Original basic research | Open | Published:
Preparation of platelet-rich plasma as a tissue adhesive for experimental transplantation in rabbits
Thrombosis Journalvolume 4, Article number: 18 (2006)
Platelet-rich plasma (PRP) is an autologous substance with adhesive properties. We aimed at developing and testing the efficacy of a method for PRP preparation in rabbits.
Materials and methods
An in vitro study was carried out to obtain PRP from forty rabbits and to analyze the number of platelets and type of substance needed to trigger platelet activation. To induce platelet activation, 5%, 10%, 25% and 50% CaCl solutions were used. Then, an in vivo study was performed in twelve rabbits to test PRP adhesiveness in lamellar corneal graft. A control group made up of six rabbits underwent corneal transplantation without using PRP.
5% CaCl was the most effective concentration in activating PRP, with a mean time of 19 minutes. An attached corneal flap was seen 3 months after surgery. A detached corneal button was seen in all controls.
Our method was able to produce rabbit-derived PRP with suitable properties for soft tissue adhesion. These results could be useful for researchers of the growing fields of tissue repair and experimental transplantation.
The platelet-rich plasma (PRP) is an autologous product that concentrates a high number of platelets in a small volume of plasma . This product mimics the last step of the coagulation cascade, leading to the formation of a fibrin clot, which consolidates and adheres to the application site in a short period of time. Evidencing hemostatic and healing properties, PRP is able to hold tissues or materials in a required configuration . Its biocompatible and biodegradable properties prevent the PRP from inducing foreign body reactions, tissue necrosis, or extensive fibrosis . Absorption of the fibrin clot is achieved during wound healing within weeks following application [4–6]. PRP has been used in humans in different kinds of transplant procedures such as dentistry [7–9], orthopedics , maxillofacial surgery , plastic surgery [12, 13] and ophthalmology . In addition, PRP may be considered a carrier for biological active agents [15, 16] and a healing substance causing less post-surgical pain [17, 18].
Although the preparation of PRP in humans is well known , its preparation in rabbits is more difficult due to the reduced volume of blood and the smaller size of this animal. A blood sample, large enough to prepare PRP but without being lethal to the animal, should be collected. Reports in the literature differ about the intensity and duration of blood centrifugation, the number of platelets present in the PRP, and the use of thrombin or other factors to activate PRP [1, 3, 20]. Investigations also disagree on the puncture site and the quantity of blood that should be drawn from rabbits [20–22]. Furthermore, published studies lack an in vivo test of PRP adhesiveness in rabbits, animal frequently used in experimental transplantation.
The aim of our study was to obtain an ideal PRP solution for tissue adhesiveness and to test the effectiveness of the compound on lamellar corneal grafts using an in-vivo rabbit eye model.
Materials and methods
The investigation was conducted in two steps. First, an in vitro study was performed to determine the best method for obtaining PRP from rabbits. An in vivo study was carried out then to evaluate the adhesiveness of the obtained PRP to soft tissue.
Forty adult New Zealand white rabbits weighing 3,000 grams each were used. Eighteen were female and twenty-two were male. The rabbits were treated in accordance with the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research .
In vitro study
Seventy-three blood samples from twenty-eight rabbits were collected. An 8.7 ml intracardiac blood sample was drawn under strict aseptic conditions. All animals received general anesthesia using a combination of midazolam 1 mg/kg intramuscular (Roche, Basel, Switzerland) and ketamine 70 mg/kg intramuscular (Fada, Buenos Aires, Argentina). The blood was aspirated with a 21 G needle. A 10 ml syringe preloaded with 1.3 ml of Anticoagulant Citrate Dextrose (ACD) solution was used to avoid coagulation. One millimeter was set apart for cell counting. The procedure was performed by two investigators (JF and SG).
Each blood sample was centrifuged for 15 minutes at 72 g at 4°C resulting in the three following layers: the inferior layer composed of red cells, the intermediate layer composed of white cells and the superior layer made up of plasma. The 6 ml plasma layer was centrifuged for another 5 minutes at 1006 g in order to obtain a two-part plasma: the upper part consisting of 5.5 ml of poor-platelet plasma (PPP) and the lower part consisting of 0.5 ml of platelet-rich plasma (PRP). The PPP was first aspirated to avoid its mixing up with the PRP. The PRP was then gently aspirated with another pipette and placed in a sterile tube. The PRP was thus prepared for activation by calcium chloride (CaCl), which inhibits the blood-thinning effect of ACD. After activation, PRP turned into a gel-like solution with adhesive properties and ready for use.
The coagulation time of PRP after CaCl instillation was evaluated in 250 μl samples placed in Eppendorf tubes with different CaCl concentrations. Coagulation was determined by the visualization of the clot at 20°C and carried out always by the same person (SG). The first analysis was performed using 5%, 10%, 25% and 50% CaCl concentrations, which were instilled into two PRP samples each from two different animals. A comparison was then made between 5% and 10% CaCl solutions in nine PRP samples, each obtained from five animals. Finally, a test was performed using 5% CaCl solution in 47 PRP samples from twenty one animals (table 1). The samples were observed for two hours and classified as non-coagulated if coagulation had not occurred during this time.
In vivo study
PRP preparation was performed in twelve rabbits using the same technique as in the in vitro study, and was activated by 5% CaCl to attach the corneal button as part of the autologous non-perforating corneal transplantation. Topical 5% iodopovidone was preoperatively instilled into the eye and over the eyelids, followed by topical 0.3% ciprofloxacin (Alcon, Sao Pablo, Brazil). Deep anterior keratectomy (one third of the stroma), using a 6-mm-diameter trephine, was done on right eyes under a ZEISS S5-Pro operating microscope (Zeiss C, Overckochen, Germany). The corneal button was excised according to Malbran's peeling off technique . The flap was then soaked in autologous PPP and kept at 4°C for about 3 minutes. In the meantime, PRP was activated, transformed into a gel and placed over the stromal surface. The corneal flap was finally replaced on the stroma. Corneal graft adherence was manually verified at the end of the procedure and 30 minutes later using a delicate forceps. A partial tarsorraphy using three 5-0 silk sutures was carried out closing two thirds of the palpebral fissure. Twenty-one percent oxygen was administered to all animals for an hour after awakening. Topical 0.3% ciprofloxacin (Alcon, San Pablo, Brazil) and 0.1% dexamethasone (Poen, Buenos Aires, Argentina) were applied 4 times daily up to 14 and 21 days, respectively. The sutures of the partial tarsorraphy were removed on day 2.
A control group made up of six rabbits underwent lamellar corneal graft. In this group, the corneal button was gently placed on the stroma without using PRP. A tarsorraphy was also performed and removed on day 2.
Follow-up examinations were daily performed during the first week and weekly thereafter, using an operating microscope, a slit-lamp and surgical loupes. Examinations were carried out with special attention to corneal adhesiveness and inflammatory reaction. These features were photographically documented using a Nikon Coolpix 5700 Digital Camera (Nikon, Tokyo, Japan) on day 2, 7, 30 and 90. Histopathological analyses of the specimens were done 2, 7, 30 and 90 days after surgery. Histological specimens were stained with hematoxylin and eosin, periodic acid-Shiff (PAS) and Masson trichrome, and examined with a Nikon Eclipse E800 microscope (Nikon, Tokyo, Japan).
In vitro study
All animals survived the blood extraction. Blood samples were free of clot. PRP was prepared in approximately 40 minutes. The number of platelets in PRP increased with respect to the number of platelets in the blood sample. In the PRP, the mean platelet concentration was 807.564 platelets/mm3 (range 622,000 – 1,350,000, SD 211,490) from an original vein concentration of 320,133 platelets/mm3 (range 280,000 – 408,000, SD 42,323). The resulting Platelet Enrichment Factor  was 152%. PRP treated with 25 % and 50% CaCl concentrations never coagulated whereas PRP treated with the 10 % CaCl concentration coagulated at 26 minutes. The mean coagulation time for 47 samples treated with the 5% CaCl concentration was 19 minutes (range 7 – 39, SD 7.4) (table 1).
In vivo study
Animals with PRP showed corneal flap adherence within 20 minutes. An attached graft was seen in all rabbits and corneal clarity was observed from day 30 onwards (Figure 1). Clinical and histopathological examination of the eyes revealed no signs of inflammation. A normal interface between the graft and the host was found at 90 days post-operatively, as well as an intact epithelium (Figure 2). No flap complications such as diffuse lamellar keratitis, wrinkles, displacements or rotation of the flap were observed. A detached corneal flap was seen in all control animals after removing the tarsorraphy.
We developed a method for making PRP in rabbits, successfully used in non-perforating corneal transplantation. A comparison of methods used for preparing PRP is shown in Table 2. Blood coagulation problems are more frequently seen in female than in male rabbits . This observation could influence rabbit PRP production. Anyhow, these differences do not seem to be relevant in humans . It should be noted that the blood sample in our investigation had to be collected intracardiacally because we were not able to avoid blood coagulation by using different puncture sites, such as the ear or femoral vein. There is a wide range of variation in the intensity and duration of centrifugation among research studies. Ideal platelet counts differ among researchers. The machine and software used seem to play a role since rabbit derived platelets are smaller than those of humans . Rabbits require specific software. That is why we carried out the platelet counts by direct observation. We thought that, by doing so, we would be able to get a more accurate count. Although some researchers have claimed that the best platelet concentration for producing PRP is 1 million/dl, this remains unclear. We tested the adhesiveness of PRP and can conclude that a Platelet Enrichment Factor of 152% seems to be useful at least for soft tissue adhesion.
Other tissue adhesives have also been used in lamellar keratoplasty in rabbits and humans [28, 29] and new synthetic glues like biodendrimer  and light-activated  adhesives are being tested in ophthalmology. Most of them have functional similarities but differ in origin and compounds. The major difference with PRP is that these commercial products are not autologous .
PRP is always autologous. Nevertheless, bovine thrombin has been used to activate PRP in previous studies without respecting the autologous aspect [22, 32–34]. In our investigation, a chemical substance (5% CaCl concentration), was found suitable for triggering PRP activation. This could avoid the use of heterologous blood components which can cause immune reactions, and in turn, lead to negative results that could be falsely ascribed to PRP.
Several variables should be carefully monitored during PRP preparation. Special care must be placed on blood extraction tools, machines for blood centrifugation and CaCl solutions to prevent contamination of the compound. The process must be kept sterile and precisely suited to separate platelets from red blood cells. Unless platelet sequestration is done carefully and without causing any damage, platelets will no longer be able to secrete growth factors actively. PRP failure in previous studies may have resulted from non-adherence to these recommendations .
PRP, as tissue adhesive, has different applications in medicine . It has also been used as carrier for pharmaceutical molecules, as antiangiogenic agents, and growth factors [32, 35, 36]. Gene therapy may benefit from PRP in future investigations since the presence of an adenoviral vector in the gel did not affect its properties . It is also worth mentioning the recent use of PRP as cell supporter in corneal limbal epithelial cell transplantation .
In this experimental study in rabbits, PRP as an adhesive was able to attach the corneal button in the setting of non-perforating corneal transplantation. The corneal flap, about one third of the corneal thickness, remained attached up to three months post-operatively. Our survey on PRP preparation disclosed several difficulties that are yet to be overcome. This study could be taken as a guideline for rabbit-derived PRP preparation. We hope that the comments and data herein prove useful to researchers of the growing fields of platelet cell and experimental transplantation. Further studies are currently underway to evaluate the corneal wound healing response in the presence of PRP adhesive.
Marx RE: Platelet-Rich Plasma: Evidence to Support Its Use. J Oral Maxillofac Surg 2004, 62: 489-96. 10.1016/j.joms.2003.12.003
Liu Y, Kalen A, Risto O, Wahlstrom O: Fibroblast proliferation due to exposure to a platelet concentrate in vitro is pH dependent. Wound Repair Regen 2002, 10: 336-40. 10.1046/j.1524-475X.2002.10510.x
Dijkstra-Tiekstra MJ, Van der Schoot CE, Pietersz RN, Reesink HW: White blood cell fragments in platelet concentrates prepared by the platelet-rich plasma or buffy-coat methods. Vox Sang 2005, 88: 275-7. 10.1111/j.1423-0410.2005.00634.x
Radosevich M, Goubran HI, Burnouf T: Fibrin sealant: scientific rationale, production methods, properties, and current clinical use. Vox Sang 1997, 72: 133-43. 10.1046/j.1423-0410.1997.7230133.x
Matras H: Fibrin seal: The state of the art. J Oral Maxillofac Surg 1985, 43: 605-11.
Dvorak HF, Harvey VS, Estrella P, Brown LF, McDonagh J, Dvorak AM: Fibrin containing gels induce angiogenesis. Implications for tumor stroma generation and wound healing. Vox Sang 1987, 57: 673-86.
Carlson NE, Roach RB Jr: Platelet-rich plasma: clinical applications in dentistry. J Am Dent Assoc 2002, 133: 1383-6.
Grageda E: Platelet-rich plasma and bone graft materials: a review and a standardized research protocol. Implant Dent 2004, 13: 301-9.
Oku da K, Tai H, Tanabe K, Suzuki H, Sato T, Kawase T, Saito Y, Wolff LF, Yoshiex H: Platelet-rich plasma combined with a porous hydroxyapatite graft for the treatment of intrabony periodontal defects in humans: a comparative controlled clinical study. J Periodontol 2005, 76: 890-8. 10.1902/jop.2005.76.6.890
Oyama T, Nishimoto S, Tsugawa T, Shimizu F: Efficacy of platelet-rich plasma in alveolar bone grafting. J Oral Maxillofac Surg 2004, 62: 555-8. 10.1016/j.joms.2003.08.023
Sammartino G, Tia M, Marenzi G, di Lauro AE, D'Agostino E, Claudio PP: Use of autologous platelet-rich plasma (PRP) in periodontal defect treatment after extraction of impacted mandibular third molars. J Oral Maxillofac Surg 2005, 63: 766-70. 10.1016/j.joms.2005.02.010
Man D, Plosker H, Winland-Brown JE: The use of autologous platelet-rich plasma (platelet gel) and autologous platelet-poor plasma (fibrin glue) in cosmetic surgery. Plast Reconstr Surg 2001, 107: 229-37. 10.1097/00006534-200101000-00037
Stuart JD, Kenney JG, Lettieri J, Spotnitz W, Baker J: Application of single-donor fibrin glue to burns. J Burn Care Rehabil 1988, 9: 619-22.
Duchesne B, Tahi H, Galand A: Use of human fibrin glue and amniotic membrane transplant in corneal perforation. Cornea 2001, 20: 230-2. 10.1097/00003226-200103000-00027
Yamada Y, Ueda M, Naiki T, Nagasaka T: Tissue-engineered injectable bone regeneration for osseointegrated dental implants. Clin Oral Implants Res 2004, 15: 589-97. 10.1111/j.1600-0501.2004.01038.x
Choi BH, Zhu SJ, Kim BY, Huh JY, Lee SH, Jung JH: Effect of platelet-rich plasma (PRP) concentration on the viability and proliferation of alveolar bone cells: an in vitro study. Int J Oral Maxillofac Surg 2005, 34: 420-4. 10.1016/j.ijom.2004.10.018
Pierce GF, Mustoe TA, Altrock BW, Deuel TF, Thomason A: Role of platelet-derived growth factor in wound healing. J Cell Biochem 1991, 45: 319-26. 10.1002/jcb.240450403
Simon D, Maneul S, Geetha V, Naik BR: Potential for osseous regeneration of platelet-rich plasma – a comparative study in mandibular third molar sockets. Indian J Dent Res 2004, 15: 133-6.
Yazawa M, Ogata H, Nakajima T, Mori T, Watanabe N, Handa M: Basic studies on the clinical applications of platelet-rich plasma. Cell Transplant 2003, 12: 509-18.
Efeoglu C, Akcay YD, Erturk S: A modified method for preparing platelet-rich plasma: an experimental study. J Oral Maxillofac Surg 2004, 62: 1403-7. 10.1016/j.joms.2004.06.047
Weibrich G, Hansen T, Kleis W, Buch R, Hitzler WE: Effect of platelet concentration in platelet-rich plasma on peri-implant bone regeneration. Bone 2004, 34: 665-71. 10.1016/j.bone.2003.12.010
Butterfield KJ, Bennett J, Gronowicz G, Adams D: Effect of platelet-rich plasma with autogenous bone graft for maxillary sinus augmentation in a rabbit model. J Oral Maxillofac Surg 2005, 63: 370-6. 10.1016/j.joms.2004.07.017
The Association for Research in Vision and Ophthalmology (ARVO): Statement for the Use of Animals in Ophthalmic and Visual Research.[http://www.arvo.org/eweb/dynamicpage.aspx?site=arvo2&webcode=AnimalsResearch]
Brecher G, Cronkite EP: Morphology and enumeration of human blood platelets. J Appl Physiol 1950, 3: 365-77.
Brecher G, Schneiderman M, Cronkite EP: The reproducibility and consistency of the platelet count. Am J Clin Pathol 1953, 23: 15-26.
Polack FM: Lamellar keratoplasty, Malbran's peeling off Technique. Arch Ophthalmo 1971, 86: 293-5.
Weibrich G, Kleis WK, Kunz-Kostomanolakis M, Loos AH, Wagner W: Correlation of platelet concentration in platlet-rich plasma to the extraction method, age, sex, and platlet count of the donor. Int J Oral Maxillofac Implants 2001, 16: 693-9.
Rosenthal AR, Harbury C, Egbert PR, Rubenstein E: Use of a platelet-fibrinogen-thrombin mixture as a corneal adhesive: experiments with sutureless lamellar kertaoplasty in the rabbit. Inv Ophthalmology 1975, 14: 872-5.
Kaufman HE, Insler MS, Ibrahim-Elzembely HA, Kaufman SC: Human fibrin tissue adhesive for sutureless lamellar keratoplasty and scleral patch adhesion. Ophthalmology 2003, 110: 2168-72. 10.1016/S0161-6420(03)00832-7
Velazquez AJ, Carnahan MA, Kristinsson J, Stinnett S: New Dendritic Adhesives for Sutureless Ophthalmic Surgical Procedures. Arch Ophthalmo 2004, 122: 867-70. 10.1001/archopht.122.6.867
Bloom JN, Duffy MT, Davis JB, McNally-Heintzelman KM: A light-activated surgical adhesive technique for sutureless ophthalmic surgery. Archives of Ophthalmology 2003, 121: 1591-5. 10.1001/archopht.121.11.1591
Hokugo A, Ozeki M, Kawakami O, Sugimoto K, Mushimoto K, Morita S, Tabata Y: Augmented bone regeneration activity of platelet-rich plasma by biodegradable gelatin hydrogel. Tissue Eng 2005, 11: 1224-33. 10.1089/ten.2005.11.1224
Ohya M, Yamada Y, Ozawa R, Ito K, Takahashi M, Ueda M: Sinus floor elevation applied tissue-engineered bone. Comparative study between mesenchymal stem cells/platelet-rich plasma (PRP) and autogenous bone with PRP complexes in rabbits. Clin Oral Implants Res 2005, 16: 622-9. 10.1111/j.1600-0501.2005.01136.x
Jung RE, Schmoekel HG, Zwahlen R, Kokovic V, Hammerle CH, Weber FE: Platelet-rich plasma and fibrin as delivery systems for recombinant human bone morphogenetic protein-2. Clin Oral Implants Res 2005, 16: 676-82. 10.1111/j.1600-0501.2005.01183.x
Slater M, Patava J, Kingham K: Involvement of platelets in stimulating osteogenic activity. J Orthop Res 1995, 13: 655-63. 10.1002/jor.1100130504
Landesberg R, Roy M, Gickman RS: Quantification of growth factor levels using a simplified method of platelet-rich plasma gel preparation. J Oral Maxillofac Surg 2000, 58: 297-300. 10.1016/S0278-2391(00)90058-2
Eggerman TL, Mondoro TH, Lozier JN, Vostal JG: Adenoviral vectors do not induce, inhibit, or potentiate human platelet aggregation. Hum Gene Ther 2002, 13: 125-8. 10.1089/10430340152712674
Gallo JE, Gimeno FL, Roldan F, Croxatto JO, Genovese J: The use of autologous platelet gel (platelet-rich-plasma) in corneal limbal epithelial cell transplantation. Invest Ophthalmol Vis Sci 2003, 44: E-Abstract 1354.
This work was supported by a grant from Austral University. We are grateful to Germán Ruffolo and Guillermo Gastón for their skilful technical assistance. We also thank Jorge Mancini and Guillermo Mazzolini for their collaboration.