ESSENTIALS OF REGENERATIVE MEDICINE IN INTERVENTIONAL PAIN MANAGEMENT
PRP: History, Mechanism of Action, Preparation and Clinical Applications
By Dr. Marko Bodor, MD; Ryan Dregalla, PhD; and Yvette Uribe, BA
Platelet Rich Plasma (PRP) was first described for clinical use in 1999 to enhance osseointegration for tooth implants well after specific individual growth factors had already been identified for healing of various wound and injury types. The therapeutic importance of platelets became clear when they became better characterized, acting as reservoirs for a wide array of bioactive factors, growth factors, adhesive proteins, coagulation factors, cytokines and chemokines released in the presence of appropriate stimuli and orchestrated in the progression of wound repair. Hence, it was logical to assume that if platelets and their bioactive factors were concentrated and delivered to an injury site, the healing process could be enhanced and expedited.
Soon after being described for oral surgery, PRP was used in veterinary and sports medicine. In 2006, Mishra and Pavelko reported on PRP as an alternative to surgery for chronic tennis elbow.In the last decade the deleterious effects of corticosteroids on tendons and cartilage have become increasingly more recognized and interest in PRP for the treatment of neuromuscular, musculoskeletal and orthopedic conditions has increased exponentially with over 700 peer-reviewed articles referenced on PubMed in 2016.Many “next generation” platelet products, including platelet-rich fibrin, leukocyte-rich or -poor PRP, platelet lysate, post-platelet degranulation serum and extracts have been developed.
Mechanism of Action
Platelets are anuclear 2-3 µm diameter fragments of megakaryocytes from the bone marrow and contain an abundance of growth, chemotactic, and clotting factors. Platelets are capable of adhering to and pulling together torn tissue using their tentacle-like filopodia with an internal network of actin and myosin. PRP is simply a platelet concentrate, platelets in plasma concentrated 3-5x or higher than in whole blood.
Platelets need to become activated in order to adhere to collagen and release their growth factors. Platelet activation depends on external stimuli such as thrombin, ADP, calcium or the presence of certain structural proteins that are not present in the endothelium such as collagen.Platelets adhere to soluble factors, extracellular matrices and other platelets. Platelet degranulation involves the fusion of alpha and dense (delta) granules to their membranes with release of growth and anabolic factors.
Platelet degranulation results in inflammation,recruitment and proliferation of several cell types including leukocytes (days 0-2),promotion of neovascularization (days 2-3), migration and division of fibroblasts, synthesis of collagens type I and III and new tissue formation (days 3-5). PRP may help repair or regenerate tissue, especially in poorly vascular regions where platelets are not already present.
Pro-Inflammatory and Catabolic Pathway
Within minutes of activation, platelets release platelet derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor beta-1 (TGF-B1), interleukin 1B (IL-1B), adenosine diphosphate (ADP) and histamine. These factors stimulate leukocytes to release inflammatory cytokines (IL-1B, TNF-α, IL-6) enhance the expression of degradative enzymes of the matrix metalloproteinase (MMP) family and prepare tissue for repair and regeneration. How much platelet-leukocyte interaction is necessary at the site of injury to optimize healing is not known. When leukocytes are increased at the site of injury, PRP will have more of an inflammatory effect. However, platelets alone may be all that is necessary to repair tissu.
Anti-Inflammatory and Anabolic Pathway
Upon degranulation platelets release growth factors over a period of days. These are later accompanied by other growth factors secreted by the leukocytes This process reduces inflammation, restores the environment to an anabolic state, and may be part of the pain-relieving effect of PRP.
Platelet related growth factors and neovascularization stimulate fibroblast homing and proliferation in tendons and ligaments Fibroblasts orient at the site of injury marked by platelet aggregates and neovessels and synthesize collagen. For hyaline cartilage injuries, neovascularization does not occur because the appropriate precursor (endothelial) cells are lacking Fibrocartilage fills these defects days to weeks after injury, but does not have the durability of hyaline cartilage.
Mechanical Functions of Platelets
Lam et al have extensively studied the mechanical properties of platelets Platelets are capable of adhering to and pulling together torn collagen using tentacle-like filopodia and an internal network of actin and myosin, the same contractile proteins in skeletal muscle. Platelets are able to generate a dynamic force of 29 nN and a static force of 70 nN, approximately 1/3rd that of slow twitch muscle fibers, Therefore it may be possible to use platelets to close small defects in tissues similar to how sutures are used in surgery.
PRP preparation begins with the collection of whole blood mixed with an anti-coagulant such as heparin, sodium citrate or citrate-dextrose, allowing cells to remain in suspension, followed by centrifugation for several minutes. There are three basic fractions of centrifuged whole blood: 1) the red blood cell fraction, always the densest, collecting at the bottom, 2) the buffy coat, containing white blood cells and possibly platelets, and 3) the plasma fraction, containing some degree of platelets. Where platelets settle depends on centrifuge time and force. The goal of most PRP preparations is 1 million platelets/µl .
Centrifugal Forces and the Composition of the Plasma and Buffy Coat Fractions
Centrifugal forces commonly used are 100 – 700 x gravity (x g with spin times on the order of 5 – 20 minutes. There is an inverse relationship between force and spin time – the higher the force the lower the spin time and vice-a-versa. A standard objective in producing PRP has been to concentrate platelets into a small volume with the count per microliter (µl) being several times (e.g. 5x) higher than in whole blood. Several years ago many preparations focused on increase over baseline concentration alone, however it has since been recognized that this is only one aspect affecting the quality and characteristics of PRP.
In systems using high centrifugal forces, leukocytes and platelets are concentrated in the buffy coat. Furthermore, this results in a high concentration of red blood cells due to the poor resolution of the interface between the buffy coat and the red blood cells. The final product is generally a red color indistinguishable from whole blood but possesses a high concentration of leukocytes and platelets (≥5x over baseline) and is known as leukocyte-rich PRP (LR-PRP).
Alternatively, lower centrifugal forces can be used over longer periods of time, allowing platelets to remain in suspension and leukocytes to collect at the red blood cell interface. In this case, aspiration of the entire plasma fraction results in PRP with a platelet concentration approximately 2x higher than in whole blood. This is due to the simple fact that red blood cells, comprising approximately 50% of whole blood, are left out. Furthermore, leukocytes within the buffy coat are also left behind, resulting in a relatively pure platelet product
However, many clinicians seek to achieve higher platelet concentrations than just 2x over baseline. To achieve this, the plasma fraction undergoes an additional centrifugation step to concentrate the platelets from the suspension into a pellet at the bottom of the container. This pellet is then re-suspended in a variable volume of plasma depending on the final desired concentration. This preparation is referred to as leukocyte-poor PRP (LP-PRP).
The differences between LR-PRP and LP-PRP are becoming better understood. These include higher inflammation and catabolic activity of LR-PRP and more anabolic and anti-inflammatory activity of LP-PRP, however, the anabolic processes of LP-PRP may favor the formation of fibrotic tissue. Both LR-PRP and LP-PRP possess anti-microbial and bactericidal properties
In an attempt to release the contents of platelets into solution, many aim to create platelet lysates. The method generally incorporates one or more freeze-thaw cycles of whole PRP at -20°C or -80°C. It has been demonstrated that PL is a viable source of growth factors above that of serum and increases cell growth when added to culture.This is viewed as an alternate version of PRP providing elevated levels of readily available growth factors that do not depend on platelet activation and degranulation.
Activation-State of Platelets
Regardless of how it was made, PRP can be injected in a “non-activated” or “activated” state. Non-activated PRP is injected as is, whereas activated PRP is injected following the addition of ADP, calcium or thrombin, or exposure to hypothermic conditions or extracellular stimuli such as collagen or glass.The latter methods serve to increase platelet sensitivity to degranulation and immobilization via adhesion. The aim of activation is to better enable platelet adhesion and delivery of growth factors to the site of injury immediately upon injection of PRP. We are unaware of in-vivo studies comparing activated versus non-activated PRP for musculoskeletal applications, although a recent study on alopecia showed no difference.
Non-activated PRP Preparations
In situations where fluidity and extended handling times may occur, PRP is injected without activation. The PRP remains as a simple concentrate and the platelets will not clot or release their growth factors right away. However, in the presence of extracellular matrices such as collagens which are major platelet activators and typically present at sites of injury, platelets will begin to degranulate. The question remains whether platelets releasing their growth factors over a period of time or right away hold any distinct advantage.
Activated PRP Preparations
Platelet activation can be achieved enzymatically, via soluble factors or by interacting with certain matrices/surfaces. Enzyme-induced activation uses thrombin at physiological or supra-physiological levels to cleave protease-activated receptors on the surface of platelets Thrombin is an irreversible, potent activator of platelets resulting in a strong intra-platelet signal for degranulation. Thrombin cleaves fibrinogen and polymerizes fibrin to produce an insoluble clot that binds and encapsulates platelets in the local vicinity. Use of thrombin to activate platelets requires that the PRP be injected immediately or else might clot and fail to pass through the needle.
Other platelet activators include calcium chloride, ADP, hypothermic conditions, and activating surfaces such as glass or collagens, but still rely on the intermediate steps of the enzymatic activation pathway, occurring downstream of thrombin-induced activation. Calcium mediates platelet granule tethering to the outer membranes and release of contents into the extracellular space ADP interacts with the P2Y1 and P2Y12 platelet surface receptors, stimulating the release of calcium from intraplatelet stores and promoting granule fusion. Calcium and ADP are thus synergistic. Hypothermia to less than 28°C has also been shown to weakly promote platelet activation, is ADP-dependent and a popular adjunct for platelet activation.Finally, collagen and negatively charged glass can be used to stimulate platelet aggregation and activation Depending on how they are activated, platelets release their growth factors differently and the optimum sequence of release is not known.
Platelet-rich fibrin (PRF) production relies on the collection of whole blood without anticoagulant, followed by a single centrifugation step for about 8 minutes at 200 x g. The result is a three-fraction blood product, as with PRP, but with the activation of clotting factors. As a result all three layers, the plasma, buffy coat and red blood cell fraction, are fixed in a fibrin matrix. The resulting PRF, consisting of the plasma fraction or plasma + buffy coat, can be extracted by cutting away the red blood cell fraction. PRF is equivalent to a 2-fold concentrated activated leukocyte-rich PRP product known to release an abundance of growth factors and which has an innate biological scaffold (fibrin PRF can easily be implanted into a surgical site, but because of its density cannot be injected unless homogenized.
Ligaments and Fibrocartilage
One of the most rewarding applications of PRP in interventional pain practice involves the treatment of ligament or fibrocartilage injuries, particularly those discovered using the skilled application of high-frequency diagnostic ultrasound. We have thus diagnosed and successfully treated chronic (and acute) hip labrum tears using LP-PRP activated with calcium chloride 10% (0.3 per 1 ml PRP) and injected into the gap of the tear (0.5 ml), above it (1 ml) and into the joint (2 ml). The calcium chloride enhances platelet aggregation in the setting of plasmin, which inhibits platelets and is in the joint fluid.
We have also had good results injecting PRP into non-displaced meniscus tears (e.g. 0.5 ml into tear, 1 ml peripheral to meniscus and 3 ml into joint, activated LP-PRP). Urzen and Fullerton reported on a case of an acute displaced bucket-handle tear reduced manually and treated with 3 injections of PRP The tear healed completely as seen on follow-up MRI and arthroscopy. Pujol et al showed improved pain, function and MRI signal in peripheral meniscus tears treated with PRP and mini-open repair versus mini-open repair alone
Podesta et al achieved high rates of return to play in 34 partial ulnar collateral ligament (elbow) injuries. Seijas et al injected PRP into partially torn anterior cruciate ligaments in a series of 19 professional soccer players, all of whom experienced improvement of stability as confirmed on KT-1000 testing and 16 returning at an average of 16 weeks after treatment It is important to note that PRP during arthroscopic surgery has not been shown to significantly improve outcomes, perhaps because circulating fluid dilutes the PRP, washed off adhesion factors on target tissues, or plasmin within the joint fluid inhibits the formation of a fibrin matrix.
In our experience, PRP is a highly effective treatment for partial tendon tears and tendinosis, but evidence-based reviews show comparable efficacy for a variety of other treatments including eccentric strengthening, needling and whole blood injections Sadoghi et al reviewed 150 studies and analyzed 14 high quality ones in animals and humans and concluded that PRP for Achilles tendinopathy has a medium to large positive effect for tendon tears but not for tendinosis.
In a double-blind randomized controlled multi-center trial, Mishra et al needled the common extensor tendons with and without PRP in 230 patients with chronic lateral epicondylitis At 6 months, 84% of the PRP and 63% of the needle only group had resolution of symptoms. Mishra’s study, like a number of others, did not separate patients with tears from those with tendinosis. For gluteus medius and minimus tendinosis, Jacobson et al found no significant difference between needling with PRP and without with 79% and 71% respective improvement at 3 months.
Fitzpatrick et al’s meta-analysis of 8 high quality studies concluded there was good evidence to support a single ultrasound-guided injection of LR-PRP for tendinopathy Fitzpatrick et al endorsed LR-PRP over LP-PRP on the basis of more studies having been done with LR-PRP as opposed to LP-PRP. The single LP-PRP study they reviewed showed improvement of pain from 7.5 to 1.2 in the 15 patients injected with LP-PRP versus 7.5 to 4.1 in 10 injected with bupivacaine at 6 months follow-up.We hope future clinical trials will compare LR-PRP to LP-PRP for tendinopathy, especially since in-vitro studies have shown that leukocytes reduce tendon matrix synthesis and promote catabolism
In our experience some tendons respond better to PRP than others depending on location and geometry. The common extensor tendon of the forearm, which has a broad attachment area, responds better than the common flexor tendon, which has a small attachment area. Tears of the rotator cuff of the hip respond better than tears of the rotator cuff of the shoulder, probably because of less tension and no adjacent joint fluid. When injecting tendons it is important to note that the needle should be visualized in two ultrasound planes to ensure injectate flows where intended. Tendons previously injected with corticosteroids or other destructive agents are less likely to respond to PRP and repeat injections might be necessary. In general, we expect significant improvement within 4-8 weeks of a single injection.
Joints and Cartilage
PRP is variably effective for osteoarthritis (OA) in our experience, in some cases resulting in long-term relief and in others not being effective at all. Typically mild to moderate improvement is seen, better and longer-lasting than with hyaluronic acid or corticosteroid, but temporary, on the order of months to a year. Results vary depending on location, with knees responding better than hips. In some cases it may not be the arthritis that is causing pain but an associated injury to the meniscus, a collateral ligament or fibrocartilage such as the hip labrum. In these cases, treating the associated injury may result in long-term resolution of symptoms and possible healing. Cartilage defects are not thought to be capable of significant healing and the primary benefit of PRP likely involves reduction of inflammation.
For knee OA, Riboh et al reviewed 6 randomized controlled trials (RCT) and 3 comparative studies on PRP LP-PRP was used in 6 studies and LR-PRP in 3; hyaluronic acid was used in 6 studies and placebo injection in 3. Riboh et al concluded that studies using LP-PRP resulted in improved pain and function compared to placebo, hyaluronic acid and corticosteroid.
Xu et al performed a meta-analysis of 10 studies on PRP for knee OA, but did not consider that LR-PRP and LP-PRP might result in different outcomes On the basis of the 2 best performed studies using LR-PRP, they concluded that PRP in general was not better than hyaluronic acid (HA). They attributed the positive outcomes of the other studies using LP-PRP to inadequate blinding of subjects and therefore a possible placebo effect.
For hip OA, Dallari et al randomized patients to PRP lysate, hyaluronic acid (HA) and PRP lysate + HA groups, showing the best outcome in the PRP lysate group, whose VAS scores improved from 3.5/10 to 1.4/10 at 3 months, rising to 2/10 at 1 year. By contrast, Battaglia et al, using PRP-lysate with an average leukocyte level of 8300/μL, showed no difference between PRP-lysate and HA and DiSante et al showed better outcomes with HA than PRP at 4 months.
For ankle OA associated with tibia vara and cross-leg sitting positions in Japan, Fukawa et al performed 3 LP-PRP lysate injections at 2 week intervals with reduction of pain from 6/10 to 2.5/10 for early and 4/10 for late stage disease at 3 months with maintenance of improvement in both groups at 6 months For moderate to advanced ankle OA, Repetto et al noted reductions of pain from 7.8+0.5 to 2.6+2.2 at an average of 17 months following a series of 4 weekly LP-PRP ankle injections For osteochondral defects of the talus, Mei-Dan et al compared LP-PRP to HA, noting improvements pain and function on the Ankle-Hindfoot scale from 66 to 98 and 66 to 78 respectively at 7 months.
For OA of the thumb, 2 intra-articular autologous conditioned plasma (2.4x LP-PRP) injections to the trapezium-metacarpal and scaphoid-trapezium-trapezoid joints spaced 4 weeks apart reduced pain from 6.2 + 1.6 to 4.0 + 2.4 at 3 months and 5.4 + 2.2 at 6 months.
Bone and Muscle
Hsu et al reviewed the evidence for PRP for spine and ankle fusions and bone in general and found no evidence that PRP is of benefit, even in conjunction with bone grafts We believe this is because bone is highly vascular, and in the case of surgery when bleeding and platelets are already present, the addition of more platelets does not add much. The same reasoning would apply to muscle. Colio et al performed a review on PRP injections for muscle and concluded that most of the published evidence, including one Level I study, indicates that PRP does not significantly benefit muscle injuries
Intervertebral Discs and Spine
Bodor et al provided a scientific rationale for PRP for intervertebral discs, given that it worked well for tendon tears and the fact that platelets have mechanical properties enabling them to pull together torn collagen. In 35 patients with lumbar and thoracic annular tears, degeneration and disc pain confirmed via anesthetic discography a week or two prior, they injected 1-2 ml of LP-PRP and noted good (reduced pain medications, improved function) to excellent (resolution of pain) outcomes in approximately two-thirds of patients at 4 to 8 weeks.13 In a double-blind RCT, Tuakli-Wosornu et al performed discography using radiographic contrast with (29 patients) and without (19 patients) LR-PRP, noting significant improvements in pain, function and patient satisfaction in the PRP group at all time points from 8 weeks to 1 year and beyond No studies have yet shown that disc height or hydration improves as a result of PRP treatment. In 46 patients with lumbar facet pain confirmed via fluoroscopically-guided intra-articular anesthetic injections, Wu et al did a RCT of LR-PRP versus corticosteroid showing improvement in both groups at 1 month but only the PRP group at 6 months.
Nerves and Neuropathic Pain
We have been surprised how often some patients (e.g. overweight 70 year old with chronic posterior ankle pain, tendinosis and tearing of the tibialis posterior) respond to PRP much faster than expected (3-5 days), with substantial improvement of pain and function before healing could have occurred. This has led us to thinking about the biologic purpose for pain. We believe there are three main reasons: 1) intrusion alert (e.g. thorn, mosquito) 2) harm avoidance (e.g. do not put hand in fire), 3) guide to healing (e.g. splint a fracture, reduce weight-bearing). With regard to the “guide to healing,” pain guides the organism to optimize the internal and external environment for healing. PRP injected at the site of injury might optimize the environment for healing. When this occurs there is no further purpose for pain, and nociceptors at the site of injury reduce or turn off the pain signal.
Kuffler has written extensively on PRP and neuropathic pain and hypothesized that PRP may help reduce neuropathic pain and neurogenic inflammation by optimizing the environment at the location of the severed nerve ends or neuroma. Kuffler et al have also been able to successfully eliminate pain in a patient with a chronic ulnar nerve injury by surgically freshening the nerve ends, placing them on both sides of a collagen tube and injecting LR-PRP within the gap Within 3 months, the patient’s chronic neuropathic pain resolved and a year later he had improvement of sensation and motor function.
In several patients with chronic neuropathy and neuropathic pain, we hydrodissected LP-PRP and LP-PRP-lysate around the ulnar nerve at the elbow without benefit. By contrast, Uzun et al found PRP to be helpful for carpal tunnel syndrome. The difference may be attributable to two primarily different etiologies, with the former being a compression neuropathy and the latter being an entrapment neuropathy caused by increased pressure within the carpal tunnel due to synovitis and swelling of flexor tendons. PRP may reduce synovitis and swelling of the tendons and pressure within the carpal tunnel and around the nerve.
Nerves have a good blood supply and in theory should not significantly benefit from additional platelets or growth factors. Further research will need to be done to determine the role of PRP for nerves and neuropathic pain.
PRP has become increasingly popular in sports, rehabilitation and pain medicine. There are at least 6 Level I studies for osteoarthritis of the knee showing improvement of pain and function following a single or a series of LP-PRP but not LR-PRP injections. There are at least 8 Level I studies showing efficacy for LR-PRP and one for LP-PRP for tendinopathies. One meta-analysis found PRP to be effective for Achilles tendon tears but not tendinopathy, but most clinical trials have not differentiated outcomes for patients with tears from those with tendinopathy.
PRP is promising for ligament and fibrocartilage injuries, especially in conjunction with precise ultrasound guidance or mini-open but not arthroscopic surgery. Muscles and bones are full of platelets (assuming an intact blood supply) and have not been shown to significantly benefit. PRP for discogenic pain is promising, with at least one Level I study showing good outcomes at 1 year and beyond.95 PRP for nerves and neuropathic pain remains investigational.
PRP procedures should ideally be performed by specialists capable of precisely identifying and guiding needles to sites of pathology using imaging guidance. Patients should avoid aspirin for 10 days and NSAIDS for 5 days. For optimum effect, PRP should not be in contact with local anesthetics and probably radiographic contrast dye. NSAIDS, which inhibit prostaglandin mediated vasodilation and have been shown to inhibit healing, should be avoided for at least 4-8 weeks following injections. While there is good evidence that PRP improves pain and function, there is limited evidence that it actually heals or regenerates tendons, discs or ligaments. Hopefully, future studies will continue to elucidate the healing and regenerative effects of PRP.
Acknowledgement: The authors would like to thank the non-profit 501(c)3 Napa Medical Research Foundation for their financial support.