"Release of growth factors in PRP activated with thrombin."

Transcription

1 "Release of growth factors in PRP activated with thrombin." Carrasco, Joaquin phd Dp. Surgery Universitat de València. Presenter Bonete, Daniel MD Hospital La Fe de València Gomar, Francisco MD Hospital Clinico Universitario Valencia Dp. Surgery Universitat de València AAOS Annual Meeting San Diego Ca. 2011

2 Conflict of Interest Statement The authors have nothing to disclose AAOS Annual Meeting San Diego Ca. 2011

3 Background Developed in the early 1990 s Platelet Gel is an autologous source of concentrated multiple growth factors.

4 Background The platelet alpha granules contain more than 30 bioactive proteins.. The more growth factors released into the wound, the more stem cells are stimulated to produce new host tissue.

5 Background Thus, one can easily see that PRP probably permits the body to heal faster and more efficiently. But, currently the clinical benefit remains questionable, because the activation of platelets is affected by many factors: the type of anticoagulant used, centrifugation speed and duration as well The amount of growth factors released

6 Objective The purpose of our study was to evaluate the release of growth factors, the quantification and its correlation with the number of platelets after the activation with thrombin and calcium in order to use it in our experimental works about consolidation of cortical bone fractures.

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8 Platelets Baseline Platelet Count: We extracted from sheep peripheral blood (anticoagulant ACD-A Citra Braintree Ma).

9 Platelets Platelet count in PRP Peripheral blood, were centrifugated: A 500 g B 600 g for 10 min... C 750 g The platelet level is newly centrifugated at 1900 g for 8 min Activated with 1000 units of thrombin (Sigma) and 10% CaCl 2 per ml.

10 Growth Factors Growth Factors Determination We have determined PDGF TGF β1 by ELISA technique (R & D system)

11 Growth Factors Level of Growth Factors in PRP Clot is formed less than 5 minutes, Aliquots were taken off the gel on the 10 ', 60' and 180 and centrifugated at 1500 g for 5 min. and frozen at - 70º C. The TGFb1 samples required activation with 1 N HCl acid medium and incubation for 24 hours at 2-8ºC. 11

12 Growth Factors Basal levels of PDGF and TGFb1 Peripheral blood was collected on ice, with EDTA 1. Centrifuged at 1000 g for 30 min. 2. second centrifugation at g for 10 min at 2-8º C

13 Statistical Analysis At least 5 determinations were done for each value Values as means ± std with variability coefficient Analysis of variance (ANOVA). Pearson correlation with significance Student s T test. Correlation studies were considered to be statistically significant at P< 0.05 and P<

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15 Platelets Basal level platelets in peripheral blood Table I. Average value of the number of platelets in peripheral blood Platelets/ml Samples of blood N=5 mean std 41719,3 v.c. 0,

16 Platelets Table II. Average value of the number of platelets determinated in PRP by one centrifugation. Platelets/ml Centrifugated 10 min A. 500 g B. 600 g C. 750 g mean std v.c ,135 Note, drag effect, a second centrifugation was then necessary

17 Platelets Table III. value of the number of platelets determinated in PRP by a 2nd centrifugation 1900 g. Platelets/ml A. 500 g B.600 g C.750 g Mean 167, , std 30, , , v. c. 0,181 0,165 0,139 We significantly improve the concentration of platelets. The best method is 1st 500 g followed by 2nd of 1900 g

18 Material Growth y Factors Método 2,5 2,0 Calibration PDGF y=0.0011x Abs nm 1,5 1,0 0,5 r=0.999 The PDGF linear regression fits ,0-0, pgr/ml PDGF pgr/ml PDGFvs Abs nm linear egression

19 Material Growth y Factors Método calibration TGFb1 1,6 1,4 1,2 y=0.0007x r= Abs nm 1,0 0,8 0,6 0,4 The TGF b1 linear regression fits ,2 0, pgr/ml PDGFvs Abs nm pgr/ml TGF b1 linear egression

20 Material Growth y Factors Método Table IV Basal level of growth factors in Peripheral blood mean std v. c. PDGF pgr/ml TGF β1 pgr/ml

21 Growth Factors kinetic of PDGF release % released In 10 min, there is % of PDGF released min 60 min 180 min Time after activation of platelets % released PDGF 21

22 Growth Factors kinetic of TGF b1 release 120 % released TGF b In 10 min, there is only 65 % of TGF b1released min 60 min 180 min time after platelet activation % released TGF b1 22

23 Growth Factors TABLE V PDGF Released A.500 g mean std v.c.. pgr/ml released % 10 min min min times B.600 g 10 min min min times C.750 g 10 min min min times 100.0

24 Growth Factors TABLE VI TGF β1 Released A.500 g mean std v.c. Pgr/ml Released % 10 min min times min B.600 g 10 min min times min C.750 g 10 min min times min

25 Growth Factors TABLA VII Pearson Correlation X(platelets) Y1(PDGF) Y2(TGFb1) Rx,y1 Rx,y2 Ry1,y2 Pearson coeficient Significance T student test P< It is (+) correlation between platelets and growth factors even between the growth factors themselves. Significance T student test P<0.01 *0.33 * * Linear regression R 2 =0.855 R 2 =0.847

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27 Discussion There is a wide variability of methods of preparation as well as different conditions of use of PRP. Are all PRP equal? 27

28 Discussion The method of concentration with a single centrifugation is not so, because you lose a lot of washed platelets by the cell stage both red and white. In the literature we can see therefore many authors that centrifuge a second time, to concentrate much more platelets. 28

29 Discussion It is asumed that the calcium participates in the activation of platelets but really calcium estimulates degranulation... Thrombin generated irreversible platelet degranulation with the loss of integrity of platelets and subsequent release of growth factors... 29

30 Discussion The lack of consensus on the composition and production of plasma concentrates makes it impossible to establish a standard that includes all the research. It s not known how many factors are being used 30

31 Discussion In our group research, a study in rabbits, by cortical defect of the tibia and fibula, the use of PRP activated only with calcium does not alter the repair and healing processes.

32 Discussion Thus, we studied three different speeds in response to the literature. So in our three methods used, the highest concentration of platelets were obtained by two centrifugations. 32

33 Discussion. We have studied the release of PDGF and TGF b1 in time. After activation with calcium and thrombin, PDGF was released practically in 10, meanwhile the release of TGF β1 was slower and sustained over time. 33

34 This method for obtaining concentrated platelets really becomes PRF (Plasma rich in factors). These results are consistent with those reported in the literature but these results allows us to standardize our method of work 34

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36 Conclusion There is a direct relationship between the activated platelets and the release of growth factors. The more platelets concentrated the more growth factors are released in the first minutes.

37 Conclusion The kinetics are different according to each growth factor probably due to their different function in the process of healing...we are ready to use the factors in a known concentration in our trials.

38 Thank you for your attention