Platelet-rich plasma for muscle injuries: A systematic review of the basic science literature

Table 1  In vitro PRP and muscle studies – summaries

Ref.	PRP preparation	Cytology findings	Study design	Outcomes measured	Results
Kelc et al[11], 2015	Whole blood in citrate dextrose anticoagulant spun 10 min at 1500 rpm. Three PRP solutions prepared at differing “growth factor concentrations” by diluting with DMEM (5%, 10% and 20%)	Not reported	Human CD56+ Myoblast cells cultured and treated with PRP (5,10,20%) and/or decorin for 4 d of treatment	Cell viability, proliferation. Myogenic differentiation, TGF-β and other fibrotic cytokine expression, MRF	PRP increased myoblast proliferation, viability, and differentiation. PRP supported myogenic shift in differentiation. Decreased TGF-β and other fibrotic cytokine expression and increased expression of MRFs
Mazzocca et al[7], 2012	PRP-LP (low platelet) – single 1500 rpm spin for 5 min. Plasma layer isolated. PRP-DS (High Platelet, high WBC) – double spin first at 1500 rpm for 5 min then again at 6300 rpm for 20 min. PRP-HP (High platelet, low WBC) – single 3200 rpm spin for 15 min	PRP-LP – Platelet (Plt) count equal to 382.0 ± 111.6 103/µL; RBC 0; WBC 0.6 ± 0.3 103/µL. PRP-HP – Plt count equal to 940.1 ± 425.8 × 103/µL; RBC 1.5± 2.5 × 103/µL; WBC 17.0 ± 5.2 × 103/µL. PRP-DS - Plt count equal to 472.6 ± 224.2 × 103/µL; RBC 0.0 ± 0.1 × 103/µL, WBC 1.5 ± 0.6 × 103/µL	Human muscles isolated from lattisimus dorsi transfer procedures cultured for 2 wk to allow for myocyte outgrowth. Myocytes treated with three PRP treatments for 96 h	Cell proliferation, growth factor concentrations (EGF, FGF2, HGF, IGF-1, PDGF, TGF-β, VEGF)	PRP-DS and PRP-LP increased cell proliferation. PRP-LP increased concentration of all growth factors except HGF, FGF, & EGF. PRP-DS increased concentration of all growth factors except FGF & HGF. PRP-HP increased concentration of all growth factors except FGF
McClure et al[1], 2016	Prepared with commercial SmartPReP® 2 system. Frozen thaw protocol to lyse platelets. Product then frozen and lyophilized to create dry PRGF	Not reported	C2C12 murine myoblasts cultured and expanded and treated with PRGF at various concentrations for 7 d	Proliferation (MTS proliferation assay), myogenic regulatory factor (MRFs) concentration, cell differentiation, skeletal muscle cell signaling, scaffold fiber alignment	PRP dose dependently increased myoblast cell proliferation, differentiation, skeletal muscle cell signaling, and concentration of MRFs (MyoD, MyoG)
Miroshnychenko et al[12], 2017	50 mL whole blood from seven volunteers processed with Pure PRP kit (EmCyte Corp) into: (1) Single spin leukocyte poor PRP; (2) Single spin mod-PRP with TGF-β1 and MSTN depletion; (3) Dual spin PRP; (4) Dual spin Mod-PRP; and (5) PPP. Second spin was 550 × g for 5 min and removed all platelets	PRP - Plt count equal to 879 ± 350.6 103/µL; WBC 1.8 ± 2.3 103/µL. PPP -Plt count equal to 9.9 ± 4.9 103/µL	Human skeletal muscle myoblast (HSMM) cell culture (CC-2580, Lanza) used to produce positive control (treated with 2% horse serum in myogenic DMEM/F-12 medium) and negative control (treated with 10% FBS in SkBM-2 basal medium). HSMM treated at varying concentrations with plasma formulations	Cell proliferation, protein production, myoblast differentiation, gene expression (MYH, MYH2, MSTN, MEF2C)	Single spin PRP & single spin Mod-PRP greatest influence on myoblast proliferation, but did not promote myogenic differentiation or formulation of myotubules. PPP and double spin PRP had little effect on proliferation, but greatest effects on promotion of myogenic differentiation and myotubule formation. PPP had a dose-dependent effect (peaking at 4%) on increasing MYH expression
Tsai et al[13], 2017	Whole blood from Sprague-Dawley rats in acid citrate-dextrose, spun at 800 × g for 30mins. Plasma isolated and spun at 3000 × g for 20 min. 10% thrombin solution added and again centrifuged at 5500 × g for 15 min. Final release filtered by 0.22 μm ultra-filtration and frozen at -20 °C	Not reported	Skeletal muscle cells isolated from Sprague-Dawley rats cultured and treated with PRP releasate. MTT assay and Immunohistochemistry with ki-67 stain also used to determine cell proliferation. Western blot used to determine changes in protein expression. Flow cytometry used to evaluate cell-cycle progression	Cell proliferation, cell viability, protein expression (cyclin A2, cyclin B1, cdk1, cdk2, PCNA)	PRP increased skeletal muscle cell viability & cell proliferation by shifting cells from the G1 phase to the S1 phase and G2/M phases. PRP increased protein expression of cyclin A2, cyclin B1, cdk1, cdk2, PCNA
Tsai et al[14], 2017	Whole blood from Sprague-Dawley rats in acid citrate-dextrose, spun at 800 × g for 30 min. Plasma isolated and spun at 3000 × g for 20 min. 10% thrombin solution added and again centrifuged at 5500 × g for 15 min. Final release filtered by 0.22 μm ultra-filtration and frozen at -20 °C	Not reported	Myocyte migration evaluated by trans-well filter migration assay and electric cell-substrate impedance sensing. Myocyte spreading evaluated microscopically. Formation of filamentous actin (F-actin) cytoskeleton assessed by immunofluorescence staining. Protein expressions of paxillin and focal adhesion kinase (FAK) assessed by Western blot analysis	Myocyte migration, spreading, FAK and Paxillin expression, F-actin formation	PRP dose-dependently promoted (1) Myocyte migration, (2) Spreading, (3) Paxillin and FAK expression, (4) F-actin formation, and (5) Wound healing

PRP: Platelet-rich plasma.

Table 2 Cytology reporting in all platelet-rich plasma muscle studies

Component	Reported studies, n (%)	Studies not reporting, n (%)
Platelet count	15 (65.2)	8 (34.8)
White blood cell count	6 (26.1)	17 (73.9)
Red blood cell count	1 (4.3)	22 (95.7)

Table 3  In vitro platelet-rich plasma and muscle studies – variables reported

Outcome	Studies reporting, n (%)	Significant increase, n	No significant change, n	Significant decrease, n
Cell viability	2 (33.3)	2	0	0
Cell proliferation	5 (83.3)	5	0	0
Proteoglycan and collagen content	0 (0)	0	0	0
Gene expression	5 (83.3)	4	0	1
Cell migration	1 (16.7)	1	0	0
Cell differentiation	3 (50)	3	0	0
Inflammatory mediation	0 (0)	0	0	0

Table 4  In vivo platelet-rich plasma and muscle studies - summaries

Ref.	PRP preparation	Cytology findings	Study design	Outcomes measured	Results
Borrione et al[9], 2014	Whole blood collected in sodium citrate spun twice first at 220 × g for 15 min and again at 1270 × g for 5 min. Pellet re-suspended and activated with 10% CaCl2 (20 mL)	Not reported	102 Wistar male adult rats had flexor sublimis muscle surgically cut and PRP immediately applied	Macroscopic evaluation, H&E changes, Leukocyte infiltration & WBC numbers, MyoD protein expression, gene expression of CD3, CD8, CD19, and CD 68, Myo D. Outcomes measured up to 7 d	PRP led to greater & earlier leukocyte infiltration (lymphocytes & monocytes) than control. PRP increased gene & MyoD protein expression
Cunha et al[2], 2014	Whole blood spun at 220 × g for 20 min at 20 °C. PRP combined with thrombin and activated with 10% CaCl2 to yield PRP gel	Not reported	20 Wistar adult male rats had vastus lateralis surgically injured. Rats randomized to treatment with and without PRP +/- exercise training	Serum lactate levels, histological analysis measuring type 1 and type 3 collagen at 3 wk after treatment	Exercise training + PRP led to greatest increase in type 3 collagen and decrease in type 1 collagen
Delos et al[3], 2014	Whole blood collected in citrate phosphate dextrose. Spun at 1000 × g for 15 min at 4 °C. Second spin under same conditions. 100 µL of PRP used for treatment	PRP – Plt count equal to 2.19 × 106 ± 2.69 × 105 µL, WBC 22.54 × 103/µL	46 male Lewis rats underwent single blunt injury to gastrocnemius muscle. Four treatment groups: Immediate PRP, Immediate saline, PRP day 1, PRP day 3	Histology, biomechanical testing (maximal isometric torque), amount of fibrosis (Masson’s trichome staining), IHC. Outcomes measured at day 15	PRP did not demonstrate any effects on outcomes including isometric torque strength, amount of fibrosis, or inflammation
Denapoli et al[15], 2016	Pure leukocyte poor PRP: Blood in 70% EDTA HEPES buffer spun at 300 × g/15 min and again 1000 × g/10 min. Platelets separated and 70% EDTA HEPES buffer added	Pure PRP- Plt count equal to 1090.0 ± 70.7 × 103/µL	30 male 10– to 12-week-old C57BL/6 wild-type mice had surgically induced blunt contusion to tibialis anterior muscle. 10 µL, pure PRP was injected at day 1, 4 and 7 d after injury. Outcomes measured at day 30	Histologic assessment of repair. Treadmill exercise for functional performance. Other PRP preparations were created and cytology & growth factor concentrations were compared between PRP groups. However, only pure leukocyte poor PRP was used in muscle contusion model	The day 7 PRP treated group had best functional performance and the most peripheral nucleated fibers on histology suggesting fastest recovery and decreased fibrosis
Dimauro et al[16], 2014	Whole blood collected in citrate phosphate dextrose. Spun at 220 × g/15 min, and again at 1270 × g/5 min. Pellet re-suspended and activated with 10% CaCl2	Averages not reported. However, PRP reported to have platelet concentration > 1250000 and WBC, neutrophils < baseline	40 Wistar male adult rats had surgical lesion made in flexor sublimus muscle. 20 rats immediately treated with PRP, other 20 untreated. Additional 10 rats anesthetized with lesion made. Outcomes measured at days 2 and 5	Gene expression of many intrinsic factors in regenerating skeletal muscle (e.g. cytokines, MRFs, growth factors), protein expression of MyoD1, Pax7, myogenin, stress response proteins, and apoptotic markers	PRP increased early expression of pro-inflammatory cytokines (e.g., TGF-β1, IL-1β. Increased expression of MRFs. No effect on VEGF. Increased ERK activation & IGF-1Eb expression
Gigante et al[23], 2012	Whole blood collected in citrate phosphate dextrose (1:5). Spun at 1000 × g/6 min. Supernatant activated with CaCl2 and then spun at 1450 × g/15 min	Not reported	Surgically induced bilateral lesions of longissimus dorsi muscle and subsequently treated with PRP matrix evaluated over 60 d	Histologic evaluation of neovascularization, muscle regeneration, fibrosis and inflammation. IMHC of myoD and Myogenin	Improved fibrosis, muscular regeneration and neovascularization. Increased expression of Myogenin
Hammond et al[6], 2009	Femoral/renal veins or intracardiac punctures on five adult male Sprague-Dawley rats (20 mL blood/each). PRP separated from whole blood (Symphony II Platelet Concentration System, DePuy). PPP used for control. Remaining PRP subjected to high frequency ultrasound (10 s). 100 µL used for injections	Not reported	72 adult male Sprague-Dawley rats had strain of tibialis anterior induced with superimposed lengthening contraction onto maximal isometric contraction using either a single repetition or multiple repetitions. Outcomes measured at days 3, 5, 7, 14, 21	Maximal isometric contraction and torque, Isometric torque, histology, and gene and protein expression of MyoD and myogenin, PDGF and IGF-1 concentrations in PRP and PPP	PRP had higher concentrations of PDGF and IGF-1. In single repetition group, PRP resulted in increased force only at day 3. No difference in return to function. For multiple repetitions, PRP improved force at multiple time points and faster return to function
Li et al[17], 2016	PRP isolated from three rats and mixed in citrate-phosphate-dextrose isolated as above. Centrifuged at 160 × g/20 min. Supernatant transferred, centrifuged 400 × g/15 min. Pellet re-suspended with remaining plasma to yield PRP	PRP - Plt count equal to 6.44 ± 0.64 × 106/µL, WBC 22.37 ± 2.25 × 103/µL	16 male Fisher rats injured with cardiotoxin injection into tibialis anterior. Four treatments: (1) Control, (2) 50 µL PRP, (3) 50 µL PRP neutralized with 280 ng/µL TGF-β1 antibody, (4) 50 µL PRP neutralized with 1400 ng/µL TGF-β1 antibody. Outcomes at 7, 14 d	Assessed muscle regeneration and collagen deposition with histology. IMHC for CD31, Alpha-SMA, Pax-7, CD68, transglutaminase-2, dystrophin to determine differentiation and mechanism for repair	PRP accelerated muscle regeneration (increased regenerating myofibers), increased angiogenesis (increased MVD-CD31, MVD-α-SMA) TGF-β1 neutralization of PRP reduced collagen deposition, PRP reduced macrophages and inflammatory response
Martins et al[19], 2016	Whole blood centrifuged 180 × g/10 min. Supernatant transferred and centrifuged at 1000 × g/10 min. Pellet re-suspended and activated with 10% calcium gluconate	PRP - Plt count equal to 4904/µL	Gastrocnemius Muscle contusion model studying the effect of PRP and reactive oxygen species over a 7-d treatment course	Reactive species byproducts (TBARS, DCFHRS), mitochondria function (MTT assay), antioxidant enzyme activities (GSH, CAT, SOD) and myeloperoxidase	PRP reduces oxidative damage and MPO enzyme, increases antioxidants
Ozaki et al[21], 2016	4mL blood from cardiac puncture combined with 0.2 mL 10% sodium citrate. Centrifuged 200 × g/15 min. Top two fractions isolated and centrifuged, at 500 × g/10 min	PRP- Plt count equal to 4999 × 103/µL	Thirty-five male Wistar rats in 5 groups (n = 7): control (C), control lesion (CL), lesion treated with low-level laser therapy (LLt), lesion treated with PRP (LP), and lesion treated with both techniques (LLtP). Muscle injury by stretching gastrocnemius muscle. PRP (100 μL) injected into distal third of tibia to be applied to gastrocnemius muscle belly	Histology for morphology, inflammatory infiltrate, oxidative stress using Raman scattering spectroscopy, collagen content	CL group had increased macrophages and oxidative stress. LP group had decreased inflammation, increased tissue organization, and increased presence of regeneration cells
Pinheiro et al[24], 2016	Intracardiac puncture -3 mL blood/each rat centrifuged 1200 × g/15 min to yield three layers. Isolated PRP/RBC layer, centrifuged 1min. PRP (0.2 mL) separated and activated with calcium gluconate (0.01 mL) to yield PRP gel	Cytology not provided	Ultrasound study following PRP therapy in a gastrocnemius muscle injury model	Pennation angle, Muscle thickness, Mean pixel intensity, claudication scores	No significant difference found
Quarteiro et al[8], 2015	Four blood samples (8 mL/rat) from five rats mixed with anticoagulant Samples centrifuged and plasma separated. Plasma centrifuged and supernatant removed leaving PRP (1mL)	PRP - Plt count equal to 1019 ± 182.25 × 103/µL	Gastrocnemius muscle injury model	Histologic assessment	No difference in collagen content at 21 days. Inflammatory process observed in groups treated with PRP
Terada et al[20], 2013	Blood obtained from intracardiac puncture. Centrifuged 800 rpm/ 15 min at 25 °C. Three PRP preparations created (rPRP, gPRP and hPRP). All three activated with 10% CaCl2 and bovine thrombin (300 IU, Fibriquik Thrombin, BioMerieux Inc., Durham, NC, United States)	PRP - Plt count equal to 208.0 ± 25.8 × 103/mL	PRP +/- Losartan in a tibialis anterior contusion model	IMHC (VEGF, CD31, Follistatin), Isometric Torque, Histological assessment (fibrosis and number of regenerating myofibers)	PRP in conjunction with losartan improved muscle recovery, reduced fibrosis. increased angiogenesis. PRP alone had similar but lesser effects
Contreras-Muñoz et al[22], 2017	3.5-4 mL whole blood obtained from intracardiac puncture, added to citrate phosphate dextrose, spun at 400 × g for 10 min. Plasma fraction extracted and spun at 800 g for 10 min	PRP - Plt count equal to 3.73 ± 0.25 × 106 platelets/ µL; WBC - 0.004 ± 0.0054 × 103 /µL	40 rats assigned to five groups: Injured rats (medial gastrocnemius injury) + single PRP injection (PRP group), daily exercise training (Exer group), or combination of single PRP injection and daily exercise training (PRP-Exer group). Untreated and intramuscular saline–injected animals were used as controls	Histologic and immunofluorescence analysis, force assessment, cross-sectional area of newly formed muscle fibers, dMHC and presence of collagen 1 in scar formation	18%, 20%, and 30% strength increase in PRP, PRP-Exer, and Exer groups. 1.5-, 2-, 2.5-fold increase in myofiber cross sectional area in PRP, PRP-Exer, and Exer groups. 20%, 34%, 41% of reduction scar formation in PRP, PRP-Exer, and Exer groups. 35% and 47% decrease in percentage of dMHC-positive regenerating fibers in PRP-Exer and Exer groups
Garcia et al[25], 2017	Cardiac puncture (4 mL) combined with 10% sodium citrate, spun at 200 × g for 15 min. Top layer + buffy coat extracted, spun at 500 × g for 10 minf	PRP – Plt count equal to 4998.676 × 103 platelets/µL	35 rats assigned to five groups: Control (C), Injury (soleus) Control (IC), injury PRP (IP), injury LLLT (ILT) and injury LLLT and PRP (ILTP)	Histologic assessment of muscle fiber morphology, collagen, inflammatory infiltrate	Intense polymorphic fibers (> 75%) in ILTP and IP groups. Lowest inflammatory infiltrate (< 20%) in ILTP compared to other injured groups. Significantly more focused collagen in ILT compared to IP and C groups

Table 5 Variables reported - in vivo muscle studies

Outcome	Studies reporting, n (%)	Significant increase, n	No significant change, n	Significant decrease, n
Cell viability	0 (0)	0	0	0
Gene expression	5 (33.3)	5	0	0
Gross appearance of muscle repair	1 (6.67)	0	1	0
Histologic assessment of muscle repair	8 (53.3)	7	1	0
Proteoglycan content	0 (0)	0	0	0
Collagen deposition	7 (46.7)	2	3	2
Muscle strength	4 (26.7)	3	1	0
Inflammatory mediation	8 (53.3)	4	1	3
Growth factors	1 (6.67)	1	0	0

Table 6  In vivo and in vitro muscle studies

Ref.	PRP preparation	Cytology findings	Study design	Outcomes measured	Results
Takase et al[27], 2017 (In vitro arm)	Whole blood extracted from male volunteers, combined with 12 mL 3.13% sodium citrate. Centrifuged 2400 rpm/10 min, again at 3600 rpm/15 min for 10 mL PRP and PPP. PRP activated by freezing at -80 °C; centrifuged again at 10000 rpm/10 min	PRP – Plt count equal to 7.2 × 105 - 9.4 × 105 platelets/mL	Murine myogenic cell line (C2C12 cells) subjected to PRP treatment. Cell morphology assessed by phase microscopy. Myotube quantification assessed by immunocytostaining. Cell proliferation assessed using water-soluble tetrazolium salt (WST) assay using a cell counting Kit-8. Oil Red-O staining used to identify lipid droplets and accumulation determined by phase contrast microscopy. rt-PCR used to quantify myogenic and adipogenic markers	Cell proliferation, myogenic differentiation (Pax7, myogenin), adipogenic differentiation [PPARγ, CCAAT/enhancer binding protein (C/EBPα)]	PRP inhibited myotube formation, decreased average area of myotubes, induced myogenic proliferation compared to myogenic group. Number of lipid droplets in PRP-adipogenic group was lower than adipogenic group. PRP suppressed expression of Pax7, myogenin, PPARγ and C/ERPα
(In vivo arm)	8 mL blood retrieved from one, 3-mo old, Sprague-Dawley rat, combined with 2 mL 3.13% sodium citrate Centrifuged at 1500 rpm for 10 min. Second spin at 3000 rpm for 10 min yielded PRP (1mL) and PPP. PRP frozen at -80 °C until needed	PRP - Plt count equal to 1.6 × 109 platelets/mL	PRP injection into subacromial space of five rat rotator cuff tear models Infraspinatus used for histology. Muscles cryosectioned, fixed with 4% PFA, stained with Oil Red-O and hematoxylin. Supraspinatus used for biochemical assays. rt-PCR to quantify genes	Oil Red-O positive lipid droplet formation, adipogenic differentiation [PPARγ, CCAAT/enhancer binding protein (C/EBPα)], and muscular atrophy [(Muscle RING Finger Protein-1 (MuRF-1) and atrogin-1]	Rotator tear groups had increased MuRF-1, atrogin-1, PPARγ and C/EBPα PRP decreased lipid droplet presence. PRP decreased expression of PPARγ and C/EBPα
Li et al[26], 2013 (In vivo arm)	Human whole blood from Central Blood Bank, Pittsburgh, PA, United States centrifuged 3000 g/ 10 min. Fraction of PRP poor supernatant transferred, PRP pellet re-suspended. PRP concentration measured by hemocytometer, activated with human thrombin (1U/mL). PRP releasate separated from cellular debris by centrifugation 3000 × g/30 min. PRP releasate stored at -80 °C	Cytology not provided	Gastrocnemius of mdx-SCID mice damaged with cardiotoxin and treated with hMDPCs treated with PRP	Histological assessment of as the number of hMHC-I-positive myofibers/1 × 105 injected cells	PRP maintained hMDPCs growth and regeneration of myofibers
(In vitro arm)	Centrifuged at 3000 × g for 10 min at RT. PRP activated with one unit per mL human thrombin. After activation, the PRP releasate obtained by centrifugation at 3000 × g for 30 min	PRP – Plt count equal to 2000/µL	hMDPCs isolated from donors cultured in PRP versus 20% FBS as a control. Assessed Proliferation, role of exogenous growth factors, gene expression, and cell differentiation	Proliferation, growth factor PDGF, VEGF, TGF-B1 RT-PCR for expression profile of stem cell markers and differentiation	PRP increased cell proliferation. PRP with anti TGF-B1 and anti VEGF did not. PRP increased the expression of BMPR1-A, BMPR1-B, BMPR2, ALDH, SOX2, Aggrecan, and Desmin. No difference in differentiation capacity

PRP: Platelet-rich plasma.

Table 7 Variables reported – combined in vivo and in vitro muscle studies

Outcome	Studies reporting, n (%)	Significant increase, n	No significant change, n	Significant decrease, n
Cell viability	0 (0)	0	0	0
Cell proliferation	1(25)	1	1
Cell differentiation	1 (25)	1	0	0
Gene expression	3 (75)	3	0	2
Gross appearance of muscle repair	0 (0)	0	0	0
Histologic assessment of muscle repair	2 (50)	2	1	1
Proteoglycan content	0 (0)	0	0	0
Collagen deposition	0 (0)	0	0	0
Muscle strength	0 (0)	0	0	0
Inflammatory mediation	0 (0)	0	0	0