. For T-PRF+MTZ gel highest IZD was recorded for Aa, later an order of Sa, Pg and Fn was recorded at 48 hours. Further T-PRF+NE gel reported an IZD order of Sa, Pg, Fn and Aa at 48 hours.(Table 4)
Regarding GFs release, in T-PRF plain there was a gradual increased release on 7th day (402.58±204.78 pg/mL) and decreased at 10th day (222.20±164.90 pg/mL) for PDGF-BB whereas there was a gradual decrease from 3rd to 10th day in case of VEGF (129.12±37.06, 107.15±23.98, 70.28±30.73 pg/mL) and IGF 1 (58.55±5.90, 16.78±9.28, 7.19±0.14 ng/mL). For T-PRF+amoxiclav gel, there was a gradual decrease release of PDGF-BB from 3rd (228.50±62.72) to 7th day (209.28±15.52) and increase release at 10th day (306.77±133.23). Whereas for VEGF there was a gradual increase release from 3rd to 10th day and for IGF 1 there was an increase from 3rd (15.57±3.85) to 7th day (21.92±7.21) and decrease at 10th day (18.30±1.87). In T-PRF+MTZ group, there was a gradual increased release of PDGF-BB from 3rd to 10th day (345.48±247.37, 324.05±54.36, 421.62±145.75 pg/mL), while VEGF showed increased release at 7th day (96.33±2.29 pg/mL) and 3rd and 10th days (85.68±22.96 pg/mL, 83.75±15.79 ng/mL) showed similar levels of release. Further for IGF 1 at 7th day (10.99±4.28 ng/mL) lower release was reported and higher levels of release were recorded at 3rd and 10th days (50.69±16.03 and 36.51±16.30 ng/mL). In case of T-PRF+NE gel group, PDGF-BB release was higher at 3rd day (355.36 pg/mL), reduced at 7th day (299.62 pg/mL) and again increased at 10th day (328.36 pg/mL), while coming to VEGF there was an increase at 7th day (108.33 pg/mL) and reduced levels were noted at 10th day (87.91 pg/mL), further in case of IGF 1 there was a decrease release reported from 3rd to 10th day (18.22 ng/mL, 18.45 ng/mL, 15.69 ng/mL).(Table 5)
Regarding FT-IR, in T-PRF injected amoxiclav gel showed varied frequency peaks at O-H stretching (3,648, 3,685 and 3,747 cm-1), C=O stretching in COOH group at 1711.54 cm-1, C=O-NH stretching in COOH group at 1,641 cm-1, C=C stretching range at 1,501-1,564 cm-1, COO-acetate at 1,391 cm-1, para substituted benzenoid band frequency shown at 772 cm-1. In case of T-PRF incorporated with MTZ gel there were peaks at 722 cm-1 for C-H bending, C-O stretching at 1,162 cm-1, N=O stretching range reported at 1,460 cm-1, CH3 bending at 1,376 cm-1 and C-H stretching peak reported at 2,922 to 2,853 cm-1. For T-PRF incorporated with NE gel C=O stretching frequency showed at 1,744 cm-1, C-H stretching, C-O stretching and CH3 bending frequencies were reported at 2,924.77-2,854.77, 1,162 and 1,376 cm-1 respectively. Further C=C and C-H stretching were recorded at 1,468 and 723 cm-1. In case of plain T-PRF, C-H stretching, O-H stretching, Amide 1 stretching band, C=C stretching, C≡N/C≡C and C-H bending were reported at 2,874-2,832 cm-1, 3,691 cm-1, 1,719-1,558 cm-1, 1,490 cm-1, 2,328-2,832 cm-1 and 724-898 cm-1.(Table 6)
IV. Discussion
Volunteers recruited for the study showed no signs of abnormality at blood draw sites. The present in vitro analysis is the first of its kind, assessing of T-PRF as a SDDS after incorporating MTZ gel, amoxiclav gel and NE individually. Proper study protocols were followed in the preparation of T-PRF clots; antibiotic and herbal extract gels procured were standardized and sterilized before use in the study. The T-PRF speed used in the current study was properly checked and established from our previous histological17,18 and human studies20. The present study utilized antibiotics and herbal extract preparations in gel forms, while most studies have used liquid forms. The use of gel form may be an easier way of handling and proper injection into T-PRF clots using syringes. Since not many studies have been performed, comparisons in the present study were made with existing literature. The specific selection of amoxiclav gel/MTZ gel in this study was due to their regular usage as line of treatment for periodontitis36. The use of NE was mainly because of its antimicrobial, astringent, antiseptic, anti-ulcer, anti-viral and antihyperglycemic properties37. It has been tried for treating gingivitis and periodontitis in the form of topical application or LDD.
In terms of drug kinetics both test and control groups showed drug release at all time frames indicating T-PRF as a SDDS. Although collagen sponge showed greater percentage of drug release than T-PRF, the inherent properties of the thicker fibrin membrane and meshwork in T-PRF helped in the gradual release of amoxiclav, MTZ and NE individually. The results of the present study results were in accordance with studies done by Indurkar and Purohit NJ38, Ercan et al.28 and Polak et al.29 where they concluded that incorporation of antibiotics didn’t change PRF physical properties and held antibiotics within the collagen frame work and releasing constantly throughout the speculated time period. Polak et al.29 also concluded that 0.5 mL of drug injection into PRF didn’t spoil the structural frame work, which was also followed in the present study helping in the constant release of amoxiclav, MTZ and NE through T-PRF and collagen sponge.
Drug release in T-PRF is slightly inferior to that of the collagen sponge used in the present study. This is not in harmony with Ercan et al.28 where they concluded that T-PRF had a greater holding capacity of doxycycline drug compared with collagen. This variation might be due to the change in type of antibiotics and type of collagen that was used in present study. However, this study, suggests that T-PRF can hold and release drug in a systematic way.
The current study results of drug kinetics of T-PRF showed similar pattern to a recent study done by Bennardo et al.39 where they used varied concentrations of vancomycin, gentamycin and linezolid incorporated in L-PRF and concluded that there was a gradual release of these drugs. Post 3rd molar extraction, the placement of these drug incorporated PRF clots at surgical sites helped in better healing without infections and lower post-operative pain. This indicates a treatment strategy to replace or enhance systemic antibiotic therapy while preserving the natural healing properties of PRF. This may be due to the inherent healing property of T-PRF, better holding of antibiotics by the fibrin network structure helped in sustained release of the drug. Moreover T-PRF also shares a similar structure with L-PRF. In the present study, the assessment of drug kinetics was done until the end of 72 hours showing the gradual release of drug. This is in accordance with previous studies done by Gessman et al.40 where they concluded that blood plasma clots could be used to deliver antibiotics. The study results also showed similarity with that of Siawasch et al.41 where MTZ was released from PRF for 3 days. This shows the autologous nature with thicker fibrin meshwork and holding the drug within the space of T-PRF clot led to constant timely release of the drug.
Regarding antimicrobial efficacy, IZD’s were recorded for amoxiclav, MTZ and NE injected in T-PRF clots against Pg, Aa, Fn and Sa. All the test groups showed antibacterial effect against these pathogens at 48 hrs. This is in accordance with recent studies done by Ercan et al.28 where they concluded that T-PRF showed antibacterial effect at the end of 7 days against Pseudomonas aeruginosa and Sa. When the current results were compared with Castro et al.35’s study, a similarity was reported. The major difference recorded was they used L-PRF and concluded an antibacterial effect against Fn, Prevotella intermedia and Aa. Whereas the present study used T-PRF injected with antibiotic and herbal extract gels which showed a great antibacterial effect. This might be due to the inherent antibacterial property of T-PRF, addition of these gels added an additional benefit to act against the pathogens.
Regarding GF release there was a gradual release of PDGF-BB, VEGF and IGF at the 3rd,7th and 10th day for all the groups. There was greater release of GF numerically but values did not vary much when comparing T-PRF plain and T-PRF injected with gels. These results were slightly superior to that of a study done by Zwittnig et al.42 where there was a constant gradual release of PDGF-BB, VEGF, TGF-beta 1 and MMP-9 in solid and liquid PRF’s. They also concluded that there were not many variations in GF’s release. Injecting antibiotic gels and NE extract didn’t affect the release of GF’s. This might be due to the thicker 3D fibrin structural frame work led to better hold of GF’s and constant release. Hence, it can also be attributed to the preservation of stimulative regenerative capacity by longer resorption time and antibacterial effect of T-PRF which resulted in superior results.
In the present study, drugs injected into T-PRF clots were compared with T-PRF plain using FT-IR spectroscopy where it was reported that peaks noted were different for test and control groups indicating the presence of drug within the clots themselves. Study graphs showed similar peaks to existing standard literature of drugs (Kumar et al.43, Foschi et al.44, and Ali et al.45) and T-PRF plain peaks showed a similar pattern with of L-PRF published literature peaks (Braga et al.46). This similarity may be due to the standard method of gel and T-PRF preparations, thicker fibrin structure and network of T-PRF clots sharing a 3D pattern with that of L-PRF, leading to similar peaks for the T-PRF plain group.
A recent study by Siawasch et al.41 assessed the antibacterial activity in L-PRF by loading different concentrations of MTZ and amoxicillin for local and systemic applications. They concluded that L-PRF showed good antibacterial activity and there was no significant difference regarding the release of growth factors with and without addition of antimicrobials. Similar results of antibacterial effect and growth factor release were recorded in the present study. This might be due to the thicker fibrin meshwork for better holding of growth factors, the inherent antibacterial property of T-PRF and addition of antimicrobials to T-PRF clots after the centrifugation process, which helped in adjunct antibacterial activity against periodontal pathogens. These gels were added after the centrifugation process, preserving the intactness of T-PRF which in turn helped in preservation of physical structural properties with gels held firmly within the space of T-PRF clot.
The current study used amoxiclav gel and MTZ gel which can be considered a standard regimen for treatment of periodontal disease while the direct usage of NE extract into periodontal disease sites directly is slightly infrequent. A study by Chatterjee et al.47 utilized NE extract as mouth wash and concluded that it can be used as an alternative treatment strategy for periodontal disease, indirectly comparable to the results of the present study which could open a new treatment strategy for increasing herbal product usage at periodontal surgical sites after cell line and animal trials. The antibacterial effect of NE injected into T-PRF results showed superior results compared to a study by Sharma S et al.48 which concluded that solanum extract did not show any effect against the pathogens like bacteroid species, Prevotella corporis, Prevotella melaninogenica and Peptostreptococcus species. The present study results were superior and showed IZD’s for major periodontal pathogens that cause destruction. This was mainly due to the usage of NE extract in gel form which had maintained a proper drug concentration with antimicrobial properties and the inherent antimicrobial property of T-PRF helped in an additive effect on the outcome of IZDs.
The results of the present study also indicate that T-PRF had a thicker 3D structure that can embed any molecules, proteins and cells. Hence, in this study, the addition of antibiotic gels or herbal extract was properly embedded into the clot, showed drug release and antimicrobial effect without hampering the release of growth factors. These results are similarity to Miron and Zhang49 where they used liquid PRF as a drug delivery system. The major difference in their study was that antibiotics were placed prior to centrifugation whereas in our study we injected them as gel forms into T-PRF clots to prevent damage to the fibrin structure. Studies have also shown that the release of growth factors can vary between various regions of PRF matrices, as well as between individuals16,18,42. For example, the release of VEGF tends to be lower in i-PRF when compared to L-PRF, while PDGF-BB and MMP-9 are higher in L-PRF as compared to other derivatives of PRF17,18,29,33. As the clots were spliced into three sections before placing them into microbial petri dishes, this effect of this apparent disparity could also explain variance in results pertaining to antibacterial efficacy as well. Study done by Sayd et al.50, where they performed a comparative study with amoxicillin+clavulanic acid (500 mg+125 mg) and MTZ (400 mg) combination thrice daily (as 1st group) vs. azithromycin (500 mg) once daily and MTZ 400 mg thrice daily combination as 2nd group in the treatment of lower mandibular 3rd molar surgical impactions and concluded that both the treatments were regularly used and neither strategy depicted superior benefits in the control of post-operative infection. Hence present in vitro study, gives a gate way for near future where when used clinically may help in sustained release of the antibiotics/herbal extract through T-PRF and reduce the usage of antibiotics systemically as a part of control of post-operative infections.
Present study results reject our null hypothesis and establish T-PRF as a SDDS where it helps in constant timely release of the antibiotics or herbal extract. Clinical implications of the current study were T-PRF had a proper contained space within the fibrin structure which accommodates the antibiotics or herbal extracts in the form of gel or liquids. Better fibrin structure and longer resorption time of T-PRF holds the growth factors and injected drugs that would help in longer time of release. These hybrid materials can be used in periodontal ST where drugs can be delivered at the surgical site. Even around the implant sites these T-PRF injected drug materials can be given which help in reduced bacterial load, stimulate osteoblastic cells and gingival cells which would help in new soft and hard tissue restoration.
Limitations of the present study include a smaller sample size (with regard to the limited sample size, random variations and greater influence of outliers: in small samples, outliers or extreme values can disproportionately affect the results and might have impacted the validity and reliability of the research findings), drugs injected after centrifugation, the use of gel forms rather than liquid, drug release checked up to 72 hours, duration of IZDs at 48 hours (the study’s goal was to assess whether T-PRF impregnation helped in antimicrobial efficacy) which was shorter and GF release checked up to 10 days whereas in the literature it was stated that T-PRF stays up to 21 days at surgical site. Hence, checking GF release for at least up to 21 days might have altered the outcome. Further animal trials and randomized controlled clinical trials with larger sample size would be helpful in assessing this new treatment strategy for regular use. In addition, in vitro studies, while valuable, do have several limitations that can impact the interpretation and applicability of our results. The lack of physiological context, simplification of biological systems, limited predictive value, artifacts and contamination can affect the accuracy and reproducibility of results7,11,17,18. Although the present study had a positive outcome regarding the usage of T-PRF as SDDS, estimating the amount of initial drug absorption, providing individual drug explanations and comparisons in near future will help improve study outcomes. Variations in the study population, T-PRF and drug preparations may have shown different outcomes in drug release.
V. Conclusion
Within the limitations of the present study, it can be concluded that T-PRF injected with amoxiclav, MTZ and NE allow for high concentrations directly at the surgical site, reducing systemic exposure and side effects. The fibrin matrix also acts as a reservoir, providing a gradual release of these agents which function as antibiotics and immunomodulators and maintain effective levels over an extended period. By eliminating or reducing bacterial contamination, these agents can create an optimal environment for the growth factors in T-PRF to promote healing. Controlling microbial presence minimizes inflammatory responses that can hinder tissue regeneration. The results of our study open the way to a new treatment strategy that eliminates the need for systemic antibiotics and can be administered locally at the site of infection. It can also be concluded that T-PRF may be a better alternative to L-PRF as it eliminates silica contamination and can be used as a continuous release antibiotic system.
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