Optimal activation methods for maximizing the concentrations of platelet-derived growth factor-BB and transforming growth factor-β1 in equine platelet-rich plasma.
Abstract: Platelet-rich plasma (PRP) therapy has been widely applied in various medical fields including humans and horses. This study aimed to establish an optimal activation method to stably and reproducibly maximize the concentrations of platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β1 (TGF-β1) contained in equine PRP. Autologous PRP was prepared from 11 Thoroughbreds. For the activation test, PRP was activated by either a single freeze-thaw cycle (Fr) or adding calcium and autologous serum containing thrombin (Ca). PDGF-BB and TGF-β1 concentrations in Fr, Ca, nonactivated (No), and platelet-poor plasma (PPP) samples were determined using ELISA and compared. For repetitive freeze-thaw test, PRP was subjected to single (Fr1), double (Fr2), triple (Fr3), or quadruple (Fr4) freeze-thaw cycles and the concentrations of both growth factors in samples were compared similarly. The PDGF-BB concentration in Ca was significantly higher than that in other preparations. The TGF-β1 concentrations in Fr and Ca were significantly higher than those in PPP and No, with no significant differences between Fr and Ca. The concentrations of both factors were significantly increased in PRP treated with multiple cycles of freeze-thaw compared with that in PRP treated with a single cycle. No significant differences were noted among Fr2, Fr3, and Fr4. Our findings suggest that activation by adding calcium and autologous serum is optimal for instant use of PRP and that double freeze-thawing is an easier and optimal activation method for cryopreserved PRP.
Publication Date: 2020-08-19 PubMed ID: 32814750PubMed Central: PMC7653321DOI: 10.1292/jvms.20-0167Google Scholar: Lookup
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- Journal Article
Summary
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The research studies the most effective methods to activate the platelet-derived growth factor-BB (PDGF-BB) and transforming growth factor-β1 (TGF-β1) in equine platelet-rich plasma (PRP). The researchers found that the addition of calcium and autologous serum as well as a double freeze-thawing process are the best activation methods.
Methods
- The researchers procured autologous PRP from 11 Thoroughbred horses.
- Two activation methods were compared: a single freeze-thaw cycle (Fr) or the addition of calcium and autologous serum that contains thrombin (Ca).
- Concentrations of PDGF-BB and TGF-β1 in activated (Fr and Ca) and non-activated (No) PRP, as well as platelet-poor plasma (PPP), were measured with ELISA.
- A repetitive freeze-thaw test was also conducted, wherein the PRP underwent single (Fr1), double (Fr2), triple (Fr3), or quadruple (Fr4) freeze-thaw cycles.
Findings
- The researchers found that the concentration of PDGF-BB was significantly higher in PRP activated with calcium and autologous serum (Ca) compared to the other preparations.
- TGF-β1 concentrations in PRP activated by a single freeze-thaw cycle (Fr) or by calcium and autologous serum (Ca) were significantly higher compared to platelet-poor plasma (PPP) and non-activated (No) samples.
- No significant differences were found between the concentrations of TGF-β1 in the single freeze-thaw (Fr) and calcium and autologous serum (Ca) activations.
- Multiple cycles of freeze-thaw significantly increased concentrations of both growth factors compared to a single freeze-thaw cycle.
- Trials showed no significant differences between the concentrations of growth factors in double, triple, and quadruple freeze-thaw cycles.
Suggested Activation Methods
- The results suggest that to maximize the concentration of growth factors, particularly PDGF-BB and TGF-β1, in PRP for immediate use, it is optimal to add calcium and autologous serum.
- On the other hand, PRP that is planned to be cryopreserved (frozen for later use) should undergo a double freeze-thaw cycle for maximal optimization.
Cite This Article
APA
Fukuda K, Kuroda T, Tamura N, Mita H, Kasashima Y.
(2020).
Optimal activation methods for maximizing the concentrations of platelet-derived growth factor-BB and transforming growth factor-β1 in equine platelet-rich plasma.
J Vet Med Sci, 82(10), 1472-1479.
https://doi.org/10.1292/jvms.20-0167 Publication
Researcher Affiliations
- Clinical Veterinary Medicine Division, Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Shimotsuke-shi, Tochigi 329-0412, Japan.
- Clinical Veterinary Medicine Division, Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Shimotsuke-shi, Tochigi 329-0412, Japan.
- Clinical Veterinary Medicine Division, Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Shimotsuke-shi, Tochigi 329-0412, Japan.
- Clinical Veterinary Medicine Division, Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Shimotsuke-shi, Tochigi 329-0412, Japan.
- The Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Shimotsuke-shi, Tochigi 329-0412, Japan.
MeSH Terms
- Animals
- Becaplermin
- Horses
- Platelet-Rich Plasma
- Thrombin
- Transforming Growth Factor beta1
- Transforming Growth Factors
References
This article includes 35 references
- Alio JL, Rodriguez AE, Abdelghany AA, Oliveira RF. Autologous platelet-rich plasma eye drops for the treatment of post-LASIK chronic ocular surface syndrome. J. Ophthalmol. 2017: 2457620.
- Barrientos S, Stojadinovic O, Golinko MS, Brem H, Tomic-Canic M. Growth factors and cytokines in wound healing. Wound Repair Regen. 16: 585–601.
- Bosch G, van Schie HT, de Groot MW, Cadby JA, van de Lest CH, Barneveld A, van Weeren PR. Effects of platelet-rich plasma on the quality of repair of mechanically induced core lesions in equine superficial digital flexor tendons: A placebo-controlled experimental study. J. Orthop. Res. 28: 211–217.
- Brossi PM, Moreira JJ, Machado TS, Baccarin RY. Platelet-rich plasma in orthopedic therapy: a comparative systematic review of clinical and experimental data in equine and human musculoskeletal lesions. BMC Vet. Res. 11: 98.
- DeRossi R, Coelho AC, Mello GS, Frazílio FO, Leal CR, Facco GG, Brum KB. Effects of platelet-rich plasma gel on skin healing in surgical wound in horses. Acta Cir. Bras. 24: 276–281.
- Dhurat R, Sukesh M. Principles and methods of preparation of platelet-rich plasma: A review and author’s perspective. J. Cutan. Aesthet. Surg. 7: 189–197.
- Everts PA, Knape JT, Weibrich G, Schönberger JP, Hoffmann J, Overdevest EP, Box HA, van Zundert A. Platelet-rich plasma and platelet gel: a review. J. Extra Corpor. Technol. 38: 174–187.
- Fukuda K, Miyata H, Kuwano A, Kuroda T, Tamura N, Kotoyori Y, Kasashima Y. Does the injection of platelet-rich plasma induce changes in the gene expression and morphology of intact Thoroughbred skeletal muscle?. J. Equine Sci. 28: 31–39.
- Giles BM, Underwood TT, Benhadji KA, Nelson DK, Grobeck LM, Lin B, Wang S, Fill JA, Man M, Pitts KR, Bamberg A. Analytical characterization of an enzyme-linked immunosorbent assay for the measurement of transforming growth factor β1 in human plasma. J. Appl. Lab. Med. 3: 200–212.
- Hammond JW, Hinton RY, Curl LA, Muriel JM, Lovering RM. Use of autologous platelet-rich plasma to treat muscle strain injuries. Am. J. Sports Med. 37: 1135–1142.
- Hauschild G, Geburek F, Gosheger G, Eveslage M, Serrano D, Streitbürger A, Johannlükens S, Menzel D, Mischke R. Short term storage stability at room temperature of two different platelet-rich plasma preparations from equine donors and potential impact on growth factor concentrations. BMC Vet. Res. 13: 7.
- Hsu WK, Mishra A, Rodeo SR, Fu F, Terry MA, Randelli P, Canale ST, Kelly FB. Platelet-rich plasma in orthopaedic applications: evidence-based recommendations for treatment. J. Am. Acad. Orthop. Surg. 21: 739–748.
- Iacopetti I, Perazzi A, Ferrari V, Busetto R. Application of platelet-rich gel to enhance wound healing in the horse: A case report. J. Equine Vet. Sci. 32: 123–128.
- Ito Y, Nakachi K, Imai K, Hashimoto S, Watanabe Y, Inaba Y, Tamakoshi A, Yoshimura T, JACC Study Group. Stability of frozen serum levels of insulin-like growth factor-I, insulin-like growth factor-II, insulin-like growth factor binding protein-3, transforming growth factor beta, soluble Fas, and superoxide dismutase activity for the JACC study. J. Epidemiol. 15Suppl 1: S67–S73.
- Jee CH, Eom NY, Jang HM, Jung HW, Choi ES, Won JH, Hong IH, Kang BT, Jeong DW, Jung DI. Effect of autologous platelet-rich plasma application on cutaneous wound healing in dogs. J. Vet. Sci. 17: 79–87.
- Kim JH, Park C, Park HM. Curative effect of autologous platelet-rich plasma on a large cutaneous lesion in a dog. Vet. Dermatol. 20: 123–126.
- Kushida S, Kakudo N, Morimoto N, Hara T, Ogawa T, Mitsui T, Kusumoto K. Platelet and growth factor concentrations in activated platelet-rich plasma: a comparison of seven commercial separation systems. J. Artif. Organs 17: 186–192.
- Lana JFSD, Purita J, Paulus C, Huber SC, Rodrigues BL, Rodrigues AA, Santana MH, Madureira JL, Jr, Malheiros Luzo ÂC, Belangero WD, Annichino-Bizzacchi JM. Contributions for classification of platelet rich plasma—proposal of a new classification: MARSPILL. Regen. Med. 12: 565–574.
- Magalon J, Chateau AL, Bertrand B, Louis ML, Silvestre A, Giraudo L, Veran J, Sabatier F. DEPA classification: a proposal for standardising PRP use and a retrospective application of available devices. BMJ Open Sport Exerc. Med. 2: e000060.
- Marx RE. Platelet-rich plasma: evidence to support its use. J. Oral Maxillofac. Surg. 62: 489–496.
- Marx RE, Carlson ER, Eichstaedt RM, Schimmele SR, Strauss JE, Georgeff KR. Platelet-rich plasma: Growth factor enhancement for bone grafts. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 85: 638–646.
- McClain AK, McCarrel TM. The effect of four different freezing conditions and time in frozen storage on the concentration of commonly measured growth factors and enzymes in equine platelet-rich plasma over six months. BMC Vet. Res. 15: 292.
- Mei-Dan O, Lippi G, Sánchez M, Andia I, Maffulli N. Autologous platelet-rich plasma: a revolution in soft tissue sports injury management?. Phys. Sportsmed. 38: 127–135.
- Navani A, Li G, Chrystal J. Platelet rich plasma in musculoskeletal pathology: A necessary rescue or a lost cause?. Pain Physician 20: E345–E356.
- Sano K, Takai Y, Yamanishi J, Nishizuka Y. A role of calcium-activated phospholipid-dependent protein kinase in human platelet activation. Comparison of thrombin and collagen actions. J. Biol. Chem. 258: 2010–2013.
- Strandberg G, Sellberg F, Sommar P, Ronaghi M, Lubenow N, Knutson F, Berglund D. Standardizing the freeze-thaw preparation of growth factors from platelet lysate. Transfusion 57: 1058–1065.
- Sundman EA, Cole BJ, Fortier LA. Growth factor and catabolic cytokine concentrations are influenced by the cellular composition of platelet-rich plasma. Am. J. Sports Med. 39: 2135–2140.
- Sutter WW, Kaneps AJ, Bertone AL. Comparison of hematologic values and transforming growth factor-β and insulin-like growth factor concentrations in platelet concentrates obtained by use of buffy coat and apheresis methods from equine blood. Am. J. Vet. Res. 65: 924–930.
- Tambella AM, Martin S, Cantalamessa A, Serri E, Attili AR. Platelet-rich plasma and other hemocomponents in veterinary regenerative medicine. Wounds 30: 329–336.
- Textor JA, Tablin F. Activation of equine platelet-rich plasma: comparison of methods and characterization of equine autologous thrombin. Vet. Surg. 41: 784–794.
- Textor JA, Norris JW, Tablin F. Effects of preparation method, shear force, and exposure to collagen on release of growth factors from equine platelet-rich plasma. Am. J. Vet. Res. 72: 271–278.
- Waselau M, Sutter WW, Genovese RL, Bertone AL. Intralesional injection of platelet-rich plasma followed by controlled exercise for treatment of midbody suspensory ligament desmitis in Standardbred racehorses. J. Am. Vet. Med. Assoc. 232: 1515–1520.
- Xie X, Zhao S, Wu H, Xie G, Huangfu X, He Y, Zhao J. Platelet-rich plasma enhances autograft revascularization and reinnervation in a dog model of anterior cruciate ligament reconstruction. J. Surg. Res. 183: 214–222.
- Yin W, Xu H, Sheng J, Zhu Z, Jin D, Hsu P, Xie X, Zhang C. Optimization of pure platelet-rich plasma preparation: A comparative study of pure platelet-rich plasma obtained using different centrifugal conditions in a single-donor model. Exp. Ther. Med. 14: 2060–2070.
- Zimmermann R, Arnold D, Strasser E, Ringwald J, Schlegel A, Wiltfang J, Eckstein R. Sample preparation technique and white cell content influence the detectable levels of growth factors in platelet concentrates. Vox Sang. 85: 283–289.
Citations
This article has been cited 8 times.- Seidel SRT, Fülber J, Barbosa ÂP, Penatti NMA, Demasi M, Baccarin RYA. Medium-term storage of platelet-derived orthobiologics: a feasible alternative for equine practice. Front Vet Sci 2025;12:1720164.
- Carmona JU, López C, Argüelles D. Addressing Heterogeneity in Equine PRP Therapies: A Scoping Review of Methods, Evidence, and Commercial Validation. Animals (Basel) 2025 Dec 13;15(24).
- Yamate T, Katagiri S. Effect of intrauterine administration of platelet-rich plasma two days before timed artificial insemination in dairy cows on endometrial thickness and conception rate. J Vet Med Sci 2026 Feb 1;88(2):272-279.
- Zhang JM, Ying L, Ying LW, Ye YZ, Yang Y, Zhou XB, Zhang QG. Arthroscopic PRP gel augmentation at the tendon-bone interface accelerates functional recovery and structural healing in rotator cuff repair: a retrospective cohort study. BMC Musculoskelet Disord 2025 Dec 3;27(1):12.
- Karimi F, Kafami M, Khakestar FG, Madahali MH, Nazemi S, Karimi S. Protective effect of exosomes derived from platelet-rich plasma during human sperm cryopreservation. Mol Cell Biochem 2025 Dec;480(12):6171-6179.
- Behera S, Mishra B, Cherian JJ, Kumar G, Mahapatra S, Mukherjee A, Roy S, Bagepally BS, Sahoo BB, Majhi MM, ShoganRaj S, Kumar V, Sarkar A, Nellaiappar P. Efficacy and safety of platelet-rich plasma as an adjunct therapy to split thickness skin graft in burn patients with granulating raw wounds: a prospective, randomized, double-blind study-study protocol. Trials 2025 Mar 12;26(1):83.
- Bolat M, Hatipoğlu H, Köroğlu M, Toptan H, Altındiş M. Use of flow cytometry method to detect contaminations of platelet suspensions. World J Microbiol Biotechnol 2024 May 30;40(7):222.
- Dang E, Chen Y, Wang W, Zhang L, An N, Yin W, Yi J, Chen Y. A comparative study of platelet storage lesion in platelet-rich plasma under cryopreservation. Ann Hematol 2024 Feb;103(2):631-643.
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