Regenerative Medicine for Muscle and Ligament Problems

Recently published in “Techniques in Regional Anesthesia and Pain Management” by Elsevier

Regenerative medicine for muscle and ligament problems: Technical aspects and evidence

By Sean Colio, MD, Matthew McAuliffe, MD, Yvette Uribe, BA, and Marko Bodor, MD

Abstract

Interest in regenerative medicine for treating musculoskeletal pathology has grown tremendously over the past decade. Part of its appeal lies in the ability to use a patient’s own cells to potentially heal acute and chronic injuries. While evidence grows supporting
its use in certain injuries, a perception exists that regenerative medicine may be a panacea. In this article,we review the evidence for platelet-rich plasma and bone marrow aspirate concentrate in treating muscle, ligament, and fibrocartilage injuries. We also offer our own practice experiences and technical considerations in the uses of these techniques.

 

PRP for muscle injuries
Muscle injuries can be classified as extrinsic due to external forces such as a laceration or direct blunt trauma to the muscle. Intrinsic muscle injuries are caused by muscular contraction during concurrent elongation resulting in over-extension of the fibers and tearing. A common location for intrinsic muscle injuries is the weak point for the muscle unit at the myotendinous junction resulting in rupturing of muscle fibers and small vessels causing bleeding and hematoma formation. The degree of muscle injury can be measured using the Peetrons ultrasonography classification: grade 0 has no visible lesion on ultrasound, grade I corresponds to minimal elongation with less than 5% of muscle involved, grade II involves partial tears involving 5%-50% of muscle volume or cross-sectional diameter and grade III lesions are full-thickness muscle tears with complete retraction.
Traditional treatment options have been directed toward reducing bleeding and swelling at the injury including rest, compression, elevation, physical therapy, ice, nonsteroidal anti-inflammatory drugs, ultrasound modalities, and time. Newer treatment options aiming to accelerate the rate of recovery, reduce recurrent injuries and minimize fibrosis are the subject of ongoing research and have led to an interest in the use of platelet-rich plasma (PRP). PRP injected at the site of muscle injury has been thought to modulate inflammation, improve angiogenesis, reduce fibrosis, and enhance muscle fiber regeneration.4-6 PRP might replace a muscle hematoma with platelets, plasma, and a fibrin scaffold to enhance hemo-stasis, secrete growth factors and guide the repair process.

Wright-Carpenter et al induced gastrocnemius muscle injuries in mice and injected autologous conditioned plasma vs saline. At 30-48 hours histological results showed accel-erated muscle satellite cell activation and increased diameter of regenerating myofibers in the PRP group vs the saline group. Quarteiro et al induced muscle injuries in the gastro-cnemius muscle of rats comparing PRP to physiologic serum injection to no treatment. Between 7 and 21 days, the PRP group showed a higher mean quantity of collagen production, but after 21 days the morphological features of muscles were the similar in all groups. Delos et al induced gastrocnemius muscle injuries in rats comparing injections of PRP vs saline. There were no differences between groups at any time point for the primary outcome measure of maximal isometric torque, nor were there any differences in the proportion of centronucleated muscle fibers and scar tissue on histological analysis after 15 days.
Hamstring muscle injuries are one of the most common injuries among athletes resulting in substantial time lost from sport. PRP has been used for these injuries with the goal of faster return to training and competition and reduced chance of reinjury. PRP is typically injected in the acute phase, 24-48 hours postinjury. Rettig et al performed a retrospective review of 10 NFL players with similar hamstring injuries, 5 of whom received PRP 24-48 hours postinjury and 5 of whom did not. The median time for return to play was 20 days in the PRP group and 17 days in the non-PRP group. A recent study by Zanon et al11 evaluated 25 professional soccer players with grade 2 hamstring injuries, all of whom received PRP injections. The average return to sport was 36.8 days, significantly longer than the 22 days reported by Ekstrand for grade 2 hamstring injuries treated without PRP.
In a double blind randomized controlled trial of PRP vs saline for acute muscle injuries in a cohort of 80 competitive and recreational athletes, Reurink et al found that PRP did not accelerate return to play or lower the reinjury rate at 1 year. Hamilton et al compared PRP vs no injection for acute grade 1 or 2 hamstring injuries among a population of professional, semiprofessional, and amateur athletes in Qatar. The median time for return to sport was 21 days in the PRP group and 25 days among those receiving no injection. Guillodo et al15 did not find faster return to play for severe (grade 3) hamstring injuries treated with a single PRP injection.
Some studies did show PRP as helping athletes return to sports more quickly. Hamid et al provided a single PRP injection of 3 mL in 14 patients with acute grade 2 hamstring injuries.16 No local anesthetic was used, the PRP was not activated, and the patients were kept supine for 10-15 minutes. The mean return to play time was 26.7 +/- 7 days in the PRP group and 42.5 +/- 20.6 days in the control group. Recently, Rossi et al17 published results on a single PRP injection for grade 2 gastrocnemius, hamstrings, and quad-riceps muscle injuries. They reported a return to play of 21.1 +/- 3.1 days for the PRP group and 25 +/- 2.8 days for the control group. No local anesthetic was used, the PRP was not activated, and the amount of PRP was correlated with the volume of the injury.
While the safety profile for PRP is appealing, based on the current evidence it is difficult to recommend its use in muscle injuries as a means for faster recovery. This should not be surprising because muscle is highly vascularized and has abundant platelets, in contrast to tendons, ligaments, and intervertebral discs. As such the administration of additional platelets would not appear to be helpful except in the case of infarcted or ischemic tissue.

Mesenchymal stem cells for muscle injuries
Hernigou et al18 published on the use of bone marrow aspirate concentrate (BMAC) in conjunction with arthroscopic rotator cuff repair in 59 patients. At time of follow-up 10 years later, 40 of the 59 did not have evidence of a re-tear or progression of fatty infiltration on magnetic resonance imag-ing (MRI), whereas the remaining 19 had an increase of 1 grade of fatty infiltration. Hernigou et al suggested that injection of BMAC into atrophied muscle may have had a positive effect, however, it is not possible to tell whether the noted improvements were attributable to a direct effect of BMAC on muscle or an indirect effect related to improved muscle activation following tendon healing or both. BMAC is being researched to treat chronic limb ischemia and infarcted myocardium. Tateishi-Yuyama et al injected BMAC into the gastrocnemius of ischemic limbs and demonstrated angio-genesis and the formation of stable capillary vessels. Beeres et al showed that BMAC injection into infarcted myocar-dium improved MRI and tissue Doppler imaging parameters of diastolic function.

PRP for ligament and fibrocartilage injuries
Ligament injuries have traditionally been classified as grades 1-3. A grade 1 injury is stretching of the ligament with no visible tear, grade 2 is an incomplete tear or rupture and grade 3 is complete tear or rupture of the ligament. The mechanism of injury involves joint distraction and tensile strain in the direction that the ligament is stabilizing.
PRP has been shown to enhance anabolic effects such as collagen synthesis, angiogenesis, proliferation of fibroblasts and anti-fibrotic effects. PRP has been shown to augment the differentiation of musculoskeletal stem cells into mature cells. In-vivo studies have assessed the effects of PRP on repair of anterior cruciate ligament (ACL) injuries, acute medial collateral ligament (MCL) injuries, meniscal tears, high ankle sprains, and ulnar collateral ligament (UCL) injuries.
Murray et al have done extensive research showing that PRP combined with a collagen composite improves the heal-ing of ACL grafts in pigs. When used for primary repair of the ACL, they found only an 11% increase in ligament strength compared to controls. They also found that high concentrations of PRP  (5) were inhibitory on proliferation of ACL ligament cells in vitro compared to low concentrations (1). In humans, Orrego et al noted that PRP had an enhancing effect on the graft maturation process evaluated by MRI signal intensity, but no significant effect on the osteoligamentous interface or tunnel widening at 6 months. Sanchez et al32 noted higher synovial coverage and graft width in patients treated with PRP as assessed arthroscopically 6-24 months following ACL reconstruction. Other studies have shown the benefits of PRP with improved graft maturation and vascularization in ACL reconstruction using autologous tendon grafts.
In a series of 19 professional soccer players with subacute partial ACL disruptions, Seijas et al injected PRP into the torn bundle during and most significantly after arthroscopy when all the irrigation fluid had been drained. All players experienced restoration of knee stability as assessed objectively by KT-1000 measurements, with 16 returning to pre-injury levels of sport at an average of 16 weeks following the procedure, 2 to lower levels of sport, and 1, who had experienced cartilage damage, not being able to return to sport.35
MCL tears generally have a good prognosis with conservative treatment with or without PRP. Foster et al noted a 27%faster return to play among those who received PRP into the MCL within 72 hours of injury. Yoshioka et al studied PRP for MCL injuries in 31 rabbits, showing higher levels of growth factors and earlier signs of repair at 3 and 6 weeks after administration.
Injuries of the UCL of the elbow can devastate the career of a throwing athlete and usually necessitate reconstructive surgery. In 34 athletes with partial UCL tears who had failed 2 months of conservative therapy, Podesta et al performed ultrasound-guided PRP injections resulting in an average return to play time of 12 weeks and 88% of patients playing without pain at 70 weeks. A recent case report by Hoffman et al demonstrated a good outcome with return to activity without pain for a patient who underwent UCL reconstruc-tion augmented by PRP and a dermal allograft.
The meniscus of the knee functions to reduce contact pressure between the femoral condyle and the tibial plateau. When the meniscus is injured, it increases the risk of osteoarthritis. Urzen and Fullerton reported on a case of a 43-year-old athlete with a displaced bucket-handle tear of his medial meniscus, which was reduced with manipulation and completely healed, as assessed by arthroscopy, following a series of 3 PRP injec-tions. The authors acknowledged that there have been several case reports documenting spontaneous healing of bucket-handle medial meniscus tears without PRP.
PRP has been studied in conjunction with arthroscopic surgery for meniscus and hip labral tears, showing no benefit.  Recognizing that irrigation fluid during arthroscopic surgery might dilute or inhibit PRP, Pujol et al used a mini-open approach to repair the meniscus with and without the addition of PRP in a case-control study. Both groups improved significantly, but the PRP group had slightly better pain, activity and quality of life scores, and 75% had resolution of increased T2 signal in the outer meniscus compared to 40% of controls at 1 year.
Injuries to the syndesmosis between the tibia and the fibula (high ankle sprains) are uncommon but can be very debilitating with a high likelihood of long-term disability. In these cases, the anterior inferior tibio-fibular ligament is often partially or completely ruptured. Using ultrasound, Laver et al diagnosed and injected PRP in 8 patients with anterior inferior tibio-fibular ligament injuries and compared them to 8 controls treated without PRP. They noted a more rapid return to play, less pain and better resolution of the gap between the tibia and fibula in the PRP compared with the control group.

Mesenchymal stem cells for ligament and fibrocartilage injuries
Kenaya et al studied partial ACL tears in rats and noted improved healing and mechanics in animals injected with cultured bone marrow mesenchymal stem cells compared with controls. Oe et al created partial ACL tears in rats and injected BMAC, cultured bone marrow mesenchymal stem cells and buffered saline. At 4 weeks the BMAC and cultured cell groups recovered 90% and 88% tensile strength compared with 50% among controls. Centeno et al used a fluoroscopi-cally guided technique to inject 10 patients with acute and subacute partial and less than 1 cm retracted complete ACL tears using a combination of BMAC, PRP, and platelet lysate. At 3 or more months, 7 of 10 patients experienced improvement of pain, function and ACL appearance on MRI. Centeno et al reported on a case of a 34-year old who experienced a 23% increase in meniscal volume 3 months following treatment with cultured bone marrow mesenchymal cells. Vangness et al compared injections of 150  (106 vs 50)  106 allogeneic cultured bone marrow mesenchymal cells 7-10 days after meniscectomy and found that 24% of the higher vs 6% of the lower vs 0% of the sodium hyaluronate groups had increased meniscal volume at 1 year. Note that typical quantities of noncultured mesenchymal cells harvested from bone or fat are several orders of magnitude smaller than cultured cells.

 

Technical considerations
In our 10 years of experience using precise ultrasound guidance to inject PRP, our highest success rates have been in treating injuries that can be visualized on ultrasound, such as intra-substance, partial or small full-thickness tears in ligaments and tendons. We have noted that the configuration of tendons or ligaments makes a difference in outcome. Flat tendons and ligaments with broad attachment areas, such as the common extensor tendon of the forearm and the MCL of the knee, tend to heal better than round tendons with short attachment areas, such as the common flexor tendon of the forearm.
Specific considerations when injecting biologic agents into tendons or ligaments includes being aware that tendons and ligaments are solid or semi-solid structures and that the spread of injectate should be seen in 3-dimensions to ensure that it reaches all areas of pathology. This requires skill and hand to eye coordination in order to precisely and efficiently rotate the ultrasound transducer back and forth between short and long axis views around a point of interest. Additional technical considerations include calculating the volume of a torn area or region of pathology before injecting and knowing when to activate PRP. Too much volume could lengthen and widen a tear while too many cells could inhibit healing. The volume of a tear or region of pathology in milliliters is calculated by multiplying its width, depth, and length in centimeters. Calcium chloride 10% should be used to activate PRP (in a 1:6 ratio) when injecting ligaments that are exposed to joint fluid in order to enable platelet adhesion and aggregation. Joint fluid contains plasmin, which inhibits platelet aggregation. Local anesthetics inhibit platelet aggre-gation and should not be mixed with PRP or BMAC.
In our experience, the success of treatment depends on the physician/surgeon having high level diagnostic ultrasound skills, enabling the discovery of problems that might have been missed resulting in a diagnosis of “chronic pain.” Furthermore, a number of ultrasound interventional techni-ques require high level ultrasound visualization skills to perform successfully, such as those recently developed for injecting the ACL and PCL.50,51 We recommend that all physicians who wish to practice regenerative medicine become proficient in diagnostic and interventional ultra-sound, as covered in several excellent texts.

Conclusion
Most of the published evidence, including one level I study, indicates that PRP and BMAC are not effective for acute muscle injuries. There is good evidence, including one level I study, that BMAC is effective in improving pain, function and perfusion in ischemic muscle. There is level II evidence indicating PRP and BMAC to be effective for some ligament and fibrocartilage injuries when done separate from arthro-scopic surgical procedures. Current best practice is for PRP and BMAC to be injected precisely to areas of identified pathology using ultrasound or other imaging guidance.

 

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