Platelet-Rich Plasma: A Study of the Variables that May Influence Its Effect on Bone Regeneration Mª del Mar Jovani-Sancho, DDS, PhD;* Chirag C Sheth, BSc, PhD;† Mariano Marqués-Mateo, MD, PhD;‡ Miguel Puche-Torres, MD, PhD§
ABSTRACT Background: Currently, the use of platelet-rich plasma in bone regeneration is a real option, although more than one opinion has alerted us to the absence of clinical benefits. Purpose: Analysis of the factors able to modify the characteristics of the platelet preparation obtained by Curasan, Plasma Rich in Growth Factors (PRGF), Platelet Concentrate Collection System (PCCS) and SmartPrep systems, relating them to the type of clinical application and the final bone regeneration achieved. Materials and Methods: A search was conducted in PubMed using the keywords “platelet-rich plasma,” “PRP,” “platelet rich growth factors,” and “oral bone regeneration.” Four widely accepted protocols for the obtention of PRP (above) were analyzed. Any clinical studies with controls, using the four preparation protocols and with a 4 to 6 weeks follow-up period were compared. The protocols were also grouped according to the type of PRP application: PRP-alone, with bone, or with bone substitutes. Results: Bone regeneration was not achieved in any of the cases using PRP obtained by Curasan and PCCS systems, whereas PRP obtained by SmartPrep achieved it only in one in three published cases and PRGF in one in six. Conclusion: Based on the poor results observed in current literature, the use of PRP in oral surgery cannot be recommended. KEY WORDS: oral bone regeneration, platelet-rich growth factors, platelet-rich plasma, PRP
INTRODUCTION
several methods to achieve it, such as guided tissue regeneration, the use of bone substitutes, or even morpho proteins. During the last two decades, a new option appeared to have been developed: the application of platelet-rich plasma preparations in the area to be regenerated. This technique described a novel technology, as it was believed that platelets only acted in tissue hemostasis, when in fact it was known that they may also play a role in wound reparation. Development of the technique employing plateletrich plasma in medical procedures was preceded by several key advancements in the field of bio-reparation of damaged tissue. Early biomaterials included fibrin (nonautologous) glue, succeeded by the development of autologous glue and ultimately to the elaboration of platelet gel. Fibrin glue was developed to satisfy the increasing demand for advanced hemostatic agents and adhesive agents used in surgical procedures. Fibrin glue may be described in broad terms as a clot consisting of a
Due to the high resilience of oral tissues, small alveolar or periodontal defects recover with little difficulty; however, if the affected area is larger, regeneration may be incomplete, which could lead to nonfunctional tissue and even necrosis.1 The need for both oral surgeons and dentists to improve bone regeneration has led to the appearance of *Head of the Department of Dentistry, Faculty of Health Sciences, Universidad CEU Cardenal Herrera, Valencia, Spain; †senior lecturer, Department of Biomedical Sciences, Faculty of Health Sciences, Universidad CEU Cardenal Herrera, Valencia, Spain; ‡associate professor, Department of Oral and Maxillofacial Surgery, Hospital Clínico Universitario, Valencia, Spain; §head of Department of Oral and Maxillofacial Surgery, Hospital Clínico Universitario, Universidad de Valencia, Valencia, Spain Corresponding Author: Prof. Mª del Mar Jovani-Sancho, C/Del pozo s/n, Alfara del Patriarca 46115, Valencia, Spain; e-mail: [email protected] © 2015 Wiley Periodicals, Inc. DOI 10.1111/cid.12361
1
2
Clinical Implant Dentistry and Related Research, Volume *, Number *, 2015
mixture of fibrinogen, factor XIII, fibronectin, thrombin, calcium chloride, and inhibitors of fibrinolysis. The source of these factors may be single or pooled donors, derived from blood banks or directly from the patient.2 In oral medicine, fibrin glue has been used as a surgical preparation, employed to improve tissue sealing, wound healing, and hemostasis.3 The increased risk of viral transmission through the use of donor blood in the formation of the fibrin clots has been described as a key factor preventing it from being used more widely.4 The trends in prevention of infectious disease transmission2 played a role in promoting the use of autologous patient blood (in spite of requiring additional visits to the hospital for blood donation prior to surgery) to create fibrin clots for use in oral surgical procedures.5 Platelet gel, first described in 1997, requires the extraction and processing of the patient’s own blood immediately prior to the operation and is formed by combining platelet-rich plasma, thrombin, and calcium chloride. Platelet gel is characterized as having higher concentrations of platelets and fibrinogen as compared with the fibrin gel described earlier.2 The advantage of the increased concentration of platelets lies in the elevated level of growth factors released following activation. Growth factors play a principal role in tissue repair by increasing mitosis, the production of collagen and promoting cellular differentiation. The release of growth factors from platelets has been shown to be related to the number of platelets present in the clot; therefore, it could be surmised that augmenting the platelet number should result in an elevation of the concentration of growth factors. Based on this premise, it may be extrapolated that increasing the number of platelets would result in more effective regeneration of damaged tissue.6 Its handling is easy due to its gelatinous consistency, and being autologous to the patient, the risk of disease transmission or immune reaction is eliminated.7 It also reduces postoperative bleeding, promotes faster healing of soft tissue, aids in graft stability, and promotes a faster vascularization.8 Recently, it has been used to enhance the regeneration of soft and hard tissues in periodontics, oral, and maxillofacial surgery.9 There are, however, a number of questions that may concern the clinician when considering applying this technique. Is greater and better bone regeneration really achieved with these preparations? Should they be mixed
with bone grafts to take effect? Which protocol gives the best results? What are the economic implications? While trying to answer these questions by means of a literature review, we uncovered contradictions in the process of preparing the PRP, type of coagulation and results obtained, and more than one opinion warns about the dearth of clinical advantages or even possible nondesirable effects. These contradictions have led to this analysis of the current state of platelet-rich plasma preparations and their effect when applied to bone regeneration in the oral cavity. Many reviews have been published on its application in the dental and maxillofacial field6,10–20 but none has taken into account the factors that can modify the characteristics of the final platelet preparation, the different forms of its application, and their relation with the final bone regeneration. The following review attempts to analyze each of these factors in depth and, if possible, recommend a PRP preparation protocol that can be applied in a nonhospital setting such as the dental clinic with a certain guarantee of success. MATERIALS AND METHODS First a PubMed search was conducted using the following keywords: “platelet-rich plasma,”“PRP,”“platelet rich growth factors,” and “oral bone regeneration.” Both in vitro and in vivo studies, case reports, and literature reviews were assessed. Following this, the references from previously selected articles were analyzed, and those relating to critical methods of obtention and application of PRP in relation to bone regeneration were also included. The articles finally accepted, ranged between 1997 and 2014, are listed in the bibliography section. RESULTS When reviewing the literature, it is surprising to see nearly as many protocols for the preparation of platelet-rich plasma as authors who use them. This high variability suggests the widespread application of nonstandardized products with different biological potentials, and it forces us to evaluate the many factors that can affect the quality of these products. The preparation of PRP involves drawing blood from a patient in tubes containing anticoagulant, its centrifugation, the separation of the plasma fraction with the highest content of platelets and the subsequent
Coagulant Growth factors concentration
4. Applicaon
Method Cell content Platelet enrichment
3. Coagulaon
Anticoagulant
2. PRP obtenon
1.Blood drawing
PRP Variables that Affect Bone Regeneration
3
PRP + autologous bone PRP + bovine bone PRP+ bone substitutes PRP alone
Figure 1 Stages and independent variables involved in the process of obtaining and manipulating platelet-rich plasma preparations as applied to bone regeneration in the oral cavity.
coagulation process leading to degranulation of the platelets, and the resulting release of growth factors. Together with the preparation method, the application procedure in the area to be regenerated must also be taken into account, as its effectiveness may vary depending on whether it is applied alone, with bone or in combination with bone substitutes (Figure 1). These differences in preparation methodology and in the placement process could explain the different results in the literature on this topic.
can be divided into two main categories: “Leucocytepoor or pure platelet-rich plasma” (P-PRP) and “Leucocyte- and platelet-rich plasma” (L-PRP)-. P-PRP is a blood fraction consisting of plasma with an undetermined volume of the buffy coat fraction (layer of leukocytes and platelets found between plasma and red blood cells once the blood has been centrifuged), whereas L-PRP contains plasma, the entire buffy coat, and residual red blood cells.16 The classification of these preparations as first described by Dohan Ehrenfest and colleagues16 are summarized in Figure 2.
Blood Extraction The first step in obtaining platelet-rich plasma is to draw blood from the patient, which must be immediately anticoagulated. Anticoagulation of the Sample. The anticoagulants commonly used in the process of drawing blood are: sodium citrate, citrate phosphate dextrose (CPD) solution, citrate phosphate dextrose solution with adenine (CPDA), citric acid, sodium citrate and dextrose (ACD), and ethylenediaminetetraacetic acid (EDTA) (Table 1). When reviewing their characteristics it can be noted that sodium citrate and ACD are the safest and that the use of ethylenediaminetetraacetic acid would not be advisable due to its potentially harmful effects on platelets. Some authors consider EDTA to be directly responsible for the lack of effectiveness of PRP.9 Centrifugation Process Methods of Obtention. Many protocols have been presented in the literature for the preparation of PRP, according to the cellular processing used and the initial volume of blood. Broadly speaking, PRP-preparations
P-PRP Obtention Protocols. The preparation of P-PRP can be further divided into two categories according to the separation technique; automatic, and manual protocols (Figure 3).
TABLE 1 Key Characteristics of Commonly Used Anticoagulants Anticoagulant
Observations, For and Against
Sodium citrate Good anticoagulant. Does not alter the membrane receptors of platelets.23 ACD Good anticoagulant. Maintains the viability of platelets.49 CPD Good anticoagulant although 10% less effective in maintaining platelet viability than ACD.49 EDTA Poor anticoagulant. Fragments platelets causing them to loose activity.21,56 Produces a high degree of platelet inhibition limiting their subsequent activation.20
4
Clinical Implant Dentistry and Related Research, Volume *, Number *, 2015
Automatic protocols
Plasmapheresis
Manual protocols
PRGF System
PRP
P-PRP
Manual Protocols: •
PCCS Automatic protocols SmartPrep
L-PRP Manual protocols
Curasan System
Figure 2 Methods for obtaining platelet-rich plasma preparations to be applied locally depending on the cellular content of the final preparation: leucocyte-poor or pure platelet-rich plasma (P-PRP), containing plasma with a buffy coat fraction, and leukocyte and platelet-rich plasma or L-PRP, containing plasma, buffy coat and residual red blood cells.
Automatic Protocols: •
Plasmapheresis by density gradient separation: Pure platelet-rich plasma is obtained from large amounts of blood (250–450 ml) days to minutes prior to surgery. It has some disadvantages such as the equipment required is expensive and large amounts of blood are needed in order to obtain even very small quantities of P-PRP,21,22 therefore its use in the outpatient setting, to which we refer in this review, is impractical.22
Plasma Rich in Growth Factors (PRGF) System, BioTechnology Institute (BTI; Vitoria, Spain): 5 to 40 ml of blood are drawn from patients in 3.8% sodium citrate-containing tubes, it is centrifuged once at 160, 270, 280, 460, or 580 × g (according to different publications) for 6 to 8 minutes.23–30 Following this, the upper part of the acellular plasma, called plasma poor in growth factors (PPGF), is separated by pipetting, and 50 μl of 10 % calcium chloride is added per ml of PRGF to obtain the clot. Some authors have also used PRGF activator® for this purpose.28,29 Fifteen to twenty minutes following the addition of calcium chloride, an unstable gel is formed that needs to be used immediately (Figure 3). After studying the evolution of this technique, inconsistencies are observed not only in centrifugation parameters, but also in what authors consider PRGF. According to the initial protocol described by Anitua in 199923 PRGF consists of the plasma portion nearest to the red blood cells by the buffy coat, whereas in subsequent applications, authors avoid this layer to prevent the inclusion of leukocytes in the preparation. Taking into account
PRGF System
PCCS, SmartPrep
Curasan System
Conventional centrifuge
High-cost centrifuges
Conventional centrifuge
Possible bacterial contamination
Unlikely bacterial contamination
Possible bacterial contamination
Simple manipulation
Complex manipulation
Simple manipulation
Slow method (15 - 25')
Slow method (15'-35')
Slow method (25')
Single centrifugation
Double centrifugation
Double centrifugation
Reasonable cost
High cost
Reasonable cost
Poor reproducibility
Reproducible
Poor reproducibility
Figure 3 Comparison of the characteristics of the systems studied for the obtention of PRP in a nonhospital setting.
PRP Variables that Affect Bone Regeneration
that this is technically imprecise and difficult to reproduce due to pipetting variability, we suggest that P-PRP as described above may in reality be described better as L-PRP poor in leukocytes.16 As we can observe, although it is an inexpensive protocol, it is imprecise and nonreproducible, and as it is a technique that uses a slow centrifuge, it will generate very low platelet concentrations. L-PRP Obtention Protocols Automatic Protocols: The two automatic protocols described below share a common base. The centrifuges used have been designed to accept a customized collection and centrifugation device, consisting of two connected compartments.16 •
•
Platelet Concentrate Coleccion System (PCCS) 3i, Implant Innovations, Palm Beach Gardens, FL (USA). The principal steps in this technique are the separation by centrifugation of blood drawn into 3.8% citrate-containing tubes in order to obtain three distinct layers (red cells, buffy coat, plateletpoor plasma [PPP]). Then, via the opening of a tubule under air pressure, the superficial layers (PPP and Buffy Coat (BC)) are transferred to the second chamber and centrifuged again but for a longer period. Finally, using the same air pressure system, most of the PPP layer is transferred back into the first compartment and thus discarded (Figure 3). The final product is rich in leucocytes.16 Harvest SmartPrep Platelet Concentrate System (SmartPrep) (Harvest Technologies, Plymouth, MA, USA): This protocol requires less manipulation than the prior technique. The two-chamber device is designed to automatically transfer the upper layers (PPP and BC) into the second chamber based on variations in weight and centrifugation speed (Figure 3).16 Small volumes of blood are processed, taking approximately 15 minutes to complete, with 5 to 6 ml of L-PRP obtained from approximately 45 to 60 ml of starting material.31,32
5
blood cells, BC, and PPP. Both BC and PPP are re-centrifuged at 2000 × g for 15 minutes and, following the removal of PPP by pipetting, L-PRP is obtained (Figure 3).33 The composition of the final preparation is unclear as, if the buffy coat is not completely included, pure platelet-rich plasma may be obtained instead of L-PRP. As this is a manual process, its success is highly operator-dependent, and the results may be poorly reproducible. Bovine thrombin and calcium chloride is finally applied to the concentrated mixture to induce coagulation.16 Cell Content of the Preparation Although Peñarrocha and colleagues31 describe the exact content of the PRP clot, which has 4% red cells, 95% platelets, and 1% leukocytes, there is no unanimity as to whether it is appropriate or not to include nonplatelet cell elements (Table 2). Dohan Ehrenfest and colleagues16 PRP classification is based on this concept, where they differentiate pure PRP from leukocyte and platelet-rich plasma. The truth is that the collection of PRP by pipette is very imprecise and commonly results in the accidental inclusion of leukocytes and red blood cells, so that, in reality, the final cell content will depend on the operator’s skills.34 This makes the evaluation of the PRP obtained in different studies complex and often we do not know if L-PRP or P-PRP was used. The main difference between the two preparations is the leukocyte content and the clinicians’ concerns because of the possible effect they may have on cell proliferation and differentiation, immunity, and possible infections. In relation to this point, it must be taken into account that L-PRP has already been used in coronary surgery, on articulations and tendon remodeling, without encountering uncontrolled immune reactions. On the contrary, it seems that it is able to reduce pain and inflammation of the treated areas. Although further studies are needed to clarify the contribution of leukocytes to the platelet concentrate, synergistic effects are being hypothesized.16
Manual Protocols:
Platelet Enrichment
•
Platelet enrichment is observed in the preparations obtained to different degrees, depending on the obtention method used and may be calculated by dividing the final platelet count by the initial count.
Curasan System (Kleinostheim, Germany): This protocol uses a two-step centrifugation procedure. A first centrifugation is made at 900 × g for 10 minutes, and the blood divided into three parts, red
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Clinical Implant Dentistry and Related Research, Volume *, Number *, 2015
TABLE 2 Possible Cellular Content of PRP in Clots of a Reddish Colour and Author’s Justification Cellular Content
L-PRP (“Red clot”): • Red cells, white cells and platelets
P-PRP (“White clot”) • White cells and platelets • Platelets
Justification
Supporting Authors
1–2 mm of red blood cells are included because platelets that have more recently been synthesized and therefore are larger and have greater activity, are mixed with the uppermost millimeter of red blood cells.
Marx and colleagues53, Anitua23, Lekovic and colleagues57, Sammartino and colleagues58, Camargo and colleagues59, Andrade and colleagues60, Gawandde and Halli61, Pradeep and colleagues62, Moghe and colleagues63, Ramanathan and Cariappa64 Dugrillon and colleagues43, Soffer and colleagues11 Anitua and colleagues26,28,29, Taschieri and colleagues65, Dragoo and colleagues66, McCarrel and colleagues67
Leukocytes are included as they contain and produce growth factors, which will influence the final number. White cells can interfere with platelet aggregation. By not including them, proinflammatory effects of proteases and acid hydrolases contained in them are avoided. Leukocytes may produce local inflammation, extending postoperative pain.
Different platelet concentrations produce different effects on bone regeneration. Very low and very high concentration preparations do not seem to increase bone regeneration substantially and may actually have a cytotoxic effect on osteoblasts. Best results are obtained by medium concentration preparations, with a very limited range, of around 1 000 000 platelets/μl (four times the platelet concentration in blood, which means a platelet increment of 400%) (Table 3). Although all protocols included in the following review obtain this increment (Table 4), it is noteworthy that within the same protocols value ranges are very broad. These variations are greater in systems in which PRP is obtained manually (Curasan System and PRGF System) and may be due to manipulation errors, which leads to a lack of
reproducibility of protocols. Systems that obtain PRP automatically (PCCS and SmartPrep) are more predictable and reliably achieve greater platelet increments. Coagulation of the Final Product Preparation of the PRP extract is followed by a coagulation step in which the coagulation of platelets will produce their degranulation and the subsequent release of growth factors (Figure 1). The key independent variable in this step is the choice of coagulant used, with the type and concentration of growth factors produced as the primary outcome variable. Coagulants. A widely accepted coagulant has been 10% calcium chloride. The calcium will intervene in the
TABLE 3 The Effects of PRP Concentration on Bone Regeneration PRP Platelet Concentration
Low Medium
High
0.5–1.5×
ABSTRACT Background: Currently, the use of platelet-rich plasma in bone regeneration is a real option, although more than one opinion has alerted us to the absence of clinical benefits. Purpose: Analysis of the factors able to modify the characteristics of the platelet preparation obtained by Curasan, Plasma Rich in Growth Factors (PRGF), Platelet Concentrate Collection System (PCCS) and SmartPrep systems, relating them to the type of clinical application and the final bone regeneration achieved. Materials and Methods: A search was conducted in PubMed using the keywords “platelet-rich plasma,” “PRP,” “platelet rich growth factors,” and “oral bone regeneration.” Four widely accepted protocols for the obtention of PRP (above) were analyzed. Any clinical studies with controls, using the four preparation protocols and with a 4 to 6 weeks follow-up period were compared. The protocols were also grouped according to the type of PRP application: PRP-alone, with bone, or with bone substitutes. Results: Bone regeneration was not achieved in any of the cases using PRP obtained by Curasan and PCCS systems, whereas PRP obtained by SmartPrep achieved it only in one in three published cases and PRGF in one in six. Conclusion: Based on the poor results observed in current literature, the use of PRP in oral surgery cannot be recommended. KEY WORDS: oral bone regeneration, platelet-rich growth factors, platelet-rich plasma, PRP
INTRODUCTION
several methods to achieve it, such as guided tissue regeneration, the use of bone substitutes, or even morpho proteins. During the last two decades, a new option appeared to have been developed: the application of platelet-rich plasma preparations in the area to be regenerated. This technique described a novel technology, as it was believed that platelets only acted in tissue hemostasis, when in fact it was known that they may also play a role in wound reparation. Development of the technique employing plateletrich plasma in medical procedures was preceded by several key advancements in the field of bio-reparation of damaged tissue. Early biomaterials included fibrin (nonautologous) glue, succeeded by the development of autologous glue and ultimately to the elaboration of platelet gel. Fibrin glue was developed to satisfy the increasing demand for advanced hemostatic agents and adhesive agents used in surgical procedures. Fibrin glue may be described in broad terms as a clot consisting of a
Due to the high resilience of oral tissues, small alveolar or periodontal defects recover with little difficulty; however, if the affected area is larger, regeneration may be incomplete, which could lead to nonfunctional tissue and even necrosis.1 The need for both oral surgeons and dentists to improve bone regeneration has led to the appearance of *Head of the Department of Dentistry, Faculty of Health Sciences, Universidad CEU Cardenal Herrera, Valencia, Spain; †senior lecturer, Department of Biomedical Sciences, Faculty of Health Sciences, Universidad CEU Cardenal Herrera, Valencia, Spain; ‡associate professor, Department of Oral and Maxillofacial Surgery, Hospital Clínico Universitario, Valencia, Spain; §head of Department of Oral and Maxillofacial Surgery, Hospital Clínico Universitario, Universidad de Valencia, Valencia, Spain Corresponding Author: Prof. Mª del Mar Jovani-Sancho, C/Del pozo s/n, Alfara del Patriarca 46115, Valencia, Spain; e-mail: [email protected] © 2015 Wiley Periodicals, Inc. DOI 10.1111/cid.12361
1
2
Clinical Implant Dentistry and Related Research, Volume *, Number *, 2015
mixture of fibrinogen, factor XIII, fibronectin, thrombin, calcium chloride, and inhibitors of fibrinolysis. The source of these factors may be single or pooled donors, derived from blood banks or directly from the patient.2 In oral medicine, fibrin glue has been used as a surgical preparation, employed to improve tissue sealing, wound healing, and hemostasis.3 The increased risk of viral transmission through the use of donor blood in the formation of the fibrin clots has been described as a key factor preventing it from being used more widely.4 The trends in prevention of infectious disease transmission2 played a role in promoting the use of autologous patient blood (in spite of requiring additional visits to the hospital for blood donation prior to surgery) to create fibrin clots for use in oral surgical procedures.5 Platelet gel, first described in 1997, requires the extraction and processing of the patient’s own blood immediately prior to the operation and is formed by combining platelet-rich plasma, thrombin, and calcium chloride. Platelet gel is characterized as having higher concentrations of platelets and fibrinogen as compared with the fibrin gel described earlier.2 The advantage of the increased concentration of platelets lies in the elevated level of growth factors released following activation. Growth factors play a principal role in tissue repair by increasing mitosis, the production of collagen and promoting cellular differentiation. The release of growth factors from platelets has been shown to be related to the number of platelets present in the clot; therefore, it could be surmised that augmenting the platelet number should result in an elevation of the concentration of growth factors. Based on this premise, it may be extrapolated that increasing the number of platelets would result in more effective regeneration of damaged tissue.6 Its handling is easy due to its gelatinous consistency, and being autologous to the patient, the risk of disease transmission or immune reaction is eliminated.7 It also reduces postoperative bleeding, promotes faster healing of soft tissue, aids in graft stability, and promotes a faster vascularization.8 Recently, it has been used to enhance the regeneration of soft and hard tissues in periodontics, oral, and maxillofacial surgery.9 There are, however, a number of questions that may concern the clinician when considering applying this technique. Is greater and better bone regeneration really achieved with these preparations? Should they be mixed
with bone grafts to take effect? Which protocol gives the best results? What are the economic implications? While trying to answer these questions by means of a literature review, we uncovered contradictions in the process of preparing the PRP, type of coagulation and results obtained, and more than one opinion warns about the dearth of clinical advantages or even possible nondesirable effects. These contradictions have led to this analysis of the current state of platelet-rich plasma preparations and their effect when applied to bone regeneration in the oral cavity. Many reviews have been published on its application in the dental and maxillofacial field6,10–20 but none has taken into account the factors that can modify the characteristics of the final platelet preparation, the different forms of its application, and their relation with the final bone regeneration. The following review attempts to analyze each of these factors in depth and, if possible, recommend a PRP preparation protocol that can be applied in a nonhospital setting such as the dental clinic with a certain guarantee of success. MATERIALS AND METHODS First a PubMed search was conducted using the following keywords: “platelet-rich plasma,”“PRP,”“platelet rich growth factors,” and “oral bone regeneration.” Both in vitro and in vivo studies, case reports, and literature reviews were assessed. Following this, the references from previously selected articles were analyzed, and those relating to critical methods of obtention and application of PRP in relation to bone regeneration were also included. The articles finally accepted, ranged between 1997 and 2014, are listed in the bibliography section. RESULTS When reviewing the literature, it is surprising to see nearly as many protocols for the preparation of platelet-rich plasma as authors who use them. This high variability suggests the widespread application of nonstandardized products with different biological potentials, and it forces us to evaluate the many factors that can affect the quality of these products. The preparation of PRP involves drawing blood from a patient in tubes containing anticoagulant, its centrifugation, the separation of the plasma fraction with the highest content of platelets and the subsequent
Coagulant Growth factors concentration
4. Applicaon
Method Cell content Platelet enrichment
3. Coagulaon
Anticoagulant
2. PRP obtenon
1.Blood drawing
PRP Variables that Affect Bone Regeneration
3
PRP + autologous bone PRP + bovine bone PRP+ bone substitutes PRP alone
Figure 1 Stages and independent variables involved in the process of obtaining and manipulating platelet-rich plasma preparations as applied to bone regeneration in the oral cavity.
coagulation process leading to degranulation of the platelets, and the resulting release of growth factors. Together with the preparation method, the application procedure in the area to be regenerated must also be taken into account, as its effectiveness may vary depending on whether it is applied alone, with bone or in combination with bone substitutes (Figure 1). These differences in preparation methodology and in the placement process could explain the different results in the literature on this topic.
can be divided into two main categories: “Leucocytepoor or pure platelet-rich plasma” (P-PRP) and “Leucocyte- and platelet-rich plasma” (L-PRP)-. P-PRP is a blood fraction consisting of plasma with an undetermined volume of the buffy coat fraction (layer of leukocytes and platelets found between plasma and red blood cells once the blood has been centrifuged), whereas L-PRP contains plasma, the entire buffy coat, and residual red blood cells.16 The classification of these preparations as first described by Dohan Ehrenfest and colleagues16 are summarized in Figure 2.
Blood Extraction The first step in obtaining platelet-rich plasma is to draw blood from the patient, which must be immediately anticoagulated. Anticoagulation of the Sample. The anticoagulants commonly used in the process of drawing blood are: sodium citrate, citrate phosphate dextrose (CPD) solution, citrate phosphate dextrose solution with adenine (CPDA), citric acid, sodium citrate and dextrose (ACD), and ethylenediaminetetraacetic acid (EDTA) (Table 1). When reviewing their characteristics it can be noted that sodium citrate and ACD are the safest and that the use of ethylenediaminetetraacetic acid would not be advisable due to its potentially harmful effects on platelets. Some authors consider EDTA to be directly responsible for the lack of effectiveness of PRP.9 Centrifugation Process Methods of Obtention. Many protocols have been presented in the literature for the preparation of PRP, according to the cellular processing used and the initial volume of blood. Broadly speaking, PRP-preparations
P-PRP Obtention Protocols. The preparation of P-PRP can be further divided into two categories according to the separation technique; automatic, and manual protocols (Figure 3).
TABLE 1 Key Characteristics of Commonly Used Anticoagulants Anticoagulant
Observations, For and Against
Sodium citrate Good anticoagulant. Does not alter the membrane receptors of platelets.23 ACD Good anticoagulant. Maintains the viability of platelets.49 CPD Good anticoagulant although 10% less effective in maintaining platelet viability than ACD.49 EDTA Poor anticoagulant. Fragments platelets causing them to loose activity.21,56 Produces a high degree of platelet inhibition limiting their subsequent activation.20
4
Clinical Implant Dentistry and Related Research, Volume *, Number *, 2015
Automatic protocols
Plasmapheresis
Manual protocols
PRGF System
PRP
P-PRP
Manual Protocols: •
PCCS Automatic protocols SmartPrep
L-PRP Manual protocols
Curasan System
Figure 2 Methods for obtaining platelet-rich plasma preparations to be applied locally depending on the cellular content of the final preparation: leucocyte-poor or pure platelet-rich plasma (P-PRP), containing plasma with a buffy coat fraction, and leukocyte and platelet-rich plasma or L-PRP, containing plasma, buffy coat and residual red blood cells.
Automatic Protocols: •
Plasmapheresis by density gradient separation: Pure platelet-rich plasma is obtained from large amounts of blood (250–450 ml) days to minutes prior to surgery. It has some disadvantages such as the equipment required is expensive and large amounts of blood are needed in order to obtain even very small quantities of P-PRP,21,22 therefore its use in the outpatient setting, to which we refer in this review, is impractical.22
Plasma Rich in Growth Factors (PRGF) System, BioTechnology Institute (BTI; Vitoria, Spain): 5 to 40 ml of blood are drawn from patients in 3.8% sodium citrate-containing tubes, it is centrifuged once at 160, 270, 280, 460, or 580 × g (according to different publications) for 6 to 8 minutes.23–30 Following this, the upper part of the acellular plasma, called plasma poor in growth factors (PPGF), is separated by pipetting, and 50 μl of 10 % calcium chloride is added per ml of PRGF to obtain the clot. Some authors have also used PRGF activator® for this purpose.28,29 Fifteen to twenty minutes following the addition of calcium chloride, an unstable gel is formed that needs to be used immediately (Figure 3). After studying the evolution of this technique, inconsistencies are observed not only in centrifugation parameters, but also in what authors consider PRGF. According to the initial protocol described by Anitua in 199923 PRGF consists of the plasma portion nearest to the red blood cells by the buffy coat, whereas in subsequent applications, authors avoid this layer to prevent the inclusion of leukocytes in the preparation. Taking into account
PRGF System
PCCS, SmartPrep
Curasan System
Conventional centrifuge
High-cost centrifuges
Conventional centrifuge
Possible bacterial contamination
Unlikely bacterial contamination
Possible bacterial contamination
Simple manipulation
Complex manipulation
Simple manipulation
Slow method (15 - 25')
Slow method (15'-35')
Slow method (25')
Single centrifugation
Double centrifugation
Double centrifugation
Reasonable cost
High cost
Reasonable cost
Poor reproducibility
Reproducible
Poor reproducibility
Figure 3 Comparison of the characteristics of the systems studied for the obtention of PRP in a nonhospital setting.
PRP Variables that Affect Bone Regeneration
that this is technically imprecise and difficult to reproduce due to pipetting variability, we suggest that P-PRP as described above may in reality be described better as L-PRP poor in leukocytes.16 As we can observe, although it is an inexpensive protocol, it is imprecise and nonreproducible, and as it is a technique that uses a slow centrifuge, it will generate very low platelet concentrations. L-PRP Obtention Protocols Automatic Protocols: The two automatic protocols described below share a common base. The centrifuges used have been designed to accept a customized collection and centrifugation device, consisting of two connected compartments.16 •
•
Platelet Concentrate Coleccion System (PCCS) 3i, Implant Innovations, Palm Beach Gardens, FL (USA). The principal steps in this technique are the separation by centrifugation of blood drawn into 3.8% citrate-containing tubes in order to obtain three distinct layers (red cells, buffy coat, plateletpoor plasma [PPP]). Then, via the opening of a tubule under air pressure, the superficial layers (PPP and Buffy Coat (BC)) are transferred to the second chamber and centrifuged again but for a longer period. Finally, using the same air pressure system, most of the PPP layer is transferred back into the first compartment and thus discarded (Figure 3). The final product is rich in leucocytes.16 Harvest SmartPrep Platelet Concentrate System (SmartPrep) (Harvest Technologies, Plymouth, MA, USA): This protocol requires less manipulation than the prior technique. The two-chamber device is designed to automatically transfer the upper layers (PPP and BC) into the second chamber based on variations in weight and centrifugation speed (Figure 3).16 Small volumes of blood are processed, taking approximately 15 minutes to complete, with 5 to 6 ml of L-PRP obtained from approximately 45 to 60 ml of starting material.31,32
5
blood cells, BC, and PPP. Both BC and PPP are re-centrifuged at 2000 × g for 15 minutes and, following the removal of PPP by pipetting, L-PRP is obtained (Figure 3).33 The composition of the final preparation is unclear as, if the buffy coat is not completely included, pure platelet-rich plasma may be obtained instead of L-PRP. As this is a manual process, its success is highly operator-dependent, and the results may be poorly reproducible. Bovine thrombin and calcium chloride is finally applied to the concentrated mixture to induce coagulation.16 Cell Content of the Preparation Although Peñarrocha and colleagues31 describe the exact content of the PRP clot, which has 4% red cells, 95% platelets, and 1% leukocytes, there is no unanimity as to whether it is appropriate or not to include nonplatelet cell elements (Table 2). Dohan Ehrenfest and colleagues16 PRP classification is based on this concept, where they differentiate pure PRP from leukocyte and platelet-rich plasma. The truth is that the collection of PRP by pipette is very imprecise and commonly results in the accidental inclusion of leukocytes and red blood cells, so that, in reality, the final cell content will depend on the operator’s skills.34 This makes the evaluation of the PRP obtained in different studies complex and often we do not know if L-PRP or P-PRP was used. The main difference between the two preparations is the leukocyte content and the clinicians’ concerns because of the possible effect they may have on cell proliferation and differentiation, immunity, and possible infections. In relation to this point, it must be taken into account that L-PRP has already been used in coronary surgery, on articulations and tendon remodeling, without encountering uncontrolled immune reactions. On the contrary, it seems that it is able to reduce pain and inflammation of the treated areas. Although further studies are needed to clarify the contribution of leukocytes to the platelet concentrate, synergistic effects are being hypothesized.16
Manual Protocols:
Platelet Enrichment
•
Platelet enrichment is observed in the preparations obtained to different degrees, depending on the obtention method used and may be calculated by dividing the final platelet count by the initial count.
Curasan System (Kleinostheim, Germany): This protocol uses a two-step centrifugation procedure. A first centrifugation is made at 900 × g for 10 minutes, and the blood divided into three parts, red
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Clinical Implant Dentistry and Related Research, Volume *, Number *, 2015
TABLE 2 Possible Cellular Content of PRP in Clots of a Reddish Colour and Author’s Justification Cellular Content
L-PRP (“Red clot”): • Red cells, white cells and platelets
P-PRP (“White clot”) • White cells and platelets • Platelets
Justification
Supporting Authors
1–2 mm of red blood cells are included because platelets that have more recently been synthesized and therefore are larger and have greater activity, are mixed with the uppermost millimeter of red blood cells.
Marx and colleagues53, Anitua23, Lekovic and colleagues57, Sammartino and colleagues58, Camargo and colleagues59, Andrade and colleagues60, Gawandde and Halli61, Pradeep and colleagues62, Moghe and colleagues63, Ramanathan and Cariappa64 Dugrillon and colleagues43, Soffer and colleagues11 Anitua and colleagues26,28,29, Taschieri and colleagues65, Dragoo and colleagues66, McCarrel and colleagues67
Leukocytes are included as they contain and produce growth factors, which will influence the final number. White cells can interfere with platelet aggregation. By not including them, proinflammatory effects of proteases and acid hydrolases contained in them are avoided. Leukocytes may produce local inflammation, extending postoperative pain.
Different platelet concentrations produce different effects on bone regeneration. Very low and very high concentration preparations do not seem to increase bone regeneration substantially and may actually have a cytotoxic effect on osteoblasts. Best results are obtained by medium concentration preparations, with a very limited range, of around 1 000 000 platelets/μl (four times the platelet concentration in blood, which means a platelet increment of 400%) (Table 3). Although all protocols included in the following review obtain this increment (Table 4), it is noteworthy that within the same protocols value ranges are very broad. These variations are greater in systems in which PRP is obtained manually (Curasan System and PRGF System) and may be due to manipulation errors, which leads to a lack of
reproducibility of protocols. Systems that obtain PRP automatically (PCCS and SmartPrep) are more predictable and reliably achieve greater platelet increments. Coagulation of the Final Product Preparation of the PRP extract is followed by a coagulation step in which the coagulation of platelets will produce their degranulation and the subsequent release of growth factors (Figure 1). The key independent variable in this step is the choice of coagulant used, with the type and concentration of growth factors produced as the primary outcome variable. Coagulants. A widely accepted coagulant has been 10% calcium chloride. The calcium will intervene in the
TABLE 3 The Effects of PRP Concentration on Bone Regeneration PRP Platelet Concentration
Low Medium
High
0.5–1.5×
