the support of bone marrow stromal cell differentiation by airbrushed nanofiber scaffolds 2013...

8172019 The Support of Bone Marrow Stromal Cell Differentiation by Airbrushed Nanofiber Scaffolds 2013 Biomaterials

httpslidepdfcomreaderfullthe-support-of-bone-marrow-stromal-cell-differentiation-by-airbrushed-nanofiber 110

The support of bone marrow stromal cell differentiation by airbrushed

nano1047297ber scaffolds

Wojtek Tutak ab Sumona Sarkar a Sheng Lin-Gibson a Tanya M Farooque a Giri Jyotsnendu abDongbo Wang a Joachim Kohn c Durgadas Bolikal c Carl G Simon Jr a

a Biosystems amp Biomaterials Division National Institute of Standards amp Technology Gaithersburg MD 20899 USAb American Dental Association Foundation National Institute of Standards amp Technology Gaithersburg MD 20899 USAc New Jersey Center for Biomaterials Rutgers University Piscataway NJ 08854 USA

a r t i c l e i n f o

Article history

Received 14 December 2012

Accepted 15 December 2012

Available online 11 January 2013

This work is dedicated to the memory of

Milenko Markovic

Keywords

Airbrushing

Bone marrow stromal cell

Cell differentiation

Electrospinning

Nano1047297ber

Stem cell

a b s t r a c t

Nano1047297ber scaffolds are effective for tissue engineering since they emulate the 1047297brous nanostructure of

native extracellular matrix (ECM) Although electrospinning has been the most common approach for

fabricating nano1047297ber scaffolds airbrushing approaches have also been advanced for making nano1047297bers

Forairbrushing compressed gasis used to blow polymer solution through a smallnozzle which shears the

polymer solution into 1047297bers Our goals were 1) to assess the versatility of airbrushing 2) to compare the

properties of airbrushed and electrospun nano1047297ber scaffolds and 3) to test the ability of airbrushed

nano1047297bers to support stem cell differentiation The results demonstrated that airbrushing could produce

nano1047297bers from a wide range of polymers and onto a wide range of targets Airbrushing was safer 10-fold

faster100-fold less expensive to set-up and ableto deposit nano1047297bers onto a broader range of targets than

electrospinning Airbrushing yielded nano1047297bers that formed loosely packedbundlesof aligned nano1047297bers

while electrospinning produced un-aligned single nano1047297bers that were tightly packed and highly

entangled Airbrushed nano1047297ber mats had larger pores higher porosity and lower modulus than elec-

trospun mats results that were likely caused by the differences in morphology (nano1047297ber packing and

entanglement) Airbrushed nano1047297ber scaffolds fabricated from 4 different polymers were each able tosupport osteogenic differentiation of primary human bone marrow stromal cells (hBMSCs) Finally the

differences in airbrushed versus electrospun nano1047297ber morphology caused differences in hBMSC shape

where cells had a smaller spread area and a smaller volume on airbrushed nano 1047297ber scaffolds These

results highlight the advantages and disadvantages of airbrushing versus electrospinning nano1047297ber

scaffolds and demonstrate that airbrushed nano1047297ber scaffolds can support stem cell differentiation

Published by Elsevier Ltd

1 Introduction

Nano1047297ber scaffolds have advanced as tissue engineering scaf-

folds since they mimic the 1047297brous nanostructure of native extra-

cellular matrix (ECM) [1e

3] Cells in vivo exist in a 3D matrixcomposed largely of collagen which forms an extensive natural

matrix of nano1047297bers with 200 nm diameter [4] Though electro-

spinning is the most common method for making nano1047297bers for

tissue engineering scaffolds [56] compressed gas can also be used

to blow polymer solutions into 1047297bers in a process that is called

solution spraying blowspinning or airbrushing Formhals [7]

patented the electrospinning method in 1934 while Norton [8]

patented the 1047297rst blowspinning device in 1936 More recently

Mattoso et al [9] reported a device with concentric nozzles where

polymer solution was injected into a stream of 1047298owing gas to

generate nano1047297bers from polystyrene (PS) poly(methyl methacry-

late) (PMMA) or poly(lactic acid)Srinivasan et al [10] found that anairbrush commonly used for painting could be loaded with PMMA

solution and used to airbrush PMMA nano1047297bers

The use of an airbrush to make nano1047297ber scaffolds is intriguing

since it is easier to use and less expensive to set up than electro-

spinning An airbrush could also be used to ldquopaintrdquo nano1047297bers onto

a broader range of targets For instance a mold of an organ that is to

be regenerated could be airbrushed with nano1047297bers to create

a nano1047297ber scaffold in the shape of the target organ In order to

advance the airbrushing technique for tissue engineering we have

assessed the ability of airbrushing to make nano1047297ber scaffolds from

a wide range of biomedical polymers Further we have compared Corresponding author Tel thorn 1 301 975 8574 fax thorn1 301 975 4977

E-mail address carlsimonnistgov (CG Simon)

Contents lists available at SciVerse ScienceDirect

Biomaterials

j o u r n a l h o m e p a g e w w w e l s e v i e r c o m l o c a t e b i o m a t e r i a l s

0142-9612$ e see front matter Published by Elsevier Ltd

httpdxdoiorg101016jbiomaterials201212020

Biomaterials 34 (2013) 2389e2398



airbrushing and electrospinning in a number of side by side tests to

determine the advantages and disadvantages of each Finally we

have tested the ability of airbrushed nano1047297ber scaffolds to support

osteogenic differentiation of primary human bone marrow stromal

cells (hBMSCs)

2 Materials amp methods

21 Airbrushed nano 1047297bers

A commercially available airbrush designed for painting (Master Airbrush

G222-SET 02 mm nozzle diameter gravitational feed) was used to fabricate

nano1047297ber scaffolds The airbrush was connected to a pressurized nitrogen tank

(241 kPa 35 pounds per square inch) The distance from the nozzle to the target was

held constant at 20 cm for all experiments For electron microscopy porosity and

mechanical tests aluminum foil was used as the target and nano1047297ber mats were

peeled off the foil for assessment For cell culture experiments tissue culture

polystyrene (TCPS) disks were used as targets TCPS disks were hot-punched from

the bottom of TCPS plates into 6 mm diameter disks that 1047297t into the bottom of

96-well plates Nano1047297bers were airbrushed from four different polymers poly-

styrene (PS taken from the bottoms of tissue culture polystyrene dishes Corning

catalog number 430599) poly(desaminotyrosyl-tyrosine ethyl ester carbonate)

(pDTEc made as described) [11] poly(DL -lactic acid) (PDLLA SurModics Pharma-

ceuticals) and poly(ε-caprolactone) (PCL SigmaeAldrich) Polymer relative molec-

ular masses solvents and polymer concentrations for airbrushing are given in

Table 1 Molecular masses were provided by the manufacturer except for PS which

was measured using gel permeation chromatography using tetrahydrofuran as

solvent

22 Electrospun nano 1047297bers

Electrospun nano1047297bers were made from PCL with a home built electrospinning

apparatusPCL solution(10mass fractionin 31volumeratiochloroformmethanol)

was loadedintoa syringe anddispensed witha syringepump at2 mLhThe positive

leadfrom thepowersupply was1047297xed tothe spinneret whichwasan 18 gaugeneedle

and the ground lead was 1047297xed to the target (aluminum foil) The distance between

needleand targetwas 15cm andvoltage was165kV Non-wovenPCL nano1047297bermats

were collected for 15 h onto an aluminum foil target Electrospun nano1047297bers were

removed from the foil for mechanical testing For cell culture samples TCPS disks

were placed on the aluminum and nano1047297bers were electrospunonto the TCPS disks

23 Deposition rate

Deposition rate for airbrushing and electrospinning was determined bymeasuring how long it took to deposit a known volume of polymer solution of

a known concentration

24 Scanning electron microscopy

For scanning electron microscopy (SEM) nano1047297ber scaffolds were sputter

coated with gold for 90 s and then imaged (Hitachi S-4700-II FE-SEM 5 kV) Fiber

diameter was determined from scanning electron micrographs from 3 1047297elds of view

using ImageJ software (NIH) to determine diameter of 30e100 nano1047297bers To assess

1047297ber diameter reproducibility PCL 1047297bers were airbrushed or electrospun on three

separate days for SEM imaging analysis For pore size several spots were imaged on

a mat and 50e120 pores were sized in scanning electron micrographs

25 Nano 1047297ber porosity

Porosity of nano1047297ber scaffolds was determined using the following equation

[12] porosity frac14 (1 [(massdensity)(width length thickness)]) Scaffolds werecut to 2 cm by 2 cm squares (width and length) Mass was determined by weighing

scaffold squares on a balance Scaffold thickness was determined by measuring the

edge thickness of the scaffold squares in SEM PS density was 105 gmL [13] pDTEc

density was 12 gmL [14] PDLLA density was 125 gmL [15] and PCL density was

11 gmL [16]

26 Mechanical analysis

Mechanical properties were evaluated using a dynamic mechanical analyzer

(RSA III instrument TA Instruments) equipped with a rectangular 1047297lm1047297ber clamp

geometry Airbrushed and electrospun nano1047297ber mats were folded twice to yielda rectangular 4-layer sheet that was 6 mm 10 mm 015 mm Samples were

weighed to insure that mass per unit area of the scaffolds was similar (within 10)

Tensile measurements on the samples were conducted with a 02 mms cross-head

speed at room temperature Modulus was calculated from the slope of the stress e

strain curve between 5 and 9 strain

27 Cell culture

Airbrushed and electrospun nano1047297ber scaffolds on TCPS disks were placed into

96-well plates and af 1047297xed to the bottoms of the wells with a small dab of silicon

vacuum grease Samples were sterilized by ethylene oxide (Anderson Products) and

degassed for 2 d under house vacuum Primary human bone marrow stromal cells

(hBMSCs Texas AampM Institute for Regenerative Medicine 24 year old female)

were cultured at 37 C with 5 by volume CO2 in a-minimum essential medium

(Invitrogen) supplemented with 165 by volume fetal bovine serum (Atlanta Bio-

logicals) and 4 mmolL L -glutamine [17] hBMSCs were passaged at 80 con1047298uency

and dissociated with 025 mass fraction trypsin containing 1 mmolL ethyl-

enediaminetetraactate (EDTA) Passage 5 hBMSCs were seeded onto scaffolds in 96-

well plates at 5000 cellswell in 02 mL of mediumwell After 24 h medium was

replaced with medium containing osteogenic supplements (10 nmolL dexametha-

sone 20 mmolL b-glycerophosphate and 005 mmolL ascorbic acid) Medium was

changed twice per week with medium containing osteogenic supplements

28 Fluorescence imaging

Cells on scaffolds were 1047297xed (37 formaldehyde massvolume in PBS (phos-

phate bufferedsaline) for 15min) washed (PBS) permeabilized (02by massTriton

X-100 for 5 min) and rinsed (PBS) Samples were stained 1 h in PBS with 20 nmolL

Alexa Fluor 546-phalloidinand 100nmolL Sytox green (Invitrogen) to stain for actin

and nuclei respectively After staining cells were washed with PBS air dried and

imaged by 1047298uorescence microscopy (Nikon Eclipse TE 300) using 4013 objectives

and 20045 hBMSCs were also imaged by confocal1047298uorescence microscopy (Leica

TCS SP5 broadband) using a 6314 oil immersion objective with a 250 nm Z-step

size

29 Picogreen DNA assay

hBMSCs grown on the nano1047297ber scaffolds were quanti1047297ed using Picogreen DNA

assay according to manufacturerrsquos protocol After cell culture scaffolds were rinsed

with PBS frozen overnight to rupture cell membranes (20 C) thawed and incu-

bated with digestion buffer (PBS with 0175 UmL papain and 145 mmolL

L -cysteine) for 16 h at 60 C Lysate (02 mL) was transferred to a clean 96-well plate

and diluted with 02 mL of Picogreen reagent (Invitrogen diluted according to

manufacturerrsquos protocol) DNA 1047298uorescence (excitation 485 nm emission 538 nm)

was measured using a platereader and calibrated using a DNA standard curve

210 Osteocalcin ELISA

ELISA assay (enzyme-linked immunosorbent assay BTI BT-460) was employed

to measure osteocalcin (OC) as a marker for osteogenic differentiation Three

scaffolds were assessed for each condition (n frac14 3) After cell culture scaffolds

were rinsed with PBS incubated with 02 mL of acetic acid (10 by volume) for1 h at 37 C and lyophilized overnight Manufacturerrsquos sample buffer was used to

Table 1

Processing conditions amp nano1047297ber scaffold properties

Fabrication Airbrushed Airbrushed Airbrushed Airbrushed Electrospun

Polymer PS pDTEc PDLLA PCL PCL

Relative molecular mass (gmol) 178000 183000 100000 80000 80000

Solvent Dimethyl-formamide Dioxane Chloroform

methanol 8515 by mass

Chloroform Chloroform

methanol 31 by mass

Concentration (mass) 8 15 6 4 10

Solution deposition rate (mLmin) 061 031 115 082 003

Scaffold deposition rate (mgmin) 186 54 90 30 6

Fiber diameter (nm)

[Mean (SD)] (n frac14 35)

1949 (643) 677 (420) 125 (88) 231 (79) 511 (185)

W Tutak et al Biomaterials 34 (2013) 2389e 23982390



resuspend the lyophilized protein before transfer to the ELISA plate ELISA plates

were processed according to manufacturerrsquos protocols A standard curve was

generated to determine osteocalcin concentration Scaffold measurements were

background subtracted using controls Controls were scaffolds that were incubated

without cells in cell culture medium with OS for 10 d (with medium changes) and

then assayed by ELISA

211 Alizarin red staining

hBMSCs on scaffolds were 1047297xed with 37 (by volume in PBS buffer) formal-

dehyde for 1 h at 37 C and then stained with Alizarin red (10 mgmL in water) for

1 h The samples were washed in deionized water and air dried Digital images of

stained scaffolds were acquired using a stereomicroscope Three scaffolds were

assessed for each condition (n frac14 3) After imaging the samples were treated with10 (by volume in water) hexadecetylpyridinumechlorideemonohydrate to extract

the Alizarin red dye Extracts from samples were collected and absorbance was

measured spectroscopically at 405 nm A standard curve of known Alizarin red

concentrations was constructed and used to 1047297t experimental data Scaffold

measurements were background subtracted using controls Controls were scaffolds

that were incubated without cells in cell culture medium with OS for 21 d (with

medium changes) stained for Alizarin red and then extracted and measured for

absorbance

212 Confocal image analysis

Confocal 1047298uorescence Z-stacks of hBMSCs cultured 1 d on airbrushed and

electrospun PCL nano1047297ber scaffolds were analyzed using ImageJ software (NIH) to

assess cell shape Sytox green staining of nuclei was used to insure that cell

morphology was assessed for single cells only (only one nucleus per object) For cell

spread area perimeter aspect ratio and roundness Z-stacks were projected into 2D

images and thresholded to result in binary images ImageJ was used to measure cellspread area and perimeter For cell volume Z-stacks were thresholded cell outlines

Fig 1 Photograph of the commercially available airbrush used to fabricate airbrushed


Fig 2 (a) Scanning electron micrographs of airbrushed nano1047297ber scaffolds made from 4 different polymers An SEM image of an electrospun PCL nano1047297ber scaffold is shown for

comparison (b) A photograph of an airbrushed PCL nano1047297ber mat is shown SEMs of boxed regions are shown on the right to demonstrate that nano 1047297ber morphology was uniform

across the airbrushed mats

W Tutak et al Biomaterials 34 (2013) 2389e 2398 2391



were created and the ImageJ plugin ldquo3D Object Counterrdquo was used to calculate cell

volumes [18]

213 Statistics

All dataare presented as means withstandard deviation The standarddeviation

(SD) is the same as the ldquocombined standard uncertainty of the meanrdquo for the

purposes of this work To test for statistically signi1047297cant differences t -test was used

for pairwise comparisons (P lt 005) and 1-way analysis of variance (ANOVA) with

Tukeyrsquos test was used for comparisons of 3 or more treatments ( P lt 005)

3 Results

31 Airbrushed nano 1047297ber scaffold characterization

Airbrushing (Fig1) was able to fabricatenano1047297ber scaffoldsfrom

all four polymers tested (PS pDTEc PDLLA and PCL) as de1047297ned in

Table 1 and as shown in SEM (Figs 2e4) For comparison PCL

nano1047297bers made byelectrospinning were also made (Figs 2e4)The

solvent systems for airbrushing and electrospinning given in Table 1

were adapted from previous information on polymer solubilities

and from work where the polymers were electrospun as described

for PS [19] pDTEc [14] PDLLA [20] and PCL [21] However trial and

error was required to identify the optimal polymer concentrationsand other airbrushing parameters reported in Table 1 When the

polymer concentration was too high the airbrush clogged When

polymer concentration was toolow the mats were gummy they did

not dry 1047297bers did not form and there were beads instead of

nano1047297bers Airbrushed nano1047297bers frequently appeared as loosely

packed bundles of aligned nano1047297bers that contained 10e100

nano1047297bers per bundle In contrast electrospun PCL nano1047297bers

were un-aligned single nano1047297bers that were tightly packed and

highly entangled Average pore sizes for airbrushed PS pDTEc

PDLLA and PCL nano1047297ber scaffolds ranged from 8 mm to 17 mm

(Fig 3a) and were larger than average pore size observed for elec-

trospun PCL nano1047297bers (3 mm) The average thickness of a PCL

nano1047297ber mat that resulted after a 43 min airbrush deposition was

249 mm (SD 269 mm n frac14 3)

When examining a mat of airbrushed PCL nano1047297bers in the SEM

a similar nano1047297ber diameter and morphology was found in

different locations in the mat indicating that airbrushed nano1047297ber

mats were uniform (Fig 2b) Airbrushing was also reproducible

since PCL nano1047297bers that were airbrushed on three different days

had similar nano1047297ber diameters (Fig 4a) The coef 1047297cient of varia-

tion of nano1047297ber diameters was similar for airbrushed and elec-

trospun nano1047297ber scaffolds (Fig 4b) demonstrating that the

variance in nano1047297ber diameter was similar for the two techniques

Airbrushed PS pDTEc PDLLA and PCL nano1047297ber scaffolds had

porosities ranging from 77 to 95 (Fig 3b) and airbrushed scaf-

folds were generally more porous than electrospun PCL nano1047297bers

(67) The difference in porosity measurements was supported by

SEM which showed that airbrushed nano1047297ber mats were loosely

packed with large voids while electrospun nano1047297bers were more

tightly packed and entangled Airbrushed PCL nano1047297ber scaffoldshad lower modulus than electrospun PCL nano1047297ber scaffolds

(Fig 4eef) The higher entanglement of individual electrospun PCL

nano1047297bers most likely caused them to have higher stiffness than

airbrushed nano1047297bers which were more loosely packed and less

entangled

Airbrushing was able to ldquopaintrdquo nano1047297bers onto irregularly

shaped objects made from a wide range of materials including

metals polymers ceramics and natural materials (Fig 5) Electro-

spinning required a power supply to set up a charge differential

between the spinnerette and target This required the target for

electrospinning to be electrically conductive Electrospinning was

also able to deposit nano1047297bers onto small non-conductive items

such as coverslips which were placed on the conductive target

(in between the charged spinnerette and the conductive target) Incontrast airbrushing was more mobile and could be easily aimed to

ldquopaintrdquo any target with nano1047297bers

32 hBMSC response to airbrushed nano 1047297ber scaffolds

Tovalidateairbrushed nano1047297bers as tissue engineering scaffolds

their ability to support osteogenic differentiation of hBMSCs was

assessed hBMSCs can differentiate down osteogenic adipogenic

and chondrogenic lineages [22] hBMSCs are the leading stem cell

candidate for skeletal tissue engineering [23] and osteogenic

differentiation is enhanced by nano1047297ber scaffolds [2124e27] Air-

brushed nano1047297ber scaffolds fabricated from 4 different polymers

(PS pDTEcPDLLA andPCL) were seededand culturedwith hBMSCs

Fluorescence staining after 1 d culture showed that hBMSCs couldadhere to airbrushed nano1047297ber scaffolds (Fig 6)

A quantitative DNA assay indicated that hBMSCs proliferated on

all 4 types of airbrushed nano1047297ber scaffolds during culture through

21 d (Fig 7a Fig S1) Osteocalcin ELISA and Alizarin red staining

for calcium demonstrated that hBMSCs synthesized osteocalcin

and deposited a calci1047297ed matrix during culture on airbrushed

nano1047297ber scaffolds (Fig 7bed Fig S1) No signi1047297cant differences

were observed between the four polymers for the DNA assay or the

osteocalcin ELISA although there were some signi1047297cant differences

between the 4 polymers for the Alizarin red absorbance measure-

ments (Fig S1) The airbrushed nano1047297ber scaffolds were stable in

culture up to 21 d They did not tear or come apart during handling

for cell culture assays Attempts were made to use scanning elec-

tron microscopy to assess the morphology of the airbrushed

0

5

10

15

20

PS pDTEc PDLLA PCL PCL

P o r e S i z e ( micro

m )

0

20

40

60

80

100


P o r o s i t

y ( )

Airbrushed Electrospun

a

b

Fig 3 (a) Pore size of nano1047297ber scaffolds determined by scanning electron microscopy

(n frac14 4) Electrospun PCL and airbrushed pDTEc were signi1047297cantly different from all

others (P lt 005 1-way ANOVA with Tukeyrsquos) (b) Porosity of nano1047297ber scaffolds

determined by scanning electron microscopy (n frac14 4) Electrospun PCL was signi1047297cantly

different than airbrushed PS airbrushed PDLLA and airbrushed PCL (P lt 005 1-way

ANOVA with Tukeyrsquos)




nano1047297bers after 21 d of cell culture however hBMSCs covered the

surface of the nano1047297bers obscuring them from view These results

show that airbrushed nano1047297ber scaffolds supported adhesion

proliferation and osteogenic differentiation of hBMSCs

Since airbrushed and electrospun nano1047297bers differed in their

structure (nano1047297ber bundling and porosity) their effect on stem

cell shape was assessed by confocal microscopy (Fig 8 Fig S2)

The role of cell shape in directing cell function is well documented

0

2

4

6

8

10

12

14

16


M o d u l u s ( M P a )

0 10 20 30 40 50 60 7000

05

10

15

20

25

30

35

40

S t r e s s ( M P a )

Strain ()

e

Airbrushed

Electrospun

50 microm

dc

a

50 m

0

5

10

15

20

25

30

35

40

45

C o e f f i c i e n t o f V a r i a t i o n ( )

A i r b r u s h e d

E l e c t r o s p u n

b

0

100

200

300

400

500

600

700

1 2 3 4 5 6

N a n o d i f i b e r D

i a m e t e r ( n m )

Airbrushed

1

2

3

1

2

3

Electrospun

f

Fig 4 (a) Comparison of the mean nano1047297ber diameters of PCL scaffolds prepared using the exact same conditions but fabricated on 3 different days using the airbrushing or

electrospinning techniques Nano1047297ber diameter was measured in SEM (n frac14 30e100) (b) Comparison of the coef 1047297cient of variation [(SD)mean] for diameter of nano1047297bers

fabricated on 3 different days by airbrushing or electrospinning techniques ( n frac14 3) The data are taken from Panel (a) There is not a statistically signi1047297cant difference between

airbrushing and electrospinning (t -test P gt 005) (c) Scanning electron micrograph (SEM) of airbrushed PCL nano1047297ber scaffold (d) SEM of electrospun PCL nano1047297ber scaffold (e)

Representative stressestrain plots for tensile testing of 3 airbrushed and 3 electrospun PCL nano1047297ber mats (f) Tensile modulus of airbrushed PCL and electrospun PCL nano 1047297ber

mats (n frac14 4) Asterisk indicates signi1047297cant difference (t -test P lt 005)




[28e31] and effects of scaffold architecture on cell shape and

function have been observed [2132] hBMSCsattained an elongated

morphology with high aspect ratio and low roundness on both

airbrushed and electrospun PCL nano1047297bers which was similar to

previous observations of hBMSCs on nano1047297ber scaffolds [21]

However hBMSCshad a smaller spread area perimeter and volume

on airbrushed nano1047297ber scaffolds than on electrospun These

results show that the differences in the structure of airbrushed and

electrospun nano1047297ber scaffolds had signi1047297cant effects on hBMSC

morphology but that both types of scaffolds drove hBMSCs into anelongated morphology

4 Discussion

Table 2 presents a comparison of the airbrushing and electro-

spinning approaches for fabricating nano1047297ber scaffolds Airbrushing

wasless expensive than electrospinning since airbrushes start at $25

while electrospinning set-ups cost $2500 for a power supply

($1000) syringe pump ($1000) and other parts ($500 glass

syringes tubing with luer 1047297ttings) Airbrushing was simpler than

electrospinning since airbrushing had fewer parts and was quicker

to set-up For airbrushing the airbrush only had to be 1) connected

to compressed gas and 2) loaded with polymer solution before use

For electrospinning the spinnerette had to be mounted the target

needed to be assembled the power supply had to be connected the

syringe pump had to be set up polymer solution had to be loadedand the tubing had to be connectedAirbrushing was also safer than

electrospinning since electrospinning required high voltage For

safety electrospinning was performed behind a plexiglass shield to

protect passers-by from the electrical hazard

Airbrushing had a 10 faster deposition rate than electro-

spinning in terms of mass of nano1047297bers fabricated per unit time

Fig 5 Demonstrating how airbrushing can ldquopaintrdquo nano1047297bers onto irregularly shaped objects made from a wide range materials (metals polymer ceramic natural materials) PCL

nano1047297bers were airbrushed onto (a) metal knee replacement for dogs (b) metal hip replacement for dogs (c) steel bolt (d) wooden tongue depressor (e) rubber hand and (f)

calcium phosphate cement screw [42]

Fig 6 Fluorescence micrographs of hBMSCs cultured on airbrushed nano1047297ber scaffolds for 1 d Nuclei are green (Sytox green) and actin is red (Alexa Fluor 546 phalloidin) Scale

bars in each row apply to all the images in the same row (For interpretation of the references to color in this 1047297

gure legend the reader is referred to the web version of this article)




pDTEc LCPALLDPSPc

1 mm ( - ) h B M S C s

( + ) h B M S C s

0

50

100

150

200

250

300

350

400

PS pDTEc PDLLA PCL

D N A ( n g S c a f f o l d

)

1 d10 d17 d21 d

a

00

01

02

03

04

05

06

07

08

PS pDTEc PDLLA PCL

O s t e o c a l c i n ( n g S c a f f o l d )

10 d

17 d

21 d

b

0

10

20

30

40

50

60

70

80

90

PS pDTEc PDLLA PCL

A

l i z a r i n R e d ( micro M )

10 d

17 d

21 d

d

Fig 7 hBMSCs cultured on airbrushed different nano1047297ber scaffolds for different times hBMSCs were cultured with osteogenic supplements (OS) starting at 24 h after cells were

seeded on scaffolds (a) hBMSC adhesion and proliferation on airbrushed nano 1047297ber scaffolds was assessed with Picogreen DNA assay Asterisks indicate signi1047297cant differences from

1 d (ANOVA with Tukeyrsquos P lt 005 n frac14 3) (b) Osteogenic differentiation of hBMSCs on airbrushed nano1047297ber scaffolds was measured by ELISA for osteocalcin protein deposition

Asterisks indicate signi1047297cant differences from 10 d (ANOVA with Tukeyrsquos P lt 005 n frac14 3) (c) Osteogenic differentiation of hBMSCs was measured by Alizarin red staining for

calcium deposition The bottom row of images is controls for non-hBMSC mediated calcium deposition where scaffolds were cultured without hBMSCs in full medium with OS and

medium changes Images were captured after 17 d culture The scale bar applies to all images (d) Alizarin red dye was extracted from stained scaffolds to quantify calcium

deposition Asterisks indicate signi1047297cant differences from 10 d (ANOVA with Tukeyrsquos P lt 005 n frac14 3)




The deposition rate for airbrushing was dependent on the viscosity

of the polymer solutions the concentration of the polymer solu-

tions and the 1047298ow rates (gas pressure) The variance in nano1047297ber

diameter for airbrushing and electrospinning was similar but the

morphology of the nano1047297bers was different Airbrushed nano1047297ber

mats had bundles of aligned nano1047297bers which crossed one another

to create larger pores and higher porosity Electrospun mats had

single nano1047297bers that were not bundled but were more tightly

packed with smaller pore size lower porosity and higher entan-

glement Srinivasan et al [10] also observed aligned nano1047297ber

bundles in SEM of airbrushed nano1047297bers These differences in

morphology likely caused the observed differences in mechanical

properties where the airbrushed nano1047297bers had a lower modulus

than electrospun The modulus of the electrospun nano1047297bers

measured in the current work [126 (08) MPa] was similar to

previous measurements of electrospun PCL nano1047297bers of 7 MPa

[33] 11 MPa [34] and 35 MPa [35]

As for versatility both airbrushing and electrospinning were

able to fabricate nano1047297bers from a wide range of polymers

Previous work showed that PMMA could be airbrushed [10] while

the current work demonstrated that PS pDTEc PDLLA and PCL

could be airbrushed Prior studies showed that these and other

polymers can be electrospun [936] In regard to targets air-

brushing was more versatile than electrospinning since airbrushing

0

200

400

600

800

1000

1200

1400

1600

AirbrushedElectrospun

A r e a ( micro m

2 )

0

100

200

300

400

500

600

700


P e r i m e t e r ( micro m )

0

1

2

3

4

5

6

7

8


A s p e c t R a t i o

00

01

02

03

04

05

06

07

08


R o u n d n e

s s

0

500

1000

1500

2000

2500

3000

3500


V o l u m e ( micro

m 3 )

20 microm


A i r b r u s h e d


A i r b r u s h e d

ElectrospunAirbrushed

a

b


A i r b r u s h e d

f

c


A i r b r u s h e d


A i r b r u s h e d

d

e

Fig 8 Confocal 1047298uorescence Z-stacks of hBMSCs cultured 1 d on airbrushed or electrospun PCL nano1047297ber scaffolds were analyzed for cell shape hBMSC actin was stained with

Alexa-Fluor-546-phalloidin (a) Fluorescence images of representatives hBMSCs cultured on an airbrushed or electrospun PCL nano 1047297ber scaffold (bef) Analysis of hBMSC (b) cell

spread area (c) perimeter (d) aspect ratio (e) roundness and (f) volume Asterisks indicate signi1047297cant differences (t -test P lt 005 n frac14 16)




was more mobile and could be aimed at any target for ldquopaintingrdquo

with nano1047297bers This could be advantageous for making nano1047297ber

scaffolds in the shape of organs when using a sacri1047297cial mold

Airbrushing might also be adapted to a portable spraycan for point-

of-care use A spray-on nano1047297ber wound dressing could be used in

1047297rst aid kits on the battle1047297eld or by 1047297rst responders [937] Elec-

trospinning afforded better control of nano1047297ber diameter than did

airbrushing Adjusting the concentration of the polymer solution

voltage pump rate spinnerette to target distance and spinnerette

diameter can enable the nano1047297ber diameter to be adjusted when

electrospinning [213839] However only the concentration of thepolymer solution gas pressure and airbrush ori1047297ce diameter can be

adjusted for airbrushing which makes it harder to control air-

brushed nano1047297ber diameter

Both airbrushed and electrospun nano1047297bers scaffolds elicit

a favorable biological response Much previous work has con1047297rmed

the ability of electrospun nano1047297ber scaffolds to support tissue

engineering applications [235621263638e41] and the current

work demonstrates that airbrushed nano1047297ber scaffolds also have

tissue engineering potential Herein hBMSCs adhered proliferated

and underwent osteogenic differentiation on airbrushed nano1047297ber

scaffolds fabricated from 4 different polymers (PS pDTEc PDLLA

PCL) The differences in alignment bundling and porosity of the

airbrushed and electrospun nano1047297ber scaffolds caused measurable

differences in hBMSC morphology Previous work has demon-strated that scaffold architecture can drive cells into shapes that

control their fate [2132] and future work may determine if differ-

ences in hBMSC morphology on airbrushed and electrospun

nano1047297bers will affect their function

5 Conclusions

An airbrushing method for making nano1047297ber scaffolds has been

compared to the more common electrospinning approach and

assessed for its ability to support stem cell differentiation When

compared to electrospinning airbrushing is 100 less expensive

easier to use safer and 10 faster Airbrushing and electrospinning

have similar reproducibility for nano1047297ber diameter both are able to

make nano1047297

bers from a wide range of polymers and both can

support cell adhesion proliferation and differentiation While air-

brushed nano1047297ber mats have larger pores and higher porosity

electrospun nano1047297ber matshave a higher modulus Airbrushed mats

have bundles of aligned nano1047297bers not found in electrospun mats

and these differences in scaffold structure cause hBMSCs to assume

a smaller size on airbrushed nano1047297bers While electrospinning

affords more control over nano1047297ber diameter the electrospinning

approach can only deposit nano1047297bers onto a targetinside thecharge

differential required for the process In contrast an airbrush can be

aimed to ldquopaintrdquo nano1047297bers onto any target Taken together these

results demonstrate that airbrushed nano1047297ber scaffolds can supportstem cell differentiation and highlight the advantages and disad-

vantages of airbrushing as compared to electrospinning

Acknowledgments

WT TMF DW and SS were supported by NRC-NIST post-

doctoral fellowships GJ was supported by an NRC-NIHNIBIB-NIST

postdoctoral fellowship We thank Kathy Flynn (NIST) for gel

permeation chromatography measurements The content is solely

the responsibility of the authors and does not necessarily represent

the of 1047297cial views of NIST This article a contribution of NIST is not

subject to US copyright Certain equipment and instruments or

materials are identi1047297ed in the paper to adequately specify the

experimental details Such identi1047297cation does not imply recom-mendation by NIST nor does it imply the materials are necessarily

the best available for the purpose The authors declare no con1047298icts

of interest

Appendix A Supplementary data

Supplementary data related to this article can be found at


References

[1] Abrams GA Goodman SL Nealey PF Franco M Murphy CJ Nanoscaletopography of the basement membrane underlying the corneal epithelium of

the rhesus macaque Cell Tissue Res 2000299(1)39e

46

Table 2

Comparison of airbrushing (AB) versus electrospinning (ES) nano1047297bers

Air-brushing a Electro-spinning a Comments

Price U $25 for an AB versus $2500 for syringe pump amp power supply to ES

Ease of use U Easier to AB than ES since AB has fewer parts amp quicker set-up

Safety U AB is safer than ES (ES requires high voltage)

Deposition rate U AB deposits nano1047297bers 10 faster than ES (by scaffold mass)

Reproducibility frac14 frac14 Variance in nano1047297ber diameter is similar for AB amp ES

Nano1047297

ber morphology Different Different AB yields bundles of aligned nano1047297

bers that are loosely packedwith large voids ES yields un-aligned single nano1047297bers that

are tightly packed and highly entangled

Pore size U AB nano1047297ber mats had a larger pore size than ES a common

criticism of ES nano1047297bers is that the pores are too small

Porosity U AB nano1047297bers have higher porosity than ES high porosity is

generally regarded as an advantage for tissue engineering

Mechanical properties U ES nano1047297ber mats have a higher modulus than AB

Versatility (polymers) frac14 frac14 Polymers Both AB amp ES can fabricate nano1047297bers from a

wide range of polymers

Versatility (target) U Target AB can be aimed to ldquopaintrdquo nano1047297bers onto any target

ES requires an electrically conductive target amp is immobile

Versatility (1047297ber diameter) U Fiber diameter ES affords better control of nano1047297ber diameter

since voltage amp pump rate can be adjusted

Cell response frac14 frac14 Both AB amp ES nano1047297ber scaffolds can support cell adhesion

proliferation amp differentiation

Cell morphology Different Different hBMSCs have an elongated morphology on both AB amp ES but

hBMSCs are smaller on AB than on ES

a A check mark(U) indicates whetherAB or EShas theadvantageAn equalsign( frac14) indicates that AB andES weresimilar forthiscategory ldquoDifferentrdquo indicates thatAB and

ES are different for this category but that neither has an advantage




[2] Li WJ Laurencin CT Caterson EJ Tuan RS Ko FK Electrospun nano1047297brousstructure a novel scaffold for tissue engineering J Biomed Mater Res 200260(4)613e21

[3] Jin G Prabhakaran MP Ramakrishna S Stem cell differentiation to epidermallineages on electrospun nano1047297brous substrates for skin tissue engineeringActa Biomater 20117(8)3113e22

[4] Elliott JT Tona A Woodward JT Jones PL Plant AL Thin 1047297lms of collagen affectsmooth muscle cell morphology Langmuir 200319(5)1506e14

[5] Li D Xia Y Electrospinning of nano1047297bers reinventing the wheel Adv Mater200414(16)1151e70

[6] Holzwarth JM Ma PX Biomimetic nano1047297brous scaffolds for bone tissueengineering Biomaterials 201132(36)9622e9

[7] Formhals A Process and apparatus for preparing arti1047297cial threads US PatentNo 1975504 1934

[8] Norton CL Method of and apparatus for producing 1047297brous or 1047297lamentarymaterial US Patent No 2048651 1933

[9] Medeiros ES Glenn GM Klamczynski AP Orts WJ Mattoso LH Solution blowspinning a new method to produce micro- and nano1047297bers from polymersolutions J Appl Polym Sci 2011113(4)2322e30

[10] Srinivasan S Chhatre SS Mabry JM Cohen RE McKinley GH Solution sprayingof poly(methyl methacrylate) blends to fabricate microtextured super-oleophobic surfaces Polymer 201152(14)3209e18

[11] Ertel SI Kohn J Evaluation of a series of tyrosine-derived polycarbonates asdegradable biomaterials J Biomed Mater Res 199428(8)919e30

[12] Chatterjee K Hung S Kumar GF Simon Jr CG Time-dependent effects of pre-aging 3D polymer scaffolds in cell culture medium on cell proliferation J FunctBiomater 20123(2)372e81

[13] Sharp DG Beard JW Size and density of polystyrene particles measure byultracentrifugation J Biol Chem 1950185(1)247e53

[14] Yang Y Bolikal D Becker ML Kohn J Zeiger DN Simon Jr CG Combinatorialpolymer scaffold libraries for screening cell-biomaterial interactions in 3DAdv Mater 200820(11)2037e43

[15] Simon CG Stephens JS Dorsey SM Becker ML Fabrication of combinatorialpolymer scaffold libraries Rev Sci Instrum 200778(7)072207

[16] Dorsey SM Lin-Gibson S Simon Jr CG X-ray microcomputed tomography forthe measurement of cell adhesion and proliferation in polymer scaffoldsBiomaterials 200930(16)2967e74

[17] Parekh SH Chatterjee K Lin-Gibson S Moore NM Cicerone MT Young MFet al Modulus-driven differentiation of marrow stromal cells in 3D scaffoldsthat is independent of myosin-based cytoskeletal tension Biomaterials 201132(9)2256e64

[18] Cordelires F Jackson J ldquo3D object counterrdquo ImageJ plugin lthttprsbwebnihgovijpluginstrackobjectshtml gt 2006

[19] Uyar T Havelund R Hacaloglu J Besenbacher F Kingshott P Functionalelectrospun polystyrene nano1047297bers incorporating a- b- and g-cyclodex-trins comparison of molecular 1047297lter performance ACS Nano 20104(9)5121e30

[20] Grafahrend D Calvet JL Klinkhammer K Salber J Dalton PD Moller M et alControl of protein adsorption on functionalized electrospun 1047297bers BiotechBioeng 2008101(3)609e21

[21] Kumar G Tison CK Chatterjee K Pine PS McDaniel JH Salit ML et al Thedetermination of stem cell fate by 3D scaffold structures through the controlof cell shape Biomaterials 201132(35)9188e96

[22] Dominici M Le Blanc K Mueller I Slaper-Cortenbach I Marini F Krause Det al Minimal criteria for de1047297ning multipotent mesenchymal stromal cellsThe international society for cellular therapy position statement Cytotherapy20068(4)315e7

[23] Robey PG Cell sources for bone regeneration the good the bad and the ugly(but promising) Tissue Eng Pt B-Rev 201117(6)423e30

[24] Smith LA Liu X Hu J Ma PX The in1047298uence of three-dimensional nano1047297brousscaffolds on the osteogenic differentiation of embryonic stem cells Bioma-terials 200930(13)2516e22

[25] Smith LA Liu X Hu J Wang P Ma PX Enhancing osteogenic differentiation of mouseembryonic stem cells bynano1047297bersTissue EngPt A 200915(7)1855e64

[26] Ruckh TT Kumar K Kipper MJ Popat KC Osteogenic differentiation of bonemarrow stromal cells on poly(epsilon-caprolactone) nano1047297ber scaffolds ActaBiomater 20106(8)2949e59

[27] Nguyen LT Liao S Chan CK Ramakrishna S Enhanced osteogenic differenti-ation with 3D electrospun nano1047297brous scaffolds Nanomedicine-UK 20127(10)1561e75

[28] Folkman J Moscona A Role of cell shape in growth control Nature 1978273(5661)345e9

[29] Chen CS Mrksich M Huang S Whitesides GM Ingber DE Geometric control of cell life and death Science 1997276(5317)1425e8

[30] McBeath R Pirone DM Nelson CM Bhadriraju K Chen CS Cell shape cyto-skeletal tension and RhoA regulate stem cell lineage commitment Dev Cell20046(4)483e95

[31] Treiser MD Yang EH Gordonov S Cohen DM Androulakis IP Kohn J et alCytoskeleton-based forecasting of stem cell lineage fates Proc Natl Acad Sci US A 2010107(2)610e5

[32] Kumar G Waters MSFarooque TMYoung MFSimonJr CGFreeformfabricatedscaffolds with roughened struts that enhance both stem cell proliferation anddifferentiation by controlling cell shape Biomaterials 201233(16)4022e30

[33] Nam J Johnson J Lannutti JJ Agarwal S Modulation of embryonic mesen-chymal progenitor cell differentiation via control over pure mechanicalmodulus in electrospun nano1047297bers Acta Biomater 20117(4)1516e24

[34] Hong S Kim G Electrospun micronano1047297brous conduits composed of poly(e-caprolactone) and small intestine submucosa powder for nervetissue regeneration J Biomed Mater Res B 201094B(2)421e8

[35] Prabhakaran MP Venugopal JR Chyan TT Hai LB Chan CK Lim AY et alElectrospun biocomposite nano1047297brous scaffolds for neural tissue engineeringTissue Eng Pt A 200814(11)1787e97

[36] Li WJ Cooper Jr JA Mauck RL Tuan RS Fabrication and characterization of sixelectrospun poly(alpha-hydroxy ester)-based 1047297brous scaffolds for tissueengineering applications Acta Biomater 20062(4)377e85

[37] Zahedi P Resaeian I Ranaei-Siadat S-O Jafari S-H Supaphol P A review onwound dressings with an emphasis on electrospun nano1047297brous polymericbandages Polym Adv Technol 201021(2)77e95

[38] Christopherson GT Song H Mao HQ The in1047298uence of 1047297ber diameter of electrospun substrates on neural stem cell differentiation and proliferationBiomaterials 200930(4)556e64

[39] Lowery JL Datta N Rutledge GC Effect of 1047297ber diameter pore size and seedingmethod on growth of human dermal 1047297broblasts in electrospun poly(epsilon-caprolactone) 1047297brous mats Biomaterials 201031(3)491e504

[40] Li WJ Danielson KG Alexander PG Tuan RS Biological response of chon-drocytes cultured in three-dimensional nano1047297brous poly(epsilon-caprolactone) scaffolds J Biomed Mater Res A 200367A(4)1105e14

[41] Baker BM Shah RP Silverstein AM Esterhai JL Burdick JA Mauck RL Sacri-1047297cial nano1047297brous composites provide instruction without impediment andenable functional tissue formation Proc Natl Acad Sci U S A 2012109(35)14176e81

[42] Friedman CD Costantino PD Takagi S Chow LC BoneSource hydroxyapatitecement a novel biomaterial for craniofacial skeletal tissue engineering andreconstruction J Biomed Mater Res 199843(4)428e32




airbrushing and electrospinning in a number of side by side tests to

determine the advantages and disadvantages of each Finally we

have tested the ability of airbrushed nano1047297ber scaffolds to support

osteogenic differentiation of primary human bone marrow stromal

cells (hBMSCs)

2 Materials amp methods

21 Airbrushed nano 1047297bers

A commercially available airbrush designed for painting (Master Airbrush

G222-SET 02 mm nozzle diameter gravitational feed) was used to fabricate

nano1047297ber scaffolds The airbrush was connected to a pressurized nitrogen tank

(241 kPa 35 pounds per square inch) The distance from the nozzle to the target was

held constant at 20 cm for all experiments For electron microscopy porosity and

mechanical tests aluminum foil was used as the target and nano1047297ber mats were

peeled off the foil for assessment For cell culture experiments tissue culture

polystyrene (TCPS) disks were used as targets TCPS disks were hot-punched from

the bottom of TCPS plates into 6 mm diameter disks that 1047297t into the bottom of

96-well plates Nano1047297bers were airbrushed from four different polymers poly-

styrene (PS taken from the bottoms of tissue culture polystyrene dishes Corning

catalog number 430599) poly(desaminotyrosyl-tyrosine ethyl ester carbonate)

(pDTEc made as described) [11] poly(DL -lactic acid) (PDLLA SurModics Pharma-

ceuticals) and poly(ε-caprolactone) (PCL SigmaeAldrich) Polymer relative molec-

ular masses solvents and polymer concentrations for airbrushing are given in

Table 1 Molecular masses were provided by the manufacturer except for PS which

was measured using gel permeation chromatography using tetrahydrofuran as

solvent

22 Electrospun nano 1047297bers

Electrospun nano1047297bers were made from PCL with a home built electrospinning

apparatusPCL solution(10mass fractionin 31volumeratiochloroformmethanol)

was loadedintoa syringe anddispensed witha syringepump at2 mLhThe positive

leadfrom thepowersupply was1047297xed tothe spinneret whichwasan 18 gaugeneedle

and the ground lead was 1047297xed to the target (aluminum foil) The distance between

needleand targetwas 15cm andvoltage was165kV Non-wovenPCL nano1047297bermats

were collected for 15 h onto an aluminum foil target Electrospun nano1047297bers were

removed from the foil for mechanical testing For cell culture samples TCPS disks

were placed on the aluminum and nano1047297bers were electrospunonto the TCPS disks

23 Deposition rate

Deposition rate for airbrushing and electrospinning was determined bymeasuring how long it took to deposit a known volume of polymer solution of

a known concentration

24 Scanning electron microscopy

For scanning electron microscopy (SEM) nano1047297ber scaffolds were sputter

coated with gold for 90 s and then imaged (Hitachi S-4700-II FE-SEM 5 kV) Fiber

diameter was determined from scanning electron micrographs from 3 1047297elds of view

using ImageJ software (NIH) to determine diameter of 30e100 nano1047297bers To assess

1047297ber diameter reproducibility PCL 1047297bers were airbrushed or electrospun on three

separate days for SEM imaging analysis For pore size several spots were imaged on

a mat and 50e120 pores were sized in scanning electron micrographs

25 Nano 1047297ber porosity

Porosity of nano1047297ber scaffolds was determined using the following equation

[12] porosity frac14 (1 [(massdensity)(width length thickness)]) Scaffolds werecut to 2 cm by 2 cm squares (width and length) Mass was determined by weighing

scaffold squares on a balance Scaffold thickness was determined by measuring the

edge thickness of the scaffold squares in SEM PS density was 105 gmL [13] pDTEc

density was 12 gmL [14] PDLLA density was 125 gmL [15] and PCL density was

11 gmL [16]

26 Mechanical analysis

Mechanical properties were evaluated using a dynamic mechanical analyzer

(RSA III instrument TA Instruments) equipped with a rectangular 1047297lm1047297ber clamp

geometry Airbrushed and electrospun nano1047297ber mats were folded twice to yielda rectangular 4-layer sheet that was 6 mm 10 mm 015 mm Samples were

weighed to insure that mass per unit area of the scaffolds was similar (within 10)

Tensile measurements on the samples were conducted with a 02 mms cross-head

speed at room temperature Modulus was calculated from the slope of the stress e

strain curve between 5 and 9 strain

27 Cell culture

Airbrushed and electrospun nano1047297ber scaffolds on TCPS disks were placed into

96-well plates and af 1047297xed to the bottoms of the wells with a small dab of silicon

vacuum grease Samples were sterilized by ethylene oxide (Anderson Products) and

degassed for 2 d under house vacuum Primary human bone marrow stromal cells

(hBMSCs Texas AampM Institute for Regenerative Medicine 24 year old female)

were cultured at 37 C with 5 by volume CO2 in a-minimum essential medium

(Invitrogen) supplemented with 165 by volume fetal bovine serum (Atlanta Bio-

logicals) and 4 mmolL L -glutamine [17] hBMSCs were passaged at 80 con1047298uency

and dissociated with 025 mass fraction trypsin containing 1 mmolL ethyl-

enediaminetetraactate (EDTA) Passage 5 hBMSCs were seeded onto scaffolds in 96-

well plates at 5000 cellswell in 02 mL of mediumwell After 24 h medium was

replaced with medium containing osteogenic supplements (10 nmolL dexametha-

sone 20 mmolL b-glycerophosphate and 005 mmolL ascorbic acid) Medium was

changed twice per week with medium containing osteogenic supplements

28 Fluorescence imaging

Cells on scaffolds were 1047297xed (37 formaldehyde massvolume in PBS (phos-

phate bufferedsaline) for 15min) washed (PBS) permeabilized (02by massTriton

X-100 for 5 min) and rinsed (PBS) Samples were stained 1 h in PBS with 20 nmolL

Alexa Fluor 546-phalloidinand 100nmolL Sytox green (Invitrogen) to stain for actin

and nuclei respectively After staining cells were washed with PBS air dried and

imaged by 1047298uorescence microscopy (Nikon Eclipse TE 300) using 4013 objectives

and 20045 hBMSCs were also imaged by confocal1047298uorescence microscopy (Leica

TCS SP5 broadband) using a 6314 oil immersion objective with a 250 nm Z-step

size

29 Picogreen DNA assay

hBMSCs grown on the nano1047297ber scaffolds were quanti1047297ed using Picogreen DNA

assay according to manufacturerrsquos protocol After cell culture scaffolds were rinsed

with PBS frozen overnight to rupture cell membranes (20 C) thawed and incu-

bated with digestion buffer (PBS with 0175 UmL papain and 145 mmolL

L -cysteine) for 16 h at 60 C Lysate (02 mL) was transferred to a clean 96-well plate

and diluted with 02 mL of Picogreen reagent (Invitrogen diluted according to

manufacturerrsquos protocol) DNA 1047298uorescence (excitation 485 nm emission 538 nm)

was measured using a platereader and calibrated using a DNA standard curve

210 Osteocalcin ELISA

ELISA assay (enzyme-linked immunosorbent assay BTI BT-460) was employed

to measure osteocalcin (OC) as a marker for osteogenic differentiation Three

scaffolds were assessed for each condition (n frac14 3) After cell culture scaffolds

were rinsed with PBS incubated with 02 mL of acetic acid (10 by volume) for1 h at 37 C and lyophilized overnight Manufacturerrsquos sample buffer was used to

Table 1

Processing conditions amp nano1047297ber scaffold properties

Fabrication Airbrushed Airbrushed Airbrushed Airbrushed Electrospun

Polymer PS pDTEc PDLLA PCL PCL

Relative molecular mass (gmol) 178000 183000 100000 80000 80000

Solvent Dimethyl-formamide Dioxane Chloroform

methanol 8515 by mass

Chloroform Chloroform

methanol 31 by mass

Concentration (mass) 8 15 6 4 10

Solution deposition rate (mLmin) 061 031 115 082 003

Scaffold deposition rate (mgmin) 186 54 90 30 6

Fiber diameter (nm)

[Mean (SD)] (n frac14 35)

1949 (643) 677 (420) 125 (88) 231 (79) 511 (185)






















absorbance

















volumes [18]

213 Statistics






3 Results























249 mm (SD 269 mm n frac14 3)




















entangled


































0

5

10

15

20



m )

0

20

40

60

80

100


P o r o s i t

y ( )


a

b


















0

2

4

6

8

10

12

14

16



0 10 20 30 40 50 60 7000

05

10

15

20

25

30

35

40


Strain ()

e

Airbrushed

Electrospun

50 microm

dc

a

50 m

0

5

10

15

20

25

30

35

40

45


A i r b r u s h e d


b

0

100

200

300

400

500

600

700

1 2 3 4 5 6



Airbrushed

1

2

3

1

2

3

Electrospun

f




















4 Discussion



























pDTEc LCPALLDPSPc

1 mm ( - ) h B M S C s

( + ) h B M S C s

0

50

100

150

200

250

300

350

400

PS pDTEc PDLLA PCL


)

1 d10 d17 d21 d

a

00

01

02

03

04

05

06

07

08

PS pDTEc PDLLA PCL


10 d

17 d

21 d

b

0

10

20

30

40

50

60

70

80

90

PS pDTEc PDLLA PCL

A


10 d

17 d

21 d

d



























[33] 11 MPa [34] and 35 MPa [35]








0

200

400

600

800

1000

1200

1400

1600


A r e a ( micro m

2 )

0

100

200

300

400

500

600

700



0

1

2

3

4

5

6

7

8



00

01

02

03

04

05

06

07

08


R o u n d n e

s s

0

500

1000

1500

2000

2500

3000

3500


V o l u m e ( micro

m 3 )

20 microm


A i r b r u s h e d


A i r b r u s h e d


a

b


A i r b r u s h e d

f

c


A i r b r u s h e d


A i r b r u s h e d

d

e


































5 Conclusions







make nano1047297














Acknowledgments











of interest




References



46

Table 2








Nano1047297






















































































absorbance

















volumes [18]

213 Statistics






3 Results























249 mm (SD 269 mm n frac14 3)




















entangled


































0

5

10

15

20



m )

0

20

40

60

80

100


P o r o s i t

y ( )


a

b


















0

2

4

6

8

10

12

14

16



0 10 20 30 40 50 60 7000

05

10

15

20

25

30

35

40


Strain ()

e

Airbrushed

Electrospun

50 microm

dc

a

50 m

0

5

10

15

20

25

30

35

40

45


A i r b r u s h e d


b

0

100

200

300

400

500

600

700

1 2 3 4 5 6



Airbrushed

1

2

3

1

2

3

Electrospun

f




















4 Discussion



























pDTEc LCPALLDPSPc

1 mm ( - ) h B M S C s

( + ) h B M S C s

0

50

100

150

200

250

300

350

400

PS pDTEc PDLLA PCL


)

1 d10 d17 d21 d

a

00

01

02

03

04

05

06

07

08

PS pDTEc PDLLA PCL


10 d

17 d

21 d

b

0

10

20

30

40

50

60

70

80

90

PS pDTEc PDLLA PCL

A


10 d

17 d

21 d

d



























[33] 11 MPa [34] and 35 MPa [35]








0

200

400

600

800

1000

1200

1400

1600


A r e a ( micro m

2 )

0

100

200

300

400

500

600

700



0

1

2

3

4

5

6

7

8



00

01

02

03

04

05

06

07

08


R o u n d n e

s s

0

500

1000

1500

2000

2500

3000

3500


V o l u m e ( micro

m 3 )

20 microm


A i r b r u s h e d


A i r b r u s h e d


a

b


A i r b r u s h e d

f

c


A i r b r u s h e d


A i r b r u s h e d

d

e


































5 Conclusions







make nano1047297














Acknowledgments











of interest




References



46

Table 2








Nano1047297





































































volumes [18]

213 Statistics






3 Results























249 mm (SD 269 mm n frac14 3)




















entangled


































0

5

10

15

20



m )

0

20

40

60

80

100


P o r o s i t

y ( )


a

b


















0

2

4

6

8

10

12

14

16



0 10 20 30 40 50 60 7000

05

10

15

20

25

30

35

40


Strain ()

e

Airbrushed

Electrospun

50 microm

dc

a

50 m

0

5

10

15

20

25

30

35

40

45


A i r b r u s h e d


b

0

100

200

300

400

500

600

700

1 2 3 4 5 6



Airbrushed

1

2

3

1

2

3

Electrospun

f




















4 Discussion



























pDTEc LCPALLDPSPc

1 mm ( - ) h B M S C s

( + ) h B M S C s

0

50

100

150

200

250

300

350

400

PS pDTEc PDLLA PCL


)

1 d10 d17 d21 d

a

00

01

02

03

04

05

06

07

08

PS pDTEc PDLLA PCL


10 d

17 d

21 d

b

0

10

20

30

40

50

60

70

80

90

PS pDTEc PDLLA PCL

A


10 d

17 d

21 d

d



























[33] 11 MPa [34] and 35 MPa [35]








0

200

400

600

800

1000

1200

1400

1600


A r e a ( micro m

2 )

0

100

200

300

400

500

600

700



0

1

2

3

4

5

6

7

8



00

01

02

03

04

05

06

07

08


R o u n d n e

s s

0

500

1000

1500

2000

2500

3000

3500


V o l u m e ( micro

m 3 )

20 microm


A i r b r u s h e d


A i r b r u s h e d


a

b


A i r b r u s h e d

f

c


A i r b r u s h e d


A i r b r u s h e d

d

e


































5 Conclusions







make nano1047297














Acknowledgments











of interest




References



46

Table 2








Nano1047297












































































0

2

4

6

8

10

12

14

16



0 10 20 30 40 50 60 7000

05

10

15

20

25

30

35

40


Strain ()

e

Airbrushed

Electrospun

50 microm

dc

a

50 m

0

5

10

15

20

25

30

35

40

45


A i r b r u s h e d


b

0

100

200

300

400

500

600

700

1 2 3 4 5 6



Airbrushed

1

2

3

1

2

3

Electrospun

f




















4 Discussion



























pDTEc LCPALLDPSPc

1 mm ( - ) h B M S C s

( + ) h B M S C s

0

50

100

150

200

250

300

350

400

PS pDTEc PDLLA PCL


)

1 d10 d17 d21 d

a

00

01

02

03

04

05

06

07

08

PS pDTEc PDLLA PCL


10 d

17 d

21 d

b

0

10

20

30

40

50

60

70

80

90

PS pDTEc PDLLA PCL

A


10 d

17 d

21 d

d



























[33] 11 MPa [34] and 35 MPa [35]








0

200

400

600

800

1000

1200

1400

1600


A r e a ( micro m

2 )

0

100

200

300

400

500

600

700



0

1

2

3

4

5

6

7

8



00

01

02

03

04

05

06

07

08


R o u n d n e

s s

0

500

1000

1500

2000

2500

3000

3500


V o l u m e ( micro

m 3 )

20 microm


A i r b r u s h e d


A i r b r u s h e d


a

b


A i r b r u s h e d

f

c


A i r b r u s h e d


A i r b r u s h e d

d

e


































5 Conclusions







make nano1047297














Acknowledgments











of interest




References



46

Table 2








Nano1047297














































































4 Discussion



























pDTEc LCPALLDPSPc

1 mm ( - ) h B M S C s

( + ) h B M S C s

0

50

100

150

200

250

300

350

400

PS pDTEc PDLLA PCL


)

1 d10 d17 d21 d

a

00

01

02

03

04

05

06

07

08

PS pDTEc PDLLA PCL


10 d

17 d

21 d

b

0

10

20

30

40

50

60

70

80

90

PS pDTEc PDLLA PCL

A


10 d

17 d

21 d

d



























[33] 11 MPa [34] and 35 MPa [35]








0

200

400

600

800

1000

1200

1400

1600


A r e a ( micro m

2 )

0

100

200

300

400

500

600

700



0

1

2

3

4

5

6

7

8



00

01

02

03

04

05

06

07

08


R o u n d n e

s s

0

500

1000

1500

2000

2500

3000

3500


V o l u m e ( micro

m 3 )

20 microm


A i r b r u s h e d


A i r b r u s h e d


a

b


A i r b r u s h e d

f

c


A i r b r u s h e d


A i r b r u s h e d

d

e


































5 Conclusions







make nano1047297














Acknowledgments











of interest




References



46

Table 2








Nano1047297




































































pDTEc LCPALLDPSPc

1 mm ( - ) h B M S C s

( + ) h B M S C s

0

50

100

150

200

250

300

350

400

PS pDTEc PDLLA PCL


)

1 d10 d17 d21 d

a

00

01

02

03

04

05

06

07

08

PS pDTEc PDLLA PCL


10 d

17 d

21 d

b

0

10

20

30

40

50

60

70

80

90

PS pDTEc PDLLA PCL

A


10 d

17 d

21 d

d



























[33] 11 MPa [34] and 35 MPa [35]








0

200

400

600

800

1000

1200

1400

1600


A r e a ( micro m

2 )

0

100

200

300

400

500

600

700



0

1

2

3

4

5

6

7

8



00

01

02

03

04

05

06

07

08


R o u n d n e

s s

0

500

1000

1500

2000

2500

3000

3500


V o l u m e ( micro

m 3 )

20 microm


A i r b r u s h e d


A i r b r u s h e d


a

b


A i r b r u s h e d

f

c


A i r b r u s h e d


A i r b r u s h e d

d

e


































5 Conclusions







make nano1047297














Acknowledgments











of interest




References



46

Table 2








Nano1047297




















































































[33] 11 MPa [34] and 35 MPa [35]








0

200

400

600

800

1000

1200

1400

1600


A r e a ( micro m

2 )

0

100

200

300

400

500

600

700



0

1

2

3

4

5

6

7

8



00

01

02

03

04

05

06

07

08


R o u n d n e

s s

0

500

1000

1500

2000

2500

3000

3500


V o l u m e ( micro

m 3 )

20 microm


A i r b r u s h e d


A i r b r u s h e d


a

b


A i r b r u s h e d

f

c


A i r b r u s h e d


A i r b r u s h e d

d

e


































5 Conclusions







make nano1047297














Acknowledgments











of interest




References



46

Table 2








Nano1047297































































































5 Conclusions







make nano1047297














Acknowledgments











of interest




References



46

Table 2








Nano1047297


































































the support of bone marrow stromal cell differentiation by airbrushed nanofiber scaffolds 2013...

Documents