International Journal of Molecular Sciences

Article The Osteogenic Differentiation Effect of the FN Type 10- on PCL Fiber

Ye-Rang Yun 1, Hae-Won Kim 2,3,4,* and Jun-Hyeog Jang 5,* ID

1 Industrial Technology Research Group, Research and Development Division, World Institute of Kimchi, Nam-Gu, Gwangju 61755, Korea; [email protected] 2 Institute of Tissue Regeneration Engineering (ITREN), Dankook University, Cheonan 330-714, Korea 3 Department of Nanobiomedical Science & BK21 PLUS NBM Global Research Center for Regenerative Medicine, Dankook University, Cheonan 330-714, Korea 4 Department of Biomaterials Science, School of Dentistry, Dankook University, Cheonan 330-714, Korea 5 Department of Biochemistry, Inha University School of Medicine, Incheon 22212, Korea * Correspondence: [email protected] (H.-W.K.); [email protected] (J.-H.J.); Tel.: +82-41-550-3081 (H.-W.K.); +82-32-890-0930 (J.-H.J.); Fax: +82-41-550-3085 (H.-W.K.); +82-32-882-1877 (J.-H.J.)

Received: 1 November 2017; Accepted: 3 January 2018; Published: 4 January 2018

Abstract: The fibronectin type 10-peptide amphiphile (FNIII10-PA) was previously genetically engineered and showed osteogenic differentiation activity on rat bone marrow stem cells (rBMSCs). In this study, we investigated whether FNIII10-PA demonstrated cellular activity on polycaprolactone (PCL) fibers. FNIII10-PA significantly increased protein production and adhesion activity on PCL fibers in a dose-dependent manner. In cell proliferation results, there was no effect on cell proliferation activity by FNIII10-PA; however, FNIII10-PA induced the osteogenic differentiation of MC3T3-E1 cells via upregulation of bone sialoprotein (BSP), collagen type I (Col I), osteocalcin (OC), osteopontin (OPN), and runt-related transcription factor 2 (Runx2) mitochondrial RNA (mRNA) levels; it did not increase the alkaline phosphatase (ALP) mRNA level. These results indicate that FNIII10-PA has potential as a new biomaterial for bone tissue engineering applications.

Keywords: FNIII10; peptide amphiphile; PCL fiber; osteogenic differentiation activity

1. Introduction Recently, various attempts have been made to find new biomaterial for tissue engineering. Among the attempts, research on the peptide amphiphile (PA) has been widely reported. PA has hydrophilic and hydrophobic components and a self-assembly ability as a peptide-based molecule. There is increased interest in the extracellular matrix (ECM) molecule mimetic PA because it can promote certain cellular activities including adhesion, proliferation, and differentiation [1–5]. Based on these characteristics, biomimetic PA is extensively utilized for tissue engineering as a new biomaterial. For example, the Arg-Gly-Asp (RGD) peptide, composed of L-arginine, glycine, and L-aspartic acid, participates in cellular attachment via integrin [6]. For this reason, numerous studies on PA-RGD have been reported in the pharmaceutical field [7–9] as well as in the tissue engineering field [10–12]. For instance, the combination of hydroxyapatite (HA) and PA-RGD enhances the osteoinductive and osteoconductive activities of the scaffold [13]. Moreover, studies on amphiphile containing fibronectin (FN) are commonly demonstrated and reported. We also previously demonstrated that the engineered fibronectin type 10-peptide amphiphile sequence (FNIII10-PA) potentiates utilization for bone tissue engineering by enhancing the adhesion, proliferation, and differentiation of rat bone marrow stem cells (rBMSCs) [14]. In another study, the tenascin-C mimetic PA (TN-C PA) showed self-assembly activity. Nanofiber containing TN-C

Int. J. Mol. Sci. 2018, 19, 153; doi:10.3390/ijms19010153 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2018, 19, 153 2 of 10

PA were shown to promote neurite outgrowth, which could potentially be used for artificial matrix therapy in neuronal regeneration [15]. PolycaprolactoneInt. J. Mol. Sci. 2018, 19, 153 (PCL) is a biomaterial that is used mostly for bone tissue engineering2 of 10 [16,17]. PCL is a biodegradable polyester with a low melting point (60 ◦C) and a low glass transition containing TN-C PA were shown to promote neurite outgrowth, which could potentially be used for temperature (−60 ◦C). Because of its biodegradation properties, PCL is regarded as a suitable artificial matrix therapy in neuronal regeneration [15]. biomaterialPolycaprolactone for long-term implantable (PCL) is a biomaterial devices [that18]. is Above used mostly all, PCL for hasbone advantages tissue engineering in tissue [16,17]. engineering applicationsPCL is fora biodegradable drug delivery polyester devices, with sutures, a low ormelting adhesion point barriers(60 °C) and without a low safety glass transition problems for the Food andtemperature Drug Administration (−60 °C). Because (FDA) of its approvalbiodegradation [19]. pr However,operties, PCL it isis difficultregarded foras PCLa suitable to influence cellularbiomaterial activity becausefor long-term it does implantable not provide devices an [18]. ECM Above type all, structure PCL has foradvantages the cells in [tissue20]. Recently, engineering applications for drug delivery devices, sutures, or adhesion barriers without safety nanofibrous PCL has been the preferred choice for bone tissue engineering applications by improving problems for the Food and Drug Administration (FDA) approval [19]. However, it is difficult for cellularPCL activities to influence and cellular mechanical activity properties. because it does PCL not isprovide indisputably an ECM type the structure most used for biomaterialthe cells [20]. in bone tissue engineeringRecently, nanofibrous research. PCL has been the preferred choice for bone tissue engineering applications by Previously,improving cellular we revealed activitiesthat and geneticallymechanical pr engineeredoperties. PCL FNIII10-PA is indisputably promoted the most cell used adhesion, proliferation,biomaterial and in differentiationbone tissue engineering of rBMSCs. research. In the present study, we investigated whether or not FNIII10-PAPreviously, with PCL we fiber revealed promoted that genetically the MC3T3-E1’s engineered cellular FNIII10-PA activities. promoted FNIII10 cell adhesion, was used as the proliferation, and differentiation of rBMSCs. In the present study, we investigated whether or not negative control in all experiments. Initially, the protein adhesion activity of FNIII10-PA was measured. FNIII10-PA with PCL fiber promoted the MC3T3-E1’s cellular activities. FNIII10 was used as the Then, thenegative effects control of FNIII10-PA in all experiments. with PCL Initially, fiber onthe MC3T3-E1protein adhesion cells activity adhesion of andFNIII10-PA proliferation was were investigated.measured. Furthermore, Then, the effects the osteogenicof FNIII10-PA differentiation with PCL fiber activity on MC3T3-E1 of FNIII10-PA cells adhesion with PCL and fibers on MC3T3-E1proliferation cells was were investigated investigated. by Furthermore, measuring the the osteogenic osteogenic differentiation differentiation activity marker of FNIII10-PA mRNA level. with PCL fibers on MC3T3-E1 cells was investigated by measuring the osteogenic differentiation 2. Resultsmarker and mRNA Discussion level.

2.1. Morphology2. Results and of PCL Discussion Fibers

PCL2.1. Morphology has been of commonly PCL Fibers used as a biomaterial in the tissue engineering field because of its non-toxic andPCL biodegradable has been commonly characteristics. used as a biomaterial On the other in the hand, tissue PCL engineering demonstrated field because a slow of degradation its rate, poornon-toxic mechanical and biodegradable properties, characteristics. and low cell On adhesion. the other Particularly, hand, PCL PCLdemonstrated shows low a slow affinity and stiffnessdegradation in cells due rate, to poor poor mechanical hydrophilicity, properties, leading and low to cell the adhesion. reduction Particularly, in cell proliferation, PCL shows low migration, differentiation,affinity and and stiffness regeneration in cells due [20 to]. poor However, hydrophilicity, nanofibrous leading PCL to the was reduction reported in cell to proliferation, improve the cellular migration, differentiation, and regeneration [20]. However, nanofibrous PCL was reported to activities in recent studies [21,22]. FNIII10 was connected to the PA sequence LLLLCCCGGDSDS improve the cellular activities in recent studies [21,22]. FNIII10 was connected to the PA sequence (L, leucine;LLLLCCCGGDSDS C, cysteine; G, (L, glycine; leucine; C, D, cysteine aspartic; G, acid; glycine; S, serine) D, aspartic to create acid; S, FNIII10-A serine) to create sequence FNIII10-A as illustrated in Figuresequence1A. PCL as illustrated fiber was in Figure fabricated 1A. PCL by fiber the electrospinningwas fabricated by technique.the electrospinning Figure technique.1B shows that the electrospunFigure 1B shows PCL that fiber the was electrospun uniform PCL with fiber straight was uniform fibers. with Average straight fibers. diameter Average of diameter PCL fibers was approximatelyof PCL fibers 1 µ Mwas (Figure approximately1C). Hence, 1 μM PCL (Figure fiber 1C). was Hence, suitable PCL fiber to investigate was suitable the to cellinvestigate adhesion the activity of FNIII10cell adhesion in combination activity of with FNIII10 biomaterial in combinat inion the with present biomaterial study. in the present study.

FigureFigure 1. Structure 1. Structure offibronectin of type type 10-peptide10-peptide amphiphile amphiphile (FNIII10-PA) (FNIII10-PA) (A) and (A morphology) and morphology of of polycaprolactone(PCL)polycaprolactone(PCL) fiber fiber (B ,(CB).,C Peptide). Peptide amphiphile amphiphile (PA) (PA) sequence, sequence, LLLLCCCGGDS LLLLCCCGGDS (L, leucine; (L, leucine; C, cysteine; G, glycine; D, aspartic acid; S, serine) was connected with fibronectin type III10. Scale is 7 mM in panel B, 50 µM in panel C (resolution 500×). Int. J. Mol. Sci. 2018, 19, 153 3 of 10

C, cysteine; G, glycine; D, aspartic acid; S, serine) was connected with fibronectin type III10. Scale is 7 Int. J. Mol.mM Sci.in panel2018, B,19 ,50 153 μM in panel C (resolution 500×). 3 of 10

2.2. Protein Adhesion Activity of FNIII10-PA on PCL Fibers 2.2. Protein Adhesion Activity of FNIII10-PA on PCL Fibers Previously, FNIII10-PA was shown to increase protein adhesion activity on tissue culture plates Previously, FNIII10-PA was shown to increase protein adhesion activity on tissue culture as compared with FNIII10. As shown in Figure 2, the protein adhesion activity of FNIII10-PA was plates as compared with FNIII10. As shown in Figure2, the protein adhesion activity of significantly increased in a dose-dependent manner (*** p < 0.001). Particularly, 5 μg·mL−1 of FNIII10-PA was significantly increased in a dose-dependent manner (*** p < 0.001). Particularly, FNIII10-PA increased protein adhesion 3-fold compared with that of FNIII10. In many studies, PCL 5 µg·mL−1 of FNIII10-PA increased protein adhesion 3-fold compared with that of FNIII10. scaffold coated with FN remarkably enhanced cell adhesion, proliferation, and differentiation In many studies, PCL scaffold coated with FN remarkably enhanced cell adhesion, proliferation, [23–25]. Drevelle et al. found that the PCL film functionalized with RGD peptide and differentiation [23–25]. Drevelle et al. found that the PCL film functionalized with RGD (Ac-CGGNGEPRGDTYRAY-NH2 derived from the bone sialoprotein) and easily adsorbed the peptide (Ac-CGGNGEPRGDTYRAY-NH derived from the bone sialoprotein) and easily adsorbed serum adhesive proteins FN and vitronectin2 (VN), leading to increased cell adhesion and spreading the serum adhesive proteins FN and vitronectin (VN), leading to increased cell adhesion and by activating focal adhesion kinase (FAK) signaling [26]. In another study, FN-immobilized spreading by activating focal adhesion kinase (FAK) signaling [26]. In another study, FN-immobilized nanobioactiveglass (nBG)/PCL (FN-nBG/PCL) scaffolds also increased cell differentiation as well as nanobioactiveglass (nBG)/PCL (FN-nBG/PCL) scaffolds also increased cell differentiation as well as cell proliferation [27]. In the present study, FNIII10-PA showed higher protein adhesion activity on cell proliferation [27]. In the present study, FNIII10-PA showed higher protein adhesion activity on PCL fibers than that of FNIII10 alone. PCL fibers than that of FNIII10 alone.

Figure 2. Protein adhesion activity of FNIII10-PA on PCL fiber.fiber. PCL fibersfibers werewere placed in 24-well plates and coatedcoated withwith 0,0, 1,1, oror 55 µμgg·mL·mL−−11 ofof FNIII10 FNIII10 or or FNIII10-PA FNIII10-PA overnight at 4 °C.◦C. Protein Protein adhesion activities are expressedexpressed asas meansmeans ±± SDSD ( (nn == 3). 3). *** *** pp << 0.001. 0.001. OD OD = =optical optical density. density.

2.3. Cell Adhesion Activity of FNIII10-PA onon PCLPCL FibersFibers As mentioned above, cells are unable to perform many cellular activities on PCL fibers,fibers, because PCL fibersfibers do do not not provide provide a structure a structure similar similar to the to natural the natural ECM. To ECM. evaluate To whetherevaluate or whether not FNIII10-PA or not enhancesFNIII10-PA cellular enhances activity cellular on PCL activi fibers,ty on cell PCL adhesion fibers, activity cell adhesi of FNIII10-PAon activity was of performed FNIII10-PA on PCLwas fibers.performed In addition, on PCL MC3T3-E1fibers. In addi celltion, spreading MC3T3-E1 on PCL cell fibers spreading by FNIII10-PA on PCL wasfibers observed by FNIII10-PA by scanning was electronobserved microscopy by scanning (SEM). electron Figure microscopy3A shows (SEM). that 5 Figureµg·mL 3A−1 ofshows FNIII10-PA that 5 μ significantlyg·mL−1 of FNIII10-PA enhanced cellsignificantly adhesion enhanced activity on cell PCL adhesion fibers (Figureactivity3A, on * PCLp < 0.05).fibers Hence,(Figure SEM3A, * analysis p < 0.05). was Hence, performed SEM inanalysis 5 µg· mLwas−1 performedof FNIII10 in or 5 FNIII10-PA μg·mL−1 of without FNIII10 a or treatment FNIII10-PA group without for comparison. a treatment In group the SEM for image,comparison. 5 µg·mL In− 1theof FNIII10-PASEM image, induced 5 μg·mL more−1 cellof spreadingFNIII10-PA and induced attachment more on cell PCL spreading fiber compared and toattachment that of FNIII10 on PCL (Figurefiber compared3B). Generally, to that PCL of FN inIII10 the (Figure nanofiber 3B). form Generally, is preferred PCL in in the bone tissue engineeringform is preferred studies, in bone because tissue PCL engineering shows low studie cellulars, because activities PCL such shows as cell low adhesion, cellular activities proliferation, such andas cell differentiation. adhesion, proliferation, For instance, Yunand et differentiation. al. reported that For PCL instance, nanofibers Yun enhanced et al. cellularreported activities that PCL by providingnanofibers aenhanced three-dimensional cellular acti structurevities by similar providing to the ECMa three-dimensional [28]. In this study, structure FNIII10-PA similar enhanced to the cellECM adhesion [28]. In this activity study, on FNIII10-PA PCL fibers, enhanced even though cell adhesion PCL was activity in the microfiber on PCL fibers, form. even Based though on these PCL results,was in the FNIII10-PA microfiber may form. improve Based theon these cell interaction results, FNIII10-PA with PCL fiber.may improve the cell interaction with PCL fiber.

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FigureFigure 3.3. Cell adhesionadhesion activity (A)) andand cellcell spreadingspreading ((BB)) ofof MC3T3-E1MC3T3-E1 onon PCLPCL fibers.fibers. PCLPCL fibersfibers −1 werewere placedplaced inin 24-well24-well platesplates andand coatedcoated withwith 0,0, 1,1, or or 5 5µ μgg·mL·mL−1 of FNIII10 or FNIII10-PA overnightovernight atat 44 ◦°C.C. MC3T3-E1MC3T3-E1 cellscells werewere culturedcultured atat aa densitydensity ofof 11× × 105 cellscells and and incubated for 30 min at 37 ◦°C.C. CellCell adhesionadhesion activitiesactivities areare expressedexpressed asas meansmeans ±± SDSD ( (nn == 3). 3). * *pp << 0.05. 0.05. For For SEM SEM analysis, analysis, 5 5μµg·mLg·mL−1− of1 ofFNIII10 FNIII10 and and FNIII10-PA FNIII10-PA were were used. used. Scale Scale is is50 50 μMµM (resolution (resolution 500×). 500× ).

2.4.2.4. Cell Proliferation ActivityActivity ofof FNIII10-PAFNIII10-PA onon PCLPCL FiberFiber WeWe previouslypreviously revealedrevealed thatthat FNIII10-PA waswas shownshown to promote rBMSCs proliferation activity. activity. However,However, MC3T3-ElMC3T3-El cellcell proliferationproliferation activity of FNIII10-PA onon PCLPCL fibersfibers waswas notnot observedobserved inin thethe presentpresent study.study. AsAs shownshown inin FigureFigure4 4,, there there were were no no differences differences in in cell cell proliferative proliferative activity activity between between FNIII10FNIII10 and FNIII10-PA. CellCell proliferativeproliferative activityactivity of FNIII10-PAFNIII10-PA couldcould bebe weakweak inin MC3T3-E1MC3T3-E1 cellscells comparedcompared to thatthat inin rBMSCrBMSC cells.cells. In the next study,study, applicationapplication of FNIII10-PAFNIII10-PA needsneeds toto confirmconfirm thethe rBMSCrBMSC proliferativeproliferative activityactivity onon PCLPCL fiber.fiber. FN was shown to have a powerfulpowerful effecteffect onon bothboth cellcell adhesionadhesion andand differentiationdifferentiation ofof stemstem cells.cells. ForFor instance,instance, Mohamadyar-ToupkanlouMohamadyar-Toupkanlou etet al.al. reportedreported thatthat nanofibrousnanofibrous PCL/HAPCL/HA scaffold scaffold coated with FN provided a suitable environment for proliferation asas wellwell asas attachmentattachment andand differentiationdifferentiation inin mousemouse MSCsMSCs (mMSCs)(mMSCs) [[23].23]. ShekaranShekaran etet al.al. foundfound thatthat ECM-coatedECM-coated PCLPCL microcarriersmicrocarriers promoted promoted human human early early mesenchymal mesenchymal stem stem cell cell growth, growth, leading leading to cell to expansioncell expansion [24]. [24]. Similarly, Similarly, Mousavi Mousavi et al. revealedet al. reve thataled three that dimensional three dimensio nanoscaffoldnal nanoscaffold coated with coated FN inducedwith FN theinduced human the cord human blood cord hematopoietic blood hematopoietic stem cell expansionstem cell expansion [25]. [25].

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Figure 4. CellCell proliferationproliferation activity activity of of FNIII10-PA FNIII10-PA on on PCL PCL fibers fibers at 0 andat 0 5and days. 5 days. PCLfibers PCL werefibers placed were placedin 24-well in 24-well plates and plates coated and with coated 5 µ gwith·mL −51 μofg·mL FNIII10−1 of orFNIII10 FNIII10-PA or FNIII10-PA overnight overnight at 4 ◦C. MC3T3-E1 at 4 °C. MC3T3-E1cells were culturedcells were at acultured density at of a 1 density× 105 cells of 1 and × 10 incubated5 cells and for incubated 5 days at for 37 ◦5C. days Cell at proliferation 37 °C. Cell proliferationactivity is expressed activity asis expressed mean ± SD as ( nmean= 3). ± SD (n = 3).

2.5. The Osteogenic Differentiation Ac Activitytivity of FNIII10- FNIII10-PAPA onon PCLPCL FibersFibers To investigate the osteogenic differentiation acti activityvity of FNIII10-PA on PCL fibers, fibers, we measured the mitochondrial RNA RNA (mRNA) (mRNA) le levelsvels of alkaline phosphatase ( (ALP)ALP),, bone bone sialoprotein sialoprotein ( (BSPBSP),), collagen type type I I ( (ColCol I I),), osteocalcin osteocalcin (OC (OC),), osteopontin osteopontin (OPN (OPN), ),and and runt-related runt-related transcription transcription factor factor 2 (2Runx2 (Runx2) genes) genes at at5 and 5 and 10 10days. days. At both At both time time points, points, the mRNA the mRNA levels levels of BSP of ,BSP Col ,ICol, OC I,, OPNOC, OPN, and, Runx2and Runx2 werewere significantly significantly upregulated upregulated by FNIII10-PA by FNIII10-PA treatment treatment (Figure (Figure 5, * 5p,* < 0.05,p < 0.05,** p < ** 0.005,p < 0.005, and ***and p*** < p0.001).< 0.001 However,). However, there there was was no noalteration alteration of ofALPALP mRNAmRNA level level at ateither either 5 5or or 10 10 days. days. Interestingly, relative mRNA mRNA level level of of ColCol I Iwaswas slightly slightly decreased decreased at at 10 10 days days compared compared with with that that atat 5 days.5 days. These These results results were were possibly possibly caused caused by by an an incr increaseease of of detached detached cells. cells. Generally, Generally, these these markers are expressed with early- or late-stage osteogenic differentiation as follows. ALP is a key marker that that is responsible for the mineralization of the ECM [[29].29]. Col I I isis the dominant collagen and and exists in many tissues such as tendons and ligaments. ALPALP andand ColCol I I areare commonly expressed expressed in in the early stage of osteogenic differentiation [30] [30] and are considered early differentiation markers. OPN is also an early marker of osteogenic osteogenic differentiation differentiation and and is is part part of of non-collagenous non-collagenous bone bone ECM ECM proteins proteins [31]. [31]. OC is associated with the late stage of osteogenic differentiation and is kn knownown as bone Gla protein (BGP), although OC is also a non-collagenous bone ECM prot proteinein [32]. [32]. As another late differentiation marker, Runx2 is a master switch for the osteogenic differentiationdifferentiation [33]. [33]. In In ou ourr results, results, FNIII10-PA FNIII10-PA significantlysignificantly inducedinduced thethe osteogenicosteogenic differentiation differentiation of of MC3T3-E1 MC3T3-E1 cells cells on on PCL PCL fibers fibers by by upregulation upregulation of ofmost most marker’s marker’s mRNA mRNA levels levels at both at 5both and 105 and days. 10 These days. results These are results similar are to similar our previous to our results previous [14]. resultsTaken together,[14]. Taken we together, reconfirmed we reconfirmed the osteogenic the differentiation osteogenic differentiation activity of FNIII10-PA activity of on FNIII10-PA PCL fibers. on PCL fibers.

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FigureFigure 5.5.The The osteogenic osteogenic differentiation differentiation activity activity of FNIII10-PAof FNIII10-PA on PCLon PCL fibers fibers at 5 daysat 5 days (A) and (A) 10 and days 10 (daysB). PCL (B). fibers PCL werefibers placed were inplaced 24-well in plates 24-well and plates coated and with coated 5 µg· mLwith−1 of5 μ FNIII10g·mL−1 orof FNIII10-PAFNIII10 or overnightFNIII10-PA at overnight 4 ◦C. MC3T3-E1 at 4 °C. cells MC3T3-E1 were cultured cells were at adensity cultured of at 5 ×a 10density4 cells of and 5 incubated× 104 cells for and 5 andincubated 10 days for at 5 and 37 ◦ C.10 days Quantitative at 37 °C. real-timeQuantitative PCR real-time results werePCR results analyzed. were * analyzed.p < 0.05, * ** p p< 0.05,< 0.005, ** p and< 0.005, *** p and< 0.001. *** p Genes:< 0.001. alkalineGenes: alkaline phosphatase phosphatase (ALP), bone (ALP) sialoprotein, bone sialoprotein (BSP), collagen (BSP), collagen type I ( Coltype I), I osteocalcin(Col I), osteocalcin (OC), osteopontin (OC), osteopontin (OPN), and(OPN runt-related), and runt-related transcription transcription factor 2 factor (Runx2 2). (Runx2).

3.3. MaterialsMaterials andand MethodsMethods

3.1.3.1. ProteinProtein ExpressionExpression andand PurificationPurification AfterAfter transformationtransformation intointo TOP10TOP10 EscherichiaEscherichia colicoli,, cellscells werewere growngrown overnightovernight atat 3737 ◦°CC inin Luria-BertaniLuria-Bertani (LB) (LB) medium medium containing containing ampicillin. ampici Inductionllin. Induction was initiated was usinginitiated 0.1% using (w/v) L0.1%-arabinose (w/v) atL-arabinose A600 = 0.6 at followed A600 = by0.6 incubation followed atby 20incubation◦C for 6 h.at Bacteria20 °C for were 6 pelletedh. Bacteria by centrifugationwere pelleted atby 6000centrifugation× g for 10 at min 6000× and g then for 10 lysed min and and sonicated. then lysed A and soluble sonicated. extract wasA soluble prepared extract by centrifugation was prepared for by 30centrifugation min at 14,000 for× g30in min a refrigerated at 14,000× g centrifuge, in a refrigerated and the centrifuge, supernatant and obtained the supernatant was purified obtained using was a nickel-nitrilotriaceticpurified using a nickel-nitrilotriacetic acid resin (Invitrogen, acid resin Carlsbad, (Invitrogen, CA, USA). Carlsbad, CA, USA).

3.2.3.2. ElectrospinningElectrospinning ofof PCLPCL FibersFibers AA PCLPCL (MW(MW == 80,000;80,000; Sigma-Aldrich,Sigma-Aldrich, St.St. Louis,Louis, MO,MO, USA)USA) solutionsolution waswas preparedprepared byby dissolvingdissolving 10%10%w w//v PCL in dichloromethanedichloromethane (DCM)(DCM) andand ethanolethanol (4:1(4:1 ratio).ratio). PCLPCL solutionssolutions werewere ultrasonicatedultrasonicated andand loadedloaded into a a 10 10 mL mL plastic plastic syringe syringe equipped equipped wi withth a 21-gauge a 21-gauge needle needle made made of stainless of stainless steel. steel. The Theneedle needle was was connected connected to a to high-voltage a high-voltage power power suppl supply.y. The The tip-to-collector tip-to-collector distance distance was was kept kept at at10 − 10cm. cm. The The voltage voltage and and injection injection rate rate were were 15 15 kV and 0.50.5 mLmL·h·h −1,, respectively. PCL solutionsolution waswas eletrospuneletrospun toto foil. After After drying, drying, PCL PCL fiber fiber (15 (15 mm mm di diameter)ameter) was was cut cut from from the the foil foil using using a punch. a punch. All Allexperiments experiments were were performed performed at room at room temperature temperature (RT). (RT). In each In each experiment, experiment, PCL PCLfibers fibers coated coated with withFNIII10 FNIII10 or FNIII10-PA or FNIII10-PA were were used. used.

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3.3. Morphology of PCL Fibers and Cells on PCL Fibers The structure of the PCL fibers was observed by SEM at an accelerating voltage of 15 kV after fixing with glutaraldehyde (2.5%), dehydrating with a graded series of ethanol (75%, 90%, 95%, and 100% (v/v) for 10 min each), treating with hexamethyldisilazane, and coating with platinum. To observe the cell spreading and expansion on PCL fibers, PCL fibers were placed in 24-well plates and were coated only once with FNIII10 or FNIII10-PA (5 µg·mL−1) overnight at 4 ◦C. Cells were seeded with 1 × 105 cells, incubated for 30 min, washed with Dulbecco’s phosphate-buffered saline (DPBS), the cells on the PCL fibers were fixed with a 3.7% (w/v) formalin solution for 15 min at RT, dehydrated with a graded series of ethanol (75%, 90%, 95%, and 100% (v/v) for 10 min each), treated with hexamethyldisilazane, and coated with platinum.

3.4. Protein Adhesion Assay To evaluate the protein adhesion activity, PCL fibers were placed in 24-well plates and were coated only once with FNIII10 or FNIII10-PA (0, 1, or 5 µg·mL−1) overnight at 4 ◦C. Each well was washed with phosphate-buffered saline (PBS) and blocked with 1% (w/v) bovine serum albumin (BSA) solution for 1 h at RT. After washing with PBS, a peroxidase conjugate of a monoclonal anti-polyhistidine antibody was added and incubated for 1 h at RT. After washing with tris-buffered saline tween (TBS-T), 200 µL Turbo TMB-enzyme-linked immunoserological assay (ELISA) was added and incubated for 30 min at RT. H2SO4 (100 µL, 2 M) was added to stop the reaction, and the absorbance was read at 450 nm. A protein adhesion assay was performed for the negative control group (un-treated), the positive control group (FNIII10), and the experimental group (FNIII10-PA). The value of the negative control was approximately equal to the value seen with FNIII10 and FNIII10-PA. Hence, results were expressed as the comparison between FNIII10 and FNIII10-PA without negative control.

3.5. Cell Culture MC3T3-E1 is a pre-osteoblastic cell line established from newborn mouse calvarias. We purchased from the American Type Culture Collection (ATCC). Cells were cultured in α-minimum essential media (α-MEM) containing 10% (v/v) heat-inactivated fetal bovine serum (FBS, WELGENE, Daegu, Korea), 100 units·mL−1 penicillin G sodium, 10 µg·mL−1 streptomycin, and 25 µg·mL−1 amphotericin ◦ B (WELGENE, Daegu, Korea) at 37 C in a humidified atmosphere with 5% CO2.

3.6. Cell Adhesion Assay Cell adhesion activity on PCL fibers was measured using the crystal violet assay. PCL fibers were placed in 24-well plates and were coated with FNIII10 or FNIII10-PA (0, 1, or 5 µg·mL−1) overnight at 4 ◦C. Each well was washed with DPBS and blocked with 1% (w/v) BSA solution for 30 min. Cells were cultured with 1 × 105 cells, incubated for 30 min, washed twice with DPBS, and fixed with 3.7% (w/v) formalin solution for 15 min at RT. Cells were then stained with 0.25% (w/v) crystal violet (Sigma, St. Louis, MO, USA) in 2% (v/v) ethanol/water for 1 h at 37 ◦C and gently washed three times with DPBS. Cells were then lysed with 2% sodium dodecyl sulfate (SDS) solution and transferred to 96-well plates. Absorbance was read at 570 nm. Results were expressed as the comparison between FNIII10 and FNIII10-PA.

3.7. Cell Proliferation Assay Cell proliferation activity was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay, according to the manufacturer’s instructions (Promega, Madison, WI, USA). FNIII10 and FNIII10-PA protein solutions were coated at 5 µg·mL−1 on 24-well plates; cells were cultured with 1 × 105 cells and incubated for 0 and 5 days at 37 ◦C. Cells were then washed three times with DPBS, and 500 µL of MTT (5 mg·mL−1 in PBS) was added to each well. After incubation for 4 h, media were removed, and formazan crystals were dissolved in Int. J. Mol. Sci. 2018, 19, 153 8 of 10

200 µL dimethyl sulfoxide (DMSO). Absorbance was read at 540 nm. The cell proliferation assay was performed in the negative control group (un-treated), the positive control group (FNIII10), and the experimental group (FNIII10-PA). After identifying the effect of FNIII10 and FNIII10-PA as compared with that of the negative control, the results were expressed as the comparison between FNIII10 and FNIII10-PA.

3.8. Quantitative Real-Time PCR Analysis Initially, PCL fibers were placed in 24-well plates and were coated with FNIII10 or FNIII10-PA (5 µg·mL−1) overnight at 4 ◦C. Cells were cultured at a density of 5 × 104 cells and incubated for 5 and 10 days at 37 ◦C. Total RNA was extracted using the Easy-spin RNA Extraction kit (iNtRON, Seoul, Korea), and circular (cDNA) was synthesized. The expression levels of the genes ALP, BSP, Col I, OC, OPN, Runx2 were confirmed by quantitative real-time PCR. All real-time PCR analyses were performed using the ABI Step One real-time PCR system. Each reaction was performed in a 20-µL reaction mixture containing 0.1 µM of each primer, 10 µL of 2× SYBR Green PCR master mix (Applied Biosystems, including AmpliTaq Gold DNA polymerase in buffer, a dNTP mix, SYBR Green I dye, Rox dye, and 10 mM MgCl2), and 1 µL of template cDNA. The Ct (cycle threshold) value for each gene was determined using the automated threshold analysis function in the ABI instrument and was normalized with respect to Ct(GAPDH) to obtain dCt (dCt = Ct(GAPDH) − Ct(specific gene)). Finally, the Ct value of FNIII10-PA was normalized by the Ct value of FNIII10. The primers used for quantitative real-time PCR are shown in Table1. Quantitative real-time PCR results were also expressed as a comparison between FNIII10 and FNIII10-PA.

Table 1. Sequences of primers used for quantitative real-time PCR.

Genes Forward Primer Reverse Primer GAPDH TCCACTCACGGCAAATTCAAC AGCCCAAGATGCCCTTCAGT ALP GGGCAATGAGGTCACATCC GTCACAATGCCCACGGACTT BSP CAGAGGAGGCAAGCGTCACT CTGTCTGGGTGCCAACACTG Col I GAGGCATAAAGGGTATCGTGG CATTAGGCGCAGGAAGGTCAGC OC CATCACTGCCACCCAGAAGAC CAGTGGATGCAGGGATGATGT OPN CCAATGAAAGCCATGACCAC CGACTGTAGGGACGATTGGA Runx2 GGCCGGGAATGATGAGAACTA GGCCCACAAATCTCAGATCGT

3.9. Statistical Analysis All experiments were conducted in triplicate. Experimental data are expressed as mean ± standard deviation (SD). The Student’s t-test with paired data sets was used to determine the significances of differences between FNIII10 and FNIII10-PA. Statistical significance was accepted for p values <0.05.

4. Conclusions Previously, we revealed that FNIII10-PA has potential in the osteogenic differentiation activity of rBMSCs. In present study, we also found FNIII10-PA enhanced cell adhesion and osteogenic differentiation activities of MC3T3-E1 cells on PCL fibers. Although the PCL microfiber form had low affinity to cells, the combination with FNIII10-PA significantly enhanced cellular activities including adhesion and differentiation. Taken together, FNIII10-PA potentiates the appropriate utilization for bone tissue engineering with biomaterials as well as working FNIII10-PA alone.

Acknowledgments: This work was supported by the Priority Research Centers Program (grant No. 2009-0093829), by the Korean Healthcare Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (HI14C0522), and by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIP) (NRF-2016R1A2B4008811). Author Contributions: Ye-Rang Yun performed the experiments and wrote the manuscript; Hae-Won Kim and Jun-Hyeog Jang organized this work and helped write the manuscript. Int. J. Mol. Sci. 2018, 19, 153 9 of 10

Conflicts of Interest: The authors have no conflicts of interest to declare.

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