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9 Articles in Volume 10, Issue #4
Chaos (Nonlinear Dynamics) and Migraine
Enhancement of Nerve Regeneration by Therapeutic Laser
Functional Capacity Evaluation (FCE)
Making Practical Sense of Cytochrome P450
Non-pharmacologic Treatment of Shingles
Pain, Neurotechnology, and the Treatment-enhancement Debate
The New Age of Prolotherapy
Treating Myofacial and Other Idiopathic Head and Neck Pain
Treatment of Painful Cutaneous Wounds

The New Age of Prolotherapy

In addition to traditional prolotherapy, platelet-rich plasma and stem cells are also available to enhance healing of musculoskeletal injuries and mitigation of pain.

We live in a technological age. With technology comes growth and enhancement of techniques and prolotherapy is no exception. In the March 2010 issue of the Mayo Clinic Health Letter, the authors talk about a new technique involving the injection of platelet rich plasma (PRP) into tendons.1 Quietly working its way through orthopaedic and sports medicine circles and disguised as a “new” treatment, PRP itself has been around since at least the early 1990s2 in surgical and dental applications, but only recently in the musculoskeletal arena. When used to treat injured tendons, ligaments or joints, PRP is simply a modern version of prolotherapy.3 Almost exactly five years ago, in the April 2005 issue of the Mayo Clinic Health Letter, the authors endorse prolotherapy and write: “In the case of chronic ligament or tendon pain that hasn’t responded to more conservative treatments such as prescribed exercise and physical therapy, prolotherapy may be helpful.”4

Now the Mayo Clinic is endorsing PRP, the “new” prolotherapy, for musculoskeletal injuries. In addition to PRP, stem cell joint injections are being used in recalcitrant cases of joint dysfunction—utilizing both bone marrow and fat tissue as stem cell repositories.5 Musculoskeletal ultrasound is also now available and gaining popularity for use in office diagnosis and guidance (notwithstanding the learning curve required for physician proficiency). This article explores these new developments and what this means for the field of prolotherapy and regenerative medicine.

Review of Prolotherapy
Introduced in the 1930s, prolotherapy is a method of injection treatment designed to stimulate healing.6 A recent definition is “the injection of growth factors or growth factor production stimulants to grow normal cells or tissue.”7 prolotherapy owes its origins to the innovation of Dr. Earl Gedney, an osteopathic physician and surgeon. In the early 1930s, Dr. Gedney caught his thumb in closing surgical suite doors thereby stretching the joint and causing severe pain and instability. After being told by his colleagues that nothing could be done for his condition and that his surgical career was over, Gedney did his own research and decided to “be his own doctor.” He knew of a group of doctors called “herniologists” that used irritating solutions to stimulate the repair of the distended connective tissue ring in hernias. He extrapolated this knowledge to inject his injured thumb and was able to fully rehabilitate it.8

In 1937, Gedney published “The Hypermobile Joint,”9 the first known article about prolotherapy (then called “sclerotherapy”) in the medical literature. The 1937 article gave a preliminary protocol and two case reports—one of a patient with knee pain and another with low back pain—with both successfully treated with this method. Gedney followed up this paper with a presentation at the February 1938 meeting of the Osteopathic Clinical Society of Philadelphia which outlined the technique.10 The solutions used then (and now) are primarily dextrose-based, although other formulas are used and can be effective.11 Prolotherapy is practiced by physicians in the U.S. and worldwide, has been shown effective in treating many musculoskeletal conditions—such as tendinopathies, ligament sprains, back and neck pain, tennis/golfers elbow, ankle pain, joint laxity and instability, plantar fasciitis, shoulder, knee pain and other joint pain.12

How Prolotherapy Works
Prolotherapy works by causing a temporary, low grade inflammation at the injection site, activating fibroblasts to the area, which, in turn, synthesize precursors to mature collagen and thus reinforce connective tissue.2 It has been well documented that direct exposure of fibroblasts to growth factors (either endogenous or exogenous) causes new cell growth and collagen deposition.13-17 Inflammation creates secondary growth factor elevation.2 The inflammatory stimulus of prolotherapy raises the level of growth factors to resume or initiate a new connective tissue repair sequence which had prematurely aborted or never started.2 Animal biopsy studies show ligament thickening, enlargement of the tendinosseous junction, and strengthening of the tendon or ligament after prolotherapy injections.18,19

Platelet Rich Plasma (PRP) Therapy

Platelet rich plasma (PRP) therapy, like prolotherapy, is a method of injection designed to stimulate healing. “Platelet rich plasma” is defined as “autologous blood with concentrations of platelets above baseline levels,”20 “which contains at least seven growth factors.”21 Cell ratios in normal blood contain only 6% platelets, however, in PRP, there is a concentration of 94% platelets (see Figures 1 and 2).22 Platelets contain a number of proteins, cytokines and other bioactive factors that initiate and regulate basic aspects of natural wound healing.23 Circulating platelets secrete growth factors, such as platelet-derived growth factor (stimulates cell replication, angiogenesis), vascular endothelial growth factor (angiogenesis), fibroblast growth factor (proliferation of myoblasts and angiogenesis), and insulin-like growth factor-1 (mediates growth and repair of skeletal muscle), among others.24 Enhanced healing is possible when platelet concentration is increased with PRP.25 Activated platelets “signal” to distant repair cells, including adult stem cells, to come to the injury site (see Figure 3). Increasing the volume of platelets accordingly increases the subsequent influx of repair and stem cells.26 Because the concentrated platelets are suspended in a small volume of plasma, the three plasma proteins fibrin, fibronectin, and vitronectin contribute to a repair matrix.27 You could compare dextrose prolotherapy and PRP this way: prolotherapy is like planting seeds in a garden; PRP therapy is planting seeds with fertilizer.

History of Platelet Rich Plasma Therapy
Beginning in the 1990s and continuing until now, “growth factors” have been a hot topic in the medical world. It is clear that growth factors play a pivotal role in all types of wound healing.28 Investigation into the use of PRP has been reported as early as the 1970s,29 but the necessary equipment was large, expensive ($40,000 in 1996), and required a large quantity of a patients blood (450 cc) and therefore limited to the operating room for large scale surgeries.30 Starting in the early 1990s, multiple reports and studies in maxillofacial dental, periodontal surgery,31,32 cosmetic surgery,33 and skin grafting showed dramatically improved healing with PRP (see Figures 4 and 5).

In the early 2000s, the use of PRP expanded into orthopedics to augment healing in bone grafts and fractures. Success there encouraged its use in sports medicine for connective tissue repair. Mishra and Pavelko, associated with Stanford University, published the first human study supporting the use of PRP for chronic tendon problems in 2006.34 This study reported a 93% reduction in pain at two year followup. Then, in 2008, Pittsburgh Steelers’ wide receiver, Hines Ward, received PRP for a knee medial collateral ligament sprain, and the Steelers went on to win SuperBowl XLII. Ward credited PRP for his ability to play in that game and his success with this treatment was discussed on national television.35

Since then, other high profile athletes—such as Takashi Saito, closing pitcher for the L.A. Dodgers, and golfer Tiger Woods—credit PRP for helping them return to their sport.36 PRP continues to gain wider acceptance in the sports world with studies continuing to validate the use of PRP for ligament and tendon injuries,37 knee osteoarthritis,38 degenerative knee cartilage,39 chronic elbow tendonosis,40 muscle strain41 and tears,42 jumpers knee,43 plantar fasciitis44 and rotator cuff tendinopathy45—albeit some skeptics and controversy remains.46,47

As the use of PRP has grown, the demand and availability for smaller, more portable and affordable machines has also grown. There are now several available models which allow the physician to create PRP from a small sample of a patients blood in the office setting (see Figure 6).48 Machines are very affordable and many companies offer a complimentary machine with a minimum purchase of PRP preparation kits over a period of time. However, not all marketed PRP devices are equal; they vary in quantity of blood required, platelet concentration, viability and number of spin cycles.49 Harvest Technologies was one of the first PRP devices to gain FDA approval.50 This system uses a floating shelf technology which preserves the viability of platelets until use. In his 2005 text, Marx rated the PReP unit by Harvest Technologies, along with PCCS by Implant Innovations, as the two most effective and practical PRP devices for physician office use, outpatient surgery centers, and wound care center treatment.51

Creation and Activation of PRP
A small amount of the patients blood is drawn (20-120 cc) into a syringe with a small amount of citrate (an anti-clotting agent) then typically spun for about 15 minutes in a special centrifugation system that separates the platelets, blood, and plasma. The plasma-poor layer is then drained off and the “buffy coat” plasma layer extracted along with a small amount of plasma and red cells. In the surgical setting, PRP is activated by the surgeon mixing in calcium chloride and/or thrombin to make a gel-like graft and then placing it where he/she wants accelerated healing. Type I collagen has also been found to be effective in activating and creating a PRP graft.52 In 2006, Murray et al demonstrated successful increase in healing of a central anterior cruciate ligament (ACL) defect in a canine ACL using a collagen-platelet rich plasma matrix graft.53 In some musculoskeletal studies, a 10% solution of calcium chloride is added to the PRP just prior to injection54,55 or is injected simultaneously via another syringe into the area being injected with PRP. Most commonly, however, connective tissue injections are given into the site where repair is needed without any additive. In that case, activation occurs by exposure to tendon-derived collagen released by the injured tissue which is being treated.56,57 “Peppering” the tissue during injection with the needle tip can help ensure endogenous thrombin release needed for activation.

Growth Factors in PRP Stimulate Repair
Growth factors present in granules are released when platelets are activated (see Figure 7).58 After activation, secretion of growth factors begins within 10 minutes. The viability of the platelets and continued release of growth factors into the tissue continues for seven days.59 Meantime, the platelets stimulate the influx of macrophages,60 stem cells and other repair cells, as discussed previously. Microtrauma created by the injection itself also stimulates influx of macrophages and growth factors as in the case of dextrose prolotherapy. Once the platelets die (average life span 7-10 days), the macro-phages continue wound healing regulation by secreting some of the same growth factors as the platelets did, as well as others.61 The amount of initial platelets present in the wound determines the rate of wound healing and explains why PRP used during a surgical procedure speeds recovery.62 This may be because PRP has a strong effect in the early phase of healing.63 Use of a “matrix” to hold the PRP material has been used—especially in the case of a large defect.

Optimum Platelet Concentration Level for PRP
Outpatient PRP preparation systems exist with the ability to concentrate platelets from two to eight times.64 There is some controversy about what the “optimum” platelet concentration should be, but a level of at least 1 million platelets per mL appears to be the “magic number.” Since the average patients platelet count is 200,000 +/- 75, a four to five times concentration appears to be the desired level.65,66 When levels are in the 5x range, the influx of adult stem cells has been noted to increase by over 200%.67 In 2008, Kajikawa et al concluded that PRP enhances the initial mobilization of “circulation-derived cells” in the early stage of tendon healing. “Circulation-derived cells” are defined as mesenchymal stem cells that have the potential to differentiate into reparative fibroblasts or tenocytes as well as macrophages.68 Under normal circumstances, circulation-derived cells last only a short time after tendon injury.69 The authors suggest this as one of the main reasons for the known low healing ability of injured tendons. If the circulation derived cells could be activated and their time-dependant decrease stalled with PRP, then the wounded tendon could more fully heal. This study found an increase in the circulation-derived cells with the PRP group, as well as increased production of types I and III collagen in the PRP group versus control.70 This finding of additional fibroblast proliferation and type I collagen production enhanced by increasing platelet concentrations concur with an earlier study by Lui et al.71 This provides evidence that PRP stimulates the chemotactic migration of human mesenchymal stem cells to the injury site in a dose-dependent manner—i.e., the more concentrated the platelets, the more stimulation.

There are also reports of less than four to five times concentration being effective, but it is possible that is a function of a higher starting baseline of platelets (i.e., the patient had a baseline of 400, thus a 2 or 3 fold expansion seemed to work well). It is also possible that studies which show the lack of effectiveness of PRP are in patients whose baseline platelet count is normally low, such that one million platelets/mL was not obtained.

Prolotherapy Versus PRP
The use of hyperosmolar dextrose (prolotherapy) has been shown to increase platelet-derived growth factor expression and up-regulate multiple mitogenic factors72 that may act as signaling mechanisms in tendon repair. Saline prolotherapy can have a similar effect.73 An interesting study published in the January 2010 JAMA compared PRP versus saline injection (basically saline prolotherapy) for chronic Achilles tendinopathy. Both groups improved “significantly” and the authors conclude there was no statistical difference between the improvement of both groups.74 Therefore, both PRP and prolotherapy have been shown to stimulate natural healing75 and both can be effective and both should be considered in the treatment plan for connective tissue repair. However, PRP may be more appropriate in some cases. When PRP is used as a prolotherapy “formula” for chronic or longstanding injuries, the PRP increases the initial healing factors and thereby the rate of healing. The prolotherapy itself (irritation, needle microtrauma) is what is “tricking” the body into initiating repair at these long forgotten sites as well as the PRP, itself, which also acts as an “irritating solution.” This is especially important with chronic injuries, degeneration and severe tendonosis, where the body has stopped recognizing that area as “something to repair.” In these cases, PRP may be more appropriate, however this determination should be made on an individual basis. PRP can also be used preferentially over dextrose prolotherapy in the case of a tendon sheath or muscle injury—areas occasionally but not typically treated with dextrose prolotherapy where the focus is the fibro-osseous junction (enthesis).76 It can also be used preferentially over dextrose prolotherapy because of patient preference (see Figure 8).

Whole Blood Injections vs PRP
Even before PRP, it was not unheard of to use whole blood as a prolotherapy solution, especially where the patient was hypersensitive to other formulas.77 A 2006 study in the British Journal of Sports Medicine studied the use of whole blood with “needling”(irritation such as with prolotherapy) and concluded that the use of autologous blood injection, combined with dry needling, “appears to be an effective treatment for medial epicondylitis.”78 Another study in that same journal in 2009 compared injections using whole blood, dextrose prolotherapy, platelet rich plasma and polidocanol (a sclerosing agent), and concluded that there is evidence to support the use of each of these agents in the treatment of connective tissue damage.79 However, there are only three known studies using whole blood, all of which were prospective case series without controls and small patient numbers.80-82 PRP studies, on the other hand, are growing not only in number, but also in quality.83,84 When examining the physiology of how activated platelets signal repair cells, it seems logical that using PRP (with higher levels of platelets per unit volume) be more effective than autologous blood although no study has yet directly compared the two.85

Whole Blood Injections Versus PRP
Even before PRP, it was not unheard of to use whole blood as a prolotherapy solution, especially where the patient was hypersensitive to other formulas.77 A 2006 study in the British Journal of Sports Medicine studied the use of whole blood with “needling”(irritation such as with prolotherapy) and concluded that the use of autologous blood injection, combined with dry needling, “appears to be an effective treatment for medial epicondylitis.”78 Another study in that same journal in 2009 compared injections using whole blood, dextrose prolotherapy, platelet rich plasma and polidocanol (a sclerosing agent), and concluded that there is evidence to support the use of each of these agents in the treatment of connective tissue damage.79 However, there are only three known studies using whole blood, all of which were prospective case series without controls and small patient numbers.80-82 PRP studies, on the other hand, are growing not only in number, but also in quality.83,84 When examining the physiology of how activated platelets signal repair cells, it seems logical that using PRP (with higher levels of platelets per unit volume) be more effective than autologous blood although no study has yet directly compared the two.85

Cortisone vs PRP
The use of cortisone in musculoskeletal injuries is controversial and the subject of various studies over the years. In February 2010, researchers in the Netherlands published the results of a well designed, two year randomized controlled blinded trial with a significant test group of 100 patients, comparing corticosteroid use to an injection of concentrated platelet rich plasma86 without ultrasound guidance. The PRP injection was given to the lateral epicondyle area of “maximum tenderness,” and a “peppering” technique was used in order to activate the thrombin release from the tendon—in this case endogenous thrombin is the activator for the injected platelet growth factors. The researchers indicate the importance of the “inflammation” phase *the first two days post treatment) during which there is a migration of macrophages to the injured tissue site. Macrophages release additional growth factors,87 and there is increased collagen synthesis on days three to five. The conclusion of the Netherlands study was that “PRP reduces pain and significantly increases function, exceeding the effect of the corticosteroid injection.”88

Safety Issues
Like prolotherapy, PRP therapy has low risk and few side effects. Concerns such as hyperplasia have been raised regarding the use of growth factors, however there have been no documented cases of carcinogenesis, hyperplasia, or tumor growth associated with the use of autologous PRP.89 PRP growth factors never enter the cell or its nucleus and act through the stimulation of external cell membrane receptors of adult mesenchymal stem cells, fibroblasts, endothelial cells, osteoblasts, and epidermal cells.90 This binding stimulates expression of a normal gene repair sequence, causing normal healing—only much faster. Therefore PRP has no ability to induce tumor formation.91 Also, because it is an autologous sample, the risk of allergy or infectious disease is considered negligible.92 Evidence also exists in studies that PRP may have an antibacterial effect.93

Is PRP “Blood Doping”?
The answer to this question is unclear and the subject of controversy. Under current rules of the World Anti-Doping Agency (WADA) for Olympic athletes, PRP is prohibited via the “intramuscular” route with other routes of administration requiring a Therapeutic Use Exemption.94 This WADA prohibition is based chiefly on the concern with the release of IGF-1 by activated platelets, although the type of IGF-1 released by platelets has too short a half-life to provide an athletic advantage, is the wrong isoform to create skeletal hypertrophy, and levels are subtherapeutic and therefore do not produce a systemic anabolic effect.95 A Consensus Meeting on the topic is planned for Spring 2010 by the Medical & Scientific Commission of the International Olympic Committee.96 Hopefully these restrictions will be lifted. While WADA regulates Olympic athletes, it does not have jurisdiction over professional sports leagues in the United States and PRP is not addressed specifically on any banned substances lists by those various leagues.

Related article: Stem Cell Prolotherapy: The Next Horizon

Musculoskeletal Ultrasound
Musculoskeletal ultrasound has been used by physicians, especially rheumatologists, in Europe for many years. Various machines exist, many are portable, and image quality has improved by light years in the past decade. Introduced to the U.S. within the last few years, musculoskeletal ultrasound allows high resolution, real time imaging of articular and periarticular—structures such as ligament, tendons, and cartilage, including tears and tendonosis—and can be used in the office setting to give quick answers and is also highly acceptable to patients.97 However, there are limitations, with one of the chief being the time it takes to learn. As stated by Dr. Rosenquist, an anesthesiologist at the University of Iowa, “It’s not something you pick up after staying at a Holiday Inn Express.”98 There is a high degree of operator variability with the technique, lack of standardization and a long learning curve.97 Musculoskeletal ultrasound is more common in Europe than the U.S. and in some European countries is part of physician training.97 The European Society of Musculoskeletal Radiology has established technical guidelines, protocols and hands-on training since 1994.99 In the U.S., there is growing demand for training in this emerging field and there are more and more courses being offered each year by various institutions.

Many prolotherapists produce spectacular results while being “low tech” without the use or necessity of musculoskeletal ultrasound. And imaging does not, nor should it, supplant the physician’s “common sense.” Imaging studies are notoriously unreliable in terms of musculoskeletal pain, with multiple studies showing a high percentage of abnormal scans in asymptomatic individuals100-103 and thus should always be correlated to the patient history and area of complaint. However, when imaging equipment is used—especially where testing can be addressed specifically to an area of complaint, along with dynamic (motion) analysis—these ultrasound studies can add useful additional information for the physician. However, a physician should avoid using it as the sole source of diagnosis but always take a good history and physical and have an understanding of the cause of a patients problem first before using imaging as a confirmation. Use of ultrasound guidance for injections may or may not be needed, depending on the specific problem being treated. Some of the PRP studies cited above did not use ultrasound guidance86 yet still obtained excellent results for the participants. Knowledge of anatomy and good technique goes a long way in the prolotherapy world and only administering injections with ultrasound guidance may limit the treatment scope, especially in a case of tendonosis where there is no discrete lesion. However, when indicated—as in the case of a discrete tear or effusion—the ability to visualize an injection under guidance, or the use of ultrasound to confirm a diagnosis, can be satisfying for the patient as well as the physician. Ultrasound can also help to objectively document change in tissue which otherwise would be purely subjective .

Conclusion
Marx and Garg write: “Surgeons do not heal tissue; they merely place it where nature can heal it.”25 With advances in science we are able to offer our patients safe, effective alternatives to surgery. Traditional prolotherapy, platelet rich plasma, and now stem cell therapy are available to enhance healing of musculoskeletal injuries and pain, along with musculoskeletal ultrasound for added diagnostic acumen. Yet, in spite of all these wonderful technological advances, there may still be times when the “low tech” approach is more practical. Technology is just a tool and should never become an obsession or violate common sense. Treating the patient in front of you and understanding what options are available for his or her condition will always be the foundation of good patient care, new age or old.

Last updated on: May 11, 2015
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