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Procedure For Autologous Thrombin Activation of Platelets in Platelet Rich Plasma (PRP)

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By: Astley E. Smith BSc, (Chem.), MSc, (Biochem.), DMD, FICOI
2006-05-01

BACKGROUND

Platelets are small anuclear granulated bodies that adhere, aggregate and form a platelet plug at sites of vascular injury to prevent blood loss. Platelet concentration in blood is approximately 300,000/ul, and they normally have a half-life of 4 days. They are formed from committed giant stem cells called megakaryocytes as they pinch off bits of their cytoplasm and extrude them into the blood circulation. Platelets have microtubules around their periphery and an extensively invaginated membrane in contact with extracellular fluid.

Their membranes contain receptors for a number of substances including collagen, adenosine diphosphate (ADP), von Willebrand factor and fibrinogen. Some of the contents of their cytoplasm have their origin in the megakaryocytes, some are internally synthesized, and some are stored after endocytosis. Among the intracellular organelles are two types of granules:

1) Dense granules, which contain the nonprotein substances, which are secreted in response to platelet activation, including serotonin, ADP/ATP, histamine, dopamine and catecholamines.1

2) *-granules, which contain secreted proteins.1-3 Among these stored proteins are mitogenic and angiogenic growth factors which are essential for hard and soft tissue regeneration and repair. Marx4 of North America and Anitua et al1 of Europe have shown that earlier and greater bone density is obtained in surgical sites treated with PRP as compared to control sites. The growth factors in platelets are platelet-derived growth factor (PDGF), transforming growth factor ß (TGF-ß), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet derived epidermal growth factor (PDEGF), and insulin-like growth factor-1 (IGF-1). These are variously involved in stimulating chemotaxis, cell proliferation and maturation in wound healing.1

In the early 1990's platelet rich plasma (PRP) was introduced as an autologous biomaterial.5,6 A variety of machines have been tailored to achieve the optimal centrifugal separation of PRP for therapeutic use from small amounts of citrated blood.7-13

In 2002, Gonshor9 developed a procedure characterized by a double spin centrifugation method using a non-automated centrifuge to sequester and concentrate platelets to a level four to eight times baseline whole blood values. The technique produced concentrations of PDGF-AB above 500% and TGFß, greater than 800%. His testing showed that the 85.1% platelet yield remained quiescent throughout the procedure, maintaining their integrity and viability, with no inadvertent activation. Bovine thrombin 5000 units and 10% Calcium chloride solution were used for platelet activation, creating a PRP gel.

In 2005 Marx and Garg15 published an excellent textbook on "Dental and Craniofacial Applications of Platelet-Rich Plasma". In this text the authors state that "growth factors have emerged as the "Holy Grail" in wound-healing" and they projected lyophilized bovine thrombin preparation as the standard for initiating clotting of PRP and activating platelets.

However a year earlier in 2004, bovine thrombin became unavailable in Canada. This event precipitated a search for an alternative to bovine thrombin. Autologous thrombin and/or recombinant human thrombin were the logical choices. Necessity therefore became the mother of invention, and a search was launched for an alternative.

SUBJECTS

This study consisted of 21 patients who required implant placements and/or bone grafting. Of the 21 patients 5 needed immediate implant placement with bone grafting, 8 needed implant placement only, and 8 needed bone grafting only.

MATERIALS AND METHODS

Materials

1) General purpose centrifuge (non-automated) with a six-position rotor, or automated centrifuge.

2) 4 -10ml yellow-top vacutainers containing acid citrate dextrose (ACD-A) solution.

3) 6 -10-ml noncoagulant red-top vacutainers.

4) 21 - gauge butterfly needle and 19 gauge butterfly needle.

5) 21/2" (63mm) blunt needle.

6) 3" (76mm) blunt needle.

7) 10% Calcium Chloride Solution.

8) 6- 5ml sterile syringes.

9) 6 - 1ml sterile syringes.

10) Sterile container for PRP.

11) Sterile container for PPP.

12) Sterile container for serum.

13) 4 sterile dappen dishes.

14) Package of sterile collagen membrane. (Colla Tape or Neo Tape)

15) Test tube rack.

Blood collection and processing

Tubes of blood were taken from the antecubital region with a 21-gauge or 19 gauge butterfly needle and collected into 4-10ml yellow-top blood collection tubes containing acid citrate dextrose (ACD-A) solution. Two additional tubes of blood were collected as described above except into 10ml red-top vacutainers with no anticoagulant. (Each of the two red-top tubes was marked with an X) (Fig. 1). The four yellow-top tubes of blood were processed to produce PRP using the Gonshor Procedure9 (Figs. 2-4). The two-redtop tubes of blood were put aside to clot.

Using the general-purpose centrifuge described in Gonshor's publication,9 the two-red-top tubes with clotted blood were centrifuged at 2000 rpm for 10 minutes along with the four red-top tubes with plasma for the second spin (Fig. 3). The serum was carefully removed from each tube with a sterile syringe and pooled, in a sterile container (Fig. 5). The containers containing PRP (4.5mls), PPP (6.5mls) and Serum (2.5 mls) were covered and placed in the surgical operatory along with a vial of 10% calcium chloride, sterile dappen dishes, 5ml and 1 ml sterile syringes (Fig. 6).

ACTIVATION OF PRP

In order to activate the PRP to get a platelet rich plasma gel, 1ml of PRP was dispensed into each of four dappen dishes (Fig. 7). 10% Calcium Chloride was added to each dappen dish in a ratio of 1 CaCl2 : 5 PRP by volume (.2 ml CaCl2 : 1 ml PRP). Serum was then added to the calcified PRP in each dappen dish in a ratio of 1 serum : 2 PRP by volume (0.5 ml serum : 1ml PRP). The complex was left to sit at room temperature and the time for PRP gel formation was recorded (Fig. 8). The above activation process was also achieved by aspirating the appropriate amounts of calcified PRP and serum into a 5mls. syringe. The activated complex could then be delivered via the syringe to any site targeted. When bone grafting was done, autogenous bone, allograft, xenograft or a composite graft was added to calcified PRP, then serum was added to produce a PRP gel in the presence of the graft material (Figs. 9 & 10). The time for PRP gel formation was recorded and the PRP gel-graft complex was then placed in situ. In all cases where resorbable membranes were used, for delivery of growth factors in situ, cell occlusivity and/or guided tissue regeneration, the membranes were dipped in calcified PRP, activated with serum then immediately placed in situ between bone and soft tissue before PRP gel was formed. The times for PRP Gel formation in situ were recorded. When Platelet Poor Plasma (PPP) was used as a tissue sealant it was also activated with serum and 10% Calcium Chloride. After dispensing 1 ml of PPP in a dappen dish Calcium Chloride was added in a ratio of 1 CaCl2 : 2.5 PPP by volume (0.4 ml CaCl2 : 1 ml PPP). Serum was then added in a ratio of 1 serum: 1 PPP by volume (1.0 ml serum: 1 ml PPP). The complex was allowed to stand at room temperature for 10 minutes then used to seal the surgical incision line and suture entry points (Fig. 11). It was left undisturbed for 5 minutes in situ. This process was also accomplished by aspirating the respective volumes of calcified PPP, 2 and serum into a 5 ml syringe and left to stand for 10 minutes before use.

RESULTS

The volume of concentrated PRP obtained amounted to an average of 1.1 ml/tube. This compared favorably with Gonshor's results.9 The total volume of PRP obtained from the 4 citrated tubes of blood was 4.5mls. The volume of serum obtained from the 2 tubes of clotted whole blood after centrifugation was 2.5 mls. The volume of PPP obtained was 6.5 ml. For the formation of platelet-rich plasma gel in vitro (Fig. 8), the time range was 5-10 minutes. The range was 30-60 seconds in situ. Complete blood processing took approximately 30 minutes. PRP gel was rapidly formed in the presence of autograft (Fig. 9) and allograft (Fig. 10). A PPP clot was formed in 10 minutes when serum was added to calcified PPP (Fig. 11). This was used as an autologous tissue sealant for the surgical incision line and suture entry points.

DISCUSSION

Physiological benefits of PRP

In order to reap the physiological benefits of PRP, it is most important that growth factors be released from viable platelets since it is during the act of granule exocytosis that the platelet completes the growth factor protein's tertiary structure.2,3,9 The tertiary structure, with its unique conformation is the most biologically active structure. The molecular conformation allows the protein to appropriately present its active site to its specific substrate so that catalysis may proceed. Anitua et al1 have listed a number of situations where guided secretion of autologous platelet products can promote healing and wound repair. The situations are: maxillofacial surgery and bone grafts, dental implant surgery, orthopaedic surgery and bone reconstruction, facial plastic and cosmetic surgery, skin ulcers, eye surgery-retinal hole repair, sports medicine-cartilage and tendon repair. To date the most widespread use of PRP is in dentistry,4,5,11,12,15 where various authors have shown that the administration of PRP in a variety of surgical sites have rapidly precipitated both hard and soft tissue regeneration and repair.1, 5-7,15,16 Others claim that the use of autologous platelets can positively affect the outcome when autografts, allografts, and xenografts are used for alveolar ridge augmentation procedures.15,16

Bone regeneration requires the recruitment, proliferation and maturation of osteoblasts which are derived from mesenchymal stem cells17,18 and one study suggests that platelet releasates also promote the migration of mesenchymal stem cells.19 Gonshor9 states that combining the growth factors PDGF and TGFß has been shown to accelerate bone repair,20 promote fibroblast proliferation, increase tissue vascularity, rate of collagen formation,21,22 mitosis of mesenchymal stem cells, endothelial cells, as well as osteoblasts, playing key roles in the rate and extent of bone formation.

In a series of 20 patients undergoing multiple extractions, PRP clot was deposited into the sockets on one side of the mouth while the other served as control. Bone biopsies were taken at extraction sites between 10 and 16 weeks. In most of the patients with PRP clot, bone regeneration was extensive and bone tissue was compact with well-organized trabeculae. In contrast, in the control group the cavity was mainly filled with connective tissue.1 By using animal models some authors have shown that, like in humans, adding PRP-clot around roughened titanium implants at the moment of their implantation in goats23 and minipigs24 improved both the extent and quality of bone regeneration around the implants.

What is initiating the clot?

It is a well-known physiological fact that blood starts to clot when it is exposed to collagen in vivo. The fact that a piece of collagen reduced activation time is evidence that an appropriate surface was provided for the initiation of the clotting cascade. The precursors and enzymes of the clotting system must therefore be present in the serum being used thereby initiating the clotting cascade. Thrombin is usually not present in serum after clotting is complete since its production is stopped by antithrombin III inhibition of factors 1Xa, Xa, XIa, and XIIa with the help of its cofactor heparin, and what is left is complexed with thrombomodulin for fibrinolysis with the help of protein C, protein S, tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA) plasminogen and plasmin.

Research needs to be done on the quantification of the clotting precursor components and enzymes of serum and their stability under varying conditions. Further studies also need to be conducted on the stability of growth factors after they have been released from platelets.

IN SITU RESULTS

The results obtained in situ are similar to results obtained by Anitua et al1 when they irrigated peri-implant surfaces with calcified PRP mixed with extruded supernatant from a retracted clot immediately after implant placement.

IN VITRO RESULTS

The longer clotting times obtained in vitro could be an indication that the concentrations of clotting precursors and enzymes may be relatively low in serum and it takes time for the reactions to develop. However, once activation was initiated subsequent clotting proceeded rapidly. This is consistent with the autocrine/ paracrine nature of intercellular communication via chemical mediators during platelet activation. Anitua et al1 states that the administration of activated platelets in fibrin clot or fibrin glue provides an adhesive support that can confine secretion to a chosen site, and the presentation of growth factors attached to platelets and/or fibrin may result in enhanced activity over recombinant proteins. Extended in vitro activation times could be advantageous when autogenous bone, allografts and xenografts are to be mixed with PRP before placement in situ for ridge augmentation grafts, ridge maintenance grafts, sinus elevation grafts, repair of schneiderian membrane as well as rapid soft tissue healing and maturation.

CONCLUSION

The procedure presented here shows how autologous thrombin in the patient's serum can be used to activate platelets. It should provide a safe alternative for therapeutic use. The ready availability of autologous PRPgel with growth factor release and autologous fibrin glue as a tissue sealant shows that autologous thrombin in the patients serum can effectively activate platelets and thereby provide an alternative to continue the stimulation of tissue regeneration, repair and maturation in dentistry and medicine.

Caution

* Growth factor levels, platelet count and activation times will vary from individual to individual as well as with age and health status.

* The aging of serum and PRP may affect activation times since the proteins are very labile in vitro.

* When this procedure is used, steps should be taken to ensure that a maximally sterile environment is provided for blood collection, PRP processing and activation.

Dr. Smith has been in private practice for 25 years in New Westminster, BC. In 2004, he invented a patient-specific procedure for autologous thrombin activation of platelets in platelet rich plasma (PRP).

Oral Health welcomes this original article.

Acknowledgments

I would like to thank Dr. Aron Gonshor of Montreal, Dr. Ross MacGillivray, Dr. Ed Pryzdial, and Dr. Dana Devine of the Centre for Blood Research, University of British Columbia, for their invaluable discussions.

Disclosure

The author claims to have no financial interest in any company or any of the products mentioned in this article.

REFERENCES

1. Eduardo Anitua, Isabel Andia, Bruno Ardanza, Paquita Nurden (3,4), Alan t. Nurden. Autologous platelets as a source of proteins for healing and tissue regeneration. Journal of Thrombosis and Haemostasis 2004; 91:4-15.

2. Rendu F, Brohard-Bohn B. The platelet release reaction: granules' constituents, secretion and function, Platelets 2001; 12:261-73.

3. Reed GL. Platelet secretion. In Platelets (ed: AD Michelson),. Elsevier Science, San Diego, 2002, pp181-95.

4. 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 1998; 85:638-646.

5. Whitman DH, Berry Rl, Green DM. Platelet gel: an autologous alternative to fibrin glue with applications in oral and maxillofacial surgery. Journal maxillofac Surg 1997; 55:1294-9.

6. 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: applications in Maxillofacial Surgery and Periodontics. Chicago: Quintessence, 1999:71-82.

7. S¿nchez Ar, Sheridan PJ, Kupp LI. Is platelet-rich plasma the perfect enhancement factor? A current review. Int J Oral Maxillofac Implants 2003; 18: 93-103.

8. Zimmermann R, Jakubietz R, Strasser E, et al. Different preparation methods to obtain platelet components as a source of growth factors for local application. Transfusion 2001; 41:1217-24.

9. Aron Gonshor, BSc, PhD, DDS, FRCD(c)* Technique for Producing Platelet-Rich Plasma and Platelet Concentrate: Background and Process. The Internal Journal of Periodontics and Restorative Dentistry. Volume 22, Number 6, 2002.

10. Sonneleitner D, Huemer P, Sullivan Dy. A simplified technique for producing platelet-rich plasma and platelet concentrate for intraoral bone grafting techniques: a technical note. Int J Oral Maxillofac Implants 2000; 15:879-882.

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; 14:529-535.

12. Lazada JL, Caplanis N, Proussaefs P, Willardsen J, Kammeyer G. Platelet-rich plasma application in sinus graft surgery: Part 1-Background and processing techniques. J Oral Implantol 2001:27:38-42.

13. Landesberg R, Roy M, Glickman RS. Quantification of growth factor levels using a simplified method of platelet-rich plasma gel preparation. J Oral Maxillofac Surg 2000;58:297-300.

14. Landesberg R, Moses M, Karpatkin M. Risk of using platelet-rich plasma gel. J Oral Maxillofac Surg 1998; 56:1116-7.

15. Robert E. Marx and Arun K. Garg. Dental and Craniofacial Applications of Platelet-Rich Plasma. Quintessence Publishing Co. Inc. 2005.

16. Tischler M. Platelet rich plasma. The use of autologous growth factors to enhance bone and soft tissue grafts. NY State Dent J 2002;68:22-4.

17. Ducy P, Schinke T, Karsenty G. The osteoblast: a sophisticated fibroblast under central surveillance. Science 2000;289:1501-4.

18. Harada S-I, Rodan GA. Control of osteoblast function and regulation of bone mass. Nature 2003;423:349-55.

19. Oprea We, Karp JM, Hosseini MM, et al., Effect of platelet releasate on bone cell migration and recruitment in vitro. J Craniofac Surg 2003;14:292-300.

20. Tang YQ, Yeaman MR, Selsted Me. Antimicrobial peptides from human platelets. Infect Immun 2002;70: 6515-7.

21. Burnstock G. Purinergic signaling and vascular cell proliferation and death. Arterioscler Thromb Vasc Biol 2002;10:271-85.

22. Schober a, Manka D, von Hundelshausen P, et al., Deposition of platelet Rantes triggering monocyte recruitment requires P-selectin and is involved in neointima formation after arterial injury. Circulation 2002;106:1523-9.

23. Anitua E, Andia Ortiz I. BTI implant system: The first implant system with a bioactive surface. Maxillaries 2001;39:2-7.

24. Zechner W, Tangl S, Tepper G, et at., Influence of platelet-rich plasma on osseous healing of dental implants: A histologic and histomorphometric study in minipigs. Int J Oral Maxillofac Implants 2003;18:15-22.

Photos

FIGURE 3--Four red top tubes with plasma and two red top tubes with whole blood ready for centrifugation.
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FIGURE 4--Platelet Poor Plasma (PPP) above Platelet Rich Plasma (PRP) after second centrifugation.
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FIGURE 5A --Serum being removed from whole blood after centrifugation.
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Caption: FIGURE 5A --Serum being removed from whole blood after ...
FIGURE 5B--Fibrin clot left behind.
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Caption: FIGURE 5B--Fibrin clot left behind.
FIGURE 6--PRP, PPP, serum and calcium cloride placed in surgical operatory for activation in dappen dishes.
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FIGURE 7--Four dappen dishes each with 1ml PRP.
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Caption: FIGURE 7--Four dappen dishes each with 1ml PRP.
FIGURE 8--PRP clot formed in second dish after activation with calcium chloride and autologous thrombin.
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FIGURE 9--PRP clot combined with autogenous bone after activation with calcium chloride and autologous thrombin.
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Caption: FIGURE 9--PRP clot combined with autogenous bone after ...
FIGURE 10--PRP clot combined with allograft after activation with calcium chloride and autologous thrombin.
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Caption: FIGURE 10--PRP clot combined with allograft after activ...
FIGURE 11--Activation of PPP with autologous thrombin.
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Caption: FIGURE 11--Activation of PPP with autologous thrombin.
FIGURE 1A--Two red top tubes with whole blood and four yellow top.
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Caption: FIGURE 1A--Two red top tubes with whole blood and four ...
FIGURE 1B--Four yellow top tubes ready for first centrifugation spin.
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Caption: FIGURE 1B--Four yellow top tubes ready for first centri...
FIGURE 2--Plasma with platelets above red blood cells after first centrifugation.
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