New Protocol to Improve the Antibacterial Activity of Platelet Rich Fibrin – In Vitro Study

Background: Plasma concentrate products have Rapidly developed during the last few years, and it had signicantly affected regenerative medicine because of its specic features which enhance healing and reduce pain after surgeries. this research aims to create an antibiotic releasing biomaterial based on the PRF. Methods: In-Vitro experimental study was conducted, 12 Healthy volunteers were included, each donated (40 ml) of blood. Chukrun’s protocol was used to prepare PRF (3000 rpm, 10m) with additional modication. Bacterial colonies and Miller-Hinton medium was prepared. Two types of bacteria were used for the culturing and incubation (Staphylococcus aureus, Enterococcus faecalis). Three Pharmaceutical forms (PF) of the antibacterial agents (Lincomycin) were incorporated with the PRF. Results: pharmaceutical form had a signicant difference on the antibacterial activity of the PRF (P=0.000), ampoule and solution forms had the highest levels of bacterial growth inhibiting, however, powder failed to inhibit bacterial growth. Moreover, no signicant difference was noticed between using 0.5ml or 1ml dose of antibiotic (P=339). Conclusion: It is possible to incorporate antibiotic with the PRF, which will improve the antibacterial activity of PRF, creating an ARB that can be used locally adjacent to minor surgery.

after surgeries (15), injectable platelet rich brin (I-PRF) had an effect on bone regeneration (16), (L-PRF) and advanced platelet rich brin (A-PRF) have a lot of application in dentistry and especially in periodontal tissue regeneration (17).
More recently, lower speeds of centrifugation are being used to create Liquid-PRF which primary composes of brinogen and thrombin that has 10-15 minutes before it converts to brin (18), and therefore, Liquid-PRF can be used as a delivery system for many types of biomolecules that can be used in regenerative medicine as Zhang proposed in 2018 (19).
Lately, attempts have been made to improve the antimicrobial activity of PRF, a recent study tested the ability of using PRF as a local antibiotic releasing device by incorporating antibiotic agent with PRF (20), Likewise, Khurshid et al added silver nanoparticles (SNP) to L-PRF to improve the antibacterial activity, and the study found that this modi cation improved both mechanical properties and antibacterial activity of the L-PRF (21), however, new strategies and combination approaches are still needed to develop an ideal local antibiotic delivery system (19).
Hence, developing advanced and improved forms of platelet concentrate products are still the main focus of biomedical research, in order to enhance tissue regeneration after surgeries and to reduce its complications, especially infection which remain the main complication after surgeries (22), therefore, in this research we aim to develop and introduce a protocol for production of antibiotic releasing biomaterial based on PRF scaffold.

Aim Of The Research:
1-To improve the antibacterial activity of PRF by adding antibiotic agents to its structure.
2-To study the effect of the pharmaceutical form and volume (dose) of the antibiotic agent on the PRF antibacterial activity.
3-To propose a uni ed protocol for the preparation of the improved PRF creating an antibiotic releasing biomaterial (ARB).

Materials And Methods:
Study design: In-vitro experimental study was conducted in the laboratory of Biology -Faculty of Science -Damascus University -Syria.

Ethical approvals:
Ethical approvals were obtained from higher committee for medical research at Damascus University and from of scienti c research committee at the faculty of Dental Medicine -Damascus university (N: 1059 t/m date: 15-12-2019), All methods were carried out in accordance with relevant guidelines and regulations.
Informed consent was obtained from all participants before participating in the research. Sample: 12 Healthy volunteers were included, each donated (40 ml) of blood, age ranged between 24-32, research procedure and expected results were explained to each volunteer, and written consent form were collected. Each patient must have been Healthy volunteers with no systematic or blood diseases, nonsmokers, and did not take any kind of antibiotics during the last month.
Research procedure: Different protocols have been found for the preparation of PRF, each protocol used different speed, relative G force and time (23). A pilot study has been conducted to test the physical properties of the PRF produced with each different protocol. Length, consistency and volume of the PRF have been assessed and the best results were noticed when using Chukrun's original protocol (24). Moreover, a small modi cation has been done to the original protocol which showed a signi cantly better results when forming ARB.
Three Pharmaceutical forms were incorporated with the fresh blood, Ampoule: Lincomycin 600 mg (0.5 ml, or 1.0 ml). Solution: one capsule of lincomycin 600 mg was added to 10 ml of saline, and two volume of solution was then added to the PRF (0.5 ml, or 1.0 ml). Powder: lincomycin 600 mg (capsule) was used, 100 and 50 mg was measured using electronic scale and added to PRF.
Measurements were made on 4-time intervals: T1: after 24 hours (one day), T2: after 48 hours (two days), T3: after 120 hours ( ve days), T4: after 240 hours (ten days). preparation protocol: The nal protocol was as the following: 1. 40 ml of blood were collected from the volunteers 2. The blood samples were divided into 4 (10 ml) plastic tubes without anticoagulant, plastic tubes can help to delay the PRF formation, therefore, it can be easily isolate the middle layer with the antibacterial agent from the tube in a liquid form (25). Each tube represents a group of pharmaceutical form of antibiotic (group A: ampoule form, B: powder, C: solution, D: control).
3. The tubes were immediately centrifuged at 3000 rpm (400 g), for 10 minutes. (Hettich EBA 200 Centrifuge, Germany) 4. Three layers are formed, erythrocytes concentrated in the lower part, brin enriched with platelets in the middle, and serum in the upper part. Immediately, the middle third from each tube was transported using a 5 ml plastic syringe to a 5 ml glass tube with a minimum time interval between each step, glass tubes can enhance the clot formation by the interaction between the silica in glass and the palates in order to create brin clot (26).
5. Fibrin clot is nally formed into the glass tube, and then it is ready to be cultured in the Miller-Hinton plates, no need to separate the PRF clot from the RBC layer with this protocol.

Preparation of bacterial colonies:
The bacteria used in this study (Staphylococcus aureus, Enterococcus faecalis), were isolated from Damascus pediatric hospital and incubated at 37ºC for 24 hours. The density of the bacterial colonies was set to 0.5 MacFarland. Mueller-Hinton agar (Himedia-India) was prepared for the culturing and incubation.
Both bacteria used in this study are Gram-positive bacterium (27) (28), and can be found in healthy humans, but can also cause life-threatening infections and diseases (29,30).
The antibiotic agent used in this research is Lincomycin, which is a Streptomyces Lincolnensis antibiotic (31). Lincomycin is a narrow-spectrum antibiotic with activity against Gram-positive bacteria including pathogenic types of Streptococcus, Staphylococcus and Mycoplasma (32).  Results: Staphylococcus: The effect of Pharmaceutical Form on inhibiting Staphylococcus growth in culture: Three forms of antibiotic agent (Lincomycin) were incorporated with the PRF,  Moreover, independent samples T-test was used to compare between the two dose, no signi cant difference was noticed in ampoule group, solution form seems to be more effective in 0.5 ml dose, with one exception in T4 (0.5 = 21.38, 1 ml = 25.5), and signi cant differences were found in T1, T4 (0.015, 0.000).   ANOVA and post hoc test results are represented in (Table N. 4). No signi cant differences were noticed between 0.5-1 ml dose of the ampoule form. Discussion: Clinicians have been using PRF and other platelet concentration products with the regenerative procedure in order to enhance the healing and to reduce the postoperative complications (34), however, studies have shown that PRF have only mild antibacterial activity against some bacterial agents like S. aureus, and it is not affective against resistance bacteria like F. Nucleatum (35,36). Moreover, the most frequent postoperative complication in minor surgery is infection due to the exposure of the ap (or membrane) and the bacterial colonization of the wound (22,37).
Therefore, this research aimed to improve the antibacterial activity of PRF by adding different forms of antibiotics and developing a protocol for the preparation of antibiotic releasing biomaterial (ARB) based on platelet rich brin (PRF), which will help to overcome the infection after minor surgery procedure.
Local ARB can be effective where infection is predictable, and can reduce the use of antibiotic especially in the eld of dental surgeries (38), and also, eliminating the side effect of using systematic antibiotic (cell toxicity, and antibiotic resistance) (39).
Several studies tried to improve the PRF, one study added silver nanoparticles (SNP) which created SNP modi ed PRF, and this study concluded that SNP modi ed PRF had an improved mechanical properties and higher antimicrobial activity (21), other study evaluated release kinetics of different antibiotic from PRF, the results showed that PRF could release antibiotics for a week (40).
The two bacteria chosen in this research are S. aureus, and E. faecalis because of their well-known roll to cause infection (41,42), also, they are a major cause of hospital wound infection (43), both bacteria were isolated from pediatric blood infection from Damascus children Hospital.
Three forms of antibiotic agent were used in this research, and the research showed that adding ampoule or solution form can signi cantly increase the antibiotic activity of PRF, compared with normal PRF (control), and ARB could release antibiotic for 10 days. on the other hand, powder form failed to improve PRF, this might be because antibiotic in powder form changed the osmotic pressure of the blood resulting in cells desolation. No signi cant difference between ampoule and solution form was noticed, however, ampoule had higher results, this can be explained because ampoule is already prepared for IV, IM injection while the solution concentration may have been diluted in the serum. No previous study had studied the effect of pharmaceutical form on the antibiotic release of PRF. 0.5 ml and 1 ml volume had similar results, however, 0.5 ml had a slightly higher antibacterial activity, this result agrees with other study, because in the protocol used in the previous research, PRF has failed to form in 1 ml volume samples (20).
The antibiotic release in our study lasted for 10 days, other study recorded that the bacterial growth inhabitation lasted between 4-7 days (20,40).
The concept of antibiotic releasing biomaterial (ARB) introduced in this research might have an important future clinical relevance with general practice, because infection of the surgical site remain the main complication after minor surgery (22). The use of ARB can have an important role in reducing infection which interfere with second intention healing after dermatologic surgery (44,45) or after open fracture (46), and will be of great interest in oral surgeries and treating of periodontal diseases and overall eliminating surgical site infection by gradual local releasing of antibiotic (47).
We recommend further research of the protocol proposed in this study using different antibiotic agents and bacteria in order to prepare this concept for the clinical application.

Conclusion:
Within the limits of this study, it can concluded that the proposed protocol in this study has succeeded to improve the antibacterial properties of PRF, creating an antibiotic releasing biomaterial (ARB) that can be used widely after minor surgery, where infection is very predictable and highly affect the survival rate of the surgery. The use of ARB can signi cantly reduce the use of systematic antibiotics which will directly reduce the complications of using systematic antibiotics, and indirectly affect the worldwide antibiotic resistance crisis. More clinical and animal research is required to verify the healing and antimicrobial properties of the ARB.

Declarations
Ethics approval and consent to participate: The research ethical approval reference number is 135\S -data: 15-12-2019, and it was obtained from the scienti c committee at the faculty of dental medicine, and the ethical committee at Damascus university Consent for publication Not applicable.

Availability of data and material
All necessary data are presented within the manuscript. All other materials and data are available upon request. For any more details regarding the data of this research please contact the corresponded author. The effect of PF* on inhibiting Staphylococcus growth in culture The effect of PF on inhibiting Enterococcus growth in culture