Using autologous thrombin in making PRP gel - case reports-

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1 60 Using autologous thrombin in making PRP gel - case reports- LivingWell Dental Hospital LivingWell Institute of Dental Research Seung-gul So, Jang-yeol Lee, Jae-bong Park, Hyoun-chull Kim, Il-hae Park, Sang-chull Lee Ⅰ. Introduction Platelet Rich Plasma(PRP) is an autologous source of transforming and growth factor (TGF, PDGF, IGF, EGF, VEGF) that seems to improve the physiological process of healing in soft and hard tissue. Through the course of making PRP, calcium chloride and commercial bovine thrombin are used to help coagulation generally. However, through many researches, the use of bovine thrombin may be associated with the development of antibodies of factor V, XI, and thrombin resulting in the risk of life-threatening coagulopathies. In Italy, bovine thrombin is limited to use for hemostasis. Batroxobin (Thrombin-like snake venom enzyme) is used instead of bovine thrombin. For resolving this problem, in our hospital, we use autologous thrombin in making PRP. The aim of this study is to introduce a method of PRP preparation. the PRP gel was made during the surgery time. Times of gel formation were been recorded. Procedures of making autologous thrombin are by following: 1. Withdraw 4-5ml of autologous blood with untreated tube. 2. Stand the tube and place calmly for 30 min. 3. Then centrifuge the tube under condition on 3600rpm for Collect the supernatant as autologous thrombin. 5. Mix autologous thrombin with 10% CaCl 2. Fig.1. the condition of centrifuge. Ⅱ. Materials and Methods We recruited 40 patients (mean age 48.3 years old, male : female = 20 : 20), that underwent local anesthesia for implant surgery. For each patient Fig ml 10% CaCl 2.

2 61 Vent(Zimmer Dental Inc, U.S.A) Fig.3. Collect the supernatant as autologous thrombin and mix it with 0.25ml 10% CaCl 2. Panorama and computer tomography was taken for analysis of residual bone and the shape of maxillary sinus. Lateral window technique and place of 3 implants were planned (Fig.6,7,8,9). During operation, we made PRP and mixed it with bone graft materials (Fig.10,11,12). Fig.6. Pre-op. Fig.7. Post-op. Fig.4. 1ml syringe filled with 0.25ml 10% CaCl 2 and 0.75ml autologous thrombin. Fig.5. Special tool for making PRP. The ratio of PRP and autologous thrombin is 10:1. Fig.8. CT image analysis on #16. Case 1 Pt: 엄 ** Age: 63 Sex: male Tx. plan: 1. left and right Maxillary sinus graft with lateral window technique 2. #16: 4.7*13mm Tapered Screw-Vent (Zimmer Dental Inc.,U.S.A) an #25, 26: 3.7*13mm,4.7*13mm Tapered Screw- Fig.9. CT image analysis on #25,26.

3 62 Fig. 10. Mixing of PRP and bone graft materials. Fig. 14. Post op. CT on #16. Fig.11. Gelation of PRP and cerasorb. Fig. 15. Post op. CT on #25. Fig.12. Gelation of PRP and autogenous bone. Fig. 16. Post op. CT on #26. The coagulated bone graft materials were easily to position on graft site.(fig. 13) Fig. 13. After bone graft. Case 2 Pt: 김 ** Age: 44 Sex: female Tx. plan: 1. #37: 4.7*13mm Tapered screw-vent (Zimmer Dental Inc., U.S.A) implant. 2. autogenous bone graft and PRP. Computer tomography and panoramic analysis were done before operation. A large bone defect were find on left lower second molar area(fig.

4 63 17,18,19).During implant operation, left lower third molar was extracted together and we got autogenous bone around extraction site using trephine bur (Fig. 20). The harvested bone mixed with PRP. When PRP and bone mixture were coagulated, we put it into the defect site (Fig. 21,22,23,24). Fig. 21. PRP+autogenous bone. Fig. 17. Pre-op. Fig. 18. Post-op. Fig. 22. Large defect between implant and bone. Fig. 19. CT image analysis on #37. Fig. 23. Coagulated PRP and bone. Fig. 20.autogenous bone is harvested by using trephine bur around extracted site. Fig. 24. Defect filled with PRP+bone.

5 64 Case 3 Pt: 장 ** Age: 38 Sex: male Tx plan: 1.#21,#22: 3.7*13mm Tapered screwvent(zimmer Dental Inc., U.S.A) implant. 2. autogenous bone graft Four months before implantation, this man had been received cyst enucleation and #21,22 extraction. After cyst was removed, autogenous bone, cerasorb, calmatrix were grafted on defect site (Fig.25,26). We planned 2 implants and bone graft on labial side additionally(fig. 28,29). When flap was performed, labial bone loss was observed. So autogenous bone from left ramus were grafted after implantation. And then bonegraft material was covered with human pericardium membrane that treated with PRP (Fig.30,31,32,33,34). Fig. 28. CT image analysis on #21. Fig. 29. CT image analysis on #22. Fig. 30. Bone loss on labial side. Fig. 25. Post-op. Fig. 26. Post-op. panoramic view after bone graft. Fig.27. Post-op. panoramic view after implantation Fig. 31. After implantation.

6 65 Fig. 32. After bone graft +PRP. Case 4 Pt: 황 ** Age: 49 Sex: female Tx.plan: 1. #11,12 ridge split and 3.7*13mm Tapered screw-vent (Zimmer Dental Inc., U.S.A) implant. 2. autogenous bone graft Fig. 33. PRP+human paricardium membrane. This woman had old crown & bridge on her maxillary anterior area. We planed change it to implant(fig. 36,37). On computer tomography analysis, the width of residual bone was too narrow for implantation, so ridge split and bone graft were planed (Fig. 38,39). When implants were placed, labial bone plate was too weak to preservation. For reinforce the labial side, PRP mixed autogenous bone was grafted and titanium mesh was used for maintain the shape of graft material (Fig. 40,41,42,43,44,45). Fig. 34. Application of membrane. Fig. 36. Pre-op. Fig.37. Post-op. Fig. 35. After 4 months, second op. was done. Fig. 38. CT image analysis on #11.

7 66 Fig. 39. CT image analysis on #21. Fig. 43. After grafted. Fig. 40. Ridge split. Fig.44. Titanium mesh for maintain the shape of graft material. Fig. 41. After implantation. Fig. 45. PRP gel for improve healing of soft tissue and hard tissue. Ⅲ. Result Fig. 42. Coagulated PRP and autogenous bone. In all 40 cases, PRP was coagulated perfectly with human thrombin. However the coagulation time had variation because of the content of thrombin were different in everyone. In almost cases coagulation time were under 5 minutes, the fastest one was 1 minute and the longest one was 15 minutes. The average coagulation time was 4.utes.

8 67 Case No. Sex/Age Operation site Coagulation time /F #16 44/M #26,27,34,37,47 48/F #12 68/F #22 63/M #16,25,26 #32,34,35,36, 47/F 37,42,43,46 63/F #27,45,46 #14,16,17,24,25,26, 47/M 27,32,36,37,43,46,47 59/M #33 45/M #46,47 10 min 5 min 5 min 5 min 3min 5min 4 min Case No. Sex/Age Operation site Coagulation time /M #35,45 31/M #36 #23,25,26,32, 69/F 42,44,45,46 40/F #21,22 69/F #17 74/F #35,36 43/F #47 38/M #37 19/M #15,35,45 66/M #14,16,17, 43,45,46,47 Average of coagulation time 1 min 5min 6 min 10 min 3min 4. Case No. Sex/Age Operation site Coagulation time /M #12,13,35,36 35/M #13,37 64/M #35,37 44/F #18,37,38,48 49/F #17,18,25,37 60/F #47 43/M #26,27 34/M #21 68/F #47 #27,35,36, 41/F 44,45,46,47 6 min 6 min 11 min 5 min 3min 1min 11 min Case No. Sex/Age Operation site Coagulation time /F #15 38/M #21,22 52/M #32,42 49/F #11,21 36/F #45,46 59/M #26,27,46,47 54/F #36,47 27/M #45 52/M #26,27 47/F #15,16,17,25 1 min 5min 15 min 3min 1min 4 min Ⅳ. Discussion Although many reports had been reported the safety of bovine thrombin using in PRP. Marx (2004) 1), Christie et al (1997) 2) persistent the safety of bovine thrombin. However Cmolik et al (1993) 3), spero et al (1993) 4), Muntean (1994) 5), Sosolic et al (1995) 6) had been reported the use of bovine thrombin may be associated with the development of antibodies to factor V, XI and thrombin. And Kassolis et al (2000) 7), Takakura et al (1994) 8), Oz et al (1992) 9), De Somer et al (2006) 10) had been reported about the benefit of autologous thrombin. Based on these data, we use autologous thrombin instead of bovine thrombin in making PRP gel. Ⅴ. Conclusion Using autologous thrombin is very safe and effective method in making PRP gel, in all cases we

9 68 got PRP gel successfully. And we don't need to worry about "anti-bovine factor V" any more. REFERENCES 1. Marx Re. Platelet rich plasma: Evidence to support its use. J oral Maxillofac Surg 2004; 62: Christie RJ, Carrington L, Alving B. Postoperative bleeding induced by topical bovine thrombin: Report of two cases. Surgery 1997; 121: Cmolik BL, Spero JA, Magovern GJ,etal. Redo cardiac surgery:late bleeding complications from topical thrombin-induced factor V deficiency. J Rhorac Cardiovasc surg 1993;105: Spero JA. Bovine thrombin-induced inhibitor of factor V and bleeding risk in postoperative neurosurgical patients. Neurosurg 1993;78: Muntean W, Zenz W, Finding K,et al. Inhibitor to factor V after exposure to fibrine\ sealant during cardiac surgery in a two-year-old child.acta Pacdiatr 1994;83: Sosolik RC, Theil KS, Brandt JT. Clinical pathology rounds, anti-bovine thrombin antibody.lab Med 1995;27: Kassolis JD, Rosen PS, Renolds MA: Alveolar ridge and sinus augmentation utilizing platelet rich plasma in combination with freeze-dried bone allograft: Case series. J periodontal 2000;71: Takakura H, Kurosawa H, Mizuno A, Tatara A, Sakamoto Y, Saitoh F. Autologous fibrin glue from concentrated platelet rich plasma: Intraoperative plasma sequestration using autotransfusion device. Rinsho Kyobu Geka 1994; 14: Oz Mc, Jeevanandam V, Smith CR, et al. Autologous fibrin glue from intraoperatively collected platelet rich plasma. Ann Thorac Surg 1992;53: De Somer F et al. Can autologous thrombin with a rest fraction of etanol be used safely for activation of concentrated autologous platelets applied on nerves. EurSpine J 2006;15(4):501-5.

10 69 Abstract PRP 제조에있어 autologous thrombin 의사용 - case reports - 소승걸, 이장렬, 박재봉, 김현철, 박일해, 이상철 리빙웰치과병원리빙웰치의학연구소 PRP 는수술시치유의결과를향상시켜주기위해사용되어왔다. 그제작과정중PRP 의응고를돕기위해 thrombin 이사용되는데, 대부분의경우에는상용화된 bovine thrombin 을사용하고있다. 하지만많은연구에서 bovine thrombin 의사용이 Bovine thrombin-induced antibodies 를만들어혈액응고를지연시킨다고보고하고있다. 심지어는심할경우생명에위험을초해할수있다고보고하고있다. 따라서본원에서는 PRP 의제조과정에서 bovine thrombin 의사용을대체하기위한방법을생각해왔으며, 현재는 PRP 제조를위해채혈한전혈을이용해 autologous thrombin 을만들어사용하고있다. Autologous thrombin 은 bovine thrombin 과마찬가지로 PRP 의응고를도왔으며. 거기에따른증례들을정리해보고자한다.