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Polymer 55 (2014) 3836e3845

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Polymer

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Enhanced bone cell functions on poly(ε-caprolactone) triacrylatenetworks grafted with polyhedral oligomeric silsesquioxanenanocages

Lei Cai 1, Camera J. Foster, Xifeng Liu, Shanfeng Wang*

Department of Materials Science and Engineering, The University of Tennessee, Knoxville, TN 37996, USA

a r t i c l e i n f o

Article history:Received 7 May 2014Received in revised form13 June 2014Accepted 18 June 2014Available online 25 June 2014

Keywords:Poly(ε-caprolactone) triacrylatePolyhedral oligomeric silsesquioxaneCell-material interactions

* Corresponding author. Tel.: þ1 865 974 7809; faxE-mail address: swang16@utk.edu (S. Wang).

1 Present address: Department of Materials SciencUniversity, Stanford, CA 94305, USA.

http://dx.doi.org/10.1016/j.polymer.2014.06.0570032-3861/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Poly(ε-caprolactone) triacrylate (PCLTA) developed in our laboratory is a photo-crosslinkable, injectable,and biodegradable polymeric biomaterial for diverse tissue engineering applications. To engineer itsphysical properties for bone regeneration, we incorporated PCLTA networks with a photo-reactivemethacryl isobutyl polyhedral oligomeric silsesquioxane (POSS), which is a silicon-based monomerwith a nano-sized cage. Homogeneous nanohybrid networks were prepared by photo-crosslinking POSSwith two PCLTAs having molecular weights of ~7000 and ~20,000 g/mol at the POSS weight composi-tions (fPOSS) of 0e20%. The lower-molecular-weight PCLTA resulted in amorphous networks while thehigher one resulted in semi-crystalline networks. POSS nanocages tethered in the PCLTA networksgreatly enhanced the mechanical and rheological properties, but did not significantly alter the surfacewettability and the capability of adsorbing serum proteins from cell culture media. Better mouse pre-osteoblastic MC3T3-E1 cell attachment, spreading, and proliferation were found on the stifferPCLTA20k networks than on the PCLTA7k ones, and on the networks with fPOSS of 10e20% than thenetworks containing no POSS. Mineralization of MC3T3-E1 cell cultured for two weeks showed asignificantly higher alkaline phosphatase activity and more mineralized nodules on the PCLTA20k net-works with fPOSS of 10e20%, in correlation with their enhanced mechanical properties. The presentresults indicated that this series of nanohybrid PCLTA/POSS networks with improved mechanical prop-erties and osteoconductivity has great potential as scaffolding materials for bone repair and regeneration.

© 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Successful bone repair and regeneration that warrant surgicalintervention often require suitable scaffold materials to treatdiverse clinical scenarios, for example, large-scale traumatic boneinjuries or fracture non-unions, where the normal physiologic re-actions cannot heal the fractures [1]. Tissue engineering strategiesusing synthetic graft substitutes are promising alternatives to thecurrent gold standard of autograft that needs a second surgery atthe site of the harvest tissue and may cause donor-site morbidity[1]. Polymeric biomaterials that can be crosslinked and hardened insitu have emerged to match the physical and mechanical propertiesof native bone tissues and possess high tunability for diverse

: þ1 865 974 4115.

e and Engineering, Stanford

applications [2]. Among them, photo-crosslinkable, injectable, andbiodegradable materials are particularly useful because they can beinjected into the injury site to fill the bone defect in a minimallyinvasive manner and then solidify with UV light [3e12]. Thiscategory of polymers includes poly(propylene fumarate) (PPF)[3,5,9], poly(ε-caprolactone) fumarate (PCLF) [11e15], poly(ε-cap-rolactone) diacrylate (PCLDA) [7], and poly(ε-caprolactone) tri-acrylate (PCLTA) [7,8,10,16]. Their biocompatibility, degradability,mechanical integrity, wettability, sterilizability, handling, andosteoconductivity have been characterized and optimized [3e12].

PCLTA is a three-arm polyester with each arm bearing a car-bonecarbon double bond end [7,8,10,16]. Compared with PCLF andPCLDA having a linear architecture, PCLTA networks have higher gelfractions because of the branched nature [7]. Using PCLTA net-works, we have fabricated bone scaffolds and nerve conduits forregulating bone and nerve cell behavior on these structures [7,8]. Bycontrolling the crosslinking density and crystallinity of PCLTAnetworks, distinct mechanical properties have been achieved atbody temperature [10]. Surface features such as concentric

L. Cai et al. / Polymer 55 (2014) 3836e3845 3837

microgrooves and honeycomb pores have also been prepared onphoto-crosslinked PCLTA substrates to regulate mouse pre-osteoblastic MC3T3-E1 cell functions [10,16]. To further expandthe range of mechanical properties for PCLTA networks, in thepresent study we utilized a methacryl isobutyl polyhedral oligo-meric silsesquioxane (POSS) that can be covalently grafted as apendent group.

POSS is a silicon-based monomer with a ~1.5 nm-sized cage andhas been blendedwith polymers tomodify their physical properties[17-21]. To avoid aggregation and attain stable molecular-leveldispersion with covalent tethering, copolymerizing POSS intopolymer systems has been employed [3,22e24]. POSS copolymerswith enhanced thermal and mechanical properties while no cyto-toxicity can be used for biomedical applications, for example, POSS-containing methacrylate-based hybrid dental materials can sup-press the volume shrinkage and enhance the biocompatibility whilethe biocompatibility of POSS-containing nanocomposites suggeststhat they may be suitable for potential cardiovascular applications[3,9,17,21,25,26]. As an alternative, direct photo-crosslinking of POSSinto a polymer network is a facile, one-step method to tune thePOSS grafting ratio and optimize properties. Previously we haveused methacryl POSS (mPOSS) with eight double bonds on thenanocage as a crosslinker to control the crosslinking density andexpedite photo-crosslinking of PPF [9]. Further biological evaluationdemonstrated that photo-crosslinked PPF/mPOSS could supportMC3T3-E1 cell attachment, proliferation, and differentiationwithout showing cytotoxicity [9]. We also synthesized PPF-co-POSScopolymers via two-step polycondensation of 1,2-propane diol,diethyl fumarate, and 1,2-propanediol isobutyl POSS to improveboth stiffness and toughness simultaneously for the crosslinkedPPF-co-POSS [3]. The mechanical properties with the non-monotonic dependence on the POSS weight composition (fPOSS)were correlated with MC3T3-E1 cell attachment, spreading, prolif-eration, mineralization, and gene expression, which all maximizedat fPOSS of 10% [3]. In this study, the POSS we chose was methacrylisobutyl POSSwith only one double bond in the structure and thus itcould be grafted as dangling nanocages in the polymer networkswithout significantly changing the crosslinking density.

Understanding cellematerial interactions is critical in tissueengineering. Tailoring material mechanical properties to match thestiffness of the related tissue can regulate cell behavior and tissueregeneration [27e30]. Our approach was to use two PCLTA net-works with distinct material properties. After crosslinking, PCLTAhaving a molecular weight of ~7000 g/mol became amorphous andelastic, while the other one with a higher molecular weight of~20,000 g/mol became semi-crystalline and mechanically tougherand stiffer. We tethered POSS at different fPOSS of 0e20% to opti-mize the material properties of these two types of PCLTA networks,especially to tune themechanical properties with minimal varianceon other surface properties. We further studied MC3T3-E1 cellattachment, spreading, proliferation, and mineralization on thesenetworks, and correlated the cell responses with the materialproperties. The goal of this study was to better interpret the majorfactors that determine the cell behavior and to optimize the PCLTA/POSS nanohybrid network properties towards bone tissue repairand regeneration.

2. Materials and methods

2.1. Photo-crosslinking of PCLTAs and PCLTA/POSS composites

Methacryl isobutyl POSS (denoted as POSS) was purchased fromHybrid Plastics (Hattiesburg, MS). PCLTAs were synthesized usingring-opening polymerization of ε-caprolactone initiated by 1,1,1-tris-(hydroxymethyl) propane, followed by acrylation using

acryloyl chloride in the presence of K2CO3 [10,31]. PCLTA7k andPCLTA20k used in this study have number-average molecularweights (Mn) of 6700 and 20,020 g/mol, and weight-average mo-lecular weights (Mw) of 8500 and 22,700 g/mol, respectively. Allother chemicals were purchased from SigmaeAldrich (Milwaukee,WI) unless noted otherwise. Photo-crosslinking of all the polymersamples was conducted using UV light (Spectroline, SB-100P; in-tensity: 4800 mw/cm2, wavelength: 315e380 nm) in the presenceof a photo-initiator, phenyl bis(2,4,6-trimethyl benzoyl)phosphineoxide (BAPO; IRGACURE 819, Ciba Specialty Chemicals, Tarrytown,NY). Briefly, 4 g mixtures of PCLTA and POSS were combined atfPOSS of 0%, 5%, 10%, and 20% in 1500 mL of CH2Cl2 with 200 mL ofBAPO/CH2Cl2 (600 mg/3 mL). After all PCLTA/POSS was dissolved inBAPO/CH2Cl2 solution, the homogeneous mixture was then trans-ferred intomolds composed of a silicone spacer (1.0 mm, thickness)or a Teflon spacer (0.37 mm, thickness) between two glass plates(2.1 mm, thickness). The mold filled with the mixture was placedunder UV light at a distance of ~7 cm from the lamp. To ensureefficient crosslinking on both sides, each side of the mold wasexposed to UV light for 10 min. Crosslinked polymer disks or sheetswere removed from the mold after being cooled to room temper-ature. Except for gel fraction and swelling ratio measurements, thecrosslinked polymer samples were soaked in acetone for two daysand then placed in vacuum for complete drying prior to physicalcharacterizations and cell studies.

2.2. Characterizations of the bulk properties of photo-crosslinkedPCLTAs and PCLTA/POSS composites

The gel fractions and swelling ratios of photo-crosslinked PCLTAsand PCLTA/POSS composites were determined by placing twopolymer disks in a glass vial with excess CH2Cl2. After one day,CH2Cl2 in each vialwas removed, and the vial containing the swollendiskwasweighed. The vial and diskswere thenplaced in vacuum forcomplete drying andweighed again. By documenting theweights ofthe original (W0), dry (Wd), and fully swollen (Ws) crosslinkedpolymer disks, their gel fractions (G) and swelling ratios (S) werecalculated using the equations of G ¼ Wd/W0 � 100% andS ¼ (Ws � Wd)/Wd � 100%, respectively. Differential ScanningCalorimetric (DSC) measurements were performed on a PerkinElmer Diamond differential scanning calorimeter in nitrogen. Thesame thermal history was applied to all the samples by first heatingup to 100 �C and cooling to �80 �C at 5 �C/min. Then a subsequentheating run was performed from �80 to 100 �C at 10 �C/min.Thermogravimetric Analysis (TGA) was performed on a TA Q50thermal analyst in nitrogen at a heating rate of 20 �C/min. Tensilemoduli (E) and compressive moduli of the crosslinked polymersamples were determined using dynamic mechanical analysis(DMTA-5, Rheometric Scientific) at 37 �C. Polymer strips(~30 mm � ~1.5 mm � ~0.3 mm, length � width � thickness) wereelongated and polymer disks (~2.5 mm � ~1.0 mm,diameter� thickness) were compressed at a strain rate of 0.001 s�1.About five specimens for each sampleweremeasured and averaged.Rheological measurements were conducted with a small strain(g ¼ 1%) using parallel plates with a diameter of 8 mm and a gap of~0.5 mm (disk thickness). Shear or storage modulus (G'), lossmodulus (G00), and viscosity h as functions of frequency weremeasured for crosslinked PCLTA/POSS disks using a strain-controlled rheometer (RDS-2, Rheometric Scientific) at 37 and 60 �C.

2.3. Surface characterization of photo-crosslinked PCLTAs andPCLTA/POSS composites

Surface morphologies of photo-crosslinked PCLTA and PCLTA/POSS composite disks were characterized using multi-mode atomic

Scheme 1. Photo-crosslinking of PCLTA/POSS. After photo-crosslinking, PCLTA7k/POSSbecomes an amorphous and compliant network, while PCLTA20k/POSS becomes asemi-crystalline and tough network.

L. Cai et al. / Polymer 55 (2014) 3836e38453838

force microscopy (AFM) with a Nanoscope V control system (VeecoInstruments, Santa Barbara, CA). AFM images (20 mm � 20 mm)were obtained using a tapping mode at room temperature and ascan rate of 0.5 Hz. With a standard silicon tapping tip on a beamcantilever, the surface topography was recorded simultaneouslyand the root mean square (rms) roughness (Rrms) was calculatedfrom height image profiles over three areas of 10 mm � 10 mm.

Frictional forces and coefficients between a stainless steel plateand photo-crosslinked PCLTA and PCLTA/POSS disks weremeasured and calculated using the same rheometer and themethod reported previously [5]. A Ram�e-Hart NRC C. A. goniometer(model 100-00-230) was used to measure the water contact angleon the polymer disks. Approximately 1 mL of distilled water(pH¼ 7.0) was injected onto the disk surface and the readings weretaken after an equilibrium time of 30 s. For each sample, threepolymer disks were used and six data points were taken forcalculating the average and standard deviation. For proteinadsorption measurements, crosslinked PCLTA and PCLTA/POSScomposite disks were submerged inMC3T3-E1 cell culturemedium(see Section 2.4) in a 24-well plate for 1 h at 37 �C. Then the diskswere washed three times using deionized water. For each washthere was 5-min gentle shaking and then the DI water wasdisposed. Two hundred 40 mL (240 mL) of 1% sodium dodecyl sulfate(SDS) solutionwas added into each well at room temperature. After30 min, the SDS was collected and this process was repeated threetimes. All the concentrations of the proteins in the SDS weredetermined on a micro-plate reader (SpectraMax Plus 384, Mo-lecular Devices, Sunnyvale, CA) using a MicroBCA protein assay kit(Pierce, Rockford, IL). A standard curve was produced by using al-bumin in SDS with eight known concentrations.

2.4. In vitro cell viability, attachment, and proliferation

Mouse MC3T3-E1 pre-osteoblastic cells (CRL-2593, ATCC,Manassas, VA) were cultured in vitro in a-Minimum Essential Me-dium (Gibco, Grand Island, NY), supplemented with 10% FBS and 1%penicillin/streptomycin (Gibco). After plating, cell suspension wasincubated with 5% CO2 and 95% relative humidity at 37 �C. Cellculture medium was changed every two days and cells were splitupon 80% confluency. Polymer disks were sterilized in 70% alcoholsolution and then dried completely in vacuum before they wereattached on the bottom of a 48-well plate using autoclaved inertsilicon-based high-temperature vacuum grease (Dow Corning,Midland, MI). MC3T3-E1 cells were seeded on the polymer disks ata density of ~15,000 cell/cm2 for evaluating cell attachment andproliferation. After culturing cells in a humidified atmosphere (5%CO2 at 37 �C) for 1, 2, and 4 days, the culture media were removedfrom the wells. The number of viable cells was determined usingthe MTS assay, a colorimetric cell metabolic assay (CellTiter 96Aqueous One Solution; Promega, Madison, WI) based on the MTStetrazolium compound, which correlated the number of viable cellsto the UV absorbance at 490 nm measured on the micro-platereader. The attached cells were also fixed in 4% paraformaldehyde(PFA) for 10 min, washed twice with PBS, and permeabilised with0.2% Triton X-100. The cells were stained using rhodamine-phalloidin for 1 h at 37 �C and DAPI at room temperature for im-aging using an Axiovert 25 light microscope (Carl Zeiss, Germany).Cell area was examined and averaged on 20 non-overlapping cellsat day 1 post-seeding using ImageJ software (National Institutes ofHealth, Bethesda, MD).

2.5. In vitro cell mineralization

MC3T3-E1 cells cultured on the polymer disks for 14 days werefixed in 4% PFA, permeabilized with 0.2% Triton X-100, and stained

with 1% (w/v) Alizarin red S aqueous solution (Ricca Chemical,Arlington, Texas) for 10 min. Phase-contrast photographs wereobtained for Alizarin-red-stained calcium deposits in mineralizedcells, as represented by positive red staining. The alkaline phos-phatase (ALP) activity and calcium content of MC3T3-E1 cells weremeasured using cell lysates. The cells were washed twice with PBS,trypsinized and washed again using a centrifuge at 1000 rpm for4 min. The residues were re-suspended in 1 mL of 0.2% Nonidet P-40 and sonicated in an ice bath for 2 min. The cell lysate was frozenat �20 �C prior to the measurements. The ALP activity of the celllysatewas determined using a fluorescence-based ALP detection kit(Sigma, St. Louis, MO) and a standard curve was constructed usingdifferent amounts of control enzyme [9]. Calcium content wasdetermined using QuantiChrom calcium assay kit (BioAssay Sys-tems, Hayward, CA) [9].

2.6. Statistical analysis

All experiments were conducted in triplicate except for themechanical testing where n ¼ 5 and contact angle measurementswhere n ¼ 6. The data were expressed as mean ± standard devia-tion. One-way analysis of variance (ANOVA) with Tukey post-hoctest was performed to assess the statistical significance (p < 0.05)of the differences between results.

3. Results and discussion

3.1. Photo-crosslinking and bulk properties

Photo-crosslinking of PCLTA/POSS was efficient to covalentlytether POSS nanocages onto the PCLTA networks (Scheme 1), asevidenced by their gel fractions of higher than 80%. Because thehigher molecular weight between double bonds in PCLTA20kresulted in a lower crosslinking density, the swelling ratio in CH2Cl2increased significantly from 3.8 ± 0.1 for crosslinked PCLTA7k to17.9 ± 0.9 for crosslinked PCLTA20k. By tethering 20% POSS to thePCLTA network, the swelling ratio increased to 4.4 ± 0.1 for cross-linked PCLTA7k/POSS (fPOSS¼ 20%) and to 23.0± 0.5 for crosslinkedPCLTA20k/POSS (fPOSS ¼ 20%), suggesting that the crosslinkingdensity was lower with more POSS in the networks because thedouble bond in POSS polymerized by itself and covalent bondingformation of POSS occupied certain volume in the polymer net-works [3].

Thermal properties of the PCLTA/POSS networks are crucial inunderstanding the effect of PCL crystallites on their mechanical

L. Cai et al. / Polymer 55 (2014) 3836e3845 3839

properties at body temperature. The DSC curves in Fig. 1a,b showedthe thermal properties of PCLTAs and PCLTA/POSS compositesbefore and after crosslinking. The pure POSS had a small pre-melting transition peak at around �20 �C and a melting peak at116 �C, indicating that the POSS cage was thermally stable betweenroom temperature and 60 �C, the range of our interest. Both peaksof POSS disappeared after crosslinking because of separation andsuppression of crosslinks at compositions of POSS up to 20%, similarto our earlier report on crosslinked PPF-co-POSS [3]. All PCLTA7k/POSS and PCLTA20k/POSS composites were semi-crystalline beforecrosslinking and showed biomodal melting peaks because of thethree-arm architecture in PCLTA, in agreement with previous re-ports [7,10,14,32]. Crystallinity cc was roughly calculated using theequation of cc ¼ DHm/[(1 � fPOSS)DHm

c ], where DHmwas the heat offusion determined from the weight-normalized integration of themelting peak(s) in the DSC curves and DHm

c of completely crystal-line PCL is 135 J/g [33]. Before crosslinking, DHm decreased withincreasing fPOSS but cc did not vary much (0.54e0.56 for PCLTA7k/

a

c

e f

d

b

Fig. 1. Thermal properties. DSC curves of (a) uncrosslinked and (b) crosslinked PCLTA/POSScrosslinked PCLTA7k/POSS, and (f) crosslinked PCLTA20k/POSS.

POSS and 0.57e0.58 for PCLTA20k/POSS). After crosslinking, onlymonomodal melting peaks existed for PCL segments because thecrystalline domains were homogenized in the networks. Cross-linked PCLTA7k/POSS composites became completely amorphousat room or body temperature crystalline peaks at ~17 �C weregreatly suppressed by the networks and cc also decreased to 0.11,0.08, 0.07, and 0.05 for fPOSS of 0%, 5%, 10%, and 20%, respectively. Incontrast, crosslinked PCLTA20k/POSS composites remained semi-crystalline with Tm at 52 �C, much higher than the body tempera-ture, and high cc values of 0.47, 0.47, 0.45, 0.42 for fPOSS ¼ 0%, 5%,10%, and 20%, respectively.

Thermal stabilities of PCLTAs and PCLTA/POSS composites wereevaluated using the TGA thermograms in Fig.1cef. POSS exhibited atwo-step weight loss process with onset thermal degradationtemperatures (Td) of 261 and 368 �C, possibly attributed to subli-mation of POSS macromers [34] and oxidation of SieCH2e groups[35], respectively. It should be noted that all the samples werestable in the temperature range for DSC measurements and thus

. TGA curves of (c) uncrosslinked PCLTA7k/POSS, (d) uncrosslinked PCLTA20k/POSS, (e)

L. Cai et al. / Polymer 55 (2014) 3836e38453840

the vinyl group was stable during the measurements. For uncros-slinked PCLTA/POSS blends, fPOSS was indicated by the weightremained at 350 �C above the Td for POSS, which decreased from96% for PCLTA7k or PCLTA20k to 84% for PCLTA7k/POSS orPCLTA20k/POSS with fPOSS of 20% (Fig. 1c,d). Nearly completedecomposition for POSS and PCLTAs was observed with minimalresidue (<2%) above 500 �C. After crosslinking, thermal stabilities ofall the composites were similar to those of crosslinked PCLTAs witha decrease in Td from 395 �C for crosslinked PCLTA7k to 380 �C forPCLTA7k/POSS (fPOSS ¼ 20%), and from 401 �C for crosslinkedPCLTA20k to 381 �C for PCLTA20k/POSS (fPOSS ¼ 20%), respectively(Fig. 1e,f). It indicated good integration of POSS nanocages in thePCLTA networks, consistent with the behavior of crosslinked PPF-co-POSS copolymers reported by us previously [3].

After obtaining the thermal properties of the polymer networks,we chose two temperatures, 25 and 60 �C, to demonstrate the effectof crystallites on composite disk morphologies. As shown in theoptical images in Fig. 2, crosslinked PCLTA7k/POSS disks were allamorphous and transparent at both temperatures because of thesuppression of PCL crystalline domains by the networks. Incontrast, crosslinked PCLTA20k/POSS remained semi-crystallineand opaque at 25 �C. At 60 �C, all disks became transparent, indi-cating that crystalline regions of PCLTA20k were largely melted.The presence of POSS aggregates was also observed as the cross-linked disks turned slightly translucent at higher fPOSS of 10% and20%.

Mechanical properties were obtained from both tensile andcompression tests. Representative tensile stressestrain curves ofthe crosslinked samples are shown in Fig. 3a, fromwhich the tensilemoduli and strains at break were achieved (Fig. 3b,c). Semi-crystalline crosslinked PCLTA20k/POSS series were stiffer andtougher than amorphous crosslinked PCLTA7k/POSS series. Asshown in Fig. 3c, for crosslinked PCLTA7k/POSS series, the strain atbreak continuously increased with increasing fPOSS up to 20%. Incontrast, crosslinked PCLTA20K/POSS demonstrated a maximumstrain at break of 328 ± 29 at fPOSS of 5%. The tensile andcompressive moduli (Fig. 3b,d) at body temperature were signifi-cantly enhanced by incorporating POSS and gradually increasedwith increasing fPOSS. Compared with the values of crosslinkedPCLTA7k, the tensile and compressive moduli of crosslinkedPCLTA7k/POSS (fPOSS¼ 10%) were 48% and 78% higher, respectively.Similarly, the tensile and compressive moduli of crosslinkedPCLTA20k/POSS (fPOSS ¼ 10%) were enhanced by 30% and 62%compared with those of crosslinked PCLTA20k. As fPOSS increasedto 20%, the stiffening effect from POSS became less prominentbecause of the decrease in the crosslinking density. At the samefPOSS, crosslinked PCLTA20k/POSS composites were much stifferand stronger than crosslinked PCLTA7k/POSS composites because

Fig. 2. Optical images of crosslinked PCLTA7k/POS

of the PCL crystallites. These results confirmed the enhancement innetwork mechanical properties from tethered POSS nanocages andthis effect could be critical in tuning cell responses (Section 3.3).

The rheological results in Fig. 4aed further characterized thedifferent properties of the elastic PCLTA7k/POSS and semi-crystalline PCLTA20k/POSS networks. Rubbery behavior wasfound for all the samples in the frequency range of 0.1e100 rad/s, asG' was independent of frequency. G0 remained the same for cross-linked PCLTA7k/POSS composites at the two temperatures, i.e., 37and 60 �C. The G' values of crosslinked PCLTA20k/POSS compositeswith enhancement from PCL crystallites were much higher thanthose of crosslinked PCLTA7k/POSS composites at 37 �C (Fig. 4a,b).When this effect was removed at 60 �C, which was higher than themelting temperature, the G' values of crosslinked PCLTA20k/POSScomposites were much smaller than those of PCLTA7k/POSS com-posites because of the lower crosslinking density (Fig. 4c,d). HigherG0 values were also found for crosslinked PCLTA/POSS disks withhigher fPOSS up to 10%, consistent with the trends in the tensile andcompressive moduli in Fig. 3b,d. At 37 �C, G0 increased by 25% fromcrosslinked PCLTA7k to crosslinked PCLTA7k/POSS (fPOSS ¼ 10%)and by 56% from crosslinked PCLTA20k to PCLTA20k/POSS(fPOSS ¼ 10%), similar to the enhancing effect of tethering POSS onthe mechanical properties.

3.2. Surface characteristics

Surface characteristics such as roughness, hydrophilicity, andthe ability to absorb serum proteins are crucial for cellematerialinteractions and osteoconductivity [36]. AFM images and the cor-responding Rrms values in Fig. 5a,b demonstrated distinct surfacemorphologies for crosslinked PCLTA/POSS disks. CrosslinkedPCLTA7k had a very smooth surface owing to its amorphous andelastic nature. Tethering POSS onto the PCLTA7k network rough-ened the surfaces from the presence of POSS nanocages on thesurface, although no composition dependence was found. Cross-linked PCLTA20k had a much rougher surface because of formationof crystalline domains, which was so dominant that the effect ofPOSS was not demonstrated. Frictional coefficient was higher forcrosslinked PCLTA7k/POSS (fPOSS ¼ 5%) because of the roughersurface, while more POSS lubricated the surface with the sameroughness, as shown in Fig. 5c. The lubricating effect of POSS wasclearly shown with a compositional dependence for crosslinkedPCLTA20k/POSS with similar roughness, where grafting POSSgradually decreased the surface friction of the network. This surfacelubricating effect of POSS was also reported in other POSS/polymercomposites such as POSS/polypropylene [37].

The water contact angle slightly increased for crosslinkedPCLTA7k/POSS with increasing fPOSS because POSS was more

S and PCLTA20k/POSS disks at 25 and 60 �C.

a b

dc

Fig. 3. Mechanical properties of crosslinked PCLTA/POSS at 37 �C. (a) Tensile stressestrain curves (Inset: enlarged area for crosslinked PCLTA7k/POSS), (b) Tensile moduli, (c) Strainsat break in tensile measurements, and (d) Compressive moduli. p < 0.05 between PCLTA7k/POSS and PCLTA20k/POSS. p < 0.05 between samples with different markers (includingthe group without a marker) in crosslinked PCLTA7k/POSS or PCLTA20k/POSS series.

a b

c d

Fig. 4. G0 vs. frequency plots of (a,c) crosslinked PCLTA7k/POSS and (b,d) crosslinked PCLTA20k/POSS at (a,b) 37 �C and (c,d) 60 �C.

L. Cai et al. / Polymer 55 (2014) 3836e3845 3841

Fig. 5. Surface properties. (a) AFM images, (b) Rrms, (c) friction coefficient, (d) water contact angle, and (e) protein adsorption of crosslinked PCLTA7k/POSS and PCLTA20k/POSSdisks.

L. Cai et al. / Polymer 55 (2014) 3836e38453842

hydrophobic than PCLTA7k, also indicating that POSS nanocagesappeared on the surface (Fig. 5d). Larger water contact angles withlittle dependence on fPOSS were found for more hydrophobiccrosslinked PCLTA20k/POSS, in agreement with the previousfinding that the surface wettability of crosslinked PPF-co-POSS wasnot influenced by POSS [3]. The capability of adsorbing serumproteins from the culture media might be influenced collectively bysurface hydrophilicity, surface roughness, and the POSS content. Asindicated in Fig. 5e, crosslinked PCLTA7k/POSS with better wetta-bility adsorbed much more proteins than crosslinked PCLTA20k/POSS but the values were almost the same after adding differentamounts of POSS, possibly because of the similar surfacewettabilityat different fPOSS.

3.3. In vitro MC3T3-E1 cell studies

To examine cellematerial interactions and explore the potentialof crosslinked PCLTA/POSS composites as scaffold materials to

promote bone regeneration, we investigated MC3T3-E1 cellattachment, spreading, proliferation, and differentiation on thepolymer disks. The fluorescence images of MC3T3-E1 cells withactin filaments stained in Fig. 6a showed spread-out phenotype onall the disks at days 1 and 4 post-seeding. MC3T3-E1 cell attach-ment values were obtained by normalizing the cell numbers on thepolymer disks to the one on tissue culture polystyrene. As shown inFig. 6b, the values on crosslinked PCLTA20k/POSS composites weresignificantly higher than those on crosslinked PCLTA7k/POSS at allfPOSS. No compositional dependence on cell attachment was foundon crosslinked PCLTA7k/POSS; however, cell spread area at day 1increased from 1710 ± 300 mm2 on crosslinked PCLTA7k to2140 ± 340 mm2 on crosslinked PCLTA7k/POSS (fPOSS ¼ 10%). Moreattached cells were found on crosslinked PCLTA20k/POSS(fPOSS ¼ 10% or 20%) with larger spread areas (2580 ± 210 or2550 ± 230 mm2) than the value (2310 ± 130 mm2) on crosslinkedPCLTA20k. As demonstrated in Fig. 6c, MC3T3-E1 cell proliferationover 4 days was also enhanced by tethering POSS in the PCLTA

Fig. 6. MC3T3-E1 cell attachment and proliferation. (a) Fluorescence images of the cells stained with rhodamine-phalloidin (red) and DAPI (blue) on crosslinked PCLTA/POSS disksat days 1 and 4 post-seeding. The scale bar of 200 mm is applicable to all the images. (b) Normalized cell attachment at 4 h and (c) cell numbers at days 1, 2, and 4. þ, p < 0.05 relativeto crosslinked PCLTA7k at each time point. *, p < 0.05 relative to crosslinked PCLTA7k/POSS and crosslinked PCLTA20k at each time point. (For interpretation of the references tocolor in this figure legend, the reader is referred to the web version of this article.)

L. Cai et al. / Polymer 55 (2014) 3836e3845 3843

networks. Consistent with the cell images, the cell numbers at day 4were significantly higher on the crosslinked PCLTA/POSS compos-ites compared with their corresponding PCLTA networks. Theproliferation rate, as indicated by the proliferation index (cellnumber at day 4 divided by the attached cell number at day 1),increased from 1.73 ± 0.15 on crosslinked PCLTA7k to 2.30 ± 0.06 oncrosslinked PCLTA7k/POSS (fPOSS ¼ 10%), and from 3.39 ± 0.12 oncrosslinked PCLTA20k to 3.61 ± 0.11 on crosslinked PCLTA20k/POSS(fPOSS ¼ 10%).

To further characterize the long-term effect of grafted POSSnanocages on MC3T3-E1 cell functions, we quantified cell miner-alization at day 14 on crosslinked PCLTA/POSS composite disksusing Alizarin red staining, ALP activity, and calcium content, asshown in Fig. 7. Interestingly, Alizarin-red-stained cell images inFig. 7a displayed much larger and darker mineral nodules on thestiffer PCLTA20k network and on nanohybrid networks at higherfPOSS of 10% and 20%, indicating that cell mineralization wassignificantly enhanced on the PCLTA networks tethered with POSSnanocages. Both ALP activity and calcium content in Fig. 7b,c, twowell-defined early osteoblastic differentiation markers, showed a

clear trend similar to that in the cell proliferation result and thatobserved from optical images of Alizarin red stains. CrosslinkedPCLTA20k consistently demonstrated significantly higher valuesthan crosslinked PCLTA7k. These two values were also significantlyupregulated in the cells cultured on PCLTA networks with fPOSS of10% and 20%, compared with those on the PCLTA networks withoutPOSS.

3.4. Discussion

Interpreting cell responses to the materials properties isimportant for optimization of material design strategies towardsachieving better cell functions and tissue regeneration. Amongnumerous material factors that influence cell behavior, the me-chanical properties or surface stiffness of the underlying polymersubstrate for supporting cells have been demonstrated to be closelycorrelated with cell functions [27e30,38]. Surface stiffness can beprobed by cells as they attach and pull on their underlying surfaces.Meanwhile, the myosin-based contractility and adhesion proteinssuch as integrins, cadherins, and focal adhesions transmit forces to

Fig. 7. Mineralization of MC3T3-E1 cells cultured for 14 days on crosslinked PCLTA/POSS disks. (a) Alizarin red stains, (b) ALP activities, and (c) calcium contents. The scale bar of200 mm is applicable to all the images. þ, p < 0.05 relative to crosslinked PCLTA7k. *, p < 0.05 relative to crosslinked PCLTA7k/POSS and crosslinked PCLTA20k.

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the substrate and receive feedback that regulates cell functionssuch as spreading, proliferation, and differentiation [27]. Moreover,actin-myosin associations also influence Ca2þ influx across the cellmembrane to trigger the cascades of mechanotransductivesignaling pathways such as mitogen-activated protein kinase(MAPK) [38]. MC3T3-E1 cells have been found to sense substratestiffness and favor relatively stiffer substrates of either hydrogels orhydrophobic surfaces with E from ~1 kPa to ~1 GPa[3,4,6,7,9,10,37,39]. For example, MC3T3-E1 cells cultured on poly-ethylene glycol-based hydrogels with tunable mechanical proper-ties showed better proliferation and differentiation on stiffersubstrates (E¼ ~420 kPa) than on softer substrates (E¼ ~14 kPa) byupregulating the MAPK pathway [37]. The difference in cellbehavior could be removed by inhibiting MAPK phosphorylation[38]. In our previous report, the elastic modulus of crosslinked PPF-co-POSS in the range of 700e1220 MPa was positively correlatedwith MC3T3-E1 cell functions and gene expression levels of celldifferentiation markers [3], consistent with the present findings.

In our approach, we employed two distinct types of PCLTAnetworks and covalently tethered POSS in one-single step to tunethe material properties over a wide range. This facile methodallowed to directly crosslink POSS nanocages in the network andenabled quick preparation of a series of nanohybrid networks withvaried fPOSS, without the need to synthesize each copolymerthrough multiple synthesis and purification steps. Only the me-chanical properties of the samples including tensile, compressive,and rheological properties showed strong dependences on fPOSS,while surface topography and wettability displayed little variance,allowing for studying the mechanosensing characteristics ofMC3T3-E1 cells. POSS nanocages were found to significantlystrengthen themechanical properties of both pure elastic networksof PCLTA7k and semi-crystalline PCLTA20k networks. Therefore, the

mechanical cue of PCLTA/POSS networks at fPOSS of 10% and 20%was the major factor to significantly improve MC3T3-E1 functions.

Besides exploring bone cell responses to the substrate stiffness,we supply two series of injectable and photo-crosslinkable PCLTA/POSS that have great potentials for bone tissue engineering becausethe resulted polymer networks have tunable mechanical propertiesin the range of trabecular bone (E ¼ 50e500 MPa) and promotedosteoconductivities [2]. This simple and successful method ofcovalently tethering POSS nanocages in the polymer networks canalso be applied to other crosslinkable polymer systems and totether other nanostructures or bioactive molecules with reactiveend groups to polymer networks. Further exploration of thesenanohybrid networks is needed, such as mechanical/tribologicalmapping on the material surfaces to determine if POSS nanocagescauses heterogeneity with local stiffening/lubrication that mayinfluence cell responses. Using the polymer systems presentedhere, various 3D structures such as porous scaffolds and micro-grooved patterns can be readily fabricated via stereolithographicmethods to study in vitro cell behavior and in vivo tissue growth inthe polymer structures [39].

4. Conclusions

We have developed two series of photo-crosslinkable polymercomposite systems by covalently tethering POSS nanocages intotwo distinct PCLTA networks at fPOSS of 0e20% for potential bonetissue engineering applications. To better understand cellematerialinteractions and material factors that promote bone regeneration,amorphous crosslinked PCLTA7k/POSS and semi-crystalline cross-linked PCLTA20k/POSS composites, as well as their correspondingPCLTA networks, have been extensively characterized in terms ofbulk characteristics such as thermal, mechanical, and rheological

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properties, and surface characteristics such as roughness, hydro-philicity, frictional coefficient, and capability of adsorbing serumproteins. Among these factors, material properties have been foundto be significantly enhanced by the crystalline domains of PCL andthe presence of tethered POSS nanocages, while other propertiesvary little at different compositions. Stiffer PCLTA20k networkshave been found to better support pre-osteoblastic MC3T3-E1 cellfunctions, including attachment, spreading, proliferation, andmineralization, than softer PCLTA7k networks. Moreover, tetheringPOSS in the network at fPOSS of 10e20% also significantly promotesthese bone cell functions than the PCLTA networks containing noPOSS, in correspondence with the enhancement in the mechanicalproperties. These two series of nanohybrid PCLTA/POSS networkswith varied mechanical properties serve as not only a good plat-form to study stiffness-dependent cell behavior, but also a simpleapproach to promote the osteoconductivity of PCLTA networks forfostering bone repair and regeneration.

Acknowledgments

This workwas supported by National Science Foundation (DMR-11-06142). CJF received partial support from the Center for Mate-rials Processing for her towork as a summer undergraduate studenton this project.

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