PRODUCT MONOGRAPH

REVIVAL PRP®

Manufactured by Xediton Pharmaceuticals Inc. 2000 Argentia Road Mississauga, Ontario L5N 1W1

October 2016

Version 1.0 October 3rd 2016.

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INDEX

1. PLATELET RICH PLASMA: STRUCTURAL AND FUNCTIONAL ROLE IN THE

PHYSIOLOGY OF JOINTS .................................................................................................. 4

1.1 Introduction ............................................................................................................................... 4 1.2 Pathophysiology and pathogenesis of OA ........................................................................... 4 1.3 PRP and its Growth Factors (GFs) ....................................................................................... 4 1.4 PRP Preparation ...................................................................................................................... 6 1.5 Role of PRP in Joint Physiology ............................................................................................ 7 1.6 Role of PRP in Bone Healing ................................................................................................. 7

2. APPLICATION OF PRP IN SPORTS MEDICINE ............................................................... 9

2.1 Role of PRP in Ligament and Tendon Repair ................................................................... 11 2.2 Rehabilitation after PRP Injections for Tendinopathy ...................................................... 12

3. AUTOLOGOUS PRP MECHANISM OF ACTION ............................................................ 13

4. THE TREATMENT OF OA: FOCUS ON PRP .................................................................. 14

4.1 Importance of Technique in PRP Injections ...................................................................... 15

5. CLINICAL EFFICACY OF PRP-BASED THERAPIES ..................................................... 16

5.1 Efficacy Assessments ........................................................................................................... 16 5.2 PRP in Osteoarthritis ............................................................................................................. 20 5.3 Use of PRP in Other Joints (e.g. Hip, Knee, Shoulder) ................................................... 21 5.4 Benefits of PRP ...................................................................................................................... 23 5.5 Conclusion: Efficacy .............................................................................................................. 23 5.6 Conclusion: Safety ................................................................................................................. 23

6. CONTRAINDICATIONS ..................................................................................................... 24

7. REVIVAL PRP® PRODUCT PROFILE .............................................................................. 25

7.1 Indications ............................................................................................................................... 25 7.2 Dosage and Administration .................................................................................................. 25 7.3 Side Effects ............................................................................................................................ 25

7.4 First Aid Measures ................................................................................................................ 25

8. CONCLUSION .................................................................................................................... 26

9. REFERENCES.................................................................................................................... 27

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1. Platelet Rich Plasma: structural and functional role in the physiology of joints 1.1 Introduction

Regenerative medicine refers to a strategy whereby the injured site is provided with the raw materials necessary for a “scarless repair”, or regeneration, to occur in situ. In regenerative medicine, the assembly of cells, growth factors and scaffold into new tissue takes place within the lesion site or in proximity to it, and is directed under local influences. The concept focuses on augmentation and optimization of the natural healing response, rather than “insertion” of an engineered product (Lana et al. 2014). Platelet Rich Plasma (PRP) is included within the field of regenerative medicine (Torricelli et al. 2011; Okabe et al. 2009; Wu et al. 2011; Sanchez-Gonzalez et al. 2012; Stellos and Gawaz 2007, as cited in Lana et al. 2014) since it can provide 2 of the 3 components (growth factors and scaffold) deemed necessary to support true tissue regeneration (Lana et al. 2014). PRP is defined as a portion of the plasma fraction of autologous blood having a platelet concentration above baseline. The goal of PRP is to enhance the body’s innate ability to repair and regenerate by concentrating platelets containing a large volume of growth factors (Dhillon et al. 2012). The seven known growth factors in PRP are: platelet derived growth factor as (PDGFaa), PDGFbb, PDGFab, transforming growth factor beta-, (TGF-b,), TGF-b 2, vascular endothelial growth factor (VEGF), and epithelial growth factor (EGF). Normal platelet counts in blood range between 150,000/μl and 350,000/μl and average about 200,000/μl. (Marx 2001).

PRP injections are used in various applications such as orthopaedics, cardiovascular surgery, cosmetics, urology and more (Dhillon et al. 2012). There are a few major variables that create differences amongst the many PRP systems and preparations including the following: gross platelet concentration, the presence or absence of leucocytes, exogenous activation requirements, and whether or not anticoagulation was performed. The variability of these parameters from one system to the next can potentially affect the magnitude of the platelets’ biological activity (Weglein et al 2014). 1.2 Pathophysiology and pathogenesis of OA

The pathogenesis of OA is multi-factorial and it affects several tissues, starting with the articular cartilage. The articular cartilage is a highly specialized structure, created and maintained by its cell element, the chondrocyte. In mature articular cartilage, chondrocytes are embedded within an extracellular matrix (ECM) containing up to 85% of water. This ECM is a translucent, opaline-transparent structure, whose “shock-absorbing / cushioning” mechanical properties are determined by the presence of highly organized macromolecular structures involving proteoglycans, collagen type II and hyaluronic acid. Healthy cartilage is a balance between ECM synthesis and degradation, whereas in OA, matrix degradation overtakes matrix synthesis. The consequent articular cartilage degradation is frequently associated with abnormal chronic joint stresses, typically involving overweight, obesity, concurrent factors such as age, metabolic diseases, inflammation and immune system disorders all leading to biochemical changes that enhance cartilage degradation.

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These factors work synergistically; the biophysical changes (mechanical stresses) and the biochemical changes (e.g. loss of inhibitors of degradation enzymes and increase in proteolytic enzymes) lead to a series of pathophysiological and ultimately degenerative events which are the causes of OA. All joint tissues become involved in OA. Subchondral bone stiffens, becomes osteoporotic, and may develop subchondral cysts. Attempts at bone repair produce subchondral sclerosis; efforts to stabilize the joints produces osteophytes at the joint margins, inflammation and thickening of the synovium ensues, producing further synovium. Periarticular tendons and ligaments become strained, resulting in tendonitis and contractures. As the joint becomes less mobile with time, surrounding muscles thin and become less supportive. From a clinical standpoint, the patient most often describes aching pain as the earliest symptom of such process. Therefore, OA begins as a disease of a single tissue (cartilage) and progresses to become a disease of an organ (synovial joint) in which cartilage is primarily affected but which involves many other tissues and structures. 1.3 PRP and its growth factors (GFs)

Platelets, or thrombocytes, are formed during hematopoiesis (which Merriam-Webster defines as the formation of blood or of blood cells in the living body) and consist of cytoplasmic fragments of large and multinucleated cells of red bone marrow (megakaryocytes). These cell fragments are found in blood plasma, i.e. the yellow liquid fraction of the blood that contains water, proteins such as albumin, globulins, clotting factors such as fibrinogen, and prothrombin (Francone et al. 1990, as cited in Lana et al. 2014). Platelets measure from 1 to 4 µm in diameter and contain organelles such as mitochondria, alpha granules, dense granules, and lysosomal granules. The alpha granules contain adhesion molecules, coagulation factors, fibrinolytic factors, antiproteases, growth factors, cytokines and antibacterial proteins. Dense granules contain adenosine diphosphate (ADP), adenosine triphosphate (ATP), calcium ions (Ca2+), serotonin, histamine, dopamine and catecholamine. (Anitua et al. 2004; Pietrzak and Eppley 2005, as cited in Lana et al. 2014). Hemostasis is the result of the combined action of three main mechanisms: vascular response, platelet activity and blood clotting. When in contact with an injured vascular endothelial surface, even of biological origin, the platelets begin an adhesion reaction to the injury location, releasing pseudopods that facilitate their aggregation. This initiates the hemostatic plug that serves as a base for aggregation factors to affix themselves to the area, resulting in the formation of the fibrin network that will obstruct the vascular injury (Souza and Elias 2005, as cited in Lana et al. 2014). The main growth factors contained in the platelet alpha-granules are: platelet derived growth factor (PDGF) in the isoforms AA, BB and AB, beta transforming growth factor (TGF-b1 and TGF-b2), vascular endothelial growth factor (VEGF), basic fibroblastic growth factor (bFGF), epidemic growth factor (EGF), insulin-like growth factor (IGF-1, IGF-2 and IGF-3), and hepatocyte growth factor (HGF), among others (Anitua et al. 2004; Pietrzak and Eppley 2005; Eppley et al. 2004a; Kubota et al. 2004; Anitua et al. 2005, as cited in Lana et al. 2014).

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PDGF is the main growth factor in the platelets, as it is the first to initiate activity at the injury and directs revascularisation, the synthesis of collagen and osteogenesis (Bittencourt et al. 2014, as cited in Lana et al. 2014). The PDGF at the point of injury triggers the target cells by adhering to the cell surface receptors and establishing tyrosine kinase protein binding. The increased concentration of this growth factor at these sites is thought to accelerate repair processes. The release of PDGF in the wound bed has a chemotactic effect on the monocytes, neutrophils, fibroblasts, mesenchymal stem cells and osteoblasts. It is also a powerful mitogenic factor for fibroblasts and smooth muscle cells and participates in the three phases of the healing cascade, particularly angiogenesis, the formation of fibrous tissue and reepithelialisation (Molloy et al. 2003, as cited in Lana et al. 2014). TGF-b is active during inflammation and influences cell migration, proliferation and replication, as well as the connection between fibronectins (Molloy et al. 2003, as cited in Lana et al. 2014). VEGF is a powerful angiogenesis stimulator produced in high concentrations after the inflammatory phase that can help with the healing of chronic wounds and endochondral ossification (Bennet et al. 2003; Maes et al. 2002, as cited in Lana et al. 2014). EGF is an important mitogenic factor for fibroblasts, endothelial cells and keratinocytes and is also involved in the healing of chronic wounds (Bennet et al. 2003, as cited in Lana et al. 2014). HGF is found in several types of tissue, such as kidney, lung, liver, several types of epithelium (Matsumoto and Nakamura 1992, as cited in Lana et al. 2014) and tumor (Boros and Miller 1995, as cited in Lana et al. 2014), and has mitogenic, morphogenic, motogenic (Matsumoto and Nakamura 1992, as cited in Lana et al. 2014), antiapoptotic (Kosai et al. 1990, as cited in Lana et al. 2014), and neurotrophic (Miyazawa et al. 1998, as cited in Lana et al. 2014) functions, which make an important contribution to tissue regeneration (Matsumoto and Nakamura 1992, as cited in Lana et al 2014). Table 1. Growth Factors and Cellular Effects. Growth Factor Cellular Effects PDGF

(Platelet Derived Growth Factor) Macrophage activation and angiogenesis

Fibroblast chemotaxis and proliferative activity

Enhances collagen synthesis

Enhances the proliferation of bone cells IGF-I

(Insulin-like Growth Factor-I) Chemotactic for myoblast and fibroblasts and stimulates

protein synthesis

Mediator in growth and repair of skeletal muscle

Enhances bone formation by proliferation and differentiation of osteoblasts

TGF-β (Transforming Growth Factor-β)

Enhances the proliferative activity of fibroblasts

Stimulates biosynthesis of Type I collagen and fibronectin

Induces deposition of bone matrix

Inhibits osteoclast formation and bone resorption

Regulation in balance between fibrosis and myocyte regeneration

PDEGF

(Platelet Derived Endothelial Growth Factor) Promotes wound healing by stimulating the proliferation of

keratinocytes and dermal fibroblasts PDAF

(Platelet Derived Angiogenic Factor) Induces vascularization by stimulating vascular endothelial

cells

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EGF (Endothelial Growth Factor)

Cellular proliferation

Differentiation of epithelial cells VEGF (Vascular Endothelial Growth Factor)

Angiogenesis

Migration and mitosis of endothelial cells

Creation of blood vessel lumen

Creation of fenestrations

Chemotactic for macrophages and granulocytes

Vasodilation (indirectly by release of nitrous oxide) HGF

(Hepatocyte Growth Factor) Stimulates hepatocyte proliferation and liver tissue

regeneration

Angiogenesis

Mitogen for endothelial cells

Antifibrotic Retrieved from Middleton, K. K., Barro, V., Muller, B., Terada, S., & Fu, F. H. (2012). Evaluation of the Effects of Platelet-Rich Plasma (PRP) Therapy Involved in the Healing of Sports-Related Soft Tissue Injuries. The Iowa Orthopaedic Journal, 32, 150–163.

1.4 PRP Preparation Depending on the preparation conditions, various formulations can be obtained which differ in type, concentration and properties of their constituents. Even with a considerable platelet concentration, the presence of white blood cells can affect the performance of treatment with PRP. The addition of a layer of leukocytes has led to the current classification (Dohan Ehrenfest et al. 2009) that distinguishes Pure Platelet-Rich Plasma (P-PRP, equal to Anitua’s PRGF—Preparation Rich in Growth Factors) and Leukocyte and Platelet-Rich Plasma (L-PRP). Also in the classification are the rich-in-fibrin gels distinguished as Pure Platelet-Rich Fibrin (P-PRF) and Leukocyte-and Platelet-Rich Fibrin (L-PRF or Choukroun’s type). The performance of PRP depends not only on composition but also on its preparation conditions. It is important to have both an appropriate concentration and the presence of intact platelets, with functionality unchanged, so that growth factors are not delivered prior to application. The inclusion of the buffy coat has positive effects for various applications; however, lymphocytes and monocytes have beneficial effects whereas neutrophils do not and must be separated prior to activation of PRP (McCarrel et al. 2009; Sundman et al. 2011). Whether or not PRP is activated prior to injection depends on the application as tissue collagen is a strong activator of platelets. However, PRP can be activated exogenously by thrombin, calcium chloride, or mechanical trauma. Once activated, a fibrin network will begin to form and the plasma will begin to solidify creating a fibrin clot or membrane. If PRP is activated too strongly, the fibrin network that forms will be a bivalent, unstable network. If it is activated in a more physiologic manner, a tetramolecular stable network will from which enhances enmeshment of cells and growth factors (Mishra et al, 2012). If calcium chloride used to activate the product (i.e. at a 10 % concentration; 0.5 cc CaCl in 5 cc PRP, 10:1 ratio) the injection of PRP must be given within 5–10 min, due to the viscous nature of the activated product (Weglein et al. 2014). PRP activation results in 70% of the stored growth factors being secreted within 10 min and close to 100 % within the first hour (Marx 2001). Platelet activation should generate fibrin-network architecture that promotes gradual release of growth factors and also an efficient interaction, proliferation, and differentiation of cells (Lana et al. 2014). 1.5 Role of PRP in Joint Physiology

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The concept that the application of PRP would result in improvement of cartilage repair is based on the physiological role of platelets in wound healing. PRP has shown an increased usage in the practice of knee surgery. The purpose of its application is to increase the efficiency of healing grafts in anterior cruciate ligament reconstructive knee surgery. Authors using PRP emphasize its benefits in reconstructive surgery compared with those in which concentrated platelets are not used. The success of the reconstruction shows the healing graft in the tunnels of the femur and tibia, as well as revascularization and a reconstruction graft in the same direction as the bone structure inside the bone channels and ligamentation inside the joint. These processes are accelerated by the use of PRP which results in more extensive rehabilitation and a lower risk of inflammatory complications (Ficek et al. 2011). Kon et al (2010) demonstrated that after administration of PRP inside the joint, patients with pain and swelling of the knee experienced significantly reduced pain, improvement in knee function and greater quality of life. 1.6 Role of PRP in Bone Healing

PRP has been shown to stimulate cell proliferation of osteoblasts and fibroblasts as well enhance regulation of osteocalcin in these cells (Graziani et al. 2006; Dolder et al. 2006, as cited in Lippross & Alini, n.d.). Osteoblasts and fibroblasts play a fundamental role in the bone healing process.

The effects of PRP at different concentrations on osteoblasts (OB) and fibroblasts (FB) at 24h are shown in Figs 1a and b, respectively. After 24h, NAP did not induce a statistically significant proliferative response compared with the negative control. However, both activated plasma (PRP-1X) and PRP-2.5X stimulated OB proliferation compared with NAP (P<0.05). In Figure 1, the maximum osteoblast (OB) proliferation was obtained with activated plasma (PRP-1X), with increasing concentrations of PRP resulting in decreased proliferation. The difference between PRP-1X and PRP-max was statistically significant (P<0.05). At 72 h, there was a statistically significant increase in OB proliferation by all preparations containing plasma (including NAP) compared with the negative control. Furthermore, PRP-1X (P<0.001), PRP-2.5X (P<0.001) and PRP-3.5X (P<0.001) induced a statistically significant increase in proliferation compared

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with the NAP. The highest cell proliferation was obtained with PRP-2.5X, with a 70% increase in cell numbers at this concentration compared with the negative control. Furthermore, the increase of proliferation induced by PRP-2.5X was statistically higher than PRP-max (P<0.001). FBs The effects of PRP concentrations on FBs at 24 and 72 h are shown in Figs 2a and b, respectively.

Two basic issues must be addressed: 1) the mechanical stability of the fracture fixation or lesion and 2) the biological aspect of the fracture/lesion healing, which involves the processes of osteogenesis, osteoconduction and osteoinduction. It is vital that revascularization through angiogenesis into the injured tissue in order for these processes to occur (Kanthan et al. 2011). PRP is found to be effective only when used together with bone graft. Platelets in PRP that are activated by the in vitro introduction of thrombin-calcium or the wound environment will release α-granules that contain numerous proteins that influence bone healing (Kanthan et al. 2011). These proteins include most of the growth factors mentioned previously. PRGF provide a bioactive condition with the addition of histones and carbohydrate side chains, which then bind to the target cells, for example osteoblasts and mesenchymal stem cells (Kanthan et al. 2011). This will lead to the activation of intracellular signalling, which directs cells to increase the cellular proliferation rate, matrix formation, collagen synthesis and cellular differentiation (Kanthan et al. 2011). From there, the effects of bone healing with be taken over by the macrophages which are attracted by the release of PDGF (Kanthan et al. 2011). It has also been suggested that the initial release of IGF from platelet granules results in the activation of mesenchymal stem cells which later transform into osteoblast-like cells that continue to provide the matrix-repair proteins (Kanthan et al. 2011). Table 2 summarizes various studies that evaluated the application of PRP in bone healing.

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Table 2: Application in bone healing Application Study Title Author(s) Type of Study Study Design Conclusion

Treatment of intrabony defects

Platelet-rich plasma combined with a porous hydroxyapatite graft for the treatment of intrabony periodontal defects in humans: a comparative controlled clinical study

Okuda K, Tai H, Tanabe K, et al (2005)

Comparative controlled clinical study

70 interproximal intrabony osseous defects were treated with PRP and a ceramic porous hydroxyapatite (HA) scaffold or HA and saline

Treatment with PRP and HA led to significantly more clinical improvement than HA and saline

Treatment of intrabony defects

Treatment of intrabony defects with bovine-derived xenograft alone and in combination with platelet-rich plasma: a randomized clinical trial

Hanna R, Trejo PM, Weltman RL (2004)

Randomized clinical trial (split mouth, double masked)

Bilateral periodontal intrabony defects were matched in 13 individuals and treated only with bovine xenograft or with PRP

PRP significantly increased the clinical periodontal response of lesions treated with xenogenic bone grafts

Treatment of infrabony defects

Surgical treatment of infrabony defects with autologous platelet concentrate or bioabsorbable barrier membrane: a prospective case series.

Papli R, Chen S (2007)

Prospective case series

Five similar bilateral paired infrabony defects were treated with autologous platelet concentrate (APC) or a bioresorbable barrier membrane (MEM)

Similar gain in clinical attachment level and probing depths in APC and MEM treated groups

Lumbar spine fusion Do autologous growth Factors enhance transforaminal lumbar interbody fusion?

Hee HT, Majd ME, Holt RT, et al (2003)

Prospective review compared to historical results

23 individuals underwent transforaminal lumbar interbody spinal fusion (TLIF) with PRP compared to historical results

2-year minimum follow-up showed faster healing in the PRP group, but no significant difference in the pseudarthrosis rate was observed

Total ankle replacement

The use of autologous concentrated growth factors to promote syndesmosis fusion in the Agility total ankle replacement. A preliminary study.

Coetzee JC, Pomeroy GC, Watts JD, et al (2005)

Comparative Study

114 and 66 agility total ankle replacements were performed with and without autologous concentrated growth factors for distal syndesmosis fusion

Autologous concentrated growth factors appeared to make a significant positive difference in the syndesmosis union rate in total ankle replacements

Treatment of mandibular continuity defects in tumour cases

Platelet-rich plasma: Growth factor enhancement for bone grafts.

Marx RE, Carlson ER, Eichstaedt RM, et al (1998)

Prospective Study

44 individuals were treated with bone graft and PRP and bone graft alone

Maturity index of bone grafts with PRP was higher than in bone grafts alone

Retrieved from: Lippross, S., & Alini, M. (n.d.) Platelet-rich plasma for bone healing—to use or not to use?

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2. Application of PRP in Sports Medicine

Basic science and pre-clinical data support the use of PRP by elite and recreational athletes for a variety of sports related injuries and disorders (Mishra et al. 2012). Athletes of all competition levels are early adopters of novel treatment methods. They are driven to find less invasive methods of injury management and faster means of returning to their sports. PRP usage is increasing in the treatment of commonly occurring enthesopathy, which is a discomfort in the area of tendons, ligaments, and articular capsule attachments. This is apparent in both professional sports as well as recreational activity (Ficek et al. 2011). Tendon injuries are very common disabling conditions in athletes which include acute or chronic degeneration as well as partial or complete tendon ruptures. Healed tendons are comprised of scar tissue which is mechanically inferior to normal tendon tissue. This results in impairment of normal tendon function and joint kinematics, predisposing the patient to further tendon injury (Middleton et al. 2012). The goal of PRP in tendon repair is to bypass the formation of non-functional scar tissue and promote healing of healthy functional tissue. Acute sports-related soft tissue injuries result from a single, traumatic event such as muscle contusion or a ligament sprain/tear. Contusions and strains are the most frequent muscular lesions, representing more than 90% of all sports related injuries. Chronic soft tissue injuries often result from repetitive mechanical stress or overuse followed by inflammation and occur over a prolonged period of time (i.e. tendinopathies such as rotator cuff tendinopathy and Achilles tendinopathy) (Middleton et al. 2012). Those most predisposed to Achilles tendon damage are runners, badminton and squash players and individuals who train in cold weather (Ficek et al. 2011). Acute ligament sprains are one of the most common orthopedic injuries. Most can be treated conservatively (i.e. isolated medial collateral ligament ruptures). Others such as the ACL need surgical reconstruction due to its relatively low healing potential (Middleton et al. 2012). Skeletal muscle injuries are common causes of severe long-term pain and physical disability, accounting for almost 55% of all sports injuries (Middleton et al. 2012). Strains and contusions are common in sports (mainly those involving contact, sprinting, jumping and acceleration activities [Kon et al. 2010]) and account for significant time loss. When a muscle is injured, there is capillary rupture and bleeding which initiates hemostasis, followed by an inflammatory reaction. The complete healing response in muscle is characterized by the degeneration inflammation phase, the regeneration phase and the fibrosis phase. The ultimate quality and function of the repair is dependent on the interplay of cytokines, cellular and growth factors. The goal of PRP therapy is to influence this process to favor the formation of functional muscle as opposed to fibrosis (Mishra et al. 2012). Occupational physical activity in sport, dance or the military increases the likelihood of Achilles tendon pathology, resulting not only from the load endured but also due to a greater likelihood of structural and functional degradation in other neighbouring and distant parts of the musculoskeletal system. Disorders of the neuromuscular control mechanism may be due to improper work habits and fatigue (Ficek et al. 2011). Mishra et al (2012) specified a Sports Medicine Platelet Rich Plasma Classification System, defined as follows:

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Table 3: Sports Medicine Classification of Platelet Rich Plasma White Blood Cells Activation Platelet Concentration

Type 1 Increased No A, 5x or > B, <5x Type 2 (aka platelet-leukocyte gel) Increased Yes A, 5x or > B, <5x Type 3 (aka platelet concentrate) Minimal or No WBCs No A, 5x or > B, <5x Type 4 (Platelet gel) Minimal or No WBCs Yes A, 5x or > B, <5x

Retrieved from Mishra, A., Harmon, K., Woodall, J., & Vieira, A. (2012). Sports Medicine Applications of Platelet Rich Plasma. Current Pharmaceutical Biotechnology CPB, 13(7), 1185-1195. doi:10.2174/138920112800624283

Due to the anatomical and functional differences between tendons, ligaments, joint capsules and muscles and their attachment to bones, the PRP injection site and the response of tissues to mechanical stimuli acting on them varies (Ficek et al. 2011). Table 4: Clinical Evidence of PRP Applications in Humans for the Treatment of Sports Lesions

Pathology Study Patients Follow Up

Results

Elbow tendinopathy

Mishra & Pavelko (2006)

20 2 years Enhanced healing and functional recovery vs bupivacaine injections

Peerbooms et al. (2010)

100 1 year Reduced pain and increased function, exceeding the effect of corticosteroid injection (better initially, then declined)

Rotator cuff tear

Maniscalco et al. (2008)

1 6 months

Pain relief and ROM recover after surgical repair; Complete integrity of the rotator cuff under the fibrin membrane by MRI

Randelli et al. (2008)

14 2 years No adverse events; good and stable clinical results after arthroscopic surgical repair

Achilles lesion/ tendinopathy

Sanchez et al. (2007)

12 32-50 months

No wound complications in surgically repaired tendons; Earlier recovery of ROM and a faster return to jogging and sport; Lower cross-sectional area

Filardo et al. (2010)

1 18 months

Fast tissue repair and return to competitive sports activity in partial tendon tear

De Vos et al. (2010)

54 24 weeks

Same results in pain and activity improvement compared with a saline injection

Patellar tendinopathy

Kon et al. (2009)

20 6 months

Marked improvement in knee function and quality of life; PRP has to be associated with physiotherapy

Filardo et al. (2010)

31 6 months

Marked clinical improvement in chronic refractory patellar tendinopathy, comparable with less severe cases; Greater improvement in the level of sports activity in PRP group

ACL tear

Orrego et al. (2008)

108 6 months

Enhancing maturation (MRI); No effect on tunnel widening

Silva et al. (2009)

40 3 months

No MRI difference compared to controls

Radice et al. (2010)

50 3-12 months

48% reduction in the time required to achieve a complete homogeneous graft signal when PRP was used for surgical ACL augmentation

Cartilage lesion/degeneration

Sanchez et al. (2003)

1 Not specified

Rapid resumption of symptom-free athletic activity after surgical treatment for knee cartilage avulsion

Sanchez et al. (2008)

60 5 weeks Better pain control and physical function improvement vs hyaluronan injections

Kon et al. (2010)

91 1 year Clinical improvement; Better results in early degeneration and younger patients; worsening from 6 to 12 months

Retrieved from Kon, E., Filardo, G., Martino, A. D., & Marcacci, M. (2010). Platelet-rich plasma (PRP) to treat sports injuries: Evidence to support its use. Knee Surgery, Sports Traumatology, Arthroscopy, 19(4), 516-527. doi:10.1007/s00167-010-1306-y

Commented [GG1]: What is this table saying? Is there a paragraph that links this table? Provide some commentary on the

table

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The use of PRP should be considered as a secondary step of treatment, occurring after or in association with physiotherapy, eccentric training and manual therapy, but prior to surgery (Ficek et al. 2011). 2.1 Role of PRP in ligament and tendon repair

The observed effects of PRP are attributed to the delivery of growth factors and pro-inflammatory mediators to damaged tissue, which is especially valuable in situations of low blood flow or hypo-cellularity, two known features of ligaments and tendons (Chao et al. 2014). In orthopedic surgery, platelet concentrates are of special interest in the management of tissues that heal slowly or not at all. Tendons and ligaments (for example the rotator cuff of the shoulder and the anterior cruciate ligament (ACL) of the knee are prime candidates (Chao et al. 2014). The use of a platelet concentration 3x or lower is recommended within the PRP and suggest allowing for leukocyte and maybe even erythrocyte ‘‘contamination’’, owing to the microbicidal effect and plentiful growth-factor release of leukocytes and the stimulation of collagen production by erythrocytes. Most importantly, it is suggested that each clinical application should have a PRP preparation tailored for it (Chao et al. 2014). PRP has also recently been introduced as an adjunctive treatment to surgical repair of ligaments and tendons with poor intrinsic healing potential including the anterior cruciate ligament (ACL), the Achilles tendon, and the rotator cuff (Chao et al. 2014). With repetitive overuse, collagen fibers in the tendon form micro tears, leading to tendonitis, also called tendinosis or tendinopathy. Tendinopathy is not considered an inflammatory condition based on lack of inflammatory cells in histological sections (Almekinders et al. 1998, as cited in Weglein et al. 2014). The injured tendons heal via scarring which adversely affects function and increases risk of re-injury. Furthermore, tendons heal at slower rates compared with other connective tissues due to poor vascularization (Sampson et al. 2008, as cited in Weglein et al. 2014). More than 90% of the damage to the Achilles tendon stretching resistance is directly proportional to its thickness and collagen fiber content (Ficek et al. 2011). Animal studies have suggested that the use of PRP could enhance the clinical outcome of Achilles tendon repair. A rat model showed earlier healing and 30 % stronger scar tissue when using PRP for surgical repair of a 3 mm Achilles tendon defect (Aspenberg 2007; Virchenko and Aspenberg 2006, as cited in Chao et al. 2014). This effect has been attributed to improved angiogenesis and cellular bioactivity (Lyras et al. 2009; Lyras et al. 2010; Tohidnezhad et al. 2011, as cited in Chao et al. 2014). Schepull et al. 2011, as cited in Lana et al. 2014). Similarly, the use of PRP in arthroscopic rotator cuff repair has shown initial, promising results from animal models and human longitudinal studies (Castricini et al. 2010; Maniscalco et al. 2008; Randelli et al. 2008; Rodeo 2007, as cited in Chao et al. 2014). In many cases, the patients will require only one PRP injection treatment. In cases of severe partial tendon tears, calcific tendinopathies, or chronic resistant tendon injuries, 1-3 PRP treatments may be needed to heal the tendon and sustain long term clinical benefit. Typically, patients are followed up between 4 and 6 weeks following initial injection to determine the need for further treatments (Weglein et al. 2014).

Commented [PS2]: Schepull is not cited by Chao, it is cited by

Lana

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It is important to note that factors such as nutritional deficiencies, hormonal deficiencies and inflammatory disorders may impair collagen synthesis and tendon-ligament healing. Therefore, it is imperative to screen for and correct conditions that may debilitate regeneration (Ko, 2010). 2.2 Rehabilitation after PRP Injections for Tendinopathy

There are three principal phases of tendon healing: the inflammatory phase, repair phase and the remodelling phase. The inflammatory phase generally lasts up to 3 days. Inflammatory cells such as neutrophils, are initially recruited to the injury site, as well as platelets and erythrocytes. Platelets are activated and a fibrin clot forms. Monocytes and macrophages arrive to the injured area in less than 24 hours, and phagocytosis of necrotic debris occurs. Cytokines and growth factors travel to the injured area. Chemotactic and vasoactive factors are released, leading to increased vascular permeability, angiogenesis, and the attraction of tenocytes to the area. The tenocytes begin to synthesize Type III collagen at this time, as opposed to the predominantly Type I collagen that is present in normal tendons (Sharma and Maffuli 2005, as cited in Peck and Mautner 2014). The repair phase begins near the end of the inflammatory phase and generally lasts for up to 6 weeks. There is proteolytic degradation of damaged tissue. During this phase, growth factors stimulate fibroblasts, which form a new extracellular matrix. Cellularity and neovascularization increase, granulation tissue is formed, and tenocytes synthesize collagen and proteoglycans at the injury site (Wang 2006, as cited in Peck and Mautner 2014). The remodeling phase begins near the end of the repair phase. The initial portion of the remodeling phase is consolidation, during which the synthesis of collagen and proteoglycans is decreased. There is decreased cellularity and neovascularization, the tissue becomes more fibrous, and the relative production of type I collagen increases, with realignment and remodeling consistent with the mechanical stress applied (Sharma and Maffuli 2005, as cited in Peck and Mautner 2014). The consolidation portion of the remodeling phase generally lasts about 4 weeks. The second portion of the remodeling phase is maturation. At this time, there is increased cross-linking of collagen fibrils. The maturation portion of the remodeling phase may last up to 1-year post-injury, during which time the injured tissue forms a scar (Lana et al. 2014). It is generally recommended for patients to avoid NSAIDs for one week prior to PNT and PRP injection(s), as well as up to six weeks’ post-procedure. Alternative analgesics may be used, such as acetaminophen, tramadol, or opiates as indicated (Peck and Mautner 2014). 3. Autologous PRP Mechanism of Action

PRP contains a number of different kinds of cells including: 1. Platelets 2. Neutrophils, which represent 40–75 % of the circulating leukocytes 3. Monocyte-macrophages, which represent 2–10 % of the circulating leukocytes

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4. Fibroblasts, which produce collagen, reticular fibers, glycosaminoglycans, and glycoproteins—these compounds are very important in the production of tendons and articular cartilage

5. Endothelial cells—these regulate permeability barriers, blood flow and vascular reactivity, act as vasodilators and constrictors, and regulate inflammation and immunity

6. Keratinocytes, which are stratified squamous epithelial cells. Their primary function is to act as a barrier Platelets contain two unique types of granules, the alpha granules and the dense granules. The alpha granules contain a variety of proteins that have various functions and release growth factors which have direct effects on the stem cells. The cell activation of the platelets causes the discharge of both alpha and dense granule contents. There are many different growth factors that are found within the platelet alpha granules. These are the growth factors that stimulate mesenchymal stem cells to help produce endothelial, fibroblastic, and osteoblastic components. They also promote the growth and differentiation of chondrocytes, fibroblasts, and osteoblasts. The most important thing that the platelet growth factors accomplish is the establishment of a blood supply which ensures that the stem cells have the ability to obtain the necessary growth factors they require (Purita 2014). An effective PRP preparation should have between 1.5–3 million platelets/µL (which is a concentration expected to support angiogenesis). A quality PRP preparation also contains high numbers of growth factors. The efficiency of a PRP product depends upon the WBC content. (Purita 2014). Once injected into the tissue, PRP will begin initially with the inflammatory phase, which includes activation of the platelets, release of growth factors, and a myriad of other reactions. The next phase is the proliferative phase. In this phase, various types of cells begin to proliferate and subsequently go onto the next phase, which is the remodeling phase. The modeling phase includes tissue repair that starts with the production and breakdown of collagen products. Typically, this phase can last for over a year (Purita 2014). Autologous PRP regulates and modulates the functions of growth factors, therefore influencing all stages of regeneration simultaneously. Growth factors are introduced into the tissues via autologous plasma injections and the high concentration is achieved by injecting a large volume of plasma. This stimulates the formation of fibroblasts and enhances their activity. It also blocks osteoclast activity and stimulates osteoblast proliferation, thus deterring further decrease of bone tissue and contributing to its regeneration. The resulting consequence is the restoration of metabolic processes, microcirculation and improvement of tissue metabolism, normalization of tissue respiration and stimulation of local immunity (Akhmerov, n.d). The actual mechanisms of action of PRP are extensive because of the release of a myriad of bioactive factors. There is a general consensus in PRP research that the injection of concentrated platelets, once activated, results in an exponential increase in numerous growth factors at the sight of injection (Middleton et al. 2012). 4. The treatment of OA: Focus on PRP

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Since there is no known cure for OA, the treatment strategy is focused on reducing pain, aiding the regeneration of tissue and/or improving joint mobility. (Spaková et al. 2012). Recent recommendations from EULAR (The European League Against Rheumatism) state that the optimum management of OA of the knee should be based on a combination of pharmacological and non-pharmacological treatments (Jordan et al. 2003). General non-pharmacological therapies include:

Healthy changes in diet for overweight patients to reduce weight and thus relief of stress on the joints

Physical therapy: low-impact exercises to help reduce weight and improve joint function (strengthening of the quadriceps, in particular, reduces strain on the knee).

Drug therapies include acetaminophen/paracetamol and non-steroidal anti-inflammatory drugs (NSAIDs) or weak opiate analgesics and injections of corticosteroids in the event of a pain flare-up or inflammation. However, the use of NSAIDs after PRP injections is not recommended due to its inhibitory properties (Reynolds et al. 1995, as cited in Peck and Mautner, 2014). Both pharmacological approaches above exert the so-called “symptom-modifying effects”, and are essential tools in treating a painful and debilitating syndrome such as OA. Ideally, however, OA treatments should also address the structural damages of cartilage and provide “structure-modifying effects”. Many oral treatments are based on glucosamine sulphate (alone or in combination with chondroitin sulphate) and are proven pharmacologic tools in reducing the cartilage structure degeneration. In particular, highly purified crystalline glucosamine sulphate (CGS) is widely considered a standard therapy in that regard. While short-term intra-articular therapy with corticosteroids is sometimes recommended for a quick control of pain and inflammation, this kind of treatment is indicated for patients having an inadequate response or a contraindication to other pharmacological therapies and where surgery (e.g. partial/total knee replacement) was inappropriate or should be delayed. PRP is assumed to restore the anabolic-catabolic imbalance found in OA joints. The cytokine Interleukin (IL)-1 beta is one of the most potent inflammatory factors in osteoarthritic joints and has been described to induce the production of destructive proteases together with the inhibition of ECM formation. PRP has been assumed to counteract IL-1 beta-induced effects on genes involved in matrix formation and degradation in osteoarthritic chondrocytes, including NFKB (which when activated, acts on various regulatory genes involved in apoptosis, inflammation and other immune responses) (Van Buul et al. 2011). 4.1 Importance of Technique in PRP Injections

PRP is prepared by centrifugation by varying the relative centrifugal force, temperature and time. Preparation procedures are also relevant, as shown by studies of the chondro-inductive and osteo-inductive potential of PRP. There are numerous protocols in the current literature that describe the optimal conditions for centrifugation, however, these various protocols have been optimized with respect to different

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variables of the process, such as volume and sampling of processed WBCs, number of spins, time period of centrifugation, and range of centrifugal acceleration (Dhurat et al. 2014). Despite these variations, all protocols follow a generic sequence that consists of blood collection, an initial centrifugation to separate RBCs, subsequent centrifugations to concentrate platelets, and other components and an activation of the sample by adding a platelet agonist. (Dhurat et al. 2014). When performing PRP injections, the amount of blood utilized depends somewhat on the underlying problem. The actual injection technique depends upon the site involved. For instance, PRP can be injected anywhere in a joint as it will spread to the involved areas on its own. Thus, it is not necessary to use any real guidance to place the PRP for a meniscus tear since the cells will travel throughout the knee. However, for a tendon it is best to inject the PRP into the tendon sheath and then into the tendon itself in a peppering fashion. A 23-gauge needle appropriate for this (Purita 2014). For tendons, it is important to correctly choose the injection zone of PRP in addition to classifying the degree of change in the structure of the tendon. These classifications emphasize the distinction between peritenon (or synovial inflammation), and increasing involvement of the tendon substance as a likely reflection of the failure to adapt to physical load. It also emphasizes the variable stress responses in the tendon structure (Sharma and Maffulli, 2005, as cited in Ficek at al. 2011). These categories are:

1) Peritenonitis (paratenonitis, tenosynovitis): inflammation of the peritenon 2) Peritenonitis with tendinosis: tendon sheath inflammation associated with

intratendinous degeneration 3) Tendinosis: degeneration in the tendon itself due to cellular hypotrophy 4) Tendinitis: asymptomatic degeneration of the tendon with disruption and

inflammatory repair response. A commonly proposed name for the tendon pain problems is tendinopathy

The various categories of structural change differentiate the potential sources of discomfort and facilitate their treatment. Locating the problem makes it easier to administer treatment at an earlier stage, before PRP is applied (Ficek et al. 2011). It is important to depend on image guidance such as MRI or ultrasound imaging for accurate placement of injections as joint injections done without image guidance are frequently inaccurate. This is especially critical when particularly important structures are in close proximity to the targeted tendon (i.e. the femoral artery to the iliopsoas tendon) (Mishra et al. 2012). Muscle injuries undergo a series of overlapping healing phases and together with the trauma-induced myofibre degeneration, capillary rupture results in a hematoma that fills the gap with the elements responsible for the subsequent phases. Cytokines and GFs released start the inflammatory phase while fibrotic tissue begins to replace the necrotic muscle gap. This provides early support for myofibres but as it becomes increasingly dense over time, it restricts myofibre regeneration. Therefore, the platelet concentrate should be administered in this time frame in order to modulate the inflammation phase and support the regeneration processes before the development of scar tissue (Kon et al. 2010).

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5. Clinical efficacy of PRP-based therapies 5.1 Efficacy Assessments

The task force of the Osteoarthritis Research Society has recommended the following pain measurement scales for assessment of OA of the knee (Altman et al., 1996).

a. Global knee pain score: visual analogue scale (VAS) or Likert scale b. Knee pain while walking (VAS or Likert scale) c. Western Ontario and McMaster Universities (WOMAC) Osteoarthritis Index: A self-

administered, disease-specific measure of health status. The WOMAC (with 24 questions including 5 assessing for pain, 2 for stiffness, and 17 for physical function) queries for symptoms related to pain, stiffness, and physical function in patients with OA of the hip and/or knee. WOMAC is a valid and reliable measure of clinically significant changes in health status following surgical or pharmacologic interventions. Each question is assigned a score between 0 and 4. Scores from the 24 questions are summed. A higher score (highest possible score is 96) corresponds to more pain, stiffness and reduced function. A lower score (lowest possible score is 0) correlates with less pain, stiffness and better joint function

d. Lequesne Index: An interview-style survey with 10 questions administered to patients

with OA of the knee: 5 deals with pain or discomfort, 1 with furthest distance walked, and 4 dealing with activities of daily living. The questionnaire is scored from 0-24 with lower scores correlating with less impairment and higher scores with more functional impairment

e. Activity related knee pain (other than walking, see b. above)

The normal structure and function of cartilage is difficult to restore when injured or degraded due to its poor blood supply and self-renewal capacity. Conventional treatments for cartilage degeneration or osteoarthritis (OA) include intra-articular injections of lubricant, anaesthetic medication or arthroplastic surgery. PRP has been widely used to promote recovery of the soft tissue lesions and has been proven effective for restoration of the impaired cartilage and/or preventing aggravation (Liu et al. 2014). Liu and colleagues (2014) conducted to compare the therapeutic effects of PRP and HA on osteoarthritis in rabbit knees. 30 rabbits were used to establish the animal models by creating a cartilage defect of 5mm in diameter on the condyles of the femurs, and were randomly divided into three groups: the PRP group, HA group and the control group and treated with the respective method of treatment. The platelet number, concentrations of growth factors of PRP and whole blood, and the IL-1β concentration in the joint fluid were investigated. Histological assessment of the cartilage was performed according to Mankin’s scoring system. The platelet concentration of PRP was found to be 6.8-fold of that in the whole blood. The IL-1β concentration in the PRP group was lower than in the HA group (p<0.01) and in the control group (p<0.01). The restoration of the defected cartilage as well as the subchondral bone was better in the PRP group than in the HA group and control group (p<0.05).

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Figure 1. Mankin’s score of the three groups at 6 weeks and 12 weeks. The score of the PRP group was lower than the other two groups at both time points. * denotes a statistically significant difference compared with the control group (p<0.05). # denotes a statistically significant difference compared with the HA group(p,0.05).

It was concluded that PRP is better than HA in promoting the restoration of the cartilage and alleviating the arthritis caused by cartilage damage. The findings demonstrated that better histological outcomes and restoration of subchondral bone were obtained with administration of P-PRP. It was also found that PRP could alleviate the inflammatory reaction in the joint cavity more than HA, as was indicated with the changes of IL-1 level in the joint fluid. The histological findings showed that an increase of chondrocytes was observed after PRP was injected into the joint cavity, which was demonstrated via HE staining. It was accompanied by increase of the cartilage extracellular matrix (ECM) production, which was confirmed via safranin-O staining. It is reasonable to relate this effect to the high concentration of the growth factors released from P-PRP, which were produced by activated platelets and can promote the restoration of the injured tissues. Moreover, it was also verified in this experiment that PRP showed explicit alleviation on the osteoarthritic changes, as was supported by the detection of IL-1b concentration in the joint fluid. Inflammatory factors such as IL-1b are indicators of the existence and severity of osteoarthritis. The anti-osteoarthritic effect of PRP may be the result from the relief of the cartilage damage, which at the same time reduced the local inflammation as well as the stimulation to the synovium, causing a drop in the IL-1b secretion. On the other hand, P-PRP has a direct influence on the synovium, as it can lower the NF-kB activity, and suppress the expression of COX-2 and CXCR4, which are the important regulatory factor in the inflammatory reactions. Meanwhile, PRP can up-regulate the expression of HGF, IL-4 and TNF-a, while HGF and TNF-a can block the expression of NF-kB to inhibit the inflammation. All these bio-active features account for the superiority of PRP over HA, (Liu et al. 2014). Another prospective, cohort study with a control group was conducted by Spaková et al. A total of 120 patients with Grade 1, 2, or 3 osteoarthritis according to the Kellgren and Lawrence grading scale were enrolled in the study. One group of patients was treated using three intra-articular applications of PRP, and the second group of patients was given three injections of hyaluronic acid. Outcome measures included the Western Ontario and McMaster Universities Osteoarthritis Index and the 11-point pain intensity Numeric Rating Scale.

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On average, a 4.5-fold increase in platelet concentration was obtained in the PRP group. No severe adverse events were observed. Statistically significantly better results in the Western Ontario and McMaster Universities Osteoarthritis Index and Numeric Rating Scale scores were recorded in a group of patients who received PRP injections after a 3- and 6-month follow-up. (Spaková et al. 2012).

There were multiple animal studies conducted that tested the efficacy of PRP on various conditions. Rodeo et al. (2007) tested the effects of growth factors on the formation of scar tissue in gaps between tendon and bone in sheep submitted to detachment of the infraspinatus tendon. The administration of osteoinductive growth factors resulted in better formation of primary bone tissue, fibrocartilage and soft tissues, with concomitant growth in the strength of tendon fixation, but the repairs were less than those obtained by treating with only a collagen sponge carrier. Lyras et al. (2009) evaluated induced injuries in the patellar tendon of rabbits. The histological and biomechanical properties were evaluated 14 and 28 days after injury. After 14 days of treatment, there was a significant increase among PRP treated groups in the load necessary for rupture and in tendon rigidity. However, after 28 days, there was no significant difference between groups regarding the histological or biomechanical properties of the patellar tendon.

Human studies that have looked at the efficacy of PRP on tendinopathies include Mishra and Pavelko (2006) who demonstrated an improvement in the pain felt by 15 patients with chronic elbow tendinosis after a single application of platelet rich plasma. These patients were compared to a control group of five patients treated with bupivacaine and were evaluated after 8 weeks, 6 months and approximately 2 years. In 93 % of the cases there was a pain reduction in the PRP-treated group.

Figure 2: A mean value of the WOMAC Osteoarthritis Index at baseline and at the 3- and 6-month follow-up in the PRP and HA groups P<0.01 between groups. WOMAC, Western Ontario and McMaster Universities; NS, nonsignificant; HA, hyaluronic acid; PRP, platelet-rich plasma

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Sampson et al. (2011) used a single application of PRP associated with physical therapy to successfully treat a severe injury to the Achilles tendon of a 71-year-old patient, thus avoiding surgical intervention. The positive results were confirmed by magnetic resonance imaging analysis, and in 24 weeks the patient showed no symptoms and was able to resume daily activities. Peerbooms et al. (2010) carried out a double-blind study, with randomized control with a level of evidence I, in favour of the use of PRP in the treatment of chronic lateral epicondylitis, when compared to injections of corticoids. The 100 patients included in the study were randomly divided to receive an injection of corticosteroid (N = 49), or an injection of the autologous platelet concentrate (N = 51). The results showed that, according to the scores of visual analogue pain, the group treated with PRP showed a significant statistic improvement at 1 year, in comparison to the group treated with corticosteroids. The corticosteroid group was better at the beginning, but after, its condition declined, while the PRP group improved progressively. Vogrin et al. (2010) carried out a prospective, randomized study to evaluate the use of platelet and leukocyte gel in ACL reconstruction with tendon graft in 25 patients. Compared to controls, there was a significant improvement in the anteroposterior stability of the knee in patients treated with the gel. De Almeida et al. (2012) selected 27 patients that were posteriorly divided at random to receive (n=12) or not to receive (n=15) PRP injections on the patellar tendon collection site during surgery for reconstruction of the ACL. The results were assessed by means of magnetic resonance (MR) of the patellar tendon after 6 months. The researchers observed that the recuperation of the opening site of the patellar tendon was significantly bigger in the PRP group than in the control group. The visual analogue scale (VAS) was also used and the post-surgery pain score was significantly lower in the group treated with PRP. It was concluded that PRP could improve the healing of the tissues on the collection site of the patellar tendon and the hypothesis was confirmed. PRP also reduced the pain after surgery. 5.2 PRP in Osteoarthritis

Articular cartilage can be defined as a hydrated tissue that functions as a load-bearing surface. Although it has superior biomechanical properties, its healing ability is poor. Untreated lesions in the cartilage can spread to involve the entire joint and cause arthritis (Gobbi et al. 2014). This condition is known as Osteoarthritis (Andia et al. 2013) which is characterized by progressive destruction, thinning and eventual wearing of articular cartilage resulting in painful joint movement (Zhu et al. 2013). Inflammation and vascular pathology in combination with cell death, meniscal changes, bone remodelling and subchondral sclerosis are all attributed to the cycle of progressive joint degradation. Chondrocyte senescence, loss of cartilage integrity and osteophyte development are all major features of osteoarthritis (Andia et al. 2013). Platelets contain numerous bioactive proteins and growth factors which regulate key processes involved in tissue repair, such as cell proliferation, chemotaxis, migration, cellular differentiation, and ECM synthesis (Molloy et al. 2003; Staudenmaier et al. 2009; Everts et al. 2006, as cited in Gobbi et al. 2014) PRGF regulates endogenous hyaluronic acid (HA)

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synthesis, thereby protecting the cartilage and lubricating the joint. It enhances the secretion of HA and induces hepatocyte growth factor (HGF) production by synovial fibroblasts isolated from arthritic patients. (Dhillon et al. 2012).

Current treatment for articular cartilage damage such as surgical intervention (i.e. microfracture, osteochondral auto- or allografts) to repair articular cartilage are often unsatisfactory and rarely restore full function (Zhu et al. 2013). The healing process of OA comprises three phases: (1) inflammation, (2) cell proliferation, and (3) remodeling. PRP theoretically augments tissue healing through the natural healing cascade. GFs are released from the granules of platelets and induce chemotaxis, cell migration, angiogenesis, proliferation, differentiation, and matrix production. PRP also enhances HA secretion and increases release of angiogenic GFs. Platelets actively participate in healing processes by delivering a broad spectrum of GFs and other active molecules (e.g., chemokines, arachidonic acid metabolites, extracellular matrix (ECM) proteins, nucleotides, ascorbic acid) to the injured site by exocytosis following adhesion or stimulation by calcium, thrombin, ADP, collagen, and magnesium. Once platelets are activated, an initial burst of GF release is followed by further sustained release, a 3- to 5-fold increase as compared with baseline. GFs secreted by platelets include platelet derived growth factor (PDGF), epidermal growth factor (EGF), insulin-like growth factor (IGF-I), transforming growth factor b-I (TGFb-I), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF). Platelet activation increases levels of anti-inflammatory cytokines because of the presence of hepatocyte GF. These GFs have a particular function in bone remodeling and wound healing as well as stimulation of cartilage matrix synthesis. These substances act in synergy on local cells inducing specific responses: promotion of proliferation, cell migration, and synthesis of ECM proteins including collagen, even changing the cell phenotype and arrangement. (Zhu et al. 2013).

PRP is used for hemostasis and for total joint arthroplasty for OA. It can be used intraoperatively in conjunction with a fibrin sealant or a gel and even alone during total knee arthroplasty. The use of PRP in knee and hip joints is associated with reduced inflammation, pain relief, improved function, and possible cartilage regeneration. (Zhu et al. 2013). Basic science, preclinical, and clinical studies collectively indicate that PRP is a promising method for treating cartilage injuries and joint pain. The application of PRP for OA in clinical trials has shown promising short-term results (1-2 years), although most of these studies were not randomized controlled trials (Zhu et al. 2013). 5.3 Use of PRP in other joints, (e.g. hip, knee, shoulder)

In general, use of PRP for various joints is common. There are several publications that report favourable data for PRP administration in hip, knee and shoulder joints. Hip:

In a study published recently by Sánchez et al. (2012b) 40 patients affected by severe unilateral hip OA received three injections of PRP, which were administered once a week. The primary end point was significant pain relief, which was described by the reduction in the intensity of the pain in at least 30 % of the base line scores, being evaluated by the WOMAC sub-scale for at least 6 months after the treatment. The visual analogue scale (VAS) and Harris’s sub-scale

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for pain on the hips were also used to verify the results. The secondary outcomes included improvement of at least 30 % in pain and incapacity. Statistically significant reductions in the scores of questionnaires for pain and function were reported. 57.5 % of the patients reported a clinically relevant reduction in pain. The study supports the safety and tolerance of the PRP injections for relief of pain and improvement of function in patients with hip OA. Knee:

Gobi et al (2012) studied the effectiveness of intra-articular PRP injections in active patients with knee OA and evaluated clinical outcomes in patients with and without previous surgical treatment for cartilage lesions. Fifty patients with knee OA were followed for a minimum of 12 months. All were treated with 2 intra-articular injections of autologous PRP. Twenty-five patients had undergone a previous operative intervention for cartilage lesions, whereas 25 had not. Operated patients had undergone either cartilage shaving or micro fracture. Multiple evaluative scores were collected at pre-treatment and at 6- and 12-month post-treatment. All patients showed significant improvement in all scores at 6 and 12 months (P < 0.01) and returned to previous activities. No significant difference in improvement was found between the evaluated subgroups (P < 0.01). The PRP treatment showed positive effects in patients with knee OA. Operated and non-operated patients showed significant improvement by means of diminishing pain and improved symptoms and quality of life.

Laudy et al (2014) performed a comprehensive, systematic literature search in computerised databases. Ten trials were included which showed intraarticular PRP injections were more effective for pain reduction (mean difference (MD) −2.45; 95% CI −2.92 to −1.98; p value <0.00001 and MD −2.07; 95% CI −2.59 to −1.55; p value <0.00001, single and double PRP injections, respectively) compared with placebo at 6 months post-injection. Intra-articular PRP injections were compared with hyaluronic acid and showed a statistically significant difference in favour of PRP on pain reduction based on the visual analogue scale and numeric rating scale (standardised mean difference −0.92; 95% CI −1.20 to −0.63; p value <0.00001) at 6 months’ post-injection. It was concluded that on the basis of the current evidence that PRP injections reduced pain more effectively than did placebo injections in OA of the knee (level of evidence: limited due to a high risk of bias). This significant effect on pain was also seen when PRP injections were compared with hyaluronic acid injections (level of evidence: moderate due to a generally high risk of bias). Additionally, function improved significantly more when PRP injections were compared with controls. Shoulder:

Rotator cuff injuries are the most common shoulder injury to occur. Current rotator cuff augmentations that surgeons have used with varying success include allograft tissue, autogenous biceps tendon and fascia lata, synthetic mesh and extracellular matrices. Currently one of the more common methods to augment tissue in rotator cuff repair is the use of extracellular or synthetic matrices. These matrices are commercially available and are derived from human or animal dermis, porcine small intestinal submucosa, equine pericardium or bovine collagen. The matrices have little inherent strength and some contain measurable amounts of DNA despite extensive processing. Although there has been some demonstrated success in animal models, there is a risk of an inflammatory reaction occurring at the site of implantation (Gamradt et al. 2007).

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A study conducted by Randelli et al (2011) evaluated 53 patients who underwent shoulder arthroscopy for the repair of a complete rotator cuff tear. Subjects were randomly assigned to two groups: the treatment group (n=26) who received intraoperative application of PRP in combination with an autologous thrombin component and the control group (n=27) who did not receive treatment. Patients were evaluated with validated outcome scores and the pain score in the treatment group was lower than the control group at 3, 7, 14 and 30 days post-surgery (p<0.05). Strength in external rotation (SER) scores were significantly higher in the treatment group as compared to the control group at 3, 6, 12 and 24 months after surgery (p<0.05). It was concluded that autologous PRP reduced pain in the first post-operative months and long term results suggest that PRP positively affected rotator cuff healing. Hyunchul Jo et al (2013) conducted a randomized control trial which evaluated the efficacy of PRP augmentation in patients undergoing arthroscopic repair for large to massive rotator cuff tears. A total of 48 patients were randomly assigned to receive either PRP-augmented (PRP group) or conventional treatment (conventional group). In the PRP group, 3 PRP gels (3 x 3 mL) were applied to each patient between the torn end and the greater tuberosity. The primary outcome measure was the re-tear rate assessed by magnetic resonance imaging (MRI) or computed tomographic arthrography (CTA) at a minimum of 9 months after surgery. Secondary outcome measures included pain, range of motion, muscle strength, overall satisfaction, functional scores, and the change in cross-sectional area (CSA) of the supraspinatus. The re-tear rate of the PRP group (20.0%) was significantly lower than that of the conventional group (55.6%) (P = 0.023). Clinical outcomes showed no statistical difference between the 2 groups (all P>0.05) except for the overall function (P = 0.043). The change in 1-year postoperative and immediately postoperative CSA was significantly different between the 2 groups: –15.54 ± –94.34 mm2 in the PRP group versus –85.62 ± 103.57 mm2 in the conventional group (P = 0.047). It was concluded that the application of PRP for large to massive rotator cuff repairs significantly improved structural outcomes, as demonstrated by a decreased re-tear rate and increased CSA of the supraspinatus compared with repairs without PRP augmentation. While there was no significant difference in clinical outcomes except the overall shoulder function after 1-year follow-up, better structural outcomes in the PRP group might suggest improved clinical outcomes at longer term follow-up. 5.4 Benefits of PRP

General benefits:

Cost effective treatment

Short recovery time

Targeted and accelerated healing

Rapid results with long-term benefits

Incredible versatility, since it can be used intraoperatively or in the clinical practice

Natural, visible improvement without artificial enhancement

High level of patient satisfaction and significant return on investment

Virtually no risk of blood-borne illness transmission or rejection, since the patient’s own blood is used to create the PRP injection

Very safe therapy with little contraindications

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Specific benefits:

In bone healing: o Improves the maturity index of bone grafts o Stimulates cell proliferation of osteoblasts and fibroblasts as well enhance

regulation of osteocalcin in these cells o Significant positive difference in the syndesmosis union rate in total ankle

replacements o Improves significantly the clinical periodontal response of lesions treated with

xenogenic bone grafts

In the treatment of cartilage lesion or degeneration: o PRP is better than HA in promoting the restoration of the cartilage and alleviating

the arthritis caused by cartilage damage. o Renders better histological outcomes and restoration of subchondral bone o Alleviates the inflammatory reaction in the joint cavity better than HA o Up-regulates the expression of HGF, IL-4 and TNF-a, while HGF and TNF-a can

block the expression of NF-kB to inhibit the inflammation

In tendinopathies: o Improves the pain felt in chronic elbow tendinosis after a single application o Better anteroposterior stability of the knee after ACL reconstruction with tendon

graft o Marked improvement in knee function and quality of life in chronic refractory

patellar tendinopathy o Substantial reduction in the time required to achieve a complete homogeneous

graft signal in ACL tear repair o Faster healing and recovery of the collection site of the patellar tendon, with a

post-surgery pain score significantly lower o Increased the strength in external rotation (SER) scores, reduces pain in the first

post-operative months and long term in rotator cuff healing o In large to massive rotator cuff repairs, achieves a lower re-tear rate, increases

cross-sectional area of the supraspinatus, improves structural outcomes and overall shoulder function after 1-year follow-up

o Earlier recovery of range of movement (ROM) and a faster return to activity in Achilles tendinopathy and rotator cuff tear

To treat Osteoarthritis: o Diminishes pain and improves symptoms and quality of life in operated and non-

operated patients with knee OA o Provides significant pain relief, improves function and less incapacity when used

to treat unilateral hip OA

In Sports-related injuries: o Rapid resumption of symptom-free athletic activity after surgical treatment for

knee cartilage avulsion, and a better pain control and physical function improvement vs hyaluronan injections

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5.5 Conclusion: Efficacy

The main advantages of PRP are its autologous nature, non-invasive collection process, and rapid preparation. PRP is generally more cost-effective and time-saving than stem cell processing and treatment and can be prepared without specialized equipment (i.e. using a standard centrifuge). It can be modified into various forms according to whether an “open” (i.e. surgical) or “closed” (i.e. percutaneous) application is desired. PRP can provide a matrix/scaffold and growth factor concentrate to enhance stem cell treatment of a lesion. Because the preparation process is rapid and requires minimal specialized equipment, PRP can be applied to a patient within hours of a treatment decision. These features make PRP extremely attractive for clinical use in a variety of settings, including not only hospitals and outpatient clinics, but also in field applications or other areas with limited medical facilities and resources. Inventory, ordering, and safe storage are not required and shelf life is not a concern, since the treatment is freshly prepared for each patient (Textor 2014). Multiple studies have demonstrated the effectiveness of PRP in restoration of the impaired cartilage and/or preventing aggravation in tendinopathies, injuries and reconstruction procedures. PRP has demonstrated superior clinical efficacy on the treatment of the OA by promoting the restoration of the cartilage; alleviating the inflammatory reaction in the joint cavity (lower IL-1 level in the joint fluid) caused by cartilage damage; increasing of the cartilage extracellular matrix (ECM) production; lowering the NF-kB activity, and suppressing the expression of COX-2 and CXCR4, which are the important regulatory factor on inflammation (Liu et al. 2014). When used to treat knee joint OA, PRP has shown statistically significant better results in the WOMAC Osteoarthritis Index and Numeric Rating Scale scores for pain after 3- and 6-month follow-up in grade 1, 2 and 3 OA (Spakova et al, 2012). Also, improved symptoms and quality of life in operated and non-operated patients with knee OA (Gobi et al, 2012). In fact, Laudy and colleagues performed in 2014 a comprehensive, systematic literature search in computerised databases and included ten trials which showed intraarticular PRP injections were more effective for pain reduction than placebo as well as when compared with hyaluronic acid injections at 6-month post-injection. Additionally, function improved significantly more when PRP injections were compared with controls. In patellar tendon regeneration, PRP showed to increase faster the strength and rigidity in the of the tendon significantly in comparison to animals that didn’t receive PRP supplementation (Lyras, 2009). De Almeida and colleagues showed in 2012 that patients had a faster surgery recovery after 6 months when treated with PRP. Patients also exhibit Visual Assessment Scores and Post-Surgery Pain Scores significantly lower that their counterparts that didn’t received PRP. In the treatment of elbow injuries, PRP demonstrated improved pain reduction in chronic elbow tendinosis as early as 8 weeks and up to 2 years compared with bupivacaine-treated patients (Mishra & Pavelko, 2006) or when compared with corticoids in chronic lateral epicondylitis (Peerbooms et al, 2010). In shoulder injuries, PRP also improved clinical outcomes in patients undergoing arthroscopic repair for large to massive rotator cuff tears. The application of PRP for large to massive rotator

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cuff repairs significantly improved structural outcomes, demonstrated by a decreased re-tear rate and increased CSA of the supraspinatus, compared with repairs without PRP augmentation (Hyunchul Jo et al, 2013). PRP also decreased pain in the first post-operative months while positively affected rotator cuff healing at long term (Randelli et al, 2011). When used to treat severe unilateral hip OA, PRP-treated group showed a clinically relevant reduction in pain evaluated by the WOMAC sub-scale, Visual analytic Scale and Harris’ sub-scale (Sanchez et al, 2012b). 5.6 Conclusion: Safety

PRP is autologous, does not provoke an immune response in the patient and is therefore perceived to have a high margin of therapeutic safety (Textor 2014). Since PRP is prepared from autologous blood, theoretically there are minimal risks for disease transmission, immunogenic reactions or cancer. Wang-Saegusa et al. 2011, as cited by Dhillon et al. 2012 reported no adverse effects following injection of PRGF into the knee joint of 800 patients at 6 months. Adverse effects are rare however there is always a small risk of injection site morbidity, infection or injury to nerves or blood vessels. Scar tissue formation and calcification at the injection site have been reported as well (Dhillon et al. 2012). Hypersensitivity to bovine thrombin used for activation has been evident and is therefore avoided in modern techniques. It is also very rare that development of antibodies against clotting factors V and IX leading to life-threatening coagulopathies have been reported however, to date, there is no compelling evidence of any systemic effects of a local PRP injection (Dhillon et al. 2012). Acting growth factors also attach to cell surfaces rather than the nucleus, which minimizes the chance of tumor formation through the use of negative feedback control (Ko 2010). 6. Contraindications

Although PRP is considered a safe treatment for patients, it is not suitable for all potential candidates. The use of PRP therapy is contraindicated in patents who are anaemic and/or have thrombocytopenia (Smith et al. 2007). Other potential contraindications are: - hemodynamic instability - severe hypovolemia - Unstable angina - Sepsis - Anticoagulant or fibrinolytic drug therapy Anti-platelet therapy has always been considered a major contraindication for PRP therapy since platelet inhibition was seen as an obstacle to the healing potential induced by PRP administration (Ko 2010). Aspirin is one of the most common forms of anti-platelet medication (Harrington et al. 2003).

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Consistent use of NSAIDs within 48 hours of procedure is not recommended as it may have a more profound impact on inhibiting platelet function and diminishing the inflammatory phase of healing. NSAIDs interfere with the chemotaxis of cells and delay normal healing in tendon injuries (Reynolds et al. 1995, as cited in Peck and Mautner, 2014). It is generally recommended for patients to avoid NSAIDs for 1 week prior to PNT and PRP injection(s), as well as up to 6 weeks post-procedure. Other contraindications include (Harmon et al. 2011): - Corticosteroid injection at treatment site within 1 month - Systemic use of corticosteroids within 2 weeks - Tobacco use - Recent fever or illness - Cancer (especially hematopoietic or of bone) - HBG < 10g/dl - Platelet count < 105/µl 7. RevivalPRP® Product Profile

Revival PRP® is a Class II medical device, a kit developed and manufactured by Xediton Pharmaceuticals Inc comprising of vacuum separation tubes and accessories for the collection of blood to help separate plasma from blood. The product has been approved since 2015 and is currently marketed throughout Canada. 7.1 Indications

Revival PRP® is indicated for the acceleration of bone and tissue healing in the following areas: Chronic wounds, orthopaedics, veterinary, general surgery, post-operative wounds, sports medicine, biological glue, aesthetics & cosmetic surgery, urology, dermatology, scalp cure & hair treatment, dental & periodontal and ophthalmology. Revival PRP® is intended only for use by a certified health care professional. 7.2 Dosage and administration

The patient is to be prepped for a standard blood draw, in which the required amount of blood is drawn into the supplied pre-filled test tube containing gel and the anticoagulant. The tube is to be inverted up and for about 5 times to mix the blood and the tube components and then centrifuged at 1500 RCF for 10 minutes. For Revival PRP® kits containing activator, the plasma is to be combined with the supplied 0.5mL of 10% calcium chloride solution prior to being injected into the patient. For Revival PRP® kits that do not contain activator, the plasma is obtained directly from the test tube and injected into the patient. 7.3 Side Effects

Some possible side effects that may occur with the use of Revival PRP® are:

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- Temporary pain, swelling or numbness from injection techniques - Infections and/or irritation, damages to blood vessels and hematoma

7.4 First Aid Measures

Contact with eyes: Rinse with cold water for at least fifteen (15) minutes. Consult a doctor. Skin contact: Wash thoroughly with cold water and soap. Ingestion: Rinse mouth with water. Consult a doctor. 8. Conclusion

The primary goal of PRP is to enhance the body’s ability to regenerate and repair by concentrating platelets that contain a large amount of growth factors. The logic behind PRP is that platelets are the first to arrive at the site of tissue injury and consequently have the potential to release these growth factors that play a critical role in mediating healing. It provides a fairly simple method to deliver a variety of these natural growth factors to the patient through a straightforward process of blood draw and centrifugation by a qualified health care professional. Preclinical evidence supports the use of PRP injections that might promote a favourable environment for joint tissue healing as well as synovial and meniscal tissue healing. Numerous studies have looked at the advantages of PRP injections over hyaluronic acid/cortisone injections, the effect of PRP in bone healing, joint repair and osteoarthritis treatment of multiple joints. PRP has demonstrated ability to counteract the inflammatory IL-1 beta-induced effects in osteoarthritic joints. The concentrated amount of growth factors stimulates the formation of fibroblasts and enhances their activity. It also blocks osteoclast activity while stimulating osteoblast proliferation. This in turn deters further decrease of bone tissue and contributes to its regeneration. The resulting consequence is the restoration of metabolic processes, microcirculation and improvement of tissue metabolism, normalization of tissue respiration and stimulation of local immunity. PRP has also widely been used in treating sports injuries. Multiple studies evaluated the effect of PRP on Achilles tendon repair, tennis elbow and other common lesions. Various protocols have been developed to establish higher concentrations of PRP, which is dependent on the specific application for which it will be used. The goal of PRP in tendon repair is to bypass the formation of non-functional scar tissue and promote healing of healthy functional tissue. PRP attempts to influence the process of the interplay of cytokines, cellular factors and growth factors to favor the formation of functional muscle as opposed to fibrosis. Studies have looked at the relationship between growth factor concentration and healing potential. Available evidence suggests that the potential efficacy of a high platelet concentration is dependant on the proposed application. Although PRP is considered a safe and effective form of treatment of several pathologies, it is not recommended for all patients. Patients should undergo a complete evaluation to rule out potential pathologies that may interfere with PRP treatment.

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Zhu JT, Xuan M, Zhang YN, Liu HW, Cai JH, Wu YH, Xiang XF, Shan GQ, Cheng B (2013) The efficacy of autologous platelet-rich plasma combined with erbium fractional laser therapy for facial acne scars or acne. Molecular Medicine Reports 8: 233-237, doi: 10.3892/mmr.2013.1455 GG observations:

1. Link charts and tables to content so that it shows relevance. Charts and tables must have a commentary explaining what the chart/table is saying

2. You may use diagrams to illustrate a point e.g. on section 2.2- Rehabilitation after PRP Injections for Tendinopathy, a diagram would be helpful

3. The benefits of PRP is shown all through the paper, but it would be good to have a section that captures all the benefits, in a strong “in your face” format. Consider a list format- eg bullets etc. DONE

4. Section on Revival can be strengthened a. Process b. IFU c. Why is our system different/ advantageous/better?

5. Using the Clinical, articles in the server, should there be more charts, tables etc. to support PRP use? Visuals reinforce message

6. We have a lot of references listed above- not sure we have tapped into these references sufficiently

7.