THE EFFECT OF GROWTH FACTORS IN PLATELET-RICH PLASMA ON THE ACTIVITY OF OSTEOBLAST CELL LINE

혈소판농축혈장 내의 성장요소가 조골세포주의 활성도에 미치는 영향

  • Jung Tae-Wook (Department of Prosthodontics, College of Dentistry, Seoul National University) ;
  • Kim Yung-Soo (Department of Prosthodontics, College of Dentistry, Seoul National University) ;
  • Kim Chang-Whe (Department of Prosthodontics, College of Dentistry, Seoul National University) ;
  • Jang Kyung-Soo (Department of Prosthodontics, College of Dentistry, Seoul National University) ;
  • Lim Young-Jun (Department of Prosthodontics, College of Dentistry, Seoul National University)
  • 정태욱 (서울대학교 치과대학 보철학교실) ;
  • 김영수 (서울대학교 치과대학 보철학교실) ;
  • 김창회 (서울대학교 치과대학 보철학교실) ;
  • 장경수 (서울대학교 치과대학 보철학교실) ;
  • 임영준 (서울대학교 치과대학 보철학교실)
  • Published : 2004.04.01

Abstract

Statement of problem: Platelet-rich plasma(PRP) is well known to be very effective method to stimulate and accelerate the healing of bone and soft tissue. However, there are few reports which deal with the mechanisms of the PRP on the activation of the osteoblasts. Purpose: This study was aimed to investigate the effect of growth factors in PRP on the activity of osteoblasts. Material and method: To evaluate the effect on human, human osteoblast cell line was cultured. PRP was extracted from the blood of a healthy volunteer. Using the recombinant growth factors of PDGF, $TGFT-\beta$, IGF-1, bFGF which are mainly found at bone matrix and their neutralizing antibody, the effect of PRP on the attachment and proliferation of osteoblasts was evaluated. To evaluate the autocrine and paracrine effects, conditioned media(CM) of PRP was made and compared with PRP. By the western blot analysis, the expression of growth factors in PRP, CM was examined. Cell morphology was compared by the light microscope. Results : 1) The effects of CM on osteoblast were similar to the effects of PRP. 2) PRP, CM, recombinant $TGF-\beta$, bFGF, IGF-1 showed significantly higher cellular attachment than control(p<0.05) in the cell attachment assay. In the cell proliferation assay, PRP, CM, recombinant $TGF-\beta$, IGF-1, bFGF, PDGF increased significantly cell proliferation(p<0.01). Among the recombinant growth factors, IGF-1 showed the highest cellular attachment and proliferation. 3) In the western blot assay, bFGF, IGF-1, PDGF weve equally expressed in PRP and CM. 4) The attachment of osteoblast cell decreased significantly after the addition of neutralizing antibody against $TGF-\beta$, IGF-1(p<0.05). In the cell proliferation assay, the addition of neutralizing antibody against $TGF-\beta$, bFGF, PDGF, IGF-1 decreased significantly the cellular proliferation(p<0.05). The amount of decreasing in the cell attachment and proliferation is the highest in at-lGF-1. 5) The cells in control group were flattened and elongated with a few cellular processes in the a light microscope. But, the cells appeared as spherical, plump cells with well developed cellular processes in experimental groups. The cells in PRP and CM had more prominent developed features than recombinant growth factor groups. Conclusions : These findings imply that PRP maximize the cellular activity in early healing period using the synergistic effect, autocrine, paracrine effects of growth factors and increase the rate and degree of bone formation.

Keywords

References

  1. Albrektsson T, Branemark PI. Hanson HA, Lindstrom J. Osseointegrated titanium implants. Acta Orthop Scand 1981:52: 155-77
  2. Wittbjer J, Rohlin M. Thorngren KG. Bone formation in demineralized bone transplants treated with biosynthetic human growth hormone. Scan J Plast Reconstr Surg 1983:17:109-18
  3. Zilch H. Noffke B. Beeinflusst der Fibrinkleber die Knonchenneobildung. Unfallheikunde 1981: 84: 363
  4. Park JB, Young SO, Kenner GH. Dental implant fixation by electrically mediated process. Biomater Med Devices Artif Organ 1978;6:291
  5. Canalis E,McCarthy T.Centrella M. Growth factors and the regulation of bone remodeling. J Clin Invest 1988:81: 277-81
  6. SN, Bostrom MPG. Lane JM. Bone growth factors. Orthop Clin North Am 2000: 31:375-87
  7. Weibrich G. Kleis WKG, Hafner G. Growth factor levels in the platelet-rich plasma producedby 2 different methods : Curasantype PRP kit versus PCCS PRP system. Int J Oral Maxillofac Implants 2002; 17: 184~90
  8. Marx RE. Platelet-rich plasma: A source of multiple autologous growth factors for bone grafts. In: Lynch SE. Genco RJ. Marx RE,eds. Tissue engineering: application in maxillofacial surgery and periodontics. Chicago: Quintessence. 1999: 71-82
  9. Marx RE. Platelet-rich plasma(PRP): what is PRP and what is not PRP? Implant dentistry 2001: 10: 225-28
  10. 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 1998:85:638-46
  11. Anitua E. Plasma rich in growth factors: Preliminary results of use in the preparation of future sites for implants. Int J Oral Maxillofac Implants 1999: 14: 529-35
  12. Rosenberg ES. Torosian J. Sinus grafting using platelet-rich plasma - initial case presentation. Pract Periodont Aesthet Dent 2000: 12: 843-50
  13. de Obarrio JJ. Arauz-Dutari JI. Chamberlain TM. Croston A. The use of autologous growth factors in periodontal surgical therapy: platelet gel biotechnology - case reports Int J Periodontics Rest- orative Dent 2000; 20 :487-97
  14. Carlson NE. Roach RB. Platelet-rich plasrna: clinical applications in dentistry. J Am Dent Assoc 2002: 133: 1383-86
  15. Maiorana C. Sommariva L. Brivio P. Sigurt'a D. Santoro F. Maxillary sinus augmentation with anorganic bovine bone(Bio-Oss) and autologous platelet-rich plasma: preliminary clinical and histologic evaluations. Int J Periodontics Restorative Dent 2003:23:227-35
  16. Babbush CA, Kevy SV. Jacobson MS. An in vitro and in vivo evaluation of autologous platelet concentrate in oral reconstruction. Implant Dent 2003: 12: 24-34
  17. Whitman DH, Berry RL. Green DM. Platelet gel: an autologous alternative to fibrin glue with application in oral and maxillofacial surgery. J Oral Maxillofac Surg 1997;55:1294-99 https://doi.org/10.1016/S0278-2391(97)90187-7
  18. Schmitz JP. Hollinger JO. The biology of platelet-rich plasma. J Oral Maxillofac Surg 2001 ;59: 1119-21 https://doi.org/10.1053/joms.2001.26801
  19. Slater M. Patava J. Kingham K. Mason RS. Involvement of platelets in stimulating osteogenic activity. J Orthop Res 1995; 13:655-63 https://doi.org/10.1002/jor.1100130504
  20. Kawase T. Okuda K. Wolff LF. Yoshie H. Platelet-rich plasma-derived fibrin clot formation stimulates collagen synthesis in periodontal ligament and osteoblastic cells in vitro. J Periodontol 2003;74:858-64 https://doi.org/10.1902/jop.2003.74.6.858
  21. Okuda K. Kawase T. Momose M. Murata M. Saito Y. Suzuki H et al. Platelet-rich plasma contains high levels of platelet-derived growth factor and transforming growth factor-$\beta$ and modulates the proliferation of periodontally related cells in vitro. J Periodontol 2003;74:849-57 https://doi.org/10.1902/jop.2003.74.6.849
  22. Pfeilschifter J. Oechsner M. Naumann A. Gronwald RGK. Minne HW. Ziegler R. Stimulation of bone matrix apposition in vitro by local growth factor: A comparison between insulin-like growth factor-L. platelet-derived growth factor. and transforming growth factor $\beta$. Endocrinology 1990; 127:69-75. https://doi.org/10.1210/endo-127-1-69
  23. Weibrich G. Kleis WKG. Hafner G. Hitzler WE. Wagner W. Comparison of platelet. leukocyte and growth factor levels in point-of-care platelet-enriched plasma. prepared using a modified Curasan kit. with preparations received from a local blood bank. Clin Oral Impl Res 2003:14:357-62
  24. Weibrich G. Kleis WKG. Hafner G. Hitzler WE. Growth factor levels in platelet-rich plasma and correlations with donor age. sex. and platelet count. J Cranio Maxillofac Surg. 2002:30:97-102
  25. Centrella M. McCarthy TL. Canalis E. Platelet-derived growth factor enhances deoxyribonucleic acid and collagen synthesis in osteoblast-enriched cultures from fetal rat parietal bone. Endocrinology 1989: 125: 13-9
  26. Moursi AM. Winnard PL. Winnard AV. Rubenstrunk JM. Mooney MP. Fibroblast growth factor 2 induces increased calvarial osteoblast proliferation and cranial suture fusion. Cleft Palate-Craniofacial Journal. 2002:39:487-96
  27. Holtrop ME. Light and electron microscopic structure of bone-forming cells. In:Hall BK. editior. The osteoblast and osteocyte. Bone. vol. 1. Caldwell (NJ): Telford Press: 1990. p.1-39