Acta Biomaterialia 46 (2016) 256–265

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Acta Biomaterialia

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Full length article

Cold atmospheric plasma (CAP) surface nanomodified 3D printedpolylactic acid (PLA) scaffolds for bone regeneration

http://dx.doi.org/10.1016/j.actbio.2016.09.0301742-7061/� 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

⇑ Corresponding author at: Department of Chemical Engineering, NortheasternUniversity, 360 Huntington Ave, Boston, MA 02115, USA.

E-mail address: th.webster@neu.edu (T.J. Webster).

Mian Wang a, Pelagie Favi a, Xiaoqian Cheng b, Negar H. Golshan a, Katherine S. Ziemer a, Michael Keidar b,Thomas J. Webster a,c,d,⇑aDepartment of Chemical Engineering, Northeastern University, 360 Huntington Ave, Boston, MA 02115, USAbDepartment of Mechanical and Aerospace Engineering, The George Washington University, Washington, DC 20052, USAcWenzhou Institute of Biomaterials and Engineering, Wenzhou Medical University, Wenzhou, ChinadCenter of Excellence for Advanced Materials Research, King Abdulaziz University, Jeddah, Saudi Arabia

a r t i c l e i n f o a b s t r a c t

Article history:Received 11 May 2016Received in revised form 23 August 2016Accepted 22 September 2016Available online 22 September 2016

Keywords:Cold atmospheric plasma (CAP)3D printingSurface modificationBone regenerationPoly-lactic acid (PLA)

Three-dimensional (3D) printing is a new fabrication method for tissue engineering which can preciselycontrol scaffold architecture at the micron-scale. However, scaffolds not only need 3D biocompatiblestructures that mimic the micron structure of natural tissues, they also require mimicking of the nano-scale extracellular matrix properties of the tissue they intend to replace. In order to achieve this, theobjective of the present in vitro study was to use cold atmospheric plasma (CAP) as a quick and inexpen-sive way to modify the nano-scale roughness and chemical composition of a 3D printed scaffold surface.Water contact angles of a normal 3D printed poly-lactic-acid (PLA) scaffold dramatically dropped afterCAP treatment from 70 ± 2� to 24 ± 2�. In addition, the nano-scale surface roughness (Rq) of the untreated3D PLA scaffolds drastically increased (up to 250%) after 1, 3, and 5 min of CAP treatment from 1.20 nm to10.50 nm, 22.90 nm, and 27.60 nm, respectively. X-ray photoelectron spectroscopy (XPS) analysisshowed that the ratio of oxygen to carbon significantly increased after CAP treatment, which indicatedthat the CAP treatment of PLA not only changed nano-scale roughness but also chemistry. Both changesin hydrophilicity and nano-scale roughness demonstrated a very efficient plasma treatment, which inturn significantly promoted both osteoblast (bone forming cells) and mesenchymal stem cell attachmentand proliferation. These promising results suggest that CAP surface modification may have potentialapplications for enhancing 3D printed PLA bone tissue engineering materials (and all 3D printedmaterials) in a quick and an inexpensive manner and, thus, should be further studied.

Statement of Significance

Three-dimensional (3D) printing is a new fabrication method for tissue engineering which can preciselycontrol scaffold architecture at the micron-scale. Although their success is related to their ability toexactly mimic the structure of natural tissues and control mechanical properties of scaffolds, 3D printedscaffolds have shortcomings such as limited mimicking of the nanoscale extracellular matrix propertiesof the tissue they intend to replace. In order to achieve this, the objective of the present in vitro study wasto use cold atmospheric plasma (CAP) as a quick and inexpensive way to modify the nanoscale roughnessand chemical composition of a 3D printed scaffold surface. The results indicated that using CAP surfacemodification could achieve a positive change of roughness and surface chemistry. Results showed thatboth hydrophilicity and nanoscale roughness changes to these scaffolds after CAP treatment played animportant role in enhancing bone cell and mesenchymal stem cell attachment and functions. Moreimportantly, this technique could be used for many 3D printed polymer-based biomaterials to improvetheir properties for numerous applications.

� 2016 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

M. Wang et al. / Acta Biomaterialia 46 (2016) 256–265 257

1. Introduction

Bone disease is a problem that affects every country, especiallythe U.S. where almost one million bone grafting procedures areperformed each year at a growth rate of almost 13% per year [1].Although bone grafting has been acceptable for small bone frac-tures, a higher success rate is necessary for large bone fracturesover longer time periods. For large bone defects, compared to tra-ditional total joint replacements which use titanium, bone tissueengineering offers a promising approach for bone repair withoutusing a permanently implanted foreign material [2]. Bone tissueengineering aims to combine cells, biomaterials, and differentgrowth factors to establish a mimetic bone structure to regeneratenew bone tissue. Ideally, engineered bone uses an extracellularmatrix (ECM) type material, cells, as well as factors to regulate cellbehavior to promote cell differentiation to calcium-depositing cellsand finally bone formation [3]. The ECM plays an important role inthe control of cell adhesion, proliferation, differentiation, and boneformation [4]. Therefore, it has now been well established that anECM which mimics both the micro- and nano-environment ofnatural bone will significantly improve biological responses.

Traditional scaffold fabrication methods, such as solvent cast-ing, particle leaching, gas foaming, phase separation, and electro-spinning have many limitations [5]. For example, for the mostpart, they lack an exact control over pore size, nano-scale rough-ness, desirable mechanical properties, and interconnectivity. Com-pared with traditional scaffold fabrication methods, 3D printingtechnology has emerged as a process than can precisely controlsuch properties and can be utilized to formulate implantable mate-rials of the exact geometry as the bone fracture at the clinical bed-side for immediate implantation [6,7]. Although 3D printingtechnology holds much promise for tissue engineering, some stud-ies have been unable to create structures that promote cellularbehavior; some believe this is because while it is easy for 3D print-ing to control micron scale roughness, nano-scale roughness hasproven more difficult. It is therefore advantageous to improveproperties of 3D printed scaffolds to fully mimic the micron- aswell as nano-topography of natural bone [8,9].

Various surface modification methods have been used toimprove the biocompatibility of orthopedic materials by imple-menting nano-structured surface features [10]. Many studies haveindicated that nano-structured surfaces are of particular impor-tance for promoting cell adhesion, proliferation, and differentiation[11]. In detail, such nano-scale structures will affect initial proteinadsorption through changes in surface energy, surface area, andwettability. Then, this nano-featured surface enhances cell adhe-sion through improved integrin binding, resulting in cell greateradhesion, clustering, spreading, proliferation, and ECM formation[4,12]. Finally, a nano-featured surface morphology has a positiveeffect on osteoblast differentiation and bone formation. Althoughmany surface modification strategies which use chemical andbiomolecular functionalization enhance biocompatibility ofimplants [8,13], those methods are often too complex (multiplesteps), expensive, and/or fragile for orthopedic applications. There-fore, a simple and more effective approach is needed to fabricatenano-features on already 3D printed scaffolds to improve theirefficacy in orthopedics.

Based on the above, in this study, cold atmospheric plasma(CAP) was selected as a method to modify 3D printed PLA scaffolds.CAP is a one-step effective material surface modification tool. CAPis carried out in the presence of helium where a very high voltageis applied to a scaffold [14]. Once the gas is ionized, CAP is formedwith a very low ion temperature, close to room temperature [15].CAP is the cluster of those ionized gases, containing reactive oxy-gen species (ROS) and reactive nitrogen species (RNS), positive/

negative ions, free radicals, electrons, and transient electrical fields[16]. While CAP has not been widely investigated for tissue engi-neering applications, it has been investigated for anti-cancer andanti-bacteria applications. It is postulated that the ROS whichCAP create, are the primary contributor of CAP-induced tumor cellapoptosis and bacteria inactivation while RNS are the primaryfactor to promote healthy cell growth [17]. These unique gascompositions and low temperatures make CAP exposure highlyeffective in several medical applications, such as sterilization,wound healing, blood coagulation, treatment of cancer, and mate-rial surface modification [14,18–20]. A previous study demon-strated that CAP surface modification will create nano-scaledsurface features of increased hydrophilicity, but, interestingly,some have also indicated the generation of a more hydrophobicsurface [16,21–24]. Either surface energy change will influence celladhesion and proliferation behavior. However, the mechanism ofthis impact is far from understood. Therefore, further investigationis needed to obtain a better correlation between plasma treatmentand cell responses. For cold plasma induced surface modification, avariety of functional factors like oxygen and nitrogen groups (i.e.,ROS and RNS) would be introduced onto substrate surfaces [25].Hence, it is hypothesized here that those CAP factors will alter3D PLA scaffold surface morphology and chemistry to improveinteractions will bone cells.

In this study, CAP parameters were modified to transform a 3Dprinted PLA scaffold to better mimic natural bone. For the firsttime, the resulting CAP modified 3D printed PLA scaffolds werecharacterized for surface roughness, chemistry, and in vitro cellbehavior.

2. Materials and methods

2.1. Cold atmospheric plasma (CAP)

The detailed description of the CAP experimental setup usedhere can be found elsewhere [26]. Briefly, CAP had a central powerelectrode 1 mm in diameter and was grounded by a counter elec-trode. Helium gas was sent through a Pyrex syringe ionized by highvoltage between the two electrodes. Optical emission spectroscopywas performed and a detection probe purchased from Stellar Net(Tampa, FL). Integration time of the collecting data was set to100 ms. A thermometer (VWR, PA) was fixed at a distance of12 mm from the syringe to measure the temperature of the plasmajet.

2.2. Fabrication of 3D printed polylactic acid (PLA) scaffolds

All 3D printed scaffolds were fabricated using a MakerBot FDMprinting system, modified with a 250 lm diameter nozzle, and aspool of 1.75 mm diameter Polylactic acid (PLA) polymer, whichwas heated in the nozzles containing the polymer. 3D printedobjects were built from 3D CAD drawing software by extrudingthe material layer by layer. This layer by layer deposition enableddirectional layer assembly.

2.3. CAP modification

Fully 3D printed PLA scaffolds were positioned downstream ofthe CAP system made by the Micro-propulsion and Nanotechnol-ogy Laboratory (MpNL) at The George Washington University.The plasma supply source was compressed purified helium gas ata flow rate of 0.5 L/min. The CAP condition used to treat PLA scaf-folds was investigated with different input voltage (9 V, 11 V) anddifferent treatment time (1, 3, and 5 min). All scaffolds were

258 M. Wang et al. / Acta Biomaterialia 46 (2016) 256–265

treated both on their top and bottom sides, and the distancebetween the PLA scaffolds and CAP was 12 mm. An untreated 3Dprinted PLA scaffold was used as the control. All scaffolds wereimmediately characterized and further sterilized under UV lightfor 0.5 h before cell seeding.

2.4. Characterization of the 3D printed PLA scaffolds

A Hitachi S-4800 high resolution field emission scanning elec-tron microscope (SEM) was used to visualize the topographical fea-tures of the untreated and CAP treated PLA scaffolds. Thetopographical studies for the CAP treated and untreated 3D PLAscaffolds were carried out using atomic force microscopy (AFM)(Parks Systems NX-10, USA). AFM images were recorded in tappingmode at room temperature. The root mean square (RMS) was ana-lyzed by XEI software. The water contact angle of CAP treated anduntreated 3D PLA scaffolds were measured by a static contact anglemeter (FTA, USA) at room temperature and 70% relative humidity(R.H.) using deionized water. A water droplet of 4 ll was placedon the scaffold surface. Triplicate measurements of the same trea-ted and untreated scaffolds were recorded and averaged. Watercontact angles were also measured at different time intervals(1 day, 3 days, 5 days, 7 days, and 10 days) after plasma treatment.

X-ray photoelectron spectroscopy (XPS) was performed using aMg/Al dual anode non-monochromated X-ray source (Phi model04-548) and a hemispherical analyzer (Phi model 10-360). The sys-tem was calibrated using Au 4f and Cu 2p, and had a minimum fullwidth half maximum (FWHM) of 1.5 eV with an 80% Gaussian/Lor-entzian distribution at a pass energy of 35.75 eV. Background sub-traction was performed using the integrated Shirley method.

2.5. Cell culture

Primary human fibroblasts, osteoblasts and bone marrow mes-enchymal stem cells (MSCs) were purchased from ATCC (PCS-201-012, CRL-11372, PCS-500-012, respectively). Fibroblasts (passageNo. 4–9) were cultured in Dulbecco’s modified Eagle’s medium(DMEM, Sigma) supplemented with 10% fetal bovine serum (FBS,Sigma), and 1% penicillin:streptomycin solution (Invitrogen).Osteoblasts (passage No. 3–8) were cultured in osteoblast medium(PromoCell) supplemented with OB GM supplemental mix (Promo-Cell). MSCs (passage No. 3–6) were cultured in complete mediacomposed of alpha minimum essential medium (Gibco) supple-mented with 16.5% fetal bovine serum (FBS) (Atlanta Biologicals),1% (v/v) L-glutamine (Invitrogen), and a 1% penicillin:streptomycinsolution (Invitrogen) and were cultured under standard cell cultureconditions (37 �C, a humidified, 5% CO2/95% air environment).

2.6. Fibroblast cell adhesion study

The purpose of the fibroblast cell adhesion study was to inves-tigate whether input voltage or treatment time play a more impor-tant role in impacting cell behavior. Samples treated with differentinput voltages with the same treatment times (9V_1 min,11V_1 min) and same treatment condition with different treat-ment times (9V_1 min, 9V_5 min) were tested with fibroblastsseeded at a density of 3 * 104 cells/ml. 3D printed PLA scaffoldsand CAP treated PLA scaffolds were placed individually into thewells of a 96-well plate, and sterilized under UV light before thecell study. They were incubated under standard cell culture condi-tions for 24 h and were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, USA) assay. Atthe end of the culture period, 15 ll of the MTT reagent was addedto each well. The plates were then returned to the incubator for4 h. Then, 100 ll of a stop solution were added to each well andwere cultured overnight. After overnight cultures, scaffolds were

removed and the 96-well plate was placed into a SpectraMax M3microplate reader (Molecular Devices) and the absorbance of theMTT solution in each well was measured at a wavelength of570 nm.

2.7. Osteoblast and MSCs cell proliferation study

The proliferation of osteoblasts and MSCs were monitored onthe 1st, 3rd, and 5th days of culture using the MTT assay asdescribed before. The CAP treated and untreated scaffolds weresterilized under UV light followed by pre-wetting with cell culturemedia. Accordingly, osteoblast and MSCs suspensions were dis-pensed onto treated and untreated scaffold and were cultured fordifferent time intervals at a concentration of 50,000 cells/ml. Sam-ples were incubated under standard culture conditions for 1, 3, and5 days and were measured by the MTT assay (Sigma, USA) asdescribed before. The absorbance of the MTT solution in each wellwas measured at a wavelength of 570 nm by a SpectraMax M3microplate reader (Molecular Devices).

2.8. SEM images of MSCs on PLA scaffolds

For cell morphology studies on PLA scaffolds, CAP treated anduntreated scaffolds were placed into 48-well plates, and werepre-wetted by cell culture media, then 50,000 cells/ml were seededinto each well and were cultured for 5 days. After culturing, scaf-folds were rinsed with phosphate buffer saline (pH 7.4) and fixedusing 2.5% glutaraldehyde for 1 h in order to observe cell morphol-ogy and cell infiltration. The specimens were subsequently dehy-drated in ethanol solutions of various concentrations (30, 50, 70,90 and 100%) for 15 min respectively prior to being criticallypoint-dried, then cells with scaffolds were sputter coated withplatinum before being observed by SEM.

2.9. Statistical analysis

All cellular experiments were run in triplicate and repeatedthree times for each substrate. Data are presented as the meanvalue standard error of the mean and were analyzed with Studentt-test as well as 2-way Analysis of Variance (ANOVA). Statisticalsignificance was considered at p < 0.05.

3. Results

3.1. CAP characterization

The spectrum of the CAP jet used for a typical operating condi-tion is shown in Fig. 1A. The spectrum presented various excitedN2, O, and He species. The main species in the CAP jet (OH at309 nm, N2

+ at 337 nm and 391 nm, He at 706 nm, and O at777 nm) are marked in the figure. The variation of the intensityof these species over time was observed in Fig. 1B. The intensityof all the species was normalized to the He (706 nm) peak. Over5 min, variation of the intensity of plasma-generated species wasvery small. Furthermore, the temperature of the plasma jetincreased from 39 to 40.5 �C in less than 2 min, then it was sus-tained at 40.5 �C for another 3 min. The consistency of the plasmajet guaranteed that the effect observed in this study was associatedwith intracellular reactions caused by CAP-generated reactive spe-cies instead of the plasma jet.

3.2. SEM

The surface topography of the CAP modified and unmodifiedPLA scaffolds were observed by eye (Fig. 2) and by SEM images

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He (706 nm)

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Fig. 1. A. Typical spectrum of CAP jet; B. The variation of main species in the CAP jet (solid lines) and the temperature of CAP jet (dash line) over time.

Fig. 2. 3D printed PLA scaffold morphology. (A) 3D printed PLA used in this study. (B) 3D printed PLA scaffold with a large diameter.

M. Wang et al. / Acta Biomaterialia 46 (2016) 256–265 259

(Fig. 3) which revealed surface morphology changes per differenttreatment times under optimal CAP modification. It can be seenthat the untreated PLA scaffold exhibited a smooth texture. Underhigher magnification, the scaffold exhibited a smooth surface withsome observed pits (Fig. 3 A3); no nano-sized features wereobserved on its surface. The surface morphology of the CAP modi-fied PLA scaffolds are displayed in Fig. 3 B3, C3, D3. As expected,the CAP modification exhibited a rough surface at the nano-scale.The average surface feature sizes observed were 80–150 nm. Inter-estingly, an increasing nano-patterned area could be observed withfurther exposure time of CAP treatment. It is believed that nano-patterning of the PLA surface after long times (1, 3, 5 min) of CAPexposure could provide more surface area for cell growth. It is pos-tulated that the CAP-induced nano-scale morphology may be aresult of charged particle–surface collision and heat accumulation

due to long term CAP treatment. As we all know, nano-scale sur-face topography has been shown to elicit beneficial cell behavior[9]. Consequently, a well-structured nano-scale surface on such3D printed PLA scaffolds can be achieved via this quick CAPtreatment.

3.3. AFM

To further quantify the surface morphology changes, AFM wasperformed. Fig. 4 and Table 1 confirmed that the surface becamerougher at the nano-scale after CAP treatment. Untreated PLA scaf-folds had a smooth surface with a root mean square (Rq) roughnessof 1.20 nm. After exposure to CAP for 1 min, 3 min and 5 min,the roughness increased to 10.50 nm, 22.90 nm, and 27.60 nm,

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Fig. 3. Low-and high-magnification scanning electron microscopy (SEM) images of unmodified 3D printed PLA scaffolds (A1-3) and CAP modified 3D printed PLA scaffolds atdifferent treatment times 1 min (B1-3), 3 min (C1-3) and 5 min (D1-3). A1-2, B1-2, C1-2, D1-2 with low magnification; A3, B3, C3, D3 with high magnification.

260 M. Wang et al. / Acta Biomaterialia 46 (2016) 256–265

respectively. The result suggests that the CAP etched the 3Dprinted PLA scaffold surface and increased roughness.

3.4. Contact angles

To determine the effect of CAP treatment on changes inhydrophilicity of the 3D printed PLA scaffold over time, the contactangles of the scaffold were measured. Under the optimized plasmaparameters, the contact angle significantly decreased after CAPtreatment, which indicated that the CAP-treated PLA scaffoldswere more hydrophilic than the untreated sample. The contactangles on the 3D PLA scaffolds before and right after CAP treatmentwith different treat times (1 min, 3 min, and 5 min) were 70 ± 3�,49 ± 1�,41 ± 2�, and 24 ± 2�, respectively. As evident from Fig. 5B,there was a time-dependent change of the contact angle for theCAP-treated PLA scaffold. When the CAP treated samples werestored (in a dry box at room temperature), the contact angles grad-ually recovered and were maintained at 40� after 3 days. This valuewas still significantly lower than that of the untreated PLA scaffold.

3.5. XPS analysis

XPS was used to investigate changes in the chemical state of the3D printed PLA surfaces without CAP treatment compared to 1, 3

and 5 min of CAP treatment. Survey scans of untreated and plasmatreated PLA scaffolds confirmed the presence of carbon and oxygenas shown in Fig. 6A. The more detailed chemical compositions arelisted in Table 2. Results showed that CAP treatment for 1 min ledto a decrease in carbon concentration on PLA 3D printed surfaceswhereas oxygen concentration increased on the surface. In addi-tion, longer CAP treatment time had no additional significant effecton carbon and oxygen concentrations. Table 2 also lists the O/Cratio, which increases from 0.5 to 0.76 after 3 min of plasma treat-ment. This suggests that the polymer chain was segmentedbetween carbon and oxygen and new bonds were created.

For a better understanding of the oxygen groups on the surface,a deeper analysis of C1s and O1s peaks were completed (Fig. 6B).The C bonding states on the untreated sample showed three com-ponents at 284.6 eV, 286.5 eV and 288.6 eV corresponding to CAC/CAH, CAO and OAC@O [27]. CAP treatment caused significant CAOand OAC@O increases, and also a new C@O peak at 290 eVemerged after 5 min of treatment (Table 3).

While the overall atomic percentage of O1s increased on thesurface for the CAP treated samples, there was no quantifiablechange in the bonding states for O1s for untreated as well as 1and 3 min of CAP treatment. However, longer plasma treatmentfor 5 min created new O1s bonding states related to the formationof new C@O groups. OAC@O is polar and, thus, the formation of

Control

CAP_3min

CAP_1min

CAP_5min

Fig. 4. AFM images of PLA 3D printed scaffolds with and without CAP treatment at different times.

Table 1The roughness of the 3D printed PLA scaffolds of interest to this study.

Surface roughness (nm) Control CAP_1 min CAP_3 min CAP_5 min

Rq 1.20 ± 0.23 10.50 ± 0.16 22.90 ± 0.26 27.60 ± 0.13

Fig. 5. A. Contact angles on 3D printed PLA scaffolds prepared at different CAP treated times (1 min, 3 min, and 5 min); B. Contact angles on untreated and CAP treated PLAscaffolds aged for different periods (1 day, 3 days, 5 days, 7 days, and 10 days).

M. Wang et al. / Acta Biomaterialia 46 (2016) 256–265 261

oxygen-containing groups on PLA surfaces can result in higherpolar surface energy as well as surface wettability. Such data con-firms the previous contact angle results which significantlydecreased after CAP treatment.

3.6. Cell adhesion results

The results of fibroblast adhesion on 3D printed PLA scaffoldstreated at different CAP treatment conditions (different input volt-

ages) and different modification times (0, 1 and 5 min) are shownin Fig. 7. Results revealed that all CAP-modified 3D PLA scaffolds sig-nificantly enhancedfibroblast cell adhesionwhen compared to scaf-foldswithout CAPmodification,which illustrates the excellent cyto-compatibility properties of CAP modified scaffolds. Cell adhesionresults at different input voltages (9 V, 11 V) for CAP-treated scaf-folds exhibited no difference. Under the same conditions, 5 min ofCAP treatment can achieve significantly higher cell attachmentwhen compared to untreated and 1 min CAP treated PLA scaffolds.

Fig. 6. XPS analysis results of CAP modification, A: XPS survey scans, B: XPS C1s and O1s tight scans of untreated and CAP treated 3D printed PLA scaffold surfaces for 1 min,3 min and 5 min.

Table 2Atomic composition of 3D printed PLA surfaces without CAP treatment compared to1, 3, and 5 min of CAP treatment.

Sample C1s [%] O1s [%] N1s [%] Si2p3 [%] Mg 2p [%] O/C

Untreated 64 31 Less than 1 4 – 0.491 min CAP 57 42 Less than 1 – – 0.743 min CAP 56 43 Less than 1 – – 0.765 min CAP 59 39 Less than 1 – 1 0.63

Table 3Relative composition of the C1s components for untreated 3D printed PLA surfacescompared to 1, 3, and 5 min of CAP treatment.

Concentration [%]

CAC/CAH CAO OAC@O C@O

Sample 284.6 eV 286.5 eV 288.6 eV 290 eVUntreated 43 10 11 –1 min CAP 25 14 18 –3 min CAP 23 16 17 –5 min CAP 17 19 14 9

262 M. Wang et al. / Acta Biomaterialia 46 (2016) 256–265

Fig. 7. Cell adhesion on 3D printed PLA scaffolds modified with different coldatmospheric plasma conditions. Data are mean ±standard error of the mean; n = 9.*p < 0.05 when compared to controls.

Day 1

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*

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*

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l Num

ber/m

l

**

Fig. 9. MSC proliferation on 3D printed PLA scaffolds modified with different CAPtreated times after 1, 3, and 5 days of culture. All samples had statistically highercell viability from 1 to 3 to 5 day. Data are mean ±standard error of the mean; n = 9.*p < 0.05 when compared to all other substrates at respective days; **p < 0.05 whencompared to the control at day 1.

M. Wang et al. / Acta Biomaterialia 46 (2016) 256–265 263

3.7. Cell proliferation study

In the adhesion study, both different input voltages and treat-ment times were employed to examine the effects of CAP treat-ment on the fibroblast cell response. The results of the adhesionstudy indicated that CAP treatment time played a more importantrole than different input voltages. Longer CAP treatment times sig-nificantly promoted fibroblast cell adhesion. Based on theseresults, different CAP treatment times were employed to examineosteoblast cell and stem cell long-term response. Fig. 8 illustratesosteoblast cell proliferation on the different CAP treated PLA scaf-folds examined in this study. The results from 1, 3 and 5 days ofosteoblast proliferation indicated that CAP treatment of the 3Dprinted PLA scaffolds improved bone cell growth. Under the sameconditions, 1 min and 3 min of CAP treatment achieved moreosteoblast cell growth when compared to untreated CAP treatedPLA scaffolds.

A human bone marrow-derived MSCs cell line was also investi-gated and characterized for cell proliferation under the same CAPsurface treatment. Based on the results in Figs. 9, 1, 3 and 5 dayMSCs proliferation studies exhibited similar trends with regardsto osteoblast proliferation results. The result demonstrated thatall of the CAP treated PLA scaffolds could offer MSCs favorable sur-faces which attracted more MSCs growth than untreated scaffoldsafter one-day of culture. Interestingly, the one-day result showedthat the highest density of MSCs occurred for 3 min CAP treatment,but for 3 and 5 days of culture, the highest MSCs density changed

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Fig. 8. Osteoblast cell proliferation on 3D printed PLA scaffolds modified withdifferent CAP treated times after 1, 3, and 5 days of culture. All samples hadstatistically higher cell viability from 1 to 3 to 5 days. Data are mean ±standarderror of the mean; n = 9. *p < 0.05 when compared to all other scaffolds at respectivedays; **p < 0.05 when compared to control at respective days; &&p < 0.05 whencompared to respective scaffolds at day 1.

to 1 min CAP treatment. It is suspected that the chemistry changedafter short treatment times which plays a more important role inmediating cell proliferation due to altered initial protein interac-tions; this will have to be the focus of future studies.

3.8. SEM images of cell morphology

In vitro, the effect of these four surfaces on the adherent mor-phology of MSCs was examined. As shown in Fig. 10, cells on thecontrol surface exhibited a flat and round shape, compared to theirregular shape with spread-out cytoplasm on the CAP modifiedPLA surface. MSCs on untreated PLA surface exhibited less flat-tened spindle-like shapes, while those on the CAP modifiednano-patterned surface, showed larger polygonal shapes, espe-cially under high magnification. In addition, the number of MSCson the CAP modified PLA scaffolds significantly increased after 5-days of culture when compared to those on the PLA control.

4. Discussion

CAP has great potential for numerous biomedical applications,such as sterilization, blood coagulation, anti-tumor, surface modi-fication and low temperature. In this study, CAP was used as a fastand effective surface modification method to improve bone regen-eration. Our results demonstrate that CAP modification can easilychange hydrophobic surface properties of the PLA scaffold to amore hydrophilic (contact angle change from 70� to 24�) surfacein simple one-step process. It is meaningful that the CAP surfacemodification could be easily controlled to avoid either damagingthe surface, or significantly impacting mechanical properties ofthe scaffold. More importantly, the CAP modified scaffold can sig-nificantly improve cell attachment resulting in increased cell pro-liferation and differentiation.

As shown in Fig. 11, the impact of CAP on PLA scaffold surfaceproperties may be attributed to two aspects: a surface roughnesschange and surface chemistry alteration. As mentioned above,CAP contains various changed species, including reactive oxygenspecies, reactive nitrogen species, and electrons. Especially impor-tant are reactive oxygen species, including peroxides, superoxide,hydroxyl radical, and atomic oxygen. When exposed to CAP, theends of the polymer chain on the PLA surface will react withcharged species within CAP. This oxidization process can effec-tively alter surface chemical groups, resulting in a more hydrophi-lic surface. We suspect that upon exposure to CAP with short times(1–3 min), the methyl end (ACH3) of the polymer at the PLA sur-face was converted to ACH2OH, ACHO, ACOOH resulting in a more

Fig. 10. SEM images of MSC cultured on 3D printed PLA scaffolds with and without CAP surface modification for 5 days (A. Control, B. 1 min CAP treatment, C. 3 min CAPtreatment, and D. 5 min CAP treatment, 1. Low magnification, 2, 3. High magnification).

Fig. 11. Proposed possible mechanisms of chemical and roughness changes for CAPtreatment of 3D printed PLA scaffolds.

264 M. Wang et al. / Acta Biomaterialia 46 (2016) 256–265

hydrophilic surface. In addition, the resulting hydroxymethylgroup might condense with a neighboring same group to form abrittle layer where carbon might be bonded to two or three oxygenatoms by the dehydrating process, which is the reason that theO@CAC and CAO bonds significantly increased after CAP treatedfor 1–3 min. In addition, the created ACH2OH and ACOOH groupsare optimal for cell adhesion and proliferation. When further treat-ing with CAP for 5 min, results of XPS from Fig. 6B showed decreas-ing O@CAO, increasing CAO, and a new O1s bonding state relatedto the formation of a O@C bond. It is proposed that the CAP processfurther oxidized the scaffold beyond the initial oxidization. AfterCAP treatment, scaffolds were exposed to the air for 7–10 days,and perhaps the new O@C bonds were easily oxidized, which isreflected by the increased contact angle.

Meanwhile, an accumulating CAP treatment will increase scaf-fold surface temperature as mentioned in the CAP characterizationsection. This accumulating temperature will partially molt andreform the polymer during CAP treatment, allowing for a conver-sion from micro-scale to nano-scale surface features. This nano-scaled surface will create higher surface area compared to amicro-scaled surface, which will promote surface energy and wet-tability once the scaffold interacts with tissue. In addition, nano-featured surfaces enhance cell adhesion due to improved integrin

M. Wang et al. / Acta Biomaterialia 46 (2016) 256–265 265

binding resulting in improved cell clustering and spreading.Finally, the nano-featured morphology has a positive effect onosteoblast cell differentiation and bone formation.

Our proliferation results further confirm the promotion of bothosteoblast and MSC proliferation on PLA scaffolds with increasingof CAP treatment time. The MSC cell proliferation results empha-sized that nano-scaled surfaces created by CAP treatment pro-moted cell attachment, proliferation and even morphologicalchanges on the scaffold surface, as observed by SEM. In addition,the slightly different response of osteoblast and MSC cell prolifer-ation may be due to different cell types having different require-ments for surface energy and roughness, and associated initialprotein adsorption. It is well known that 3D printing can fabricatescaffolds with ideal structures and required mechanical properties.However, printing scaffolds with desirable high resolution at thenanoscale is still a significant limitation. In order to address thislimitation, many possible approaches have been investigated suchas incorporating scaffolds surfaces with protein, nanoparticles,etching by chemicals, and plasma deposition. But most of thosemodification methods are costly, ineffective, and may damagethe original scaffold polymer. Our results indicated that CAP treat-ment is an effective tool for a one-step, low cost, effective surfacemodification process for all kinds of scaffold polymers, promotingcell responses. This new tool should be a promising surface modi-fication method for not only 3D printing biomedical scaffolds, butalso for numerous additional biomedical applications.

5. Conclusion

This study used CAP to treat 3D printed PLA scaffolds forimproving bone cell attachment and function. The CAP modifica-tion process showed that water contact angles, surface roughness,and oxygen to carbon ratios were improved under an optimizedCAP condition. The changes in those properties showed that bothhydrophilicity and nano-scale roughness altered which played animportant role in enhancing bone cell and MSCs attachment andfunctions. Those promising results suggest that CAP surface modi-fication may have potential applications not previously studied forenhancing numerous bone tissue engineering applications.

Conflict of interest

All authors have no conflict of interest.

Acknowledgements

The authors would like to acknowledge Northeastern Universityfor funding, Dr. William H. Fowle of Northeastern University forhelp with SEM images, Belinda Slakman from the Chemical Engi-neering Writing Center for help revising the paper, and the GeorgeJ. Kostas Center for help with AFM images.

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