Download PDF
Vet Comp Orthop Traumatol 2016; 29(02): 115-124
DOI: 10.3415/VCOT-15-01-0011
Original Research
Schattauer GmbH

Implications of anticoagulants and gender on cell counts and growth factor concentration in platelet-rich plasma and platelet-rich gel supernatants from rabbits

Juan C. González
1   Grupo de Investigación Terapia Regenerativa, Departamento de Salud Animal, Universidad de Caldas, Manizales, Colombia
,
Catalina López
1   Grupo de Investigación Terapia Regenerativa, Departamento de Salud Animal, Universidad de Caldas, Manizales, Colombia
,
Jorge U. Carmona
1   Grupo de Investigación Terapia Regenerativa, Departamento de Salud Animal, Universidad de Caldas, Manizales, Colombia
› Author Affiliations
Financial support: This project was supported by the grant No. 0092311 of the Vicerrectoría de Investigaciones y Postgrados of the Universidad de Caldas, Manizales, Colombia.
Further Information

Publication History

Received: 15 January 2015

Accepted: 18 January 2015

Publication Date:
19 December 2017 (online)

Also available at
   Permissions and Reprints

Summary

Objectives: Our objectives were as follows: 1) to validate a protocol for producing rabbit platelet-rich plasma (PRP); 2) to determine the influence of two anticoagulants, sodium citrate and acid-citrate-dextrose solution A, and gender on cell count in PRP and growth factor concentration in pure platelet-rich gel supernatants; 3) to correlate the variables evaluated.

Methods: Whole blood from 18 New Zealand rabbits (9 males and 9 females) was obtained with sodium citrate and acid- citrate-dextrose solution A for processing PRP fractions (A and B), which were evaluated for haematology. The PRP fractions were either activated with calcium gluconate or lysated with a detergent. The concentrations of transforming growth factor beta 1 and platelet-derived growth factor BB were assayed by ELISA.

Results: The sodium citrate PRP-B had significantly higher counts of platelets in comparison to PRP-A and whole blood obtained with the same anticoagulant and the homologous acid-citrate-dextrose solution A PRP fraction. The sodium citrate PRP-A had a significantly higher count of leukocytes compared to the homologous acid-citrate-dextrose solution A fraction. All the PRP fractions had a significant leuko-reduction when compared to whole blood. The sodium citrate PRP-A fraction from female rabbits had significantly lower platelet counts and significantly higher leukocyte counts than the same acid-citrate-dextrose solution A fraction. Growth factor concentration was not affected by the type of anticoagulant or gender.

Clinical significance: The type of anticoagulant and gender affected the cell counts in PRP, but they did not influence the growth factor concentration. More complete rabbit PRP studies should be performed before evaluating this type of substance in models of disease.

Keywords

Animal model - platelet concentrates - platelet-derived growth factor BB - rabbit - transforming growth factor beta 1 - platelet-rich plasma
 

  • References

  • 1 Eggermont LHP, Leveille SG, Shi L. et al. Pain characteristics associated with the onset of disability in older adults: The maintenance of balance, independent living, intellect, and zest in the elderly boston study. J Am Geriatr Soc 2014; 62: 1007-1016.
    CrossrefPubMedSearch in Google Scholar
  • 2 Smoljanovic T, Bojanic I, Hannafin JA. et al. Traumatic and overuse injuries among international elite junior rowers. Am J Sports Med 2009; 37: 1193-1199.
    CrossrefPubMedSearch in Google Scholar
  • 3 Davies M. Geriatric screening in first opinion practice - results from 45 dogs. J Small Anim Pract 2012; 53: 507-513.
    CrossrefPubMedSearch in Google Scholar
  • 4 Rossdale PD, Hopes R, Digby NJ. et al. Epidemiological study of wastage among racehorses 1982 and 1983. Vet Rec 1985; 116: 66-69.
    CrossrefPubMedSearch in Google Scholar
  • 5 Dyson PK, Jackson BF, Pfeiffer DU. et al. Days lost from training by two- and three-year-old Thoroughbred horses: A survey of seven UK training yards. Equine Vet J 2008; 40: 650-657.
    CrossrefPubMedSearch in Google Scholar
  • 6 Carmona JU, Prades M. Pathophisiology of osteoarthritis. Comp Equine 2009; 4: 28-40.
    PubMedSearch in Google Scholar
  • 7 Carmona JU, López C. Superficial digital flexor tendon tendinopathy and suspensory ligament desmopathy in horses: Pathophysiology and regenerative therapies. Arch Med Vet 2011; 43: 203-214.
    CrossrefPubMedSearch in Google Scholar
  • 8 Goodrich LR, Nixon AJ. Medical treatment of osteoarthritis in the horse - A review. Vet J 2006; 171: 51-69.
    CrossrefPubMedSearch in Google Scholar
  • 9 Baek SH, Kim SY. Pharmacologic treatment of osteoarthritis. J Korean Med Assoc 2013; 56: 1123-1131.
    CrossrefPubMedSearch in Google Scholar
  • 10 Habib GS, Saliba W, Nashashibi M. Local effects of intra-articular corticosteroids. Clin Rheumatol 2010; 29: 347-356.
    CrossrefPubMedSearch in Google Scholar
  • 11 Niemansburg SL, Van Delden JJM, Öner FC. et al. Ethical implications of regenerative medicine in orthopedics: An empirical study with surgeons and scientists in the field. Spine J 2014; 14: 1029-1035.
    CrossrefPubMedSearch in Google Scholar
  • 12 Smith RKW, Garvican ER, Fortier LA. The current ‘state of play' of regenerative medicine in horses: What the horse can tell the human. Reg Med 2014; 9: 673-685.
    PubMedSearch in Google Scholar
  • 13 Lynch SE. Methods for evaluation of regenerative procedures. J Periodontol 1992; 63: 1085-1092.
    CrossrefPubMedSearch in Google Scholar
  • 14 Carmona JU, López C, Sandoval JA. Review of the currently available systems to obtain platelet related products to treat equine musculoskeletal injuries. Rec Pat Reg Med 2013; 3: 148-159.
    PubMedSearch in Google Scholar
  • 15 Silva RF, Carmona JU, Rezende CMF. Intra-articular injections of autologous platelet concentrates in dogs with surgical reparation of cranial cruciate ligament rupture. Vet Comp Orthop Traumatol 2013; 26: 285-290.
    Thieme ConnectPubMedSearch in Google Scholar
  • 16 Bashir J, Sherman A, Lee H. et al. Mesenchymal stem cell therapies in the treatment of musculoskeletal diseases. PM R 2014; 6: 61-69.
    CrossrefPubMedSearch in Google Scholar
  • 17 Harmon KG, Rao AL. The use of platelet-rich plasma in the nonsurgical management of sports injuries: hype or hope?. Hematology Am Soc Hematol Educ Program 2013; 2013: 620-626.
    CrossrefPubMedSearch in Google Scholar
  • 18 Vannini F, Di Matteo B, Filardo G. et al. Platelet-rich plasma for foot and ankle pathologies: A systematic review. Foot Ankle Surg 2014; 20: 2-9.
    CrossrefPubMedSearch in Google Scholar
  • 19 Kon E, Filardo G, Di Martino A. et al. Platelet-rich plasma (PRP) to treat sports injuries: Evidence to support its use. Knee Surg Sports Traumatol Arthrosc 2011; 19: 516-527.
    CrossrefPubMedSearch in Google Scholar
  • 20 Metcalf KB, Mandelbaum BR, McIlwraith CW. Application of platelet-rich plasma to disorders of the knee joint. Cartilage 2013; 4: 295-312.
    CrossrefPubMedSearch in Google Scholar
  • 21 Marques LF, Stessuk T, Camargo ICC. et al. Platelet-rich plasma (PRP): Methodological aspects and clinical applications. Platelets 2015; 26: 101-113.
    CrossrefPubMedSearch in Google Scholar
  • 22 Nguyen RT, Borg-Stein J, McInnis K. Applications of platelet-rich plasma in musculoskeletal and sports medicine: An evidence-based approach. PM R 2011; 3: 226-250.
    CrossrefPubMedSearch in Google Scholar
  • 23 Dohan Ehrenfest DM, Bielecki T, Mishra A. et al. In search of a consensus terminology in the field of platelet concentrates for surgical use: platelet-rich plasma (PRP), platelet-rich fibrin (PRF), fibrin gel polymerization and leukocytes. Curr Pharm Biotechnol 2012; 13: 1131-1137.
    CrossrefPubMedSearch in Google Scholar
  • 24 Dohan Ehrenfest DM, Andia I, Zumstein MA. et al. Classification of platelet concentrates (Platelet-Rich Plasma-PRP, Platelet-Rich Fibrin-PRF) for topical and infiltrative use in orthopedic and sports medicine: current consensus, clinical implications and perspectives. Muscles Ligaments Tendons J 2014; 4: 3-9.
    PubMedSearch in Google Scholar
  • 25 DeLong JM, Russell RP, Mazzocca AD. Platelet-rich plasma: the PAW classification system. Arthroscopy 2012; 28: 998-1009.
    CrossrefPubMedSearch in Google Scholar
  • 26 Mishra A, Harmon K, Woodall J. et al. Sports medicine applications of platelet rich plasma. Curr Pharm Biotechnol 2012; 13: 1185-1195.
    CrossrefPubMedSearch in Google Scholar
  • 27 Krueger CA, Wenke JC, Masini BD. et al. Characteristics and impact of animal models used for sports medicine research. Orthopedics 2012; 35: e1410-e1415.
    CrossrefPubMedSearch in Google Scholar
  • 28 Bõsze Z, Houdebine LM. Application of rabbits in biomedical research: A review. World Rabbit Sci 2006; 14: 1-14.
    PubMedSearch in Google Scholar
  • 29 Huh SW, Shetty AA, Kim SJ. et al. The effect of platelet rich plasma combined with microfracture for the treatment of chondral defect in a rabbit knee. Tissue Eng Reg Med 2014; 11: 178-185.
    PubMedSearch in Google Scholar
  • 30 Chung SW, Song BW, Kim YH. et al. Effect of platelet-rich plasma and porcine dermal collagen graft augmentation for rotator cuff healing in a rabbit model. Am J Sports Med 2013; 41: 2909-2918.
    CrossrefPubMedSearch in Google Scholar
  • 31 Kollitz KM, Parsons EM, Weaver MS. et al. Platelet-rich plasma for zone II flexor tendon repair. Hand 2014; 9: 217-224.
    CrossrefPubMedSearch in Google Scholar
  • 32 Lane JG, Healey RM, Chase DC. et al. Use of platelet-rich plasma to enhance tendon function and cellularity. Am J Orthop 2013; 42: 209-214.
    PubMedSearch in Google Scholar
  • 33 Deng D, Wang W, Wang B. et al. Repair of Achilles tendon defect with autologous ASCs engineered tendon in a rabbit model. Biomaterials 2014; 35: 8801-8809.
    CrossrefPubMedSearch in Google Scholar
  • 34 Kanazawa T, Soejima T, Noguchi K. et al. Tendon-to-bone healing using autologous bone marrow-derived mesenchymal stem cells in ACL reconstruction without a tibial bone tunnel -A histological study. Muscles Ligaments Tendons J 2014; 4: 201-206.
    PubMedSearch in Google Scholar
  • 35 González JC, López C, Carmona JU. Evaluation of a manual method for producing pure-platelet rich plasma (P-PRP) in rabbits: Hematological study. Arch Med Vet 2013; 45: 267-272.
    CrossrefPubMedSearch in Google Scholar
  • 36 Burt DW, Paton IR. Evolutionary origins of the transforming growth factor-β gene family. DNA Cell Biol 1992; 11: 497-510.
    CrossrefPubMedSearch in Google Scholar
  • 37 Kazakos K, Lyras DN, Thomaidis V. et al. Application of PRP gel alone or in combination with guided bone regeneration does not enhance bone healing process: An experimental study in rabbits. J Craniomaxillofac Surg 2011; 39: 49-53.
    CrossrefPubMedSearch in Google Scholar
  • 38 Yoshioka T, Kanamori A, Washio T. et al. The effects of plasma rich in growth factors (PRGF-Endoret) on healing of medial collateral ligament of the knee. Knee Surg Sports Traumatol Arthrosc 2013; 21: 1763-1769.
    CrossrefPubMedSearch in Google Scholar
  • 39 Woo SH, Jeong HS, Kim JP. et al. Favorable vocal fold wound healing induced by platelet-rich plasma injection. Clin Exp Otorhinolaryngol 2014; 7: 47-52.
    CrossrefPubMedSearch in Google Scholar
  • 40 Jeon YR, Kang EH, Yang CE. et al. The effect of platelet-rich plasma on composite graft survival. Plast Reconstr Surg 2014; 134: 239-246.
    CrossrefPubMedSearch in Google Scholar
  • 41 Ishida K, Kuroda R, Miwa M. et al. The regenerative effects of platelet-rich plasma on meniscal cells in vitro and its in vivo application with biodegradable gelatin hydrogel. Tissue Eng 2007; 13: 1103-1112.
    CrossrefPubMedSearch in Google Scholar
  • 42 Sun Y, Feng Y, Zhang CQ. et al. The regenerative effect of platelet-rich plasma on healing in large osteochondral defects. Int Orthop 2010; 34: 589-597.
    CrossrefPubMedSearch in Google Scholar
  • 43 Lee AJ, Chung WH, Kim DH. et al. Anterior cruciate ligament reconstruction in a rabbit model using canine small intestinal submucosa and autologous platelet-rich plasma. J Surg Res 2012; 178: 206-215.
    CrossrefPubMedSearch in Google Scholar
  • 44 McCarrel TM, Minas T, Fortier LA. Optimization of leukocyte concentration in platelet-rich plasma for the treatment of tendinopathy. J Bone Joint Sur Am 2012; 94: e143-1-8.
    PubMedSearch in Google Scholar
  • 45 Kisiday JD, McIlwraith CW, Rodkey WG. et al. Effects of platelet-rich plasma composition on anabolic and catabolic activities in equine cartilage and meniscal explants. Cartilage 2012; 3: 245-254.
    CrossrefPubMedSearch in Google Scholar
  • 46 Boswell SG, Schnabel LV, Mohammed HO. et al. Increasing platelet concentrations in leukocyte-reduced platelet-rich plasma decrease collagen gene synthesis in tendons. Am J Sports Med 2014; 42: 42-49.
    CrossrefPubMedSearch in Google Scholar
  • 47 Giraldo CE, López C, Álvarez ME. et al. Effects of the breed, sex and age on cellular content and growth factor release from equine pure-platelet rich plasma and pure-platelet rich gel. BMC Vet Res 2013; 9.
    PubMedSearch in Google Scholar
  • 48 Adamou D, Laiou E. Utility of platelet indices in clinical practice. Acta Microbiol Hell 2011; 56: 5-15.
    PubMedSearch in Google Scholar
  • 49 Kingston JK, Bayly WM, Sellon DC. et al. Effects of sodium citrate, low molecular weight heparin, and prostaglandin E1 on aggregation, fibrinogen binding, and enumeration of equine platelets. Am J Vet Res 2001; 62: 547-554.
    CrossrefPubMedSearch in Google Scholar
  • 50 Macey M, Azam U, McCarthy D. et al. Evaluation of the anticoagulants EDTA and citrate, theophylline, adenosine, and dipyridamole (CTAD) for assessing platelet activation on the ADVIA 120 hematology system. Clin Chem 2002; 48: 891-899.
    PubMedSearch in Google Scholar
  • 51 Argüelles D, Carmona JU, Pastor J. et al. Evaluation of single and double centrifugation tube methods for concentrating equine platelets. Res Vet Sci 2006; 81: 237-245.
    CrossrefPubMedSearch in Google Scholar
  • 52 Wen D, He W, Wang H. et al. Effect of Sheshang capsule on platelet function of rabbits bitten by Trimeresurus stejnegeri and its mechanism. Zhonghua Wei Zhong Bing Ji Jiu Yi Xue 2014; 26: 585-588.
    PubMedSearch in Google Scholar
  • 53 Lei H, Gui L, Xiao R. The effect of anticoagulants on the quality and biological efficacy of platelet-rich plasma. Clin Biochem 2009; 42: 1452-1460.
    CrossrefPubMedSearch in Google Scholar
  • 54 Giraldo CE, Álvarez ME, Carmona JU. Effects of sodium citrate and acid citrate dextrose solutions on cell counts and growth factor release from equine pure-platelet rich plasma and pure-platelet rich gel. BMC Vet Res 2015; 11: 60.
    CrossrefPubMedSearch in Google Scholar
  • 55 Silva RF, Álvarez ME, Ríos DL. et al. Evaluation of the effect of calcium gluconate and bovine thrombin on the temporal release of transforming growth factor beta 1 and platelet-derived growth factor isoform BB from feline platelet concentrates. BMC Vet Res 2012; 8: 212.
    CrossrefPubMedSearch in Google Scholar
  • 56 Silva RF, Carmona JU, Rezende CMF. Comparison of the effect of calcium gluconate and batroxobin on the release of transforming growth factor beta 1 in canine platelet concentrates. BMC Vet Res 2012; 8: 121.
    CrossrefPubMedSearch in Google Scholar
  • 57 Masago H, Shibuya Y, Munemoto S. et al. Alveolar ridge augmentation using various bone substitutes -A web form of titanium fibers promotes rapid bone development. Kobe J Med Sci 2007; 53: 257-263.
    PubMedSearch in Google Scholar
  • 58 Findikcioglu K, Findikcioglu F, Yavuzer R. et al. Effect of platelet-rich plasma and fibrin glue on healing of critical-size calvarial bone defects. J Craniofac Surg 2009; 20: 34-40.
    CrossrefPubMedSearch in Google Scholar
  • 59 Wei HY, Ding ZN, Shi HW. et al. Qualitative analysis of platelet rich plasma prepared by acute plateletpheresis in patients undergoing heart surgery. Zhongguo Shi Yan Xue Ye Xue Za Zhi 2014; 2: 521-524.
    PubMedSearch in Google Scholar