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EuropeanJ Vun et al. Cells and Materials Vol. 41 2021 (pages 269-315) Effects DOI: of 10.22203/eCM.v041a19platelet products on bio-physiology ISSN of BM-MSCs1473-2262

THE IN VITRO EFFECTS OF PRODUCTS ON THE BIOPHYSIOLOGICAL FUNCTIONS OF HUMAN MARROW MESENCHYMAL STROMAL CELLS: A SYSTEMATIC REVIEW

J. Vun1,2,3,*, M. Panteli1,2,3, E. Jones2 and P.V. Giannoudis1,2,3,4

1 Academic Department of Trauma and Orthopaedics, School of Medicine, University of Leeds, Leeds, UK 2 Leeds Institute of Rheumatic and Musculoskeletal Medicine, University of Leeds, Leeds, UK 3 Leeds Orthopaedic and Trauma Sciences, Leeds General Infirmary, University of Leeds, Leeds, UK 4 NIHR Leeds Biomedical Research Centre, Chapel Allerton Hospital, Leeds, UK

Abstract

Platelet products (PP) and bone-marrow aspirate are popular sources of osteoinductive signalling molecules and osteogenic mesenchymal stromal cells (BM-MSCs) used in the treatment of impaired bone healing. However, the combined use of PP and BM-MSCs in clinical studies has reported mixed results. Understanding the cellular and molecular interactions between PP and BM-MSCs plays the important role of guiding future research and clinical application. This systematic review investigates the effects of PP on the biophysiological functions of BM-MSCs in in vitro human studies, including (i) proliferation, (ii) migration, (iii) differentiation, (iv) growth factor/cytokine/ expression, (v) immunomodulation, (vi) chemotactic effect on haematopoietic stem cells, (vii) response to apoptotic stress, and (viii) gene expression.In vitro studies in human have demonstrated the multi-faceted ‘priming effect’ of PP on the biophysiological functions of BM-MSCs. PP has been shown to improve proliferation, migration, osteogenic differentiation, reaction to apoptotic stress as well as immunomodulatory, pro-angiogenic and pro-inflammatory capacities of BM- MSCs. Several factors are highlighted that restrict the transferability of these findings into clinical practice. Therefore, more collaborative in vitro research in humans modelled to reflect clinical practice is required to better understand the effects of PP exposure on the biophysiological function(s) of BM-MSCs in human.

Keywords: Bone marrow mesenchymal stem (s), mesenchymal (s), platelet-rich plasma, , platelet concentrate, platelet releasate, platelet gel, platelet-rich , bone healing, non-union.

*Address for correspondence: Dr James Shen Hwa Vun, Clinical Research Fellow, Academic Department of Trauma and Orthopaedics, School of Medicine, University of Leeds, Clarendon Wing, Level A, Great George Street, Leeds, LS1 3EX, West Yorkshire, UK. Telephone number: +44 1133922750 Fax number: +44 113392329 Email: [email protected]

Copyright policy: This article is distributed in accordance with Creative Commons Attribution Licence (http://creativecommons.org/licenses/by-sa/4.0/).

List of Abbreviations BMP bone morphogenetic protein CEPBA CCAAT/enhancer-binding protein α αMEM alpha-minimum essential medium CFU colony-forming unit aPRP activated PRP CM conditioned medium ACAN aggrecan COL1 collagen type 1 ACD-A acid-citrate dextrose solution A COL1A1 collagen type 1 α1 ADIPOQ adiponectin, C1Q and collagen COL2A1 collagen type 2 α1 domain containing COMP cartilage oligomeric matrix protein aFGF acidic fibroblast growth factor COX-2 cyclooxygenase-2 ALP alkaline phosphatase CPD citrate phosphate dextrose APOE apolipoprotein E DAPI 4′,6-diamidino-2-phenylindole bFGF basic fibroblast growth factor DMEM Dulbecco′s modified Eagle′s medium BMA bone marrow aspirate EDTA ethylenediamine tetraacetic acid BMAC BMA concentrate FABP4 fatty acid binding protein 4 BM-MSCs bone marrow mesenchymal stromal FBS foetal bovine cells fPL filtered PL

269 www.ecmjournal.org J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs

FTPL frozen-thawed PL RUNX2 runt-related transcription factor 2 G-CSF colony-stimulating SDF-1 stroma-derived factor-1 factor SEM scanning electron GLUT4 transporter type 4 SNPL PL obtained by sonification HG-DMEM high-glucose DMEM SOX SRY-box transcription factor HGF growth factor SPARC secreted protein acidic and cysteine HPLF human platelet lysate from fresh rich platelet concentrates SREBP1 sterol regulatory element-binding HPLO human platelet lysate from expired transcription factor 1 platelet concentrates TERT telomerase reverse transcriptase HSCs haematopoietic stem cells TGF transforming growth factor IL ucPRP umbilical cord PRP IP-10 interferon-γ-inducible protein 10 uPA urokinase-type plasminogen iNOS inducible synthase activator ITS insulin--selenium VEGF vascular endothelial growth factor KO-DMEM knock out-DMEM receptor L-PRP leukocyte-rich PRP L-PDGS lipocalin-type prostaglandin D synthase LG-DMEM low-glucose DMEM Introduction LPL lipoprotein lipase LXRα X receptor alpha Bone healing is a unique biological process leading MCP-1 chemoattractant protein-1 to bone repair and restoration of bone function to MMP matrix metalloproteinases its pre-injury level (Einhorn and Gerstenfeld, 2015). MNC mononuclear cell According to the ‘diamond concept’, bone healing MIP inflammatory protein involves the orchestrated interaction between naPPP non-activated PPP multiple factors at the molecular, physiological and naPRP non-activated PRP biomechanical level (Giannoudis et al., 2007). NF-κB nuclear factor kappa-light-chain- The three fundamental molecular components enhancer of activated B cells of bone healing are: (a) osteogenic progenitor cells, NM nutrient medium such as BM-MSCs; (b) osteoinductive signalling NO nitric oxide molecules, such as growth factors and cytokines; OM osteogenic differentiation medium (c) osteoconductive scaffold/matrix (Calori and PAI-1 plasminogen activator inhibitor-1 Giannoudis, 2011). In addition, other essential PBMC peripheral mononuclear cells factors at the physiological and biomechanical levels PD population doubling crucial to successful bone healing include the host PDGF platelet-derived growth factor (i.e. patient) physiology, mechanical stability and PG platelet gel vascularity of the affected local bone environment PG-CM platelet gel conditioned-medium (Giannoudis et al., 2008). PGE2 prostaglandin E2 Although every attempt has been made to PL platelet lysate optimise the physiological and biomechanical factors PLGF placental growth factor during primary surgery, impaired bone healing (e.g. PLIN perlipin delayed union and non-union) remains common, POU5F1 POU class 5 homeobox 1 occurring in 5-10 % of all acute fractures (Ekegren et PP platelet product al., 2018; Mills and Simpson, 2013). Impaired bone PPARG peroxisome proliferator-activated healing is challenging to treat and poses a significant receptor gamma burden on the patient, socioeconomic and healthcare PPP platelet-poor plasma systems (Bishop et al., 2012; Hak et al., 2014). Multiple P-PRP leukocyte-poor PRP operations are often required to achieve treatment PRISMA Preferred Reporting Items for success, which exposes patient to further risks of Systematic Reviews and Meta- peri-operative complications, prolonged periods of Analyses rehabilitation and delay in returning to work. From PR platelet releasate a socioeconomic perspective, the direct treatment PRC platelet-rich concentrate costs have been estimated to be between £7,000 and PRGF plasma rich in growth factors £79,000 per person depending on complexity (Bishop PRF platelet-rich fibrin et al., 2012). In European healthcare systems the PRP platelet-rich plasma indirect costs through productivity losses have been PRP-CM PRP-conditioned culture medium estimated to be 10 times higher (Hak et al., 2014). PTGES prostaglandin E synthase In an effort to optimise bone healing, surgeons RANTES regulated on activation, normal T have recently attempted a ‘polytherapy’ approach cell expressed and secreted involving the simultaneous application of all the

www.ecmjournal.org 270 J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs three fundamental molecular components of bone terminology that allows for the effective, accurate healing, as mentioned previously (Calori et al., 2011). description of these PPs, reflective of its contents and Autologous sources of these biological components, physical properties (e.g. liquid, gel, fibrin membrane). which guarantees histocompatibility, minimal Clinical studies investigating the use of PPs morbidity and ease of access during the harvesting either alone or in combination with BM-MSCs have process, are ultimately desirable. This has made reported mixed results (Andia and Maffulli, 2019; autologous iliac crest bone graft, PPs and BMA/ Bielecki et al., 2008; Calori et al., 2008; Chiang et al., BMAC popular sources of osteoconductive scaffolds, 2007; Etulain, 2018; Galasso et al., 2008; Golos et al., osteoinductive molecules and osteogenic BM-MSCs, 2014; Malhotra et al., 2015; Mariconda et al., 2008; respectively (Calori and Giannoudis, 2011; Gianakos Roffi et al., 2017; Sanchez et al., 2009; Say et al., 2014; et al., 2017; Roffi et al., 2017). Verboket et al., 2018). The pooling of fractures from PPs or platelet derivatives are terminologies often different anatomical sites (e.g. tibia, femur, humerus) used interchangeably to represent haemoderivatives (Bielecki et al., 2008; Calori et al., 2008; Chiang et al., containing and growth factors, cytokines 2007; Galasso et al., 2008; Golos et al., 2014; Malhotra and molecules beneficial for bone healing. PPs, which et al., 2015; Mariconda et al., 2008; Roffi et al., 2017; are commonly used in the clinical and laboratory Sanchez et al., 2009; Say et al., 2014), coupled with settings, include PRP, PL, PRC, PR, PG and PRF. the heterogeneity in techniques used to harvest and Briefly, when harvested in the presence of prepare PPs and BM-MSCs (Etulain, 2018; Roffi et anticoagulants, peripheral can be centrifuged al., 2017) made comparison between studies difficult, to produce two distinct fractions: (i) PRP (also hindering the in-depth scientific understanding of referred to as platelet concentrates) and (ii) PPP. On these biological therapies. the other hand, of blood harvested in A systematic review on in vitro studies in human the absence of anticoagulants induces coagulation, will provide an improved scientific understanding of producing PRF. Finally, when peripheral blood how PPs interacts with and affects the biophysiological is allowed to naturally coagulate and then is functions of BM-MSCs at the cellular level. This has centrifuged, this produces autologous serum. far-reaching importance, serving to guide future Inducing the release of growth factors from PRP research and the clinical application of BM-MSCs and ex vivo could be achieved through two common PPs when treating patients with or at risk of impaired methods: (i) physical disruption/ and (ii) bone healing. chemical activation. Physical disruption of PRP, The main objective of the present systematic review such as repeated freeze-thaw cycles and ultrasound was to investigate the biophysiological functions of sonification, results in platelet lysis and, therefore, BM-MSCs influenced by PPs. Additionally, the acellular PL rich in growth factors. Chemical different methods to harvest, prepare and culture activation of PRP (commonly with calcium salts or BM-MSCs and PPs were also evaluated. thrombin) leads to the production of two distinct products, due to two distinct processes that occur during activation: (i) platelet degranulation and the Materials and Methods release of a plasmatic fraction rich in growth factors, known as platelet releasate or PRGF or platelet Protocol released supernatants; (ii) fibrinogenesis leading to This systematic review was conducted in accordance formation of a platelet-rich clot, also known as PG with the key principles recommended in PRISMA (Soares et al., 2020). statement (Hutton et al., 2015; Moher et al., 2009). Several classification systems for platelet concentrates and PRP have been developed in the last Information sources and search strategy decade (DeLong et al., 2012; Dohan Ehrenfest et al., A comprehensive systematic bibliographic search 2009; Lana et al., 2017; Magalon et al., 2016; Mautner was performed in the following databases on the et al., 2015; Mishra et al., 2012). The strengths of these 8th of September 2020: PubMed, Medline (via Ovid), classifications lie in their call for a comprehensive Embase (via Ovid), Scopus, Google Scholar and the documentation of centrifugation and preparation Cochrane Library. No restriction was applied to the protocol, cellular constituents (platelet concentration, date of publication. The following search terms were red count, count) and used: ‘bone’ or ‘bone marrow’ or ‘mesenchymal stem method of platelet activation. However, the choice cell(s)’ or ‘mesenchymal stromal cell(s)’ or ‘MSC’ or of using the term PRP for PP that have undergone ‘platelet product’ or ‘platelet rich plasma’ or ‘PRP’ exogenous chemical activation (which strictly or ‘platelet lysate’ or ‘platelet rich concentrate’ or speaking should be called PG) in these classification ‘platelet releasate’ or ‘platelet rich fibrin’ or ‘platelet systems creates further confusion. Consequently, gel’ or ‘platelet released growth factors’. The choice PRP is often used loosely as a universal term of studies was limited to human studies only. encompassing all forms of platelet-rich preparations Bibliographies of all identified studies were manually with variable preparation protocols and composition. searched for any additional eligible studies. Therefore, there still remains a lack in standardised

271 www.ecmjournal.org J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs

Figure 1. PRISMA flowchart on study selection.

Eligibility criteria discrepancies or disagreement between reviewers Studies were original research articles that fulfilled were resolved by consensus. the following inclusion criteria: (i) in vitro studies in human; (ii) studies that included comparison Data extraction between BM-MSCs versus PPs and BM-MSCs. Studies Relevant information on author, year of publication, involving use of scaffold (in addition to PPs and BM- patient demographics, site of harvest, processing MSCs) were excluded, since the presence of another methods (e.g. centrifugation), characteristics, osteoconductive ± osteoinductive element (i.e. the culture method, incubation period, laboratory scaffold itself) complicates the interpretation on how , biophysiological functions of BM-MSCs were PP affects the biophysiological function of BM-MSCs. carefully extracted. The biophysiological functions of Furthermore, in vitro studies combining a xenogeneic interest were proliferation, migration, differentiation, component (PPs or BM-MSCs) with human BM-MSCs cytokine/protein/growth factor expression, gene or PPs, clinical studies and case reports were also expression, immunomodulation and reaction to excluded. apoptotic stress.

Study selection Data analysis All retrieved records were first uploaded into Outcomes of interest were entered into an electronic EndNote followed by removal of duplicate records. database. The following were compared across Then, retrieved records were independently screened different studies: (i) harvesting process, (ii) by two reviewers (JV, MP) in a blinded, standardised centrifugation technique/preparatory steps, (iii) manner. Title and abstract sift was conducted tissue culturing and incubation period of PPs first, followed by review of full text. Only studies and BM-MSCs, (iv) effect of PP exposure on the fulfilling the eligibility criteria were included. Any biophysiological functions of BM-MSCs.

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Results commercial grade PL: n = 1), all other studies used PL processed using buffy coats or platelet / Study selection concentrate acquired from a . A total of 904 citations were identified during the Only a small number of studies reported on searches, followed by removal of duplicate records harvest volume (n = 12). The health (n = 12) and age (n = 281). At title and abstract sift, 569 citations not (n = 8) of volunteers/patients were only reported in meeting the inclusion criteria were excluded. Full text some studies (Table 2a,b). sifting of the remaining citations (n = 58) excluded a further 25 citations, yielding a total of 33 citations for BM-MSCs the final analysis (Fig. 1). In most of the studies BM-MSCs were harvested from BMA (n = 20) and bone marrow harvested Study characteristics during surgery (n = 5).The remaining studies used Table 1 summarises the main study characteristics of either commercially purchased BM-MSCs (n = 4) or the 33 studies included in the analyses and the BM- cryopreserved BM-MSCs (n = 4) (Table 3a,b). MSC functions assessed. The choice of PPs used was The commonest harvest site was iliac crest (n = 11), as follows: PRP-CM (n = 1); PL (n = 17); PR (n = 8) ; PRC followed by long (n = 3). Other sources of (n = 2); PG-CM (n = 2) and PRF (n = 3). The effect of PPs BM-MSCs include femoral head (n = 1) (Agis et al., on the proliferation of BM-MSCs was most commonly 2009) and maxilla (n = 1) (Dohan Ehrenfest et al., assessed (n = 27), followed by differentiation n( = 23); 2010); whilst 17 studies did not specify harvest site gene expression (n = 14); migration (n = 8) ; cytokine/ of the BM-MSCs used. The volume of bone marrow growth factor release (n = 7) and immunomodulation harvested was only reported in 6 studies. Likewise, (n = 6). Other functions less commonly assessed were the age (n = 16) and health (n = 19) of volunteers/ the response of BM-MSCs towards apoptotic stress patients were only reported in some studies. (n = 1) (Yin et al., 2016) and stimulation of fibrinolysis and plasminogen activity (n = 1) (Agis et al., 2009). Preparation and processing of harvested samples The majority of the studies (21 out of total 33) PPs – centrifugation, anticoagulant, platelet activation reported on the sample sizes for both PPs and BM- and concentrations of platelet and leukocytes MSCs. Only 6 of these 21 studies had a sample size of As illustrated in Table 4a-e, the centrifugation n ≥ 10 for both components (PPs and BM-MSCs) (Ben protocol was very variable among studies, most of Azouna et al., 2012; Jenhani et al., 2011; Lucarelli et al., which used a laboratory centrifugation system. 2003; Moisley et al., 2019; Verrier et al., 2010; Yin et al., Similarly, the use of anticoagulants was variable 2016); with the remaining having a small sample size and dependant on (i) protocol, (ii) source of platelet of n ≤ 6 for one or both components. Discrepancy in used (e.g. peripheral blood, platelet apheresis sample size between PPs and BM-MSCs within the concentrate, commercial PL) and (iii) the choice of same study was also observed (Amable et al., 2014; PPs intended for the experiment. Anticoagulants, Ben Azouna et al., 2012; Bernardi et al., 2017; Bernardi such as ACD-A (n = 8), heparin (n = 3), CPD (n = 3) et al., 2013; Goedecke et al., 2011; Gottipamula et al., and sodium citrate (n = 1) were used if the first step 2012; Infante et al., 2017; Jenhani et al., 2011; Kasten et involved producing PRP, before subjecting the al., 2008; Parsons et al., 2008; Perut et al., 2013; Prins et sample to other processes, such as freeze-thawing al., 2009; Vogel et al., 2006; Yin et al., 2016) (Table 2a,b or activation. and 3a,b). Activation of platelet to release the alpha-granules (Marx, 2004) was performed in studies assessing Harvesting process PR, PG-CM and PRF. Activation was achieved by With the exception of two studies (Dohan Ehrenfest exposing PPs to in 5 studies, to et al., 2010; Verrier et al., 2010), PPs and BM-MSCs thrombin in 4 and to both thrombin and calcium used in most of these in vitro studies were harvested gluconate in 1 study. from different donors, explaining the discrepancy in Leukocyte and platelet concentrations of PPs were sample size between PPs and BM-MSCs seen in some not reported in most studies. Leukocyte concentration studies (Table 2a,b and 3a,b). was only documented in 4 studies (Infante et al., 2017; Moisley et al., 2019; Perut et al., 2013; Yin et al., 2016), PPs whereas platelet concentration was reported in more PRP-CM (Yin et al., 2016), PRC (Parsons et al., 2008; studies (n = 15). Only 3 studies compared the effects of Samuel et al., 2016), PG-CM (Perut et al., 2013; Schar leukocyte content on the biophysiological functions et al., 2015) and PRF (Dohan Ehrenfest et al., 2010; of BM-MSCs (Moisley et al., 2019; Perut et al., 2013; Lucarelli et al., 2010; Moradian et al., 2017) were Yin et al., 2016). all harvested from peripheral blood. For studies assessing PR, only half (n = 4) used PR processed from BM-MSCs – centrifugation isolation method, fresh vs. peripheral blood; whilst the rest were from platelet subcultured cells apheresis/concentrates. As for studies using PL, with Most of the studies used a laboratory system to the exception of 3 studies (peripheral blood: n = 2; isolate mononuclear cells rich in BM-MSCs from the

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Table 1. Study characteristics. O = osteogenesis; A = adipogenesis; C = chondrogenesis.

Cytokine/ protein Gene Author (year) Proliferation Differentiation Migration expression expression Immunomodulation Others PRP-CM Cell viability/ Yin et al., 2016 ü O ü ü ü ü apoptosis PL Karadjian ü O et al., 2020 Moisley et al., 2019 ü ü Skific et al., 2018 ü O and A Infante et al., 2017 ü ü ü Muraglia ü et al., 2015 Muraglia et al., 2014 ü (lyophilised PL) Jonsdottir-Buch ü O, A and C ü ü et al., 2013 Bernardi et al., 2013 ü O, A and C ü Murphy et al., 2012 ü ü Lange et al., 2012 A ü Gottipamula ü O, A and C ü ü ü et al., 2012 Ben Azouna ü O, A and C ü ü et al., 2012 Jenhani et al., 2011 ü ü Goedecke ü O and A ü ü et al., 2011 Verrier et al., 2010 O ü ü Prins et al., 2009 ü O, A and C Vogel et al., 2006 O, A and C PR Nguyen et al., 2019 ü ü Liou et al., 2018 ü C ü Bernardi et al., 2017 (assessed PR and ü O, A and C ü PL) Kosmacheva O ü et al., 2014 Amable et al., 2014 ü O, A and C ü ü Stimulation of fibrinolysis and Agis et al., 2009 plasminogen activity Gruber et al., 2004 ü O ü ü Lucarelli et al., 2003 ü O ü PRC Samuel et al., 2016 ü O ü Parsons et al., 2008 ü O ü PG-CM Schar et al., 2015 ü Perut et al., 2013 ü O PRF Moradian ü et al., 2017 Lucarelli et al., 2010 ü Dohan Ehrenfest ü O et al., 2010

www.ecmjournal.org 274 J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs bone marrow harvested. This was achieved by using commonest. The increase in proliferation of BM- a density-gradient centrifugation system (n = 19), MSCs demonstrated a positive correlation with PP and laboratory-grade centrifugation into pellets and concentrations up to 10 %. Only 2 studies assessed manual isolation by nucleated before the effect of PP concentrations over 10 % (Amable et seeding (n = 3). 5 studies using clinically harvested al., 2014; Lucarelli et al., 2010), with opposing findings. bone marrow and 5 studies using commercially/tissue Amable et al. (2014) demonstrated that PR-CM with bank acquired BM-MSCs did not describe the method PR concentrations over 10 % had an inhibitory effect used to isolate mononuclear cells. towards BM-MSC proliferation, whereas Lucarelli et With the exception of 2 studies that strictly al. (2010) demonstrated that PRF-CM at 20 % induced used freshly aspirated BM-MSCs only in all of their stronger proliferation when compared to 10 % PRF- laboratory assays (Muraglia et al., 2014; Perut et al., CM. A dose-dependent increase in proliferation was 2013), the majority either used fresh BM-MSCs in their also observed in a study comparing single and double proliferation assays only (n = 3) or subcultured BM- PRF membranes (Dohan Ehrenfest et al., 2010). MSCs (BM-MSCs transferred from previous culture The effects of leukocyte content in PP was only into a new vessel for continued growth in fresh assessed by 3 studies, each using a different PP culture medium) (n = 31). Huge variation exists in (Moisley et al., 2019; Perut et al., 2013; Yin et al., tissue culture medium used for cultivation/expansion 2016). When compared against its leukocyte-poor and the passage number of BM-MSCs used for assays counterparts, BM-MSCs incubated in leukocyte- (median: passage 3, range: passage 0 to 6) (Table 5a-c). rich PG-CM demonstrated greater proliferative Cellular detachment of subcultured BM-MSCs was capacity (Perut et al., 2013); whereas L-PRP-CM performed by enzymatic methods (porcine trypsin, was found to be inferior (Yin et al., 2016). However, n = 22; recombinant corn-derived enzyme TrypZean, leukocyte content in PL was not found to influence n = 2). proliferation (Moisley et al., 2019). Increasing platelet concentrations in PR was found to result in improved Incubation of BM-MSCs with PPs BM-MSC proliferation (Gruber et al., 2004). All studies were conducted by exposing BM-MSCs to The effect of incubation time on PPs was only a continuous dose of PPs. As previously mentioned, assessed by 1 study (Parsons et al., 2008), whereby except for 2 donor-matched studies (Dohan Ehrenfest BM-MSCs incubated with 2.5 % PRC showed more et al., 2010; Verrier et al., 2010), BM-MSCs and PPs proliferation in the first 24 h only, with proliferation used in all other in vitro studies were not harvested equal to that of the control group at 48 h and inferior from the same donor(s). Most studies (70.9 %, n = 22 to control group at 72 h. out of total 31) with more than one donor reported pooling PPs in experiments (Table 6a-c). Effects of PP on migration of BM-MSCs Incubation period of BM-MSCs with PPs was The chemotactic effects of PP on the migratory variable among studies and dependent on the capacity of BM-MSCs was only assessed by 8 physiological function of BM-MSCs assessed: (i) studies (Table 9). PRP-CM, PR, PRF-CM and PG- migration: 4 to 48 h; (ii) proliferation: 2 h to 14 d; (iii) CM demonstrated positive chemotactic effects on differentiation: 8 to 21 d; (iv) gene expression: 5 to the migratory capacity of BM-MSC in vitro (Gruber 21 d; (v) cytokine, protein, growth factor expression: et al., 2004; Nguyen et al., 2019; Schar et al., 2015; Yin 3 to 14 d; (v) cell viability/apoptosis: 7 d; (vi) fibrin et al., 2016). Most PL studies reported on its positive dissolution assay: 24 h (Table 6a-c). The choices of chemotactic effects (Infante et al., 2017; Moisley et al., tissue culture medium and seeding density used 2019; Murphy et al., 2012). Migratory capacity of BM- for each assay were reported in all studies. Notably, MSCs improved with increasing PP concentration the choices of tissue culture medium, culture dish/ (up to 10 %) in most studies that compared between well plates and seeding density for assays were very different PP concentrations [PL: n = 2 (Goedecke et variable among studies (Table 7a-c). al., 2011; Murphy et al., 2012) and PR: n = 2 (Gruber et al., 2004; Nguyen et al., 2019)]. However, no studies Effects of PPs on proliferation of BM-MSCs have compared PP concentrations over 10 %. The Table 8a-c summarises the choice of laboratory assays, effects of leukocyte content in PP on the migratory PPs used, comparative groups and their findings. The capacity of BM-MSCs was assessed by 2 studies, presence of PPs was found to significantly increase with different conclusions (Moisley et al., 2019; Yin proliferation of BM-MSCs in most of the studies, et al., 2016). Whilst Yin et al. (2016) demonstrated when compared to controls: PRP-CM (n = 1), PL that P-PRP-CM performed significantly better than (n = 13), PR (n = 3), PRC (n = 1), PG-CM (n = 1), PRF L-PRP-CM, Moisley et al. (2019) demonstrated that (n = 2). leukocyte depletion do not affect BM-MSC migration. The effects of (i) concentration/dose of PPs, (ii) leukocyte content, and (iii) incubation time on Effects of PPs on differentiation of BM-MSCs proliferation of BM-MSCs have been assessed in Table 10a-c summarises the effects of PPs on the some studies. In terms of concentrations of PPs in differentiation potential of BM-MSCs into different PP-conditioned medium, variable concentrations mesenchymal lineages. Osteogenesis was most have been used, with 5 % to 10 % being the commonly assessed (n = 18), whilst adipogenesis

275 www.ecmjournal.org J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs n/a PRP mL per 45 mL per 45 mL

donor donor 100 mL ± ±

Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated buffy coats 40 mL each bag) Harvest volume Harvest 10-20 × buffy 10-20 × bags (30- 50 mL of pooled 7-13 pooled 450 450 bank bank bank Source buffy coat Peripheral blood Peripheral blood Peripheral blood Peripheral Aachen, Germany) PRP from apheresis units Buffy coat units from blood bank Buffy coat units from blood bank Buffy coat units from blood bank Buffy coat units from blood bank Buffy coat units from blood bank Platelet concentrate from blood bank Platelet Commercial human PL (PL BioScience, Commercial human PL (PL BioScience, bank blood from products platelet Pooled from rich concentrates derived Platelet apheresis collections from blood Platelet apheresis collections from blood Platelet apheresis concentrate from blood Platelet bank blood from products platelet Pooled n/a Age Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated 21-45 years 21-45 years 22-58 years 28-79 years (average: 49 years) (average: PL PRP-CM Healthy Healthy Healthy Healthy Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Patient’s health Patient’s iliac crest exposure patient undergoing routine patient undergoing routine involving surgery orthopaedic ) n 1 2 5 5 10 11 20 28 30 16 ≥ 10 ≥ 10 Not stated Not stated Not stated Not stated Not stated Sample size ( (5-10 buffy coats) purchased human PL) (10-20 buffy coat used) Not stated (commercially , 2015 , 2014 , 2013 , 2012 , 2019 , 2011 , 2017 , 2010 , 2012 , 2006 , 2018 , 2009 , 2016 et al. et al. , 2020 , 2013 , 2012 , 2012 , 2011 et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. Karadjian Goedecke et al. et al. et al. et al. et al. Ben Azouna Gottipamula Author (year) Author Yin Jonsdottir-Buch Prins (lyophilised PL) Skific Vogel Lange Verrier Infante Jenhani Moisley Murphy Muraglia Muraglia Bernardi Table 2a. Harvesting process of PPs. 2a. Harvesting Table

www.ecmjournal.org 276 J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs 9 mL 25 mL 80 mL 65 mL 18 mL 200 mL Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Harvest volume Harvest Inc) Source Peripheral blood Peripheral blood Peripheral blood Peripheral blood Peripheral blood Peripheral blood Peripheral blood Peripheral blood Peripheral blood Peripheral blood Peripheral device, Haemonetics) Platelet apheresis concentrate Platelet Whole blood from bank Platelet concentrate (> 5 doses) Platelet Platelet apheresis (PR: Haemonetics, MA; Platelet Caridian BCT separator, Accel Trimal PL: (MCS+ concentrates apheresis Platelet 54 Age Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated 20-30 years 24-39 years 27-38 years 26-38 years (mean: 32.6 years) PR PRF PRC PG-CM Healthy Healthy Healthy Healthy Healthy Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Patient’s health Patient’s tooth replacement Healthy, non-smokers Patient undergoing implant implant undergoing Patient molar first for maxilla in placement ) n 3 6 4 6 3 1 15 18 10 11 11 10 Not stated Not stated Not stated Sample size ( , 2003 , 2010 , 2017 , 2019 , 2014 , 2008 , 2016 , 2004 , 2015 , 2013 , 2018 , 2009 , 2014 , 2017 , 2010 et al. et al. et al. et al. et al. PL et al. et al. et al. et al. et al. et al. et al. Moradian et al. et al. et al. Kosmacheva Author (year) Author Agis Liou Perut Schar Dohan Ehrenfest Gruber Samuel Parsons Amable Nguyen Bernardi Assessed PR and Lucarelli Lucarelli Table 2b. Harvesting process of PPs. 2b. Harvesting Table

277 www.ecmjournal.org J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs 4 mL 60 mL 3-5 mL aspirate Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Volume of specified Iliac crest Iliac crest Iliac crest Iliac crest Iliac crest Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Harvest site Harvest Proximal femur Anterior iliac crest filtered before culture Long bone not clearly Greater trochanter of femur fracture patients; bone debris BMA BMA BMA BMA BMA BMA BMA BMA BMA BMA BMA centre centre Source Commercial Lonza Commercial Lonza Bone marrow samples Bone marrow washouts Passage 3-6 cryopreserved Passage During orthopaedic surgery National Bone marrow Graft National Bone marrow Graft BM-MSC prepared from BMA 25.5) years ± Age 4-74 years Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated 25-48 years 17-69 years 50-78 years PL 8-52 (median 22) years 29-73 (median 46) years 28-79 (average: 49) years 28-79 (average: 16-41 (mean 33 ± 2) years PRP-CM 12-75 (mean: 40.7 Not stated Not stated Not stated Not stated Not stated Not stated Not stated Fit and well Healthy donors Healthy donors Healthy donors Healthy donors Patient’s health Patient’s harvest procedures harvest orthopaedic surgery Healthy young volunteers replacement or spinal surgery ‡: same study cohort and methodology. Healthy patient undergoing elective Healthy patient undergoing elective Healthy donors undergoing total hip surgery involving iliac crest exposure Patient undergoing routine orthopaedic Patient Patient undergoing hip operations or Patient Patient undergoing surgery of proximal femur Patient ) n 3 4 4 5 9 6 14 10 11 13 13 13 16 Not stated Not stated Not stated Sample size ( = 3 for differentiation = 5 for gene expression n n 3 commercially available ‡ ‡ , 2017 , 2010 , 2012 , 2006 , 2018 , 2009 , 2016 , 2020 , 2019 , 2015 , 2014 , 2013 , 2013 , 2012 , 2012 , 2011 , 2012 , 2011 et al. et al. et al. et al. et al. et al. et al. Jenhani Moisley Murphy Bernardi Muraglia Muraglia Karadjian Goedecke et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. Author year Author Ben Azouna Gottipamula Yin Jonsdottir-Buch Prins Skific Vogel Lange Verrier Infante Table 3a. Harvesting process of BM-MSCs. 3a. Harvesting Table

www.ecmjournal.org 278 J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs Not Not Not 2 mL 5 mL 10 mL aspirate applicable applicable applicable Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Volume of Maxilla Iliac crest Iliac crest Iliac crest Iliac crest Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated Harvest site Harvest Femoral head Posterior iliac crest BMA BMA BMA BMA Source Cell bank group Ltd Bone marrow Bone marrow Bone marrow Bone marrow biopsy from site of bone defect trabecular bone marrow Trabecular bone marrow Commercially purchased Bone marrow samples from passage 3 human BM-MSCs discarded material collected created by drilling for future Bone marrow/cells harvested Bone marrow/cells harvested implant placement at maxilla during reconstructive surgery during reconstructive Commercially acquired Lonza 54 Age Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not stated 65-68 years 60-87 years 33-54 years 19-45 years 55, 75 and 65 years PR PRF PRC PG-CM 10-33 (mean: 18.3 ± 6.9) years union surgery Not stated Not stated Not stated Not stated Not stated Not stated Not stated arthroplasty Healthy donors Healthy donors Healthy donors Patient’s health Patient’s ‡: same study cohort and methodology. Patients undergoing arthroplasty Patients Patients undergoing total knee/ hip Patients maxilla for first molar tooth replacement Patients undergoing elective orthopaedic undergoing elective Patients Patient undergoing implant placement in Patient Not clearly stated; patients with fracture non- ) n 3 3 4 6 4 4 3 3 1 10 Not stated Not stated Not stated Not stated Not stated Sample size (

, , 2004 , 2015 , 2013 , 2018 , 2009 et al. , 2019 , 2014 , 2014 , 2003 , 2016 , 2008 , 2017 , 2010 , 2010 et al. 2017 et al. et al. et al. et al. Samuel Parsons Amable Nguyen Lucarelli Lucarelli Moradian et al. et al. et al. et al. et al. et al. et al. et al. et al. Author year Author Kosmacheva Bernardi Agis Liou Perut Schar Dohan Ehrenfest Gruber Table 3b. Harvesting process of BM-MSCs. 3b. Harvesting Table

279 www.ecmjournal.org J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs

(n = 8) and chondrogenesis (n = 7) were less commonly CM (p < 0.001). With regards to angiogenesis, PR investigated. resulted in an increased release of thrombospondin Osteogenic differentiation of BM-MSCs following (Amable et al., 2014), PLGF (Amable et al., 2014), aFGF PP exposure was found to be increased in most of the (Amable et al., 2014), VEGF-D (Amable et al., 2014) studies (66.7 %, n = 12 out of 18 studies) (Amable et al., by BM-MSCs (Amable et al., 2014). However, VEGF 2014; Dohan Ehrenfest et al., 2010; Gottipamula et al., release was found to be equivocal [PR: upregulated 2012; Karadjian et al., 2020; Kosmacheva et al., 2014; (n = 1) (Amable et al., 2014); PL: less than control (n = 2) Lucarelli et al., 2003; Parsons et al., 2008; Perut et al., (Ben Azouna et al., 2012; Jenhani et al., 2011)]. On the 2013; Samuel et al., 2016; Skific et al., 2018; Verrier et other hand, the release of adipogenic molecules al., 2010; Yin et al., 2016). In contrast, PP exposure did L-PGDS (Lange et al., 2012) and leptin (Ben Azouna not improve the adipogenic potential (87.5 %, n = 7 et al., 2012) by BM-MSCs following PP exposure was out of 8 studies) (Amable et al., 2014; Ben Azouna found to be weaker than control group. et al., 2012; Goedecke et al., 2011; Gottipamula et al., PPs increased the secretion of catabolic MMPs 2012; Lange et al., 2012; Prins et al., 2009; Vogel et al., (MMP1, MMP3, MMP7, MMP8 and MMP13) and 2006) and chondrogenic potential (66.7 %, n = 4 out extracellular matrix and other molecules of 6 studies) (Ben Azouna et al., 2012; Liou et al., 2018; (heparan sulphate, elastin, , collagens I and Prins et al., 2009; Vogel et al., 2006) of BM-MSCs in III) (Amable et al., 2014). Amable et al. (2014) studied the majority of the studies. also the release of other growth factors and found that Only Lucarelli et al. (2003) investigated the effect PP increases the secretion of HGF, G-CSF, TGF- 1 of PP withdrawal on BM-MSC differentiation. and TGF- 2; whereas it downregulates PDGF-BB They reported that despite PP withdrawal, BM- secretion. β MSCs previously exposed to PPs retained their β improved osteogenic and chondrogenic capability, Effects of PPs on other BM-MSC functions when compared against controls. Finally, only 2 The effect of PPs on the immunomodulatory function studies investigated the effect of leukocyte content of BM-MSCs was assessed by 4 studies (Bernardi et on osteogenic differentiation of BM-MSCs, with al., 2017; Gottipamula et al., 2012; Jonsdottir-Buch et opposing findings (Perut et al., 2013; Yin et al., 2016). al., 2013; Yin et al., 2016) (Table 12). Whilst L-PRP- CM was found to induce the activation of the NF- Effects of PPs on growth factor/cytokine/protein κB signalling pathway, which could activate genes expression of BM-MSCs responsible for and other immune Table 11a,b summarises the findings of studies that responses (Yin et al., 2016), the immunomodulatory have investigated the effect of PP on the expression effects of BM-MSCs exposed to PL seemed to be of growth factors, cytokines, proteins and enzymes equivocal. Jonsdottir-Buchet al. (2013) demonstrated by BM-MSCs. Overall, PPs were found to have a PL to have no effect on the ability of BM-MSCs to chemotactic, pro-inflammatory, pro-osteogenic and reduce mononuclear cell proliferation; whilst 2 other pro-angiogenic effect on BM-MSCs. Chemotactic studies demonstrated BM-MSCs cultured with PL to factors such as RANTES (Ben Azouna et al., 2012; have weaker inhibitory effects on PBMCs (Bernardi Jenhani et al., 2011), SDF-1α (Goedecke et al., 2011), et al., 2017) and T-cell proliferation (Gottipamula et eotaxin (Amable et al., 2014), IP-10 (Amable et al., al., 2012) when compared to controls. 2014), MIP-1β (Amable et al., 2014) and MCP-1 Other biophysiological function of BM-MSCs (Amable et al., 2014) were upregulated in BM-MSCs assessed were (i) response towards apoptotic stress, exposed to PPs. (ii) chemotactic effect on HSCs and (iii) capacity to Pro-inflammatory cytokines (PGE2, NO, IL-2R, stimulate plasminogen activation. Yin et al. (2016) IL-6, IL-7, IL-8, IL-12 and IL-15) were universally found that both L-PRP- and P-PRP-CM are able to upregulated by PPs in all in vitro studies (Amable et inhibit -induced apoptosis of BM-MSCs al., 2014; Ben Azouna et al., 2012; Jenhani et al., 2011; and, therefore, enhance its viability, with L-PRP- Yin et al., 2016). Comparing the effects of leukocyte CM having the strongest effect. The chemotactic content in PRP-CM on the release of pro-inflammatory effect of BM-MSCs in terms of inducing migration cytokines PGE2 and NO by BM-MSCs, Yin et al. (2016) of HSCs was only assessed by 1 study, whereby demonstrated that only L-PRP-CM upregulates the the chemotactic effect of BM-MSCs cultured in production of these pro-inflammatory cytokines 5 % and 10 % PL was demonstrably inferior when significantly when compared to P-PRP-CM and compared to control (Goedecke et al., 2011). Agis et controls; whereas no difference was observed when al. (2009) found that BM-MSCs pre-incubated with comparing P-PRP-CM to control (FBS). PR demonstrated a stronger capacity to stimulate Exposure to PPs was found to significantly plasminogen activation and fibrinolysis. upregulate most pro-osteogenic proteins by BM- MSCs. These include osteocalcin (Samuel et al., 2016; Gene expression of BM-MSCs following PP Yin et al., 2016), RUNX2 (Yin et al., 2016) and BMP2 exposure (Verrier et al., 2010). Comparing the effect of leukocyte Table 13a-c summarises the effect PP has on gene concentration, Yin et al. (2016) reported stronger expression of BM-MSCs, based on the biophysiological upregulation in P-PRP-CM in comparison to L-PRP- functions of BM-MSCs. Samples were taken from BM-

www.ecmjournal.org 280 J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs Not used Activator Activator applicable Not stated Not stated Not stated Not stated Not applicable Not stated Not stated Not stated Not stated Activation for 15 min g for 30 min g t residual supernatant Processing (steps) for 10 min for 10 min th g g for 10 min g for 15 min for 7 min g extraction of supernatant 2,700 × resuspension in 1/5 g centrifugation: on Separated plasma. 250 × centrifugation: centrifugation: 160 × centrifugation: 400 × nd st st nd 2 Extraction of supernatant Precipitated platelets resuspended in residual plasma ≥ P-PRP Laboratory-grade centrifuge 1 Automated clinical-grade centrifuge, BioCUE™ device, Zimmer Biomet Automated Laboratory-grade centrifuge 1 2 3 freeze-thaw cycles of cPRP, PRP and fPRP Automated clinical grade centrifuge Automated Group Medical Polymer Products Co., Ltd) Shandong Weigao (WeGo, - - - Laboratory-grade centrifuge Freeze − 80 °C, thawing 37 °C of freshly collected platelet apheresis product Heparin added to avoid gel formation 3 centrifugation cycles at 900 × Pooled as culture supplemen Laboratory-grade centrifuge 570 × collected layer Plasma PL: 3 freeze-thaw cycles of P-PRP at − 20 °C/37 °C Sterile filtration: 0.22 µ m filter Not stated 1,100 × PRP passed through white blood cell syringe filter (Acrodisc) PL • • • L-PRP: P-PRP: • • • • • • cPRP: • • PRP: • • • fPRP: • cPL, PL and fPL: • • • • • • • • • • PRP-CM ACD-A ACD-A Not stated Heparin (5,000 UI) at blood collection Anticoagulant used Acid citrate dextrose /L (P-PRP) /L (L-PRP) 9 9 /L (L-PRP) /L (P-PRP) 9 9 /μL (PRP) ‡: same study cohort and methodology. /μL (cPRP) /μL (PRP) platelets 5 /μL (cPRP) 3 3 5 11 (2×)” Platelet: Platelet: Platelet: Not stated Leukocyte: Leukocyte: 5 × 10 1.8 × 10 Platelet: not stated Platelet: 6.3 × 10 15.9 × 10 20.6 × 10 Leukocyte: not stated Leukocyte: no leukocytes 0.18 ± × 10 Platelet: not stated clearly; Platelet: 34.58 ± 8.48 × 10 Apheresis product containing 1461.80 ± 189.14 × 10 1436.70 ± 257.97 × 10 Leukocyte/platelet concentration “moderate enrichment of platelets (cPRP) P-PRP product PRP (PRP) Not stated grade PRP (fPRP) Laboratory-grade Platelet apheresis Platelet Type of PRP used Clinical grade L-PRP Filtrated Laboratory- Both P-PRP and L-PRP , , , , et al. et al. et al. et al. , 2020 2016 2019 2018 2017 (year) Author Author Yin Yin Karadjian et al. Skific Infante Moisley Table 4a. Preparation and processing of PPs. Table

281 www.ecmjournal.org J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs Not used Activator Activator applicable Not stated Not stated Not stated Not stated Not stated Not stated Not stated Not applicable Not stated Not stated Not stated Not stated Not stated Not stated Not stated Activation platelets/ µ L 6 min to remove platelet for 15 min to remove g for 15 min g (1,100 rpm for 10 min for 15 min to produce P-PRP g for 15 min to separate RBC fraction g for 20 min PRP and centrifugation at high-speed for for 10 min for 10 min g Processing (steps) g g for 6 min at 22 °C g for 20 min g min of frozen/thawed PRC for 20 min of frozen/thawed g 4,975 × Supernatant filtration: 40 µ m cell strainer Further filtration: 0.45 µ m stericups Laboratory centrifugation of buffy coat units soft spin at 341 × autostop), frozen at − 30 °C In-line filtration to deplete WBC (PALL Freeze-thaw cycles of PRP Laboratory centrifugation: 4,000 × centrifugation: 4,975 × centrifugation of PRP: 2600 rpm for 20 min centrifugation: 1,600 × centrifugation: centrifugation: 200-300 × st nd nd st nd - - - - - Buffy coats (free of red blood cells and plasma: automated clinical grade system; Compomat G3) P-PRP: PL: - - - PRP: Laboratory-grade centrifugation of pooled thrombocyte concentrates 200 × PL: Freeze thawing of PRP 5 freeze-thaw cycles of pooled platelet concentrates Laboratory-grade centrifugation 4,000 × fragments Laboratory-grade centrifuge 230 g × 15 min, room temperature of plasma and buffy layers Removal Further centrifugation: 1,100 × PL: freeze-thaw cycles of P-PRP Laboratory-grade centrifuge 1 2 Filtration and freezing at − 80 °C Centrifugation at 4,975 × Supernatants used as culture medium supplements Laboratory-grade centrifuge PRP: centrifugation of buffy coat 2 PL: 3 freeze-thaw cycles of 20 min, room temperature Laboratory-grade centrifuge 1 2 of platelet poor plasma Removal P-PRP: resuspension of platelet pellet, standardised to 10 PL: freeze-thaw cycles of P-PRP Not applicable PL • • • • • • • • • • • • • • • • • • • • • • • • • • • • • Heparin Not stated Not stated Not stated Not stated Not stated Not stated Not stated at blood collection Anticoagulant used platelets/μL platelets/μL 6 6 ‡: same study cohort and methodology. platelets/μL 6 Platelet: not stated Platelet: not stated Platelet: not stated Platelet: not stated Platelet: not stated Platelet: Leukocyte: not stated Leukocyte: not stated Leukocyte: not stated Leukocyte: not stated Leukocyte: not stated Leukocyte: not stated Leukocyte: not stated Leukocyte: not stated Platelet: 10 Platelet: Platelet: 10 × Platelet: 10 × Platelet: Leukocyte/platelet concentration Not stated Not stated Not stated Not stated Not stated Not stated Not stated thaw cycle PL produced by Type of PRP used sonification of PRP PL following freeze- P-PRP used to produce , , et al. et al. , 2015 , 2014 , 2013 , 2012 , 2012 , 2012 2013 2012 (year) Author Author Murphy Bernardi Muraglia Muraglia et al. et al. et al. et al. et al. et al. Jonsdottir- Buch Lange Ben Azouna Gottipamula Table 4b. Preparation and processing of PPs. Table

www.ecmjournal.org 282 J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs 2 2 2 used CaCL CaCL PL: no Activator Activator PR: CaCl Not stated Not stated Not stated Not stated Not stated Yes Yes PL: no PR: Yes Not stated Not stated Not stated Not stated Not stated Activation for 10 min g for 10 min to eliminate platelet g for 20 min g for 45 min at 4 °C for 10 min g g or 5 min for 5 min g 2 2 Processing (steps) (2012) rpm 20 min

et al. for 15 min, room temperature for 5 min (room temperature) g g for 30 min (room temperature) g Ben Azouna 2× freeze (− 80 °C)-thaw cycles and centrifugation of platelet apheresis product 1,600 × Supernatants pooled and filtered at 70 µ m cell strainer Stored at − 20 °C 200 × 2,000 × Activated platelet apheresis incubated at 40 °C for 60 min until Activated complete clot formation Centrifugation at 2,200 × PR collected and stored at − 80 °C centrifugation: 3,500 rpm f centrifugation: 2,000 st nd - - All supernatants discarded Resuspension of platelet pellet in sterile PBS at 1 : 10 Subsequent freeze-thaw cycles Freeze (− 80 °C)-thawing of platelet apheresis concentrates Laboratory-grade centrifugation: 750 × fragments P-PRP: laboratory-grade centrifugation of buffy coat, 205 × Plus) Leukocyte filtration (Fresenius Bio P P-PRP frozen at − 80 °C until use Laboratory-grade centrifugation 1 of supernatant Removal 2 CaCL activation: Platelet Laboratory-grade centrifuge 480 × CaCL activation: Platelet incubation at 37 °C Overnight Further centrifugation at 2,000 × Laboratory-grade centrifuge PR: - - - PL: Supernatant obtained after discarding fibrin clot = PRP releasate Laboratory-grade centrifugation (2 steps): - - - - Freeze-thawing (− 20 °C/56 °C) of platelet apheresis concentrates Laboratory centrifugation: 5,000 × Filtration: 40 µ m cell filter Same as PR • • • • • • • • • • • • • • • • • • • • • • • • • • (2012) Ben Azouna CPDA dextrose specified Not stated Not stated Not stated Not stated et al. Yes, but choice of Yes, anticoagulant not Citrate-phosphate- at blood collection Anticoagulant used Same as 2012) μL / 6 / µ L et al. ( 6 platelets/μL platelets/mL 6 9 ‡: same study cohort and methodology. Ben Azouna Platelet: 1 × 10 Platelet: Platelet: not stated Platelet: not stated Platelet: Platelet: not stated Platelet: Platelet: 1.56 × 10 Platelet: Leukocyte: not stated Leukocyte: not stated Leukocyte: not stated Leukocyte: not stated Leukocyte: not stated Leukocyte: not stated Leukocyte: not stated Platelet: 1 × 10 Platelet: Platelet: 10 × Platelet: Same as Leukocyte/platelet concentration P-PRP Not stated Not stated Not stated Not stated Not stated Not stated thaw cycle Type of PRP used PL following freeze- P-PRP used to produce , , , , ,

‡ et al. et al. et al. et al. et al. , 2019 , 2017 , 2011 2010 2009 2006 2018 2011 (year) and PL Author Author Nguyen Bernardi Goedecke et al. et al. et al. Liou Prins Vogel Verrier Verrier Assessed PR Jenhani Table 4c. Preparation and processing of PPs. Table

283 www.ecmjournal.org J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs 2 n/a n/a and used CaCL Human Human calcium (for PR) thrombin thrombin Activator Activator gluconate Thrombin Thrombin No No Yes Yes Yes Yes PL: no PR: yes Activation for 5 min g 10 min for g × cells/mL) for 20 min 9 10 min g for g for 20 min for 17 min g × g at 37 °C for 1 h for 10 min 2 Processing (steps) for 10 min g g for 10 min 80 °C g − °C to remove red blood cells for 15 min at 20 °C to remove for 10 min at 20 °C to obtain PRP g g 1,400 × Resuspension of pellets in serum-free α-MEM Activation of platelet suspension (human thrombin) Activation 30 min at room temperature Centrifugation and collection of supernatants (PR) centrifugation: 800 × centrifugation: 200 × st nd Laboratory-grade centrifugation (2 steps) 1,000 × 3,000 × Laboratory-grade centrifuge Centrifugation of platelet concentrates: 1,400 Human thrombin activation Further centrifugation: 1,400 Sterile filtration of supernatants (PR) Stored at leading to PG formation Thrombin activation Filtration and supernatant stored (PR at – 80 °C) Laboratory-grade centrifugation of pooled platelet concentrate to produce platelet concentrate (5 × 10 20 min incubation at °C Thrombin activation: Clot centrifugation at 3,500 × Supernatant: PR collected and stored at − 20 °C Laboratory-grade centrifugation of peripheral blood: 300 × Further centrifugation: 700 × CaCL activation: Platelet incubation at 4 °C Overnight PR: centrifugation of clot at 3,000 × Laboratory-grade centrifuge 1 2 Resultant platelet pellets isolated, resuspended in sterile PBS ≥ PRC Centrifuge device not used Utilised CAPTION device (Smith and Nephew relying on filtration technology) - - - Laboratory-grade centrifugation of leukocyte-depleted platelet apheresis concentrate aliquots - PR: PL: repeated freeze-thaw cycles of platelet suspension (in absence thrombin) filtered sterile and Centrifugation of supernatants from PR and PL were stored at − 40 °C - PR • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • PRC ACD ACD-A ACD-A dextrose Not stated Not stated Citrate-phosphate- at blood collection Anticoagulant used Acid citrate dextrose cells/mL 9 platelets/μL 6 platelets/mL 8 ‡: same study cohort and methodology. cells/mL following 9 Platelet: not stated Platelet: Platelet: not stated Platelet: not stated Platelet: first centrifugation Leukocyte: depleted Leukocyte: depleted Leukocyte: not stated Leukocyte: not stated Leukocyte: not stated Leukocyte: not stated Leukocyte: not stated Platelet: 1 × 10 Platelet: Platelet: 2 × 10 Platelet: Platelet: 10 × Platelet: Leukocyte/platelet concentration Platelet: 5 × 10 Platelet: PR PR PR Not stated Not stated Not stated Not stated Type of PRP used P-PRP used to produce P-PRP used to produce P-PRP used to produce , , , , , et al. et al. et al. et al. et al. , 2003 , 2014 2016 2008 2004 2014 2009 (year) Author Author Lucarelli et al. et al. Agis Kosmacheva Kosmacheva Gruber Samuel Parsons Parsons Amable Table 4d. Preparation and processing of PPs. Table

www.ecmjournal.org 284 J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs 2 used 2 Not PRP 10 % used CaCL TPD™ reagent Activator Activator Thrombin to activate to activate applicable Not stated CaCl No yes Yes Yes PRP gel: Not stated Activation 1 mol/L 2 min ?? for g disposable tube containing TPD™ ® for min, 25 °C to form PRF matrix g Processing (steps) containing glass bottle (0.25 mL CacL 2 for 15 min at room temperature) g for 12 min, room temperature for 8 min for 15 min g g g for 6 min to obtain PRP centrifugation: 3,800 g × 10 min of resultant plasma and buffy g nd for 10 min blood with ACD-A, centrifuged in GPSIII (Biomet Inc separation blood with ACD-A in Clotalyst Blood with Laboratory-grade centrifuge Blood in glass-coated plastic tube 400 × 460 × Supernatant collected = P-PRP sample in 21 mL sodium citrate 150 mL venous 730 × 2 coat Supernatant collected = L-PRP g centrifugation: 4,500 × L-PRP: clinical-grade centrifuge L-PRP clot/gel formed by mixing: - - L-PRF: - - - tube at 1,900 × Thrombin reagent; centrifuged at 1,900 × nd min to allow for 3-layer separation Laboratory-grade centrifuge for 12 min to allow 3-layer (PRF clot in middle layer) with pliers PRF clot harvested Soft compression of PRF clot with sterile gauze into a membrane week PRF membranes remained in culture plate for first Laboratory-grade centrifuge P-PRP: - - L-PRP: - - - - to produce PG Both P-PRP and L-PRP then undergo activation Clinical grade centrifuge (FIBRINET®, Cascade Medical Enterprises, USA) Wayne, 1,100 × PRP transferred to CaCl and gently swirled) 2 Laboratory-grade centrifuge 400 × Compression of fibrin clot • • • PRF • • • • • • • • • • • • • • • PG-CM process L-PRP Sodium citrate Not applicable Not applicable Not applicable ACD-A used to at blood collection Anticoagulant used 3 ‡: same study cohort and methodology. /μL (L-PRP) 3 /μL (P-PRP) 3 Platelet: Platelet: Leukocyte: platelets/μL (P-PRP) platelets/μL (L-PRP) 3 3 0 × 10 Platelet: not stated Platelet: not stated Platelet: not stated Platelet: 2,600 ± 547.72/mm 5.7 × 10 Leukocyte: not stated Leukocyte: not stated Leukocyte: not stated Leukocyte: not stated 194 × 10 912 × 10 Leukocyte/platelet concentration Platelet count (on residual serum): Platelet PG L-PRP gel Not applicable Not applicable Not applicable Both P-PRP and Type of PRP used L-PRP used to produce L-PRP used to produce , , et al. et al. , 2017 , 2010 , 2010 PRF 2015 2013 (year) Dohan Author Author Lucarelli Assessed Ehrenfest Moradian et al. et al. et al. Perut Perut Schar PRP-gel and Table 4e. Preparation and processing of PPs. Table

285 www.ecmjournal.org J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs Not stated Not applicable (enzymatic or mechanical) (trypsin-EDTA, Invitrogen) (trypsin-EDTA, Enzymatic by trypsinisation Enzymatic by trypsinisation Enzymatic by trypsinisation Enzymatic by trypsinisation Enzymatic by trypsinisation Enzymatic by trypsinisation Enzymatic by TrypLE™ Select Method of cellular detachmentMethod + 10 % FBS medium (Lonza) 50 mmol/L + 4 μg/L FGF2 equivalent at medium change) equivalent Product ATMP-Ready) (DMEM (changed to serum-free medium acids solution + 2-mercaptoethanol 25 mmol/L HG-DMEM + penicillin/ DMEM Advanced Therapy Medical Advanced DMEM α-MEM + 10 % FBS 1 antibiotics + 100 IU/mL penicillin mg/mL Coon’s-modified Ham’s F-12 medium Coon’s-modified Ham’s F-12 medium DMEM GlutaMAX™ + 1 % antibiotics streptomycin 100 mg/L + L-glutamine α-MEM + 2 mmol/L L-glutamine 1 % streptomycin + 2 mmol/L L- glutamine Standard Mesenchymal Stem Cell Basal MSC expansion media (Miltenyi Biotec) 200 mmol/L + MEM non-essential amino mL streptomycin + 2 mmol/L L-glutamine + 10 %FBS 100 IU/mL penicillin μg/ penicillin-streptomycin- solution Tissue culture medium used for expansion (penicillin G and streptomycin) + 10 % FBS passages Passage 5 Passage 1 Passage passage 4 Passage 4-5 Passage all 7 passages Not applicable passages 1 to 4 Passage number Passage expression: passage 3 Differentiation assays: Proliferation: total of 6 Proliferation: passage 0 Osteogenesis: passage 1 Differentiation and gene Proliferation: throughout Proliferation: throughout Differentiation: passage 2 Differentiation: passage 7 PL PRP-CM Yes Yes Yes Yes Yes Yes Yes Yes used in laboratory assays cells rich in BM-MSCs No fresh mononuclear Subcultured cells used ‡: same study cohort and methodology. medium medium Not stated Not stated Not stated Not stated Not stated Healthcare) Ficoll-Premium Ficoll-Paque™ Plus (GE Plus Ficoll-Paque™ % methyl violet in (with 0.1 % methyl Centrifugation condition(s) PBS; nucleated cells counted PBS; nucleated cells counted % methyl violet in (using 0.1 % methyl 0.1 mol/L citric acid as nuclear 0.1 mol/L citric acid as nuclear Density gradient centrifugation: stain) before culturing in culture stain) before culturing in culture Bone marrow washed twice with Bone marrow washed twice with Bone marrow washed used Not stated Not stated Not stated Laboratory Laboratory Laboratory centrifugation centrifugation centrifugation pellet, manual pellet, manual pellet, manual prior to seeding prior to seeding prior to seeding mononuclear cells mononuclear cells mononuclear cells centrifugation into centrifugation into centrifugation into Laboratory density- Laboratory density- Laboratory density- counting of nucleated counting of nucleated counting of nucleated Centrifugation system Centrifugation system , , , , , , , et al. , 2013 et al. et al. , 2016 et al. et et al. et al. et al. 2020 2019 2018 2017 2015 2014 2013 et al. et al. Author (year) Author Yin Karadjian Moisley Skific Infante Muraglia Muraglia Jonsdottir- Buch Bernardi Table 5a. Preparation and processing of BM-MSCs. Table

www.ecmjournal.org 286 J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs solution] Not stated Not stated Not stated from corn) [0.05 % (vol/vol) (0.25 %trypsin/EDTA) (0.05 % trypsin/EDTA) Enzymatic by TrypZean (trypsin/EDTA solution) (trypsin/EDTA mmol/L EDTA trypsin/1 mmol/L EDTA (enzymatic or mechanical) Enzymatic by trypsinisation Enzymatic by trypsinisation Enzymatic by trypsinisation Enzymatic by trypsinisation Enzymatic by trypsinisation Enzymatic by trypsinisation Enzymatic by trypsinisation (recombinant enzyme derived (recombinant enzyme derived Method of cellular detachmentMethod mol/L −4 nonessential + ng/mL basic-FGF at

FBS day 5 ± PRP penicillin/streptomycin penicillin/streptomycin penicillin streptomycin + 10 % pre ‐ selected FBS + 0.025 mg/mL fungizone mol/L dexamethasone + 10 + 0.025 mg/mL amphotericin B U/mL); followed by addition of fresh followed U/mL); −9

ascorbic acid 2-phosphate + 100 U + linoleic acid bovine serum mL streptomycin + 2 µ m L-glutamine LG-DMEM + 10 %FBS or PRP for 48 h amino-acids + Glutamax and LG- DMEM or DMEM-KO + 2 mmol/L penicillin + 1,000 U streptomycin 2 % αMEM + 100 U/mL penicillin 0.1 mg/ αMEM + 100 U/mL penicillin 0.1 mg/ h; followed by serum-free media for 24 h; followed + 100 μg/mL streptomycin IU/mL α-MEM + 10 % FBS with 1 antibiotics 60 % LG-DMEM, 40 MCDB-201 + ITS α-MEM + 1 % v/v antibiotic-antimycotic + 10 LG-DMEM + 1 % penicillin/streptomycin DMEM: nutrient mixture F-12 + 10 % FBS (100 medium containing 5 mL streptomycin + 2 mmol/L L-glutamine αMEM + 100 U/mL penicillin μg/mL IMDM containing 10 % FBS Tissue culture medium used for expansion 3 to 5 specified Passage 1 Passage 4 Passage 4 Passage applicable applicable Not stated Passage 3-6 Passage Passage 2 to 3 Passage (cryopreserved) Passage number Passage Passage 2-4 used Passage Proliferation: not Proliferation: not Other assays: passage 2 Other assays: passage 2 Proliferation: passages 1 Other assays: not clearly Differentiation: not stated Proliferation: until passage PL PR Yes Yes Yes Yes Yes Yes Yes Yes clearly specified Proliferation: no Proliferation: no Proliferation: yes Other assays: yes Other assays: yes Other assays: not in laboratory assays Subcultured cells used

g 3 ‡: same study cohort and methodology. for 30 min for 20 min, g g Not stated Not stated Biosciences) centrifugation Ficoll 1.077 g/cm Not clearly stated room temperature at room temperature ‐ gradient density Percoll Ficoll Hypaque solution Ficoll (Histopaque-1077), purchased BM-MSCs used Centrifugation condition(s) g/mL) gradient (density 1.068 g/mL) Not applicable; commercially for 20 min at room temperature Ficoll-Paque™ Plus (Amersham Plus Ficoll-Paque™ Density gradient centrifugation: centrifuged at 800 × centrifugation: 900 × Biocoll solution density gradient Ficoll Hypaque solution at 800 × used used specified Not stated commercially centrifugation centrifugation centrifugation centrifugation centrifugation centrifugation centrifugation Not applicable; Yes, but type not Yes, Not clearly stated Laboratory density- Laboratory density- Laboratory density- Laboratory density- Laboratory density- Laboratory density- Laboratory density- purchased BM-MSCs Centrifugation system Centrifugation system , , , , , , , , ‡ et ‡ et al. et al. et al. et al. et al. et al. et al. et al. , 2012 , 2012 , 2011 2012 2012 2010 2009 2006 2019 2018 2011 al. et al. Liou Prins Vogel Lange et al. Goedecke Ben Azouna Verrier Gottipamula Jenhani Nguyen Author (year) Author Murphy Table 5b. Preparation and processing of BM-MSCs. Table

287 www.ecmjournal.org J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs Not stated Not stated Not stated Not applicable (0.25 % trypsin) TrypLE™ Express (0.25 % trypsin/EDTA) (enzymatic or mechanical) Enzymatic by trypsinisation Enzymatic by trypsinisation Enzymatic by trypsinisation Enzymatic by trypsinisation Enzymatic by trypsinisation Enzymatic by trypsinisation Enzymatic by trypsinisation Method of cellular detachmentMethod media’ + 10 % FBS ciprofloxacin + 1 % Glutamax-1 α-MEM + 20 % FBS mmol/L dexamethasone sulphate + 20 % FBS -8 + 2 mmol/L glutamine + streptomycin (100 μg/mL) + ultraglutamine (2 mmol/L) acid-2 phosphate + 10 % FBS ± 10 mmol/L L-ascorbic acid phosphate α-MEM + 10 % FBS μg/mL μg/mL) + streptomycin (100 μg/mL) mL) -4 α-MEM + 20 % FBS 100 units/mL mg/L) + 10 % FBS LG-DMEM (1,000 mg/L) DMEM/F-12 + 10 % FBS 100 U/mL Not clearly specified ‘recommended penicillin + 100 mg/mL streptomycin penicillin + 100 mg/mL streptomycin α-MEM + 10 % FBS penicillin (100 U/ L-DMEM supplemented with 10 % FBS α-MEM + 100 U/mL penicillin 0.1 mg/ HG-DMEM + 1 % antibiotics (penicillin- mL streptomycin + 100 mmol/L ascorbic mL penicillin + 100 μg/mL streptomycin + 10 α-MEM, 2 mmol/L L-glutamine + 100 U/ mmol/L) streptomycin) + glutamine (200 mmol/L) U/mL) + 1 % penicillin/streptomycin (100 U/mL) magnesium salt n-hydrate when indicated U/mL) α-MEM + 10 % FBS penicillin (100 U/mL) Tissue culture medium used for expansion not

stated Passage 2 Passage 2 Passage 3 Passage 3 Passage 3 Passage passage 6 Not stated Not applicable Passage number Passage Not clearly specified Differentiation assay: Proliferation assay: Osteogenesis: passage 2 Between passages 2 and 5 Between Not more than 10 passages PR PRF PRC PG-CM Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No; fresh mononuclear cells in laboratory assays rich in BM-MSCs used Subcultured cells used

g

g ), g g 3 ‡: same study cohort and methodology. for 25 min g for 15 min g for 15 min for 10 min for 25 min for 30 min Not stated Not clearly stated Not clearly stated Not clearly stated at 338 × Not applicable; BM-MSC purchased from cell bank purchased BM-MSCs used Ficoll-Paque PLUS density Ficoll-Paque Centrifugation condition(s) Not applicable; commercially Histopaque® (Sigma) Density gradient centrifugation: 700 × gradient centrifugation: 600 × Ficoll-verograffin (1.077 g/cm Ficoll-verograffin centrifuged at 450 × Cells were resuspended in 5 mL Cells were of α-MEM, centrifuged at 135 × Ficoll (lymphoprep), centrifuged Ficoll-Paque Premium of density Ficoll-Paque 1.073 g/mL, centrifuged at 360 × used used sorting cell bank commercially centrifugation centrifugation centrifugation centrifugation centrifugation centrifugation centrifugation centrifugation Not applicable; Not clearly stated centrifugation and Laboratory density- Laboratory density- Laboratory density- Laboratory density- Laboratory density- Laboratory density- Laboratory density- Laboratory density- Laboratory density- Not applicable; BM- purchased BM-MSCs MSCs purchased from magnetic-activated cell magnetic-activated Centrifugation system Centrifugation system , , , , , , , , , et et al. et al. et al. et al. et al. et al. et al. et al. et al. , 2014 , 2017 , 2010 2014 2009 2004 2003 2016 2008 2015 2013 2010 Dohan al. Moradian et al. et al. Agis Perut Schar Ehrenfest Kosmacheva Gruber Samuel Parsons Amable Author (year) Author Lucarelli Lucarelli Table 5c. Preparation and processing of BM-MSCs. Table

www.ecmjournal.org 288 J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs Not stated Not stated Not stated Not applicable (0.25 % trypsin) TrypLE™ Express (0.25 % trypsin/EDTA) (enzymatic or mechanical) Enzymatic by trypsinisation Enzymatic by trypsinisation Enzymatic by trypsinisation Enzymatic by trypsinisation Enzymatic by trypsinisation Enzymatic by trypsinisation Enzymatic by trypsinisation Method of cellular detachmentMethod d d d d h h d d d h d d d d d d d d d d d d d d d d d d h h h h ; cell counting for PD: not stated media’ d in PRP-CM + 10 % FBS d ciprofloxacin Osteogenesis: 14 Adipogenesis: 21 Migration: 24 Migration: 24 Migration: 24 Migration: 18 14 + 1 % Glutamax-1 Proliferation: 7 Proliferation: 5 Proliferation: 14 Proliferation: 10 Proliferation: 10 α-MEM + 20 % FBS Osteogenesis: 21 Osteogenesis: 14 mmol/L dexamethasone Adipogenesis: 14 Adipogenesis: 14 Adipogenesis: 14 sulphate + 20 % FBS Differentiation: 14 Differentiation: 21 Differentiation: 28 -8 + 2 mmol/L glutamine Chondrogenesis: 28 Chondrogenesis: 21 Gene expression: 14 Proliferation: 4 and 7 Proliferation: not stated Proliferation: up to 96 Protein expression: 14 Osteogenesis: 7 and 14 Immunomodulation: 48 Immunomodulation: 72 Protein expression: day 14 + streptomycin (100 μg/mL) + ultraglutamine (2 mmol/L) acid-2 phosphate + 10 % FBS Cell viability/apoptosis: 7 ± 10 mmol/L L-ascorbic acid phosphate α-MEM + 10 % FBS μg/mL μg/mL) + streptomycin (100 μg/mL) mL) -4 Cytokine expression: day 3 or 4 Cytokine expression: day 3 or 4 α-MEM + 20 % FBS 100 units/mL mg/L) + 10 % FBS LG-DMEM (1,000 mg/L) DMEM/F-12 + 10 % FBS 100 U/mL Not clearly specified ‘recommended penicillin + 100 mg/mL streptomycin penicillin + 100 mg/mL streptomycin Proliferation: throughout 7 passages α-MEM + 10 % FBS penicillin (100 U/ L-DMEM supplemented with 10 % FBS α-MEM + 100 U/mL penicillin 0.1 mg/ HG-DMEM + 1 % antibiotics (penicillin- mL streptomycin + 100 mmol/L ascorbic mL penicillin + 100 μg/mL streptomycin + 10 α-MEM, 2 mmol/L L-glutamine + 100 U/ mmol/L) streptomycin) + glutamine (200 mmol/L) U/mL) + 1 % penicillin/streptomycin (100 U/mL) Incubation period with platelet product magnesium salt n-hydrate when indicated U/mL) α-MEM + 10 % FBS penicillin (100 U/mL) Tissue culture medium used for expansion Gene/cytokine/growth factor expression: 14 Osteogenesis/adipogenesis/chondrogenesis: 21 Proliferation (MTT and clonogenic assays): 10-14 Proliferation: CFU-F 14 not

Gene expression: day 0, 14 or 21, dependant on type of differentiation stated Passage 2 Passage 2 Passage 3 Passage 3 Passage 3 Passage passage 6 Not stated Not applicable Passage number Passage Not clearly specified Differentiation assay: Proliferation assay: Osteogenesis: passage 2 Between passages 2 and 5 Between Not more than 10 passages PR PL PRF PRC PG-CM PRP-CM Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No; fresh Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes product mononuclear cells in laboratory assays Not stated Not stated rich in BM-MSCs used Subcultured cells used

g

Pooling of platelet g ), g g 3 for 25 min g for 15 min g No No No No No No No No No No No No No No for 15 min for 10 min for 25 min for 30 min Not stated same donor Not clearly stated Not clearly stated Not clearly stated at 338 × Not applicable; BM-MSC purchased from cell bank purchased BM-MSCs used Ficoll-Paque PLUS density Ficoll-Paque Centrifugation condition(s) PP and BM-MSCs from Not applicable; commercially Histopaque® (Sigma) Density gradient centrifugation: 700 × gradient centrifugation: 600 × Ficoll-verograffin (1.077 g/cm Ficoll-verograffin centrifuged at 450 × Cells were resuspended in 5 mL Cells were of α-MEM, centrifuged at 135 × Ficoll (lymphoprep), centrifuged Ficoll-Paque Premium of density Ficoll-Paque 1.073 g/mL, centrifuged at 360 × ) 3) 3) 4) 5) 4) 14) 10) 11) 13) 13) 13) n = = n n n = n = n = 10) n = n = n = n = n = n = -MSCs 11) 20) 28) 30) 10) 10)

≥ ≥ = n = n = n n = n = n n not stated) not stated) not stated) not stated) used used not stated) not stated) not stated) sorting cell bank n = n = n = n = PL ( PL ( PL ( PL ( PL ( PL ( PRP ( n = n = n = commercially ( ( ( centrifugation centrifugation centrifugation centrifugation centrifugation centrifugation centrifugation centrifugation Sample size ( Not applicable; BM-MSCs ( BM-MSCs ( BM-MSCs ( BM-MSCs ( BM-MSCs ( PL and BM-MSCs PL and BM-MSCs PL and BM BM-MSCs ( BM-MSCs ( BM-MSCs ( BM-MSCs ( BM-MSCs ( BM-MSCs ( PL ( PL ( PL ( PL ( Not clearly stated centrifugation and Laboratory density- Laboratory density- Laboratory density- Laboratory density- Laboratory density- Laboratory density- Laboratory density- Laboratory density- Laboratory density- Not applicable; BM- purchased BM-MSCs MSCs purchased from magnetic-activated cell magnetic-activated Centrifugation system Centrifugation system , , , , , , ,

, , , , , , , , , , et et al. et al. et al. et al. et al. et al. et al. , 2016 et al. et al. et al. et al. et al. et al. et al. et al. et al. , 2014 , 2017 et al. , 2020 , 2015 , 2014 , 2012 , 2012 , 2010 2014 2009 2004 2003 2016 2008 2015 2013 2010 2019 2018 2017 2013 2013 2012 2012 2011 Dohan al. et al. Moradian et al. et al. Agis Muraglia Muraglia Karadjian Perut Schar et al. et al. et al. et al. et al. Jonsdottir- Buch Ehrenfest Skific Lange Kosmacheva Gruber Samuel Ben Azouna Parsons Amable Infante Gottipamula Author (year) Author Jenhani Lucarelli Lucarelli Moisley Author (year) Author Murphy Yin Bernardi Table 6a. Incubation period of PPs with BM-MSCs. 6a. Incubation Table

289 www.ecmjournal.org J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs (45Ca2+ incorporation) d h d h d h h d d d d d d d d d d d d d d h d d d d d d d h h (ALP activity), 21 d Not stated Migration: 3 Proliferation: 6 Migration: 20 Proliferation: 9 Proliferation: 6 Osteogenesis: 5 Proliferation: 24 Proliferation: 14 Proliferation: 10 Proliferation: 14 Proliferation: 14 Osteogenesis: 10 Osteogenesis: 21 Osteogenesis: 21 Adipogenesis: 21 Adipogenesis: 21 Differentiation: 14 Gene expression: 5 Chondrogenesis: 24 Chondrogenesis: 21 Chondrogenesis: 28 Differentiation: 14 -21 Gene expression: 21 Gene expression: 21 BMP2 expression: 23 Migration: 0, 24 and 48 Casein zymography: 24 Western blot analysis: 24 Western Differentiation: 3, 6 and 9 Fibrin dissolution assay: 25.5 Immunomodulation: not stated Chemotaxis: until culture confluence Incubation period with platelet product qPCR-uPA, PAI-1 and UPAr levels: not stated levels: and UPAr PAI-1 qPCR-uPA, (von Kossa ), 18 d Osteogenesis: 14 PL PR No Yes Yes Yes Yes Yes Yes Yes product Not stated Not stated Not stated Not stated Pooling of platelet No No No No No No No No No No No No same donor PP and BM-MSCs from 6) 16) ) n = 5) 9) 3) 3) 4) 4) n = 10) 6) n n = n = n = n = n = n = 3) 4) 2) 5) 5) n = 15) 18) 10) n = 15) n = n = n = n = n = n = n = n = n = not stated) not stated) PL ( PL ( PL ( PR ( PR ( PL ( PR ( PR ( PR ( n = n = ( ( M-MSCs ( Sample size ( BM-MSCs ( BM-MSCs ( BM-MSCs ( BM-MSCs ( BM-MSCs ( BM-MSCs ( PR and BM-MSCs PR and BM-MSCs BM-MSCs ( PR and BM-MSCs ( PL and BM-MSCs ( , ,

, , , , , , , , et al. et al. et al. et al. et al. et al. et al. et al. et al. et al. , 2011 , 2014 2010 2009 2006 2019 2018 2017 2014 2009 2004 2003 and PL Goedecke et al. et al. Agis Liou Prins Vogel Verrier Assessed PR Kosmacheva Gruber Amable Nguyen Author (year) Author Bernardi Lucarelli Table 6b. Incubation period of PPs with BM-MSCs. Table

www.ecmjournal.org 290 J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs h ested at different time points h h d d d d d d h 1, 2 and 3 d , h onwards mineralisation: 23 Migration: 4 Proliferation: 6 Proliferation: 12 Proliferation: 28 Osteogenesis: 28 Differentiation: 14 In vitro Proliferation: 24, 48 and 72 Proliferation: 0 Osteogenesis: day 8, 16, and 24 Proliferation: day 0, 8, 16 and 24 Gene expression: day 0, 8, 16 and 24 Gene expression: 0, 6, 12, 24, and 48 Incubation period with platelet product ) were used for incubation with BM-MSCs in migration chambers 4 ) were d , 1, 3, 7, 14, and 28 h (8 Supernatants of all 3 platelet products (L-PRP gel, L-PRF, blood clot) harv PRF Note: PRF membranes were removed at from culture plates day 7; authors used PRF-CM 7 removed Note: PRF membranes were PRC PG-CM Yes Yes Yes donor) product Not stated Not stated Not stated Pooling of platelet Not applicable (single No No No No No No Yes same donor PP and BM-MSCs from ) 4) 3) 3) n 3) 6) 11) n = n = n = 10) n = not stated) not stated) 1) 11) n = n = n = n = n = n = n = ( ( Not stated PRC ( PRP ( PRC ( PRF-CM ( Sample size ( BM-MSCs ( BM-MSCs ( BM-MSCs ( PRP/PRF/blood clot PRF-CM and BM-MSCs BM-MSCs ( BM-MSCs ( , - , , , , et al. , 2010 et al. et al. et al. et al. , 2017 2016 2008 2015 2013 2010 et al. Moradian et al. Perut Schar Samuel Parsons Author (year) Author Dohan Ehren fest Lucarelli Table 6c. Incubation period of PPs with BM-MSCs. Table

291 www.ecmjournal.org J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs

MSCs incubated in PRP-CM (n = 1), PL (n = 6) and This systematic review has identified 33 in vitro PRC (n = 2). studies in humans that compared the combined PP was found to upregulate the expression use of PP and BM-MSCs to BM-MSCs alone. It was of BM-MSC gene markers for pluripotency and evident that there is a lack of consistent terminology proliferation (POU5F1, SOX2) (Amable et al., 2014); amongst these studies. For instance, several studies immunomodulation/inflammation COX-2( , iNOS) used the term PRP, which upon closer look at their (Yin et al., 2016) and matrix metalloproteinases methodology, were in fact using frozen-thawed PRP (MMP13) (Verrier et al., 2010). (Goedecke et al., 2011; Infante et al., 2017; Muraglia The effect of PP on expression of osteogenic gene et al., 2014; Murphy et al., 2012; Vogel et al., 2006). markers was variable, dependent upon the gene The freeze-thawing process of the PRP in these marker measured and choice of PP used. Osteogenic studies would have resulted in lysis of platelets. genes such as BMP2 (Amable et al., 2014; Samuel et al., Thus, PL would have been a more appropriate term 2016; Verrier et al., 2010), osteocalcin (Gottipamula et to accurately reflect the acellularity and contents of al., 2012; Yin et al., 2016), osteopontin (Jonsdottir-Buch the PP used in these studies. Therefore, the present et al., 2013; Kosmacheva et al., 2014; Samuel et al., study attempted to clearly categorise these studies 2016), bone sialoprotein 2 (Samuel et al., 2016; Verrier into PRP-CM, PL, PR, PRC, PG-CM and PRF to reflect et al., 2010), osterix (Gottipamula et al., 2012) and (i) the specific type of PP used, (ii) its consistency i.e.( COL1 (Samuel et al., 2016) were upregulated in most liquid, solid) and (iii) its contents (e.g. cellularity, studies. On the other hand, PP exposure resulted in lysed platelets, leukocyte). Hopefully, this would aid equivocal expression of ALP (Jonsdottir-Buch et al., comparison between the important findings of these 2013; Parsons et al., 2008; Samuel et al., 2016; Verrier in vitro studies with future laboratory work and aid et al., 2010) and RUNX2 (Amable et al., 2014; Gruber in its transferability into clinical work. et al., 2004; Jonsdottir-Buch et al., 2013; Parsons et al., The majority of these studies had a small 2008; Samuel et al., 2016; Yin et al., 2016) as well as sample size (n ≤ 6) (Agis et al., 2009; Amable et al., downregulation of SPARC (Amable et al., 2014) and 2014; Bernardi et al., 2017; Bernardi et al., 2013; osteonectin (Samuel et al., 2016). Dohan Ehrenfest et al., 2010; Goedecke et al., 2011; Effects of PP on expression of adipogenic gene Gottipamula et al., 2012; Gruber et al., 2004; Infante markers of BM-MSCs were variable: ADIPOQ et al., 2017; Liou et al., 2018; Lucarelli et al., 2010; (Amable et al., 2014), CEPBA (Amable et al., 2014) and Parsons et al., 2008; Perut et al., 2013; Prins et al., 2009; PPARG (Amable et al., 2014) were upregulated; whilst Vogel et al., 2006). Key aspects such as the health L-PGDS (Lange et al., 2012), LPL (Ben Azouna et al., and demographics of patients/volunteers, harvest 2012), FABP4 (Lange et al., 2012), PLIN (Lange et al., volume of BM-MSCs and PPs as well as the leukocyte 2012), GLUT4 (Lange et al., 2012) and APOE (Lange and platelet concentration in PP were not reported et al., 2012) were downregulated when compared to by most studies, which would have otherwise controls. Similarly, expression of chondrogenic gene substantiated the correct interpretation of the results markers were variable: COMP (Infante et al., 2017) reported. Harvested samples were processed using and COL1A1 (Verrier et al., 2010) were upregulated; different centrifugation systems, mostly being COL2A1 downregulated (Infante et al., 2017); ACAN laboratory grade (Agis et al., 2009; Amable et al., (Amable et al., 2014; Infante et al., 2017; Liou et al., 2014; Ben Azouna et al., 2012; Bernardi et al., 2017; 2018) and SOX9 (Amable et al., 2014; Infante et al., Dohan Ehrenfest et al., 2010; Goedecke et al., 2011; 2017) equivocal. No effect on COL2 was observed Gottipamula et al., 2012; Gruber et al., 2004; Infante (Liou et al., 2018). et al., 2017; Jenhani et al., 2011; Jonsdottir-Buch et al., 2013; Kosmacheva et al., 2014; Lange et al., 2012; Liou et al., 2018; Muraglia et al., 2014; Muraglia et al., 2015; Discussion Murphy et al., 2012; Perut et al., 2013; Prins et al., 2009; Samuel et al., 2016; Schar et al., 2015; Skific et al., 2018; PPs and BM-MSCs are increasingly used in the Verrier et al., 2010; Vogel et al., 2006). Comparisons current treatment of impaired bone healing. However, between PL produced using a laboratory and clinical the clinical effectiveness of their combined use is grade centrifugation system on proliferation and difficult to conclude, due to differing clinical results migration of BM-MSCs have been attempted (Moisley and the huge heterogeneity in terms of harvesting, et al., 2019), but not yet on the other categories of PPs. preparation techniques, contents and type of PPs used Furthermore, the practice of pooling harvested PPs in these studies. Pre-clinical studies using animal was common practice within the majority of in vitro models have their limitations, since animal cells do studies analysed (Amable et al., 2014; Ben Azouna not always resemble human results. Systematically et al., 2012; Bernardi et al., 2017; Bernardi et al., 2013; assessing the results of in vitro studies in humans Goedecke et al., 2011; Gottipamula et al., 2012; Infante should provide the best collective evidence which can et al., 2017; Jenhani et al., 2011; Jonsdottir-Buch et al., guide clinical practice. This review represents the first 2013; Karadjian et al., 2020; Kosmacheva et al., 2014; systematic review in the literature that specifically Lange et al., 2012; Liou et al., 2018; Lucarelli et al., 2010; assessed the effects of PPs on the biophysiological Moisley et al., 2019; Muraglia et al., 2014; Muraglia et functions of BM-MSCs in human. al., 2015; Parsons et al., 2008; Prins et al., 2009; Samuel

www.ecmjournal.org 292 J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs et al., 2016; Skific et al., 2018; Vogel et al., 2006). This is Expansion of harvested BM-MSCs and the not reflective of clinical practice, where patients are subsequent use of subcultured (or passaged) BM- commonly treated with autologous PP and BM-MSCs MSCs were common in most studies (Agis et al., 2009; (i.e. donor-matched) processed using a clinical-grade Amable et al., 2014; Bernardi et al., 2017; Bernardi automated centrifugation system (Lee et al., 2014; et al., 2013; Dohan Ehrenfest et al., 2010; Goedecke Martin et al., 2013). et al., 2011; Gottipamula et al., 2012; Gruber et al., Being a biological reservoir containing a panel of 2004; Infante et al., 2017; Jonsdottir-Buch et al., 2013; potent biological factors, it is unsurprising that most Kosmacheva et al., 2014; Lange et al., 2012; Liou et in vitro studies have been focused upon harnessing al., 2018; Lucarelli et al., 2003; Lucarelli et al., 2010; these platelet secretomes (e.g. cytokines, growth Moisley et al., 2019; Moradian et al., 2017; Muraglia factors, ) either through physical lysis et al., 2015; Murphy et al., 2012; Nguyen et al., 2019; (e.g. freeze-thawing) or through exogenous chemical Parsons et al., 2008; Prins et al., 2009; Samuel et al., activation (e.g. CaCl2, thrombin). Noteworthy, these 2016; Schar et al., 2015; Skific et al., 2018; Verrier et al., methods in essence lead to the immediate secretion 2010; Vogel et al., 2006; Yin et al., 2016), since larger of the potent platelet secretomes upon delivery quantities of BM-MSCs were desired for laboratory (Marx, 2001; 2004). Therefore, it is unsurprising that assays. However, studies have demonstrated the most in vitro studies utilised PL (Agis et al., 2009; Ben ‘internalisation’ of bovine proteins in BM-MSCs Azouna et al., 2012; Bernardi et al., 2017; Bernardi et al., expanded in culture medium containing FBS, a 2013; Goedecke et al., 2011; Gottipamula et al., 2012; commonly used animal serum (Spees et al., 2004). Gruber et al., 2004; Infante et al., 2017; Jenhani et al., The stemness of passaged BM-MSCs and, therefore, 2011; Jonsdottir-Buch et al., 2013; Kosmacheva et al., how closely it reflects fresh BM-MSCs used in clinical 2014; Lange et al., 2012; Moisley et al., 2019; Muraglia practice is questionable. Furthermore, in vitro passage et al., 2014; Muraglia et al., 2015; Murphy et al., 2012; of BM-MSCs could also result in the loss of homing Prins et al., 2009; Skific et al., 2018; Vogel et al., 2006), (Rombouts and Ploemacher, 2003), migration (Bustos PR (Agis et al., 2009; Amable et al., 2014; Bernardi et et al., 2014; Choumerianou et al., 2010), proliferation al., 2017; Gruber et al., 2004; Kosmacheva et al., 2014; (Ganguly et al., 2017) and differentiation (Kim et al., Liou et al., 2018; Lucarelli et al., 2003; Nguyen et al., 2012) capabilities of BM-MSCs. The use of fresh or 2019) and PG-CM (Perut et al., 2013; Schar et al., minimally cultivated BM-MSCs would have been 2015), all of which were induced to release platelet more representative of clinical practice. secretomes through its manufacturing process. Incubation time with PP reflects the duration Notably, a significantly larger number of studies which BM-MSCs were exposed to the inductive used PL, traditionally regarded as a stable, xeno-free molecules contained in PP, such as growth factors product, with less homogeneity when compared to and cytokines (Di Matteo et al., 2015; Roffi et al., 2017). FBS and vastly used in the mass ex vivo expansion BM-MSCs in most of the studies were exposed to a of BM-MSCs for clinical applications (Schallmoser continuous dose of PP through prolonged periods et al., 2020). However, a recent study by Scherer et of co-incubation. Differently, in clinical practice, al. (2012) comparing activated and non-activated autologous PPs and BM-MSCs are commonly platelets demonstrated better tissue healing and harvested on the day of surgery and incubated for a angiogenesis in the non-activated platelet group. The short duration prior to implantation (Lee et al., 2014; authors suggested that natural activation occurring Martin et al., 2013). In vitro studies reflecting the short intrinsically ‘on demand’ may be more effective than incubation periods seen in clinical settings would a bolus of exogenously activated platelets (Scherer et have been more informative, as it will elucidate al., 2012). As yet, no in vitro studies have compared whether short PP exposure is effective at optimising the effects of activated and non-activated PPs on the potent biophysiological functions of BM-MSCs the biophysiological functions of human BM-MSCs, necessary for advantageous bone repair. which could guide clinical practice on whether these Despite the inherent weaknesses limiting PPs should be activated ex vivo prior to delivery. transferability into clinical practice, these in vitro Another important aspect to consider is whether studies in humans have nonetheless demonstrated leukocyte content in PP would influence the promising findings. Mostin vitro studies focused on biophysiological behaviour of BM-MSCs. P-PRP- the impact of PP on proliferation and differentiation CM was found to result in stronger osteogenic gene of BM-MSCs (Amable et al., 2014; Ben Azouna et expression in BM-MSCs (Yin et al., 2016). However, al., 2012; Bernardi et al., 2017; Bernardi et al., 2013; the effects of leukocyte content on proliferation, Dohan Ehrenfest et al., 2010; Goedecke et al., 2011; differentiation and migration remain unclear, due Gottipamula et al., 2012; Gruber et al., 2004; Infante to the conflicting findings from the small number of et al., 2017; Jenhani et al., 2011; Jonsdottir-Buch et al., studies available (Moisley et al., 2019; Perut et al., 2013; 2013; Karadjian et al., 2020; Kosmacheva et al., 2014; Yin et al., 2016). Furthermore, the effect of leukocyte Lange et al., 2012; Liou et al., 2018; Lucarelli et al., 2003; content on other functions of BM-MSCs, such as Lucarelli et al., 2010; Moisley et al., 2019; Moradian et cytokine expression and immunomodulation, were al., 2017; Muraglia et al., 2014; Muraglia et al., 2015; not assessed. Murphy et al., 2012; Nguyen et al., 2019; Parsons et al.,

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2008; Perut et al., 2013; Prins et al., 2009; Samuel et al., et al., 2019; Schar et al., 2015; Yin et al., 2016). The 2016; Skific et al., 2018; Verrier et al., 2010; Vogel et al., effects of PP concentration were compared up to 10 % 2006; Yin et al., 2016). The huge focus thrown upon only, whereby increasing PP concentrations enhances these two biophysiological functions were perhaps the migratory capacity of BM-MSCs (Goedecke et al., unsurprising, given that the successful proliferation 2011; Gruber et al., 2004; Murphy et al., 2012; Nguyen of BM-MSCs and its subsequent differentiation et al., 2019). into the desired mesenchymal lineage are key Comminuted, segmental and open fractures determinants for successful bone repair. Overall, represent injuries with a higher risk of impaired bone PP has been shown to increase cellular proliferation healing. This is because the zone of injury in these (Amable et al., 2014; Ben Azouna et al., 2012; Bernardi high-risk fractures are subjected to immense amounts et al., 2013; Dohan Ehrenfest et al., 2010; Goedecke et of inflammatory, oxidative and apoptotic stressors al., 2011; Gottipamula et al., 2012; Gruber et al., 2004; (Wang et al., 2017). Despite the paucity of research on Infante et al., 2017; Jenhani et al., 2011; Jonsdottir- immunomodulation and response towards apoptotic Buch et al., 2013; Karadjian et al., 2020; Lucarelli et stress, findings from Yinet al. (2016) were encouraging. al., 2003; Moradian et al., 2017; Muraglia et al., 2014; Yin et al. (2016) demonstrated PRP-CM to (i) have a Muraglia et al., 2015; Murphy et al., 2012; Nguyen et protective effect and preserved the viability of BM- al., 2019; Perut et al., 2013; Prins et al., 2009; Samuel MSCs in the face of apoptotic stress and (ii) improve et al., 2016; Skific et al., 2018; Yin et al., 2016) and immunomodulatory functions in BM-MSCs. Despite osteogenic differentiation of BM-MSCs in the majority the use of subcultured BM-MSCs and PPs that were of these studies (Amable et al., 2014; Dohan Ehrenfest not donor-matched, it has nonetheless uncovered et al., 2010; Gottipamula et al., 2012; Karadjian et al., important biophysiological functions of BM-MSCs 2020; Kosmacheva et al., 2014; Lucarelli et al., 2003; primed by PPs relevant to improving bone repair in Parsons et al., 2008; Perut et al., 2013; Samuel et al., high-risk fractures. Therefore, further research using 2016; Skific et al., 2018; Verrier et al., 2010; Yin et fresh donor-matched BM-MSCs and PP is warranted al., 2016). The use of passaged BM-MSCs, known given it bears closer resemblance to clinical practice. to affect differentiation potential (Kim et al., 2012), An important question to ask when applying could explain these mixed findings in the remaining these biologics during surgery is whether the priming studies [no difference n( = 3) (Ben Azouna et al., 2012; effect of PPs on BM-MSCs observed inin vitro studies Goedecke et al., 2011; Prins et al., 2009); reduced would be lost following surgery, since it is subjected (n = 1) (Vogel et al., 2006); equivalent (n = 2) (Gruber to the homeostatic and dilutional effects of circulation et al., 2004; Jonsdottir-Buch et al., 2013)], therefore, once inside the . Albeit not assessed by warranting further research using fresh or minimally many studies, results from Lucarelli et al. (2003) were cultured BM-MSCs. On the other hand, chondrogenic encouraging, where BM-MSCs were demonstrated to and adipogenic differentiation were not improved by retain their improved osteogenic and chondrogenic PPs in most studies (Amable et al., 2014; Ben Azouna capability despite PP withdrawal. This suggested et al., 2012; Goedecke et al., 2011; Gottipamula et al., that the priming effect of PPs is perhaps not transient 2012; Lange et al., 2012; Liou et al., 2018; Prins et al., and limited to the incubation period only, as could 2009; Vogel et al., 2006). Increasing PP concentrations be expected. up to 10 % have also been demonstrated to improve In the attempt to uncover the underlying cellular proliferation (Amable et al., 2014; Bernardi et al., and molecular mechanisms explaining the effects 2017; Bernardi et al., 2013; Goedecke et al., 2011; of PP on the bio-physiology of BM-MSCs, several Gottipamula et al., 2012; Jenhani et al., 2011; Karadjian studies have investigated the release of cytokines, et al., 2020; Lucarelli et al., 2003; Muraglia et al., 2014; growth factors and proteins. Following PP exposure, Muraglia et al., 2015; Murphy et al., 2012; Nguyen et BM-MSCs were found to upregulate their release al., 2019; Perut et al., 2013; Prins et al., 2009; Skific et al., of (i) pro-inflammatory cytokines (IL-2R, IL-6, IL-7, 2018; Yin et al., 2016). However, no studies have yet IL-8, IL-12, IL-15, PGE2, NO) (Amable et al., 2014; investigated the correlation between PP concentration Ben Azouna et al., 2012; Jenhani et al., 2011; Yin et and the differentiation potential of BM-MSCs. al., 2016); (ii) chemokines (eotaxin, IP-10, MIP-1 , The ability to migrate is a potent biophysiological MCP-1, RANTES, SDF-1α) (Amable et al., 2014; Ben function possessed by BM-MSCs, allowing it to be Azouna et al., 2012; Goedecke et al., 2011; Jenhani et al.β, signalled and recruited to areas requiring more 2011); (iii) pro-angiogenic factors (thrombospondin, osteogenic cells (Su et al., 2018). Hence, being able to PLGF, aFGF, VEGF-D) (Amable et al., 2014) and (iv) influence its migratory capacity is crucial, thereby osteogenic proteins (osteocalcin, RUNX2 and BMP2) improving the chances of successful bone repair. PP (Samuel et al., 2016; Verrier et al., 2010; Yin et al., 2016). represents such a promising chemotactic stimulus All of these are important for successful bone repair, since it possesses multiple osteoinductive molecules since higher levels of pro-inflammatory cytokines (Di Matteo et al., 2015; Roffi et al., 2017; Schallmoser et advantageous for mediating the early inflammatory al., 2020). This was further confirmed by the improved phase of fracture healing, whilst inducing an migratory capacity in BM-MSCs exposed to PP angiogenic and chemotactic state in BM-MSCs, lead (Goedecke et al., 2011; Gruber et al., 2004; Infante et al., to improved vascularity and recruitment of non- 2017; Moisley et al., 2019; Murphy et al., 2012; Nguyen resident BM-MSCs, cytokines and growth factors to

www.ecmjournal.org 294 J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs acid L-glutamine Proliferation: Osteogenesis: DMEM GlutaMAX™ indomethacin, 5 μg/mL insulin Tissue culture medium (base)* Differentiation Medium (Lonza) Immunomodulation: RPMI 1640 medium Migration: DMEM GlutaMAX™ + 2 % FBS Proliferation assay: α-MEM + 1 % antibiotics Osteogenesis: OsteoDiff Media (Miltenyi Biotec) Adipogenesis: AdipoDiff Media (Miltenyi Biotec) Adipogenesis: Adipogenesis: StemPro adipogenic differentiation kit Migration assay: 250 μL serum-free medium (DMEM) Osteogenesis: StemPro osteogenesis differentiation kit Chondrogenesis: ChondroDiff Media (Miltenyi Biotec) Chondrogenesis: Chondrogenic differentiation medium acids solution, 50 mmol/L 2-bercaptoethanol, 4 μg/L FGF2 ascorbic acid-2-phosphate, 10 mmol/L β-glycerophosphate Osteogenesis: MSC Osteogenic Differentiation Media (Lonza) Proliferation/cells from 2D constructs used for gene expression: h, followed by DMEM Proliferation: MSC expansion medium for 24 h, followed *used as basal medium before addition of PP/other. Proliferation: DMEM/F12 media + 1 % penicillin/streptomycin, 4 IU/mL heparin α-MEM containing 10 % FBS and 1 antibiotics (penicillin G streptomycin) Proliferation: DMEM Advanced Therapy Medical Product (DMEM ATMP-Ready) Therapy Medical Product (DMEM Advanced Proliferation: DMEM PL Proliferation: α-MEM + 2 mmol/L L-glutamine, 1% penicillin-streptomycin-neomycin solution PRP-CM Adipogenesis: StemProH Adipogenesis Differentiation medium (Gibco) Chondrogenesis: MSC Chondrocyte Adipogenesis: StemProH mmol/L 3-isobutyl-1-mehylxanthine, 50 μmol/L Adipogenesis: basal medium + 1 μmol/L dexamethasone, 0.5 mmol/L 3-isobutyl-1-mehylxanthine, Serum-free Coon’s-modified Ham’s F-12 medium + 100 IU/mL penicillin, μg/mL, streptomycin, 2 mmol/L Coon’s-modified Ham’s F-12 medium + 100 IU/mL penicillin, mg/mL streptomycin, 2 mmol/L L-glutamine 25 mmol/L HG-DMEM + L-glutamine, 100mg/mL penicillin/streptomycin, 0.1 μmol/L dexamethasone, 2.5 mg/L 25 mmol/L HG-DMEM + 100 mg/L penicillin/streptomycin, 200 L-glutamine, MEM non-essential amino Osteogenesis: basal medium + 0.1 μmol/L dexamethasone, 10 mmol/L 2-phosphate, 0.2 L-ascorbic tissue 2 (6-well (6-well (24-well (12-well (12-well 2 2 2 2 2 cells/well (6-well (6-well cells/well 5 /dish (60 mmol/L (225 cm 5 2 (24-well plates) (24-well cells/cm 2 5 cells/cm 5 (96-well plate) (96-well 2 cells (15 mL culture tube) cells 5 4 to 6 × 10 5 Seeding density of BM-MSCs Proliferation assay: 500 cells/well (96-well plate) (96-well Proliferation assay: 500 cells/well Differentiation: not stated Migration: not stated Proliferation: 1 × 10 culture flasks) Osteogenesis and adipogenesis: 3,000 cells/cm plate) Proliferation: 4,000 cells/cm Migration: 12,000 cells/chamber [m-Slide Chemotaxis 3D chamber (Ibidi GmbH, Martinsried, Germany)] 2D cultures (gene expression): 2 × 10 plate) Proliferation (clonogenic assay): 1 × 10 dish) Petri plate) (24-well Proliferation (MTT assay): 5,000 cells/well Osteogenic differentiation: 3,000 cells/cm Chondrogenic differentiation: 250,000 cells/pellet Adipogenic differentiation: 10,000 cells/cm Immunomodulation: 25,000 MSC/cm Proliferation: not stated Osteogenesis and adipogenesis: 1,500 cells/cm plate) Chondrogenesis: 2.5 × 10 Proliferation assay: 4,000 cells/well (96-well plate) (96-well Proliferation assay: 4,000 cells/well Cell viability and apoptosis assay: 1 × 10 plate) Migration assay: 2.1 × 10 Differentiation and gene expression: not stated plate) (24-well Osteogenesis: 35,000 cells/well Proliferation assay: plate) (24-well 2,000 BM-MSC/well Migration assay: 50,000 BM-MSC/transwell Proliferation assay: 5,000 cells/well (24-well plate) (24-well Proliferation assay: 5,000 cells/well • • • • • • • • • • • • • • • • • • • • • • • • • • • , et al. , 2020 , 2015 , 2014 , 2013 , 2012 , 2019 , 2017 , 2018 , 2016 et al. et al. et al. et al. et al. et al. et al. 2013 et al. et al. Author (year) Author Yin *lyophilised PL Skific Infante Moisley Murphy Bernardi Muraglia Muraglia Karadjian Jonsdottir-Buch Table 7a. Seeding density of BM-MSCs and choice culture medium used for assays. Table

295 www.ecmjournal.org J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs

10 mmol/L ,

0.1 mmol/L ascorbic acid-2-phosphate , mol/L dexamethasone, 30 μg ascorbic acid/mL, 10 mmol/L -8 Osteogenesis: Adipogenesis: amphotericin B specified) × 14 d Chondrogenesis: 10 ng/mL TGF-β3 b-glycerophosphate β-glycerophosphate Migration: DMEM for 20 h Tissue culture medium (base)* L-glutamine, 0.025 mg/mL fungizone insulin/mL, 100 μmol/L indomethacin Chondrogenesis: NH Chondrodiff medium Osteogenesis: NH Osteodiff human medium Chemotaxis: DMEM until culture confluence isobutylmethylxanthine, 60 μmol/L indomethacin isobutylmethylxanthine, Proliferation: 14 d in medium (not clearly specified) b-glycerolphosphate, 100 μmol/L ascorbate-2-phosphate nonessential amino acids 2-phosphate, 350 μmol/L proline, 1× ITS, 10 ng/mL TGF-b1 Growth medium: LG-DMEM + 1 % penicillin/streptomycin , *used as basal medium before addition of PP/other. of addition before medium basal as *used Proliferation: LG-DMEM or DMEM-KO + 2 mmol/L, penicillin/streptomycin methyl xanthine, 100 units/mL penicillin, 100 μg/mL streptomycin ‐ 1 methyl isobutyl Chondrogenesis: Stempro (Invitrogen) chondrogenesis differentiation medium mmol/L isobutyl methyl xanthine, 0.2 mmol/L Indomethacin, 10 ng/mL insulin methyl 0.5 mmol/L isobutyl Proliferation: αMEM supplemented with 100 U/mL penicillin + μg/mL streptomycin PL IMDM + 10 % FBS Adipogenesis: LG-DMEM (with 1 g/L glucose) + 20 % FBS, μmol/L dexamethasone, 0.5 mmol/L Immunosuppression: RPMI (Rose-well Park Memorial Institute) medium ± phytohaemaggultinin Park Immunosuppression: RPMI (Rose-well Osteogenesis: HG-DMEM (with 4.5 g/L glucose) + 2 % FBS, 0.1 μmol/L dexamethasone mmol/L Proliferation and expansion media - αMEM + 100 U/mL penicillin 0.1 mg/mL streptomycin, 2 µ m HG-DMEM + 5 μg/mL insulin transferrin, selenous acid, 0.1 μmol/L dexamethasone, 500 µ L osteogenic induction medium containing HG-DMEM + 10 % FBS, 100 IE/mL penicillin, g/mL d culture in DMEM followed by ‘adipogenic medium or osteogenic medium’ (not clearly Differentiation: 4 d culture in DMEM followed Proliferation: αMEM + 100 U/mL penicillin, 0.1 mg/mL streptomycin, 2 mmol/L L-glutamine, 0.025 mmol/L sodium pyruvate, 0.35 mmol/L proline, 1.25 mg/mL BSA, 0.17 mmol/L ascorbic acid-2 ‐ phosphate, 1 sodium pyruvate, Osteogenesis: 10 % FBS + 2 mmol/L Glutamax, mmol/L isobutyl-methylxanthine, 100 μmol/L Adipogenesis: Growth medium + 1 μmol/L dexamethasone, 0.5 mmol/L isobutyl-methylxanthine, streptomycin, 0.1 µ M dexamethasone, 10 mmol/L β-glycerol-phosphate, 0.17 ascorbic acid-2-phosphate HG-DMEM + 10 % FBS, 0.01 mg/mL insulin, 1 μmol/L dexamethasone, 0.2 mmol/L indomethacin, 0.5 3 ‐ d, followed by 1 d of growth medium + insulin as maintenance indomethacin, 10 μmol/L insulin for 2-3 d, followed Adipogenesis: DMEM + 10 % FBS, 1 penicillin/streptomycin, 2 mmol/L L-glutamine, μmol/L dexamethasone, mmol/L sodium pyruvate + 170 μmol/L ascorbic acid- Chondrogenesis: HG-DMEM + 0.1 μmol/L dexamethasone, 1 mmol/L sodium pyruvate mmol/L isobutylmethylxanthine, 1 μg Adipogenesis: 10 % FBS + 2 mmol/L Glutamax, 1 μmol/L dexamethasone, 0.5 isobutylmethylxanthine, 2 cm 2 cells/25 cells 6 6 dish 2 cells/cm 3 2 (6-well plate) (6-well 2 mm/L cells/cm cells/well (24-well plate) (24-well cells/well 3 cells/well (96-well plate) (96-well cells/well cells/well (24-well plate) (24-well cells/well 4 5 4 mL pelleted in 15 mL cells were 5 cells cells/well (24-well plate) (24-well cells/well 5 3 (12-well plate) (12-well 2 cells/well (24-well plate) (24-well cells/well Not stated cells/well (24-well plate) (24-well cells/well 4 cells (T25 flasks) cells (T25 flasks) 4 5 5 were seeded in 24-well or 12-well plates or 12-well seeded in 24-well were 2 Seeding density of BM-MSCs Proliferation: 100 BM-MSCs on 100 on BM-MSCs 100 Proliferation: Differentiation: not stated Immunosuppression: 2 × 10 Proliferation: various density: 0.5, 1, and 2 × 10 Proliferation: various Osteogenesis: 4 × 10 Adipogenesis: 5 × 10 Chondrogenesis: 2.5 × 10 polypropylene tubes polypropylene Chondrogenesis assay: micromasses of 1 × 10 Osteogenesis assay: 3.5 × 10 Adipogenesis assay: 3.5 × 10 Differentiation assay: 5,000 cells/cm plate) (6-well Proliferation assay: 100 cells/well Migration assay: 5 × 10 ELISA: 5,000 cells/cm Proliferation: 1 × 10 Osteogenesis and adipogenesis: 1 × 10 Chondrogenesis: 3 × 10 Proliferation: 1 × 10 for cytokine: 1 × 10 Passaged 30,000 cells/cm • • • • • • • • • • • • • • • • • • • • , , , 2011 et al. , 2011 et al. , 2010 , 2012 , 2006 , 2009 et al. et al. et al. et al. 2012 2012 et al. et al. Author (year) Author Prins Vogel Lange Verrier Ben Azouna Jenhani Gottipamula Goedecke Table 7b. Seeding density of BM-MSCs and choice of culture medium used for assays. of culture medium used choice and of BM-MSCs density 7b. Seeding Table

www.ecmjournal.org 296 J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs 0.169 UI/mL human μmol/L L-ascorbic acid 2-phosphate, acid L-ascorbic μmol/L (Sigma-Aldrich), adipogenic differentiation kit chondrogenic differentiation kit osteogenic differentiation kit ® ® ® 10 % FBS (0.1mmol/L) Osteogenesis: Adipogenesis: Chondrogenesis: αMEM containing: Serum free α-MEM Not clearly specified mol/L/L dexamethasone penicillin/streptomycin -6 10 mmol/L beta-glycerophosphate, 50 beta-glycerophosphate, mmol/L Immunomodulation: not stated Tissue culture medium (base)* Migration: αMEM and antibiotics Proliferation: αMEM and antibiotics insulin, 6.25 μg/mL human transferrin Gene expression: αMEM and antibiotics Osteogenesis: StemPro Proliferation: DMEM + 1 % v/v antibiotic-antimycotic Adipogenesis: StemPro Chondrogenesis: StemPro 50 μg/mL ascorbic acid, 10 ng/mL recombinant human TGF-β3 αMEM + 10 % FBS, 100 U/mL penicillin G, μg/mL streptomycin U/mL), 1 % Glutamax-1 L-DMEM + 1 % penicillin/streptomycin (100 U/mL), Proliferation: LG-DMEM + penicillin/streptomycin, heparin 30 U/mL DMEM/F-12 + 10 % FBS, 100 U/mL penicillin, mg/mL streptomycin DMEM/F-12 + 10 % FBS, 100 μg/mL streptomycin, IU/ mL penicillin Osteogenesis: αMEM + antibiotics, 50 μmol/L ascorbic acid-2 phosphate *used as basal medium before addition of PP/other. 10 μmol/L Humulin-N, 0.2 mmol/L indomethacin, penicillin/streptomycin 20 % FBS, 100 units/mL penicillin, mg/mL streptomycin, 2 mmol/L glutamine nmol/L dexamethasone, 10 dexamethasone, nmol/L 10 PR PRF PRC PG-CM αMEM + 10 % FBS, mmol/L β-glycerophosphate, 50 μg ascorbic acid, 0.1 dexamethasone mmol/L 3-isobutyl-1-methylxanthine + 10 mg/mL ciprofloxacin, LG-DMEM + 1 μmol/L dexamethasone, 0.5 mmol/L 3-isobutyl-1-methylxanthine LG-DMEM + 50 μg/mL L-ascorbic acid 2-phosphate, 10 ng/mL TGF-β3 mmol/L), dexamethasone B-glycerophosphate (10 mmol/L), C (50 μg/mL), Differentiation: standard medium + Vitamin Chondrogenesis: DMEM + 1 % v/v antibiotic-antimycotic, 1× ITS, 0.1 μmol/L dexamethasone, 40 μg/mL proline, αMEM supplemented with 100 U/mL penicillin, 0.1 mg/mL streptomycin, μmol/L ascorbic acid-2 phosphate mmol/L), Proliferation: standard medium – HG-DMEM + 1 % antibiotics (penicillin-streptomycin), glutamine (200 mmol/L), Osteogenesis and adipogenesis: LG-DMEM + 1 M/L hydrocortisone, 0.05 g/L ascorbic acid, indomethacin, LG-DMEM + LG-DMEM cells/well cells/well cells/well cells/well 4 4 4 10 ) in T25 flasks

2 ×

2 (96-well plate) (96-well (96-well plate) (96-well (24-well plate) (24-well 2 2 (24-well plate) (24-well 2 cells/ cm 2 4 (24-well plates) (24-well 2 μL (24-well plate) cells in 100 μL (24-well 4 cells cells/well (6-well plate) (6-well cells/well 5 4 cells/well (24-well plate) (24-well cells/well 3 cells/cm cells/well (96-well plate) (96-well cells/well mL (24-well plate) cells/ mL (24-well cells/cm 4 3 4 4 cells/mL (48-well Boyden chamber)cells/mL (48-well 5 2 (24-well plate) (24-well 2 Seeding density of BM-MSCs cells/cm 5 cells/ ml in each group plate) (96-well cells/well 5 3 Boyden chamber plate) (24-well Proliferation: 1,000 cells/well (96-well) Proliferation: 1,000 cells/well Differentiation: 20,000 cells/plate (60 mm Nunc culture plates) Cell counting assay (proliferation assay): 1 × 10 plate) (96-well (6-well CFU-F assay (proliferation assay): 500 cells/well plate) Migration assay: 2 × 10 Proliferation: 6 × 10 Adipogenic differentiation: 26,000 cells/mL Osteogenic differentiation: 10,000 cells/mL Chondrogenic differentiation: pellets containing 10,000 cells Proliferation: 2 × 10 Differentiation assay: 2 × 10 cm Petri dish Proliferation: 100 cells/10 cm Petri Chondrogenesis: 2 × 10 Proliferation: 200 cells in 10 mmol/L diameter culture dishes Osteogenesis: 4,000 cells/cm Adipogenesis: 4,000 cells/cm Chondrogenesis: 10 × Immunomodulation: not stated Proliferation: 5 × 10 Migration: 5 × 10 Osteogenesis: 5 × 10 Gene expression: not stated Proliferation: 1.5 × 10 Osteogenesis: 800 cells/cm Gene expression: not stated mmol/L Petri dishes or 40 mmol/L Petri 5 × 10 Osteogenesis: 40,000 cells/mL (8,000 cells/cm 5000 cells/cm chamber of 20,000 calcein-AM stained BM-MSCs in lower Proliferation and differentiationassay: 1 2 × 10 5 × 10 • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • , et , 2017 , 2003 , 2010 , 2017 , 2019 et al. , 2014 , 2008 , 2016 , 2004 , 2015 , 2013 , 2018 , 2009 et al. et al. et al. et al. et al. et al. et al. et al. et al. , 2010 2014 et al. et al. et al. et al. al. Author (year) Author Agis Liou Perut Schar Gruber Samuel Kosmacheva Parsons Amable Nguyen Dohan Ehrenfest Bernardi Lucarelli Lucarelli Moradian *Assessed PR and PL Table 7c. Seeding density of BM-MSCs and choice culture medium used for assays. Table

297 www.ecmjournal.org J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs 0.05) 10 % vs. 0.05) p < 0.001) p < p < 0.05) L-PRP ( p < vs. 0.05) 0.01) controls, p < vs. p < 0.001) p < 10 % FBS) Findings vs. 0.05) p < 10 % FBS 0.05 vs. not stated) not stated) HPLF/HPLO proliferated faster than FBS PD shorter in HPLF/HPLO > FBS, generation time between Average p < HPLF and HPLO in terms of PD No difference between generation time Morphology of MSCs in HPL-supplemented medium shows denser cell bodies and more spindle-shaped cells than FBS group p p PRP promotes proliferation BM-MSCs significantly ( in presence P-PRP Highest proliferation observed CFU-F assay: colony numbers in 5 % PL (stored at − 80 °C) > (4 °C) > 10 % FBS ( times higher than control MTT assay: proliferation in PL-CM several ( 1 % PL: similar performance compared to 20 FBS 10 % PL: more cell proliferation than 1 PL, FBS and 20 ( to FBS alternative PPP: found to be effective (10 % aPPP = 10 FBS in performance) 10 % PL > FBS cPL ( Leukocyte content found not to influence proliferation PD: 10 % PL > 5 FBS ( Cumulative Rate of decline in number colonies with increased passages is less PL Colonies in 5 % and 10 PL are larger contain more cells FBS colonies (all ALP positive PL/PPP > controls in terms of (a) total and concentrations) and (b) middle/large-size colonies with increasing PL Both proliferation and PD of BM-MSCs improve concentration 2.5 % PL alone > 10 FBS in proliferation (0.5 and 1 has capacity similar proliferative PL/PPP combination superior to FBS at all concentrations ( PD time (hours): 10 % SN PL (PD: 65) > FT 67.6) FBS (PD: 137.5) at passage 7 PD of FTPL: perform better than control Cumulative PD and time of SNPL: SN PL concentrations ≥ 5 % Cumulative performs better than control Significant increase in proliferation with PL ( TGF- β1 has no effect on proliferation and does not modify potential of PL proliferative 10 % PL demonstrated superior PD > control ( • • • • • • • • • • • • • • • • • • • • • • • • • • • mol/L 7

− PL PRP-CM ) Groups compared Ve control) 10 % FBS (− Ve 10 % FBS + 1 ng/mL FGF-2 (+ Ve control) PL (0.5 %; 1 2.5 %) PL/PPP (0.5 %/4.5 %; 1 %/4 2.5 %/2.5 % 10 % FBS (control) 5 % PL (from PRP-CM) 10 % FBS (control) HPLF HPLO 10 % FBS (control) 10 % L-PRP-CM 10 % P-PRP-CM 10 % FBS (control) 10 % PL 10 % FBS (control) 10 % FT PL concentrations: 10 %, SN PL (various 7.5 %, 5 2.5 %) 10 % FBS (control) 20 % FBS (control) 1 % PL 10 % PL 10 % FBS (control) 10 % cPL 10 % PL 10 % FBS (control) 10 % PL 5 % PL ITS (control) PL PL + 10 ng/mL TGF-β1 dexamethasone + 50 µ g/mL ascorbic acid • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • PD time PD assay PD assay MTT assay MTT assay Kit, Roche) CFU-F assay CFU-F assay Assay used XTT assay (Roche) count PD rate of 7 d Cell counting to assess PD quantification kit (Invitrogen) Counting Kit-8 (CCK-8; Dojindo) Cell counting using Quant-iT PicoGreen Cell counting on days 1, 3, 5 and 7 using Cell counting (Neubauer haemocytometer) to Cell counting to assess PD, cumulative PD and Cell counting to assess PD, cumulative XTT colorimetric assay (Cell Proliferation Assay XTT colorimetric assay (Cell Proliferation , et al. , 2020 , 2015 , 2014 , 2013 , 2012 , 2019 , 2017 , 2018 , 2016 et al. et al. et al. et al. et al. et al. et al. 2013 et al. et al. Author (year) Author Yin Skific Infante Moisley Murphy Bernardi Muraglia Muraglia Karadjian Jonsdottir-Buch Table 8a. Effects of PPs on proliferation BM-MSCs. Table

www.ecmjournal.org 298 J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs 0.007) control = 0.001) p = 0.03) vs.

p = PL) all groups: p vs. vs. Ve control: − Ve 10 % FBS,

vs. 5 % PL >

10 % FBS)

0.05) 0.008) 0.01; 1 % PR Findings not stated) p < LG-PL (36.5 h) 0.05)

p p = p < p < PL ( h) >

5 % PL >

PL (

PR > 0.05)

1 % PR: p < vs. FBS >

FBS alone /FBS + FGF2

0.0007) p < 0.05)

0.0007) < 0.05; 2 % PR p ≤ 0.001) CFU-F: LG-PL significantly higher > KO-PL, LG-FBS, OR KO-FBS ( PD time: LG-FBS (66.4 PD: PL > FBS ( not stated) p < Both 5 % and 10 PL had significantly higher median yield of MSCs as compared to 10 % FBS (10 %PL > PD: faster BM-MSC proliferation in PL > FBS (median PD of PL: 2-fold of 10 % FBS; PL > all Median number of colonies not statistically significant between groups p p Colony counts: no difference between groups Colony counts: no difference between small spindle- Colony morphology: larger, denser colonies with very shaped cells in PL-supplemented expansion culture media medium PD time: cultures containing FBS longer than PL-supplemented medium ( CFU-F assay: no clear difference in colony counts (FBS PD time: PL > ( Ve control group 1 % PR: similar proliferation compared to + Ve 2 % PR and 5 PR: prolonged proliferation period of BM-MSCs ( Cell counting assay at day 9: cell number larger in 5 % PR > 2 control (5 % PR control > 1 % PRP group − Ve > + Ve p < PD assay: PR > CFU-F assay: FBS > Colony numbers: PL > FBS ( BM-MSCs cultured in PL-CM: increased yield at each passage PD by passage 3: PL > FBS ( Time to reach passage 3: PL significantly shorter than FBS ( 10 % PR sustains highest proliferation rate and shortest doubling time ( PR concentrations higher than 10 % inhibits cell growth BM-MSCs Increased duration of treatment with PR does not affect proliferation • • • • • • • • • • • • • • • • • • • • • • • PL PR Groups compared KO-DMEM 10 % FBS + 1 ng/mL FGF2 10 % FBS + 5 PL 10 % PL 5 % PL 10 % FBS + PL 10 % FBS 10 % FBS (control) 5 % PL 10 % PL 10 % FBS (control) 5 % PL 10 % FBS in DMEM/F12 medium (+ Ve control) DMEM/F12 medium alone (− Ve control) 1 % PR 2 % PR 5 % PR TGF-b3 (control) 10 % PR 10 % FBS (control) 7.5 % PL 7.5 % PR 10 % FBS (control) concentrations (1 %; PR at various 2.5 %; 5 10 20 30 40 % and 50 %) 10 % FBS (control) 10 % PL 10 % FBS + 1 ng/mL FGF2 10 % FBS + 5 PL 10 % PL 5 % PL In 2 different media: LG-DMEM and • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • PD assay CFU assay PD assay PD assay PD assay PD assay CFU assay CFU assay CFU assay CFU assay CFU assay CFU assay CFU-F assay Assay used • • extrapolate proliferation curve Cell-number counting with haemocytometer Cell counting (Neubauer haemocytometer) to , 2012 , 2012 , 2011 , 2017 , 2019 , 2014 , 2011 , 2009 , 2018 et al. et al. et al. et al. et al. et al. et al. et al. et al. Author (year) Author Liou Prins Jenhani Amable Nguyen Bernardi Goedecke Ben Azouna Gottipamula Table 8b. Effects of PPs on proliferation BM-MSCs. Table

299 www.ecmjournal.org J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs h d of of d

P-PRP < 0.01) P-PRP p all other 2 PD in in PD 2 0.01) vs. vs. vs. vs. p < 0.001) 0.001); no significant no 0.001); p < Ve) Ve) at all time points p < 0.014; fL-PRP 0.01) h ( h Ve/− p = not stated) p < p 1.8-fold over controls ( 1.8-fold over

h, significantly decreased after 72 serum-free α-MEM: statistically < 0.01), disappearing beyond day 14 ±

p 0.05) vs. P-PRP gels, d ( d p < vs. Findings PPP gels efficiency in terms of BM-MSC control cultures (+ % PR twice that in control (4 PD PD (4 in control that twice PR % 0.001) control, P-PRP > % FBS up to 48 (p < vs. 0.05] % FBS significantly up to 48 to up significantly FBS % 0.05) 20 20 0.01) p < p < 1 PRF for first 14 first for PRF 1 p < fL-PRP > 0.027) p = 0.001) 0.01) % PRF-CM = % PRF-CM > PRF-CM % PR improves cellular proliferation of BM-MSCs; increase in PR improves is dose dependent (10 % > 1 control) ( Induction of proliferation by PR is maintained throughout the entire period of testing (up to 9 d) Serum is not necessarily required for PR-mediated induction of BM- MSC proliferation in 10 BM-MSCs of PD control at day 6) 6 within PD) (2 proliferation regular to return BM-MSCs withdrawing PR L-PRP > cellular proliferation (L-PRP gels, bFGF release: faster in both forms of L-PRP gel (fresh/frozen), higher with concentrations than P-PRP gel [L-PRP (both forms) and PPP gels, BM-MSC proliferation is significantly correlated with amount of bFGF released p < p < Earlier onset of proliferation (24 h): PRC > serum ( 48 h: no difference 72 h: serum > PRC Highest concentration (PR 1), 10.0 proliferation/mitogenic in PR) of (platelets/mL increase Dose-dependent activity PRF-CM (all concentrations) significant different proliferation at all time points 5 % PRF-CM < 20 FBS at all time points ( 10 ( 20 difference after 72 h 1 or 2 PRF membranes > ( Standard medium: dose-dependent effect in number of PRF membrane > PRF 2 used: Differentiation medium: dose-dependent effect in number of PRF Differentiation medium: dose-dependent effect in number of PRF membrane used: 2 PRF > 1 throughout whole experiment ( proliferation 2 PRF more 14 onwards: day Proliferation from conditions ( days 8 and 16 ( incubation period ( PRC significantly increases cell proliferation as compared to control at 12-d the throughout significantly BM-MSCs of proliferation increases PRF • • • • • • • • • • • • • • • • • • • • • • • BM- vs. BM-MSCs BM-MSCs BM-MSCs BM-MSCs vs. vs. % FBS FBS % PR PRF PRC 20 PG-CM Groups compared /mL (PR4) 6 /mL (PR1) /mL (PR2) /mL (PR3) 8 7 6 BM-MSCs + 1 % PR + 10 % PR + BM-MSCs PPP gel (control) L-PRP gel fL-PRP gel P-PRP gel 10 % FBS (control) PRF control) Serum free α-MEM (− Ve control) 20 % FBS + α-MEM (+ Ve 5 % PRF-CM 10 % PRF-CM + 20 % FBS 10 PR BM-MSCs + medium 10 % PR MSCs + 10 % PR alone control) Standard medium (− Ve PRF + standard medium (PRF) medium 2 PRF membranes + standard (2PRF) control) Differentiation medium (+ Ve PRF + differentiation medium (DPRF) medium (D2PRF) 10 % FBS (control) 15 % PRC 2.5 % serum (control) 2.5 % PRC Serum-free medium (control) concentrations: platelet different with PR 20 % PRF-CM differentiation 2 PRF membranes + differentiation 3 comparisons: • • • • • • • • • • • • • • • • • at 24, 48 and 72 h • • 2 × 10 4 × 10 8 × 10 1.6 × 10 • • • • • • • from culture removed PRF membranes were plates at day 7 . analyzer MTT assay MTT-assay MTT assay assay at days 0, 8, 16, 24 proliferation Assay used ® labelled with [3H] thymidine AlamarBlue Liquid scintillation counting of cells pulse AlamarBlue test: to assess cell viability and Methylene blue assay and NucleoCounter Methylene Pico Green reagent and Fluroskan fluorometer Cell counting using Coulter LH 750 , et al. , 2017 , 2003 , 2010 , 2008 , 2016 , 2004 , 2013 et al. et al. et al. et al. et al. et al. 2010 et al. Author (year) Author Perut Gruber Samuel Parsons Lucarelli Lucarelli Moradian Dohan Ehrenfest Table 8c. Effects of PPs on proliferation BM-MSCs. Table

www.ecmjournal.org 300 J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs FBS h vs. d of of d

P-PRP < 0.01) P-PRP p all other 2 PD in in PD 2 0.01) vs. vs. vs. vs. p < 0.0006; cPL

0.001) 0.001) =

0.001); no significant no 0.001); p 0.05) p < p < Ve) Ve) at all time points p < 0.014; fL-PRP p < FBS 0.01) h ( h Ve/− p = not stated) vs. p < p 0.05)

<

p 1.8-fold over controls ( 1.8-fold over h, significantly decreased after 72 serum-free α-MEM: statistically < 0.01), disappearing beyond day 14 0.01) ± p controls 0.05) not stated) < vs. P-PRP gels, p d ( d vs. p p < vs. Findings PPP gels efficiency in terms of BM-MSC 0.04)

) Ve correlation with migration Ve = control cultures (+ -10 − p % PR twice that in control (4 PD PD (4 in control that twice PR % % PR and control groups ( 0.001) Ve control group Ve % FBS 10

control, × P-PRP > % FBS up to 48 (p < vs. %PL ( 0.05] % FBS significantly up to 48 to up significantly FBS %

0.05) Findings 1.02

20 20 = 10 0.01) p < p < 1 PRF for first 14 first for PRF 1 p vs. Ve correlation with migration Ve p <

%FBS fL-PRP > L-PRP at days 3, 7 and 14 (

0.027) 0.8-fold over controls ( 0.8-fold over >

10 ± %FBS p =

vs. 0.001) 0.01) % PRF-CM = % PRF-CM > PRF-CM % PR improves cellular proliferation of BM-MSCs; increase in PR improves is dose dependent (10 % > 1 control) ( Induction of proliferation by PR is maintained throughout the entire period of testing (up to 9 d) Serum is not necessarily required for PR-mediated induction of BM- MSC proliferation in 10 BM-MSCs of PD control at day 6) 6 within PD) (2 proliferation regular to return BM-MSCs withdrawing PR L-PRP > cellular proliferation (L-PRP gels, bFGF release: faster in both forms of L-PRP gel (fresh/frozen), higher with concentrations than P-PRP gel [L-PRP (both forms) and PPP gels, BM-MSC proliferation is significantly correlated with amount of bFGF released p < p < Earlier onset of proliferation (24 h): PRC > serum ( 48 h: no difference 72 h: serum > PRC 0.03) Highest concentration (PR 1), 10.0 proliferation/mitogenic in PR) of (platelets/mL increase Dose-dependent activity PRF-CM (all concentrations) significant different proliferation at all time points 5 % PRF-CM < 20 FBS at all time points ( 10 ( 20 difference after 72 h 1 or 2 PRF membranes > ( Standard medium: dose-dependent effect in number of PRF membrane > PRF 2 used: Differentiation medium: dose-dependent effect in number of PRF Differentiation medium: dose-dependent effect in number of PRF membrane used: 2 PRF > 1 throughout whole experiment ( proliferation 2 PRF more 14 onwards: day Proliferation from conditions ( days 8 and 16 ( incubation period ( PRC significantly increases cell proliferation as compared to control at 12-d the throughout significantly BM-MSCs of proliferation increases PRF 0.06); 10

p < 0.01) =

• • • • • • • • • • • • • • • • • • • • • • • p not stated) p < % FBS in terms of migration capacity ( p

20

% aPL (

%PL ( > BM-

% PL: comparable FBS (control): L-PRF % PL (

10 % PR: no difference 5

% PL ( vs. 1

1 PL

> vs.

% PR: BM-MSC migration better than 1 5 5 >

vs.

% vs. >

vs. BM-MSCs BM-MSCs BM-MSCs BM-MSCs 1

> 0.0008) vs. 0.05)

vs. =

% ucPL % % PL % FBS % PL

p fPL more migratory towards % ucPL % PR: no difference in migration compared to + % and 5 % PR p < for BM-MSCs

Both L-PRP-CM and P-PRP-CM significantly promoted migration of BM-MSCs (compared to FBS; P-PRP-CM promoted more BM-MSC migration, compared to L-PRP-CM ( Both PL and cPL induce significantly more migration than FBS (PL significantly still BM-MSCs with slightly, response migratory reduces fPL in seen leukocytes of Removal Only PRP is chemotactic for BM-MSCs ( chemotactic not cells: culture to needed FBS of amount reduce to supplement medium basal as used ITS, 10 1 10 10 higher in 10 Migration capacity of BM-MSCs was 10 10 1 2 2 Highest concentration (PR 1), 6.7 increase of cell migration Dose-dependent (platelets/mL) BM-MSC migration: occurred mainly at day 1 (L-PRP gel and blood clot) 3 (L-PRF) Migration IL-1 β secreted by platelet products: + PDGF-AB and IGF-1 secreted by platelet products: VEGF secreted by platelet products: no influence on migration PL PR • • % FBS FBS % • • • • • • • • • • • • • • • • • • • • • PR PRF PG-CM PRC PRP-CM 20 PG-CM Groups compared h, days

Ve control) Ve

/mL (PR4) 6 /mL (PR1) /mL (PR2) /mL (PR3) 8 7 6 BM-MSCs + 1 % PR + 10 % PR + BM-MSCs PPP gel (control) L-PRP gel fL-PRP gel P-PRP gel 10 % FBS (control) PRF control) Serum free α-MEM (− Ve control) 20 % FBS + α-MEM (+ Ve 5 % PRF-CM 10 % PRF-CM + 20 % FBS 10 PR BM-MSCs + medium 10 % PR MSCs + 10 % PR alone control) Standard medium (− Ve PRF + standard medium (PRF) medium 2 PRF membranes + standard (2PRF) control) Differentiation medium (+ Ve PRF + differentiation medium (DPRF) medium (D2PRF) 10 % FBS (control) 15 % PRC 2.5 % serum (control) 2.5 % PRC Serum-free medium (control) concentrations: platelet different with PR 20 % PRF-CM differentiation 2 PRF membranes + differentiation Ve control) Ve ) − 3 comparisons: • • • • • • • • • • • • • • • • • at 24, 48 and 72 h • • 2 × 10 4 × 10 8 × 10 1.6 × 10 • • • • • • • from culture removed PRF membranes were plates at day 7 Ve control Ve − Groups compared Ve control) Ve

/mL (PR4) 6 /mL (PR1) /mL (PR2) /mL (PR3) 8 7 6 % FBS (control) 10

10 10 10 % FBS (control) % FBS (control) % PL × % FBS (control) % PL % ucPL % aPPP % FBS (control) % FBS in DMEM/F12 medium (+ medium DMEM/F12 in FBS % L-PRP-CM P-PRP-CM 10

% PL % PR % PR % PR % PL % ucPL × × ×

cPL PL 10 10 5 10 1 2 5 10 Serum-free medium (control) PR with different platelet concentrations: 2 4 8 1.6 10 L-PRP L-PRF Blood clot ITS/PL PL PL/PL (+ DMEM/DMEM ( 1 1 10 10 10 20 DMEM/F12 medium alone ( • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 1. 2. 3. 4. Supernatants collected from: differentwere time compared points (8 At 1, 3, 7, 14 and 28) • analyzer MTT assay MTT-assay MTT assay assay at days 0, 8, 16, 24 proliferation Assay used ® labelled with [3H] thymidine AlamarBlue Liquid scintillation counting of cells pulse AlamarBlue test: to assess cell viability and Methylene blue assay and NucleoCounter Methylene assay chemotaxis 3D Pico Green reagent and Fluroskan fluorometer (Inucyte) Cell counting using Coulter LH 750 Hematology Assay used chamber (Ibidi) WimScratch assay WimScratch , µ Slide Scratch wound-healing Boyden Chamber assay Boyden Chamber assay Transwell migration assay Transwell migration assay Transwell migration assay Transwell et al. , 2017 , 2003 , 2010 , 2008 , 2016 , , 2004 , , , , 2013 , et al. et al. et al. et al. , 2015 , 2018 et al. et al. et al. 2010 et al. , 2016 et al. et al. et al. et al. 2019 2012 2011 2019 2004 et al. et al. Author (year) Author et al. Perut Gruber Samuel Parsons Lucarelli Lucarelli Gruber Moradian Moisley Nguyen Author (year) Author Murphy Yin Dohan Ehrenfest Goedecke Skific Schar Table 9. Effects of PPs on migration BM-MSCs. Table

301 www.ecmjournal.org J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs vs. not p FTPL 0.005) % PL:

= 0.00007) p p < FBS ( 0.05) not stated) > p p < FBS, vs. not stated) not stated) p p not stated) p control. PR was 10- and 10- and PR was > Dexa control. unsupplemented control ( control unsupplemented > 0.01) > control 0.05) not stated) and control groups ( Findings p p < p < Dexa FBS (control) of same concentration at any > ≥ % PL: statistically significant on days 1 and 7. 10 ALP activity: Dexa > PL ( PL assay: 45Ca2+ incorporation 400-fold more than Dexa ( respectively PL staining: Kossa von Osteogenesis: PL > FBS in differentiation ( PL and FBS. ( Adipogenesis: similar between Chondrogenesis: PL > FBS in differentiation ( ALP activity in hPL time point. 1 statistically significant on days 7 and 21 faster differentiate PL with incubated BM-MSCs staining: red Alizarin than controls (PL reached maximum calcium expression at day 14 day 21 in FBS). 1 % PL > FBS at all time points. 10 two groups at days 1, 7 and 14. End calcium content similar between activity: day 7: FBS > HPLO HPLF; 14: HPLF ALP Alizarin red staining: slightly more mineralisation in HPLF and HPLO > FBS Authors on adipogenesis and chondrogenesis. made No comparisons reported on ability of BM-MSCs to differentiate similar control More calcium nodules in groups treated with PRP-CM ( P-PRP-CM > L-PRP-CM ( potential stated) reported that and Authors “BM-MSC FBS. were able to differentiate in all three lineages” Significant reduction in accumulation in PL group ( group PL in accumulation lipid in reduction Significant groups No difference between No difference observed in the osteogenic and adipogenic differentiation Numbers of differentiated adipocytes and osteoblasts in PL No comparison made in terms of differentiation between SNPL, • • • • • • • • • • • • • • • • • • PL PRP-CM nmol/L dexamethasone Groups compared LG-PL LG-FBS (control) KO-PL KO-FBS (control) 10 % FBS + 1 ng/mL FGF2 10 % FBS + 5 PL 10 % PL 5 % PL 10 % FBS (control) 5 % PL 10 % PL IMDM/10 % FBS (control) 10 (Dexa) 10 % PL 10 % FBS (control) 10 % FT PL 10 % SN PL FBS (control) PL FBS (control) HPLF HPLO 10 % FBS (control) L-PRP-CM P-PRP-CM 1 % FBS (control) 10 % FBS (control) 1 % PL 10 % PL 10 % FBS (control) 5 % PL 10 % PL • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • staining ALP activity Sudan red staining ALP activity assay von Kossa staining Alizarin red staining toluidine blue staining and von Kossa staining Adipogenesis: oil red O Adipogenesis: oil red O Adipogenesis: oil red O 45Ca2+ incorporation assay Chondrogenesis: safranine O Adipogenesis: Nile red staining Chondrogenesis: toluidine blue Adipogenesis: oil red O staining Chondrogenesis: alcian blue and Osteogenesis: von Kossa staining Osteogenesis: von Kossa staining Staining used/variables measured Staining used/variables in osteogenic and adipogenic medium Alizarin red staining and ALP staining Alizarin red staining and Chondrogenesis: Carazzi haematoxylin Osteogenesis: alizarin red staining and Osteogenesis: Nuclear Fast Red Solution Microscopic analysis following culturing Osteogenesis: von Kossa and Alizarin red Osteogenesis: von Kossa and Type of assessed Osteogenesis Osteogenesis Osteogenesis Osteogenesis Osteogenesis Osteogenesis Osteogenesis Osteogenesis Osteogenesis Adipogenesis Adipogenesis Adipogenesis Adipogenesis Adipogenesis Adipogenesis Adipogenesis differentiation Chondrogenesis Chondrogenesis Chondrogenesis Chondrogenesis

, , , , , 2012 , 2018 et al. et al. et al. , 2016 et al. , 2012 , 2012 , 2013 et al. 2011 2010 2020 2013 et al. et al. et al. et al. et al. Ben Azouna Verrier Gottipamula Author (year) Author Yin Bernardi Karadjian Goedecke Jonsdottir-Buch Skific Lange Table 10a. Effects of PPs on differentiation BM-MSCs. Table

www.ecmjournal.org 302 J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs FBS. 0.05) ≤

p 0.05) 0.05) p > p > PL on calcification at day 14 ( 14 day at calcification on PL not stated) p > Degree of calcium ossification is directly . Findings Ve control) + Ve vs. Ve controls + Ve vs. Chondrogenesis: no difference between groups ( Chondrogenesis: no difference between groups ( Adipogenesis: no difference between duration = inhibition of Duration of treatment with 10 % PR: increased chondrogenesis (decreased deposition of cartilage specific extracellular matrix production). FBS > PR throughout slight reduction in staining intensity. PR concentration: only 20 % showed 1 %, 5 % and 10 control No difference between Alizarin red staining: PR leads to pronounced accumulation of calcium ossificates dependent on PR concentration added in differentiation medium control and nutrient medium + PR groups: no calcium ossificates − Ve ALP synthesis slightly when added into ALP staining: PR increases osteogenic medium ( Adipogenesis: reduced by PR Chondrogenesis: increased by PR Osteogenesis: increased by PR ( Osteogenic differentiation: FBS differentiation: Osteogenic despite PR withdrawal P1 and PR2 < FBS BM-MSCs able to maintain differentiation potential No comparisons made PR 3 > Only PR. concentrations of with increased decrease activity: ALP ability chondrogenic and PR retaine osteogenic in 10 % cultured BM-MSCs No difference in differentiation potential (osteo/adipo/chondrogenesis) • • • • • • • • • • • • • • • • R Ve control) Ve PL PR /mL (PR1) /mL (PR2) /mL (PR3) 8 7 6 Groups compared 20 % FBS 10 % PR TGF-b3 (control) PR 10 % FBS (control) 7.5 % PL 7.5 % PR-SRGF 10 % FBS (control) 1 % PR Serum free medium (control) platelet PR with different concentrations: 2 × 10 4 × 10 8 × 10 10 % FBS (control) 5 % PL 2 % FBS (control) 3 % PL (− medium Nutrient (+ Ve medium Osteogenic control) Nutrient medium + 2.5 % PR P + 2.5 % Osteogenic medium • • (1) (2) (3) • • Note: compared the effect of PR on osteogenesis and withdrawal chondrogenesis of each group • • • • • • Compared effect of: duration (with 10 % PR: days 1, 3, 7, 21) and concentration (1 %, 5 10 20 %) of PR • • • • • • • • • acid ALP staining NBT substrate ALP activity assay Alizarin red staining Safranin O/fast green Chondrogenesis: DAPI Adipogenesis: oil red O Adipogenesis: oil red O Adipogenesis: oil red O Osteogenesis: ALP staining Osteogenesis: alizarin red S Osteogenesis: alizarin red S Alcian blue/Nuclear Fast Red Chondrogenesis: toluidine blue Chondrogenesis: toluidine blue Adipogenesis: oil red O solution Osteogenesis: von Kossa staining Osteogenesis: alizarin red staining Staining used/variables measured Staining used/variables Collagen II immunohistochemistry Chondrogenesis: von Kossa staining Chondrogenesis: alcian blue 1 % acetic Osteogenesis: ALP staining with BCIP/ Osteogenesis: Type of assessed Osteogenesis Osteogenesis Osteogenesis Osteogenesis Osteogenesis Osteogenesis Osteogenesis Adipogenesis Adipogenesis Adipogenesis Adipogenesis differentiation Chondrogenesis Chondrogenesis Chondrogenesis Chondrogenesis Chondrogenesis Chondrogenesis , , , , , 2006 , 2009 , 2018 et al. et al. et al. et al. , 2014 2017 2014 2004 2003 et al. et al. et al. et al. Kosmacheva Gruber Amable Author (year) Author Bernardi Lucarelli Liou Prins Vogel Table 10b. Effects of PPs on differentiation BM-MSCs. Table

303 www.ecmjournal.org J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs 1 1 > < 0.01) 0.05) p not stated) p < p 0.001) % FBS; significant P-PRP and PPP gels) p < osteogenic osteogenic medium; 10 0.05) at all time points; > vs. > p < osteogenic medium. Absent % % PRC 15 other conditions at all time points % PRC = > 15 Findings osteogenic medium > not stated) % PRC p = 0.01; standard medium up to day 21; differentiation medium Ve Ve control: no differentiation nodule. In presence of PRF: <

p not stated) < 0.01) p p at all time points) SEM: − numerous module evident (differentiation > standard medium) Alizarin red staining (23 d): PRC > control during first 5 d ( ALP activity (after day 3): PRC > control (2.5-fold, osteoblast inducing at effective more gel fL-PRP and gel L-PRP ( BM-MSCs of mineralisation and differentiation ( ability mineralisation to correlated positively significantly is release bFGF of BM-MSCs ( L-PRP in numbers platelet by influenced not was mineralisation BM-MSC and fL-PRP gels ALP activities: 1 or 2 PRF > other conditions at all time points ( von Kossa staining: 1 or 2 PRF ( Dose-dependent effect of PRF on BM-MSC differentiation (2PRF PRF, Alizarin red staining: 15 % PRC > 10 FBS ( 15 % PRC comparable to osteogenic medium ALP activity: 15 increase on days 8 and 16 (15 % PRC > osteogenic medium, Immunohistochemistry: RUNX2 − osteocalcin and osteopontin − staining for all markers in 10 % FBS • • • • • • • • • • • • ® Ve Ve differentiation medium PRC PRF PG-CM Groups compared differentiation medium medium differentiation Standard medium (− control) PRF + standard medium (PRF) 2 PRF membranes + standard medium (2PRF) Differentiation medium (+Ve control) PRF + (DPRF) 2 PRF membranes + (D2PRF) Ve control) 10 % FBS (− Ve Osteogenic medium, StemPro control) (+ Ve 15 % PRC 2.5 % FBS (control) 2.5 % PRC PPP (control) L-PRP gel fL-PRP gel P-PRP gel • • • • • • • • • • • • • • • SEM ALP activity ALP activity von Kossa staining Alizarin red staining Alizarin red staining osteopontin: days 8, 16 and 24. stain) for RUNX2, Osteocalcin, ALP activity: days 8, 16 and 24 Staining used/variables measured Staining used/variables Alizarin red staining: days 8, 16 and 24 Immunohistochemistry (using Hoechst Type of assessed Osteogenesis Osteogenesis Osteogenesis Osteogenesis differentiation

, , , 2013 et al. et al. , 2010 2016 2008 et al. et al. Samuel Parsons Author (year) Author Perut Dohan Ehrenfest Table 10c. Effects of PPs on differentiation BM-MSCs. Table

www.ecmjournal.org 304 J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs

p 0.05 p < PL, 0.05) + p < 0.05) PL group control/FBS > p < vs. %) Findings % and 10 and % 0.05) p < , IL-2 and IL-17 g FBS or P-PRP-CM) vs. 0.001) 0.05; p < p < Both L-PRP-CM and P-PRP-CM upregulate expression Both L-PRP-CM and P-PRP-CM upregulate expression of OC and RUNX2 significantly when compared against FBS (control) ( Upregulation stronger in P-PRP-CM > L-PRP-CM ( 10 % FBS + 5 PL = control group in expression Expression decreased in 10 % PL and 5 a clear increase in BMP-2 protein levels Only PL shows time over 10-fold by upregulated significantly levels protein BMP-2 at day 23 compared to 1 ( in control groups Undetectable BMP-2 levels L-PRP-CM upregulates production of PGE2 and NO L-PRP-CM upregulates production of PGE2 and NO ( P-PRP-CM and FBS No significant difference between (5 PL IL-8: and IL-6 GM-CSF: undetectable PR increases pro-inflammatory profile of BM-MSCs (in particular cytokines IL-2R, IL-6, IL-7, IL-12 and IL-15) ( not stated) IL-1β, GM-CSF, cytokines: proinflammatory Undetectable TNF-α, IFN- Most anti-inflammatorycytokines were undetectable. anti- of release on PR of effect the on conclusion clear No inflammatory cytokines 10 % FBS + 5 PL = control group in expression Expression decreased in 10 % PL and 5 15 % PRC > osteogenic medium 10 FBS ( of L-PGDS in FBS Higher levels • • • • • • • • • • • • • • • • • • ELISA ELISA Assay used Western blot Western Western blot Western blot Western Western blot: RUNX2 Western Multiplex bead-based ELISA: to measure OC cytokine assay system) immunoassay (Bioplex Commercial luminex kit ELISA: to measure PGE2 sssay kit: to measure NO Griess reaction using NO ng/mL FGF2 ng/mL FGF2 1ng/mL 1ng/mL FGF2 1 1 Osteogenesis Inflammation Adipogenesis + + +

Ve control) (+ Ve ® nmol/L dexamethasone % % FBS % FBS % FBS Groups compared Culture medium (un- Culture medium supplemented, as control) 10 (control) 10 % PL FBS (control) 10 % FBS (control) L-PRP-CM P-PRP-CM 10 (control) 10 % FBS + 5 PL 10 % PL 5 % PL 10 % FBS (control) 10 % PR 10 % FBS (control) L-PRP-CM P-PRP-CM 10 (control) 10 % FBS + 5 PL 10 % PL 5 % PL control) 10 % FBS (− Ve Osteogenic medium, StemPro 15 % PRC 10 (control) 10 % FBS + 5 PL 10 % PL 5 % PL PL • • • • • • • • • • • • • • • • • • • • • • • • • • • • NO IL-6 IL-8 PGE2 IL-17 Leptin BMP-2 RUNX2 L-PGDS GM-CSF assessed CaSR, PTHR Osteocalcin (OC) Osteocalcin (OC) Pro-inflammatory cytokines: Anti-inflammatory cytokines: IL-1RA, IL-4, IL-5, IL-10, IL-13 and IFN-α GM-CSF, IL-1β, IL-2, IL-2R, IL-6, IL-7, IL- 8, TNF- α, IFN-g, IL-12p40/p70, IL-15 and Cytokines/growth factors/proteins/MMPs PL PL PL PL PL PR PRC Platelet Platelet PRP-CM product PRP-CM , , , ; , 2010 , 2012 , 2016 , 2016 et al. et al. et al. , 2012 , 2012 , 2012 et al. et al. 2011 2014 2016 et al. et al. et al. et al. et al. Author year Author Ben Azouna Ben Azouna Ben Azouna Samuel Jenhani Amable Yin Yin Lange Verrier Table 11a. Effects of PP on growth factor/cytokine/protein expression BM-MSCs. Table

305 www.ecmjournal.org J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs not p not stated) p not stated) p 0.01) Findings p < not stated) p not stated) p = 0.04) p not stated) p PR activates heparan sulphate, elastin, laminin, collagen PR activates I and III ( II collagens and fibronectin decorin, perlecan, Aggrecan, and IV not detectable in all groups PR increases chemokines IP-10, MIP-1β and MCP-1, mild MCP-1, and MIP-1β IP-10, chemokines increases PR increase with eotaxin ( conditions all in undetectable MIP-1α and RANTES MIG, pro-angiogenic factors in BM-MSCs PR activates (thrombospondin, PLGF, aFGF, VEGF-D, VEGF) ( stated) Angiogenin, endostatin and angiopoietin-1 undetectable in both groups PR increases HGF, G-CSF, TGF-β1 and TGF-β2 secretion ( PR downregulates PDGF-BB secretion ( not IGF-1 and PDGF-AB PDGF-AA, TGF-β3, bFGF, EGF, secreted in both groups medium; low concentrations in control medium MSCs, compared to PL-cultured MSCs (5 % and 10 PRP) ( MMP13 ( No difference between groups No difference between Present at high concentrations with all PL-supplemented SDF-1α levels are significantly higher in FBS-cultured VEGF: control > PL ( PR increases secretion of MMP1, MMP3, MMP7, MMP8 and Undetectable • • • • • • • • • • • • • • • ELISA ELISA Assay used Western blot Western 24, 48 and 72 h ELISA: PDGF-AB Multiplex bead-based Multiplex bead-based Multiplex bead-based cytokine assay system) cytokine assay system) cytokine assay system) immunoassay (Bioplex immunoassay (Bioplex immunoassay (Bioplex Commercial luminex kit Commercial luminex kit Commercial luminex kit Commercial luminex kit Supernatants collected at MMPs ng/mL FGF2 ng/mL ng/mL FGF2 ng/mL FGF2 ng/mL FGF2 1 1 1 1 Chemotaxis + Angiogenesis + + + Other growth factors Vascular smooth muscle Vascular % FBS % % FBS % FBS % FBS 10 % FBS (control) 10 % PR 10 (control) 10 % FBS + 5 PL 10 % PL 10 % FBS (control) 10 % PR 5 % PL Groups compared 10 (control) 10 % FBS + 5 PL 10 % PL 5 % PL 10 (control) 10 % FBS + 5 PL 10 % PL 10 (control) 10 % FBS + 5 PL 10 % PL 5 % PL 10 % FBS (control) 10 % PR 10 % FBS (control) 5 % PL 10 % PL 10 % FBS (control) 10 % PR 10 % FBS (control) 10 % PR 5 % PL Extracellular matrix proteins and molecules • • • • • • • • • • • • • • • • • • • • • • • • • • • • • VEGF SDF-1α RANTES assessed and IGF-1 FGF2, G-CSF 1β and RANTES ASMA and SM22a collagens I, II, III and IV MMP-1, -3, -7, -8 and -13 Heparan sulphate, aggrecan, decorin, endostatin, aFGF, thrombospondin-2, angiopoietin-1, angiogenin and PLGF elastin, laminin, perlecan, fribronectin, Pro-angiogenic factors: VEGF, VEGF-D, TGF-β3, PDGF-AA, PDGF-AB, PDGF-BB eotaxin, IP-10, MCP-1, MIG, MIP-1α, MIP- EGF, HGF, bFGF, G-CSF, TGF-β1, TGF-β2, Cytokines/growth factors/proteins/MMPs PL PL PL PL PL PR PR PR PR PR Platelet Platelet product , , , , , , , , , ; ; ; et al. et al. et al. et al. et al. et al. et al. et al. et al. , 2012 , 2012 , 2012 , 2012 2011 2011 2014 2014 2014 2011 2014 2014 2011 et al. et al. et al. et al. Author year Author Ben Azouna Ben Azouna Ben Azouna Ben Azouna Jenhani Jenhani Jenhani Amable Amable Amable Amable Amable Goedecke Table 11b. Effects of PP on growth factor/cytokine/protein expression BM-MSCs. Table

www.ecmjournal.org 306 J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs T-cells : MNCs alone MNCs alone = 0.003) p vs. < 0.05) p < 0.05) p not stated) < 0.05) HSCs, when compared to 5 % + Findings p p 1 and PR (all ratios) significantly ratios) (all PR and 1 : < 0.01) p = 0.002) and 10% PL ( p % FBS induces a stronger chemoattraction stronger a induces FBS % ≤ 0.03) < 0.05) p p in PL at 20 at PL in han FBS ( lower Co-culture with BM-MSCs reduces Co-culture with BM-MSCs reduces proliferation of MNCs ( PL and control do not affect ability of BM- MSCs to reduce MNC proliferation BM-MSCs pre-incubated with PR cause large and assay dissolution film fibrin in zone lysis increased caseinolysis Fibrin and casein zymographies: increased in BM-MSCs plasminogen activation exposed to PR of BM-MSCs increased levels and PAI-1 uPA by PR ( Apoptotic stress: both L-PRP-CM- and Apoptotic stress: both L-PRP-CM- and P-PRP-CM-inhibited camptothecin-induced apoptosis to enhance viability of BM-MSCs ( Apoptotic stress: L-PRP-CM > P-PRP-CM in inhibiting apoptosis ( Immunomodulation: L-PRP-CM induces activation of NF-κB signalling pathway, evidenced by the translocation and accumulation of NF-κB p65 in the nucleus of BM-MSCs ( BM-MSCs cultured in PL show dose- dependent (ratio of BM-MSCs : PBMCs) T-cell inhibition on mitogen-activated proliferation ( Cells grown in FBS > PL terms of immunosuppression proportional to the ratio of BM-MSCs of the ratio to proportional seeded (significant at 1 : 5) Immunomodulation of BM-MSCs expanded Suppression of proliferation is 10 • • • • • • • • • • CD34 towards PL ( • • • h Groups compared HSCs in response to: + KO-FBS LG-FBS L-PRP-CM P-PRP-CM 10 % FBS (control) 10 % FBS L-PRP-CM P-PRP-CM vs. vs. 1. 2. 3. 10 % FBS (control) 7.5 % PR ratios 20 : 1, 10 1 and 7.5 % PL at PBMC : BM-MSC 5 : 1 BM-MSCs (FBS) + MNCs + MNCs BM-MSCs (PL) MNCs alone BM-MSCs pre-incubated with PR BM-MSCs incubated in serum-free medium Nearly confluent BM-MSCs serum-starved for 24 and treated with: 1. 2. 3. control) SDF-1 (+ Ve BM-MSCs + 10 % FBS BM-MSCs + 5 % PL BM-MSCs + 10 % PL Apoptotic stress: serum-starved BM-MSCs treated BM-MSCs treated Apoptotic stress: serum-starved camptothecin- before medium, in following 7 d for induced apoptosis: different ratios with T-cells No controls used BM-MSCs expanded in 10 % SN PL and seeded at (0, 1 : 1,000, 100, 10 5) LG-PL KO-PL • • • • • • • • • • • • • Migration of CD34 • • • • • • markers) MTT assay Assay used Western blotting Western 7AAD cell by BM-MSCs proliferation assay Fibrin zymography Casein zymography + released by BM-MSCs 5-bromo-2-deoxyuridine Transwell migration assay Transwell Fibrin film dissolution assay V-FITC/PI staining (apoptotic qPCR – uPA and PAI-1 levels levels and PAI-1 qPCR – uPA Flow cytometry using annexin CD45 PC7m, 7-AAD) on inhibition of Mixed lymphocyte reaction assays Flow cytometry enumeration (CD44 7AAD + HSCs assays capacity assessed by BM-MSCs apoptotic stress peripheral blood – through afibrin zymography/qPCR zymography/casein BM-MSC function Immunomodulation Immunomodulation Immunomodulation, Immunomodulation, Immunomodulation of T-cell proliferation Chemotactic effect on effect of PL expanded Capacity of BM-MSCs BM-MSCs on inhibition to activate plasminogen to activate mononuclear cell subset inhibition of CD45 Stimulation of fibrinolytic Response of BM-MSCs to PL PL PL PL PL PR PR and Platelet Platelet product PRP-CM

, , , et al. et al. , 2017 , 2013 , 2012 , 2011 et al. 2016 2013 2009 (year) Author Author Bernardi Bernardi Yin Goedecke et al. et al. et al. et al. Agis Jonsdottir- Buch Gottipamula Table 12. Effects of PPs on other BM-MSC functions. Table

307 www.ecmjournal.org J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs 0.05) , p ≤ 0.05) and decreased osteopontin p < 0.001) , ( p < ALP RUNX2 Osterix and ALP bone sialoprotein II 0.001 (5 % PR)] not stated p < p 0.05), , p < ( Findings RUNX2 0.001) in PL BMP2 p < and were higher in PL were 0.05 (2.5 % PR); Osterix expression p < SPARC Osterix BMP2 NM increases levels of osteopontin expression, with a significant NM increases levels and : no differences between groups at all time points : no differences between 0.01); displays trend toward downregulation of 0.01); displays trend toward higher than SPP1 p < ( : increased expression HPLO > HPLF FBS at both day 7 and 14 ( downregulation of as a tendency toward as well not modified by PL osteocalcin and PR upregulates PR downregulates Day 0 (before differentiation): expression of both upon addition of PL Day 14: not stated RUNX2 ALP Both Osteocalcin PL upregulates expression of MMP13 RUNX2 Dexa group: same trend as PL observed 2.5 to 5 % PR in increase noted in 2.5 % PR ( of osteopontin expression significantly 2.5 to 5 % PR in OM increases levels [compared to controls, Upregulation of both genes by P-PRP-CM > L-PRP-CM FBS ( RUNX2 • • • • • • • • • • • • • • mol/L Osteogenesis −7 Groups compared 1 10 % FBS (control) L-PRP-CM 10 % FBS (control) 10 % PR FBS (control) PL P-PRP-CM ITS (control) PL PL + 10 ng/mL TGF-b1 dexamethasone + 50 µ g/mL ascorbic acid, day 14 and 2 10 % FBS + 1 ng/mL FGF2 10 % FBS + 5 PL 10 % PL 5 % PL Culture medium (un-supplemented, as control) 10 nmol/L dexamethasone (Dexa) 10 % PL Ve control) No PR (in NM, as − Ve 1 % PR (NM) 2.5 % PR (NM) 5 % PR (NM) control) No PR (in OM, as + Ve 1 % PR (OM) 2.5 % (OM) 5 % PR (OM) FBS (control) HPLF HPLO • • • • • • • • • • • • • • • • • taken before (day 0) and after induction of differentiation (day 14) • • • • • • • • • • •

, SPP1 , Osterix , Osterix RUNX2 , , RUNX2 , RUNX2 RUNX2 bone sialoprotein RUNX2 , , Osteopontin ALP Genes assessed osteopontin Osteocalcin , ALP Osteocalcin 2 BMP2 BMP2, RUNX2, SPARC PL PL PL PL PL PR PR Platelet Platelet product PRP-CM , , , et al. , 2014 et al. et al. , 2017 , 2010 , 2016 , 2013 et al. et al. et al. 2012 2012 2014 et al. et al. Author (year) Author Yin Jonsdottir-Buch Verrier Infante Ben Azouna Gottipamula Kosmacheva Amable Table 13a. Gene expression of BM-MSCs following PP exposure. following of BM-MSCs expression Gene 13a. Table

www.ecmjournal.org 308 J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs : : (3- SOX9 COMP peaked at RUNX2 0.019); (6-fold) and MMP13 BMP2 p < = 0.050); p BMP2 and COL2 , throughout whole : FBS > PL (5-100-fold higher 0.05); level of 0.05); level p < : 4-fold at day 21 ( ACAN APOE : 3-fold at day 14 ( L-PDGS 0.05) not stated 0.05)] 0.05) and p p < 0.001)] 0.05) ( ACAN COL2A1 p = p < p < 0.05) p < Findings 0.05) not stated GLUT4 p < p , , p < COL1A1 PLIN , ACAN 0.028); levels low in 0.028); levels 0.024) and 21 ( SOX9 p = : reduced by PL [ : increased by PL [ p = FABP4 , osteogenic medium): higher expression of ALP (1-fold) and osteogenic medium): higher expression of osteogenic medium): higher expression of osteogenic medium): higher upregulation in PRC for SOX9 vs. vs. vs. (2-fold) at day 8 ( COMP (4-fold) in PRC group ( undetectable in all conditions 0.05) CEBPA and : FBS > PL ( : unaltered by PL , at 12 h: PRC > control ( and up to 24 h: PRC > control at 6, 12, h ( at 0 and 48 h: no difference COL1 p < : significantly induced in PL h: no difference between groups at 24 h: no difference between aPRP upregulates aPRP downregulates COL2A1 PPARg3 expression) LXRα SREBP1 L-PGDS differentiation period Day 8 (PRC fold), Day 16 (PRC osteopontin (5-fold) in PRC group ( Day 24 (PRC osteopontin Osteonectin: persistently upregulated in osteogenic medium when compared to PRC ( ALP at 12 h: PRC > control (PRC 8.2-fold higher than serum) ALP BMP2 12 h BMP2 RUNX2 COL2A1 2-fold at days 14 ( ACAN 3-fold at day 14 and 6-fold 21 ( PR concentrations do not alter expression of Duration of treatment with 10 % PR: increased duration PR exposure = inhibition of chondrogenesis Expression of both genes: control > PR, PL upregulates expression of • • • • • • • • • • • • • • • • • • • • • • (control) ® mol/L dipogenesis -7 Osteogenesis A Chondrogenesis /mL (platelets/mL) Groups compared 8 10 % PL Culture medium (un-supplemented, as control) 10 nmol/L dexamethasone (Dexa) TGF-b3 (control) PR 10 % FBS (control) 10 % PR-CM 2.5 % FBS (control) 2.5 % PRC ITS (control) PL PL + 10 ng/mL TGF-b1 dexamethasone + 50 µ g/mL ascorbic acid), days 14 and 21 FBS (control) PL Serum free medium (control) PR at 2 × 10 Osteogenic medium, StemPro 15 % PRC • • • • • • • • • • • • • • Compared effect of duration (with 10 % PR: days 1, 3, 7, 21) and PR concentration (1 %, 5 10 20 %) • • • •

,

,

and and , and COMP , COLI , RUNX2 , COL2 GLUT4

, , osteopontin osteocalcin PPARG , , SOX9 APOE CEPBA RUNX2 L-PGDS COL1A1 BMP2 , COL2A1 BMP2 PLIN SREBP1, LXRα , , , ACAN Genes assessed Adipocyte master ALP RUNX-2 Adipocyte markers: ALP regulator: Lipocyte transcription osteonectin ACAN, COL2A1, SOX9 ACAN FABP4 factors: PL PL PL PR PR PR PRC PRC Platelet Platelet product , 2014 , 2008 , 2016 , 2004 , 2017 , 2010 , 2012 , 2018 et al. et al. et al. et al. et al. et al. et al. et al. Author (year) Author Liou Lange Verrier Infante Gruber Samuel Parsons Amable Table 13b. Gene expression of BM-MSCs following PP exposure. Table

309 www.ecmjournal.org J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs MMP13 and COL10 not stated p , not stated p 0.01) p < ( POU5F1 Findings and MMP13 : not modified by PL SOX2 0.001) significantly upregulated by L-PRP-CM when compared with compared when L-PRP-CM by upregulated significantly p < COL10A1 iNOS and and VEGF , not detectable COX-2 PR slightly upregulates TERT Day 0 (before differentiation): expression of both PPARg decreased upon addition upon decreased PPARg both of expression differentiation): (before 0 Day of PL in 10 % Day 14 (after osteogenic induction): decreased expression of PPARg FBS + 5 % PL; effect on other culture conditions not stated FBS or P-PRP-CM ( Changes in concentrations of PR do not affect expression All adipogenic markers upregulated by PR, Both PL upregulated expression of COL1A1 • • • • • • • • • mol/L mol/L −7 10 + MMP dipogenesis Hypertrophy A 50 µ g/mL ascorbic acid), day Pluripotency and proliferation Pluripotency + Immunomodulation/inflammation ng/mL ng/mL TGF-b1 Groups compared 10 +

10 % FBS (control) L-PRP-CM P-PRP-CM 10 % FBS + 1 ng/mL FGF2 10 % FBS + 5 PL 10 % PL 5 % PL 10 % FBS (control) 10 % PR 10 % FBS (control) 10 % PR-CM dexamethasone 14 and 21 TGF-b3 (control) 10 % PR Culture medium (un-supplemented, as control 10 nmol/L dexamethasone (Dexa) 10 % PL ITS (control) PL PL • • • • • • • • • • • • • taken before and after induction of differentiation • • • • • • T COL10A1 , MMP13 , MMP13 VEGF , PPARG, LPL PPARG, COX-2, iNOS COL10 Genes assessed POU5F1, SOX2, TER ADIPOQ, CEPBA, PPARG COL1A1 PL PL PL PR PR PR Platelet Platelet product PRP-CM , , 2014 , 2014 et al. , 2017 , 2010 , 2018 , 2016 et al. et al. et al. et al. 2012 et al. et al. Author (year) Author Yin Liou Verrier Infante Ben Azouna Amable Amable Table 13c. Gene expression of BM-MSCs following PP exposure. following of BM-MSCs expression Gene 13c. Table

www.ecmjournal.org 310 J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs the zone of injury – further guaranteeing successful of BM-MSCs to PPs, as seen commonly in clinical bone repair. Moreover, the release of growth factors practice, has not been investigated. Therefore, further (HGF, G-CSF, TGF- 1, TGF- 2), MMPs (MMP1, collaborative multi-centre in vitro research in humans MMP3, MMP7, MMP8, MMP13) and extracellular modelled to reflect clinical practice is required to matrix molecules (heparanβ sulphate,β elastin, laminin, better understand the effects of PP exposure on the collagen I and III), with multiple regulatory roles that biophysiological function(s) of BM-MSCs in human. enhance bone regeneration, was likewise larger in BM-MSCs exposed to PP (Amable et al., 2014). Taken together, these results agreed with findings from Conflicts of interest and source of funding functional assays. BM-MSC gene expression has also been assessed None of the authors has any conflict of interest by several studies. Proliferative gene markers in relevant to the study and no funding was received BM-MSCs were found to be consistently upregulated for the completion of this project. following PP exposure, confirming the findings from differentiation assays (Amable et al., 2014). The effects of PP on the expression of BM-MSC gene markers References for osteogenesis, chondrogenesis, adipogenesis and immunomodulation were rather variable and Agis H, Kandler B, Fischer MB, Watzek G, Gruber dependant on the gene assessed. Alterations to R (2009) Activated platelets increase fibrinolysis of the physiological behaviour and characteristics of mesenchymal progenitor cells. J Orthop Res 27: 972- subcultured BM-MSCs following multiple passages 980. using highly variable media (Ganguly et al., 2019) Amable PR, Teixeira MV, Carias RB, Granjeiro could potentially explain the inconsistency in gene JM, Borojevic R (2014) Mesenchymal stromal cell expression observed. proliferation, gene expression and protein production The present systematic review highlights the in human platelet-rich plasma-supplemented usefulness of PPs, many of which could be harvested media. PLoS One 9: e104662. DOI: 10.1371/journal. and delivered autologously. Until research would pone.0104662. advance in a manner whereby the array of most Andia I, Maffulli N (2019) New biotechnologies potent growth factors, cytokines and proteins could for musculoskeletal injuries. Surgeon 17: 244-255. be commercially produced and compliant with Ben Azouna N, Jenhani F, Regaya Z, Berraeis L, Ben the Good Manufacturing Practice, PPs remain a Othman T, Ducrocq E, Domenech J (2012) Phenotypical useful adjunct to bone healing. To ensure that the and functional characteristics of mesenchymal stem strengths of these PPs are put to the best use and cells from bone marrow: comparison of culture using not risk them being disregarded in the history of different media supplemented with human platelet medicine as another ‘mythic wonder drug’ (Wang lysate or fetal bovine serum. Stem Cell Res Ther 3: 6. and Avila, 2007), there are several aspects that the DOI: 10.1186/scrt97. clinical, scientific community and the industry are Bernardi M, Agostini F, Chieregato K, Amati E, responsible for, including: (1) standardised use of Durante C, Rassu M, Ruggeri M, Sella S, Lombardi terminology; (2) standardised preparation protocols; E, Mazzucato M, Astori G (2017) The production (3) standardised protocols for clinical application; (4) method affects the efficacy of platelet derivatives to consistent description of source, volume, method of expand mesenchymal stromal cells in vitro. J Transl harvesting/processing and contents of these PPs used. Med 15: 90. DOI: 10.1186/s12967-017-1185-9. Only then, further progress and breakthrough would Bernardi M, Albiero E, Alghisi A, Chieregato be made in the fields of bone regeneration and tissue K, Lievore C, Madeo D, Rodeghiero F, Astori G engineering. (2013) Production of human platelet lysate by use of ultrasound for ex vivo expansion of human bone marrow-derived mesenchymal stromal cells. Conclusion Cytotherapy 15: 920-929. Bielecki T, Gazdzik TS, Szczepanski T (2008) In vitro studies in human have demonstrated Benefit of percutaneous injection of autologous the multi-faceted ‘priming effect’ of PPs on the platelet-leukocyte-rich gel in patients with delayed biophysiological functions of BM-MSCs. PPs have union and nonunion. Eur Surg Res 40: 289-296. been shown to improve proliferation, migration, Bishop JA, Palanca AA, Bellino MJ, Lowenberg osteogenic differentiation, immunomodulation, DW (2012) Assessment of compromised fracture reaction to apoptotic stress as well as pro-angiogenic healing. J Am Acad Orthop Surg 20: 273-282. and pro-inflammatory capacities of BM-MSCs. The Bustos ML, Huleihel L, Kapetanaki MG, Lino- lack of standardised terminology and protocols Cardenas CL, Mroz L, Ellis BM, McVerry BJ, Richards surrounding the use of PPs was highlighted, along TJ, Kaminski N, Cerdenes N, Mora AL, Rojas M with other factors that unfortunately restrict the (2014) Aging mesenchymal stem cells fail to protect transferability of these findings into clinical practice. because of impaired migration and antiinflammatory Furthermore, the impact of short-term exposure response. Am J Respir Crit Care Med 189: 787-798.

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Murphy MB, Blashki D, Buchanan RM, Yazdi IK, release growth factors and induce cell migration in Ferrari M, Simmons PJ, Tasciotti E (2012) Adult and vitro. Clin Orthop Relat Res 473: 1635-1643. umbilical cord blood-derived platelet-rich plasma for Scherer SS, Tobalem M, Vigato E, Heit Y, mesenchymal stem cell proliferation, chemotaxis, and Modarressi A, Hinz B, Pittet B, Pietramaggiori G cryo-preservation. Biomaterials 33: 5308-5316. (2012) Nonactivated versus thrombin-activated Nguyen ATM, Tran HLB, Pham TAV (2019) In vitro platelets on wound healing and fibroblast-to- evaluation of proliferation and migration behaviour myofibroblast differentiationin vivo and in vitro. Plast of human bone marrow-derived mesenchymal stem Reconstr Surg 129: 46e-54e. cells in presence of platelet-rich plasma. Int J Dent Skific M, Golemovic M, Crkvenac-Gornik K, 2019: 9639820. DOI: 10.1155/2019/9639820. 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Schallmoser K, Henschler R, Gabriel C, Koh MBC, Burnouf T (2020) Production and quality requirements of human platelet lysate: a position Discussion with Reviewer statement from the working party on cellular therapies of the International Society of Blood Reviewer: In addition to the variations due to many Transfusion. Trends Biotechnol 38: 13-23. preparation protocols, it would be interesting to Schar MO, Diaz-Romero J, Kohl S, Zumstein MA, have the authors’ opinion on the inter-individual Nesic D (2015) Platelet-rich concentrates differentially variations. Prepared in similar conditions, PPs from

www.ecmjournal.org 314 J Vun et al. Effects of platelet products on bio-physiology of BM-MSCs different patients will have different properties (in components used (e.g. only growth factors in PL/ term of efficacy). Do you think that the preparation PR; combination of both cells and growth factors of PPs has more influence on its outcome than the in PRP) as well as its preparation and incubation patient of origin? period with BM-MSCs are all flexible and remains at Authors: Factors affecting BM-MSC functions are the clinician’s disposal when treating patients with multifactorial. Both PP preparation method and autologous PPs. donor play equally important roles in determining the outcomes when using PPs clinically to optimise BM-MSC function. However, whilst patient’s biology Editor’s note: The Scientific Editor responsible for is often not-modifiable, the choice of PPs and its this paper was Martin Stoddart.

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