The application of regenerative therapies for the treatment of musculoskeletal conditions has emerged over the last decade with recent acceleration. These include prolotherapy, platelet-rich plasma, and mesenchymal stem cell therapy. These strategies augment the body’s innate physiology to heal pathologic processes. This article presents an overview of platelet-rich plasma and mesenchymal stem cell therapy for the treatment of musculoskeletal injuries. A brief literature review is included, as are techniques for the use of ultrasound guidance to assist with these procedures.
Key points
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Regenerative medicine techniques, including platelet-rich plasma (PRP) and mesenchymal stem cell (MSC) therapy, are becoming increasingly popular for the treatment of surgical and nonsurgical musculoskeletal injuries.
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PRP and MSCs, collectively referred to as orthobiologics, work by augmenting natural healing processes and have been found effective at increasing tissue regeneration.
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Orthobiologics are becoming increasingly preferred over conventional treatments, including anti-inflammatory medications and corticosteroid injections, which have been shown to have adverse effects.
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Ultrasound guidance can assist in the performance of the various procedures used in regenerative medicine, ranging from venipuncture to bone marrow and adipose aspiration.
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Further high-quality research on PRP and MSCs will serve to further define the most appropriate and effective applications for these treatments.
Introduction
Historical and recent evidence increasingly refute the use of corticosteroid injections for most sports injuries, especially tendinopathies. Coombes and colleagues studied lateral epicondylalgia and found poorer long-term outcomes with local corticosteroid injections compared with placebo. The application of regenerative therapies for the treatment of musculoskeletal conditions has emerged over the last decade with recent acceleration. These include prolotherapy, platelet-rich plasma (PRP), and mesenchymal stem cell (MSC) therapy. In brief, these strategies augment the body’s innate physiology to heal pathologic processes. This article focuses on some of the specific issues related to PRP and adipose and bone marrow MSC procedures, often collectively referred to as orthobiologics. Discussed are issues related to PRP and MSC injection procedures not addressed elsewhere in this issue. We also review specific factors related to patient and target tissue selection for the use of biologic agents for tendon, ligament, joint, and spinal pathologies.
Introduction
Historical and recent evidence increasingly refute the use of corticosteroid injections for most sports injuries, especially tendinopathies. Coombes and colleagues studied lateral epicondylalgia and found poorer long-term outcomes with local corticosteroid injections compared with placebo. The application of regenerative therapies for the treatment of musculoskeletal conditions has emerged over the last decade with recent acceleration. These include prolotherapy, platelet-rich plasma (PRP), and mesenchymal stem cell (MSC) therapy. In brief, these strategies augment the body’s innate physiology to heal pathologic processes. This article focuses on some of the specific issues related to PRP and adipose and bone marrow MSC procedures, often collectively referred to as orthobiologics. Discussed are issues related to PRP and MSC injection procedures not addressed elsewhere in this issue. We also review specific factors related to patient and target tissue selection for the use of biologic agents for tendon, ligament, joint, and spinal pathologies.
Platelet-rich plasma
Background
Wound healing naturally occurs in three phases: (1) the inflammatory phase, (2) the proliferative phase, and (3) the remodeling phase. Platelets, nonnucleated blood components formed from megakaryocytes, play an integral role in this process. Alpha granules within platelets include many growth factors, including insulin-like growth factor 1, platelet-derived growth factor, vascular endothelial growth factor, and transforming growth factor (TGF)-β1. These growth factors, among others released by platelets, influence chemotaxis and induce angiogenesis and extracellular matrix production, eventually leading to tissue repair.
Definition
PRP is autologous blood containing a higher than physiologic concentration of platelets. Considering this definition, no two samples of PRP are identical. The efficacy of PRP is therefore likely impacted by the composition of the PRP itself. It is challenging to compare the efficacy of PRP across clinical studies if the composition of the PRP used is not reported in detail. Several classification systems for PRP have been proposed, although none have been widely accepted. The Platelet Count, Leukocyte Presence, Red Blood Cell Presence, and Use of Activation classification system identifies PRP samples by platelet concentration (absolute number of platelets), leukocyte concentration (including concentration of neutrophils), red blood cell concentration, and activation by exogenous agents. This classification has been suggested as more specifically categorizing the type of PRP injected especially when reported in clinical studies.
Mechanism of Action
The concept supporting the clinical use of PRP is the augmentation of healing processes in tissues with low intrinsic healing potential. Several studies have investigated the mechanism in which this occurs. Kajikawa and colleagues found that PRP activated circulation-derived cells (which play an important role in the healing processes of tissues) in wounded rat patellar tendons. PRP has also been shown to increase levels of growth factors that promote angiogenesis, increasing the blood supply to the injured area. This then allows for circulating factors to reach the injured area to promote healing and remodeling.
Growth factors in PRP also give it anabolic properties; it increases tenocyte proliferation, matrix gene expression, protein production, and chemotaxis of bone marrow cells. Furthermore, TGF-β1, found in PRP, inhibits the expression and release of multiple catabolic growth factors including interleukin (IL)-1β and tumor necrosis factor (TNF)-α. The anti-inflammatory effects of PRP are evident in its use in osteoarthritis (OA). It was found to decrease expression of nuclear factor kappa-light-chain-enhancer of activated B cells, a major inflammation regulator.
A PRP injection usually requires 60 to 100 mL of whole blood obtained via peripheral venipuncture. The whole blood undergoes processing as described later for PRP to be harvested. Although peripheral venipuncture is most often done in other settings without imaging guidance, it is challenging for clinicians who do not otherwise routinely perform venipuncture in their practice. Ultrasound guidance is commonly used during the placement of central venous catheters in emergency department and intensive care unit settings. However, recent studies have also found benefit of ultrasound guidance in small vessel, peripheral venous access.
Peripheral veins can often be visualized (in patients with fair skin or prominent veins) or palpated. Deeper veins are difficult to identify without imaging guidance and thus can be more difficult to access. Ultrasound guidance has been shown to increase overall success of peripheral venous catheterization while decreasing time needed for cannulation. Another important factor to consider is the number of skin punctures or attempts needed to catheterize a vein. Multiple skin punctures increase the risk of complications (infection and bleeding) and patient discomfort. Ultrasound guidance provides the advantage of a higher likelihood of successful venous access on the first attempt.
When using ultrasound guidance to assist in venipuncture for PRP, veins of the forearm are used. Veins can be differentiated from arteries using color or power Doppler imaging. Also, veins are easily compressible with probe pressure, whereas arteries are less compressible. Nerves often travel along blood vessels; thus, ultrasound may assist the operator in avoiding nerves and other adjacent structures during venipuncture. The vein is usually visualized in a transverse view, “out of plane” to the approaching needle. With this view, the ultrasound operator can visualize the tip of the needle as it approaches the lumen of the vein ( Figs. 1 and 2 ). Given the relatively large volume of blood needed, ultrasound visualization is maintained throughout the blood draw to ensure the needle stays within the lumen of the vein.
PRP is prepared by centrifugation of anticoagulated whole blood. It is initially separated into three layers based on specific gravity: (1) plasma (top layer), (2) platelets and leukocytes (middle layer, termed the “buffy coat”), and (3) red blood cells (bottom layer). The bottom layer is typically discarded; the buffy coat and top layer are then often subjected to a second centrifugation to separate PRP from platelet-poor plasma. PRP may then be activated with calcium chloride or thrombin to cause the release of growth factors from alpha granules to occur more rapidly, although this additional step is not universally performed. The senior author does not recommend activation with the addition of an agent, but rather natural activation of platelets through contact with degenerative tissue during subsequent injection. Different harvesting and centrifugation kits yield different volumes and concentrations of platelets. It has even been shown that PRP samples produced from the same patient, using the same centrifugation protocol and equipment, may lead to PRP of varying composition.
Evidence for its Application
Although there has been an overall increase in the application of and research regarding the use of PRP, there is still a paucity of randomized control trials producing Level 1 evidence. Lateral epicondylosis is an overuse injury, which in the past was typically treated with a corticosteroid injection. Although there may be inflammation acutely, in chronic tendon injury, there is generally no evidence of inflammatory cells but rather degenerative changes within the tendon. Therefore, the use of anti-inflammatory agents, such as corticosteroids, becomes less logical, and furthermore, has been shown to be harmful to the tendon and clinical outcomes. In contrast, the use of such agents as PRP to stimulate tissue healing would seem to be a more reasonable choice. The current literature seems to support the efficacy of PRP in the treatment of chronic injuries. A comprehensive review of the scientific evidence of PRP for the treatment of all tendinopathies and OA is beyond the scope of this article and has recently been reviewed by Malanga and Nakamura. The authors concluded that there is currently Level 1 evidence to support the use of PRP for treatment of tendinopathies of the elbow and OA of the knee. Additional studies seem to demonstrate efficacy in other tendons and ligaments, although Level 1 evidence is lacking for the use of PRP for the treatment of muscle strain, acute ligamentous sprains, patellar tendinosis, Achilles tendinosis, and rotator cuff injuries, and the authors additionally suggest that additional investigation is needed for the use of PRP in these conditions.
Mesenchymal stem cells
Background
MSCs are found abundantly in adipose tissue and bone marrow, and have been used for orthopedic applications given their therapeutic effects that potentiate tissue healing. Their use for clinically relevant therapies has evolved, particularly in musculoskeletal medicine, including cartilage disorders and soft tissue injuries.
Definition
Adult MSCs are derived from perivascular cells called pericytes. Once a free pericyte, dissociated from the basal lamina of a blood vessel, it is exposed to the chemotactic profile of the soft tissue environment and becomes an MSC. The Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy has proposed three criteria to define MSCs: (1) MSCs must be plastic adherent when maintained in standard culture conditions; (2) they must express CD105, CD73, and CD90 and lack expression of CD34, CD45, CD14 or CD11b, CD79a or CD19, and HLA-DR in culture; and (3) MSCs must have the potential to differentiate into osteoblasts, adipocytes, and chondrocytes in vitro.
Mechanism of Action
MSCs have multiple mechanisms that include anti-inflammatory, immunomodulatory, and paracrine effects. In an inflammatory environment that includes IL-1, IL-2, IL-12, TNF-α, and interferon (INF)-γ, MSCs secrete an array of growth factors and anti-inflammatory proteins with complex feedback mechanisms among the many types of immune cells. The key immunomodulatory cytokines include prostaglandin E 2 , TGF-β1, hepatocyte growth factor, stromal cell-derived factor 1, nitrous oxide, indoleamine 2,3-dioxygenase, IL-4, IL-6, IL-10, IL-1 receptor antagonist, and soluble TNF-α receptor.
MSCs inhibit many inflammatory cells including T cells, natural killer cells, B cells, monocytes, macrophages, and dendritic cells. MSCs promote the transition of T-helper 1 to T-helper 2 cells by reducing INF-γ and increasing IL-4 and IL-10. The restored T-helper 1/T-helper 2 balance has been shown to improve tissue regeneration in cartilage, muscle, and other injuries. Reducing INF-γ and production of IL-4 promotes macrophage conversion from M1 (encourage inflammation) to M2 (decrease inflammation). This transition promotes a shift from proinflammatory, antiangiogenic, tissue growth inhibition to anti-inflammatory, proremodeling, and tissue healing. The paracrine behavior of secreted bioactive growth factors, cytokines, and chemokines is responsible for the many functions of the MSC immune response and healing potential.
Evidence for Mesenchymal Stem Cell Therapies
Osteoarthritis
The anti-inflammatory and immunomodulator properties of MSCs make them effective in the treatment of articular cartilage defects. A case study done by Goldring demonstrated this regenerative potential examining the use of MSCs in a patient with meniscus and cartilage injuries. Twenty-four weeks after percutaneous injection into the affected knee joint, there was a significant increase in cartilage and meniscus volume, as seen on MRI. The patient attained increased range of motion and decreased pain scores. In addition, several pilot studies have demonstrated improved patient pain scores and functional status after cartilage defect treatment with cultured bone marrow stromal cells. Emadedin and colleagues studied six women who were candidates for joint arthroplasty. They underwent cultured bone marrow MSC injection. MRI in three of the six patients demonstrated decreased edematous subchondral patches. Initial improvements in pain and function were reported; however, 6 months postinjection, all patients presented with recurring pain.
An institutional review board approved registry (Regenexx, IORG0002115) was started in 2005 and is currently collecting outcome and adverse effects data from more than 2500 patients who have received bone marrow concentrate (BMC) injection treatment of various orthopedic conditions. Preliminary, unpublished data collected from 539 cases of BMC application in the knee demonstrate positive results. Of the patients who have returned for follow-up to date (145 patients at 1 month, 98 patients at 1 year, 30 patients at 2 years, and 11 patients at 3 years), improvement in symptoms was demonstrated in 40%, 52%, 60%, and 68%, respectively (J.R. Schultz, unpublished data, 2014).
Sampson and colleagues reported preliminary data with favorable outcomes in 125 patients receiving hip, knee, shoulder, ankle, or cervical zygapophyseal joint BMC injections. There was a 71% reduction in overall pain at a median follow-up of 148 days postinjection in patients with complete data available. When comparing data from 87 patients with both preprocedural and postprocedural pain scores (complete) versus 38 patients with incomplete data, there was no evidence of selection bias, because both groups had similar characteristics (ie, age, body mass index, follow-up time, satisfaction). Knee injections had the largest improvement in pain scores compared with the other joints. Satisfaction with the procedure was reported by 92% of patients, and 95% would recommend the procedure to a friend. Contrary to prior reports in the literature, age had no correlation with outcomes in this cohort of patients; those aged up to 79 years reported positive results. BMC therapy has also been used as an adjunct postoperatively to accelerate healing after procedures, such as arthroscopic debridement, meniscal transplantation, and subchondroplasty.
Adipose-derived MSCs have also shown clinical promise. Koh and Choi performed MSC injection in conjunction with arthroscopic lavage in elderly patients with knee OA. Two years later, 14 of the 16 patients underwent second look arthroscopy, which showed improved or maintained cartilage. They also reported improved Knee Injury and Osteoarthritis Outcome Scores, Visual Analog Scale pain scores, and Lysholm scores at the 2-year follow-up. Jo and colleagues examined 18 patients with knee OA and found decreased cartilage defect size, improved knee function, and decreased pain after injection of adipose-derived MSCs.
Osteonecrosis of the femoral head
The quality of outcome studies on stem cell therapy for osteonecrosis is improving. Gangji and colleagues showed a reduction in mean volume of femoral necrotic lesions after BMC injection compared with standard care alone at 24 months. A 5-year follow-up study showed patients with early femoral head osteonecrosis were less likely to deteriorate to fractural stage if injected with BMC. Those patients also had a longer survivorship compared with control subjects (52.2 months vs 26.5 months). Hernigou and colleagues reported 69% resolution and 50% decreased mean volume in 342 patients with stage 1 or 2 avascular necrosis. Zhao and colleagues found BMC improved Harris Hip scores and Femoral Head Low Signal Intensity scores compared with core decompression. Mao and colleagues found that, 5 years after BMC injection, 72 out of 78 hips with osteonecrosis of the femoral head achieved a satisfactory clinical result, whereas only 6 of 78 required a total hip arthroplasty.
Nonunion fractures
A recent study examined 86 patients with diabetes with ankle nonunion treated with BMC and compared results with matched subject with diabetes who underwent standard iliac crest autograft. In the BMC-treated group, 82.1% healed with no major complications. Conversely, 62.3% of the standard care group healed; major complications in that group included 5 amputations, 11 instances of osteonecrosis of fracture wound edge, and 17 infections. Overall, the BMC group showed lower morbidity with greater healing rate.
Anterior cruciate ligament injuries
There is also evidence that BMC injection has beneficial effects in anterior cruciate ligament tears. In a case series by Centeno and colleagues, 10 patients with ACL tears with less than 1 cm retraction underwent an intraligamentous injection of BMC under fluoroscopic guidance. Seven of the 10 patients showed improvement with evidence of improved ligament integrity and increased lower extremity functional scores.
Tendinopathy
Among other soft tissue injuries, there is a greater available volume of studies on tendon injury. Pascual-Garrido and colleagues studied eight patients with refractory patellar tendinopathy for 5 years after bone marrow–derived MSC injections. Of the eight patients, seven were completely satisfied. Statistically significant improvement was seen for most clinical scores including the Knee Injury and Osteoarthritis Outcome Scores activity of daily living, symptom, and sport subscores.
Recent evidence is in favor of the efficacy of stem cells to augment surgical repair of rotator cuff tears. Ellera Gomes and colleagues used mononuclear autologous stem cells to augment conventional rotator cuff repair in 14 patients. MRI analysis after a 12-month follow-up period demonstrated tendon integrity in all cases. Hernigou and colleagues examined 90 patients 10 years after routine arthroscopy with or without BMC injection and found substantial improvement in tendon integrity. In the BMC-treated group, 87% of patients had intact rotator cuffs compared with 44% in the control group.
Safety
PRP and MSC are autologous procedures. Because the cells are removed from and replaced into the same patient, there is no risk of a transfusion reaction or other immune-modulated reactions to the substances when they are reinjected. Risk of disease transmission, such as from an allogenic tissue donor, is also not of concern with these autologous procedures. Wakitani and colleagues studied the safety of bone marrow–derived MSCs for treatment of articular cartilage defects. They monitored 41 patients who received 45 bone marrow MSC injections for 5 to 137 months postprocedure and found no occurrences of infection or tumor growth. Feisst and colleagues cited several phase I and II trials assessing the safety of adipose-derived stem cells without any identifiable significant adverse events. There continues to be ongoing trials assessing the safety and efficacy of bone marrow– and adipose-derived MSCs with promising results.
Further Research
Although the evidence for stem cell application has shown some promise, there remains a need for more robust studies. To date, there are only two studies regarding cartilage treatments with MSCs that are prospective, observational cohorts, one case series with histologic markings and one randomized control trial. With the publication of higher quality research, the appropriate application for stem cell therapies in musculoskeletal medicine will be clarified.
Procedures
Bone marrow
The posterior superior iliac spine is the most commonly accessed landmark for bone marrow aspiration (BMA). This is done either manually with axial force applied to an 11-gauge trephine needle or with a manual drill using a smaller gauge needle. Using ultrasound guidance, a linear-array transducer is typically used (a curvilinear-array transducer may be necessary because of patient body habitus) ( Fig. 3 ). The posterior superior iliac spine should be identified with the medial aspect of the probe fixed on it while the lateral portion of the probe is swept between the greater trochanter and the anterior superior iliac spine. Within this arc, the aspirate needle is guided in plane from lateral to medial to the ilium, about 1 to 2 cm from the iliac crest surface for marrow entry ( Fig. 4 ). It can then be guided cranially for additional sites (within zone 1 as shown in Fig. 5 ).
