Biological Effect of the Surface Modification of the FibrousPoly(L-lactic acid) Scaffolds by Radio Frequency MagnetronSputtering of Different Calcium-Phosphate Targets

S. I. Goreninskii1 & N. N. Bogomolova2 & A. I. Malchikhina2 & A. S. Golovkin3&

E. N. Bolbasov2 & T. V. Safronova4 & V. I. Putlyaev4 & S. I. Tverdokhlebov2

# Springer Science+Business Media New York 2017

Abstract Biodegradable materials, in particular poly(L-lacticacid), are widely used in medicine and tissue engineering.Electrospinning is one of the most promising methods forthe fabrication of scaffolds for tissue and organ regeneration.Due to their fibrous structure, high surface-to-volume ratioand great adjustability of electrospinning parameters suchscaffolds are able to mimic the topology of the extracellularmatrix (ECM) of a native human tissue. This paper demon-strates the effect of radio frequency magnetron sputtering(RFMS) modification of the poly(L-lactic acid) fibrous scaf-folds on their structure and cell adhesion and proliferation.RFMS modification was performed using four different tar-gets: hydroxyapatite (HAP), tricalcium phosphate (TCP),amorphous calcium pyrophosphate (CPP) and dicalciumphosphate dihydrate (DCPD). Biodegradable fibrous mate-rials with maximum Ca/P ratio on the surface at 0.542 wereobtained. It was observed that prolonged time of the treatmentleads to destruction of the fibers on the surface layer of thescaffold. Moreover, we have indicated that all obtained

materials demonstrate cytotoxic activity due to the formationof the toxic compounds on the material surface.

Keywords Poly(lactic) acid . Scaffold . Biomaterial .

Magnetron sputter deposition . Surface modification .

Calcium-phosphate

1 Introduction

Fibrous scaffolds, made of different polymers, are well-knownmaterials for regenerative medicine. Their unique architectureprovides necessary interactions between functional and sup-port cells, thus creating an environment for tissue formationand regeneration [1]. An electrospinning technique is one ofthe common methods for production of such structures [2].

Biologically resorbable polymers, such as poly(ε-caprolactone), poly(lactic acid) and poly(glycolic acid), arebeing actively utilized in modern medicine due to their bio-compatibility and degradability, making them promising ma-terials for implants, drug delivery systems and other biomed-ical devices [3]. However, these materials demonstrate no bi-ological activity. For tailoring of the surface and bulk proper-ties of biodegradable materials for specified applications, anumber of modification methods were proposed [4, 5].

Radio frequency magnetron sputtering (RFMS) is one ofthe methods, successfully applied for coating of biologicallyand chemically inert materials with a wide range of coatings,including calcium phosphate (CaP) [6], yttria-stabilized zirco-nia [7] and aluminium-doped zinc oxide [8]. Among the listedcoatings, calcium phosphate provides improved biocompati-bility of the material and enhances bone tissue formation [9,10]. It is stated that three-dimensional fibrous structures, com-bined from a biocompatible polymer and such inorganic

* S. I. Tverdokhlebovtverd@tpu.ru

1 The Department of Biotechnology and Organic Chemistry, NationalResearch Tomsk Polytechnic University, 30 Lenin av.,Tomsk, Russian Federation 634050

2 The Department of Experimental Physics, National Research TomskPolytechnic University, 30 Lenin av., Tomsk, Russian Federation634050

3 Federal Almazov Medical Research Centre, 2 Akkuratova st., St.Petersburg, Russian Federation 197341

4 The Chemistry Department, Lomonosov Moscow State University,GSP-1, Leninskie Gory, Moscow, Russian Federation 119991

BioNanoSci.DOI 10.1007/s12668-016-0383-x

compounds as calcium phosphates, are needed for bone tissueregeneration [11].

Previously, our group reported modification of poly(L-lac-tic acid) (PLLA) surface by RFMS method that leads to en-hanced cell adhesion and viability [12]. At the same time, itwas found that at a small period of time, CaP coating was notformed [13]. It was proposed that the needed CaP layer will beachieved with an increase of the treatment time.

By Ozeki et al. [14], it was shown that target selection af-fects such properties of the CaP coating as its crystal structure,formation rate and Ca/P molar ratio. Thus, for the optimizationof RFMS modification, various targets should be tested.

As during RFMS process, only surface layers of the scaf-fold are being bombarded with atoms and ions from the target;lower layers of the scaffold are not being modified and do notchange their mechanical properties. The aim of this work wasto investigate biological effect of poly(L-lactic acid) scaffoldRFMS modification.

2 Materials and Methods

Poly(L-lactic acid) scaffolds with the size of 60 × 70 × 1 mmwere used as substrates for sputtering of CaP coatings. Thenanofibrous scaffolds were produced by electrospinning of3% (w/w) PLLA (Purasorb PL 38, Carbion, Amsterdam, theNetherlands) polymer solution in chloroform (Ekos-1,Moscow, Russia) using NANON-01A electrospinning equip-ment (MECC Co., Fukuoka, Japan). Preparation of the PLLAsolution was carried out in a sealed glass reactor at a roomtemperature with continuous stirring until a homogeneousclear solution was obtained. The electrospun scaffolds werecollected on a target drum rotating collector at 50 rpm placedat a distance of 15 cm from the syringe tip. The flow rate of thePLLA solution was 4 mL/h with an applied voltage of 25 kV.In order to remove residual solvents, the samples were placedin a vacuum chamber at a pressure of 10−3 Pa at room tem-perature until constant mass was achieved.

Four powder calcium phosphate targets with different com-positions and structures were used for RF magnetronsputtering: artificial hydroxyapatite (HAP) Ca10(PO4)6(OH)2(Institute of Solid State Chemistry and Mechanochemistry,Siberian Branch of the Russian Academy of Science,Novosibirsk, Russia), β-tricalcium phosphate (TCP)Ca3(PO4)2 (Ekos-1, Moscow, Russia), amorphous calciumpyrophosphate (CPP) Ca2P2O7 × 2(H2O) (ChemistryDepartment, Lomonosov Moscow State University,Moscow, Russia) and dicalcium phosphate dihydrate(DCPD) CaHPO4 × 2(H2O) (Reachem,Moscow, Russia) withthe a Ca/P ratio of 1.67, 1.5, 1 and 1, respectively. The targetwas presented as a powder, filled in a cathode holder with thearea of 220 cm2.

Coating deposition was carried out using the universalmagnetron sputtering system (developed and assembled inHybrid Materials Laboratory of the Tomsk PolytechnicUniversity [12], Tomsk, Russia) under the following condi-tions: generator frequency—13.56 MHz, target-to-substratedistance—60 mm, pre-pressure in the chamber—3 × 10−3 Pa, working gas—argon (Ar) 99.999%, depositionpressure—4–4.5 × 10−1 Pa, power applied to the target—320 W (power density—1.45 W × cm−2 ), target size—220 cm2, deposition process duration—5 h. The substrateholder temperature was controlled by a thermocouple anddid not exceed 50 °C.

Morphology of the scaffold surface was studied with thescanning electron microscope Quanta 200 3D (FEI Company,Hillsboro, USA) in low vacuum with an electron beam accel-erating voltage of 20 kV with magnification of 5000× and20,000×. Distribution of the scaffold fiber diameter was cal-culated using ImageJ 1.49 [15] and Origin 8.1 (OriginLab,Northampton, USA) software packages.

Chemical composition of the scaffold surfaces was inves-tigated by X-ray fluorescence analysis (XRF) using a 1800XRF spectrometer (Shimadzu Corp., Kioto, Japan) at an ac-celerating voltage of 40 kV, scanning speed of 8°/min andscanning step of 0.1°. Sample scanning has been performedvia calcium (Ca), phosphorus (P), carbon (C), oxygen (О) andchlorine (Cl) channels.

For the investigations of cell adhesion and viability, fat-derived multipotent mesenchymal stem cells (MMSC) wereused. Cells were collected from healthy donors andimmunophenotyped with a flow cytometer GuavaEasyCyte6 (Merck Milipore, Darmstadt, Germany) usingCD19, CD34, CD45, CD73, CD90 and CD105 monoclonalantibodies (Becton, Dickinson and Company, Franklin Lakes,USA) as previously described [16]. The study was performedaccording to Helsinki declaration, and approval was obtainedfrom the local Ethics Committees in Almazov FederalMedical Research Centre. Written informed consent was ob-tained from all subjects prior to fat tissue biopsy. The cellswere maintained in alpha-MEM medium (PanEco, Moscow,Russia) supplemented with 10% fetal calf serum (HyCloneLaboratories, South Logan, USA), 50 units/mL penicillinand 50 μg/mL streptomycin (Invitrogen, Waltham, USA) at37 °C and 5% CO2.

Material samples were cocultivated with MMSC into thewells of a 24-well plate for 72 h. Cells were seeded at a densityof 40,000 cells per well. For measurement of adhesion levelafter cocultivation, samples were washed with phosphate-

�Fig. 1 SEM images of PLLA scaffolds before (a, b) and after (c -j)modification by RFMS method with sputter deposition of the followingtargets: Hydroxyapatite target (с, d); Tricalcium phosphate target (e, f);.Calcium pyrophosphate target (g, h); Dicalcium phosphate dehydratetarget (i, j). Left column—5000× magnification; right column—20,000×magnification

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Average fiber diameter 2.5±0.3 µm

Average fiber diameter 1.8±0.4 µm

Average fiber diameter 1.7±0.5 µm

Average fiber diameter 2.0±0.5 µm

Average fiber diameter 2.0±0.5 µm

a b

c d

e f

g h

i j

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buffered saline (PBS); all seeded cells were fixed with 4%paraformaldehyde (PFA) and stained with nucleus dye DAPI(4,6-diamidino-2-phenylindole). Stained samples were visual-ized using a fluorescence microscope Axio Observer Z1 (CarlZeiss, Oberkochen, Germany). To verify viability, cells weretrypsinized from the sample surface, stained with Annexin VFITC and propidium iodide, followed by flow cytometry anal-ysis on GuavaEasyCyte6 (Merck Milipore, Darmstadt,Germany). Cells cultivated in the plate wells without sampleswere set as a control. All tests were performed in triplicates.

Cocultivation samples were washed trice with 1.0 mL ofPBS with shaking for 5 min. Washing PBS was collected andexposed to MTT testing. All washed samples were tested foradhesion and viability level as described above. MTT test wasperformed with MMSC in a 96-well plate. Cells were seededat a density of 10,000 cells per well for 24 h to reach asubconfluent layer. After that, culture medium was evacuatedand sample washing PBS was added to the wells with 100 μLof 1, 10 and 100% dilutions. One hundred percent means thatnon-diluted washing PBS was used. MTT was added to eachwell in concentration of 5 mg/mL. After 3 h of cultivation,supernatant was evacuated and 100 μL of dimethyl sulfoxidewas added to each well. After dissolution of formazan, theintensity was measured colorimetrically at 550 nm using aSmartSpec Plus spectrophotometer (BioRad, Hercules,USA). Distilled water and pure PBS were used as controls.All tests were performed in triplicates.

Statistical analysis was performed using Statistica 7.0 soft-ware (Statsoft, Tulsa, USA). Data is presented as Mean ± errorofmean. The significance of differencewas calculated using one-way ANOVA test and the Mann-Whitney U-test. Differencesbetween groups were stated as significant in p < 0.05.

3 Results and Discussion

The SEM images of the biodegradable scaffold surface areshown in Fig. 1.

In comparison with original PLLA scaffolds (Fig. 1a, b),the average fiber diameter was decreased. There are fracturesand breaks in the structure of the scaffold fibers after RFMSmodification (Fig. 1c–j), caused by bombardment with parti-cles of different natures such as neutral atoms and ions,sputtered from the target surface (CaO, PO4

2−, OH−, Ca+,Ca2+), reflected argon ions, neutral atoms, electrons, photonsand UV radiation (Fig. 1).

Typical fluorescence spectra for calcium (Ca), phosphorus(P), oxygen (O), carbon (C) and chlorine (Cl) channels beforeand after modification of scaffolds by RFMS methods areshown in Fig. 2. Quantitative values of elemental analysisfor the samples before and after RFMS treatment are shownin Table 1. The amount of chlorine was decreased by 10 timesafter plasma treatment. This is probably due to changes in thepolymer fiber structure during the process of RF magnetron

Fig. 2 XRF of scaffolds for the following elements before and after modification by RFMS method with sputter deposition of different CaP targets.Channels of C (a), O (b), Ca (c), P (d) and Cl (e)

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sputtering. This allows the residual solvent (dichloromethane)diffuse out of the sample volume, and chlorine evaporatesthrough the vacuum system [12].

The highest content of calcium and phosphorus was obtain-ed from the PLLA scaffold by sputtering of DCPD target(Table 1). It may be said that DCPD target demonstrates the

Fig. 3 Modified samples afterwashing with PBS with cellsstained with nucleus dye DAPI. aTricalcium phosphate target. bHydroxyapatite target. c PLLA. dCalcium pyrophosphate target. eDicalcium phosphate dehydratetarget

Table 1 The content of the chemical elements (С, O, Ca, P, Cl) in the samples before and after modification by RFMSmethod with sputter depositionof different CaP targets

Sample/the sputtering target The content of the chemical element, at.%

С O Ca P Cl Ca/P

PLLA/– 62.39 37.46 Background noise level Background noise level 0.130 –

PLLA/hydroxyapatite 64.35 35.34 0.013 0.024 0.013 0.542

PLLA/tricalcium phosphate 63.91 36.00 Background noise level 0.030 0.040 –

PLLA/calcium pyrophosphate 64.71 35.10 0.025 0.119 0.034 0.210

PLLA/dicalcium phosphate dehydrate 64.05 35.81 0.018 0.074 0.037 0.243

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highest sputtering coefficient. The stoichiometric calcium-to-phosphorus ratio in all obtained materials is below one. Theamount of phosphorus in the surface layer of modified scaf-folds was more than that of calcium due to the presence ofoxygen in the composition of poly(L-lactic acid) scaffolds.Phosphorus readily forms chemical bonds with oxygenforming phosphorus pentoxide [17], pyrophosphates [18]and other phosphorus-containing compounds [19].

Results of the XRF analysis demonstrate that the largestCa/P ratio was achieved by sputtering of hydroxyapatite tar-get. But in comparison with CPP and DCPD targets, hydroxy-apatite target had the lowest sputtering coefficient, resulting inrelatively low calcium and phosphate content.

According to the results of XRF and SEM studies, the mostsuitable target for sputtering by RFMSmethod on PLLA scaf-fold is dicalcium phosphate dihydrate. The average diameterof the scaffold fibers after sputtering of DCPD target is closestto the values of the untreated PL38 scaffold. This demon-strates that when using the DCPD target, fiber scaffold struc-ture is less prone to change due to radiation and bombardmentby particles of various natures. At the same time, target has thehighest sputtering coefficient that confirmed the contents Caand P in the samples.

During the first experiments of cell viability and adhesiontesting, all samples demonstrated very low number oftrypsinized cells (less than 30 cells/μL). Besides in all

samples, the rate of viable cells was significantly lower thanthat in the control (Table 2). The rest of cells from TCP, HAP,CPP and DCPD samples were presented in late apoptosis ornecrosis stage. Only in the case of PL 38, level of early apo-ptosis was significantly higher than that in the control. Thus,all samples demonstrated cytotoxic properties, meanwhile PL38 was less cytotoxic.

Adhesion level was very low (Table 3). These resultsproved high cytotoxic properties of all the modified surfaces.

We hypothesized that continuous RFMS modification leadsto formation of cytotoxic compounds. That is why we tried towash the samples to increase biocompatibility of their surfaces.After that, adhesion level and cell viability after cocultivationwere measured again. Number of adhered cells increased, butanyway, an adhesion property of modified surfaces was low(Table 4, Fig. 3). It was controverted with our previous resultswhen the adhesion level was higher by several orders [12].

Washing PBS from the samples was used to perform MTTtest to prove the presence of cytotoxic compounds in thewashing solutions from the tested samples.

It is known that optical density (OD) of the solutionafter performing MTT-test correlates with number of livingcells and their activity. Cells with low metabolic activitycould convert low level of MTT into formazan.Meanwhile, cells with a high proliferation rate are veryactive in this conversion.

Table 2 Cell viability aftercocultivation with obtainedsamples

Sample/the sputtering target Viable, % Late apoptosis, necrosis, % Early apoptosis, %

Control 89.0 ± 0.6 4.4 ± 0.2 3.1 ± 0.2

PLLA/– 60.7 ± 4.4b 13.8 ± 1.6 22.0 ± 3.9a

PLLA/hydroxyapatite 22.8 ± 2.4a 42.5 ± 5.1a 2.8 ± 0.8

PLLA/tricalcium phosphate 18.3 ± 3.3a 47.1 ± 7.2a 1.3 ± 0.3

PLLA/calcium pyrophosphate 38.0 ± 4.5a 41.7 ± 2.7a 2.5 ± 0.3

PLLA/dicalcium phosphate dehydrate 43.3 ± 20.8a 24.8 ± 10.7c 2.0 ± 0.6

a p < 0.01 comparing to controlb p = 0.04 comparing to controlc p = 0.03 comparing to control

Table 3 Amount of MMSC on1 mm2 of the surface Sample/the sputtering target Without washing After sample washing

PLLA/– 36.1 ± 5.2a,b 59.7 ± 8.3a,b,c

PLLA/hydroxyapatite 33.3 ± 4.7a,b 65.9 ± 8.0a,b

PLLA/tricalcium phosphate 35.9 ± 6.2a,b 86.0 ± 9.0a

PLLA/calcium pyrophosphate 22.1 ± 2.5a 120.8 ± 19.7a

PLLA/dicalcium phosphate dehydrate 5.4 ± 1.9b 27.0 ± 7.3b

a p < 0.05 comparing to dicalcium phosphate dehydrate targetb p < 0.05 comparing to calcium pyrophosphate targetc p < 0.05 comparing to tricalcium phosphate target

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It was shown that there were no differences in OD betweenpure PBS (control) and washing PBS in 1 or 10% concentra-tion (Table 5). Meanwhile, the addition of 100%washing PBSleads to the significant decrease of OD in TCP (p = 0.02) andHAP (p = 0.02) samples comparing to the control. Washesfrom these two types of samples had the highest cytotoxicproperties to the cells.

By means of XRF and SEM, it was stated that the mosteffective target for prolonged RFMS modification of poly(L-lactic acid) fibrous scaffolds was dicalcium phosphate dehy-drate. Using that target at applied conditions, maximum Ca/Pratio which is the nearest to the natural one in human bonewasachieved [20] and the average diameter of the scaffold fiberswas less affected. But cell studies of the modified materialsdemonstrated cytotoxic activity of the obtained samples. MTTtest confirmed the formation of toxic compounds on the scaf-fold surface.

It may be suggested that poly(L-lactic acid) fibrous scaf-folds, modified by RFMS method at the applied parame-ters, are not suitable for bone tissue engineering. Fibrousstructures, obtained by Prabhakaran et al. [21] byelectrospinning of the solution of PLLA and hydroxyapa-tite, maintained high adhesion, proliferation and minerali-zation of osteoblasts, being promising materials for suchapplications. Cell studies of the PLLA electrospun scaf-folds, coated with calcium phosphates by electrodeposition

and exposure to simulated body fluid, demonstrated en-hanced proliferation and osteoblastic differentiation ofMC3T3-E1 cells [22]. Thus, there are a number of tech-niques, developed and more successfully applied for theproduction of CaP-PLLA composite fibrous structures.

Among the methods of fabrication of polymer structureswith biologically active coatings, a Blayer-by-layer^ techniquemust be noticed. From the point of biocompatibility and pro-cess efficiency, this method seems more preferable than con-tinuous RFMSmodification, as it allows obtaining wide rangeof materials with tailored mechanical and surface properties[23, 24] and biological activity [25, 26]. Incorporation of suchinorganic compounds as calcium phosphates and carbonnanotubes [27] for stimulation of osteoblast adhesion and pro-liferation rate with this approach may be promising solutionfor bone tissue engineering.

4 Conclusion

The effect of magnetron sputtering of the different calciumphosphate targets on the surface of biodegradable nanofibrousscaffolds made of poly(L-lactic acid) was investigated. It wasfound that continuous plasma treatment leads to crucial chang-es in the architecture of the scaffold surface layers. Moreover,

Table 5 Results of MTT testwith washing PBS from differentsamples

Sample/the sputtering target 1% washed PBS 10% washed PBS 100% washed PBS

Control (pure PBS) 0.829 ± 0.038

PLLА/– 0.765 ± 0.064 0.818 ± 0.081 0.668 ± 0.048b,d

PLLА/tricalcium phosphate 0.768 ± 0.035 0.799 ± 0.049 0.476 ± 0.029a

PLLА/hydroxyapatite 0.809 ± 0.046 0.826 ± 0.028 0.474 ± 0.037a

PLLА/calcium pyrophosphate 0.841 ± 0.070 0.948 ± 0.134 0.678 ± 0.027c,d

PLLА/dicalcium phosphate dehydrate 0.994 ± 0.120 0.847 ± 0.126 0.628 ± 0.088

a p = 0.02 comparing to controlb p = 0.04 comparing to tricalcium phosphate targetc p = 0.03 comparing to tricalcium phosphate targetd p = 0.03 comparing to hydroxyapatite target

Table 4 Cell viability aftercocultivation with washedsamples

Sample/the sputtering target Viable, % Late apoptosis, necrosis, % Early apoptosis, %

Control 85.4 ± 1.1 6.5 ± 0.5 3.9 ± 0.4

PLLА/– 87.4 ± 1.1 5.5 ± 0.5 4.1 ± 0.4

PLLA/tricalcium phosphate 70.2 ± 1.1a 12.7 ± 0.9a 9.4 ± 0.5a

PLLA/hydroxyapatite 78.3 ± 1.2a 8.1 ± 0.8 9.4 ± 0.5a

PLLA/calcium pyrophosphate 77.4 ± 1.1a 8.4 ± 0.6 5.0 ± 0.1

PLLA/dicalcium phosphate dehydrate 76.0 ± 1.4a 11.0 ± 1.0a 5.3 ± 0.2a

a p < 0.01 comparing to control

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it was found that extended time of RFMS modification leadsto the increase of cytotoxicity of the material.

Acknowledgements This research was funded by the Russian ScienceFoundation (project no. 16-13-10239) and performed in TomskPolytechnic University.

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