| ObjectiveExplore the antimicrobial activity of platelet rich plasma (PRP) against Propionibacterium acnes in vitro and evaluate its clinical curative effect on treating inflammatory acne via the animal experiments and the observation of clinical curative effect combined with fractional laser, so that lay the foundation for the clinical application of PRP.Methods1. Antimicrobial activity of PRP on Propionibacterium acnes:(1) Preparation of PRP:① Collect the whole blood samples:EDTA-K2 sterile vacuum tubes were used to take a suitable amount of venous blood from healthy subjects, and the tubes were inverted five times to ensure proper mixing with the anticoagulant avoiding blood clothing or hemolysis in the tubes. Routine blood test was measured in another tube of whole blood (1ml). ② Separate the platelet from the whole blood:Centrifugation conditions for the first time:centrifugal speed was selected as 1500 rpm/min, centrifugal time was selected as 10min. After centrifugation, the plasma (PRP) was separated by lml sterile syringes under sterile condition. ③Adjust the concentrations of platelet in PRP:PRP obtained in first centrifugal was divided into two parts according to the test results. One part of the PRP was reserved for heavy suspended platelets, the other part would be carried on second times of centrifugal and the speed and time were selected as 3000rpm/min and 20min. Under sterile condition, the supernatant was transferred into a clean tube, and it was labeled as poor platelet plasma (PPP).The remaining platelet precipitation was suspended with the indwelling PRP and sufficient mixed, platelets were counted, then adjusted the concentration of platelet to 1000 × 109/L, which will be used in the subsequent experiment(2) Preparation of drug sensitive piece of paper:Two pieces of Erythromycin Enteric-coated Tablets (0.125g) were dissolved in 100ml distilled water, blending with magnetic stirrer, removed the coat of the pills, initial erythromycin solution was autoclaved and then made into Erythromycin solution of 1.7umol/l, placed in the 4℃ refrigerator, until ready for use. Selecting the Xinhua lqualitative filter paper that absorbent strong and uniform texture, then made it into some circular paper of 6 mm diameter with the puncher, the circular paper was autoclaved before use. Under sterile condition, the filter papers with a diameter of 6 mm were immersed in the solution of PRP, PPP and erythromycin for 30 min, respectively. Thus, we got the drug sensitive pieces of PRP, PPP and erythromycin.(3) Bacteriostatic experiment of PRP on Propionibacterium acnes (ATCC6919):Propionibacterium acnes standard strains was inoculated in anaerobic brain heart infusion agar plates, then cultured in the 37℃ with 10%CO2, H2,10% and 10% N2 for 48h. Picked the propionibacterium acnes that anaerobic culture for 48 h with a disposable sterile swab and soaked in sterile saline solution, prepared as bacteria suspension. The concentration of bacteria fluid was adjusted to 0.5 McFarland standard(1.5×108 CFU/ml) by McIntosh turbidimetric instrument, until ready for use. Leaching the bacteria liquid with disposable sterile swabs and evenly coated onanaerobic brain heart infusion agar, drying 2-3 min. Drug sensitive pieces of prp, ppp and erythromycin were placed on the anaerobic brain heart infusion agar coating with bacteria respectively, then cultured in the 37℃ constant temperature and humidity incubator for 48h and observed the results.2、Therapeutic effect of PRP on the animal model of inflammatory acne:(1) Establish the animal model of rabbit ear acne:In this experiment, 26 ripe New Zealand White rabbits were divided into 2 groups.3 rabbits received no intervention as normal control group, the remaining 23 rabbits as model group.0.25 mL of 50% oleic acid was drawed into the external ear way of the rabbits by 0.5 mL syringe every day for treatment.50ul of propionibacterium acne bacteria liquid (1.5×108 CFU/ml) was injected into the ear skin lesions of the rabbits at 7 days after modeling. On the 14 day after bacteria inoculation,2 New Zealand white rabbit were selected from the normal control group and model group separately, and skin tissue specimens (full-thickness skin) were harvested from the same ear area of the rabbits for hematoxylin-eosin (HE) staining to identify the pathological changes.(2) Treatment of rabbit ear acne:21 rabbits with ear acne were divided into 3 groups for therapy. Group 1 was set as the normal saline group, applyed 0.3mL physiological saline to the inflammatory acne lesions of the rabbits’right ear; group 2 was set as treatment group,0.3mL PRP(platelet count is 1000×109/L) was applied to the inflammatory acne lesions of the rabbits’right ear; group 3 was set as positive control group of Vitamin A acid cream,0.025% vitamin A acid cream [2.8 × 10~5 g/(kg · d)] was applied to the inflammatory acne lesions of the rabbits’right ear. All the groups above were smeared medicine once a day for total 14 days.24 hours later after the last administration, skin tissue specimens (full-thickness skin) were harvested from the same administration site in the right ear of the New Zealand white rabbits from each group for the biopsy. Fixed in 4% neutral formalin, made to paraffin section and hematoxylin-eosin (HE) staining, and the tissue structure was observed under microscope.3. Clinical observation of the treatment on inflammatory acne by PRP combined with fractional laser:(1) Methods:A total of 16 patients with mild-to-moderate inflammatory acne were treated with PRP combined with the fractional laser in the Laser Plastic Surgery Center of our hospital between May,2013 and February,2014. 40ml blood were extracted in each patient by sterile anticoagulant tubes before treatment, autologous platelet rich plasma (APRP) was prepared by centrifuge. The APRP was smeared thoroughly on the treatment area after the treatment on the patients’facial acne area with lattice bait laser. The patients were requested to have a follow-up visit every four weeks to observe and record the changes of skin lesionsand and the adverse reaction over 6 months.(2) Criteria of curative effects:The curative effect was judged through eyeballing and the reduction of facial lesions. The decrease rate of skin lesions= (Number of skin lesions before the treatment-Number of skin lesions after the treatment)/Number of skin lesions before the treatment X 100%. Basically cures:The decrease rate of skin lesions≥90%; Apparent effect: The decrease rate of skin lesions was rate from 60% to 89%; effective:The decrease rate of skin lesions was rate from 20% to 59%; Invalid:The decrease rate of skin lesions less than 20% or worse. Total effective rate= cure rate +efficiency rate.Results1. Inhibitory effect of PRP on Propionibacterium acnes (ATCC6919):Platelet rich plasma is sensitive to Propionibacterium acnes, the inhibitory zone diameter in the disk diffusion test is (15.37±0.7470)mm 2. Therapeutical effect of PRP on the animal model of inflammatory acne:After treatment with oleic acid for 2 week, the skin of rabbits’ears looked like acne changes, similar to the acne of human from its appearance and histological observation. After treatment with platelet rich plasma, lesions were attenuated significantly.3. Clinical observation of the treatment on inflammatory acne by PRP combined with fractional laser:The result showed that the inflammation of patients’face caused by acne was attenuated after PRP and fractional laser treatment. Acne area of skin wound healing significantly faster than a single fractional laser treatment. In addition, combined therapy can reduce adverse effects compared to laser treatment alone, and it also avoid the risk of resistance caused by taking oral medications such as retinoic acid or antibiotics.Conclusion1. Platelet rich plasma has good antibacterial effect on acne Propionibacterium which is the main pathogenic bacteria of the acne;2. Inflammatory acne in rabbit model was operational and reproducible. PRP can reduce the abnormal follicular keratosis of inflammatory acne in rabbit model, inhibit the hyperplasia of sebaceous glands, and significantly reduce inflammatory response of dermis; 3. The inflammation of patients’face caused by acne was attenuated after PRP and fractional laser treatment.The patient with combined therapy has better prognosis compared to patients with laser treatment alone. |
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