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Research ArticleLipogems Product Treatment Increasesthe Proliferation Rate of Human Tendon Stem Cells withoutAffecting Their Stemness and Differentiation Capability

Pietro Randelli12 Alessandra Menon1 Vincenza Ragone1 Pasquale Creo1

Sonia Bergante1 Filippo Randelli1 Laura De Girolamo3 Umberto Alfieri Montrasio3

Giuseppe Banfi34 Paolo Cabitza1 Guido Tettamanti1 and Luigi Anastasia12

1 IRCCS Policlinico San Donato San Donato Milanese 20097 Milan Italy2Department of Biomedical Sciences for Health University of Milan 20133 Milan Italy3IRCCS Istituto Ortopedico Galeazzi 20161 Milan Italy4Universita Vita-Salute San Raffaele 20132 Milan Italy

Correspondence should be addressed to Pietro Randelli pietrorandelliunimiit and Luigi Anastasia luigianastasiaunimiit

Received 7 August 2015 Revised 30 October 2015 Accepted 11 November 2015

Academic Editor Li Jin

Copyright copy 2016 Pietro Randelli et alThis is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

Increasing the success rate of rotator cuff healing remains tremendous challenge Among many approaches the possibility ofactivating resident stem cells in situ without the need to isolate them from biopsies could represent valuable therapeutic strategyAlong this line it has been recently demonstrated that lipoaspirate product Lipogems contains and produces growth-factorsthat may activate resident stem cells In this study human tendon stem cells (hTSCs) from the rotator cuff were cocultured in atranswell system with the Lipogems lipoaspirate product and compared to control untreated cells in terms of cell proliferationmorphology stem cell marker and VEGF expression and differentiation and migration capabilities Results showed that theLipogems product significantly increases the proliferation rate of hTSCs without altering their stemness and differentiationcapability Moreover treated cells increase the expression of VEGF which is crucial for the neovascularization of the tissue duringthe healing process Overall this study supports that directly activating hTSCs with the Lipogems lipoaspirate could represent anew practical therapeutic approach In fact obtaining a lipoaspirate is easier safer and more cost-effective than harvesting cellsfrom tendon or bone marrow biopsies expanding them in GMP facility and then reinjecting them in the patient

1 Introduction

Rotator cuff tears represent the vast majority of shoulderinjuries in adult patients and are a common contributingfactor to shoulder pain and occupational disability whoseprevalence is rising due to the increase of the world popu-lation age [1] Although surgical procedures for rotator cuffrepair have evolved and improved over the past decadesa high rate of retear is still observed [2ndash4] This is mainlycaused by a failure of tendon healing especially in the caseof the supraspinatus tendon [5ndash9] Therefore increasing thesuccess rate of rotator cuff healing remains a tremendous

challenge for orthopedic surgeons which encourages thedevelopment of new alternative therapies [10ndash15] In factonce injured tendons do not completely regain the normalstructural and biomechanical properties resulting in theformation of scar tissue adhesions fatty infiltration andmatrix disorganization which increase the risk of retearAmong several factors tendon poor vascularization reducesthe availability of oxygen growth-factors and other nutrientsnecessary for tissue regeneration and significantly affects thequality and speed of the tendon healing response Thereforein considering new strategies for tendon engineering thegoal of promoting neoangiogenesis is vital to accelerate

Hindawi Publishing CorporationStem Cells InternationalArticle ID 4373410

2 Stem Cells International

the healing process Moreover several studies have shownthat different types of stimuli could activate the nor-mal growth-factor-mediated healing cascades [16] Alongthis line it has been recently demonstrated that lipoaspi-rates contain and produce growth-factors such as platelet-derived growth-factor (PDGF) fibroblast growth-factor(FGF) transforming growth-factor beta (TGF-120573) and vas-cular endothelial growth-factor (VEGF) which are known toplay important regulatory roles in cellular functions includ-ing adhesion chemotaxis proliferation migration matrixsynthesis differentiation and angiogenesis [16ndash18] The cellfraction responsible for growth-factor production and regu-lation is mainly the stromal vascular fraction which couldhave acceleratory effect on the healing process of injuredtendons [19] Given the tissue availability as well as theeasy and minimally invasive access to tissue sources adiposetissue may in fact represent a potential choice for tendonrepair and regeneration [20] Among many approaches aninnovative technology Lipogems provides a nonexpandedready-to-use and microfragmented adipose tissue that isexpected to have the peculiar advantage of maintaining anintact stromal vascular niche harboring cellular elementswith mesenchymal stem cell and pericyte characteristics [21]This system works through a mild mechanical tissue cluster-size reduction in a completely closed system avoiding the useof any enzyme additives and other additional manipulations(ie centrifugation and subfractional harvesting) [21]

In a previous study on the Lipogems product Bianchiet al speculated that the action of the Lipogems product dueto an intact stromal vascular niche could involve the secre-tion of trophic mediators delivering instructive messagesthat functionally promote a more compliant regenerativeenvironment within the recipient tissue [21] In particularthe lipoaspirate could stimulate the self-healing processesby activating resident stem cells through paracrine mecha-nismsTherefore the Lipogems product may be instrumentalfor activating resident progenitor cells to proliferate andcontribute to the tendon healing process Actually it hasbeen recently demonstrated that also the human rotatorcuff contains a reservoir of progenitor cells which can beisolated and expanded in vitro [22ndash26] Thus the availabilityof a completely closed disposable system for adipose tissueprocessing could allow the procedure to be readily safelyand economically performed in clinical settings to affordsignificant tissue repair overcoming the difficulty of an exvivo expansion and the complexity of the current GoodManufacturing Practice (GMP) requirements for preparingcells for a therapeutic use [27]

On these bases the aim of this study was to assessthe effects of the Lipogems product on primary cultures ofhuman tendon stem cells (hTSCs)

2 Materials and Methods

21 Cell Cultures Human tendon stem cells (hTSCs) wereisolated from supraspinatus tendon specimens collectedduring arthroscopic rotator cuff repair according to ourprevious procedure [22] The isolated hTSCs were cul-tured in normal growth medium composed of 120572-Minimal

Essential Medium (120572-MEM) (Sigma-Aldrich) supplementedwith 2mM glutamine (Euroclone) 1 antibiotic-antimycoticmixture (Euroclone) and 20 (vv) fetal bovine serum (FBS)(HyClone Thermo Fisher Scientific) at 37∘C in a humidifiedatmosphere of 5 CO

2 The medium was changed every 2-3

days All experiments were carried out with cells at passagefour after isolation

22 Processing of Adipose Tissue with the Lipogems DeviceHuman subcutaneous adipose tissue samples were obtainedwith patient informed consent from abdominal lipoaspira-tion procedures performed before arthroscopic rotator cuffrepair and processed by using the Lipogems device accordingto the manufacturerrsquos instructions previously described [21]Avoiding the presence of air the lipoaspirate tissue wassubjected to a first cluster reduction obtained by pushingthe lipoaspirate from the syringe into the device through thefirst large filter (blue cap) and allowing the correspondingquantity of saline to exit towards the wasting bag The fivestainless steel beads contained in the device were essential toobtain a temporary emulsion between oil blood and salinewhich could be washed away against density following thecurrent of saline moved by gravity in the wasting bag Afterthis washing step (the flowing solution appears clear andthe lipoaspirate yellow) the saline flux was stopped and thedevice was reversed (gray cap up) leading to the secondadipose cluster reduction Such reduction was obtained bypushing the floating adipose clusters through the secondcutting hexagonal filter (grey cap) pushing fluid from belowwith a 60mL syringe The reduced cluster was collected inanother 60mL syringe placed above and positioned to gentlydecant the Lipogems product by gravity in order to removeexcessive saline solution Amean of 50mL of lipoaspirate wascollected and processed with the Lipogems device to obtainan amount of 20mL of the final Lipogems product whichwastransferred to several 10mL syringes to be reinjected in thesame patient or used in our experiments

23 Lipogems Coculture Experiments of hTSCs with theLipogems Product hTSCs were seeded on the bottom of 6-well plates in normal growth medium Twenty-four hoursafter seeding a volume of 250 120583L of the freshly processedLipogems product was transferred to the transwell polycar-bonate microporous inserts (04120583m membrane pore sizeFalcon BD Figure 1) above hTSCs monolayers allowing forpotential diffusion of soluble mediators but preventing cell-cell contact Control cells were cultured under the sameconditions without the Lipogems product The medium wasreplaced every 48 h in all experiments To study the effectsof the Lipogems treatment on multilineage differentiationpotential of hTSCs cells were cultured with the Lipogemsproduct in osteogenic and adipogenic differentiation mediaas described below

24 Cell Morphology and Proliferation For analysis of cellviability hTSCs were plated at a concentration of 26 times 103cellscm2 and exposed to the Lipogems product for 96 has described above Cell morphology was examined daily

Stem Cells International 3

Physiologicalsaline solution

Solutions waste

The Lipogems product iscollected and transferred to

the transwell insert

Transwell insert containing Lipogemsproduct (250120583L) on microporous

membrane (04 120583m pore size)hTSCs are seeded on the well below


Figure 1 Schematic illustration of the Lipogems device and the transwell coculture system of hTSCs with the Lipogems product Thelipoaspirate was processed using the Lipogems device to obtain the final Lipogems product A transwell coculture systemwas used to study theeffects of the Lipogems product on primary cultures of hTSCs Cells were seeded on the bottom of a 6-well plate in normal growth mediumTwenty-four hours after plating the Lipogems product was transferred in the upper well of a transwell cell culture system (250120583Ltranswellinsert) separated from hTSCs in the lower well by a 04 120583m microporous polycarbonate membrane hTSCs and Lipogems product weremaintained in coculture for successive analyses

with a phase-contrast microscope (Axiovert 40 CFL Zeissequipped with a Moticam 2300 camera Motic) to assess theeffects of the Lipogems product on hTSCs phenotype Cellgrowth curves were prepared by harvesting with Trypsin-EDTA solution (Sigma-Aldrich) and then counting witha Countess Cell Counter (Invitrogen Life Technologies)according to the manufacturerrsquos procedure Cell viability wasdetermined by trypan blue dye exclusion assay All assayswere carried out in triplicate for each sample

25 Cell Apoptosis Apoptosis was measured by flow cytome-try on Lipogems-treated and control cells at 0 48 and 96 hof treatment using Annexin V-FITC Apoptosis DetectionKit (Enzo Life Sciences) according to the manufacturerrsquosprotocol Briefly adherent cells were trypsinized washedin PBS by gentle shaking and resuspended with 200 120583L ofa specific Binding Buffer (10mM HEPESNaOH pH 74140mMNaCl 25mMCaCl

2) containing 5120583L of Annexin V-

FITC After incubation for 10min in the dark at room tem-perature cells were washed in PBS resuspended in 200120583L ofBinding Buffer and then stainedwith 10120583LPropidium Iodide(20120583gmL) Samples were acquired with a Navios FlowCytometer (Beckman Coulter) and analyzed using Kaluza 12software (Beckman Coulter)

26 Flow Cytometric Analysis After a four-day treatmentLipogems-treated and control cells were characterizedby flow cytometry for the expression of key stem cell

markers Flow cytometry analysis was performed on 1 times 105cellssample Briefly aspecific binding sites were blockedwith a blocking solution (50 1x PBS 50 FBS) for 30minat room temperature and washed twice with PBS at 4∘CCells were stained with fluorochrome-conjugated mouseantihuman antibodies at the optimal concentration (1 20dilution) in PBS for 10min at 4∘C and washed twice withPBS at 4∘C Cell characterization was performed usingthe following antibodies 120572CD9 FITC 120572CD73 FITC120572HLA-DR FITC 120572CD13 PE 120572CD29 PE 120572CD44 PE120572CD45 PE 120572CD71 PE 120572CD90 PE 120572CD105 PE 120572CD106PE 120572CD34 PerCP-eFluor710 120572CD166 PerCP-eFluor710120572HLA-ABC FITC 120572NG2 PE and 120572SSEA-4 PE (all fromeBioscience) 120572Lineage Cocktail FITC 120572CD18 PE 120572CD140aPE 120572CD140b APC 120572CD146 PE and 120572Stro-1 Alexa Fluor 647(all from BioLegend) and 120572CD117 PE (Miltenyi Biotec) Therespective isotype antibodies were used as controls Sampleswere acquired with a Navios Flow Cytometer (BeckmanCoulter) and data were processed with Kaluza software(Beckman Coulter)

27 Adipogenic Differentiation To assess the effects of theLipogems product on the adipogenic differentiation capacityof hTSCs cells were plated at a concentration of 3 times 104cellscm2 and preconditioned with the Lipogems productfor 96 h in normal growth medium and then switched toDMEM-low glucose (Sigma-Aldrich) 10 FBS (HyClone


Thermo Fisher Scientific) 4mM L-glutamine (Euroclone)and 1 antibiotic-antimycotic mixture (Euroclone) withthe addition of the mesenchymal stem cell adipogenesiskit (Millipore) for 21 days according to the manufacturerrsquosinstructions At day 21 Oil Red O solution (Millipore) wasused to stain lipid droplets of derived adipocytes accordingto themanufacturerrsquos procedures All photomicrographswereacquired with Axiovert 40 microscope (Zeiss) equipped witha Moticam 2300 camera (Motic) The adipogenic mediumwas changed every 2-3 days

28 Osteogenic Differentiation To assess the effects of theLipogems product on the osteogenic differentiation capacityof hTSCs cells were plated at a concentration of 3 times 104cellscm2 and preconditioned with the Lipogems productfor 96 h in normal growth medium and then switchedto the osteogenesis induction medium which was con-stituted of DMEM-low glucose (Sigma-Aldrich) 10 FBS(HyClone Thermo Fisher Scientific) 4mM L-glutamine(Euroclone) and 1 antibiotic-antimycotic mixture (Euro-clone) supplemented with 01120583Mdexamethasone 50 120583gmLL-ascorbic acid-2-phosphate and 10mM120573-glycerophosphate(all reagents from Sigma-Aldrich) for 17 days At day 17Alizarin Red solution (Millipore) was used to detect calciumdeposition in derived osteoblasts according to the manufac-turerrsquos instruction All photomicrographs were acquired withAxiovert 40 microscope (Zeiss) equipped with a Moticam2300 camera (Motic) The osteogenic medium was changedevery 2-3 days

29 RNA Extraction and Real-Time PCR Total RNA wasisolated using TRIzol Reagent (Ambion Life Technologies)and 1 120583g of extracted RNA was reverse transcribed to cDNAusing the iScript cDNA synthesis kit (BioRad) according tothe manufacturerrsquos instructions Real-Time PCR was per-formed in a 96-well plate with 10 ng of cDNA as template02 120583M primers and 1x Power SYBR Green PCR MasterMix (Applied Biosystems Life Technologies) in 20120583L finalvolume per well using a StepOnePlus Real-Time PCR System(Applied Biosystems) The mRNA levels of Tenomodulin(TNMD) collagen type I alpha-1 (COL1A1) Tenascin CNanog homeobox (Nanog) octamer-binding transcriptionfactor 4 (Oct4) kruppel-like factor 4 (KLF4) vascularendothelial growth-factor (VEGF) peroxisome proliferator-activated receptor-120574 (PPAR-120574) lipoprotein lipase (LPL) alka-line phosphatase (ALP) myogenin (MYOG) and myogenicdifferentiation 1 (MYOD) were assessed Ribosomal pro-tein S14 (S14) was used as reference gene in quantitativeanalysis We performed a series of validation experimentsconfirming that S14 is constitutively and stably expressedin all cell samples with an amplification efficiency of 978and linear response in the expression range studied (Supple-mentary Figure 1 in Supplementary Material available onlineat httpdxdoiorg10115520164373410) Primer sequencesare as follows TNMD forward 51015840-TGTATTGGATCAATC-CCACTCTAAT-31015840 and reverse 51015840-TTTTTCGTTGGCAGG-AAAGT-31015840 COL1A1 forward 51015840-GGGATTCCCTGGACC-TAAAG-31015840 and reverse 51015840-GGAACACCTCGCTCTCCA-31015840

Tenascin C forward 51015840-CGGGGCTATAGAACACCAGT-31015840 and reverse 51015840-AACATTTAAGTTTCCAATTTCAGG-TT-31015840 Nanog forward 51015840-GGTCCCAGTCAAGAAACA-GA-31015840 and reverse 51015840-GAGGTTCAGGATGTTGGAGA-31015840Oct4 forward 51015840-AGGAGAAGCTGGAGCAAAA-31015840 andreverse 51015840-GGTCGAATACCTTCCCAAA-31015840 KLF4 for-ward 51015840-GACTTCCCCCAGTGCTTC-31015840 and reverse 51015840-CGTTGAACTCCTCGGTCTC-31015840 VEGF forward 51015840-CAA-CATCACCATGCAGATTATGC-31015840 and reverse 51015840-TCG-GCTTGTCACATTTTTCTTGT-31015840 PPAR-120574 forward 51015840-TTCCTTCACTGATACACTGTCTGC-31015840 and reverse 51015840-GGAGTGGGAGTGGTCTTCCATTAC-31015840 LPL forward51015840-AGAGAGAACCAGACTCCAATG-31015840 and reverse 51015840-GGCTCCAAGGCTGTATCC-31015840 ALP forward 51015840-CGC-ACGGAACTCCTGACC-31015840 and reverse 51015840-GCCACCACC-ACCATCTCG-31015840MYOG forward 51015840-AAGAAGGGGAGA-GGAACAGC-31015840 and reverse 51015840-GCAACTTCAGCACAG-GAGAC-31015840 MYOD forward 51015840-GCTAGGTTCAGCTTT-CTCGC-31015840 and reverse 51015840-CACCTGCTACATTTGGGACC-31015840 and S14 forward 51015840-GTGTGACTGGTGGGATGAAGG-31015840 and reverse 51015840-TTGATGTGTAGGGCGGTGATAC-31015840

Amplification Protocol An initial denaturation at 95∘C for3min followed by 40 cycles of 5 s each at 95∘C and 30 s at57∘C Relative quantification of target genes was performed intriplicate analyzed using the 2minusΔΔCt method and normalizedto the corresponding S14 values

210 Cell Migration by Wound-Healing Assay Wound-healing assay was performed as previously described [28]hTSCs were grown to confluence in 6-well plates and treatedwith the Lipogems product or cultured in the growthmediumalone A sterile P200 pipet tip was used to create a scratchacross the cell monolayer Then cultures were washed oncewith 1mL of growth medium to remove the damaged anddetached cells After replacing the medium hTSCs wereallowed to grow for 48 h At different time points cell cultureswere examined with a phase-contrast microscope (Axiovert40 CFL Zeiss equippedwith aMoticam 2300 cameraMotic)and images of the same scratch fields were acquired at time0 and after 17 20 25 30 and 42 h from the scratch Thegap area between the cells was calculated in each acquiredimage using software ImageJ The migration rate was basedon the measure of the recovered wound area (experimentaldata expressed in percentage) All assays were carried out intriplicate for each sample

211 Statistical Analysis Statistical analysis was performedusing GraphPad Prism v 60 software (GraphPad SoftwareInc) Data were typical results from three replicate experi-ments for each of the three patients-derived cell lines andwere expressed as mean plusmn standard deviation (SD) Pairedcomparisons were performed by two-tailed 119905-test The signif-icance level was set at 119901 value lower than 005

3 Results

To assess the effects of the Lipogems product on hTSCscells were cocultured for up to 96 h with freshly obtained


hTSCs (control) hTSCs + Lipogems

(a)

96

Cell growth curve1500

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1000

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72

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0

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)

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lowast

lowastlowast

lowastlowast

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Figure 2 Effects of the Lipogems product on hTSCs morphology and proliferation (a) Phase-contrast microphotographs (originalmagnification times10) and (b) cell growth curves of hTSCs during 96 h treatment with the Lipogems product in normal growth medium andcompared to control cells All experiments were performed in triplicate Error bars show the mean plusmn SD of three different experiments 119901values were calculated using Studentrsquos 119905-test Only 119901 values lt 005 are indicated lowast119901 lt 005 lowastlowast119901 lt 001 as compared to control cells

Lipogems product in a transwell cell culture system asdescribed in the Methods and graphically outlined inFigure 1 Control hTSCs were cultured using the same tran-swell system but without adding the Lipogems product

31 Effects on Cell Morphology and Proliferation Cell mor-phology analysis revealed no significant differences betweenLipogems-treated and control cells during 96 h treatment(Figure 2(a) cell morphology at 48 h after treatment) Cellgrowth analysis revealed a statistically significant increase incell proliferation of Lipogems-treated cells as compared tocontrols the effect being statistically significant after 48 h Inparticular hTSCs showed a significant increase of 24 (119901 lt001) 97 (119901 lt 005) and 5 (119901 lt 001) in the presenceof the Lipogems product at 48 72 and 96 h respectively ascompared to control cells (Figure 2(b))

32 Effects on Cell Apoptosis Apoptosis was measured byflow cytometry on hTSCs at 0 48 and 96 h of treatment withthe Lipogems product using Annexin V-FITC and comparedto control cells at the same time points (Figure 3) Results

revealed no significant apoptosis in all tested samples (alwaysbelow 05) with no significant differences between controland Lipogems-treated cells

33 Effects on Cell Phenotype by Flow Cytometry The expres-sion of key stem cell markers was evaluated by flow cytom-etry in hTSCs after a 96 h treatment with the Lipogemsproduct and compared to control untreated cells Results ofcomparative flow cytometry analyses showed no significantdifferences between the two groups for all tested markers(Table 1)

34 Effects on Tenogenic Adipogenic Osteogenic andMyogenic Differentiation Promotion We assessed whetherLipogems product treatment would induce hTSCs todifferentiate into other cell phenotypes We found that 96 hexposure to Lipogems product did not increase nor inducethe expression of mRNA levels of tenogenic (TenomodulinCOL1A1 Tenascin C) (Figure 4(a)) adipogenic (PPAR-120574 andLPL) osteogenic (ALP) and myogenic (MYOD MYOG)markers (Supplementary Figure 2)


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PIPI

PI PIPI

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Tim

e (h)

Annexin V

Annexin V Annexin V

Annexin VAnnexin V


Figure 3 Effects of the Lipogems product on hTSCs apoptosis Flow cytometric analysis of hTSCs survival rate at 0 48 and 96 h after thetreatmentwith the Lipogems product (right panel) as compared to control cells (left panel) throughdouble stainingwithAnnexinV-FITCandPI Early apoptotic cells (Annexin V-positivePI-negative) are localized in the lower right region late apoptotic and necrotic cells (AnnexinV-positivePI-positive) are localized in the upper regions and vital cells (double negative) are localized in the lower left region

35 Effects on Cell Plasticity To assess the effects of theLipogems product on the multidifferentiation capacity ofhTSCs cells preconditioned with the Lipogems product for96 h were induced to differentiate in vitro toward adipocytesand osteoblasts Figures 4(b) and 4(c) see Methods

Results showed no noticeable differences in the adipogenic(Figure 4(b)) and osteogenic (Figure 4(c)) differentiationof hTSCs suggesting no significant effects of Lipogemstreatment on the differentiation capacity towards these cellphenotypes


TNMD

hTSCs (control)

COL1A1

05

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NE NERQ05

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(a) Tendon marker expression

hTSCs + AM+LipogemshTSCs (control) + AM

AM = adipogenic medium

(b) Adipogenic differentiation

hTSCs + OM+LipogemshTSCs (control) + OM

OM = osteogenic medium

(c) Osteogenic differentiation

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KLF4

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(d) Stem cell marker expression

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Figure 4 Continued



hTSCs (control)hTSCs + Lipogems

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e (

)

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(f) Wound-healing assay

Figure 4 Effects of the Lipogems product on tendon marker expression (a) Gene expression of TNMD Tenascin C and COL1A1 by Real-Time PCR at 96 h of treatment with the Lipogems product Values are expressed as fold-changes relative to control cells Effects of theLipogems product on the in vitro differentiation of hTSCs toward the adipogenic and the osteogenic phenotypes Adipogenic and osteogenicdifferentiation ability of hTSCs cultured with the Lipogems product in the appropriate differentiation medium was evaluated by Oil Red O(b) and Alizarin Red-S (c) staining respectively (b) Lipid intracellular droplets (red) in the adipocytes were stained with Oil Red O solution(c) Alizarin Red-S staining revealed the presence of calcium deposits (yellowish-brown) Typical results are shown Original magnificationtimes10 Effect of Lipogems treatment on stem cells marker (Nanog Oct4 and KLF4) (d) and VEGF (e) expression by Real-Time PCR at 96 h oftreatment with the Lipogems product Values are expressed as fold-changes relative to control cells Effect of the Lipogems product on hTSCsmigration (f) Representative time-lapse migration images of control and Lipogems-treated cells Images were acquired at 0 and 25 h in invitro wound-healing assay Original magnification times5 The migration rate was measured by quantifying the total area of the wounded regionlacking cells The average percentages of recovered area obtained from three different experiments at 17 20 25 30 and 42 h of treatment withthe Lipogems product as compared to control cells All quantitative data are expressed as the means plusmn SD of three different experiments 119901values were calculated using Studentrsquos 119905-test Only 119901 values lt 005 are indicated lowastlowast119901 lt 001 compared to control cells

36 Effects on Stem Cell Marker and VEGF Expression Toevaluate the effects of Lipogems treatment on hTSCs stem-ness the expression of Nanog Oct4 and KLF4wasmeasuredby Real-Time PCR and compared to that of control untreatedcells (Figure 4(d)) Results revealed no significant differencesin stem cell marker expression between Lipogems-treatedand control cells Analysis of the VEGF a key markerinvolved in vasculogenesis and angiogenesis by Real-TimePCR revealed a significantly higher mRNA expression levelabout 13-fold (119901 lt 001) in Lipogems-treated cells ascompared to control cells (Figure 4(e))

37 Effects on Cell Migration In order to evaluate the effectsof the Lipogems product on the repairing capacity of hTSCsan in vitro wound-healing assay was performed The woundwas completely closed in all conditions within 45ndash48 h andLipogems-treated and control cells showed a similar rate ofwound closure (a representative image of Lipogems-treatedand control hTSCs moving into the wound space is shownin (Figure 4) for both groups at 0 and 25 h after scratching)Quantitative analyses indicated no significant differences incellmigration velocity between Lipogems-treated and controlcells at all time points (119901 lt 005 Figure 4(f))

4 Discussion

The stromal vascular fraction which is mainly responsiblefor growth-factors production and regulation has been suc-cessfully used in orthopedic practice [19 29ndash31] However aprecise chemical identification of these factors is still missingNonetheless many reports support the notion that a definedcombination of thesemolecules could greatly improve rotatorcuff tendon healing via autocrine and paracrine signaling [1617] For instance according to the results recently reported byDoornaert et al it is likely that the lipoaspirate provides localsignals to stimulate the self-healing processes by activatingresident stem cells through paracrine mechanisms [16] Therecent discovery that also the human rotator cuff possessesa population of resident progenitor stem cells [22 23] thatcan be easily isolated and cultured in vitro allowed the designof this study with the ultimate goal of better understandingthe effects of the Lipogems product on adult stem cells Akey question that needed to be addressed was to ascertainwhether the beneficial effects of the Lipogems product weredue to soluble factors released by the lipoaspirate Thereforethe use of a transwell system allowed studying the effectsof Lipogems product without a direct contact between thelipoaspirate and the stem cell population These types of


Table 1 Summary of the surface markers by cytofluorimetry ofLipogems-treated and control hTSCs Numbers show the expressionpercentage for each cell surface protein

Markers hTSCs (control) () hTSCs + Lipogems ()CD9 5531 6591CD13 100 100CD18 0 0CD29 9827 9677CD34 0 0CD44 100 100CD45 0 0CD71 6228 3725CD73 100 100CD90 100 100CD105 100 100CD106 398 230CD117 0 0CD140a 0 0CD140b 4715 346CD146 135 0CD166 8472 8699HLA-ABC 100 100HLA-DR 0 0Lineage 023 0NG2 0 0SSEA-4 0 0Stro-1 0 0

indirect coculturing systems have been previously used tosuccessfully direct the differentiation of stem cells and topromote cell survival and expansion in vitro [32] Thus inall reported experiments we could assume that the observedeffects were due to soluble factors released in the mediumby the lipoaspirate Results showed that hTSCs coculturedwith the Lipogems product significantly increased theirproliferation capability with no appreciable cytotoxic effectsIndeed finding new ways to activate stem cells is vital foran effective treatment as stem cell progenitors are usuallypresent in very low numbers in adult tissues Attempts tocircumvent the problem by harvesting adult progenitors frombiopsies expanding them in vitro and then reinjecting intothe patients have many drawbacks In fact isolating andculturing stem cells at GMP level are very troublesomeand expensive Moreover to date all methods for culturingstem cells in vitro cannot prevent their tendency to losepotency during passages and eventually to become senescentTherefore local injection of products like Lipogems couldrepresent a more practical way of activating resident stemcells without the need to isolate them from biopsies Ourresults are in agreement with those reported on HumanUmbilical Vein Endothelial Cells (HUVECs) where amarkedproliferation increase by Lipogems product addiction to thecultures was observed [33] Interestingly in our study wedid not observe any loss of stemness when hTSCs were

cultured in the presence of Lipogems product as no signif-icant changes in key stem cells markers could be observedMoreover treatment with Lipogems product alone did notinduce tenogenesis adipogenesis osteogenesi nor myogen-esis These findings support the notion that the lipoaspiratereleases factors that increase stem cell proliferation withoutaltering their potency These results were confirmed also bydifferentiation experiments where Lipogems-treated hTSCscould be induced to differentiate towards osteoblasts andadipocytes with the same efficiency as untreated controls

Finally we observed that Lipogems exposure moderatelyincreases hTSC expression of VEGF which has been shownto play a central role in the neovascularization process beingthe main regulator of angiogenesis [17 34ndash39] While atthis stage we cannot predict whether the observed VEGFincrease could have biological effects angiogenesis is to beconsidered an essential factor in designing novel approachesfor tissue engineering as the restoration of blood flow wouldprovide a source of nutrients and oxygen and metabolicsubstrates and also the access for circulating cells that canhelp supporting tissue regeneration [19 40] Although nostudies have directly evaluated the role of VEGF in rotatorcuff repair some reported results demonstrate an improvedtensile strength in animal tendon healing models with VEGFaugmentation [17 41] Moreover increase in VEGF mRNAexpression levels in a rat model of supraspinatus tendonoveruse injury was reported [42] Clearly at this stageit cannot be excluded that the Lipogems product releasesparacrine angiogenic and antiapoptotic factors other thanVEGF It has been documented that intact human adiposestem cells (hASCs) which can be found in the Lipogemsproduct are significantlymore responsive than enzymaticallydissociated hASCs to a mixture of hyaluronic butyric andretinoic acid previously shown to induce stem cell expressionof several vasculogenic genes Therefore the peculiarity andeffectiveness of the Lipogems productmay reside in the easilyobtainable source of living hASCs that once injected couldrelease a variety of soluble factors able to activate residenthTSCs

5 Conclusions

Herein we reported the effects of the Lipogems lipoaspirateon human tendon stem cells from the rotator cuff Cocultureof these progenitor cells with Lipogems released factors sig-nificantly increases cell proliferation without affecting stemcell marker expression and differentiation capability Overallthese results support that activating resident progenitor cellswith the Lipogems product without the need to isolate stemcells from a biopsy expanding them in vitro and then re-injecting them into the patient could be a practical andcost-effective new therapeutic approach for increasing rotatorcuff tendon healing Although the Lipogems product releasedfactors have yet to be identified preliminary results in ourlaboratories revealed that the effects are not limited to tendonstem cells In fact preliminary increase in proliferation couldbe observed also on stem cells from the cardiovascular system(data not shown) Therefore understanding the chemicalcomposition of the Lipogems product could allow chemical


synthesis of these factors and using them instead of thelipoaspirate Further studies in this direction are currentlyongoing in our laboratories

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Pietro Randelli andAlessandraMenon equally contributed tothis paper

References

[1] J S Sher J W Uribe A Posada B J Murphy and M BZlatkin ldquoAbnormal findings on magnetic resonance images ofasymptomatic shouldersrdquoThe Journal of Bone amp Joint SurgerymdashAmerican Volume vol 77 no 1 pp 10ndash15 1995

[2] M Chen W Xu Q Dong Q Huang Z Xie and Y MaoldquoOutcomes of single-row versus double-row arthroscopic rota-tor cuff repair a systematic review andmeta-analysis of currentevidencerdquo Arthroscopy vol 29 no 8 pp 1437ndash1449 2013

[3] Y G Rhee N S Cho and J H Yoo ldquoClinical outcome andrepair integrity after rotator cuff repair in patients older than 70years versus patients younger than 70 yearsrdquo Arthroscopy vol30 no 5 pp 546ndash554 2014

[4] M D McElvany E McGoldrick A O Gee M B Neradilekand F A Matsen ldquoRotator cuff repair published evidence onfactors associated with repair integrity and clinical outcomerdquoThe American Journal of Sports Medicine vol 43 no 2 pp 491ndash500 2015

[5] C-F Liu L Aschbacher-Smith N J Barthelery N DymentD Butler and C Wylie ldquoWhat we should know before usingtissue engineering techniques to repair injured tendons adevelopmental biology perspectiverdquo Tissue Engineering Part BReviews vol 17 no 3 pp 165ndash176 2011

[6] R B Evans ldquoManaging the injured tendon current conceptsrdquoJournal of HandTherapy vol 25 no 2 pp 173ndash190 2012

[7] S Hoppe M Alini L M Benneker S Milz P Boileau and MA Zumstein ldquoTenocytes of chronic rotator cuff tendon tearscan be stimulated by platelet-released growth factorsrdquo Journalof Shoulder and Elbow Surgery vol 22 no 3 pp 340ndash349 2013

[8] K DWeeks III J S Dines S A Rodeo and A Bedi ldquoThe basicscience behind biologic augmentation of tendon-bone healinga scientific reviewrdquo Instructional course lectures vol 63 pp 443ndash450 2014

[9] A M Abtahi E K Granger and R Z Tashjian ldquoFactorsaffecting healing after arthroscopic rotator cuff repairrdquo WorldJournal of Orthopaedics vol 6 no 2 pp 211ndash220 2015

[10] P Randelli F Randelli V Ragone et al ldquoRegenerative medicinein rotator cuff injuriesrdquo BioMed Research International vol2014 Article ID 129515 9 pages 2014

[11] I G Petrou A Grognuz N Hirt-Burri W Raffoul and L ALaurent-Applegate ldquoCell therapies for tendons old cell choicefor modern innovationrdquo Swiss Medical Weekly vol 144 ArticleID w13989 2014

[12] R A McCormack M Shreve and E J Strauss ldquoBiologicaugmentation in rotator cuff repairmdashshould we do it who

should get it and has it workedrdquoBulletin of theHospital for JointDisease vol 72 no 1 pp 89ndash96 2013

[13] M VMoraM A R Iban J D Heredia et al ldquoStem cell therapyin the management of shoulder rotator cuff disordersrdquo WorldJournal of Stem Cells vol 7 no 4 pp 691ndash699 2015

[14] Z Ahmad F Henson J Wardale A Noorani G Tytherleigh-Strong and N Rushton ldquoReview article regenerative tech-niques for repair of rotator cuff tearsrdquo Journal of OrthopaedicSurgery vol 21 no 2 pp 226ndash231 2013

[15] W D Murrell A W Anz H Badsha W F Bennett R EBoykin and A I Caplan ldquoRegenerative treatments to enhanceorthopedic surgical outcomerdquo PMampR vol 7 no 4 supplementpp S41ndashS52 2015

[16] M A J Doornaert H Declercq F Stillaert et al ldquoIntrinsicdynamics of the fat graft in vitro interactions between themaincell actorsrdquo Plastic and Reconstructive Surgery vol 130 no 5 pp1001ndash1009 2012

[17] A Bedi T Maak C Walsh et al ldquoCytokines in rotator cuffdegeneration and repairrdquo Journal of Shoulder andElbow Surgeryvol 21 no 2 pp 218ndash227 2012

[18] N Pallua M Serin and T P Wolter ldquoCharacterisation ofangiogenetic growth factor production in adipose tissue-derivedmesenchymal cellsrdquo Journal of Plastic Surgery andHandSurgery vol 48 no 6 pp 412ndash416 2014

[19] M Behfar F Sarrafzadeh-Rezaei R Hobbenaghi N Delirezhand B Dalir-Naghadeh ldquoAdipose-derived stromal vascularfraction improves tendon healing in rabbitsrdquo Chinese Journal ofTraumatology vol 14 no 6 pp 329ndash335 2011

[20] A I GoncalvesM T Rodrigues S-J Lee et al ldquoUnderstandingthe role of growth factors in modulating stem cell tenogenesisrdquoPLoS ONE vol 8 no 12 Article ID e83734 2013

[21] F Bianchi M Maioli E Leonardi et al ldquoA new nonenzymaticmethod and device to obtain a fat tissue derivative highlyenriched in pericyte-like elements by mild mechanical forcesfrom human lipoaspiratesrdquo Cell Transplantation vol 22 no 11pp 2063ndash2077 2013

[22] P Randelli E Conforti M Piccoli et al ldquoIsolation andcharacterization of 2 new human rotator cuff and long headof biceps tendon cells possessing stem cell-like self-renewaland multipotential differentiation capacityrdquo American Journalof Sports Medicine vol 41 no 7 pp 1653ndash1664 2013

[23] H Utsunomiya S Uchida I Sekiya A Sakai K Morideraand T Nakamura ldquoIsolation and characterization of humanmesenchymal stem cells derived from shoulder tissues involvedin rotator cuff tearsrdquo American Journal of Sports Medicine vol41 no 3 pp 657ndash668 2013

[24] C-C Tsai T-FHuangH-LMa E-RChiang and S-CHungldquoIsolation of mesenchymal stem cells from shoulder rotatorcuff a potential source for muscle and tendon repairrdquo CellTransplantation vol 22 no 3 pp 413ndash422 2013

[25] N Song A D Armstrong F Li H Ouyang and C NiyibizildquoMultipotentmesenchymal stem cells fromhuman subacromialbursa potential for cell based tendon tissue engineeringrdquoTissueEngineeringmdashPart A vol 20 no 1-2 pp 239ndash249 2014

[26] A F Steinert M Kunz P Prager et al ldquoCharacterization ofbursa subacromialis-derived mesenchymal stem cellsrdquo StemCell Research ampTherapy vol 6 p 114 2015

[27] C Tremolada G Palmieri and C Ricordi ldquoAdipocyte trans-plantation and stem cells plastic surgery meets regenerativemedicinerdquo Cell Transplantation vol 19 no 10 pp 1217ndash12232010


[28] C-C Liang A Y Park and J-L Guan ldquoIn vitro scratchassay a convenient and inexpensive method for analysis of cellmigration in vitrordquo Nature Protocols vol 2 no 2 pp 329ndash3332007

[29] A J Nixon L A Dahlgren J L Haupt A E Yeager and DL Ward ldquoEffect of adipose-derived nucleated cell fractions ontendon repair in horses with collagenase-induced tendinitisrdquoAmerican Journal of Veterinary Research vol 69 no 7 pp 928ndash937 2008

[30] N H Riordan T E Ichim W-P Min et al ldquoNon-expandedadipose stromal vascular fraction cell therapy for multiplesclerosisrdquo Journal of Translational Medicine vol 7 article 292009

[31] F Bacou R Boubaker El Andalousi P-A Daussin et al ldquoTrans-plantation of adipose tissue-derived stromal cells increasesmass and functional capacity of damaged skeletal musclerdquo CellTransplantation vol 13 no 2 pp 103ndash111 2004

[32] K Rubina N Kalinina A Efimenko et al ldquoAdipose stromalcells stimulate angiogenesis via promoting progenitor celldifferentiation secretion of angiogenic factors and enhancingvessel maturationrdquo Tissue Engineering Part A vol 15 no 8 pp2039ndash2050 2009

[33] F Bianchi E Olivi M Baldassarre et al ldquoLipogems a newmodality of fat tissue handling to enhance tissue repair inchronic hind limb ischemiardquo CellR4 vol 2 no 6 Article IDe1289 2014

[34] I Linero and O Chaparro ldquoParacrine effect of mesenchymalstem cells derived from human adipose tissue in bone regener-ationrdquo PLoS ONE vol 9 no 9 Article ID e177001 2014

[35] R J Griffeth D Garcıa-Parraga M Mellado-Lopez et alldquoPlatelet-rich plasma and adipose-derived mesenchymal stemcells for regenerative medicine-associated treatments in bot-tlenose dolphins (Tursiops truncatus)rdquo PLoS ONE vol 9 no 9Article ID e108439 2014

[36] W Petersen F Unterhauser T Pufe T Zantop N Sudkampand A Weiler ldquoThe angiogenic peptide vascular endothelialgrowth factor (VEGF) is expressed during the remodelingof free tendon grafts in sheeprdquo Archives of Orthopaedic andTrauma Surgery vol 123 no 4 pp 168ndash174 2003

[37] W Petersen T Pufe F Unterhauser T Zantop R Mentleinand A Weiler ldquoThe splice variants 120 and 164 of the angio-genic peptide vascular endothelial cell growth factor (VEGF)are expressed during Achilles tendon healingrdquo Archives ofOrthopaedic and Trauma Surgery vol 123 no 9 pp 475ndash4802003

[38] M Bidder D A Towler R H Gelberman and M I BoyerldquoExpression of mRNA for vascular endothelial growth factorat the repair site of healing canine flexor tendonrdquo Journal ofOrthopaedic Research vol 18 no 2 pp 247ndash252 2000

[39] M I Boyer J T Watson J Lou P R Manske R H Gel-berman and S Rong Cai ldquoQuantitative variation in vascularendothelial growth factor mRNA expression during early flexortendon healing an investigation in a canine modelrdquo Journal ofOrthopaedic Research vol 19 no 5 pp 869ndash872 2001

[40] M Behfar F Sarrafzadeh-Rezaei R Hobbenaghi N Delirezhand B Dalir-Naghadeh ldquoEnhanced mechanical properties ofrabbit flexor tendons in response to intratendinous injection ofadipose derived stromal vascular fractionrdquo Current Stem CellResearch andTherapy vol 7 no 3 pp 173ndash178 2012

[41] F Zhang H Liu F Stile et al ldquoEffect of vascular endothelialgrowth factor on rat Achilles tendon healingrdquo Plastic andReconstructive Surgery vol 112 no 6 pp 1613ndash1619 2003

[42] SM Perry S EMcIlhennyMCHoffman and L J SoslowskyldquoInflammatory and angiogenic mRNA levels are altered ina supraspinatus tendon overuse animal modelrdquo Journal ofShoulder and Elbow Surgery vol 14 no 1 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of


Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology


Molecular Biology International

GenomicsInternational Journal of


The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014


BioinformaticsAdvances in

Marine BiologyJournal of



Signal TransductionJournal of


BioMed Research International

Evolutionary BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014


Genetics Research International


Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


the healing process Moreover several studies have shownthat different types of stimuli could activate the nor-mal growth-factor-mediated healing cascades [16] Alongthis line it has been recently demonstrated that lipoaspi-rates contain and produce growth-factors such as platelet-derived growth-factor (PDGF) fibroblast growth-factor(FGF) transforming growth-factor beta (TGF-120573) and vas-cular endothelial growth-factor (VEGF) which are known toplay important regulatory roles in cellular functions includ-ing adhesion chemotaxis proliferation migration matrixsynthesis differentiation and angiogenesis [16ndash18] The cellfraction responsible for growth-factor production and regu-lation is mainly the stromal vascular fraction which couldhave acceleratory effect on the healing process of injuredtendons [19] Given the tissue availability as well as theeasy and minimally invasive access to tissue sources adiposetissue may in fact represent a potential choice for tendonrepair and regeneration [20] Among many approaches aninnovative technology Lipogems provides a nonexpandedready-to-use and microfragmented adipose tissue that isexpected to have the peculiar advantage of maintaining anintact stromal vascular niche harboring cellular elementswith mesenchymal stem cell and pericyte characteristics [21]This system works through a mild mechanical tissue cluster-size reduction in a completely closed system avoiding the useof any enzyme additives and other additional manipulations(ie centrifugation and subfractional harvesting) [21]

In a previous study on the Lipogems product Bianchiet al speculated that the action of the Lipogems product dueto an intact stromal vascular niche could involve the secre-tion of trophic mediators delivering instructive messagesthat functionally promote a more compliant regenerativeenvironment within the recipient tissue [21] In particularthe lipoaspirate could stimulate the self-healing processesby activating resident stem cells through paracrine mecha-nismsTherefore the Lipogems product may be instrumentalfor activating resident progenitor cells to proliferate andcontribute to the tendon healing process Actually it hasbeen recently demonstrated that also the human rotatorcuff contains a reservoir of progenitor cells which can beisolated and expanded in vitro [22ndash26] Thus the availabilityof a completely closed disposable system for adipose tissueprocessing could allow the procedure to be readily safelyand economically performed in clinical settings to affordsignificant tissue repair overcoming the difficulty of an exvivo expansion and the complexity of the current GoodManufacturing Practice (GMP) requirements for preparingcells for a therapeutic use [27]

On these bases the aim of this study was to assessthe effects of the Lipogems product on primary cultures ofhuman tendon stem cells (hTSCs)

2 Materials and Methods

21 Cell Cultures Human tendon stem cells (hTSCs) wereisolated from supraspinatus tendon specimens collectedduring arthroscopic rotator cuff repair according to ourprevious procedure [22] The isolated hTSCs were cul-tured in normal growth medium composed of 120572-Minimal

Essential Medium (120572-MEM) (Sigma-Aldrich) supplementedwith 2mM glutamine (Euroclone) 1 antibiotic-antimycoticmixture (Euroclone) and 20 (vv) fetal bovine serum (FBS)(HyClone Thermo Fisher Scientific) at 37∘C in a humidifiedatmosphere of 5 CO

2 The medium was changed every 2-3

days All experiments were carried out with cells at passagefour after isolation

22 Processing of Adipose Tissue with the Lipogems DeviceHuman subcutaneous adipose tissue samples were obtainedwith patient informed consent from abdominal lipoaspira-tion procedures performed before arthroscopic rotator cuffrepair and processed by using the Lipogems device accordingto the manufacturerrsquos instructions previously described [21]Avoiding the presence of air the lipoaspirate tissue wassubjected to a first cluster reduction obtained by pushingthe lipoaspirate from the syringe into the device through thefirst large filter (blue cap) and allowing the correspondingquantity of saline to exit towards the wasting bag The fivestainless steel beads contained in the device were essential toobtain a temporary emulsion between oil blood and salinewhich could be washed away against density following thecurrent of saline moved by gravity in the wasting bag Afterthis washing step (the flowing solution appears clear andthe lipoaspirate yellow) the saline flux was stopped and thedevice was reversed (gray cap up) leading to the secondadipose cluster reduction Such reduction was obtained bypushing the floating adipose clusters through the secondcutting hexagonal filter (grey cap) pushing fluid from belowwith a 60mL syringe The reduced cluster was collected inanother 60mL syringe placed above and positioned to gentlydecant the Lipogems product by gravity in order to removeexcessive saline solution Amean of 50mL of lipoaspirate wascollected and processed with the Lipogems device to obtainan amount of 20mL of the final Lipogems product whichwastransferred to several 10mL syringes to be reinjected in thesame patient or used in our experiments

23 Lipogems Coculture Experiments of hTSCs with theLipogems Product hTSCs were seeded on the bottom of 6-well plates in normal growth medium Twenty-four hoursafter seeding a volume of 250 120583L of the freshly processedLipogems product was transferred to the transwell polycar-bonate microporous inserts (04120583m membrane pore sizeFalcon BD Figure 1) above hTSCs monolayers allowing forpotential diffusion of soluble mediators but preventing cell-cell contact Control cells were cultured under the sameconditions without the Lipogems product The medium wasreplaced every 48 h in all experiments To study the effectsof the Lipogems treatment on multilineage differentiationpotential of hTSCs cells were cultured with the Lipogemsproduct in osteogenic and adipogenic differentiation mediaas described below

24 Cell Morphology and Proliferation For analysis of cellviability hTSCs were plated at a concentration of 26 times 103cellscm2 and exposed to the Lipogems product for 96 has described above Cell morphology was examined daily



Solutions waste






















3 Results




(a)

96


hTSCs (control)

1000

hTSCs + Lipogems

72

500

0

0

Cel

l num

ber (

)

24 48

Time (h)

lowast

lowastlowast

lowastlowast

(b)









100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

PIPI

PI PIPI

0

48

96

Tim

e (h)

Annexin V

Annexin V Annexin V

Annexin VAnnexin V






TNMD

hTSCs (control)

COL1A1

05

10

00

Tenascin C

NE NERQ05

15

10

00

RQ

05

15

10

00

RQ










05

15

10

00

RQ

05

15

10

00

RQ

05

15

10

00

RQ

Nanog Oct4

KLF4




05

15

10

00

RQ


VEGFlowastlowast

(e) VEGF expression

Figure 4 Continued




0

25

Tim

e (h)

Wou

nd cl

osur

e (

)

120

100

80

60

40

20

15 450 30

Time (h)





4 Discussion








5 Conclusions








References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



Solutions waste






















3 Results




(a)

96


hTSCs (control)

1000

hTSCs + Lipogems

72

500

0

0

Cel

l num

ber (

)

24 48

Time (h)

lowast

lowastlowast

lowastlowast

(b)









100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

PIPI

PI PIPI

0

48

96

Tim

e (h)

Annexin V

Annexin V Annexin V

Annexin VAnnexin V






TNMD

hTSCs (control)

COL1A1

05

10

00

Tenascin C

NE NERQ05

15

10

00

RQ

05

15

10

00

RQ










05

15

10

00

RQ

05

15

10

00

RQ

05

15

10

00

RQ

Nanog Oct4

KLF4




05

15

10

00

RQ


VEGFlowastlowast

(e) VEGF expression

Figure 4 Continued




0

25

Tim

e (h)

Wou

nd cl

osur

e (

)

120

100

80

60

40

20

15 450 30

Time (h)





4 Discussion








5 Conclusions








References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology









3 Results




(a)

96


hTSCs (control)

1000

hTSCs + Lipogems

72

500

0

0

Cel

l num

ber (

)

24 48

Time (h)

lowast

lowastlowast

lowastlowast

(b)









100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

PIPI

PI PIPI

0

48

96

Tim

e (h)

Annexin V

Annexin V Annexin V

Annexin VAnnexin V






TNMD

hTSCs (control)

COL1A1

05

10

00

Tenascin C

NE NERQ05

15

10

00

RQ

05

15

10

00

RQ










05

15

10

00

RQ

05

15

10

00

RQ

05

15

10

00

RQ

Nanog Oct4

KLF4




05

15

10

00

RQ


VEGFlowastlowast

(e) VEGF expression

Figure 4 Continued




0

25

Tim

e (h)

Wou

nd cl

osur

e (

)

120

100

80

60

40

20

15 450 30

Time (h)





4 Discussion








5 Conclusions








References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



(a)

96


hTSCs (control)

1000

hTSCs + Lipogems

72

500

0

0

Cel

l num

ber (

)

24 48

Time (h)

lowast

lowastlowast

lowastlowast

(b)









100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

PIPI

PI PIPI

0

48

96

Tim

e (h)

Annexin V

Annexin V Annexin V

Annexin VAnnexin V






TNMD

hTSCs (control)

COL1A1

05

10

00

Tenascin C

NE NERQ05

15

10

00

RQ

05

15

10

00

RQ










05

15

10

00

RQ

05

15

10

00

RQ

05

15

10

00

RQ

Nanog Oct4

KLF4




05

15

10

00

RQ


VEGFlowastlowast

(e) VEGF expression

Figure 4 Continued




0

25

Tim

e (h)

Wou

nd cl

osur

e (

)

120

100

80

60

40

20

15 450 30

Time (h)





4 Discussion








5 Conclusions








References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

100

101

102

103

PIPI

PI PIPI

0

48

96

Tim

e (h)

Annexin V

Annexin V Annexin V

Annexin VAnnexin V






TNMD

hTSCs (control)

COL1A1

05

10

00

Tenascin C

NE NERQ05

15

10

00

RQ

05

15

10

00

RQ










05

15

10

00

RQ

05

15

10

00

RQ

05

15

10

00

RQ

Nanog Oct4

KLF4




05

15

10

00

RQ


VEGFlowastlowast

(e) VEGF expression

Figure 4 Continued




0

25

Tim

e (h)

Wou

nd cl

osur

e (

)

120

100

80

60

40

20

15 450 30

Time (h)





4 Discussion








5 Conclusions








References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


TNMD

hTSCs (control)

COL1A1

05

10

00

Tenascin C

NE NERQ05

15

10

00

RQ

05

15

10

00

RQ










05

15

10

00

RQ

05

15

10

00

RQ

05

15

10

00

RQ

Nanog Oct4

KLF4




05

15

10

00

RQ


VEGFlowastlowast

(e) VEGF expression

Figure 4 Continued




0

25

Tim

e (h)

Wou

nd cl

osur

e (

)

120

100

80

60

40

20

15 450 30

Time (h)





4 Discussion








5 Conclusions








References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




0

25

Tim

e (h)

Wou

nd cl

osur

e (

)

120

100

80

60

40

20

15 450 30

Time (h)





4 Discussion








5 Conclusions








References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







5 Conclusions








References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







References




















































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology
























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

research article lipogems product treatment increases the ... · were acquired with a navios flow...

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