Platelet-Rich Plasma in Tendon Models: A Systematic Review of Basic Science Literature

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1 Systematic Review Platelet-Rich Plasma in Tendon Models: A Systematic Review of Basic Science Literature Nikolas Baksh, B.S., Charles P. Hannon, B.S., Christopher D. Murawski, B.S., Niall A. Smyth, M.D., and John G. Kennedy, M.D., M.Ch., M.M.Sc., F.R.C.S.(Orth) Purpose: To perform a systematic review of the basic science literature on the use of platelet-rich plasma (PRP) in tendon models. Methods: We searched the PubMed/Medline and Embase databases in June 2012 using the following parameters: ((tenocytes OR tendon OR tendinitis OR tendinosis OR tendinopathy) AND (platelet rich plasma OR PRP OR autologous conditioned plasma OR ACP)). The inclusion criteria for full-text review were in vivo and in vitro studies examining the effects of PRP on tendons and/or tenocytes. Clinical studies were excluded. Only studies published in peerreviewed journals that compared PRP directly with a control were included. Data were extracted based on a predefined data sheet, which included information on PRP preparation, study methods, and results. Studies were analyzed for trends, comparing and contrasting the reported effects of PRP. Results: The search yielded 31 articles for inclusion in our review. Of the studies, 22 (71%) reported platelet concentrations in the PRP; 6 (19%) reported cytology. Eight in vivo studies found decreased tendon repair time, increased fiber organization, or both with PRP treatment. Eight in vitro studies reported that PRP treatment increased cell proliferation; 7 reported an increase in growth factor expression. Three in vivo studies found increased vascularity, and 4 found increased tensile strength with PRP treatment. Conclusions: In the basic science studies evaluated, it appears that PRP confers several potential effects on tendon models compared with a control. However, the literature is inconsistent with regard to reporting the methods of preparation of PRP and in reporting platelet concentrations and cytology. Clinical Relevance: Establishing proof of concept for PRP may lead to further high-quality clinical studies in which the appropriate indications can be defined. Tendon injuries account for approximately 30% to 50% of all sports-related injuries. 1 Lesion severity can range from a mild sprain to complete rupture of the tendon, with resultant degeneration and chronic instability. 2 Tendons are poorly vascularized and heal slowly when compared to other soft-tissues. As a result, treatment tends to be lengthy, outcomes tend to be variable, and reinjury is common. 2-5 There is also relatively little known regarding the pathophysiology of acute and chronic tendon pathologies, resulting in a paucity of successful evidence-based treatment paradigms. 2,5,6 It is unsurprising, therefore, that novel approaches to the treatment of such From the Hospital for Special Surgery, New York, New York, U.S.A. The authors report that they have no conflicts of interest in the authorship and publication of this article. Received July 11, 2012; accepted October 17, Address correspondence to John G. Kennedy, M.D., F.R.C.S.(Orth), Hospital for Special Surgery, 523 E. 72nd St., Suite 507, New York, NY, U.S.A. kennedyj@hss.edu Ó 2013 by the Arthroscopy Association of North America /12459/$ conditions are the subject of active basic science and clinical research. One such novel treatment modality is the use of platelet-rich plasma (PRP). 2,3,5,7-23 PRP is an autologous blood product centrifuged to produce a concentrated platelet product with numerous bioactive molecules and growth factors, including vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), fibroblast growth factor, platelet-derived growth factor (PDGF), transforming growth factor (TGF) b, and epidermal growth factor (EGF). 2,5,10,16,24,25 PRP has been used in other medical fields such as oral and maxillofacial surgery to enhance bone and soft-tissue healing, and it has recently gained attention in orthopaedics and sports medicine as a biological adjunct for the treatment of various pathologies, including bone, cartilage, tendon, and ligament pathologies. 5,14,23,24,26 It is hypothesized that the growth factors and other bioactive molecules present in PRP have the potential to augment cellular migration, proliferation, angiogenesis, and matrix deposition in tendon healing. 14 Presently, the Level I clinical evidence for the use of PRP in tendon healing is minimal, with mixed results. 1, Arthroscopy: The Journal of Arthroscopic and Related Surgery, Vol 29, No 3 (March), 2013: pp

2 Table 1. Data Extraction for In Vitro Studies Author PRP Preparation Methods Outcomes Measured Results Anitua et al., Anitua et al., Carofino et al., de Mos et al., de Mos et al., Jo et al., McCarrel and Fortier, Morizaki et al., Human whole blood double spun and activated. Platelet concentration 200% and 400% greater than whole blood. Human whole blood single spun and activated. Platelet values higher in PRP. Two preparations made from whole blood. Single-spun preparation had 2.6 greater platelet concentration. Double-spun preparation had 3.3 greater platelet concentration and 10 fewer WBCs than whole blood. Human whole blood, 500 ml, spun 3 and activated. PRP had 2.55 platelet concentration and decreased WBCs and RBCs. Human whole blood, 500 ml, spun 3 and activated. Platelet count 2.8 greater than whole blood. PRP had 5 greater platelet count than whole blood. WBC count reduced in PRP from 6.11 to 0.01 and RBC reduced from 4.48 to PRP had 5.54 greater platelet concentration than whole blood. WBC count was similar to whole blood. Whole blood spun and activated. PRP had 5.4 greater platelet count than whole blood. Schnabel et al., Whole blood activated. PRP had 3.77 greater platelet count than whole blood. WBC count was not different than that of whole blood, and RBC count decreased in PRP compared with whole blood. Fibroblasts cultured and treated for 3 d with PRP. Immunostaining for morphology and protein expression. Tendons from ACL reconstruction were cultured with PRP for 6 d. Outcomes measured at multiple time points. Bicep tenocytes were obtained from male donors treated with single- or double-spun PRP with or without anesthetic. Hamstring tendons cultured and treated with PRP for 14 d. Outcomes measured at days 4, 7, and 14. PRP tested with ELISA for VEGF and PDGF expression. Achilles tendons obtained from tendinopathy and 5 healthy Achilles tendons as controls. Cultured on cell or chondrogenic media for 14 d and then PRP for 7 d. Twenty-oneeday control culture. RNA extracted. Rotator cuff tendons with degenerative ends used for cell culture. PRP added and outcomes measured at 7 and 14 d. Equine tendons cultured and treated with PRP for 96 h. Outcomes measured at 0, 24, and 96 h. Horse flexor digitorum profundus tendons lacerated, repaired, and left in cell culture for 2 or 4 wk with PRP treatment. Equine superficial digital flexor tendon cultured for 3 d and treated with PRP at 100%, 50%, and 0% concentrations. HA; procollagen type I C-peptide, VEGF, and HGF; and cell morphology and proliferation. Cell proliferation; IGF, TGF, PDGF, VEGF, HGF, and EGF concentrations; and platelet counts. Tenocyte proliferation (radioactive thymidine assay) and cell viability (luminescence assay). Cell proliferation (DNA assay); total collagen (hydroxyproline assay); and COL1, COL3, MMP1, MMP3, MMP13, TGF, and VEGF. Chondrogenic gene expression (SOX9, COL2A1, COL10A1), GAG (thionin stain), and H&E staining. Tenocyte proliferation (WST assay), matrix gene expression (RT PCR), total collagen (Sircol assay [Biocolor, Newtonabbey, Ireland]), and GAG (Blyscan [Biocolor] DMMB). TGF and PDGF expression tested with ELISA. Gene expression measured with RT PCR. Cell viability (scanning confocal microscope), histologic changes, and mechanical strength tested. IGF, TGF, and PDGF levels in PRP quantified with ELISA. Gene expression (RT PCR), cell proliferation (DNA assay), and total collagen (Sircol assay) assessed. PRP increased proliferation and VEGF and HA expression. PRP increased proliferation and PDGF, TGF, VEGF and HGF concentrations; EGF concentration decreased in PRPtreated group. IGF expression remained the same. PRP showed significant increases in cell proliferation. Treatment with local anesthetic and PRP decreased cell proliferation and viability. The PRP-treated group had increased cell proliferation and VEGF and TGF expression. Total collagen increased 3.3 by day 14. No change occurred in COL1:COL3. COL1 and COL3 decreased in PRP group. Tendinotic tissue had greater expression of chondrogenic markers. The PRP-treated group had decreased expression of chondrogenic markers. Tenocyte proliferation, matrix gene expression (COL1 and COL3), and GAG were increased in PRP-treated group. The relation was dose dependent. PRP-treated group had increased TGF, PDGF, and COL1:COL3 ratio. PRPtreated group had decreased COL3, MMP3, and MMP13. PRP-treated group had increased tendon strength and stiffness. PRP-treated group had increased TGF and PDGF expression in addition to increased COL1, COL3, and MMP3. COL1:COL3 was only increased in treated group with 100% PRP. PRPtreated group had no difference in cell proliferation. PRP-treated group had decreased MMP13. (continued) PLATELET-RICH PLASMA IN TENDON HEALING 597

3 Table 1. Continued Author PRP Preparation Methods Outcomes Measured Results Tohidnezhad et al., Visser et al., Visser et al., Zargar Baboldashti et al., Zhai et al., Zhang and Wang, Whole blood obtained from Wistar rats, double spun, resuspended, and activated. PRP had 5 to 10 increase in platelet concentration. 18 ml of canine whole blood double spun. Platelet resuspended in 2 ml of plasma and activated. Platelet-rich fibrin matrix and plateletrich fibrin membrane (PRP fibrin constructs) produced from canine whole blood. 55 ml of whole blood single spun. Platelet count range was 353 to platelets/ml. Whole blood, 6 ml, double spun. PRP platelet count 3- to 10-fold greater than whole blood. Rabbit whole blood, 7-9 ml, double spun and activated. Average platelet count 3.25 greater than whole blood. Rat Achilles tenocytes cultured. PRP applied for 10 min or 6 h. Canine patellar tendons cultured and seeded onto scaffold and treated with PRP or control. Constructs cultured and eluents collected for growth factors at 1, 3, 5, and 7 d. Canine patellar tendon fibroblasts cultured for 24 h and then growth factors added for 48 h. Human hamstring tenocytes cultured and treated with PRP, control, ciprofloxacin and PRP, or dexamethasone and PRP. Tenocytes cultured from healthy male adult hamstring tendons. Cultured with or without PRP and with or without osteoblasts for 10 d. Patellar stem cells from NZ white rabbits cultured for 10 d and treated with PRP of varying concentrations. Scratch assay assessed cell motility; cell proliferation (CyQuant molecular probes [Eugene, OR]; reagent) and viability (WST), antioxidant gene (Luciferase assay), and tenocyte growth also assessed. Cell proliferation and viability assessed (MTT assay). Culture immunostaining for TGF. Total collagen (hydroxyproline) and GAG (methylene blue) measured. TGF concentration in eluents (immune-absorbent assay) and cell proliferation and viability (MTT colorimetric assay). Constructs stained for fibrin. Tenocyte viability (AlamarBlue assay [AbD Serotec, Kidlington, England]) and senescence (b-galactosidase stain). Cell proliferation (optical density), COL1, vimentin, COL3, VCAM-1, and nucleus (immunostaining). Stem cell differentiation (morphology, nucleostemin stain), collagen expression (Sircol assay, Western blot), gene expression for differentiation (RT PCR), and cell proliferation. PRP increased tenocyte proliferation, viability, migratory capability, antioxidant gene, and expression of tenomodulin. PRP-treated group had increased TGF concentration (4.7), cell density, total collagen, and GAG. Platelet-rich fibrin matrix and plateletrich fibrin membrane increased concentration of TGF and fibrin. PRP increased cell viability of cells, including those treated with ciprofloxacin and dexamethasone (dose dependent). Decreased senescence found in dexamethasone and PRP group. PRP increased growth rates and decreased inhibition of cell growth through cytokines. Stem cells treated with 2%-10% PRP increased cell size elongation, COL1, COL3, and total collagen. PRP-treated group had decreased nucleostemin (dose dependent). Tenocyte genes were highly expressed; those related to other cell types were minimally expressed. DMMB, 1,9-dimethylmethylene blue; ELISA, enzyme-linked immunosorbent assay; GAG, glycosaminoglycan; HA, hyaluronic acid; HGF, hepatocyte growth factor; MMP, matrix metalloproteinase; MTT, tetrazolium dye colorimetric assay; NZ, New Zealand; PCR, polymerase chain reaction; RBC, red blood cell; RT, reverse transcriptase; VCAM, vascular cell adhesion molecule; WBC, white blood cell; WST, water soluble tetrazolium salt. 598 N. BAKSH ET AL.

4 Table 2. Data Extraction for In Vivo Studies Author PRP Preparation Methods Outcomes Measured Results Bosch et al., Whole blood single spun. PRP platelet concentration 5.54 greater than whole blood. Lesion created in horse flexor digitorum profundus tendons and treated with 3 ml of PRP at 1 wk postoperatively with evaluation at 24 wk. Bosch et al., Characteristics of PRP not described. Lesion created in horse flexor digitorum profundus tendons and treated with 3 ml of PRP at 1 wk postoperatively with evaluation at 24 wk. Bosch et al., PRP had platelet count 3.8 greater than plasma. Lesion created in horse flexor digitorum profundus tendons and treated with 3 ml of PRP at 1 wk postoperatively. Dragoo et al., Leukocyte-rich PRP prepared from 27 ml of whole blood. Leukocyte-poor PRP prepared from 9 ml of whole blood. Leukocyte-rich PRP approximately 4 greater platelet count than leukocytepoor PRP and approximately 5 greater than whole blood. 2 ml of PRP (leukocyte rich and leukocyte poor) injected into mature NZ white rabbits. Saline solution and whole blood injected into control rabbits. Tendons harvested at 5 and 14 d, stained with H&E, and evaluated at 40 and 400 magnification. Harris et al., Kajikawa et al., Lyras et al., Lyras et al., Whole blood, 50 ml, double spun and activated to create 3 ml of platelet gel. Platelet count for PRP to for whole blood. Rat whole blood, 60 ml, double spun and resuspended. PRP had 8.8 greater platelet concentration than whole blood. Also had 10.8 and 7.7 greater TGF and PDGF, respectively. 2 ml of PRP prepared from 8 ml whole blood. PRPFast protocol. 2 ml of PRP prepared from 8 ml of whole blood. PRPFast protocol. Male NZ white rabbits injected with 0.5 ml of platelet gel in Achilles, with control on contralateral side. Tendons harvested at 2 wk and 6 wk. Last group reinjected at 6 wk and harvested at 12 wk. Bilateral lesions created in patellar tendon of bone marrow GFP chimeric rats; 20 ml of PRP injected. Achilles tendon of NZ white rabbits surgically ruptured and treated with 1 ml of PRP or control. Achilles tendon of NZ white rabbits surgically ruptured and treated with 1 ml of PRP or control. H&E changes; clinical evaluation; TGF, PDGF, and IGF expression; GAG (DMMB assay); collagen content (mass spectrometry); and mechanical load to failure. Blood flow (color Doppler ultrasonography) and angiogenesis (factor VIII immunostaining). Computerized ultrasonography and H&E analysis with PRP treatment at 24 wk. H&E changes and presence of neutrophils, lymphocytes, and macrophages. Vascularity, fibrosis, and cell count also assessed. H&E changes, inflammatory cells (azure and trichrome stain), neovascularization, collagen formation, and calcium deposition (von Kossa and alizarin red stains). GFP analysis, H&E changes, COL1 and COL3 levels (immunohistochemistry), macrophages, cell proliferation (BrdU), and collagen production (HSP47). H&E changes and TGF expression (immunohistochemistry). H&E changes and vascularization (CD31 marker). PRP-treated tendons had increased collagen, GAG, cellularity, load to failure, and elastic modulus. H&E showed PRPtreated group had improved structural organization. PRP-treated group had increased blood flow except at week 5 compared with control. Total number of blood vessels increased in PRP-treated group and had improved structural organization. H&E showed improved fiber arrangement with PRP. PRP-treated group had increased fibrillogenesis and collagen formation. Fluid and cellular accumulation decreased in PRP-treated group. Platelet-WBC ratio was higher in leukocyte-poor PRP than leukocyte-rich PRP. Leukocyte-poor PRP had decreased inflammatory response. Leukocyte-rich PRP had higher vascularity, WBCs, fibrosis, and fiber disruption scores at day 5. No difference in cell count at day 14. PRP-treated group had monocytic and lymphocytic infiltration at 2 wk. New collagen bundles were present in PRP group. PRP-treated group had increased collagen production and density of macrophages 3 d postoperatively. Macrophages proliferated 2 faster in PRP-treated group. PRP-treated group had increased TGF expression at weeks 1 and 2. Tenocytes more organized in PRP group at 3 wk, but TGF levels decreased. Tendon completely healed in PRP-treated group at 4 wk, unlike control. PRP-treated group had increased vascularization (CD31) at weeks 1 and 2. Tenocyte organization greater in PRP group at week 3. Vascularization decreased at week 4. Tendon healed at week 4 in PRP-treated group and almost healed in control at 4 wk. (continued) PLATELET-RICH PLASMA IN TENDON HEALING 599

5 Table 2. Continued Author PRP Preparation Methods Outcomes Measured Results Lyras et al., ml of PRP prepared from 8 ml of whole blood. Platelet concentration 8 greater in PRP than whole blood. Patellar tendon of NZ white rabbits resected to create defect and filled with PRP or left untreated. Lyras et al., Lyras et al., Lyras et al., Spang et al., Virchenko and Aspenberg, ml of PRP prepared from 8 ml of whole blood. PRPFast protocol. 2 ml of PRP prepared from 8 ml of whole blood. PRPFast protocol. 2 ml of PRP prepared from 8 ml of whole blood. PRPFast protocol. Whole blood from Wistar rat, 9 ml, single spun. Two PRP treatment groups with platelet concentrations increased 0%-50% and increases >51%. Whole blood, 4-6 ml, double spun platelets/l in PRP. PRP gel created with thrombin and CaCl 2 added to 50 ml of PRP. Patellar tendon of NZ white rabbits resected to create defect and filled with PRP or left untreated. Patellar tendon of NZ white rabbits resected to create defect and filled with PRP or left untreated. NZ white rabbits with Achilles tendon ruptured and treated with 0.5 ml PRP or control. Tendons harvested at 1, 2, 3, and 4 wk for evaluation. Tendon removed from inferior pole of patella and reattached. Treated with 0.5 ml of PRP or saline solution. 3-mm portion of Achilles tendon removed and left unsutured in Sprague- Dawley rats. 50-mL control, PRP gel, or PRP added to site. One group had Botox [Allergan, Irvine, CA]-induced paralysis postoperatively. One group was kept in activity cages and one group in normal cages. H&E assessment (with trichrome stain), mechanical assessment, load to failure, stiffness, and energy uptake (pulled at 1 mm/s). H&E changes and angiogenesis (CD31 immunostaining). H&E assessment and IGF levels (immunostaining). H&E changes and IGF levels (immunostaining). H&E changes, load to failure, stiffness, and energy absorbed (pulled at 0.1 mm/s). Load to failure, stiffness, energy uptake, transverse area, and stress at failure (pulled at rate 1 mm/s). At 28 d, PRP tendon completely healed; control tendon almost healed. PRP had increased number of fibroblasts with distinct orientation, as well as increased collagen deposition. At day 14, PRP group had significantly increased load to failure, stress, and stiffness. PRP increased neovascularization at weeks 1 and 2. PRP group had decreased vessel density but more compact, organized tissue with elongated tenocytes. Tendon completely healed at week 4 with no CD31 expression at that time. PRP group had denser, more mature tenocytes with more longitudinally organized fibers at week 3. PRP increased expression of IGF during weeks 1-3. Tendon completely healed by week 4. PRP group had elongated tenocytes with longitudinal organization at week 3. Tendon completely healed at week 4 unlike control with little inflammation. PRP group had increased expression of IGF. Treated tendons ruptured at higher force at site of original transection. Load to failure and energy absorbed were increased in PRP group with >51% platelet concentration. PRP increased material properties of tendon. Platelets lost stimulatory effect with Botox unloading. Activity increased size of tendon, force of failure, and energy; not synergistic with PRP. CaCl 2, calcium chloride; DMMB, 1,9-dimethylmethylene blue; GAG, glycosaminoglycan; GFP, green fluorescent protein; HGF, hepatocyte growth factor; H&E, hematoxylin-eosin; NZ, New Zealand; PRPFast, PRP preparation protocol (Bioteck, Vicenza, Italy). 600 N. BAKSH ET AL.

6 PLATELET-RICH PLASMA IN TENDON HEALING 601 Despite this, however, the use of PRP has advanced rapidly without extensive data supporting its safety or clinical efficacy. 10 For PRP to gain widespread acceptance as an evidence-based treatment modality for tendon pathology, large-scale randomized controlled clinical trials of high methodologic quality must first be conducted. Before such trials are performed, however, proof of concept must be shown through in vitro and in vivo basic science studies. The purpose of this systematic review was to evaluate the available basic science evidence for the use of PRP in tendon models. It was hypothesized that the basic science literature would show that PRP confers multiple potential effects on tendon models when compared with a control. Methods A systematic review of the basic science literature published on the use of PRP in tendon models was conducted. We searched the PubMed/Medline and Embase databases using a defined set of search parameters in June The search criteria were as follows: ((tenocytes OR tendon OR tendinitis OR tendinosis OR tendinopathy) AND (platelet rich plasma OR PRP OR autologous conditioned plasma OR ACP)). We included both in vitro and in vivo studies that examined the effects of PRP on tendons and/or tenocytes. Only studies that compared PRP directly with a control group (saline solution, no treatment, or control cell medium) were selected for review. Clinical studies including randomized controlled trials and case studies were excluded. Animal studies that observed the effects of PRP on traumatic, rather than surgically induced, tendon injuries were excluded. Studies examining the effect of PRP on intra-articular tendon grafts were excluded because of the potential for confounding variables associated with transplanting the tendon into an intra-articular environment. In studies that contained additional treatment variables, only the portion of the experiment that compared PRP directly with the control were evaluated. Only studies written in English and published in a peer-reviewed journal were included in this review. Various preparations and treatment modalities for PRP were included for review, such as single- and twice-centrifuged PRP, calcium chloride- and thrombin-activated PRP, PRP injections, PRP gels, and releasates from PRP clots. An abstract review was performed preliminarily for selection of manuscripts for full-text review. Relevant data were then extracted from the selected literature based on a predefined data sheet, which included information on PRP preparation and cytology, study design and methods, study subjects, outcomes measured, and results (Tables 1-3). Once the data collection process was complete, data were analyzed for Table 3. Data Extraction for Combined In Vitro and In Vivo Studies Author PRP Preparation Methods Outcomes Measured Results Anitua et al., In vitro In vivo Wang et al., In vitro Human whole blood spun and activated. Platelet count 2 greater in PRP than whole blood. Sheep whole blood of PRP prepared in same way as in vitro. Whole blood, 50 ml, double spun and activated. Platelet count in PRP was 4 greater than whole blood. Human tendons from ACL reconstruction obtained and cells plated on wells coated with PRP matrices. Skeletally mature sheep injected with 2 ml of PRP, platelet-poor plasma, and saline solution weekly for 4 wk. Tenocytes obtained from digested hamstring tendon biopsy specimens and cultured with 1%, 5%, or 10% PRP for 7 d. In vivo Tenocytes cultured in 10% PRP or 10% FBS (control) in diffusion chambers implanted into 15 mice. Synthesis of TGF, VEGF, and HGF; cell proliferation; and type I collagen. H&E changes in cell density with PRP treatment. Tenocyte proliferation, viability, and collagen production and messenger RNA expression of tenocyte markers SCX, COL-I, COL-III, and DCN. Immunohistochemical, H&E, and TEM analysis performed; differences in COL1, COL3, SCX, DCN, and collagen deposition measured. PRP-treated group had increased cell proliferation (dose dependent) as well as increased VEGF, HGF, and TGF. No difference in type I collagen production. PRP-treated group had increased cell density and neovascularization and more organization of fibers. There were differences in cell proliferation between 10% PRP-treated group and control at day 7. 10% PRP-treated group had greatest collagen production and greatest expression of tenocyte markers. PRP-treated group had more collagen fibrils but no difference in gene expression compared with control. DCN, decorin; FBS, fetal bovine serum; H&E, hematoxylin-eosin; HGF, hepatocyte growth factor; SCX, scleraxis; TEM, transmission electron microscope.

7 602 N. BAKSH ET AL. trends in outcomes, comparing and contrasting the effects of PRP reported in the literature. Results Search and Literature Selection The search parameters yielded 174 results on PubMed/Medline and 205 results from Embase (Fig 1). Duplicates were excluded, and 37 studies fit the inclusion criteria for systematic review. The fulltext review yielded 31 articles that fit the inclusion criteria. 2-22,24,32-40 We excluded 3 studies because they were not available in the English language, 1 because it studied the effect of PRP on an intra-articular tendon, and 2 because they studied several variables without an emphasis on PRP alone against a control. Of the included studies, 14 were in vivo 3,4,9,10,12,13,17,18,24,32-36 and 15 were in vitro. 2,5-8,11,14-16,19,21,22,37-39 Two studies had both in vitro and in vivo arms. 20,40 In Vitro Of the 15 in vitro studies, 14 studied tenocytes or tendon stem cells 2,5,6,8,11,14-16,19,21,22,38,39 and 1 studied fibroblasts cultured from tendons 7 (Table 1). In the former group, 7 articles studied human tenocytes 5,6,8,11,21,22,39 whereas the remaining 7 used animal cell cultures (canine, rabbit, rat, and equine). 2,14-16,19,37,38 Human tendons were collected from the hamstring (3 studies), 5,21,22 bicep (1 study), 8 Achilles (1 study), 6 and rotator cuff (1 study). 11 One study reported that tendons were harvested during ACL reconstruction but did not specify the tendon used. 39 Of the 15 studies, 13 (86.7%) reported the platelet concentration in PRP either as an absolute value or as a value relative to whole blood (Table 4). All reported increases in platelet concentration, ranging from approximately 2.5 to 10 times that of whole blood (Table 1). 2,5-8,11,14-16,21,22,37,39 Variability existed in the volume of whole blood drawn for PRP preparation (Table 1). Five studies reported cytometry for PRP preparations, with 3 describing reduced white blood cell counts in PRP compared with whole blood and 2 reporting no significant difference (Table 4). 5,8,11,14,16 Of the 15 studies, 9 (60%) measured cell proliferation in response to PRP treatment. 2,5,7,8,11,16,19,22,39 Of those, a total of 8 (88.9%) showed significantly increased cell proliferation in the presence of PRP. 2,5,7,8,11,19,22,39 Furthermore, 6 studies (40%) examined cell viability, 2,8,15,19,21,38 and of those, 4 (66.7%) reported an increase with PRP treatment. 2,19,21,38 The remaining studies showed no significant difference in cell viability with PRP treatment compared with control. 8,15 In 8 studies (53%) the authors observed the effects of PRP on the expression of growth factors, 5-7,14,16,19,38,39 which included IGF-1, TGF-b1, PDGF, VEGF, hepatocyte growth factor, and EGF. TGF-b1 was the most Fig 1. PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) flow diagram for literature selection.

8 PLATELET-RICH PLASMA IN TENDON HEALING 603 Table 4. PRP Reporting In Vivo and In Vitro Parameter Reported Not Reported Platelet concentration in PRP n ¼ 22 n ¼ 9 Cytology of PRP n ¼ 6 n ¼ 25 commonly measured, and it was assessed in 6 of 8 studies (75%). 5,14,16,19,38,39 Of these 8 studies, 7 (87.5%) showed significantly increased levels of growth factor with PRP treatment, 5-7,14,16,38,39 although 1 of these showed decreases in PDGF and EGF with concomitant increases in VEGF and hepatocyte growth factor. 39 Although the eighth study reported increased TGF in the PRP treatment group, the authors failed to state whether it was significantly different by comparison to the control. 19 Five studies reported on the effects of PRP on collagen synthesis, 5,14,16,37,38 of which 4 (80%) reported increased collagen expression with PRP treatment. 14,16,37,38 The remaining study showed increased total collagen but decreased gene expression of type I collagen (COL1) and type III collagen (COL3). 5 In Vivo Of the 14 in vivo studies, 7 (50%) reported platelet concentrations (Table 4). 9,10,12,18,33-35 All showed platelet levels that were significantly (1.5 to 10 times) higher than whole blood. 9,10,12,18,33-35 Only 1 of the studies reported cytometry for PRP 9 (Table 4), and only 1 analyzed the PRP for growth factors, which included PDGF, TGF-b1, VEGF, and EGF 12 (Table 2). The majority of studies (57%) used a New Zealand white rabbit model. 4,9,10,13,24,32,35,36 Of the rabbit studies, 4 investigated the Achilles tendon 4,10,13,24 and 4 studied the patellar tendon. 9,32,35,36 An equine model was used in 3 articles (21.4%), studying the superficial digital flexor tendon in each. 3,33,34 Three studies used a rat model (21.4%), 12,17,18 evaluating the patellar tendon in 2 12,17 and the Achilles tendon in Of the 14 studies, 13 (92.8%) performed a histologic assessment of the treated and control tendons, using hematoxylin-eosin stain, immunostaining, or both. 3,4,9,10,12,13,17,24,32-36 Of these 13 studies, 8 (61.5%) showed either earlier tendon healing, increased longitudinal organization and structure, or both (Table 1). 4,13,24,32-36 The remaining studies showed no significant differences between the PRP and control groups. 3,9,10,12,17 Three studies examined the effect of PRP on growth factors, and all 3 showed an initial significant increase in the level of growth factors in the PRP-treated group compared with the control group. 13,24,32 PRP-treated groups showed subsequent decreases in growth factor levels below control groups at later time points. 13,24,32 The growth factors measured were IGF and TGF-b1. Three studies investigated the effect of PRP on angiogenesis during lesion repair, and all 3 showed increased vascularity and/or blood flow in the PRPtreated groups. 3,4,36 Four studies reported the effects of PRP on collagen deposition, and each showed an increase in collagen deposition and content. 12,33-35 Four studies investigated the mechanical strength of tendons treated with PRP by measuring load to failure. 17,18,34,35 In this regard, 3 studies (75%) showed a significant increase in load to failure with PRP treatment. 17,34,35 One additional study described a trend toward increased load to failure with PRP treatment, but this did not reach statistical significance. 18 In Vitro and In Vivo The 2 studies that examined both in vivo and in vitro effects of PRP used human tenocytes for the in vitro component of the study (Table 3). 20,40 For the in vivo component, 1 studied sheep Achilles tendons 40 and the other studied human tenocytes cultured in diffusion chambers and implanted in mice. 20 Both studies reported increased platelet counts in the PRP, but neither reported cytometry. Increases in cell proliferation, collagen production, growth factor expression, and fiber organization were found with PRP treatment in both studies. 20,40 Discussion The basic science literature has shown that PRP may have several potential effects on tendon models in a laboratory environment compared with a control. One such effect is increased expression of growth factors, such as PDGF and TGF, 5-7,13,14,16,24,32,38-40 which are essential to the healing process. 27,41 Traumatic injury results in the formation of a platelet-rich hematoma, which releases growth factors and initiates the recruitment of inflammatory cells. 41 These inflammatory cells then release additional growth factors and cytokines that continue the healing process. 42 Kajikawa et al. 12 found that PRP treatment significantly increased the levels of circulation-derived cells, primarily macrophages, at the site of the wound. In theory, this could function to further augment the concentration of growth factors at the injury site. A growth factor present in PRP and a critical signaling molecule in the angiogenesis pathway is VEGF. 3,4,36,43,44 Each of the in vivo studies that investigated the effects of PRP on angiogenesis found increased vascularization in the treatment group. 3,4,36 In this regard, PRP may facilitate the healing of tendon tissue by increasing angiogenesis at the injury site. In vitro PRP was found to significantly increase tenocyte proliferation. 2,5,7,8,11,19,22,39 Zhang and Wang 37 found that PRP preferentially upregulated tenocyte-related genes such as COL1, COL3, and tenascin C when applied to tendon-derived stem cells while simultaneously downregulating genes that may compromise the tendinous structure. This suggests that

9 604 N. BAKSH ET AL. PRP promotes tenocyte proliferation by affecting both the mature and undifferentiated cells in the tendon. It should be acknowledged, however, that the effects of increased proliferation, whether positive or negative, have not yet been investigated clinically. PRP was also found to affect collagen expression in tenocytes. The COL3-COL1 ratio is thought to be an important factor in tendon repair, because an overproduction of COL3 results in a fibrotic, structurally inferior tendon. 11 A total of 9 studies showed increases in total collagen synthesis with PRP treatment. 12,14,16,33-35,37,38,40 Three studies reported the COL3-COL1 ratio, 5,14,16 of which 2 showed a decrease in the COL3-COL1 ratio compared with control. 14,16 McCarrel and Fortier 14 reported that PRP treatment decreased COL3 levels and increased COL1 levels, whereas Schnabel et al. 16 reported increases in both, with a greater increase in COL1. de Mos et al. 5 reported no change in the COL3-COL1 ratio, increased total collagen, and decreased gene expression of COL3 and COL1. Further research is necessary to clarify the effects PRP on the expression of COL3 and COL1 and the clinical implications of such. Several in vivo studies have reported that PRP-treated tendons healed at an earlier time point and were of superior quality to control tendons, with better organization of fibroblasts and collagen bundles. 4,24,32-36,40 The PRP treatment groups also had earlier regression of granulation tissue than the control groups, indicating an increased rate of repair. 4,24,32-36,40 All studies that observed tensile strength and mechanical load to failure found an increased load to failure in the PRP-treated group. 17,18,34,35 This may be beneficial in the clinical setting with regard to rehabilitation of a tendon injury. Earlier weight bearing and mobilization are associated with fewer adhesions, increased postoperative range of motion, earlier return to activity, and greater patient satisfaction. 45,46 Furthermore, Gelberman et al. 47 showed that immobilization impairs the repair of the injured tendon, delaying remodeling of the new collagen fibrils. The data suggest that PRP as an adjunct to surgical repair would increase the tensile strength at the surgical site. A clinical study by Sánchez et al. 48 reported that patients receiving adjuvant PRP treatment with Achilles reconstruction had a significantly earlier return to activity, sports, and full range of motion. Caution should be used, however, when translating these models clinically until they are fully and rigorously investigated with high-quality studies. Limitations Although the data have shown that PRP had several potential effects on tendon models, there were several limitations. Rat models are not treated with autologous blood product because of a lack of sufficient circulating volume; therefore, when rats were used in vivo, 1 or more rats were sacrificed to harvest blood for PRP preparation. 12,17,18 As a result, there was the confounding factor of variation between platelet levels in the whole blood used to create PRP and the whole blood of the treatment rats. In addition, this does not truly mimic clinical practice, because it is, by definition, an allogeneic PRP preparation and may potentially cause an increased immunogenic response. Several studies failed to report platelet levels in PRP. Previous research has documented that there is interindividual and intraindividual variability in platelet concentrations, and its effect on clinical outcomes is poorly understood. 49 Therefore it is important that authors report the platelet concentrations of PRP so that they can be compared systematically for variation and outcome. In this regard, DeLong et al. 50 recently developed the PAW classification system for reporting PRP composition. It is based on the platelet concentration, the activation method used, and the white cell count of the PRP, yielding the acronym PAW. Of the 23 studies that they cited, they could only classify 1 with the PAW system, showing a lack of reporting of these important data. We agree that a classification system should be used to report PRP preparations so that the effects of PRP can be compared between studies. This is imperative, because Mazzocca et al. 49 reported that varying preparations of PRP had different in vitro effects on proliferation and growth factor expression in tenocytes, osteoblasts, and myocytes. Furthermore, additional research investigating the effects of varying platelet concentrations on different cell types should be pursued. Few studies provided full cytology for their PRP preparations. The effect of leukocyte concentration on tendon models is poorly understood. Only 1 study 9 investigated the differences between leukocyte-rich and leukocyte-poor PRP, and it reported increased inflammation, fibrosis, and fiber disruption in the leukocyterich group at an early time point. At a later time point, there was no significant difference between the 2 treatment groups. The study, however, did not address clinical outcomes or test the treatments on an injury model. Therefore the effect of leukocyte content in the clinical setting is still unknown and is an important area for further study. Another limitation of the basic science literature is that the majority of in vivo studies analyzed the effects of PRP on surgically created acute lesions in the tendon. In many cases the lesions were immediately treated with PRP before closure. 4,12,13,17,18,32,35,36 Clinically, however, chronic tendon pathologies are more common, and even acute injuries may present days to weeks after the initial trauma. 51,52 Although creating chronic tendon pathology in an animal model is more difficult than creating an acute lesion, successful attempts have been documented in the literature. 53 Future in vivo research on PRP and chronic tendon pathologies is warranted.

10 PLATELET-RICH PLASMA IN TENDON HEALING 605 To date, the available clinical research shows inconsistent results with the use of PRP. 54,55 Barber et al. 54 published a case series of patients undergoing arthroscopic rotator cuff repair with PRP treatment. The patients treated with PRP had lower rates of retear on magnetic resonance imaging based on Rowe scores. In contrast, Rodeo et al. 56 evaluated the effect of a platelet-rich fibrin matrix on rotator cuff tendon healing in a prospective randomized clinical trial (Level of Evidence II) of 79 patients. They found no differences in tendon vascularity, muscle strength, and clinical rating scales, with the platelet-rich fibrin matrix showing a potentially negative effect on tendon healing. In addition, Chahal et al. 55 conducted a systematic review on PRP use in arthroscopic rotator cuff repair and found no significant differences in retear rates with PRP treatment. PRP has also been investigated clinically in cases involving the Achilles tendon, with no benefit shown in several highlevel studies. 1,29,57 The benefits of PRP use in the clinical setting remain unclear. Conclusions In this descriptive systematic review of basic science studies, it appears that PRP confers several effects on tendon models compared with a control. The hypothesis of this study is therefore supported. However, the literature is inconsistent with regard to reporting the methods of preparation of PRP and in reporting platelet concentrations and cytology. These data are imperative, and investigators must take care to report them in a standard fashion. Care should also exercised when translating these results to a clinical setting, in which randomized controlled clinical trials of high methodologic quality must be conducted to determine the appropriate indications for PRP. 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