Use of an Antifibrotic Agent Improves the Effect of Platelet-Rich Plasma on Muscle Healing After Injury

Terada, Satoshi MD; Ota, Shusuke MD, PhD; Kobayashi, Makoto MD; Kobayashi, Tetsuo MD, PhD; Mifune, Yutaka MD, PhD; Takayama, Koji MD, PhD; Witt, Michelle MS; Vadalà, Gianluca MD; Oyster, Nick BS; Otsuka, Takanobu MD, PhD; Fu, Freddie H. MD; Huard, Johnny PhD

Journal of Bone & Joint Surgery - American Volume:
doi: 10.2106/JBJS.L.00266
Scientific Articles
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.

Author Information

1Stem 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

2Department of Orthopaedic and Trauma Surgery, University Campus Bio-Medico of Rome, University of Rome, Via Alvaro Portillo 200, 00128 Rome, Italy

3Department of Orthopaedic Surgery, Nagoya City University, Kawasumi Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan

4Department of Orthopaedic Surgery, 3471 Fifth Avenue, University of Pittsburgh Medical Center, Pittsburgh, PA 15213

Article Outline

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.

Platelet-rich plasma (PRP) is an autologous blood-derived product that possesses increased concentrations of growth factors and secretory proteins1-3. It has been reported that PRP can influence myoblast proliferation4 and the proliferation and migration of fibroblasts5 in vitro. Several studies have indicated that PRP can enhance angiogenesis because of the numerous growth factors that it contains6,7. Researchers have investigated the potential use of PRP to treat pathological conditions in articular cartilage, tendon, ligament, bone, and skeletal muscle2,8-10. However, the presence of high concentrations of transforming growth factor (TGF)-β1 in the PRP could potentially promote fibrosis in injured skeletal muscle, although an increase in fibrosis development can accelerate the healing process after tendon and ligament injuries11,12.

An increase in fibrosis after muscle injury contributes to the risk of reinjury13. We have previously investigated the use of antifibrotic agents such as decorin14, relaxin15, gamma interferon16, and suramin17 to obtain better healing after muscle injury, and we demonstrated that these can enhance muscle regeneration and inhibit fibrosis by blocking TGF-β1 after skeletal muscle injury. Unfortunately, some of these compounds cannot be readily translated to clinical practice because of the lack of an oral formulation, the lack of U.S. Food and Drug Administration (FDA) approval, and/or a relatively severe side-effect profile.

Losartan was the first orally active, commercially available, non-peptide angiotensin II type 1 receptor (AT1) blocker. It is a well-known antihypertensive drug that is widely used for the treatment of hypertension and congestive heart failure18. TGF-β1 signaling is divided into Smad-dependent and Smad-independent pathways. The Smad-dependent pathway leads to the phosphorylation of Smad2 and Smad3 (pSmad2/3), which is then translocated into the nucleus where it regulates TGF-β1 transcription19. Losartan inhibits the Smad-dependent pathway through the blockade of AT112. Its anti-TGF-β1 effect is clinically relevant in many fibrotic disease states such as renal disease, pulmonary fibrosis, cardiomyopathy, and aortic aneurysm20-23. In skeletal muscle, TGF-β1 has been reported to have a negative effect on satellite cell differentiation during muscle regeneration24. It has also been reported that blocking TGF-β1 with losartan can increase myoblast proliferation and fusion in vitro25. Cohn et al. reported that losartan improved muscle morphology, reduced TGF-β1 signaling, and enhanced muscle regeneration in a mouse model of Marfan syndrome26. Moreover, our research group has demonstrated that losartan can reduce muscle fibrosis, increase the number of regenerating myofibers, and enhance the physiological function of injured muscle through the inhibition of TGF-β127.

Follistatin, a myostatin-binding protein and a member of the TGF-β superfamily, has been reported to enhance myogenic differentiation and accelerate the maturation of myotubes in a dose-dependent manner in vitro28,29. Furthermore, our group has shown that the oral administration of the recommended safe human equivalent dose of losartan (10 mg/kg/day) for three or seven days after a contusion injury can dramatically improve healing of skeletal muscle by enhancing the expression of follistatin in vivo30.

In the current study, we hypothesized that combining PRP and losartan treatments could substantially improve healing of skeletal muscle after a contusion injury (by stimulating muscle regeneration and angiogenesis and by preventing fibrosis) compared with either PRP or losartan treatment alone.

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Materials and Methods

Isolation and Characterization of PRP

Whole blood was collected from twenty adult female wild-type mice (C57BL/6J; Jackson Laboratory, Bar Harbor, Maine) by means of cardiac puncture, pooled, and transferred into tubes containing 3.2% sodium citrate. PRP was then isolated as described previously by Plachokova et al.31. The platelet concentration of the PRP was determined with use of a previously described method recommended by the International Committee for Standardization in Hematology32.

An enzyme-linked immunosorbent assay (mouse TGF-beta 1 DuoSet ELISA Development Kit; R&D Systems, Minneapolis, Minnesota) was performed according to the manufacturer’s protocol to assess the concentration of TGF-β1 in the newly isolated PRP.

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Animal Model

The animal experimentation policies and procedures followed in the study were in accordance with those detailed by the U.S. Department of Health and Human Services and the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All experimental protocols were approved by the University of Pittsburgh’s Institutional Animal Care and Use Committee.

Eighty-seven wild-type female mice (C57BL/6J; Jackson Laboratory), eight to ten weeks old and weighing 23.4 to 28.2 g, were used in the study. A muscle contusion injury was created in both tibialis anterior muscles of eighty-four of the mice as described previously17,33,34. The muscle contusion involved a high-energy blunt injury that resulted in a large hematoma followed by massive muscle regeneration, simulating the healing process seen in humans35. Thirty-two mice underwent physiological testing at two or four weeks (four mice in each of the four groups at each time point). The three uninjured mice also underwent physiologically testing as a normal control group. Thirty-six of the mice that did not undergo physiological testing were killed at one, two, or four weeks for histological analysis (three mice per group per time point), and the remaining sixteen mice were killed at one or two weeks and used in the polymerase chain reaction (PCR) analysis (two mice per time point per group).

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Administration of Losartan and Injection of PRP (Fig. 1)

All injured mice were randomly assigned to one of four groups. The control and PRP groups (n = 19 mice each) were supplied with normal drinking water from day 1 after injury until the end point of the experiment. The losartan and PRP + losartan groups (n = 19 mice each) received commercially available losartan (COZAAR, Merck) dissolved in their drinking water at the dosage level of 10 mg/kg/day, which is equivalent to the clinical dosage given to treat human patients with high blood pressure (50 mg/day)36, from day 3 after injury until the end point of the experiment (Fig. 1). The losartan concentration in the drinking water was calculated on the basis of the mean body weight of the mice and their expected mean daily fluid intake (derived from data acquired previously by our laboratory30). The mice were caged individually and were allowed access to water or the losartan solution ad libitum from the time of injury. One day after injury, 20 μL of PRP was injected directly into the contusion-injured region of the mice in the PRP and PRP + losartan groups as described previously37,38; the other two groups received an injection of PBS (phosphate-buffered saline) solution.

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Histological Evaluation of Muscle Regeneration and Fibrosis Development

The tibialis anterior muscles were isolated at one, two, or four weeks after injury and were flash-frozen in 2-methylbutane precooled with liquid nitrogen. Serial transverse cryosections (thickness, 8 μm) were stained with hematoxylin and eosin to evaluate the number of regenerating myofibers within the injury sites at two and four weeks after injury. The total number of centronucleated myofibers, which are known to be the regenerating myofibers39 within the injury site, were quantified by selecting ten random ×200 high-power fields (hpf) from each sample14,16,17,37,40.

A Masson Modified IMEB Trichrome Stain Kit (IMEB, Chicago, Illinois) was used to assess areas of fibrillar collagen deposition within the injury sites at four weeks after injury. After staining, the ratio of the fibrotic area to the total cross-sectional area of ten randomly selected sections was calculated to estimate the percentage of fibrosis development as described previously34,37.

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Physiological Evaluation of Muscle Strength Recovery

In vivo functional tests were performed at two and four weeks after injury as described previously30. Briefly, the anterior crural muscles were stimulated via the sciatic nerve, and twitch and tetanic torques were measured with use of an in situ muscle physiology test apparatus (Model 809B; Aurora Scientific, Aurora, Ontario, Canada). The twitch function is reported as the mean of ten peak twitch torque measurements, and the tetanic torque is reported as the maximum of two to three peak tetanic torque measurements. The mice were then killed and the muscles were removed and weighed prior to being flash-frozen. Functional analysis was also performed in three normal mice that represented an uninjured normal comparison group.

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Immunofluorescent Staining and RNA Analysis

At one week after injury, immunohistochemistry was performed using antibodies against vascular endothelial growth factor (VEGF) and against CD31, according to previously described protocols (see Appendix), to detect the expression of these compounds at the injury site. At two weeks after injury, pSmad2/3 and follistatin expression levels were quantified by means of immunohistochemistry as described previously17,34,37,41.

At one and two weeks after injury, RNA was isolated and real-time RT-PCR (reverse transcription-PCR) analysis was performed to determine the messenger RNA (mRNA) expression of mouse VEGF, CD31, and follistatin in the injured tibialis anterior muscle. RNA samples from multiple mice were not pooled. The sequences, amplicon positions, and gene access numbers of the PCR primers are listed in the Appendix.

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Statistical Analysis

All results are expressed as the mean and the standard deviation. All histological and physiological results were analyzed with use of the Kruskal-Wallis test followed by a Scheffe post hoc analysis. A p value of <0.05 was considered significant. All analyses were performed with use of SPSS software (IBM, Armonk, New York).

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Source of Funding

Funding support was provided in part by the Department of Defense (grants W81XWH-06-1-0406 and W81XWH-08-2-0032 [AFIRM] awarded to J.H.), the William F. and Jean W. Donaldson Endowed Chair at Children’s Hospital of Pittsburgh, and the Henry J. Mankin Endowed Chair at the University of Pittsburgh. Support from the Italian Minister of Foreign Affairs was also received by Dr. Gianluca Vadalà in the form of an Italy-USA scientific and technological cooperation research grant.

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Results

Platelet Count and TGF-β1 Concentration

The platelets in the PRP used in the study exhibited normal morphology. The mean platelet concentration was 208.0 ± 25.8 × 104/mL in the PRP, which represented a 5.5-fold increase over the concentration of 38.0 ± 4.5 × 104/mL in the whole blood. The concentration of TGF-β1 in the freshly isolated PRP was 3998.5 ng/mL, which was 16.5 times higher than the concentration of 242.1 ng/mL in the platelet-poor plasma).

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PRP Treatment Accelerated Muscle Regeneration After Injury (Fig. 2)

At two weeks after injury, the injured muscles in both PRP-treated groups contained numerous centronucleated, regenerating myofibers; the number of regenerating myofibers per high-power field was significantly higher (p < 0.001) in both the PRP group (124.9 ± 20.7) and the PRP + losartan group (140.4 ± 19.1) than in the control group (54.9 ± 14.5). No difference was observed between the PRP group and the PRP + losartan group.

At four weeks after injury, the number of regenerating myofibers was significantly lower in the control group than in the other groups (p = 0.01 to <0.001). Moreover, the number of regenerating myofibers per high-power field was significantly higher in the PRP + losartan group (151.3 ± 42.5) than in the control group (54.3 ± 11.5, p < 0.001), the losartan group (107.0 ± 35.2, p = 0.01), and the PRP group (132.8 ± 26.2, p = 0.033).

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Losartan and PRP Treatment Reduced the Development of Muscle Fibrosis (Fig. 3)

At four weeks after injury, the area of fibrotic scar tissue was significantly greater (p < 0.001) in the control group than in the other groups. Fibrotic areas were significantly less prevalent in the losartan group (4.1% ± 2.4%) and the PRP + losartan group (2.1% ± 1.3%) than in the control group (21.4% ± 6.1%; p < 0.001 and p < 0.001, respectively) and the PRP group (9.8% ± 3.4%; p = 0.027 and p < 0.001).

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Combined PRP and Losartan Treatment Improved Muscle Strength (Fig. 4)

At two weeks after injury, the specific peak twitch torque in N-mm/kg was significantly greater in the PRP group (5.6 ± 1.0, p = 0.049) and the PRP + losartan group (6.1 ± 2.3, p = 0.011) than in the control group (3.3 ± 1.1). Similarly, the specific tetanic torque in N-mm/kg was significantly greater in the PRP group (9.5 ± 1.6, p = 0.040) and the PRP + losartan group (10.4 ± 2.6, p = 0.007) than in the control group (5.5 ± 2.3).

At four weeks after injury, the specific peak twitch torque in N-mm/kg was significantly greater in the PRP + losartan group (10.5 ± 2.0) than in the control group (3.3 ± 0.6, p < 0.001), the losartan group (7.2 ± 1.9, p = 0.017), and the PRP group (7.5 ± 2.9, p = 0.036). Similarly, the specific tetanic torque in N-mm/kg was significantly greater in the PRP + losartan group (13.7 ± 3.1) than in the control group (4.9 ± 1.9, p < 0.001), the losartan group (9.2 ± 3.5, p = 0.025), and the PRP group (9.4 ± 2.3, p = 0.032). Moreover, there were no significant differences between the PRP + losartan group and the uninjured normal group (twitch, 11.4 ± 2.2 N-mm/kg, p = 0.93; tetanic, 13.2 ± 4.9 N-mm/kg, p = 0.99).

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PRP Treatment Enhanced VEGF Expression and Angiogenesis in Injured Muscle (Fig. 5)

At one week after injury, VEGF expression in the injured tibialis anterior muscles was significantly greater in the PRP group (4.0% ± 2.5%) and the PRP + losartan group (4.5% ± 3.1%) than in the control group (0.12% ± 0.2%; p = 0.002 and p = 0.001, respectively) and the losartan group (0.5% ± 0.7%; p = 0.007 and p = 0.002) (Figs. 5-A and 5-B).

At one week after injury, CD31-expressing areas in the injured tibialis anterior muscles were significantly more prevalent (p < 0.001) in the PRP group (4.2% ± 2.4%) and the PRP + losartan group (5.2% ± 1.6%) than in the control group (0.9% ± 0.8%) and the losartan group (1.4% ± 1.3%) (Figs. 5-C and 5-D). At one and two weeks after injury, real-time PCR revealed that the expression of CD31 was significantly greater (p = 0.001 to 0.046) in both PRP-treated groups than in the control group and the losartan group (Table I).

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Losartan Treatment Suppressed pSmad2/3 Expression in Injured Muscle (Fig. 6)

At two weeks after injury, pSmad2/3-positive areas in the injured tibialis anterior muscles were significantly less prevalent in the losartan group (0.8% ± 1.2%) and the PRP + losartan group (0.9% ± 0.5%) than in the control group (4.2% ± 1.6%, p < 0.001 for both) and the PRP group (3.2% ± 0.6%, p = 0.023 and p = 0.041, respectively). Moreover, pSmad2/3-positive areas were less prevalent in the PRP group than in the injured but untreated control group.

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Combined PRP and Losartan Treatment Enhanced Follistatin Expression in Injured Muscle (Fig. 7)

At two weeks after injury, follistatin-expressing areas in the injured tibialis anterior muscles were significantly more prevalent in the PRP + losartan group (6.0% ± 2.7%) than in the control group (0.5% ± 0.3%, p <0.001), the losartan group (2.6% ± 1.8%, p = 0.001), and the PRP group (3.9% ± 1.7%, p = 0.030). Furthermore, at one and two weeks after injury, real-time PCR analysis revealed that follistatin expression was significantly greater (p <0.001 to p = 0.039) in the PRP + losartan group than in the other groups (Table I).

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Discussion

We evaluated the combined use of PRP injection and oral administration of losartan for treating muscle contusion injuries in a mouse model. We hypothesized that (1) combining these treatments would lead to greater improvements in muscle regeneration and muscle strength compared with administration of PRP alone, and (2) blocking TGF-β1 by administering losartan in addition to PRP would reduce fibrosis development in injured muscle and further enhance the functional healing of the muscle.

During the healing of injured skeletal muscle, the release of growth factors regulates muscle cell proliferation and differentiation to promote muscle regeneration24,37. Several studies have indicated that injection of PRP can deliver many physiological growth factors and cytokines, such as VEGF, bFGF (basic fibroblast growth factor), PDGF (platelet-derived growth factor), and HGF (hepatocyte growth factor), which can aid in the healing of injured skeletal muscle by activating satellite cells in vivo3,42. VEGF, in particular, plays an important role in tissue healing through induction of angiogenesis9 and contributes to the dynamic process of capillary formation and muscle regeneration after injury43,44. Deasy et al. reported that the implantation of VEGF-overexpressing stem cells into injured skeletal muscle led to an increase in angiogenesis and a reduction in fibrosis43. Our group recently demonstrated that the intramuscular transplantation of muscle-derived stem cells accelerated skeletal muscle healing after a contusion injury by increasing the expression of VEGF37. Since PRP contains numerous beneficial growth factors including VEGF, we hypothesized that it could aid in the healing of injured skeletal muscle via the enhancement of angiogenesis; however, it is known that PRP also contains TGF-β1, which could potentially have detrimental effects on skeletal muscle healing because TGF-β1 is a key factor in the development of fibrosis in the kidneys, liver, lungs, and skeletal muscle45-47.

We have previously shown that losartan can inhibit fibrosis development after skeletal muscle injury by blocking TGF-β127; however, the effect of PRP on fibrosis development in injured skeletal muscle is still unclear48. In the current study, both the PRP group and the PRP + losartan group had a reduction in the development of fibrosis compared with the control group despite the high concentration of TGF-β1 present in the PRP. Several studies have also indicated that follistatin can stimulate angiogenesis in vitro and in vivo and can decrease fibrosis development29,49,50, suggesting that the injection of PRP into injured skeletal muscle could decrease fibrosis development via an enhancement of angiogenesis resulting from increased expression of follistatin and not simply by increasing levels of VEGF and PDGF.

The functional recovery of injured muscle is the most important outcome measure for patients who have a severe muscle injury51. Improvements in muscle regeneration and the reduction of fibrosis have been correlated with enhanced muscle function13. Hammond et al. showed that the injection of PRP into injured rat muscle could reduce the length of time required to achieve full recovery after an injury because of its positive effect on myogenesis3. In the current study, the histological results in the PRP, PRP + losartan, and losartan groups all demonstrated significantly greater improvements in muscle healing compared with the control group at two and four weeks after injury. However, the PRP + losartan group showed almost complete functional recovery by four weeks and significantly superior histological and physiological results compared with all of the other groups.

Our group has previously reported on several different therapeutic modalities for the treatment of muscle contusion injuries. These modalities included the direct injection of suramin17, high doses of orally ingested losartan27,30, and the intramuscular transplantation of muscle-derived stem cells37, and each of these modalities was shown to be effective for increasing muscle regeneration and reducing fibrosis. In the current study, the results obtained in the PRP + losartan group appeared histologically equivalent to those seen in the previous studies; however, those other treatments are not currently clinically available because of a lack of an oral formulation, a lack of FDA approval, and/or a relatively severe side-effect profile. The current study, on the other hand, provides evidence that the combined use of PRP and losartan, which are both currently clinically available, could be an efficacious option for the treatment of muscle contusions.

Several limitations of this study should be noted. First, we have not examined the optimal timing, quantity, or frequency of PRP administration. However, we believe that it would be quite simple to optimize the timing and dosage of a combined PRP + losartan therapy regimen for the treatment of skeletal muscle injuries in the near future. Second, we did not optimize the preparative technique for obtaining the best growth factor formulation in the PRP. Our evaluation of ten PRP samples isolated from unidentified human patients revealed that the concentrations of TGF-β1, for example, were very high and that the overall growth factor concentrations were highly variable among the different samples (data not shown). These findings are in general agreement with the data reported in the literature52; thus, it will be important to standardize PRP preparation procedures in the future. Third, the PRP used in the study was isolated from pooled whole blood samples obtained from twenty female mice, which prevented us from analyzing the variability of growth factor concentrations among the individual mice. All of the mice were injected with PRP that possessed the same concentration of growth factors. The use of pooled (and therefore allogeneic) PRP that has had its growth factor concentrations determined in advance could be useful as a way of standardizing the PRP53; however, this would eliminate the advantage of using autologous PRP.

In summary, we have demonstrated in an animal model that a combined PRP + losartan treatment regimen can improve overall skeletal muscle healing after a muscle contusion injury by increasing the speed of revascularization of the injured muscle, enhancing muscle regeneration, and inhibiting the development of fibrosis. This combined PRP + losartan treatment accelerated the functional recovery of the muscle after injury and, more importantly, this treatment could be readily applied clinically.

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Appendix Cited Here...

Tables showing immunohistochemical staining protocols and the sequences and gene access number of the primers are available with the online version of this article as a data supplement at jbjs.org.

NOTE:The authors thank Jessica Tebbets and Burhan Gharaibeh for their technical assistance. They also thank James H. Cummins for his editorial assistance in the preparation of the manuscript.

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.

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