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Investigating the microenviron-mental effects of scaffoldchemistry and topology inhuman mesenchymal stromalcell/polymeric hollowmicrofiber constructsClaudio Ricci,1 Luisa Trombi,1Ilaria Soriga,2 Fabio Piredda,2Mario Milazzo,3 Cesare Stefanini,3,4Luca Bruschini,1,5 Giuseppe Perale,6Gianni Pertici,6 Serena Danti1,3,71OtoLab, Azienda Ospedaliero-Universitaria Pisana, Pisa; 2Departmentof Chemistry, Materials and ChemicalEngineering, Politecnico di Milano, Milan;3Creative Engineering Design Area,Biorobotics Institute, Scuola SuperioreSant’Anna, Pontedera (PI), Italy;4Department of Biomedical Engineering,Khalifa University, Abu Dhabi, UAE;5Department of Surgical, Medical,Molecular Pathology and EmergencyMedicine, University of Pisa, Pisa, Italy;6Department of Innovative Technologies,University of Applied Sciences and Artsof Southern Switzerland, Manno,Switzerland; 7Department of Civil andIndustrial Engineering, University of Pisa,Pisa, Italy

Abstract

Tissue engineering scaffolds have shown anintrinsic ability to provide cellular stimulation,thus behaving as physically active microenvi-ronments. This study reports on the interac-tion between human mesenchymal stromalcells (hMSCs) and dry-wet spun polymermicrofiber meshes. The following scaffoldingparameters were tested: i) polymer type: poly-L-lactide (PLLA) vs poly-e-caprolactone (PCL);ii) non-solvent type: ethanol (Et-OH) vs iso-propanol/gelatin; iii) scaffold layout: patternedvs random microfiber fabrics. After two cultureweeks, the effects on metabolic activity, scaf-fold colonization and function of undifferenti-ated hMSCs were assayed. In our study, thepolymer type affected the hMSC metabolicactivity timeline, and the metabolic picksoccurred earlier in PLLA (day 6) than in PCL(day 9) scaffolds. Instead, PLLA vs PCL had noendpoint effect on alkaline phosphatase (ALP)activity expression. On average, the hMSCsgrown on all the random microfiber fabricsshowed an ALP activity statistically superior tothat detected on patterned microfiber fabrics,with the highest in Et-OH random subtypes.

Such findings are suggestive of enhancedosteogenic potential. The understanding ofscaffold-driven stimulation could enable envi-ronmental hMSC commitment, paving the wayfor new regenerative strategies.

Introduction

About two decades ago, tissue engineeringwas proposed as an emerging trans-discipli-nary field to generate three-dimensional (3D)tissue substitutes ex vivo aimed at accom-plishing unmet clinical needs and enablingnew regenerative therapies.1 Nowadays, viable3D cellular constructs have been successfullyobtained in the laboratory by culturing cellsunder extrinsic stimulation factors on 3D bio-material-based scaffolds. These are used to actas temporary templates for 3D organizationand differentiation of stem cells (SCs), leadingto translational approaches.2,3 Among the adultSCs, the mesenchymal stem (or stromal) cells(MSCs) provide a very useful and accessiblecell source and are employed as an effectivetool to obtain several differentiated cellulartypes, in particular those deriving from meso-dermal tissues, such as osteoblasts, chondrob-lasts, myoblasts and adipoblasts.4 Indeed, mus-culoskeletal tissues are often affected bypathologies for which surgical tissue replace-ments are broadly needed, thus making MSC-based therapies highly appealing.5 The use ofundifferentiated cells has posed the challengeof finely tuning and controlling their differen-tiative profiles; this is usually obtained in vitroby providing the constructs with chemical (e.g.,soluble factors) or, more recently, mechanical(e.g., stresses via fluid dynamics) stimulithrough the culture media (CM).6

Nonetheless, one of the most recent frontiersin controlling SC behavior is represented bythe application of specific physical stimuli,inherently induced by the scaffold as amicroenvironment.7 Indeed, interfacing SCswith defined biomaterial cues (e.g. certaintypes of surface topology and material stiff-ness) has been reported to affect their biology,thus accrediting the scaffold not only as amerely passive substrate for cellular organiza-tion, but rather as an active component of tis-sue-engineered systems.8

A number of scaffolding technologies havebeen developed over the last two decades withthe primary intent of efficiently housing thecells within proper 3D settings.9 Fiber-basedscaffolds offer highly customizable features,ranging from nano- to micro-scale, owing tothe many possibilities offered for tailoringfibers and fabrics.10 Among them, meshes ofultrafine (<10 μm) fibers produced via electro-spinning are probably the best investigated intissue engineering.11 However, the architectur-

al features and thickness limitations of elec-trospun scaffolds have restricted their applica-tions to replace the bi-dimensional defects ofsoft connective tissues.12,13 Instead, large diam-eter (>100 μm) microfiber scaffolds, producedvia 3D printing or other spinning apparatuses,do not show any limitations of thickness, mak-ing them true 3D scaffolds. The hollowmicrofibers, also referred to as microtubules,belong to the large-diameter microfiber classand can be manufactured via dry-wetspinning.14 This technique exploits ternary (ormulti-) component miscibility to producefibers from solutions. Basically, in order to bespun, the (co)polymer is dissolved inside a sol-vent (or solvent mixture), while a non-solvent(or solvent mixture) is concomitantly used to

Correspondence: Serena Danti, OtoLab, AziendaOspedaliero-Universitaria Pisana, via Paradisa 2,56124 Pisa, Italy. Tel: +39.050.997882 - Fax: +39.050.997495.E-mail: s.danti@med.unipi.it

Key words: Poly(L-lactic) acid; Poly-caprolactone;Dry-wet spinning; Viability; Tissue engineering.

Acknowledgments: the authors gratefullyacknowledge Dr. Delfo D’Alessandro and Dr.Sabrina Danti (University of Pisa) for their pre-cious contribution to electron microscopy andstatistical analyses, respectively. SimoneMaccagnan [Gimac, Castronno (VA), Italy] isacknowledged for his fundamental technical sup-port to set-up the dry-wet spinning apparatus.Many thanks are due to Prof. Stefano Berrettini(University of Pisa) for supporting this research.

Contributions: CR, LT, IS, FP, MM, performedexperiments; MM and SD analyzed data; GP, GPand SD designed the study; CS, LB, GP and GPprovided materials and equipment; CR, GP, GPand SD wrote the manuscript; all the authorsgave final approval.

Conflict of interest: GP and GP are stockholdersof Industrie Biomediche Insubri S/A (Mezzovico-Vira, Switzerland). All other authors declare nopotential conflict of interest.

Funding: this study was supported by the ItalianMinistry of University and Research grant (PRIN2010S58B38) and was part of the graduation the-sis defense of IS and FP at Politecnico di Milano(year 2014).

Received for publication: 7 December 2015.Revision received: 9 May 2016.Accepted for publication: 30 May 2016.

This work is licensed under a Creative CommonsAttribution 4.0 License (by-nc 4.0).

©Copyright C. Ricci, et al., 2016Licensee PAGEPress, ItalyBiomedical Science and Engineering 2016; 2:10doi:10.4081/bse.2016.10

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obtain the fiber cavity. Upon drying, the wetfiber solidifies under a ternary (or multi-)component system, whose miscibility gapsultimately tailor the fiber morphology.Although very appealing in terms of manufac-turing, the necessity for solvents and non-sol-vents poses potential cytotoxicity issues forbiomedical use. So far, regenerative opportuni-ties offered by hollow microfiber scaffolds havebeen the subject only of a limited number ofstudies.14-19 A decade ago, Lazzeri et al. report-ed on the fabrication of bioresorbable dry-wetspunmicrofibers emphasizing their versatilityof incorporating nanoparticle-loaded drugsuseful for tissue engineering applications.14

Later on, Ellis et al. produced microfiber mem-branes by phase inversion and tested themusing an immortalized osteoblast cell line.These authors envisioned that hollowmicrofiber scaffolds could be interesting bonesubstitutes for centimeter-size defects, withfiber cavities possibly accommodating capillaryand small vasculature infiltration.15 Other invitro studies, mostly performed by the authorsof this research, have shown that hollowmicrofiber bonded scaffolds were capable ofsupporting osteoblast, fibroblast and myoblastcell lines, as well as primary chondrocytes,umbilical cord and bone marrow MSCs, thusappearing widely suitable for musculoskeletaltissue regeneration.16-19 However, no lineagespecification driven by dry-wet spun scaffoldshas been assessed so far. Recently, somereports have suggested that other fibrous scaf-folds can play biomimetic roles on cellularorganization.20,21 The microenvironmentaleffects intrinsically induced by the scaffoldsbecome particularly important when undiffer-entiated cells, like the SCs, are used, sincetopological stimuli can affect, on multi-scaleorders, cell attachment, growth up to differen-tiation.7,8 Owing to its strategic relevance, theunderstanding of scaffold-induced cellularstimulation deserves additional and systematicinvestigative efforts in regenerative therapies.This study was aimed at fabricating 3D dry-

wet spun microfiber scaffolds for tissue engi-neering applications. Therefore, the influenceplayed by polymer, non-solvent and fabric lay-out upon human MSC (hMSC) culture, wasstudied. The poly-L-lactic acid (PLLA) and thepoly-e-caprolactone (PCL), both approved forclinical practice, were tested as polymers,while ethanol (Et-OH) and isopropanol/gelatin(IP-OH/G) water solutions were used as non-solvents. The obtained hollow microfiberswere assembled either orderly or randomly, soas to provide different topologic cues for cellu-lar organization. The investigation of biologi-cal effects on hMSC behavior possibly causedby the abovementioned parameters was car-ried out by assaying metabolic activity, viabili-ty, 3D organization and expression of alkalinephosphatase (ALP) activity, the latter as a con-

stitutive marker of these cells that is addition-ally imputed in their proneness to osteogenicdifferentiation.Insight into fabrication parameters affect-

ing hMSC behavior on microtubule meshescould enable new knowledge and ultimatelydefine novel routes to control hMSC fate viaarchitectural and chemical parameters.

Materials and methods

MaterialsFor scaffold manufacturing, the following

materials were purchased and used withoutmodifications: PLLA, Mw≈200,000 and iv=1.4(Purac Biochem, Gorkum, Netherlands); PCL,Mn≈80,000 (Sigma-Aldrich, St. Louis, MO,USA); dichloromethane (Sigma-Aldrich); Et-OH, 95% vol. and IP-OH, 50% vol. [Carlo ErbaReagenti, Cornaredo (MI) Italy]; gelatin (G),CAS Number 9000-70-8 (Merck-Millipore,Darmstadt, Germany).For in vitro experiments, the following

materials were purchased and used withoutmodifications: Lymphoprep (Axis-Shield,Dundee, UK); low-glucose Dulbecco’s ModifiedEagle Medium (D-MEM), L-glutamine, peni-cillin-streptomycin, trypsin, phosphatebuffered saline (PBS), trypan blue, neutral red,methylene blue, P-nitrophenol, 1.5 M 2-amino-2-methyl-1-propanol, 4-nitrophenyl phosphatedisodium salt hexahydrate, NaOH (all fromSigma-Aldrich); heat-inactivated fetal bovine(FBS) (Invitrogen, Carlsbad, CA, USA);PicoGreen kit (Quant-iTTM; Thermo FisherScientific Inc., Waltham, MA, USA); tissue-cul-ture polystyrene flasks, and ultra-low attach-ment (Corning, St. David’s Park, UK); flucona-zole (Pfizer, New York, NY, USA); absolute Et-OH (Bio-Optica, Milan, Italy); alamarBlue®

(Serotec Ltd., Kidlington, UK).

Scaffold manufacturingHollow microfibers of PLLA and PCL were

produced via dry-wet spinning, using a propri-etary machine (Gimac, Castronno, Italy) locat-ed at Industrie Biomediche Insubri S/A(Mezzovico, Switzerland). This is a three-axismoving apparatus designed to co-spin, via acomputer-controlled motor unit, two solutionsthroughout a double-annulus spinneret of sub-millimeter diameters, thus leading to a contin-uous hollow microfiber (Figure 1A). Specifically, polymer solution and non-sol-

vent solution, placed inside two separatesyringes, converge to the spinneret passingthrough the outer and the inner annuli, withdiameters 860 μm and 300 μm, respectively(Figure 1B). The wet fiber, as generated at itssprout, is thus collected by a Teflon rotatingdrum placed in air environment, designed to

allow evaporation of solvents. The whole proce-dure is thus considered a dry-wet spinningprocess. Indeed, the formation of themicrofiber occurs under a ternary (or multi-)phase system chemistry, which depends on themiscibility gaps of the polymer(s), solvent(s),and non-solvent(s) employed. In this study,PLLA and PCL were used as polymers, anddichloromethane as a solvent. Polymers weredissolved in dichloromethane at 1:3.5 ratio(w/v). Finally, either Et-OH or IP-OH solutioncontaining 0.5% (w/v) G were chosen as non-solvents. The latter non-solvent solution wasobtained by mixing equal volumes of IP-OHand G aqueous solution, achieved by dissolving1% (w/v) G in double-distilled water at 50°Cunder stirring. After collection, the microfiberswere either orderly or disorderly assembledinto non-woven meshes by means of the fiberbonding technique, finally giving rise to eitherpatterned or random fabric layouts. Briefly,predefined amounts of fibers, as produced bythe dry-wet spinning process, were placed onTeflon nets and heated, under compression, tobond the microfibers together via contactpoints generated by controlled melting.Specifically, PLLA and PCL meshes were heat-ed at 150°C for 1 h and 60°C for about 3 min,respectively. After bonding, the meshes werepuncher-cut into discs of 15 mm diameter withabout 2 mm thickness.

Microfiber and scaffold characteri-zation At their sprout from the spinneret, the

microfibers were photographed to assess theeffectiveness of manufacturing. After layoutassembly, the scaffolds were mounted on alu-minum stumps and sputter-coated with gold(Sputter Coater Emitech K550; QuorumTechnologies Ltd., Lewes, UK) for 4 min.Scaffold and fiber morphology was observedvia scanning electron microscopy (SEM)(Zeiss EVO MA10; Zeiss, Oberkochen,Germany) under an accelerating voltage of 5-10 kV. To assess maintenance of hollow mor-phology in the microfibers after mesh assem-bly, the scaffolds underwent fracture at -81°C.The microfiber diameters of the scaffold sur-faces were measured on serial micrographsacquired at 100× magnification (n=14).Finally, the sputter-coated scaffold surfaceswere photographed by means of a cameraequipped for digital macro (Canon Ixus;Canon, Tokyo, Japan).

Isolation and culture of humanmesenchymal stromal cellsHMSCs were isolated from bone marrow

aspirates of patients admitted to our hospitalfor orthopedic surgery. Samples were collectedafter informed consent, treated anonymouslyand in conformity to the principles expressed

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by the Declaration of Helsinki. Briefly, thebone marrow aspirate was diluted 1:4 in sterilesaline and layered on Lymphoprep as a densitygradient inside sterile tubes. The mononuclearcell layer, selected via centrifugation at 400×gfor 25 min, was suspended in CM, containinglow-glucose D-MEM, 2 mM L-glutamine, 100U/mL penicillin, 100 mg/mL streptomycin and10% (v%) heat-inactivated FBS. The mononu-clear cells were plated at a density of 0.2·105

cells/cm2 in tissue-culture polystyrene flasks.After 24 h, hMSCs were purified from othernon-adherent mononuclear cells via washingwith sterile saline. When the cultures reachedabout 80% confluence, the hMSCs weredetached by using 0.25% trypsin and replatedat a density of 103 cells/cm2 to allow theirexpansion. Cell cultures were carried out inincubator under standard conditions, namely37°C, 95% relative humidity, and 5% CO2/95%air environment. An extensive characteriza-tion and multi-lineage differentiation capabili-ty of hMSCs is reported in our previous stud-ies.22

Culture of human mesenchymalstromal cell/scaffold constructsThe microfiber scaffolds (n=4) were steril-

ized by overnight soaking in absolute Et-OHunder ultraviolet irradiation for 1 h. Afterwashing 10 min with a 3×antibiotics/flucona-zole solution in sterile saline, the scaffoldswere imbued with FBS for 30 min to favor celladhesion. Excess FBS was removed from thescaffolds before cell seeding. HMSCs weredetached with trypsin, counted under 0.2% try-pan blue staining for viability evaluation, sus-pended in 20 μL of CM and seeded on the scaf-folds at a density of 5×105 viable cells per scaf-fold. After seeding, the hMSC/scaffold con-structs were placed in the incubator for 1 h topermit cell adhesion and were finally coveredwith CM at 5 mL/sample inside 6-well plates.Cell/scaffold constructs were cultured understandard culture conditions for 16 days, replac-ing CM every 3 days.

Metabolic activity of human mes-enchymal stromal cell/scaffoldconstructsThe metabolic activity of the cells grown on

the scaffolds was monitored along the culturetime using the alamarBlue® assay. This bioas-say incorporates a REDOX indicator resultingin color change of the CM according to cellmetabolism. Moreover, it can be performedmultiple times on the same samples onaccount of its negligible toxicity. The data wereacquired according to the manufacturer’sinstructions and expressed as percentage ofreduced alamarBlue® (%ABred). Briefly, sam-ples (n=3) and negative controls (n=3), name-ly, media plus dye without cells, by using equa-

tion #1 from the manufacturer, were incubatedfor 3 h at 37°C with the alamarBlue® dye dilut-ed in 3 mL of CM for each scaffold, accordingto the manufacturer’s recommendations. Thismethod for calculation against negative con-trols is useful to assess %ABred increase of thesame samples along a timeline. The totalquantity of dye solution per well was chosen intheoretical excess, in order to accomplish anymetabolic activity increases all the sampleswithin 16-day cultures. Indeed, exceeding100% of dye reduction would affect reactionkinetics, thus making any comparisons unreli-able. Viability tests were performed every 3days after seeding. At each time-point, 100 μLof supernatant from sample or control wasloaded into 96-well plates; then, excess super-natant was removed from the cultures andreplaced with fresh CM. The absorbance (l) ofsupernatants was measured with a spec-trophotometer (Victor 3; PerkinElmer,Waltham, MA, USA) under a double wave-length reading (570 nm and 600 nm). Finally,%ABred was calculated by correlating theabsorbance values and the molar extinctioncoefficients of the dye at the selected wave-lengths, following the protocol provided by themanufacturer. The equation applied (eq. 1) isshown below, in which, l=absorbance, s=sam-ple, and c=negative control:

Scaffold colonization by humanmesenchymal stromal cellsColonization of the scaffold surfaces by

viable hMSCs was qualitatively investigatedusing a non-disruptive assay based on NeutralRed dye, which stains lysosomes of live cells inred. Briefly, at the endpoint, one specimen pertype (n=1) was incubated with 50 μg/mL CMsolution for 1 h. Samples were rinsed in sterilePBS and their surface was observed by meansof a camera equipped for digital macro (CanonIxus; Canon). Methylene Blue is a stain used to visualize

cells against their background, which is usefulto evaluate cell adhesion and colonization on

fixed cell/polymer scaffold specimens. OneMSC/scaffold construct of each type was fixedin 4% neutral buffered formalin/PBS solutionat 4°C overnight. Samples were rinsed in PBS,soaked in a 0.05% Methylene Blue solution for5 min, rinsed with double-distilled water, air-dried at room temperature. All the stainedsamples were observed under 3D digitalmicroscopy (KH-7700; Hirox-USA,Hackensack, NJ, USA). In addition, one construct per type (n=1)

was processed for SEM analysis. Briefly it wasformalin-fixed dehydrated in a graded series ofethanol aqueous solutions up to absolute Et-OH. Thereafter, dehydration of the hMSC/scaf-fold constructs was completed in a vacuumoven at 37°C overnight. Samples were mount-ed on aluminum stubs, sputter-coated withgold (Emitech K550), and observed via SEM(Zeiss EVO MA10) under an accelerating volt-age of 5-10 kV. Micrographs were acquired at302× magnifications.

Construct cellularityAt the endpoint, the double-stranded (ds)

DNA (ds-DNA) content in hMSC/scaffold con-structs was measured using the PicoGreenassay. On day 16, the CM was removed fromeach construct and replaced with 2 mL of dou-ble-distilled sterile water. Construct lysates(n=2) were obtained following two freeze (at -81°C for 24 h)/thawing (at 37°C for 20 min)cycles. Thawing was performed in ultrasonicbath (Falc; Progen Scientific, London, UK) setat 47 kHz frequency and 20 W output power.Standard solutions of DNA in double-distilledwater at concentrations ranging in 0-6 μg/mLwere prepared and 50 μL of standard or samplewas loaded in a 96-well black microplate.Working buffer and PicoGreen dye solutionswere prepared according to the manufacturer’sinstructions and added at 100 and 150 μL perwell, respectively. After a 10-min incubation inthe dark at room temperature, the fluores-cence intensity of the samples was measuredon a plate reader (Victor3; PerkinElmer),under an excitation wavelength of 485 nm andan emission wavelength of 535 nm. Ds-DNAconcentration in cell lysates was thus calculat-ed against the standard curve.

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Table 1. Fiber diameters at the scaffold surfaces.

PLLA PCL

Patterned Et-OH 427±101 242±60Random Et-OH 296±88 514±167Patterned IP-OH/G 312±141 544±90Random IP-OH/G 556±245 274±65PLLA, poly-L-lactic acid; PCL, poly-e-caprolactone; Et-OH, ethanol; IP-OH, isopropanol; G, gelatin. Values are reported in μm, as mean±standarddeviation.

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Alkaline phosphatase activity inhuman mesenchymal stromalcell/scaffold constructsIntracellular ALP activity was evaluated in

cascade on the same construct lysates assayedfor cellularity (n=2) using a colorimetric end-point assay based on P-nitrophenol. Standardsof P-nitrophenol were prepared in concentra-tions ranging from 0 to 250 μM. As necessary,the samples were diluted in double-distilledwater to stay within the detection range of theassay. Therefore, individual wells of a 96-wellplate were loaded as follows: 80 μL/well of stan-dard or sample, 20 μL/well of alkaline buffersolution at pH 10.3 (1.5 M 2-amino-2-methyl-1-propanol), and 100 μL/well of 4 mg/mL ALPsubstrate solution (4-nitrophenyl phosphatedisodium salt hexahydrate in double-distilledwater). The microplate was incubated at 37°Cfor 1 h and 100 μL/well of 0.3 M NaOH wasfinally added to stop the reaction. Theabsorbance was then measured at 405 nm on aplate reader (Victor3; PerkinElmer). Finally,ALP activity was normalized by cellularity.

Statistical analysisIn hMSC/scaffold constructs, quantitative

data were presented as mean±standard devia-tion. Differences were investigated by analysisof variance (ANOVA) with post-hoc t test.Significance was set at P<0.05.

Results

Scaffold fabrication and characteri-zationUsing the dry-wet spinning apparatus

shown in Figure 1, hollow microfibers basedon PLLA and PCL were successfully producedusing 96% vol. Et-OH or 50% vol. IP-OH/G solu-tion as non-solvents (Figure 2A). After produc-tion, the microfibers showed hollow sectionswith main diameters ranging in about 250-500μm. PLLA microfibers had a larger diameterand thinner wall (Figure 2 A1-A2) than the PCLmicrofibers, which showed a smaller diameterand thicker wall (Figure 2 A3-A4), independ-ently of the non-solvent used. Upon scaffoldassembly, the microfibers underwent compres-sion and heating. Thereafter, the diameters ofmicrofibers at the scaffold surface wereimaged and measured via SEM. The morpholo-gy of scaffold layouts and microfibers is shownin Figures 2B and 3. Micrographs of PLLA arereported in Figure 3A, while those of PCL inFigure 3B. For each scaffold type, preservationof fiber cavities after compression and heatingwas confirmed via SEM (Figure 3, lens),although random PCL scaffolds were the mostaltered (Figure 3 B2, lens). Microfiber wall

thickness was measured in a one representa- tive SEM micrograph per sample and ranged in

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Figure 1. Scheme of the dry-wet spinning system and process: A) 3D drawing showing thespinning system and its moving axes; B) sketch representing the spinning process and keycomponents.

Figure 2. Photographs of hollow microfibers and scaffold surfaces. A) Hollow microfibersat their sprout from the spinneret and further identified by polymer non-solvent couples:A1) PLLA Et-OH; A2) PLLA IP-OH/G; A3) PCL Et-OH; and A4) PCL IP-OH/G. Scalebar ¼ mm. B) Photographs of scaffold surfaces sputter coated with gold, obtained viaeither random (left column) or ordered (right column) arrangement of microfibers: B1)PLLA Et-OH; B2) PLLA IP-OH/G; B3) PCL Et-OH; and B4) PCL IP-OH/G. Originalmagnification 4×, scale bar=1 mm. PLLA, poly-L-lactide; Et-OH, ethanol; IP-OH/G, iso-propanol/gelatin; PCL, poly-ε-caprolactone.

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20-70 μm in PLLA, while 40-90 μm in PCL scaf-folds.At their surfaces, orderly assembled scaf-

folds showed tight microfiber bonding, thusresulting in lower interspaced layouts thanrandomly assembled fabrics. In the patternedscaffolds, the outer microfibers usually had flatmorphology with evidence of intra-fiber melt-ing and topography artefacts, such as micro-grooves in PLLA and striping in PCL, probablyimprinted by the Teflon nets during heating(Figures 3A and B). Differently, randomlyassembled scaffolds showed best-preservedmicrofiber topography (Figures 3A and B). PCLmicrofibers were more affected by fusion andflattening induced via the fiber bondingmethod than PLLA ones, which, conversely,could be more easily identified and measuredas single microfibers. Microfiber diametersmeasured at the scaffold surfaces are reportedin Table 1. Owing to the artefacts generated bypost-spinning processing, microfiber diame-ters displayed very broad sizes, ranging from274±65 μm in IP-OH/G random PCL, to556±245 μm in IP-OH/G random PLLA. Noapparent trend could be observed among allthe scaffolds subtypes.

Human mesenchymal stromal cellcolonization of microfiber meshscaffoldsUsing SEM analysis, adherent cells were

found to be layering the microfiber surfaces atthe top surface of all the scaffolds, althoughsometimes rarely visible because of their flatmorphology and tight connections forminghomogeneous cell sheets (Figure 4).Incubation with Neutral Red and staining withMethylene Blue at the endpoint allowed theviable cells colonizing the scaffolds to be easilyvisualized (Figures 5 and 6). Viable hMSCswere also detected on the inner microfibers ofthe scaffolds, which resulted to be the best col-onized, as confirmed by the more intense redcolor. From a qualitative evaluation, cellularcolonization was most pronounced in IP-OH/Grandom and Et-OH random PLLA (Figure 5A),and in Et-OH random PCL constructs (Figure5B). Methylene Blue staining performed onfixed samples allowed an easier handling ofthe scaffolds and a better contrast against thebackground than those obtained in neutral redstained samples, so as to enable enhancedvisualization of cells also in the inner layers(Figure 6).

Metabolic activity of human mes-enchymal stromal cell/scaffoldconstructsThe alamarBlue® assay performed at

sequential time-points up to 15 days allowedhMSC metabolism along the culture time to bemonitored (Figure 7A and B). Under the

applied experimental conditions of this assay,all the constructs showed initially low metabol-ic activity ranging from 5.28±0.85% (in PLLAEt-OH random) to 11.80±0.81% (in PCL Et-OHrandom), the latter being the highest with sta-tistical significance among the hMSC/PCL sub-types (P<0.05). All the hMSC/PLLA constructsshowed metabolic increases 6 days after seed-ing, followed by a slow decrease, which wassteadily maintained until the endpoint (Figure7A). In hMSC/PLLA constructs, the highestABred% value averagely detected was18.03±3.21% in the IP-OH/G random subtypeon day 6, although with no statistical signifi-cance with respect to other hMSC/PLLA sub-types. On average, the PLLA IP-OH/G randomconstructs had a superior metabolic activity atany assayed time-point; in particular, it was

the highest among the PLLA constructs fromday 12 until the endpoint (14.62±1.23%,P<0.05). On the other hand, all the hMSC/PCLconstructs displayed picks in their metabolicactivity on day 9; this was followed by a dropand a slowly decreasing trend up to the end-point (Figure 7B). Among PCL constructs, thehighest metabolic activity was found in the Et-OH random subtype, with statistical signifi-cance in the PCL series (P<0.05). Additionally,this value was the highest among all the con-structs (29.87±5.61%, P<0.05). Finally, themetabolic activity of hMSC/PCL constructsranged from 6.75±0.25% (Et-OH patterned) to11.10±1.17% (Et-OH random). The latter sub-type showed an averagely superior metabolicactivity at all assayed time-points.

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Figure 3. Scanning electron microscope (SEM) micrographs showing scaffold surfaces atthe endpoint: A) poly-L-lactide (PLLA), and B) poly-ε-caprolactone (PCL). Images aregrouped by non-solvent type and layout sub-type; scale bar=200 μm, 100× magnification.Lenses show SEM micrographs of hollow microfibers after scaffold fabrication; scalebar=100 μm, 302× magnification.

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Human mesenchymal stromal cellfunction in microfiber mesh scaffoldsIntracellular ALP activity was quantified at

the endpoint to assess cell function and osteo-differentiative potential on different scaffoldsubtypes; ALP values were normalized by cellu-larity (Figure 7C). ALP activity ranged from2017±324 μM/(h.μg of ds-DNA) (Et-OH pat-terned) to 2986±295 μM/(h.μg of ds-DNA) (Et-OH random) in PLLA scaffold series, while itranged from 1887±103 μM/(h.μg of ds-DNA)(Et-OH patterned) to 3291±483 μM/(h.μg ofds-DNA) (Et-OH random) in PCL scaffoldseries. Considering that the ds-DNA of ahuman diploid cell is 7.18 pg/cell, the abovementioned results mean that, at intracellularlevel, single hMSCs produced active ALPenzyme ranging from 0.226±0.007 nM/min.cell(Et-OH patterned PCL) to 0.394±0.033nM/min�cell (Et-OH random PCL), which wereaveragely the highest and lowest values foundamong all the scaffolds subtype. Unpaired t-student tests were applied to check signifi-cance between homologous subgroups differ-ing only according to layout type. Statisticalfindings showed that ALP activity values of Et-OH random were significantly superior tothose of Et-OH patterned subtypes, both forPLLA (P=0.00029) and PCL (P=0.0000039). Nodifferences were similarly detected among theIP-OH/G samples. ANOVA was used to compareALP activity under the following variables:polymer, non-solvent, and layout type.Differences in ALP activity due to the polymertype resulted to be non-significant(F(1,40)=0.30, P=0.585). Differently, both non-solvent and layout types led to a statisticallysignificant difference in ALP activity expres-sion. Specifically, there were statistically sig-nificant effects of the variables non-solvent(F(1,40)=8.86, P=0.0049) – being the values ofEt-OH averagely higher than those of the IP-OH/G subgroups – and layout (F(1,40)=20.25,P=0.000057) – since the values of randomwere averagely higher than those of patternedsubgroups.

Discussion

Tissue engineering scaffolds have beeninvoked to act as cell-conditioning microenvi-ronments.7,8 This phenomenon has recentlybeen disclosed as not being a prerogative ofmaterials provided with ad hoc chemical mod-ifications. In fact, the chemo-physical featuresof the scaffolds have been demonstrated toenable topological hints which SCs canrespond with behavioral changes.8 Therefore,the intrinsic stimulations induced by the scaf-

fold offer the intriguing possibility of address-ing SC differentiation pathways, which is thesubject of innovative researches.8

Due to the limited literature on dry-wetspun scaffolds, this investigation is very novelin the tissue-engineering scenario and can bemainly compared to other fibrous scaffold out-comes. This study was aimed at identifyingsome fabrication parameters in dry-wet spunmicrofiber scaffolding, which can affecthMSCs. In particular, polymer (PLLA vs PCL),non-solvent (Et-OH vs IP-OH/G), and fabric lay-out (patterned vs random) were tested asinherent scaffold features having possibleimplications for metabolic activity, 3D colo-nization, and function of hMSCs. Our findings showed that the polymer influ-

enced the metabolic activity timeline ofhMSCs, while microfiber layout enhanced ALP

activity in randomly orientated scaffolds.Finally, Et-OH as a non-solvent, in combinationwith random microfiber assembly, showed thehighest activity values for this enzyme.Therefore, the obtained results are suggestiveof osteogenic lineage specification induced byrandomly oriented dry-wet spun microfiberscaffolds, which could disclose possible spill-overs for bone tissue engineering applications.The first step of this study consisted in man-

ufacturing hollow microfibers via dry-wet spin-ning. The main advantages of this scaffoldingtechnique over other fiber-based methods relyon the possibility to manufacture polymericmicrofibers exploiting polymer solubility, with-out having to work from the molten state.16 Inthis way, the chemical properties of the poly-mer are most preserved. There are other spin-ning techniques used to fabricate fibers from

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Figure 4. Scanning electron microscope micrographs showing human mesenchymal stro-mal cells (hMSCs) on scaffold surfaces at the endpoint: A) poly-L-lactide (PLLA), and B)poly-ε-caprolactone (PCL). Images are grouped by non-solvent type and layout sub-type.Scale bar=100 μm, 301× magnifications.

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polymeric solutions, such as wet-spinning andelectrospinning.12,23,24 However, wet-spunfibers have reduced possibility for drug incor-poration if compared to that of dry-wet spunfibers.14 Finally, unlike electrospun fibers, indry-wet spun fibers no theoretical limits existto reach the desired thickness.15 In this study,scaffolds with about 2 mm thickness wereassembled. The methodology reported in thispaper allowed the fabrication of hollowmicrofibers based on two bioresorbable FDA-approved polymers, namely, PLLA and PCL.Both polymers belong to the linear aliphaticpolyester family, but they display some chemi-cal differences. For example, the PCL isremarkably hydrophobic. In particular, PLLAand PCL greatly differ for their mechanicalproperties: PLLA is the stiffest, while PCL isthe most extensible.25

The mechanical properties of the materials,such as rigidity, are increasingly recognized ascritical regulators in MSC biology, however,their mechanism of action has been only par-tially elucidated.26 It has been reported that, onstiff surfaces, MSCs enhance tension andadhesiveness, which is ultimately hypothe-sized to modulate their lineage specification.7

In our experiments, the polymer type had adocumented effect on the metabolic activity.Specifically, metabolism vs time curves ofhMSCs growing on same-polymer scaffoldsshowed analogous trends. Metabolic raisesoccurred at time-points that were earlier inPLLA (day 6) than in PCL (day 9). In tissue-engineered constructs, the overall metabolicactivity of cells is a useful tool to encode anumber of biological information, like cytotox-icity/cytocompatibility, viability and prolifera-tion. Of the many tests commercially available,alamarBlue® allows the metabolism of thesame samples to be monitored over time withgood correlation with 3D cellularity, dependingon construct permeability and culture time.27

Specifically, fibrous scaffolds offer open-porestructures which can overcome metabolite dif-fusion barriers in 3D constructs.28 In our spec-imens, ABred% averagely ranged from 5.23% onday 3 (the lowest value) to 14.62% on day 15(the highest value). Endpoint results werequalitatively double-checked with the neutralred assay, proving the presence and density ofviable cells on the meshes. Within a 2-weektimeframe, the hMSC/scaffold constructsincreased their metabolism at time-points thatwere typical for each polymer-groups, showingvalues up to 18.03% (PLLA, the highest valueon day 6) and 29.87% (PCL, the highest valueon day 9). From day 9 (PLLA) and 12 (PCL)onwards, the metabolic activity of the con-structs reached a plateau, suggesting that thatpost-proliferative culture conditions could bereached.27 Being the cultures carried out con-comitantly, with identical test specificationsand cellular batch, the trend of the metabolism

curve was attributed to a peculiar influence ofthe polymer type on hMSC biochemistry. Bothconstruct colonization and metabolic activityresulted best on PLLA IP-OH/G random andPCL Et-OH random scaffold subtypes, confirm-ing that, in our scaffolds and time-points, nei-

ther the polymer nor the non-solvent exerted apreferential role on hMSC metabolism. A dedi-cated study will be necessary to assess whichproperties (e.g., chemical, physical or mechan-ical) of the polymers are possibly responsiblefor this metabolic response.

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Figure 5. Photographs showing neutral red-stained human mesenchymal stromal cells(hMSCs)/scaffolds at the endpoint: A) poly-L-lactide (PLLA), and B) poly-ε-caprolactone(PCL). Main images: magnification 4×, scale bar=1 mm. Lens: digitally zoomed-in micro-graphs showing cell details, original magnification 35×.

Figure 6. Stereomicroscopy images showing methylene blue-stained human mesenchymalstromal cells (hMSCs)/scaffolds at the endpoint: A) poly-L-lactide (PLLA), and B) poly-ε-caprolactone (PCL). Magnification 50×, scale bar as printed by the microscope 2000 μm.

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Once the microfibers had been producedfrom both polymers, the fiber bonding methodwas applied to link them either orderly or ran-domly as 3D-layered structures. After spinning,fiber size ranged in 250-500 μm, showing onlyqualitative differences between the two poly-mers. These results are in line with other stud-ies, reporting dry-wet spun PLA-(co)polymermicrofibers.14-19 The morphological analysisperformed on the scaffold via SEM showed thatthe surface microfibers were often flattened,sometimes melted together and carrying printsfrom the grid used during heating, eventhough they maintained hollow morphology.Their average size was inhomogeneous, but itremained close to that of the fabrication range.Size differences could not be correlated to anyscaffold parameters chosen for this study. Theonly appreciable diversity was that the randomsubtypes partially exposed on their surfacessecond layer microfibers, which appeared mor-phologically preserved. Canha-Gouveia andcolleagues recently showed that hierarchicalfeatures in fibrous scaffolds produced alternat-ing orderly (3D printed) and random (electro-spun) fiber layers, and had an osteogeniceffect on SCs derived from Wharton’s jelly.29

In our study, the microfiber arrangementaffected the intracellular ALP activity withspecificity. Many cell types are known to con-stitutively express ALP under different isoen-zymes. However, the ALP expression in SCshas denoted particular implications, accordingto their hierarchical potency. For example, theALP expression correlated very well with stem-ness in embryonic-like SCs, while it correlatedwith differentiation in adult SCs.30 In adulthMSCs derived from the bone marrow, ALP hasbeen recognized as a reliable marker to assessfurther bone forming capacity.31 Indeed, theALP levels induced by extrinsic cellular stimu-lation have shown to be predictive for the invivo osteogenic differentiation capacity ofthese hMSCs. The study of Prins and cowork-ers defined the significance of this marker byassaying homogeneous subpopulations ofbone marrow hMSCs, selected from one het-erogeneous population.31 In our experiments,the same hMSC batch was equally divided intoall the different scaffold types, each type bro-ken up into multiple numbers. No differentia-tive supplements were added in the CM, sothat any significant difference in ALP activityexpression is likely to be the result of a physi-cal conditioning of the scaffold as a microenvi-ronment. The detected differences betweenALP activity expressed by hMSCs in patternedand random scaffolds showed remarkable sta-tistical significance under the ANOVA test(P=0.000057), indicating potential forosteogenic lineage specification driven by ran-dom layout geometry. According to publishedpapers, the obtained ALP values are compara-ble to those of trabecular bone fragments or

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Figure 7. Bar graphs showing the results of quantitative tests: A-B) metabolic activity overculture time of human mesenchymal stromal cells (hMSCs) grown in poly-L-lactide(PLLA) and poly-ε-caprolactone (PCL) scaffolds; and C) expression of intracellular alka-line phosphatase (ALP) activity per cell at the endpoint. *P<0.05.

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bone marrow cultured in vitro with osteogenicsupplements for 15 days, while they are higherthan those usually detected in MSCs or bonecells grown on culture plastics.32 It is worthnoting that the expression of ALP, like othermarkers, results to be frequently increased in3D-culture systems. Finally, some specific influence of the non-

solvent was detected upon ALP activity expres-sion. The microfibers spun by using Et-OH as anon-solvent showed, on average, significantlyhighly values (P=0.0049) of ALP activity versusthose produced with IP-OH/G. This statisticaldifference was less notable than that calculat-ed by comparing random versus patterned lay-outs. The combination of random and Et-OHthus showed the highest ALP activity values. Inthis study, two volatile non-solvents, Et-OH andIP-OH, were used at concentrations that werechosen to balancing their potential toxicity.33

The microfibers were dried upon collection,and post-processed in oven, which allowed sol-vent and non-solvent evaporation. The absenceof cytotoxic effects, as from metabolic and via-bility assays discussed earlier, corroboratedthe effective removal of any toxic componentsfrom the scaffolds and are in line with observa-tions conducted with dry-wet spun PLLA(co)polymers.14-19 From a technological stand-point, the polymer/solvent/non-solvent thermo-dynamic system determines the miscibilitygaps and the equilibrium phases controllingthe morphological features of the fiber.14,16

Under the applied conditions, phase separa-tion did not cause fiber nanoporosity. In theirstudy on spongy scaffolds, Viswanathan andcolleagues highlighted that SC adhesion wasdependent on topology and chemistry, andindependent of porosity. They also showed thattopology, architecture and interconnectivityaffected the SC osteogenic differentiationpotential.8 At this early stage of our investiga-tion it is not possible to hypothesize any valu-able reasons explaining the role played by thenon-solvent in these fibrous scaffolds. Onlyfurther studies could ascertain hMSC/randommicrofiber scaffold bone formation capacity invitro and in vivo and whether the non-solventmakes a true difference. It can be hypothe-sized that other physical characteristics of thematerials, such as surface roughness andhydrophilicity, may affect hMSC behavior. Tuning scaffold features intelligently to

address SC fate will enable the development ofsmart scaffolds that could catalyze the desiredtissue formation from immature cells, helpingto discourage SC trans-differentiation and tis-sue overgrowth, which are the actual concernsof translational therapies.

Conclusions

In this study we investigated the influenceof polymer, non-solvent and scaffold layout ofdry-wet microfiber scaffolds on metabolicactivity, colonization and ALP activity expres-sion of bone marrow hMSCs. The polymer typeaffected the timeline trend of metabolic activi-ty. Both construct colonization and metabolicactivity resulted to be independent of otherscaffold subtypes. Instead, hMSCs grown onrandom microfiber fabrics averagely showedstatistically superior intracellular ALP activityper cell to that detected on patterned fabrics.The highest ALP values were detected in theEt-OH random subtypes. Although the underly-ing mechanisms of cell-substrate interactionleading to ALP increase and metabolic behav-ior still need to be elucidated, the outcomesreported in this study propose new insightsinto fibrous scaffolds like SC conditioningmicroenvironments, which could concur topave the way for future strategic directions inSC tissue engineering.

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