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Scientific Articles   |    
Use of an Antifibrotic Agent Improves the Effect of Platelet-Rich Plasma on Muscle Healing After Injury
Satoshi Terada, MD1; Shusuke Ota, MD, PhD1; Makoto Kobayashi, MD1; Tetsuo Kobayashi, MD, PhD1; Yutaka Mifune, MD, PhD1; Koji Takayama, MD, PhD1; Michelle Witt, MS1; Gianluca Vadalà, MD2; Nick Oyster, BS1; Takanobu Otsuka, MD, PhD3; Freddie H. Fu, MD4; Johnny Huard, PhD1
1 Stem Cell Research Center, Department of Orthopaedic Surgery, University of Pittsburgh Medical Center, Bridgeside Point II, Suite 206, 450 Technology Drive, Pittsburgh, PA 15219. E-mail address for J. Huard: jhuard@pitt.edu
2 Department of Orthopaedic and Trauma Surgery, University Campus Bio-Medico of Rome, University of Rome, Via Alvaro Portillo 200, 00128 Rome, Italy
3 Department of Orthopaedic Surgery, Nagoya City University, Kawasumi Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan
4 Department of Orthopaedic Surgery, 3471 Fifth Avenue, University of Pittsburgh Medical Center, Pittsburgh, PA 15213
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Investigation performed at the Stem Cell Research Center, Department of Orthopaedic Surgery, University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania

A commentary by Christopher H. Evans, PhD, DSc, is linked to the online version of this article at jbjs.org.



Disclosure: One or more of the authors received payments or services, either directly or indirectly (i.e., via his or her institution), from a third party in support of an aspect of this work. In addition, one or more of the authors, or his or her institution, has had a financial relationship, in the thirty-six months prior to submission of this work, with an entity in the biomedical arena that could be perceived to influence or have the potential to influence what is written in this work. No author has had any other relationships, or has engaged in any other activities, that could be perceived to influence or have the potential to influence what is written in this work. The complete Disclosures of Potential Conflicts of Interest submitted by authors are always provided with the online version of the article.

Copyright © 2013 by The Journal of Bone and Joint Surgery, Inc.
J Bone Joint Surg Am, 2013 Jun 05;95(11):980-988. doi: 10.2106/JBJS.L.00266
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Abstract

Background: 

Muscle contusions are a common type of muscle injury and are frequently encountered in athletes and military personnel. Although these injuries are capable of healing in most instances, incomplete functional recovery often occurs because of the development of fibrosis in the muscle. We hypothesized that a combination of platelet-rich plasma (PRP) injection and oral administration of losartan (an antifibrotic agent) could enhance muscle healing by stimulating muscle regeneration and angiogenesis and by preventing fibrosis in contusion-injured skeletal muscle.

Methods: 

Contusion injuries were created in the tibialis anterior muscles of mice. Two treatments were tested, alone and in combination: 20 μL of PRP injected into the contusion site one day after injury, and 10 mg/kg/day of losartan administered beginning three days after injury and continuing until the end point of the experiment. Muscle regeneration and fibrosis development were evaluated by histological analysis, and functional recovery was measured by physiological testing.

Results: 

Muscle regeneration and muscle function were significantly promoted in the combined PRP + losartan treatment group compared with the other groups. Combined PRP + losartan treatment significantly decreased the expression of phosphorylated Smad2/3 and the development of fibrosis compared with PRP treatment alone, and it increased vascular endothelial growth factor (VEGF) expression and the number of CD31-positive structures compared with losartan treatment alone. Follistatin, a positive regulator of muscle growth, was expressed at a higher level in the PRP + losartan group compared with the other groups.

Conclusions: 

PRP + losartan combinatorial therapy improved overall skeletal muscle healing after muscle contusion injury by enhancing angiogenesis and follistatin expression and by reducing the expression of phosphorylated Smad2/3 and the development of fibrosis. These results suggest that blocking the expression of transforming growth factor (TGF)-β1 with losartan improves the effect of PRP therapy on muscle healing after a contusion injury.

Clinical Relevance: 

These findings could contribute to the development of biological treatments that aid in the healing of skeletal muscle after injury.

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    References

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    These activities have been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Academy of Orthopaedic Surgeons and The Journal of Bone and Joint Surgery, Inc. The American Academy of Orthopaedic Surgeons is accredited by the ACCME to provide continuing medical education for physicians.
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    Satoshi Terada, MD
    Posted on November 04, 2013
    Author Response to Previous Comment
    Stem Cell Research Center, Department of Orthopaedic Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA, USA

    We appreciate Dr. Kelc's thoughtful comments and have addressed each of them below, point by point.

    In the in vivo study by Terada et al., losartan, an angiotensin receptor blocker, was used in combination with PRP because of its inhibitory effect on TGF- β activity (1). The study is well-designed and methodically sophisticated with extremely promising and important results. However, we would like to express two major concerns although losartan has been previously shown to have a positive effect in muscle regeneration after injury (2).

    First of all, the idea to concentrate platelets and inject them locally at the site of injury is based on the need of their local effect as well as the absence of unwanted systemic effects and potential performance enhancement in athletes. Losartan is a drug available in enteral form and as such acts systemically. Although it is a relatively safe drug with only a few reported unwanted effects, its use in professional athletes should be considered from a safety point of view as they are frequently physiologically hypotensive with hypertrophic heart remodulation.

    As you mention, it is still unclear if the use of losartan in athletes with hypertrophic hearts is safe; however, the dosage of losartan we used in our study is the clinical dosage given to treat human patients with high blood pressure and interestingly, in the study referenced below, they found that losartan treatment was able to prevent cardiac hypertrophy induced by swim training. It was also reported that hypertensive patients treated with losartan did not have an increase in the prevalence of arrhythmias compared with trained athletes and had a reduction in left ventricular hypertrophy. So we believe that its use in professional athletes should be safe and hope additional studies will be performed in a greater number of more patients in the future.

    Secondly, the concentration of TGF-β, a cytokine responsible for tissue fibrosis and assumed to be involved in muscle regeneration has, in the study by Terada et al., only been determined in a PRP sample where its level is already known to be increased. As this information is generally important, its amount in a tissue sample would be of an even greater value in order to provide additional information about the molecular background of the regeneratory enhancement.

    In the current study, instead of measuring TGF-β1 we measured p-Smad2/3, which is a signaling mediator of TGF-β1 which is translocated into the nucleus where it regulates TGF-β1 transcription; however, as you comment, it would be interesting to measure the amount of TGF-β1 itself in the muscle tissue. We hope to do so in future studies.

    Furthermore, the information about tissue concentration of myostatin (MSTN), a skeletal muscle specific TGF-β superfamily member, would be even more important as it is widely accepted that it probably plays the largest role in fibrosis development in an injured muscle. If regeneratory enhancement by PRP and losartan predominantly affects only TGF-β activity without significantly regulating MSTN expression, it would be appropriate to consider combining PRP with a certain other antifibrotic agent to achieve the desired effect on both cytokines and simultaneously use it as for intramuscular administration.

    However, the study represents a major step forward in the regeneratory therapy of skeletal muscle after injury. Hopefully, further studies of this kind will provide additional information and insight into the molecular background of therapy and its functional outcomes and will ideally lead to new therapeutic strategies as an addition to the conservative means by R.I.C.E. principle.

    Our group previously reported that losartan can inhibit the expression of MSTN. In addition, it has been shown that PRP contains follistatin, which is an important regulator of muscle growth, and is an antagonist of MSTN(3). ; however, it remains unclear whether the combinatorial use of PRP with losartan affects the expression of MSTN. We hope future studies will be performed regarding this in the future.

    REFERENCES
    1) Oliveira EM, Sasaki MS, Cerêncio M: Local renin-angiotensin system regulates left ventricular hypertrophy induced by swimming training independent of circulating renin: a pharmacological study. J Renin Angiotensin Aldosterone Syst. 2009 Mar;10(1):15-23.

    2) Zakynthinos E, Pierrutsakos Ch, Daniil Z, Papadogiannis D: Losartan controlled blood pressure and reduced left ventricular hypertrophy but did not alter arrhythmias in hypertensive men with preserved systolic function. Angiology, 2005 Jul-Aug;56(4):439-49.

    3) Kobayashi T, Uehara K, Ota S: The timing of administration of a clinically relevant dose of losartan influences the healing process after contusion induced muscle injury. J Appl Physiol (1985). 2013 Jan 15;114(2):262-73.

    4) Witt, M. et al.: Platelet Rich Plasma May Contribute to Muscle Healing by Elevated Levels of Decorin and Follistatin, presented as a Poster at the 2010 Orthopaedic Research Society annual meeting.

    Robi Kelc, Matjaz Vogrin
    Posted on August 01, 2013
    Is losartan truly safe and the best antifibrotic agent to be combined with a PRP therapy for muscle injuries?
    Department of Orthopaedic Surgery, University Medical Center Maribor, Ljubljanska ulica 5, 2000 Maribor, Slovenia

    The complex regeneration process of skeletal muscle after injury or disease is limited by fibrous scar formation, slow healing time, and a high recurrence rate. A therapy based on platelet-rich plasma (PRP) has become a promising lead in recent years as an alternative to current medical practices; however, concerns have been raised that PRP derived TGF-β could contribute to even greater fibrosis.

    In the an in vivo study by Terada et al., losartan, an angiotensin receptor blocker, was used in combination with PRP because of its inhibitory effect on TGF- β activity (1). The study is well-designed and methodically sophisticated with extremely promising and important results. However, we would like to express two major concerns although losartan has been previously shown to have a positive effect in muscle regeneration after injury (2).

    First of all, the idea to concentrate platelets and inject them locally at the site of injury is based on the need of their local effect as well as the absence of unwanted systemic effects and potential performance enhancement in athletes. Losartan is a drug available in enteral form and as such acts systemically. Although it is a relatively safe drug with only a few reported unwanted effects, its use in professional athletes should be considered from a safety point of view as they are frequently physiologically hypotensive with hypertrophic heart remodulation.

    Secondly, the concentration of TGF-β, a cytokine responsible for tissue fibrosis and assumed to be involved in muscle regeneration has, in the study by Terada et al., only been determined in a PRP sample where its level is already known to be increased. As this information is generally important, its amount in a tissue sample would be of an even greater value in order to provide additional information about the molecular background of the regeneratory enhancement.

    Furthermore, the information about tissue concentration of myostatin (MSTN), a skeletal muscle specific TGF-β superfamily member, would be even more important as it is widely accepted that it probably plays the largest role in fibrosis development in an injured muscle. If regeneratory enhancement by PRP and losartan predominantly affects only TGF-β activity without significantly regulating MSTN expression, it would be appropriate to consider combining PRP with a certain other antifibrotic agent to achieve the desired effect on both cytokines and simultaneously use it as for intramuscular administration.

    However, the study represents a major step forward in the regeneratory therapy of skeletal muscle after injury. Hopefully, further studies of this kind will provide additional information and insight into the molecular background of therapy and its functional outcomes and will ideally lead to new therapeutic strategies as an addition to the conservative means by R.I.C.E. principle.

    1. Terada S OS, Kobayashi M, Kobayashi T, Mifune Y, Takayama K, Witt M, Vadalà G, Oyster N, Otsuka T, Fu FH, Huard J. Use of an antifibrotic agent improves the effect of platelet-rich plasma on muscle healing after injury. J Bone Joint Surg Am. 2013;95(11):980-8.

    2. Bedair HS, Karthikeyan T, Quintero A, Li Y, Huard J. Angiotensin II receptor blockade administered after injury improves muscle regeneration and decreases fibrosis in normal skeletal muscle. Am J Sports Med. 2008 Aug;36(8):1548-54.

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